1,052 research outputs found
Highly efficient methods for the one-pot synthesis of b-substituted enones
A mild and practically-convenient one-pot procedure for the direct b-substitution of enones has been developed using a conjugate addition–oxidation strategy with a full range of copper-based reagents and N-tert-butylphenylsulfinimidoyl chloride; alkyl- and aryl-substituted enones are delivered in good to excellent yields
Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.
Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency
Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.
Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics
DNA hybridization to mismatched templates: a chip study
High-density oligonucleotide arrays are among the most rapidly expanding
technologies in biology today. In the {\sl GeneChip} system, the reconstruction
of the target concentration depends upon the differential signal generated from
hybridizing the target RNA to two nearly identical templates: a perfect match
(PM) and a single mismatch (MM) probe. It has been observed that a large
fraction of MM probes repeatably bind targets better than the PMs, against the
usual expectation from sequence-specific hybridization; this is difficult to
interpret in terms of the underlying physics. We examine this problem via a
statistical analysis of a large set of microarray experiments. We classify the
probes according to their signal to noise () ratio, defined as the
eccentricity of a (PM, MM) pair's `trajectory' across many experiments. Of
those probes having large () only a fraction behave consistently with
the commonly assumed hybridization model. Our results imply that the physics of
DNA hybridization in microarrays is more complex than expected, and they
suggest new ways of constructing estimators for the target RNA concentration.Comment: 3 figures 1 tabl
Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.
Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism
Effective affinities in microarray data
In the past couple of years several studies have shown that hybridization in
Affymetrix DNA microarrays can be rather well understood on the basis of simple
models of physical chemistry. In the majority of the cases a Langmuir isotherm
was used to fit experimental data. Although there is a general consensus about
this approach, some discrepancies between different studies are evident. For
instance, some authors have fitted the hybridization affinities from the
microarray fluorescent intensities, while others used affinities obtained from
melting experiments in solution. The former approach yields fitted affinities
that at first sight are only partially consistent with solution values. In this
paper we show that this discrepancy exists only superficially: a sufficiently
complete model provides effective affinities which are fully consistent with
those fitted to experimental data. This link provides new insight on the
relevant processes underlying the functioning of DNA microarrays.Comment: 8 pages, 6 figure
The Structure of the Big Bang from Higher-Dimensional Embeddings
We give relations for the embedding of spatially-flat
Friedmann-Robertson-Walker cosmological models of Einstein's theory in flat
manifolds of the type used in Kaluza-Klein theory. We present embedding
diagrams that depict different 4D universes as hypersurfaces in a higher
dimensional flat manifold. The morphology of the hypersurfaces is found to
depend on the equation of state of the matter. The hypersurfaces possess a
line-like curvature singularity infinitesimally close to the
3-surface, where is the time expired since the big bang. The family of
timelike comoving geodesics on any given hypersurface is found to have a
caustic on the singular line, which we conclude is the 5D position of the
point-like big bang.Comment: 11 pages, 5 figures, revtex4, accepted in Class. Quant. Gra
Preparation of anti-vicinal amino alcohols: asymmetric synthesis of D-erythro-Sphinganine, (+)-spisulosine and D-ribo-phytosphingosine
Two variations of the Overman rearrangement have been developed for the highly selective synthesis of anti-vicinal amino alcohol natural products. A MOM-ether directed palladium(II)-catalyzed rearrangement of an allylic trichloroacetimidate was used as the key step for the preparation of the protein kinase C inhibitor D-erythro-sphinganine and the antitumor agent (+)-spisulosine, while the Overman rearrangement of chiral allylic trichloroacetimidates generated by asymmetric reduction of an alpha,beta-unsaturated methyl ketone allowed rapid access to both D-ribo-phytosphingosine and L-arabino-phytosphingosine
Solving the riddle of the bright mismatches: hybridization in oligonucleotide arrays
HDONA technology is predicated on two ideas. First, the differential between
high-affinity (perfect match, PM) and lower-affinity (mismatch, MM) probes is
used to minimize cross-hybridization. Second, several short probes along the
transcript are combined, introducing redundancy. Both ideas have shown problems
in practice: MMs are often brighter than PMs, and it is hard to combine the
pairs because their brightness often spans decades. Previous analysis suggested
these problems were sequence-related; publication of the probe sequences has
permitted us an in-depth study of this issue. Our results suggest that
fluorescently labeling the nucleotides interferes with mRNA binding, causing a
catch-22 since, to be detected, the target mRNA must both glow and stick to its
probe: without labels it cannot be seen even if bound, while with too many it
won't bind. We show that this conflict causes much of the complexity of HDONA
raw data, suggesting that an accurate physical understanding of hybridization
by incorporating sequence information is necessary to perfect microarray
analysis.Comment: 4 figure
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