Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-ß-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5ß1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates.

BACKGROUND: One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms. RESULTS: Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter. CONCLUSION: In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

[Incapacity to work in prehospital German emergency medical services staff]. / Arbeitsunfähigkeit bei präklinischen Rettungskräften in Deutschland: Ergebnisse der EMS-Health-Studie.

Background: The determination of incapacity to work is a central approach for analyses of absence due to sickness. Nevertheless, no data are yet available for incapacity to work and associated factors in the German prehospital emergency medical services (EMS) staff. Objective: The aim of this analysis was to identify the proportion of EMS staff with at least one incapacity for work (AU) in the previous 12 months and associated factors. Material and methods: This was a nationwide survey study with rescue workers. Factors associated with work disability were identified using multivariable logistic regression, calculating odds ratios (OR) and associated 95% confidence intervals (95% CI). Results: Included in this analysis were 2298 employees of the German emergency medical services (female 42.6%, male 57.2%). Overall, 60.10% of female participants and 58.98% of male participants reported an incapacity for work in the previous 12 months. Incapacity for work was significantly associated with having a high school diploma (high school diploma: OR: 0.51, 95% CI 0.30; 0.88, p = 0.016; reference: secondary school diploma), working in a rural environment (OR: 0.65, 95% CI 0.50; 0.86, p = 0.003) or urban environment (OR: 0.72, 95% CI: 0.53; 0.98, p = 0.037). Furthermore, hours worked per week (OR: 1.01, 95% CI: 1.00; 1.02, p = 0.003) and 5-< 10 years of service (OR: 1.40, 95% CI: 1.04; 1.89, p = 0.025) were associated with higher odds of work disability. Neck and back pain, depression, osteoarthritis, and asthma in the previous 12 months also showed a significant association with work disability in the same time period. Conclusion: This analysis shows that chronic diseases, educational attainment, area of assignment, years of service, and hours worked per week, among others, were associated with incapacity for work in the previous 12 months in German EMS staff.

Dexamethasone-mediated inhibition of Notch signalling blocks the interaction of leukaemia and mesenchymal stromal cells.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by a poor prognosis. Bone marrow mesenchymal stromal cells (BM MSCs) support leukaemic cells in preventing chemotherapy-induced apoptosis. This encouraged us to investigate leukaemia-BM niche-associated signalling and to identify signalling cascades supporting the interaction of leukaemic cells and BM MSC. Our study demonstrated functional differences between MSCs originating from leukaemic (AML MSCs) and healthy donors (HD MSCs). The direct interaction of leukaemic and AML MSCs was indispensable in influencing AML cell proliferation. We further identified an important role for Notch expression and its activation in AML MSCs contributing to the enhanced proliferation of AML cells. Supporting this observation, overexpression of the intracellular Notch domain (Notch ICN) in AML MSCs enhanced AML cells' proliferation. From a therapeutic point of view, dexamethasone treatment impeded Notch signalling in AML MSCs resulting in reduced AML cell proliferation. Concurrent with our data, Notch inhibitors had only a marginal effect on leukaemic cells alone but strongly influenced Notch signalling in AML MSCs and abrogated their cytoprotective function on AML cells. In vivo, dexamethasone treatment impeded Notch signalling in AML MSCs leading to a reduced number of AML MSCs and improved survival of leukaemic mice. In summary, targeting the interaction of leukaemic cells and AML MSCs using dexamethasone or Notch inhibitors might further improve treatment outcomes in AML patients.

Insights in the Complex DegU, DegS, and Spo0A Regulation System of Paenibacillus polymyxa by CRISPR-Cas9-Based Targeted Point Mutations.

Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated, the single mutants DegU Q218*, DegS L99F, and Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild-type and the engineered strains revealed differences in swarming behavior, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365. IMPORTANCE To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate several regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.

The effect of hearing aid dynamic range compression on speech intelligibility in a realistic virtual sound environment.

Measures of "aided" speech intelligibility (SI) for listeners wearing hearing aids (HAs) are commonly obtained using rather artificial acoustic stimuli and spatial configurations compared to those encountered in everyday complex listening scenarios. In the present study, the effect of hearing aid dynamic range compression (DRC) on SI was investigated in simulated real-world acoustic conditions. A spatialized version of the Danish Hearing In Noise Test was employed inside a loudspeaker-based virtual sound environment to present spatialized target speech in background noise consisting of either spatial recordings of two real-world sound scenarios or quadraphonic, artificial speech-shaped noise (SSN). Unaided performance was compared with results obtained with a basic HA simulator employing fast-acting DRC. Speech reception thresholds (SRTs) with and without DRC were found to be significantly higher in the conditions with real-world background noise than in the condition with artificial SSN. Improvements in SRTs caused by the HA were only significant in conditions with real-world background noise and were related to differences in the output signal-to-noise ratio of the HA signal processing between the real-world versus artificial conditions. The results may be valuable for the design, development, and evaluation of HA signal processing strategies in realistic, but controlled, acoustic settings.

Airway Administration of Flagellin Regulates the Inflammatory Response to Pseudomonas aeruginosa.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

A method for realistic, conversational signal-to-noise ratio estimation.

The analysis of real-world conversational signal-to-noise ratios (SNRs) can provide insight into people's communicative strategies and difficulties and guide the development of hearing devices. However, measuring SNRs accurately is challenging in everyday recording conditions in which only a mixture of sound sources can be captured. This study introduces a method for accurate in situ SNR estimation where the speech signal of a target talker in natural conversation is captured by a cheek-mounted microphone, adjusted for free-field conditions and convolved with a measured impulse response to estimate its power at the receiving talker. A microphone near the receiver provides the noise-only component through voice activity detection. The method is applied to in situ recordings of conversations in two real-world sound scenarios. It is shown that the broadband speech level and SNR distributions are estimated more accurately by the proposed method compared to a typical single-channel method, especially in challenging, low-SNR environments. The application of the proposed two-channel method may render more realistic estimates of conversational SNRs and provide valuable input to hearing instrument processing strategies whose operating points are determined by accurate SNR estimates.

Speech intelligibility in a realistic virtual sound environment.

In the present study, speech intelligibility was evaluated in realistic, controlled conditions. "Critical sound scenarios" were defined as acoustic scenes that hearing aid users considered important, difficult, and common through ecological momentary assessment. These sound scenarios were acquired in the real world using a spherical microphone array and reproduced inside a loudspeaker-based virtual sound environment (VSE) using Ambisonics. Speech reception thresholds (SRT) were measured for normal-hearing (NH) and hearing-impaired (HI) listeners, using sentences from the Danish hearing in noise test, spatially embedded in the acoustic background of an office meeting sound scenario. In addition, speech recognition scores (SRS) were obtained at a fixed signal-to-noise ratio (SNR) of -2.5 dB, corresponding to the median conversational SNR in the office meeting. SRTs measured in the realistic VSE-reproduced background were significantly higher for NH and HI listeners than those obtained with artificial noise presented over headphones, presumably due to an increased amount of modulation masking and a larger cognitive effort required to separate the target speech from the intelligible interferers in the realistic background. SRSs obtained at the fixed SNR in the realistic background could be used to relate the listeners' SI to the potential challenges they experience in the real world.

Guided ecological momentary assessment in real and virtual sound environments.

Ecological momentary assessment (EMA) outcome measures can relate people's subjective auditory experience to their objective acoustical reality. While highly realistic, EMA data often contain considerable variability, such that it can be difficult to interpret the results with respect to differences in people's hearing ability. To address this challenge, a method for "guided" EMA is proposed and evaluated. Accompanied and instructed by a guide, normal-hearing participants carried out specific passive and active listening tasks inside a real-world public lunch scenario and answered EMA questionnaires related to aspects of spatial hearing, listening ability, quality, and effort. In situ speech and background noise levels were tracked, allowing the guided EMA task to be repeated inside two acoustically matched, loudspeaker-based laboratory environments: a 64-channel virtual sound environment (VSE) and a three-channel audiology clinic setup. Results showed that guided EMA provided consistent passive listening assessments across participants and conditions. During active listening, the clinic setup was found to be less challenging than the real-world and the VSE conditions. The proposed guided EMA approach may provide more focused real-world assessments and can be applied in realistic laboratory settings to aid the development of ecologically valid hearing testing.

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line.

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.

The impact of noise power estimation on speech intelligibility in cochlear-implant speech coding strategies.

The advanced combination encoder (ACE™) is an established speech-coding strategy in cochlear-implant processing that selects a number of frequency channels based on amplitudes. However, speech intelligibility outcomes with this strategy are limited in noisy conditions. To improve speech intelligibility, either noise-dominant channels can be attenuated prior to ACE™ with noise reduction or, alternatively, channels can be selected based on estimated signal-to-noise ratios. A noise power estimation stage is, therefore, required. This study investigated the impact of noise power estimation in noise-reduction and channel-selection strategies. Results imply that estimation with improved noise-tracking capabilities does not necessarily translate into increased speech intelligibility.

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation.

The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near-surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC-flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC-expressing bacteria outcompeted FljB-expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.

Proliferation status defines functional properties of endothelial cells.

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

The impact of exploiting spectro-temporal context in computational speech segregation.

Computational speech segregation aims to automatically segregate speech from interfering noise, often by employing ideal binary mask estimation. Several studies have tried to exploit contextual information in speech to improve mask estimation accuracy by using two frequently-used strategies that (1) incorporate delta features and (2) employ support vector machine (SVM) based integration. In this study, two experiments were conducted. In Experiment I, the impact of exploiting spectro-temporal context using these strategies was investigated in stationary and six-talker noise. In Experiment II, the delta features were explored in detail and tested in a setup that considered novel noise segments of the six-talker noise. Computing delta features led to higher intelligibility than employing SVM based integration and intelligibility increased with the amount of spectral information exploited via the delta features. The system did not, however, generalize well to novel segments of this noise type. Measured intelligibility was subsequently compared to extended short-term objective intelligibility, hit-false alarm rate, and the amount of mask clustering. None of these objective measures alone could account for measured intelligibility. The findings may have implications for the design of speech segregation systems, and for the selection of a cost function that correlates with intelligibility.

Murine alveolar epithelial cells and their lentivirus-mediated immortalisation.

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air-blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800Ω ·cm². Transport across the air-blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω·cm²) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

Preserving spatial perception in rooms using direct-sound driven dynamic range compression.

Fast-acting hearing-aid compression systems typically distort the auditory cues involved in the spatial perception of sounds in rooms by enhancing low-level reverberant energy portions of the sound relative to the direct sound. The present study investigated the benefit of a direct-sound driven compression system that adaptively selects appropriate time constants to preserve the listener's spatial impression. Specifically, fast-acting compression was maintained for time-frequency units dominated by the direct sound while the processing of the compressor was linearized for time-frequency units dominated by reverberation. This compression scheme was evaluated with normal-hearing listeners who indicated their perceived location and distribution of sound images in the horizontal plane for virtualized speech. The experimental results confirmed that both independent compression at each ear and linked compression across ears resulted in broader, sometimes internalized, sound images as well as image splits. In contrast, the linked direct-sound driven compression system provided the listeners with a spatial perception similar to that obtained with linear processing that served as the reference condition. The independent direct-sound driven compressor created a sense of movement of the sound between the two ears, suggesting that preserving the interaural level differences via linked compression is advantageous with the proposed direct-sound driven compression scheme.