Linear Fragments Research Articles

Effects of vicariant barriers, habitat stability, population isolation and environmental features on species divergence in the south‐western Australian coastal reptile community

Identifying explicit hypotheses regarding the factors determining genetic structuring within species can be difficult, especially in species distributed in historically dynamic regions. To contend with these challenges, we use a framework that combines species distribution models, environmental data and multi-locus genetic data to generate and explore phylogeographic hypotheses for reptile species occupying the coastal sand-dune and sand-plain habitats of the south-western Australian biodiversity hotspot, a community which has both a high diversity of endemics and has varied dramatically in spatial extent over time. We use hierarchical amova, summary statistic and distance-based analyses to explicitly test specific phylogeographic hypotheses. Namely, we test if biogeographic vicariance across barriers, habitat stability, population isolation along a linear habitat or fragmentation across different environments can explain genetic divergence within five co-distributed squamate reptile species. Our results show that patterns of genetic variation reflect complex and species-specific interactions related to the spatial distribution of habitats present currently and during repeated glacial minima, as opposed to being associated with historical factors such as habitat stability between glacial and inter-glacial periods or vicariant barriers. We suggest that the large impact of habitat characteristics over time (i.e. relative levels of habitat connectivity, climatic gradients and spatial heterogeneity of soil types) reflects the ecological restrictions of the sand-dune and sand-plain reptile communities and may explain the lack of concordance across taxa. The study demonstrates the general utility of the approach for assemblage-level, as well as single species, phylogeographic study, including its usefulness for exploring biologically informed hypotheses about what factors have influenced patterns of genetic variation.

Defying Entropy: Forming Large Head‐to‐Tail Macrocycles in the Gas Phase

Spectral fingerprints: Collision-induced dissociation (CID) of protonated peptides in the gas phase results in linear fragment ions with a five-membered oxazolone ring on their C-terminal side. Infrared spectroscopy confirms that smaller fragments adopt oxazolone structures. Conversely, in mid-sized and larger fragments an isomerization to "head-to-tail" macrocycles is observed (see picture).

An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same

The Gateway(®) recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7kb in size. Using this approach, the yield of colony PCR positive destination clones was 100% for genes of various sizes tested in our experiments.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3–18) and its analog [Phe8]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.

Epitope mapping of BP230 leading to a novel enzyme‐linked immunosorbent assay for autoantibodies in bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by circulating autoantibodies against BP180 and BP230. For BP180, the NC16A domain has previously been identified as the main antigenic target in BP, while data about the diagnostic value of epitopes on BP230 were inconclusive. To identify the most appropriate epitopes on BP230 to be applied in a simple, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for routine detection of serum autoantibodies. Ten overlapping linear fragments covering the whole length of BP230 were expressed in Escherichia coli. Based on Western blot analysis with sera from patients with BP (n = 49) and healthy controls (n = 94), the diagnostic performance of the fragments was compared by receiver operating characteristics curve analysis. The BP230-C3 fragment comprising the C-terminal portion (amino acids 2326-2649) was subsequently applied in a novel ELISA. The operating characteristics of this ELISA were analysed by probing sera from patients with BP (n = 118), pemphigus vulgaris (n = 50), rheumatoid arthritis and other inflammatory arthritides (n = 170), and systemic lupus erythematosus (n = 56), and from healthy blood donors (n = 483). Among all the fragments, BP230-C3 provided the best efficiency in serologically diagnosing BP by Western blot. An ELISA employing BP230-C3 revealed a diagnostic sensitivity of 56·8% and specificity of 97·6%. Its diagnostic added value amounted to 4·2% compared with the anti-BP180-NC16A-4X ELISA alone. Recombinant BP230-C3 is a suitable target antigen for the detection of serum autoantibodies against BP230.

Self-similarity in a general aggregation–fragmentation problem. Application to fitness analysis

We consider the linear growth and fragmentation equation:∂∂tu(x,t)+∂∂x(τ(x)u)+β(x)u=2∫x∞β(y)κ(x,y)u(y,t)dy, with general coefficients τ, β and κ. Under suitable conditions (see Doumic Jauffret and Gabriel, 2010 [1]), the first eigenvalue represents the asymptotic growth rate of solutions, also called the fitness or Malthus coefficient in population dynamics. This value is of crucial importance in understanding the long-time behavior of the population. We investigate the dependence of the dominant eigenvalue and the corresponding eigenvector on the transport and fragmentation coefficients. We show how it behaves asymptotically depending on whether transport dominates fragmentation or vice versa. For this purpose we perform a suitable blow-up analysis of the eigenvalue problem in the limit of a small/large growth coefficient (resp. fragmentation coefficient). We exhibit a possible non-monotonic dependence on the parameters, in contrast to what would have been conjectured on the basis of some simple cases.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Modelling the distribution of the invasive Roesel’s bush-cricket (Metrioptera roeselii) in a fragmented landscape

The development of conservation strategies to mitigate the impact of invasive species requires knowledge of the species ecology and distribution. This is, however, often lacking as collecting biological data may be both time-consuming and resource intensive. Species distribution models can offer a solution to this dilemma by analysing the species-environment relationship with help of Geographic information systems (GIS). In this study, we model the distribution of the non-native bush-cricket Metrioptera roeselii in the agricultural landscape in mid-Sweden where the species has been rapidly expanding in its range since the 1990s. We extract ecologically relevant landscape variables from Swedish CORINE land-cover maps and use species presence-absence data from large-scale surveys to construct a species distribution model (SDM). The aim of the study is to increase the knowledge of the species range expansion pattern by examining how its distribution is affected by landscape composition and structure, and to evaluate SDM performance at two different spatial scales. We found that models including data on a scale of 1 × 1 km were able to explain more of the variation in species distribution than those on the local scale (10 m buffer on each side of surveyed road). The amount of grassland in the landscape, estimated from the area of arable land, pasture and rural settlements, was a good predictor of the presence of the species on both scales. The measurements of landscape structure – linear elements and fragmentation - gave ambivalent results which differed from previous small scaled studies on species dispersal behaviour and occupancy patterns. The models had good predictive ability and showed that areas dominated by agricultural fields and their associated grassland edges have a high probability being colonised by the species. Our study identified important landscape variables that explain the distribution of M. roeselii in Mid-Sweden that may also be important to other range expanding orthopteran species. This work will serve as a foundation for future analyses of species spread and ecological processes during range expansion.

Quantitative Structure–Property Relationship (QSPR) Modeling of Normal Boiling Point Temperature and Composition of Binary Azeotropes

Quantitative structure–property relationship (QSPR) modeling of normal boiling point temperature (Taz) and the composition (weight fraction, X1w) of 176 binary azeotropic mixtures was performed using ensemble multiple linear regression analysis and fragment descriptors implemented in ISIDA software. The models have been validated in external 5-fold cross-validations procedure and on an additional test set of 24 azeotropes. The prediction errors (3–4 K for Taz and 10–14 wt % for X1w) are comparable with the noise in experimental data. A simple empirical relationship linking Taz with boiling points of two molecular components of azeotrope has been suggested.

Synthesis and structure of coordination polymer compounds of AgReO4 and AgPF6 with piperazine

Coordination polymers [Ag(C4H10N2)]ReO4 (I) and [Ag(C4H10N2)]PF6 (II) (C4H10N2 is piperazine, Ppz) were synthesized and their structures were determined. Crystals of compound I are monoclinic, space group P21/c, a = 6.207(1) A, b = 12.533(1) A, c = 11.386(1) A, β = 93.41(1)°, V = 884.2(2) A3, ρcalc = 3.337 g/cm3, Z = 4. Crystals of II are monoclinic, space group C2/m, a = 8.723(1) A, b = 9.083(1) A, c = 5.797(1) A, β = 95.07(1)°, V = 457.5(1) A3, ρcalc = 2.548 g/cm3, Z = 2. Structure I contains polymer chains [Ag(Ppz)]∞+. The silver atom is linked with two nitrogen atoms of the adjacent Ppz ligands to form a nearly linear fragment; the Ag-Nav distance is 2.173 A, and the NAgN angle is 169.4(3)°. The chains are linked with each other by weak interactions Ag…O(ReO4) (2.643(8) A) and N-H…O hydrogen bonds. The structure of compound II also contains cationic polymer chains [Ag(Ppz)]∞+. The Ag+ ion is located in the inversion center and has a linear coordination (Ag-N distance is 2.171(9) A). The central P atom of the octahedral fluorophos-phate ion is also located in the inversion center; the anion is slightly distorted and has no contacts with silver ions at a distance <3.4 A.

High mobility group B1 protein interacts with its receptor RAGE in tumor cells but not in normal tissues

The high mobility group box 1 (HMGB1) protein is an abundant non-histone component of chromatin well known for its two DNA binding domains, HMG box A and HMG box B. The main characteristics of the HMGB1 protein as an 'architectural' factor are its ability to recognize and bind with high affinity to distorted DNA and its ability to induce kinks in linear DNA fragments. The HMGB1 protein has been correlated to cancer progression. An elevated expression of HMGB1 occurred in certain types of primary tumor, including melanoma and colon, prostate, pancreatic and breast cancers, and in the majority of cases HMGB1 is associated with invasion and metastasis. The main signaling pathway is activated through the interaction of HMGB1 with its Receptor for Advanced Glycation End products (RAGE). Certain data indicate that an elevated expression of RAGE and HMGB1 is not always a prerequisite of poor prognosis of tumor development. The cellular localization of the ligand/receptor pair also requires consideration. The data concerning the expression of HMGB1 protein and its receptor RAGE in various tissues and tumor cells reflect the overall production of the proteins. However, they do not refer to their cellular localization and there is no direct evidence for the formation of a stable complex between them. In the present study, we investigated the subcellular distribution of HMGB1 and its receptor RAGE in various rat organs compared to Guerin ascites tumor cells. In the normal tissues the proteins exist in their soluble form, whereas in the tumor cells they are insoluble and membrane-bound. HMGB1 forms a stable complex with RAGE only in the protein extract derived from the cancer cells predominantly in the membrane fraction.

Disulfide and amide-bridged cyclic peptide analogues of the VEGF81–91 fragment: Synthesis, conformational analysis and biological evaluation

The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the β-hairpin fragment VEGF81–91 are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central β-turn of the VEGF81–91 β-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction.

E46K Human α-Synuclein Transgenic Mice Develop Lewy-like and Tau Pathology Associated with Age-dependent, Detrimental Motor Impairment

Synucleinopathies are a group of neurodegenerative disorders associated with the formation of aberrant amyloid inclusions composed of the normally soluble presynaptic protein α-synuclein (α-syn). Parkinson disease is the most well known of these disorders because it bears α-syn pathological inclusions known as Lewy bodies (LBs). Mutations in the gene for α-syn, including the E46K missense mutation, are sufficient to cause Parkinson disease as well as other synucleinopathies like dementia with LBs. Herein, we describe transgenic mice expressing E46K human α-syn in CNS neurons that develop detrimental age-dependent motor impairments. These animals accumulate age-dependent intracytoplasmic neuronal α-syn inclusions that parallel disease and recapitulate the biochemical, histological, and morphological properties of LBs. Surprisingly, the morphology of α-syn inclusions in E46K human α-syn transgenic mice more closely resemble LBs than the previously described transgenic mice (line M83) that express neuronal A53T human α-syn. E46K human α-syn mice also develop abundant neuronal tau inclusions that resemble neurofibrillary tangles. Subsequent studies on the ability of E46K α-syn to induce tau inclusions in cellular models suggest that both direct and indirect mechanisms of protein aggregation are probably involved in the formation of the tau inclusions observed here in vivo. Re-evaluation of presymptomatic transgenic mice expressing A53T human α-syn reveals that the formation of α-syn inclusions in mice must be synchronized; however, inclusion formation is diffuse within affected areas of the neuroaxis such that there was no clustering of inclusions. Collectively, these findings provide insights in the mechanisms of formation of these aberrant proteinaceous inclusions and support the notion that α-syn aggregates are involved in the pathogenesis of human diseases.

English

&nbsp; Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be more than 70% and the quality of the recovered DNA is proved to be good enough for future research. Here, we provided&nbsp;another good method to recover DNA from agarose gel besides using commercial kits. &nbsp; Key words:&nbsp;Trap method, recovery of DNA, agarose gel electrophoresis.

Control of mechanic properties of 3D network polymers based on vinyl and hydride-containing siloxanes

The catalytic hardening of siloxane oligomers containing equivalent amount of hydride and vinyl groups in the linear and cyclic fragments of the oligomers was investigated and the effect of the position of these groups on the strength of the 3D network polymers was demonstrated.

Human Mre11/Human Rad50/Nbs1 and DNA Ligase IIIα/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

Engineering multiple genomic deletions in Gram‐negative bacteria: analysis of the multi‐resistant antibiotic profile of Pseudomonas putida KT2440

The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-negative bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates - perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini. With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms.

Effect of hydrogen on the formation of the atomic structure of linear carbon chains: An ab initio approach

An ab initio study of the features of the formation of the atomic structure of carbon chains, which are structural parts of films of linear carbon chains, has been performed using the electron density functional theory. It has been shown that the formation and stabilization of experimentally observed kinks of carbon chains require the presence of hydrogen impurities in the chamber during the synthesis of linear carbon chains. The kinks of a carbon chain are formed in the process of the adsorption of hydrogen atoms on a chain. The optimal kink angle of carbon chains has been determined. The stability of kinks of carbon chains has been estimated as a function of the length of linear fragments. An additional mechanism has been proposed for the formation of kinks in carbon chains owing to the joining of short carbon chains with hydrogen ends.

Effect of Cisplatin on the Flexibility of Linear DNA

With the aid of an atomic force microscope (AFM), we study the interaction between linear DNA fragment and cisplatin. For different cisplatin concentrations, the AFM used to observe the conformation of DNA has a gradual change. The contour length, the end-to-end distance and the local bend angles of the linear DNA fragment can be accurately measured. The persistence length of DNA interacting with cisplatin is decreased with the increasing cisplatin concentration. Furthermore, it is demonstrated that the local bend angles of DNA chains are increased by the binding interaction of cisplatin.