Interdependence with Our Most Forgetful Elders: Alzheimer\u27s in the Anthropocene

This dissertation uses autoethnography and critical psychological and philosophical theories to explore what people with Alzheimer’s disease teach us about being, forgetting, and dying in the Anthropocene. The author collected personal memory data from her lived experience of being with her mother while she had Alzheimer’s disease, and organized these memories into a series of vignettes. Each vignette was analyzed with critical psychological and philosophical theories to illuminate intersubjective themes of denial, things, ancestors, place, dying, and time. These themes connected the personal to the epochal and articulated the wisdom that our most forgetful elders can share in the Anthropocene, an era that reproduces itself around the fulcrum of forgetfulness about the interdependence of humans with the rest of the biosphere. A number of themes emerged which were elaborated in the form of wisdom being passed down from our most forgetful elders, including: denial is part of the journey, and needs to be worked through; listen for the animacy of things and other beings; you can call upon your ancestors for support and guidance; did you know that you can time travel?; agency in death is difficult to discern; be a steward of chronic grief; and care is political. It was found that our most forgetful elders remind us of our interdependence and show us ways of resisting capitalist and colonial ontological pressures, which in turn can help us navigate the climate chaos brought about by the Anthropocene

Contribution of the genetic background to the immune response of broilers vaccinated or challenged with LPAI H9N2

<p>Abstract</p> <p>Background</p> <p>The knowledge on the immune responses to LPAI is limited. The purpose of this study was to investigate the immune responses of two divergently selected lines of broilers, a line responding with high antibody response to antigens (HH), and a line responding with low antibody titers (LL) to antigen.</p> <p>Methods</p> <p>Day old chicks from each line were divided in two groups, one vaccinated with inactivated H9N2 vaccine and one non-vaccinated. At 21 days of age all the chicks were challenged with field isolate of H9N2, 1X10^6.5 ELD₅₀ per chick by drops to the eye, nose and beak. Twenty four hours and 14 days post challenge (PC), the chickens were weighed blood spleen and lungs were taken and leukocytes were isolated. The leukocytes were stained with monoclonal antibodies for surface markers and analyzed by flow cytometry. We used Elispot assay to identify the number of antibody producing cells in each of the organs. mRNA was extracted using TRIsol reagent in order to assess the cytokine production level by qRT-PCR using the SYBR green methods.</p> <p>Results</p> <p>Our results showed that LL-vaccinated group gained more weight than any of the other group. Using IDEXX kit, no antibody titers could be identified in vaccinated chicks 21 days post vaccination while 14 days PC vaccinated HH chickens demonstrated the highest average antibody titers. LL vaccinated chickens demonstrated higher average antibody titer than non-vaccinated LL. Using the Elispot assay no difference were found between the groups either cells producing IgA, IgM or IgY beside of a high number of IgY producing cells in the lungs of vaccinated HH birds.</p> <p>Conclusions</p> <p>Further data on leukocytes subpopulations using flow cytometry, cytokines production (IFNγ, IL-6, IL-18, IL-2 and IL-4) isotype specific antibody responses and number and functionality of NK cells are in process.</p

Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation.

The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to 'activation' of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. However, in contradistinction to the heme-activation mechanism, the amino-artemisinins are effective in vitro against non-heme-degrading apicomplexan parasites including T. gondii, with IC 50 values of 50-70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 μM) of these two compounds within few days. Thus, T. gondii strains that were resistant against artemisone and artemiside were generated by treating the T. gondii reference strain ME49 with stepwise increasing amounts of these compounds, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Interestingly, eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and - to a lesser extent - in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. These findings suggest that amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours

Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains.

In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B, and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. In addition to this protein, 20 other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57, exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene

Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis for patients with high-grade and metastatic disease is still very poor, and survivors are burdened with long-lasting side effects. Therefore, more effective and less toxic therapies are needed. Surface proteins are ideal targets for antibody-based therapies, like bispecific antibodies, antibody-drug conjugates, or chimeric antigen receptor (CAR) T-cells. Specific surface targets for RMS are scarce. Here, we performed a surfaceome profiling based on differential centrifugation enrichment of surface/membrane proteins and detection by LC-MS on six fusion-positive (FP) RMS cell lines, five fusion-negative (FN) RMS cell lines, and three RMS patient-derived xenografts (PDXs). A total of 699 proteins were detected in the three RMS groups. Ranking based on expression levels and comparison to expression in normal MRC-5 fibroblasts and myoblasts, followed by statistical analysis, highlighted known RMS targets such as FGFR4, NCAM1, and CD276/B7-H3, and revealed AGRL2, JAM3, MEGF10, GPC4, CADM2, as potential targets for immunotherapies of RMS. L1CAM expression was investigated in RMS tissues, and strong L1CAM expression was observed in more than 80% of alveolar RMS tumors, making it a practicable target for antibody-based therapies of alveolar RMS

Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis for patients with high-grade and metastatic disease is still very poor, and survivors are burdened with long-lasting side effects. Therefore, more effective and less toxic therapies are needed. Surface proteins are ideal targets for antibody-based therapies, like bispecific antibodies, antibody-drug conjugates, or chimeric antigen receptor (CAR) T-cells. Specific surface targets for RMS are scarce. Here, we performed a surfaceome profiling based on differential centrifugation enrichment of surface/membrane proteins and detection by LC-MS on six fusion-positive (FP) RMS cell lines, five fusion-negative (FN) RMS cell lines, and three RMS patient-derived xenografts (PDXs). A total of 699 proteins were detected in the three RMS groups. Ranking based on expression levels and comparison to expression in normal MRC-5 fibroblasts and myoblasts, followed by statistical analysis, highlighted known RMS targets such as FGFR4, NCAM1, and CD276/B7-H3, and revealed AGRL2, JAM3, MEGF10, GPC4, CADM2, as potential targets for immunotherapies of RMS. L1CAM expression was investigated in RMS tissues, and strong L1CAM expression was observed in more than 80% of alveolar RMS tumors, making it a practicable target for antibody-based therapies of alveolar RMS