Large-scale spontaneous self-organization and maturation of skeletal muscle tissues on ultra-compliant gelatin hydrogel substrates.

Cellular self-organization is the fundamental driving force behind the complex architectures of native tissue. Yet, attempts at replicating native tissue architectures in vitro often involve complex micro-fabrication methods and materials. While impressive progress has been made within engineered models of striated muscle, the wide adaptation of these models is held back by the need for specific tools and knowhow. In this report, we show that C2C12 myoblasts spontaneously organize into highly aligned myotube tissues on the mm to cm scale, when cultured on sufficiently soft yet fully isotropic gelatin hydrogel substrates. Interestingly, we only observed this phenomenon for hydrogels with Young's modulus of 6 kPa and below. For slightly more rigid compositions, only local micrometer-scale myotube organization was observed, similar to that seen in conventional polystyrene dishes. The hydrogel-supported myotubes could be cultured for multiple weeks and matured into highly contractile phenotypes with notable upregulation of myosin heavy chain, as compared to myotubes developed in conventional petri dishes. The procedure for casting the ultra-soft gelatin hydrogels is straight forward and compatible with standardized laboratory tools. It may thus serve as a simple, yet versatile, approach to generating skeletal muscle tissue of improved physiological relevance for applied and basic research.

3D Printed Stackable Titer Plate Inserts Supporting Three Interconnected Tissue Models for Drug Transport Studies.

Current in vitro drug screening methods often rely on single-cell models and are therefore imprecise in predicting drug absorption, distribution, metabolism, excretion, and toxicity. This study presents a method to fabricate 3D printed inserts that are compatible with commercially available titer plates. Hydrogels can be casted into the inserts and cells can be cultured either in or on the hydrogels. Once individual cell cultures are fully differentiated, the three different cell cultures are stacked on top of each other for biological experiments. To show the possibilities of this approach, three tissue models representing the first pass metabolism is used. The three tissue models are based on gelatin hydrogels and Caco-2, HUVEC, and HepG2 cells to simulate the small intestine, vascular endothelium, and liver, respectively. The device is simple to fabricate, user friendly, and an alternative to microfluidic-based organ on a chip systems. The presented first pass metabolism study allows for gaining information on drug absorption, distribution, metabolism, and, in the future, excretion in one compact device complying the micro titer plate format.

Transglycosylating ß-d-galactosidase and α-l-fucosidase from Paenibacillus sp. 3179 from a hot spring in East Greenland.

Thermal springs are excellent locations for discovery of thermostable microorganisms and enzymes. In this study, we identify a novel thermotolerant bacterial strain related to Paenibacillus dendritiformis, denoted Paenibacillus sp. 3179, which was isolated from a thermal spring in East Greenland. A functional expression library of the strain was constructed, and the library screened for ß-d-galactosidase and α-l-fucosidase activities on chromogenic substrates. This identified two genes encoding a ß-d-galactosidase and an α-l-fucosidase, respectively. The enzymes were recombinantly expressed, purified, and characterized using oNPG (2-nitrophenyl-ß-d-galactopyranoside) and pNP-fucose (4-nitrophenyl-α-l-fucopyranoside), respectively. The enzymes were shown to have optimal activity at 50°C and pH 7-8, and they were able to hydrolyze as well as transglycosylate natural carbohydrates. The transglycosylation activities were investigated using TLC and HPLC, and the ß-d-galactosidase was shown to produce the galactooligosaccharides (GOS) 6'-O-galactosyllactose and 3'-O-galactosyllactose using lactose as substrate, whereas the α-l-fucosidase was able to transfer the fucose moiety from pNP-fuc to lactose, thereby forming 2'-O-fucosyllactose. Since enzymes that are able to transglycosylate carbohydrates at elevated temperature are desirable in many industrial processes, including food and dairy production, we foresee the potential use of enzymes from Paenibacillus sp. 3179 in the production of, for example, instant formula.

Microcontainers for oral insulin delivery - In vitro studies of permeation enhancement.

Oral delivery of peptides is challenging due to their low uptake through the small intestinal epithelium. Tight junctions, connecting the enterocytes, impede permeability, often necessitating the use of permeation enhancers in the formulation. Loading of peptide and permeation enhancer into micro-scale devices, such as microcontainers, can potentially confine the effective absorptive area through unidirectional release and thereby enhance absorption. This concept is investigated by in vitro permeation studies of insulin across Caco-2 cell and Caco-2/HT29-MTX-E12 co-culture monolayers mimicking the intestinal absorption barrier. The importance of proximity between the microcontainers and the barrier is assessed, by keeping the amounts of insulin and sodium caprate fixed throughout all experiments, while collectively orienting the unidirectional release towards the cell monolayers. Increasing the distance is observed to have a negative effect on insulin permeation matching a one-phase exponential decay function, while no difference in insulin transport is observed between Caco-2 and co-culture monolayers. Although there are no signs of cytotoxicity caused by the microcontainer material, reversible cell deterioration, as a consequence of high local concentrations of sodium caprate, becomes evident upon qualitative assessment of the cell monolayers. These results both suggest a potential of increasing oral bioavailability of peptides by the use of microcontainers, while simultaneously visualising the ability of regaining monolayer integrity upon local permeation enhancer induced toxicity.

Effects of Hearing Loss and Fast-Acting Compression on Amplitude Modulation Perception and Speech Intelligibility.

OBJECTIVE: The purpose was to investigate the effects of hearing-loss and fast-acting compression on speech intelligibility and two measures of temporal modulation sensitivity. DESIGN: Twelve adults with normal hearing (NH) and 16 adults with mild to moderately severe sensorineural hearing loss were tested. Amplitude modulation detection and modulation-depth discrimination (MDD) thresholds with sinusoidal carriers of 1 or 5 kHz and modulators in the range from 8 to 256 Hz were used as measures of temporal modulation sensitivity. Speech intelligibility was assessed by obtaining speech reception thresholds in stationary and fluctuating background noise. All thresholds were obtained with and without compression (using a fixed compression ratio of 2:1). RESULTS: For modulation detection, the thresholds were similar or lower for the group with hearing loss than for the group with NH. In contrast, the MDD thresholds were higher for the group with hearing loss than for the group with NH. Fast-acting compression increased the modulation detection thresholds, while no effect of compression on the MDD thresholds was observed. The speech reception thresholds obtained in stationary noise were slightly increased in the compression condition relative to the linear processing condition, whereas no difference in the speech reception thresholds obtained in fluctuating noise was observed. For the group with NH, individual differences in the MDD thresholds could account for 72% of the variability in the speech reception thresholds obtained in stationary noise, whereas the correlation was insignificant for the hearing-loss group. CONCLUSIONS: Fast-acting compression can restore modulation detection thresholds for listeners with hearing loss to the values observed for listeners with NH. Despite this normalization of the modulation detection thresholds, compression does not seem to provide a benefit for speech intelligibility. Furthermore, fast-acting compression may not be able to restore MDD thresholds to the values observed for listeners with NH, suggesting that the two measures of amplitude modulation sensitivity represent different aspects of temporal processing. For listeners with NH, the ability to discriminate modulation depth was highly correlated with speech intelligibility in stationary noise.

Characterization of thin gelatin hydrogel membranes with balloon properties for dynamic tissue engineering.

Cell or tissue stretching and strain are present in any in vivo environment, but is difficult to reproduce in vitro. Here, we describe a simple method for casting a thin (about 500 µm) and soft (about 0.3 kPa) hydrogel of gelatin and a method for characterizing the mechanical properties of the hydrogel simply by changing pressure with a water column. The gelatin is crosslinked with mTransglutaminase and the area of the resulting hydrogel can be increased up 13-fold by increasing the radial water pressure. This is far beyond physiological stretches observed in vivo. Actuating the hydrogel with a radial force achieves both information about stiffness, stretchability, and contractability, which are relevant properties for tissue engineering purposes. Cells could be stretched and contracted using the gelatin membrane. Gelatin is a commonly used polymer for hydrogels in tissue engineering, and the discovered reversible stretching is particularly interesting for organ modeling applications.

Investigating time-efficiency of forward masking paradigms for estimating basilar membrane input-output characteristics.

It is well known that pure-tone audiometry does not sufficiently describe individual hearing loss (HL) and that additional measures beyond pure-tone sensitivity might improve the diagnostics of hearing deficits. Specifically, forward masking experiments to estimate basilar-membrane (BM) input-output (I/O) function have been proposed. However, such measures are very time consuming. The present study investigated possible modifications of the temporal masking curve (TMC) paradigm to improve time and measurement efficiency. In experiment 1, estimates of knee point (KP) and compression ratio (CR) of individual BM I/Os were derived without considering the corresponding individual "off-frequency" TMC. While accurate estimation of KPs was possible, it is difficult to ensure that the tested dynamic range is sufficient. Therefore, in experiment 2, a TMC-based paradigm, referred to as the "gap method", was tested. In contrast to the standard TMC paradigm, the maker level was kept fixed and the "gap threshold" was obtained, such that the masker just masks a low-level (12 dB sensation level) signal. It is argued that this modification allows for better control of the tested stimulus level range, which appears to be the main drawback of the conventional TMC method. The results from the present study were consistent with the literature when estimating KP levels, but showed some limitations regarding the estimation of the CR values. Perspectives and limitations of both approaches are discussed.

Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases.

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography-tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin-like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20 °C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor.

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.

Draft genome sequences of two protease-producing strains of arsukibacterium, isolated from two cold and alkaline environments.

Arsukibacterium ikkense GCM72(T) and a close relative, Arsukibacterium sp. MJ3, were isolated from two cold and alkaline environments as producers of extracellular proteolytic enzymes active at high pH and low temperature. This report describes the two draft genome sequences, which may serve as sources of future industrial enzymes.

Refining a model of hearing impairment using speech psychophysics.

The premise of this study is that models of hearing, in general, and of individual hearing impairment, in particular, can be improved by using speech test results as an integral part of the modeling process. A conceptual iterative procedure is presented which, for an individual, considers measures of sensitivity, cochlear compression, and phonetic confusions using the Diagnostic Rhyme Test (DRT) framework. The suggested approach is exemplified by presenting data from three hearing-impaired listeners and results obtained with models of the hearing impairment of the individuals. The work reveals that the DRT data provide valuable information of the damaged periphery and that the non-speech and speech data are complementary in obtaining the best model for an individual.

Effects of tonotopicity, adaptation, modulation tuning, and temporal coherence in "primitive" auditory stream segregation.

The perceptual organization of two-tone sequences into auditory streams was investigated using a modeling framework consisting of an auditory pre-processing front end [Dau et al., J. Acoust. Soc. Am. 102, 2892-2905 (1997)] combined with a temporal coherence-analysis back end [Elhilali et al., Neuron 61, 317-329 (2009)]. Two experimental paradigms were considered: (i) Stream segregation as a function of tone repetition time (TRT) and frequency separation (Δf) and (ii) grouping of distant spectral components based on onset/offset synchrony. The simulated and experimental results of the present study supported the hypothesis that forward masking enhances the ability to perceptually segregate spectrally close tone sequences. Furthermore, the modeling suggested that effects of neural adaptation and processing though modulation-frequency selective filters may enhance the sensitivity to onset asynchrony of spectral components, facilitating the listeners' ability to segregate temporally overlapping sounds into separate auditory objects. Overall, the modeling framework may be useful to study the contributions of bottom-up auditory features on "primitive" grouping, also in more complex acoustic scenarios than those considered here.

Simulating psychophysical tuning curves in listeners with dead regions.

OBJECTIVE: This study investigates the relation between diagnosis of dead regions based on the off-frequency psychophysical tuning curve (PTC) tip and the frequency and level of the probe tone. DESIGN: A previously developed functional model of auditory processing was used to simulate the complete loss of inner hair cells (IHC), dysfunction of outer hair cells (OHC), complete loss of IHCs in combination with OHC dysfunction, and IHC insensitivity. The model predictions were verified through comparison with experimental data. STUDY SAMPLE: This study compares PTC data of five normal-hearing listeners and six hearing-impaired listeners with model-simulated PTC data. RESULTS: It was shown that OHC activity and IHC insensitivity may significantly alter the shift of PTC tips with increasing probe level. CONCLUSIONS: Model results suggest that OHC activity and IHC insensitivity can change the outcome of dead region diagnosis using PTCs. Supplementary to PTC dead region diagnostic information, model results may provide additional information regarding the edge frequency of a dead region and OHC function.

Quantum dot-based nanosensors for diagnosis via enzyme activity measurement.

Enzymes are essential in the human body, and the disorder of enzymatic activities has been associated with many different diseases and stages of disease. Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have garnered great attention in molecular diagnostics. Owing to their superior optical properties, tunable and narrow emissions, stable brightness and long lifetime, QD-based enzyme activity measurement has demonstrated improved detection sensitivity, which is considered particularly valuable for early disease diagnosis. Recent studies have also shown that QD-based nanosensors are capable of probing multiple enzyme activities simultaneously. This review highlights the current development of QD-based nanosensors for enzyme detection. The enzyme-QD hybrid system, equipped with unique electronic, optical and catalytic properties, is envisioned as a potential solution in addressing challenges in diagnostics and therapeutics.

DNA-based sensor for real-time measurement of the enzymatic activity of human topoisomerase I.

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

Characterizing auditory processing and perception in individual listeners with sensorineural hearing loss.

This study considered consequences of sensorineural hearing loss in ten listeners. The characterization of individual hearing loss was based on psychoacoustic data addressing audiometric pure-tone sensitivity, cochlear compression, frequency selectivity, temporal resolution, and intensity discrimination. In the experiments it was found that listeners with comparable audiograms can show very different results in the supra-threshold measures. In an attempt to account for the observed individual data, a model of auditory signal processing and perception [Jepsen et al., J. Acoust. Soc. Am. 124, 422-438 (2008)] was used as a framework. The parameters of the cochlear processing stage of the model were adjusted to account for behaviorally estimated individual basilar-membrane input-output functions and the audiogram, from which the amounts of inner hair-cell and outer hair-cell losses were estimated as a function of frequency. All other model parameters were left unchanged. The predictions showed a reasonably good agreement with the measured individual data in the frequency selectivity and forward masking conditions while the variation of intensity discrimination thresholds across listeners was underestimated by the model. The model and the associated parameters for individual hearing-impaired listeners might be useful for investigating effects of individual hearing impairment in more complex conditions, such as speech intelligibility in noise.

Effects of pulsing of the target tone on the audibility of partials in inharmonic complex tones.

The audibility of partials was measured for complex tones with partials uniformly spaced on an ERB(N)-number scale. On each trial, subjects heard a sinusoidal "probe" followed by a complex tone. The probe was mistuned downwards or upwards (at random) by 3% or 4.5% from the frequency of one randomly selected partial in the complex (the "target"). The subject indicated whether the target was higher or lower in frequency than the probe. The probe and the target were pulsed on and off and the ramp times and inter-pulse intervals were systematically varied. Performance was better for longer ramp times and longer inter-pulse intervals. In a second experiment, the ability to detect which of two complex tones contained a pulsed partial was measured. The pattern of results was similar to that for experiment 1. A model of auditory processing including an adaptation stage was able to account for the general pattern of the results of experiment 2. The results suggest that the improvement in ability to hear out a partial in a complex tone produced by pulsing that partial is partly mediated by a release from adaptation produced by the pulsing, and does not result solely from reduction of perceptual confusion.

A computational model of human auditory signal processing and perception.

A model of computational auditory signal-processing and perception that accounts for various aspects of simultaneous and nonsimultaneous masking in human listeners is presented. The model is based on the modulation filterbank model described by Dau et al. [J. Acoust. Soc. Am. 102, 2892 (1997)] but includes major changes at the peripheral and more central stages of processing. The model contains outer- and middle-ear transformations, a nonlinear basilar-membrane processing stage, a hair-cell transduction stage, a squaring expansion, an adaptation stage, a 150-Hz lowpass modulation filter, a bandpass modulation filterbank, a constant-variance internal noise, and an optimal detector stage. The model was evaluated in experimental conditions that reflect, to a different degree, effects of compression as well as spectral and temporal resolution in auditory processing. The experiments include intensity discrimination with pure tones and broadband noise, tone-in-noise detection, spectral masking with narrow-band signals and maskers, forward masking with tone signals and tone or noise maskers, and amplitude-modulation detection with narrow- and wideband noise carriers. The model can account for most of the key properties of the data and is more powerful than the original model. The model might be useful as a front end in technical applications.