Stimulation parameters differ between current anti-modiolar and peri-modiolar electrode arrays implanted within the same child.

OBJECTIVE: To compare stimulation parameters of peri-modiolar and anti-modiolar electrode arrays using two surgical approaches. METHODS: Impedance, stimulation thresholds, comfortably loud current levels, electrically evoked compound action potential thresholds and electrically evoked stapedial reflex thresholds were compared between 2 arrays implanted in the same child at 5 time points: surgery, activation/day 1, week 1, and months 1 and 3. The peri-modiolar array was implanted via cochleostomy in all children (n = 64), while the anti-modiolar array was inserted via a cochleostomy in 43 children and via the round window in 21 children. RESULTS: The anti-modiolar array had significantly lower impedance, but required higher current levels to elicit thresholds, comfort, electrically evoked compound action potential thresholds and electrically evoked stapedial reflex thresholds than the peri-modiolar array across all time points, particularly in basal electrodes (p < 0.05). The prevalence of open electrodes was similar in anti-modiolar (n = 5) and peri-modiolar (n = 3) arrays. CONCLUSION: Significant but clinically acceptable differences in stimulation parameters between peri-modiolar and anti-modiolar arrays persisted four months after surgery in children using bilateral cochlear implants. The surgical approach used to insert the anti-modiolar array had no overall effect on outcomes.

Clostridium perfringensα-toxin interaction with red cells and model membranes.

The effects of Clostridium perfringensα-toxin on host cells have previously been studied extensively but the biophysical processes associated with toxicity are poorly understood. The work reported here shows that the initial interaction between the toxin and lipid membrane leads to measurable changes in the physical properties and morphology of the membrane. A Langmuir monolayer technique was used to assess the response of different lipid species to toxin. Sphingomyelin and unsaturated phosphatidylcholine showed the highest susceptibility to toxin lypolitic action, with a two stage response to the toxin (an initial, rapid hydrolysis stage followed by the insertion and/or reorganisation of material in the monolayer). Fluorescence confocal microscopy on unsaturated phosphatidylcholine vesicles shows that the toxin initially aggregates at discrete sites followed by the formation of localised "droplets" accumulating the hydrolysis products. This process is accompanied by local increases in the membrane dipole potential by about 50 (±42) mV. In contrast, red blood cells incubated with the toxin suffered a decrease of the membrane dipole potential by 50 (±40) mV in areas of high toxin activity (equivalent to a change in electric field strength of 10(7) V m(-1)) which is sufficient to affect the functioning of the cell membrane. Changes in erythrocyte morphology caused by the toxin are presented, and the early stages of interaction between toxin and membrane are characterised using thermal shape fluctuation analysis of red cells which revealed two distinct regimes of membrane-toxin interaction.

Veterinary and human biobanking practices: enhancing molecular sample integrity.

Animal models have historically informed veterinary and human pathophysiology. Next-generation genomic sequencing and molecular analyses using analytes derived from tissue require integrative approaches to determine macroanalyte integrity as well as morphology for imaging algorithms that can extend translational applications. The field of biospecimen science and biobanking will play critical roles in tissue sample collection and processing to ensure the integrity of macromolecules, aid experimental design, and provide more accurate and reproducible downstream genomic data. Herein, we employ animal experiments to combine protein expression analysis by microscopy with RNA integrity number and quantitative measures of morphologic changes of autolysis. These analyses can be used to predict the effect of preanalytic variables and provide the basis for standardized methods in tissue sample collection and processing. We also discuss the application of digital imaging with quantitative RNA and tissue-based protein measurements to show that genomic methods augment traditional in vivo imaging to support biospecimen science. To make these observations, we have established a time course experiment of murine kidney tissues that predicts conventional measures of RNA integrity by RIN analysis and provides reliable and accurate measures of biospecimen integrity and fitness, in particular for time points less than 3 hours post-tissue resection.

The frequency and impact of ROS1 rearrangement on clinical outcomes in never smokers with lung adenocarcinoma.

BACKGROUND: To determine the frequency and predictive impact of ROS1 rearrangements on treatment outcomes in never-smoking patients with lung adenocarcinoma. PATIENTS AND METHODS: We concurrently analyzed ROS1 and ALK rearrangements and mutations in the epidermal growth factor receptor (EGFR), and KRAS in 208 never smokers with lung adenocarcinoma. ROS1 and ALK rearrangements were identified by fluorescent in situ hybridization. RESULTS: Of 208 tumors screened, 7 (3.4%) were ROS1 rearranged, and 15 (7.2%) were ALK-rearranged. CD74-ROS1 fusions were identified in two patients using reverse transcriptase-polymerase chain reaction. The frequency of ROS1 rearrangement was 5.7% (6 of 105) among EGFR/KRAS/ALK-negative patients. Patients with ROS1 rearrangement had a higher objective response rate (ORR; 60.0% versus 8.5%; P = 0.01) and a longer median progression-free survival (PFS; not reached versus 3.3 months; P = 0.008) to pemetrexed than those without ROS1/ALK rearrangement. The PFS to EGFR-tyrosine kinase inhibitors in patients harboring ROS1 rearrangement was shorter than those without ROS1/ALK rearrangement (2.5 versus 7.8 months; P = 0.01). CONCLUSIONS: The frequency of ROS1 rearrangements in clinically selected patients is higher than that reported for unselected patients, suggesting that ROS1 rearrangement is a druggable target in East-Asian never smokers with lung adenocarcinoma. Given the different treatment outcomes to conventional therapies and availability of ROS1 inhibitors, identification of ROS1 rearrangement can lead to successful treatment in ROS1-rearranged lung adenocarcinomas.

The effect of oxidative stress on the membrane dipole potential of human red blood cells.

The membrane dipole potential (ψ(d)) is an important biophysical determinant of membrane function and a sensitive indicator of lipid organisation. In this study we have used the environmentally sensitive probe di-8-anepps to explore the effects of oxidative stress on the membrane dipole potential of human erythrocytes. Cells suspended in 0.15mM phosphate buffered saline containing 0.1mg/ml albumin maintained a mean value for ψ(d) of 270 (±20) mV over the course of 1hour. In the presence of 0.4mM cumene hydroperoxide there was an increase in ψ(d) of 14 (±7)%, accompanied by a decrease in cell diameter of ~14 (±2)%. Exposure of the cells to 0.4mM hydrogen peroxide caused ψ(d) to decrease by 13 (±8)% at the centre of the cell and 8 (±5)% at the edge whilst the diameter remained constant. In both cases the changes were equivalent to a change in transmembrane electric field of a magnitude of ~10MVm(-1), sufficient to influence membrane function. Raman microspectrometry supported the conclusion that cumene exerts its effect primarily on membrane lipids whilst hydrogen peroxide causes the formation of spectrin-haemoglobin complexes which stiffen the membrane.

PTEN and PIK3CA gene copy numbers and poor outcomes in non-small cell lung cancer patients with gefitinib therapy.

METHODS: Fluorescent in situ hybridisation analyses of PTEN, PIK3CA, EGFR and CEN7 were performed on tumour specimens from patients treated on the expanded access gefitinib trial. Progression-free survival (PFS) and overall survival (OS) were correlated with outcomes in all patients and EGFR wild-type patients. RESULTS: Progression-free survival (hazard ratio=2.54, P<0.001) and OS (hazard ratio=4.04, P<0.001) were significantly shorter in patients whose tumours had all of the following molecular patterns: CEN7 <4 copies per cell, PTEN loss (<2 copies in at least 20% of cells), and PIK3CA gain (>2 copies in at least 40% of cells) both in all and EGFR wild-type only patients. CONCLUSION: The combination of low CEN7 copy number, PTEN loss, and PI3KCA gain may be useful for identifying NSCLC patients unlikely to benefit from treatment with EGFR (TKIs), specifically in wild-type EGFR cases.

Use it or lose it? Lessons learned from the developing brains of children who are deaf and use cochlear implants to hear.

In the present paper, we review what is currently known about the effects of deafness on the developing human auditory system and ask: Without use, does the immature auditory system lose the ability to normally function and mature? Any change to the structure or function of the auditory pathways resulting from a lack of activity will have important implications for future use through an auditory prosthesis such as a cochlear implant. Data to date show that deafness in children arrests and disrupts normal auditory development. Multiple changes to the auditory pathways occur during the period of deafness with the extent and type of change being dependent upon the age and stage of auditory development at onset of deafness, the cause or type of deafness, and the length of time the immature auditory pathways are left without significant input. Structural changes to the auditory nerve, brainstem, and cortex have been described in animal models of deafness as well in humans who are deaf. Functional changes in deaf auditory pathways have been evaluated by using a cochlear implant to stimulate the auditory nerve with electrical pulses. Studies of electrically evoked activity in the immature deaf auditory system have demonstrated that auditory brainstem development is arrested and that thalamo-cortical areas are vulnerable to being taken over by other competitive inputs (cross-modal plasticity). Indeed, enhanced peripheral sight and detection of visual movement in congenitally deaf cats and adults have been linked to activity in specific areas of what would normally be auditory cortex. Cochlear implants can stimulate developmental plasticity in the auditory brainstem even after many years of deafness in childhood but changes in the auditory cortex are limited, at least in part, by the degree of reorganization which occurred during the period of deafness. Consequently, we must identify hearing loss rapidly (i.e., at birth for congenital deficits) and provide cochlear implants to appropriate candidates as soon as possible. Doing so has facilitated auditory development in the thalamo-cortex and allowed children who are deaf to perceive and use spoken language.

Multiple effects of childhood deafness on cortical activity in children receiving bilateral cochlear implants simultaneously.

OBJECTIVE: Auditory development is disrupted without normal hearing but might proceed to some extent depending on the type and onset of deafness. We therefore hypothesized that activity in the auditory cortex would be highly variable in children who are deaf. METHODS: To answer this, activity in the deaf brain was evoked by electrical pulses from newly provided bilateral cochlear implants (CIs) in 72 children (n=144 responses). RESULTS: Responses were categorized by visual inspection into 3 main types which were validated by principal component cluster analyses; 49% had a negative amplitude wave similar to that previously reported in pre-term infants, 26% were dominated by a positive peak typical of responses in young normal hearing children and experienced paediatric CI users, 25% were novel multi-peaked responses. No significant demographic differences, including duration and onset of deafness, were found between response types. However, children with severe biallelic mutations of GJB-2 showed predominately negative peak type responses (79%) as compared with their peers without these mutations who had a more equal distribution between cortical response types. CONCLUSION: Cortical development in children who are deaf is heterogeneous but can be better predicted when the genotype is known to be a GJB-2 mutation. SIGNIFICANCE: Remediation of childhood deafness seeks to restore normal development and function of central auditory functions and thus may need to be tailored to account for effects specific to the aetiology of deafness.

Flow-driven transition and associated velocity profiles in a nematic liquid-crystal cell.

The alignment properties and distribution of flow speed during Poiseuille flow through a microchannel of a nematic liquid crystal in a cell with homeotropic surface alignment has been measured using a combination of conoscopy, fluorescence confocal polarizing microscopy, and time-lapse imaging. Two topologically distinct director profiles, with associated fluid velocity fields, are found to exist with the preferred state dictated by the volumetric flow rate of the liquid crystal. The results show excellent agreement with model data produced using the Ericksen-Leslie nematodynamics theory.

Optical imaging of the effect of in-plane fields on cholesteric liquid crystals.

The effects of in-plane electric fields on the director structure of cholesteric liquid crystals has been imaged in three dimensions using fluorescence confocal polarizing microscopy. The results show that a liquid crystal lying outside the electrode gap can be significantly affected by stray fields occurring above the electrode surface, resulting in a 90 degrees rotation of the cholesteric helix. Distinct differences between the behavior of cholesterics with positive and negative dielectric anisotropies are observed.

Polarization rotator using a hybrid aligned nematic liquid crystal cell.

Using both direct mathematical analysis and numerical modeling based on the predictions by Jones [1] it is shown that if the director in a liquid crystal cell is in a plane which lies at 45 degrees to the incident polarization, then, for normally incident light, the transmission signal which conserves polarization will always have a phase difference of pi/2 from the transmission signal of the orthogonal polarization. This is independent of the director profile in the plane, the cell thickness, the anisotropy of the liquid crystal refractive index and the optical parameters of other isotropic layers in the cell. Based on this realization a hybrid aligned nematic liquid crystal cell has been tested as a thresholdless voltage-controlled polarization rotator. By using a quarter-wave plate to compensate for the phase difference between the two orthogonal output polarizations a simple liquid crystal spatial light modulator has been realized.

Dynamic response of a dual-frequency chiral hybrid aligned nematic liquid-crystal cell.

The dynamic response of a chiral dual-frequency hybrid aligned nematic liquid-crystal cell to a multiple frequency pulse has been characterized using a time-resolved fully leaky guided-mode optical characterization technique. On application of a low-frequency voltage the cell is found to switch to homeotropic alignment, effectively destroying the inherent twist in the cell. When this voltage is immediately followed by a high-frequency voltage the structure is driven into a homogeneously aligned twisted structure. Analysis of the response of the director to this change in effective dielectric anisotropy of the material reveals a form of backflow. This arises due to the combination of the coupling between the rotation and flow of the director, the constraining effect of the pitch on the twist in the cell, and the driving of the director into homogeneous alignment. The measured director profiles have been compared to model profiles generated using the Leslie-Eriksen-Parodi nematodynamics theory, and the viscosity coefficients for the material have been determined.

Optical characterization of a dual-frequency hybrid aligned nematic liquid crystal cell.

The dielectric anisotropy of a highly dispersive dual-frequency nematic liquid crystal (MDA-00-3969 (Merck KGa)) has been determined using the optical fully-leaky guided-mode technique. A 4Vrms sinusoidal voltage was applied across a 5microm hybrid aligned nematic (HAN) cell at various frequencies in both the positive and negative dielectric anisotropy regime. Optical data was collected at each frequency enabling the director profile in each case to be determined using a multi-layer optics model in combination with a liquid crystal free-energy minimization routine. The thresholdless response of the HAN cell combined with the extreme sensitivity of the optical characterization technique has allowed subtle changes in dielectric permittivity with frequency to be observed. The resulting measured dispersion shows excellent agreement with a single Debye-type relaxation model.

Smooth muscle hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen.

BACKGROUND: Airway smooth muscle hypertrophy is closely associated with the pathophysiology of hyper-reactive airways in allergic asthma. OBJECTIVE: To determine whether repeated exposure to allergens during postnatal lung development promotes remodelling of airway smooth muscle. METHODS: Infant, male rhesus monkeys (30-day-old) were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol periodically over 5 months. Smooth muscle mass and bundle size and abundance in conducting airways were measured and compared with age-matched control (filtered air-exposed) monkeys. RESULTS: Total smooth muscle mass and average bundle size were significantly greater in the conducting airways of monkeys exposed to HDMA. Smooth muscle bundle abundance was not affected by exposure to HDMA. CONCLUSION: Repeated cycles of allergen exposure alter postnatal morphogenesis of smooth muscle, affecting both total mass and bundle size, in conducting airways of infant monkeys.

Three-dimensional mapping of smooth muscle in the distal conducting airways of mouse, rabbit, and monkey.

Airway smooth muscle remodeling is implicated in a number of constrictive pulmonary diseases such as asthma and may include changes in smooth muscle orientation and abundance. Both factors were compared in the normal distal bronchioles of the mouse, rabbit, and rhesus monkey (respiratory bronchioles included). Airway smooth muscle was measured by using a three-dimensional approach employing confocal microscopy and whole-mount cytochemistry with fluorochrome-conjugated phalloidin, a probe for polymerized actin. Smooth muscle orientation had a wide range of angles along the airway, but the distribution was conserved among species and among distal airway generations. At the bifurcation of proximal bronchioles, smooth muscle was nearly parallel to the longitudinal axis of the airway. Smooth muscle abundance was significantly different between species (abundance was less in the monkey compared with the mouse and rabbit), and there was a trend for abundance to decrease with each more distal airway generation. This study defines the normal distribution of smooth muscle in three test species and provides a basis for future comparisons with the diseased state.

Evaluation of TGF-alpha and EGFR expression in oral leukoplakia and oral submucous fibrosis by quantitative immunohistochemistry.

OBJECTIVE: Oral leukoplakia (OL) and oral submucous fibrosis (OSMF) are clinically distinct preneoplastic states that precede the development of oral squamous cell carcinoma. Recently, attempts are being made to identify specific molecular event(s) as prognostic markers to identify oral precancerous lesions with higher malignant potential. The goal of the present study was to evaluate the expression of EGFR and its ligand TGF-alpha in OL with dysplasia and OSMF as intermediate markers of malignancy by quantitative immunohistochemistry. METHODS: Oral tissues were stained with monoclonal antibodies to TGF-alpha and EGFR. The results were analyzed with Photoquant image analysis software. RESULTS: The expression of TGF-alpha and EGFR was upregulated in OL, OSMF and oral squamous cell carcinomas relative to normal oral mucosa. The area and intensity of staining of TGF-alpha in the proliferative layers (stratum germinativum) increased linearly in OL with mild, moderate and severe dysplasia as compared to control mucosa (p < 0.05). EGFR levels increased linearly in the stratum spinosum in OL with increasing degrees of dysplasia (p < 0.05). In general, the expression of both proteins in OSMF was similar to that in OL with moderate dysplasia. CONCLUSIONS: EGFR and TGF-alpha represent early markers of malignancy in OL with dysplasia. Quantitative measurement of these proteins may provide intermediate endpoints in prospective chemopreventive trials.

Quantitative estimation of PCNA, c-myc, EGFR and TGF-alpha in oral submucous fibrosis--an immunohistochemical study.

The objectives of the present study were to evaluate the expression of three proliferation markers, viz., epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-alpha) and proliferating cell nuclear antigen (PCNA) and one genomic marker, c-myc in OSMF. Oral tissues were stained with monoclonal antibodies to PCNA, c-myc, TGF-alpha and EGFR. The results were analyzed with Photoquant image analysis software. The expression of PCNA, c-myc, TGF-alpha and EGFR was higher in oral submucous fibrosis (OSMF) than in normal control oral mucosa. The oral epithelium was divided into a proliferative compartment (stratum germinativum) and a differentiated compartment (stratum spinosum). A differential pattern of expression of PCNA and c-myc was observed in OSMF. While the intensity of staining decreased, the percent area of expression of PCNA and c-myc increased in stratum germinativum in OSMF (P<0.05). This suggests that greater proportions of cells exhibit PCNA and c-myc immunoreactivity and are in the proliferative pool in OSMF. TGF-alpha levels increased in the proliferative layers and EGFR levels increased in the differentiated layers (P<0.05) of the oral epithelium in OSMF. Quantitative measurement of these oncoproteins substantiates the precancerous nature of OSMF and may provide intermediate end-points in prospective chemopreventive trials.

Factors modulating the epithelial response to toxicants in tracheobronchial airways.

As one of the principal interfaces between the organism and the environment, the respiratory system is a target for a wide variety of toxicants and carcinogens. The cellular and architectural complexity of the respiratory system appears to play a major role in defining the focal nature of the pulmonary response to environmental stressors. This review will address the biological factors that modulate the response of one of the major target compartments within the respiratory system, the tracheobronchial airway tree. Individual airway segments respond uniquely to toxic stress and this response involves not only the target cell population, e.g. epithelium, but also other components of the airway wall suggesting a trophic interaction within all components of the airway wall in maintaining steady state and responding to injury. A number of biological factors modulate the nature of the response, including: (1) metabolic potential at specific sites for activation and detoxification; (2) the nature of the local inflammatory response; (3) age of the organism at the time of exposure; (4) gender of the exposed organism; (5) history of previous exposure; and (6) species and strain of the organism exposed.