GSK2245035, a TLR7 agonist, Does Not Increase Pregnancy Loss in Cynomolgus Monkeys.

GSK2245035, a small molecule Toll-like Receptor 7 (TLR7) agonist developed for immunomodulatory treatment for allergic airways disease, aimed to reduce Th2 and enhance Th1/Treg responses to aeroallergens via the local induction of type I interferons (IFNs). GSK2245035 demonstrated selectivity for potent release of type I IFNs compared to TNF-α and IL-6, with dose dependent increases in the interferon inducible chemokine, IP-10, in the nasal compartment. Implantation and parturition require pro-inflammatory processes including IFNs, Interferon Stimulated Genes, TNFα and IP-10 while pregnancy requires immune regulation to maintain maternal fetal immune tolerance, and recombinant type I IFNs induced abortions in monkeys. Due to its mechanism of action, GSK2245035 was studied at pharmacologically and clinically relevant doses in a monkey pregnancy model. Monkeys received 0, 3 or 30 ng/kg/week GSK2245035 intranasally once weekly, from Day 20 postcoitum through Day 63 postpartum. Although systemic IFN-α and IP-10 levels were approximately 14.8 or 40 -fold (respectively) above predose levels at 3 or 30 ng/kg/week, respectively, there were no effects on pregnancy and infant outcome. Non-adverse effects included increased incidence of nasal discharge, increased maternal body temperature at 30 ng/kg/week and dose-dependent increases in maternal IP-10 and IFN-α and decreased infant anti-KLH IgM and IgG titers following KLH immunization at ≥3 ng/kg/week, relative to controls. Potentially, lower IFN-α and IP-10 levels as well as once-weekly intranasal dosing vs daily subcutaneous or intramuscular dosing with recombinant type I IFNs could explain the lack of pregnancy effects; however, there was an undesired impact on offspring immune function.

Harderoporphyria: Case of lifelong photosensitivity associated with compound heterozygous coproporphyrinogen oxidase (CPOX) mutations.

A 78-year-old man with a history of neonatal anemia and jaundice and life-long photosensitivity was found to have harderoporphyria, as evidenced by increased porphyrins in urine, plasma, erythrocytes and feces including large amounts of harderoporphyrin in feces and erythrocytes. Two previously undescribed coproporphyrinogen oxidase (CPOX) mutations were identified, including a deletion of four amino acids in a region of the enzyme mutated in 7 of the 8 previously reported cases. This case increases the molecular heterogeneity of this rare porphyria, and illustrates that it should be considered as a cause of chronic photosensitivity and porphyrin elevation at any age.

Probing the wound healing potential of biogenic silver nanoparticles.

OBJECTIVE: Silver nanoparticles (AgNPs) are known for their antimicrobial profile and wound healing activities. However, cytotoxicity and cosmetic abnormalities associated with silver pose a major challenge in their translation for therapeutic applications. Our objective was to develop biogenic AgNPs, using a single-step green synthesis, and to investigate their in vitro and in vivo behaviour as wound-healing agents. METHOD: AgNPs were prepared using the green synthesis approach with aqueous Bryonia laciniosa leaves extract. The AgNPs were then evaluated for physicochemical properties, stability, and antimicrobial and in vivo wound healing activities. RESULTS: Stable AgNPs with characteristic absorption at 408nm and 15±3nm particle size were generated via the active involvement of Bryonia laciniosa. No loss of stability was detected after 6 months at room temperature. Antibacterial activity was observed against both Gram-negative and Gram-positive bacteria with no cytotoxicity observed in vitro at a concentration of 200 µg/mL and effective cytokine modulation. In vivo wound healing experiments showed improved wound contracting ability in rats where, after 14 days, wound alleviation was 47.1±2.2% in the control groups, compared with 78.1±1.4% and 92.6±6.7% for a silver-based marketed cream and the AgNPs, respectively. CONCLUSION: The developed AgNPs proved to be superior wound healing agents owing to scarless healing with insignificant inflammation and toxicity. DECLARATION OF INTEREST: There were no external sources of funding for this study. The authors have no conflicts of interest to declare.

Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library.

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF- clones may have a selective advantage over the clones having ORFs.

Identification of an allosteric binding site on the transcription factor p53 using a phage-displayed peptide library.

Monoclonal antibody PAb1620 recognizes a conformational epitope on the transcription factor p53 and, upon binding, allosterically inhibits p53 binding to DNA. A highly diverse (1.5 x 10(10) members) phage-displayed library of peptides containing 40 random amino acids was used to identify the PAb1620 binding site on p53. Panning this library against PAb1620 resulted in three unique peptides which have statistically significant sequence identities with p53 sufficient to identify the binding site as being composed of amino acids 106-113 and 146-156. Based on these results, we propose a mechanism by which PAb1620 can allosterically inhibit p53 binding to DNA through an indirect interaction between the antibody binding site and the L1 loop (amino acids 112-124) of p53, which is a component of the DNA binding region.

E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.

Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties.