Reassembly Of Fragments Research Articles

Catastrophic cellular events leading to complex chromosomal rearrangements in the germline.

Although complex chromosomal rearrangements were thought to reflect the accumulation of DNA damage over time, recent studies have shown that such rearrangements frequently arise from 'all-at-once' catastrophic cellular events. These events, designated chromothripsis, chromoanasynthesis, and chromoanagenesis, were first documented in the cancer genome and subsequently observed in the germline. These events likely result from micronucleus-mediated chromosomal shattering and subsequent random reassembly of DNA fragments, although several other mechanisms have also been proposed. Typically, only one or a few chromosomes of paternal origin are affected per event. These events can produce intrachromosomal deletions, duplications, inversions, and translocations, as well as interchromosomal translocations. Germline complex rearrangements of autosomes often result in developmental delay and dysmorphic features, whereas X chromosomal rearrangements are usually associated with relatively mild clinical manifestations. The concept of these catastrophic events provides novel insights into the etiology of human genomic disorders. This review introduces the molecular characteristics and phenotypic outcomes of catastrophic cellular events in the germline.

基于轮廓线特征点的交互式文物拼接

基于轮廓线特征点的交互式文物拼接

Bimolecular fluorescence complementation based on the red fluorescent protein FusionRed

The aim of this work is to construct split variants of the red fluorescent protein FusionRed, each of which consists of two separate polypeptides, nonfluorescent parts of FusionRed, that can form functional fluorescent proteins upon reassociation. At the first stage, various circularly permuted FusionRed variants have been created (in circular permutants the protein polypeptide chain is divided into two parts, which change places so that the C-terminal part is followed by the N-terminal part). Two variants with the highest rate of chromophore maturation (fluorescence development) have been selected out of 23 tested permutation points. These proteins called cpFR76-73 and cpFR189-188 (the first number indicates the last amino acid residue of the N-terminal part; the second number, the first residue of the C-terminal part) are spectrally similar to parental FusionRed but possess lower fluorescence quantum yields. Split variants corresponding to these two circular permutants have been tested in mammalian cells. For reassembly of the fluorescent protein fragments, heterodimerizing leucine zippers have been used. It has been shown that split variant FR189-188 matures at 37°C and possesses fluorescence brightness similar to that of FusionRed. Consequently, FR189-188 is potentially suitable for a wide range of applications, for example, the study of protein–protein interactions or visualization of cell populations, in which two target gene promoters are simultaneously active.

A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.

Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m-2) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.

A new lightweight support for the restoration and presentation of a large Roman mosaic

Abstract This paper presents a new technique employed in the construction of a lightweight backing for the Roman floor mosaic XIII.8 from Emona (Ljubljana, Slovenia). The rather large mosaic did not remain in situ but was instead lifted in 1997 before being restored and reassembled during a long and demanding conservation process between 2013 and 2014. Due to the size of the mosaic and subsequent demands associated with its presentation, as well as the need for easy handling when carrying and assembling the restored fragments, a necessity arose to develop a lightweight, compatible and easily removable support. Hence with an investigation of mechanical properties of lightweight mortars based on natural hydraulic lime was carried out. A low mortar density was obtained via the use of a lightweight aggregate composed of recycled glass beads. Conservation–restoration processes included documentation, cleaning, application of the new support, retouching and reassembly of the mosaic fragments.

Effects of high pressure processing (hydrostatic high pressure and ultra-high pressure homogenisation) on whey protein native state and susceptibility to tryptic hydrolysis at atmospheric pressure

Dispersion of whey protein isolate (WPI) prepared at 10% proteins (w/w) and pH6.5, was pressure-processed using high hydrostatic pressure (HHP) at 300MPa and 25°C for 15min, or ultra-high pressure homogenisation (UHPH) at 300MPa and initial fluid temperature (Tin) of 24°C. UHPH-processing was followed (or not) by rapid cooling of the processed fluid at the immediate HP-valve outlet. Short-time thermal treatment (STTT) at 75°C for 10s, 43s or 110s was studied for comparison. Processing-induced effects were investigated in terms of (i) protein denaturation by differential scanning calorimetry (DSC) and ammonium sulphate (AS) precipitation, (ii) susceptibility to tryptic proteolysis at atmospheric pressure after processing, (iii) protein particle sizes from light scattering measurement during the hydrolysis process and, (iv) residual proteins by SDS-PAGE after 135min of hydrolysis. UHPH followed by efficient cooling at the HP-valve outlet, limited significantly (p=0.05) protein denaturation as assessed by DSC and AS precipitation, while increasing notably protein susceptibility to tryptic attack, comparing with the non-processed sample. In opposite, neglecting the cooling step at the HP-valve outlet, led to significantly (p=0.05) lower residual protein native state, then tryptic hydrolysis efficiency. HHP treatment led to intermediate results. The slight protein unfolding that pre-processing could induced, favoured thus the further trypsin attack. Generally speaking, high pressure (avoiding overheating) offers this potential. Process-induced protein aggregation, but also reassembly of protein fragments and peptides released during hydrolysis may affect the proteolysis efficiency.

Functional assembly of protein fragments induced by spatial confinement.

Natural proteins are often confined within their local microenvironments, such as three-dimensional confinement in organelles or two-dimensional confinement in lipid rafts on cytoplasmic membrane. Spatial confinement restricts proteins' entropic freedom, forces their lateral interaction, and induces new properties that the same proteins lack at the soluble state. So far, the phenomenon of environment-induced protein functional alteration still lacks a full illustration. We demonstrate here that engineered protein fragments, although being non-functional in solution, can be re-assembled within the nanometer space to give the full activity of the whole protein. Specific interaction between hexahistidine-tag (His-tag) and NiO surface immobilizes protein fragments on NiO nanoparticles to form a self-assembled protein "corona" on the particles inside the nanopores of mesoporous silica. Site-specific assembly forces a shoulder-by-shoulder orientation and promotes fragment−fragment interaction; this interaction together with spatial confinement of the mesopores results in functional re-assembly of the protein half fragments. To our surprise, a single half fragment of luciferase (non-catalytic in solution) exhibited luciferase activity when immobilized on NiO in the mesopores, in the absence of the complimentary half. This shows for the first time that spatial confinement can induce the folding of a half fragment, reconstitute the enzyme active site, and re-gain the catalytic capability of the whole protein. Our work thereby highlights the under-documented notion that aside from the chemical composition such as primary sequence, physical environment of a protein also determines its function.

Secondary correction of post-traumatic craniofacial deformities.

Despite advances in the acute management of craniofacial fractures, some patients will present with secondary deformities. In an attempt to formulate treatment guidelines, we present a review of 152 cases of post-traumatic craniofacial deformities treated by us using different surgical techniques. Analysis of the underlying bony and soft tissue deficits was followed by stepwise reassembly of the skeletal fragments and re-establishment of facial contour and symmetry. The postoperative outcomes were evaluated in terms of aesthetic and functional parameters. Though all patients had considerable improvement after secondary correction of these deformities, soft tissue deficits and time elapsed till secondary reconstruction remain the major factors in achieving an ideal aesthetic outcome.

Data fragment forensics for embedded DVR systems

A recent increase in the prevalence of embedded systems has led them to become a primary target of digital forensic investigations. Embedded systems with DVR (Digital Video Recorder) capabilities are able to generate multimedia (video/audio) data, and can act as vital pieces of evidence in the field of digital forensics.To counter anti-forensics, it is necessary to derive systematic forensic techniques that can be used on data fragments in unused (unallocated) areas of files or images. Specifically, the techniques should extract meaningful information from various types of data fragments, such as non-sequential fragmentation and missing fragments overwritten by other data.This paper proposes a new digital forensic system for use on video data fragments related to DVRs. We demonstrate in detail special techniques for the classification, reassembly, and extraction of video data fragments, and introduce an integrated framework for data fragment forensics based on techniques described in this paper.

Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir)

The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb’s free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the GH1, GH3, GH5, GH9 and GH10 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

Chromothripsis: how does such a catastrophic event impact human reproduction?

The recent discovery of a new kind of massive chromosomal rearrangement, baptized chromothripsis (chromo for chromosomes, thripsis for shattering into pieces), greatly modifies our understanding of molecular mechanisms implicated in the repair of DNA damage and the genesis of complex chromosomal rearrangements. Initially described in cancers, and then in constitutional rearrangements, chromothripsis is characterized by the shattering of one (or a few) chromosome(s) segments followed by a chaotic reassembly of the chromosomal fragments, occurring during one unique cellular event. The diversity and the high complexity of chromothripsis events raise questions about their origin, their ties to chromosome instability and their impact in pathology. Several causative mechanisms, involving abortive apoptosis, telomere erosion, mitotic errors, micronuclei formation and p53 inactivation, have been proposed. The remarkable point is that all these mechanisms have been identified in the field of human reproduction as causal factors for reproductive failures and chromosomal abnormalities. Consequently, it seems important to consider this unexpected catastrophic phenomenon in the context of fertilization and early embryonic development in order to discuss its potential impact on human reproduction.

Prevalence and clinical implications of chromothripsis in cancer genomes

A variety of mutational mechanisms shape the landscape of somatic mutations in cancer genomes. Although the contribution of single nucleotide mutations is well studied, getting a hold of structural genomic rearrangements is more difficult due to their complexity and diversity in sizes and classes. Here, we discuss the incidence of complex genomic rearrangements and their impact on cancer development and progression. Catastrophic genome rearrangements have recently been described in various cancer genomes. Such complex genomic rearrangements may be a result of local shattering of chromosomes followed by reassembly of DNA fragments, a process termed chromothripsis. In addition, DNA replication errors may lead to complex genomic rearrangements in cancer. Complex reshuffling of chromosomes can cause formation of gene fusions, disruption of tumor suppressors, and amplification of oncogenes. Furthermore, the occurrence of chromothripsis has been associated with poor prognosis in neuroblastoma, melanoma, and multiple myeloma. Complex genomic rearrangements, such as chromothripsis, may affect cancer gene function and thereby have a major impact on cancer progression, prognosis, and therapy response.

The position of Madagascar within Gondwana and its movements during Gondwana dispersal

A reassembly of the Precambrian fragments of central Gondwana is presented that is a refinement of a tight reassembly published earlier. Fragments are matched with conjugate sides parallel as far as possible and at a distance of 60–120km from each other. With this amount of Precambrian crust now stretched into rifts and passive margins, a fit for all the pieces neighbouring Madagascar – East Africa, Somalia, the Seychelles, India, Sri Lanka and Mozambique – may be made without inelegant overlap or underlap. This works less well for wider de-stretched margins on such small fragments. A model of Gondwana dispersal is also developed, working backwards in time from the present day, confining the relative movements of the major fragments – Africa, Antarctica and India – such that ocean fracture zones collapse back into themselves until each ridge-reorganisation is encountered. The movements of Antarctica with respect to Africa and of India with respect to Antarctica are defined in this way by a limited number of interval poles to achieve the Gondwana ‘fit’ situation described above. The ‘fit’ offers persuasive alignments of structural and lithologic features from Madagascar to its neighbours. The dispersal model helps describe the evolution of Madagascar’s passive margins and the role of the Madagascar Rise as a microplate in the India–Africa–Antarctica triple junction. Intrusions, extrusions and dykes observed in Madagascar and its neighbours, largely from aeromagnetic survey data, are related to the outbreak of the Karoo/Bouvet mantle plume at ∼182Ma, the Marion mantle plume at ∼88Ma and the Reunion mantle plume at ∼66Ma. The dispersal model may be viewed and downloaded as an animation at: http://www.reeves.nl/gondwana.

The PprA protein is required for accurate cell division of γ-irradiated Deinococcus radiodurans bacteria

Deinococcus radiodurans, one of the most radioresistant organisms known to date is able to reconstruct an intact genome from hundreds of DNA fragments. Here, we investigate the in vivo role of PprA, a radiation-induced Deinococcus specific protein. We report that DNA double strand break repair in cells devoid of PprA and exposed to 3800Gy γ-irradiation takes place efficiently with a delay of only 1h as compared to the wild type, whereas massive DNA synthesis begins 90min after irradiation as in the wild type, a phenotype insufficient to explain the severe radiosensitivity of the ΔpprA mutant. We show that the slow kinetics of reassembly of DNA fragments in a ΔpprA ΔrecA double mutant was the same as that observed in a ΔrecA single mutant demonstrating that PprA does not play a major role in DNA repair through RecA-independent pathways. Using a tagged PprA protein and immunofluorescence microscopy, we show that PprA is recruited onto the nucleoid after γ-irradiation before DNA double strand break repair completion, and then is found as a thread across the septum in dividing cells. Moreover, whereas untreated cells devoid of PprA displayed a wild type morphology, they showed a characteristic cell division abnormality after irradiation not found in other radiosensitive mutants committed to die, as DNA is present equally in the two daughter cells but not separated at the division septum. We propose that PprA may play a crucial role in the control of DNA segregation and/or cell division after DNA double strand break repair.

Microwave and traditional solvothermal syntheses, crystal structures, mass spectrometry and magnetic properties of CoII4O4 cubes

We report a study of the comparative solvothermal syntheses, using traditional and microwave ovens, of two complexes [Co4(μ3-OMe)4(L)4(MeOH)4], where (HL = Hhmb, 2-hydroxy-3-methoxy-benzaldehyde (1) or Hheb = 2-hydroxy-3-ethoxy-benzaldehyde (2)), their crystal structures and magnetic properties in the solid state and the ESI-MS of the solutions. The microwave heating is more efficient in time and quality of crystals than the traditional one where what is achieved per minute by the former takes an hour by the latter. The structures consist of stacks of high-symmetry [Co4(μ3-OMe)4] cubanes in tetragonal space groups. The inner cores of the clusters have almost the same geometry but the distances between them are longer for the ethoxy than the methoxy derivative. The magnetic interaction within the cluster is ferromagnetic in both cases but spin-orbit competes with it. The different isothermal magnetizations at low temperatures may be due to different intercluster coupling. Electrospray ionization mass spectrometry (ESI-MS) from methanol solution reveals an exchange of methoxide for hydroxide and in ethanol of methoxide for ethoxide at different proportions suggesting a probable "step by step" substitution. Interestingly, besides the [Co4] cubane a number of higher nuclearity species [Co5] and [Co7] were also observed but are not expected under electrospray conditions. This can easily happen by the re-assembly of the smaller fragments produced by ionization process. This unique feature opens up new possibilities for the systematic screening of reactions in search for new cluster architectures, which may then be realised in the solid state.

Characterization of conformation-dependent prion protein epitopes.

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

HistoStitcher ©: An interactive program for accurate and rapid reconstruction of digitized whole histological sections from tissue fragments

We present an interactive program called HistoStitcher © for accurate and rapid reassembly of histology fragments into a pseudo-whole digitized histological section. HistoStitcher © provides both an intuitive graphical interface to assist the operator in performing the stitch of adjacent histology fragments by selecting pairs of anatomical landmarks, and a set of computational routines for determining and applying an optimal linear transformation to generate the stitched image. Reconstruction of whole histological sections from images of slides containing smaller fragments is required in applications where preparation of whole sections of large tissue specimens is not feasible or efficient, and such whole mounts are required to facilitate (a) disease annotation and (b) image registration with radiological images. Unlike manual reassembly of image fragments in a general purpose image editing program (such as Photoshop), HistoStitcher © provides memory efficient operation on high resolution digitized histology images and a highly flexible stitching process capable of producing more accurate results in less time. Further, by parameterizing the series of transformations determined by the stitching process, the stitching parameters can be saved, loaded at a later time, refined, or reapplied to multi-resolution scans, or quickly transmitted to another site. In this paper, we describe in detail the design of HistoStitcher © and the mathematical routines used for calculating the optimal image transformation, and demonstrate its operation for stitching high resolution histology quadrants of a prostate specimen to form a digitally reassembled whole histology section, for 8 different patient studies. To evaluate stitching quality, a 6 point scoring scheme, which assesses the alignment and continuity of anatomical structures important for disease annotation, is employed by three independent expert pathologists. For 6 studies compared with this scheme, reconstructed sections generated via HistoStitcher © scored higher than reconstructions generated by an expert pathologist using Photoshop.

The GAL genetic switch: visualisation of the interacting proteins by split‐EGFP bimolecular fluorescence complementation

A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.

Reassembly of 2D Fragments in Image Reconstruction

Digital images may be considered as collection of pixels. If a single image is divided into more than one part, then these subparts are treated as fragments for an image.Joining of 2D fragments of an in image means we have to reassemble these image’s fragments. The joining of fragments to reconstruct images and objects is a problem associated in several applications, like archeology, medicine, art restoration, and forensics.We mainly focused on 2D Image Reconstruction by joining two 2D fragments. This approach is based on the information generated from the boundary and from the color contents of the two fragments. Local curvature is calculated to obtain transformation independent coordinates. Based on this information comparison is done to obtain maximum matching parts among fragments. Finally longest matching parts can be joined to obtain single image.

Kinetics and Reaction Coordinates of the Reassembly of Protein Fragments Via Forward Flux Sampling

We studied the mechanism of the reassembly and folding process of two fragments of a split lattice protein by using forward flux sampling (FFS). Our results confirmed previous thermodynamics and kinetics analyses that suggested that the disruption of the critical core (of an unsplit protein that folds by a nucleation mechanism) plays a key role in the reassembly mechanism of the split system. For several split systems derived from a parent 48-mer model, we estimated the reaction coordinates in terms of collective variables by using the FFS least-square estimation method and found that the reassembly transition is best described by a combination of the total number of native contacts, the number of interchain native contacts, and the total conformational energy of the split system. We also analyzed the transition path ensemble obtained from FFS simulations using the estimated reaction coordinates as order parameters to identify the microscopic features that differentiate the reassembly of the different split systems studied. We found that in the fastest folding split system, a balanced distribution of the original-core amino acids (of the unsplit system) between protein fragments propitiates interchain interactions at early stages of the folding process. Only this system exhibits a different reassembly mechanism from that of the unsplit protein, involving the formation of a different folding nucleus. In the slowest folding system, the concentration of the folding nucleus in one fragment causes its early prefolding, whereas the second fragment tends to remain as a detached random coil. We also show that the reassembly rate can be either increased or decreased by tuning interchain cooperativeness via the introduction of a single point mutation that either strengthens or weakens one of the native interchain contacts (prevalent in the transition state ensemble).