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.     

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.     

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.     

Occurrence of Disinfection By-Products in Swimming Pools in the Area of Thessaloniki,Northern Greece. Assessment of Multi-Pathway Exposure and Risk

This study investigated the occurrence of disinfection by-products (DBPs) (trihalomethanes (THMs), haloacetic acids (HAAs), halonitriles (HANs), halonitromethane (TCNM) and haloketones (HKs)) in different type of swimming pools in the area of Thessaloniki, northern Greece by employing the EPA methods 551.1 and 552.3. Moreover, general water quality parameters (pH, residual chlorine, dissolved organic carbon, UV₂₅₄ absorption, total nitrogen, alkalinity and conductivity) were also measured. The concentrations of DBPs showed great variability among swimming pools as well as within the same pool between sampling campaigns. HAAs exhibited the highest concentrations followed by THMs, HANs, TCNM and HKs. Exposure doses for four age groups (3–<6 y, 6–<11 y, 11–<16 y and adults) were calculated. Route-specific exposures varied among DBPs groups. Inhalation was the dominant exposure route to THMs and TCNM (up to 92–95%). Ingestion and dermal absorption were the main exposure routes to HAAs (40–82% and 18–59%, respectively), depending on the age of swimmers. HANs contributed up to 75% to the calculated cytotoxicity of pool water. Hazard indices for different exposure routes were <1, suggesting non-carcinogenic risk. Inhalation posed the higher carcinogenic risk for THMs, whereas risk via oral and dermal routes was low. Ingestion and dermal contact posed the higher risk for HAAs. Risk management strategies that minimise DBPs exposure without compromising disinfection efficiency in swimming pools are necessary.     

Quality Parameters of Wheat Bread with the Addition of Untreated Cheese Whey

Τhe present study was carried out to evaluate wheat bread of three different flour compositions prepared by replacing water with untreated cheese whey (WCB). Bread prepared with water was taken as the control (CB). All breads were stored at 24 ± 1 °C for up to 6 days. Microbiological, physicochemical, and sensory analyses were determined as a function of storage time. WCB had lower total viable counts (TVC) (3.81 log cfu/g for CB and 2.78 log cfu/g for WCB on day 2 of storage) and showed delayed mold growth by 1 day (day 4 for CB and day 5 for WCB). WCB also had lower pH (5.91 for CB and 5.71 for WCB on day 0), higher titratable acidity values (TTA) (2.5–5.2 mL NaOH/10 g for CB and 4.5–6.8 mL NaOH/ 10 g for WCB), and higher protein content (PC) (PC 7.68% for CB and 8.88% for WCB). WCB was characterized by a more intense flavor, reduced hardness but similar cohesiveness, springiness, and adhesiveness compared to CB. Based primarily on sensory (appearance/mold formation) data, the shelf life of WCB was 4–5 days compared to 3–4 days for CB stored at 24 ± 1 °C. The proposed use of whey in bread preparation contributes decisively to the environmentally friendly management of whey.     

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.     

Megistoquinones I and II, two quinoline alkaloids with antibacterial activity from the bark of Sarcomelicope megistophylla

Two alkaloids, megistoquinone I (1) and megistoquinone II (2), were isolated from the bark of Sarcomelicope megistophylla. Their structures have been elucidated on the basis of MS and NMR data. Both belong to quinoline alkaloid series and should be considered as oxidation products of a furo[2,3-b]quinoline precursor. The two alkaloids showed antibacterial properties with minimum inhibitory concentration (MIC) ranging from 2.35 to 5.25 mg/ml for 1 and 0.73 to 1.23 mg/ml for 2.     

Electrochemical study of some non-steroidal anti-inflammatory drugs: solvent effect and antioxidant activity

The electrochemical behavior of 12 non-steroidal anti-inflammatory drugs (NSAIDs) was studied by means of cyclic voltammetry at a glassy carbon electrode. The underlying solvent had a considerable effect to the oxidation potentials of the investigated NSAIDs due to the alteration of their polar intermediates’ solvation. Oxicams were more capable of electrochemical oxidation, and the influence of both specific and non-specific solute–solvent interactions in their reactivity was confirmed by means of Kamlet–Taft’s analysis. Oxicams were further studied by chronoamperometry at the potentials of 300, 500, and 800 mV. The results obtained by the employed electroanalytical techniques were compared with the reactivity of oxicams towards 1,1- diphenyl-2-dipicrylhydrazyl (DPPH). The study showed a correlation of oxicams’ amperometric signal at 800 mV with their absolute reaction rate, z with DPPH.