Enzyme functionalized microgels enable precise regulation of dissolved oxygen and anaerobe culture.

Anaerobes are a major constituent of the gut microbiome and profoundly influence the overall health of humans. However, the lack of a simple, cost-effective, and scalable system that mimics the anaerobic conditions of the human gut is hindering research on the gut microbiome and the development of therapeutics. Here, we address this gap by using glucose oxidase and catalase containing gelatin microparticles (GOx-CAT-GMPs) to precisely regulate dissolved oxygen concentration [O2] via GOx-mediated consumption of oxygen. Fluorescence images generated using conjugated polymer afterglow nanoparticles showed that [O2] can be tuned from 257.9 â€‹± â€‹6.2 to 0.0 â€‹± â€‹4.0 â€‹µM using GOx-CAT-GMPs. Moreover, when the obligate anaerobe Bacteroides thetaiotaomicron was inoculated in media containing GOx-CAT-GMPs, bacterial growth under ambient oxygen was comparable to control conditions in an anaerobic chamber (5.4 â€‹× â€‹105 and 8.8 â€‹× â€‹105 colony forming units mL-1, respectively). Finally, incorporating GOx-CAT-GMPs into a bioreactor that permitted continuous radial diffusion of oxygen and glucose generated a gut-mimetic [O2] gradient of 132.4 â€‹± â€‹2.6 â€‹µM in the outer ring of the reactor to 7.9 â€‹± â€‹1.7 â€‹µM at the core. Collectively, these results indicate that GOx-CAT-GMPs are highly effective oxygen-regulating materials. These materials can potentially be leveraged to advance gut microbiome research and fecal microbiota transplantation, particularly in low-resource settings.

APPL proteins modulate DNA repair and radiation survival of pancreatic carcinoma cells by regulating ATM.

Despite intensive multimodal therapies, the overall survival rate of patients with ductal adenocarcinoma of the pancreas is still poor. The chemo- and radioresistance mechanisms of this tumor entity remain to be determined in order to develop novel treatment strategies. In cancer, endocytosis and membrane trafficking proteins are known to be utilized and they also critically regulate essential cell functions like survival and proliferation. On the basis of these data, we evaluated the role of the endosomal proteins adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif (APPL)1 and 2 for the radioresistance of pancreatic carcinoma cells. Here, we show that APPL2 expression in pancreatic cancer cells is upregulated after irradiation and that depletion of APPL proteins by small interfering RNA (siRNA) significantly reduced radiation survival in parallel to impairing DNA double strand break (DSB) repair. In addition, APPL knockdown diminished radiogenic hyperphosphorylation of ataxia telangiectasia mutated (ATM). Activated ATM and APPL1 were also shown to interact after irradiation, suggesting that APPL has a more direct role in the phosphorylation of ATM. Double targeting of APPL proteins and ATM caused similar radiosensitization and concomitant DSB repair perturbation to that observed after depletion of single proteins, indicating that ATM is the central modulator of APPL-mediated effects on radiosensitivity and DNA repair. These data strongly suggest that endosomal APPL proteins contribute to the DNA damage response. Whether targeting of APPL proteins is beneficial for the survival of patients with pancreatic adenocarcinoma remains to be elucidated.

Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking.

We demonstrate an enzyme stabilization approach whereby a model enzyme is PEGylated, followed by controlled chemical modification with glutaraldehyde. Using this stabilization strategy, size increases and aggregation due to intermolecular crosslinking are avoided. Immediately following synthesis, the PEGylated enzyme with and without glutaraldehyde modification possessed specific activities of 372.9 ± 20.68 U/mg and 373.9 ± 15.14 U/mg, respectively (vs. 317.7 ± 19.31 U/mg for the native enzyme). The glutaraldehyde-modified PEGylated enzyme retains 73% original activity after 4 weeks at 37 °C (vs. 2% retention for control).

In vitro evaluation of chondrosarcoma cells and canine chondrocytes on layer-by-layer (LbL) self-assembled multilayer nanofilms.

Short-term cell-substrate interactions of two secondary chondrocyte cell lines (human chondrosarcoma cells, canine chondrocytes) with layer-by-layer self-assembled multilayer nanofilms were investigated for a better understanding of cellular-behaviour dependence on a number of nanofilm layers. Cell-substrate interactions were studied on polyelectrolyte multilayer nanofilms (PMNs) of eleven different biomaterials. Surface characterization of PMNs performed using AFM showed increasing surface roughness with increasing number of layers for most of the biomaterials. LDH-L and MTT assays were performed on chondrosarcoma cells and canine chondrocytes, respectively. A major observation was that 10-bilayer nanofilms exhibited lesser cytotoxicity towards human chondrosarcoma cells than their 5-bilayer counterparts. In the case of canine chondrocytes, BSA enhanced cell metabolic activity with increasing number of layers, underscoring the importance of the multilayer nanofilm architecture on cellular behaviour.

Nogo-A knockdown inhibits hypoxia/reoxygenation-induced activation of mitochondrial-dependent apoptosis in cardiomyocytes.

Programmed cell death of cardiomyocytes following myocardial ischemia increases biomechanical stress on the remaining myocardium, leading to myocardial dysfunction that may result in congestive heart failure or sudden death. Nogo-A is well characterized as a potent inhibitor of axonal regeneration and plasticity in the central nervous system, however, the role of Nogo-A in non-nervous tissues is essentially unknown. In this study, Nogo-A expression was shown to be significantly increased in cardiac tissue from patients with dilated cardiomyopathy and from patients who have experienced an ischemic event. Nogo-A expression was clearly associated with cardiomyocytes in culture and was localized predominantly in the endoplasmic reticulum. In agreement with the findings from human tissue, Nogo-A expression was significantly increased in cultured neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation. Knockdown of Nogo-A in cardiomyocytes markedly attenuated hypoxia/reoxygenation-induced apoptosis, as indicated by the significant reduction of DNA fragmentation, phosphatidylserine translocation, and caspase-3 cleavage, by a mechanism involving the preservation of mitochondrial membrane potential, the inhibition of ROS accumulation, and the improvement of intracellular calcium regulation. Together, these data demonstrate that knockdown of Nogo-A may serve as a novel therapeutic strategy to prevent the loss of cardiomyocytes following ischemic/hypoxic injury.

Opsoclonus myoclonus: a non-epileptic movement disorder that may present as status epilepticus.

We describe two cases of a non-epileptic florid movement disorder presenting as status epilepticus. Both patients presented with florid jerking of the limbs and eyes. Convulsive status epilepticus related to presumed meningitis or encephalitis was suspected in both cases. The patients received treatment for seizures, without resolution of the abnormal movements, resulting ultimately in anaesthetic, intubation and ventilation. EEGs showed no epileptic discharges. The diagnosis was opsoclonus myoclonus syndrome in both. One patient was treated with adrenocorticotropic hormone (40 IU/day), the other with prednisolone (4 mg/kg/day) with rapid resolution of symptoms. Neither patient had an underlying neoplasm or infectious agent identified. To date, neither patient has suffered a relapse of symptoms nor does either show any sign of developmental delay. These cases show that the movements in opsoclonus myoclonus syndrome can be sufficiently florid to mimic convulsive status epilepticus. Video footage of both patients at the time of diagnosis is presented online.

Polyelectrolyte-coated alginate microspheres as drug delivery carriers for dexamethasone release.

Alginate microspheres loaded with dexamethasone were prepared by the droplet generator technique. Important parameters affecting drug release, including initial drug content, the type of polyelectrolyte coating, and a combination of different ratios of coated and uncoated microspheres were investigated to achieve in vitro dexamethasone delivery with approximately zero order release kinetics, releasing up to 100% of entrapped drug within 1 month, wherein dexamethasone released at a steady rate of 4.83 microg/day after an initial burst release period. These findings imply that these polyelectrolyte-coated alginate microspheres show promise as release systems to improve biocompatibility and prolong lifetime of implantable glucose sensors.

Transcallosal resection of hypothalamic hamartoma for gelastic epilepsy.

INTRODUCTION: Hypothalamic hamartomas (HHs) are commonly associated with severe epilepsy resistant to anticonvulsant therapy. Historically, surgical resection of HHs resulted in considerable morbidity. DISCUSSION: Two series of patients who successfully underwent resection using a transcallosal approach have now been published; we report the first UK experience of this technique in a series of five patients with HHs and gelastic epilepsy resistant to anticonvulsant therapy. Patients were assessed pre- and postoperatively for seizure activity, endocrine function, ophthalmology, and neurocognitive function. Two patients had precocious puberty and all had evidence of developmental delay and behavioral problems. Postoperatively, all children experienced at least a 50% reduction in seizure frequency with abolition of major seizure types; one child remains seizure-free. One child developed a mild postoperative right hemiparesis and one developed transient diabetes insipidus. CONCLUSION: There were no adverse developmental effects of surgery. Transcallosal resection of HHs ameliorates resistant epilepsy syndromes associated with HH.

Expansion of the first PolyA tract of ARX causes infantile spasms and status dystonicus.

BACKGROUND: ARX is a paired-type homeobox gene located on the X chromosome that contains five exons with four polyalanine (PolyA) tracts, a homeodomain, and a conserved C-terminal aristaless domain. Studies in humans have demonstrated remarkable pleiotropy: malformation phenotypes are associated with protein truncation mutations and missense mutations in the homeobox; nonmalformation phenotypes, including X-linked infantile spasms (ISS), are associated with missense mutations outside of the homeobox and expansion of the PolyA tracts. OBJECTIVE: To investigate the role of ARX, we performed mutation analysis in 115 boys with cryptogenic ISS. This included two pairs of brothers. RESULTS: We found an expansion of the trinucleotide repeat that codes for the first PolyA tract from 10 to 17 GCG repeats (c.333_334ins[GCG]7) in six boys (5.2%) ages 2 to 14, from four families, including the two pairs of brothers. In addition to ISS, all six boys had severe mental retardation and generalized dystonia that appeared around the age of 6 months and worsened, eventually leading to stable severe quadriplegic dyskinesia within age 2 years. Three children experienced recurrent, life-threatening status dystonicus. In four children brain MRI showed multiple small foci of abnormal cavitation on T1 and increased signal intensity on T2 in the putamina, possibly reflecting progressive multifocal loss of tissue. CONCLUSION: The phenotype of infantile spasms with severe dyskinetic quadriparesis increases the number of human disorders that result from the pathologic expansion of single alanine repeats. ARX gene testing should be considered in boys with infantile spasms and dyskinetic cerebral palsy in the absence of a consistent perinatal history.

Application of self-assembled ultra-thin film coatings to stabilize macromolecule encapsulation in alginate microspheres.

Alginate-based hydrogels have several unique properties that have enabled them to be used as a matrix for the entrapment of a variety of enzymes, proteins and cells for applications in bioprocessing, drug delivery and chemical sensing. However, control over release rates or, in some cases, stable encapsulation remains a difficult goal, especially for small particles with high surface-area-to-volume ratios. In this work, the potential to limit diffusion of macromolecules embedded in alginate spheres with nanofilm coatings was assessed. Alginate microspheres were fabricated using an emulsification process with high surfactant concentration to form beads in the size range of 2-10 microm. Using calcium chloride for ionotropic gelation, dextran was encapsulated in the gel phase by mixing with the alginate in solution. The exterior surface was then modified with polyelectrolyte coatings using the layer-by-layer self assembly technique. Leaching studies to assess retention of dextran with varying molecular weights confirmed that the application of multi-layer thin films to the alginate microspheres was effective in reducing leaching rate and total loss of the encapsulated material from the microspheres. For the best case, the rate of release for dextran of 2,000,000 Dalton molecular weight decreased from 1% h(-1) in bare microspheres to 0.1% h(-1) in polyelectrolyte-coated microspheres. The effectiveness of nanofilms reducing loss of the encapsulated macromolecules was found to vary between different polycation materials used. These studies support the feasibility of using these microsystems for development of long-term stable encapsulated systems, such as implantable biosensors.

Quenching Properties of a Self-Referenced Fluorescence Oxygen Nanosensor under a Wide-Field Intrinsic Optical Signal Imaging System.

Wide-field intrinsic optical signal (IOS) imaging is an excellent method to map in vivo neurological metabolic changes during brain activity. It has a spatial resolution of 50-100µm and a wide imaging field of several cm2that cannot be achieved by regular microscopic imaging. A nano-encapsulated fluorescence dye sensor that is specifically sensitive to oxygen level was fabricated by using electrostatic layer-by-layer (LbL) self-assembly technique. Fluorescence images of the nanosensor solution were collected by a wide-field IOS imaging system under different oxygen concentrations. Mathematical models for nanosensor fluorescence were developed based on the imaging results. Correlations between the nanosensor fluorescence and oxygen concentration models indicate an inverse quenching relationship between the nanosensor fluorescence and oxygen concentration.

Comparison of selective attachment and growth of smooth muscle cells on gelatin- and fibronectin-coated micropatterns.

Tissue engineering research has been on going for many years, people are making all the effort to explore the cell functions in cellular level and even in molecular level. Making the cells functional in an in vitro environment is a preliminary goal for the implantation and repair of complicated tissues/organs. Fabricating artificial ECM to mimic the in vivo environment is an essential approach in tissue engineering. The work in this paper is to study how rat aorta smooth muscle cells (RASMCs) behave in two engineered cell culture scaffolds: gelatin- and fibronectin (FN)-coated micropatterns. The investigation on the initial attachment and further growth of SMCs cultured on gelatin- and FN-coated micropatterns was addressed. This study focused on both the characterization of gelatin and fibronectin assembly properties and cell responses to these two protein-coated micropatterns. Thin film patterns with gelatin and fibronectin coatings were fabricated on microscope glass slides using photolithography, electrostatic layer-by-layer self-assembly and lift-off (LbL-LO) technologies. In this work, the scaffolds were built up by commonly used polyelectrolyte materials and proteins through LbL process, containing cationic poly(diallyldimethylammonium chloride) (PDDA), poly(allylamine hydrochloride) (PAH), anionic poly(sodium 4-styrenesulfonate) (PSS), gelatin and fibronectin. The resulting polyelectrolyte thin films were characterized by contact angle (CA), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and fluorescence microscopy. CA measurement shows the consistent hydrophylicity of gelatin surfaces in different number of layers with LbL deposition method. Different from our previous QCM measurement of gelatin, fibronectin does not show high electrostatic attraction to either positively or negatively charged polyelectrolytes, although it can be weakly assembled to both polyelectrolyte surfaces. AFM images show Gelatin- and FN-coated micropatterns are around 50-60 nm thick. RASMCs were cultured on these gelatin- and FN-coated micropatterns. It was observed that, for the cells cultured on gelatin-coated micropatterns, they initially landed on the gelatin-coated surface, not on the PDDA-coated surface in between. But further growth of the cells was affected by the shape of the patterns: strip pattern limited cell growth beyond the patterns, but square patterns could not. While, it was found interestingly, for the cells cultured on FN-coated micropatterns, SMCs initially landed on PDDA-coated surface, and then migrated to FN-coated both square and strip patterns. These findings indicate that both gelatin and fibronectin are adhesive proteins, but they have different effects on the initial attachment and later growth for SMCs.

Pyruvate dehydrogenase deficiency presenting as dystonia in childhood.

Two individuals with pyruvate dehydrogenase (PDH) deficiency due to missense mutations in the gene for the E1alpha subunit (PDHA1) presented during childhood with dystonia. The first patient, a male, presented at age 4 years with dystonia affecting the lower limbs, which responded to treatment with combined carbidopa and levodopa. The second patient, a female, was first investigated at age 6 years because of a dystonic gait disorder. In both patients, the main clue to the biochemical diagnosis was a raised concentration of lactate in the cerebrospinal fluid. PDH activity was significantly reduced in cultured fibroblasts in both cases. Dystonia is a previously unrecognized major manifestation of PDH deficiency and is of particular interest as the mutations in the PDHA1 gene in these patients have both been identified previously in individuals with typical presentations of the condition.

Micropatterning of nanoengineered surfaces to study neuronal cell attachment in vitro.

Methods for producing protein patterns with defined spatial arrangement and micro- and nanoscale features are important for studying cellular-level interactions, including basic cell-cell communications, cell signaling, and mechanisms of drug action. Toward this end, a straightforward, versatile procedure for fabricating micropatterns of bioactive nanofilm coatings as multifunctional biological testbeds is demonstrated. The method, based on a combination of photolithography and layer-by-layer self-assembly (LbL), allows for precise construction of nanocomposite films of potentially complex architecture, and patterning of these films on substrates using a modified lift-off (LO) procedure. As a first step in evaluating nanostructures made with this process, "comparison chips," comprising two coexisting regions of square patterns with relevant proteins/polypeptides on a single substrate, were fabricated with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Using neuronal cells as a model biological system, comparison chips were produced with secreted phospholipase A2 (sPLA2), a known membrane-active enzyme for neurons, for direct comparison with gelatin, poly-l-lysine (PLL), or bovine serum albumin (BSA). Fluorescence microscopy, surface profilometry, and atomic force microscopy techniques were used to evaluate the structural properties of the patterns on these chips and show that the patterning technique was successful. Preliminary cell culture studies show that neurons respond and bind specifically to the sPLA2 enzyme embedded in the polyelectrolyte thin films and present as the outermost layer. These findings point to the potential for this method to be applied in developing test substrates for a broad array of studies aimed at identifying important biological structure-function relationships.

RET nanobiosensors using affinity of an apo-enzyme toward its substrate.

Fluorescent biosensors can be highly specific and sensitive, and may be engineered as implantable devices for metabolic monitoring. Commonly-used systems for glucose monitoring based on resonance energy transfer (RET) and competitive binding involve Concanavalin A (Con A), which is known to be toxic, and has problems of aggregation and irreversible binding. This work presents an improved RET assay wherein Con A was replaced by apo-glucose oxidase (apo-GOx). Fluorescence measurements confirm that the apo-GOx/dextran complexes are highly sensitive to glucose, observed as an increase in the donor peak relative to acceptor with the stepwise addition of glucose solution. The assay showed strong signals and excellent repeatability, with a sensitivity of 9x10/sup -4/ (ratio units)/mM over the range of 0-90 mM glucose. If properly encapsulated, these sensors can potentially be used for in vivo sensing without the concern of toxicity associated with Con A.

Using electrostatic self-assembled gradient nanosensor phantoms for calibration of an optical intrinsic signal imaging system.

Wide-field optical intrinsic signal (OIS) imaging has been used in functional cortex mapping for its excellent spatial resolution. To compensate for its low temporal resolution, extrinsic dye signals have been introduced. Fluorescence spectroscopy in the form of nanoengineered sensors has also been used to detect biochemical signals of molecular interactions. In this paper, oxygen-sensitive dye Ru(dpp)/sub 3//sup 2+/ was immobilized into nano-sized spheres by electrostatic layer-by-layer (LbL) self-assembly, and then deposited on glass slides as intensity gradients. By comparing gradient ratios and ratios of function dye Ru(dpp)/sub 3//sup 2+/ and reference dye between epi-fluorescence microscope and an OIS imaging system, the feasibility and efficiency of nano-sized oxygen sensors in OIS imaging were studied.

Optimal design of nanoengineered implantable optical sensors using a genetic algorithm.

A genetic algorithm as a design tool for optimized optical glucose sensors is presented. These proposed sensors are fabricated by assembling ultrathin polyelectrolyte films on the surface of calcium alginate microspheres containing glucose oxidase and an oxygen-quenched ruthenium fluorophore. The sensors are rendered ratiometric by inclusion of a complementary reference fluorophore via polyelectrolyte-dye conjugates. The genetic algorithm, in conjunction with a computational model of the chemical sensor, selects the optimal values for diffusivities of glucose and oxygen in the polyelectrolyte films, the enzyme concentration, microsphere radius, and film thickness that give the optimum sensor response. The values given by the genetic algorithm will be used to design future sensor prototypes.

Brain haemorrhage in five infants with coagulopathy.

Most intracranial haemorrhages in infants after the neonatal period are secondary to non-accidental injury. Occasionally brain haemorrhages in non-mobile infants are due to an inherited coagulopathy. This may often be diagnosed with a coagulation screen on admission. Little is known about the neurological outcome of infants in the latter group. Five infants are described who presented with acute spontaneous brain haemorrhage secondary to an inherited coagulopathy (n = 3) and vitamin K deficiency in alpha(1) antitrypsin deficiency (n = 1) and Alagille's syndrome (n = 1). Despite the critical clinical presentation and the severe imaging findings, these five infants made a good neurological recovery. Infants presenting with spontaneous ICH due to a significant (inherited) coagulopathy are usually easy to differentiate from non-accidental shaking injury; their bleeding pattern within the brain seems different from non-accidental shaking injury and neurodevelopment outcome appears better.