Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL8:CXCR1 complex

Protein-capture agents are widely used for the detection, immobilization and isolation of proteins and are the foundation for the development of in vitro diagnostic chips. The chemokine CXCL8 is an interesting protein target due to its involvement in the human inflammatory response. We constructed a novel structural model of CXCL8 interaction with its G-protein coupled receptor CXCR1, taking into account previously reported experimental data. From this CXCL8:CXCR1 model complex, the interaction of CXCL8 with residues near the extracellular domains 3 and 4 of CXCR1 were used as a scaffold for the rational design of a peptide capture agent called 'IL8RPLoops'. A molecular dynamics simulation of IL8RPLoops indicates a stable helical conformation consistent with the CXCR1 structure from which it was derived. CXCL8 capture in fluorescence-based assays on beads and on glass demonstrates that IL8RPLoops is an effective capture agent for CXCL8. Additionally, we found IL8RPLoops to be a potent inhibitor of CXCL8-induced neutrophil migration and CXCL8:CXCR1 association. A theoretical binding model for IL8RPLoops:CXCL8 is proposed, which shows the peptide predominantly interacting with CXCL8 via electrostatic contacts with the ELR motif at the CXCL8 N-terminus

Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O6-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg−1 protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O6-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O6-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O6-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner

Technology generation to dissemination:lessons learned from the tef improvement project

Indigenous crops also known as orphan crops are key contributors to food security, which is becoming increasingly vulnerable with the current trend of population growth and climate change. They have the major advantage that they fit well into the general socio-economic and ecological context of developing world agriculture. However, most indigenous crops did not benefit from the Green Revolution, which dramatically increased the yield of major crops such as wheat and rice. Here, we describe the Tef Improvement Project, which employs both conventional- and molecular-breeding techniques to improve tef\u2014an orphan crop important to the food security in the Horn of Africa, a region of the world with recurring devastating famines. We have established an efficient pipeline to bring improved tef lines from the laboratory to the farmers of Ethiopia. Of critical importance to the long-term success of this project is the cooperation among participants in Ethiopia and Switzerland, including donors, policy makers, research institutions, and farmers. Together, European and African scientists have developed a pipeline using breeding and genomic tools to improve the orphan crop tef and bring new cultivars to the farmers in Ethiopia. We highlight a new variety, Tesfa, developed in this pipeline and possessing a novel and desirable combination of traits. Tesfa\u2019s recent approval for release illustrates the success of the project and marks a milestone as it is the first variety (of many in the pipeline) to be released

Enhanced West Nile virus surveillance in the North Kent marshes, UK

Background As part of efforts to more fully understand the potential risks posed by West Nile virus (WNV) and Usutu virus (USUV) in the UK, and following on from previous reports of a potential bridge vector Culex modestus for these viruses, at wetland sites in North Kent, mosquito surveillance was undertaken more widely across the Isle of Sheppey, the Hoo Peninsula and the Kent mainland. Methods Larval surveys were conducted and Mosquito Magnet® adult traps were used to collect adult mosquitoes. Pools of female mosquitoes were tested for the presence of WNV using real-time reverse transcriptase polymerase chain reaction. A subset of samples was tested for USUV. Results Culex modestus was found in both the pre-imaginal and imago stage at all five locations surveyed, accounting for 90% of adult mosquitoes collected. WNV or USUV were not detected in any sample. Conclusions Although no mosquitoes have been shown to be virus positive, the field survey data from this study demonstrated the dominance of an important bridge vector species for WNV in this region. Its wide geographical distribution highlights the need to update risk assessments on WNV introduction, and to maintain vigilance for WNV in the South East of England

Evaluating the risk for Usutu virus circulation in Europe : comparison of environmental niche models and epidemiological models

Abstract Background Usutu virus (USUV) is a mosquito-borne flavivirus, reported in many countries of Africa and Europe, with an increasing spatial distribution and host range. Recent outbreaks leading to regional declines of European common blackbird (Turdus merula) populations and a rising number of human cases emphasize the need for increased awareness and spatial risk assessment. Methods Modelling approaches in ecology and epidemiology differ substantially in their algorithms, potentially resulting in diverging model outputs. Therefore, we implemented a parallel approach incorporating two commonly applied modelling techniques: (1) Maxent, a correlation-based environmental niche model and (2) a mechanistic epidemiological susceptible-exposed-infected-removed (SEIR) model. Across Europe, surveillance data of USUV-positive birds from 2003 to 2016 was acquired to train the environmental niche model and to serve as test cases for the SEIR model. The SEIR model is mainly driven by daily mean temperature and calculates the basic reproduction number R0. The environmental niche model was run with long-term bio-climatic variables derived from the same source in order to estimate climatic suitability. Results Large areas across Europe are currently suitable for USUV transmission. Both models show patterns of high risk for USUV in parts of France, in the Pannonian Basin as well as northern Italy. The environmental niche model depicts the current situation better, but with USUV still being in an invasive stage there is a chance for under-estimation of risk. Areas where transmission occurred are mostly predicted correctly by the SEIR model, but it mostly fails to resolve the temporal dynamics of USUV events. High R0 values predicted by the SEIR model in areas without evidence for real-life transmission suggest that it may tend towards over-estimation of risk. Conclusions The results from our parallel-model approach highlight that relying on a single model for assessing vector-borne disease risk may lead to incomplete conclusions. Utilizing different modelling approaches is thus crucial for risk-assessment of under-studied emerging pathogens like USUV

Drivers of Political Participation: The Role of Partisanship, Identity, and Incentives in Mobilizing Zambian Citizens

Scholars and policymakers widely view identity as a key driver of African citizens’ political engagement. In doing so, however, they have emphasized ethnicity and largely sidelined other identities, including gender, local origin, shared residency, and partisanship. In this paper, we explore which identities drive political engagement and why they do so. We employ an original survey experiment that includes various identities and other incentives that may drive citizens’ participation around Zambia’s 2021 national elections. We find that partisanship most influences individuals’ stated willingness to campaign for a candidate or meet with an MP, while ethnicity and social incentives play less significant roles. Finally, we explore the mechanisms underpinning these results and find that citizens anticipate sanctions if they fail to support a co-partisan but not a co-ethnic candidate. These findings have important implications for understanding political engagement and democratic development throughout the region.</jats:p

Crystallization and preliminary crystallographic analysis of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease

Trypanothione reductase from Trypanosoma cruzi is the most promising target molecule for the rational design of a specific drug against Chagas' disease. The recombinant protein was purified in a single Chromatographie step and crystallized. Two crystal forms suitable for X-ray diffraction analysis were obtained. Tetragonal crystals (a = b = 87.4 Å, c = 152.3 Å) were grown from 30% polyethylene glycol (average M r = 8,000) in the presence of 0.2% β-n-octylglucoside (space group either P42 with one dimer or P4222 with one monomer in the asymmetric unit). Monoclinic crystals (space group P2, a = 136.3 Å, b = 91.1 Å, c = 126.0 Å, β = 94°) were grown from 1.2 M sodium citrate in the presence of 2% octanoyl-N-methyl-glucamide. They contain two dimers of the enzyme in the asymmetric unit; both crystal forms diffract to 3 Å resolution

Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has proven its clinical utility in hematological malignancies. Optimization is still required for its application in solid tumors. Here, the lack of cancer-specific structures along with tumor heterogeneity represent a critical barrier to safety and efficacy. Modular CAR T cells indirectly binding the tumor antigen through CAR-adaptor molecules have the potential to reduce adverse events and to overcome antigen heterogeneity. We hypothesized that a platform utilizing unique traits of clinical grade antibodies for selective CAR targeting would come with significant advantages. Thus, we developed a P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human antibodies containing P329G L234A/L235A (LALA) mutations for Fc silencing. METHODS: A single chain variable fragment-based second generation P329G-targeting CAR was retrovirally transduced into primary human T cells. These CAR T cells were combined with IgG1 antibodies carrying P329G LALA mutations in their Fc part targeting epidermal growth factor receptor (EGFR), mesothelin (MSLN) or HER2/neu. Mesothelioma, pancreatic and breast cancer cell lines expressing the respective antigens were used as target cell lines. Efficacy was evaluated in vitro and in vivo in xenograft mouse models. RESULTS: Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, P329G-targeting CAR T cells revealed specific effector functions only when combined with antibodies carrying P329G LALA mutations in their Fc part. P329G-targeting CAR T cells cannot be activated by an excess of human IgG. P329G-directed CAR T cells combined with a MSLN-targeting P329G-mutated antibody mediated pronounced in vitro and in vivo antitumor efficacy in mesothelioma and pancreatic cancer models. Combined with a HER2-targeting antibody, P329G-targeting CAR T cells showed substantial in vitro activation, proliferation, cytokine production and cytotoxicity against HER2-expressing breast cancer cell lines and induced complete tumor eradication in a breast cancer xenograft mouse model. The ability of the platform to target multiple antigens sequentially was shown in vitro and in vivo. CONCLUSIONS: P329G-targeting CAR T cells combined with antigen-binding human IgG1 antibodies containing the P329G Fc mutation mediate pronounced in vitro and in vivo effector functions in different solid tumor models, warranting further clinical translation of this concept