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Pompe disease is a rare autosomal recessive disorder caused by the deficiency of acid α-glycosidase resulting in accumulation of glycogen in the lysosomes. The late-onset form of the disease (LOPD) causes primarily progressive muscle weakness and respiratory insufficiency. Enzyme replacement therapy (ERT) introduced in 2006, showed mild improvement or stabilization of the symptoms although long-term data are limited. Aim of the study was to describe the progression of body composition and walking ability in LOPD patients receiving ERT consistently for 9 years.Lean body mass, bone mineral density, body fat and 6 min walking distance were assessed in three male and three female LOPD patients (height 165.8 ± 11.2 cm, age 42.3 ± 11.8yrs, body mass 71.1 ± 20.8 kg, at study entry), every three years, for 9 years since ERT initiation (T0, T3, T6, T9).Total body and upper extremities' lean mass remained unchanged (The current data show that enzyme replacement therapy may preserve lean body mass, bone mineral density and walking capacity in LOPD patients.

Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including the production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by the full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5.19% genome reduction. DEL2 can be characterized by faster growth and inability to produce three main native metabolites: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to observe a formation of a blue halo, indicating a potential production of actinorhodin by both DEL2 and a wild type.

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host. Keywords: Natural products, Synthetic biology, Aromatic polyketides, CRISPR/Cas9, Polyketide, Production platform in eukaryotes, Metabolic engineering, Biotechnology

Five series of cheese spreads were prepared and pasteurized at either 92 or 98 °C. The basic ingredient of the samples was a blend of traditional greek cheeses i.e. Myzithra, a whey cheese and Feta cheese in proportion 0.9/0.1. The first series of samples contained only this mixture whereas the other series consisted of samples to which apart from the mixture of the cheeses, either UHT milk or water or olive oil were added in various proportions. The flow properties of the samples were studied by performing lubricated squeeze flow experiments. The results indicated that the elongational viscosity values exhibited, under steady conditions, by the samples were considerably lower to that of the sample contained only the blend of the two cheeses which was not heat treated. It was found that the various ingredients added to the samples affected their flow behavior in various degrees with the olive oil to cause the most noticeable changes. Creep experiments performed using the same samples supported, in general, the findings from the elongational flow measurements.

Parvalbumin (PV+) interneurons constitute a small proportion of the total neuronal population (less than 2% in the hippocampus), yet they possess crucial roles in shaping neuronal network activity (Freund and Buzsáki, 1996; Jonas et al., 2004; Pelkey et al., 2017). PV+ interneurons inhibit their postsynaptic targets efficiently by applying fast perisomatic inhibition and have been directly implicated in the generation of network activity at the gamma (γ) band frequency (30–80 Hz) (Antonoudiou et al., 2020; Cardin et al., 2009; Hájos et al., 2004; Mann et al., 2005; Sohal et al., 2009). Network oscillations at γ-band frequency are believed to facilitate information transmission through circuit synchronization and local gain control that may be instrumental in multiple cognitive processes such as attention, learning, and memory (Akam and Kullmann, 2010; Fries, 2015; Howard et al., 2003; Montgomery and Buzsaki, 2007; Sohal, 2016). Importantly, these oscillations are thought to be metabolically very costly, and it has therefore been postulated that PV+ interneurons require substantial amounts of energy via ATP hydrolysis to sustain the high firing rate and dissipate ion gradients during neuronal transmission (Attwell and Laughlin, 2001; Kann, 2011; Kann, 2016; Kann and Kovács, 2007; Kann et al., 2014). Thus, it is crucial to understand the metabolic expenditure and the involvement of mitochondria in PV+ interneurons. Indeed, electron microscopy, histochemical, and transcriptomic approaches have revealed that PV+ interneurons have a higher density of energy-producing mitochondria and elevated expression levels of electron transport chain components (Adams et al., 2015; Gulyás et al., 2006; Nie and Wong-Riley, 1995; Paul et al., 2017). The spatiotemporal organization of mitochondria is essential for the precise provision of ATP and Ca2+-buffering for neuronal transmission and communication (Devine and Kittler, 2018; MacAskill and Kittler, 2010). Miro1 is a mitochondrial adaptor protein, responsible for coupling mitochondria to the cytoskeleton and for their bidirectional trafficking in axons and dendrites (Birsa et al., 2013; Guo et al., 2005; López-Doménech et al., 2016; López-Doménech et al., 2018; Macaskill et al., 2009; Nguyen et al., 2014; Saotome et al., 2008; Wang and Schwarz, 2009). Global deletion of Miro1 (encoded by the Rhot1 gene) is perinatal lethal, while the conditional removal of Miro1 from cortical and hippocampal pyramidal cells alters the occupancy of dendritic mitochondria due to impairment in trafficking, resulting in dendritic degeneration and cell death (López-Doménech et al., 2016). In contrast, the significance of mitochondrial trafficking and distribution in PV+ interneurons, and the role of Miro1, is completely unexplored and especially interesting as their axon is highly branched with a cumulative length reaching up to 50 mm in the hippocampus (Hu et al., 2014). In this study, we generated a transgenic mouse line where mitochondria are fluorescently labelled in PV+ interneurons. We crossed this line with the Miro1 floxed mouse (Rhot1flox/flox), to generate a model where Miro1 was conditionally knocked-out exclusively in PV+ interneurons. Using two-photon live-imaging of ex vivo organotypic brain slices, we demonstrated a reduction in mitochondrial trafficking in the absence of Miro1 in PV+ interneurons in the hippocampus. The impairment in Miro1-directed mitochondrial transport led to an accumulation of mitochondria in the soma and their depletion from axonal presynaptic terminals in acute hippocampal brain slices. Loss of Miro1 resulted in alterations in axonal but not dendritic branching in PV+ interneurons. While the ability of PV+ interneurons to apply long-lasting inhibition to post-synaptic targets remained intact, the changes in Miro1-dependent mitochondrial dynamics were accompanied by an increased frequency of γ-oscillations in hippocampal brain slices and a reduction in anxiety-related emotional behavior. Thus, we show that Miro1-dependent mitochondrial positioning is essential for correct PV+ interneuron function, network activity, and anxiolytic animal behavior.

Streptomyces griseofuscusDSM 40191 is a fast growingStreptomycesstrain that remains largely underexplored as a heterologous host. Here, we report the genome mining ofS. griseofuscus, followed by the detailed exploration of its phenotype, including production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering ofS. griseofuscuswere explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Using CRISPR-BEST technology, core genes of 4 biosynthetic gene clusters (BGCs) that are situated on the chromosome arms were inactivated and the outcomes of the inactivations were tested. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strainS. griseofuscusDEL1. DEL1 was further modified by full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5,19% genome reduction. Sequencing confirmed that DEL2 does not bear any crucial off-target effects or rearrangements in its genome. It can be characterized by faster growth and inability to produce three main native metabolites ofS. griseofuscus: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to confirm the production of actinorhodin by bothS. griseofuscuswild type and DEL2. We believe that this strain will serve as a good chassis for heterologous expression of BGCs.ImportanceThe rise of antibacterial resistance calls on the development of the next generation of antibiotics, majority of which are derived from natural compounds, produced by actinomycetes. The manipulation, refactoring and expression of BGCs coding for such natural products is a promising approach in secondary metabolite discovery. Thus, the development of a versatile panel of heterologous hosts for the expression of BGCs is essential. We believe that first-to-date systematic, detailed characterisation ofS. griseofuscus, a highly promising chassis strain, will not only facilitate the further development of this particular strain, but also will set a blueprint for characterisation of other potential hosts.

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

Matrix/fiber interfacial bonding, is one of the most significant issues that needed to be addressed, for the wider acceptance of natural fiber reinforced composites to outdoor applications. The hydrophilic nature of the specific fibers is a major obstacle for the bonding with the hydrophobic polymer matrices , thus the formation of poor interface between the two components is inevitable. In this study, unidirectional flax fiber reinforced Polylactic Acid (PLA) composites (Flax/PLA)were fabricated through “film stacking method”, resulting to panel samples. Flax fibers were treated with amino-silane coupling agent, at different silane content values, in order to optimize the composite properties regarding this parameter. The effect of the amount of treatment onPLA/Flax composites has been studied in terms of their flexural response, both static and creep, in order to evaluate their long-term performance as a function of the silane treatment. Results about the effectiveness of silane-treated flax fibers on the mechanical performance of the bio-composite under investigation have been obtained. It has been found that silane loading over a specific number, results to deterioration of the composite’s properties.

Background Nintedanib represents an antifibrotic compound able to slow down disease progression of patients with idiopathic pulmonary fibrosis (IPF). Objective To investigate the safety and efficacy of nintedanib in patients with IPF in a real-life setting. Methods This was a multicentre, retrospective, observational, real-life study for patients with IPF receiving nintedanib between October 2014 and October 2016. Results We identified 94 patients with IPF receiving nintedanib (72 males, mean age±SD: 73.8 ± 7.5, mean%FVC±SD = 68.1 ± 18.3, mean%DLCo±SD = 44.4 ± 14.5). Diarrhea (n = 52, 55.3%) was the most commonly reported adverse event. Twenty patients (21.2%) had to permanently discontinue nintedanib due to severe adverse events. In the 6-months follow-up, median decline in %FVC predicted and %DLCO predicted were 1.36 (95%Cl: 0 to 2.97) and 4.00 (95%Cl: 2.01 to 6.20), respectively, when deaths were censored and excluded from the analysis. At 12 months, mean%FVC±SD and mean%DLCo±SD were 64.5 ± 19.1 and 43.7 ± 15.4, respectively. With regards to mortality, 17 patients (18.1%) died over a study period of 730 days. Conclusion Nintedanib demonstrated an acceptable safety and efficacy profile in our real-world observational study. Prospective observational studies in the context of registries that collect well-defined supporting data over time are sorely needed to answer residual questions on drug's performance.

A simple and selective HPLC method, using a monolithic column, was developed for the simultaneous determination of the histamine H2-receptor antagonists: famotidine, cimetidine and nizatidine, in the presence of sulfadimethoxine as internal standard. The separation was performed on a Chromolith Performance RP-18 column (100 mm x 4.6 mm i.d.) with an isocratic mobile phase consisting of 0.05 mol/L acetate buffer (adjusted to pH 6.5 with triethylamine)/methanol/acetonitrile (85:10:5, v/v/v). The wavelength was set at 230 nm. Linearity was obtained for concentrations between 0.2 to 50 μg/mL and limits of detection were in the range 0.07-0.17 μg/mL. Full validation with respect to linearity, selectivity, detection and quantification limits, accuracy, precision and robustness, the latter using the Youden’s test, was carried out. The method was successfully applied to the determination of the drugs in human serum and urine following solid phase extraction. Average recoveries between 88.0 to 104.4% and 88.0 to 108.0% in serum and urine samples, respectively, were obtained.

Gamma-frequency oscillations (30-120 Hz) can be separated into fast (>60 Hz) and slow oscillations, with different roles in neuronal encoding and information transfer. While synaptic inhibition is important for synchronization across the gamma-frequency range, the role of distinct interneuronal subtypes in fast and slow gamma states remains unclear. Here, we used optogenetics to examine the involvement of parvalbumin (PV+) and somatostatin (SST+) expressing interneurons in gamma oscillations in the mouse hippocampal CA3 ex vivo. Disrupting either PV+ or STT+ interneuron activity, via either photo-inhibition or photo-excitation, led to a decrease in the power of cholinergically-induced slow gamma oscillations. Furthermore, photo-excitation of SST+ interneurons induced fast gamma oscillations, which depended on both synaptic excitation and inhibition. Our findings support a critical role for both PV+ and SST+ interneurons in slow hippocampal gamma oscillations, and further suggest that STT+ interneurons are capable of switching the network between slow and fast gamma states.

In the present study, three series of natural fiber-polymeric composite materials have been prepared and experimentally studied, in terms of Scanning Electron Microscopy, Differential Scanning Calorimetry, Tensile-Flexural testing, Dynamic mechanical analysis and Creep. The materials are unidirectional flax fiber composites, prepared with the “film stacking method”, based on three types of biodegradable polymeric matrices. The addition of flax fibers with this method leads to a significant toughening of the polymers under investigation. In order to further improve the composites thermomechanical performance, three different surface treatments of flax fibers, namely silanization, plasticization and treatment with maleic anhydride, were employed. The effect of the fibers treatment on the mechanical properties of the composites has been comparatively studied and discussed. The surface treatment with maleic anhydride resulted to the highest Young's modulus increment, whereas silanization and plasticization improved both tensile and flexural properties. Similar effect was obtained for storage modulus increment, as well creep resistance of the composites with modified flax fibers.

The K+/Cl− cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl− levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.

Natural or artificially manufactured peptides attract scientific interest worldwide owing to their wide array of pharmaceutical and biological activities. X-ray structural studies are used to provide a precise extraction of information, which can be used to enable a better understanding of the function and physicochemical characteristics of peptides. Although it is vulnerable to disassociation, one of the most vital human peptide hormones, somatostatin, plays a regulatory role in the endocrine system as well as in the release of numerous secondary hormones. This study reports the successful crystallization and complete structural model of octreotide, a stable octapeptide analogue of somatostatin. Common obstacles in crystallographic studies arising from the intrinsic difficulties of obtaining a suitable single-crystal specimen were efficiently overcome as polycrystalline material was employed for synchrotron and laboratory X-ray powder diffraction (XPD) measurements. Data collection and preliminary analysis led to the identification of unit-cell symmetry [orthorhombic,P212121,a= 18.5453 (15),b= 30.1766 (25),c= 39.798 (4) Å], a process which was later followed by complete structure characterization and refinement, underlying the efficacy of the suggested (XPD) approach.

The short-time creep behavior at tensile and single cantilever mode of deformation for a series of biodegradable composites was thoroughly studied. The composites were based on a biodegradable polymer matrix consisted a blend of poly(butylene adipate-terephthalate) (PBAT) copolyester, produced by non-renewable resources, and Polylactic acid (PLA). The matrix was reinforced with three different wood fiber types, at 20 and 30 wt%. The experimental data were analyzed in terms of Findley's and Burger's viscoelastic models. The effect of stress and temperature and wood fiber type on the material's creep response was analytically studied, while the Burger's model parameters were related to the composites morphology. In all cases, the wood fibers improved the creep resistance of the composites.

Hemicelluloses have been extracted from wheat straw with an alkaline peroxide solution. Biodegradable films intended for food packaging have been produced from hemicelluloses mixed with glycerol and different concentrations of citric acid (as a crosslinking agent), with or without sodium hypophosphite (catalyst to the crosslinking reaction). A curing treatment at 150 °C has been carried out on the dried films in order to promote formation of ester bonds between citric acid and hemicelluloses. The crosslinking reaction, evidenced by FTIR spectra, improved water resistance and water vapor barrier properties of the films. On the other hand, the citric acid effects on film tensile properties were more consistent with those of a plasticizer than of a crosslinker, which might be ascribed to a flexible crosslinking. Sodium hypophosphite (SHP) did not affect the properties of crosslinked films. FTIR spectra indicated that some crosslinking reaction occurred even in a non-cured film.

In this work multiwall carbon nanotubes (MWCNTs) dispersed in a polymer matrix have been used for strain sensing of the resulting nanocomposite under tensile loading. This was achieved by measuring the relative electrical resistance change (Δ R / R 0 ) in conductive polyvinylidenefluoride (PVDF)/MWCNTs nanocomposites prepared by melt-mixing with varying filler content from 0.5 wt.% to 8 wt.%. Two main parameters were systematically studied. The PVDF/MWCNTs mixing procedure that results in a successful MWCNTs dispersion, and the effect of MWCNTs content on material’s sensing behaviour. The samples were subjected to tensile loading and the longitudinal strain was monitored together with the longitudinal electrical resistance. The results showed that MWCNTs dispersed in insulating PVDF matrix have the potential to be used as a sensitive network to monitor the strain levels in polymer/carbon nanotube nanocomposites as the deformation level of each sample was being reflected by the resistance changes.

Electrical and dielectric measurements are a powerful tool for following in real time physical and chemical processes, evolution of morphology and mechanical integrity in complex polymeric systems. Here we employ electrical dc measurements and broadband dielectric spectroscopy (DS) to follow the state of polymerization and curing processes in polymer/clay nanocomposites and the quality of filler dispersion, alignment and integrity of carbon nanotubes (CNTs) in various polymer/CNT nanocomposites, focusing on fundamental aspects of the various processes involved.

Purpose To evaluate the anatomical and functional outcomes of patients treated with ocriplasmin for vitreomacular traction syndrome (VMT) or macular holes (MH) combined with VMT in a tertiary retina center. Methods Eleven eyes of ten patients (8 females, 2 males) with VMT (8 with VMT alone and 3 with MH combined with VMT) were included in the study. The patients were treated with a single ocriplasmin injection and examined at day 1, 7 and 28 post-injection. Age, gender, phakic lens status, vitreomacular adhesion diameter, presence of epiretinal membrane, macular hole size, cystoid macular oedema and the status of the ellipsoid zone were recorded. Best-corrected visual acuity (BCVA) and spectral-domain-optical coherence tomography (SD-OCT) were performed at baseline and at each examination during the follow up period. Adverse effects were also recorded. Results Six eyes (54,5%) presented VMT resolution, while one out of three patients presented MH closure. All patients experienced VMT release by 7 days. Patients with VMT resolution had an increase in BCVA of +0.5 logMAR. . Conclusions Ocriplasmin may be considered as an effective treatment option for VMT and macular holes with VMT.

In this work a series of polylactic acid/SiO2 nanocomposites have been prepared by a melt mixing procedure. The dispersion quality was examined by scanning electron microscopy. To study the degradation behavior of the polylactic acid/nanocomposites prepared, the samples were immersed in a buffer solution at a temperature of 37℃ with a pH of 7.4 for a time period of up to 23 weeks. These conditions simulate those in the human body, appropriate in medical applications. In order to assess their suitability in biomedical applications, we investigated the biocompatibility of these materials in terms of cell viability, growth, and morphology. A good initial cell adhesion has been detected, supporting their potential use in bone tissue engineering applications. The hydrolytic degradation of polylactic acid, under the prescribed conditions, was studied by the molecular weight reduction in terms of size exclusion chromatography, whereas the progress of thermal stability of polylactic acid and polylactic acid/nanocomposites during aging was tested by thermogravimetric analysis. The evolution of the materials’ thermomechanical properties during aging was studied by differential scanning calorimetry, dynamic mechanical analysis, and tensile testing. The crystallization behavior in polylactic acid and the way it is affected by the presence of nanofillers during degradation procedure has been studied and values of 44% crystallinity increment have been found. At the specific aging conditions studied, silica nanoparticles accelerate the degradability of polylactic acid, having a higher impact on Young’s modulus, under the specified aging conditions, for 7 weeks and hereafter this acceleration is retarded, due to the crystallinity increment, as a result of the molecular weight reduction.

The effect of type and content of fillers on the thermal, mechanical, and rheological behavior of the commercial biodegradable polyester product, Ecovio® (BASF) is analytically studied. Ecovio® is basically a blend of poly(butylene adipate-terephthalate) (PBAT) copolyester (Ecoflex®, BASF) and polylactide (PLA). Two different types of fillers (nanosilica particles and micro-sized wood-flour), at various weight fractions were used for this purpose. The role of these fillers on the thermomechanical performance of Ecovio® was investigated in terms of several experimental techniques including scanning electron microscopy (SEM), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), creep, tensile testing, and single cantilever bending. The rheological behavior has been systematically analyzed, providing additional evidence about the dispersion quality and the interfacial effects between nanofillers and matrix. One of the findings of this work is that the presence of PBAT in the blend (PLA/PBAT) enhances the compatibility of the polymer matrix with both fillers and their dispersion quality. POLYM. COMPOS., 35:1140–1149, 2014. © 2013 Society of Plastics Engineers

BACKGROUND Pneumatic tube system (PTS) is a widely used method of transporting blood samples in hospitals. The aim of this study was to evaluate the effects of the PTS transport in certain routine laboratory parameters as it has been implicated with hemolysis. METHODS A systematic review and a meta-analysis were conducted. PubMed and Scopus databases were searched (up until November 2016) to identify prospective studies evaluating the impact of PTS transport in hematological, biochemical and coagulation measurements. The random-effects model was used in the meta-analysis utilizing the mean difference (MD). Heterogeneity was quantitatively assessed using the Cohran's Q and the I2 index. Subgroup analysis, meta-regression analysis, sensitivity analysis, cumulative meta-analysis and assessment of publication bias were performed for all outcomes. RESULTS From a total of 282 studies identified by the searching procedure, 24 were finally included in the meta-analysis. The meta-analysis yielded statistically significant results for potassium (K) [MD=0.04 mmol/L; 95% confidence interval (CI)=0.015-0.065; p=0.002], lactate dehydrogenase (LDH) (MD=10.343 U/L; 95% CI=6.132-14.554; p

alpha-D-glucose is a weak inhibitor (Ki = 1.7 mM) of glycogen phosphorylase (GP) and acts as physiological regulator of hepatic glycogen metabolism; it binds to GP at the catalytic site and stabilizes the inactive T state of the enzyme promoting the action of protein phosphatase 1 and stimulating glycogen synthase. The three-dimensional structures of T state rabbit muscle GPb and the GPb-alpha-D-glucose complex have been exploited in the design of better regulators of GP that could shift the balance between glycogen synthesis and glycogen degradation in favour of the former. Close examination of the catalytic site with alpha-D-glucose bound shows that there is an empty pocket adjacent to the beta-1-C position. beta-D-glucose is a poorer inhibitor (Ki = 7.4 mM) than alpha-D-glucose, but mutarotation has prevented the binding of beta-D-glucose in T state GP crystals. A series of beta-D-glucose analogues has been designed and tested in kinetic and crystallographic experiments. Several compounds have been discovered that have an increased affinity for GP than the parent compound.

A theoretical approach for the prediction of creep rupture time of polymers and polymer composites is analyzed in the present work. This analysis takes into account the viscoelastic path at small strains and the viscoplastic path at higher stresses. The calculation of the rate of creep strain is based on a thermally activated rate process, while the emergence and growth of plastic strain, with increasing creep time, is also taken into account. When the accumulated strain attains values, high enough to lead to failure, its slope versus time exhibits an abrupt change. At this specific time, the creep rate function in respect to time appears a minimum. The creep failure time is defined as the time where the creep rate takes its minimum value. The model has been tested for various types of polymeric materials, as well as for polymer composites. Once the model parameters are estimated from short time creep strain data, then it was proved to successfully predict the creep failure time at a variety of stress levels, for all material types examined.

Glycogen phosphorylase is a molecular target for the design of potential hypoglycemic agents. Structure-based design pinpointed that the 3′-position of glucopyranose equipped with a suitable group has the potential to form interactions with enzyme's cofactor, pyridoxal 5′-phosphate (PLP), thus enhancing the inhibitory potency. Hence, we have investigated the binding of two ligands, 1-(β-d-glucopyranosyl)5-fluorouracil (GlcFU) and its 3′-CH 2OH glucopyranose derivative. Both ligands were found to be low micromolar inhibitors with K i values of 7.9 and 27.1μm, respectively. X-ray crystallography revealed that the 3′-CH 2OH glucopyranose substituent is indeed involved in additional molecular interactions with the PLP γ-phosphate compared with GlcFU. However, it is 3.4 times less potent. To elucidate this discovery, docking followed by postdocking Quantum Mechanics/Molecular Mechanics - Poisson-Boltzmann Surface Area (QM/MM-PBSA) binding affinity calculations were performed. While the docking predictions failed to reflect the kinetic results, the QM/MM-PBSA revealed that the desolvation energy cost for binding of the 3′-CH 2OH-substituted glucopyranose derivative out-weigh the enthalpy gains from the extra contacts formed. The benefits of performing postdocking calculations employing a more accurate solvation model and the QM/MM-PBSA methodology in lead optimization are therefore highlighted, specifically when the role of a highly polar/charged binding interface is significant.

C5 halogen substituted glucopyranosyl nucleosides (1-(β-D-glucopyranosyl)-5-X-uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective K(i) values of 1.02, 3.27, and 1.94 μM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ-hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1-(β-D-glucopyranosyl)uracil (K(i) =12.39 μM), as revealed by X-ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM-PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell.

In this work multiwall carbon nanotubes (MWCNTs) dispersed in a polymer matrix have been used for strain sensing of the resulting nanocomposite under tensile loading. This was achieved by measuring the relative electrical resistance change (ΔR/R0) in conductive PVDF (Polyvinylidenefluoride)/MWCNT nanocomposites prepared by melt-mixing with varying filler content from 1.25wt% to 8wt%. The samples were subjected to tensile loading and the longitudinal strain was monitored together with the longitudinal electrical resistance. The results showed that CNTs dispersed in an insulating polymer matrix have the potential to be used as a sensitive network to monitor or predict the damage in polymer/carbon nanotube nanocomposites.

This study examines the effect of type and content of various nanofillers on the aging behavior of polylactic acid (PLA)/nanocomposites compared with that of pristine PLA, under specific environmental conditions, namely 80% relative humidity, 40°C temperature, and exposure time up to 6 months. Two different types of nanosized fillers (silica and montmorillonite, MMT) at three different weight fractions as well their mixtures were used for this purpose. The role of the various nanofillers on the aging of pristine PLA and its nanocomposites was investigated in terms of several experimental techniques including Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), Dynamic Mechanical Analysis (DMA), and tensile testing. All studied samples, except those of PLA/MMT/Si nanocomposites, exhibited the same trend in properties during aging. In particular, no significant changes are noted after 1 month of aging. The materials after 3 months of aging experienced a dramatic lowering of the stress–strain curve, whereas at 6 months a reverse effect was observed. Comparing the effect of nanofiller types on the degradation, one could conclude that PLA/MMT nanocomposites exhibit a more homogeneous degradation effect, having a higher impact on yield stress in comparison with silica. In contrast, silica has a greater effect in Young's modulus and strain at break, compared with MMT. The peculiar behavior of the PLA/MMT/Si nanocomposites during aging is explained by the hindering action of the mixture of the two nanofillers on the molecular reordering of the PLA chains in the amorphous region. POLYM. COMPOS., 2012. © 2011 Society of Plastics Engineers

This study compares the effects of two different types of nanosized fillers (silica and montmorillonite) at three different weight fractions as well their mixtures on the thermomechanical properties of polylactide (PLA). The role of aggregation and interphase was investigated in terms of several experimental techniques, including scanning electron microscopy, wide-angle X-ray scattering (WAXS), thermogravimetric analysis, differential scanning calorimetry, dynamic mechanical analysis (DMA), and tensile measurements. The experimental results clearly suggest that silica and montmorillonite have different reinforcing and toughening effects on PLA, while the combination of the two different nanofillers has a detrimental effect on the tensile properties of the material. Four micromechanics models describing the Young's modulus of the nanocomposites were used to study the different matrix–nanofiller interactions. The best fit of the experimental results was obtained with a model that assumes the presence of an interphase surrounding each nanoparticle. The increase of the nanofiller content above a certain amount was not accompanied by a corresponding increase of the interphase, because the total nanofiller surface area was counterbalanced by the creation of aggregates. It is postulated that the aggregation effect acts antagonistically to the interphase effect. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

In this study, we examined the role of environmental parameters and physical structure in the aging process of poly(lactic acid) (PLA). The role of heating history on the aging behavior of the material was also investigated. PLA samples with a D-content of 4.25% were exposed to a relative humidity of 80% at three different temperatures, 20, 40, and 50°C (below the glass-transition temperature of the material), at various aging periods of 30, 60, 80, 100, and 130 days. Selected samples were subjected to two consecutive heating runs. The stability of PLA was monitored by a number of techniques, including size exclusion chromatography, differential scanning calorimetry, dynamic mechanical analysis, and tensile measurements. The initial thermal processing (150°C) of the material resulted in an overall molecular weight reduction. A substantial lowering of properties was observed for PLA samples aged at 20°C for 30 days. No further loss of properties was observed for samples aged up to 40°C for several time intervals. A major portion (80–90%) of the induced changes in the tensile properties could be reversed after drying. At 50°C and 100 days of aging, a sharp decrease in the overall properties was noticed. The results seem to confirm the earlier finding that PLA degradation driven by hydrolysis needs a higher temperature (>50°C) in combination with ample time to take place. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

The function of the central nervous system largely depends on growth and differentiation (neurite outgrowth) of neural cells and it is well established that growth factors, especially nerve growth factor NGF stimulate neurite outgrowth. However, additional factors are implicated in this process notably the redox state of the cells. For the first time we could demonstrate that the application of recombinant thioredoxin stimulates neurite outgrowth of PC12 cells to the same extend as NGF. Thioredoxin, a small redox protein is a major player in the cellular protein reduction system. An increased expression and secretion of thioredoxin is achieved by the application of the novel sialic acid precursor N-propionylmannosamine (ManNProp). From earlier studies it is known that this N-acylmannosamine analog stimulates significantly the neurite outgrowth in cell cultures. This finding would give new insights into the mechanism of the nerve-stimulatory action of ManNProp and demonstrates the novel role of thioredoxin during the regulation of nerve growth, encouraging further studies.

Sialic acids usually represent the terminal monosaccharide of glycoconjugates and are directly involved in many biological processes. The cellular concentration of their nucleotide-activated form is one pacemaker for the highly variable sialylation of glycoconjugates. Hence, the determination of CMP-sialic acid levels is an important factor to understand the complex glycosylation machinery of cells and to standardize the production of glycotherapeutics. We have established a highly sensitive strategy to quantify the concentration of nucleotide-activated sialic acid by a combination of reduction and fluorescent labeling using the fluorophore 1,2-diamino-4,5-methylenedioxybenzene (DMB). The labeling with DMB requires free keto as well as carboxyl groups of the sialic acid molecule. Reduction of the keto group prior to the labeling process precludes the labeling of nonactivated sialic acids. Since the keto group is protected against reduction by the CMP-substitution, labeling of nucleotide-activated sialic acids is still feasible after reduction. Subsequent combination of the DMB-high-performance liquid chromatography (HPLC) application with mass spectrometric approaches, such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and electrospray-ionization (ESI)-MS, allows the unambiguous identification of both natural and modified CMP-sialic acids and localization of potential substituents. Thus, the described strategy offers a sensitive detection, identification, and quantification of nucleotide-activated sialic acid derivatives in the femtomole range without the need for nucleotide-activated standards.

In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5'-phosphate (U5P) and uridine 5'-diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with K(i) values of 4.0 and 0.65 mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purine-binding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily.

Time- and dose-dependent measurements of metabolites of galactose (with glucose as control) in various organs of rats are discussed. Not only the liver but especially the brain and to a lesser extent the muscles also have the capacity to take up and metabolize galactose. Primarily, the concentrations of UDP-galactose, a pivotal compound in the metabolism of galactose, and UDP-glucose are measured. An important feature lies in the demonstration that galactose and glucose are metabolized to amino acids and that the only increases observed in the brain appear in the concentrations of glutamate, glutamine, GABA measured after acute galactose loads. In addition the increase in the amino acid concentrations after galactose has been administered persists for longer periods of time than after glucose administration. This conversion of hexoses, especially galactose, to amino acids requires the consumption of ammonia equivalents in the brain ; this finding might stimulate the use of galactose as a new means of removal of this neurotoxic compound from the brain in patients suffering from hepatic encephalopathy or Alzheimer's disease.

In this work, yielding and post yielding effects are analyzed using the idea of the inhomogeneous distribution of strain in polymers under deformation. This idea is directly connected with the free volume concept, as created in the polymeric bulk during the frozen in process, and or the density fluctuations combined with other type defects. A simple strain density distribution function is assumed, following experimental results available from techniques such as positron annihilation lifetime spectroscopy (PALS). Hereafter, a functional form of the rate of plastic deformation is extracted, which will be combined with a given kinematic formulation. The proposed analysis is tested with experimental data of polystyrene (PS) and PS/SiO 2 nanocomposites. The incorporation of nanosized fillers into the polymeric bulk, strongly affects the free volume distribution and/or the distribution of defects related with density fluctuations. This fact is reflected in the model parameter values, and their variation in respect to the different material types. With the proposed analysis, it has been proved that all features of yield process, including strain softening and strain hardening effect are easily described.

Fanconi anemia is a fatal, hereditary chromosome instability syndrome of early childhood with progressive pancytopenia and cancer-proneness. Hypersensitivity to alkylating agents points to DNA repair inefficiency. Excess reactive oxygen intermediates and hypersensitivity to oxygen, all features of Fanconi anemia cells, give evidence for a disturbed oxidative metabolism. Here, we report that expression of the inducible heme oxygenase-1, an essential antioxidative defense protein, is impaired in Fanconi anemia cells and can be reinstated with the transfection of Fanconi A wild-type cDNA. A causative interaction of Fanconi anemia proteins with transcription of selected proteins is indicated. The results enlighten the oxygen sensitivity in Fanconi anemia.

A large number of works deal with material synthesis and characterization of polymer nanocomposites due to their improved thermo-mechanical properties, but the fundamental mechanisms for mechanical property enhancement are not yet completely defined. In particular, a special class of polymer/organoclay nanocomposites has been observed to exhibit an impressive improvement in different types of properties, physical and chemical ones. In the present work, a model is presented and applied to formulate the elastoplastic response of epoxy/clay nanocomposites, experimentally tested elsewhere. The model based on Mori–Tanaka theory, for the estimation of the elastic stiffness tensor for composite materials, is combined with the self-consistent model of Budiansky and Wu, valid for crystal plasticity. Then the macroscopic plastic response of the heterogeneous material is linked with the microstructural parameters, i.e., the plastic behaviour of the effective particle. The model was proved to successfully describe the tensile response of the epoxy/clay nanocomposites with varying clay weight fraction.

Sialic acid precursors are mediators of the sialic acid pathway. In this manuscript we present evidence that the application of sialic acid a precursor modulates gene expression and cell differentiation. The concept that sugars are involved in cellular transcription was first proposed by Jacob and Monod nearly 40 years ago studying the regulation of the lac-operon in prokaryotes. Surprisingly, these findings have never been transferred to eukaryotic systems. For our studies we have chosen PC12 cells. PC12-cells differentiate after application of NGF into a neuron-like phenotype. It is shown that treatment of PC12 cells with two different sialic acid precursors N-acetyl- or N-propanoylmannosamine, without application of NGF also induces neurite outgrowth. Moreover, the PC12 cells show the same morphology as the NGF-treated cells. Surprisingly, after application of both sialic acid precursors the phosphorylation and translocation of erk1/2 into the nucleus are activated, thus influencing the expression of genes involved in the differentiation of cells, such as the transcription factor c-Jun or TOAD-64/Ulip/CRMP (Turned ON After Division, 64 kd/ unc-33-like phosphoprotein/Collapsin Response Mediator Protein). These are the first experimental data showing that the sialic acid metabolism is closely associated with signal transduction and regulation of neuronal differentiation.

The advantages of cyanate esters (CEs) versus competitor systems such as epoxies and polyimides, as well as the great reinforcing potential of organoclays properly dispersed into a polymeric matrix, have been examined in a series of polycyanurate (PCN)/montmorillonite (MMT) nanocomposites prepared under appropriate polymerization conditions. The curing schedule applied resulted in gradual propagation of polymerization. Through this procedure, the intragallery curing rate becomes comparable to the extragallery one, allowing intercalation before gelation. Systems with clay loadings from 1 to 3% per weight were synthesized, and their morphology and mechanical properties were studied by means of scanning electron microscopy (SEM), atomic force microscopy (AFM), wide angle X-ray scattering (WAXS), dynamic mechanical analysis (DMA), and tensile tests. Microscopy investigations revealed better dispersion for the 3 wt % system compared to smaller concentrations, in which aggregation and, in some cases, agglomeration were the conspicuous features. Roughness and area analyses revealed more homogeneous dispersion for this nanocomposite. Topology and 3D-phase images further suggested considerable reduction of the average particle diameters. WAXS analysis showed that the interlayer spacing of nanocomposites was increased compared to pristine MMT, indicating the formation of intercalated structures. On the other hand, tensile strength and elongation at break values displayed abrupt diminution with MMT addition, while Young's modulus exhibited a slight but systematic increment with MMT content. The decreasing glass transition tendency observed for small clay loadings was reversed in the case of 3 wt %, while secondary transitions were practically unaffected by the presence of MMT. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1036–1049, 2008

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.