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Radiographic stress shielding is a common finding in uncemented convertible short-stem shoulder arthroplasty (UCSSSA). The distal filling ratio (DFR) has been described as a predictor for the occurrence of stress shielding. A DFR 70% was mentioned as a risk factor for the occurrence of stress shielding for some UCSSSA. However, measurements were only performed on conventional radiographs and no validation exists for 3D automated planning tools.DFR was manually measured on postoperative true ap radiographs of 76 shoulder arthroplasties using a standardized protocol and were compared to preoperative CT scans with an automated calculation of the DFR after virtual implantation of the stem.The mean DFR measured on X-rays was 75.9% (SD = 8.7; 95% CI = 74-78) vs. 78.9% (SD = 9.1; 95% CI = 76.8-83) automatically measured on CT scans. This difference was significant (p 0.001). In 7 out of 76 cases (9%) the difference between manual measurement on radiographs and computerized measurement on CT scans was 10%.Manual measurement of the DFR is underestimated on conventional radiographs compared to automated calculation on CT scans be a mean of 3%. Therefore, automated measurement of the DFR on CT scans seems to be beneficial, especially in cases with osteopenic cortices. Manual measurement of the DFR on conventional ap radiographs in cases without CT scans, however, is still a viable alternative.Level IV, retrospective study.

Three-dimensional surgical planning software provides virtual reconstructions of the shoulder with automated joint indices for a preoperative case assessment. The aim of this single center study was to evaluate the concordance between the preoperatively selected humeral components and the final implants used in shoulder arthroplasty.129 cases who had undergone anatomic (n = 16) or reverse shoulder arthroplasty (n = 117) using the same type of uncemented short stem implant and were included for review in this study. The type of arthroplasty, stem size, stem inclination, tray-offset and liner-thickness were noted preoperatively and compared to the final implant specifications used in surgery.The type of arthroplasty matched the surgical plan in 99.2% of cases, as one case was converted from RSA to TSA. The concordance of planned to implanted stem size was 44.2% and the planned size was in range of one adjacent size in 87.6% of cases. Stem inclination in TSA matched the surgical plan in 50% of cases. Tray offset in RSA was predicted correctly in 65% and liner-thickness matched the surgical plan in 98.3% of cases.Despite a low degree of concordance of planned to implanted stem sizes of 44.2%, the choice of stem size was found to be in range of one adjacent size in 87.6% of cases. Further investigations of other contributing factors are necessary to increase the accuracy of the preoperative selection of humeral implants.level IV, retrospective case study.

Background Several short-stemmed press-fit humeral components have been developed in recent years for anatomic total shoulder arthroplasty (TSA) as well as reverse shoulder arthroplasty (RSA). Varying radiographic outcomes have been reported, with some studies reporting concerning rates of aseptic loosening. This study analyzed the radiographic findings of a press-fit convertible short-stemmed humeral component in both TSA and RSA. Methods There were 150 anatomic TSAs (group 1) and 77 RSAs (group 2) analyzed radiographically at a minimum follow-up of 2 years postoperatively. Plain radiographs were reviewed for stem loosening, alignment, signs of stress shielding, and the filling ratio. Results At final follow-up, 49% of group 1 and 65% of group 2 had no evidence for radiographic changes. In those with radiographic changes, low bone adaptions were found in 83% and high adaptions in 17% in both groups. Larger stem sizes with higher filling ratios were associated with high radiographic adaptions in both groups (P = .02). The overall filling ratios were higher in group 2 (P = .002). Cortical contact of the stem led to higher bone adaptions (P = .014). Conclusions The short humeral component analyzed in this study showed encouraging survival rates without aseptic loosening. Radiographic changes are associated with a higher filling ratio and cortical contact of the stem. Surgeons should aim to achieve fixation with the minimal required canal filling to minimize radiographic changes with the uncemented humeral component used in this study.

Background: The radiological phenomenon of subsidence following the implantation of uncemented short-stem reverse prostheses (USSP) has not yet been described. The purpose of this study was to describe the rate and potential risk factors for subsidence. We hypothesized that subsidence may be a frequent finding and that a subsidence of &gt, 5 mm (mm) is associated with an inferior clinical outcome. Methods: A total of 139 patients with an average age of 73 &plusmn, 9 years were included. The clinical and radiological outcome was evaluated at a minimum follow-up (FU) of 12 months. Results: No humeral component loosening was present at a mean FU of 18 (range, 12&ndash, 51) months. Mean Constant Score (CS) and Subjective Shoulder Value (SSV) improved significantly from 34.3 &plusmn, 18.0 points and 37.0 &plusmn, 19.5% preoperatively to 72.2 &plusmn, 13.4 points and 80.3 &plusmn, 16.5% at final FU (p &lt, 0.001). The average subsidence of the USSP was 1.4 &plusmn, 3.7 mm. Subsidence of &gt, 5 mm was present in 15 patients (11%). No association between a subsidence &gt, 5 mm and CS or SSV was found (p = 0.456, p = 0.527). However, a subsidence of &gt, 5 mm resulted in lower strength at final FU (p = 0.022). Complications occurred in six cases (4.2%), and the revision rate was 3.5% (five cases). Conclusions: Although subsidence of USSP is a frequent radiographic finding it is not associated with loosening of the component or a decrease in the clinical outcome at short term FU. Level of evidence: Level 4, retrospective study.

The sorption properties of metal–organic frameworks (MOFs) can be influenced by introducing covalently attached functional side chains, which make this subclass of porous materials promising for applications as diverse as gas storage and separation, catalysis, and drug delivery. The incorporation of side groups usually comes along with disorder, as the synthesis procedures rarely allow for one specific position among a larger group of equivalent sites to be selected. For a series of isoreticular CAU-1 frameworks, chosen as model compounds, one out of four positions at every linker is modified with equal probability. Here, we investigate the influence of this disorder on Ar sorption and 129Xe nuclear magnetic resonance spectroscopy using hyperpolarized 129Xe gas. Models used for predicting the pore dimensions as well as their distributions were derived from the unfunctionalized framework by replacing one proton at every linker with either an amino, an acetamide, or a methyl urea functionality. The resultin...

The aim of the study was to evaluate the short-term clinical results of anatomic total shoulder arthroplasty with a short-stem prosthesis in primary osteoarthritis.65 shoulders with a mean age of 70 years (range 47-85 years) were available for minimum follow-up of 24 months. Clinical outcome was determined by range of motion, Constant score (CS) age and sex-adjusted Constant score (CS%), and subjective shoulder value (SSV). The influence of six different factors (high bone adaptations, age 65 years, female gender, dominant side, atrophy of the supraspinatus tendon ≥ grade 2, glenoid type B2/B3) on the clinical outcome was assessed.At mean follow-up of 37 months (range 24-58 months), the CS improved from 36 ± 8 to 75 ± 12 (p 0.001). The shoulder flexion (100° ± 21° to 159° ± 19°) as well as the external rotation (3° ± 11° to 43° ± 18°) improved significantly (p 0.001). Three complications were noted (transient neuropraxia of the radial nerve, subjective instability, hematoma with superficial wound infection) leading to one revision surgery (wound debridement). No stem loosening was observed. High bone adaptation was present in 19 out of 65 shoulders (29%). The clinical outcome was not influenced by high bone adaptations (p ≥ 0.095). Age 65 years (n = 44) and female gender (n = 38) were associated with worse clinical outcome (p ≤ 0.043).In the short term, the clinical results of this anatomical short-stem shoulder prosthesis are encouraging. A low prevalence of high bone adaptations was found without any influence on the clinical outcome and stem loosening was not observed.

This study analyzed the radiographic results of a cemented all-polyethylene keeled glenoid component available in different sizes and multiple backside radiuses of curvature.The study group consisted of 118 cases (114 patients). There were 63 women and 51 men. Mean age at the time of arthroplasty was 68 years (range, 51-85 years). True anterior-posterior radiographs obtained postoperatively and at the final follow-up were analyzed for implant seating and the occurrence of radiolucent lines. Glenoid morphology and fatty infiltration of the rotator cuff muscles were examined using computed tomography scans. Mean follow-up was 38 months (range, 24-70 months).The mean radiolucent line score after surgery was 0.54 points (range, 0-3 points), and 90% had no or only 1 radiolucent line. At the final follow-up, the mean score was 1.06 points (range, 0-3 points), and 74% had no or only 1 radiolucent line. The score increased significantly over time (P .001). No component was at risk for loosening. No correlation was found between patient age, sex, hand dominance, glenoid morphology, or fatty infiltration of the rotator cuff muscles and the occurrence of radiolucent lines.In the short-term, the glenoid component analyzed in this study showed promising radiographic results, with a low number of radiolucent lines without failure. However, the mean radiolucent line score increased significantly over time, and long-term observations are necessary to confirm a possible advantage compared with older component designs.

Mutations in the ATP-binding cassette subfamily A member 3 (ABCA3) gene are the most common monogenetic cause of surfactant dysfunction disorders in newborns and interstitial lung diseases in children and young adults. Although the effect of mutations resulting in truncated or incomplete proteins can be predicted, the consequences of missense variants cannot be as easily. Our aim was to investigate the intracellular handling and disturbance of the cellular surfactant system in a stable cell model with several different clinically relevant ABCA3 missense mutations. We found that the investigated missense mutations within the ABCA3 gene affect surfactant homeostasis in different ways: first by disrupting intracellular ABCA3 protein localization (c. 643C > A, p. Q215K; c. 2279T > G, p. M760R), second by impairing the lipid transport of ABCA3 protein (c. 875A > T, p. E292V; c. 4164G > C, p. K1388N), and third by yet undetermined mechanisms predisposing for the development of interstitial lung diseases despite correct localization and normal lipid transport of the variant ABCA3 protein (c. 622C > T, p. R208W; c. 863G > A, p. R288K; c. 2891G > A, p. G964D). In conclusion, we classified cellular consequences of missense ABCA3 sequence variations leading to pulmonary disease of variable severity. The corresponding molecular pathomechanisms of such ABCA3 variants may specifically be addressed by targeted treatments.

The ABCA3 lipid transporter is located in the limiting membrane of lamellar bodies (LBs) in type-II-pneumocytes. Mutations within the ABCA3 gene may functionally impair the transporter, causing lung diseases in newborns, children and adults. Assays to quantify volume and lipid filling of the LBs on the level of the vesicular structures and thereby assess the function of ABCA3 are still lacking. In the present study human influenza haemagglutinin- (HA-) tagged wild type and mutant ABCA3 proteins were stably expressed in lung A549 cells. Fluorescently-labelled TopFluor phosphatidylcholine (TopF-PC) incorporated in surfactant-like liposomes was delivered to the cells and visualized by confocal microscopy. Subsequently, a comprehensive image analysis method was applied to quantify volume and fluorescence intensity of TopF-PC in ABCA3-HA-positive vesicles. TopF-PC accumulated within the vesicles in a time and concentration-dependent manner, whereas the volume remained unchanged, suggesting active transport into preformed ABCA3 containing vesicles. Furthermore, this finding was supported by a decrease of the fluorescence intensity within the vesicles when either the ATPase of the transporter was inhibited by vanadate, or when a disease-causing mutation (K1388N) close to the ABCA3-nucleotide binding domain 2 was introduced. Conversely, a mutation (E292V) located in the first cytoplasmic loop of ABCA3 did not significantly affect lipid transport, but rather resulted in smaller vesicles. In addition to these findings, the assay used in this work for analysing the PC-lipid transport into ABCA3 positive vesicles will be useful to screen for compounds susceptible to restore function in mutated ABCA3 protein.

Introduction Preoperative 3D planning programs for anatomic (TSA) and reverse total shoulder arthroplasty (RSA) allow the analysis of glenohumeral joint pathoanatomy and templating for implant size selection and placement. The aim of this multicenter study was to compare the preoperative glenoid implant type and size planned to the final glenoid implant type and size used intraoperatively. Methods Two hundred patients (100 TSA and 100 RSA) with a mean age of 72 years who had undergone preoperative planning and subsequent shoulder arthroplasty (100 TSA and 100 RSA) were included. All preoperative plans were saved and were analyzed for arthroplasty type (TSA vs. RSA), implant type (augment vs. nonaugment), and size (ie, polyethylene size, polyethylene radius of curvature, glenoid baseplate diameter, baseplate post length, and baseplate lateralization). The preoperative plan was available during surgery and was compared to the final implants inserted by the surgeon. Results There were no intraoperative conversions of TSA to RSA or vice versa. In patients planned for a TSA, complete concordance between the preoperative plan and final implant selection was 85%. A complete mismatch for TSA glenoid size, backside radius of curvature, and augmentation occurred in 2%. For RSA, complete concordance was found in 90% of cases. A complete mismatch for implant type, size, post length, and glenosphere size occurred in 3%. Conclusion A high concordance was found between preoperative 3D planning implant selection and the glenoid component inserted at surgery for TSA and RSA. This high concordance may assist with surgical preparedness, implant stocks, and possibly future implant production.

Shorter humeral reverse total shoulder arthroplasty (RTSA) stems may reduce stress shielding, however, potentially carry the risk of varus/valgus malalignment. This radiographic study’s purpose was to measure the incidence of stem malalignment and thus the realized neck-shaft angle (NSA). The hypothesis was that malalignment of the stem is a frequent postoperative radiographic finding. Radiographs of an uncemented curved short stem RTSA with a 145° NSA were reviewed. The study group included 124 cases at a mean age of 74 (range 48–91) years. The humeral stem axis was measured and defined as neutral if the value fell within ± 5° of the longitudinal humeral axis. Angular values > 5° were defined as malaligned in valgus or varus. The filling ratio of the implant within the humeral shaft was measured at the level of the metaphysis (FRmet) and diaphysis (FRdia). The average humeral stem axis angle was 4 ± 3° valgus, corresponding to a true mean NSA of 149 ± 3°. Stem axis was neutral in 73% (n = 90) of implants. Of the 34 malaligned implants, 82% (n = 28) were in valgus (NSA = 153 ± 2°) and 18% (n = 6) in varus (NSA = 139 ± 1°). The average FRmet and FRdia were 0.68 ± 0.11 and 0.72 ± 0.11, respectively. A low positive association was found between stem diameter and filling ratios (r = 0.39; p 5° malaligned. The majority of malaligned components (86%) were implanted in valgus, corresponding to an NSA of > 150°. As such, surgeons must be aware that shorter and smaller stems may lead to axial malalignment influencing the true SA. Level IV, retrospective study.

Background Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort. Methods We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015. Results Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects. Conclusions Overall long-term (>5 years) survival of subjects with two disease-causing ABCA3 mutations was

ABCA3 is a surfactant lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ATP-binding cassette, sub-family A (ABC1), member 3 (ABCA3) gene cause respiratory distress syndrome in newborns, and chronic interstitial lung disease in children and adults. ABCA3 belongs to the class of full ABC transporters, which are supposed to be functional in their monomeric forms. Although other family members e.g., ABCA1 and ABCC7 have been shown to function as oligomers, the oligomerization state of ABCA3 is unknown. In the present study, the oligomerization of ABCA3 was investigated in cell lysates and crude membrane preparations from transiently and stably transfected 293 cells using blue native PAGE (BN-PAGE), gel filtration and co-immunoprecipitation. Additionally, homooligomerization was examined in vivo in cells using bioluminescence resonance energy transfer (BRET). Using BN-PAGE and gel filtration, we demonstrate that non-denatured ABCA3 exists in different oligomeric forms, with monomers (45%) and tetramers (30%) being the most abundant forms. Furthermore, we also show the existence of 20% dimers and 5% trimers. BRET analyses verified intermolecular interactions in vivo. Our results also demonstrated that the arrest of ABCA3 in the endoplasmic reticulum (ER), either through drug treatment or induced by mutations in ABCA3, inhibited the propensity of the protein to form dimers. Based on our results, we suggest that transporter oligomerization is crucial for ABCA3 function and that a disruption of oligomerization due to mutations represents a novel pathomechanism in ABCA3-associated lung disease.

The ABCA3 gene encodes a lipid transporter in type II pneumocytes critical for survival and normal respiratory function. The frequent ABCA3 variant R288K increases the risk for neonatal respiratory distress syndrome among term and late preterm neonates, but its role in children’s interstitial lung disease has not been studied in detail. In a retrospective cohort study of 228 children with interstitial lung disease related to the alveolar surfactant system, the frequency of R288K was assessed and the phenotype of patients carrying a single R288K variant further characterized by clinical course, lung histology, computed tomography and bronchoalveolar lavage phosphatidylcholine PC 32:0. Cell lines stably transfected with ABCA3-R288K were analyzed for intracellular transcription, processing and targeting of the protein. ABCA3 function was assessed by detoxification assay of doxorubicin, and the induction and volume of lamellar bodies. We found nine children with interstitial lung disease carrying a heterozygous R288K variant, a frequency significantly higher than in the general Caucasian population. All identified patients had neonatal respiratory insufficiency, recovered and developed chronic interstitial lung disease with intermittent exacerbations during early childhood. In vitro analysis showed normal transcription, processing, and targeting of ABCA3-R288K, but impaired detoxification function and smaller lamellar bodies. We propose that the R288K variant can underlie interstitial lung disease in childhood due to reduced function of ABCA3, demonstrated by decelerated detoxification of doxorubicin, reduced PC 32:0 content and decreased lamellar body volume.

The sorption properties of metal-organic frameworks (MOFs) can be influenced by introducing covalently attached functional side chains, which make this subclass of porous materials promising for applications as diverse as gas storage and separation, catalysis, and drug delivery. The incorporation of side groups usually comes along with disorder, as the synthesis procedures rarely allow for one specific position among a larger group of equivalent sites to be selected. For a series of isoreticular CAU-1 frameworks, chosen as model compounds, one out of four positions at every linker is modified with equal probability. Here, we investigate the influence of this disorder on Ar sorption and

BACKGROUND: Children's interstitial lung diseases (chlLD) comprise a broad spectrum of diseases. Besides the genetically defined surfactant dysfunction disorders, most entities pathologically involve the alveolar surfactant region, possibly affecting the surfactant proteins SP-B and SP-C. Therefore, our objective was to determine the value of quantitation of SP-B and SP-C levels in bronchoalveolar lavage fluid (BALF) for the diagnosis of chlLD. METHODS: Levels of SP-B and SP-C in BALF from 302 children with chlLD and in controls were quantified using western blotting. In a subset, single-nucleotide polymorphisms (SNPs) in the SFTPC promoter were genotyped by direct sequencing. RESULTS: While a lack of dimeric SP-B was found only in the sole subject with hereditary SP-B deficiency, low or absent SP-C was observed not only in surfactant dysfunction disorders but also in patients with other diffuse parenchymal lung diseases pathogenetically related to the alveolar surfactant region. Genetic analysis of the SFTPC promoter showed association of a single SNP with SP-C level. CONCLUSION: SP-B levels may be used for screening for SP-B deficiency, while low SP-C levels may point out diseases caused by mutations in TTF1, SFTPC, ABCA3, and likely in other genes involved in surfactant metabolism that remain to be identified. We conclude that measurement of levels of SP-B and SP-C was useful for the differential diagnosis of chlLD, and for the precise molecular diagnosis, sequencing of the genes is necessary.

Diffuse parenchymal lung diseases (DPLDs) are characterized by chronic inflammation and fibrotic remodeling of the interstitial tissue. A small fraction of DPLD cases can be genetically defined by mutations in certain genes, with ABCA3 being the gene most commonly affected. However, the pathomechanisms underlying ABCA3-induced DPLD are far from clear. To investigate whether ABCA3 plays a role in cellular cholesterol homeostasis, phospholipids, free cholesterol, and cholesteryl esters were quantified in cells stably expressing ABCA3 using mass spectrometry. Cellular free cholesterol and lipid droplets were visualized by filipin or oil red staining, respectively. Expression of SREBP regulated genes was measured using qPCR. Cell viability was assessed using the XTT assay. We found that wild type ABCA3 reduces cellular free cholesterol levels, induces the SREBP pathway, and renders cells more resistant to loading with exogenous cholesterol. Moreover, ABCA3 mutations found in patients with DPLD interfere with this protective effect of ABCA3, resulting in free cholesterol induced cell death. We conclude that ABCA3 plays a previously unrecognized role in the regulation of cellular cholesterol levels. Accumulation of free cholesterol as a result of a loss of ABCA3 export function represents a novel pathomechanism in ABCA3-induced DPLD.

Understanding host–guest interactions is one of the key requirements for adjusting properties in metal–organic frameworks (MOFs). In particular, systems with coordinatively unsaturated Lewis acidic metal sites feature highly selective adsorption processes. This is attributed to strong interactions with Lewis basic guest molecules. Here we show that a combination of 13C MAS NMR spectroscopy with state-of-the-art density functional theory (DFT) calculations allows one to unravel the interactions of water, 2-aminopyridine, 3-aminopyridine, and diethylamine with the open metal sites in Cr-MIL-101. The 13C MAS NMR spectra, obtained with ultrafast magic-angle spinning, are well resolved, with resonances distributed over 1000 ppm. They present a clear signature for each guest at the open metal sites. Based on competition experiments this leads to the following binding preference: water < diethylamine ≈ 2-aminopyridine < 3-aminopyridine. Assignments were done by exploiting distance sum relations derived from spin...

Designing shuttles that help transit riders complete the last mile of their trip (from a transit station or stop to their destination) plays an important role in leveraging rail capacity and growing a regional transit market. Unavailable or imprecise data have made it difficult to conduct effective design for shuttle routes on the basis of a defined potential market. This study developed a methodology for determining the size and location of the untapped market of commuters who drove long distances for work but could have made the same trip with commuter rail and last-mile shuttle service. The analysis was conducted with U.S. Census Longitudinal Employer–Household Dynamics data, a relatively new public data set of statewide workplace and residence pairs at the census block level. As a case study, the analysis was applied to the potential for last-mile shuttle connections between employment and 14 FrontRunner commuter rail stations outside downtown Salt Lake City, Utah. For the shuttle market to be captured, the workplace and residence pairs in this methodology were selected on the basis of three criteria: ( a) a residence within the FrontRunner park-and-ride shed, ( b) a workplace within 2 mi of a FrontRunner station, and ( c) an overall trip distance of 15 mi or greater. The study identified stations with the highest number of trips that met all three criteria and designed shuttle routes on the basis of the associated employment locations. Although this analysis focused on nontraditional commuting patterns, the same methodology would be useful for many other analyses of commuter markets.

Adenosine triphosphate (ATP)-binding cassette subfamily A member 3 (ABCA3), a phospholipid transporter in lung lamellar bodies (LBs), is essential for the assembly of pulmonary surfactant and LB biogenesis. Mutations in the ABCA3 gene are an important genetic cause for respiratory distress syndrome in neonates and interstitial lung disease in children and adults, for which there is currently no cure. The aim of this study was to prove that disease causing misfolding ABCA3 mutations can be corrected in vitro and to investigate available options for correction. We stably expressed hemagglutinin (HA)-tagged wild-type ABCA3 or variants p.Q215K, p.M760R, p.A1046E, p.K1388N or p.G1421R in A549 cells and assessed correction by quantitation of ABCA3 processing products, their intracellular localization, resembling LB morphological integrity and analysis of functional transport activity. We showed that all mutant proteins except for M760R ABCA3 were rescued by the bithiazole correctors C13 and C17. These variants were also corrected by the chemical chaperone trimethylamine N-oxide and by low temperature. The identification of lead molecules C13 and C17 is an important step toward pharmacotherapy of ABCA3 misfolding-induced lung disease.

Introduction: ABCA3 is a lipid transporter expressed in alveolar type II pneumocytes. It is involved in the transport of phospholipids into lamellar bodies (LBs) and is therefore essential for the assembly of pulmonary surfactant and LB biogenesis. Mutations in the ABCA3 gene display a common genetic cause for respiratory distress syndrome in newborns and interstitial lung disease in children. Aim: As ABCA3 shares several features with CFTR (ABCC7), small molecules that are able to correct mutated CFTR proteins were tested for ABCA3 mutations. Methods: A549 cells stably expressing HA-tagged wildtype ABCA3 or variants Q215K, M760R, A1046E, K1388N, or G1421R, were treated with a range of different correctors to identify molecules that are able to restore ABCA3 processing and function. Cells were analyzed in regard to ABCA3 protein pattern in Western blotting and immunofluorescence. Activity of corrected ABCA3 protein was quantified using a lipid transport assay with TopFluor-conjugated phosphatidylcholine. Results: Two of the tested correctors restored processing of all ABCA3 variants except for M760R and led to a WT-like appearance of mutated protein in Western blotting and immunofluorescence. Lipid transport into ABCA3-HA-positive vesicles was increased by corrector treatment for WT ABCA3 and all variants except for M760R. Conclusion: The identification of molecules with the ability to correct processing and function of mutated ABCA3 proteins are an important step towards the development of drugs for the treatment of children suffering from respiratory distress syndrome or interstitial lung disease.

The resistance of metal-organic frameworks towards water is a very critical issue concerning their practical use. Recently, it was shown for microporous MOFs that the water stability could be increased by introducing hydrophobic pendant groups. Here, we demonstrate a remarkable stabilisation of the mesoporous MOF Al-MIL-101-NH2 by postsynthetic modification with phenyl isocyanate. In this process 86 % of the amino groups were converted into phenylurea units. As a consequence, the long-term stability of Al-MIL-101-URPh in liquid water could be extended beyond a week. In water saturated atmospheres Al-MIL-101-URPh decomposed at least 12-times slower than the unfunctionalised analogue. To study the underlying processes both materials were characterised by Ar, N2 and H2 O sorption measurements, powder X-ray diffraction, thermogravimetric and chemical analysis as well as solid-state NMR and IR spectroscopy. Postsynthetic modification decreased the BET equivalent surface area from 3363 to 1555 m(2) g(-1) for Al-MIL-101-URPh and reduced the mean diameters of the mesopores by 0.6 nm without degrading the structure significantly and reducing thermal stability. In spite of similar water uptake capacities, the relative humidity-dependent uptake of Al-MIL-101-URPh is slowed and occurs at higher relative humidity values. In combination with (1) H-(27) Al D-HMQC NMR spectroscopy experiments this favours a shielding mechanism of the Al clusters by the pendant phenyl groups and rules out pore blocking.

Fault-hiding control reconfiguration aims at hiding a fault from the nominal controller while the reconfigured closed-loop system possesses admissible behaviour. The necessary degrees of freedom are created by placing a reconfiguration block between nominal controller and faulty plant. We aim at a guaranteed non-conflicting, complete and controllable behaviour of the self-reconfiguring closed-loop system, in particular for an arbitrary solution to the nominal control problem. Thereby, the nominal controller design and the design of the reconfiguration block are completely decoupled. This is desirable from a practical perspective, since in this way additional fault-tolerant control capabilities can be retrofit to an existing control system. In this paper, we propose a self-reconfiguring control architecture, state our reconfiguration problem in terms of finite languages and address the synthesis of discrete event dynamic reconfiguration blocks. To illustrate our results, we provide a running example.

Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort.We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015.Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects.Overall long-term (5 years) survival of subjects with two disease-causing ABCA3 mutations was20%. Response to therapies needs to be ascertained in randomised controlled trials.

Fault-tolerant control systems with discrete-event dynamics allow for differing sets of design requirements, that specify the system's behaviour during nominal operation and in the case of component degradation or component malfunction. This paper is concerned with the design of fault-tolerant control algorithms for discrete event systems in the framework of supervisory control theory. Its main contribution is a modelling framework that describes the evolution of systems which are potentially subject to faults. These models are called fault-accommodating models. Within this context, the occurrence of faults is modelled by means of unobservable and uncontrollable events. Hence, the design problem of fault-tolerant controllers is transformed to a supervisory control problem under partial observation. Consequently, there is no need for explicitly diagnosing the occurrence of faults and relaxed diagnosability conditions are applied. Finally, the paper provides extensive examples in order to illustrate the application of all derived methodological results. All computations needed for controller design and system analysis were implemented using a freely available software toolbox.

This paper addresses abstraction-based supervisory control for plant and specification behaviours that are not necessarily ω-closed, i.e. plant behaviours that exhibit eventuality properties and specifications that impose eventuality properties on the closed loop. Technically, the core idea is to combine results from previous work on abstraction-based supervision of input-output behaviours with results on supervisory control of ω-languages. As our main result, we identify a controllability condition for the plant, that ensures a nonblocking closed-loop behaviour with a controller that has been obtained for a plant abstraction.

Interstitial lung diseases (ILD) comprise disorders of mostly unknown cause. Among the few molecularly defined entities, mutations in the gene encoding the ATP-binding cassette (ABC), subfamily A, member 3 (ABCA3) lipid transporter represent the main cause of inherited surfactant dysfunction disorders, a subgroup of ILD. Whereas many cases are reported, specific methods to functionally define such mutations are rarely presented.In this study, we exemplarily utilized a set of molecular tools to characterize the mutation K1388N, which had been identified in a patient suffering from ILD with lethal outcome. We also aimed to correlate in vitro and ex vivo findings.We found that presence of the K1388N mutation did not affect protein expression, but resulted in an altered protein processing and a functional impairment of ABCA3. This was demonstrated by decreased dipalmitoyl-phosphatidylcholine (PC 32:0) content and malformed lamellar bodies in cells transfected with the K1388N variant as compared to controls.Here we present a set of tools useful for categorizing different ABCA3 mutations according to their impact upon ABCA3 activity. Knowledge of the molecular defects and close correlation of in vitro and ex vivo data will allow us to define groups of mutations that can be targeted by small molecule correctors for restoring impaired ABCA3 transporter in the future. Pediatr Pulmonol. 2016;51:1284-1294. © 2016 Wiley Periodicals, Inc.

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

Background: ABCA3 is an ATP-dependent lipid transporter involved in surfactant production and is located in the limiting membrane of lamellar bodies (LB) in type II pneumocytes. Mutations within the ABCA3-gene cause respiratory distress syndrome in newborns and chronic interstitial lung diseases in children and adults. In order to develop correctors that can rescue mutant ABCA3, a reliable method is needed to measure ABCA3 transport activity. Methods: Liposomes of surfactant-like composition were prepared and fluorescent Top-Fluor-Phosphatidylcholine (TopF-PC) was incorporated. A549 cells stably expressing ABCA3-HA were incubated with these liposomes. ATP-dependence of transport was evaluated using an ATPase-inhibitor. Cells were imaged using confocal laser scanning microscopy and fluorescence intensity was analysed using the Fiji-Plugin Particle_in_cell-3D , which has been modified for the analysis of vesicular transport. Results: We show that TopF-PC fluorescence can be used to estimate the amount of lipids present in single LBs. Moreover, we found differences in the TopF-PC fluorescence intensity between wild type-cells and cells expressing mutations, which are supposed to be functional ones. We were able to demonstrate an increase of fluorescence in the LBs over different time slots from 0 to 72 hours. In concordance with active transport of TopF-PC by ABCA3, we found a decrease in ABCA3-function after treatment with the ATPase-inhibitor. Summary: This method presents an important step towards a functional assay to analyse transport of lipids by ABCA3 and its impairment by mutations. The assay will enable us to identify correctors, which can improve lipid transport function of mutated ABCA3.

Introduction: ABCA3 is a lipid transporter found in alveolar type II cells, where it localizes to the outer membrane of lamellar bodies which store surfactant. Mutations in the ABCA3 gene are the most common known genetic cause of respiratory distress syndrome in newborns and of late onset interstitial lung disease in children. Objectives and methods: WT ABCA3 cDNA fused to a HA-tag was cloned into the pT2HB SB expression vector. Clinical relevant mutations R208W, Q215K, R288K, E292V, M760R, G964D and K1388N were inserted by site-directed mutagenesis into the WT ABCA3 gene. Vectors were stably transfected into A549 cells with the “Sleeping Beauty” system based on transposition. The level of ABCA3 expression and processing was examined by quantitative PCR and immunoblot analysis. Subcellular protein localization was analyzed by immunofluorescence. Changes in total lipid composition within the different cell lines were investigated by mass spectrometry. Results: Immunoblot analyses of mutated ABCA3 revealed changes in protein processing compared to the wildtype. Furthermore we found that ABCA3 is localized mainly at the limiting membrane of CD63-positive vesicles, whereas Q215K and M760R showed a different localization pattern. Mass spectrometry analyzed indicated alteration of lipid composition. Conclusion: We have prepared and characterized cellular models with ABCA3 mutations in order to investigate specific mechanisms for restoring its wildtype function. Supported by “Deutsches Zentrum fur Lungenforschung“ (DZL) and „Deutsche Forschungsgemeinschaft“ (DFG).

Background Aim of this study was to verify a systematic and practical categorization system that allows dynamic classification of pediatric DPLD irrespective of completeness of patient data. Methods The study was based on 2322 children submitted to the kids-lung-register between 1997 and 2012. Of these children 791 were assigned to 12 DPLD categories, more than 2/3 belonged to categories manifesting primarily in infancy. The work-flow of the pediatric DPLD categorization system included (i) the generation of a final working diagnosis, decision on the presence or absence of (ii) DPLD and (iii) a systemic or lung only condition, and (iv) the allocation to a category and subcategory. The validity and inter-observer dependency of this workflow was re-tested using a systematic sample of 100 cases. Results Two blinded raters allocated more than 80 % of the re-categorized cases identically. Non-identical allocation was due to lack of appreciation of all available details, insufficient knowledge of the classification rules by the raters, incomplete patient data, and shortcomings of the classification system itself. Conclusions This study provides a suitable workflow and hand-on rules for the categorization of pediatric DPLD. Potential pitfalls were identified and a foundation was laid for the development of consensus-based, international categorization guidelines. Electronic supplementary material The online version of this article (doi:10.1186/s13023-015-0339-1) contains supplementary material, which is available to authorized users.