involves karyotyping, whereas in the Netherlands, patients who undergo amniocentesis have a more limited assessment only for trisomies 13, 18, and 21 and the sex chromosomes; thus, the tradeoff is a bit different.In addition, cfDNA is provided as a screening test for trisomies 13, 18, and 21, not just for Down syndrome. However, the performance characteristics of cfDNA for trisomies 13 and 18 are not as favorable as for T21, with a higher rate of falsenegative and false-positive results. In addition, a percentage of patients-somewhere between 1.5% and 8%-fail to obtain a result, usually because of insufficient fetal DNA. Such "low fetal fraction" is associated with obesity, which is a significant problem affecting a high percentage of reproductive-aged women in the United States. It is estimated that 20% to 50% of cfDNA tests fail to provide adequate fetal DNA in obese women. In addition, low fetal fraction is also associated with aneuploidy; therefore, women with cfDNA test failure should be considered high risk and offered follow-up with diagnostic testing (as well as a second attempt at cfDNA). When these potential outcomes are all considered, the performance characteristics of cfDNA versus traditional screening are not as clearly superior.Like the authors of this abstracted paper, several other authors and experts (Prenat Diagn 2013;33 (7):636-642) have suggested a contingent approach, using multiple marker screening as an initial screening tool, and then offering NIPT to intermediate-risk patients and either cfDNA or invasive testing to the highest-risk patients. Before completely changing the current standard of care, we need to understand this tradeoff. These authors consider comparative costs, but do not really provide incremental cost-effectiveness ratios, which are the best way to compare these strategies. While cfDNA is a better test if we are looking at a very precise test for a single disorder, only in patients in whom the test is successful at providing a result current screening may be preferable if we are looking to screen the entire population for a broad range of birth defects. Cost utility analyses, conducted by independent investigators and considering all important outcomes, are clearly needed before our approach completely changes. -MEN)

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

Knowledge of how the brain achieves its diverse central control of basic physiology is severely limited by the virtual absence of appropriate cell models. Isolation of clonal populations of unique peptidergic neurons from the hypothalamus will facilitate these studies. Herein we describe the mass immortalization of mouse primary hypothalamic cells in monolayer culture, resulting in the generation of a vast representation of hypothalamic cell types. Subcloning of the heterogeneous cell populations resulted in the establishment of 38 representative clonal neuronal cell lines, of which 16 have been further characterized by analysis of 28 neuroendocrine markers. These cell lines represent the first available models to study the regulation of neuropeptides associated with the control of feeding behavior, including neuropeptide Y, ghrelin, urocortin, proopiomelanocortin, melanin-concentrating hormone, neurotensin, proglucagon, and GHRH. Importantly, a representative cell line responds appropriately to leptin stimulation and results in the repression of neuropeptide Y gene expression. These cell models can be used for detailed molecular analysis of neuropeptide gene regulation and signal transduction events involved in the direct hormonal control of unique hypothalamic neurons, not yet possible in the whole brain. Such studies may contribute information necessary for the strategic design of therapeutic interventions for complex neuroendocrine disorders, such as obesity.