Divergent access to a novel 3,4-diaminophytosphingosine-like ceramide via sequential Overman rearrangement.

A straightforward approach to a novel phytosphingosine-like ceramide has been accomplished. The cornerstone features of this divergent synthesis are a cascade Overman rearrangement of tris(imidate) to introduce three desired stereogenic centres via sequential chirality transfer and an effective olefin cross-metathesis to install a long side chain. The final unusual phytoceramides were evaluated for their capacity to inhibit the proliferation of cancer cell lines. The preliminary results revealed that compound 21 exhibits promising anticancer activity against HeLa and HCT-116 cells as well as the excellent selectivity in cytotoxicity (malignant vs non-malignant cell lines).

Dynamic study of small toxic hydrophobic proteins PepA1 and PepG1 of Staphylococcus aureus.

Toxin-antitoxin (TA) systems are small genetic elements which encode toxin proteins that interfere with vital cellular functions. PepA1 and PepG1 toxin proteins, known also as SprA1 and SprG1, are type I TA. In Staphylococcus aureus (S. aureus), their expression without the antitoxin counterparts (SprA1AS and SprF1), is lethal to the pathogen. Molecular Dynamics (MD) simulation was performed for PepA1 and PepG1 to understand their dynamic state, conformational changes, and their toxicity. The protein structures were constructed and used for MD simulation and the conformational changes, stability, flexibility, fluctuations, hydrophobicity, and role of their dynamic state on function prediction were studied extensively by GROMACS MD simulation analysis tools. In silico study indicated that the PepA1 and PepG1 proteins change their structural conformation from an open to closed state where PepA1 conformational changes were faster (10 ns) than PepG1 (20 ns) while PepG1 exerted more stability and flexibility than PepA1. According to SASA values, PepG1 is more hydrophobic than the PepA1 and forms fewer hydrogen bonds than PepA1. The in vivo study with PepA1 and PepG1 proteins provided evidence that both the conformation changes between the open and closed states and the amino acid sequence are crucial for peptide toxicity.

Micro-hub location selection for sustainable last-mile delivery.

Sustainable Last-Mile Delivery (LMD) is one of the key phases in city logistics. Micro-hubs in cities are new emerging solutions for an easier and viable last-mile delivery process. The important question in smart and modern cities is the determination of the best micro-hub location for the LMD. This paper solves the micro-hub location selection for sustainable LMD using the multi-criteria decision-making (MCDM) techniques. The main reason for solving the micro-hub location selection is to make the last-mile delivery process in Pardubice as easier and effortless as possible. The Best-Worst Method (BWM), Criteria Importance Through Intercriteria Correlation (CRITIC) method, and Weighted Aggregated Sum Product Assessment (WASPAS) method are coupled to solve the micro-hub location selection for sustainable LMD. First, five criteria and alternatives are identified and discussed with the experts. Second, the hybrid criteria importance is determined by combining the BWM and CRITIC methods. Third, the obtained hybrid weights are integrated within the WASPAS method to rank the micro-hub locations. The findings of the Hybrid BWM-CRITIC-WASPAS model show the Alternative 2 ("Hurka") as the best possible location for Pardubice in the context of the LMD. In addition, a comparative analysis with some of the existing MCDM approaches is conducted for the same problem and its results show a high level of matching with the applied hybrid BWM-CRITIC-WASPAS method, which means that Alternative 2 ("Hurka") is strongly recommended as a micro-hub location for sustainable LMD in Pardubice.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

Ribosome-Mediated Attenuation of vga(A) Expression Is Shaped by the Antibiotic Resistance Specificity of Vga(A) Protein Variants.

Vga(A) protein variants confer different levels of resistance to lincosamides, streptogramin A, and pleuromutilins (LSAP) by displacing antibiotics from the ribosome. Here, we show that expression of vga(A) variants from Staphylococcus haemolyticus is regulated by cis-regulatory RNA in response to the LSAP antibiotics by the mechanism of ribosome-mediated attenuation. The specificity of induction depends on Vga(A)-mediated resistance rather than on the sequence of the riboregulator. Fine tuning between Vga(A) activity and its expression in response to the antibiotics may contribute to the selection of more potent Vga(A) variants because newly acquired mutation can be immediately phenotypically manifested.

Rapid approach to complex boronic acids.

The compatibility of free boronic acid building blocks in multicomponent reactions to readily create large libraries of diverse and complex small molecules was investigated. Traditionally, boronic acid synthesis is sequential, synthetically demanding, and time-consuming, which leads to high target synthesis times and low coverage of the boronic acid chemical space. We have performed the synthesis of large libraries of boronic acid derivatives based on multiple chemistries and building blocks using acoustic dispensing technology. The synthesis was performed on a nanomole scale with high synthesis success rates. The discovery of a protease inhibitor underscores the usefulness of the approach. Our acoustic dispensing-enabled chemistry paves the way to highly accelerated synthesis and miniaturized reaction scouting, allowing access to unprecedented boronic acid libraries.

Isocyanide-Based Multicomponent Reactions of Free Phenylboronic Acids.

Boronic acids are amongst the most useful synthetic intermediates, frequently used by modern drug design. However, their access and fast synthesis of libraries are often problematic. We present a methodology on the synthesis of drug-like scaffolds via IMCRs with unprotected phenylboronic acids. To demonstrate an application of our approach, we also performed one-pot Suzuki couplings on the primary MCR scaffolds. Moreover, we performed a thorough data-mining of the Cambridge Structural Database, revealing interesting geometrical features.

Differentially expressed miRNAs in trisomy 21 placentas.

OBJECTIVE: Molecular pathogenesis of Down syndrome (DS) is still incompletely understood. Epigenetic mechanisms, including miRNAs gene expression regulation, belong to potential influencing factors. The aims of this study were to compare miRNAs expressions in placentas with normal and trisomic karyotype and to associate differentially expressed miRNAs with concrete biological pathways. METHODS: A total of 80 CVS samples - 41 with trisomy 21 and 39 with normal karyotype - were included in our study. Results obtained in the pilot study using real-time PCR technology and TaqMan Human miRNA Array Cards were subsequently validated on different samples using individual TaqMan miRNA Assays. RESULTS: Seven miRNAs were verified as upregulated in DS placentas (miR-99a, miR-542-5p, miR-10b, miR-125b, miR-615, let-7c and miR-654); three of these miRNAs are located on chromosome 21 (miR-99a, miR-125b and let-7c). Many essential biological processes, transcriptional regulation or apoptosis, were identified as being potentially influenced by altered miRNA levels. Moreover, miRNAs overexpressed in DS placenta apparently regulate genes involved in placenta development (GJA1, CDH11, EGF, ERVW-1, ERVFRD-1, LEP or INHA). CONCLUSION: These findings suggest the possible participation of miRNAs in Down syndrome impaired placentation and connected pregnancy pathologies. © 2016 John Wiley & Sons, Ltd.

Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach.

Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).

cis-trans Isomerization of silybins A and B.

Methods were developed and optimized for the preparation of the 2,3-cis- and the 10,11-cis-isomers of silybin by the Lewis acid catalyzed (BF3∙OEt2) isomerization of silybins A (1a) and B (1b) (trans-isomers). The absolute configuration of all optically pure compounds was determined by using NMR and comparing their electronic circular dichroism data with model compounds of known absolute configurations. Mechanisms for cis-trans-isomerization of silybin are proposed and supported by quantum mechanical calculations.

A case of an epignathus with intracranial extension appearing as a persistently open mouth at 16 weeks and subsequently diagnosed at 20 weeks of gestation.

We report a rare case of oral mass (epignathus) with intracranial extension originally suspected antenatally at 16 weeks' gestation because of a persistent open mouth. Postmortem MRI and pathologic examination of the fetus confirmed an oral teratoma with bilateral ventricular dilatation, corpus callosum agenesis, and a neuroepithelial intracranial cyst. The relevant literature regarding this anomaly is reviewed.

Distribution of antidepressants between plasma and red blood cells.

OBJECTIVE: The distribution of different antidepressants between plasma and red blood cells (RBCs) or between water and erythrocyte membranes (ghosts) has not been sufficiently compared so far. MATERIALS AND METHODS: Distribution of seven antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, didesmethylimipramine, dothiepin, and citalopram) was measured in vitro in small volumes of blood or erythrocyte membrane suspension using radiolabeled drugs. Blood samples were taken from healthy subjects. RESULTS: The distribution of antidepressants between plasma and RBCs is strongly affected by temperature; however, it does not depend on the antidepressant concentration in the range of their therapeutic concentrations. The data analysis proved that the ratio of RBCs to plasma volume concentrations is the suitable parameter characterizing antidepressant distribution in whole blood. Significantly higher ratios of RBCs to plasma concentrations were found for demethylated metabolites of tricyclic antidepressants and in the case of citalopram. Citalopram showed the highest accumulation in intact RBCs and at the same time the lowest binding to isolated membranes. The binding of drugs to isolated erythrocyte membranes was much higher than in whole blood. CONCLUSION: The concentration ratio of antidepressant in RBCs and in plasma is sensitive not only to the binding properties of plasma proteins and cell membranes, but also to changes in drug molecule, both in aminopropyl chain and in aromatic rings. This ratio is to a large extent characteristic of a particular antidepressant.