Porous Frameworks for Biocatalysis Applications

Biocatalysis is a growing field, which involves the use of enzymes or other biological materials to speed up chemical reactions. Often, biocatalytic pathways are superior to traditional chemical synthesis, owing to the high selectivity, efficiency, and non-toxicity of enzymes. However, due to their structural complexity, biomolecules are unstable to the harsh conditions often used in industrial synthesis. To provide protection to enzymes, metal-organic frameworks (MOFs) and hydrogen-bonded organic frameworks (HOFs) have arisen as promising materials for enzyme immobilization. The most widely utilized MOF for enzyme encapsulation is ZIF-8, which can protect enzymes from harsh conditions including organic solvents, heating, and proteolysis. However, certain enzymes, such as catalase, lose activity when immobilized in/on ZIF-8, due to unfolding upon contact with the hydrophobic framework surface. Single enzyme nanogels and polymer-enzyme conjugates were used as methods of tuning the enzyme-MOF interface, aiming to prevent this detrimental surface interaction. Neither method allowed catalase activity to be retained, suggesting that additional factors may contribute to enzyme unfolding. Re-evaluation of the system revealed gradual decomposition of ZIF-8 when stored as an aqueous suspension, leading to the release of free 2-methylimidazole. This was detrimental to catalase activity, either by direct interaction between the linker and catalase, or indirectly by pH effects on the enzyme structure. This indicated that physical barriers would be an ineffective method to protect catalase from denaturation upon encapsulation in ZIF-8. To overcome the limitations of MOFs, HOFs may be used for enzyme encapsulation. However, there are few studies that investigate the spatial distribution of proteins in HOFs, which is critical to their eventual application as it can impact how effective the HOF is at protecting protein. Ferritin was determined to be a convenient model protein for studying the localization of proteins in HOFs, as its iron core can be visualized using transmission electron microscopy (TEM). Combined with a washing procedure of 10% SDS, it allowed individual protein macromolecules encapsulated within the framework to be identified. Various methods were trialed to increase the loading of protein in BioHOF-1, including slowed framework growth by use of a modulator, surface modification of protein, and incubation with a guanidinium-decorated polymer. All methods showed either marginal impact on protein loading or were not broadly applicable to other proteins. To offer additional protection to surface-bound protein, layer-by-layer encapsulation of BioHOF-1 was performed. This was able to protect surface-bound catalase from proteolysis by a Trypsin enzyme. With further work, this system could be employed to create high-loading enzyme@HOF biocomposites with enhanced protective properties.Thesis (MPhil) -- University of Adelaide, School of Physics, Chemistry and Earth Sciences, 202

Final report of the 2018 Census External Data Quality Panel

At the time this report was submitted for publication in January 2020, Stats NZ was finalizing its release schedule for census products that allow users to produce their own tabulations and statistical analyses using census data (see Appendix 2 for a summary of release dates for 2018 Census products). In early 2020, Stats NZ will release 2018 Census data for use in the Data Lab, a distributed set of secure computer sites where users can access, with approval, microdata for research purposes. By March 2020, 2018 Census data will be in the Integrated Data Infrastructure (IDI) and available for use by approved researchers and policy analysts who wish to negotiate access to the IDI.https://www.stats.govt.nz/assets/Uploads/Reports/Final-report-of-the-2018-Census-External-Data-Quality-Panel/Downloads/Final-report-of-the-2018-Census-External-Data-Quality-Panel-corrected.pd

The expression of ovine placental lactogen, StAR and progesterone-associated steroidogenic enzymes in placentae of overnourished growing adolescent ewes.

Overnourishing pregnant adolescent sheep promotes maternal growth but reduces placental mass, lamb birth weight and circulating progesterone. This study aimed to determine whether altered progesterone reflected transcript abundance for StAR (cholesterol transporter) and the steroidogenic enzymes (Cyp11A1, Hsd3b and Cyp17). Circulating and placental expression of ovine placental lactogen (oPL) was also investigated. Adolescent ewes with singleton pregnancies were fed high (H) or moderate (M) nutrient intake diets to restrict or support placental growth. Experiment 1: peripheral progesterone and oPL concentrations were measured in H (n=7) and M (n=6) animals across gestation (days 7-140). Experiment 2: progesterone was measured to mid- (day 81; M: n=11, H: n=13) or late gestation (day 130; M: n=21, H: n=22), placental oPL, StAR and steroidogenic enzymes were measured by qPCR and oPL protein by immunohistochemistry. Experiment 1: in H vs M animals, term placental (

Identification of a proteasome-targeting arylsulfonamide with potential for the treatment of Chagas' disease

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas’ disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between β4 and β5 subunits that catalyze chymotrypsin-like activity. A mutation in the β5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the β4/β5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery

Toolkit of Approaches To Support Target-Focused Drug Discovery for Plasmodium falciparum Lysyl tRNA Synthetase

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages

Anti-Müllerian Hormone Serum Concentrations of Women with Germline BRCA1 or 2 BRCA2 Mutations

Study funding/competing interest(s): kConFab is supported by a grant from the Australian National Breast Cancer Foundation, and previously by the National Health and Medical Research Council (NHMRC), the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. KAP is an Australian National Breast Cancer Foundation Practitioner Fellow. JLH is a NHMRC Senior Principal Research Fellow. MH is a NHMRC Practitioner Fellow. RA reports personal fees from Roche Diagnostics & Beckman Coulter outside the submitted work and CS reports other from Melbourne IVF outside the submitted work. The remaining authors have nothing to declare and no conflicts of interest.Study question: Do women with BRCA1 and BRCA2 mutations have reduced ovarian reserve, as measured by circulating anti-müllerian hormone (AMH) concentration?  Summary answer: Women with a germline mutation in BRCA1 have reduced ovarian reserve as measured by AMH.  What is known already: The DNA repair enzymes encoded by BRCA1 and BRCA2 are implicated in reproductive aging. Circulating AMH is a biomarker of ovarian reserve and hence reproductive lifespan.  Study design, size, duration: Cross-sectional study of AMH concentrations of 693 women at the time of enrolment into the Kathleen Cuningham Foundation Consortium for research into Familial Breast Cancer (kConFab) cohort study (recruitment from 19/08/1997 until 18/9/2012). AMH was measured on stored plasma samples between November 2014 and January 2015 using an electrochemiluminescence immunoassay platform.  Participants/materials, setting, methods: Eligible women were from families segregating BRCA1 or BRCA2 mutations and had known mutation status. Participants were aged 25 to 45 years, had no personal history of cancer, retained both ovaries and were not pregnant or breastfeeding at the time of plasma storage. Circulating AMH was measured for 172 carriers and 216 non-carriers from families carrying BRCA1 mutations, and 147 carriers and 158 non-carriers from families carrying BRCA2 mutations. Associations between plasma AMH concentration and carrier status were tested by linear regression, adjusted for age at plasma storage, oral contraceptive use, body mass index and cigarette smoking.  Main results and the role of chance: Mean AMH concentration was negatively associated with age (P < 0.001). Mutation carriers were younger at blood draw than non-carriers (P ≤ 0.031). BRCA1 mutation carriers had, on average, 25% (95% CI: 5% - 41%, P = 0.02) lower AMH concentrations than non-carriers and were more likely to have AMH concentrations in the lowest quartile for age (OR 1.84, 95% CI: 1.11-303, P=0.02). There was no evidence of an association between AMH concentration and BRCA2 mutation status (P = 0.94).  Limitations, reasons for caution: The clinical implications of the lower AMH concentrations seen in BRCA1 mutation carriers cannot be assessed by this study design.  Wider implications of the findings: Women with a germline mutation in BRCA1 may have reduced ovarian reserve. This is consistent with other smaller studies in the literature and has potential implications for fertility and reproductive lifespan. Publisher PDFPeer reviewe

Case study for model validation : assessing a model for thermal decomposition of polyurethane foam.

A case study is reported to document the details of a validation process to assess the accuracy of a mathematical model to represent experiments involving thermal decomposition of polyurethane foam. The focus of the report is to work through a validation process. The process addresses the following activities. The intended application of mathematical model is discussed to better understand the pertinent parameter space. The parameter space of the validation experiments is mapped to the application parameter space. The mathematical models, computer code to solve the models and its (code) verification are presented. Experimental data from two activities are used to validate mathematical models. The first experiment assesses the chemistry model alone and the second experiment assesses the model of coupled chemistry, conduction, and enclosure radiation. The model results of both experimental activities are summarized and uncertainty of the model to represent each experimental activity is estimated. The comparison between the experiment data and model results is quantified with various metrics. After addressing these activities, an assessment of the process for the case study is given. Weaknesses in the process are discussed and lessons learned are summarized

Repositioning of a diaminothiazole series confirmed to target the cyclin-dependent kinase CRK12 for use in the treatment of African animal trypanosomiasis

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12