Additional Functional Elements Research Articles

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing

Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis.

Minor structural changes, major functional impacts: posttranslational modifications and drug targets.

Posttranslational modifications (PTMs) are essential mechanisms that provide chemical diversity to proteins. The additional functional and structural elements can be introduced to exceed the primary amino acid composition. PTMs impact key biological and physiological processes including cell signaling, metabolism, protein degradation and influences interactions with other macromolecules. However, characterization of the structural and functional signatures of modified proteins has been historically limited. Since defects in PTMs are linked to numerous disorders and diseases, PTMs and their modifying enzymes are considered as potential drug targets. This has fueled new initiatives to determine how PTMs affect protein structure and function. In this review, I summarize some of the major, well-studied protein PTMs and related drug targets. Since PTMs are widely used for therapeutic targets or disease markers, highlighting structural changes after PTM provides new frontiers in understanding the detailed mechanism and related drug developments.

An asymmetric flow-focusing droplet generator promotes rapid mixing of reagents

Nowadays droplet microfluidics is widely used to perform high throughput assays and for the synthesis of micro- and nanoparticles. These applications usually require packaging several reagents into droplets and their mixing to start a biochemical reaction. For rapid mixing microfluidic devices usually require additional functional elements that make their designs more complex. Here we perform a series of 2D numerical simulations, followed by experimental studies, and introduce a novel asymmetric flow-focusing droplet generator, which enhances mixing during droplet formation due to a 2D or 3D asymmetric vortex, located in the droplet formation area of the microfluidic device. Our results suggest that 2D numerical simulations can be used for qualitative analysis of two-phase flows and droplet generation process in quasi-two-dimensional devices, while the relative simplicity of such simulations allows them to be easily applied to fairly complicated microfluidic geometries. Mixing inside droplets formed in the asymmetric generator occurs up to six times faster than in a conventional symmetric one. The best mixing efficiency is achieved in a specific range of droplet volumes, which can be changed by scaling the geometry of the device. Thus, the droplet generator suggested here can significantly simplify designs of microfluidic devices because it enables both the droplet formation and fast mixing of the reagents within droplets. Moreover, it can be used to precisely estimate reaction kinetics.

Molecular Dynamics Simulations of Mitochondrial Uncoupling Protein 2.

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

Modified Metal–Organic Frameworks as Efficient Catalysts for Lignocellulosic Biomass Conversion

Biomass, a promising replacement for fossil fuels, can be used to produce eco‐friendly liquid fuels and chemicals. Various studies are investigating catalysts for lignocellulosic biomass conversion to valuable chemicals and fuels. Metal–organic frameworks (MOFs) are important catalysts because of their well‐ordered porous structures and large surface areas. Although MOFs can be applied directly, four modification strategies can be used to alter their catalytic properties and improve catalytic performance. In the first strategy, coordinatively unsaturated sites are created by changing the bonding state of the metal node. In the second approach, organic linkers with additional functional groups or active elements are used. In the third strategy, MOFs and other active elements are combined. In the final approach, MOFs are carbonized to produce carbon‐supported metal catalysts. We review the applications of modified MOFs for the catalytic conversion of biomass derivatives and discuss the factors that contribute to their improved catalytic performance.

Mandible Aseptic Osteonecrosis Caused by admission of the Narcotic Substances Containing Aminophosphonic Impurities: Levelling with Trilon B

The aim of our study was to correct osteosclerotic changes (aseptic osteonecrotic lesion) of mandibular bones in laboratory white rats. Changes were caused by 1 and 3 monthly intragastric administration of amino phosphonic impurity. It was formed during artisanal manufacture of meta-amphetamine. A dose was 63 mg/kg 1 time per day, and followed by exposure to 5% water solution of Trilon B at a dose of 250 mg/kg. Mandible structure was researched by light microscopy. Three additional functional indices and elements of node-strut analysis were enrolled. Indices and their abbreviations were used in accordance to Committee on Histomorphometry Nomenclature (ASBMR).
 It was found that intragastric administration of Trilon B at a dose of 250 mg/kg after one-month- and three-month influence of amino phosphonic impurity (by-product of methamphetamine artisanal synthesis) at a dose of 63 mg/kg led to improvements of mandible bone remodeling. Also, enhancement of processes that removed osteosclerotic changes as well as an involution of myelofibrosis took place.

Design norms and codes for educational facilities of a new format

ntroduction. The issue of early occupational guidance of children and adolescents is associated with a creation of a new educational environment and the necessity for design of new and refurbishment of existing facilities of extended education (EE) and further vocational education (FVE). The architectural shaping of the educational environment requires consideration of numerous factors, including the arrangement of accessible environment for people with limited mobility (PLM) to provide for development and education of handicapped students. Materials and methods. An analysis of current enactments and design codes for education facilities was made. Based on polling conducted by the authors among teachers, psychologists, speech pathologists and students, non-compliances of the existing functional groups of premises and their dimensions with contemporary methods of education and teaching were found out. By analysis of domestic and foreign design practice, additional functional layout elements (FLEs) were determined. Results. Scientifically based proposals were given, as to the selection of additional FLEs, links were identified to other functional groups and their basic specifications, also proposals were made as to the alteration of regulatory requirements to FLEs. Conclusions. A hypothesis of the demand for new functional areas of education facilities (FLEs of studio spaces) was confirmed. Amendments were proposed to the existing regulatory design basis of EE and FVE facilities, as well as recommendations for issuing of technical design assignments for architects and designers. The scientific novelty comprises new design principles of the architectural environment of EE and FVE facilities under recognition of the revealed factors based on the studies of a range of requirements to the shaping of education facilities of the new type.

Theory and design of Schwarzschild scan objective for Optical Coherence Tomography.

Optical coherence elastography (OCE) is one form of multi-channel imaging that combines high-resolution optical coherence tomography (OCT) imaging with mechanical tissue stimulation. This combination of structural and functional imaging can require additional space to integrate imaging capabilities with additional functional elements (e.g., optical, mechanical, or acoustic modulators) either at or near the imaging axis. We address this challenge by designing a novel scan lens based on a modified Schwarzchild objective lens, comprised of a pair of concentric mirrors with potential space to incorporate additional functional elements and minimal compromise to the available scan field. This scan objective design allows perpendicular tissue-excitation and response recording. The optimized scan lens design results in a working distance that is extended to ~140 mm (nearly 2x the focal length), an expanded central space suitable for additional functional elements (>15 mm in diameter) and diffraction-limited lateral resolution (19.33 μm) across a full annular scan field ~ ± 7.5 mm to ± 12.7 mm.

Elaborating a coiled-coil-assembled octahedral protein cage with additional protein domains.

De novo design of protein nano-cages has potential applications in medicine, synthetic biology, and materials science. We recently developed a modular, symmetry-based strategy for protein assembly in which short, coiled-coil sequences mediate the assembly of a protein building block into a cage. The geometry of the cage is specified by the combination of rotational symmetries associated with the coiled-coil and protein building block. We have used this approach to design well-defined octahedral and tetrahedral cages. Here, we show that the cages can be further elaborated and functionalized by the addition of another protein domain to the free end of the coiled-coil: in this case by fusing maltose-binding protein to an octahedral protein cage to produce a structure with a designed molecular weight of ~1.8 MDa. Importantly, the addition of the maltose binding protein domain dramatically improved the efficiency of assembly, resulting in ~ 60-fold greater yield of purified protein compared to the original cage design. This study shows the potential of using small, coiled-coil motifs as off-the-shelf components to design MDa-sized protein cages to which additional structural or functional elements can be added in a modular manner.

Microfluidic Generation of Bioinspired Spindle-knotted Graphene Microfibers for Oil Absorption.

Graphene materials have a demonstrated value in water treatment. Efforts to promote these materials are focused on the generation of functional graphene adsorbents for effectively removing contaminants from water. Here, inspired by the conformation of spider silks, we present a novel graphene microfiber material with spindle-knotted microstructures by using a microfluidic emulsification and spinning collaborative technology. The size and spacing of the spindle-knots were highly controllable by adjusting the flow rates of microfluidics during the generation process of the microfibers. The generated microfibers could adsorb oil from a water-oil mixed environment due to their hydrophobic surface chemistry. Because of the surface energy curvature gradient and the difference in Laplace pressure, the collected oil tended to form droplets and move from joints between the spindle-knots to the knots. In addition, by encapsulating additional functional elements, such as magnetic nanoparticles, the graphene microfiber with the ability to control and facilitate the collection of oily contaminants can also be achieved. These features greatly prove the promising values of the spindle-knotted graphene microfibers in the protection of the environment.

Combinatorial Optimization of Sequence-Defined Oligo(ethanamino)amides for Folate Receptor-Targeted pDNA and siRNA Delivery

Cationic polymers present a versatile platform for the nonviral delivery of therapeutic nucleic acids. In order to achieve effective nucleic acid transfer, polymeric carriers ought to comprise multiple functionalities. Precise chemistries for site-specific placements of the different delivery modules within the carriers present the basis for uncovering structure-activity relationships required for further optimization. Here we present the design and systematic evaluation of a library of 42 sequence-defined oligo(ethanamino)amides generated by solid-phase assisted syntheses. The carriers contained two- or four-arm topologies of different artificial oligoamino acid domains for nucleic acid complexation, terminated by cysteines for disulfide-triggered polyplex stabilization, linked with monodisperse polyethylene glycol (PEG) for surface shielding and terminal folic acid for receptor specific cellular uptake. Additional functional elements included histidines for endosomal escape and/or tyrosine trimers for enhanced hydrophobic polyplex stabilization. In vitro screening of the oligomer library identified a folate-PEG-linked two-arm oligocation structure comprising histidines and tyrosine trimers as the most effective class of carriers for the delivery of pDNA and siRNA.

Experimental and Numerical Studies on Integration Thermoplastic Composite Compatible Piezoelectric Ceramics for Actuatory Application of Cylindrical Hollow Structures

Regarding the economical use of fiber-reinforced plastics (FRP) as a construction material for structural components whose additional value caused not only in their high specific mechanical properties. Due to the layerwise structure definition of continuous fiber reinforced composites the corresponding production technologies offers a high potential for integration of additional functional elements. Past efforts to the integration of functions in fiber-reinforced composites usual provide in front of a passive use of the piezoelectric effect (eg. structural-health monitoring). Through efficient and structurally defined using of piezoceramic actuators, the planar structure topology of cylindrical hollow FRP profiles can be actively influenced. Based on experimental studies on the definition of basic concepts for the integration of thermoplastic compatible piezoceramic modules (TPM) in fiber composite tubular segments, this paper deals with the understanding and performance capabilities of such actuarical hollow frp structures. The selective excitation and manipulation of the vibration behavior of such rotationally symmetric structures serves for generation of wave effects with radial translational characteristic. The performed experimental studies on the structural behavior of active piezo integral pipe segments are abstracted and compared by means of numerical simulations using multi-physical elements.

Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes.

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.

Technology Pushed Process and Product Innovation – Joining by Linear Flow Splitting

In order to take full advantage of manufacturing technologies, designers have to use technology specific manufacturing-induced properties. An adequate selection and optimisation of the manufacturing technology and a consequent combination of product and process development can lead to product innovations. Linear flow splitting yields ultra fine grained (UFG) microstructures which are characterised by high hardness, ductility and fatigue strength. These manufacturing-induced properties are particularly suited for rolling contact areas of linear guiding systems. Multi-functional linear systems require an integration of additional functional elements. For the hereby necessary joining, using the mechanisms acting in the linear flow splitting process seems attractive. The paper introduces a mechanical joining process following this technology push approach. Thereby, manufacturing-induced properties and mechanisms induced during the linear flow splitting process are utilised to integrate additional functional elements. Necessary joining operations can be integrated into the continuous flow production, maintaining the technological potential to manufacture large quantities at low costs. Extensive experiments show the applicability of various joined elements and materials for process integrated mechanical joining. The potential for product innovations is illustrated by an innovative linear system. Its core component is an integrated rack as part of a rack and pinion drive of a facade cleaner.

Vibration roller as an automatic system for soil density equalization

A soil cultivating roller equipped with a number of additional functional elements is developed, allowing to transform a passive roller into the monitoring system of soil density automatic regulation.

Decoding the human genome

Interpreting the human genome sequence is one of the major scientific endeavors of our time. In February 2001, when the human genome reference sequence was initially released (Lander et al. 2001), our understanding of the encoded contents was surprisingly limited. It was perplexing to many in the scientific community when we realized that the human genome contains only ∼21,000 distinct protein-coding genes (Claverie 2001; Hollon 2001; Pennisi 2003; Clamp et al. 2007), as other less complex species like the nematode Caenorhabditis elegans were known to have a similar number of protein-coding genes (Hillier et al. 2005). It quickly became apparent that the developmental and physiological complexity of humans would not be explained solely by the number of protein-coding genes, and the quest to understand the contents of the human genome began full force. The Encyclopedia of DNA Elements (ENCODE) Project was launched in September of 2003 with the daunting task of identifying all the functional elements encoded in the human genome sequence. To accomplish this task, the National Human Genome Research Institute (NHGRI) organized The ENCODE Project Consortium, which consists of an international group of scientists with diverse expertise in experimental and computational methods for generating and analyzing high-throughput genomic data (The ENCODE Project Consortium 2004). During the initial four years, the consortium conducted a pilot project which focused on annotating functional elements in a defined 1% of the human genome consisting of ∼30 Mb divided among 44 genomic regions. On June 14, 2007, a report summarizing the findings of the pilot project revealed pervasive transcription of the human genome, with the majority of nucleotides represented in transcripts in at least a limited number of cell types at some time (The ENCODE Project Consortium 2007). Many of these transcripts comprised novel noncoding RNA genes. Importantly, The ENCODE Pilot Project assigned function to 60% of the evolutionarily constrained bases in the 44 genomic regions and identified many additional functional elements seemingly unconstrained across mammalian evolution. Integration of the various experimental data generated by The ENCODE Pilot Project provided further insights into connections between chromatin structure (modifications and accessibility) and gene expression (The ENCODE Project Consortium 2007; Koch et al. 2007; Thurman et al. 2007; Zhang et al. 2007) and the timing of replication (Karnani et al. 2007). Armed with increased knowledge about the types of functional elements contained within the human genome sequence and with the advent of massively parallel sequencing, in 2007 the ENCODE project was expanded to study the entire human genome. This month, Nature published a paper entitled “An integrated encyclopedia of DNA elements in the human genome,” which reports the production and initial analysis of 1640 data sets focused on two major classes of annotations: genes (both coding and noncoding) along with their corresponding RNA transcripts, and transcriptional regulatory regions. This paper (The ENCODE Project Consortium 2012), along with companion papers in Nature, Genome Research, and Genome Biology, provides much more than a mere inventory of sequence elements but rather presents an integrated analysis providing important insights into the functional organization of the human genome. In alignment with the tradition of large consortia sponsored by the NHGRI, the ENCODE project has made all data and derived results available through a freely accessible database (Rosenbloom et al. 2010). The following sections describe some of the highlights of the ENCODE project, including technical accomplishments, high quality data sets, and integrated analyses with other resources, such as disease-associated variants identified through genome-wide association studies.

Comparative and Joint Analysis of Two Metagenomic Datasets from a Biogas Fermenter Obtained by 454-Pyrosequencing

Biogas production from renewable resources is attracting increased attention as an alternative energy source due to the limited availability of traditional fossil fuels. Many countries are promoting the use of alternative energy sources for sustainable energy production. In this study, a metagenome from a production-scale biogas fermenter was analysed employing Roche's GS FLX Titanium technology and compared to a previous dataset obtained from the same community DNA sample that was sequenced on the GS FLX platform. Taxonomic profiling based on 16S rRNA-specific sequences and an Environmental Gene Tag (EGT) analysis employing CARMA demonstrated that both approaches benefit from the longer read lengths obtained on the Titanium platform. Results confirmed Clostridia as the most prevalent taxonomic class, whereas species of the order Methanomicrobiales are dominant among methanogenic Archaea. However, the analyses also identified additional taxa that were missed by the previous study, including members of the genera Streptococcus, Acetivibrio, Garciella, Tissierella, and Gelria, which might also play a role in the fermentation process leading to the formation of methane. Taking advantage of the CARMA feature to correlate taxonomic information of sequences with their assigned functions, it appeared that Firmicutes, followed by Bacteroidetes and Proteobacteria, dominate within the functional context of polysaccharide degradation whereas Methanomicrobiales represent the most abundant taxonomic group responsible for methane production. Clostridia is the most important class involved in the reductive CoA pathway (Wood-Ljungdahl pathway) that is characteristic for acetogenesis. Based on binning of 16S rRNA-specific sequences allocated to the dominant genus Methanoculleus, it could be shown that this genus is represented by several different species. Phylogenetic analysis of these sequences placed them in close proximity to the hydrogenotrophic methanogen Methanoculleus bourgensis. While rarefaction analyses still indicate incomplete coverage, examination of the GS FLX Titanium dataset resulted in the identification of additional genera and functional elements, providing a far more complete coverage of the community involved in anaerobic fermentative pathways leading to methane formation.

Human genetic variation recognizes functional elements in noncoding sequence

Noncoding DNA, particularly intronic DNA, harbors important functional elements that affect gene expression and RNA splicing. Yet, it is unclear which specific noncoding sites are essential for gene function and regulation. To identify functional elements in noncoding DNA, we characterized genetic variation within introns using ethnically diverse human polymorphism data from three public databases-PMT, NIEHS, and SeattleSNPs. We demonstrate that positions within introns corresponding to known functional elements involved in pre-mRNA splicing, including the branch site, splice sites, and polypyrimidine tract show reduced levels of genetic variation. Additionally, we observed regions of reduced genetic variation that are candidates for distance-dependent localization sites of functional elements, possibly intronic splicing enhancers (ISEs). Using several bioinformatics approaches, we provide additional evidence that supports our hypotheses that these regions correspond to ISEs. We conclude that studies of genetic variation can successfully discriminate and identify functional elements in noncoding regions. As more noncoding sequence data become available, the methods employed here can be utilized to identify additional functional elements in the human genome and provide possible explanations for phenotypic associations.

Elements of a cybernetic epistemology: Design rules for complex goal‐orientated systems

AbstractFeedback systems have often been considered as relevant for an understanding of the brain. But obviously most biological systems have a much more complicated structure. Surprisingly the functional and structural necessities determining the route from simple feedback systems to complex goal‐orientated systems like the brain have never been investigated. Therefore this paper studies systematically the possibilities for adding functional elements like sensors, memory, effectors etc. to a feedback system. Then it investigates which combinations of functional elements are necessary to get to working goal‐orientated systems. Based on that necessities and options of data organization and the resulting cognitive possibilities are analysed. This abstract analysis delivers design rules for complex—technical and biological—goal‐orientated systems and basic epistemological facts how additional functional elements can add to their cognitive abilities. Finally, a ‘design for a simple brain’ illustrates the application of these design rules. Copyright © 2009 John Wiley & Sons, Ltd.

Mutations Causing DOK7 Congenital Myasthenia Ablate Functional Motifs in Dok-7

Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.