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

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A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase.

SNi-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 SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus ß-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a ß-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a ß-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'ß-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.

Expanding Maize Genetic Resources with Predomestication Alleles: Maize-Teosinte Introgression Populations.

Teosinte ( subsp. H. H. Iltis & Doebley) has greater genetic diversity than maize inbreds and landraces ( subsp. ). There are, however, limited genetic resources to efficiently evaluate and tap this diversity. To broaden resources for genetic diversity studies in maize, we developed and evaluated 928 near-isogenic introgression lines (NILs) from 10 teosinte accessions in the B73 background. Joint linkage analysis of the 10 introgression populations identified several large-effect quantitative trait loci (QTL) for days to anthesis (DTA), kernel row number (KRN), and 50-kernel weight (Wt50k). Our results confirm prior reports of kernel domestication loci and identify previously uncharacterized QTL with a range of allelic effects enabling future research into the genetic basis of these traits. Additionally, we used a targeted set of NILs to validate the effects of a KRN QTL located on chromosome 2. These introgression populations offer novel tools for QTL discovery and validation as well as a platform for initiating fine mapping.

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. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.

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. Two typical marks for active chromatin, H3K79(me) and H4K16(ac) decrease the affinity of Sir3 for chromatin, yet only H4K16(ac) affects chromatin structure, as measured by nuclease accessibility. Surprisingly, we found that the Sir2-4 subcomplex, unlike Sir3, has higher affinity for chromatin carrying H4K16(ac). NAD-dependent deacetylation of H4K16(ac) promotes binding of the SIR holocomplex but not of the Sir2-4 heterodimer. This function of H4K16(ac) cannot be substituted by H3K56(ac). We conclude that acetylated H4K16 has a dual role in silencing: it recruits Sir2-4 and repels Sir3. Moreover, the deacetylation of H4K16(ac) by Sir2 actively promotes the high-affinity binding of the SIR holocomplex.

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. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs to be site-specifically incorporated 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. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.

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. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.

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. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.

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. The extent of ganglioside synthesis was found to be proportional to enzyme concentration and length of incubation time. As a test of the finalized screen efficacy, individual colonies from a saturation mutagenesis library of nucleophile mutants of an endoglycoceramidase were screened to identify the most active enzyme for ganglioside synthesis. This screen should find general application in assaying both glycolipid biosynthesis and glycolipid hydrolysis, as it is highly sensitive and can be used with crude cell extracts.

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. METHODS: Thirty-one patients with multi-system trauma (age range, 15 to 61; mean 31.4 years) were seen after motor vehicle accidents between January 1, 2003 and July 1, 2007. Chest x-ray findings warranted thoracic CT scans, which revealed disruptions of the thoracic aorta. Intra-operative arteriograms in all patients and intravascular ultrasound (IVUS) (n = 17) delineated the extent of the aortic injuries. The aortic length from the subclavian artery to the injury averaged 2.5 cm (range, 1.5 to 4.0 cm). The repairs were performed with Gore (W.L. Gore & Associates, Flagstaff, Ariz) (n = 15), Aneuryx (Medtronic, Santa Rosa, Calif) (n = 15), and Zenith (Cook, Inc., Bloomington, Ind) (n = 1) Aortic Extension Cuffs. A femoral artery approach was used in 27 patients and a supra-inguinal retroperitoneal iliac approach in four. All patients underwent thoracic CT scans during follow-up. RESULTS: In all patients, the stent-graft cuffs successfully excluded the TTAD: 21 patients had 2 cuffs, 9 had 3 cuffs, and 1 had 4 cuffs. The aorta adjacent to the injury mean diameter was 18.5 mm (range, 17-24 mm). No subclavian arteries were covered. Two patients required an additional cuff for exclusion of the Type I endoleaks at the distal attachment site within 6 weeks of initial endograft repair. There were no procedure-related deaths; 2 patients died of other injuries. At follow-up, extending to 41 months (range, 3 to 41 months), two pseudo-aneurysms occurred which required open operative repair: 1 due to infection (4 months) and a leaking pseudoaneurysm (14 months). A CT scan in all other survivors demonstrated no device-related complications, endoleaks, or cuff migrations. CONCLUSION: Stent-graft repair of TTAD is technically feasible. The technique of "stacked" aortic cuffs provides an acceptable option when urgent therapy is needed, when patients are deemed high-risk for open operative repair, or until thoracic endografts are designed which can safely treat focal, smaller aortic diameter injuries.

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. Filter retrieval was performed in the catheterization laboratory under sterile conditions from a right internal jugular vein approach. After IVCF retrieval, repeat vena cavography was performed to evaluate the IVC for contrast extravasation, intraluminal defects, or IVC narrowing. RESULTS: Twelve (10.3%) filters were not retrieved as the patients died of their injuries (no deaths related to IVCF placement or retrieval). Forty-one (35.0%) filters had dwell times >180 days (mean 261.5 days, range 182-403). Of these, 31 (76%) were uneventfully retrieved; 10 were left in place permanently. Pre-retrieval vena cavography identified filter tilting in 13 filters: 9 had a mild tilt < or =10 degrees, and 4 had severe tilting > or =25 degrees. All of the 10 filters that could not be retrieved were tilted (4 severe and 6 mild). In comparison to the 64 (54.7%) filters in place for <180 days (mean 51 days, range 42-180), 4 (6.2%) could not be retrieved (p = 0.367). No filter had trapped thrombus identified by vena cavography at the time of retrieval. None of the retrieved filters had structural fracture or collapse, and none had migrated. Post-retrieval vena cavograms demonstrated no contrast extravasation, intraluminal defects, or impingement on adjacent organs. CONCLUSION: If retrieval of a Gunther Tulip filter with an dwell time >180 days is considered, the patient should be ambulatory and a candidate for anticoagulation if indicated; notably, the filter should have a <25 degrees tilt. Under these circumstances, retrieval of the Günther Tulip filter after 180 days of dwell time appears justified and safe.

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. (1964) J. Lipid Res. 5, 145-155)), and trapped as a glycosyl-enzyme intermediate. These snapshots provide the first molecular insight into enzyme recognition and association with gangliosides, revealing the structural adaptations necessary for glycosidase-catalyzed hydrolysis and detailing a novel ganglioside binding topology. Consistent with the chemical duality of the substrate, the active site of endo-glycoceramidase is split into a wide, polar cavity to bind the polyhydroxylated oligosaccharide moiety and a narrow, hydrophobic tunnel to bind the ceramide lipid chains. The specific interactions with the ceramide polar head group manifest a surprising aglycone specificity, an observation substantiated by our kinetic analyses. Collectively, the reported structural and kinetic data provide insight toward rational redesign of the synthetic glycosynthase mutant of endo-glycoceramidase to enable facile synthesis of nonnatural, therapeutically useful gangliosides.

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. A consideration for endovascular coiling of the aneurysm was entertained, but no proximal "neck" on the aneurysm was present. The patient, therefore, underwent an uneventful resection of the aneurysm with end-to-end anastomosis. Histopathologic examination of the aneurysm demonstrated atherosclerotic degeneration with endothelial disruption, medial necrosis, and fibrous proliferation. The cause of a saphenous vein graft aneurysm is rare and unknown. The management of vein graft aneurysms, however, should be subject to the same criteria that apply to other aneurysms because once vein graft dilation occurs, it is followed by a rapid increase in size, which may lead to possible rupture.