RAVER1 hinders lethal EMT and modulates miR/RISC activity by the control of alternative splicing.

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.

[Automatic hearing screening for better monitoring of hearing health using the example of the German armed forces]. / Automatisiertes Hörscreening zur verbesserten Überwachung der Hörgesundheit am Beispiel der Bundeswehr.

In the present study, the concept of a systematic automated screening of temporary soldiers was evaluated based on the example of the ENT Department of the Bundeswehr Central Hospital Koblenz. From 2014 to 2017, anonymized data of 169 individuals were collected from the setting of the Bundeswehr Central Hospital. Included in the data are results from measurements with automated pure-tone audiometry (APTA; e.g., [3]), from measurements with the digit triple test for determination of the speech discrimination threshold in noise (e.g., [20]), and from interviews with questionnaires (Hearing-Dependent Daily Activities [HDDA], e.g., [14]; HearCom questionnaire, e.g., [15]). There was an initial publication from this project evaluating the questionnaires in terms of their suitability for detecting hearing loss [14]. In the following (from March 2015), only the HDDA, which was described as more sensitive, was used for measurements at the hearing screening measurement station. A complete run with the three procedures took approximately 22 min. Approximately 17% of the examined participants had abnormal findings in at least one of the procedures at the screening station. The results of the respective methods taken together detect more than any method alone and can be assumed to be complementary. Deviations between APTA with level monitor and manual tone audiometry were within the measurement accuracy. In the range between 1 and 4 kHz, hearing thresholds are somewhat underestimated with APTA. The threshold for the HDDA questionnaire with an HDDA sum ≥ 19 was confirmed. Automated hearing screening offers a good opportunity to check hearing ability on a regular basis in a standardized and reliable manner, while keeping personnel requirements low.

[Hearing test results of 18- to 20-year old men preceding military conscription from 2008-2010]. / Hörtestergebnisse bei 18- bis 20-jährigen Männern aus Kreiswehrersatzämtern von 2008­2010.

OBJECTIVE: High-frequency hearing loss is known to increase with age, whereas the amount and rate of increase of hearing loss in younger people is still unclear. A large proportion of young age groups is expected to preserve normal hearing. Therefore, the requirements for screening methods are particularly high and the motivation to participate is low. The obligatory examinations preceding military conscription include a pure-tone audiogram and thus allow the analysis of the hearing status of young male adults. MATERIAL AND METHODS: The prevalence of hearing impairment was determined using air-conduction thresholds of 18- to 20-year old men, from 54 German registration offices (KWEA), measured in 2008 to 2010. The criterion was based on candidates exceeding a threshold of 20 dB HL for one of the frequencies 3, 4, or 6 kHz in at least one ear. This very strict criterion was compared to other definitions of hearing impairment. RESULTS: The prevalence of hearing impairment was, on average, 15.3 % and thus in the same range as in other studies. However, the results of single KWEA differed by up to a factor of 10. CONCLUSIONS: Due to high fluctuations in measurement quality in the KWEA, regional differences in hearing thresholds of young men are not resolvable and it remains unclear whether the hearing loss has increased in comparison to earlier studies. The high variability of measurements near thresholds requires permanent quality inspections. However, hearing thresholds derived from screening cannot be evaluated applying the same criteria as for hearing tests in audiological centers or studies.

The Dark Side of the Epitranscriptome: Chemical Modifications in Long Non-Coding RNAs.

The broad application of next-generation sequencing technologies in conjunction with improved bioinformatics has helped to illuminate the complexity of the transcriptome, both in terms of quantity and variety. In humans, 70-90% of the genome is transcribed, but only ~2% carries the blueprint for proteins. Hence, there is a huge class of non-translated transcripts, called long non-coding RNAs (lncRNAs), which have received much attention in the past decade. Several studies have shown that lncRNAs are involved in a plethora of cellular signaling pathways and actively regulate gene expression via a broad selection of molecular mechanisms. Only recently, sequencing-based, transcriptome-wide studies have characterized different types of post-transcriptional chemical modifications of RNAs. These modifications have been shown to affect the fate of RNA and further expand the variety of the transcriptome. However, our understanding of their biological function, especially in the context of lncRNAs, is still in its infancy. In this review, we will focus on three epitranscriptomic marks, namely pseudouridine (Ψ), N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We will introduce writers, readers, and erasers of these modifications, and we will present methods for their detection. Finally, we will provide insights into the distribution and function of these chemical modifications in selected, cancer-related lncRNAs.

NANOS1 restricts oral cancer cell motility and TGF-ß signaling.

Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer of the head and neck area accounting for approx. 377,000 new cancer cases every year. The epithelial-to-mesenchymal transition (EMT) program plays an important role in OSCC progression and metastasis therefore contributing to a poor prognosis in patients with advanced disease. Transforming growth factor beta (TGF-ß) is a powerful inducer of EMT thereby increasing cancer cell aggressiveness. Here, we aimed at identifying RNA-binding proteins (RBPs) that affect TGF-ß-induced EMT. To this end we treated oral cancer cells with TGF-ß and identified a total of 643 significantly deregulated protein-coding genes in response to TGF-ß. Of note, 19 genes encoded RBPs with NANOS1 being the most downregulated RBP. Subsequent cellular studies demonstrated a strong inhibitory effect of NANOS1 on migration and invasion of SAS oral cancer cells. Further mechanistic studies revealed an interaction of NANOS1 with the TGF-ß receptor 1 (TGFBR1) mRNA, leading to increased decay of this transcript and a reduced TGFBR1 protein expression, thereby preventing downstream TGF-ß/SMAD signaling. In summary, we identified NANOS1 as negative regulator of TGF-ß signaling in oral cancer cells.

Automatic intra-subject registration and fusion of multimodal cochlea 3D clinical images.

BACKGROUND: The postoperative imaging assessment of Cochlear Implant (CI) patients is imperative. The main obstacle is that Magnetic Resonance imaging (MR) is contraindicated or hindered by significant artefacts in most cases with CIs. This study describes an automatic cochlear image registration and fusion method that aims to help radiologists and surgeons to process pre-and postoperative 3D multimodal imaging studies in cochlear implant (CI) patients. METHODS AND FINDINGS: We propose a new registration method, Automatic Cochlea Image Registration (ACIR-v3), which uses a stochastic quasi-Newton optimiser with a mutual information metric to find 3D rigid transform parameters for registration of preoperative and postoperative CI imaging. The method was tested against a clinical cochlear imaging dataset that contains 131 multimodal 3D imaging studies of 41 CI patients with preoperative and postoperative images. The preoperative images were MR, Multidetector Computed Tomography (MDCT) or Cone Beam Computed Tomography (CBCT) while the postoperative were CBCT. The average root mean squared error of ACIR-v3 method was 0.41 mm with a standard deviation of 0.39 mm. The results were evaluated quantitatively using the mean squared error of two 3D landmarks located manually by two neuroradiology experts in each image and compared to other previously known registration methods, e.g. Fast Preconditioner Stochastic Gradient Descent, in terms of accuracy and speed. CONCLUSIONS: Our method, ACIR-v3, produces high resolution images in the postoperative stage and allows for visualisation of the accurate anatomical details of the MRI with the absence of significant metallic artefacts. The method is implemented as an open-source plugin for 3D Slicer tool.

Characterization of Escherichia coli causing cellulitis in broilers.

The aim of the study was to investigate cellulitis caused by Escherichia coli which has been responsible for economic and welfare problems in Danish broiler production between 2014 and 2016. The study included 13 flocks with unusually high condemnation rates due to cellulitis during a period of approximately one year. From six flocks, 126 condemned carcasses were collected at a Danish slaughterhouse. Further 272 broilers dead on their own were collected on nine broiler farms from flocks with increased mortality and cellulitis (2 farms included both birds from the rearing period and broilers subsequently condemned). All broilers were subjected to post mortem investigation including bacteriology and 247 E. coli isolates were obtained in pure culture from typical lesions of cellulitis. Two-hundred-thirty six E. coli isolates were investigated by pulsed field gel electrophoresis for clonality and 21 selected strains representing major clones were subsequently multi locus sequence typed allowing comparison to sequence types (ST) in the databases. One dominating PFGE type (A) was found to cause cellulitis on all 13 flocks (67% of all isolates). The clone belonged to ST117, which is well described as a pathogen in poultry, and was the primary agent responsible for cellulitis. Whole genome sequencing of eight E. coli isolates confirmed the close genetic relationship between isolates from the outbreaks and showed the presence of genes predicted to encode for the autotransporter proteins aatA, pic and upaG, reported to be of importance for adhesion of E. coli to eukaryotic cells.

Autoantibodies to the inhibitor of apoptosis protein survivin in patients with brain tumors.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is frequently expressed in cancers, including meningiomas and gliomas. Survivin may be associated with tumor progression and poor prognosis of patients with brain tumors. Using ELISA and immunoblot analysis we asked whether survivin is capable of eliciting a humoral immune response in patients with meningiomas and gliomas. Survivin-specific antibodies were detected in 5 of 42 (11.9%) patients with meningiomas and 3 of 35 (8.6%) patients with malignant gliomas of the WHO grades 3 and 4, but not in healthy controls. Tumors of patients with detectable anti-survivin antibodies demonstrated survivin expression in at least 20% of the tumor cells as assessed by immunohistochemistry. We conclude that patients with meningiomas and malignant gliomas can mount a high-titer IgG immune response against the 'universal' tumor-associated antigen survivin. Anti-survivin antibodies may represent attractive tools for diagnosis and follow-up of brain tumors.

Minichromosome maintenance protein 3 elicits a cancer-restricted immune response in patients with brain malignancies and is a strong independent predictor of survival in patients with anaplastic astrocytoma.

PURPOSE: The identification of new molecular markers in astrocytic tumors may help to understand the biology of these tumors in more detail. Informative tumor markers may represent prognostic factors for response to therapy and outcome as well as potential targets for novel anticancer therapies. EXPERIMENTAL DESIGN: Tumor-associated antigens were identified by immunoscreening of a human glioma cDNA expression library with allogeneic sera from patients with diffuse astrocytoma (WHO grades 2-4). The expression of one of the identified antigens, the replication licensing factor minichromosome maintenance protein 3 (MCM3), was analyzed by immunohistochemistry in 142 primary and 27 recurrent astrocytomas (WHO grades 2-4). In addition, 98 serum specimens from patients with primary and secondary brain malignancies and 30 serum specimens from healthy controls were examined by serologic immunoscreening for immunoreactivity with MCM3. RESULTS: MCM3 is overexpressed in human astrocytic tumors and elicits a cancer-restricted humoral immune response in 9.3% (9 of 97) of patients with brain tumors (n = 95) and brain metastases (n = 2) but not in healthy controls. Expression of MCM3 in diffuse astrocytoma is significantly associated with age (P < 0.001), histologic grade (P < 0.001), time to recurrence (P = 0.01), and expression of the proliferation marker Ki-67 (P < 0.001) but not with sex (P = 0.800). Univariate and multivariate Cox regression analysis confirmed MCM3 expression as an independent predictor of poor outcome in astrocytoma patients (P < 0.001 for both). CONCLUSIONS: MCM3 may represent a glioma-associated antigen with significant prognostic role as well as have some potential as a target for cancer-directed therapy.

Specific activation of operculum 3 (OP3) brain region during provoked tinnitus-related phantom auditory perceptions in humans.

The phantom sound perception mechanism by which a sound perception occurs without any external sound source is still enigmatic. According to our previous fMRI study, a small region in the parietal operculum 3 was hyperactivated as a function of tinnitus periodicity in subjects with acoustic trauma tinnitus sequelae. This region was localized in the vicinity of neural correlates of middle-ear tympano-ossicular chain movements due to pressure variations. Disturbed proprioceptors are known to trigger illusory perceptions; therefore, we hypothesized that a disturbance of middle-ear proprioceptors may originate phantom sound perceptions. We designed an fMRI study that aimed to stimulate middle-ear proprioceptors by repetitive vibrations using various rates of click trains. In this study, we report that exposure to specific rates of stimuli for a few minutes at comfortable intensity level in healthy subjects distinctly triggered transient tinnitus-like aftereffects. The fMRI neural correlates of the aftereffects were unequivocally localized in the same parietal region as in acoustic trauma tinnitus sufferers. Our results strongly suggest that a middle-ear kinesthetic/proprioceptive illusion exists at the origin of acoustic trauma tinnitus via a somatosensory pathway encompassing the trigeminal system.

Analysis of cytogenetic aberrations in esthesioneuroblastomas by comparative genomic hybridization.

Esthesioneuroblastoma (ENB) are rare tumors originating from the olfactory epithelium of the superior nasal cavity. This lesion is morphologically closely related to Ewing sarcoma and other peripheral primitive neuroectodermal tumors (pPNET). The affiliation of ENB to the pPNET family is still under discussion. Only very limited and contradictory cytogenetic data are available on ENB and only one patient has been analyzed by comparative genomic hybridization (CGH), so far. In the present study, genomic imbalances of three ENB were analyzed by CGH to evaluate (1) a recurrent pattern of imbalances, and (2) its relation to the pPNET family. The CGH analysis of three ENB revealed multiple recurrent aberrations including DNA overrepresentations of chromosomal material of the entire chromosome 19, partial gains of the long arms of chromosomes 8, 15, and 22, and deletions of the entire long arm of chromosome 4. Beside these common aberrations, several single gains and losses occurred, that is, gains on 6p, 10q, 1p, 9q, and 13q. We confirmed the former observation of amplified genetic material on chromosome 8 and found several new, currently not described recurrent genetic aberrations distinct from those described for pPNET. Our findings give evidence that ENB is not part of the pPNET family. We suggest that the combined gain of genetic material on 15q, 22q, and chromosome 8 might be indicative for ENB. To verify our findings and to define prognosis-related aberrations, a larger number of cases needs to be studied.

Preliminary speech recognition results after cochlear implantation in patients with unilateral hearing loss: a case series.

INTRODUCTION: Cochlear implants known to provide support in individuals with bilateral hearing loss may also be of great benefit for individuals with unilateral hearing loss. This case report demonstrates the positive effects of cochlear implantation on speech understanding in noise conditions in patients with unilateral hearing loss and normal hearing on the contralateral side. To the best of our knowledge, the data presented here are from the first few cases to receive a cochlear implant for unilateral hearing loss. CASE PRESENTATION: Four Caucasian German men, two aged 48 and the others aged 51 and 57 years old, with post-lingual unilateral hearing loss and normal hearing on the contralateral side were implanted with a cochlear implant. All our patients were members of the German army. Before and after implantation, they were given a battery of speech tests in different hearing conditions to assess the effect of unilateral cochlear implantation on speech understanding in noise conditions. Test results showed that all patients benefited from unilateral cochlear implantation, particularly in terms of speech understanding in noise conditions. CONCLUSIONS: Unilateral cochlear implantation might be a successful treatment method for patients with unilateral hearing loss not benefiting from alternative treatment options. The results of this case report open up the field of cochlear implantation for expanded criteria and new areas of research.

MGMT activity in mucosal epithelium and squamous cell carcinoma of the head and neck.

UNLABELLED: Smoking and alcohol abuse cause squamous cell carcinoma of the head and neck (SCCHN) through smoke-induced mutations, which are counteracted by O(6)-methylguanine-DNA methyltransferase (MGMT). This study aimed at elucidating the role of MGMT in SCCHN and its precursor lesions (SIN). MGMT was also determined in the normal mucosa (NM) and blood lymphocytes (PBLCs). RESULTS: a) MGMT was lower in NM than in PBLCs. b) Smoking reduced MGMT in NM but had no effect in PBLCs. c) MGMT activity increased in the sequence NM

A redshifted codon-optimized firefly luciferase is a sensitive reporter for bioluminescence imaging.

Bioluminescence imaging has evolved as a powerful tool for monitoring biological processes in vivo. As transmission efficiency of light through tissue increases greatly for wavelengths above 600 nm we examined whether a redshifted codon-optimized firefly luciferase (lambdamax=615 nm) could be successfully employed as a sensitive reporter in mammalian cells. To this end, unmodified codon-optimized luciferase (lambdamax=557 nm) as well as the red-emitting S284T mutant luciferase were expressed simultaneously in human glioma cells in vitro as well as in quadriceps muscles of mice in vivo. We show here that activity of the redshifted enzyme in human glioma cell culture approached approximately one-fourth of that seen with the unmodified enzyme. In contrast, light emission by the red-emitting luciferase in vivo was generally more efficient than that produced by its unmodified counterpart, most likely due to reduced absorption of red light by tissue. The mean ratio of light emission produced by the redshifted luciferase to that of the unmodified enzyme in vivo was approximately 3. Application of this new redshifted luciferase together with other optical reporters may be of considerable importance to biological research as it allows for imaging of deeper tissues as well as simultaneous monitoring of two molecular events in vitro and in vivo if appropriate filter sets are employed.

Comparative analysis of the epithelium stroma interaction of acquired middle ear cholesteatoma in children and adults.

In the clinical setting, pediatric cholesteatomas frequently behave more aggressively than similar lesions in adults. The reason for the difference in behavior is still unclear. The purpose of the present study was to investigate the cell to cell and epithelial-stroma interaction of acquired cholesteatoma in adults and children and search for differences on the cellular level, which might explain the different behavior of these lesions. Operative specimens of 54 patients [40 adults (average age of 39.7 years), 14 children (average age of 8.3 years)] who underwent primary surgery for an acquired cholesteatoma of the middle ear were examined by histopathology and DNA-image cytometry (DNA-ICM). Immunohistochemical investigations included expression of proliferation markers (proliferation cell nuclear antigen and MIB-1) along with cell surface markers reflecting the cell-to-cell interaction (i.e. alpha1beta6-integrin, E-cadherin, I-CAM = CD54), and the epithelial to stroma interaction (i.e. alphav and beta3 intergin chains, V-CAM = CD106, CD44v6 and fibronectin). Pediatric cholesteatomas demonstrated higher incidence of acute inflammation and more extensive disease relative to those from the adults. Indices of DNA-ICM, however, revealed normal diploid DNA content in both groups. Higher proliferation scores occurred in the pediatric group compared to adult cholesteatoma. Cell surface markers and cell adhesion molecules were equally expressed in both groups except alpha1beta6-integrin and fibronectin, which were over expressed in pediatric cholesteatomas. Statistically, however, these differences showed only a trend towards significance. According to the results of the present study, pediatric and adult cholesteatomas do not show any marked differences on the cellular level. Thus the observed clinical more aggressive behavior of pediatric cholesteatoma is likely due to other secondary factors such as more intense inflammation, disturbed middle ear ventilation or the diminished calcium salt content of pediatric bone.

Molecular biologic characteristics of seven new cell lines of squamous cell carcinomas of the head and neck and comparison to fresh tumor tissue.

BACKGROUND: Squamous cell carcinoma (SCC) is the most frequent malignant tumor of the upper aerodigestive tract. Cell lines of these tumors facilitate the investigation of various tumor biological parameters. This study was conducted to compare molecular biologic characteristics between cell lines and fresh tumor tissue. METHODS: In seven SCC-derived cell lines, cytokeratin 5/6 and cytokeratin 19 expression, DNA content, chromosome aberrations and tumorigenicity were assessed in nude rats. Unbalanced numerical and structural chromosomal aberrations were investigated by comparative genomic hybridization (CGH), and results were compared to those obtained in fresh tumor tissues of the same patients. RESULTS: All cell lines expressed cytokeratins 5/6 and 19, indicating their epidermoid origin. Tumor growth after transplantation into nude rats occurred in five of seven cell lines. Routine histology and immunohistochemical examinations confirmed SCC. Aneuploidy was detected in all cell lines, with a 2c deviation index ranging from 1.9 through 9.5 and a 5c exceeding rate ranging from 2.6 through 36.7%. The most frequent chromosomal aberrations in cell lines were overrepresentations of chromosomal material on chromosomes 15q, 7p (5 cases each), 3q, 5p (4 cases each), and 11q and 17q (3 cases each) and losses of chromosomal material on chromosomes 3p, 18q (3 cases each), and 19p and 7q (2 cases each). Comparing these results to CGH analysis of fresh tumor tissue from the same patients, overrepresentations of chromosomal material on 10q, 20q and 21q, along with loss of chromosomal material on 4q was detected more frequently in primary tumors, whereas overrepresentations on 7p and loss of chromosomal material on 7q were more frequently detected in cell lines. Nevertheless, there was a high degree of similarity of chromosomal alterations in cell lines and corresponding fresh tumor tissue. CONCLUSION: The data suggest a high degree of genetic similarity between tumor cells of cell lines and the tumors from which they were derived. Therefore, these cell lines can serve as an accurate model to investigate cell biology of SCC in vitro.

Investigations for fine mapping of amplifications in chromosome 3q26.3-28 frequently occurring in squamous cell carcinomas of the head and neck.

OBJECTIVE: Overrepresentations of chromosomal material on the long arm of chromosome 3 frequently occur in squamous cell carcinoma of the head and neck. This experimental study was conducted for further fine mapping of these overrepresentations by interphase fluorescence in situ hybridization (FISH) of tumor cells in cell lines. METHODS: Seven cell lines derived from squamous cell carcinomas of the head and neck were investigated by comparative genomic hybridization to analyze unbalanced chromosomal aberrations. Overrepresentations of chromosomal material on the telomeric part of the long arm of chromosome 3 were further analyzed by interphase FISH using YAC contig clones. RESULTS: Chromosomal aberrations which frequently occurred were overrepresentations on 5p (n = 4), 7p (n = 5), 11q13 (n = 3), 15q (n = 5), 17q (n = 3), 19q (n = 2), 20q (n = 2) and 22q (n = 3). Reoccurring losses of chromosomal material were found in 3p (n = 3), 7q (n = 2), 18q (n = 3) and 19p (n = 2). Gains of chromosomal material on chromosome 3q were found in 4 out of 7 cell lines, with a high copy number of amplifications occurring in the chromosomal region of 3q26.3-28. Further experiments revealed a physical mapping of this amplification to a narrow band of 13.8 Mbp on chromosome 3q, whose amplification borders were represented by the YAC clones 754_f_3 centomeric and 955_b_2 telomeric. CONCLUSIONS: By FISH, the amplification of chromosomal material on 3q could be fine mapped on a narrow band on 3q26.3-27. This aberration can be considered as a breakpoint in tumorigenesis. Putative candidate oncogenes and tumor suppressor genes located in this region might be a target for mutations leading to tumor progression.