Deafness gene screening based on a multilevel cascaded BPNN model.

Sudden sensorineural hearing loss is a common and frequently occurring condition in otolaryngology. Existing studies have shown that sudden sensorineural hearing loss is closely associated with mutations in genes for inherited deafness. To identify these genes associated with deafness, researchers have mostly used biological experiments, which are accurate but time-consuming and laborious. In this paper, we proposed a computational method based on machine learning to predict deafness-associated genes. The model is based on several basic backpropagation neural networks (BPNNs), which were cascaded as multiple-level BPNN models. The cascaded BPNN model showed a stronger ability for screening deafness-associated genes than the conventional BPNN. A total of 211 of 214 deafness-associated genes from the deafness variant database (DVD v9.0) were used as positive data, and 2110 genes extracted from chromosomes were used as negative data to train our model. The test achieved a mean AUC higher than 0.98. Furthermore, to illustrate the predictive performance of the model for suspected deafness-associated genes, we analyzed the remaining 17,711 genes in the human genome and screened the 20 genes with the highest scores as highly suspected deafness-associated genes. Among these 20 predicted genes, three genes were mentioned as deafness-associated genes in the literature. The analysis showed that our approach has the potential to screen out highly suspected deafness-associated genes from a large number of genes, and our predictions could be valuable for future research and discovery of deafness-associated genes.

The contribution of inner electron excitation to the electronic stopping power of palladium for protons.

The electronic stopping power of palladium (Pd) for protons is investigated based on time-dependent density functional theory combined with Ehrenfest molecular dynamics simulations. The electronic stopping power of Pd with explicitly considering inner electrons for protons is calculated and the excitation mechanism for the inner electrons of Pd is revealed. The velocity proportionality of the low-energy stopping power of Pd is reproduced. Our study verified that the inner electron excitation contributes significantly to the electronic stopping power of Pd in the high energy range, which is strongly dependent on the impact parameter. The electronic stopping power obtained from the off-channeling geometry is in quantitative agreement with the experimental data in a wide velocity range, and the discrepancy around the stopping maximum is further reduced by considering the relativistic correction on the binding energy of inner electrons. The velocity dependence of the mean steady-state charge of protons is quantified, and the results showed that the participation of 4p-electrons reduces the mean steady-state charge of protons, and consequently decreases the electronic stopping power of Pd in the low energy range.

An Update on Temporal Bone Paragangliomas.

OPINION STATEMENT: Temporal bone paragangliomas (TBPs) are indolent, classically benign and highly vascular neoplasms of the temporal bone. There are two types of TBPs, tympanomastoid paragangliomas (TMPs) and tympanojugular paragangliomas (TJPs). The most common symptoms are hearing loss and pulsatile tinnitus. Diagnostic workup, besides conventional physical and laboratory examinations, includes biochemical testing of catecholamine and genetic testing of SDHx gene mutations as well as radiological examination. Although surgery is traditionally the mainstay of treatment, it is challenging due to the close proximity of tumor to critical neurovascular structures and thus the high risk of complications, especially in patients with advanced lesions. Radiotherapy and active surveillance have been increasingly recommended for selected patients. Decision on treatment should be made comprehensively. Curative effect depends on various factors. Long-term follow-up with clinical, laboratory, and radiological examinations is essential for all patients.

Treatment Strategies for Malignancies of the External Auditory Canal.

OPINION STATEMENT: Malignant tumors of the external auditory canal (EAC) are rare tumors in the head and neck. Delayed diagnosis is not uncommon because the symptoms of early tumors are nonspecific. Various surgical and oncological treatment modalities have been reported. Decision-making depends on pathological feature and stage of the lesions, patient's general condition and preference, and physician's experience and skill. Radical surgery is widely accepted as the primary treatment of choice. Postoperative radiotherapy is used more often to improve local and regional control of the disease. Chemotherapy is usually recommended for advanced disease, residual disease, and metastasis. Prognosis is affected by multiple factors such as TNM stage, surgical margin, pathological type and differentiation of tumor, involvement of facial nerve, and so on. Although the survival rate is improved significantly over the past several decades with the development of skull base surgery, neuroradiology, anesthesiology, and oncology, it remains challenging to diagnose and treat EAC malignancies due to the rarity, the local anatomical complexity of temporal bone, and the lack of standard TNM staging system.

Effects of Acute Exposure to 3500 MHz (5G) Radiofrequency Electromagnetic Radiation on Anxiety-Like Behavior and the Auditory Cortex in Guinea Pigs.

Numerous studies have shown that radiofrequency electromagnetic radiation (RF-EMR) may negatively affect human health. We detected the effect of 3500 MHz RF-EMR on anxiety-like behavior and the auditory cortex (ACx) in guinea pigs. Forty male guinea pigs were randomly divided into four groups and exposed to a continuous wave of 3500 MHz RF-EMF at an average specific absorption rate (SAR) of 0, 2, 4, or 10 W/kg for 72 h. After exposure, malondialdehyde (MDA) levels, antioxidant enzyme activity, anxiety-like behavior, hearing thresholds, cell ultrastructure, and apoptosis were detected. Our results revealed that hearing thresholds and basic indexes of animal behavior did not change significantly after exposure (P > 0.05). However, the MDA levels of ACx were increased (P < 0.05), and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) activities were decreased (P < 0.05) in the exposure groups compared to the sham group. Ultrastructural changes of ACx, including swollen mitochondria and layered myelin sheaths, were observed. Cytochrome-c relocalization, caspase-9, and cleaved caspase-3 activation were detected in the exposure groups. In conclusion, these results suggest that oxidative stress is an important mechanism underlying the biological effects of RF-EMR, which can induce ultrastructural damage to the ACx and cell apoptosis through a mitochondria-dependent mechanism. Moreover, oxidative stress, apoptosis induction and ultrastructural damage increase in a SAR-dependent manner. However, RF-EMR does not increase hearing thresholds or induce anxiety. Bioelectromagnetics. 43:106-118, 2022. © 2021 Bioelectromagnetics Society.

The Effects of Mobile Phone Radiofrequency Radiation on Cochlear Stria Marginal Cells in Sprague-Dawley Rats.

To investigate the possible mechanisms for biological effects of 1,800 MHz mobile radiofrequency radiation (RFR), the radiation-specific absorption rate was applied at 2 and 4 W/kg, and the exposure mode was 5 min on and 10 min off (conversation mode). Exposure time was 24 h short-term exposure. Following exposure, to detect cell DNA damage, cell apoptosis, and reactive oxygen species (ROS) generation, the Comet assay test, flow cytometry, DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining, and a fluorescent probe were used, respectively. Our experiments revealed that mobile phone RFR did not cause DNA damage in marginal cells, and the rate of cell apoptosis did not increase (P > 0.05). However, the production of ROS in the 4 W/kg exposure group was greater than that in the control group (P < 0.05). In conclusion, these results suggest that mobile phone energy was insufficient to cause cell DNA damage and cell apoptosis following short-term exposure, but the cumulative effect of mobile phone radiation still requires further confirmation. Activation of the ROS system plays a significant role in the biological effects of RFR. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

Loss of NFBD1/MDC1 disrupts homologous recombination repair and sensitizes nasopharyngeal carcinoma cells to PARP inhibitors.

BACKGROUND: Nasopharyngeal carcinoma (NPC), a highly invasive tumor, exhibits a distinctive racial and geographic distribution. As options of agents for effective combination chemoradiotherapy for advanced NPC are limited, novel therapeutic approaches are desperately needed. Here the potential of silencing NFBD1 in combination with PARP inhibition as a novel therapeutic strategy for NPC was investigated. METHODS: To investigate the function of NFBD1, we created NFBD1-depleted NPC cell lines via lentivirus mediated shRNA, and the colony formation, MTS assay, comet assay and apoptosis analysis were used to evaluate the sensitivity of NFBD1 knockdown on PARP inhibition. The signaling change was assessed by western blot, Immunofluorescence and flow cytometry. Furthermore, Xenografts model was used to evaluate the role of silencing NFBD1 in combination with PARP inhibition. RESULTS: We find that silencing NFBD1 in combination with PARP inhibition significantly inhibits the cell proliferation and cell cycle checkpoint activity, and increases the apoptosis and DNA damage. Mechanistic studies reveal that NFBD1 loss blocks olaparib-induced homologous recombination repair by decreasing the formation of BRCA1, BRCA2 and RAD51 foci. Furthermore, the xenograft tumor model demonstrated significantly increases sensitivity towards PARP inhibition under NFBD1 deficiency. CONCLUSIONS: We show that NFBD1 depletion may possess sensitizing effects of PARP inhibitor, and consequently offers novel therapeutic options for a significant subset of patients.

Sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation will increase in lipopolysaccharide-induced inflammation in vitro model.

BACKGROUND: With the increasing popularity of mobile phones, the potential hazards of radiofrequency electromagnetic radiation (RF-EMR) on the auditory system remain unclear. Apart from RF-EMR, humans are also exposed to various physical and chemical factors. We established a lipopolysaccharide (LPS)-induced inflammation in vitro model to investigate whether the possible sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation (at specific absorption rates: 2, 4 W/kg) will increase. METHODS: Spiral ganglion neurons (SGN) were obtained from neonatal (1- to 3-day-old) Sprague Dawley® (SD) rats. After the SGN were treated with different concentrations (0, 20, 40, 50, 100, 200, and 400 µg/ml) of LPS, the Cell Counting Kit-8 (CCK-8) and alkaline comet assay were used to quantify cellular activity and DNA damage, respectively. The SGN were treated with the moderate LPS concentrations before RF-EMR exposure. After 24 h intermittent exposure at an absorption rate of 2 and 4 W/kg, DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autophagy markers LC3-II and Beclin1 were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay. RESULTS: LPS (100 µg/ml) induced DNA damage and suppressed cellular activity (P < 0.05). LPS (40 µg/ml) did not exhibit cellular activity changes or DNA damage (P > 0.05); therefore, 40 µg/ml was used to pretreat the concentration before exposure to RF-EMR. RF-EMR could not directly induce DNA damage. However, the 4 W/kg combined with LPS (40 µg/ml) group showed mitochondria vacuoles, karyopyknosis, presence of lysosomes and autophagosome, and increasing expression of LC3-II and Beclin1. The ROS values significantly increased in the 4 W/kg exposure, 4 W/kg combined with LPS (40 µg/ml) exposure, and H2O2 groups (P < 0.05, 0.01). CONCLUSIONS: Short-term exposure to radiofrequency electromagnetic radiation could not directly induce DNA damage in normal spiral ganglion neurons, but it could cause the changes of cellular ultrastructure at special SAR 4.0 W/kg when cells are in fragile or micro-damaged condition. It seems that the sensitivity of SGN to damage caused by mobile phone electromagnetic radiation will increase in a lipopolysaccharide-induced inflammation in vitro model.

The Role of Asymmetry Values, Gain, and Pathological Saccades of the Video Head Impulse Test (vHIT) in Sudden Sensorineural Hearing Loss.

OBJECTIVE: The purpose of this study was to evaluate the value of asymmetry values, gain, and pathological saccades of the video head impulse test (vHIT) in sudden sensorineural hearing loss (SSNHL). STUDY DESIGN: Retrospective study. SETTING: Tertiary referral center. PATIENTS: A total of 226 individuals diagnosed with unilateral definite SSNHL were hospitalized. The assessment included a comprehensive evaluation of medical history, pure-tone test, acoustic impedance, positional test, video nystagmography (VNG), vHIT, vestibular evoked myogenic potentials (VEMPs) and magnetic resonance. INTERVENTIONS: vHIT, VNG, cVEMP, oVEMP. Statistical analysis was performed with SPSS version 22.0 for Windows. MAIN OUTCOME MEASURES: The asymmetry values, gain, and pathological saccades of the vHIT. RESULTS: The abnormal gain of vHIT in anterior, horizontal, and posterior canal in SSNHL patients with vertigo were revealed in 20 of 112 (17.9%), 24 of 112 (21.4%), and 60 of 112 (53.6%), respectively. The vHIT pathological saccades (overt + covert) of anterior, horizontal, and posterior canal in SSNHL patients with vertigo were observed in 5 of 112 (4.6%), 52 of 112 (46.4%), and 58 of 112 (51.8%), respectively. Multivariate analysis indicated that the prognosis of patients with vertigo was correlated with vHIT gain of posterior canal, pathological saccade in horizontal canal, asymmetric ratio of horizontal canal gain, asymmetric ratio of posterior canal gain, Canal paresis (%) on caloric test and spontaneous nystagmus. CONCLUSION: In the vHIT of patients with SSNHL with vertigo, the posterior canal is most easily affected. Reduced gain of posterior canal, pathological saccade of horizontal canal, and larger asymmetric gain of posterior canal and horizontal canal may be negative prognostic factors.

Large-scale mutational analysis identifies UNC93B1 variants that drive TLR-mediated autoimmunity in mice and humans.

Nucleic acid-sensing Toll-like receptors (TLR) 3, 7/8, and 9 are key innate immune sensors whose activities must be tightly regulated to prevent systemic autoimmune or autoinflammatory disease or virus-associated immunopathology. Here, we report a systematic scanning-alanine mutagenesis screen of all cytosolic and luminal residues of the TLR chaperone protein UNC93B1, which identified both negative and positive regulatory regions affecting TLR3, TLR7, and TLR9 responses. We subsequently identified two families harboring heterozygous coding mutations in UNC93B1, UNC93B1+/T93I and UNC93B1+/R336C, both in key negative regulatory regions identified in our screen. These patients presented with cutaneous tumid lupus and juvenile idiopathic arthritis plus neuroinflammatory disease, respectively. Disruption of UNC93B1-mediated regulation by these mutations led to enhanced TLR7/8 responses, and both variants resulted in systemic autoimmune or inflammatory disease when introduced into mice via genome editing. Altogether, our results implicate the UNC93B1-TLR7/8 axis in human monogenic autoimmune diseases and provide a functional resource to assess the impact of yet-to-be-reported UNC93B1 mutations.

Pregnant Patients with Sudden Sensorineural Hearing Loss: Treatments and Efficacy.

BACKGROUND: The aim of this study was to study the safety and effectiveness of oral and tympanic hormone injection in the treatment of sudden sensorineural hearing loss during pregnancy. METHODS: Data were collected via prospective method. A total of 102 pregnant women with sensorineural hearing loss as experimental group and another 102 patients of sensorineural hearing loss without pregnancy as control group were simultaneously included in the study. Pure tone audiometry test was examined at pre- and posttreatment in 1 week, 2 weeks, and 12 weeks. The experimental group received oral and tympanic hormones, while the control group was treated with the Clinical Practice Guideline: Sudden Hearing Loss (2019) of USA. Recovery rate and hearing gain were assessed by the Clinical Practice Guidelines. RESULTS: After treatment, the effects of the experimental group and the control group were compared at the 1st, 2nd, and 12th week after treatment. It was found that at the 12th week after treatment, the curative effect of the experimental group was significantly different from that of the control group, and the difference was statistically significant. CONCLUSION: The pregnant women with sensorineural hearing loss were more serious than nonpregnant women, and the treatment efficacies were worse than control group. For pregnancy patients with sudden deafness, oral steroids and tympanic cavity injection is an effective, safe first-line treatment options.

Sudden sensorineural hearing loss during pregnancy: etiology, treatment, and outcome.

OBJECTIVE: To analyze the etiologies, treatments, and outcomes of sensorineural hearing loss (SSNHL) during pregnancy. STUDY DESIGN: Retrospective chart review of 25 pregnant patients treated for SSNHL between January 2012 and September 2019. Forty-nine age matched non-pregnant women with severe and profound hearing loss diagnosed with SSNHL during the same period served as controls. Data were recorded on age, symptoms, onset of hearing loss, audiometric results, treatments, and outcomes. RESULTS: The mean age was 29.6 years (range 23-38 years). Intratympanic steroids (ITS) were administered in 15 (60.0%) pregnant women with SSNHL. Three women were treated with postauricular steroids only, while another woman was treated with intravenous ginkgo leaf extract and dipyridamole. The remaining six women received no medications. More than half (8/15, 53.3%) of pregnant women with SSNHL receiving ITS experienced hearing improvement. Pregnant women with profound hearing loss who received no medication had no hearing improvement. Most pregnant women with SSNHL (12/15, 80.0%) had higher fibrinogen levels than controls (mean values 3.77±0.71 g/L and 2.54±0.48 g/L, respectively). CONCLUSION: Fibrinogen could be a risk factor for SSNHL during pregnancy. ITS may benefit pregnant women with severe and profound SSNHL.

The Homologous Recombination Repair Pathway is Associated with Resistance to Radiotherapy in Nasopharyngeal Carcinoma.

Radiotherapy plays a major role in the management of nasopharyngeal carcinoma (NPC). However, the radioresistant cells limit its efficiency and drive recurrence inside the irradiated tumor volume leading to poor outcome for patients. To illuminate the signal pathway involved in the radioresistance and evaluate the potential for predicting NPC response to radiotherapy, we established the radioresistant NPC cell line (CNE2-RR) derived from NPC cell line CNE2 by gradually increased the radiation dose (total 60 Gy), and the radioresistance of CNE2-RR cells was evaluated by the colony formation, FCM and comet assays. Furthermore, comparison of established CNE2-RR cell line to parental cell line found the homologous recombination repair (HRR) proteins differences involved in NPC radioresistance. In addition, the differentially expressed proteins were further validated by western blotting, immunofluorescence and IHC in tumor xenografs and radioresistant NPC tissues. Furthermore, the correlation of HRR proteins expression levels with NPC radioresistance were evaluated. The results showed that the upregulation of HRR proteins were significantly correlated with NPC radioresistance. In addition, using the Youden Index cutoff value, a panel of the HRR proteins analyses revealed a sensitivity of 70%, specificity of 72%. Furthermore, silencing NFBD1 enhanced the radiosensitivity of CNE2-RR cells by impairing IR-inducing γ-H2AX and HR proteins foci formation. These results suggest that controlling the HRR signaling pathway may hold promise to overcome NPC radioresistance.

Homologous recombination enhances radioresistance in hypopharyngeal cancer cell line by targeting DNA damage response.

BACKGROUND: Radiotherapy is a central treatment option for hypopharyngeal squamous cell carcinoma, but the prognoses of patients treated with radiotherapy only are not satisfactory due to radioresistance. The underlying molecular mechanisms remain largely elusive, and mechanism-derived predictive markers of radioresistance are currently unavailable. METHODS: In this study, we first established a specifically radioresistant FaDu cell line by repeated exposure to ionizing radiation with a total dose of 60 Gy (FaDu-RR). The validation of FaDu-RR cells was performed by clonogenic cell survival assay and cell proliferation assay. Microarrays and bioinformatics were analyzed to determine the differentially expressed mRNAs and their functions. DNA-repair capabilities were tested by cell cycle analysis and comet assay. The expressions of four key proteins in homologous recombination pathways, including BRCA1, BRCA2, RPA1, and Rad51, were detected both in FaDu-RR cells and radioresistant xenograft. RESULTS: We established the specifically radioresistant FaDu cell line. Through microarrays and bioinformatics, homologous recombination pathways were suggested to play important roles in radioresistant mechanisms. High expression levels of key proteins in homologous recombination pathways were then detected both in FaDu-RR cells and radioresistant xenograft. Silencing RPA1 could reduce the radioresistance of FaDu-RR cells. CONCLUSION: Our results provided strong evidence that homologous recombination enhances the radioresistance in hypopharyngeal carcinoma. Proteins in homologous recombination pathways may be potential biomarkers to predict hypopharyngeal carcinoma response to radiotherapy, establishing a basis for their utility in clinical practice.

Depletion of NFBD1/MDC1 Induces Apoptosis in Nasopharyngeal Carcinoma Cells Through the p53-ROS-Mitochondrial Pathway.

NFBD1, a signal amplifier of the p53 pathway, is vital for protecting cells from p53-mediated apoptosis and the early phase of DNA damage response under normal culture conditions. Here we investigated its expression in patients with nasopharyngeal carcinoma (NPC), and we describe the biological functions of the NFBD1 gene. We found that NFBD1 mRNA and protein were more highly expressed in NPC tissues than in nontumorous tissues. To investigate the function of NFBD1, we created NFBD1-depleted NPC cell lines that exhibited decreased cellular proliferation and colony formation, an increase in their rate of apoptosis, and an enhanced sensitivity to chemotherapeutic agents compared with in vitro controls. However, N-acetyl cysteine (NAC) and downregulation of p53 expression could partially reverse the apoptosis caused by the loss of NFBD1. Further analysis showed that loss of NFBD1 resulted in increased production of intracellular reactive oxygen species (ROS) depending on p53, which subsequently triggered the mitochondrial apoptotic pathway. Using a xenograft model in nude mice, we showed that silencing NFBD1 also significantly inhibited tumor growth and led to apoptosis. Taken together, our data suggest that inhibition of NFBD1 in NPC could be therapeutically useful.

Relevant In Vitro Predictors of Human Acellular Dermal Matrix-Associated Inflammation and Capsule Formation in a Nonhuman Primate Subcutaneous Tissue Expander Model.

Objective: Benchtop methods were evaluated for preclinical inflammation/capsule formation correlation following implantation of human acellular dermal matrices. Methods: Dermal matrices were compared with native dermis for structure (histology, scanning electron microscopy), collagen solubility (hydroxyproline), enzymatic susceptibility (collagenase), and thermal stability (differential scanning calorimetry). Results were compared with implantation outcomes in a primate tissue expander model. Results: Native dermis, electron beam-sterilized, and freeze-dried human acellular dermal matrices had equivalent morphology, acid-soluble collagen (60.5% ± 6.3%, 65.3% ± 3.2%, and 63.3% ± 2.4%, respectively), and collagenase resistance. Implant results showed minimal inflammation/matrix degradation, lack of capsule formation, insignificant elastic modulus change (57.65 ± 20.24 MPa out-of-package/44.84 ± 23.87 MPa in vivo), and low antibody induction (2- to 8-fold increase) for electron beam-sterilized matrix. Similar results for freeze-dried dermal matrix were previously observed. γ-Irradiated, γ-irradiated/freeze-dried, and ethanol-stored dermal matrices were statistically different from native dermis for acid-soluble collagen (82.4% ± 5.8%, 72.2% ± 6.2%, and 76.8% ± 5.0%, respectively) and collagenase digestion rate, indicating matrix damage. γ-Irradiated matrix-implanted animals demonstrated elevated inflammatory response, foreign body giant cells, capsule formation at the tissue expander junction, and robust matrix metalloproteinase-1 staining with significant elastic modulus decrease (37.43 ± 7.52 MPa out-of-package/19.58 ± 1.16 MPa in vivo). Antibody increase (32- to 128-fold) was observed 6 to 10 weeks following γ-irradiated matrix implantation. Ethanol-stored dermal matrix elicited an acute antibody response (4- to 128-fold increase, 2-4 weeks) and macrophage-concentrated synovial-like hyperplasia at the tissue expander junction, moderate matrix metalloproteinase-1 staining, and significant elastic modulus decrease (61.15 ± 9.12 MPa out-of-package/17.92 ± 4.02 MPa in vivo) by 10 weeks implantation. Conclusion: Demonstrated loss of collagen integrity in vitro may be predictive of inflammation/capsule formation in primate tissue expander models. These results may be further predictive of clinical observations.

Decreased Poly(ADP-Ribose) Polymerase 1 Expression Attenuates Glucose Oxidase-Induced Damage in Rat Cochlear Marginal Strial Cells.

Oxidative damage to the inner ear is responsible for several types of sensorineural deafness. Cochlear stria marginal cells (MCs) are thought to be vulnerable to such oxidative stress. Activated poly(ADP-ribose) polymerase 1 (PARP1) has been implicated in several diseases, but the effect of PARP1 on MCs subjected to oxidative stress remains elusive. In this study, we established an in vitro cellular oxidative stress model using glucose oxidase (GO) and attempted to explore the role that PARP1 plays in the oxidative damage of MCs. In this study, PARP1 and poly-ADP-ribose (PAR) were highly expressed in GO-treated MCs, and this was accompanied by loss of MC viability, excessive generation of reactive oxygen species (ROS), collapse of mitochondria membrane potential (ΔΨm), and redistribution of the mitochondrial downstream pathway-related molecules Bax and cytochrome c, eventually causing MC death. These effects were almost completely counteracted by suppressing PARP1 expression with small interfering RNA (siRNA). We also found that caspase-3 activation was a downstream event of PARP activation and that apoptosis of MCs was suppressed, although not completely, by pretreatment with the pan-caspase inhibitor z-VAD-fmk. The suppression was less than that when PARP1 expression was inhibited. We conclude that GO treatment induces activation of PARP1, which causes MC damage via mitochondrial mediation. PARP1 plays a pivotal role in GO-induced MC death, at least in part, via the caspase-3 cascade. Our study might provide a new cellular and molecular approach for the treatment of oxidative stress-related sensorineural deafness.

A porcine-derived acellular dermal scaffold that supports soft tissue regeneration: removal of terminal galactose-alpha-(1,3)-galactose and retention of matrix structure.

Sub-optimal clinical outcomes after implantation of animal-derived tissue matrices may be attributed to the nature of the processing of the material or to an immune response elicited in response to xenogeneic epitopes. The ability to produce a porcine-derived graft that retains the structural integrity of the extracellular matrix and minimizes potential antigenic response to galactose-alpha-(1,3)-galactose terminal disaccharide (alpha-Gal) may allow the scaffold to support regeneration of native tissue. Dermal tissue from wild-type (WT-porcine-derived acellular dermal matrix [PADM]) or Gal-deficient (Gal(-/-) PADM) pigs was processed to remove cells and DNA while preserving the structural integrity of the extracellular matrix. In addition, the WT tissue was subjected to an enzymatic treatment to minimize the presence of alpha-Gal (Gal-reduced PADM). Extracellular matrix composition and integrity was assessed by histological, immunohistochemical (IHC), and ultrastructural analysis. In vivo performance was evaluated by implantation into the abdominal wall of Old World primates in an exisional repair model. Anti-alpha-Gal activity in the serum of monkeys implanted subcutaneously was assessed by ELISA. Minimal modification to the extracellular matrix was assessed by evaluation of intact structure as demonstrated by staining patterns for type I and type VII collagens, laminin, and fibronectin similar to native porcine skin tissues. Explants from the abdominal wall showed evidence of remodeling, notably fibroblast cell repopulation and revascularization, as early as 1 month. Serum ELISA revealed an initial anti-alpha-Gal induction that decreased to baseline levels over time in the primates implanted with WT-PADM, whereas no or minimal anti-Gal activity was detected in the primates implanted with Gal(-/-) PADM or Gal-reduced PADM. The combination of a nondamaging process, successful removal of cells, and reduction of xenogeneic alpha-Gal antigens from the porcine dermal matrix are critical for producing a material with the ability to remodel and integrate into host tissue and ultimately support soft tissue regeneration.

Identification of network-level coding units for real-time representation of episodic experiences in the hippocampus.

To examine the network-level organizing principles by which the brain achieves its real-time encoding of episodic information, we have developed a 96-channel array to simultaneously record the activity patterns of as many as 260 individual neurons in the mouse hippocampus during various startling episodes. We find that the mnemonic startling episodes triggered firing changes in a set of CA1 neurons in both startle-type and environment-dependent manners. Pattern classification methods reveal that these firing changes form distinct ensemble representations in a low-dimensional encoding subspace. Application of a sliding window technique further enabled us to reliably capture not only the temporal dynamics of real-time network encoding but also postevent processing of newly formed ensemble traces. Our analyses revealed that the network-encoding power is derived from a set of functional coding units, termed neural cliques, in the CA1 network. The individual neurons within neural cliques exhibit "collective cospiking" dynamics that allow the neural clique to overcome the response variability of its members and to achieve real-time encoding robustness. Conversion of activation patterns of these coding unit assemblies into a set of real-time digital codes permits concise and universal representation and categorization of discrete behavioral episodes across different individual brains.

Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain.

By integrating convergent protein engineering and rational inhibitor design, we have developed an in vivo conditional protein knockout andor manipulation technology. This method is based on the creation of a specific interaction interface between a modified protein domain and sensitized inhibitors. By introducing this system into genetically modified mice, we can readily manipulate the activity of a targeted protein, such as alpha-Ca(2+)calmodulin-dependent protein kinase II (alphaCAMKII), on the time scale of minutes in specific brain subregions of freely behaving mice. With this inducible and region-specific protein knockout technique, we analyzed the temporal stages of memory consolidation process and revealed the first postlearning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.