The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles.

Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCE Like all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genus Enterovirus The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

EAACI Position paper on the standardization of nasal allergen challenges.

Nasal allergen challenge (NAC) is an important tool to diagnose allergic rhinitis. In daily clinical routine, experimentally, or when measuring therapeutic success clinically, nasal allergen challenge is fundamental. It is further one of the key diagnostic tools when initiating specific allergen immunotherapy. So far, national recommendations offered guidance on its execution; however, international divergence left many questions unanswered. These differences in the literature caused EAACI to initiate a task force to answer unmet needs and find a consensus in executing nasal allergen challenge. On the basis of a systematic review containing nasal allergen challenges of the past years, task force members reviewed evidence, discussed open issues, and studied variations of several subjective and objective assessment parameters to propose a standardized way of a nasal allergen challenge procedure in clinical practice. Besides an update on indications, contraindications, and preparations for the test procedure, main recommendations are a bilaterally challenge with standardized allergens, with a spray device offering 0.1 mL per nostril. A systematic catalogue for positivity criteria is given for the variety of established subjective and objective assessment methods as well as a schedule for the challenge procedure. The task force recommends a unified protocol for NAC for daily clinical practice, aiming at eliminating the previous difficulty of comparing NAC results due to unmet needs.

Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-ß-lactamase.

In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear ß-lactamase, CphA from Aeromonas hydrophila. Significant different transition state structures are obtained for the hydrolysis of both antibiotics: while the TS of the CEF is an ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study sheds some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MßL inhibitors against a broad spectrum of bacterial pathogens. We have finally probed that a re-parametrization of semiempirical methods should be done to properly describe the behavior the metal cation in active site, Zn(2+), when used in QM/MM calculations.

Long-term surgical and functional outcomes of the intact canal wall technique for middle ear cholesteatoma in the paediatric population.

In this paper, we report the postoperative outcomes in canal wall up procedures with second stage surgery in 40 children undergoing intervention for cholesteatoma of the middle ear. The residuals, recurrences and the hearing results were analysed. All 40 patients had a follow-up of at least five years. Of the 39 patients who underwent two staged surgery, 18 (46.1%) had a residual lesion that was identified and excised during the second surgery. Over a five year follow-up period, there were five (12.5%) patients with recurrences, all belonging to the group in whom a residual cholesteatoma was identified during the second staged surgery. The rate of residual cholesteatoma tends to decrease as age increases. The type of cholesteatoma, acquired or congenital middle ear, were not statistically related to the incidence of residual cholesteatoma. Hearing analysis showed that hearing recovery was excellent with canal wall up procedures and remained stable over five years.

Stoichiometry of coagulation revisited.

The roles of particles and natural organic matter (NOM) in determining coagulant (alum) doses in potable water treatment were investigated at two pH conditions (6 and 7). The concentrations of NOM and colloidal silica particles in raw water were systematically varied separately and in combination, and the impacts of these two classes of contaminants on the minimum effective alum doses were investigated using observations of turbidity and dissolved organic carbon (DOC) in laboratory jar tests. At both pHs, coagulant requirements for the removal of these contaminants by sedimentation and filtration were dominated by the DOC concentration in the raw water. The presence of low NOM concentrations (0.75-1.5 mg of C/L) decreased the minimum effective alum dose dramatically for waters low in silica particles, possibly by promoting the precipitation of aluminum hydroxide and/or Al-NOM solids, whose removal would otherwise be limited by low collision opportunities. Strong stoichiometric relationships were observed between DOC and coagulant demand at both pHs regardless of silica particle concentration. Silica contributed to coagulant demand only at very high particle concentrations.

Pretreatment chemistry for dual media filtration: model simulations and experimental studies.

Laboratory dual media filtration experiments were conducted (a) in direct filtration mode using model raw water moderate in turbidity and low in DOC, and (b) in conventional filtration mode treating water moderate in turbidity and high in DOC. Model simulations of filter performance for the removal of particles provided hypotheses for the experimental studies of dual media filtration. An increase in alum dose in direct filtration mode, while improving filter performance, also showed some disadvantages, including rapid development of head loss. Suboptimal dose in direct filtration significantly impaired the filter performance. In conventional mode, the effect of alum dose on the filter performance, while obvious, was not as dramatic as in direct filtration. Ripening indicated by particle counts occurred earlier than by turbidity and breakthrough of particle counts started earlier than breakthrough of turbidity, suggesting that turbidity can be used as a more conservative monitor of filter performance during the ripening period to minimise the risk of passage of small particles, while particle counts can be considered a more sensitive indicator of deteriorating filter performance during the breakthrough period. The lower sand layer served as a multiple barrier for particle when the performance of the anthracite layer was not effective.

Compression and compaction properties of plasticised high molecular weight hydroxypropylmethylcellulose (HPMC) as a hydrophilic matrix carrier.

The compression and compaction properties of plasticised high molecular weight USP2208 HPMC were investigated with the aim of improving tablet formation in HPMC matrices. Experiments were conducted on binary polymer-plasticiser mixtures containing 17 wt.% plasticiser, and on a model hydrophilic matrix formulation. A selection of common plasticisers, propylene glycol (PG) glycerol (GLY), dibutyl sebacate (DBS) and triacetin (TRI), were chosen to provide a range of plasticisation efficiencies. T(g) values of binary mixtures determined by Dynamic Mechanical Thermal Analysis (DMTA) were in rank order PG>GLY>DBS>TRI>unplasticised HPMC. Mean yield pressure, strain rate sensitivity (SRS) and plastic compaction energy were measured during the compression process, and matrix properties were monitored by tensile strength and axial expansion post-compression. Compression of HPMC:PG binary mixtures resulted in a marked reduction in mean yield pressure and a significant increase in SRS, suggesting a classical plasticisation of HPMC analogous to that produced by water. The effect of PG was also reflected in matrix properties. At compression pressures below 70 MPa, compacts had greater tensile strength than those from native polymer, and over the range 35 and 70 MPa, lower plastic compaction values showed that less energy was required to produce the compacts. Axial expansion was also reduced. Above 70 MPa tensile strength was limited to 3 MPa. These results suggest a useful improvement of HPMC compaction and matrix properties by PG plasticisation, with lowering of T(g) resulting in improved deformation and internal bonding. These effects were also detectable in the model formulation containing a minimal polymer content for an HPMC matrix. Other plasticisers were largely ineffective, matrix strength was poor and axial expansion high. The hydrophobic plasticisers (DBS, TRI) reduced yield pressure substantially, but were poor plasticisers and showed compaction mechanisms that could be attributed to phase separation. The effect of different plasticisers suggests that the deformation characteristics of this HPMC in the solid state is dominated by hydroxyl mediated bonding, rather than by hydrophobic interactions between methoxyl-rich regions.

A simple, high throughput method for the quantification of sodium alginates on oesophageal mucosa.

Sodium alginate is a potential bioadhesive, but the lack of a convenient and suitable method for its quantification on the mucosal surface complicates the evaluation of its mucosal retentive properties. This paper develops and evaluates a spectrophotometric method for the rapid quantification of a range of sodium alginates differing in chemical composition, and investigates how quantification was influenced by the presence of oesophageal mucosa. The method, based on dye complexation with 1,9-dimethyl methylene blue (DMMB) was sensitive to alginate molecular weight and uronic acid composition, however, no significant correlations between assay performance and alginate molecular characteristics were demonstrated. The assay was also influenced by complexation time, calcium ions and mucin, but was unaffected by the presence of oesophageal tissue scrapings. The assay proved to be capable of quantifying sodium alginate with excellent linearity (r = 0.999), reproducibility (CV < 3%) and sensitivity (0.3 g l(-1)) and proved to be a precise, high-throughput method that may be used for quantifying the retention of sodium alginate on oesophageal mucosa.

A novel application of NMR microscopy: measurement of water diffusion inside bioadhesive bonds.

The self-diffusion coefficient of water (D) inside bioadhesive bonds formed by dry and prehydrated hydrophilic matrices has been spatially resolved using nuclear magnetic resonance (NMR) microscopy. One-dimensional profiles showing the variation of D inside bioadhesive bonds were calculated from nine diffusion-weighted profiles obtained immediately after bond formation and every 5 min for 30 min. The resulting data indicated that the hydration state of a hydrophilic matrix can significantly and dramatically influence the dynamics of water movement inside a bioadhesive bond.

First order release rate from porous PLA microspheres with limited exit holes on the exterior surface.

We applied the finite element method (FEM) to calculate release profiles from computer simulated slabs, one with a limited number of exit holes on the exterior surface, and the other with uniform structure. The former slab showed a first order release rate, and a nearly uniform drug concentration distribution within the device during the release process. It was concluded that circulation of the drug molecules within the slab resulted in the uniform concentration and consequently first order release rate. This theoretical work was used to explain the first order release rate of an active ingredient (flourescin-4-isothiocyanate-dextran, M(W)=71000 Da) from porous PLA (poly(D,L)-lactic acid) microspheres, which by canning electron microscopy (SEM) examination showed only a few exit holes on their exterior surface. Calculations indicated that the internal surface adsorption of the active ingredient, or the pore size distribution of the microspheres, could not influence the mechanism for the first order release rate, and the small number of exit holes on the exterior surface was likely to be the rate-determining factor. The exit holes could be observed by SEM and their size and number is consistent with our interpretations.

Peer reviewed: making watershed management work.

New York City's watershed management strategy is unprecedented in its scope, scale, and cost.

A method for quantifying differential expansion within hydrating hydrophilic matrixes by tracking embedded fluorescent microspheres.

A method is described for quantifying the pattern of deformation within a matrix and is demonstrated by analyzing the expansion of polymer hydrophilic matrix tablets. The fundamental features of the method are the incorporation of nondiffusing markers into the matrix and the subsequent tracking of these markers during deformation. Since the markers are too large to diffuse, their individual movement reflects the translocation of the surrounding matrix, and the separation between pairs of markers reveals any perturbation in the intervening area. By tracking many markers, the pattern of deformation within a matrix can be ascertained. The method was demonstrated on hydrating hydrophilic matrix tablets, using fluorescent microspheres as nondiffusing markers which were observed with a confocal laser scanning microscope. Analysis of the tracks showed a wave of expansion moving from the exterior toward the core, with the greatest and earliest expansion found in the outer regions. The results also showed that even as deeper layers started to expand the outer layers continued to swell.

Determination of the internal morphology of poly (D,L-lactide) microspheres using stereological methods.

The morphological characteristics of the internal structure of poly (D,L-lactide) microspheres have been determined by stereological methods in two different formulations of microspheres, with different internal structures, prepared by using a double emulsion method. In one formulation the internal emulsion was produced by homogenisation at 3000 rpm, whilst the other was prepared at 11000 rpm. As expected the formulation prepared at the lower speed contained larger and more broadly distributed pores than that prepared at the higher speed. The porosity, pore size distribution and total internal surface area of the microspheres were obtained by stereological methods from electron microscopic measurements of the sectioned microspheres. It was found that whilst the porosity of the microspheres was 0.6 in both formulations, the preparation method gave rise to large differences in their pore size distribution characteristics. The pore size distribution was simulated by computer modelling to validate and compare alternative stereological algorithms. It was found that the Saltykov unfolding method predicts the measured pore size distribution more accurately than the Cruz-Orive unfolding method (at significance level alpha=0.1). This finding was attributed to the violation of one of the basic assumptions of the Cruz-Orive unfolding method.

Self-diffusion and molecular mobility in PVA-based dissolution-controlled systems for drug delivery.

Nuclear magnetic resonance (NMR) microscopy has been used to monitor the hydration of poly(vinyl alcohol) (PVA) samples of varying molecular weight. One-dimensional profiles weighted to predominantly show the variation of water concentration were acquired every 3 min during the first 30 min of hydration and subsequently at 1 and 2 h. Diffusion-weighted profiles obtained after 30 min and 1 and 2 h were used to calculate the spatial variation of the water self-diffusion coefficient. The resulting data provide supporting evidence for the hypothesis that phenomena such as reptation are important near the glassy/rubbery interface of polymers during dissolution, while the diffusion gradually changes to Zimm type near the rubbery/solvent interface.

Starch granule surface imaging using low-voltage scanning electron microscopy and atomic force microscopy.

High resolution imaging of wheat and potato starch granule surfaces has been performed using low-voltage scanning electron microscopy and atomic force microscopy. The complimentary images of uncoated granules demonstrate that the two starch types possess substantially different surface topologies; potato starch has many protrusions (100-300 nm in diameter), above a flatter surface containing 20-50 nm structures, whilst wheat starch possesses far fewer protrusions and generally has a smoother surface composed of approximately 20 nm structures. The protrusions are believed to be carbohydrate in nature and thus represent the ends of amylopectin side-chain clusters at the granule surface.

Uptake of drugs by catheters: the influence of the drug molecule on sorption by polyurethane catheters.

The sorption of drugs by indwelling intravenous catheters may have clinical consequences both by alteration of the dose received by the patient and by physically affecting the catheter materials themselves which may lead to changes in mechanical properties and biocompatibility. Studies of drug sorption to new catheter materials are therefore important. Pellethane, a polyurethane increasingly used in vascular access catheters, is as yet little studied in terms of its capacity for drug sorption. In this work a range of drugs known to be sorbed by PVC infusion sets were studied with respect to their sorption by Pellethane catheters. Standard lengths of catheter were incubated with solutions of drugs and samples of the solution were taken at intervals, assayed spectrophotometrically and compared with control solutions incubated without catheter. Losses from solution of up to 93% were found after 24 h. A series of highly sorbing and clinically relevant drugs was identified and their uptake was studied until equilibrium had been reached. A correlation was evident between the octanol/water partition coefficient and the fraction of drug taken up from solution at equilibrium, with the more hydrophobic drugs being taken up to a greater extent by the catheter.

Structure and behavior in hydrophilic matrix sustained release dosage forms: 4. Studies of water mobility and diffusion coefficients in the gel layer of HPMC tablets using NMR imaging.

PURPOSE: The purpose of this study was to characterise the water mobility in the gel layer of hydrating HPMC tablets. Water mobility in the gel layer of different HPMCs was studied. METHODS: NMR imaging, a non-invasive technique, has been used to measure the spatial distribution of self-diffusion coefficient (SDC) and T2 relaxation times across the gel layer. RESULTS: It has been shown that there is a water mobility gradient across the gel layer of HPMC tablets. Although SDC and T2 relaxation times in the outer parts of the gel layer approached that of free water, in the inner parts they decreased progressively. Water mobility and SDC in the gel layer of different HPMCs appeared to vary with degree of substitution of the polymer and the lowest values were obtained across the gel layer of K4M tablets. CONCLUSIONS: Water mobility varies across the gel layer of hydrating HPMC tablets and it is dependent on the degree of substitution of the polymer.