rRNA Operon Copy Number Can Explain the Distinct Epidemiology of Hospital-Associated Methicillin-Resistant Staphylococcus aureus.

The distinct epidemiology of original hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and early community-associated MRSA (CA-MRSA) is largely unexplained. S. aureus carries either five or six rRNA operon copies. Evidence is provided for a scenario in which MRSA has adapted to the hospital environment by rRNA operon loss (six to five copies) due to antibiotic pressure. Early CA-MRSA, in contrast, results from wild-type methicillin-susceptible S. aureus (MSSA) that acquired mecA without loss of an rRNA operon. Of the HA-MRSA isolates (n = 77), 67.5% had five rRNA operon copies, compared to 23.2% of the CA-MRSA isolates (n = 69) and 7.7% of MSSA isolates (n = 195) (P < 0.001). In addition, 105 MSSA isolates from cystic fibrosis patients were tested, because these patients are repeatedly treated with antibiotics; 32.4% of these isolates had five rRNA operon copies. For all subsets, a correlation between resistance profile and rRNA copy number was found. Furthermore, we showed that in vitro antibiotic pressure may result in rRNA operon copy loss. We also showed that without antibiotic pressure, S. aureus isolates containing six rRNA copies are more fit than isolates with five copies. We conclude that HA-MRSA and cystic fibrosis isolates most likely have adapted to an environment with high antibiotic pressure by the loss of an rRNA operon copy. This loss has facilitated resistance development, which promoted survival in these niches. However, strain fitness decreased, which explains their lack of success in the community. In contrast, CA-MRSA isolates retained six rRNA operon copies, rendering them fitter and thereby able to survive and spread in the community.

A Randomised Controlled Trial of Consent Procedures for the Use of Residual Tissues for Medical Research: Preferences of and Implications for Patients, Research and Clinical Practice.

BACKGROUND: Despite much debate, there is little evidence on consequences of consent procedures for residual tissue use. Here, we investigated these consequences for the availability of residual tissue for medical research, clinical practice, and patient informedness. METHODS: We conducted a randomised clinical trial with three arms in six hospitals. Participants, patients from whom tissue had been removed for diagnosis or treatment, were randomised to one of three arms: informed consent, an opt-out procedure with active information provision (opt-out plus), and an opt-out procedure without active information provision. Participants received a questionnaire six weeks post-intervention; a subsample of respondents was interviewed. Health care providers completed a pre- and post-intervention questionnaire. We assessed percentage of residual tissue samples available for medical research, and patient and health care provider satisfaction and preference. Health care providers and outcome assessors could not be blinded. RESULTS: We randomised 1,319 patients, 440 in the informed consent, 434 in the opt-out plus, and 445 in the opt-out arm; respectively 60.7%, 100%, and 99.8% of patients' tissue samples could be used for medical research. Of the questionnaire respondents (N = 224, 207, and 214 in the informed consent, opt-out plus, and opt-out arms), 71%, 69%, and 31%, respectively, indicated being (very) well informed. By questionnaire, the majority (53%) indicated a preference for informed consent, whereas by interview, most indicated a preference for opt-out plus (37%). Health care providers (N = 35) were more likely to be (very) satisfied with opt-out plus than with informed consent (p = 0.002) or opt-out (p = 0.039); the majority (66%) preferred opt-out plus. CONCLUSION: We conclude that opt-out with information (opt-out plus) is the best choice to balance the consequences for medical research, patients, and clinical practice, and is therefore the most optimal consent procedure for residual tissue use in Dutch hospitals. TRIAL REGISTRATION: Dutch Trial Register NTR2982.

Factors affecting the gene expression of in vitro cultured human preimplantation embryos.

STUDY QUESTION: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? SUMMARY ANSWER: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture. WHAT IS KNOWN ALREADY: Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking. STUDY DESIGN, SIZE, DURATION: Donated, good quality, day 4 cryopreserved human preimplantation embryos (N = 89) were randomized to be cultured in one of two culture media (G5 medium or HTF medium) and one of two oxygen concentrations (5% or 20%), with stratification for maternal age. Next to these variables, developmental stage after culture was taken into account in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37). MAIN RESULTS AND THE ROLE OF CHANCE: Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. LIMITATIONS, REASONS FOR CAUTION: Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RT-PCR validation was not performed due to scarcity of the material. WIDER IMPLICATIONS OF THE FINDINGS: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines. STUDY FUNDING/COMPETING INTERESTS: This study received no external funding and there were no competing interests.

Comparison of Timothy grass pollen extract- and single major allergen-induced gene expression and mediator release in airway epithelial cells: a meta-analysis.

BACKGROUND: Seasonal allergic rhinitis (AR) is a global health problem and its prevalence has increased considerably in the last decades. As the allergic response with its clinical manifestations is triggered by only a few proteins within natural extracts, there is an increasing tendency for single-component-resolved diagnosis and immunotherapy. OBJECTIVE: As natural exposure is not to single proteins, but to complex mixtures of molecules, we were interested in comparing the activation of respiratory epithelial cells induced by the purified major allergen Phl p 1 with the induction caused by a complete extract of Timothy grass pollen (GPE). METHODS: NCI-H292 cells were exposed to GPE or Ph1 p 1 for 24 h, isolated RNA and cell culture supernatants were used for microarray analysis, multiplex enzyme-linked immunosorbant assay (ELISA) and subsequent analysis. RESULTS: We found 262 genes that showed a GPE-induced change of at least 3-fold, whereas Ph1 p 1-stimulation resulted in 71 genes with a fold induction of more than 3-fold. Besides genes that were regulated by both stimuli, we also detected genes displaying an opposite response after stimulation, suggesting that GPE might be more than purified major allergens with regard to induced immune responses. CONCLUSIONS AND CLINICAL RELEVANCE: Additional components within GPE and the resulting modulation of general processes affecting gene transcription and signalling pathways might be crucial to maintain/overcome the diseased phenotype and to induce the influx of cells contributing to late-phase allergic responses. When the initial process of sensitization is the matter of interest or late-phase allergic responses, one might miss important immune modulatory molecules and their interaction with allergens by applying single components only.

Purified Timothy grass pollen major allergen Phl p 1 may contribute to the modulation of allergic responses through a pleiotropic induction of cytokines and chemokines from airway epithelial cells.

By definition, allergens are proteins with the ability to elicit powerful T helper lymphocyte type 2 (Th2) responses, culminating in immunoglobulin (Ig)E antibody production. Why specific proteins cause aberrant immune responses has remained largely unanswered. Recent data suggest that there may be several molecular paths that may affect allergenicity of proteins. The focus of this study is the response of airway epithelium to a major allergen from Phleum pratense Phl p 1. Instead of focusing on a few genes and proteins that might be affected by the major allergen, our aim was to obtain a broader view on the immune stimulatory capacity of Phl p 1. We therefore performed detailed analysis on mRNA and protein level by using a microarray approach to define Phl p 1-induced gene expression. We found that this allergen induces modulation and release of a broad range of mediators, indicating it to be a powerful trigger of the immune system. We were able to show that genes belonging to the GO cluster 'cell communication' were among the most prominent functional groups, which is also reflected in cytokines and chemokines building centres in a computational model of direct gene interaction. Further detailed comparison of grass pollen extract (GPE)- and Phl p 1-induced gene expression might be beneficial with regard to the application of single components within diagnosis and immunotherapy.

Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors.

BACKGROUND: Spontaneous in vitro transition of undifferentiated spermatogonia into the pluripotent cell state has been achieved using neonatal and adult mouse testis tissue. In an effort to establish an analogous source of human patient-specific pluripotent stem cells, several research groups have described the derivation of embryonic stem cell-like cells from primary cultures of human testis. These cells are characterized in all studies as growing in compact colonies, expressing pluripotency-associated markers and possessing multilineage differentiation capabilities in vitro, but only one study claimed their ability to induce teratomas. This controversy initiated a debate about the pluripotent state and origin of human testis-derived ES-like cells (htES-like cells). METHODS: htES-like cell colonies were obtained from primary testicular cultures of three individuals and selectively expanded using culture conditions known to support the propagation of blastocyst-derived human embryonic stem cells (ESCs), mouse epiblast stem cells and 'naïve' human ESCs. The stem cell properties of htES-like cells were subsequently assessed by testing the expression of ESC-specific markers, differentiation abilities in vitro and in vivo, and microarray profiling. RESULTS: The expression of pluripotency-associated markers in htES-like cells and their differentiation abilities differed significantly from those of ESCs. Gene expression microarray analysis revealed that htES-like cells possess a transcriptome distinct from human ESCs and fibroblasts, but closely resembling the transcriptome of mesenchymal stem cells (MSCs). The similarity to MSCs was confirmed by detection of SSEA4/CD146 expressing cells within htES-like colonies and efficient in vitro differentiation toward three mesodermal lineages (adipogenic, osteogenic, chondrogenic). CONCLUSIONS: Taken together, these results indicate that htES-like cells, in contrast to pluripotent stem cells derived from adult mouse testis, are not pluripotent and most likely not of germ cell but of mesenchymal origin.

Timothy grass pollen extract-induced gene expression and signalling pathways in airway epithelial cells.

BACKGROUND: Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics. Our understanding of the epithelial contribution to immune responses is limited as most studies focus on only a few individual genes or proteins. OBJECTIVE: To describe in detail the Timothy grass pollen extract (GPE)-induced gene expression in AECs. METHODS: NCI-H292 cells were exposed to GPE for 24 h, and isolated RNA and cell culture supernatants were used for microarray analysis and multiplex ELISA, respectively. RESULTS: Eleven thousand and seven hundred fifty-eight transcripts were affected after exposure to GPE, with 141 genes up-regulated and 121 genes down-regulated by more than threefold. The gene ontology group cell communication was among the most prominent categories. Network analysis revealed that a substantial part of regulated genes are related to the cytokines IL-6, IL-8, IL-1A, and the transcription factor FOS. After analysing significantly regulated signalling pathways, we found, among others, epidermal growth factor receptor 1, IL-1, Notch-, and Wnt-related signalling members. Unexpectedly, we found Jagged to be down-regulated and an increased release of IL-12, in line with a more Th1-biased response induced by GPE. CONCLUSION AND CLINICAL RELEVANCE: Our data show that the stimulation of AECs with GPE results in the induction of a broad response on RNA and protein level by which they are able to affect the initiation and regulation of local immune responses. Detailed understanding of GPE-induced genes and signalling pathways will allow us to better define the pathogenesis of the allergic response and to identify new targets for treatment.

The effect of calcium on the transcriptome of sporulating B. subtilis cells.

Bacterial spores formed in the presence of high concentrations of minerals are a major problem in the food industry because of their extreme heat resistance. In order to enhance our insight in the molecular mechanisms underlying this phenomenon we have performed a detailed time-resolved analysis of the genome-wide transcriptome pattern of Bacillus subtilis sporulated both in the absence and presence of high calcium concentrations. The data was analysed in two ways. First, we determined the influence of the presence of high calcium levels during sporulation on the expression of gene groups as defined in Subtilist and KEGG pathways database. Second, we assessed the differential expression at the level of individual genes. When analysing groups and pathways, we found that those annotated as being involved in sporulation were significantly affected. Also, groups and pathways involved in flagella formation and biofilm matrix production were affected by the presence of calcium in the sporulation medium. When we analysed the behaviour of individual genes we found 305 genes influenced by calcium, including all known spore coat polysaccharide biosynthesis genes (10 induced and 1 repressed). A number of the calcium affected genes were also involved in biofilm formation. Minimal overlap with other stress outputs like sigma B activation and weak acid stress response was noted. Those genes that did overlap were unique to that combination which corroborates the notion that the cells sense these conditions differently.

Comparative transcriptomic responses to chronic cadmium, fluoranthene, and atrazine exposure in Lumbricus rubellus.

Transcriptional responses of a soil-dwelling organism (the earthworm Lumbricus rubellus) to three chemicals, cadmium (Cd), fluoranthene (FA), and atrazine (AZ), were measured following chronic exposure, with the aim of identifying the nature of any shared transcriptional response. Principal component analysis indicated full or partial separation of control and exposed samples for each compound but not for the composite set of all control and exposed samples. Partial least-squares discriminant analysis allowed separation of the control and exposed samples for each chemical and also for the composite data set, suggesting a common transcriptional response to exposure. Genes identified as changing in expression level (by the least stringent test for significance) following exposure to two chemicals indicated a substantial number of common genes (> 127). The three compound overlapping gene set, however, comprised only 25 genes. We suggest that the low commonality in transcriptional response may be linked to the chronic concentrations (approximately 10% EC50) and chronic duration (28 days) used. Annotations of the three compound overlapping gene set indicated that genes from pathways most often associated with responses to environmental stress, such as heat shock, phase I and II metabolism, antioxidant defense, and cation balance, were not represented. The strongest annotation signature was for genes important in mitochondrial function and energy metabolism.

Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm.

INTRODUCTION: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics. METHODS: Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays. RESULTS: When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed. CONCLUSION: Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens.

Comparison of expression profiles induced by dust mite in airway epithelia reveals a common pathway.

BACKGROUND: Airway epithelial cells have shown to be active participants in the defense against pathogens by producing signaling and other regulatory molecules in response to the encounter. METHODS: In previous manuscripts, we have studied the effect of house dust mite (HDM) extract on both an epithelial cell-line (H292) and primary nasal epithelial cell. When we compare these responses we conclude that the H292 cells more closely resemble nasal epithelium of healthy controls (share 107 probe-sets) than of allergic individuals (share 17 probe-sets). RESULTS: Interestingly, probably because of an absent intraindividual variation between samples, more probe-sets (8280) change expression significantly in H292 than in either healthy (555) or allergic (401) epithelium. CONCLUSIONS: A direct comparison of all the responses in these epithelial cells reveals a core-response to HDM of just 29 genes. These genes (CCL20, IL-8, CXCL2, CXCL1, IL-1B, AREG, TNFAIP3, HBEGF, PTGS2, BMP2, LDLR, PLAUR, PLAU, NFKB2, NFKB1, JUN, ATF3, EGR1, NPC1, TICAM1, EPHA2, CTGF, DUSP1, SPRY1, TLR-3, complement factor C3, IVNS1ABP, SerpinB3, and PSAT1) have described links with allergy or inflammation and may even describe the well-established relationship between viral infections and allergic exacerbations or allergy development.

[Nitrous-oxide sedation indispensable in the dental care of anxious people and the mentally impaired]. / Lachgassedatie onmisbaar bij de tandheelkundige behandeling van gehandicapten en angstigen.

The long-term occupational exposure to high concentrations of nitrous oxide in health-care workers in a Dutch hospital, with probable adverse effects on their offspring, has triggered a discussion about the benefits and risks of nitrous oxide. In Dutch dentistry, nitrous-oxide sedation is a valuable and indispensable aid in the treatment of patients with challenging behaviour, such as those with mental impairment or in extremely-anxious adults and children. Nitrousoxide sedation can be used without risk to the dental team, provided that measures are taken for sufficient scavenging and room ventilation. A possible ban on nitrous oxide would be a major setback for specialty-care dentistry in the Netherlands.

The oedema-protective effect of Lycra support stockings.

BACKGROUND: Oedema may be an early sign of chronic venous insufficiency (CVI), but swelling of the lower legs is a common phenomenon in many people. The distinction between physiological swelling and CVI is not clear. There is a gradual transition between healthy legs and the early stages of CVI. In case of CVI, medical elastic compression stockings are used in order to prevent oedema completely. In case of healthy people without demonstrable CVI, no medical stockings are required in the prevention of oedema but stockings exerting less compression. OBJECTIVE: The aim of this study was to investigate the effect of mild compression on the development of swelling of the legs and the effect on subjective complaints in healthy subjects. METHODS: The diurnal volume change (DVC) of the lower legs during full working days was monitored with an optical leg volume meter in 118 healthy volunteers (60 males, 58 females) without objective symptoms of CVI. The DVCs after wearing two kinds of class I support stockings (X: average pressure at the ankle of almost 14 mm Hg; Y: almost 18 mm Hg) were compared with the DVC after wearing a control stocking (Z: almost 6 mm Hg). Also, the effect on subjective feelings of the legs was noted. RESULTS: It appeared that healthy people have a mean daily volume increase of the legs of 2.3% in females and 1.6% in males. Mild compression stockings reduced this daily increase with 31 and 18% in males and females, respectively, by stocking X and 37 and 32% by stocking Y. Subjective feelings occurred in 57% of all cases. A beneficial effect on subjective feelings, in particular of tired and swollen legs, was found. A difference in this beneficial effect between stocking X and Y was not obvious. CONCLUSIONS: Mild compression stockings reduce diurnal oedema and unpleasant feelings of the legs in healthy individuals.

The outcome of an additional patch-test reading on days 6 or 7.

In a retrospective study (period 1997-1998), the usefulness of an additional patch-test reading on day (D) 6 or 7 was investigated. In 62 out of 760 patients (8.2%), 77 late-positive reactions, those manifest after D3, were seen. 4 late reactions of allergens of which a related allergen reacted earlier (at D2 or D3) in the same patient were not incorporated in the study as late-positive. Allergens most involved in producing late-positive reactions were nickel sulfate (20 reactions), neomycin sulfate (7), tixocortol-21-pivalate (5), p.t. butylphenol formaldehyde resin (5) and Cl+ Me isothiazolinone (5). Special attention is paid to ?+ reactions at D6 or D7, because these reactions could add extra information to the outcome of patch-test readings, considering that this might be a group of allergens that has been positive between D3 and D6/7 or become positive later on. When this group is included, the total number of late-positive reactions is 105 in 88 out of 760 patients (11.6%). By doing D3 readings in combination with D6 or D7 readings only, one would miss 24 positive reactions. However, we concluded that it was preferable to have a reading on D2 and D3. An extra reading on D6 or D7 is very useful as it gives additional information in 8.2% of patch-tested patients.

Species-specific responses to metals in organically enriched river water, with emphasis on effects of humic acids.

Invertebrate communities in polluted rivers are often exposed to a wide variety of compounds. Due to complex interactions, 'pollution tolerant' species are not necessarily the most tolerant species to toxicants tested under standard laboratory conditions. It was hypothesized that the distribution of species in polluted rivers is not only dependent on the tolerance of species to toxicants, but also on species-specific capacities to modify or compensate for negative effects of toxicants. To test this hypothesis, species-specific responses to metals in organically enriched river water were studied under controlled conditions. The zebra mussel Dreissena polymorpha and the midge Chironomus riparius were exposed to metal-polluted water from the River Dommel. Additionally, the (interactive) effects of metals and humic acids (HA) on both species were evaluated. In spite of a lower tolerance of Chironomus riparius to metals in laboratory studies, the midge was the most tolerant of the two test species to metal-polluted site water. The results indicated that the sensitivities of the two test species determined in laboratory tests were inversely related to their sensitivities to polluted river water. In accordance with these results, midge larvae were protected from copper (Cu) toxicity by HA, while metal toxicity was not reduced (Cu) or even amplified (cadmium) by HA for the zebra mussel. Thus, the presence of (naturally occurring) HA in site water may partly account for discrepancies between responses of species to bioassays and toxicity tests. It is suggested that these differences in responses to metals in site water are strongly influenced by species-specific preferences for organic compounds (like HA). It is concluded that the response to organic compounds present in site water largely determines whether a species is classified as 'pollution tolerant' or 'pollution sensitive'.

Polymorphism of C4 and CYP21 genes in various primate species.

To study the genetic heterogeneity of the C4 and CYP21 genes in selected primate species we used the technique of restriction fragment length polymorphism (RFLP). Genomic DNA was digested using several restriction endonucleases and filters were hybridized with a 500 bp BamHI/KpnI fragment derived from the 5' section of a human C4-cDNA and with a 1700 bp BamHI obtained from a human CYP21 gene. Abundant RFLP heterogeneity was observed for the C4 genes within a rhesus monkey population but not for the chimpanzee colony analyzed. Duplicated C4-CYP21 clusters can be traced back in humans, but also in chimpanzees, orang-utans and rhesus monkeys. Thus, duplication of the basic C4-CYP21 cluster in primates may have happened more than 30 million years ago. Non-duplicated C4-CYP21 regions were found for the gorilla and orang-utan. Apart from this, shortened C4A and C4B genes were observed in chimpanzees, orang-utans and rhesus monkeys, whereas the so-called long variety of the C4A gene appears to be present in humans and orang-utans. This ancestral modification, resulting from an insertion of a 6.5 kb intron in the C4A gene, therefore predates at least speciation of human and orang-utan which is estimated to have taken place more than 12 million years ago.