Nine enzymes are required for assembly of the pacidamycin group of peptidyl nucleoside antibiotics

Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis that to date has not been clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage as well as a 3'-deoxyuridine nucleoside attached to DABA(3) of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood because of the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. This work has identified a minimum set of enzymes needed for generation of the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. On the basis of the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogues were produced using in vitro total biosynthesis.     

Attachment of Pseudomonas putida onto differently structured kaolinite minerals: a combined ATR-FTIR and 1H NMR study

The attachment of Pseudomonas (P.) putida onto well (KGa-1) and poorly (KGa-2) crystallized kaolinite was investigated in this study. Batch experiments were carried out to determine the attachment isotherms of P. putida onto both types of kaolinite particles. The attachment process of P. putida onto KGa-1 and KGa-2 was adequately described by a Langmuir isotherm. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy and Nuclear Magnetic Resonance were employed to study the attachment mechanisms of P. putida. Experimental results indicated that KGa-2 presented higher affinity and attachment capacity than KGa-1. It was shown that electrostatic interactions and clay mineral structural disorders can influence the attachment capacity of clay mineral particles.     

The first measurable cardinal can be the first uncountable regular cardinal at any successor height

We use techniques due to Moti Gitik to construct models in which for an arbitrary ordinal ?, is both the least measurable and least regular uncountable cardinal.     

Synthesis,characterisation and photoswitchability of a new [2]rotaxane of α-cyclodextrin with a diazobenzene containing π-conjugated molecular dumbbell

In this work, a new [2]rotaxane consisted of a diazobenzene containing π-conjugated linear compartment, including the 4,4′-bipyridyl moiety and α-cyclodextrin (α-CD) as the macrocyclic compartment, was synthesised with yields of nearly 57% and fully characterised. α-CD easily assembled with the linear compartment and suitable bulky ends (stoppers) in water to give a new [2]rotaxane. The characterisation of this supramolecular compound was accomplished using several spectroscopic techniques such as ¹H NMR, ¹³C NMR and 2D NMR spectroscopy, powder X-ray diffraction, fourier transform infrared spectroscopy, induced circular dichroism and UV–vis spectrophotometry, as well as scanning electron microscopy and Energy Dispersive X-ray. Furthermore, the reversible E–Z photoisomerisation of both [2]rotaxane and its molecular dumbbell was investigated by irradiation with UV light.     

Interactive bicriterion decision support for a large scale industrial scheduling system

In this paper we develop an interactive decision analysis approach to treat a large scale bicriterion integer programming problem, addressing a real world assembly line scheduling problem of a manufacturing company. This company receives periodically a set of orders for the production of specific items (jobs) through a number of specialised production (assembly) lines. The paper presents a non compensatory approach based on an interactive implementation of the ε-constraint method that enables the decision maker to achieve a satisfactory goal for each objective separately. In fact, the method generates and evaluates a large number of non dominated solutions that constitute a representative sample of the criteria ranges. The experience with a specific numerical example shows the efficiency and usefulness of the proposed model in solving large scale bicriterion industrial integer programming problems, highlighting at the same time the modelling limitations.     

QoS-aware service evaluation and selection

Making the provision of services QoS-aware is to the advantage of both clients and providers in the e-business domain. This paper studies the problem of providers that receive multiple concurrent requests for services demonstrating different QoS properties. It introduces the “Selective Multiple Choice Knapsack Problem” that aims to identify the services, which should be delivered in order to maximise the provider’s profit, subject to maximum bandwidth constraints. This problem is solved by a proposed algorithm that has been empirically evaluated via numerous experiments.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

Phosphonopeptide K-26 biosynthetic intermediates in Astrosporangium hypotensionis

Precursors and advanced intermediates for phosphonopeptide K-26 biosynthesis were synthesized and incorporation studies in Astrosporangium hypotensionis suggest a new mechanism of C-P bond formation in aromatic phosphonates.     

A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The “GELFrEE” (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5–25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.     

Biosynthetic origins of C-P bond containing tripeptide K-26

[reaction: see text] Primary metabolic precursors for K-26, a naturally occurring tripeptide phosphonic acid from Actinomyces sp. K-26, are investigated by heavy-atom isotope labeled substrate incorporation experiments. A highly sensitive selected reaction monitoring (SRM)-based method for isotopic incorporation estimation in natural products is reported. The incorporation of heavy-atom isotope labeled tyrosine compounds into the (R)-1-amino-2-(4-hydroxyphenyl)-ethylphosphonic acid moiety of compound K-26 suggests a new mechanism of biosynthesis of phosphonate functionality in natural products.