Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols.

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

(123)I-FP-CIT SPECT imaging in early diagnosis of dementia in patients with and without a vascular component.

Alzheimer's disease (AD) and vascular dementia (VaD) are the most common cause of dementia. Cerebral ischemia is a major risk factor for development of dementia. (123)I-FP-CIT SPECT (DaTScan) is a complementary tool in the differential diagnoses of patients with incomplete or uncertain Parkinsonism. Additional application of DaTScan enables the categorization of Parkinsonian disease with dementia (PDD), and its differentiation from pure AD, and may further contribute to change the therapeutic decision. The aim of this study was to analyze the vascular contribution towards dementia and mild cognitive impairment (MCI). We evaluated the utility of DaTScan for the early diagnosis of dementia in patients with and without a clinical vascular component, and the association between neuropsychological function, vascular component and dopaminergic function on DaTScan. One-hundred and five patients with MCI or the initial phases of dementia were studied prospectively. We developed an initial assessment using neurologic examination, blood tests, cognitive function tests, structural neuroimaging and DaTScan. The vascular component was later quantified in two ways: clinically, according to the Framingham Risk Score (FRS) and by structural neuroimaging using Wahlund Scale Total Score (WSTS). Early diagnosis of dementia was associated with an abnormal DaTScan. A significant association was found between a high WSTS and an abnormal DaTScan (p < 0.01). Mixed AD was the group with the highest vascular component, followed by the VaD group, while MCI and pure AD showed similar WSTS. No significant associations were found between neuropsychological impairment and DaTScan independently of associated vascular component. DaTScan seems to be a good tool to discriminate, in a first clinical assessment, patients with MCI from those with established dementia. There was bigger general vascular affectation observable in MRI or CT in patients with abnormal dopaminergic uptake seen on DaTScan.

HIV-associated neurocognitive disorders.

Currently, neuropsychological impairment among HIV+ patients on antiretroviral therapy leads to a reduction in the quality of life and it is an important challenge due to the high prevalence of HIV-associated neurocognitive disorders and its concomitant consequences in relation to morbidity and mortality- including those HIV+ patients with adequate immunological and virological status. The fact that the virus is established in CNS in the early stages and its persistence within the CNS can help us to understand HIV-related brain injury even when highly active antiretroviral therapy is effective. The rising interest in HIV associated neurocognitive disorders has let to development new diagnostic tools, improvement of the neuropsychological tests, and the use of new biomarkers and new neuroimaging techniques that can help the diagnosis. Standardization and homogenization of neurocognitive tests as well as normalizing and simplification of easily accessible tools that can identify patients with increased risk of cognitive impairment represent an urgent requirement. Future efforts should also focus on diagnostic keys and searching for useful strategies in order to decrease HIV neurotoxicity within the CNS.

Quantitation method of N,N '-disalicylidene-1,2-propanediamine by comprehensive two-dimensional gas chromatography coupled to a nitrogen chemiluminescence detector.

Metal deactivator additives (MDAs) have been used for over 60 years to prevent metal catalyzed reactions in petroleum products; a commonly used metal deactivator is N,N'-disalicylidene-1,2-propanediamine. The quantitation of low MDA concentrations in fuels is challenging due to the complexity of the sample matrix. In this work, this difficulty was overcome using GC × GC hyphenated with a nitrogen chemiluminescence detector. The high resolution power of GC × GC avoided co-elution between the MDA and other sample matrix compounds; while the enhanced sensitivity of GC × GC and the use of a nitrogen chemiluminescence detector supplied a high sensitivity and specificity for nitrogen compounds. For the analysis, the MDA additive was derivatized with the silylation agent N,O-bis (trimethylsilyl)trifluoroacetamide at room temperature and its quantitation was based on an external calibration curve; good linear response was obtained in the 1.4-8.6 ppm range.

1,1-ethylenedithiolato complexes of palladium(II) and platinum(II) with isocyanide and carbene ligands.

The reactions of [Tl(2)[S(2)C=C[C(O)Me](2)]](n) with [MCl(2)(NCPh)(2)] and CNR (1:1:2) give complexes [M[eta(2)-S(2)C=C[C(O)Me](2)](CNR)(2)] [R = (t)Bu, M = Pd (1a), Pt (1b); R = C(6)H(3)Me(2)-2,6 (Xy), M = Pd (2a), Pt (2b)]. Compound 1b reacts with AgClO(4) (1:1) to give [[Pt(CN(t)Bu)(2)](2)Ag(2)[mu(2),eta(2)-(S,S')-[S(2)C=C[C(O)Me](2)](2)]](ClO(4))(2) (3). The reactions of 1 or 2 with diethylamine give mixed isocyanide carbene complexes [M[eta(2)-S(2)C=C[C(O)Me](2)](CNR)[C(NEt(2))(NHR)]] [R = (t)Bu, M = Pd (4a), Pt (4b); R = Xy, M = Pd (5a), Pt (5b)] regardless of the molar ratio of the reagents. The same complexes react with an excess of ammonia to give [M[eta(2)-(S,S')-S(2)C=C[C(O)Me](2)](CN(t)Bu)[C(NH(2))(NH(t)Bu)]] [M = Pd (6a), Pt (6b)] or [M[eta(2)-(S,S')-S(2)C=C[C(O)Me](2)][C(NH(2))(NHXy)](2)] [M = Pd (7a), Pt (7b)] probably depending on steric factors. The crystal structures of 2b, 4a, and 4b have been determined. Compounds 4a and 4b are isostructural. They all display distorted square planar metal environments and chelating planar E,Z-2,2-diacetyl-1,1-ethylenedithiolato ligands that coordinate through the sulfur atoms.