Distinct pathways drive anterior hypoblast specification in the implanting human embryo.

Development requires coordinated interactions between the epiblast, which generates the embryo proper; the trophectoderm, which generates the placenta; and the hypoblast, which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent, as in the mouse. However, while BMP inhibits anterior signalling centre specification in the mouse, it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally, we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies.

Targeting Cross-Presentation as a Route to Improve the Efficiency of Peptide-Based Cancer Vaccines.

Cross-presenting dendritic cells (DC) offer an attractive target for vaccination due to their unique ability to process exogenous antigens for presentation on MHC class I molecules. Recent reports have established that these DC express unique surface receptors and play a critical role in the initiation of anti-tumor immunity, opening the way for the development of vaccination strategies specifically targeting these cells. This study investigated whether targeting cross-presenting DC by two complementary mechanisms could improve vaccine effectiveness, in both a viral setting and in a murine melanoma model. Our novel vaccine construct contained the XCL1 ligand, to target uptake to XCR1+ cross-presenting DC, and a cell penetrating peptide (CPP) with endosomal escape properties, to enhance antigen delivery into the cross-presentation pathway. Using a prime-boost regimen, we demonstrated robust expansion of antigen-specific T cells following vaccination with our CPP-linked peptide vaccine and protective immunity against HSV-1 skin infection, where vaccine epitopes were natively expressed by the virus. Additionally, our novel vaccination strategy slowed tumor outgrowth in a B16 murine melanoma model, compared to adjuvant only controls, suggesting antigen-specific anti-tumor immunity was generated following vaccination. These findings suggest that novel strategies to target the antigen cross-presentation pathway in DC may be beneficial for the generation of anti-tumor immunity.

Tumor penetrating peptides inhibiting MYC as a potent targeted therapeutic strategy for triple-negative breast cancers.

Overexpression of MYC oncogene is highly prevalent in many malignancies such as aggressive triple-negative breast cancers (TNBCs) and it is associated with very poor outcome. Despite decades of research, attempts to effectively inhibit MYC, particularly with small molecules, still remain challenging due to the featureless nature of its protein structure. Herein, we describe the engineering of the dominant-negative MYC peptide (OmoMYC) linked to a functional penetrating 'Phylomer' peptide (FPPa) as a therapeutic strategy to inhibit MYC in TNBC. We found FPPa-OmoMYC to be a potent inducer of apoptosis (with IC50 from 1-2 µM) in TNBC cells with negligible effects in non-tumorigenic cells. Transcriptome analysis of FPPa-OmoMYC-treated cells indicated that the fusion protein inhibited MYC-dependent networks, inducing dynamic changes in transcriptional, metabolic, and apoptotic processes. We demonstrated the efficacy of FPPa-OmoMYC in inhibiting breast cancer growth when injected orthotopically in TNBC allografts. Lastly, we identified strong pharmacological synergisms between FPPa-OmoMYC and chemotherapeutic agents. This study highlights a novel therapeutic approach to target highly aggressive and chemoresistant MYC-activated cancers.

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery.

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.

ß-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides.

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 ß-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length ß-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of ß-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split ß-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the ß-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Radiation dose from common radiological investigations and cumulative exposure in children with cystic fibrosis: an observational study from a single UK centre.

OBJECTIVES: Cumulative radiation exposure is associated with increased risk of malignancy. This is important in cystic fibrosis (CF) as frequent imaging is required to monitor disease progression and diagnose complications. Previous estimates of cumulative radiation are outdated as the imaging was performed on older equipment likely to deliver higher radiation. Our objectives were to determine the radiation dose delivered to children during common radiological investigations using modern equipment and to identify the number of such investigations performed in a cohort of children with CF to calculate their cumulative radiation exposure. DESIGN, SETTING AND PARTICIPANTS: Data including age at investigation and radiation exposure measured as estimated effective dose (EED) were collected on 2827 radiological studies performed on children at one UK paediatric centre. These were combined with the details of all radiological investigations performed on 65 children with CF attending the same centre to enable calculation of each child's cumulative radiation exposure. RESULTS: The mean EED for the common radiological investigations varied according to age. The range was 0.01-0.02 mSv for chest X-rays, 0.03-0.11 mSv for abdominal X-rays, 0.57-1.69 mSv for CT chest, 2.9-3.9 mSv for abdominal and pelvic CT, 0.20-0.21 mSv for sinus CT and 0.15-0.52 mSv for fluoroscopy-guided procedures. The mean EED was three to five times higher for helical compared with axial chest CT scans. The mean annual cumulative EED for our cohort of children with CF was 0.15 mSv/year with an estimated cumulative paediatric lifetime EED (0-18 years) of 3.5 mSv. CONCLUSIONS: This study provides up-to-date estimations of the radiation exposure when using common radiological investigations. These doses and the estimates of cumulative radiation exposure in children with CF are lower than previously reported. This reflects the reduced EED associated with modern equipment and the use of age-specific scanning protocols.

GFP-complementation assay to detect functional CPP and protein delivery into living cells.

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.

Sensitizing and Th2 adjuvant activity of cysteine protease allergens.

BACKGROUND: Innate properties that enhance immune responses might increase the propensity of certain allergens to induce allergic sensitization. Either a direct adjuvant effect or the increased immune response to the allergen could then increase allergic responses to bystander antigens. Here, we report on a model that does not use Th2-skewing adjuvants and yet achieves sensitization solely via the nasal mucosa. METHODS: Animals were sensitized with either enzymatically active, inactive or non-activated cysteine proteases via the nasal mucosa. Following two sensitization phases, mice were challenged with a higher dose of allergen. For bystander sensitization, mice received recombinant Der p 2 at sensitization in conjunction with the cysteine protease and were challenged with rDer p 2 alone. Sensitization was determined by measuring allergen-specific antibody responses and cytokine and cellular infiltrates into the lungs following challenge. RESULTS: Sensitization for Th2-type lung hypersensitivity for both the cysteine protease and bystander antigens was readily achieved and both were dependent on the proteolytic activity of the allergen. Bystander adjuvant activity was demonstrated for mice that were low IgE responders to the cysteine protease, showing a response independent from the immune response to the enhancing cysteine protease. Airway hyperreactivity was induced in the susceptible NOD strain of mouse, and mice subjected to prolonged administration of papain maintained the ability to produce lung hypersensitivity and Th2-type responses. CONCLUSIONS: These experiments demonstrate that cysteine protease activity at low doses can be an adjuvant for respiratory Th2 responses for themselves and bystander antigens in the absence of another adjuvant.

Promoter control over foreign antigen expression in a murine cytomegalovirus vaccine vector.

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Acyclic Stereoselection in the Ortho Ester Claisen Rearrangement.

The ortho ester Claisen rearrangement of trisubstituted allylic alcohols exhibits significant levels of diastereoselection. In E allylic alcohols, a 1,3-diaxial interaction develops in the chairlike transition state leading to the anti isomer, rendering the reaction syn selective by a factor of 3-5 to 1. In Z allylic alcohols, the 1,3-diaxial interaction develops in the transition state leading to the syn isomer, generating an anti:syn selectivity of 6-15 to 1. The relative stereochemistry of the syn isomer was confirmed independently by the synthesis of the mycotoxin botryodiplodin.