Defining cell culture dynamics in response to growth factor provision for efficient optimization of cell based therapies

The design of processes to manufacture Cell Based Therapies (CBTs) is particularly challenging due to the dynamic cell culture environment and the complex response of the cell product. An approach to process design needs to achieve a balance between resource spent and process knowledge gained to meet manufacturing goals around cost, quality and risk. In order to address this, we developed a parsimonious mathematical modelling framework designed to allow representation of common mechanisms in cell culture, and associated process operations, that drive cell culture outcomes(1). We propose that this framework can be applied to generate widely applicable ‘template models’ and experimental strategies for process optimization. In this case we have demonstrated the application to define provision rates of elements that support cell growth in a hematopoietic culture system (erythroblasts). Building on a model that defined the influence of environmental change (incorporated as a single non-specific variable linked to cell growth and density) on population growth(2), we have defined the cell culture dynamics in response to specific elements such as growth factor provision. We have shown how achieving the correct combination of delivery rates of growth factors with other factors (e.g. metabolic) can ensure that each is used with maximal efficiency, significantly driving down volume use (more than 3-fold) and process supplementation costs. The models also define the corollary of such intensification in terms of increasingly demanding process control with respect to volume handling and process timing; however, an advantage of the level of intensification achieved is that the system oxygen demand increases to a level that oxygen input rates can be used to identify cell growth-inhibition in advance of reduced viability. This work demonstrates that models that appropriately represent common process dynamics will be a key tool to achieve the risk and cost reduction required for CBT manufacture. (1) AJ Stacey, EA Cheeseman, KE Glen, RLL Moore, RJ Thomas. Experimentally integrated dynamic modelling for intuitive optimization of cell-based processes and manufacture. Biochem Eng J. 2018. 132: 130-138 (2) KE Glen, EA Cheeseman, AJ Stacey, RJ Thomas. A mechanistic model of erythroblast growth inhibition providing a framework for optimization of cell therapy manufacturing. Biochem Eng J. 2018. 133:28-38

Application of quality by design tools to upstream processing of platelet precursor cells to enable in vitro manufacture of blood products

Annually 4.5 million platelet units are transfused in Europe and the United States. These are obtained solely from allogeneic donations and have a shelf life of 5-7 days. To address the corresponding supply challenge, Moreau et al.1 devised a novel process for producing megakaryocytes (MKs, the platelet precursor cell) in vitro. A transcription-factor driven, forward-programming (FOP) approach converts human pluripotent stem cells into MKs. This strategy has the unique advantage of generating high yields of pure MKs in chemically defined medium which could lead to the production of a consistent, reliable supply of platelets which overcomes the logistical, financial and biosafety challenges for health organisations worldwide. Here we follow a Quality by Design (QbD) approach to enable improvements to the upstream processing of FOPMKs. Firstly, we created a process flow diagram for production of in vitro platelets for transfusion, which segregated processes into individual unit operations for control and optimisation. Next, we developed a Quality Target Product Profile (QTPP) and identified Critical Quality Attributes (CQAs) for each stage. We conducted a range of experiments utilising Design of Experiments (DOE) and mechanistic modelling2 tools to link Critical Process Parameters (CPPs) to CQAs. For adherent culture, we identified a productivity limit related to surface area available for growth and a cell loss phase which was dependent on cell seeding density, RhoK inhibitor usage and seed density. Using suspension cultures of FOPMK. We noted that TPO and Doxycycline concentration were CPPs as these impacted cell net growth rate and phenotype trajectory. Furthermore, we noted that medium exhaustion led to a 30% loss of viable cells over 8 hours. Proof of concept studies also showed that FOPMKs can be cultured in scaled-down suspension systems (ambr-15 and spinner flask culture) whilst retaining CQAs. 1. Moreau, T. et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nat. Commun. 7, 1–15 (2016). 2. Stacey, A. J., Cheeseman, E. A., Glen, K. E., Moore, R. L. L. & Thomas, R. J. Experimentally integrated dynamic modelling for intuitive optimisation of cell-based processes and manufacture. Biochem. Eng. J. 132, 130–138 (2018)

Titration of C-5 Sterol Desaturase Activity Reveals Its Relationship to Candida albicans Virulence and Antifungal Susceptibility Is Dependent upon Host Immune Status

Mutations that completely inactivate Erg3p enable the prevalent human pathogen C. albicans to endure the azole antifungals in vitro . However, such null mutants are less frequently identified in azole-resistant clinical isolates than other resistance mechanisms, and previous studies have reported conflicting outcomes regarding antifungal resistance of these mutants in animal models of infection

The Astropy Problem

The Astropy Project (http://astropy.org) is, in its own words, "a community effort to develop a single core package for Astronomy in Python and foster interoperability between Python astronomy packages." For five years this project has been managed, written, and operated as a grassroots, self-organized, almost entirely volunteer effort while the software is used by the majority of the astronomical community. Despite this, the project has always been and remains to this day effectively unfunded. Further, contributors receive little or no formal recognition for creating and supporting what is now critical software. This paper explores the problem in detail, outlines possible solutions to correct this, and presents a few suggestions on how to address the sustainability of general purpose astronomical software

CD8+ T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8+ T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8+ T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2550–558-specific CD8+ T cells being cross-reactive between IAV and IBV. Memory CD8+ T cells towards these specificities are present in blood (CD27+CD45RA− phenotype) and tissues (CD103+CD69+ phenotype) of healthy individuals, and effector CD27−CD45RA−PD-1+CD38+CD8+ T cells in IAV/IBV patients. Our data show influenza-specific CD8+ T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8+ T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Global Carbon Budget 2023

Accurate assessment of anthropogenic carbon dioxide (CO2) emissions and their redistribution among the atmosphere, ocean, and terrestrial biosphere in a changing climate is critical to better understand the global carbon cycle, support the development of climate policies, and project future climate change. Here we describe and synthesize data sets and methodology to quantify the five major components of the global carbon budget and their uncertainties. Fossil CO2 emissions (EFOS) are based on energy statistics and cement production data, while emissions from land-use change (ELUC), mainly deforestation, are based on land-use and land-use change data and bookkeeping models. Atmospheric CO2 concentration is measured directly, and its growth rate (GATM) is computed from the annual changes in concentration. The ocean CO2 sink (SOCEAN) is estimated with global ocean biogeochemistry models and observation-based f CO2 products. The terrestrial CO2 sink (SLAND) is estimated with dynamic global vegetation models. Additional lines of evidence on land and ocean sinks are provided by atmospheric inversions, atmospheric oxygen measurements, and Earth system models. The resulting carbon budget imbalance (BIM), the difference between the estimated total emissions and the estimated changes in the atmosphere, ocean, and terrestrial biosphere, is a measure of imperfect data and incomplete understanding of the contemporary carbon cycle. All uncertainties are reported as ±1σ. For the year 2022, EFOS increased by 0.9 % relative to 2021, with fossil emissions at 9.9 ± 0.5 Gt C yr−1 (10.2 ± 0.5 Gt C yr−1 when the cement carbonation sink is not included), and ELUC was 1.2 ± 0.7 Gt C yr−1, for a total anthropogenic CO2 emission (including the cement carbonation sink) of 11.1 ± 0.8 Gt C yr−1 (40.7±3.2 Gt CO2 yr−1). Also, for 2022, GATM was 4.6±0.2 Gt C yr−1 (2.18±0.1 ppm yr−1; ppm denotes parts per million), SOCEAN was 2.8 ± 0.4 Gt C yr−1, and SLAND was 3.8 ± 0.8 Gt C yr−1, with a BIM of −0.1 Gt C yr−1 (i.e. total estimated sources marginally too low or sinks marginally too high). The global atmospheric CO2 concentration averaged over 2022 reached 417.1 ± 0.1 ppm. Preliminary data for 2023 suggest an increase in EFOS relative to 2022 of +1.1 % (0.0 % to 2.1 %) globally and atmospheric CO2 concentration reaching 419.3 ppm, 51 % above the pre-industrial level (around 278 ppm in 1750). Overall, the mean of and trend in the components of the global carbon budget are consistently estimated over the period 1959–2022, with a near-zero overall budget imbalance, although discrepancies of up to around 1 Gt C yr−1 persist for the representation of annual to semi-decadal variability in CO2 fluxes. Comparison of estimates from multiple approaches and observations shows the following: (1) a persistent large uncertainty in the estimate of land-use changes emissions, (2) a low agreement between the different methods on the magnitude of the land CO2 flux in the northern extra-tropics, and (3) a discrepancy between the different methods on the strength of the ocean sink over the last decade. This living-data update documents changes in methods and data sets applied to this most recent global carbon budget as well as evolving community understanding of the global carbon cycle. The data presented in this work are available at https://doi.org/10.18160/GCP-2023 (Friedlingstein et al., 2023)

Global urban environmental change drives adaptation in white clover

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale