A compartmentalized approach to the assembly of physical maps

<p>Abstract</p> <p>Background</p> <p>Physical maps have been historically one of the cornerstones of genome sequencing and map-based cloning strategies. They also support marker assisted breeding and EST mapping. The problem of building a high quality physical map is computationally challenging due to unavoidable noise in the input fingerprint data.</p> <p>Results</p> <p>We propose a novel compartmentalized method for the assembly of high quality physical maps from fingerprinted clones. The knowledge of genetic markers enables us to group clones into clusters so that clones in the same cluster are more likely to overlap. For each cluster of clones, a local physical map is first constructed using FingerPrinted Contigs (FPC). Then, all the individual maps are carefully merged into the final physical map. Experimental results on the genomes of rice and barley demonstrate that the compartmentalized assembly produces significantly more accurate maps, and that it can detect and isolate clones that would induce "chimeric" contigs if used in the final assembly.</p> <p>Conclusion</p> <p>The software is available for download at <url>http://www.cs.ucr.edu/~sbozdag/assembler/</url></p

Sequencing of 15 622 Gene-bearing BACs Clarifies the Gene-dense Regions of the Barley Genome

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant

A Graph-Theoretical Approach to the Selection of the Minimum Tiling Path from a Physical Map

The problem of computing the minimum tiling path (MTP) from a set of clones arranged in a physical map is a cornerstone of hierarchical (clone-by-clone) genome sequencing projects. We formulate this problem in a graph theoretical framework, and then solve by a combination of minimum hitting set and minimum spanning tree algorithms. The tool implementing this strategy, called FMTP, shows improved performance compared to the widely used software FPC. When we execute FMTP and FPC on the same physical map, the MTP produced by FMTP covers a higher portion of the genome, and uses a smaller number of clones. For instance, on the rice genome the MTP produced by our tool would reduce by about 11 percent the cost of a clone-by-clone sequencing project. Source code, benchmark data sets, and documentation of FMTP are freely available at \u3ehttp://code.google.com/p/fingerprint-based-minimal-tiling-path/ under MIT license

Cholesterol modulates acetylcholine receptor diffusion by tuning confinement sojourns and nanocluster stability

Translational motion of neurotransmitter receptors is key for determining receptor number at the synapse and hence, synaptic efficacy. We combine live-cell STORM superresolution microscopy of nicotinic acetylcholine receptor (nAChR) with single-particle tracking, mean-squared displacement (MSD), turning angle, ergodicity, and clustering analyses to characterize the lateral motion of individual molecules and their collective behaviour. nAChR diffusion is highly heterogeneous: subdiffusive, Brownian and, less frequently, superdiffusive. At the single-track level, free walks are transiently interrupted by ms-long confinement sojourns occurring in nanodomains of ~36 nm radius. Cholesterol modulates the time and the area spent in confinement. Turning angle analysis reveals anticorrelated steps with time-lag dependence, in good agreement with the permeable fence model. At the ensemble level, nanocluster assembly occurs in second-long bursts separated by periods of cluster disassembly. Thus, millisecond-long confinement sojourns and second-long reversible nanoclustering with similar cholesterol sensitivities affect all trajectories; the proportion of the two regimes determines the resulting macroscopic motional mode and breadth of heterogeneity in the ensemble population.Fil: Mosqueira, Alejo. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Camino, Pablo A.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Towards a matrix mechanics framework for dynamic protein network

Protein–protein interaction networks are currently visualized by software generated interaction webs based upon static experimental data. Current state is limited to static, mostly non-compartmental network and non time resolved protein interactions. A satisfactory mathematical foundation for particle interactions within a viscous liquid state (situation within the cytoplasm) does not exist nor do current computer programs enable building dynamic interaction networks for time resolved interactions. Building mathematical foundation for intracellular protein interactions can be achieved in two increments (a) trigger and capture the dynamic molecular changes for a select subset of proteins using several model systems and high throughput time resolved proteomics and, (b) use this information to build the mathematical foundation and computational algorithm for a compartmentalized and dynamic protein interaction network. Such a foundation is expected to provide benefit in at least two spheres: (a) understanding physiology enabling explanation of phenomenon such as incomplete penetrance in genetic disorders and (b) enabling several fold increase in biopharmaceutical production using impure starting materials

Creating complex protocells and prototissues using simple DNA building blocks

Building synthetic protocells and prototissues hinges on the formation of biomimetic skeletal frameworks. Recreating the complexity of cytoskeletal and exoskeletal fibers, with their widely varying dimensions, cellular locations and functions, represents a major material hurdle and intellectual challenge which is compounded by the additional demand of using simple building blocks to ease fabrication and control. Here we harness simplicity to create complexity by assembling structural frameworks from subunits that can support membrane-based protocells and prototissues. We show that five oligonucleotides can anneal into nanotubes or fibers whose tunable thicknesses and lengths spans four orders of magnitude. We demonstrate that the assemblies' location inside protocells is controllable to enhance their mechanical, functional and osmolar stability. Furthermore, the macrostructures can coat the outside of protocells to mimic exoskeletons and support the formation of millimeter-scale prototissues. Our strategy could be exploited in the bottom-up design of synthetic cells and tissues, to the generation of smart material devices in medicine

Reaching Out to Tribal Communities: Lessons Learned and Approaches to Consider

When transportation safety decision-making is desired, the involvement and engagement with a community is essential. A streamlined delivery of a project or program is more likely to occur when active dialogue and an exchange of ideas occurs in advance and occurs frequently. This is particularly important in tribal communities, who value sustained relationships and represent the focus population of this study. The research team, on six separate occasions, met with local and regional tribal leaders to explore and discuss transportation safety needs within and outside tribal communities, as well as discern the recommended approaches to foster ongoing dialogue about these needs. In all cases these discussions closely correlated with existing research studies or activities; transportation safety and equity is not seen as separate from other tribal foci and community needs. Specific recommendations to consider, in no particular order, included the following: invest respectfully enough time for people to talk; tribes think long-term and consider the impact of any decision from a long-term viewpoint so an iterative process and re-sharing of ideas is critical; the power of decision is in the hands of the tribe and its members; do not lump tribes together as each tribe is sovereign and unique and every community should be expected to think differently; all tribes are unique as is the environmental and social context; to disseminate information widely and iteratively, do so when there is a large group or event; be sure to understand the Tribal governance, decision making, and organizational structure; know who is the tribal Chairman or Chairwoman; and develop an emic and etic understanding of the community

Scalable production of 3D microtissues using novel microfluidic technologies

Tissue engineering approaches are widely studied with the goal to replace or repair human tissues. However, while studies are often promising in a laboratory environment, there remain difficulties in the translation of laboratory-based studies towards clinical applications due to low in vivo efficiency and/or complex impractical procedures.An interesting strategy for improving therapy effectiveness is by evolving from conventional 2D cell culture to more biomimetic 3D cell culture approaches. While therapy efficiency can be greatly improved using 3D cell culture, current 3D microtissue production techniques are often non-scalable batch processes, limiting clinical and industrial translation. A continuous production method is needed in order to improve the microtissue production rate and improve the feasibility of clinical application.Microfluidics offers the possibility to evolve microtissue production towards a continuous process. Using conventional on-chip microfluidics, microtissues can be produced in a controlled and continuous manner by cell encapsulation in hollow microcapsules. However, conventional on-chip microfluidics offers challenges such as complex multistep processes, the use of potentially harmful oils and surfactants and often low throughputs, which are currently hampering widespread clinical and industrial translation of microfluidically produced microtissues. There is therefore a need to evolve microfluidics towards a clean, fast and single step scalable approach to fulfill the clinical requirements for tissue engineering approaches that take advantage of 3D microtissues.This thesis describes multiple microfluidic solutions that focus on overcoming these challenges hampering the widespread clinical and industrial use of microtissues. A reusable, cleanroom-free, multifunctional microfluidic device is developed using standard cutting and abrasion technology, which allows the production of microtissue-laden microcapsules in a single step-manner. This on-chip process is then evolved towards an off-chip jetting approach which allows for the production of microtissue-laden microcapsules in an ultra-high throughput manner (&gt;10 ml/min) without the need of potentially harmful oils and surfactants. This in-air microfluidic approach is also utilized for mass production of microtissues in larger compartmentalized hydrogels, which are used for the production of large clinical-sized tissues. A multitude of microtissues are formed using these described microfluidic technologies such as human mesenchymal stem cell spheroids, chondrocyte spheroids, fibroblast spheroids, cholangiocyte and cholangiocarcinoma organoids, lumen-forming embryoid bodies, contracting cardiospheres, and clinical sized cartilage tissues.To summarize, this thesis introduces multiple microfluidic systems for scalable microcapsule and microtissue production with the aim to remove the hurdles towards clinical and industrial translation of 3D microtissues.<br/

Catalytic processing in ruthenium-based polyoxometalate coacervate protocells

The development of programmable microscale materials with cell-like functions, dynamics and collective behaviour is an important milestone in systems chemistry, soft matter bioengineering and synthetic protobiology. Here, polymer/nucleotide coacervate micro-droplets are reconfigured into membrane-bounded polyoxometalate coacervate vesicles (PCVs) in the presence of a bio-inspired Ru-based polyoxometalate catalyst to produce synzyme protocells (Ru4PCVs) with catalase-like activity. We exploit the synthetic protocells for the implementation of multi-compartmentalized cell-like models capable of collective synzyme-mediated buoyancy, parallel catalytic processing in individual horseradish peroxidase-containing Ru4PCVs, and chemical signalling in distributed or encapsulated multi-catalytic protocell communities. Our results highlight a new type of catalytic micro-compartment with multi-functional activity and provide a step towards the development of protocell reaction networks. \ua9 2020, The Author(s)