Solid-Phase Synthesis as a Tool to Create Exactly Defined, Branched Polymer Vectors for Cell Membrane Targeting.

Modern drug formulations often require, besides the active drug molecule, auxiliaries to enhance their pharmacological properties. Tailor-made, biocompatible polymers covalently connected to the drug molecule can fulfill this function by increasing its solubility, reducing its toxicity, and guiding it to a specific target. If targeting membrane-bound proteins, localization of the drug close to the cell membrane and its target is beneficial to increase drug efficiency and residence time. In this study, we present the synthesis of highly defined, branched polymeric structures with membrane-binding properties. One to three hydrophilic poly(ethylene oxide) or poly(2-ethyloxazoline) side chains were connected via a peptoid backbone using a two-step iterative protocol for solid-phase peptoid synthesis. Additional groups, e.g., a hydrophobic anchor for membrane attachment, were introduced. Due to the nature of solid-phase synthesis, the number and order of the side chains and additional units can be precisely defined. The method proved to be versatile for the generation of multifunctional, branched polymeric structures of molecular weights up to approximately 7000 g mol-1. The behavior of all compounds towards biological membranes and cells was investigated using liposomes as cell membrane models, HEK293 and U251-MG cell lines, and red blood cells, thereby demonstrating their potential value as drug auxiliaries with cell membrane affinity.

The interacting effect of climate change and herbivory can trigger large-scale transformations of European temperate forests.

In many regions of Europe, large wild herbivores alter forest community composition through their foraging preferences, hinder the forest's natural adaptive responses to climate change, and reduce ecosystem resilience. We investigated a widespread European forest type, a mixed forest dominated by Picea abies, which has recently experienced an unprecedented level of disturbance across the continent. Using the forest landscape model iLand, we investigated the combined effect of climate change and herbivory on forest structure, composition, and carbon and identified conditions leading to ecosystem transitions on a 300-year timescale. Eight climate change scenarios, driven by Representative Concentration Pathways 4.5 and 8.5, combined with three levels of regeneration browsing, were tested. We found that the persistence of the current level of browsing pressure impedes adaptive changes in community composition and sustains the presence of the vulnerable yet less palatable P. abies. These development trajectories were tortuous, characterized by a high disturbance intensity. On the contrary, reduced herbivory initiated a transformation towards the naturally dominant broadleaved species that was associated with an increased forest carbon and a considerably reduced disturbance. The conditions of RCP4.5 combined with high and moderate browsing levels preserved the forest within its reference range of variability, defining the actual boundaries of resilience. The remaining combinations of browsing and climate change led to ecosystem transitions. Under RCP4.5 with browsing effects excluded, the new equilibrium conditions were achieved within 120 years, whereas the stabilization was delayed by 50-100 years under RCP8.5 with higher browsing intensities. We conclude that forests dominated by P. abies are prone to transitions driven by climate change. However, reducing herbivory can set the forest on a stable and predictable trajectory, whereas sustaining the current browsing levels can lead to heightened disturbance activity, extended transition times, and high variability in the target conditions.

Phototriggered Cytotoxicity of Ferrocene-Based Micelles - A Nonconventional Approach to Phototherapy.

We report the design, synthesis, and in vitro evaluation of stimuli-responsive nanoscale micelles that can be activated by light to induce a cytotoxic effect. Micelles were assembled from amphiphilic units made of a photoactivatable ferrocenyl linker, connected on one side to a lipophilic chain, and on the other side to a hydrophilic pegylated chain. In vitro experiments indicated that pristine micelles ("off" state) were nontoxic to MCF-7 cancer cells, even at high concentrations, but became potent upon photoactivation ("on" state). The illumination process led to the dissociation of the micelles and the concomitant release of iron species, triggering cytotoxicity.

Insights into localization, energy ordering, and substituent effect in excited states of azobenzenes from coupled cluster calculations of nuclear spin-induced circular dichroism.

Nuclear spin-induced circular dichroism (NSCD) is a molecular effect of differential absorption of left- and right-circularly polarized light due to nuclear spins in the molecule. In this work, new tools for its calculation are presented. Specifically, analytic expressions for the computation of the K term of NSCD have been derived and implemented for the second-order coupled cluster singles and doubles (CC2) model. NSCD results obtained thereby for three derivatives of azobenzenes have been compared with results from time-dependent density functional theory (TD-DFT). The complementary information that could be obtained from NSCD measurements compared to NMR for these three species is discussed.

Characteristic nuclear spin-induced optical rotation in oxygen-containing organic molecules.

Nuclear spin-induced optical rotation (NSOR) is a nuclear magneto-optic effect that manifests itself as a rotation of the plane of polarization of linearly polarized light. The effect is induced by ordered nuclear magnetic moments within a molecule. NSOR is sensitive to specific, localized interactions. Hence, the connection between the local chemical environment and the corresponding NSOR signal is crucial to understand. Despite the fact that contributions to better understand the connection have been made, the general systematics still remain unknown. In this paper, NSOR in oxygen compounds is investigated systematically to better understand the impact of oxygen atoms on the NSOR signal. NSOR signals are computed using density-functional theory methods for five different classes of oxygen compounds. The ability of NSOR to distinguish different molecules and individual nuclei in the molecules is studied and the information provided by NSOR is compared to conventional NMR spectroscopy. The results reveal that NSOR is capable of chemical distinction between nuclei and molecules, and by using NMR and NSOR together it is possible to distinguish nuclei near the oxygen atom.

Tuning polymer-blood and polymer-cytoplasm membrane interactions by manipulating the architecture of poly(2-oxazoline) triblock copolymers.

Bioactive moieties designed to bind to cell membrane receptors benefit from coupling with polymeric carriers that have enhanced affinity to the cell membrane. When bound to the cell surface, such carriers create a "2D solution" of a ligand with a significantly increased concentration near a membrane-bound receptor compared to a freely water-soluble ligand. Bifunctional polymeric carriers based on amphiphilic triblock copolymers were synthesized from 2-pent-4-ynyl oxazoline, 2-nonyl oxazoline and 2-ethyl oxazoline. Their self-assembly and interactions with plasma proteins and HEK 293 cells were studied in detail. The affinity of these triblock copolymers to HEK 293 cell membranes and organ tissues was tunable by the overall hydrophobicity of the polymer molecule, which is determined by the length of the hydrophobic and hydrophilic blocks. The circulation time and biodistribution of three representative triblock copolymers were monitored after intravenous administration to C57BL/6 albino mice. A prolonged circulation time was observed for polymers with longer hydrophobic blocks, despite their molecular weight being below the renal threshold.

Amphiphilic (di-)gradient copoly(2-oxazoline)s are potent amyloid fibril formation inhibitors.

MOTIVATION: Amyloidoses are diseases caused by the accumulation of normally soluble proteins in the form of insoluble amyloids, leading to the gradual dysfunction and failure of various organs and tissues. Inhibiting amyloid formation is therefore an important therapeutic target. HYPOTHESIS: We hypothesized that mono- and di-gradient amphiphilic copolymers of hydrophilic 2-(m)ethyl-2-oxazoline and hydrophobic 2-aryl-2-oxazolines may inhibit amyloid fibril formation. EXPERIMENTS: In the model system with hen egg white lysozyme (HEWL) as amyloidogenic protein we determined the effect of these polymers on the amyloid formation by making use of the thioflavin T fluorescence, transmission electron microscopy, isothermal titration calorimetry, and dynamic light scattering. FINDINGS: We found that some gradient copolymers possess very potent concentration-dependent inhibitory effects on HEWL amyloid formation. Structure-activity relationship revealed that copolymers with higher ratios of aromatic monomeric units had stronger amyloid suppression effects, most plausibly due to the combination of hydrophobic and π-π interactions. The measurements also revealed that the polymers that inhibit amyloid formation most plausibly do so in the form of micelles that interact with the growing amyloid fibril ends, not with isolated HEWL molecules in solution. These findings suggest the potential use of these gradient copolymers as therapeutic agents for amyloidoses.

Plasmonic Ag/Cu/PEG nanofluids prepared when solids meet liquids in the gas phase.

Since the time of Faraday's experiments, the optical response of plasmonic nanofluids has been tailored by the shape, size, concentration, and material of nanoparticles (NPs), or by mixing different types of NPs. To date, water-based liquids have been the most extensively investigated host media, while polymers, such as poly(ethylene glycol) (PEG), have frequently been added to introduce repulsive steric interactions and protect NPs from agglomeration. Here, we introduce an inverse system of non-aqueous nanofluids, in which Ag and Cu NPs are dispersed in PEG (400 g mol-1), with no solvents or chemicals involved. Our single-step approach comprises the synthesis of metal NPs in the gas phase using sputtering-based gas aggregation cluster sources, gas flow transport of NPs, and their deposition (optionally simultaneous) on the PEG surface. Using computational fluid dynamics simulations, we show that NPs diffuse into PEG at an average velocity of the diffusion front of the order of µm s-1, which is sufficient for efficient loading of the entire polymer bulk. We synthesize yellow Ag/PEG, green Cu/PEG, and blue Ag/Cu/PEG nanofluids, in which the color is given by the position of the plasmon resonance. NPs are prone to partial agglomeration and sedimentation, with a slower kinetics for Cu. Density functional theory calculations combined with UV-vis data and zeta-potential measurements prove that the surface oxidation to Cu2O and stronger electrostatic repulsion are responsible for the higher stability of Cu NPs. Adopting the De Gennes formalism, we estimate that PEG molecules adsorb on the NP surface in mushroom coordination, with the thickness of the adsorbed layer L < 1.4 nm, grafting density σ < 0.20, and the average distance between the grafted chains D > 0.8 nm. Such values provide sufficient steric barriers to retard, but not completely prevent, agglomeration. Overall, our approach offers an excellent platform for fundamental research on non-aqueous nanofluids, with metal-polymer and metal-metal interactions unperturbed by the presence of solvents or chemical residues.

Landslides associated with recent road constructions in the Río Lucma catchment, eastern Cordillera Blanca, Peru.

Extensive road construction works recently took place in the remote eastern part of the Peruvian Cordillera Blanca, aiming at a better connection of isolated mountain communities with regional administrative centres. Here we document and characterize landslides associated with these road construction efforts in the Río Lucma catchment, Peru. We show that a total area of 321,332 m2 has been affected by landslides along the 47.1 km of roads constructed between 2015 and 2018. While landslides downslope the roads (48.2%) and complex landslides crossing the roads (46.4%) were the most frequent landslide types in relation to the position of the road; slide-type movement (60.7%) prevails over the flow-type movement (39.3%). Timewise, we found that 75.0% of landslides were observed simultaneously with road construction work, while the remaining 25.0% occurred up to seven months after the roads had been constructed. We plotted the lagged occurrence of these subsequent landslides against precipitation data, showing that 85.7% of them were observed during the wet season (November to April). We conclude that the majority of mapped landslides were directly associated with road constructions and that the road constructions also may set preconditions for landslides, which mainly occurred during the subsequent wet season.

Unexpected NMR shieldings of sp- and sp2-hybridized carbon atoms in graphyne systems.

Graphynes (GYs) are two-dimensional alloptropic forms of carbon consisting of periodically arranged sp- and sp2-hybridized carbon atoms in a planar structure. Graphynes can be formally created from graphene by inserting sp-hybridized carbon links into selected points of the graphene lattice. Depending on where the links are introduced, several forms of graphynes have been proposed with properties that make them potential candidates for new generation electronics or for applications in chemical processes. Since the applications of each form of GY depend on its structure, it is of interest to experimentally distinguish different forms of graphynes. In this paper we propose nuclear magnetic resonance (NMR) as a potential method of choice for such distinction. We computationally investigate on the DFT level the 13C-NMR chemical shifts for α-, ß-, γ-, rhombic, and 6,6,12-graphynes, and α- and γ-graphdiynes. We perform the calculations both in periodic systems and with approximate finite models. The results show that NMR chemical shifts in graphynes are dependent on the structure and reflect the local bonding around the carbon nucleus. Interestingly, NMR shifts of several graphynes show anomalous values, differing significantly from shifts found in typical sp2-hybridized systems. We analyze these results in terms of local structural parameters and qualitatively investigate the possible origins of these anomalous NMR shifts. The results show that NMR is a viable method for determining the structure of graphynes and their finite precursor molecules.

Protein coronas coating polymer-stabilized silver nanocolloids attenuate cytotoxicity with minor effects on antimicrobial performance.

Silver nanoparticles are versatile platforms with a variety of applications in the biomedical field. In this framework, their presence in biological media inevitably leads to the interaction with proteins thus conducting to the formation of biomolecular coronas. This feature alters the identity of the nanomaterial and may affect many biological events. These considerations motivated the investigation of protein adsorption onto the surface of polymer-stabilized AgNPs. The metallic colloids were coated by polyethyleneimine (PEI), polyvinylpyrrolidone (PVP), and poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PEO-b-P2VP), and nanoparticle-protein interaction was probed by using a library of analytical techniques. The experimental data revealed a higher extent of protein adsorption at the surface of AgNPs@PVP whereas PEO-b-P2VP coating conducted to the least amount. The main component of the protein coronas was evidenced to be bovine serum albumin (BSA), which is indeed the protein at the highest abundancy in the model biological media. We have further demonstrated reduced cytotoxicity of the silver colloids coated by biomolecular coronas as compared to the pristine counterparts. Nevertheless, the protein coatings did not notably reduce the antimicrobial performance of the polymer-stabilized AgNPs. Accordingly, although the protein-repelling property is frequently targeted towards longer in vivo circulation of nanoparticles, we herein underline that protein coatings, which are commonly treated as artifacts to be avoided, may indeed enhance the biological performance of nanomaterials. These findings are expected to be highly relevant in the design of polymer-stabilized metallic colloids intended to be used in healthcare.

Changes in forest nitrogen cycling across deposition gradient revealed by δ15N in tree rings.

Tree rings provide valuable insight into past environmental changes. This study aimed to evaluate perturbations in tree ring width (TRW) and δ15N alongside soil acidity and nutrient availability gradients caused by the contrasting legacy of air pollution (nitrogen [N] and sulphur [S] deposition) and tree species (European beech, Silver fir and Norway spruce). We found consistent declines of tree ring δ15N, which were temporarily unrelated to the changes in the TRW. The rate of δ15N change in tree rings was related to the contemporary foliar carbon (C) to phosphorus (P) ratio. This observation suggested that the long-term accumulation of 15N depleted N in tree rings, likely mediated by retained N from deposition, was restricted primarily to stands with currently higher P availability. The shifts observed in tree-ring δ15N and TRW suggest that acidic air pollution rather than changes in stand productivity determined alteration of N and C cycles. Stable N isotopes in tree rings provided helpful information on the trajectory of the N cycle over the last century with direct consequences for a better understanding of future interactions among N, P and C cycles in terrestrial ecosystems.

Lipid Nanomaterials for Targeted Delivery of Dermocosmetic Ingredients: Advances in Photoprotection and Skin Anti-Aging.

Despite the health benefits of the sun, overexposure to solar radiation without proper precautions can cause irreversible damage to exposed skin. In the search for balance between the risks and benefits of exposure to solar radiation in human health, a technological alternative was found, the incorporation of photoprotective products in lipid nanoparticulate systems for topical application. These nanometric systems have demonstrated several advantages when used as adjuvants in photoprotection compared to chemical and/or physical sunscreens alone. The increase in the sun protection factor (SPF), photostability and UV action spectrum are parameters that have benefited from the application of these systems in order to increase the effectiveness and safety of photoprotective formulations containing organic and/or inorganic sunscreens.

Acute Pneumonia Caused by Clinically Isolated Legionella pneumophila Sg 1, ST 62: Host Responses and Pathologies in Mice.

Legionnaires' disease is a severe form of lung infection caused by bacteria belonging to the genus Legionella. The disease severity depends on both host immunity and L. pneumophila virulence. The objective of this study was to describe the pathological spectrum of acute pneumonia caused by a virulent clinical isolate of L. pneumophila serogroup 1, sequence type 62. In A/JOlaHsd mice, we compared two infectious doses, namely, 104 and 106 CFU, and their impact on the mouse status, bacterial clearance, lung pathology, and blood count parameters was studied. Acute pneumonia resembling Legionnaires' disease has been described in detail.

Chelators for Treatment of Iron and Copper Overload: Shift from Low-Molecular-Weight Compounds to Polymers.

Iron and copper are essential micronutrients needed for the proper function of every cell. However, in excessive amounts, these elements are toxic, as they may cause oxidative stress, resulting in damage to the liver and other organs. This may happen due to poisoning, as a side effect of thalassemia infusion therapy or due to hereditary diseases hemochromatosis or Wilson's disease. The current golden standard of therapy of iron and copper overload is the use of low-molecular-weight chelators of these elements. However, these agents suffer from severe side effects, are often expensive and possess unfavorable pharmacokinetics, thus limiting the usability of such therapy. The emerging concepts are polymer-supported iron- and copper-chelating therapeutics, either for parenteral or oral use, which shows vivid potential to keep the therapeutic efficacy of low-molecular-weight agents, while avoiding their drawbacks, especially their side effects. Critical evaluation of this new perspective polymer approach is the purpose of this review article.

pH-responsive polymersome-mediated delivery of doxorubicin into tumor sites enhances the therapeutic efficacy and reduces cardiotoxic effects.

The delivery of therapeutics into sites of action by using cargo-delivery platforms potentially minimizes their premature degradation and fast clearance from the bloodstream. Additionally, drug-loaded stimuli-responsive supramolecular assemblies can be produced to respond to the inherent features of tumor microenvironments, such as extracellular acidosis. We report in this framework the use of pH-responsive polymersomes (PSs) manufactured using poly([N-(2-hydroxypropyl)] methacrylamide)35-b-poly[2-(diisopropylamino)ethyl methacrylate]75 as the building unit (PHPMA35-b-PDPA75). The self-assemblies were produced with desired size towards long circulation time and tumor accumulation (hydrodynamic diameter - DH ~ 100 nm), and they could be successfully loaded with 10% w/w DOX (doxorubicin), while maintaining colloidal stability. The DOX loaded amount is presumably mainly burst-released at the acidic microenvironment of tumors thanks to the pH-switchable property of PDPA (pKa ~ 6.8), while reduced drug leakage has been monitored in pH 7.4. Compared to the administration of free DOX, the drug-loaded supramolecular structures greatly enhanced the therapeutic efficacy with effective growth inhibition of EL4 lymphoma tumor model and 100% survival rate in female C57BL/6 black mice over 40 days. The approach also led to reduced cardiotoxic effect. These features highlight the potential application of such nanotechnology-based treatment in a variety of cancer therapies where low local pH is commonly found, and emphasize PHPMA-based nanomedicines as an alternative to PEGylated formulations.

N-Alkylmorpholines: Potent Dermal and Transdermal Skin Permeation Enhancers.

Transdermal drug delivery is an attractive non-invasive method offering numerous advantages over the conventional routes of administration. The main obstacle to drug transport is, however, the powerful skin barrier that needs to be modulated, for example, by transdermal permeation enhancers. Unfortunately, there are still only a few enhancers showing optimum properties including low toxicity and reversibility of enhancing effects. For this reason, we investigated a series of new N-alkylmorpholines with various side chains as potential enhancers in an in vitro permeation study, using three model permeants (theophylline, indomethacin, diclofenac). Moreover, electrical impedance, transepidermal water loss, cellular toxicity and infrared spectroscopy measurements were applied to assess the effect of enhancers on skin integrity, reversibility, toxicity and enhancers' mode of action, respectively. Our results showed a bell-shaped relationship between the enhancing activity and the hydrocarbon chain length of the N-alkylmorpholines, with the most efficient derivatives having 10-14 carbons for both transdermal and dermal delivery. These structures were even more potent than the unsaturated oleyl derivative. The best results were obtained for indomethacin, where particularly the C10-14 derivatives showed significantly stronger effects than the traditional enhancer Azone. Further experiments revealed reversibility in the enhancing effect, acceptable toxicity and a mode of action based predominantly on interactions with stratum corneum lipids.

Does polysaccharide glycogen behave as a promoter of amyloid fibril formation at physiologically relevant concentrations?

We investigated the influence of glycogen (GG), phytoglycogen (PG), mannan (MAN) and cinnamoyl-modified GG (GG-CIN) on amyloid fibril formation. We used hen egg-white lysozyme (HEWL) as a model system and amyloid beta peptide (1-42) (Aß1-42) as an Alzheimer's disease-relevant system. For brief detection of fibrils was used thioflavin T (ThT) fluorescence assay and the results were confirmed by transmission electron microscopy (TEM). We also deal with the interaction of polysaccharides and HEWL with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). We found that all polysaccharides accelerated the formation of amyloid fibrils from both HEWL and Aß1-42. At high but physiologically relevant concentrations of GG, amyloid fibril formation was extremely accelerated for HEWL. Therefore, on the basis of the herein presented in vitro data, we hypothesize, that dietary d-glucose intake may influence amyloid fibril formation not only by influencing regulatory pathways, but also by direct glycogen-amyloid precursor protein molecular interaction, as glycogen levels in tissues are highly dependent on d-glucose intake.

Chemically modified glycogens: how they influence formation of amyloid fibrils?

The formation of amyloid fibrils from certain proteins stays behind a number of pathologies, so-called amyloidoses. Glycosaminoglycans are polysaccharides and are known natural constituents of amyloids in vivo. However, little is known about the effect of other naturally abundant polysaccharides, and even less is known about the effect of chemically modified polysaccharides on the formation of amyloid fibrils. In the case of low-molecular weight compounds, aromatic substances are known to often influence amyloid formation significantly. We investigated the influence of glycogen (GG) and several modifications of GG with cinnamoyl groups, benzoyl groups and phenylacetyl groups. As model systems, hen egg-white lysozyme (HEWL) and amyloid beta peptide (1-42) (Aß1-42), which is an Alzheimer disease-relevant system, were used. The fluorescence of thioflavin-T (ThT) was used for the rapid detection of fibrils, and the fluorescence results were confirmed by transmission electron microscopy (TEM). Other techniques, such as isothermal titration calorimetry (ITC) and dynamic light scattering (DLS), were employed to determine the interactions between HEWL and the modifications. We achieved similar results with both model systems (HEWL and Aß1-42). We showed that π-π interactions played an important role in the process of amyloid fibril formation because fundamental changes were observed in this process even with a very small number of groups containing an aromatic ring. It was found that almost all GG modifications accelerated the formation of amyloid fibrils in both model systems, HEWL and Aß1-42, except for GG-Ph1 (1.6 mol% phenylacetyl groups), which had a retarding effect compared to all other modifications.

Climate warming induced synchronous growth decline in Norway spruce populations across biogeographical gradients since 2000.

Covering large parts of Europe, Norway spruce (Picea abies L Karst.) plays an important role in the adaptation strategy of forest services to future climate change. Although dendroecology can provide valuable information on the past relationships between tree growth and climate, most previous studies were biased towards species-specific distribution limits, where old individuals grow slowly under extreme conditions. In the present study, we investigated the growth variability and climate sensitivity of 2851 Norway spruce trees along longitudinal (E 12-26°), latitudinal (N 45-51°), and elevation (118-1591 m a.s.l.) gradients in central-eastern Europe. We reveal that summer weather significantly affects the radial growth of spruce trees, but the effects strongly vary along biogeographical gradients. Extreme summer heatwaves in 2000 and 2003 reduced the growth rates by 10-35%, most pronounced in the southern Carpathians. In contrast to the population in the Czech Republic, climate warming induced a synchronous decline in the growth rates across biogeographical gradients in the Carpathian arc. By demonstrating the increased vulnerability of Norway spruce under warmer climate conditions, we recommended that the forest services and conservation managers replace or admix monocultures of this species with more drought-resilient mixtures including fir, beech and other broadleaved species.