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Summary: Objectives: Dutch guidelines recommend contact precautions in patients with multidrug resistant microorganisms cultured during the previous 12 months. To evaluate this policy, duration of carriage of multidrug resistant Enterobacterales was assessed among discharged hospital patients and patients attending their general practitioner (GP). Additionally, we assessed factors associated with clearance. Methods: From January 2013 until May 2016, rectal or faecal samples accompanied by questionnaires on patient characteristics were obtained at time of study inclusion and 3, 6 and 12 months later, in 72 patients with multidrug resistant Enterobacterales. Clearance was defined as one or more negative cultures without a subsequent positive culture at 12 months after study inclusion. The percentage of clearance, intermittent carriage and persistence was determined and associated factors were assessed by logistic regression analysis. Results: Clearance was found in 31 patients (43.1% [95%CI: 32.3–54.6]) of which 23 patients had two or more subsequent negative cultures. Twelve patients were classified as intermittent carriers (16.7% [95%CI: 9.8–26.9]) and 29 patients (40.3% [95% CI: 29.7–51.8]) as persistent carriers. Of the intermittent carriers, the majority (n=9) had two negative cultures during the study period. There was no difference in clearance between discharged hospitalized patients and GP patients. The only factor associated with clearance at 12 months in both univariable and multivariable analyses was not traveling to a foreign country (OR=3.5 [95%CI: 1.0–12.4]). Conclusion: Active screening for clearance of multidrug resistant Enterobacterales in patients within the health care setting is probably not beneficial due to high levels of intermittent and persistent carriage.

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation-fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.

The objectives and outcomes of market-based policy instruments in electricity industry restructuring, adopted by Australia are analyzed. The need for greater care in framing such schemes to ensure desired results for greenhouse gas regulations is addressed.

Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs) were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs) than for Hydrozoa FPs derived from Aequorea victoria (avFPs). Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2) and Tag red fluorescent protein (TagRFP)) or to a pKa shift of the chromophore (FP variant mCherry). [ABSTRACT FROM AUTHOR]

Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205. This packaging resulted in deviating spectroscopic features of the encapsulated EGFP molecules. Here we show that for monomeric EGFP variant (mEGFP) micellar encapsulation affects the absorption and fluorescence properties to a much lesser extent, and that changes in circular dichroism characteristics are specific for encapsulated EGFP. Time-resolved fluorescence anisotropy of encapsulated (m)EGFP established the occurrence of homo-FRET (Förster resonance energy transfer) with larger transfer correlation times in the case of EGFP. Together, these findings support that EGFP dimerizes whereas the mEGFP mainly remains as a monomer in the densely packed C3Ms. We propose that dimerization of encapsulated EGFP causes a reorientation of Glu222, resulting in a pKa shift of the chromophore, which is fully reversible after release of EGFP from the C3Ms at a high ionic strength. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation—fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction. [ABSTRACT FROM AUTHOR]

Protein encapsulation with polymershas a high potential for drugdelivery, enzyme protection and stabilization. Formation of such structurescan be achieved by the use of polyelectrolytes to generate so-calledcomplex coacervate core micelles (C3Ms). Here, encapsulation of enhancedgreen fluorescent protein (EGFP) was investigated using a cationic-neutraldiblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205andpoly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlationspectroscopy (FCS) revealed a preferred micellar composition (PMC)with a positive charge composition of 0.65 for both diblock copolymersand micellar hydrodynamic radii of approximately 34 nm. FCS data showthat at the PMC, C3Ms are formed above 100 nM EGFP, independent ofpolymer length. Mixtures of EGFP and nonfluorescent GFP were usedto quantify the amount of GFP molecules per C3M, resulting in approximately450 GFPs encapsulated per micelle. This study shows that FCS can besuccessfully applied for the characterization of protein-containingC3Ms. [ABSTRACT FROM AUTHOR]

Postprandial oxidation of dietary free amino acids or egg white protein was studied using the [13CO2] breath test in rats, as well as in humans. Thirty-eight male rats were assigned to four dietary test groups. Two diets only differed in their protein fraction. Diet I contained 21% egg white protein. For the breath test egg white protein, intrinsically labelled with [1-13C]-leucine, was used as a substrate. Diet II contained the same amino acids as diet I, though not as egg white protein but in free form. Free [1-13C]-leucine was used to label this diet. In addition, two 1:1 mixtures of both diets were used. During the breath test either the free amino acid or the protein fraction was labelled as in diets I or II. The animals were breath-tested following short-term (day 5) and long-term adaptation (day 20) to their experimental diet. For all diets, including the mixed diets, postprandial oxidative losses on day 5 were significantly higher for the free leucine compared with the protein-derived leucine. Differences between free and protein-derived leucine oxidation had, however, largely disappeared on day 20. The human subjects were breath-tested without any adaptation period to the diets. The oxidative losses of free leucine were also higher than those of protein-derived leucine. None of the studies showed any indication for an interaction between the oxidation of protein-derived amino acids and free amino acids. It is concluded that free and protein-derived amino acids in the diet are mainly metabolized independently. [ABSTRACT FROM AUTHOR]

In the present study, the effect of free amino acid (FAA) diets on the intestinal absorption rate of methionine and leucine was studied ‘ ex vivo’ with rats adapted for different periods of time to the diets, using the everted sac method. The adaptation period to the 21% FAA diet with an amino acid content based on casein was either, 0 (no adaptation, N-ADA), 5 (short-term adaptation, ST-ADA), or 26–33 days (long-term adaptation, LT-ADA). Within the ST-ADA and the LT-ADA groups, three different levels of methionine were included: 50%, 100% and 200% of the level normally present in casein. All diets were iso-nitrogenous and iso-caloric. After the adaptation period (0, 5, or 26–33 days), intestinal everted sacs were prepared. Methionine or leucine was added to the medium as transport substrate. The methionine absorption rate of the rats of the LT-ADA groups was higher than that of the N-ADA groups. Furthermore, adaptation to 200% dietary methionine levels caused a significantly slower leucine absorption compared to the 100%, and 50% group. Methionine absorption was similar in the 100% and 200% groups, but the absorption of methionine in the 50% group was enhanced in the distal part of the intestines. We concluded that in response diets with 21% FAAs as only amino acid source, amino acid absorption is decreased to avoid toxic effects of high levels of methionine in the circulation. [ABSTRACT FROM AUTHOR]

This study examined, whether the postprandial fate of dietary amino acids from different amino acid sources is regulated by the responses of insulin, glucagon, corticosterone and growth hormone (GH). Male Wistar rats were cannulated in the vena jugularis and assigned to dietary groups. The diets contained 21% casein or the same amino acids in free form. In the free amino acid diets, methionine level was varied between the groups. The feed was supplied in two distinct meals. In previous experiments it was established that oxidative amino acid losses of the free amino acid diets and protein diets were different. After 3 weeks on those diets, it appeared that the differences in postprandial oxidative losses had been diminished. GH was measured every 12 min, from 144 min before the start of the experimental meal over the following 144 min. Insulin and corticosterone were measured six times from the start of the meal until 270 min after the meal. No differences have been observed between the hormonal responses to both meals at day 5 and at day 26. In conclusion, it has been found that the differences in the oxidative losses between protein and free amino acid meals are not mediated by the combined action of the insulin, glucagon, corticosterone and GH. Postprandial catabolism of amino acids is most probably regulated by substrate induction. [ABSTRACT FROM AUTHOR]

The fast growing desalination industry is facing two major challenges such as cost reduction and reduction in the environmental impact. The usage of fossil-based chemicals (like most antiscalants) has a negative influence on both challenges. Most often these chemicals are not environmentally friendly and the cost for these chemicals are increasing rapidly due to the rising prices of raw materials (e.g. crude oil and phosphate rock). Most of the currently available environmentally friendly scale inhibitors have some drawbacks related to cost and/or performance. Carboxymethyl inulin is an environmentally friendly antiscalant which has been proven to be cost-effective in RO processes and therefore, could have a positive contribution to both of the above-mentioned challenges. [ABSTRACT FROM AUTHOR]

Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores., (© 2022 IOP Publishing Ltd.)

The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium) 128 - b-poly(ethylene-oxide) 477 . Förster resonance energy transfer (FRET) allowed us to determine the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength ( I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of experimental and theoretical considerations, we propose that C3Ms relax to their final state by association and dissociation of near-neutral soluble protein-polymer complexes. To obtain protein-containing C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.