Proteomics and microscopy tools for the study of antimicrobial resistance and germination mechanisms of bacterial spores.

Bacterial spores are ubiquitous in nature and can withstand both chemical and physical stresses. Spores can survive food preservation processes and upon outgrowth cause food spoilage as well as safety risks. The heterogeneous germination and outgrowth behavior of isogenic spore populations exacerbates this risk. A major unknown factor of spores is likely to be the inherently heterogeneous spore protein composition. The proteomics methods discussed here help in broadening the knowledge about spore structure and identification of putative target proteins from spores of different spore formers. Approaches to synchronize Bacillus subtilis spore formation, and to analyze spore proteins as well as the physiology of spore germination and outgrowth are also discussed. Live-imaging and fluorescence microscopy techniques discussed here allow analysis, at single cell level, of the 'germinosome', the process of spore germination itself, spore outgrowth and the spore intracellular pH dynamics. For the latter, a recently published improved pHluorin (IpHluorin) under control of the ptsG promoter is applicable. While the data obtained from such tools offers novel insight in the mechanisms of bacterial spore awakening, it may also be used to probe candidate antimicrobial compounds for inhibitory effects on spore germination and strengthen microbial risk assessment.

Determination of n-alkanes in C. annuum (bell pepper) fruit and seed using GC-MS: comparison of extraction methods and application to samples of different geographical origin.

An efficient extraction and analysis method was developed for the isolation and quantification of n-alkanes from bell peppers of different geographical locations. Five extraction techniques, i.e., accelerated solvent extraction (ASE), ball mill extraction, ultrasonication, rinsing, and shaking, were quantitatively compared using gas chromatography coupled to mass spectrometry (GC-MS). Rinsing of the surface wax layer of freeze-dried bell peppers with chloroform proved to be a relatively quick and easy method to efficiently extract the main n-alkanes C27, C29, C31, and C33. A combined cleanup and fractionation approach on Teflon-coated silica SPE columns resulted in clean chromatograms and gave reproducible results (recoveries 90-95 %). The GC-MS method was reproducible (R(2) = 0.994-0.997, peak area standard deviation = 2-5%) and sensitive (LODs, S/N = 3, 0.05-0.15 ng/µL). The total main n-alkane concentrations were in the range of 5-50 µg/g dry weight. Seed extractions resulted in much lower total amounts of extracted n-alkanes compared to flesh and surface extractions, demonstrating the need for further improvement of pre-concentration and cleanup. The method was applied to 131 pepper samples from four different countries, and by using the relative n-alkane concentration ratios, Dutch peppers could be discriminated from those of the other countries, with the exception of peppers from the same cultivar. Graphical Abstract Procedure for pepper origin determination.

Identification and differential gene expression of adhesin-like wall proteins in Candida glabrata biofilms.

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

[Infraorbital lesion after scuba diving with chronic sinusitis: diagnosis and treatment of reverse block]. / Infraorbitalisläsion nach Sporttauchen bei chronischer Sinusitis: Diagnostik und Therapie der Umkehrblockierung.

A 32-year-old diver repeatedly reported pain in the right side of the face after diving. He felt strong pain on pressure over the right face combined with hypaesthesia of the infraorbital nerve. In addition, he reported nasal breathing obstruction and problems regulating pressure in the middle ear. ENT consultation revealed a septal deviation to the right side of the nasal cavity. CT diagnosis showed the typical picture of chronic pansinusitis with total transfer of the right frontal sinus, subtotal transfer of the right ethmoid and subtotal transfer of the right maxillary sinus. Following successful functional sinus surgery (FESS), combined with septal surgery, the patient was able to dive again symptom-free.

Early hydroxychloroquine but not chloroquine use reduces ICU admission in COVID-19 patients.

BACKGROUND: The global push for the use of hydroxychloroquine (HCQ) and chloroquine (CQ) against COVID-19 has resulted in an ongoing discussion about the effectivity and toxicity of these drugs. Recent studies report no effect of (H)CQ on 28-day mortality. We investigated the effect of HCQ and CQ in hospitalized patients on the non-ICU COVID-ward. METHODS: A nationwide, observational cohort study was performed in The Netherlands. Hospitals were given the opportunity to decide independently on the use of three different COVID-19 treatment strategies: HCQ, CQ, or no treatment. We compared the outcomes between these groups. The primary outcomes were 1) death on the COVID-19 ward, and 2) transfer to the intensive care unit (ICU). RESULTS: The analysis included 1064 patients from 14 hospitals: 566 patients received treatment with either HCQ (n = 189) or CQ (n = 377), and 498 patients received no treatment. In a multivariate propensity-matched weighted competing regression analysis, there was no significant effect of (H)CQ on mortality on the COVID ward. However, HCQ was associated with a significantly decreased risk of transfer to the ICU (hazard ratio (HR) = 0.47, 95% CI = 0.27-0.82, p = 0.008) when compared with controls. This effect was not found in the CQ group (HR = 0.80, 95% CI = 0.55-1.15, p = 0.207), and remained significant after competing risk analysis. CONCLUSION: The results of this observational study demonstrate a lack of effect of (H)CQ on non-ICU mortality. However, we show that the use of HCQ - but not CQ - is associated with a 53% reduction in risk of transfer of COVID-19 patients from the regular ward to the ICU. Recent prospective studies have reported on 28-day, all-cause mortality only; therefore, additional prospective data on the early effects of HCQ in preventing transfer to the ICU are still needed.

Characterization of a maternal type VI collagen in Xenopus embryos suggests a role for collagen in gastrulation.

We characterized a novel extracellular matrix element that is present in the earliest developmental stages of Xenopus laevis, and is recognized by an mAb 3D7. Based on amino acid composition, breakdown patterns by bacterial collagenases, and the molecular weights of the components of the antigen (240, 200, and 140 kD), we found it very similar to mammalian collagen type VI. The antigen is evenly distributed in unfertilized eggs. Shortly after fertilization, it becomes localized intracellularly in the periphery of the cleaving embryo as well as in the extracellular spaces. During gastrulation, the antigen was localized in the cells lining the blastopore and in the extracellular space between the two cell layers, in the presumptive archenteron. When Fab elements of the 3D7 antibody were added to the culture medium, gastrulation was blocked, suggesting a role for the antigen in gastrulation movements.

Comparison of adult oral health in Australia, the USA, Germany and the UK.

BACKGROUND: Australian adults reportedly have poor oral health when compared to 28 other OECD countries. The Australian ranking was based on edentulism and caries experience data from selected age groups that apparently were collected in 1987-88. The objective of this study was to compare the oral health of Australian adults with that of three other western countries that have comprehensive oral health survey data. METHODS: Published data were obtained from the NHANES 2003-2004, the Fourth German Oral Health Study 2005 and the UK Adult Dental Health Survey 1998. Data from the Australian NSAOH 2004-06 were analysed to generate comparable age-specific estimates using nine dental clinical indicators, two measures of oral hygiene behaviour and two of dental attendance. RESULTS: Australia had the best oral health based on two clinical indicators, was equal first on three indicators and ranked second in the remaining clinical indicators. Australia ranked first or second based on dental flossing, use of mouthwash and frequency of dental attendance. CONCLUSIONS: The oral health of the Australian adult population was among the best of the four nations studied.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene-diene rubber using gas chromatography and mass spectrometry II. Addition and combination reactions.

The dicumyl-peroxide-initiated addition and combination reactions of mixtures of alkanes (n-octane, n-decane) and alkenes [5,6-dihydrodicyclopentadiene (DCPDH), 5-ethylidene-2-norbornane (ENBH) and 5-vinylidene-2-norbornane (VNBH)] were studied to mimic the peroxide cross-linking reactions of terpolymerised ethylene, propylene and a diene monomer (EPDM). The reaction products of the mixtures were separated by both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GCxGC). The separated compounds were identified from their mass spectra and their GC and GCxGC elution pattern. Quantification of the various alkyl/alkyl, alkyl/allyl and allyl/allyl combination products shows that allylic-radicals comprise approximately 60% of the substrate radicals formed. The total concentration of the products formed by combination is found to be independent of the concentration and the type of alkene. The total concentration of the products formed by addition to the alkene increases with increasing concentration of alkene. In addition, the total concentration of the formed addition products depends strongly on the type of the alkene used, viz. VNBH>ENBH approximately DCPDH, which is a consequence of differences in steric hindrance of the unsaturation. The peroxide curing efficiency, defined as the number of moles of cross-linked products formed per mol of peroxide, is 173% using 9% (w/w) 5-vinylidene-2-norbornane (VNBH). This indicates that the addition reaction is recurrent. All these findings are consistent with experimental studies on peroxide curing of EPDM rubber. In addition, the present results provide more-detailed structural information, increasing the understanding of the mechanism of peroxide curing of EPDM. The described approach to use low-molecular-weight model compounds followed by GC-mass spectrometry (MS) and GCxGC-MS analysis is proven to be a very powerful tool to study the cross-linking of EPDM.

Characterisation of UV-cured acrylate networks by means of hydrolysis followed by aqueous size-exclusion combined with reversed-phase chromatography.

UV-cured networks prepared from mixtures of di-functional (polyethylene-glycol di-acrylate) and mono-functional (2-ethylhexyl acrylate) acrylates were analysed after hydrolysis, by aqueous size-exclusion chromatography coupled to on-line reversed-phase liquid-chromatography. The mean network density and the fraction of dangling chain ends of these networks were varied by changing the concentration of mono-functional acrylate. The amount and the molar-mass distribution of the polyethylene-glycol chains between cross-links (M(XL)) and polyacrylic acid (PAA) backbone chains (the so-called kinetic chain length (kcl)) in the different acrylate networks were determined quantitatively. The molar-mass distribution of kcl revealed an almost linear dependence on the concentration of mono-functional acrylate. Analysis of the starting materials showed a significant concentration of mono-functional polyethylene-glycol acrylate. In combination with the analysis of the extractables of the UV-cured networks (polymers not attached to the network, impurities that originate from the photo-initiator and unreacted monomers), more insight in the total network structure was obtained. It was shown that the UV-cured networks contain only small fractions of residual compounds. With these results, the chemical network structure for the different UV-cured acrylate polymers was expressed in network parameters such as the number of PAA units which are cross-linked, the degree of cross-linking, and the network density, which is the molar concentration of effective network chains between cross-links per volume of the polymers. The mean molar mass of chains between chemical network junctions (M(C)) was calculated and compared with results obtained from solid-state NMR and DMA. The mean molar mass of chains between network junctions as determined by these methods was similar.

Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium.

In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.

Perp is required for tissue-specific cell survival during zebrafish development.

The tumor suppressor p53 has two alternative effects, causing either cell cycle arrest or apoptosis. These different effects are supposed to be mediated by the transcriptional activation of different target genes. perp, encoding a transmembrane protein of the Pmp22 family, is a transcriptional p53 target exclusively upregulated in apoptotic cells. However, its role during normal development had remained largely unclear. Here, we report the isolation and characterization of a zebrafish perp homolog. Upon overexpression in early zebrafish embryos, perp induces apoptosis. In addition, it contributes to p53-dependent and UV-induced cell death. However, during normal zebrafish development, perp displays a p53-independent and spatially restricted expression in specific cell types and tissues. Antisense-mediated loss of Perp function leads to increased apoptosis in perp-expressing cells of the developing skin and notochord. We conclude that, in contrast to its proapoptotic function in stressed cells, Perp plays an antiapoptotic role during normal zebrafish development to regulate tissue-specific cell survival.

The use of δ(2)H and δ(18)O isotopic analyses combined with chemometrics as a traceability tool for the geographical origin of bell peppers.

Two approaches were investigated to discriminate between bell peppers of different geographic origins. Firstly, δ(18)O fruit water and corresponding source water were analyzed and correlated to the regional GNIP (Global Network of Isotopes in Precipitation) values. The water and GNIP data showed good correlation with the pepper data, with constant isotope fractionation of about -4. Secondly, compound-specific stable hydrogen isotope data was used for classification. Using n-alkane fingerprinting data, both linear discriminant analysis (LDA) and a likelihood-based classification, using the kernel-density smoothed data, were developed to discriminate between peppers from different origins. Both methods were evaluated using the δ(2)H values and n-alkanes relative composition as variables. Misclassification rates were calculated using a Monte-Carlo 5-fold cross-validation procedure. Comparable overall classification performance was achieved, however, the two methods showed sensitivity to different samples. The combined values of δ(2)H IRMS, and complimentary information regarding the relative abundance of four main alkanes in bell pepper fruit water, has proven effective for geographic origin discrimination. Evaluation of the rarity of observing particular ranges for these characteristics could be used to make quantitative assertions regarding geographic origin of bell peppers and, therefore, have a role in verifying compliance with labeling of geographical origin.

High resolution end group determination of low molecular weight polymers by matrix-assisted laser desorption ionization on an external ion source fourier transform ion cyclotron resonance mass spectrometer.

Matrix-assisted laser desorption ionization was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer equipped with a 7-T superconducting magnet to analyze end groups of synthetic polymers in the mass range from 500 to 5000 u. Native, perdeutero methylated, propylated, and acetylated polyethylene glycol and polyvinyl pyrrolidone with unknown end-group elemental composition were investigated in the mass range up to 5000 u by using a 2,5-dihydroxybenzoic acid matrix. A small electrospray setup was used for the deposition of the samples. Two methods to process data were evaluated for the determination of end groups from the measured masses of the component molecules in the molecular weight ranges: a regression method and an averaging method. The averaging method is demonstrated to allow end-group mass determinations with an accuracy within 3 mu for the molecular weight range from 500 to 1400 and within 20 mu for the molecular weight range from 3400 to 5000. This is sufficient to identify the elemental composition of end groups in unknown polymer samples.

In-source decay of hyperbranched polyesteramides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hyperbranched polyesteramides (DA2), prepared from hexahydrophthalic anhydride (D) and diisopropanolamine (A) have been characterized, by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), field desorption (FD)-MS, and electrospray ionization (ESI)-MS. MALDI of polyesteramides produces protonated molecules. The spectra show a complex chemical composition distribution and end-group distribution which are mainly composed of two series of homologous oligomers DnA(n)+1 - mH2O and DnA(n) - mH2O, where m = 1-2. Signals from protonated molecules DnAn+1 and DnAn are almost absent in the MALDI spectrum, whereas these ions are responsible for the base peak of DnA(n)+1 - mH2O and DnA(n) - mH2O (m = 1-2) clusters in the ESI spectrum. The absence of -OH end-groups signals in the MALDI spectrum is due to a metastable decay of protonated DnA(n)+1 and DnAn ions in the ion source of the MALDI mass spectrometer prior to ion extraction. In-source decay results in the formation of protonated lower DnA(n)+1 - mH2O and DnA(n) - mH2O oligomers and their corresponding neutrals, leading to wrong conclusions concerning the relative end-group distribution as a function of the degree of polymerization and the chemical composition.

Renal perfusion imaging using contrast-enhanced phase-inversion ultrasound.

AIMS: We evaluated different techniques of contrast-enhanced phase-inversion ultrasound to visualize renal perfusion in native kidneys and kidney transplants. MATERIALS AND METHODS: Contrast-enhanced phase inversion ultrasound with different levels of mechanical index and frame rate was performed in 20 kidneys of 13 healthy volunteers. In addition, five dysfunctioning kidneys of patients with chronic renal failure, five functionally intact kidney transplants, three kidney transplants with compensated renal failure, and two kidney transplants with acute rejection were studied. Analysis using a software algorithm for time-resolved perfusion imaging was compared to single-image analysis performed by three independent radiologists. RESULTS: Optimal depiction of renal perfusion was achieved only by using a mechanical index, which was high enough to destroy the microbubbles of the contrast agent (burst imaging) combined with a low frame rate (0.5 images/second). Renal cortex and medulla showed a homogeneous enhancement in kidneys of healthy volunteers and functionally intact renal transplants. Dysfunctioning kidneys of patients with chronic renal failure as well as kidney transplants with compensated renal failure or acute rejection showed a significantly reduced level of enhancement. Computer-assisted time-resolved perfusion analysis was not superior to single-image analysis. CONCLUSION: Renal perfusion patterns of normal and abnormal tissue can be visualized using contrast-enhanced phase-inversion ultrasound imaging.

Changes in the redox state and composition of the quinone pool of Escherichia coli during aerobic batch-culture growth.

Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.

Differentiation-dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins.

AIM: A thorough understanding of the mechanisms of adipocyte differentiation and metabolism is important for the prevention and/or treatment of obesity and its complications, including type 2 diabetes mellitus. A complex role for prostaglandins (PGs) in adipogenesis is suggested. We examined the expression and cellular localization of enzymes in the cyclooxygenase (COX) cascade that synthesize PGs as well as the PG profile as a function of differentiation status in 3T3-L1 cells. METHODS: Murine 3T3-L1 preadipocytes were used as a model for studies of adipocyte differentiation induced by a hormone cocktail and compared with the parental fibroblastic line NIH 3T3. Both cell lines were incubated in maintenance medium or differentiation medium. Nine days after differentiation, the expression of enzymes in the COX cascade was evaluated by immunoblot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry, and PG formation was examined using enzyme immunoassay. RESULTS: A differentiation-dependent diminution of COX-1 and COX-2 mRNA and cognate proteins in 3T3-L1 cells was observed. PG release, including PGE(2), 6-keto PGF(1alpha), PGD(2) and 15d-PGJ(2), significantly decreased following differentiation in 3T3-L1 cells (anova/Tukey, p < 0.05). However, microsomal PGE synthase (mPGES) and lipocalin-type PGD synthase (L-PGDS) were selectively upregulated. Immunocytochemistry revealed that COX-1 and COX-2 became intracellularly more diffuse upon differentiation, whereas mPGES was redistributed to the nuclear compartment. CONCLUSIONS: Regulation of PG formation and COX-2 expression in 3T3-L1 cells is differentiation-dependent and involves changes in the levels of gene expression of the individual isoforms as well as redistribution of the enzymes within cellular compartments.