Registration of the Chickpea Germplasm PHREC-Ca-Comp. #1 with Enhanced Resistance to Ascochyta Blight

The chickpea or garbanzo bean (Cicer arietinum L.) germplasm PHREC-Ca-Comp. #1 (Reg. No. GP-282, PI 659664) was developed by the former Alternative Crops Breeding Program at the University of Nebraska Agricultural Research Division and was released in 2010. It was bred specifically for adaptation to growing conditions in Nebraska and for enhanced resistance to Ascochyta blight, a major disease of chickpea caused by Ascochyta rabiei (Pass.) Labr. PHREC-Ca- Comp. #1 is a composite of PI 315797, PI 343014, PI 379217, PI 471915, PI 598080, and W6 17256. The composite was developed in the fall of 2002 and was evaluated in six irrigated and four dryland environments at Scottsbluff, Sidney, and Alliance, NE, from 2004 to 2009. Across irrigated environments, PHREC-Ca-Comp. #1 had the lowest severity rating for Ascochyta blight and a higher yield under both irrigated and dryland conditions than ‘Sierra’, ‘Dwelley’, ‘Dylan’, and ‘Troy’. PHREC-Ca-Comp. #1 is a small, round, cream-colored kabuli-type chickpea. It exhibits an upright, indeterminate growth habit. Plants average 66 cm in height and have excellent resistance to lodging. PHREC-Ca-Comp. #1 has a fern leaf structure and white flowers and blooms 44 d after planting. It is a midseason bean, maturing 116 d after planting. Although its seed size does not meet commercial standards, PHREC-Ca-Comp. #1 has value in breeding programs as a source of resistance to Ascochyta blight and because of its high yield potential

Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis

The guanylate binding protein (GBP) family of interferon-inducible GTPases promotes antimicrobial immunity and cell death. During bacterial infection, multiple mouse Gbps, human GBP2, and GBP5 support the activation of caspase-1-containing inflammasome complexes or caspase-4 which trigger pyroptosis. Whether GBPs regulate other forms of cell death is not known. The apicomplexan parasite Toxoplasma gondii causes macrophage death through unidentified mechanisms. Here we report that Toxoplasma-induced death of human macrophages requires GBP1 and its ability to target Toxoplasma parasitophorous vacuoles through its GTPase activity and prenylation. Mechanistically, GBP1 promoted Toxoplasma detection by AIM2, which induced GSDMD-independent, ASC-, and caspase-8-dependent apoptosis. Identical molecular determinants targeted GBP1 to Salmonella-containing vacuoles. GBP1 facilitated caspase-4 recruitment to Salmonella leading to its enhanced activation and pyroptosis. Notably, GBP1 could be bypassed by the delivery of Toxoplasma DNA or bacterial LPS into the cytosol, pointing to its role in liberating microbial molecules. GBP1 thus acts as a gatekeeper of cell death pathways, which respond specifically to infecting microbes. Our findings expand the immune roles of human GBPs in regulating not only pyroptosis, but also apoptosis

Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii

Guanylate binding proteins (GBPs) are a family of large interferon‐inducible GTPases that are transcriptionally upregulated upon infection with intracellular pathogens. Murine GBPs (mGBPs) including mGBP1 and 2 localize to and disrupt pathogen‐containing vacuoles (PVs) resulting in the cell‐autonomous clearing or innate immune detection of PV‐resident pathogens. Human GBPs (hGBPs) are known to exert antiviral host defense and activate the NLRP3 inflammasome, but it is unclear whether hGBPs can directly recognize and control intravacuolar pathogens. Here, we report that endogenous or ectopically expressed hGBP1 fails to associate with PVs formed in human cells by the bacterial pathogens Chlamydia trachomatis or Salmonella typhimurium or the protozoan pathogen Toxoplasma gondii. While we find that hGBP1 expression has no discernible effect on intracellular replication of C. trachomatis and S. typhimurium, we observed enhanced early Toxoplasma replication in CRISPR hGBP1‐deleted human epithelial cells. We thus identified a novel role for hGBP1 in cell‐autonomous immunity that is independent of PV translocation, as observed for mGBPs. This study highlights fundamental differences between human and murine GBPs and underlines the need to study the functions of GBPs at cellular locations away from PVs

Differential regulation of effector- and central-memory responses to Toxoplasma gondii infection by IL-12 revealed by tracking of Tgd057-specific CD8+ T cells

10.1371/journal.ppat.1000815PLoS Pathogens6

UV-induced ligand exchange in MHC class I protein crystals

High-throughput structure determination of protein−ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, “conditional”, peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This “in crystallo” exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC β2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families

Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

Researching underwater: a submerged study

This chapter explores the unknown territory of a lost project: an ethnography of a public swimming pool. The discussion is contextualised within my broader sociological theory of ‘nothing’, as a category of unmarked, negative social phenomena, including no-things, no-bodies, no-wheres, non-events and non-identities. These meaningful symbolic objects are constituted through social interaction, which can take two forms: acts of commission and acts of omission. I tell the story of how this project did not happen, through the things I did not do or that did not materialise, and how I consequently did not become a certain type of researcher. I identify three types of negative phenomena that I did not observe and document – invisible figures, silent voices and empty vessels – and, consequently, the knowledge I did not acquire. However, nothing is also productive, generating new symbolic objects as substitutes, alternatives and replacements: the somethings, somebodies and somewheres that are done or made instead. Thus finally, I reflect on how not doing this project led me to pursue others, cultivating a different research identity that would not otherwise have existed

Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization

Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been broadly applied to protozoan parasites, which have unique genomic structures and virulence strategies compared with viral and bacterial pathogens. Here, we present a CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. Using libraries of barcoded T. gondii, we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes representing a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. These data establish a cellular barcoding approach for protozoa and evidence that the blood-brain barrier is not a major bottleneck to colonization by T. gondii

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome

International audienc

Rethinking therapeutic strategies in cancer: wars, fields, anomalies and monsters

This article argues that the excessive focus on cancer as an insidious living defect that needs to be destroyed has obscured the fact that cancer develops inside human beings. Therefore, in order to contribute to debates about new cancer therapies, we argue that it is important to gain a broader understanding of what cancer is and how it might be otherwise. First, in order to reframe the debate, we utilize Pierre Bourdieu’s field analysis in order to gain a stronger understanding of the structure of the (sub)field of cancer research. In doing so, we are able to see that those in a dominant position in the field, with high levels of scientific capital at their disposal, are in the strongest position to determine the type of research that is carried out and, more significantly, how cancer is perceived. Field analysis enables us to gain a greater understanding of the complex interplay between the field of science (and, more specifically, the subfield of cancer research) and broader sources of power. Second, we draw attention to new possible ways of understanding cancer in its evolutionary context. One of the problems facing cancer research is the narrow time frame within which cancer is perceived: the lives of cancer cells are considered from the moment the cells initially change. In contrast, the approach put forward here requires a different way of thinking: we take a longer view and consider cancer as a living entity, with cancer perceived as anomalous rather than abnormal. Third, we theorize the possibility of therapeutic strategies that might involve the redirection (rather than the eradication) of cancer cells. This approach also necessitates new ways of perceiving cancer