Self-assembled lipid and membrane protein polyhedral nanoparticles

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.The functions of many membrane proteins are intimately coupled to the generation, utilization, and/or sensing of transmembrane gradients (1). Despite advances in the structure determination of membrane proteins (2), the high-resolution structural analysis of membrane proteins in a biological membrane is uncommon and in the presence of a functionally relevant gradient remains an as-yet unrealized experimental challenge. This stems from the fact that the primary 2D- and 3D ordered specimens used in structural studies of membrane proteins by X-ray crystallography and electron microscopy lack closed membrane surfaces, thus making it impossible to establish physiologically relevant transmembrane gradients.As an alternative, we have been developing methodologies for the self-assembly of lipids and membrane proteins into closed polyhedral structures that can potentially support transmembrane gradients for structural and functional studies. The possibility of generating polyhedral arrangements of membrane proteins in proteoliposomes was motivated by the existence of polyhedral capsids of membrane-enveloped viruses (3, 4), the ability of surfactant mixtures to self-assemble into polyhedral structures (5, 6), and the formation of proteoliposomes from native membranes containing bacteriorhodopsin (7, 8) and light-harvesting complex II (LHCII) (9). Significantly, the high-resolution structure of LHCII was determined from crystals of icosahedral proteoliposomes composed of protein subunits in chloroplast lipids (10). Whereas detergent solubilized membrane proteins and lipid mixtures can self-assemble to form 2D-ordered crystalline sheets or helical tubes favorable for structure determination by electron microscopy (11–14), simple polyhedral ordered assemblies have only been described to form from select native membranes (7–9). To expand the repertoire of membrane protein structural methods, we have prepared membrane protein polyhedral nanoparticles (MPPNs) of the bacterial mechanosensitive channel of small conductance (MscS) (15, 16) from detergent solubilized protein and phospholipids, and demonstrated that they are amenable to structural analysis using electron microscopy.Conditions for generating MPPNs were anticipated to resemble those for other types of 2D-ordered bilayer arrangements of membrane proteins, particularly 2D crystals, in that membrane protein is mixed with a particular phospholipid at a defined ratio, followed by dialysis to remove the solubilizing detergent (17). The main distinction is that because MPPNs are polyhedral, conditions are sought that will stabilize highly curved surfaces of polyhedra rather than the planar (flat) specimens desired for 2D crystals. We used the Escherichia coli MscS as a model system. MscS is an intrinsically stretch-activated channel identified by Booth and coworkers (15) that confers resistance to osmotic downshock in E. coli. MscS forms a heptameric channel with 21 transmembrane helices (3 from each subunit) and a large cytoplasmic domain with overall dimensions of ∼8 × ∼12 nm parallel and perpendicular to the membrane plane; structures have been reported in both nonconducting (16, 18) and open-state conformations (19). Different phospholipids were added to purified the E. coli MscS solubilized in the detergent Fos-Choline 14 and the system was allowed to reach equilibrium by dialysis at different temperatures. To gain insight into the biophysical parameters that govern MPPN formation, we investigated the role of lipid head group, alkyl chain length, pH, and protein construct. Table S1 shows the observed influence of these various factors on our ability to form uniform MPPNs (as opposed to disordered aggregates or polydisperse proteoliposomes). The optimal conditions for MPPN formation used 1,2-dimyristoyl-sn-glycero-3-phosphocholine [added to ∼1:0.1 (wt/wt) protein:phospholipid] at pH 7 with the His-tagged MscS that is anticipated to be positively charged under these conditions. The biophysical properties of the protein are important as the best results were achieved using a His-tagged construct and the presence of a FLAG tag at the C terminus of MscS interfered with MPPN formation, even though the tag is ∼10 nm from the membrane-spanning region of MscS.To monitor MPPN formation, dynamic light scattering (DLS) was used. Under optimal conditions, we observed (Fig. 1A) the complete transition of solubilized MscS particles with a narrow distribution centered around a mean radii of 4.5 nm to MPPNs with a narrow distribution centered around a mean radii of 20 nm. We further characterized these particles using negative-stain electron microscopy. Fig. 1B is a field view negative-stain electron micrograph of a solution of detergent-solubilized MscS and lipid before initiation of the self-assembly process. Fig. 1C is a field view negative-stain electron micrograph of the same sample after the self-assembly process. We observed the incorporation of MscS into highly uniform polyhedra with mean radii of ∼20 nm (90%) and ∼17 nm (10%) by negative-stain electron microscopy. To gain more insight into the biophysical properties of these particles, we performed protein and phosphorus analysis on multiple samples to determine the lipid:protein ratio (Fig. S1). The observed lipid:protein ratio of the MscS MPPNs was 11 ± 1 (mole lipid:mole protein subunit) and consistent with a single layer of lipids forming a bilayer surrounding each protein. This ratio is comparable to the observed lipid to protein ratio found in 2D crystals of membrane proteins such as bacteriorhodopsin (lipid:protein ratio of 10; refs. 20 and 21) and aquaporin (lipid:protein ratio of 9; ref. 22).Open in a separate windowFig. 1.Preparation of MscS MPPNs. (A) DLS analysis of particles before dialysis and after completion of dialysis when MPPNs are formed. The observed radius of MscS alone was 4.5 nm and the particle radius at the end of dialysis was observed to be 20 nm. In both cases 99% of the scattering mass was observed in the distributions centered at 4.5 nm and 20 nm, respectively. (B) Negative-stain electron microscopy analysis of MscS before dialysis. Individual MscS proteins can be observed as small doughnut-shaped particles. (C) Negative-stain electron microscopy analysis of MPPNs following dialysis of the sample in B. MPPNs can be clearly observed and appear as uniform assemblies of individual MscS molecules. (Scale bars, 100 nm.)To further elucidate the structural nature of these particles and to unambiguously determine the symmetry, we performed electron cryotomography with image reconstruction using IMOD (23) combined with Particle Estimation for Electron Tomography (PEET) program (ref. 24 and SI Materials and Methods). In principle, electron tomography provides a complete 3D map of the particles and would allow us to unambiguously determine the MPPN symmetry. However, the alignment process was highly biased by the missing wedge phenomenon (24) due to poor signal:noise and resulted in an incomplete map (Fig. 2A). To overcome this alignment bias, we assigned random initial orientation values to all particles and constrained possible angular shifts to less than 30° to achieve a more uniform distribution of orientations (Fig. S2). This strategy resulted in a much improved density map (Fig. 2B) that revealed individual molecules with a size and shape that are in good agreement to the known molecular structure of MscS (Fig. 2C and Fig. S3). Building on the analysis of Haselwandter and Phillips (25), a systematic analysis was conducted (Table S2) of the symmetry relationships between MscSs in MPPNs that identified the arrangement corresponding to the snub cuboctahedron (dextro), an Archimedean solid. The snub cuboctahedron has cubic (octahedral) symmetry which, as recognized by Crick and Watson (26), provides an efficient way to pack identical particles in a closed, convex shell. In this particular arrangement, 24 MscS molecules are related by the 432-point group symmetry axes that pass through the faces, but not the vertices, of the snub cuboctahedron. Because the MscS molecules are positioned on the vertices of this chiral polyhedron, they occupy general positions that permit the ordered packing of the heptamers of a biomacromolecule (or indeed any type of particle). This is an important observation as it means that the individual MscS molecules with sevenfold symmetry are capable of packing into a symmetric assembly that is amenable to averaging. Whereas 24 objects can be arranged with identical environments in a snub cuboctahedron, certain integer multiples of this number can also be accommodated using the principles of quasiequivalence (27, 28) to form larger closed shells.Open in a separate windowFig. 2.Cryotomography of MscS MPPNs. (A) The PEET isosurface derived from 162 individual particles selected from eight single-tilt tomograms. The strong bias due to the missing wedge is observed along the lower part of the surface, but individual MscS heptamers are still discernible in the image. (B) The corresponding PEET isosurface, following introduction of randomized starting Euler angles to minimize missing-wedge bias. (C) The use of randomized starting Euler angles results in a much-improved map with apparent octahedral (432) symmetry that could be fit with 24 molecules of the MscS crystal structure. Isosurface renderings of the volume averages were generated using Chimera (31).Using the symmetry derived by electron cryotomography, we proceeded to collect high-resolution single-particle electron cryomicroscopy data. Samples prepared identically for cryotomography were imaged under low-dose conditions and a total of 4,564 particles were processed using the Electron Micrograph Analysis 2 (EMAN2) software package (SI Materials and Methods) (29). The final map had a resolution of 9 Å by Fourier shell correlation (Fig. S4) and allowed us to model the inner and outer helices of the transmembrane pore (Fig. 3). The arrangement of the helices more closely resembles the nonconducting conformation (16, 18) than the open-state structure (19), although some differences in the positioning of the outer helices relative to the nonconducting structure are indicated in sections 2 and 3 of Fig. 3. These results demonstrate that membrane proteins are capable of assembling into MPPNs that are amenable to high-resolution structure analysis by single-particle electron cryomicroscopy. Higher resolution data will be required, however, to detail the precise conformational differences between MscS in the phospholipid environment of MPPNs compared with those in the detergent-solubilized state used in the X-ray crystal structure analyses.Open in a separate windowFig. 3.Single-particle image analysis reconstructed from 4,564 particles processed with EMAN2 and subsequently the density surrounding a single MscS heptamer was extracted and sevenfold averaged as described in SI Materials and Methods. (Left) A cross-section through the electron density revealing the translocation pathway and cytoplasmic vestibule, and showing the overall fit of the closed structure of MscS (red ribbons) fit to the map (cyan). (Right) Stereoviews of cross-sections in the density map normal to the sevenfold axis at sections 1, 2, and 3. The closed-structure coordinates (red ribbons) of MscS were fit to the map using rigid body refinement in Chimera (31) showing the position of the transmembrane helices.In these promising initial studies we used traditional dialysis methods to screen conditions for MPPN formation. These methods are time consuming and require substantial quantities of a sample. To more efficiently screen conditions for MPPN formation with a variety of membrane proteins, we designed and fabricated a free interface diffusion microfluidic device (30) (Fig. 4A and Fig. S5) This device greatly simplifies the screening process and minimizes the amount of sample required for determining suitable conditions for MPPN formation. Using this device, we were able to produce MPPNs from MscS but more importantly from several other proteins that had previously failed to produce MPPNs using traditional dialysis. Fig. 4 B and C shows the results of using this device for the mechanosensitive channel of large conductance (MscL) and the connexon Cx26, respectively, where polyhedra were only observed in the presence of the target protein. Intriguingly, several different particles sizes could be observed for both MscL and Cx26 and we hypothesize that the variable-sized polyhedra may correspond to different packing arrangements similar to triangulation numbers observed in viral polyhedral assemblies. This microfluidic device will provide rapid screening of conditions for the formation of MPPNs and it is hoped will expedite membrane protein structural analysis in native lipid environments.Open in a separate windowFig. 4.Preparation of MPPNs using a microfluidics-based free interface diffusion system. (A) Schematic illustration of the device used for lipid–protein nanoparticle formation. From left to right, molecules in the center flow diffuse into the outer flow by the concentration gradient, with small molecules (larger diffusion coefficient) moving more quickly than larger molecules. Specifically, monomer detergents are removed through interfacial diffusion, whereas larger membrane proteins remain in the center flow, forming nanoparticles. Both the ratio of input:buffer and the flow rate influence particle formation. (B). Negative-stain electron microscopy images of MPPNs of MscL and (C) Cx26 formed using the microfluidic device from A. (Scale bar, 100 nm.) Insets show 2.5× magnification of a select region of interest.The self-assembly of membrane proteins into polyhedral nanoparticles demonstrates a potentially powerful method for studying the structure and function of membrane proteins in a lipid environment. MPPNs represent a novel form of lipid–protein assemblies which lie between single particles and large crystalline sheets or tubes. We have demonstrated that conditions favorable for MPPN formation can be identified and have elucidated the structure, symmetry, and potential application to membrane protein structure analysis. In addition we have designed and fabricated microfluidic devices for high-throughput screening of conditions for MPPN formation. MPPNs may allow a variety of perturbations to be achieved such as pH, voltage, osmotic, concentration gradients, etc. that cannot be achieved with other membrane protein assemblies and will potentially allow us to activate various types of gated channels and receptors so that active conformational states can be structurally investigated. The potential of such materials for targeted drug delivery with precisely controlled release mechanisms offers an intriguing avenue for future biomedical applications.     

Charge transport and rectification in molecular junctions formed with carbon-based electrodes

Molecular junctions formed using the scanning-tunneling-microscope–based break-junction technique (STM-BJ) have provided unique insight into charge transport at the nanoscale. In most prior work, the same metal, typically Au, Pt, or Ag, is used for both tip and substrate. For such noble metal electrodes, the density of electronic states is approximately constant within a narrow energy window relevant to charge transport. Here, we form molecular junctions using the STM-BJ technique, with an Au metal tip and a microfabricated graphite substrate, and measure the conductance of a series of graphite/amine-terminated oligophenyl/Au molecular junctions. The remarkable mechanical strength of graphite and the single-crystal properties of our substrates allow measurements over few thousand junctions without any change in the surface properties. We show that conductance decays exponentially with molecular backbone length with a decay constant that is essentially the same as that for measurements with two Au electrodes. More importantly, despite the inherent symmetry of the oligophenylamines, we observe rectification in these junctions. State-of-art ab initio conductance calculations are in good agreement with experiment, and explain the rectification. We show that the highly energy-dependent graphite density of states contributes variations in transmission that, when coupled with an asymmetric voltage drop across the junction, leads to the observed rectification. Together, our measurements and calculations show how functionality may emerge from hybrid molecular-scale devices purposefully designed with different electrodes beyond the so-called “wide band limit,” opening up the possibility of assembling molecular junctions with dissimilar electrodes using layered 2D materials.Recent interest in understanding charge transport in molecular-scale devices and at metal/organic interfaces has led to innovations in both experimental and theoretical techniques designed to probe such devices (1, 2). Molecular junctions in a metal–molecule–metal motif using a variety of metals including Au, Ag, Pt, Al, and Cu have been studied extensively (3–7), contributing significantly to our understanding of the fundamental principles required to realize molecular-scale electronic components such as rectifiers or switches (8–14). However, the nanogap electrodes using such metals are mechanically unstable due to the high atomic mobility of metal atoms (15–18) and all except for Au oxidize easily under ambient conditions (6). Furthermore, the electrode density of states near the Fermi energy is typically nearly energy independent. This results in molecular junctions formed with metals having rather smooth and featureless transmission probabilities around the Fermi energy, limiting their applications. Carbon-based electrodes such as graphite have remarkable mechanical strength as well as a nonconstant highly dispersive density of states near its Fermi energy (19). In addition, molecules can be bonded covalently to carbon-based materials and can also bind through a van der Waals-based π–π stacking interaction (20). However, to date, such materials have not been used to create molecular junctions using the scanning-tunneling-microscope–based break-junction technique (STM-BJ). All-carbon electrodes have been used in the past, including carbon nanotubes and graphene (21–23); however, such devices are not easy to fabricate and characterize electronically with a statistically significant method. Moreover, there have been no computational studies on such junctions aimed at understanding the relation between charge transport and electrode properties.Here, we measure the conductance of a series of graphite/amine-terminated oligophenyl/Au molecular junctions using the STM-BJ technique (4). We show that the conductance of this series decays exponentially with molecular backbone length with a decay constant that is essentially the same as that for measurements with Au electrodes. We show further that these molecular junctions rectify (14, 24), due to an asymmetry in the coupling of the molecule with the Au and graphite electrodes. The nature and magnitude of the rectification is directly connected to the nonconstant density of states of graphite near the Fermi level. The trends from self-energy–corrected density functional theory calculations are in agreement with our experimental results; specifically, we find that junction conductance decreases as the junction is elongated, as the angle between the molecule and the graphite substrate increases. These measurements and calculations together demonstrate new classes of molecular junctions with dissimilar electrodes using layered 2D electrodes.     

Fundamentals of Endoscopic Surgery cognitive examination: development and validity evidence

Background

Flexible endoscopy is an integral part of surgical care. Exposure to endoscopic procedures varies greatly in surgical training. The Society of American Gastrointestinal and Endoscopic Surgeons has developed the Fundamentals of Endoscopic Surgery (FES), which serves to teach and assess the fundamental knowledge and skills required to practice flexible endoscopy of the gastrointestinal tract. This report describes the validity evidence in the development of the FES cognitive examination.

Methods

Core areas in the practice of gastrointestinal endoscopy were identified through facilitated expert focus groups to establish validity evidence for the test content. Test items then were developed based on the content areas. Prospective enrollment of participants at various levels of training and experience was used for beta testing. Two FES cognitive test versions then were developed based on beta testing data. The Angoff and contrasting group methods were used to determine the passing score. Validity evidence was established through correlation of experience level with examination score.

Results

A total of 220 test items were developed in accordance with the defined test blueprint and formulated into two versions of 120 questions each. The versions were administered randomly to 363 participants. The correlation between test scores and training level was high (r = 0.69), with similar results noted for contrasting groups based on endoscopic rotation and endoscopic procedural experience. Items then were selected for two test forms of 75 items each, and a passing score was established.

Conclusions

The FES cognitive examination is the first test with validity evidence to assess the basic knowledge needed to perform flexible endoscopy. Combined with the hands-on skills examination, this assessment tool is a key component for FES certification.     

Liver–kidney transplantation to cure atypical HUS: still an option post-eculizumab?

Patients with end-stage renal disease (ESRD) due to atypical HUS (aHUS) now have several potential options that can enable successful kidney transplantation. This editorial addresses these options by considering key factors that are important when making an individual treatment decision.     

A selective allosteric potentiator of metabotropic glutamate (mGlu) 2 receptors has effects similar to an orthosteric mGlu2/3 receptor agonist in mouse models predictive of antipsychotic activity

Recent studies suggest that agonists of group II metabotropic glutamate (mGlu) receptors (mGlu2/3) have potential utility as novel therapeutic agents for treatment of psychiatric disorders such as anxiety and schizophrenia. Agonists of mGlu2/3 receptors block amphetamine- and phencyclidine (PCP)-induced hyperlocomotor activity in rodents, two actions that may predict potential antipsychotic activity of these compounds. We now report that LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], a recently described selective allosteric potentiator of mGlu2 receptor, has behavioral effects similar to mGlu2/3 receptor agonists. LY487379 and LY379268 [(-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate], an ortho-steric mGlu2/3 receptor agonist, induced similar dose-dependent reductions in PCP- and amphetamine-induced hyperlocomotor activity in C57BL6/J mice at doses that did not significantly alter spontaneous locomotor activity. These effects were blocked by the mGlu2/3 receptor antagonist LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid]. LY487379 had a short duration of action compared with LY379268. Furthermore, unlike the mGlu2/3 agonist, LY487379 reversed amphetamine-induced disruption of prepulse inhibition of the acoustic startle reflex. When LY379268 was given chronically, it failed to block amphetamine- and PCP-induced hyperlocomotor activity. The finding that the effects of an orthosteric mGlu2/3 receptor agonist in these models can be mimicked by a selective allosteric potentiator of mGlu2 suggests that these effects are mediated by the mGlu2 receptor subtype. Furthermore, these data raise the possibility that a selective allosteric potentiator of mGlu2 receptor could have utility as a novel approach for the treatment of schizophrenia.     

Melanoma Screening by Means of Complete Skin Exams for All Patients in a Dermatology Practice Reduces the Thickness of Primary Melanomas at Diagnosis

Objective: Previous studies have shown that dermatologists detect thinner melanomas than both non-dermatologists and patients in high incidence areas. The authors report depths of melanomas in a central New York practice where the incidence is low, hypothesizing that incidental melanomas detected by a dermatologist will be thinner than melanomas that are part of the chief complaint. Design: A retrospective chart review examining melanoma depth to determine the importance of universal full skin exams. Setting: Private dermatology clinic in Auburn, New York, employing one board-certified dermatologist and two physician extenders. Participants: Men and women who attended the clinic between 2003 and 2013 who had 235 biopsy-proven melanomas. Total patient visits in this time period was 50,699. Measurements: Office notes were reviewed to determine the chief complaint, patient demographics, and depth of the tumor. The authors noted if the melanoma was discovered by the patient, a referring physician, dermatology physician extender, or the dermatologist. Results: More than 45 percent of melanomas were an incidental finding on full skin exam. The dermatologist detected statistically thinner melanomas than melanomas that presented as the chief complaint. The dermatologist tended to detect thinner melanomas than referring physicians and patients. Conclusion: A significant portion of melanomas are incidentally found on full skin exam, and thinner melanomas are detected by dermatologists. Universal skin cancer screening takes little additional time, and appropriate use of physician extenders can greatly increase access to dermatological care. Full skin exams increase melanoma detection, decreases overall thickness at diagnosis, and decreases patient morbidity and mortality.Melanoma is the most deadly type of skin cancer with limited treatment for deep tumors. The average age adjusted melanoma death was 2.73 per 100,000 in the United States between 2006 and 2010.1 Thus, early detection of melanoma offers the best hope for a cure. Cutaneous melanoma presents a unique opportunity for intervention compared to other malignancies, as screening is simple, noninvasive, and takes little time. Clinical full skin exams have been shown to decrease the number of deep melanomas,2 which is important because the depth of invasion for malignant melanoma is the most important prognostic factor. However, even with the availability of such an easy screening test, the guidelines of when and whom to screen are unclear.3 The effect of screening on patient mortality has not been studied in a randomized trial, and formal guidelines are lacking. Previous studies have attempted to determine who detects melanomas and how they present in their clinics.2,4–6 Several of these studies were performed in areas of higher incidence of melanoma, such as Australia2 and Florida.6 The aim of this study is to report the percentage of the authors’ patients incidentally found to have melanoma as part of a full skin exam and to compare the depth of invasion with patients whose melanoma was related to their chief complaint. The authors compare their data from a private practice near Syracuse, New York, which has a relatively low incidence of melanoma in the United States,1 to some of the previously reported studies and make recommendations for future screening.     

Epidemiology of treatment-associated mucosal injury after treatment with newer regimens for lymphoma, breast, lung, or colorectal cancer

Goals of work: Oral and gastrointestinal (GI) mucositis are frequent complications of chemotherapy and radiotherapy for cancer, contributing to not only the morbidity of treatment but its cost as well. The risk associated with specific chemotherapeutic agents, alone and in combination, has been characterized previously. In the current study, we sought to estimate the risk associated with newer regimens for the treatment of non-Hodgkin’s lymphoma (NHL) and common solid tumors. Methods: We reviewed published studies reporting phase II and III clinical trials of dose-dense regimens for breast cancer and NHL, TAC (docetaxel, adriamycin, cyclophosphamide) chemotherapy for breast cancer, and infusional 5-fluorouracil-based regimens for colorectal cancer. Platinum-, gemcitabine-, and taxane-based regimens for lung cancer, either alone or in combination with radiotherapy, were also considered. Using modified meta-analysis methods, we calculated quality-adjusted estimates of the risk for oral and GI mucositis by tumor type and regimen. Case reports are used to emphasize the relevance of the findings for patient care. Main results: Our findings demonstrate that mucosal toxicity remains an important complication of cancer treatment. Moreover, innovations in drug combinations, scheduling, or mode of administration significantly modulate the risk for both oral and GI mucositis. Conclusions: Ongoing review of the clinical trial experience will remain important as newer, targeted agents enter standard clinical practice.     

Safety features of budesonide inhalation suspension in the long-term treatment of asthma in young children

Early inhaled corticosteroid treatment improves symptom control and pulmonary function in children with asthma; however, long-term safety data are limited in infants and young children. This study assessed the long-term safety of budesonide inhalation suspension (BIS) in young children with persistent asthma. To continue to provide BIS to children who needed it—prior to US Food and Drug Administration approval—children 8 years of age or younger with mild, moderate, or severe persistent asthma who previously completed a 52-week open-label study of BIS were enrolled in an additional multicenter, open-label study that was to be concluded upon BIS approval. Patients already receiving BIS continued their current regimens. Patients younger than 4 years and those 4 years of age or older not receiving BIS at baseline started with total daily doses of 0.5 and 1.0 mg, respectively. BIS doses were adjusted throughout the study based on individual response. Adverse events and changes in laboratory parameters, vital signs, and physical examination findings were assessed. Of 198 enrolled patients, 152 (76.8%), 68 (34.3%), and 31 (15.7%) completed 1, 2, and 3 years of BIS treatment (mean daily dose 0.62±0.32 mg), respectively. One hundred sixty-six (83.8%) patients experienced an adverse event, of which 8.6% were considered by the investigator to be drug related. Adverse events were those typically occurring in a pediatric asthma population, with respiratory infection (49.0%) and sinusitis (25.3%) occurring at the greatest incidence. Only 2 patients withdrew due to adverse events. Mean changes in laboratory test results and physical examination findings were not clinically important throughout the study. Long-term BIS treatment is well tolerated in young children with persistent asthma, with a safety profile similar to that of short-term administration.     

Population pharmacokinetics and pharmacodynamics of cefpirome in critically ill patients against Gram-negative bacteria

Objectives To develop a population pharmacokinetics model for cefpirome in ICU patients, to assess pharmacokinetic-pharmacodynamic profiles vs. MIC distribution of likely ICU pathogens, and to assess their expected cumulative fraction of response (CFR). Design and setting Prospective observational study in a multidisciplinary ICU. Measurements and results Twelve patients received 2 g cefpirome intravenously over 12 h. Thirteen blood samples were taken on two occasions. Demographic and creatinine clearance data were collected. Based on the final covariate model obtained using NONMEM, Monte Carlo simulations were undertaken to simulate free-drug concentrations for two administration methods: intermittent bolus administration (IBA) and continuous infusion (CI) with a loading dose of 0.5 g. Concentration-time profiles were evaluated by the probability of achieving free-drug concentrations above the MIC for more than 65% of dosing interval. Using MIC distributions from the EUCAST programme the CFR for each method was evaluated. A three-compartment model with zero-order input best described the concentration-time data. The CFR for Escherichia coli and Klebsiella spp. was greater than 97% in all IBA and CI doses but for Pseudomonas aeruginosa, and Acinetobacter spp. achieved target concentrations of 56% and 46%, respectively. High-dose CI cefpirome (6 g/day) for P. aeruginosa and Acinetobacter spp. was required to achieve CFR of 89%. Conclusion Measured creatinine clearance appears to be a good marker of cefpirome clearance and potentially could be used to individualise cefpirome therapy. When given as IBA or CI for E. coli and Klebsiella spp., cefpirome should be successful. Cefpirome fails to achieve the bactericidal target even when administered at high-doses such as 6 g/day for P. aeruginosa and Acinetobacter spp. Prospective clinical studies are needed to conclusively validate these findings.