Temporal evolution of strut light intensity after implantation of bioresorbable polymeric intracoronary scaffolds in the ABSORB cohort B trial-an application of a new quantitative method based on optical coherence tomography.

BACKGROUND Quantitative light intensity analysis of the strut core by optical coherence tomography (OCT) may enable assessment of changes in the light reflectivity of the bioresorbable polymeric scaffold from polymer to provisional matrix and connective tissues, with full disappearance and integration of the scaffold into the vessel wall. The aim of this report was to describe the methodology and to apply it to serial human OCT images post procedure and at 6, 12, 24 and 36 months in the ABSORB cohort B trial. METHODS AND RESULTS In serial frequency-domain OCT pullbacks, corresponding struts at different time points were identified by 3-dimensional foldout view. The peak and median values of light intensity were measured in the strut core by dedicated software. A total of 303 corresponding struts were serially analyzed at 3 time points. In the sequential analysis, peak light intensity increased gradually in the first 24 months after implantation and reached a plateau (relative difference with respect to baseline [%Dif]: 61.4% at 12 months, 115.0% at 24 months, 110.7% at 36 months), while the median intensity kept increasing at 36 months (%Dif: 14.3% at 12 months, 75.0% at 24 months, 93.1% at 36 months). CONCLUSIONS Quantitative light intensity analysis by OCT was capable of detecting subtle changes in the bioresorbable strut appearance over time, and could be used to monitor the bioresorption and integration process of polylactide struts.

Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri.

BACKGROUND The free-living amoeba Naegleria fowleri is the causative agent of the rapidly progressing and typically fatal primary amoebic meningoencephalitis (PAM) in humans. Despite the devastating nature of this disease, which results in > 97% mortality, knowledge of the pathogenic mechanisms of the amoeba is incomplete. This work presents a comparative proteomic approach based on an experimental model in which the pathogenic potential of N. fowleri trophozoites is influenced by the compositions of different media. RESULTS As a scaffold for proteomic analysis, we sequenced the genome and transcriptome of N. fowleri. Since the sequence similarity of the recently published genome of Naegleria gruberi was far lower than the close taxonomic relationship of these species would suggest, a de novo sequencing approach was chosen. After excluding cell regulatory mechanisms originating from different media compositions, we identified 22 proteins with a potential role in the pathogenesis of PAM. Functional annotation of these proteins revealed, that the membrane is the major location where the amoeba exerts its pathogenic potential, possibly involving actin-dependent processes such as intracellular trafficking via vesicles. CONCLUSION This study describes for the first time the 30 Mb-genome and the transcriptome sequence of N. fowleri and provides the basis for the further definition of effective intervention strategies against the rare but highly fatal form of amoebic meningoencephalitis.

Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.

Cardiac tissue engineering approaches can deliver large numbers of cells to the damaged myocardium and have thus increasingly been considered as a possible curative treatment to counteract the high prevalence of progressive heart failure after myocardial infarction (MI). Optimal scaffold architecture and mechanical and chemical properties, as well as immune- and bio-compatibility, need to be addressed. We demonstrated that radio-frequency plasma surface functionalized electrospun poly(ɛ-caprolactone) (PCL) fibres provide a suitable matrix for bone-marrow-derived mesenchymal stem cell (MSC) cardiac implantation. Using a rat model of chronic MI, we showed that MSC-seeded plasma-coated PCL grafts stabilized cardiac function and attenuated dilatation. Significant relative decreases of 13% of the ejection fraction (EF) and 15% of the fractional shortening (FS) were observed in sham treated animals; respective decreases of 20% and 25% were measured 4 weeks after acellular patch implantation, whereas a steadied function was observed 4 weeks after MSC-patch implantation (relative decreases of 6% for both EF and FS).

Impacts of a word-picture training on reading in youth with mixed intellectual disabilities. A waiting-list control group comparison

Individuals with intellectual disabilities (ID) often struggle with learning how to read. Reading difficulties seem to be the most common secondary condition of ID. Only one in five children with mild or moderate ID achieves even minimal literacy skills. However, literacy education for children and adolescents with ID has been largely overlooked by researchers and educators. While there is little research on reading of children with ID, many training studies have been conducted with other populations with reading difficulties. The most common approach of acquiring literacy skills consists of sophisticated programs that train phonological skills and auditory perception. Only few studies investigated the influence of implicit learning on literacy skills. Implicit learning processes seem to be largely independent of age and IQ. Children are sensitive to the statistics of their learning environment. By frequent word reading they acquire implicit knowledge about the frequency of single letters and letter patterns in written words. Additionally, semantic connections not only improve the word understanding, but also facilitate storage of words in memory. Advances in communication technology have introduced new possibilities for remediating literacy skills. Computers can provide training material in attractive ways, for example through animations and immediate feedback .These opportunities can scaffold and support attention processes central to learning. Thus, the aim of this intervention study was to develop and implement a computer based word-picture training, which is based on statistical and semantic learning, and to examine the training effects on reading, spelling and attention in children and adolescents (9-16 years) diagnosed with mental retardation (general IQ  74). Fifty children participated in four to five weekly training sessions of 15-20 minutes over 4 weeks, and completed assessments of attention, reading, spelling, short-term memory and fluid intelligence before and after training. After a first assessment (T1), the entire sample was divided in a training group (group A) and a waiting control group (group B). After 4 weeks of training with group A, a second assessment (T2) was administered with both training groups. Afterwards, group B was trained for 4 weeks, before a last assessment (T3) was carried out in both groups. Overall, the results showed that the word-picture training led to substantial gains on word decoding and attention for both training groups. These effects were preserved six weeks later (group A). There was also a clear tendency of improvement in spelling after training for both groups, although the effect did not reach significance. These findings highlight the fact that an implicit statistical learning training in a playful way by motivating computer programs can not only promote reading development, but also attention in children with intellectual disabilities.

Energy and charge transfer dynamics in DNA based light harvesting antennae

DNA can serve as a versatile scaffold for chromophore assemblies. For example, light-harvesting antennae have been realized by incorporating phenanthrene and pyrene building blocks into DNA strands. It was shown that by exciting at 320 nm (absorption of phenanthrene), an emission at 450 nm is observed which corresponds to a phenanthrene-pyrene exciplex. The more phenanthrenes are added into the DNA duplex, the higher is the fluorescence intensity with no significant change in quantum yield. This shows that phenanthrene acts as a donor and efficiently transfers the excitation energy to the pyrene. Up to now, the mechanism of this energy transfer and exciplex formation is not known. Therefore, we first aim at studying the photo-cycle of such DNA assemblies through transient absorption spectroscopy. Based on the results, we will explore ways to manipulate the energy transfer by application of intense THz fields. Ground as well as excited state Stark effect dynamics will be investigated.

An educational review of cartilage repair: precepts & practice - myths & misconceptions - progress & prospects.

OBJECTIVE The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

One-pot heterogeneous synthesis of Δ(3)-tetrahydrocannabinol analogues and xanthenes showing differential binding to CB(1) and CB(2) receptors

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive cannabinoid in hemp (Cannabis sativa L.) and responsible for many of the pharmacological effects mediated via cannabinoid receptors. Despite being the major cannabinoid scaffold in nature, Δ(9)-THC double bond isomers remain poorly studied. The chemical scaffold of tetrahydrocannabinol can be assembled from the condensation of distinctly substituted phenols and monoterpenes. Here we explored a microwave-assisted one pot heterogeneous synthesis of Δ(3)-THC from orcinol (1a) and pulegone (2). Four Δ(3)-THC analogues and corresponding Δ(4a)-tetrahydroxanthenes (Δ(4a)-THXs) were synthesized regioselectively and showed differential binding affinities for CB1 and CB2 cannabinoid receptors. Here we report for the first time the CB1 receptor binding of Δ(3)-THC, revealing a more potent receptor binding affinity for the (S)-(-) isomer (hCB1Ki = 5 nM) compared to the (R)-(+) isomer (hCB1Ki = 29 nM). Like Δ(9)-THC, also Δ(3)-THC analogues are partial agonists at CB receptors as indicated by [(35)S]GTPγS binding assays. Interestingly, the THC structural isomers Δ(4a)-THXs showed selective binding and partial agonism at CB2 receptors, revealing a simple non-natural natural product-derived scaffold for novel CB2 ligands.

Functionalization of β-caryophyllene generates novel polypharmacology in the endocannabinoid system

The widespread dietary plant sesquiterpene hydrocarbon β-caryophyllene (1) is a CB2 cannabinoid receptor-specific agonist showing anti-inflammatory and analgesic effects in vivo. Structural insights into the pharmacophore of this hydrocarbon, which lacks functional groups other than double bonds, are missing. A structure-activity study provided evidence for the existence of a well-defined sesquiterpene hydrocarbon binding site in CB2 receptors, highlighting its exquisite sensitivity to modifications of the strained endocyclic double bond of 1. While most changes on this element were detrimental for activity, ring-opening cross metathesis of 1 with ethyl acrylate followed by amide functionalization generated a series of new monocyclic amides (11a, 11b, 11c) that not only retained the CB2 receptor functional agonism of 1 but also reversibly inhibited fatty acid amide hydrolase (FAAH), the major endocannabinoid degrading enzyme, without affecting monoacylglycerol lipase (MAGL) and α,β hydrolases 6 and 12. Intriguingly, further modification of this monocyclic scaffold generated the FAAH- and endocannabinoid substrate-specific cyclooxygenase-2 (COX-2) dual inhibitors 11e and 11f, which are probes with a novel pharmacological profile. Our study shows that by removing the conformational constraints induced by the medium-sized ring and by introducing functional groups in the sesquiterpene hydrocarbon 1, a new scaffold with pronounced polypharmacological features within the endocannabinoid system could be generated. The structural and functional repertoire of cannabimimetics and their yet poorly understood intrinsic promiscuity may be exploited to generate novel probes and ultimately more effective drugs.

New nucleoside analogues for the recognition of pyrimidine-purine inversion sites

A new nucleoside designed to enhance triplex stability has been synthesised in 15 steps starting from sugar 2. This pathway contains the sugar derivative 9 which is a useful intermediate for the introduction of other natural and unnatural bases into the 2'-aminoethoxy nucleoside containing scaffold

Synthesis of cyclopentane amide DNA (cpa-DNA) and its pairing properties

We recently reported on the synthesis and pairing properties of the DNA analogue bicyclo[3.2.1]amide DNA (bca-DNA). In this analogue the nucleobases are attached via a linear, 4-bond amide-linker to a structurally preorganized sugar-phosphate backbone unit. To define the importance of the degree of structural rigidity of the bca-backbone unit on the pairing properties, we designed the structurally simpler cyclopentane amide DNA (cpa-DNA), in which the bicyclo[3.2.1]-scaffold was reduced to a cyclopentane unit while the base-linker was left unchanged. Here we present a synthetic route to the enantiomerically pure cpa-DNA monomers and the corresponding phosphoramidites containing the bases A and T, starting from a known, achiral precursor in 9 and 12 steps, respectively. Fully modified oligodeoxynucleotides were synthesized by standard solid-phase oligonucleotide chemistry, and their base-pairing properties with complementary oligonucleotides of the DNA-, RNA-, bca-DNA-, and cpa-DNA-backbones were assessed by UV melting curves and CD-spectroscopic methods. We found that cpa-oligoadenylates form duplexes with complementary DNA that are less stable by -2.7 degrees C/mod. compared to DNA. The corresponding cpa-oligothymidylates do not participate in complementary base-pairing with any of the investigated backbone systems except with its own (homo-duplex). As its congener bca-DNA, cpa-DNA seems to prefer left-handed helical duplex structures with DNA or with itself as indicated by the CD spectra

Identification of a new genomic hot spot of evolutionary diversification of protein function.

Establishment of phylogenetic relationships remains a challenging task because it is based on computational analysis of genomic hot spots that display species-specific sequence variations. Here, we identify a species-specific thymine-to-guanine sequence variation in the Glrb gene which gives rise to species-specific splice donor sites in the Glrb genes of mouse and bushbaby. The resulting splice insert in the receptor for the inhibitory neurotransmitter glycine (GlyR) conveys synaptic receptor clustering and specific association with a particular synaptic plasticity-related splice variant of the postsynaptic scaffold protein gephyrin. This study identifies a new genomic hot spot which contributes to phylogenetic diversification of protein function and advances our understanding of phylogenetic relationships.

Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

Rolling Circle Amplification (RCA) is an isothermal enzymatic method generating single-stranded DNA products consisting of concatemers containing multiple copies of the reverse complement of the circular template precursor. Little is known on the compatibility of modified nucleoside triphosphates (dN*TPs) with RCA, which would enable the synthesis of long, fully modified ssDNA sequences. Here, dNTPs modified at any position of the scaffold were shown to be compatible with rolling circle amplification, yielding long (>1 kb), and fully modified single-stranded DNA products. This methodology was applied for the generation of long, cytosine-rich synthetic mimics of telomeric DNA. The resulting modified oligo-nucleotides displayed an improved resistance to fetal bovine serum.

The effect of enamel proteins on erosion.

Enamel proteins form a scaffold for growing hydroxyapatite crystals during enamel formation. They are then almost completely degraded during enamel maturation, resulting in a protein content of only 1% (w/v) in mature enamel. Nevertheless, this small amount of remaining proteins has important effects on the mechanical and structural properties of enamel and on the electrostatic properties of its surface. To analyze how enamel proteins affect tooth erosion, human enamel specimens were deproteinated. Surface microhardness (SMH), surface reflection intensity (SRI) and calcium release of both deproteinated and control specimens were monitored while continuously eroding them. The deproteination itself already reduced the initial SMH and SRI of the enamel significantly (p < 0.001 and p < 0.01). During the course of erosion, the progression of all three evaluated parameters differed significantly between the two groups (p < 0.001 for each). The deproteinated enamel lost its SMH and SRI faster, and released more calcium than the control group, but these differences were only significant at later stages of erosion, where not only surface softening but surface loss can be observed. We conclude that enamel proteins have a significant effect on erosion, protecting the enamel and slowing down the progression of erosion when irreversible surface loss starts to occur.

Bridged bicyclic peptides as potential drug scaffolds: synthesis, structure, protein binding and stability

Double cyclization of short linear peptides obtained by solid phase peptide synthesis was used to prepare bridged bicyclic peptides (BBPs) corresponding to the topology of bridged bicyclic alkanes such as norbornane. Diastereomeric norbornapeptides were investigated by 1H-NMR, X-ray crystallography and CD spectroscopy and found to represent rigid globular scaffolds stabilized by intramolecular backbone hydrogen bonds with scaffold geometries determined by the chirality of amino acid residues and sharing structural features of β-turns and α-helices. Proteome profiling by capture compound mass spectrometry (CCMS) led to the discovery of the norbornapeptide 27c binding selectively to calmodulin as an example of a BBP protein binder. This and other BBPs showed high stability towards proteolytic degradation in serum.