A completely automated sample preparation instrument and consumable device for isolation and purification of nucleic acids

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.     

Noncovalent Functionalization of Pnictogen Surfaces: From Small Molecules to 2D Heterostructures

Beyond graphene, 2D pnictogen polymers are rapidly growing among the family of 2D materials. Due to their unique properties, this group has received considerable interest in recent years. Those properties include tunable electronic band gaps, high charge carrier mobility, and in‐plane anisotropic properties. This Review covers the noncovalent functionalization of pnictogen surfaces considering experimental and theoretical studies. Noncovalent functionalization is of great importance for effective modulation of the electronic structure of these materials as well as improvement of their stability toward surface oxidation. This Review highlights their noncovalent modification by organic molecules, in which enhanced surface stability of phosphorene and generated functionalized materials for applications in biomedical, supercapacitors, energy storage, and biosensors. Moreover, the noncovalent interactions with small molecules show its significance for sensing applications. Lastly, the interactions of pnictogen sheets with other 2D materials and their applications for van der Waals heterostructure formation are discussed. Current state‐of‐the‐art as well as future perspectives in this field are covered.     

(Un)expected Similarity of the Temporary Adhesive Systems of Marine,Brackish, and Freshwater Flatworms

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano, the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum, representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.     

A Human Pan-Cancer System Analysis of Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 3 (PLOD3)

The overexpression of the enzymes involved in the degradation of procollagen lysine is correlated with various tumor entities. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) expression was found to be correlated to the progression and migration of cancer cells in gastric, lung and prostate cancer. Here, we analyzed the gene expression, protein expression, and the clinical parameters of survival across 33 cancers based on the Clinical Proteomic Tumor Analysis Consortium (CPTAC), function annotation of the mammalian genome 5 (FANTOM5), Gene Expression Omnibus (GEO), Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA) and The Cancer Genome Atlas (TCGA) databases. Genetic alteration, immune infiltration and relevant cellular pathways were analyzed in detail. PLOD3 expression negatively correlated with survival periods and the infiltration level of CD8⁺ T cells, but positively correlated to the infiltration of cancer associated fibroblasts in diverse cancers. Immunohistochemistry in colon carcinomas, glioblastomas, and soft tissue sarcomas further confirm PLOD 3 expression in human cancer tissue. Moreover, amplification and mutation accounted for the largest proportion in esophageal adenocarcinoma and uterine corpus endometrial carcinoma, respectively; the copy number alteration of PLOD3 appeared in all cancers from TCGA; and molecular mechanisms further proved the effect of PLOD3 on tumorigenesis. In particular, PLOD3 expression appears to have a tumor immunological effect, and is related to multiple immune cells. Furthermore, it is also associated with tumor mutation burden and microsatellite instability in various tumors. PLOD3 acts as an inducer of various cancers, and it could be a potential biomarker for prognosis and targeted treatment.     

A quantitative multi-phase field model of polycrystalline alloy solidification

A multi-phase field model for quantitative simulations of polycrystalline solidification of binary alloys is introduced. During the free-growth stage of solidification, the model exploits the thin-interface analysis developed by Karma [3] in order to realistically capture bulk phase diffusion and the sharp interface corrections predicted by traditional models of solidification. During grain boundary coalescence, the model is constructed to reproduce the properties of repulsive grain boundaries described by Rappaz et al. [29]. The model provides a very simple mechanism for decoupling of solute and concentration fields at steady state, an important feature for calculating grain boundary energies.     

Solid Lipid Curcumin Particles Protect Medium Spiny Neuronal Morphology,and Reduce Learning and Memory Deficits in the YAC128 Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is a genetic neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by massive neuronal degeneration in the striatum. In this study, we utilized solid lipid curcumin particles (SLCPs) and solid lipid particles (SLPs) to test their efficacy in reducing deficits in YAC128 HD mice. Eleven-month-old YAC128 male and female mice were treated orally with SLCPs (100 mg/kg) or equivalent volumes of SLPs or vehicle (phosphate-buffered saline) every other day for eight weeks. Learning and memory performance was assessed using an active-avoidance task on week eight. The mice were euthanized, and their brains were processed using Golgi-Cox staining to study the morphology of medium spiny neurons (MSNs) and Western blots to quantify amounts of DARPP-32, brain-derived neurotrophic factor (BDNF), TrkB, synaptophysin, and PSD-95. We found that both SLCPs and SLPs improved learning and memory in HD mice, as measured by the active avoidance task. We also found that SLCP and SLP treatments preserved MSNs arborization and spinal density and modulated synaptic proteins. Our study shows that SLCPs, as well as the lipid particles, can have therapeutic effects in old YAC128 HD mice in terms of recovering from HD brain pathology and cognitive deficits.     

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement**

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.     

A context-aware middleware for real-time semantic enrichment of distributed multimedia metadata

This paper investigates the problem of the real-time integration and processing of multimedia metadata collected by a distributed sensor network. The discussed practical problem is the efficiency of the technologies used in creating a Knowledge Base in real-time. Specifically, an approach is proposed for the real-time, rule-based semantic enrichment of lower level context features with higher-level semantics. The distinguishing characteristic is the provision of an intelligent middleware-based architecture on which low level components such as sensors, feature extraction algorithms, data sources, and high level components such as application-specific ontologies can be plugged. Throughout the paper, Priamos, a middleware architecture based on Semantic Web technologies is presented, together with a stress-test of the system’s operation under two test case scenarios: A smart security surveillance application and a smart meeting room application. Performance measurements are conducted and corresponding results are exposed.     

Spacing characterization in Al–Cu alloys directionally solidified under transient growth conditions

We study spacing selection in directional solidification of Al–Cu alloys under transient growth conditions. New experimental results are presented which reveal that the mean dendritic spacing vs. solidification front speed exhibits plateau-like regions separated by regions of rapid change, consistent with previous experiments of Losert and co-workers. Quantitative phase-field simulations of directional solidification with dynamical growth conditions approximating those in the experiments confirm this behavior. The mechanism of this type of change in mean dendrite arm spacing is consistent with the notion that a driven periodically modulated interface must overcome an energy barrier before becoming unstable, in accord with a previous theory of Langer and co-workers.