Microfluidic flow cell for sequential digestion of immobilized proteoliposomes

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 μg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 μg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.     

Interaction of colloidal nanoparticles with their local environment: the (ionic) nanoenvironment around nanoparticles is different from bulk and determines the physico-chemical properties of the nanoparticles

The physico-chemical properties of colloidal nanoparticles (NPs) are influenced by their local environment, as, in turn, the local environment influences the physico-chemical properties of the NPs. In other words, the local environment around NPs has a profound impact on the NPs, and it is different from bulk due to interaction with the NP surface. So far, this important effect has not been addressed in a comprehensive way in the literature. The vicinity of NPs can be sensitively influenced by local ions and ligands, with effects already occurring at extremely low concentrations. NPs in the Hückel regime are more sensitive to fluctuations in the ionic environment, because of a larger Debye length. The local ion concentration hereby affects the colloidal stability of the NPs, as it is different from bulk owing to Debye Hückel screening caused by the charge of the NPs. This can have subtle effects, now caused by the environment to the performance of the NP, such as for example a buffering effect caused by surface reaction on ultrapure ligand-free nanogold, a size quenching effect in the presence of specific ions and a significant impact on fluorophore-labelled NPs acting as ion sensors. Thus, the aim of this review is to clarify and give an unifying view of the complex interplay between the NP''s surface with their nanoenvironment.     

Bouldering: an alternative strategy to long-vertical climbing in root-climbing hortensias

In the Neotropics, the genus Hydrangea of the popular ornamental hortensia family is represented by climbing species that strongly cling to their support surface by means of adhesive roots closely positioned along specialized anchoring stems. These root-climbing hortensia species belong to the nearly exclusive American Hydrangea section Cornidia and generally are long lianescent climbers that mostly flower and fructify high in the host tree canopy. The Mexican species Hydrangea seemannii, however, encompasses not only long lianescent climbers of large vertical rock walls and coniferous trees, but also short ‘shrub-like’ climbers on small rounded boulders. To investigate growth form plasticity in root-climbing hortensia species, we tested the hypothesis that support variability (e.g. differences in size and shape) promotes plastic responses observable at the mechanical, structural and anatomical level. Stem bending properties, architectural axis categorization, tissue organization and wood density were compared between boulder and long-vertical tree-climbers of H. seemannii. For comparison, the mechanical patterns of a closely related, strictly long-vertical tree-climbing species were investigated. Hydrangea seemannii has fine-tuned morphological, mechanical and anatomical responses to support variability suggesting the presence of two alternative root-climbing strategies that are optimized for their particular environmental conditions. Our results suggest that variation of some stem anatomical traits provides a buffering effect that regulates the mechanical and hydraulic demands of two distinct plant architectures. The adaptive value of observed plastic responses and the importance of considering growth form plasticity in evolutionary and conservation studies are discussed.     

Engineering magnetic nanostructures with inverse hysteresis loops

Top-down lithography techniques allow the fabrication of nanostructured elements with novel spin configurations,which provide a new route to engineer and manipulate the magnetic response of sensors and electronic devices and understand the role of fundamental interactions in materials science.In this study, shallow nanostructure-pattemed thin films were designed to present inverse magnetization curves,i.e.,an anomalous magnetic mechanism characterized by a negative coercivity and negative remanence.This procedure involved a method for manipulating the spin configuration that yielded a negative coercivity after the patterning of a single material layer.Patterned NiFe thin films with trench depths between 15％-25％ of the total film thickness exhibited inverse hysteresis loops for a wide angular range of the applied field and the trench axis.A model based on two exchange-coupled subsystems accounts for the experimental results and thus predicts the conditions for the appearance of this magnetic behavior.The findings of the study not only advance our understanding of patterning effects and confined magnetic systems but also enable the local design and control of the magnetic response of thin materials with potential use in sensor engineering.     

Microwave effect: analyzing citations from classic theories and their reinventions—a case study from a classic paper in aquatic ecology—Brooks & Dodson, 1965

Citations play an essential role in creating a knowledge network and recognizing relevant contributions during the process of scientific production. Despite the citations establishing the links between new evidence and the preceding ideas, classic articles may not be cited adequately. Our aim is to identify if classic studies are cited over time and if the recent studies are producing new knowledge or just “giving a new look” to pre-existing ideas. We evaluated whether the theory proposed by Brooks and Dodson (Science 150(3692): 28–35, 1965)-Size-efficiency Hypothesis was referenced in studies on the subject since its publication. Through the analysis of 1480 scientific papers, we quantified—from 1965 to 2018—the citation index (CI) of the original article considering the number of articles produced on the topic per year and the number of citations to other authors (intermediaries). We observed that 60% of the papers and 59% of the intermediaries do not refer to the original article. The CI was low and negatively affected by the age of the original article, showing that the frequency of citation was lower than the rate by which articles on the topic were published. There is a tendency to cite more recent articles and articles that corroborate their own findings. Our data illustrated the microwave effect, in which pre-existing ideas and theories are “reheated” by more recent articles where little of the original idea is modified. The microwave effect can create the impression of scientific advancement when there is little being added to the knowledge already produced.

     

High-Yield Separation of Extracellular Vesicles Using Programmable Zwitterionic Coacervates

Programmable coacervates based on zwitterionic polymers are designed as dynamic materials for ion exchange bioseparation. These coacervates are proposed as promising materials for the purification of soft nanoparticles such as liposomes and extracellular vesicles (EVs). It is shown that the stimulus-responsiveness of the coacervates and the recruitment of desired molecules can be independently programmed by polymer design. Moreover, the polymeric coacervates can recruit and release intact liposomes, human EVs, and nanoalgosomes in high yields and separate vesicles from different types of impurities, including proteins and nucleic acids. This approach combines the speed and simplicity of precipitation methods and the programmability of chromatography with the gentleness of aqueous two-phase separation, thereby guaranteeing product stability. This material represents a promising alternative for providing a low-shear, gentle, and selective purification method for EVs.     

Electroconductive Biohybrid Collagen/Pristine Graphene Composite Biomaterials with Enhanced Biological Activity

Electroconductive substrates are emerging as promising functional materials for biomedical applications. Here, the development of biohybrids of collagen and pristine graphene that effectively harness both the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching native cardiac tissue) obtainable with pristine graphene is reported. As well as improving substrate physical properties, the addition of pristine graphene also enhances human cardiac fibroblast growth while simultaneously inhibiting bacterial attachment (Staphylococcus aureus). When embryonic‐stem‐cell‐derived cardiomyocytes (ESC‐CMs) are cultured on the substrates, biohybrids containing 32 wt% graphene significantly increase metabolic activity and cross‐striated sarcomeric structures, indicative of the improved substrate suitability. By then applying electrical stimulation to these conductive biohybrid substrates, an enhancement of the alignment and maturation of the ESC‐CMs is achieved. While this in vitro work has clearly shown the potential of these materials to be translated for cardiac applications, it is proposed that these graphene‐based biohybrid platforms have potential for a myriad of other applications—particularly in electrically sensitive tissues, such as neural and neural and musculoskeletal tissues.     

Determination of cadmium at ultra-trace levels by CPE–molecular fluorescence combined methodology

A highly sensitive micelle-mediated extraction methodology for the preconcentration and determination of trace levels of cadmium by molecular fluorescence has been developed. Metal was complexed with o-phenanthroline (o-phen) and eosin (eo) at pH 7.6 in buffer Tris medium and quantitatively extracted into a small volume of surfactant-rich phase of PONPE 7.5 after centrifugating. The chemical variables affecting cloud point extraction (CPE) were evaluated and optimized. The RSD for six replicates of cadmium determinations at 0.84 μg L⁻¹ level was 1.17%. The linearity range using the preconcentration system was between 2.79 × 10⁻³ μg L⁻¹ and 2.81 μg L⁻¹ with a correlation coefficient of 0.99. Under the optimal conditions, it obtained a LOD of 8.38 × 10⁻⁴ μg L⁻¹ and LOQ of 2.79 × 10⁻³ μg L⁻¹. The method presented good sensitivity and selectivity and was applied to the determination of trace amounts of cadmium in commercially bottled mineral water, tap water and water well samples with satisfactory results. The proposed method is an innovative application of CPE-luminescence to metal analysis comparable in sensitivity and accuracy with atomic spectroscopies.     

Motivational interviewing in drug abuse services: A randomized trial.

Motivational interviewing (MI) is a directive, client-centered brief intervention to elicit behavior change by helping clients explore and resolve ambivalence. In this clinical trial, 152 outpatients and 56 inpatients entering public agencies for treatment of drug problems were randomly assigned to receive or not receive a single session of manual-guided MI. Drug use was assessed by self-report, urine toxicology, and collateral reports from significant others at baseline, 3, 6, 9, and 12 months. Contrary to prior reports, MI showed no effect on drug use outcomes when added to inpatient or outpatient treatment, although both groups showed substantial increases in abstinence from illicit drugs and alcohol. (PsycINFO Database Record (c) 2010 APA, all rights reserved)