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Objective The main purpose of the present study was to assess the risk for major revision surgery after perichondrium transplantation (PT) at a minimum of 22 years postoperatively and to evaluate the influence of patient characteristics. Design Primary outcome was treatment success or failure. Failure of PT was defined as revision surgery in which the transplant was removed, such as (unicondylar) knee arthroplasty or patellectomy. The functioning of nonfailed patients was evaluated using the International Knee Documentation Committee (IKDC) score. In addition, the influence of patient characteristics was evaluated. Results Ninety knees in 88 patients, aged 16 to 55 years with symptomatic cartilage defects, were treated by PT. Eighty knees in 78 patients were eligible for analysis and 10 patients were lost to follow-up. Twenty-eight knees in 26 patients had undergone major revision surgery. Previous surgery and a longer time of symptoms prior to PT were significantly associated with an increased risk for failure of cartilage repair. Functioning of the remaining 52 patients and influence of patient characteristics was analyzed using their IKDC score. Their median IKDC score was 39.08, but a relatively young age at transplantation was associated with a higher IKDC score. Conclusions This 22-year follow-up study of PT, with objective outcome parameters next to patient-reported outcome measurements in a unique group of patients, shows that overall 66% was without major revision surgery and patient characteristics also influence long-term outcome of cartilage repair surgery.

Four years after its first report, expansion microscopy (ExM) is now being routinely applied in laboratories worldwide to achieve super-resolution imaging on conventional fluorescence microscopes. By chemically anchoring all molecules of interest to the polymer meshwork of an expandable hydrogel, their physical distance is increased by a factor of ∼4-5× upon dialysis in water, resulting in an imprint of the original sample with a lateral resolution up to 50-70 nm. To ensure a correct representation of the original spatial distribution of the molecules, it is crucial to confirm that the expansion is isotropic, preferentially in all three dimensions. To address this, we present an approach to evaluate the local expansion factor within a biological sample and in all three dimensions. We use photobleaching to introduce well-defined three-dimensional (3D) features in the cell and, by comparing the size and shape pre- and postexpansion, these features can be used as an intrinsic ruler. In addition, our method is capable of pointing out sample distortions and can be used as a quality control tool for expansion microscopy experiments in biological samples. ispartof: ACS OMEGA vol:5 issue:12 pages:6792-6799 ispartof: location:United States status: published

Methyltransferasen (MTasen) bilden eine grose Familie von Enzymen, die vielfaltige Zielstrukturen methylieren, von DNA, RNA und Proteinen bis zu niedermolekularen Verbindungen. Die meisten MTasen nutzen S-Adenosyl-l-methionin (AdoMet) als Methylquelle. In den letzten Jahren wurde intensiv an der Entwicklung von AdoMet-Analoga gearbeitet, mit dem Ziel, andere Einheiten als einfache Methylgruppen zu ubertragen. Derzeit gibt es zwei Hauptklassen von AdoMet-Analoga – doppelt aktivierte Molekule und Molekule auf Aziridinbasis – die jeweils einen anderen Ansatz zur Transalkylierung des Zielmolekuls verfolgen. Dieser Aufsatz erortert die Methoden zur Markierung und Funktionalisierung von Biomolekulen unter Verwendung von AdoMet-abhangigen MTasen und AdoMet-Analoga. Er behandelt die Synthesewege hin zu AdoMet-Analoga, die Stabilitat von AdoMet-Analoga in biologischen Umgebungen und ihre Anwendung in Transalkylierungen. Schlieslich werden einige Perspektiven fur die Anwendung von AdoMet-Analoga in der biologischen Forschung, Genetik oder Epigenetik und Nanotechnologie aufgezeigt.

The development of novel molecular sieves opens opportunities in the development of more sensitive analytical devices. In this paper, metal organic frameworks (MOFs), specifically ZIF-8 and ZIF-93, are grown on fiber optic based surface plasmon resonance (FO-SPR) sensors. FO-SPR has enabled sensitive sensing capabilities in biomedical settings and the addition of an MOF coating opens the way for the sensing of volatile organic compounds (VOCs) in gaseous media. FO-SPR probes were homogeneously functionalized with ZIF-8 and ZIF-93 in each case using two different precursor solutions to obtain a sequential nucleation and growth phase. The difference in MOF nucleation and growth kinetics of the two solutions was directly monitored by the FO-SPR system. The two established MOF-FO-SPR sensors were then subjected to sensing experiments with several alcohol vapors to establish their sensing capabilities. Vapors with mPa partial pressures, ppm concentrations, could successfully be detected, e.g., an LOD of 2.5 ppm for methanol detection was acquired. The difference in recognition behavior of the hydrophobic ZIF-8 and more hydrophilic ZIF-93 recognition layers can be exploited to yield qualitative information regarding the vapor composition.

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by hypertrophic differentiation of the engineered cartilaginous tissue caused by endochondral ossification. Since a healthy cartilage matrix contains high amounts of Aggrecan and COMP, we hypothesized that their supplementation in the biogel used in the generation of subperiosteal cartilage mimics the composition of the cartilage extracellular matrix environment, with beneficial properties for the engineered cartilage. Supplementation of COMP or Aggrecan was studied in vitro during chondrogenic differentiation of rabbit periosteum cells and periosteum-derived chondrocytes. Low melting agarose was supplemented with bovine Aggrecan, human recombinant COMP or vehicle and was injected between the bone and periosteum at the upper medial side of the tibia of New Zealand white rabbits. Generated subperiosteal cartilage tissue was analyzed for weight, GAG and DNA content and ALP activity. Key markers of different phases of endochondral ossification were measured by RT-qPCR. For the in vitro experiments, no significant differences in chondrogenic marker expression were detected following COMP or Aggrecan supplementation, while in vivo favorable chondrogenic marker expression was detected. Gene expression levels of hypertrophic markers as well as ALP activity were significantly decreased in the Aggrecan and COMP supplemented conditions compared to controls. The wet weight and GAG content of the in vivo generated subperiosteal cartilage tissue was not significantly different between groups. Data demonstrate the potential of Aggrecan and COMP to favorably influence the subperiosteal microenvironment for the in vivo generation of cartilage for the optimization of cartilage regenerative approaches.

Single-molecule DNA mapping has the potential to serve as a powerful complement to high-throughput sequencing in metagenomic analysis. Offering longer read lengths and forgoing the need for complex library preparation and amplification, mapping stands to provide an unbiased view into the composition of complex viromes and/or microbiomes. To fully enable mapping-based metagenomics, sensitivity and specificity of DNA map analysis and identification need to be improved. Using detailed simulations and experimental data, we first demonstrate how fluorescence imaging of surface stretched, sequence specifically labeled DNA fragments can yield highly sensitive identification of targets. Second, a new analysis technique is introduced to increase specificity of the analysis, allowing even closely related species to be resolved. Third, we show how an increase in resolution improves sensitivity. Finally, we demonstrate that these methods are capable of identifying species with long genomes such as bacteria with high sensitivity. ispartof: NAR GENOMICS AND BIOINFORMATICS vol:2 issue:1 ispartof: location:England status: published

Strain-stabilized perovskites The perovskite materials used for solar cells and light-emitting diodes (which are black in color) are generally less stable at room temperature than the electronically inactive nonperovskite phases (which are yellow in color). Steele et al. show that for CsPbI 3 , strain induced in a thin film after annealing the material to 330°C and then rapidly cooling it to room temperature kinetically trapped the black phase. Grazing-incidence wide-angle x-ray scattering revealed the crystal distortions and texture formation created by interfacial strain. Science , this issue p. 679

Photocatalytic reactions occur at the crystal-solution interface, and hence specific crystal facet expression and surface defects can play an important role. Here we investigate the structure-related photoreduction at zinc oxide (ZnO) microparticles via integrated light and electron microscopy in combination with silver metal photodeposition. This enables a direct visualization of the photoreduction activity at specific crystallographic features. It is found that silver nanoparticle photodeposition on dumbbell-shaped crystals mainly takes place at the edges of O-terminated (0001̅) polar facets. In contrast, on ZnO microrods photodeposition is more homogeneously distributed with an increased activity at {101̅1̅} facets. Additional time-resolved measurements reveal a direct spatial link between the enhanced photoactivity and increased charge carrier lifetimes. These findings contradict previous observations based on indirect, bulk-scale experiments, assigning the highest photocatalytic activity to polar facets. The presented research demonstrates the need for advanced microscopy techniques to directly probe the location of photocatalytic activity. ispartof: JOURNAL OF PHYSICAL CHEMISTRY LETTERS vol:8 issue:2 pages:340-346 ispartof: location:United States status: published

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim An integrated optical-electron microscope is presented that enables the in situ study of dynamic processes on photoactive materials. Here, the deposition of metallic silver nanostructures at ZnO photocatalyst particles is monitored in real time under ambient conditions by means of scanning electron microscopy. Zinc oxide crystals are immobilized on an electron transparent silicon nitride window. By passing UV light through an opposing optically transparent window, the zinc oxide is illuminated resulting in the photocatalytic formation of silver nanostructures. Both windows are part of a specially designed liquid cell filled with a dilute aqueous silver nitrate solution. Using the presented system, different electron detectors are evaluated for their ability to provide detailed images despite the interference caused by the liquid surrounding the sample. Special care has to be taken since direct silver reduction from solution induced by the electron beam interferes with the photocatalytic process. Oxygen gas, produced during the photocatalytic reaction, is also shown to complicate the imaging of the dynamic nanoscale processes in the scanning electron microscope. Nevertheless, the integrated approach allows to directly establish structure–activity relationships and to unravel optically induced processes at nanostructured materials. ispartof: PARTICLE & PARTICLE SYSTEMS CHARACTERIZATION vol:33 issue:7 pages:412-418 status: published

The impressive optoelectronic performance and low production cost of metal halide perovskites have inspired applications well beyond efficient solar cells. Herein, we widen the materials engineering options available for the efficient and selective photocatalytic oxidation of benzylic alcohols, an industrially significant reaction, using formamidinium lead bromide (FAPbBr(3)) and other perovskite-based materials. The best performance was obtained using a FAPbBr(3)/TiO2 hybrid photo catalyst under simulated solar illumination. Detailed optical studies reveal the synergetic photophysical pathways arising in FAPbBr(3)/TiO2 composites. An experimentally supported model rationalizing the large conversion enhancement over the pure constituents shows that this strategy offers new prospects for metal halide perovskites in photocatalytic applications. Research Foundation-Flanders (FWO) [G.0962.13, G0B39.15, ZW15_09 GOH6316N, G.0B49.15]; KU Leuven Research Fund [C14/15/053]; Flemish government through long term structural funding Methusalem (CASAS2) [Meth/15/04]; Hercules foundation [HER/11/14]; European Union (Horizon) Marie Sklodowska-Curie innovation program [722591, 641887]; NSFC [21773031]

Organic-inorganic halide perovskites (OIHPs) have demonstrated outstanding energy conversion efficiency in solar cells and light-emitting devices. In spite of intensive developments in both materials and devices, electronic traps and defects that significantly affect their device properties remain under-investigated. Particularly, it remains challenging to identify and to resolve traps individually at the nanoscopic scale. Here, photo-active traps (PATs) are mapped over OIHP nanocrystal morphology of different crystallinity by means of correlative optical differential super-resolution localization microscopy (Delta-SRLM) and electron microscopy. Stochastic and monolithic photoluminescence intermittency due to individual PATs is observed on monocrystalline and polycrystalline OIHP nanocrystals. Delta-SRLM reveals a heterogeneous PAT distribution across nanocrystals and determines the PAT density to be 1.3 x 10(14) and 8 x 10(13) cm(-3) for polycrystalline and for monocrystalline nanocrystals, respectively. The higher PAT density in polycrystalline nanocrystals is likely related to an increased defect density. Moreover, monocrystalline nanocrystals that are prepared in an oxygen and moisture-free environment show a similar PAT density as that prepared at ambient conditions, excluding oxygen or moisture as chief causes of PATs. Hence, it is conduded that the PATs come from inherent structural defects in the material, which suggests that the PAT density can be reduced by improving crystalline quality of the material.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints. ispartof: JOURNAL OF PHYSICS D-APPLIED PHYSICS vol:51 issue:44 ispartof: location:England status: published

The methyltransferase enzymes can be applied to deliver a range of modifications to pre-determined sites on large DNA molecules with exceptional specificity and efficiency. To date, however, a limited number of modifications have been delivered in this way because of the complex chemical synthesis that is needed to produce a cofactor analogue carrying a specific function, such as a fluorophore. Here, we describe a method for the direct transfer of a series of functional compounds (seven fluorescent dyes, biotin and polyethylene glycol) to the DNA duplex. Our approach uses a functional cofactor analogue, whose final preparative step is performed alongiside the DNA modification reaction in a single pot, with no purification needed. We show that fluorophore conjugation efficiency in these mixtures is significantly improved compared to two-step labeling approaches. Our experiments highlight the remarkable malleability and selectivity of the methyltransferases tested. Additional analysis using high resolution localization of the fluorophore distribution indicates that target sites for the methyltransferase are predominantly labeled on a single strand of their palindromic site and that a small and randomly-distributed probability of off-site labeling exists. ispartof: Nucleic Acids Research vol:46 issue:11 pages:1-8 ispartof: location:England status: Published online

The performance of zeolites as solid acid catalysts is strongly influenced by the accessibility of active sites. However, synthetic zeolites typically grow as complex aggregates of small nanocrystallites rather than perfect single crystals. The structural complexity must therefore play a decisive role in zeolite catalyst applicability. Traditional tools for the characterization of heterogeneous catalysts are unable to directly relate nanometer scale structural properties to the corresponding catalytic performance. In this work, an innovative correlative super-resolution fluorescence and scanning electron microscope is applied and the appropriate analysis procedures are developed to investigate the effect of small-port H-mordenite (H-MOR) morphology on the catalytic performance, along with the effects of extensive acid leaching. These correlative measurements revealed catalytic activity at the interface between intergrown H-MOR crystallites that was assumed inaccessible, without compromising the shape selective properties. Furthermore, it was found that extensive acid leaching led to an etching of the originally accessible microporous structure, rather than the formation of an extended mesoporous structure. The associated transition of small-port to large-port H-MOR therefore did not render the full catalyst particle functional for catalysis. The applied characterization technique allows a straightforward investigation of the zeolite structure-activity relationship beyond the single-particle level. We conclude that such information will ultimately lead to an accurate understanding of the relationship between the bulk scale catalyst behavior and the nanoscale structural features, enabling a rationalization of catalyst design. ispartof: ACS Catalysis vol:7 issue:8 pages:5234-5242 ispartof: location:United States status: published

Osteoarthritis (OA) is a big burden of disease worldwide and one of the most common causes of disability in the adult population. Currently applied therapies consist of physical therapy, oral medication, intra-articular injections, and surgical interventions, with the main goal being to reduce pain and improve function and quality of life. Intra-articular (IA) administration of drugs has potential benefits in OA treatment because it minimizes systemic bioavailability and side effects associated with oral administration of drugs without compromising the therapeutic effect in the joint. However, IA drug residence time is short and there is a clinical need for a vehicle that is able to provide a sustained release long enough for IA therapy to fulfill its promise. This review summarizes the use of different polymeric systems and the incorporated drugs for IA drug delivery in the osteoarthritic joint with a primary focus on clinical needs and opportunities.

Many studies have reported on the effects of cyclooxygenase-2 (COX-2) inhibition on osteogenesis. However, far less is known about the effects of COX-2 inhibition on chondrogenic differentiation. Previous studies conducted by our group show that COX-2 inhibition influences in vitro chondrogenic differentiation. Importantly, this might have consequences on endochondral ossification processes occurring in vivo, such as bone fracture healing, growth plate development and ectopic generation of cartilage. The goal of our study was to investigate, in vivo, the effect of COX-2 inhibition by celecoxib on the cartilaginous phase of three different endochondral ossification scenarios.10 mg/kg/d celecoxib or placebo were orally administered for 25 d to skeletally-immature New Zealand White rabbits (n = 6 per group). Endochondral ossification during fracture healing of a non-critical size defect in the ulna, femoral growth plate and ectopically-induced cartilaginous tissue were examined by radiography, micro-computed tomography (mu-CT), histology and gene expression analysis.Celecoxib treatment resulted in delayed bone fracture healing, alterations in growth plate development and progression of mineralisation. In addition, chondrogenic differentiation of ectopically-induced cartilaginous tissue was severely impaired by celecoxib. In conclusion, we found that celecoxib impaired the chondrogenic phase of endochondral ossification.

Affinity probe capillary electrophoresis (APCE) is potentially one of the most versatile technologies for protein diagnostics, offering an excellent balance between robustness, analysis speed and sensitivity. Combining the immunosensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification, aptamers can further push the analytical limits of APCE to offer ultrasensitive, multiplexed detection of protein biomarkers, even when differences in electrophoretic mobility between the different aptamer-target complexes are limited. It is demonstrated how, through careful selection of experimental parameters, simultaneous detection of picomolar levels of three target proteins can be achieved even with aptamers that were initially selected under very different conditions and further taking into account that the aptamers need to be modified to allow successful PCR amplification. Aptamer-enhanced APCE offers limits of detection that are orders of magnitude lower than those that can be achieved through traditional capillary electrophoresis-based immunosensing. With recent developments in aptamer selection that for the first time realise the promise of aptamers as easily accessible, high affinity recognition molecules, it can therefore be envisioned that aptamer-enhanced APCE on parallel microfluidic platforms can be the basis for a truly high-throughput multiplexed proteomics platform, rivalling genetic screening for the first time.

Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist's toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

Organometal halide perovskites show promising features for cost-effective application in photovoltaics. The material instability remains a major obstacle to broad application because of the poorly understood degradation pathways. Here, we apply simultaneous luminescence and electron microscopy on perovskites for the first time, allowing us to monitor in situ morphology evolution and optical properties upon perovskite degradation. Interestingly, morphology, photoluminescence (PL), and cathodoluminescence of perovskite samples evolve differently upon degradation driven by electron beam (e-beam) or by light. A transversal electric current generated by a scanning electron beam leads to dramatic changes in PL and tunes the energy band gaps continuously alongside film thinning. In contrast, light-induced degradation results in material decomposition to scattered particles and shows little PL spectral shifts. The differences in degradation can be ascribed to different electric currents that drive ion migration. Moreover, solution-processed perovskite cuboids show heterogeneity in stability which is likely related to crystallinity and morphology. Our results reveal the essential role of ion migration in perovskite degradation and provide potential avenues to rationally enhance the stability of perovskite materials by reducing ion migration while improving morphology and crystallinity. It is worth noting that even moderate e-beam currents (86 pA) and acceleration voltages (10 kV) readily induce significant perovskite degradation and alter their optical properties. Therefore, attention has to be paid while characterizing such materials using scanning electron microscopy or transmission electron microscopy techniques. ispartof: Journal of Physical Chemistry Letters vol:7 issue:3 pages:561-566 ispartof: location:United States status: published

Supported metal nanoparticle catalysts are commonly obtained through deposition of metal precursors onto the support using incipient wetness impregnation. Typically, empirical relations between metal nanoparticle structure and catalytic performance are inferred from ensemble averaged data in combination with high-resolution electron microscopy. This approach clearly underestimates the importance of heterogeneities present in a supported metal catalyst batch. Here we show for the first time how incipient wetness impregnation leads to 10-fold variations in silver loading between individual submillimeter-sized silica support granules. This heterogeneity has a profound impact on the catalytic performance, with 100-fold variations in hydrogenation performance at the same level. In a straightforward fashion, optical microscopy interlinks single support particle level catalytic measurements to structural and compositional information. These detailed correlations reveal the optimal silver loading. A thorough consideration of catalyst heterogeneity and the impact thereof on the catalytic performance is indispensable in the development of catalysts. ispartof: ACS Catalysis vol:5 issue:11 pages:6690-6695 ispartof: location:United States status: published

Spatial restrictions around catalytic sites, provided by molecular-sized micropores, are beneficial to reaction selectivity but also inherently limit diffusion. The molecular transport can be enhanced by introducing meso- and macropores. However, the impact of this extraframework porosity on the local nanoscale reactivity is relatively unexplored. Herein we show that the area of enhanced reactivity in hierarchical zeolite, examined with super-resolution fluorescence microscopy, is spatially restricted to narrow zones around meso- and macropores, as observed with focused ion-beam-assisted scanning electron microscopy. This comparison indicates that reagent molecules efficiently reach catalytic active sites only in the micropores surrounding extraframework porosity and that extensive macroporosity does not warrant optimal reactivity distribution throughout a hierarchical porous zeolite. ispartof: ChemCatChem vol:7 issue:22 pages:3646-3650 ispartof: location:Germany status: published

© The Royal Society of Chemistry 2015. We report an investigation on the effect of stabilization agents and surface curvatures on oxidative etching of three classes of anisotropically shaped gold nanoparticles namely, rods, bipyramids and prisms. In particular, the dual role of the stabilizing agent CTAB in the etching process is explored, showing how it acts both as a source of bromine ions, accelerating etching and as a protection agent, resulting in anisotropic reshaping. ispartof: RSC Advances vol:5 issue:9 pages:6829-6833 status: published

Detection of allergens and micro bacteria on a fiber optic SPR sensor is performed by means of an oligonucleotide bio-recognition surface. In this work, the DNA surface is optimized by mixing with PEG monolayers to suppress unspecific and enhance specific detection. Furthermore, due to the surface characteristic of DNA functionalized gold nanoparticles the bioassay sensitivity is increased and a mean of real-time multiplexing is introduced.

Detection of single nucleotide polymorphisms (SNPs) is a crucial challenge in the development of a novel generation of diagnostic tools. Accurate detection of SNPs can prove elusive, as the impact of a single variable nucleotide on the properties of a target sequence is limited, even if this sequence consists of only a few nucleotides. New, accurate and facile strategies for the detection of point mutations are therefore absolutely necessary for the increased adoption of point-of-care molecular diagnostics. Currently, PCR and sequencing are mostly applied for diagnosing SNPs. However these methods have serious drawbacks as routine diagnostic tools because of their labour intensity and cost. Several new, more suitable methods can be applied to enable sensitive detection of mutations based on specially designed hybridization probes, mutation recognizing enzymes and thermal denaturation. Here, an overview is presented of the most recent advances in the field of fast and sensitive SNP detection assays with strong potential for integration in point-of-care tests. ispartof: The Analyst vol:139 issue:2 pages:353-370 ispartof: location:England status: published

Structural and temporal inhomogeneities can have a marked influence on the performance of inorganic and biocatalytic systems alike. While these subtle variations are hardly ever accessible through bulk or ensemble averaged activity screening, insights into the molecular mechanisms underlying these diverse phenomena are absolutely critical for the development of optimized or novel catalytic systems and processes. Fortunately, state-of-the-art fluorescence microscopy methods have allowed experimental access to this intriguing world at the nanoscale. In this tutorial review we will first provide a broad overview of key concepts and developments in the application of single molecule fluorescence spectroscopy to (bio)catalysis research. In the second part topics specific to both bio and heterogeneous catalysis will be reviewed in more detail. ispartof: Chemical Society Reviews vol:43 issue:4 pages:990-1006 ispartof: location:England status: published

Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughtput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of `personalized medicine'. Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart mainly because traditional, antibody-based technologies struggle to meet the stringent sensitivity- and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given. ispartof: Sensors vol:13 issue:1 pages:898-930 ispartof: location:Switzerland status: published

The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 ± 82 nM, 419 ± 63 nM, and 450 ± 60 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.

Skeletogenesis and bone fracture healing involve endochondral ossification, a process during which cartilaginous primordia are gradually replaced by bone tissue. In line with a role for cyclooxygenase-2 (COX-2) in the endochondral ossification process, non-steroidal anti-inflammatory drugs (NSAIDs) were reported to negatively affect bone fracture healing due to impaired osteogenesis. However, a role for COX-2 activity in the chondrogenic phase of endochondral ossification has not been addressed before. We show that COX-2 activity fulfils an important regulatory function in chondrocyte hypertrophic differentiation. Our data reveal essential cross-talk between COX-2 and bone morphogenic protein-2 (BMP-2) during chondrocyte hypertrophic differentiation. BMP-2 mediated chondrocyte hypertrophy is associated with increased COX-2 expression and pharmacological inhibition of COX-2 activity by NSAIDs (e.g., Celecoxib) decreases hypertrophic differentiation in various chondrogenic models in vitro and in vivo, while leaving early chondrogenic development unaltered. Our findings demonstrate that COX-2 activity is a novel factor partaking in chondrocyte hypertrophy in the context of endochondral ossification and these observations provide a novel etiological perspective on the adverse effects of NSAIDs on bone fracture healing and have important implications for the use of NSAIDs during endochondral skeletal development.

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH 2 )-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer–IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly. While single pTDI molecules could be visualized with excellent resolution (35 nm), pPDI molecules bleached rapidly under similar conditions. Femtosecond transient absorption measurements proved that the overlap between the stimulated-emission band and the excited-state absorption band is the main reason for the observed difference. Thus, assessment of the excited-state absorption band provides a rational means of dye selection and determination of the optimal wavelength for STED.

We have selected aptamers binding to lysozyme from a DNA library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment. During the selection process the dissociation constant of the ssDNA pool decreased from the micromolar to the low nanomolar range within five rounds of selection. The final aptamer had a dissociation constant of 2.8 +/- 0.3 nM, 6.1 +/- 0.5 nM, and 52.9 +/- 9.1 nM as determined by fluorescence anisotropy, surface plasmon resonance and affinity capillary electrophoresis respectively. The aptamers were successfully challenged for specificity against other egg white proteins. The high affinity aptamers open up possibilities for the development of aptamer based food and medical diagnostics.

Nowadays, an increasing number of minimal invasive treatment alternatives to hysterectomy may be offered to the patient. In determining the appropriate treatment option, the patient has a distinct dilemma if a minimal invasive treatment with lesser effect than hysterectomy should be chosen or if a hysterectomy should be chosen which is a major surgery and requires longer recovery than the minimal invasive alternative. Quality-of-life (QoL) questionnaires that take subjective health perception into account are currently used to assess the treatment effects. The objective of this literature study is to determine and discuss the role of QoL as an outcome in randomized controlled trials (RCT) or systematic reviews of RCTs that study the treatment effect of hysterectomy compared to that of minimal invasive alternatives. A systematic literature search was performed in the PubMed database and in the Cochrane database to find randomized trials and systematic reviews of randomized trials, comparing hysterectomy with minimal invasive or conservative treatment options with sufficient follow-up using satisfaction, health status, and quality of life as outcomes. The results were based on nine randomized trials and two systematic reviews. The differences are mostly in favor of hysterectomy. In two out of four studied treatment alternatives, the satisfaction or health status is different in favor of hysterectomy while the QoL is equivalent. After 2 years of follow-up, differences between both groups have disappeared, possibly because of the crossover effect. Possible reasons for the lesser response of QoL compared to satisfaction or health status are discussed. The fundamental question if patients have a better quality of life at all times if they choose for a minimal invasive alternative of hysterectomy remains unresolved. Information, individualization, and freedom of choice before surgery probably best serve the sense of well being and quality of life thereafter.

In this paper we present a fiber optic surface plasmon resonance (SPR) sensor as a reusable, cost-effective and label free biosensor for measuring DNA hybridization and DNA-protein interactions. This is the first paper that combines the concept of a fiber-based SPR system with DNA aptamer bioreceptors. The fibers were sputtered with a 50nm gold layer which was then covered with a protein repulsive self-assembled monolayer of mixed polyethylene glycol (PEG). Streptavidin was attached to the PEG's carboxyl groups to serve as a versatile binding element for biotinylated ssDNA. The ssDNA coated SPR fibers were first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for a random 37-mer ssDNA. This single stranded DNA showed a 15 nucleotides overlap with the receptor ssDNA on the SPR fiber. A linear calibration curve was observed in 0.5-5 microM range. A negative control test did not reveal any significant non-specific binding, and the biosensor was easily regenerated. In a second assay the fiber optic SPR biosensor was functionalized with ssDNA aptamers against human immunoglobulin E. Limits of detection (2nM) and quantification (6nM) in the low nanomolar range were observed. The presented biosensor was not only useful for DNA and protein quantification purposes, but also to reveal the binding kinetics occurring at the sensor surface. The dissociation constant between aptamer and hIgE was equal to 30.9+/-2.9nM. The observed kinetics fully comply with most data from the literature and were also confirmed by own control measurements.

Methyltransferases (MTases) compose a large family of enzymes which have the ability to methylate a diverse set of targets, ranging from the three major biopolymers DNA, RNA and protein to small molecules. Most of these MTases use the cofactor S-Adenosyl-L-Methionine (AdoMet) as their methyl source. Given the important biological role of methylation, e.g. in epigenetic regulation of gene activity and the vast potential of targeted functionalization in biology, diagnostics and nanotechnology, it should come as no surprise that recent years have seen significant efforts in the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist- the doubly-activated and aziridine based molecules- each of which employs a different approach for transalkylation, as opposed to transmethylation, of the target molecule. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet-dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology. ispartof: Angewandte Chemie - International Edition vol:56 issue:19 pages:5182-5200 ispartof: location:Germany status: published