One-Pot Phosphate-Mediated Synthesis of Novel 1,3,5-Trisubstituted Pyridinium Salts: A New Family of S. aureus Inhibitors

Polysubstituted pyridinium salts are valuable pharmacophores found in many biologically active molecules. Their synthesis typically involves the use of multistep procedures or harsh reaction conditions. Here, we report water-based phosphate mediated reaction conditions that promote the condensation of arylacetaldehydes with amines to give 1,3,5-pyridinium salts. The reaction, carried out at pH 6, provides conditions suitable for the use of less stable aldehydes and amines in this Chichibabin pyridine condensation. The evaluation of selected 1,3,5-trisubstituted pyridinium salts highlighted that they can inhibit the growth of S. aureus in the low μg/mL range. The synthetic accessibility of these compounds and preliminary growth inhibition data may pave the way towards the discovery of new anti-bacterials based on the 1,3,5-trisubstituted pyridinium scaffold

A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase

A new colorimetric method has been developed to screen transaminases using an inexpensive amine donor. The assay is sensitive, has a low level of background coloration, and can be used to identify and profile transaminase activities against aldehyde and ketone substrates in a high-throughput format. Significantly it is also amendable to solid phase colony screening

Pleistocene uplift and palaeoenvironments of Macquarie Island: evidence from palaeobeaches and sedimentary deposits

Macquarie Island (54°30'S, 159°00'E) is an emergent part of the Macquarie Ridge Complex composed of ocean-floor rocks of Miocene age now 4000 m above the ocean floor. A number of landforms, including palaeobeaches now above sea level (a.s.l.)on Macquarie Island, were formed by marine erosion during uplift of the island. During the last Pleistocene period of low sea level (c. 20 ka) the island was three times larger than now. Thermoluminescence (TL) dating of two palaeobeaches indicates Pleistocene ages: 172 ± 40 ka for one at 100 m a.s.l. and 340 ± 80 ka for another at 263 m a.s.l. Matching the altitude sequence of palaeobeaches on Macquarie Island with the pattern of peaks in world sea level determined from deep sea cores allows an independent estimate of beach ages. Comparison of the altitude and sea level sequences most plausibly places the 100 m palaeobeach in Oxygen Isotope Stage 5e (130-125 ka) and the 263 m palaeobeach in Stage 9 (340-330 ka), matching reasonably with the TL dates. Other palaeobeaches at about 50 m and 170-190 m a.s.l. then correlate with high sea levels. We calculate an average rate of uplift forthe island of 0.8 mma-I . At this rate, 4000 m of Macquarie Ridge uplift would have taken about five million years and the top of the island may first have emerged some 700 to 600 ka. During the six Pleistocene glacial-interglacial cycles since then, there has been periglacial rather than glacial activity on cold uplands, but conditions suitable for vegetation of the present type persisted close to sea level

Integral field spectroscopy of massive young stellar objects in the N113 H II region in the Large Magellanic Cloud

The Spitzer Surveying the Agents of Galaxy Evolution (SAGE) survey has allowed the identification and analysis of significant samples of Young Stellar Object (YSO) candidates in the Large Magellanic Cloud (LMC). However, the angular resolution of Spitzer is relatively poor meaning that at the distance of the LMC, it is likely that many of the Spitzer YSO candidates in fact contain multiple components. We present high-resolution K-band integral field spectroscopic observations of the three most prominent massive YSO candidates in the N113 H II region using Very Large Telescope/Spectrograph for INtegral Field Observations in the Near Infrared (VLT/SINFONI). We have identified six K-band continuum sources within the three Spitzer sources and we have mapped the morphology and velocity fields of extended line emission around these sources. Br γ, He I and H2 emission is found at the position of all six K-band sources; we discuss whether the emission is associated with the continuum sources or whether it is ambient emission. H2 emission appears to be mostly ambient emission and no evidence of CO emission arising in the discs of YSOs has been found. We have mapped the centroid velocities of extended Br γ emission and He I emission and found evidence of two expanding compact H II regions. One source shows compact and strong H2 emission suggestive of a molecular outflow. The diversity of spectroscopic properties observed is interpreted in the context of a range of evolutionary stages associated with massive star formation

Combined tissue and fluid proteomics with Tandem Mass Tags to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer's Disease case study

RATIONALE: Ideal biomarkers are present in readily accessible samples including plasma and cerebrospinal fluid (CSF), and are directly derived from diseased tissue, therefore likely to be of relatively low abundance. Traditional unbiased proteomic approaches for biomarker discovery have struggled to detect low-abundance markers due to the high dynamic range of proteins, the predominance of hyper-abundant proteins, and the use of data-dependent acquisition mass spectrometry (MS). To overcome these limitations and improve biomarker discovery in peripheral fluids, we have developed TMTcalibrator™; a novel MS workflow combining isobarically labelled diseased tissue digests in parallel with an appropriate set of labelled body fluids to increase the chance of identifying low-abundance, tissue-derived biomarkers. METHODS: A disease relevant cell line was labelled with TMT® in a range of concentrations generating a multi-point calibration curve. Peripheral biofluid samples were labelled with the remaining tags and quantitative analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer with a Top10 CID-HCD MS3 synchronous precursor selection (SPS) method. SPS allowed direct analysis of non-depleted, unfractionated CSF samples with complete profiling of six individual samples requiring only 15 hours of MS time, equivalent to 1.5 h per sample. RESULTS: Using the TMTcalibrator™ workflow allowed the identification of several markers of microglia activation that are differentially quantified in the CSF of patients with Alzheimer's disease (AD). We report peptides from 41 proteins that have not previously been detected in the CSF, that appear to be regulated by at least 60% in AD. CONCLUSIONS: This study has demonstrated the benefits of the new TMTcalibrator™ workflow and the results suggest this is a suitable and efficient method of detecting low-abundance peptides within biological fluids. The use of TMTcalibrator™ in further biomarker discovery studies should be considered to overcome some of the limitations commonly associated with more conventional approaches

VSGdb: a database for trypanosome variant surface glycoproteins, a large and diverse family of coiled coil proteins

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Background: Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long alpha-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only similar to 20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for a helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome- specific questions, we have created VSGdb, a database of all known sequences.|Description: VSGdb contains fully annotated VSG sequences from the genome sequencing project, with which it shares all identifiers and annotation, and other available sequences. The database can be queried in various ways. Sequence retrieval, in FASTA format, can deliver protein or nucleotide sequence filtered by chromosomes or contigs, gene type ( functional, pseudogene, etc.), domain and domain sequence family. Retrieved sequences can be stored as a temporary database for BLAST querying, reports from which include hyperlinks to the genome project database ( GeneDB) CDS Info and to individual VSGdb pages for each VSG, containing annotation and sequence data. Queries (text search) with specific annotation terms yield a list of relevant VSGs, displayed as identifiers leading again to individual VSG web pages.|Conclusion: VSGdb http://www.vsgdb.org/is a freely available, web-based platform enabling easy retrieval, via various filters, of sets of VSGs that will enable detailed analysis of a number of general and trypanosome- specific questions, regarding protein structure potential, epitope variability, sequence evolution and recombination events

Furfurylamines from biomass: Transaminase catalysed upgrading of furfurals

Furfural is recognised as an attractive platform molecule for the production of solvents, plastics, resins and fuel additives. Furfurylamines have many applications as monomers in biopolymer synthesis and for the preparation of pharmacologically active compounds, although preparation via traditional synthetic routes is not straightforward due to by-product formation and sensitivity of the furan ring to reductive conditions. In this work transaminases (TAms) have been investigated as a mild sustainable method for the amination of furfural and derivatives to access furfurylamines. Preliminary screening with a recently reported colorimetric assay highlighted that a range of furfurals were readily accepted by several transaminases and the use of different amine donors was then investigated. Multistep synthetic routes were required to synthesise furfurylamine derivatives for use as analytical standards, highlighting the benefits of using a one step biocatalytic route. To demonstrate the potential of using TAms for the production of furfurals, the amination of selected compounds was then investigated on a preparative scale

One-pot, two-step transaminase and transketolase synthesis of L-gluco-heptulose from L-arabinose

The use of biocatalysis for the synthesis of high value added chemical building blocks derived from biomass is becoming an increasingly important application for future sustainable technologies. The synthesis of a higher value chemical from L-arabinose, the predominant monosaccharide obtained from sugar beet pulp, is demonstrated here via a transketolase and transaminase coupled reaction. Thermostable transketolases derived from Deinococcus geothermalis and Dei nococcus radiodurans catalysed the synthesis of L-gluco-heptulose from L-arabinose and β-hydroxypyruvate at elevated temperatures with high conversions. β-Hydroxypyruvate, a commercially expensive compound used in the transketolase reaction, was generated in situ from L-serine and α-ketoglutaric acid via a thermostable transaminase, also from Deinococcus geothermalis. The two steps were investigated and implemented in a one-pot system for the sustainable and efficient production of L-gluco-heptulose

ω-Transaminases for the amination of functionalised cyclic ketones

The potential of a number of enantiocomplementary ω-transaminases (ω-TAms) in the amination of cyclic ketones has been investigated. After a preliminary screening of several compounds with increasing complexity, different approaches to shift the equilibrium of the reaction to the amine products were studied, and reaction conditions (temperature and pH) optimised. Interestingly, 2-propylamine as an amine donor was tolerated by all five selected ω-TAms, and therefore used in further experiments. Due to the higher conversions observed and interest in chiral amines studies then focused on the amination of α-tetralone and 2-methylcyclohexanone. Both ketones were aminated to give the corresponding amine with at least one of the employed enzymes. Moreover, the amination of 2-methylcyclohexanone was investigated in more detail due to the different stereoselectivities observed with TAms used. The highest yields and stereoselectivities were obtained using the ω-TAm from Chromobacterium violaceum (CV-TAm), producing 2-methylcyclohexylamine with complete stereoselectivity at the (1S)-amine position and up to 24 : 1 selectivity for the cis : trans [(1S,2R) : (1S,2S)] isomer