Phosphodiesterase 4 inhibition attenuates persistent heart and lung injury by neonatal hyperoxia in rats

Phosphodiesterase (PDE) 4 inhibitors are potent anti-inflammatory drugs with antihypertensive properties, and their therapeutic role in bronchopulmonary dysplasia (BPD) is still controversial. We studied the role of PDE4 inhibition with piclamilast on normal lung development and its therapeutic value on pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) in neonatal rats with hyperoxia-induced lung injury, a valuable model for premature infants with severe BPD. The cardiopulmonary effects of piclamilast treatment (5 mg·kg(-1)·day(-1)) were investigated in two models of experimental BPD: 1) daily treatment during continuous exposure to hyperoxia for 10 days; and 2) late treatment and injury-recovery in which pups were exposed to hyperoxia or room air for 9 days, followed by 9 or 42 days of recovery in room air combined with treatment started on day 6 of oxygen exposure until day 18. Prophylactic piclamilast treatment reduced pulmonary fibrin deposition, septum thickness, arteriolar wall thickness, arteriolar vascular smooth muscle cell proliferation and RVH, and prolonged survival. In the late treatment and injury-recovery model, hyperoxia caused persistent aberrant alveolar and vascular development, PH, and RVH. Treatment with piclamilast in both models reduced arteriolar wall thickness, attenuated RVH, and improved right ventricular function in the injury recovery model, but did not restore alveolarization or angiogenesis. Treatment with piclamilast did not show adverse cardiopulmonary effects in room air controls in both models. In conclusion, PDE4 inhibition attenuated and partially reversed PH and RVH, but did not advance alveolar development in neonatal rats with hyperoxic lung injury or affect normal lung and heart development.     

CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200^−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.     

Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: Data from a cohort study

ABSTRACT: BACKGROUND: Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes. METHODS: We performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007-2010. Clinical, molecular, and enzymatic data were integrated. RESULTS: Eighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8%) when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%). Patients with (fatal) neonatal presentations before diagnosis displayed residual MCAD enzyme activities <1%. After diagnosis and initiation of treatment, residual MCAD enzyme activities <10% were associated with an increased risk of hypoglycaemia and carnitine supplementation. The prevalence of MCAD deficiency upon screening was 1/8,750 (95% CI 1/7,210-1/11,130). CONCLUSIONS: Determination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities <10% should be considered to have the same risks as patients with classical ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and 10% residual MCAD enzyme activity.     

Prevention and treatment of tail biting in weaned piglets

The aims of this study were to evaluate four preventive measures and two curative treatments of tail biting. The preventive measures were: chain, rubber hose, straw rack (5 g/pig/day) and the provision of straw on the floor twice daily by hand (2 × 10 g/pig/day). The two curative treatments, which were applied following the onset of tail biting in a pen were: straw twice daily (as in the fourth preventive measure) and the removal of the biter. In total, 960 undocked weaned piglets (10 piglets per pen) were observed during 5 weeks. Tail lesions (none, bite marks and wounds) were recorded daily. The incidence of pens with wounded pig tails was significantly lower when straw was provided twice daily (8% of pens) compared to the chain (58% of pens) and rubber hose (54% of pens) treatment, but did not differ significantly from the straw rack treatment (29% of pens). Tails with bite marks were significantly less common in pens with twice daily straw (16% of pens) compared to chain (88% of pens), rubber hose (79% of pens) and straw rack (75% of pens). No significant difference was found between the curative treatments. Both treatments showed a reduced incidence of red fresh blood on the tails at days 1–9 following curative treatment, compared to day 0. However, neither curative treatment eliminated tail biting entirely. In conclusion, this study indicates that tail biting is best prevented with a small amount of straw, provided twice daily, and to a lesser extent with a straw rack, compared to providing a chain or a rubber hose. Once tail biting has occurred, providing a small amount of straw twice daily and removing the biter appears to be equally effective.     

Shifts in Bacterial Communities of Eggshells and Antimicrobial Activities in Eggs during Incubation in a Ground-Nesting Passerine

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components—pH, lysozyme and ovotransferrin—during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon’s α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment.     

Plasmin reduces fibronectin deposition by mesangial cells in a protease-activated receptor-1 independent manner

BackgroundProtease-activated receptor-1 (PAR-1) potentiates diabetic nephropathy (DN) as evident from reduced kidney injury in diabetic PAR-1 deficient mice. Although thrombin is the prototypical PAR-1 agonist, anticoagulant treatment does not limit DN in experimental animal models suggesting that thrombin is not the endogenous PAR-1 agonist driving DN.ObjectivesTo identify the endogenous PAR-1 agonist potentiating diabetes-induced nephropathy.MethodsUnbiased protease expression profiling in glomeruli from human kidneys with DN was performed using publically available microarray data. The identified prime candidate PAR-1 agonist was subsequently analysed for PAR-1-dependent induction of fibrosis in vitro.ResultsOf the 553 proteases expressed in the human genome, 247 qualified as potential PAR-1 agonists of which 71 were significantly expressed above background in diabetic glomeruli. The recently identified PAR-1 agonist plasmin(ogen), together with its physiological activator tissue plasminogen activator, were among the highest expressed proteases. Plasmin did however not induce mesangial proliferation and/or fibronectin deposition in vitro. In a PAR-1 independent manner, plasmin even reduced fibronectin deposition.ConclusionExpression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN.     

The Holocene treeline in the northern Andes (Ecuador): First evidence from soil charcoal

Indications for the speed and timing of past altitudinal treeline shifts are often contradictory. Partly, this may be due to interpretation difficulties of pollen records, which are generally regional rather than local proxies. We used pedoanthracology, the identification and dating of macroscopic soil charcoal, to study vegetation history around the treeline in the northern Ecuadorian Andes. Pedoanthracology offers a complementary method to pollen-based vegetation reconstructions by providing records with high spatial detail on a local scale. The modern vegetation is tussock grass páramo (tropical alpine vegetation) and upper montane cloud forest, and the treeline is located at ca. 3600 m. Charcoal was collected from soils in the páramo (at 3890 and 3810 m) and in the forest (at 3540 m), and represents a sequence for the entire Holocene.The presence of páramo taxa throughout all three soil profiles, especially in combination with the absence of forest taxa, shows that the treeline in the study area has moved up to its present position only late in the Holocene (after ca. 5850 cal years BP). The treeline may have been situated between 3600 m and 3800 m at some time after ca. 4900 cal years BP, or it may never have been higher than it is today. The presence of charcoal throughout the profiles also shows that fires have occurred in this area at least since the beginning of the Holocene.These results contradict interpretations of palaeological data from Colombia, which suggest a rapid treeline rise at the Pleistocene–Holocene transition. They also contradict the hypothesis that man-made fires have destroyed large extents of forest above the modern treeline. Instead, páramo fires have probably contributed to the slowness of treeline rise during the Holocene.     

Trafficking and function of the tetraspanin CD63

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.     

Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation

Two solvent-tolerant Pseudomonas putida S12 strains, originally designed for phenol and p-coumarate production, were engineered for efficient production of p-hydroxystyrene from glucose. This was established by introduction of the genes pal and pdc encoding l-phenylalanine/l-tyrosine ammonia lyase and p-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite l-tyrosine into p-hydroxystyrene, via p-coumarate. Degradation of the p-coumarate intermediate was prevented by inactivating the fcs gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM p-hydroxystyrene at a yield of 6.7% (C-mol of p-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h⁻¹. At this concentration, growth and production were completely halted due to the toxicity of p-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract p-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h⁻¹) and a final total p-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter⁻¹) was obtained in the 1-decanol phase. Thus, a P. putida S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound p-hydroxystyrene.The demand for so called “green” production of chemicals is rapidly increasing due to the declining availability of fossil fuels and the urgency to reduce CO₂ emissions (10, 30). However, this bioproduction may be hindered by the toxicity of the product of interest, such as substituted aromatics, to the production host (1, 2, 12, 29). One way to cope with this product toxicity is to deploy solvent-tolerant microorganisms as biocatalysts (5, 28). Of special interest among these solvent-tolerant hosts are Pseudomonas putida strains that have been engineered to produce a variety of compounds such as p-hydroxybenzoate (25, 33), p-coumarate (19), and (S)-styrene oxide (22). In our laboratory, we study and employ the solvent-tolerant P. putida S12. This strain is well suited for the production of substituted aromatic chemicals (18, 19, 33, 38) thanks to its extreme solvent tolerance (5, 35) and metabolic versatility toward aromatics (14, 16, 34).An example of an industrially relevant but extremely toxic aromatic is p-hydroxystyrene (4-vinyl phenol) (23). This compound is widely used as a monomer for the production of various polymers that are applied in resins, inks, elastomers, and coatings. Ben-Bassat et al. (2, 3, 23) reported p-hydroxystyrene production from glucose in Escherichia coli. In this strain, phenylalanine/tyrosine ammonia lyase (PAL/TAL; encoded by pal) from Rhodotorula glutinis and p-coumaric acid decarboxylase (PDC; encoded by pdc) from Lactobacillus plantarum were introduced for the conversion of l-tyrosine into p-hydroxystyrene via p-coumarate. The maximum concentration of p-hydroxystyrene was limited to 3.3 mM due to the toxicity of the product to the E. coli host (3, 23). To alleviate product toxicity, a two-phase fermentation with 2-undecanone as the extractant was performed. This approach resulted in a modest 14.2 mM p-hydroxystyrene in the organic phase and 0.5 mM p-hydroxystyrene in the water phase (2). Toxicity-related adverse effects on p-hydroxystyrene production may also be avoided by dividing the whole process into three stages: production of l-tyrosine from glucose by E. coli, conversion of l-tyrosine into p-coumarate by immobilized PAL-overexpressing E. coli cells, and chemical decarboxylation of p-coumarate into p-hydroxystyrene (29).In this report, we address and strongly enhance the bio-based production of p-hydroxystyrene from glucose by employing the solvent-tolerant P. putida S12 as a host. Previously, two strains, P. putida S12 C3 (19) and P. putida S12 TPL3 (38), have been constructed for the production of the l-tyrosine-derived aromatics p-coumarate and phenol, respectively. These strains were highly optimized for aromatics production, resulting in a heavily increased metabolic flux toward l-tyrosine. Therefore, they are suitable platform strains for the production of other l-tyrosine-derived aromatics (33). The bifunctional enzyme PAL/TAL (EC 4.3.1.25) from Rhodosporidium toruloides and the enzyme PDC (EC 4.1.1.-) from L. plantarum were introduced into these strains to allow the conversion of l-tyrosine into p-hydroxystyrene (Fig. ​(Fig.1).1). These minor modifications resulted in an efficient biocatalyst for the production of the value-added compound p-hydroxystyrene from glucose.Open in a separate windowFIG. 1.Schematic overview of the biochemical pathway for p-hydroxystyrene production. TAL, tyrosine ammonia lyase; FCS, feruloyl-coenzyme A synthetase. The cross indicates the disruption of fcs, disabling p-coumarate degradation.