Recessive LAMC3 mutations cause malformations of occipital cortical development

The biological basis for regional and inter-species differences in cerebral cortical morphology is poorly understood. We focused on consanguineous Turkish families with a single affected member with complex bilateral occipital cortical gyration abnormalities. By using whole-exome sequencing, we initially identified a homozygous 2-bp deletion in LAMC3, the laminin γ3 gene, leading to an immediate premature termination codon. In two other affected individuals with nearly identical phenotypes, we identified a homozygous nonsense mutation and a compound heterozygous mutation. In human but not mouse fetal brain, LAMC3 is enriched in postmitotic cortical plate neurons, localizing primarily to the somatodendritic compartment. LAMC3 expression peaks between late gestation and late infancy, paralleling the expression of molecules that are important in dendritogenesis and synapse formation. The discovery of the molecular basis of this unusual occipital malformation furthers our understanding of the complex biology underlying the formation of cortical gyrations.     

Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate

Biotherapeutics are subject to immune surveillance within the body, and anti-biotherapeutic immune responses can compromise drug efficacy and patient safety. Initial development of targeted antidrug immune memory is coordinated by T cell recognition of immunogenic subsequences, termed “T cell epitopes.” Biotherapeutics may therefore be deimmunized by mutating key residues within cognate epitopes, but there exist complex trade-offs between immunogenicity, mutational load, and protein structure–function. Here, a protein deimmunization algorithm has been applied to P99 beta-lactamase, a component of antibody-directed enzyme prodrug therapies. The algorithm, integer programming for immunogenic proteins, seamlessly integrates computational prediction of T cell epitopes with both 1- and 2-body sequence potentials that assess protein tolerance to epitope-deleting mutations. Compared to previously deimmunized P99 variants, which bore only one or two mutations, the enzymes designed here contain 4–5 widely distributed substitutions. As a result, they exhibit broad reductions in major histocompatibility complex recognition. Despite their high mutational loads and markedly reduced immunoreactivity, all eight engineered variants possessed wild-type or better catalytic activity. Thus, the protein design algorithm is able to disrupt broadly distributed epitopes while maintaining protein function. As a result, this computational tool may prove useful in expanding the repertoire of next-generation biotherapeutics.     

Reestablishment of the inactive X chromosome to the ground state through cell fusion-induced reprogramming

The restricted gene expression pattern of a differentiated cell can be reversed by fusion of the somatic cell with a more developmentally potent cell type, such as an embryonic stem (ES) cell. During this reprogramming process, somatic cells obtain most of the characteristics of pluripotent cells. Reactivation of an inactive X chromosome (Xi) is an important epigenetic marker confirming the pluripotent reprogramming of somatic cells. Female somatic cells contain one active X chromosome (Xa) and one Xi, and following the fusion of these cells with male ES cells, the Xi becomes activated, resulting in XaXaXaY fusion hybrid cells. To monitor Xi reactivation, transgenic female neural stem cells (fNSCs) carrying a green fluorescent protein (GFP) reporter gene expressed on the Xa (X-GFP), but not on the Xi, were used for reprogramming. XaXi^GFP NSCs, whose GFP reporter was silenced, were fused with HM1 ES cells (XY) to induce pluripotent reprogramming. The Xi^GFP of NSCs were found to be activated on day 4 post-fusion, indicating reactivation of the Xi. Hybrid cells showed pluripotent cell-specific characteristics cells including inactivation of the NSC marker Nestin, DNA demethylation of Oct4, DNA methylation of Nestin, and reactivation of the Xi. Following differentiation of the (GFP-positive) hybrid cells through embryoid body formation, the proportion of GFP-negative cells was found to be approximately 26?%, indicating that there was random inactivation of one of the three Xas. Here, we showed that the Xi of somatic cells is reprogrammed to the Xa state and that cellular differentiation occurs randomly, i.e., regardless of the Xa or Xi state, indicating that the memory of the Xi of somatic cells has been erased and reset to the ground state (i.e., inner cell mass-like state), indicating that random X-chromosome inactivation occurs upon differentiation.     

Genome-wide functional analysis of pathogenicity genes in the rice blast fungus

Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.     

Mapping photonic entanglement into and out of a quantum memory

Developments in quantum information science rely critically on entanglement-a fundamental aspect of quantum mechanics that causes parts of a composite system to show correlations stronger than can be explained classically. In particular, scalable quantum networks require the capability to create, store and distribute entanglement among distant matter nodes by means of photonic channels. Atomic ensembles can play the role of such nodes. So far, in the photon-counting regime, heralded entanglement between atomic ensembles has been successfully demonstrated through probabilistic protocols. But an inherent drawback of this approach is the compromise between the amount of entanglement and its preparation probability, leading to intrinsically low count rates for high entanglement. Here we report a protocol where entanglement between two atomic ensembles is created by coherent mapping of an entangled state of light. By splitting a single photon and performing subsequent state transfer, we separate the generation of entanglement and its storage. After a programmable delay, the stored entanglement is mapped back into photonic modes with overall efficiency of 17%. Together with improvements in single-photon sources, our protocol will allow 'on-demand' entanglement of atomic ensembles, a powerful resource for quantum information science.     

Brain metabolism dictates the polarity of astrocyte control over arterioles

Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue. When oxygen availability is lowered and astrocyte calcium concentration is elevated, astrocyte glycolysis and lactate release are maximized. External lactate attenuates transporter-mediated uptake from the extracellular space of prostaglandin E(2), leading to accumulation and subsequent vasodilation. In conditions of low oxygen concentration extracellular adenosine also increases, which blocks astrocyte-mediated constriction, facilitating dilation. These data reveal the role of metabolic substrates in regulating brain blood flow and provide a mechanism for differential astrocyte control over cerebrovascular diameter during different states of brain activation.     

Optimization of Casting Process for Heat and Abrasion Resistant Large Gray Iron Castings

Numerical simulations were used to optimize the casting design and conditions for large cast iron castings for marine engines, Simulations of the mold filling and solidification sequences were used to analyze the problems of previous casting conditions with marked improvements for large cylinder liner parts, The amount and positions of chills were optimized to improve the mechanical properties and to minimize the shrinkage and micro porosity in the castings. Ultra sonic testing, penetration testing, and mechanical property testing show no defects in the castings with the productivity significantly increased.