Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.

BACKGROUND: The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. RESULTS: Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. CONCLUSION: We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

One century after: a reappraisal of the gnathos (sensu Pierce, 1914) in Larentiinae (Lepidoptera: Geometridae).

One century ago, F.N. Pierce in his well-known monograph on the genitalia of Geometridae (Lepidoptera), divided the family into two major subdivisions, the Gnathoi and the Agnathoi, depending on the presence or absence of the gnathos in males. In his study, Pierce assigned the Larentiinae to the Agnathoi based on the apparent absence of the gnathos in this subfamily. A re-examination of the male genitalic characters of numerous larentiine species representing 14 different tribes provided, contrary to Pierce's results, evidence for the presence of the gnathos in Larentiinae. Illustrations of the gnathos (or its remnants) in male genitalia of selected species are provided and the value of the uncus and gnathos for inferring phylogenetic relationships is discussed.

Determination of organ-specific anemia tolerance.

OBJECTIVE: Utilization of anemia tolerance reduces the need for and risks of perioperative transfusion. Recent publications indicate that the critical limit for oxygen supply might not be the same for each organ system. Therefore, we investigated the effects of acute dilutional anemia on heart, brain, kidneys, liver, small intestine, and skeletal muscle to quantify organ-specific tolerance of different levels of acute anemic hypoxia. We hypothesized that, in some organs, tissue hypoxia occurs before the critical limits of systemic oxygen supply are reached. DESIGN: Laboratory animal experiments. SETTING: Animal research laboratory at university medical school. SUBJECTS: A total of 18 domestic pigs of either sex (average weight: 19.6 kg). INTERVENTIONS: Animals were anesthetized, ventilated, and randomized into three groups and then hemodiluted by exchange of 6% hydroxyethyl starch (130,000:0.4) for whole blood to the group-specific endpoint: Sham (no hemodilution), Hb4 (hemoglobin 4.3 g/dL), Hbcrit (2.7 g/dL). Subsequently, 10 mg/kg pimonidazole (which forms protein adducts in hypoxic cells) was injected. One hour after injection, tissue samples were collected and analyzed for pimonidazole-protein adduct quantification (dot blot) and as a surrogate for transcriptional activation during hypoxia the expression of vascular endothelial growth factor messenger RNA. Relevant hemodynamic and metabolic parameters were collected. MEASUREMENTS AND MAIN RESULTS: Hemodynamics, metabolic parameters, or oxygen consumption did not indicate that tissue oxygenation was restricted before reaching Hbcrit. However, kidneys and skeletal muscle showed enhanced pimonidazole binding and vascular endothelial growth factor expression at Hb4. By contrast, liver oxygenation was actually improved at Hb4. Heart, brain, and liver showed no signs of tissue hypoxia at Hb4. CONCLUSIONS: Heart, brain, kidneys, liver, small intestine, and skeletal muscle experience tissue hypoxia at different degrees of acute anemia, as assessed by the pimonidazole method and vascular endothelial growth factor expression. Further studies are needed to elucidate the mechanisms that determine organ-specific anemia tolerance.

The structure and musculature of male terminalia in the tribe Xanthorhoini Pierce and related tribes (Lepidoptera: Geometridae: Larentiinae), with particular reference to the Palaearctic and Australian regions.

The musculature of the male genitalia was reviewed for the tribe Xanthorhoini and related tribes (Lepidoptera, Geometridae, Larentiinae). The genitalia morphology of males of 11 species was discussed and illustrated, and nine paired and unpaired genital muscles identified. Muscles m1, m2(10), m5(7), m6(5), m7(6), m8(3) and m21 have similar position in all species considered in the paper. Comparative morphology of the male terminalia and position of extensors of the valvae m3(2) and flexors m4 confirmed the previously uncertain separation of Euphyiini and Scotopterygini. Cataclysmini share musculature characters with the tribe Xanthorhoini. The generic affiliation of Xanthorhoe biriviata (Borkhausen) is questionable considering an unusual location of muscles m4. Generally, the places of attachment of the muscles m3(2) and m4 to the sclerites afford valuable characters for the higher classification of this group.

Review of the species of Visiana Swinhoe from the Papua New Guinea region (Lepidoptera: Geometridae: Larentiinae).

The genus isiana Swinhoe which belongs to the subfamily Larentiinae (Lepidoptera: Geometridae) is widely distributed within the Indo-Australian region. The present study is the first extensive review of historic Visiana specimens from the Papua New Guinea region deposited in the Natural History Museum (London, UK) and Australian National Insect Collection (Canberra, Australia). The species examined are as follows: V. hyperctenista (Prout) from the Bismarck Archipelago, V. vinosa (Warren) from the eastern part of Papua New Guinea, V. ranensis (Prout), stat. nov. from the Moluccas (Indonesia), and four newly described species V. horista, sp. nov. from Bougainville Island, V. grandivinosa sp. nov. from New Britain, V. variovinosa, sp. nov. from the eastern part of Papua New Guinea, and V. morobensis, sp. nov. from the Morobe province. The lectotype is designated for V. hyperctienista. Redescriptions of V. hyperctenista, V. vinosa and V. ranensis are provided, the four new species are described, figures of all seven species are shown. The species V. hyperctenista and V. horista are treated as a sister-group, the diagnostic characters are defined. A checklist of the known Papuan species of the genus Visiana is provided.

Comparison of the structure and musculature of male terminalia in the genera Chloroclystis Hübner, Sigilliclystis Galsworthy, and Ziridava Walker (Lepidoptera: Geometridae: Larentiinae).

The structure and musculature of the male terminalia are described in three type species of the eupitheciine genera Chloroclystis Hübner, Sigilliclystis Galsworthy, and Ziridava Walker (Lepidoptera: Geometridae: Larentiinae). The following traits are characteristic for all three genera: (1) the tergal flexors of the valvae, muscles m4 running to the medial part of the transtilla or membrane anterior to the transtilla; (2) the sternal extensors of the valvae, muscles m8(3), broadly running to the inner wall of the saccus, with long lateral bundles; (3) the twisted muscles m7(6) running from the lower margin of the saccus. The peculiar structure of the transtilla is described and illustrated.

Abundant, diverse, unknown: Extreme species richness and turnover despite drastic undersampling in two closely placed tropical Malaise traps.

Arthropods account for a large proportion of animal biomass and diversity in terrestrial systems, making them crucial organisms in our environments. However, still too little is known about the highly abundant and megadiverse groups that often make up the bulk of collected samples, especially in the tropics. With molecular identification techniques ever more evolving, analysis of arthropod communities has accelerated. In our study, which was conducted within the Global Malaise trap Program (GMP) framework, we operated two closely placed Malaise traps in Padang, Sumatra, for three months. We analyzed the samples by DNA barcoding and sequenced a total of more than 70,000 insect specimens. For sequence clustering, we applied three different delimitation techniques, namely RESL, ASAP, and SpeciesIdentifier, which gave similar results. Despite our (very) limited sampling in time and space, our efforts recovered more than 10,000 BINs, of which the majority are associated with "dark taxa". Further analysis indicates a drastic undersampling of both sampling sites, meaning that the true arthropod diversity at our sampling sites is even higher. Regardless of the close proximity of both Malaise traps (< 360 m), we discovered significantly distinct communities.

Notes on the Indo-Australian genus Ziridava Walker (Lepidoptera: Geometridae: Larentiinae), with description of two new species.

The present review summarizes the current state ofknowledge of the genus Ziridava Walker by presenting images of available genitalia slides of the holotypes and paratypes and identifying research gaps for future studies. Two new species, namely Z. mauriakra sp. nov. from the Philippines and Z. orchidensis sp. nov. from the Lanyu Island (Taiwan), are described and illustrated. They share the characteristics of the genus Ziridava, including the uncus reduced to a short trident, the long, slender valvae with costa distinctly broadened for basal one-fourth, the broad juxta constricted medially and the saccus rather rectangular with rounded corners in males and the square-shaped papillae anales, the ductus seminalis arising from a lateral lobe of corpus bursa, with a signum comprised of scobinate patches on the dorsal and ventral surface of corpus bursa in females.

Contributions to the biodiversity of Vietnam - Results of VIETBIO inventory work and field training in Cuc Phuong National Park.

VIETBIO [Innovative approaches to biodiversity discovery and characterisation in Vietnam] is a bilateral German-Vietnamese research and capacity building project focusing on the development and transfer of new methods and technology towards an integrated biodiversity discovery and monitoring system for Vietnam. Dedicated field training and testing of innovative methodologies were undertaken in Cuc Phuong National Park as part and with support of the project, which led to the new biodiversity data and records made available in this article collection. VIETBIO is a collaboration between the Museum für Naturkunde Berlin - Leibniz Institute for Evolution and Biodiversity Science (MfN), the Botanic Garden and Botanical Museum, Freie Universität Berlin (BGBM) and the Vietnam National Museum of Nature (VNMN), the Institute of Ecology and Biological Resources (IEBR), the Southern Institute of Ecology (SIE), as well as the Institute of Tropical Biology (ITB); all Vietnamese institutions belong to the Vietnam Academy of Science and Technology (VAST). The article collection "VIETBIO" (https://doi.org/10.3897/bdj.coll.63) reports original results of recent biodiversity recording and survey work undertaken in Cuc Phuong National Park, northern Vietnam, under the framework of the VIETBIO project. The collection consist of this "main" cover paper - characterising the study area, the general project approaches and activities, while also giving an extensive overview on previous studies from this area - followed by individual papers for higher taxa as studied during the project. The main purpose is to make primary biodiversity records openly available, including several new and interesting findings for this biodiversity-rich conservation area. All individual data papers with their respective primary records are expected to provide useful baselines for further taxonomic, phylogenetic, ecological and conservation-related studies on the respective taxa and, thus, will be maintained as separate datasets, including separate GUIDs also for further updating.

The structure and musculature of male terminalia in the Palaearctic and Indo-Australian species of the eupitheciine genera Eupithecia Curtis, Gymnoscelis Mabille, and Pasiphila Meyrick (Lepidoptera: Geometridae: Larentiinae).

Eupitheciini is a diverse cosmopolitan tribe of larentiine moths. The structure and musculature of the male terminalia were described in six Palaearctic and Indo-Australian eupitheciine species from the genera Eupithecia Curtis, Gymnoscelis Mabille, and Pasiphila Meyrick (Lepidoptera: Geometridae: Larentiinae). The tergal flexors of the valvae, muscles m4, running to the medial part of the transtilla or to the membrane dorsad of the transtilla are identified as a potential synapomorphy for the tribe Eupitheciini. The tergal extensors of the valvae, muscles m3(2), running deep into the costa valvae is a possible synapomorphy for the genus Gymnoscelis. The genus Pasiphila does not share all eupitheciine characters, with the tergal flexors of the valvae, muscles m4, extending from the dorsal parts of the vinculum. The Eupithecia haworthiata species-group differs from other species-groups by the shape of the uncus and the sternal extensors of the valvae, muscles m8(3), with lateral fibres longer than the medial ones.

Australasian genus Crasilogia Warren (Lepidoptera: Geometridae: Larentiinae).

The Australasian genus Crasilogia Warren comprising the species C. dispar Warren, C. flavipennis Warren, C. fulvitincta Joicey Talbo, C. fumipennis Warren, C. gressitti Holloway, C. simplex (Prout), C. parasimplex, sp. nov., and C. diversisimplex, sp. nov. is reviewed based on the museum specimens, including primary types. Lectotype is designated for C. simplex.

Using Malaise traps for collecting Lepidoptera (Insecta), with notes on the preparation of Macrolepidoptera from ethanol.

The present paper deals with the potential of employing Malaise traps for collecting butterflies and moths for morphological analysis and presents a protocol for preparing Macrolepidoptera from Malaise trap samples that were preserved in ethanol. About 80 specimens of Lepidoptera, including Nymphalidae, Geometridae, Hesperiidae, Erebidae, Noctuidae, Pyralidae and Tortricidae, were mounted, following the protocol. All specimens with robust wings and contrasting wing patterns were well suited for the study of external morphology, regardless of the family. The specimens used in this study were collected in highland forest areas of central Vietnam with a little known entomofauna, as part of the German-Vietnamese biodiversity project 'VIETBIO'. The study offers new methodological approaches in an attempt to make the most of the material that was obtained using Malaise traps.

DNA barcoding data release for Coleoptera from the Gunung Halimun canopy fogging workpackage of the Indonesian Biodiversity Information System (IndoBioSys) project.

We present the results of a DNA barcoding pipeline that was established as part of the German-Indonesian IndobioSys project - Indonesian Biodiversity Information System. Our data release provides the first large-scale diversity assessment of Indonesian coleoptera obtained by canopy fogging. The project combined extensive fieldwork with databasing, DNA barcode based species delineation and the release of results in collaboration with Indonesian counterparts, aimed at supporting further analyses of the data. Canopy fogging on 28 trees was undertaken at two different sites, Cikaniki and Gunung Botol, in the south-eastern area of the Gunung Halimun-Salak National Park in West Java, Indonesia. In total, 7,447 specimens of Coleoptera were processed, of which 3,836 specimens produced DNA barcode sequences that were longer than 300 bp. A total of 3,750 specimens were assigned a Barcode Index Number (BIN), including 2,013 specimens from Cikaniki and 1,737 specimens from Gunung Botol. The 747 BINs, that were obtained, represented 39 families of Coleoptera. The distribution of specimens with BINs per tree was quite heterogeneous in both sites even in terms of the abundance of specimens or diversity of BINs. The specimen distribution per taxon was heterogeneous as well. Some 416 specimens could not be identified to family level, corresponding to 72 BINs that lack a family level identification. The data have shown a large heterogeneity in terms of abundance and distribution of BINs between sites, trees and families of Coleoptera. From the total of 747 BINs that were recovered, 421 (56%) are exclusive from a single tree. Although the two study sites were in close proximity and separated by a distance of only about five kilometres, the number of shared BINs between sites is low, with 81 of the 747 BINs. With this data release, we expect to shed some light on the largely hidden diversity in the canopy of tropical forests in Indonesia and elsewhere.

Contribution to the knowledge of the genus Visiana Swinhoe (Lepidoptera: Geometridae: Larentiinae), with the description of two new species from Indonesia.

The present study contributes to the knowledge of the Indo-Pacific genus Visiana Swinhoe based on historic specimens collected in Indonesia and deposited in the collections of the Natural History Museum (London, UK) and Naturalis Biodiversity Center (Leiden, the Netherlands). Two new species V. cyclopsensis, sp. nov. from the Cyclops Mountains (West Papua) and V. minahasensis, sp. nov. from Minahasa (Sulawesi) are described and illustrated. These belong to two different species groups. An updated checklist of the genus Visiana, comprising 18 species is included.

The Mt Halimun-Salak Malaise Trap project - releasing the most species rich DNA Barcode library for Indonesia.

The Indonesian archipelago features an extraordinarily rich biota. However, the actual taxonomic inventory of the archipelago remains highly incomplete and there is hardly any significant taxonomic activity that utilises recent technological advances. The IndoBioSys project was established as a biodiversity information system aiming at, amongst other goals, creating inventories of the Indonesian entomofauna using DNA barcoding. Here, we release the first large scale assessment of the megadiverse insect groups that occur in the Mount Halimun-Salak National Park, one of the largest tropical rain-forest ecosystem in West Java, with a focus on Hymenoptera, Coleoptera, Diptera and Lepidoptera collected with Malaise traps. From September 2015 until April 2016, 34 Malaise traps were placed in different localities in the south-eastern part of the Halimun-Salak National Park. A total of 4,531 specimens were processed for DNA barcoding and in total, 2,382 individuals produced barcode compliant records, representing 1,195 exclusive BINs or putative species in 98 insect families. A total of 1,149 BINs were new to BOLD. Of 1,195 BINs detected, 804 BINs were singletons and more than 90% of the BINs incorporated less than five specimens. The astonishing heterogeneity of BINs, as high as 1.1 exclusive BIN per specimen of Diptera successfully processed, shows that the cost/benefit relationship of the discovery of new species in those areas is very low. In four genera of Chalcidoidea, a superfamily of the Hymenoptera, the number of discovered species was higher than the number of species known from Indonesia, suggesting that our samples contain many species that are new to science. Those numbers shows how fast molecular pipelines contribute substantially to the objective inventorying of the fauna giving us a good picture of how potentially diverse tropical areas might be.

Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications.

BACKGROUND: Biosafety is a key aspect in the international Genetically Engineered Machine (iGEM) competition, which offers student teams an amazing opportunity to pursue their own research projects in the field of Synthetic Biology. iGEM projects often involve the creation of genetically engineered bacterial strains. To minimize the risks associated with bacterial release, a variety of biosafety systems were constructed, either to prevent survival of bacteria outside the lab or to hinder horizontal or vertical gene transfer. MAIN BODY: Physical containment methods such as bioreactors or microencapsulation are considered the first safety level. Additionally, various systems involving auxotrophies for both natural and synthetic compounds have been utilized by iGEM teams in recent years. Combinatorial systems comprising multiple auxotrophies have been shown to reduced escape frequencies below the detection limit. Furthermore, a number of natural toxin-antitoxin systems can be deployed to kill cells under certain conditions. Additionally, parts of naturally occurring toxin-antitoxin systems can be used for the construction of 'kill switches' controlled by synthetic regulatory modules, allowing control of cell survival. Kill switches prevent cell survival but do not completely degrade nucleic acids. To avoid horizontal gene transfer, multiple mechanisms to cleave nucleic acids can be employed, resulting in 'self-destruction' of cells. Changes in light or temperature conditions are powerful regulators of gene expression and could serve as triggers for kill switches or self-destruction systems. Xenobiology-based containment uses applications of Xeno-DNA, recoded codons and non-canonical amino acids to nullify the genetic information of constructed cells for wild type organisms. A 'minimal genome' approach brings the opportunity to reduce the genome of a cell to only genes necessary for survival under lab conditions. Such cells are unlikely to survive in the natural environment and are thus considered safe hosts. If suitable for the desired application, a shift to cell-free systems based on Xeno-DNA may represent the ultimate biosafety system. CONCLUSION: Here we describe different containment approaches in synthetic biology, ranging from auxotrophies to minimal genomes, which can be combined to significantly improve reliability. Since the iGEM competition greatly increases the number of people involved in synthetic biology, we will focus especially on biosafety systems developed and applied in the context of the iGEM competition.

Microvascular dysfunction in rheumatoid arthritis assessed by laser Doppler anemometry: relationship to soluble adhesion molecules and extraarticular manifestations.

In search of a noninvasive diagnostic test for rheumatoid vasculitis (RV), this study addressed the questions whether changes in capillary blood cell velocity (CBV) detected by laser Doppler anemometry in patients with rheumatoid arthritis (RA) were correlated with the levels of soluble adhesion molecules and whether cutaneous flow abnormalities may reflect extraarticular manifestations in RA. In 31 RA patients and 20 patients with osteoarthritis (OA), CBV was measured in the skin above the left ring finger at rest and after 3-min arterial occlusion. Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), and soluble P-selectin (sP-selectin) were assessed by enzyme linked immunosorbent assay. Peak CBV was reduced in RA patients compared to OA patients (0.42 +/- 0.07 mm/s vs. 0.70 +/- 0.13 mm/s; P = 0.013). Both CBV during rest and reactive hyperemia were not correlated with the levels of soluble adhesion molecules. There were no significant differences in resting or peak CBV between RA patients with or without extraarticular manifestations. The lack of an inverse correlation between the levels of soluble adhesion molecules and CBV during rest and reactive hyperemia contradicts the assumption that inflammatory vascular damage indicated by increased levels of soluble adhesion molecules was the main reason for the impairment of microcirculation. The present results do not suggest that cutaneous flow abnormalities may reflect extraarticular manifestations in RA.

Comparison of the structure and musculature of male terminalia in the tribes Operophterini, Phileremini and Triphosini (Lepidoptera: Geometridae: Larentiinae).

The skeleton and musculature of the male terminalia are described and illustrated for the tribes Operophterini Packard, Phileremini Pierce and Triphosini Tutt (Lepidoptera, Geometridae, Larentiinae), with Solitaneini Leraut included in the discussion. Nine genital muscles, typical of the subfamily, were identified. The previously uncertain separation of Triphosini from Rheumapterini Herbulot is confirmed, based on the morphology of male terminalia, including the position of extensors and flexors of the valvae. Based on the morphology, a close relationship of the Phileremini, Solitaneini, Triphosini and also Operophterini is suggested. Additionally, some notes on the most recent molecular phylogeny of the Larentiinae are included.

A streamlined collecting and preparation protocol for DNA barcoding of Lepidoptera as part of large-scale rapid biodiversity assessment projects, exemplified by the Indonesian Biodiversity Discovery and Information System (IndoBioSys).

Here we present a general collecting and preparation protocol for DNA barcoding of Lepidoptera as part of large-scale rapid biodiversity assessment projects, and a comparison with alternative preserving and vouchering methods. About 98% of the sequenced specimens processed using the present collecting and preparation protocol yielded sequences with more than 500 base pairs. The study is based on the first outcomes of the Indonesian Biodiversity Discovery and Information System (IndoBioSys). IndoBioSys is a German-Indonesian research project that is conducted by the Museum für Naturkunde in Berlin and the Zoologische Staatssammlung München, in close cooperation with the Research Center for Biology - Indonesian Institute of Sciences (RCB-LIPI, Bogor).

Identification of sawflies and horntails (Hymenoptera, 'Symphyta') through DNA barcodes: successes and caveats.

The 'Symphyta' is a paraphyletic assemblage at the base of the order Hymenoptera, comprising 14 families and about 8750 species. All have phytophagous larvae, except for the Orussidae, which are parasitoids. This study presents and evaluates the results of DNA barcoding of approximately 5360 specimens of 'Symphyta', mainly adults, and 4362 sequences covering 1037 species were deemed of suitable quality for inclusion in the analysis. All extant families are represented, except for the Anaxyelidae. The majority of species and specimens are from Europe, but approximately 38% of the species and 13% of the specimens are of non-European origin. The utility of barcoding for species identification and taxonomy of 'Symphyta' is discussed on the basis of examples from each of the included families. A significant level of cryptic species diversity was apparent in many groups. Other attractive applications include the identification of immature stages without the need to rear them, community analyses based on metabarcoding of bulk samples and association of the sexes of adults.