Curation and reporting of pathogenic genome-wide copy-number variants in a prenatal cell-free DNA screen.

Purpose: Advances in fetal fraction amplification in prenatal cell-free DNA screening now allow for high-resolution detection of copy-number variants (CNVs). However, approaches to interpreting CNVs as part of a primary screen are still evolving and require consensus. Here, we present a conservative, patient-centered framework for reporting fetal CNVs.

Correction: Inaccuracies and shortcomings in "Adherence of cell-free DNA noninvasive prenatal screens to ACMG recommendations".

This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY-NC-ND 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.

A front-face 'Si synthase' engineered from a retaining 'double-S2' hydrolase.

Si-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since Si-like, S1 and S2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double S2 substitution, changed its mode to Si-like.

Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog.

Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible.

Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells.

We demonstrate the evolution of the PylRS/tRNA(CUA) pair for genetically encoding photocaged cysteine. By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, we establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. We demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.

A dual role of H4K16 acetylation in the establishment of yeast silent chromatin.

Discrete regions of the eukaryotic genome assume heritable chromatin structure that is refractory to transcription. In budding yeast, silent chromatin is characterized by the binding of the Silent Information Regulatory (Sir) proteins to unmodified nucleosomes. Using an in vitro reconstitution assay, which allows us to load Sir proteins onto arrays of regularly spaced nucleosomes, we have examined the impact of specific histone modifications on Sir protein binding and linker DNA accessibility.

Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair.

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA(CUA Pyl) in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast.

Regulation of cellular metabolism by protein lysine acetylation.

Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue.

A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation.

Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine.

Designer enzymes for glycosphingolipid synthesis by directed evolution.

Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities.

High-throughput screening of cell lysates for ganglioside synthesis.

A high-throughput screen to detect the synthesis of natural and non-natural gangliosides by cell lysates has been developed and automated. Utilizing the binding specificity of cholera toxin B-subunit for the oligosaccharide moiety of the ganglioside G M1, the synthesis of sugar-sphingolipid glycosidic linkages was detected using a modified enzyme-linked immunosorbent assay (ELISA)/enzyme-linked lectin assay (ELLA). The screen was optimized and validated for high-throughput screening of cell lysates by evaluating different vectors, promoters, substrates and detection strategies.

Endovascular repair of traumatic thoracic aortic disruptions with "stacked" abdominal endograft extension cuffs.

Objective: Endovascular stent graft repair of a traumatic thoracic aortic disruption (TTAD) is rapidly becoming an accepted alternative to open surgical repair. The use of currently approved thoracic stent grafts especially in younger patients with small, "steep," tapered aortas, remains a concern due to the acute thoracic endograft collapse and enfolding. The objective of this study, the largest report to date, was to evaluate the mid-term results of TTAD treated with abdominal aortic "stacked" extension cuffs, with follow-up extending to 41 months.

Retrievability of the Günther Tulip vena cava filter after dwell times longer than 180 days in patients with multiple trauma.

Purpose: To evaluate the retrieval feasibility of the Günther Tulip inferior vena cava filter (IVCF) after dwell times >180 days in patients with multiple trauma.

Methods: A retrospective study was conducted of 117 multiple-trauma patients (70 men; mean age 36 years, range 17 to 64) who underwent prophylactic placement of Günther Tulip retrievable IVCFs between December 1, 2003 and October 1, 2006. Prior to IVCF retrieval, all patients had vena cavography to identify possible IVCF thrombus entrapment.

Structural and mechanistic analyses of endo-glycoceramidase II, a membrane-associated family 5 glycosidase in the Apo and GM3 ganglioside-bound forms.

endo-Glycoceramidase, a membrane-associated family 5 glycosidase, deviates from the typical polysaccharide substrate specificity of other soluble members of the family, preferentially hydrolyzing glycosidic linkages between the oligosaccharide and ceramide moieties of gangliosides. Here we report the first x-ray crystal structures of an endo-glycoceramidase from Rhodococcus sp., in the apo form, in complex with the ganglioside G(M3) (Svennerholm ganglioside nomenclature (Svennerholm, L.

Atherosclerotic aneurysm formation in a lower extremity saphenous vein graft.

Saphenous vein is the most widely used conduit for arterial bypass procedures and aneurysms of the vein graft are rare. We report a true aneurysm of a reversed femoropopliteal saphenous vein graft implanted nine years earlier. Duplex ultrasonography identified an aneurysm of the saphenous vein graft and arteriography confirmed this finding.

Engineering of glycosidases and glycosyltransferases.

In recent years, substantial advances have been made in the engineering of glycosidases and glycosyltransferases for the synthesis and degradation of glycan structures. Key developments include improvement of the thermostability of xylanase through comprehensive saturation mutagenesis, creation of the first glycosynthase derived from an inverting glycosidase and the emergence of a new class of modified glycosidases capable of efficiently synthesizing thioglycosidic linkages. Of particular note is the increased use of random mutagenesis and directed evolution tactics for tailoring glycosidase activity.

The synthesis of water soluble decalin-based thiols and S-nitrosothiols--model systems for studying the reactions of nitric oxide with protein thiols.

The syntheses of three decalin-based tert-thiols displaying varying degrees of solubility in aqueous milieu are described. The S-nitroso derivatives of these compounds have also been prepared and the structures of two of these determined by single crystal X-ray diffraction. These compounds have been designed for studying the interaction of nitric oxide (NO) with thiols under physiological conditions.

Developing promiscuous glycosidases for glycoside synthesis: residues W433 and E432 in Sulfolobus solfataricus beta-glycosidase are important glucoside- and galactoside-specificity determinants.

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification.

Naturally occurring Ehrlichia chaffeensis infection in two prosimian primate species: ring-tailed lemurs (Lemur catta) and ruffed lemurs (Varecia variegata).

A naturally occurring infection of Ehrlichia chaffeensis in lemurs is described. DNA of Ehrlichia chaffeensis was identified by polymerase chain reaction in peripheral blood from six of eight clinically ill lemurs. Organisms were cultured from the blood of one lemur exhibiting clinical and hematologic abnormalities similar to those of humans infected with E.

Molecular evidence supporting Ehrlichia canis-like infection in cats.

Currently, the pathogenic role of Ehrlichia canis in cats has been proposed predominantly on the basis of the serologic evidence of natural infection and the infrequent detection of morulae-like structures within the cytoplasm of leukocytes in cats. The purpose of this report was to provide molecular evidence supporting E. canis-like infection in 3 cats that had clinical manifestations consistent with canine ehrlichiosis but lacked antibodies to E.