Atypical fibroxanthoma (AFX) is a rare mesenchymal tumor with predominance in older male patients located mainly in chronically UV-exposed skin. Differentiation from clinically more aggressive pleomorphic dermal sarcoma (PDS) is still under debate and immunohistochemical markers are not available yet. An immunohistochemical study, including 41 cases of AFX was conducted to investigate the expression of 3q encoded oncogene SEC62 in AFX and determine the associations with histomorphologic, clinical and viral parameters. Our cohort displayed a mean of 79.9 years at the onset of the disease. In total, 90.2% (37/41) AFXs were located in the head and neck area, whereas, four were located at the extremities (9.7%). Tumor diameter ranged between 0.06 and 40 cm2 with a mean of 5.7 cm2. SEC62 expression was markedly increased in lesional tissue compared with the adjacent healthy squamous epithelium. We found significantly higher expression of SEC62 in cases of AFX with tumor necrosis. Tendency of higher Sec62-IRS-scores were found for tumors with higher Clark levels and a tumor size >5 cm2. Sec62 is involved in endoplasmic reticulum stress tolerance and cell migration, and has been identified as a novel prognostic marker for non-small cell lung cancer as well as head and neck squamous cell carcinoma. For the first time, to the best of our knowledge, we suggest a role of 3q oncogene SEC62 in AFX and discuss a potential prognostic relevance in cases of disputable AFX with unfavorable histomorphologic features and may initiate a discussion on Sec62 serving as discriminating marker between AFX and PDS.

Human IgA antibodies effectively engage myeloid cells for the FcαRI-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Established methods to investigate ADCC are the 51chromium and Calcein release assays. Their critical limitations are the end-point measurement, the unspecific release of the probes, the requirement of target cells in suspension and thus do not reflect physiologic conditions of adherently growing cells. Here we report the label-free real-time monitoring of granulocyte-mediated ADCC using an impedance-based method. We investigated the efficacy of an engineered epidermal growth factor receptor (EGFR)-directed IgA2 antibody to engage neutrophils for ADCC against a panel of adherently growing EGFR-expressing cancer cell lines majorly head and neck squamous cell carcinoma (HNSCC). The impedance assay allowed the documentation of the IgA-neutrophil-and FcαRI-signaling dependent ADCC of adherently growing target cells. While at a short-term it provided comparable results to release assays, in the long run real time monitoring also revealed cell-line specific kinetics and long-term efficacy. Although short-term results may depend on EGFR expression, long-term efficacy did not correlate with the surface level of EGFR nor of the myeloid checkpoint CD47 pointing to additional critical parameters to predict the treatment efficacy. Real-time monitoring of neutrophil-mediated ADCC allowed documenting effector cell activity and exhaustion. Along with excess expression of Mac-1 ligands, which may explain the target cell resistance, this eventually leads to tumor cell outgrowth at later time points. In conclusion, the impedance assay provides valuable information on the kinetics, effector cell performance, efficacy and critical parameters of IgA-dependent granulocyte-mediated cytotoxicity and is expected to become an important tool in its evaluation.

Pressure sensitive adhesives based on silicone materials are used particularly for skin adhesion, e.g., the fixation of electrocardiogram (ECG) electrodes or wound dressings. However, adhesion to sensitive tissue structures is not sufficiently addressed due to the risk of damage or rupture. We propose an approach in which a poly-(dimethylsiloxane) (PDMS)-based soft skin adhesive (SSA) acts as cellular scaffold for wound healing. Due to the intrinsically low surface free energy of silicone elastomers, functionalization strategies are needed to promote the attachment and spreading of eukaryotic cells. In the present work, the effect of physical adsorption of three different proteins on the adhesive properties of the soft skin adhesive was investigated. Fibronectin adsorption slightly affects adhesion but significantly improves the cellular interaction of L929 murine fibroblasts with the polymeric surface. Composite films were successfully attached to explanted tympanic membranes. This demonstrates the potential of protein functionalized SSA to act as an adhesive scaffold in delicate biomedical applications.

Profound knowledge exists about the clinical, morphologic, genomic, and transcriptomic characteristics of most lymphoma entities. However, information is currently lacking on the dynamic behavior of malignant lymphomas. This pilot study aimed to gain insight into the motility of malignant lymphomas and bystander cells in 20 human lymph nodes. Generally, B cells were faster under reactive conditions compared with B cells in malignant lymphomas. In contrast, PD1-positive T cells did not show systematic differences in velocity between reactive and neoplastic conditions in general. However, lymphomas could be divided into two groups: one with fast PD1-positive T cells (e.g., Hodgkin lymphoma and mantle cell lymphoma; means 8.4 and 7.8 µm/min) and another with slower PD1-positive T cells (e.g., mediastinal grey zone lymphoma; mean 3.5 µm/min). Although the number of contacts between lymphoma cells and PD1-positive T cells was similar in different lymphoma types, important differences were observed in the duration of these contacts. Among the lymphomas with fast PD1-positive T cells, contacts were particularly short in mantle cell lymphoma (mean 54 s), whereas nodular lymphocyte-predominant Hodgkin lymphoma presented prolonged contact times (mean 6.1 min). Short contact times in mantle cell lymphoma were associated with the largest spatial displacement of PD1-positive cells (mean 12.3 µm). Although PD1-positive T cells in nodular lymphocyte-predominant Hodgkin lymphoma were fast, they remained in close contact with the lymphoma cells, in line with a dynamic immunological synapse. This pilot study shows for the first time systematic differences in the dynamic behavior of lymphoma and bystander cells between different lymphoma types.

The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uni- and multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives.

SEC62 oncogene located at chromosomal region 3q26 encodes for a transmembrane protein of the endoplasmic reticulum (ER) and is expressed at high levels in numerous human malignancies. SEC62 overexpression has been associated with worse prognosis and high risk for lymphatic and distant metastases in head and neck cancer, cervical cancer, hepatocellular cancer, and lung cancer. However, its role in the development and tumor biology of melanocytic lesions has not been investigated so far. An immunohistochemical study including 209 patients with melanocytic lesions (malignant melanoma (MM), n = 93; melanoma metastases (MET), n = 28; Spitz nevi (SN), n = 29; blue nevi (BN), n = 21; congenital nevi (CN), n = 38) was conducted and SEC62 expression was correlated with clinical data including patient survival and histopathological characteristics. SN showed the highest SEC62 expression levels followed by MET, MM, CN, and BN. High SEC62 expression correlated with a shorter overall and progression-free survival in MM patients. Additionally, high Sec62 levels correlated significantly with higher tumor size (T stage), the presence of tumor ulceration, and the presence of lymph node as well as distant metastases. Strikingly, SEC62 expression showed a strong correlation with Clark level. Taken together, these data demonstrate that SEC62 is a promising prognostic marker in MM and has the potential to predict biological behavior and clinical aggressiveness of melanocytic lesions.

Optoacoustic stimulation offers an alternative stimulation strategy for the hearing organ. To serve as the base for a novel auditory prosthesis, the optoacoustic stimulation must be biocompatible and energy-saving.

Chromosome 3 amplification affecting the 3q26 region is a common genomic alteration in cervical cancer, typically marking the transition of precancerous intraepithelial lesions to an invasive phenotype. Though potential 3q encoded target genes of this amplification have been identified, a functional correlation of potential oncogenic function is still missing. In this study, we investigated copy number changes and the expression level of SEC62 encoded at 3q26.2 as a new potential 3q oncogene in dysplastic cervical lesions and analyzed its role in cervical cancer cell biology.

Hearing impairment affects ∼460 million people worldwide. Conservative therapies, such as hearing aids, bone conduction systems, and middle ear implants, do not always sufficiently compensate for this deficit. The optical stimulation is currently under investigation as an alternative stimulation strategy for the activation of the hearing system. To assess the biocompatibility margins of this emerging technology, we established a method applicable in whole-mount preparations of murine tympanic membranes (TM). We irradiated the TM of anesthetized mice with 532-nm laser pulses at an average power of 50, 89, 99, and 125 mW at two different locations of the TM and monitored the hearing function with auditory brainstem responses. Laser-power-dependent negative side effects to the TM were observed at power levels exceeding 89 mW. Although we did not find any significant negative effects of optical stimulation on the hearing function in these mice, based on the histology results further studies are necessary for optimization of the used parameters.

Inhibitory glycine receptors containing the α3 subunit (GlyRα3) regulate sensory information processing in the CNS and retina. In previous work, we demonstrated the presence of postsynaptic GlyRα3 immunoreactivity at efferent synapses of the medial and lateral olivocochlear bundle in the organ of Corti; however, the role of these α3-GlyRs in auditory signalling has remained elusive. The present study analyzes distortion-product otoacoustic emissions (DPOAEs) and auditory brainstem responses (ABRs) of knockout mice with a targeted inactivation of the Glra3 gene (Glra3(-/-)) and their wildtype littermates (Glra3(+/+)) before and seven days after acoustic trauma (AT; 4-16 kHz, 120 dB SPL, 1 h). Before AT, DPOAE thresholds were slightly, but significantly lower, and DPOAE amplitudes were slightly larger in Glra3(-/-) as compared to Glra3(+/+) mice. While click- and f-ABR thresholds were similar in both genotypes before AT, threshold-normalized click-ABR wave I amplitudes were smaller in Glra3(-/-) mice as compared to their wildtype littermates. Following AT, both the decrement of ABR wave I amplitudes and the delay of wave I latencies were more pronounced in Glra3(-/-) than Glra3(+/+) mice. Accordingly, correlation between early click-evoked ABR signals (0-2.5 ms from stimulus onset) before and after AT was significantly reduced for Glra3(-/-) as compared to Glra3(+/+) mice. In summary, these results show that loss of α3-GlyRs compromises suprathreshold auditory nerve activity, but not outer hair cell function.

Cav1.3 L-type Ca(2+)-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca(2+)- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca(2+)-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRD(HA/HA)). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca(2+)-dependent inactivation of Ca(2+)-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability.

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

Chromosome 3q26 amplification represents a frequent alteration in head and neck squamous cell carcinomas (HNSCCs). Overexpression of 3q26 encoded genes SEC62 and SOX2 was detected in various cancers, including HNSCCs, indicating their potential function as oncogenes. In our study, we elucidated the function of SEC62 and SOX2 in HNSCC patients, with a main focus on their effect on lymphatic metastasis and patient survival. We analyzed SEC62 and SOX2 expression in tissue specimens from 65 HNSCC patients and 29 patients with cervical cancer of unknown primary (CUP); a higher SEC62 and lower SOX2 expression was observed in the lymph node metastases from HNSCC patients compared with the respective primary tumor. Lymph node metastases from CUP patients showed higher SEC62 and lower SOX2 expression compared with lymph node metastases from HNSCC patients. When proceeding from the N1 to the N3 stage, SEC62 expression in the lymph node metastases showed an increase and SOX2 expression showed a decrease. Moreover, both genes showed a highly significant relevance as prognostic biomarkers, with the worst prognosis for patients with high SEC62 and low SOX2 expression levels. In functional analyses, knockdown of SEC62 resulted in an inhibition of HNSCC cell migration while, conversely, SEC62 and SOX2 overexpression stimulated cell migration. Taken together, our study showed that the expression of the 3q oncogenes SEC62 and SOX2 affects lymphatic metastasis and cell migration in HNSCC and CUP patients and has a high prognostic relevance in these diseases.

Inner hair cell (IHC) Cav1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Cav1.3-deficient (Cav1.3-/-) mice stay immature because they fail to up-regulate voltage- and Ca2+-activated K+ (BK) channels but persistently express small conductance Ca2+-activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Cav1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Cav1.3-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Cav1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFP flex/flex and Pax2::cre;Cacna1d-eGFP flex/-. Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFP flex/- mice recapitulated the phenotype of systemic Cav1.3-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Cav1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFP flex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2-driven Cre recombinase in switching two rather than one Cacna1d-eGFPflex allele lead us to study Pax2::cre;Cacna1d-eGFP flex/- mice. Notably, control Cacna1d-eGFPflex/- IHCs showed a significant reduction in Cav1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required.

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve-glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-2'-deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA.

Perineuronal nets (PNNs) are specialized aggregations of extracellular matrix (ECM) molecules surrounding specific neurons in the central nervous system (CNS). PNNs are supposed to control synaptic transmission and are frequently associated with neurons firing at high rates, including principal neurons of auditory brainstem nuclei. The origin of high-frequency activity of auditory brainstem neurons is the indefatigable sound-driven transmitter release of inner hair cells (IHCs) in the cochlea.

Worldwide, ∼460  million people suffer from disabling hearing impairment. Many of these patients are still not sufficiently supplied with currently available auditory technologies. Optical stimulation of the hearing organ offers a promising alternative for a new generation of auditory prostheses.