Activation of phosphatase and tensin homolog on chromosome 10 mediates the inhibition of FcgammaR phagocytosis by prostaglandin E2 in alveolar macrophages

PGE2 has important inhibitory effects on the macrophage host defense functions of phagocytosis and killing, yet the molecular mechanisms involved remain to be fully elucidated. PGE2 causes an elevation of cAMP in alveolar macrophages (AMs), which in turn activates the cAMP effector targets, protein kinase A and the exchange protein activated by cAMP (Epac)-1. We now report that FcgammaR-induced PI3K/Akt and ERK-1/2 activation are inhibited by PGE2 in AMs. By specifically inhibiting the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in AMs, we attenuated the inhibitory effects of both PGE2 and a specific Epac-1 agonist (8-pCPT-2'-O-Me-cAMP) on FcgammaR-mediated phagocytosis and Akt/ERK-1/2 activation; PTEN inhibition also decreased PGE2-induced suppression of bacterial killing by AMs. Moreover, PGE2 and the Epac-1 agonist induced an increase in PTEN lipid phosphatase activity, and this was associated with decreased tyrosine phosphorylation on PTEN-a mechanism known to regulate PTEN activity. Using a pharmacological approach, we demonstrated a role for Src homology 2-containing protein tyrosine phosphatase-1 in the PGE2-induced tyrosine dephosphorylation of PTEN. Collectively, these data reveal that PGE2, via Epac-1 activation, enhances SHP-1 activity, resulting in increased PTEN activity. We suggest that this mechanism contributes to the ability of PGE2 to inhibit PI3K-dependent innate immune signaling in primary macrophages.     

Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo

Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC(50) increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V(max) and K(m)) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.     

Making use of multiple surveys: Estimating breeding probability using a multievent‐robust design capture–recapture model

Increased environmental stochasticity due to climate change will intensify temporal variance in the life‐history traits, and especially breeding probabilities, of long‐lived iteroparous species. These changes may decrease individual fitness and population viability and is therefore important to monitor. In wild animal populations with imperfect individual detection, breeding probabilities are best estimated using capture–recapture methods. However, in many vertebrate species (e.g., amphibians, turtles, seabirds), nonbreeders are unobservable because they are not tied to a territory or breeding location. Although unobservable states can be used to model temporary emigration of nonbreeders, there are disadvantages to having unobservable states in capture–recapture models. The best solution to deal with unobservable life‐history states is therefore to eliminate them altogether. Here, we achieve this objective by fitting novel multievent‐robust design models which utilize information obtained from multiple surveys conducted throughout the year. We use this approach to estimate annual breeding probabilities of capital breeding female elephant seals (Mirounga leonina). Conceptually, our approach parallels a multistate version of the Barker/robust design in that it combines robust design capture data collected during discrete breeding seasons with observations made at other times of the year. A substantial advantage of our approach is that the nonbreeder state became “observable” when multiple data sources were analyzed together. This allowed us to test for the existence of state‐dependent survival (with some support found for lower survival in breeders compared to nonbreeders), and to estimate annual breeding transitions to and from the nonbreeder state with greater precision (where current breeders tended to have higher future breeding probabilities than nonbreeders). We used program E‐SURGE (2.1.2) to fit the multievent‐robust design models, with uncertainty in breeding state assignment (breeder, nonbreeder) being incorporated via a hidden Markov process. This flexible modeling approach can easily be adapted to suit sampling designs from numerous species which may be encountered during and outside of discrete breeding seasons.     

Modeling Tumor Phenotypes In Vitro with Three-Dimensional Bioprinting

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Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.     

Antimicrobial function of short amidated peptide fragments from the tick‐derived OsDef2 defensin

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram‐positive and Gram‐negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3–12) and Os(11–22) exhibit activity when screened against Gram‐positive and Gram‐negative bacteria. Carboxyamidation of both peptides increased membrane‐mediated activity, although carboxyamidation of Os(11–22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3–12)NH₂ and Os(11–22)NH₂, have minimum bactericidal concentrations of 3.3 μM against Escherichia coli. Killing was reached within 10 minutes for Os(3–12)NH₂ and only during the second hour for Os(11–22)NH₂. In an E. coli membrane liposome system, both Os and Os(3–12)NH₂ were identified as membrane disrupting while Os(11–22)NH₂ was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3–12)NH₂ did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3–12 fragment relies on a membrane disruptive mechanism while the 11–22 fragment involves additional target mechanisms. The salt‐resistant potency of Os(3–12)NH₂ identifies it as a promising candidate for further development.