Stochastic association of neighboring replicons creates replication factories in budding yeast

Peer reviewedPublisher PD

Long term geological record of a global deep subsurface microbial habitat in sand injection complexes

Peer reviewedPublisher PD

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein

Segregation of object and background motion in the retina

An important task in vision is to detect objects moving within a stationary scene. During normal viewing this is complicated by the presence of eye movements that continually scan the image across the retina, even during fixation. To detect moving objects, the brain must distinguish local motion within the scene from the global retinal image drift due to fixational eye movements. We have found that this process begins in the retina: a subset of retinal ganglion cells responds to motion in the receptive field centre, but only if the wider surround moves with a different trajectory. This selectivity for differential motion is independent of direction, and can be explained by a model of retinal circuitry that invokes pooling over nonlinear interneurons. The suppression by global image motion is probably mediated by polyaxonal, wide-field amacrine cells with transient responses. We show how a population of ganglion cells selective for differential motion can rapidly flag moving objects, and even segregate multiple moving objects

Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.Harvard University. Society of FellowsNational Human Genome Research Institute (U.S.) (Center for Excellence in Genomics Science P50 HG003170)United States. Dept. of Energy (Genomes to Life DE-FG02-02ER63445)United States. Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.)Wyss Institute of Biologically Inspired EngineeringNational Institutes of Health (U.S.) (Transformative R01 (R01 NS073124-01))European School of Molecular Medicine (predoctoral fellowship

γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor

Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine—the immediate glutathione precursor—to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor

The taper of cast post preparation measured using innovative image processing technique

<p>Abstract</p> <p>Background</p> <p>No documentation in the literature about taper of cast posts. This study was conducted to measure the degree of cast posts taper, and to evaluate its suitability based on the anatomy aspects of the common candidate teeth for post reconstruction.</p> <p>Methods</p> <p>Working casts for cast posts, prepared using Gates Glidden drills, were collected. Impressions of post spaces were made using polyvinyl siloxan putty/wash technique. Digital camera with a 10' high quality lens was used for capturing two digital images for each impression; one in the Facio-Lingual (FL) and the other in the Mesio-Distal (MD) directions. Automated image processing program was developed to measure the degree of canal taper. Data were analyzed using Statistical Package for Social Sciences software and One way Analysis of Variance.</p> <p>Results</p> <p>Eighty four dies for cast posts were collected: 16 for each maxillary anterior teeth subgroup, and 18 for each maxillary and mandibular premolar subgroup. Mean of total taper for all preparations was 10.7 degree. There were no statistical differences among the total taper of all groups (P = .256) or between the MD and FL taper for each subgroup. Mean FL taper for the maxillary first premolars was lower significantly (P = .003) than the maxillary FL taper of the second premolars. FL taper was higher than the MD taper in all teeth except the maxillary first premolars.</p> <p>Conclusions</p> <p>Taper produced did not reflect the differences among the anatomy of teeth. While this technique deemed satisfactory in the maxillary anterior teeth, the same could not be said for the maxillary first premolars. Careful attention to the root anatomy is mandatory.</p

Alternative Splicing Events Are a Late Feature of Pathology in a Mouse Model of Spinal Muscular Atrophy

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT–PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival

A mini-review on the development of Si-based thin film anodes for Li-ion batteries

This review provides a summary of the progress in research on various Si-based thin films as anode materials for lithium-ion batteries. The lithiation mechanism models, different types of materials from pure monolithic Si thin film to Si-based three-dimensional structured composite thin films, the effect of liquid and solid-state electrolytes on the performance of Si were considered and various available preparation techniques were discussed. A table summarizing important information on such systems including the thin film features, preparation methods and conditions, electrochemical test conditions and obtained results in order to elucidate the approaches used to prepare a stable thin film anode with high capacity and long cycle life is provided. We believe that this review will help the researchers to find some answers and induce some new ideas