Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.

The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunits (approximately 30% reduction within 25 min, maximum after 40 min) independent of the PKC phosphorylation site (T15A/S16A). In addition to this major effect of PMA, which was independent of the intracellular sodium concentration and was prevented by the PKC inhibitor bisindolylmaleimide GF 109203X (BIM), another BIM-resistant, PKC site-independent decrease was observed when the Ip was measured at low sodium concentrations (total reduction approximately 50% at 5 mM sodium). Using ouabain binding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatment stimulated fluid phase endocytosis sevenfold and decreased by 16.5% the basolateral cell surface area measured by transepithelial capacitance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, to a withdrawal of sodium pumps from the basolateral cell surface independent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area.     

Mould biodiversity in homes II. Analysis of mattress dust

Fungal species from 130 samples of mattress dust have been identified. The number of moulds varies from one mattress to another with extreme values ranging from less than 1000 to more than 70 000 000 CFU/g of dust (median value of 30 000 CFU/g).Eurotium repens is undoubtedly the most frequent species detected in mattress dust.Aureobasidium pullulans, Alternaria alternata, Penicillium chrysogenum, Aspergillus penicilloides andAspergillus restrictus, which were frequently isolated and/or revealed important concentrations in dust samples, are the other main occurring species. Fungal flora in mattresses are more numerous in winter, the peak period being December-January. The most important contaminations by xerophilic species occurred during that peak period, whereas non-osmophilous species were constantly present during the year. The qualitative conditions of the habitat have an influence on the abundance of moulds in mattresses. Mattresses from damp houses could be a source forAlternaria throughout the year. No significant difference has been detected between the contamination level of men and women's mattresses (P=0.651). Children's mattresses are less contaminated than adults (P=0.025). The level of contamination could be affected by perspiration.     

The impact of the neisserial DNA uptake sequences on genome evolution and stability

Background  

Efficient natural transformation in Neisseria requires the presence of short DNA uptake sequences (DUSs). Doubts remain whether DUSs propagate by pure selfish molecular drive or are selected for 'safe sex' among conspecifics.     

Synthesis and in vitro pharmacological studies of C(4) modified salvinorin A analogues

Salvinorin A is the most potent naturally occurring opioid agonist with a high selectivity and affinity for kappa-opioid receptor. To explore its structure-activity relationships, modifications at the C(4) position have been studied and a series of salvinorin A derivatives were prepared. These C(4)-modified salvinorin A analogues were screened for binding and functional activities at the human kappa-opioid receptor and several potent new agonists have been identified.     

A multicenter study confirms CD226 gene association with systemic sclerosis-related pulmonary fibrosis

Idiopathic inflammatory myopathies (IIMs) comprise a group of autoimmune diseases that are characterized by symmetrical skeletal muscle weakness and muscle inflammation with no known cause. Like other autoimmune diseases, IIMs are treated with either glucocorticoids or immunosuppressive drugs. However, many patients with an IIM are frequently resistant to immunosuppressive treatments, and there is compelling evidence to indicate that not only adaptive immune but also several non-immune mechanisms play a role in the pathogenesis of these disorders. Here, we focus on some of the evidence related to pathologic mechanisms, such as the innate immune response, endoplasmic reticulum stress, non-immune consequences of MHC class I overexpression, metabolic disturbances, and hypoxia. These mechanisms may explain how IIM-related pathologic processes can continue even in the face of immunosuppressive therapies. These data indicate that therapeutic strategies in IIMs should be directed at both immune and non-immune mechanisms of muscle damage.     

Conversion of t11t13 CLA into c9t11 CLA in Caco-2 cells and inhibition by sterculic oil

Background

Conjugated linoleic acids (CLA), and principally c9t11 CLA, are suspected to have numerous preventive properties regarding non-infectious pathologies such as inflammatory diseases, atherosclerosis and several types of cancer. C9t11 CLA is produced in the rumen during biohydrogenation of linoleic acid, but can also be synthesized in mammalian tissues from trans-vaccenic acid (C18:1 t11) through the action of delta-9 desaturase (D9D). For several years, it is also known that c9t11 CLA can be synthesized from conjugated linolenic acids (CLnA), i.e. c9t11c13 CLnA and c9t11t13 CLnA. This study aimed at investigating to which extent and by which route c9t11 CLA can be produced from another isomer of CLA, the t11t13 CLA that is structurally very similar to c9t11t13 CLnA, in Caco-2 cells.

Methodology/Principal Findings

Caco-2 cells were incubated for 24 h with 20 µmol/l of t11t13 CLA in the absence or presence of sterculic oil used as an inhibitor of D9D. Caco-2 cells were able to convert t11t13 CLA into c9t11 CLA, and c9t11t13 CLnA was formed as an intermediate compound. In the presence of sterculic oil, the production of this intermediate was decreased by 46% and the formation of c9t11 CLA was decreased by 26%. No other metabolite was detected.

Conclusions/Significance

These results not only highlight the conversion of t11t13 CLA into c9t11 CLA but demonstrate also that this conversion involves first a desaturation step catalysed by D9D to produce c9t11t13 CLnA and then the action of another enzyme reducing the double bond on the Δ13 position.     

Viunalikeviruses are environmentally common agents of horizontal gene transfer in pathogens and biocontrol bacteria

Bacteriophages have been used as natural biocontrol and therapeutic agents, but also as biotechnological tools for bacterial engineering. We showed recently that the transducing bacteriophage ϕMAM1 is a ViI-like phage and a member of the new genus, ‘Viunalikevirus''. Here, we show that four additional ViI-like phages and three new environmentally isolated viunalikeviruses, all infecting plant and human pathogens, are very efficient generalised transducers capable of transducing chromosomal markers at frequencies of up to 10⁻⁴ transductants per plaque-forming unit. We also demonstrate the interstrain transduction of plasmids and chromosomal markers, including genes involved in anabolism, genes for virulence and genes encoding secondary metabolites involved in biocontrol. We propose that all viunalikeviruses are likely to perform efficient horizontal gene transfer. Viunalikeviruses therefore represent useful agents for functional genomics and bacterial engineering, and for chemical and synthetic biology studies, but could be viewed as inappropriate choices for phage therapy.Combined morphological, genomic and phylogenetic analyses have recently led to the proposed creation of a new phage genus, ‘Viunalikevirus'', within the Myoviridae family (Adriaenssens et al., 2012a). The first member of this proposed genus, Salmonella phage ViI, was isolated in the 1930s (Craigie and Yen, 1938) and multiple viunalikeviruses have been sequenced and characterised since 2010 (Pickard et al., 2010; Anany et al., 2011; Hooton et al., 2011; Kutter et al., 2011; Matilla and Salmond, 2012; Park et al., 2012; Adriaenssens et al., 2012a, 2012b; Hsu et al., 2013; Luna et al., 2013; Shahrbabak et al., 2013). Viunalikeviruses are characterised as virulent (lytic) phages showing similar genome size, extensive DNA homology, strong gene synteny and a complex adsorption apparatus, which uses tail spike proteins as host-recognition determinants (Adriaenssens et al., 2012a).We recently isolated the ViI-like phage, ϕMAM1, that infects several environmental and clinical isolates belonging to Serratia and Kluyvera genera (Matilla and Salmond, 2012). During the characterisation of ϕMAM1, we showed that it mediates highly efficient generalised transduction (Matilla and Salmond, submitted for publication). These observations were consistent with a previous report, that the Salmonella phage ViI was also capable of transduction (Cerquetti and Hooke, 1993) and we have confirmed that phage ViI can transduce chromosomal markers and plasmids at frequencies of up to 4.6 × 10⁻⁵ transductants per plaque-forming unit (p.f.u.; Figure 1a; Supplementary Table 1).Open in a separate windowFigure 1Transduction capabilities of viunalikeviruses. (a) Transduction frequencies of LIMEstone1, LIMEstone2, ViI and CBA120 phages. The graph also shows transduction efficiencies of LIMEstone phages within and between Dickeya solani strains. Transduction efficiency was defined as the number of transductants obtained per p.f.u. In all cases, error bars represent the standard deviations (n=3). (b) Skimmed milk agar plates showing protease production in the wild-type (wt) Dickeya solani strains MK10, MK16 and IPO 2222. LIMEstone1- (LS1) and LIMEstone2- (LS2) mediated transduction of the spp::Km marker from the protease negative mutant strain MK10P1 to the wild-type strains MK10, MK16 and IPO 2222 result in a protease-negative phenotype. (c–e) LIMEstone-mediated transduction of the oocN::Km marker from the oocydin A-negative mutant strain MK10oocN to the wild-type strains MK10 (c), MK16 (d) and IPO 2222 (e) results in an oocydin A-negative phenotype and, consequently, in the generation of strains defective in their antimicrobial activity against the plant pathogenic oomycete, Pythium ultimum. The anti-oomycete assays were performed as described previously (Matilla et al., 2012).Most generalised transducers utilise a headful packing strategy where phage terminases recognise specific sequences (pac sites) in the DNA and perform cycles of packing that result in mature phage particles (Fineran et al., 2009a). Indeed, phage terminases with reduced specificity for pac sequences may lead to the evolution of efficient transducing phages (Schmeiger, 1972). Based on the high similarity between the terminases of ϕMAM1, ViI and those of other previously sequenced viunalikeviruses, we hypothesised that all of these ViI-like phages should be capable of transduction in their respective bacterial hosts. To test this hypothesis, we investigated three additional viunalikeviruses, Escherichia coli phage CBA120 (Kutter et al., 2011), and Dickeya phages LIMEstone1 and LIMEstone2 (Adriaenssens et al., 2012b). All the bacteriophages, bacterial strains, plasmids and primers used in this study are listed in the Supplementary Tables 2 and 3. Experimental procedures are presented as Supplementary Material.The LIMEstone phages specifically infect some strains of the emerging plant pathogen, Dickeya solani (Adriaenssens et al., 2012b), and here we showed that they also infect the recently sequenced D. solani strains MK10, MK16 and IPO 2222. As predicted, we confirmed that the LIMEstone phages effected efficient transduction of various auxotrophic markers between Dickeya solani strains (Figure 1a; Supplementary Table 4). To our knowledge, only one Dickeya transducing phage, ϕEC2, has been isolated previously (Resibois et al., 1984). Additional mutant strains were constructed and the generalised nature of the transduction was confirmed by transfer of multiple chromosomal markers, including mutations in the gene cluster encoding biosynthesis of the anti-oomycete haterumalide, oocydin A (Matilla et al., 2012) and in the locus for synthesis and secretion of protease virulence factors. Transduction frequency was higher at an multiplicity of infection (m.o.i.) of 0.1 and 0.01 with efficiencies of up to 10⁻⁴ transductants per p.f.u. (Figure 1a; Supplementary Tables 4 and 5).We also demonstrated transduction of a kanamycin resistance-marked plasmid pECA1039-Km3 between strains MK10, MK16 and IPO 2222 at frequencies of up to 8.6 × 10⁻⁵ (Supplementary Table 4). Plasmid pECA1039 (originally isolated from the phytopathogen, Pectobacterium atrosepticum) encodes a bifunctional type III Toxin-Antitoxin (TA) system, ToxIN, with abortive infection capacity. Although ToxIN aborts infection of various enterobacteria by diverse phages (Fineran et al., 2009b) it did not protect against infection by the tested viunalikeviruses, ϕMAM1, ViI, CBA120, LIMEstone1 or LIMEstone2 (not shown). Furthermore, another type III TA system, TenpIN, from the insect pathogen, Photorhabdus luminescens (Blower et al., 2012), failed to protect against any of the five ViI-like phages (not shown).In addition, we also tested the transduction capacity of the E. coli phage, CBA120, and confirmed transduction of plasmid-borne antibiotic resistances at a frequency of up to 10⁻⁴ transductants per p.f.u. (Figure 1a; Supplementary Table 6).We decided to test our hypothesis that the viunalikeviruses may all be generalised transducers by first isolating new viunalikeviruses from the environment. From treated sewage effluent, we isolated three new bacteriophages infecting Dickeya solani, ϕXF1, ϕXF3 and ϕXF4, as defined initially by their very characteristic ViI-like morphology in electron microscopy (Figures 2a–c). As predicted, all of these new phages were able to transduce chromosomal markers and plasmids at frequencies of up to 3 × 10⁻⁶ transductants per p.f.u. (Figure 2e; Supplementary Table 7). Sequencing of structural and non-structural protein-encoding genes of ϕXF1, ϕXF3 and ϕXF4 showed high nucleotide homology (between 80% and 100%) with the corresponding orthologs in LIMEstone1 (Supplementary Figure 1), indicating that these virgin environmental isolates also clade within the Viunalikevirus genus.Open in a separate windowFigure 2Environmental isolation and characterisation of new viunalikeviruses with generalised transduction functionality. Transmission electron micrographs of phages ϕXF1 (a), ϕXF3 (b), ϕXF4 (c) and ϕXF28 (d) are shown. As an internal control, ϕXF28 was an example of a new lytic phage isolated from the same environment but showing no transduction capabilities. Bars, 50 nm. (e) Transduction frequencies of the new viunalikeviruses ϕXF1, ϕXF3 and ϕXF4. Transduction experiments were performed using 10⁹ cells with ϕXF1, ϕXF3, ϕXF4 at an m.o.i. of 0.01. Transduction efficiency was defined as the number of transductants obtained per p.f.u. Error bars represent the standard deviations (n=3).Although we did not have access to other ViI-like Salmonella phages SFP10 (Park et al., 2012), ϕSH19 (Hooton et al., 2011) and Marshall (Luna et al., 2013), Escherichia phage PhaxI (Shahrbabak et al., 2013), Shigella phage ϕSboM-AG3 (Anany et al., 2011) and Klebsiella phage 0507-KN2-1 (Hsu et al., 2013), our results allow us to predict that all of these phages will mediate generalised transduction. Importantly, these phages would be expected to contribute to the horizontal gene transfer of virulence factors and antimicrobial-resistance determinants in diverse environments.Viunalikeviruses do not seem to be limited to the enterobacteria as bacteriophages showing ViI-like morphology have been isolated in Acinetobacter (Ackermann et al., 1994), Bordetella (Adriaenssens et al., 2012b) and Sinorhizobium (Werquin et al., 1988). Furthermore, another ViI-like morphotype phage (ϕM12 of Sinorhizobium meliloti) has also been shown to be an efficient transducer (Finan et al., 1984). Taken together, these results suggest that, even in the absence of strongly predictive comparative genomic detail, a characteristically discrete ViI-like morphology in electron microscopy may be sufficient to identify new phages as strong candidates for possession of generalised transduction capacity.The emergence and dissemination of antibiotic-resistant pathogens coupled with low discovery rates for new antimicrobials, plus increasing legal constraints on the use of chemical pesticides, have (re)focussed attention on the potential use of bacteriophages for ‘natural biocontrol'' of human, animal and plant pathogens. Several viunalikeviruses have been proposed as candidate therapeutic agents for the control of bacterial infections (Anany et al., 2011; Hooton et al., 2011; Park et al., 2012; Hsu et al., 2013; Shahrbabak et al., 2013) and the LIMEstone phages have been used in successful field trials for biocontrol of D. solani infections (Adriaenssens et al., 2012b). However, their efficient transduction capacities could provide a route for dissemination of virulence factors, such as proteases (Marits et al., 1999). In fact, we have demonstrated the interstrain transduction of plasmids and oocydin A, auxotrophy and protease markers between three different D. solani strains, at high frequencies (Figures 1 and ​and2;2; Supplementary Tables 4 and 7). Also, the irregular distribution of the oocydin A gene cluster within the Dickeya genus and the fact that its genomic context varies between strains raises the possibility of phage-mediated horizontal gene transfer between bacterial strains. These results emphasize strongly that when considering the genomics of phages for ‘phage therapy'' the absence of genes readily defined as playing roles in lysogeny or bacterial virulence may be insufficient to inspire confidence that use of a particular therapeutic phage presents no risk–particularly among the high efficiency-transducing viunalikeviruses.     

Accelerated adipogenic differentiation of hMSCs in a microfluidic shear stimulation platform

The use of transplanted adipose tissue to repair crucial defects is clinically interesting for surgical reconstruction. Terminally differentiated adipocytes are utilized to promote the healthy regeneration of defective tissue. Use of differentiated mesenchymal stem cells, capable of differentiation into adipocytes, is advantageous because of their regenerative properties. Conventionally, the differentiation of hMSCs toward adipocytes occurs through chemical stimulation. We designed a microfluidic system, consisting of plastic tubing and a syringe pump, to create an environment of shear to accelerate this differentiation process. This system employed a flow rate equivalent to the accelerated flow rates found within the arterial system in order to promote and activate intracellular and extracellular proteins associated with the adipogenic lineage. Confirmation of sustained viability following shear exposure was obtained using a fluorescent live‐dead assay. Visualization of intracellular lipid accumulation was achieved via Oil Red O staining. When placed into culture, shear stimulated hMSCs were further induced toward brown adipose tissue, as evidenced by a greater quantity of lipid triglycerides, relative to unstimulated hMSCs. qRT‐PCR analysis validated the phenotypic changes observed when the hMSCs were later cultured in adipogenic differentiation media. Additionally, increased fold change for adipogenic markers such as LPL1, CFL1, and SSP1 were observed as a result of shear stimulation. The significance of this work lies in the demonstration that transient fluid shear exposure of hMSCs in suspension can influence differentiation into adipocytes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:440–446, 2016     

Spontaneous and granulocyte–colony-stimulating factor-enhanced marrow response and progenitor cell mobilization in mice after myocardial infarction

Background aimsHematopoietic (HPC), mesenchymal (MPC) and/or endothelial (EPC) progenitor cells are being studied to repair the myocardium after acute or chronic ischemia. We examined marrow response to myocardial infarction (MI) and the ability of granulocyte–colony-stimulating factor (G-CSF) to enhance mobilization of HPC, MPC and EPC in peripheral blood (PB) and bone marrow (BM) of MI mice.MethodsWe induced MI in C57Bl/6 mice, while sham-operated (SO) animals were similarly operated on but without coronary artery ligation. Animals were treated with either saline or G-CSF, from day ?5 to day +5 after MI or from day 0 to day +5. Progenitor cell numbers in PB and BM were evaluated by fluorescence-activated cell sorting (FACS) analysis and cell culture.ResultsWhite blood cells (WBC) decreased in BM and increased in PB after MI; G-CSF amplified this effect in BM but not in PB. HPC numbers decreased in BM after MI, while HPC and granulocyte–macrophage colony-forming units (GM-CFU) increased in PB only after G-CSF treatment, and more prominently so in MI than in SO mice. MPC and fibroblast–colony-forming units (F-CFU) as well as EPC were mobilized into the PB after MI and further after G-CSF treatment. Plasma troponin T concentrations decreased after G-CSF treatment.ConclusionsBM is globally affected by acute MI, but not simple body injury, with intense mobilization of marrow MPC and EPC into the PB but inhibition of HPC. Progenitor cell entry into the PB may be paralleled by depletion of their BM pools. G-CSF is required for HPC mobilization and enhances MPC and EPC entry into the PB.