Chondrocyte redifferentiation and construct mechanical property development in single-component photocrosslinkable hydrogels

Hydrogels are promising materials for cartilage repair, but the properties required for optimal functional outcomes are not yet known. In this study, we functionalized four materials that are commonly used in cartilage tissue engineering and evaluated them using in vitro cultures. Gelatin, hyaluronic acid, polyethylene glycol, and alginate were functionalized with methacrylic anhydride to make them photocrosslinkable. We found that the responses of encapsulated human chondrocytes were highly dependent on hydrogel type. Gelatin hydrogels supported cell proliferation and the deposition of a glycosaminoglycan rich matrix with significant mechanical functionality. However, cells had a dedifferentiated phenotype, with high expression of collagen type I. Chondrocytes showed the best redifferentiation in hyaluronic acid hydrogels, but the newly formed matrix was highly localized to the pericellular regions, and these gels degraded rapidly. Polyethylene glycol hydrogels, as a bioinert control, did not promote any strong responses. Alginate hydrogels did not support the deposition of new matrix, and the stiffness decreased during culture. The markedly different response of chondrocytes to these four photocrosslinkable hydrogels demonstrates the importance of material properties for chondrogenesis and extracellular matrix production, which are critical for effective cartilage repair.

High-resolution GNSS-tracked drifter for studying surface dispersion in shallow water

The use of GNSS tracked Lagrangian drifters allows more realistic quantification of fluid motion and dispersion coefficients than Eulerian techniques because such drifters are analogues of particles that are relevant to flow field characterisation and pollutant dispersion. Using the fast growing Real Time Kinematic (RTK) positioning technique derived from Global Satellite Navigation Systems (GNSS), drifters are developed for high frequency (10 Hz) sampling with position estimates to centimetre accuracy. The drifters are designed with small size and less direct wind drag to follow the sub-surface flow which characterizes dispersion in shallow waters. An analysis of position error from stationary observation indicates that the drifter can efficiently resolve motion up to 1 Hz. The result of the field deployments of the drifter in conjunction with acoustic Eulerian devices shows higher estimate of the drifter streamwise velocities. Single particle statistical analysis of field deployments in a shallow estuarine zone yielded dispersion coefficients estimate comparable to those of dye tracer studies. The drifters capture the tidal elevation during field studies in a tidal estuary.

The influence of flight speed on the ranging performance of bats using frequency modulated echolocation pulses

Many species of bat use ultrasonic frequency modulated (FM) pulses to measure the distance to objects by timing the emission and reception of each pulse. Echolocation is mainly used in flight. Since the flight speed of bats often exceeds 1% of the speed of sound,Doppler effects will lead to compression of the time between emission and reception as well as an elevation of the echo frequencies, resulting in a distortion of the perceived range. This paper describes the consequences of these Doppler effects on the ranging performance of bats using different pulse designs. The consequences of Doppler effects on ranging performance described in this paper assume bats to have a very accurate ranging resolution, which is feasible with a filterbank receiver. By modeling two receiver types, it was first established that the effects of Doppler compression are virtually independent of the receiver type. Then, used a cross-correlation model was used to investigate the effect of flight speed on Doppler tolerance and range–Doppler coupling separately. This paper further shows how pulse duration, bandwidth, function type, and harmonics influence Doppler tolerance and range–Doppler coupling. The influence of each signal parameter is illustrated using calls of several bat species. It is argued that range–Doppler coupling is a significant source of error in bat echolocation, and various strategies bats could employ to deal with this problem, including the use of range rate information are discussed.

Acoustic identification of twelve species of echolocating bat by discriminant function analysis and artificial neural networks

We recorded echolocation calls from 14 sympatric species of bat in Britain. Once digitised, one temporal and four spectral features were measured from each call. The frequency-time course of each call was approximated by fitting eight mathematical functions, and the goodness of fit, represented by the mean-squared error, was calculated. Measurements were taken using an automated process that extracted a single call from background noise and measured all variables without intervention. Two species of Rhinolophus were easily identified from call duration and spectral measurements. For the remaining 12 species, discriminant function analysis and multilayer back-propagation perceptrons were used to classify calls to species level. Analyses were carried out with and without the inclusion of curve-fitting data to evaluate its usefulness in distinguishing among species. Discriminant function analysis achieved an overall correct classification rate of 79% with curve-fitting data included, while an artificial neural network achieved 87%. The removal of curve-fitting data improved the performance of the discriminant function analysis by 2 %, while the performance of a perceptron decreased by 2 %. However, an increase in correct identification rates when curve-fitting information was included was not found for all species. The use of a hierarchical classification system, whereby calls were first classified to genus level and then to species level, had little effect on correct classification rates by discriminant function analysis but did improve rates achieved by perceptrons. This is the first published study to use artificial neural networks to classify the echolocation calls of bats to species level. Our findings are discussed in terms of recent advances in recording and analysis technologies, and are related to factors causing convergence and divergence of echolocation call design in bats.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

The primacy of vital signs – Acute care nurses’ and midwives’ use of physical assessment skills: A cross sectional study

BACKGROUND: Registered nurses and midwives play an essential role in detecting patients at risk of deterioration through ongoing assessment and action in response to changing health status. Yet, evidence suggests that clinical deterioration frequently goes unnoticed in hospitalised patients. While much attention has been paid to early warning and rapid response systems, little research has examined factors related to physical assessment skills. OBJECTIVES: To determine a minimum data set of core skills used during nursing assessment of hospitalised patients and identify nurse and workplace predictors of the use of physical assessment to detect patient deterioration. DESIGN: The study used a single-centre, cross-sectional survey design. SETTING and PARTICIPANTS: The study included 434 registered nurses and midwives (Grades 5-7) involved in clinical care of patients on acute care wards, including medicine, surgery, oncology, mental health and maternity service areas, at a 929-bed tertiary referral teaching hospital in Southeast Queensland, Australia. METHODS: We conducted a hospital-wide survey of registered nurses and midwives using the 133-item Physical Assessment Skills Inventory and the 58-item Barriers to Registered Nurses’ Use of Physical Assessment scale. Median frequency for each physical assessment skill was calculated to determine core skills. To explore predictors of core skill utilisation, backward stepwise general linear modelling was conducted. Means and regression coefficients are reported with 95% confidence intervals. A p value < .05 was considered significant for all analyses. RESULTS: Core skills used by most nurses every time they worked included assessment of temperature, oxygen saturation, blood pressure, breathing effort, skin, wound and mental status. Reliance on others and technology (F = 35.77, p < .001), lack of confidence (F = 5.52, p = .02), work area (F = 3.79, p = .002), and clinical role (F = 44.24, p < .001) were significant predictors of the extent of physical assessment skill use. CONCLUSIONS: The increasing acuity of the acute care patient plausibly warrants more than vital signs assessment; however, our study confirms nurses’ physical assessment core skill set is mainly comprised of vital signs. The focus on these endpoints of deterioration as dictated by early warning and rapid response systems may divert attention from and devalue comprehensive nursing assessment that could detect subtle changes in health status earlier in the patient's hospitalisation.

Single voxel Magnetic Resonance Spectroscopic (1H-MRS) analysis of deep brain structures after traumatic prefrontal brain injury (TBI) in rat

Chronic difficulties arising from mild brain injury (TBI) are difficult to predict because the processes underlying changes after TBI are poorly understood. In mild brain injury the extent of neuropsychiatric and cognitive symptoms correspond poorly to overt tissue loss (Barth 1983; Liu 2010). Cellular, immune and hormonal cascades occurring after injury and continuing during the healing process may impact uninjured brain regions sensitive to the effects of physiological and emotional stress, which receive projections from the injury site. Changes in these most basic properties due to injury or disease have profound implications for virtually every aspect of brain function through disruption of neurotransmitter, neuroendocrine and metabolic systems. In order to screen for changes in transmitter and metabolic activity, in this study we developed Single voxel proton Magnetic Resonance Spectroscopy (1H-MRS) for use in both injured and control animals. We first evaluated if 1H-MRS could be used to evaluate in vivo, alterations in brain metabolism and catabolism of the prefrontal cortex, amygdala and ventral hippocampus in both control and injured animals after controlled cortical impact injury to the rat prefrontal cortex. We found that metabolite measurements for Myo-Inositol, Choline, creatine, Glutamate+Glutamine, and N-acetyl-acetate are attainable in deep brain structures in vivo in injured and controls rats. We next seek to evaluate longitudinally, in vivo, alterations in brain metabolism and catabolism of the prefrontal cortex, amygdala and ventral hippocampus during the first month after controlled cortical impact injury to the rat prefrontal cortex. These ongoing studies will provide data on the changes in transmitters and metabolites over time in injured and non-injured subjects. These studies address some of the fundamental questions about how mild brain injury has such diverse effects on overall brain health and function.

Soft-switching single inductor current-fed push-pull converter for PV applications

A switching control strategy is proposed for single inductor current-fed push-pull converter with a secondary side active voltage doubler rectifier or a voltage rectifier used in photovoltaic (PV) grid interfacing. The proposed switching control strategy helps to turn-on and turn-off the primary side power switches with zero-voltage and zero-current switching. The operation of the push-pull converter is analyzed for two modes of operation. The feasibility of the proposed switching control strategy is validated using simulation and experimental results.

Real-time assessment of GNSS observation noise with single receivers

Stochastic modelling is critical in GNSS data processing. Currently, GNSS data processing commonly relies on the empirical stochastic model which may not reflect the actual data quality or noise characteristics. This paper examines the real-time GNSS observation noise estimation methods enabling to determine the observation variance from single receiver data stream. The methods involve three steps: forming linear combination, handling the ionosphere and ambiguity bias and variance estimation. Two distinguished ways are applied to overcome the ionosphere and ambiguity biases, known as the time differenced method and polynomial prediction method respectively. The real time variance estimation methods are compared with the zero-baseline and short-baseline methods. The proposed method only requires single receiver observation, thus applicable to both differenced and un-differenced data processing modes. However, the methods may be subject to the normal ionosphere conditions and low autocorrelation GNSS receivers. Experimental results also indicate the proposed method can result on more realistic parameter precision.

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

Experimental and numerical investigation of strain-rate dependent mechanical properties of single living cells

The objective of this project is to investigate the strain-rate dependent mechanical behaviour of single living cells using both experimental and numerical techniques. The results revealed that living cells behave as porohyperlastic materials and that both solid and fluid phases within the cells play important roles in their mechanical responses. The research reported in this thesis provides a better understanding of the mechanisms underlying the cellular responses to external mechanical loadings and of the process of mechanical signal transduction in living cells. It would help us to enhance knowledge of and insight into the role of mechanical forces in supporting tissue regeneration or degeneration.

Frequency of convergence and accommodative disorders in a clinical population of Mashhad, Iran

Purpose To investigate the frequency of convergence and accommodation anomalies in an optometric clinical setting in Mashhad, Iran, and to determine tests with highest accuracy in diagnosing these anomalies. Methods From 261 patients who came to the optometric clinics of Mashhad University of Medical Sciences during a month, 83 of them were included in the study based on the inclusion criteria. Near point of convergence (NPC), near and distance heterophoria, monocular and binocular accommodative facility (MAF and BAF, respectively), lag of accommodation, positive and negative fusional vergences (PFV and NFV, respectively), AC/A ratio, relative accommodation, and amplitude of accommodation (AA) were measured to diagnose the convergence and accommodation anomalies. The results were also compared between symptomatic and asymptomatic patients. The accuracy of these tests was explored using sensitivity (S), specificity (Sp), and positive and negative likelihood ratios (LR+, LR−). Results Mean age of the patients was 21.3 ± 3.5 years and 14.5% of them had specific binocular and accommodative symptoms. Convergence and accommodative anomalies were found in 19.3% of the patients; accommodative excess (4.8%) and convergence insufficiency (3.6%) were the most common accommodative and convergence disorders, respectively. Symptomatic patients showed lower values for BAF (p = .003), MAF (p = .001), as well as AA (p = .001) compared with asymptomatic patients. Moreover, BAF (S = 75%, Sp = 62%) and MAF (S = 62%, Sp = 89%) were the most accurate tests for detecting accommodative and convergence disorders in terms of both sensitivity and specificity. Conclusions Convergence and accommodative anomalies are the most common binocular disorders in optometric patients. Including tests of monocular and binocular accommodative facility in routine eye examinations as accurate tests to diagnose these anomalies requires further investigation.