Deimmunizing substitutions in Pseudomonas exotoxin domain III perturb antigen processing without eliminating T-cell epitopes

© 2019 Moss et al. Effective adaptive immune responses depend on activation of CD4 T cells via the presentation of antigen peptides in the context of major histocompatibility complex (MHC) class II. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity of peptides that are presented to T cells. A recombinant immunotoxin, comprising exotoxin A domain III (PE-III) from Pseudomonas aeruginosa and a cancer-specific antibody fragment, has been developed to manage cancer, but its effectiveness is limited by the induction of neutralizing antibodies. Here, we observed that this immunogenicity is substantially reduced by substituting six residues within PE-III. Although these substitutions targeted T-cell epitopes, we demonstrate that reduced conformational stability and protease resistance were responsible for the reduced antibody titer. Analysis of mouse T-cell responses coupled with biophysical studies on single-substitution versions of PE-III suggested that modest but comprehensible changes in T-cell priming can dramatically perturb antibody production. The most strongly responsive PE-III epitope was well-predicted by a structure-based algorithm. In summary, single-residue substitutions can drastically alter the processing and immunogenicity of PE-III but have only modest effects on CD4 T-cell priming in mice. Our findings highlight the importance of structure-based processing constraints for accurate epitope prediction

Competition law and policy and the food value chain

This On-Topic revisits the complex issues rising in the food sector and its value chain. Both the European Union and the US competition authorities have scrutinized relationships between food chain actors. The increasing market concentration raises new challenges for competition enforcement authorities dealing with the creation of new powerful actors at the distribution but also at the factor of production (input) levels. The concept of superior bargaining power has played a key role, sometimes criticised, in order to assess these relationships. The papers also discuss the critical intersection of competition law with public policy, with the aim to preserve sustainability, food safety and the stability of agricultural markets

Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications

Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC) lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain

Increased apical Na+ permeability in cystic fibrosis is supported by a quantitative model of epithelial ion transport.

Cystic fibrosis (CF) is caused by mutations in the Cystic Fibrosis Trans-membrane conductance Regulator (CFTR) gene, which encodes an anion channel. In the human lung CFTR loss causes abnormal ion transport across airway epithelial cells. As a result CF individuals produce thick mucus, suffer persistent bacterial infections and have a much reduced life expectancy. Trans-epithelial potential difference (Vt) measurements are routinely carried out on nasal epithelia of CF patients in the clinic. CF epithelia exhibit a hyperpolarised basal Vt and a larger Vt change in response to amiloride (a blocker of the epithelial Na(+) channel, ENaC). Are these altered bioelectric properties solely a result of electrical coupling between the ENaC and CFTR currents, or are they due to an increased ENaC permeability associated with CFTR loss? To examine these issues we have developed a quantitative mathematical model of human nasal epithelial ion transport. We find that while the loss of CFTR permeability hyperpolarises Vt and also increases amiloride-sensitive Vt, these effects are too small to account for the magnitude of change observed in CF epithelia. Instead, a parallel increase in ENaC permeability is required to adequately fit observed experimental data. Our study provides quantitative predictions for the complex relationships between ionic permeabilities and nasal Vt, giving insights into the physiology of CF disease that have important implications for CF therapy

Selective inhibition of T suppressor-cell function by a monosaccharide

Interactions between regulatory T lymphocytes and other cells are assumed to occur at the level of the cell surface. T cells which suppress the generation of specifically effector cells have been described as having antigenic, idiotypic, allotypic and I-region specificity1−4. Other T suppressor cells generated by in vitro cultivation with or without mitogenic stimulation5,6 have suppressive activity for T and B cells but no specificity can be assigned to them. These T suppressor cells (Ts) inhibit various lymphoid functions—this either reflects their polyclonal origin or indicates that the structures recognized by the Ts receptors must be common for many cell types. Carbohydrates on cell membrane-inserted glycoproteins or glycolipids might function as specific ligands for recognition by cellular receptors or soluble factors. Almost all cell-surface proteins of mammalian cells are glycosylated. There is evidence for lectin-like carbohydrate binding proteins not only in plants7 but also in toxins8, viruses9, prokaryotic cells10 and even mammalian cells, including T cells11. A functional role for these lectin-like proteins has been described for slime moulds and suggested for the selective association of embryonic cells12,13. We report here that addition of a monosaccharide can counteract the effect of T suppressor cells during the generation of alloreactive cytotoxic T cells (CTLs) in vitro

An ASKAP Search for a Radio Counterpart to the First High-significance Neutron Star-Black Hole Merger LIGO/Virgo S190814bv

We present results from a search for a radio transient associated with the LIGO/Virgo source S190814bv, a likely neutron star-black hole (NSBH) merger, with the Australian Square Kilometre Array Pathfinder. We imaged a 30 deg2 field at ΔT = 2, 9, and 33 days post-merger at a frequency of 944 MHz, comparing them to reference images from the Rapid ASKAP Continuum Survey observed 110 days prior to the event. Each epoch of our observations covers 89% of the LIGO/Virgo localization region. We conducted an untargeted search for radio transients in this field, resulting in 21 candidates. For one of these, AT2019osy, we performed multiwavelength follow-up and ultimately ruled out the association with S190814bv. All other candidates are likely unrelated variables, but we cannot conclusively rule them out. We discuss our results in the context of model predictions for radio emission from NSBH mergers and place constrains on the circum-merger density and inclination angle of the merger. This survey is simultaneously the first large-scale radio follow-up of an NSBH merger, and the most sensitive widefield radio transients search to-date

A Homolog of the Vaccinia Virus D13L Rifampicin Resistance Gene is in the Entomopoxvirus of the Parasitic wasp, Diachasmimorpha longicaudata

The parasitic wasp, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), introduces an entomopoxvirus (DlEPV) into its Caribbean fruit fly host, Anastrepha suspensa. (Loew) (Diptera: Tephritidae), during oviposition. DlEPV has a 250–300 kb unipartite dsDNA genome, that replicates in the cytoplasm of the host's hemocytes, and inhibits the host's encapsulation response. The putative proteins encoded by several DlEPV genes are highly homologous with those of poxviruses, while others appear to be DlEPV specific. Here, a 2.34 kb sequence containing a 1.64 kb DlEPV open reading frame within a cloned 4.5 kb EcoR1 fragment (designated R1–1) is described from a DlEPV EcoRI genomic library. This open reading frame is a homolog of the vaccinia virus rifampicin resistance (rif) gene, D13L, and encodes a putative 546 amino acid protein. The DlEPV rif contains two EcoRV, two HindIII, one XbaI, and one DraII restriction sites, and upstream of the open reading frame the fragment also contains EcoRV, HindII, SpEI, and BsP106 sites. Early poxvirus transcription termination signals (TTTTTnT) occur 236 and 315 nucleotides upstream of the consensus poxvirus late translational start codon (TAAATG) and at 169 nucleotides downstream of the translational stop codon of the rif open reading frame. Southern blot hybridization of HindIII-, EcoRI-, and BamH1-restricted DlEPV genomic DNA probed with the labeled 4.5 kb insert confirmed the fidelity of the DNA and the expected number of fragments appropriate to the restriction endonucleases used. Pairwise comparisons between DlEPV amino acids and those of the Amsacta moorei, Heliothis armigera, and Melanoplus sanguinipes entomopoxviruses, revealed 46, 46, and 45 % similarity (identity + substitutions), respectively. Similar values (41–45%) were observed in comparisons with the chordopoxviruses. The mid portion of the DlEPV sequence contained two regions of highest conserved residues similar to those reported for H. armigera entomopoxvirus rifampicin resistance protein. Phylogenetic analysis of the amino acid sequences suggested that DlEPV arose from the same ancestral node as other entomopoxviruses but belongs to a separate clade from those of the grasshopper- infecting M. sanguinipes entomopoxvirus and from the Lepidoptera-infecting (Genus B or Betaentomopoxvirus) A. moorei entomopoxvirus and H. armigera entomopoxvirus. Interestingly, the DlEPV putative protein had only 3–26.4 % similarity with RIF-like homologs/orthologs found in other large DNA non-poxviruses, demonstrating its closer relationship to the Poxviridae. DlEPV remains an unassigned member of the Entomopoxvirinae (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm) until its relationship to other diptera-infecting (Gammaentomopoxvirus or Genus C) entomopoxviruses can be verified. The GenBank accession number for the nucleotide sequence data reported in this paper is EF541029

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Introduction Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. Methods We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Results Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Conclusion Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo

Solar-type dynamo behaviour in fully convective stars without a tachocline

In solar-type stars (with radiative cores and convective envelopes), the magnetic field powers star spots, flares and other solar phenomena, as well as chromospheric and coronal emission at ultraviolet to X-ray wavelengths. The dynamo responsible for generating the field depends on the shearing of internal magnetic fields by differential rotation. The shearing has long been thought to take place in a boundary layer known as the tachocline between the radiative core and the convective envelope. Fully convective stars do not have a tachocline and their dynamo mechanism is expected to be very different, although its exact form and physical dependencies are not known. Here we report observations of four fully convective stars whose X-ray emission correlates with their rotation periods in the same way as in Sun-like stars. As the X-ray activity - rotation relationship is a well-established proxy for the behaviour of the magnetic dynamo, these results imply that fully convective stars also operate a solar-type dynamo. The lack of a tachocline in fully convective stars therefore suggests that this is not a critical ingredient in the solar dynamo and supports models in which the dynamo originates throughout the convection zone.Comment: 6 pages, 1 figure. Accepted for publication in Nature (28 July 2016). Author's version, including Method