Structure-function Studies of Kinetoplastid Proteins

The class kinetoplastida include parasites responsible for devastating diseases like African sleeping sickness, Chagas’s disease and Leishmaniases, mainly effecting people in the developing world. Current treatments are highly toxic and inefficient, leading to an urgent need of novel anti-parasitic compounds. This thesis focuses on the structural characterisation of potential drug targets against these parasites, namely adenosine kinase from Trypanosoma brucei (TbAK), thymidine kinases from T. brucei (TbTK) and Leishmania major (LmTK) and the leucyl aminopeptidases from T. brucei (TbLAP-A), T. cruzi (TcLAP-A) and L. major (LmLAP-A). Structures of TbAK were solved in two conformations, open (apo) and closed (in complex with adenosine and ADP), both to 2.6 Å. Comprised of a big α/β-domain and a small lid domain, the structures confirm the large conformational change of the lid domain upon substrate binding. The structures of C-terminally truncated versions of LmTK and TbTK were determined as ligand-bound complexes with resolutions up to 2.4 Å and 2.2 Å, respectively. They show high similarity to structures of homologues in the PDB. The structures solved in this thesis give valuable information about ligand binding and aid rational drug design. Leucyl aminopeptidase (LAP-A) was evaluated as a potential drug target in T. brucei parasites. It is not essential for T. brucei parasites grown in vitro, shown by generation and analysis of LAP-A-depleted parasites. Although this does not support LAP-A as a drug target in T. brucei, no conclusions can be drawn about the potential in T. cruzi and L. major. Several structures of the LAP-As were solved, the highest resolution ones to 2.3 Å, 2.3 Å and 2.5 Å for TbLAP-A, TcLAP-A and LmLAP-A, respectively. These enzymes are hexameric and show the typical two-domain architecture of M17 LAPs. Although the physiological function remains elusive, the work in this thesis provides a firm basis for future studies

Caring Calls: A Weekly Phone Call Intervention and the Correlation with Loneliness in Rural Dwelling Adults

Purpose: This pilot study’s aim was to examine the impact of weekly phone calls from interprofessional students on loneliness in rural dwelling adults. Design: A pretest and posttest quasi-experimental design was utilized. Setting: The intervention was based in southeastern Minnesota, although the participants could have lived anywhere. Zoom for Healthcare © was utilized, and students were able to make the phone calls from their homes, therefore, no travel was necessary. Subjects: The subjects were rural dwelling adults (over the age 18 years old). Intervention: Interprofessional students made weekly social phone calls to rural dwelling adults over ten weeks. Measures: The University of California, Los Angeles (UCLA) Loneliness Scale was used to compare the level of loneliness prior to the intervention to after the intervention. Results: A Wilcoxon non-parametric test was conducted to analyze results. The overall average of the scores was -0.39, showing there was a decrease in loneliness over the ten weeks among participants, although not statistically significant (p = 0.25) Conclusion: Although there was a decrease in loneliness among the participants, the sample size was small, and the results were not statistically significant. More research is needed on this topic to determine if phone calls from students can decrease loneliness in rural-dwelling adults

Stability of Hydroxo/Oxo/Fluoro Zirconates vs. Hafniates - A DFT Study

We performed density functional theory (DFT) calculations on binary and ternary oxo/fluoro crystals of the geochemical twin pair zirconium and hafnium to evaluate and compare their stabilities. This is the first DFT study on bulk ZrF4 or HfF4, as well as on a hypothetical ZrOF2 or HfOF2 bulk crystal. For α-MO2, β-MF4 and MOF2, we have found significantly higher cohesive energies for the respective hafnium species. This suggests a considerable gap in affinity toward fluorine and oxygen between the twin pair in the solid state. In agreement with experimental findings, this gap is slightly more pronounced for fluorine. This study is also the first to evaluate the theoretical, endothermic mono-hydroxylation of the respective fluorides or oxyfluorides to model the difference in affinity toward fluoride versus hydroxide. For these, we could also find a slight energetic preference for the hafnium compound

Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major

Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2’-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5’-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.Peer reviewe

Sexual selection and the evolution of condition-dependence: an experimental test at two resource levels

Stronger condition-dependence in sexually selected traits is well-documented, but how this relationship is established remains unknown. Moreover, resource availability can shape responses to sexual selection, but resource effects on the relationship between sexual selection and condition-dependence are also unknown. In this study, we directly test the hypotheses that sexual selection drives the evolution of stronger-condition-dependence and that resource availability affects the outcome, by evolving fruit flies (Drosophila melanogaster) under relatively strong or weak sexual selection (through varied sex ratios) and at resource-poor or resource-rich adult diets. We then experimentally manipulated condition via developmental diet and assessed condition-dependence in adult morphology, behavior, and reproduction. We observed stronger condition-dependence in female size in male-biased populations and in female ovariole production in resource-limited populations. However, we found no evidence that male condition-dependence increased in response to sexual selection, or that responses depended on resource levels. These results offer no support for the hypotheses that sexual selection increases male condition-dependence or that sexual selection's influence on condition-dependence is influenced by resource availability. Our study is, to our knowledge, the first experimental test of these hypotheses. If the results we report are general, then sexual selection's influence on the evolution of condition-dependence may be less important than predicted

Avoiding Drug Resistance by Substrate Envelope-Guided Design: Toward Potent and Robust HCV NS3/4A Protease Inhibitors

Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve \u3e 95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets

Standardisation of clinical assessment, management and follow-up of acute hospitalised exacerbation of copd: A europe-wide consensus

Background: Despite hospitalization for exacerbation being a high-risk event for morbidity and mortality, there is little consensus globally regarding the assessment and management of hospitalised exacerbations of COPD. We aimed to establish a consensus list of symptoms, physiological measures, clinical scores, patient questionnaires and investigations to be obtained at time of hospitalised COPD exacerbation and follow-up. Methods: A modified Delphi online survey with pre-defined consensus of importance, feasibility and frequency of measures at hospitalisation and follow-up of a COPD exacerbation was undertaken. Findings: A total of 25 COPD experts from 18 countries contributed to all 3 rounds of the survey. Experts agreed that a detailed history and examination were needed. Experts also agreed on which treatments are needed and how soon these should be delivered. Experts recommended that a full blood count, renal function, C-reactive protein and cardiac blood biomarkers (BNP and troponin) should be measured within 4 hours of admission and that the modified Medical Research Council dyspnoea scale (mMRC) and COPD assessment test (CAT) should be performed at time of exacerbation and follow-up. Experts encouraged COPD clinicians to strongly consider discussing palliative care, if indicated, at time of hospitalisation. Interpretation: This Europe-wide consensus document is the first attempt to standardise the assessment and care of patients hospitalised for COPD exacerbations. This should be regarded as the starting point to build knowledge and evidence on patients hospitalised for COPD exacerbations

Sexual selection and the evolution of condition-dependence: an experimental test at two resource levels

Stronger condition-dependence in sexually selected traits is well-documented, but how this relationship is established remains unknown. Moreover, resource availability can shape responses to sexual selection, but resource effects on the relationship between sexual selection and condition-dependence are also unknown. In this study, we directly test the hypotheses that sexual selection drives the evolution of stronger-condition-dependence and that resource availability affects the outcome, by evolving fruit flies (Drosophila melanogaster) under relatively strong or weak sexual selection (through varied sex ratios) and at resource-poor or resource-rich adult diets. We then experimentally manipulated condition via developmental diet and assessed condition-dependence in adult morphology, behavior, and reproduction. We observed stronger condition-dependence in female size in male-biased populations and in female ovariole production in resource-limited populations. However, we found no evidence that male condition-dependence increased in response to sexual selection, or that responses depended on resource levels. These results offer no support for the hypotheses that sexual selection increases male condition-dependence or that sexual selection’s influence on condition-dependence is influenced by resource availability. Our study is, to our knowledge, the first experimental test of these hypotheses. If the results we report are general, then sexual selection’s influence on the evolution of condition-dependence may be less important than predicted

Defining the target population to make marine image-based biological data FAIR

Marine imaging studies have unique constraints on the data collected requiring a tool for defining the biological scope to facilitate data discovery, quality evaluation, sharing and reuse. Defining the ‘target population’ is way of scoping biological sampling or observations by setting the pool of organisms to be observed or sampled. It is used in survey design and planning, to determine statistical inference, and is critical for data interpretation and reuse (both images and derived data). We designed a set of attributes for defining and recording the target population in biological studies using marine photography, incorporating ecological and environmental delineation and marine imaging method constraints. We describe how this definition may be altered and recorded at different phases of a project. The set of attributes records the definition of the target population in a structured metadata format to enhance data FAIRness. It is designed as an extension to the image FAIR Digital Objects metadata standard, and we map terms to other biological data standards where possible. This set of attributes serves a need to update ecological metadata to align with new remotely-sensed data, and can be applied to other remotely-sensed ecological image data