Characterising novel, humanised and physiological mouse models of FUS-ALS

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative condition, with mutations in the Fused in Sarcoma (FUS) gene accounting for particularly severe, early onset forms of the disease. Despite extensive research into ALS, there is still no cure, and almost nothing in the way of efficacious treatments. This may be in part due to a lack of physiologically relevant models. Mouse models of ALS, and other neurodegenerative diseases, have historically been transgenic and as such, the phenotypes they display may represent artefacts of overexpression, rather than disease relevant mechanisms. In this thesis I will present data characterising genomically humanised knock-in mouse models of FUS-ALS. In these knock-in mice, the mouse Fus gene has been replaced with the human FUS sequence at the endogenous mouse Fus locus. The humanised knock-in FUS mice express the human FUS gene, from the ATG start codon through to the 3’UTR and including all introns and exons, in a physiologically relevant pattern of expression. Initial characterisation of humanised FUS wildtype knock-in mice reveals minimal disruption of molecular, cellular, and overt phenotypes resulting from humanising FUS. Heterozygous humanised FUS-ALS mice show disease-relevant phenotypes, including progressive late-onset reduction in muscle strength from one year, hyperactivity and metabolic impairments. This is alongside cellular and molecular changes from 4 months, as shown by RNA sequencing data from the spinal cord and tibialis anterior muscle, and primary motor neuron culture studies. Two distinct FUS mutations are then compared to try and understand what drives different disease severity seen in FUS-ALS patients. The phenotypes displayed by the FUS-ALS models highlight the potential of fully humanised knock-in mice to aid in unravelling early disease mechanisms, and ultimately produce therapies targeted towards the human gene and protein, in the context of, but not limited to, ALS

Case-Informed Lessons for Scaling Innovation at Community and Technical Colleges

This evaluation report of Achieving the Dream's Catalyst Fund builds on the emergent research on scale,and its reconceptualization from replication to transformation.In Section One of this report, we provide a brief overview on the importance of scale in the context of a national movement to increase college completion, including a review of the most salient literature on sustainability and scale that informed our evaluation.In Section Two, we describe the Catalyst Fund initiative to support four community colleges to scale an innovative practice to serve most of their students, followed by an overview of our evaluation approach.In Sections Three and Four, we discuss our evaluation findings, and offer illustrative examples of the key factors that appear necessary to achieve scale. Finally, we conclude the report with recommendations for colleges and other stakeholders that wish to scale innovation –and transform their organizational culture –in service of student success and the college completion agenda

The meaningful vote explained in sticky notes

What does the Commons vote on a meaningful vote mean? Joelle Grogan (Middlesex University, left) and Georgia Price explain

Public Benefits and Community Colleges: Lessons from the Benefits Access for College Completion Evaluation

This Final Evaluation Report provides the lessons learned from the Benefits Access for College Completion demonstration (BACC) demonstration project at five of the seven community colleges over the past three years. From the onset of BACC, the evaluation was focused on documenting and learning how the participating colleges approached this work, and how and why they made adjustments during the demonstration. This evaluation approach was intended to provide useful formative feedback to the colleges during the demonstration, but it also was intended to help answer the overarching evaluation question posed by the funders: What are the most promising models for community colleges to increase benefits access for their students, and how can these models be integrated into community college operations?During the course of our evaluation, we observed three key findings that emerged from the BACC demonstration. Colleges converged on the need for a centralized hub to deliver benefits access services, and also began moving toward an opt-out model of pre-screening and screening for benefits access by connecting this initial step in the application process to existing student support services like financial aid and advising. Cutting across these two findings is the critical importance of leadership and commitment to benefits access – up and down the administrative hierarchy and across departments and divisions, but especially for student services. In the following sections, we first present an overview of the BACC demonstration and the various approaches colleges explored at the onset. In Section 2, we provide a detailed discussion of the three main findings from our evaluation, including how the model for delivering benefits access services changed during the demonstration, highlighting specific examples from the five colleges. In Section 3, we discuss the impact analysis at one college where quantitative student data were matched with state administrative data on the receipt of public benefits. We conclude the report by summarizing our core findings, and pointing to additional research that is needed to better understand how benefits access services can be implemented and sustained on a college campus, and the impact of these benefits on student academic outcomes.

Caregiver perspectives of scoliosis surgery for children with cerebral palsy : a qualitative study

Purpose: To explore the perspectives of primary caregivers of children with cerebral palsy (CP) who had spinal surgery for scoliosis. Materials and Methods: A qualitative study was conducted using semi-structured interviews and guided by qualitative description methodology. Participants were caregivers of children with CP aged 5–18, who had undergone spinal surgery for scoliosis in Australia. The research team included a parent with lived experience. Results: Fourteen participants (8 biological mothers), aged 40–49 years, completed online semi-structured interviews. Four themes were identified emerged. Life with a child with CP underpinned all experiences which were founded on familiarity with their child, medical procedures, and hospitalisation. Three subthemes were parents are the experts in knowing their child, children are vulnerable, and impact on caregivers. Theme 2 involved the significance of decision making to proceed with surgery. Theme 3 underscored a need to be prepared for the surgical journey and, in Theme 4, participants spoke of needing to expect the unexpected. Conclusion: The findings highlight the importance of understanding caregiver experiences and can help inform health professionals and other families in the decision-making process, preparing for and navigating spinal surgery

Multivariate and network meta-analysis of multiple outcomes and multiple treatments: rationale, concepts, and examples

Organisations such as the National Institute for Health and Care Excellence require the synthesis of evidence from existing studies to inform their decisions—for example, about the best available treatments with respect to multiple efficacy and safety outcomes. However, relevant studies may not provide direct evidence about all the treatments or outcomes of interest. Multivariate and network meta-analysis methods provide a framework to address this, using correlated or indirect evidence from such studies alongside any direct evidence. In this article, the authors describe the key concepts and assumptions of these methods, outline how correlated and indirect evidence arises, and illustrate the contribution of such evidence in real clinical examples involving multiple outcomes and multiple treatment

Exploring the role of anti-solvent effects during washing on active pharmaceutical product purity

Washing is a key step in pharmaceutical isolation to remove unwanted crystallisation solvent, rich in impurities, (mother liquor) from the Active Pharmaceutical Ingredient (API) filter cake. This study looks at strategies for optimal wash solvent selection, minimising dissolution of API product crystals while preventing precipitation of product or impurities. Selection of wash solvent to avoid both these phenomena can be challenging but is essential to maintain yield, purity, and particle characteristics throughout the isolation process. An anti-solvent screening methodology has been developed to quantitatively evaluate the propensity for precipitation of APIs and their impurities of synthesis during washing. This is illustrated using paracetamol and two typical impurities of synthesis during the washing process. The solubility of paracetamol in different binary wash solutions was measured to provide a basis for wash solvent selection. A map of wash solution composition boundaries for precipitation for the systems investigated was developed to depict where anti-solvent phenomena will take place. For some crystallisation and wash solvent combinations investigated, as much as 90% of the dissolved paracetamol and over 10% of impurities present in the paracetamol saturated mother liquor was shown to precipitate out. Such levels of uncontrolled crystallisation during washing in a pharmaceutical isolation process can have drastic effect on the final product purity. Whilst precipitation of both product and impurities from the mother liquor can be avoided by using a solvent in which the API has a solubility similar to that in the mother liquor, for example use of acetonitrile as a wash solvent does not result in any precipitation of the paracetamol API or its impurities. However, the high solubility of paracetamol in acetonitrile, would result in noticeable dissolution of API during washing and would lead to agglomeration during the subsequent drying step. Conversely, use of n-heptane as wash solvent for a paracetamol crystal slurry resulted in the highest amount of precipitation amongst the solvent pairings evaluated. This can be mitigated by designing a multi-stage washing strategy where wash solutions of differing wash solvent concentration are used to minimise step changes in solubility when mother liquor and wash solvent come into contact

Understanding and mitigating the consequences of undesired crystallisation taking place during washing of active pharmaceuticals

Washing is a key step in pharmaceutical isolation to remove the unwanted crystallization solvent (mother liquor) from the Active Pharmaceutical Ingredient (API) filter cake. The mother liquor is typically replaced with a miscible solvent in which the API has lower solubility, to prevent any product loss, and lower boiling point to allow for easy removal during drying. However, precipitation of API and the associated impurities of synthesis in the mother liquor may occur during washing and can affect the purity of the isolated product. In addition, formation of crystal bridges in the cake leads to agglomeration, which affects the particle size distribution and powder flow properties.1 An anti-solvent screening methodology is developed to quantitatively analyse the propensity for precipitation of paracetamol and its impurities during the washing process. Aim of this work was to validate the notion that the precipitation of API and its impurities occurs during the washing process. This analysis was conducted on paracetamol crystalized from three different solvents; ethanol, isopropanol and isoamyl alcohol. Three different wash solvents were evaluated; heptane, acetonitrile and isopropyl acetate. The solubility of paracetamol in different binary wash solutions was measured to support the wash solvent selection. A map of wash solution composition boundaries for the systems investigated was developed to depict where anti-solvent phenomena will take place. For some crystallization and wash solvent systems investigated, as much as 90% of paracetamol and over 10% of impurities present in the paracetamol saturated mother liquor was found to precipitate out. Similar level of uncontrolled crystallization during washing in a pharmaceutical process can have drastic effect on final product purity. The use of n-heptane as wash solvent always resulted in precipitation of both paracetamol and related impurities, for any given crystallization solvent. n-Heptane used to wash paracetamol crystallized from ethanol was found to produce the highest amount of precipitation. This is consistent with the largest difference in solubility of paracetamol between the crystallization and wash solvents. By using a mixture of heptane and ethanol as the initial wash solvent this effect could be minimized preventing precipitation of the API and its impurities. Use of acetonitrile as a wash solvent does not result in any precipitation of the API or impurity. However, the high solubility paracetamol in acetonitrile, would result in dissolution of API during the washing process. Wash solvents with high solubility should therefore be used cautiously to prevent any reduction in yield. Also the presence of a wash solvent in which the API has appreciable solubility in a deliquored / damp cake can lead to the formation of crystal bridges, in between particles, during drying and will result in agglomeration. X-Ray Powder Diffraction (XRPD) analysis was carried out on the paracetamol deposited API crystallizing out, this showed the presence of metastable form 1 (monoclinic) form. Therefore, no change in polymorphism was encountered due to this unwanted precipitation of API in the system investigated. Future research involves deliberately wetting the API cake with selected wash solvent and controlling the rate of washing to aid both displacement and dilution washing mechanism. References [1] Ottoboni, S., Price, C., Steven, C., Meehan, E., Barton, A., Firth, P., Mitchell, P., Tahir, F., 2018. Development of a novel continuous filtration unit for pharmaceutical process development and manufacturing. J Pharm Sci, 1

Exploring the role of anti-solvent effects during washing on active pharmaceutical ingredient purity

Washing is a key step in pharmaceutical isolation to remove the unwanted crystallization solvent (mother liquor) from the active pharmaceutical ingredient (API) filter cake. This study looks at strategies for optimal wash solvent selection, which minimizes the dissolution of API product crystals while preventing the precipitation of product or impurities. Selection of wash solvents to avoid both these phenomena can be challenging but is essential to maintain the yield, purity, and particle characteristics throughout the isolation process. An anti-solvent screening methodology has been developed to quantitatively evaluate the propensity for precipitation of APIs and their impurities of synthesis during washing. This is illustrated using paracetamol (PCM) and two typical impurities of synthesis during the washing process. The solubility of PCM in different binary wash solutions was measured to provide a basis for wash solvent selection. A map of wash solution composition boundaries for precipitation for the systems investigated was developed to depict where anti-solvent phenomena will take place. For some crystallization and wash solvent combinations investigated, as much as 90% of the dissolved PCM and over 10% of impurities present in the PCM saturated mother liquor were found to precipitate out. Such levels of uncontrolled crystallization during washing in a pharmaceutical isolation process can have a drastic effect on the final product purity. Precipitation of both the product and impurities from the mother liquor can be avoided by using a solvent in which the API has a solubility similar to that in the mother liquor; for example, the use of acetonitrile as a wash solvent does not result in precipitation of either the PCM API or its impurities. However, the high solubility of PCM in acetonitrile would result in noticeable dissolution of API during washing and would lead to agglomeration during the subsequent drying step. Contrarily, the use of n-heptane as a wash solvent for a PCM crystal slurry resulted in the highest amount of precipitation among the solvent pairs evaluated. This can be mitigated by designing a multi-stage washing strategy where wash solutions of differing wash solvent concentrations are used to minimize step changes in solubility when the mother liquor and the wash solvent come into contact