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Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.

Renal scarring after pyelonephritis is common in infancy. In this experimental study performed on tissue from 10-d-old infant and 40-d-old pubertal rats, several aspects of the renal innate immune response to a pyelonephritogenic strain of alpha-hemolysin-expressing Escherichia coli were compared. The kidney typically responds to urinary tract infection with release of proinflammatory cytokines, e.g. IL-6. Basal production of IL-6 from 10-d-old renal cortical tissue was approximately 20% of that from 40-d-old tissue. Six-hour incubation in the presence of supernatant from the E. coli culture caused an approximately 15-fold increase of IL-6 release in 10-d-old tissue and a 5-fold increase in 40-d-old tissue. The absolute level of IL-6 release in stimulated tissue was, however, significantly lower at 10 d than at 40 d. Lipopolysaccharide, the most immunogenic component of E. coli, signals via Toll-like receptor 4. Reverse transcriptase PCR performed on outer renal cortex indicated that expression of Toll-like receptor 4 mRNA was similar in both ages. Microdissection studies revealed that Toll-like receptor 4 mRNA was expressed in proximal tubules but not in glomeruli. The exotoxin alpha-hemolysin, expressed by a majority of uropathogenic E. coli isolates, stimulates IL-6 release via an alternative pathway that signals via intracellular calcium oscillations. We conclude that the higher susceptibility to pyelonephritic scarring is unlikely related to immaturity of innate immune system, as measured by cellular release of IL-6. Instead, the underlying mechanisms for pyelonephritic scarring are most likely multifactorial and may be mainly attributed to anatomic immaturity of the urinary tract.

Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability ( Pf) was measured in individual LLC-PK1cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH2terminus with green fluorescent protein. Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased Pfof cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the Pfin transfected cells. The effect was abolished by the PKC inhibitor Ro 31–8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser180, a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser180and that the effect is likely mediated by gating of the channel.

We investigated sex differences in the motor responses to the full and selective dopamine D1-like receptor agonist, (+/-)-6-chloro-7,8-dihydroxyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297; 0.3, 3, and 10 mg/kg, s.c.), in non-habituated adult rats. In general, SKF-81297 produced a biphasic effect on motor activity (including locomotion, rearing and exploratory activity) which consisted of an initial short inhibition followed by a long-lasting stimulation. These effects were dose- and sex-dependent. The inhibitory phase was more pronounced in males than females while the opposite was true for the stimulatory phase. Importantly, the motor inhibitory effects of SKF-81297 were not due to an increase in stereotypy (e.g., grooming activity). These biphasic effects on several motor parameters suggest the presence of two distinct dopamine D1 receptor populations which have opposite effects on motor activity and which are, in part, sexually dimorphic.

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.

The prosperity of a country is closely related to its level of education to fuel research and innovation. Doctoral graduates have attained the highest education level and should be the key players in research and innovation. The number of doctoral graduates is increasing rapidly in most/many countries, but is less well correlated to changes in prosperity of a country.The innovative medicines initiative (IMI) was established to help Europe strengthen its position in biomedical research and development. During its planning stage IMI observed large gaps in the scientific interaction between academia and industry in Europe, and that undergraduate students were not realizing the career opportunities within biomedical R&D. A major objective for the education and training section of IMI, the European Medicines Research Training Network (EMTRAIN, http://www.emtrain.eu), has therefore been to work out a framework for public private partnership PhD (PPP-PhD) and to create a cohort of networking, industry-aware scientists.

Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na+-K+-ATPase, that partial inhibition of Na+-K+-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na+-K+-ATPase, but not in cells expressing ouabain-insensitive Na+-K+-ATPase, whereas inhibition of Na+-K+-ATPase by lowering extracellular K+concentration decreased attachment in all three cell types. Thirty percent inhibition of Na+-K+-ATPase significantly attenuated attachment. Na+-K+-ATPase inhibition caused a sustained increase in the intracellular Ca2+concentration that obscured Ca2+transients observed in untreated cells during attachment. Inhibitors of Ca2+transporters significantly decreased attachment, but inhibition of Na+/H+exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na+-K+-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca2+.

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.

The classical view of neuronal protein synthesis is that proteins are made in the cell body and then transported to their functional sites in the dendrites and the dendritic spines. Indirect evidence, however, suggests that protein synthesis can directly occur in the distal dendrites, far from the cell body. We are developing protocols for dual labeling of RNA and proteins using 15N-uridine and 18O- or 13C-leucine pulse chase in cultured neurons to identify and localize both protein synthesis and fate of newly synthesized proteins. Pilot experiments show discrete localization of both RNA and newly synthesized proteins in dendrites, close to dendritic spines. We have for the first time directly imaged and measured the production of proteins at the subcellular level in the neuronal dendrites, close to the functional sites, the dendritic spines. This will open a powerful way to study neural growth and synapse plasticity in health and disease.

Our group has studied the origin of Ca2+ oscillations triggered by the cardiotonic steroid ouabain, a highly specific ligand of Na+-K+-ATPase. It has been shown that these low frequency oscillation...

The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca 2+ ] i ) oscillations that elicit activation of the transcription factor, NF-κB. Partial inhibition of Na,K-ATPase using low extracellular K + and depolarization of cells did not have these effects. Incubation of cells in Ca 2+ -free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca 2+ ] i oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca 2+ ] i oscillations prevented activation of NF-κB. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca 2+ ] i oscillations involved in transcriptional regulation in mammalian cells.

In this study, coreceptor usage of HIV-1 other than subtype B in relation to HIV-1 transmission from mother to child was investigated. Repeated sampling of 42 HIV- 1-seropositive, asymptomatic women in Cameroon during the second and third trimesters of pregnancy, at delivery, and 6 months postpartum were performed. Env subtyping was carried out from uncultured peripheral blood mononuclear cells (PBMCs) by heteroduplex mobility assay and, whenever necessary, by DNA sequencing. Virus isolates were tested for coreceptor usage on human cell lines-U87.CD4 and GHOST(3)-engineered to express stably CD4 and the chemokine receptors CCR1, CCR2b, CCR3, CCR5, or CXCR4, or the orphan receptors BOB/gpr15 or Bonzo/STRL33/TYMSTR. Transmission rate was 11.9%. Viruses were predominantly envelope subtype A and used CCR5 as coreceptor and, surprisingly, 4 of 28 (14.2%) isolates from mothers and I of 3 isolates from children used the orphan receptor Bonzo as well. In 2 transmitting mothers from whom sequential HIV-1 isolates were available, viral coreceptor usage evolved from CCR5 monotropic to CCR5/Bonzo dual tropic during pregnancy, and in 1 case transmission of this virus could be documented. Our data suggest that evolution of HIV-1 coreceptor usage to dual (or multi-) tropism may occur during pregnancy.

The precision by which sodium balance is regulated suggests an intricate interaction between modulatory factors released from intra- and extrarenal sources. Intrarenally produced dopamine has a central role in this interactive network. Dopamine, produced in renal tubular cells acts as an autocrine and paracrine factor to inhibit the activity of Na+,K+-ATPase as well as of a number of sodium influx pathways. The natriuretic effect of dopamine is most prominent under high salt diet. The antinatriuretic effects of noradrenaline, acting on alpha-adrenoceptors and angiotensin II are opposed by dopamine as well as by atrial natriuretic peptide (ANP). Several lines of evidence have suggested that ANP acts via the renal dopamine system and recent studies from our laboratory have shown that this effect is attributed to recruitment of silent D1 receptors from the interior of the cell towards the plasma membrane. Taken together, the observations suggest that dopamine coordinates the effects of antinatriuretic and natriuretic factors and indicate that an intact renal dopamine system is of major importance for the maintenance of sodium homeostasis and normal blood pressure.

Maintenance of a normal blood pressure requires a precise and fine-tuned regulation of salt metabolism. This is accomplished by a bidirectional regulation of renal tubular sodium transporters by natriuretic and antinatriuretic hormones. Dopamine, produced in the renal proximal tubular cells, plays an important role in this interactive system. Dopamine inhibits the activity of Na+,K+ ATPase as well as of many important sodium influx pathways in the nephron. These effects of dopamine are particularly pronounced in situation of sodium loading. There is an abundance of evidence suggesting that the natriuretic effects of ANP are to a large extent mediated via renal dopamine 1 like receptors. The renal tubular dopamine 1 like receptors are, under basal conditions, mainly located intracellularly. ANP and its second messenger, cGMP, cause a rapid translocation of the dopamine 1 like receptors to the plasma membrane. This phenomenon may explain how ANP and dopamine act in concert to regulate sodium metabolism. Regulation of sodium metabolism and blood pressure is critically dependent on a normal function of the renal dopamine system. Hence, abnormalities in the interaction between dopamine and ANP may predispose to hypertension.

It is well documented that dopamine and dopamine D1 agonists convert the protein phosphatase-1 inhibitor, DARPP-32, from its dephosphorylated, inactive form into its Thr34-phosphorylated, active form, and that these effects on DARPP-32 constitute essential components of the mechanism by which dopamine and D1 agonists achieve their biological effects. In contrast to dopamine and D1 agonists, dopamine D2 agonists dephosphorylate and inactivate DARPP-32. Here we have examined the possibility that the biological effects of dopamine D2 receptor agonists might also involve DARPP-32. For this purpose, we have examined regulation of the activity of the electrogenic ion pump Na+,K+-ATPase, an established target for dopamine signalling. We have found that dopamine D1 agonists and dopamine D2 agonists inhibit Na+,K+-ATPase activity in dissociated cells from the mouse neostriatum and that, in each case, the effect is abolished in cells from mice deficient in DARPP-32. We conclude that DARPP-32 may play an obligatory role in dopaminergic signalling mediated both by D1 receptors and by D2 receptors.

Rare diseases aff ect a limited number of individuals (defi ned as no more than one in 2000 individuals in the European Union and no more than about one in 1250 in the USA), but the number of disorders that fi t this defi nition is very large (>5000 according to WHO). Therefore, the number of patients aff ected by a rare disease could be about 30 million in Europe and 25 million in North America. The true burden of rare diseases in Europe and elsewhere is diffi cult to estimate, since epidemiological data for most of these diseases are not available. Rare diseases are an important public-health issue and a challenge for the medical community. They are called health orphans, because rare diseases were neglected for many years. Similarly, before the USA passed the Orphan Drug Act in 1983, the pharmaceutical industry had neglected the development of treatments for rare diseases, hence the name orphan drugs. Public awareness about the diffi culties of patients with rare diseases was fi rst raised by the report of the National Commission on Orphan Disease of the US Government in 1989. The Commission’s hearings with hundreds of stakeholders highlighted issues that aff ected patients’ care, such as little information on rare diseases, diffi culties of fi nancing research, drawbacks of providing adequate health insurance and coverage of medical expenses, and the limited availability of eff ective treatments. The International Classifi cation of Diseases (ICD) that is used in most countries is not convenient for rare diseases. The absence of a universally recognised coding system is an obstacle for reliable registration of patients in national or international databases, preventing assessment of the economic and social eff ects of rare diseases. For some disorders, national or international registries are available, which have been set up and maintained by researchers, patients’ associations, public institutions, or drug companies. The European Rare Disease Task Force of the Health and Consumers Protection Directorate General of the European Commission has set up a working group to collaborate with WHO on ICD-10, and is considering all other existing classifi cations to provide the rare-diseases community with a uniform system. Assessment of the prevalence of rare diseases was attempted by the European Organization for Rare Diseases (Eurordis), and Orphanet, with the support of the European Commission. This study not only provided an estimate of the prevalence of several rare diseases (table), but also showed the absence of reliable data, low consistency between sources of information, and poor methodological quality of epidemiological studies. Additionally, facilities for biochemical or genetic testing are scarce. We use the collective term of rare diseases to include a very heterogeneous group of disorders that can aff ect any system. Most rare diseases are genetic disorders, which are often severely disabling, substantially aff ect life expectancy, and impair physical and mental abilities. These disabilities result in reduced quality of life, and aff ect an individual’s potential for education and earning capabilities. One example is inborn errors of metabolism, most of which are rare (prevalence between 1 in 1400 and 1 in 5000 livebirths). An Italian prospective study (1985–97) on patients aged 0–17 years revealed that, of the 1935 newborn babies with inborn errors of metabolism identifi ed by the study, only 11% reached adulthood. Rare diseases also pose a considerable burden on the aff ected families. This burden was assessed by sending a questionnaire to 2500 patients with chronic diseases (8·2% of which were rare diseases). Patients with rare disorders had the worst exper ience in terms of loss of social and economic oppor tunities, and of medical care. Patients with rare diseases face diagnostic delays. This issue was shown in a survey of eight rare diseases (Crohn’s disease, cystic fi brosis, Duchenne muscular dystrophy, Ehlers-Danlos syndrome, Marfan’s syndrome, Prader-Willi syndrome, tuberous sclerosis, and Lancet 2008; 371: 2039–41

Activation by protein kinase A by forskolin phosphorylates and inactivates Na + ,K + -ATPase in COS-7 cells (Cheng et al. 1997b). In this study we show, using [ 3 H]ouabain binding, that forskolin-induced inhibition of Na + ,K + -ATPase activity is not because of internalization of the enzyme. The effect of forskolin on Na + ,K + -ATPase activity was examined by two independent methods, ouabain-sensitive 86 Rb + uptake in intact cells and ATP hydrolysis in microsomal preparations from cells. The change in number of functional pumps on cell surface before and after protein kinase A activation was assessed by [ 3 H]ouabain binding measured under equilibrium conditions. Cells, which had been ATP-depleted by antimycin A and 2-deoxyglucose treatment, served as a positive control for the internalization of Na + ,K + -ATPase. Activation of protein kinase A with forskolin in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine, inhibited Na + ,K + -ATPase activity, but this treatment had no effect on specific ouabain binding. No change in ouabain binding was found following activation of protein kinase C by phorbol ester or diacyl glycerol analogue treatment in cells. These data suggest that protein kinase A phosphorylation and inhibition of Na + ,K + -ATPase activity does not lead to any internalization of the enzyme in COS-7 cells.

Renal sympathetic nerves play a central role in the regulation of tubular Na+reabsorption. Norepinephrine (NE) and neuropeptide Y (NPY) are colocalized in renal sympathetic nerve endings. The purpose of this study is to examine the integrated effects of these neurotransmitters on the regulation of Na+-K+-ATPase, the enzyme responsible for active Na+reabsorption in renal tubular cells. Studies were performed on proximal tubular segments, which express adrenergic α- and β-receptors, as well as NPY-Y2receptors. It was found that α- and β-adrenergic agonists had opposing effects on Na+-K+-ATPase activity. β-Adrenergic agonists induced a dose-dependent inhibition of the Na+-K+-ATPase activity, whereas α-adrenergic agonists stimulated the enzyme. NPY abolished β-agonist-induced deactivation of Na+-K+-ATPase and enhanced α-agonist-induced activation of Na+-K+-ATPase. The β-adrenergic agonist appeared to inhibit Na+-K+-ATPase activity via a cAMP pathway. NPY antagonized β-agonist-induced accumulation of cAMP. In our preparation, NE alone had no net effect but stimulated the Na+-K+-ATPase activity in the presence of β-adrenergic antagonists, as well as in the presence of NPY. The results indicate that, in renal tissue, NPY determines the net effect of its colocalized transmitter, NE, by its ability to attenuate the β- and enhance the α-adrenergic effect.

Background: Norbin is a neuron-specific, cytosolic protein that interacts with the metabotropic glutamate receptor 5 (mGluR5) and has a profound impact on mGluR5 signaling. Yet, little is known about its synaptic distribution. Results: Here we have analyzed the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), actin and mGluR5 in spines using super-resolution microscopy. Norbin was found to have a high degree of colocalization with actin and a lower degree of colocalization with PSD-95. Co-immunoprecipitation studies confirmed that interaction occurs between Norbin and actin, but not between Norbin and PSD-95. Norbin was also found to have a high degree of colocalization with the perisynaptically located mGluR5. Findings based on structured illumination microscopy (3D-SIM) of exogenous expressed Norbin-GFP were confirmed by stimulated emission depletion microscopy (STED) of immunolabeled endogenous Norbin. Conclusions: Norbin associates with actin rather than with PSD-95 in dendritic spines. Results regarding protein localization and colocalization performed with conventional confocal microscopy must be interpreted with great caution. The now available super-resolution microscopy techniques provide more accurate information about sub-cellular protein localization than previously was possible.

Background: One out of ten newly diagnosed patients in Europe was infected with a virus carrying a drug resistant mutation. We analysed the patterns over time for transmitted drug resistance mutations (TDRM) using data from the European Spread program.Methods: Clinical, epidemiological and virological data from 4317 patients newly diagnosed with HIV-1 infection between 2002 and 2007 were analysed. Patients were enrolled using a pre-defined sampling strategy.Results: The overall prevalence of TDRM in this period was 8.9% (95% CI: 8.1-9.8). Interestingly, significant changes over time in TDRM caused by the different drug classes were found. Whereas nucleoside resistance mutations remained constant at 5%, a significant decline in protease inhibitors resistance mutations was observed, from 3.9% in 2002 to 1.6% in 2007 (p = 0.001). In contrast, resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) doubled from 2.0% in 2002 to 4.1% in 2007 (p = 0.004) with 58% of viral strains carrying a K103N mutation. Phylogenetic analysis showed that these temporal changes could not be explained by large clusters of TDRM.Conclusion: During the years 2002 to 2007 transmitted resistance to NNRTI has doubled to 4% in Europe. The frequent use of NNRTI in first-line regimens and the clinical impact of NNRTI mutations warrants continued monitoring. © 2014 Frentz et al. licensee BioMed Central Ltd. 14 Cited By :16

Background: One out of ten newly diagnosed patients in Europe was infected with a virus carrying a drug resistant mutation. We analysed the patterns over time for transmitted drug resistance mutations (TDRM) using data from the European Spread program.Methods: Clinical, epidemiological and virological data from 4317 patients newly diagnosed with HIV-1 infection between 2002 and 2007 were analysed. Patients were enrolled using a pre-defined sampling strategy.Results: The overall prevalence of TDRM in this period was 8.9% (95% CI: 8.1-9.8). Interestingly, significant changes over time in TDRM caused by the different drug classes were found. Whereas nucleoside resistance mutations remained constant at 5%, a significant decline in protease inhibitors resistance mutations was observed, from 3.9% in 2002 to 1.6% in 2007 (p = 0.001). In contrast, resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) doubled from 2.0% in 2002 to 4.1% in 2007 (p = 0.004) with 58% of viral strains carrying a K103N mutation. Phylogenetic analysis showed that these temporal changes could not be explained by large clusters of TDRM.Conclusion: During the years 2002 to 2007 transmitted resistance to NNRTI has doubled to 4% in Europe. The frequent use of NNRTI in first-line regimens and the clinical impact of NNRTI mutations warrants continued monitoring. © 2014 Frentz et al.; licensee BioMed Central Ltd.

1. At birth, rapid removal of lung liquid from potential airspaces is required to establish pulmonary gas exchange. To investigate the role for water channels, aquaporins (AQP) and ion transporters in this process, the mRNA expression of AQP, Na+,K(+)-ATPase and the amiloride-sensitive Na+ channel (ENaC) were studied in the fetal and postnatal rat lung. 2. The mRNA expression of all transporters studied increased postnatally. 3. The following water channels were expressed in the lung, AQP1, 4 and 5. The most specific perinatal induction pattern was observed for AQP4. A sharp and transient increase of AQP4 mRNA occurred just after birth coinciding with the time course for clearance of lung liquid. This transient induction of AQP4 mRNA at birth was lung-tissue specific. Around birth there was a moderate increase in AQP1 mRNA, which was not transient. AQP5 increased continuously until adulthood. 4. Fetal lung AQP4 mRNA was induced by both beta-adrenergic agonists and glucocorticoid hormone, which are factors that have been suggested to accelerate the clearance of lung liquid. 5. Immunocytochemistry revealed that AQP4 was located in the basolateral membranes of bronchial epithelia in newborn rats, consistent with the view that this is the major site for perinatal lung liquid absorption. 6. The Na+,K(+)-ATPase alpha 1 subunit and ENaC alpha-subunit mRNA also increased around birth, suggesting that they co-operatively facilitate lung liquid clearance at birth. 7. These data indicate that removal of lung liquid at birth is associated with pronounced and well-synchronized changes in the expression of AQP and the ion transporters studied. The transient perinatal induction of AQP4, which could be prenatally induced by beta-adrenergic agonists, and the localization of this water channel strongly suggest that it plays a critical role for removal of lung liquid at the time of birth.

Pyelonephritis provokes growth retardation and apoptosis in infant rat renal cortex. Childhood pyelonephritis is a common cause of renal cortical scarring and hypoplastic kidneys. To understand the mechanisms underlying the cortical lesions, urinary tract infection was induced in three-week-old rats by an intravesical infusion of E. coli , type 06 K13 HI, a rat nephropathogenic strain. Four days after infection, histopathological examination showed marked infiltration of leukocytes in the medullary tissue adjoining the calyces and pelvis. In the cortex, signs of inflammation were found only in the cortical zone adjacent to the pelvis. No cells indicative of inflammation were observed in other parts of the cortex. Immunohistochemistry for endogenous proliferating cell nuclear antigen (PCNA) demonstrated a marked decrease in immunoreactivity in proximal tubular (PT) cells. The mitotic response of PT cells, assessed by 3 H-thymidine autoradiography, showed a highly significant decrease during the first four days after induction of the infection. Four days after infection, a transient increase in apoptotic cells was observed in cortical cells outside the inflammatory areas. No increase in apoptotic cells was detected in the cortex 10 days after infection. Only a few apoptotic cells were detected in the control kidneys. In conclusion, the data indicate that inhibition of cell proliferation and enhancement of apoptosis may contribute to the renal parenchymal loss after childhood pyelonephritis.

The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3´, 5´-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an “aggregated” manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32’s possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.

Background: International travel plays a role in the spread of HIV-1 across Europe. It is, however, not known whether international travel is more important for spread of the epidemic as compared to endogenous infections within single countries. In this study, phylogenetic associations among HIV of newly diagnosed patients were determined across Europe.Results: Data came from the SPREAD programme which collects samples of newly diagnosed patients that are representative for national HIV epidemics. 4260 pol sequences from 25 European countries and Israel collected in 2002-2007 were included.We identified 457 clusters including 1330 persons (31.2% of all patients). The cluster size ranged between 2 and 28. A number of 987 patients (74.2%) were part of a cluster that consisted only of patients originating from the same country. In addition, 135 patients (10.2%) were in a cluster including only individuals from neighboring countries. Finally, 208 patients (15.6%) clustered with individuals from countries without a common border. Clustering with patients from the same country was less prevalent in patients being infected with B subtype (P-value, SCOPUS: ar.j, info:eu-repo/semantics/published