Genome-scale engineering for systems and synthetic biology

Genome-modification technologies enable the rational engineering and perturbation of biological systems. Historically, these methods have been limited to gene insertions or mutations at random or at a few pre-defined locations across the genome. The handful of methods capable of targeted gene editing suffered from low efficiencies, significant labor costs, or both. Recent advances have dramatically expanded our ability to engineer cells in a directed and combinatorial manner. Here, we review current technologies and methodologies for genome-scale engineering, discuss the prospects for extending efficient genome modification to new hosts, and explore the implications of continued advances toward the development of flexibly programmable chasses, novel biochemistries, and safer organismal and ecological engineering

Quantum SU(2) faithfully detects mapping class groups modulo center

The Jones-Witten theory gives rise to representations of the (extended) mapping class group of any closed surface Y indexed by a semi-simple Lie group G and a level k. In the case G=SU(2) these representations (denoted V_A(Y)) have a particularly simple description in terms of the Kauffman skein modules with parameter A a primitive 4r-th root of unity (r=k+2). In each of these representations (as well as the general G case), Dehn twists act as transformations of finite order, so none represents the mapping class group M(Y) faithfully. However, taken together, the quantum SU(2) representations are faithful on non-central elements of M(Y). (Note that M(Y) has non-trivial center only if Y is a sphere with 0, 1, or 2 punctures, a torus with 0, 1, or 2 punctures, or the closed surface of genus = 2.) Specifically, for a non-central h in M(Y) there is an r_0(h) such that if r>= r_0(h) and A is a primitive 4r-th root of unity then h acts projectively nontrivially on V_A(Y). Jones' [J] original representation rho_n of the braid groups B_n, sometimes called the generic q-analog-SU(2)-representation, is not known to be faithful. However, we show that any braid h not= id in B_n admits a cabling c = c_1,...,c_n so that rho_N (c(h)) not= id, N=c_1 + ... + c_n.Comment: Published by Geometry and Topology at http://www.maths.warwick.ac.uk/gt/GTVol6/paper18.abs.html Version 4: Sentence added to proof of lemma 4.1, page 536, lines 7-

Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome

MRF4 (herculin/Myf-6) is one of the four member MyoD family of transcription factors identified by their ability to enforce skeletal muscle differentiation upon a wide variety of nonmuscle cell types. In this study the mouse germline MRF4 gene was disrupted by targeted recombination. Animals homozygous for the MRF4bh1 allele, a deletion of the functionally essential bHLH domain, displayed defective axial myogenesis and rib pattern formation, and they died at birth. Differences in somitogenesis between homozygous MRF4bh1 embryos and their wild-type littermates provided evidence for three distinct myogenic regulatory programs (My1-My3) in the somite, which correlate temporally and spatially with three waves of cellular recruitment to the expanding myotome. The first program (My1), marked initially by Myf-5 expression and followed by myogenin, began on schedule in the MRF4bh1/bh1 embryos at day 8 post coitum (E8). A second program (My2) was highly deficient in homozygous mutant MRF4 embryos, and normal expansion of the myotome failed. Moreover, expression of downstream muscle-specific genes, including FGF-6, which is a candidate regulator of inductive interactions, did not occur normally. The onset of MyoD expression around E10.5 in wild-type embryos marks a third myotomal program (My3), the execution of which was somewhat delayed in MRF4 mutant embryos but ultimately led to extensive myogenesis in the trunk. By E15 it appeared to have largely compensated for the defective My2 program in MRF4 mutants. Homozygous MRF4bh1 animals also showed improper rib pattern formation perhaps due to the absence of signals from cells expressing the My2 program. Finally, a later and relatively mild phenotype was detected in intercostal muscles of newborn animals

Changes in the Protein Profile of Streptomyces griseus during a Cycloheximide Fermentation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75202/1/j.1749-6632.1987.tb23846.x.pd

Differential neuroproteomic and systems biology analysis of spinal cord injury

Acute spinal cord injury (SCI) is a devastating condition with many consequences and no known effective treatment. Although it is quite easy to diagnose traumatic SCI, the assessment of injury severity and projection of disease progression or recovery are often challenging, as no consensus biomarkers have been clearly identified. Here rats were subjected to experimental moderate or severe thoracic SCI. At 24h and 7d postinjury, spinal cord segment caudal to injury center versus sham samples was harvested and subjected to differential proteomic analysis. Cationic/anionic-exchange chromatography, followed by 1D polyacrylamide gel electrophoresis, was used to reduce protein complexity. A reverse phase liquid chromatography-tandem mass spectrometry proteomic platform was then utilized to identify proteome changes associated with SCI. Twenty-two and 22 proteins were up-regulated at 24 h and 7 day after SCI, respectively; whereas 19 and 16 proteins are down-regulated at 24 h and 7 day after SCI, respectively, when compared with sham control. A subset of 12 proteins were identified as candidate SCI biomarkers - TF (Transferrin), FASN (Fatty acid synthase), NME1 (Nucleoside diphosphate kinase 1), STMN1 (Stathmin 1), EEF2 (Eukaryotic translation elongation factor 2), CTSD (Cathepsin D), ANXA1 (Annexin A1), ANXA2 (Annexin A2), PGM1 (Phosphoglucomutase 1), PEA15 (Phosphoprotein enriched in astrocytes 15), GOT2 (Glutamic-oxaloacetic transaminase 2), and TPI-1 (Triosephosphate isomerase 1), data are available via ProteomeXchange with identifier PXD003473. In addition, Transferrin, Cathepsin D, and TPI-1 and PEA15 were further verified in rat spinal cord tissue and/or CSF samples after SCI and in human CSF samples from moderate/severe SCI patients. Lastly, a systems biology approach was utilized to determine the critical biochemical pathways and interactome in the pathogenesis of SCI. Thus, SCI candidate biomarkers identified can be used to correlate with disease progression or to identify potential SCI therapeutic targets

Fluorescent Si Nanoparticle-Based Electrode for Sensing Biomedical Substances

We have been studying the miniaturization of silicon crystals and the transition from the solid state to the atomistic state. We demonstrated the existence of “sweet spots” in cluster size in the range 1–3nm that have enhanced chemical, structural, and photo stability. The particles are produced by an electrochemical etching process as dispersion in liquid, and they are reconstituted in films, patterns, alloys, or spread on chips to produce super chips. Unlike bulk, these Si nanoparticle configurations have a spectacular ability to glow in distinct RGB colors. In this paper we describe an electrode sensor built by decorating metal or heavily doped silicon electrode with nanoparticles. We demonstrated amperometric response of the electrode to glucose and compared the response to that of heavily doped silicon wafer decorated with GOx. The all silicon electrode shows improved sensitivity, selectivity and stability. Light induced modulation of the response allows phase sensitive detection. The device is suitable for miniaturization, which may enable in vivo use

Universal manifold pairings and positivity

Gluing two manifolds M_1 and M_2 with a common boundary S yields a closed manifold M. Extending to formal linear combinations x=Sum_i(a_i M_i) yields a sesquilinear pairing p= with values in (formal linear combinations of) closed manifolds. Topological quantum field theory (TQFT) represents this universal pairing p onto a finite dimensional quotient pairing q with values in C which in physically motivated cases is positive definite. To see if such a "unitary" TQFT can potentially detect any nontrivial x, we ask if is non-zero whenever x is non-zero. If this is the case, we call the pairing p positive. The question arises for each dimension d=0,1,2,.... We find p(d) positive for d=0,1, and 2 and not positive for d=4. We conjecture that p(3) is also positive. Similar questions may be phrased for (manifold, submanifold) pairs and manifolds with other additional structure. The results in dimension 4 imply that unitary TQFTs cannot distinguish homotopy equivalent simply connected 4-manifolds, nor can they distinguish smoothly s-cobordant 4-manifolds. This may illuminate the difficulties that have been met by several authors in their attempts to formulate unitary TQFTs for d=3+1. There is a further physical implication of this paper. Whereas 3-dimensional Chern-Simons theory appears to be well-encoded within 2-dimensional quantum physics, eg in the fractional quantum Hall effect, Donaldson-Seiberg-Witten theory cannot be captured by a 3-dimensional quantum system. The positivity of the physical Hilbert spaces means they cannot see null vectors of the universal pairing; such vectors must map to zero.Comment: Published by Geometry and Topology at http://www.maths.warwick.ac.uk/gt/GTVol9/paper53.abs.htm

A library of infectious hepatitis C viruses with engineered mutations in the E2 gene reveals growth-adaptive mutations that modulate interactions with scavenger receptor class B type I

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. IMPORTANCE Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability

Observability of pulsar beam bending by the Sgr~A* black hole

According to some models, there may be a significant population of radio pulsars in the Galactic center. In principle, a beam from one of these pulsars could pass close to the supermassive black hole (SMBH) at the center, be deflected, and be detected by Earth telescopes. Such a configuration would be an unprecedented probe of the properties of spacetime in the moderate- to strong-field regime of the SMBH. We present here background on the problem, and approximations for the probability of detection of such beams. We conclude that detection is marginally probable with current telescopes, but that telescopes that will be operating in the near future, with an appropriate multiyear observational program, will have a good chance of detecting a beam deflected by the SMBH.Comment: 18 pages, 16 figure