Development of a quantitative assay to measure expression of transforming growth factor β (TGF-β) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.     

The developmental biogeography of hawksbill sea turtles in the North Pacific

High seas oceanic ecosystems are considered important habitat for juvenile sea turtles, yet much remains cryptic about this important life‐history period. Recent progress on climate and fishery impacts in these so‐called lost years is promising, but the developmental biogeography of hawksbill sea turtles (Eretmochelys imbricata) has not been widely described in the Pacific Ocean. This knowledge gap limits the effectiveness of conservation management for this globally endangered species. We address this with 30 years of stranding observations, 20 years of bycatch records, and recent simulations of natal dispersal trajectories in the Hawaiian Archipelago. We synthesize the analyses of these data in the context of direct empirical observations, anecdotal sightings, and historical commercial harvests from the insular Pacific. We find hawksbills 0–4 years of age, measuring 8–34 cm straight carapace length, are found predominantly in the coastal pelagic waters of Hawaii. Unlike other species, we find no direct evidence of a prolonged presence in oceanic habitats, yet satellite tracks of passive drifters (simulating natal dispersal) and our small sample sizes suggest that an oceanic phase for hawksbills cannot be dismissed. Importantly, despite over 600 million hooks deployed and nearly 6000 turtle interactions, longline fisheries have never recorded a single hawksbill take. We address whether the patterns we observe are due to population size and gear selectivity. Although most sea turtle species demonstrate clear patterns of oceanic development, hawksbills in the North Pacific may by contrast occupy a variety of ecosystems including coastal pelagic waters and shallow reefs in remote atolls. This focuses attention on hazards in these ecosystems – entanglement and ingestion of marine debris – and perhaps away from longline bycatch and decadal climate regimes that affect sea turtle development in oceanic regions.     

Effects of remote ischemic preconditioning in high-risk patients undergoing cardiac surgery (Remote IMPACT): a randomized controlled trial

Background:Remote ischemic preconditioning is a simple therapy that may reduce cardiac and kidney injury. We undertook a randomized controlled trial to evaluate the effect of this therapy on markers of heart and kidney injury after cardiac surgery.Methods:Patients at high risk of death within 30 days after cardiac surgery were randomly assigned to undergo remote ischemic preconditioning or a sham procedure after induction of anesthesia. The preconditioning therapy was three 5-minute cycles of thigh ischemia, with 5 minutes of reperfusion between cycles. The sham procedure was identical except that ischemia was not induced. The primary outcome was peak creatine kinase–myocardial band (CK-MB) within 24 hours after surgery (expressed as multiples of the upper limit of normal, with log transformation). The secondary outcome was change in creatinine level within 4 days after surgery (expressed as log-transformed micromoles per litre). Patient-important outcomes were assessed up to 6 months after randomization.Results:We randomly assigned 128 patients to remote ischemic preconditioning and 130 to the sham therapy. There were no significant differences in postoperative CK-MB (absolute mean difference 0.15, 95% confidence interval [CI] −0.07 to 0.36) or creatinine (absolute mean difference 0.06, 95% CI −0.10 to 0.23). Other outcomes did not differ significantly for remote ischemic preconditioning relative to the sham therapy: for myocardial infarction, relative risk (RR) 1.35 (95% CI 0.85 to 2.17); for acute kidney injury, RR 1.10 (95% CI 0.68 to 1.78); for stroke, RR 1.02 (95% CI 0.34 to 3.07); and for death, RR 1.47 (95% CI 0.65 to 3.31).Interpretation:Remote ischemic precnditioning did not reduce myocardial or kidney injury during cardiac surgery. This type of therapy is unlikely to substantially improve patient-important outcomes in cardiac surgery. Trial registration: ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT01071265","term_id":"NCT01071265"}}NCT01071265.Each year, 2 million patients worldwide undergo cardiac surgery. For more than 25% of these patients, the surgery is complicated by myocardial infarction (MI) and/or acute kidney injury, both of which are strongly associated with morbidity and mortality.^1–3 Preventing MI and acute kidney injury after cardiac surgery would improve survival.An important cause of MI and acute kidney injury in patients undergoing cardiac surgery is ischemia–reperfusion injury.^4,5 This type of injury begins as ischemia, which is then exacerbated by a systemic inflammatory response upon restoration of organ perfusion.⁶ Remote ischemic preconditioning may mitigate ischemia–reperfusion damage. It is accomplished by inducing, before surgery, brief episodes of ischemia in a limb, which lead to widespread activation of endogenous cellular systems that may protect organs from subsequent severe ischemia and reperfusion.^7–9Small randomized controlled trials evaluating the efficacy of remote ischemic preconditioning have had mixed results.^10–17 Interpretation of their data is difficult because of small sample sizes and heterogeneity in the preconditioning procedures and patient populations (e.g., few trials have evaluated patients at high risk of organ injury and postoperative death). Whether remote ischemic preconditioning effectively mitigates ischemia–reperfusion injury therefore remains uncertain. We undertook the Remote Ischemic Preconditioning in Cardiac Surgery Trial (Remote IMPACT) to determine whether this procedure reduces myocardial and kidney injury. We proposed that a large trial to determine the effect on clinically important outcomes would be worthwhile only if a substantial effect on myocardial or kidney injury, or both, were observed in the current study.     

Simultaneous determination of abacavir and zidovudine from rat tissues using HPLC with ultraviolet detection

A simple high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of abacavir and zidovudine (AZT) in rat plasma, amniotic fluid, fetal, and placental tissues. Extraction of abacavir, AZT, and the internal standard, azidouridine (AZDU) in amniotic fluid was carried out by protein precipitation. Extraction from plasma, fetal and placental homogenates was achieved by using a salting out technique. Chromatographic separation was performed using a C8 column (150 mm x 4.6 mm, 5 microm). The mobile phase consisted of 12% acetonitrile in 25 mM sodium phosphate buffer (adjusted to pH 7 with sodium hydroxide) for the fetus, placenta, plasma and amniotic fluid samples at a flow rate of 0.8 mL/min. The method was validated over the range from 0.05 to 50 microg/mL for both abacavir and AZT in the four biological matrices. The absolute recovery of abacavir ranged from 79 to 94%, while AZT recoveries ranged from 79 to 90% in the different biological matrices. The internal standard recovery ranged from 90 to 92%. Acceptable intra- and inter-day assay precision (<10% R.S.D.) and accuracy (<10% error) were observed over 0.05-50 microg/mL for all four matrices.     

Regulation of a Phage Endolysin by Disulfide Caging

In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz_P1, of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz₁₀₃, the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz₁₀₃ does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz₁₀₃ is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz_P1 and Lyz₁₀₃ define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz₁₀₃ is the first enzyme found to be regulated by disulfide bond caging of its active site.In infections by double-stranded DNA phages, host lysis requires degradation of the peptidoglycan by a phage-encoded endolysin (17). By far the most intensively studied endolysin is the T4 lysozyme E (EC 3.2.1.17), which attacks the glycosidic bonds between GlcNAc and MurNAc in the murein (1). During the latent period, canonical endolysins are produced as fully active enzymes sequestered in the cytoplasm, thereby preventing premature lysis. Another phage protein, the holin, terminates the infection cycle by suddenly forming extremely large, nonspecific holes in the membrane that allow the endolysin to escape and attack the murein layer. Recently, studies of the lysis system of bacteriophage P1 have revealed that phage-encoded endolysins are not always dependent upon holins for export (19, 20). Although it is an ortholog of T4 E, the P1 lysozyme, Lyz_P1, is translocated across the cytoplasmic membrane by the host sec system by virtue of an N-terminal transmembrane domain (TMD) that is absent in E (Fig. ​(Fig.11 A). Since this transmembrane domain is not removed by signal peptidase, nascent Lyz_P1 remains tethered to the membrane with its catalytic residues already present in the periplasm. The Lyz_P1 TMD exits the membrane and becomes part of the soluble, periplasmic form of the protein, either at a low spontaneous rate or, more efficiently, when the holin triggers to depolarize the membrane (11). Because of the unique ability to direct sec-mediated export and membrane insertion and to support release into the periplasm from the bilayer, the TMD of Lyz_P1 was designated a signal anchor release (SAR) domain. More recently, other SAR endolysins have been identified and characterized (15, 16). In fact, bioinformatic analysis suggests that most members of the T4 lysozyme family, recognizable by the Glu-8aa-(Asp/Cys)-5aa-Thr catalytic triad (Fig. ​(Fig.1A),1A), are SAR endolysins (43 of 58 entries in the GenBank protein database) (16).Open in a separate windowFIG. 1.Sequence alignments. (A) Alignment of T4E, Lyz_P1, and Lyz₁₀₃. Catalytic residues are highlighted in blue, Cys residues in pink, and SAR domains in yellow. Locations of Leu substitutions are indicated below the corresponding Gly residue (green). Inactivating/caging disulfides for Lyz_P1 and Lyz₁₀₃, respectively, are shown as arrows connected with a black line above the participating Cys residues. Disulfides resulting from isomerization are shown below the participating Cys residues as arrows connected with a brown line. (B) Alignments of Lyz_P1→103 and Lyz_103→P1 conversion mutants. Catalytic residues are highlighted in blue, Cys residues in pink, and SAR domains in yellow. Locations of Cys substitutions are indicated below the corresponding residue.Since the lyz_P1 gene is expressed well before progeny P1 phage have been assembled, there must be a mechanism to ensure that the membrane-tethered form of the protein is kept enzymatically inactive so that premature lysis is avoided. A key to the regulation of Lyz_P1 is the fact that the P1 enzyme has a catalytic cysteine residue, Cys₅₁ (Fig. ​(Fig.1A),1A), in the central position of the catalytic triad, in contrast to E and most of its orthologs, which have an Asp residue in this position (20). Genetic, biochemical, and structural analysis of Lyz_P1 demonstrated that the membrane-tethered form is inactive for two reasons: first, the entire catalytic domain is misfolded, so the active-site cleft is completely missing, and second, the catalytic Cys₅₁ is covalently occupied in a disulfide bond with another Cys at position 44. This led to a model for activation in which a thiol (Cys₁₃) present in the SAR domain becomes unmasked upon membrane release and triggers a disulfide bond isomerization, liberating the thiol of the catalytic Cys₅₁. This model was confirmed by crystal structures showing the alternative disulfide linkages in the inactive and active forms of Lyz_P1 (19).Lyz_P1 became the prototype of a class of SAR endolysins recognizable by the Asp→Cys substitution in the catalytic triad and the presence of activating Cys in the N-terminal SAR domain. However, most SAR endolysins belong to a second major class, represented by R₂₁, the endolysin of the lambdoid phage 21 (20). These enzymes have the canonical Glu-8aa-Asp-5aa-Thr catalytic triad and no Cys residue in the SAR domain. Instead, genetic and structural analysis revealed that in the inactive form, the catalytic domain has nearly the correct fold, except for a displacement of the active-site Glu, but is subject to steric hindrance by the proximity of the bilayer in which the SAR domain is embedded. In the soluble, active form, the SAR domain of R₂₁ has refolded into the main body of the enzyme, providing a floor to the active site and repositioning the catalytic glutamate to its proper place (16). Thus, the R₂₁ regulatory scheme is markedly different from that of Lyz_P1, where the released SAR domain provides only the free thiol for the disulfide bond rearrangement and makes few contacts with the enzyme itself.Here, we examine the regulation of the endolysin Lyz₁₀₃ of the Erwinia amylovora phage ERA103 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF160123","term_id":"121621816","term_text":"EF160123"}}EF160123), which seems to have characteristics of both of these major classes: it has a Cys residue in an N-terminal hydrophobic sequence but retains the canonical Asp residue in the catalytic triad. The results are discussed in terms of a model for SAR-dependent disulfide bond isomerization distinct from that of Lyz_P1 and its homologs but which nevertheless confers a covalent constraint on premature activation of the muralytic activity.     

Adiponectin Promotes Macrophage Polarization toward an Anti-inflammatory Phenotype

It is established that the adipocyte-derived cytokine adiponectin protects against cardiovascular and metabolic diseases, but the effect of this adipokine on macrophage polarization, an important mediator of disease progression, has never been assessed. We hypothesized that adiponectin modulates macrophage polarization from that resembling a classically activated M1 phenotype to that resembling alternatively-activated M2 cells. Peritoneal macrophages and the stromal vascular fraction (SVF) cells of adipose tissue isolated from adiponectin knock-out mice displayed increased M1 markers, including tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 and decreased M2 markers, including arginase-1, macrophage galactose N-acetyl-galactosamine specific lectin-1, and interleukin-10. The systemic delivery of adenovirus expressing adiponectin significantly augmented arginase-1 expression in peritoneal macrophages and SVF cells in both wild-type and adiponectin knock-out mice. In culture, the treatment of macrophages with recombinant adiponectin protein led to an increase in the levels of M2 markers and a reduction of reactive oxygen species and reactive oxygen species-related gene expression. Adiponectin also stimulated the expression of M2 markers and attenuated the expression of M1 markers in human monocyte-derived macrophages and SVF cells isolated from human adipose tissue. These data show that adiponectin functions as a regulator of macrophage polarization, and they indicate that conditions of high adiponectin expression may deter metabolic and cardiovascular disease progression by favoring an anti-inflammatory phenotype in macrophages.     

Alkaline phosphatase activation and collagen synthesis in human skin fibroblasts in culture

It has been noted in regenerating wounds that alkaline phosphatase activity in fibroblasts reaches a maximum when the collagen production is greatest. Tissue culture studies were carried out to show that prednisolone phosphate, while increasing the specific activity of alkaline phosphatase in human diploid skin fibroblasts, did not affect accumulation of collagen-hydroxyproline in monolayers or media. Addition of sodium ascorbate, resulted in rapid accumulation of hydroxyproline in the culture over a 13-day interval, while alkaline phosphatase activity increased only slightly over the last 6 days. When prednisolone and ascorbate were added in combination, alkaline phosphatase activity was significantly increased: but accumulation of hydroxyproline was no greater than in cultures to which ascorbate alone was added. Activation of alkaline phosphatase induced by prednisolone phosphate does not appear to be directly related to the biosynthesis of collagen in human skin fibroblasts in tissue culture.