The importance of significant event analysis

Significant event analysis (SEA) is a structured quality improvement activity that is well established in general practice. Participation in SEA prompts primary care teams to reflect on their clinical reasoning, to highlight exemplary care, and to identify any potential improvements in both practice and wider healthcare systems. This article provides an overview of the SEA process and the events surrounding a SEA meeting. Cancer care examples are used to demonstrate how SEA can be used to enhance team-based learning and improve future patient care

Computational methods for cell culture media optimization and product quality control

Biologics development leading to approval can take decades. Acceleration of this timeline is necessary to bring safe and efficacious drugs to patients as early as possible. One research focus to reduce development time is the cell culture process development and optimization. In this study, we will present two computational strategies that cover: (1) enhancing cell culture media by amino acid optimization using Orthogonal-Partial Least Squares (OPLS) regression, and (2) modulating protein glycosylation by altering small molecule compound concentrations based on the Concentration Impact Factor. Disproportionate nutrient balance in cell culture medium can have a negative impact on cell culture performance. In our study, OPLS regression was used to explain cell growth and monoclonal antibody (mAb) production dynamics from Chinese Hamster Ovary (CHO) cells as a function of amino acid (AA) stoichiometric balances. The OPLS model was trained on metabolic data from 24 concurrent 14-day fed-batch cultures. Metabolic fluxes and respective stoichiometric balances were then generated by calculating the difference between the theoretical biomass demand of each AA and the actual AA usage towards mAb production and experimental consumption. As a result, highly weighted stoichiometric balances represented those AA that could potentially enhance the previous feed medium and aim to achieve a higher intracellular catabolic activity. Accordingly, we used our computational model to generate varied amino acid additions to either a platform feed or a low nutrient feed by means of a 16-run mixture design. The experimental results showed that addition of model generated key AA resulted in a ~55% increase in peak cell density and ~90% increase in mAb production, respectively. Appropriately glycosylated therapeutic mAb are critical for the proper molecular folding, stability, and in-vivo efficacy of the expressed proteins. Cell culture process conditions and medium compositions have been demonstrated to affect the expression of various glycosylation species. In this study, we evaluated a set of selected small compound for their potential in modifying glycosylation levels in mAb expressed in three different proprietary CHO cell lines. These small molecule compounds were first tested on one cell line to establish a baseline. To quantitate the glycosylation modifications, we have developed a mathematical correlation of a dimensionless number, termed Concentration Impact factor (Cf), to describe the degree changes in glycosylation species. Using the Cf algorithm established for the 1st cell line, we subsequently tested with other two cell lines, and were able to modulate and confirmed the level of glycan expression. This indicates that Cf correlation may serve as a tool to provide early assessment of final glycosylation profiles and levels on therapeutic proteins due to small molecule supplementations. Overall, the two computational methods presented here are aimed to enhance biologics development speed as well as ensure product quality control

Successful treatment of recurrent renal stones with Cinacalcet in a patient with primary hyperparathyroidism

A man aged 72 years with long-standing primary hyperparathyroidism (HPT), a background of recurrent bilateral renal stones and failed parathyroid surgery is described. During the 27 months preceding treatment, episodes of renal colic became increasingly frequent and he required multiple surgical interventions. Given the lack of medical therapies to definitively treat his symptoms, he was started on a trial of the calcimimetic, Cinacalcet. Cinacalcet has previously been shown to reduce hypercalcaemia in patients with primary HPT. Despite this, there is a paucity of evidence to suggest that its use is associated with a long-term reduction in urinary calcium excretion and renal stone recurrence. In our case, within 4 months of starting treatment, serum and urinary calcium had normalised and parathyroid hormone concentrations were within reference ranges. To date, over a 50-month treatment period, there has been a complete cessation in stone formation, and no further urological intervention has been required

Peanut agronomy experiments with five varieties in the Bundaberg and Kingaroy regions in the 2021-22 season

This report summarises the results of the peanut agronomy experiments conducted at Bundaberg and Kingaroy during the 2021-22 season as part of the above project. The experiments were laid out in a split-plot design with three replications at each location. Five peanut varieties, including Holt, Alloway, Kairi, Wheeler and P85-p112-151 (P85), were assigned to main plots, and four plant populations, 6, 12, 18, and 24 plants per m2, were assigned to subplots. All varieties were runner types except Wheeler, which represented a 'Virginia' type. Planting was done by the precision planting 20/20® and vSet® electronic seed metering system. The experiments were irrigated using irrigation scheduling software Aquaman via the web-based 'Yield Prophet'

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis

Learning from new colorectal cancers: a qualitative synthesis of significant event reports

Background Colorectal cancer is the second leading cause of cancer-related mortality in the United Kingdom and a significant contributor to morbidity and mortality worldwide. Early diagnosis provides opportunities for intervention and improved survival. Significant Event Analysis (SEA) is a well-established quality improvement method for learning from new cancer diagnoses. Aim This study aimed to provide additional insights into diagnostic processes for colorectal cancer and to identify areas for improvement in patient care pathways. Design & setting 53 general practices across Pennine Lancashire, England, submitted one or more SEA reports as part of an incentivised scheme. Method A standardised data collection form was used to collate learning points and recommendations for improvements. 161 reports were analysed using an inductive framework analysis approach. Results There was an overarching theme of building vigilance and collaboration between and within general practices and secondary care. Four main sub-themes were also identified including education, individualised and flexible care, ownership and continuity, and communication. Conclusion These findings provide additional insights into colorectal cancer pathways from a primary care perspective. Practices should be supported in developing protocols for assessment and follow-up of patients with varying presentations. Screening and access to investigations are paramount for improving early diagnosis, however, a flexible diagnostic approach is required according to the individual circumstances of each patient

Systems biology approach in the development of chemically-defined media for production of protein therapeutics in Chinese hamster ovary cells

Cell culture medium plays a critical role on mammalian cell growth, protein expression and quality. Typical cell culture medium formulations consist of \u3e50 components which include amino acids, vitamins, trace metals, lipids and proteins. Chinese Hamster Ovary (CHO) cells that produce biotherapeutics are propagated in specific cell culture media to ensure robust productivity and product quality. Systems biology has been applied to multiple areas of biological research to gain a better understanding of disease origins and to identify potential new drug targets. Although CHO cells are simpler systems, they share similar biochemistry and cellular pathways. Therefore, leveraging the systems biology knowledge from animal systems and applying these strategic systems biological tools to bioprocess development can be valuable in gaining better understanding of CHO cell culture performance, optimizing cell culture media, and subsequently resulting in better control of the overall production processes. In this presentation, we will present several case studies of various ‘omics tools applied to (1) optimize cell culture medium formulation for improve cell growth and productivity via metabolomics, (2) understand effects of medium components on cellular gene expression via transcriptomics, and on product quality via glycomics, and (3) identify potential cellular protein targets that are affected by stress imposed during production process via proteomics. The development of a statistical model that aims to highlight key metabolites and a machine learning model that identifies significantly important genes which are involved in monoclonal antibody production will also be discussed

Activation of the Receptor Tyrosine Kinase, RET, improves long-term Hematopoietic Stem Cell outgrowth and potency

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche, but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor, RET, at the cell surface of HSCs, in mediating sustained cellular growth, resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex, GDNF/GFRa1, show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally, we demonstrate that RET drives a multi-faceted intracellular signalling pathway, including key signalling intermediates AKT, ERK1/2, NFkB and p53, responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions

Gold–Oligonucleotide Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and oligonucleotides to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral nucleic acid sequence (23 bases), which was identified through in silico screening. Oligonucleotides were designed to demonstrate specific complementarity towards the target enteroviral nucleic acid to produce aggregated gold–oligonucleotide nanoconstructs. The conserved target enteroviral nucleic acid sequence (≥1 × 10−7 M, ≥1.4 × 10−14 g/mL) initiates gold–oligonucleotide nanoconstruct disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow assays that utilise gold–oligonucleotide nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence