Protected Fragments Research Articles

On-resin peptide ligation via C-terminus benzyl ester

Here, we report a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and C-terminal peptide ligation on a Rink Amide resin. On-resin native chemical ligation and auxiliary-assisted peptide ligation were successfully achieved. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor water-soluble peptide ligation.

Heard but not seen: Comparing bat assemblages and study methods in a mosaic landscape in the Western Ghats of India.

We used capture (mist‐netting) and acoustic methods to compare the species richness, abundance, and composition of a bat assemblage in different habitats in the Western Ghats of India. In the tropics, catching bats has been more commonly used as a survey method than acoustic recordings. In our study, acoustic methods based on recording echolocation calls detected greater bat activity and more species than mist‐netting. However, some species were detected more frequently or exclusively by capture. Ideally, the two methods should be used together to compensate for the biases in each. Using combined capture and acoustic data, we found that protected forests, forest fragments, and shade coffee plantations hosted similar and diverse species assemblages, although some species were recorded more frequently in protected forests. Tea plantations contained very few species from the overall bat assemblage. In riparian habitats, a strip of forested habitat on the river bank improved the habitat for bats compared to rivers with tea planted up to each bank. Our results show that shade coffee plantations are better bat habitat than tea plantations in biodiversity hotspots. However, if tea is to be the dominant land use, forest fragments and riparian corridors can improve the landscape considerably for bats. We encourage coffee growers to retain traditional plantations with mature native trees, rather than reverting to sun grown coffee or coffee shaded by a few species of timber trees.

IMPACT OF LINEAR ELEMENTS PROTECTION ON STABILITY OF PIPELINE TRANSPORT SYSTEMS

[For the English abstract and full text of the article please see the attached PDF-File (English version follows Russian version)].ABSTRACT Protection of individual linear elements (pipelines) is performed using technical, technological and other measures that ensure their operability in the presence of a process of progressive damage, that is, an emergency scenario where the pipelines of the system fail in a random sequence. With the help of the simulation method it is established that the greatest protective effect is observed if a group of pipelines is located near the source of the product, and all unprotected consumers are present in the protected fragment. It is shown that the topology of a «line» or «tree» type is preferable for the protected fragment of the system (simplex). Keywords: pipeline transport, system, safety, protection, emergency situation, progressive damage, simulation modeling.

DNA Accessibility by MNase Digestions.

Micrococcal nuclease (MNase) digestion of chromatin cuts linker DNA between neighboring nucleosomes and in this way generates mononucleosomes. The protected fragments can then be analyzed by genome-wide sequencing techniques or by quantitative PCR to obtain information about the positions of nucleosomes in the chromatin. Nucleosomes are differentially sensitive to MNase digestion, which means that titrations of MNase should be performed to obtain a comprehensive map of the nucleosome positions of a chromatin region or genome.

Forest edge disturbance increases rattan abundance in tropical rain forest fragments

Human-induced forest fragmentation poses one of the largest threats to global diversity yet its impact on rattans (climbing palms) has remained virtually unexplored. Rattan is arguably the world’s most valuable non-timber forest product though current levels of harvesting and land-use change place wild populations at risk. To assess rattan response to fragmentation exclusive of harvesting impacts we examined rattan abundance, demography and ecology within the forests of northeastern, Australia. We assessed the community abundance of rattans, and component adult (>3 m) and juvenile (≤3 m) abundance in five intact forests and five fragments (23–58 ha) to determine their response to a range of environmental and ecological parameters. Fragmented forests supported higher abundances of rattans than intact forests. Fragment size and edge degradation significantly increased adult rattan abundance, with more in smaller fragments and near edges. Our findings suggest that rattan increase within fragments is due to canopy disturbance of forest edges resulting in preferential, high-light habitat. However, adult and juvenile rattans may respond inconsistently to fragmentation. In managed forest fragments, a rattan abundance increase may provide economic benefits through sustainable harvesting practices. However, rattan increases in protected area forest fragments could negatively impact conservation outcomes.

RNA substrate length as an indicator of exosome interactions in vivo.

Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the "central channel" and "direct access" routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads.

Mitochondrial Ribosome (Mitoribosome) Profiling for Monitoring Mitochondrial Translation In Vivo.

Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole-cell lysate and the protected mRNA fragments are isolated. These fragments are then converted to sequencing libraries or analyzed by northern blot hybridization to reveal the distribution of mitoribosomes across the mitochondrial transcriptome. As information about RNA abundance is required to resolve translational from RNA effects, we also present an RNA sequencing approach that can be performed in parallel. Accurately capturing the biologically relevant distribution of mitoribosome positions depends on several critical parameters that are discussed. Application of mitoribosome profiling can reveal mechanisms of mitochondrial translational control that were not previously possible to uncover. © 2017 by John Wiley & Sons, Inc.

RNA substrate length as an indicator of exosome interactions in vivo

Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads.

Termite (Isoptera) Diversity in a Gallery Forests Relict in the Colombian Eastern Plains

Termites are a key group for the dynamics of gallery forests in the Colombian Orinoco region, but have been little studied. Richness and relative abundance of termites found in protected gallery forest fragments within commercial forest plantations were quantified. Sampling was conducted at three sites surrounding the creeks “Huerta La Grande”, “Caño Claro” and “Caño Los Micos”, along linear transects 50 m long and divided into five 5 × 2 m plots; there were nine transects and 45 plots. Termite sampling involved the examination of fallen branches, pieces of wood in contact with the soil, arboreal termitaria, epigeal nests and soil samples 20 cm deep (four per plot). Thirty morphospecies, mostly xylophages from the families Termitidae (Apicotermitinae, Termitinae, Nasutitermitinae, Syntermitinae) and Rhinotermitidae (Heterotermitinae), were found. No association was found between richness or termite abundance and tree size, soil pH, soil organic carbon content or accumulation depth of leaf litter on the soil. The termite diversity and abundance found in fragments of gallery forest highlights the importance of maintaining this type of vegetation to preserve the ecosystem services derived from the biological activity of termites. Keywords: Heterotermes convexinotatus, Microcerotermes, Conservation, Orinoquia, Species richness

RNA substrate length as an indicator of exosome interactions in vivo

Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads.

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis

Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity.

Examining a mechanism of generating the fragments of protective film in the trybological system "epoxycomposite–steel"

The influence of multifunctional fillers on wear resistance of epoxycomposites, operated under conditions of friction without lubrication at elevated slip velocity and loadings was explored. Introduction of these fillers in the optimum amount provides high cohesive strength and gives epoxycomposites tribotechnical properties. The developed composition of materials allows the implementation of the effect of selective transfer during friction interaction. Wear intensity of epoxycomposites under different loading and velocity modes was determined, which made it possible to establish favourable mode of formation of a protective film. The analysis of structural elements of tribosurfaces of materials and a counter was carried out and chemical composition of fragments of the protective film on tribosufraces was defined. It proved a hypothesis on the formation of a copper protective film at tribosurfaces as a result of selective transfer. The reasons for initiation of the process of generation of protective film fragments on surfaces of tribobodies was established and the sequence of stages of its formation in highly filled epoxycomposites was defined. The shape, dimensions and the area of fragments of a protective film, formed at tribosurfaces of the system epoxycomposite – a steel counter body system, were determined. The formed film in the form of fragments of elongated shape in direction of the friction process stabilizes the process of tribointeraction due to the capability of self-restoring. Using the developed epoxycomposites, which operate under the mode of selective transfer, decreases friction coefficient and wear intensity due to the formation of laminar and porous structure of a film. Accordingly, it will allow controlling the process of friction, reducing material and economic costs in the process of maintenance and repair of equipment, which solves topical scientific and practical tasks of modern tribomaterials science. Feasibility of using the protective copper films in tribonodes was established, which will make it possible to expand the area of using the triboproducts in machine building, instrument engineering, chemical and light industries.

Nest Sites of Middle Spotted WoodpeckersLeiopicus mediusin a Primeval Forest

Forest management usually reduces the diversity and amount of substrates in which woodpeckers can excavate holes. In such conditions the recorded patterns of nest site selection could reflect a more reduced array of substrates than the species' nest site preferences. We analysed new data on nest sites of Middle Spotted Woodpeckers Leiopicus medius collected during 27 breeding seasons (1987–2013) in the strictly protected fragments of the Bialowieza National Park in Poland. The birds could use diverse excavation substrates, of various statures, that varied in condition from living and healthy to dead and completely rotten. Middle Spotted Woodpeckers used a wide array of tree species, with little overlap in the species used in the riverine habitat (mostly Alnus glutinosa, and Fraxinus excelsior) and oak—hornbeam habitats (Carpinus betulus and Quercus robur). The birds preferred to excavate in Q. robur but avoided Tilia cordata and Picea abies. They placed holes in large trees (median girth at breast height:...

Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate.

Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.

Cultural viability of reintroducing the ecologically extinct Alagoas Curassow (Pauxi mitu Linnaeus, 1766) to Northeast Brazil

The Alagoas Curassow (Pauxi mitu) became extinct in the wild in the 1980's through a combination of loss/degradation of its Atlantic Forest habitat and over-hunting. Ambitious plans are now underway to reintroduce captive-reared Curassows using a patchwork of protected forest fragments on private lands. Although the planned reintroduction sites are broadly ecologically suitable, it is not clear that the threats from hunting and habitat disturbance have been removed. In other words, the cultural (as opposed to biological) suitability and viability of these sites is unknown. We used a semi-structured social survey of 402 residents who live near three proposed reintroduction sites to evaluate the cultural suitability of the reintroduction. Specifically, we adopted a multimodel inference approach to identify socio-demographic characteristics (e.g., age, gender, education level, duration of residence), knowledge (e.g., of local biodiversity and hunting practices) and behaviors (e.g., bushmeat consumption and firewood collection) that influence support for the reintroduction. Respondents were generally positive to bringing the Curassow back, though our data indicates that hunting is still a major part of the local culture. Support for the reintroduction was most strongly associated with older and, especially, better educated residents. Residents from different reintroduction sites also differed in their professed levels of support. Our results highlight the importance of focused environmental educational programmes to improve the cultural feasibility of this flagship reintroduction for Northeast Brazil.

Induction of protective immunity against Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina infections using multivalent epitope DNA vaccines

Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria.

The awesome power of ribosome profiling.

Ribosome profiling has proved to be a fantastic innovation which has revealed so much about mRNA translation that had previously gone unrecognized. The RNA community owes Jonathan Weissman and Nick Ingolia a deep sense of gratitude for conceiving the approach in the first place, and for their detailed optimization of the methodology. The origins of the approach can be traced back to the discovery of polysomes over 50 years ago, and the finding that treatment with endoribonucleases converted them to monomeric ribosomes that were each bound to a short protected mRNA fragment. This was first exploited by Joan Steitz and Marilyn Kozak to identify and sequence the translation initiation sites of prokaryotic bacteriophage RNA and eukaryotic reovirus RNAs, respectively; and extended by Sandra Wolin and Peter Walter to reveal ribosome pausing at the initiation and termination codons of preprolactin mRNA, as well as at the point at which SRP interacts with the nascent protein. Because of the limitations of sequencing methods available in that era, these experiments necessarily used cell-free translation systems programmed preferably with a single mRNA species (or a maximum of 4 reovirus mRNAs).

Unusual Course of the Reaction of Allyl Phosphine Oxides with the Grundmann Ketone

This article describes efficient preparation of isomeric allyl phosphine oxides possessing a protected cyclohexanediol fragment. Their base-catalyzed interconversions are examined and reactions with the Grundmann ketone provide an adduct containing the rearranged vinyl phosphine oxide moiety, instead of 19-norvitamin D3 analogs, the expected products of the Horner–Wittig process.

The synthesis of a glucoconjugate of the peptidic fragment of cryptophycin-24

A novel glucoconjugate of the peptidic fragment of cryptophycin-24 was prepared through the replacement of the leucic acid residue with L-leucine and the functionalisation of tyrosine residue with glucose.Those modifications lead to a novel protected tripeptide fragment of cryptophycin-24.

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.