Dynamic reconfiguration of pro-apoptotic BAK on membranes.

BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.

[Adenoids-diagnosis and treatment: the new German S2k guideline]. / Adenoide Vegetationen ­ Diagnostik und Therapie ­ die neue S2k-Leitlinie.

Hyperplasia of the pharyngeal tonsils is to be considered pathologic when nasopharyngeal symptoms of mechanical obstruction and/or chronic inflammation occur. Chronic Eustachian tube dysfunction can result in various middle ear diseases such as conductive hearing loss, cholesteatoma, and recurrent acute otitis media. During examination, attention should be paid to the presence of adenoid facies (long face syndrome), with a permanently open mouth and visible tip of the tongue. In the case of severe symptoms and/or failure of conservative treatment, adenoidectomy is usually performed on an outpatient basis. Conventional curettage remains the established standard treatment in Germany. Histologic evaluation is indicated for clinical evidence of mucopolysaccharidoses. Due to the risk of hemorrhage, the preoperative bleeding questionnaire, which is obligatory before every pediatric surgery, is referred to. Recurrence of adenoids is possible despite correct adenoidectomy. Before discharge home, otorhinolaryngologic inspection of the nasopharynx for secondary bleeding should be performed and anesthesiologic clearance obtained.

Adenoid hypertrophy-​diagnosis and treatment: the new S2k guideline.

Hyperplasia of the pharyngeal tonsils is to be considered pathologic when nasopharyngeal symptoms of mechanical obstruction and/or chronic inflammation occur. Chronic Eustachian tube dysfunction can result in various middle ear diseases such as conductive hearing loss, cholesteatoma, and recurrent acute otitis media. During examination, attention should be paid to the presence of adenoid facies (long face syndrome), with a permanently open mouth and visible tip of the tongue. In the case of severe symptoms and/or failure of conservative treatment, adenoidectomy is usually performed on an outpatient basis. Conventional curettage remains the established standard treatment in Germany. Histologic evaluation is indicated for clinical evidence of mucopolysaccharidoses. Due to the risk of hemorrhage, the preoperative bleeding questionnaire, which is obligatory before every pediatric surgery, is referred to. Recurrence of adenoids is possible despite correct adenoidectomy. Before discharge home, otorhinolaryngologic inspection of the nasopharynx for secondary bleeding should be performed and anesthesiologic clearance obtained.

The Bak core dimer focuses triacylglycerides in the membrane.

Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.

Quantification of mesenchymal stem cell growth rates through secretory and excretory biomolecules in conditioned media via Fresnel reflection.

An efficient and low cost optical method for directly measuring the concentration of homogenous biological solutes is proposed and demonstrated. The proposed system operates by Fresnel reflection, with a flat-cleaved single-mode fiber serving as the sensor probe. A laser provides a 12.9 dBm sensor signal at 1,550 nm, while a computer-controlled optical power meter measures the power of the signal returned by the probe. Three different mesenchymal stem cell (MSC) lines were obtained, sub-cultured and trypsinized daily over 9 days. Counts were measured using a haemocytometer and the conditioned media (CM) was collected daily and stored at -80 °C. MSCs release excretory biomolecules proportional to their growth rate into the CM, which changes the refractive index of the latter. The sensor is capable of detecting changes in the number of stem cells via correlation to the change in the refractive index of the CM, with the measured power loss decreasing approximately 0.4 dB in the CM sample per average 1,000 cells in the MSC subculture. The proposed system is highly cost-effective, simple to deploy, operate, and maintain, is non-destructive, and allows reliable real-time measurement of various stem cell proliferation parameters.

Homozygous null mutations in ZMPSTE24 in restrictive dermopathy: evidence of genetic heterogeneity.

Restrictive dermopathy (RD) results in stillbirth or early neonatal death. RD is characterized by prematurity, intrauterine growth retardation, fixed facial expression, micrognathia, mouth in the 'o' position, rigid and tense skin with erosions and denudations and multiple joint contractures. Nearly all 25 previously reported neonates with RD had homozygous or compound heterozygous null mutations in the ZMPSTE24 gene. Here, we report three new cases of RD; all died within 3 weeks of birth. One of them had a previously reported homozygous c.1085dupT (p.Leu362PhefsX19) mutation, the second case had a novel homozygous c.1020G>A (p.Trp340X) null mutation in ZMPSTE24, but the third case, a stillborn with features of RD except for the presence of tapering rather than rounded, bulbous digits, harbored no disease-causing mutations in LMNA or ZMPSTE24. In the newborn with a novel ZMPSTE24 mutation, unique features included butterfly-shaped thoracic 5 vertebra and the bulbous appearance of the distal clavicles. Skin biopsies from both the stillborn fetus and the newborn with c.1020G>A ZMPSTE24 mutation showed absence of elastic fibers throughout the dermis. This report provides evidence of genetic heterogeneity among RD and concludes that there may be an additional locus for RD which remains to be identified.

Ceramic encapsulation with polymer via co-axial electrohydrodynamic jetting.

Co-flowing media of a polymeric solution (30 wt% polymethylsilsesquioxane in ethanol) and a ceramic suspension (10 wt% alumina in glycerol) were subjected to an electric field. The flow rates of the media (10-30 microL min(-1)) and the applied voltage (0-11 kV) were varied systematically during the experimentation by making gradual increments to each variable, which enabled the construction of a mode selection map. Under co-flowing conditions, with the flow rate of polymer solution (outer needle) twice that of the ceramic suspension (inner needle), encapsulated droplets of polymer-coated alumina were produced within stable cone-jet mode. These were collected in a thin film of water and the resultant particle size varied between 1 and 38 microm. Encapsulation was confirmed with scanning electron microscopy and element analysis.

Electrohydrodynamic jetting behaviour of polyhedral oligomeric silsesquioxane nanocomposite.

In this paper, we investigate in detail the electrohydrodynamic spraying of a nonbiodegradable nanocomposite polyhedral oligomeric silsesquioxane polymer developed in our laboratories and currently being explored for coating metallic stent materials. Different concentrations of the polymer have been dissolved to prepare, characterise, and electrohydrodynamically deposit the polymer on stainless steel. From the experiments, the solution containing 15 wt% polymer was selected for further investigation. The variation of film/coating thickness as a function of spraying time was studied and the structural features of the film were assessed using microscopy. Films were also tensile tested. This study has identified a process and conditions which can be used in our stent coating research.

Generation of multilayered structures for biomedical applications using a novel tri-needle coaxial device and electrohydrodynamic flow.

In this short communication, we describe the scope and flexibility of using a novel device containing three coaxially arranged needles to form a variety of novel morphologies. Different combinations of materials are subjected to controlled flow through the device under the influence of an applied electric field. The resulting electrohydrodynamic flow allows us to prepare double-layered bubbles, porous encapsulated threads and nanocapsules containing three layers. The ability to process such multilayered structures is very significant for biomedical engineering applications, for example, generating capsules for drug delivery, which can provide multistage controlled release.

Preparation of polymeric and ceramic porous capsules by a novel electrohydrodynamic process.

The preparation of capsules for medical and industrial use can be achieved via several conventional routes, yielding either hard or soft receptacles, depending on the type and the content of the material to be encapsulated. Together with tablets, capsules are amongst the most commonly used means of administering medication and this makes progress in capsule preparation technology a key area of drug delivery research. Here we uncover new technology for the preparation of capsules with porous chambers. The novelty is signified in the use of an electrohydrodynamic process engineering route and its potential is elucidated using a polymeric material; polymethylsilsesquioxane, which can be converted into an identical ceramic form by means of simple pyrolysis. Thus, both polymeric and ceramic capsules have been prepared. The effects of process control parameters such as the applied voltage and flow rate, on the characteristics of the capsules prepared are discussed.

Numbness of the lower lip does not adversely affect quality of life or patients' satisfaction after mandibular orthognathic surgery.

Measures of patient-reported quality of life (QoL) are increasingly being used to tailor services that are funded by Clinical Commissioning Groups (CCG) in England. Mandibular osteotomies may result in altered sensation of the lower lip, but we know of limited evidence about the resulting effect on QoL. The modified Bristol orthognathic patient outcomes questionnaire was given to patients who had mandibular osteotomies at the Queen Elizabeth Hospital, Birmingham, between March 2006 and April 2016. Questionnaires were collected at the final orthognathic postoperative appointment. The significance of the difference in QoL between those who had altered sensation of the lower lip and those who did not was compared using a two-tailed t test. During this period 170 patients had mandibular orthognathic operations. Completed questionnaires were received from 117 of those patients (69%) during this period, after a follow up of about six months. We found no significant difference between the perceived benefits of treatment between the 41 who had altered sensation and the 74 who did not (p=0.30). Only 5/41 who reported residual numbness six months postoperatively stated that they would not choose to have the same treatment again. In conclusion, orthognathic surgery results in an appreciable improvement in QoL and should continue to be funded by CCG in England. Contrary to the perception of some clinicians, those patients with residual numbness of the lip did not have significantly poorer QoL. Future interpretation of the data will be improved if they are collected both before and after the operation.

Synthesis and evaluation on pH- and temperature-responsive chitosan-p(MAA-co-NIPAM) hydrogels.

In this study, chitosan-poly(methacrylic acid-co-N-isopropylacrylamide) [chitosan-p(MAA-co-NIPAM)] hydrogels were synthesized by emulsion polymerization. In order to be used as a carrier for drug delivery systems, the hydrogels had to be biocompatible, biodegradable and multi-responsive. The polymerization was performed by copolymerize MAA and NIPAM with chitosan polymer to produce a chitosan-based hydrogel. Due to instability during synthesis and complexity of components to produce the hydrogel, further study at different times of reaction is important to observe the synthesis process, the effect of end product on swelling behaviour and the most important is to find the best way to control the hydrogel synthesis in order to have an optimal swelling behaviour for drug release application. Studied by using Fourier transform infra-red (FTIR) spectroscopy found that, the synthesized was successfully produced stable chitosan-based hydrogel with PNIPAM continuously covered the outer surface of hydrogel which influenced much on the stability during synthesis. The chitosan and PMAA increased the zeta potential of the hydrogel and the chitosan capable to control shrinkage above human body temperature. The chitosan-p(MAA-co-NIPAM) hydrogels also responses to pH and temperature thus improved the ability to performance as a drug carrier.

Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors.

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Polymeric Based Therapeutic Delivery Systems Prepared Using Electrohydrodynamic Processes.

The development of therapeutic dosage (e.g. pharmaceutical) systems is an ongoing process which, in recent times has incorporated several emerging disciplines and themes at timely intervals. While the concepts surrounding dosage forms have developed and evolved, many polymeric excipients remain as the preferred choice of materials over existing counterparts, serving functions as matrix materials, coatings and providing other specific functional properties (e.g. adhesion, controlled release and mechanical properties). There have been, however, developments in the deployment of synthetic polymeric materials (e.g. polycaprolactone, poly lactic co-glycolic acid) when compared to naturally occurring materials (e.g. lactose, gelatin). Advances in pharmaceutical process technologies have also provided novel engineering platforms to develop a host of exciting structure based materials ranging from the nanometer to the macro scales. Some of these structure enabling technologies include spray drying, super critical processing, microfluidics and even wet chemical methods. More recently electrohydrodynamic (EHDA) engineering methods have emerged as robust technologies offering potential to fabricate a plethora of generic structures (e.g. particles, fibres, bubbles and pre-determined patterns) on a broad scale range. This review focuses on key developments using various EHDA technologies for the pharmaceutical and biomaterial remits when selecting synthetic and/or naturally occurring polymers as pharmaceutical (and therapeutic) excipients. In addition, the underlying EHDA process principles are discussed along with key parameters and variables (both materials and engineering). EHDA technologies are operational at ambient conditions and recent developments have also demonstrated their viability for large scale production. These are promising technologies which have potential in established (e.g. films, dressings and microparticles) and emerging scientific themes (e.g. nanomedicines and tissue engineering).

Comparison of the microbiological milieu of patients randomized to either hydrophilic or conventional PVC catheters for clean intermittent catheterization.

INTRODUCTION: Control of bacteriuria is problematic in patients who perform clean intermittent catheterization for management of neurogenic bladder. This population is often burdened with multiple urinary tract infections (UTIs), placing them at increased risk of end-stage renal disease. Hydrophilic catheters are a potential way to improve smooth and clean insertion, reduce disruption of the urothelium, and reduce bacterial colonization. OBJECTIVE: The goal of the study was to compare the type and virulence of microorganisms recovered from the urine of patients that use either a hydrophilic or conventional polyvinyl chloride (PVC) catheter. METHODS: Fifty patients with an underlying diagnosis of myelomeningocele were recruited for a 12-month prospective, randomized, investigator-blinded study. Twenty-five patients were allocated to the hydrophilic catheter intervention, and 25 continued use of a PVC catheter. Cultures were performed on urine obtained by catheterization at enrollment, and 3, 6, and 12 months. Bacterial species were assigned a designation as either potentially pathogenic or non-pathogenic. Escherichia coli isolates were the most predominant and were serotyped to further stratify the pathogenicity of the strains. Lastly, patients were surveyed at enrollment, and at the two later time points evaluating their current catheter for satisfaction. RESULTS: A total of 232 different bacterial isolates were obtained from the 182 collected urine cultures. In addition, seven species were recovered from the two UTI reported during the study period. Bacterial growth was not detected in 29 of the samples (16%). Although not statistically significant, collectively there was a 40% decrease in the average number of potentially pathogenic species recovered from those patients using hydrophilic catheters (0.81 per urine sample) compared with PVC catheter use (1.24 per urine sample). Since E. coli species can be either pathogenic or non-pathogenic, we examined 14 of the most commonly implicated serotypes associated with uropathogenic E. coli (UPEC). We identified the serotype of 57% of E. coli strains recovered. There was a trend for the recovery of fewer UPEC serotypes from the hydrophilic group (54% hydrophilic verses 64% PVC), further suggesting that the catheter type may influence the microbiological milieu. Although no significant differences were reported in patient satisfaction, almost half of the patients from the hydrophilic catheter cohort continue use of this type of catheter. CONCLUSIONS: There was a trend for reduced recovery of potentially pathogenic bacteria with the use of hydrophilic catheters. The reduction in potentially pathogenic species will reduce antibiotic exposures and some patients may prefer the comfort hydrophilic catheters provide.

Smart microneedle coatings for controlled delivery and biomedical analysis.

The work presented demonstrates an unconventional approach in the preparation of smart microneedle (MN) coatings utilising electrohydrodynamic atomisation (EHDA) principles. Stainless steel (600-900 µm in height) MNs were coupled to a ground electrode (in the EHDA coating set-up) with the deposition distance and collecting methodology varied for an ethanol:methanol (50:50) vehicle system. The preparation of nano- and micrometre-scaled pharmaceutical coatings was achieved. Fluorescein dye (serving as potential drug, sensory materials or disease state markers) and polyvinylpyrrolidone (PVP, polymer matrix system) formed the remaining components of the coating formulation. Based on these excipients and by varying the coating process, particles (100 nm to 3 µm) and fibres (400 nm to 1 µm) were deposited directly on MNs in controlled and selectable fashion (flow rates variable ∼ 5-50 µL/min, applied voltage variable 6-19 kV). These demonstrated options for multiple targeting and analysis applications. The underlying EHDA process permits room temperature fabrication, controlled output and scale-up potential for emerging MN devices as drug systems or lab-chip testing devices.

Preparation and evaluation of cerium oxide-bovine hydroxyapatite composites for biomedical engineering applications.

The fabrication and characterization of bovine hydroxyapatite (BHA) and cerium oxide (CeO2) composites are presented. CeO2 (at varying concentrations 1, 5 and 10wt%) were added to calcinated BHA powder. The resulting mixtures were shaped into green cylindrical samples by powder pressing (350MPa) followed by sintering in air (1000-1300°C for 4h). Density, Vickers microhardness (HV), compression strength, scanning electron microscopy (SEM) and X-ray diffraction (XRD) studies were performed on the products. The sintering behavior, microstructural characteristics and mechanical properties were evaluated. Differences in the sintering temperature (for 1wt% CeO2 composites) between 1200 and 1300°C, show a 3.3% increase in the microhardness (564 and 582.75HV, respectively). Composites prepared at 1300°C demonstrate the greatest compression strength with comparable results for 5 and 10wt% CeO2 content (106 and 107MPa) which are significantly better than those for 1wt% and those that do not include any CeO2 (90 and below 60MPa, respectively). The results obtained suggest optimal parameters to be used in preparation of BHA and CeO2 composites, while also highlighting the potential of such materials in several biomedical engineering applications.

Stratified scaffolds for osteochondral tissue engineering applications: electrospun PDLLA nanofibre coated Bioglass®-derived foams.

This work focuses on designing bilayered constructs by combining electrospun poly-DL-Lactide (PDLLA) fibers and Bioglass®-derived scaffolds for development of osteochondral tissue replacement materials. Electrospinning was carried out using a solution of 5 wt/v% PDLLA in dimethyl carbonate. The PDLLA layer thickness increased from 2 to 150 µm with varying electrospinning time. Bioactivity studies in simulated body fluid showed that HA mineralization decreased as the thickness of the PDLLA layer increased. A preliminary in vitro study using chondrocyte cells (ATDC5) showed that cells attached, proliferated and migrated into the fibrous network, confirming the potential applicability of the bilayered scaffolds in osteochondral defect regeneration.

Antimicrobial properties of electrically formed elastomeric polyurethane-copper oxide nanocomposites for medical and dental applications.

With the rapidly advancing field of nanotechnology having an impact in several areas interfacing life and physical sciences, the potential applications of nanoparticles as antimicrobial agents have been realized and offer great opportunities in addressing several viral and bacterial outbreak issues. Polyurethanes (PUs) are a diverse class of polymeric materials which also have applications in several areas of biomedical science ranging from blood contact devices to implantable dental technologies. In this report, copper oxide (CuO) nanoparticles (mean size ∼50 nm) are embedded into a PU matrix via two electrical fabrication processes. To elucidate the antimicrobial activity, a range of different loading compositions of CuO within the PU matrix (0%, 1%, 5%, and 10% w/w) are electrospun to form thin porous films (thickness < 10 µm). After washing, the films are tested for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA). Significant reduction of populations was demonstrated with 10% w/w CuO over a 4-h period. This approach demonstrates the potential of generating tailored antimicrobial structures for a host of applications, such as designer filters, patterned coatings, breathable fabrics, adhesive films (as opposed to sutures), and mechanically supporting structures.