Rapid Exchange of Stably Bound Protein and DNA Cargo on a DNA Origami Receptor.

Biomolecular complexes can form stable assemblies yet can also rapidly exchange their subunits to adapt to environmental changes. Simultaneously allowing for both stability and rapid exchange expands the functional capacity of biomolecular machines and enables continuous function while navigating a complex molecular world. Inspired by biology, we design and synthesize a DNA origami receptor that exploits multivalent interactions to form stable complexes that are also capable of rapid subunit exchange. The system utilizes a mechanism first outlined in the context of the DNA replisome, known as multisite competitive exchange, and achieves a large separation of time scales between spontaneous subunit dissociation, which requires days, and rapid subunit exchange, which occurs in minutes. In addition, we use the DNA origami receptor to demonstrate stable interactions with rapid exchange of both DNA and protein subunits, thus highlighting the applicability of our approach to arbitrary molecular cargo, an important distinction with canonical toehold exchange between single-stranded DNA. We expect this study to benefit future studies that use DNA origami structures to exploit multivalent interactions for the design and synthesis of a wide range of possible kinetic behaviors.

Single-molecule detection on a portable 3D-printed microscope.

Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson's disease, with an improved sensitivity of >100,000-fold over bulk measurements.

Kistimonas alittae sp. nov., a gammaproteobacterium isolated from the marine annelid Alitta succinea.

A novel Gram-stain-negative, rod-shaped, motile, non-spore-forming, facultatively anaerobic marine bacterium was isolated from the gastrointestinal tract of the sandworm Alitta succinea collected from Grice Cove, South Carolina, USA. The strain was arginine dihydrolase-positive, and oxidase- and catalase-positive. Growth occurred between 10 and 37 °C, with optimal growth occurring between 30 and 32 °C. Comparative 16S rRNA gene sequence analysis showed its nearest neighbours are members of the genus Kistimonas of the family Hahellaceae, which is found in the order Oceanospirillales, class Gammaproteobacteria. The closest related species was Kistimonas asteriae KMD 001T with 16S rRNA gene sequence similarity of 99.0 %. However, DNA-DNA hybridization between these strains revealed less than 70 % DNA-DNA relatedness, supporting the novel species status of the strain. The major fatty acids were C16 : 0, C18 : 0, C18 : 1ω7c and a summed feature that contained C16 : 1ω6c/C16 : 1ω7c. The major respiratory quinone was ubiquinone-9 and the predominant polar lipids were phosphatidylserine, phosphoethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content was 52.5 mol%. Based on the data presented, strain BGP-2T is considered to represent a novel member of the genus Kistimonas, for which the name Kistimonas alittae sp. nov. is proposed. The type strain is BGP-2T (=CCUG 65711T=JCM 30010T).

Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88.

BACKGROUND: Higher-order self-assembly of proteins, or "prion-like" polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level. RESULTS: We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88. CONCLUSION: We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital "all-or-none" responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.

Kinetic barriers to α-synuclein protofilament formation and conversion into mature fibrils.

Oligomeric and protofibrillar aggregates that are populated along the pathway of amyloid fibril formation appear generally to be more toxic than the mature fibrillar state. In particular, α-synuclein, the protein associated with Parkinson's disease, forms kinetically trapped protofibrils in the presence of lipid vesicles. Here, we show that lipid-induced α-synuclein protofibrils can convert rapidly to mature fibrils at higher temperatures. Furthermore, we find that ß-synuclein, generally considered less aggregation prone than α-synuclein, forms protofibrils at higher temperatures. These findings highlight the importance of energy barriers in controlling the de novo formation and conversion of amyloid fibrils.

The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity.

Proteostasis, or protein homeostasis, encompasses the maintenance of the conformational and functional integrity of the proteome and involves an integrated network of cellular pathways. Molecular chaperones, such as the small heat shock proteins (sHsps), are key elements of the proteostasis network that have crucial roles in inhibiting the aggregation of misfolded proteins. Failure of the proteostasis network can lead to the accumulation of misfolded proteins into intracellular and extracellular deposits. Deposits containing fibrillar forms of α-synuclein (α-syn) are characteristic of neurodegenerative disorders including Parkinson's disease and dementia with Lewy bodies. Here we show that the sHsp Hsp27 (HSPB1) binds to α-syn fibrils, inhibiting fibril growth by preventing elongation. Using total internal reflection fluorescence (TIRF)-based imaging methods, we show that Hsp27 binds along the surface of α-syn fibrils, decreasing their hydrophobicity. Binding of Hsp27 also inhibits cytotoxicity of α-syn fibrils. Our results demonstrate that the ability of sHsps, such as Hsp27, to bind fibrils represents an important mechanism through which they may mitigate cellular toxicity associated with aberrant protein aggregation. Fibril binding may represent a generic mechanism by which chaperone-active sHsps interact with aggregation-prone proteins, highlighting the potential to target sHsp activity to prevent or disrupt the onset and progression of α-syn aggregation associated with α-synucleinopathies.

ß-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces.

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson's disease through the processes involved in the formation of amyloid fibrils. α and ß-synuclein are homologous proteins found at comparable levels in presynaptic terminals but ß-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and ß-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that ß-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that ß-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of α-synuclein.

Intracellular α-synuclein deposits, known as Lewy bodies, have been linked to a range of neurodegenerative disorders, including Parkinson's disease. α-Synuclein binds to synthetic and biological lipids, and this interaction has been shown to play a crucial role for both α-synuclein's native function, including synaptic plasticity, and the initiation of its aggregation. Here, we describe the interplay between the lipid properties and the lipid binding and aggregation propensity of α-synuclein. In particular, we have observed that the binding of α-synuclein to model membranes is much stronger when the latter is in the fluid rather than the gel phase, and that this binding induces a segregation of the lipids into protein-poor and protein-rich populations. In addition, α-synuclein was found to aggregate at detectable rates only when interacting with membranes composed of the most soluble lipids investigated here. Overall, our results show that the chemical properties of lipids determine whether or not the lipids can trigger the aggregation of α-synuclein, thus affecting the balance between functional and aberrant behavior of the protein.

Modeling the Thermoproteaceae RNase P RNA.

The RNA component of the RNase P complex is found throughout most branches of the tree of life and is principally responsible for removing the 5' leader sequence from pre-tRNA transcripts during tRNA maturation. RNase P RNA has a number of universal core features, however variations in sequence and structure found in homologs across the tree of life require multiple Rfam covariance search models to detect accurately. We describe a new Rfam search model to enable efficient detection of the diminutive archaeal Type T RNase P RNAs, which are missed by existing Rfam models. Using the new model, we establish effective score detection thresholds, and detect four new RNase P RNA genes in recently completed genomes from the crenarchaeal family Thermoproteaceae.

Discovery of a minimal form of RNase P in Pyrobaculum.

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5' leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533-1540]. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5' tRNA leaders. The "missing" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5' pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5' tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.

The small nucleolar ribonucleoprotein (snoRNP) database.

Small nucleolar ribonucleoproteins (snoRNPs) are widely studied and characterized as guide RNAs for sequence-specific 2'-O-ribose methylation and psuedouridylation of ribosomal RNAs. In addition, snoRNAs have also been shown to interact with some tRNAs and direct alternative splicing in mRNA biogenesis. Recent advances in bioinformatics have resulted in new algorithms able to rapidly identify noncoding RNAs generally and snoRNAs specifically in genomic and metagenomic sequences, resulting in a rapid increase in the number and diversity of identified snoRNA sequences. The snoRNP database is a web-based collection of snoRNA and snoRNA-associated protein sequences from a wide range of species. The database currently contains 8994 snoRNA sequences from Bacteria, Archaea, and Eukaryotes and 589 snoRNA-associated protein sequences. The snoRNP database can be found at: http://evolveathome.com/snoRNA/snoRNA.php.

The RNase P family.

Ribonuclease P (RNase P) is a ribonucleoprotein comprised of a catalytic RNA subunit and one or several protein subunits. RNase P is best known for its role in 5'-processing of tRNA precursors. RNase P enzymes from almost all forms of life, including protein-synthesizing organelles, contain an RNase P with a conserved, homologous RNA. Five distinct structure classes of RNase P RNAs have been identified in bacteria and archaea; eukaryotic RNase P RNAs are not yet sufficiently well surveyed for structure classes to be defined. Here we will examine the structure variations in RNase P RNAs in bacteria, archaea, eukaryotes, plastids and mitochondria with special emphasis on the functional roles these unique secondary structures perform.

The RNA structure alignment ontology.

Multiple sequence alignments are powerful tools for understanding the structures, functions, and evolutionary histories of linear biological macromolecules (DNA, RNA, and proteins), and for finding homologs in sequence databases. We address several ontological issues related to RNA sequence alignments that are informed by structure. Multiple sequence alignments are usually shown as two-dimensional (2D) matrices, with rows representing individual sequences, and columns identifying nucleotides from different sequences that correspond structurally, functionally, and/or evolutionarily. However, the requirement that sequences and structures correspond nucleotide-by-nucleotide is unrealistic and hinders representation of important biological relationships. High-throughput sequencing efforts are also rapidly making 2D alignments unmanageable because of vertical and horizontal expansion as more sequences are added. Solving the shortcomings of traditional RNA sequence alignments requires explicit annotation of the meaning of each relationship within the alignment. We introduce the notion of "correspondence," which is an equivalence relation between RNA elements in sets of sequences as the basis of an RNA alignment ontology. The purpose of this ontology is twofold: first, to enable the development of new representations of RNA data and of software tools that resolve the expansion problems with current RNA sequence alignments, and second, to facilitate the integration of sequence data with secondary and three-dimensional structural information, as well as other experimental information, to create simultaneously more accurate and more exploitable RNA alignments.

Characterization of diazotrophs containing Mo-independent nitrogenases, isolated from diverse natural environments.

Molybdenum-independent nitrogenases were first described in the nitrogen-fixing bacterium Azotobacter vinelandii and have since been described in other diazotrophic bacteria. Previously, we reported the isolation of seven diazotrophs with Mo-independent nitrogenases from aquatic environments. In the present study, we extend these results to include diazotrophs isolated from wood chip mulch, soil, "paraffin dirt," and sediments from mangrove swamps. Mo-deficient, N-free media under both aerobic and anaerobic conditions were used for the isolations. A total of 26 isolates were genetically and physiologically characterized. Their phylogenetic placement was determined using 16S rRNA gene sequence analysis. Most of the isolates are members of the gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria. Two other isolates, AN1 and LPF4, are closely related to Enterobacter spp. and Paenibacillus spp., respectively. PCR and/or Southern hybridization were used to detect the presence of nitrogenase genes in the isolates. PCR amplification of vnfG and anfG was used to detect the genetic potential for the expression of the vanadium-containing nitrogenase and the iron-only nitrogenase in the isolates. This study demonstrates that diazotrophs with Mo-independent nitrogenases can be readily isolated from diverse natural environments.

Is Alba an RNase P subunit?

It has been suggested that Alba, a well-established chromatin protein in Archaea, is also a subunit of the archaeal RNase P holoenzyme, based on the observation that the homolog of this protein in humans has been shown to be associated with RNase P activity. Using the same biochemical methods we used previously to show that four other proteins homologous to eukaryotic RNase P proteins are bona fide RNase P subunits in Archaea, we could not detect any association of the Alba homolog in Methanothermobacter thermoautotrophicus (Mth1483p) with the RNase P holoenzyme. In addition, the presence of Mth1483p did not enhance the activity of RNase P holoenzyme reconstituted from recombinant subunits. In conclusion, we find no evidence that Alba is an RNase P subunit.

The RNA Ontology Consortium: an open invitation to the RNA community.

The aim of the RNA Ontology Consortium (ROC) is to create an integrated conceptual framework-an RNA Ontology (RO)-with a common, dynamic, controlled, and structured vocabulary to describe and characterize RNA sequences, secondary structures, three-dimensional structures, and dynamics pertaining to RNA function. The RO should produce tools for clear communication about RNA structure and function for multiple uses, including the integration of RNA electronic resources into the Semantic Web. These tools should allow the accurate description in computer-interpretable form of the coupling between RNA architecture, function, and evolution. The purposes for creating the RO are, therefore, (1) to integrate sequence and structural databases; (2) to allow different computational tools to interoperate; (3) to create powerful software tools that bring advanced computational methods to the bench scientist; and (4) to facilitate precise searches for all relevant information pertaining to RNA. For example, one initial objective of the ROC is to define, identify, and classify RNA structural motifs described in the literature or appearing in databases and to agree on a computer-interpretable definition for each of these motifs. To achieve these aims, the ROC will foster communication and promote collaboration among RNA scientists by coordinating frequent face-to-face workshops to discuss, debate, and resolve difficult conceptual issues. These meeting opportunities will create new directions at various levels of RNA research. The ROC will work closely with the PDB/NDB structural databases and the Gene, Sequence, and Open Biomedical Ontology Consortia to integrate the RO with existing biological ontologies to extend existing content while maintaining interoperability.

Radiopacity of nonmetallic root canal posts.

PURPOSE: This study determined the radiopacity of a group of nonmetallic posts. MATERIALS AND METHODS: Four specimens were cut from 7 posts, and tooth sections were cut from extracted teeth. Radiographic images of all specimens along with an aluminum step wedge were obtained on occlusal films. Optical density readings for each specimen image were determined with a transmission densitometer. Radiopacity values were subsequently calculated as equivalents of aluminum thickness. RESULTS: Analysis of variance revealed significant differences in radiopacity values among the posts (P < .001). One nonmetallic post that was made of zirconium had a radiopacity value significantly greater than that of enamel. Another, made of glass fibers, acrylic resin, and fillers, had a radiopacity that was greater than that of dentin but smaller than that of enamel. The remaining 3 nonmetallic posts had radiopacity values lower than dentin. CONCLUSION: The 3 posts with radiopacity that was lower than dentin cannot be considered sufficiently radiopaque.

Structural implications of novel diversity in eucaryal RNase P RNA.

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.

Bartonella quintana in cynomolgus monkey (Macaca fascicularis).

We identified a Bartonella quintana strain by polymerase chain reaction amplification, cloning, and sequencing of DNA extracted from lysed erythrocytes and cultured colonies grown from peripheral blood collected from a captive-bred cynomolgus monkey (Macaca fascicularis). This report describes naturally acquired B. quintana infection in a nonhuman primate.