Manufacturing solutions for robust cell therapy expansion and harvest

The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that ease scalability, utilize animal origin-free materials and are compatible with limited downstream processing steps. As more cell therapeutics progress through clinical testing, current in vitro culture methods are proving cumbersome to scale and lack robustness. Moreover, high quality animal origin-free reagents and downstream processing devices support the future implementation of large scale manufacturing solutions that will be required following clinical success. Here, we describe the implementation of single use bioreactors and high quality media for expansion of cell therapies. We focus on autologous T cells and will review solutions addressing animal origin-free expansion of cells within the context of different upstream process development steps as well as scaling with good cell quality, high recovery, high viability and good activity. We also compare the effect of donor variability on performance. Start to finish solutions for expansion, including high quality reagents, are key enabling technologies for success in commercializing cell therapies

Health empowerment scripts : simplifying social/green prescriptions

Social prescriptions are one term commonly used to describe non-pharmaceutical approaches to healthcare and are gaining popularity in the community, with evidence highlighting psychological benefits of reduced anxiety, depression and improved mood and physiological benefits of reduced risk of cardiovascular disease and reduced hypertension. The relationship between human health benefits and planetary health benefits is also noted. There are, however, numerous barriers, such as duration and frequencies to participate in activities, access, suitability, volition and a range of unpredictable variables (such as inclement weather, shifting interests and relocating home amongst others) impeding a comprehensive approach to their use on a wider scale. From a multidisciplinary perspective, this commentary incorporates a salutogenic and nature-based approach to health, we also provide a range of recommendations that can be undertaken at the patient level to assist in shifting the acknowledged systemic barriers currently occurring. These include using simple language to explain the purpose of health empowerment scripts, ensuing personal commitment to a minimum timeframe, enabling ease of access, co-designing a script program, providing ongoing motivational support and incorporating mindfulness to counter unexpected disruptions. Copyright © 2022 Lawson, Wissing, Henderson-Wilson, Snell, Chambers, McNeil and Nuttman

Process development approaches for expansion of adherent stem cells in microcarrier-based bioreactor culture

Industry trends in regenerative medicine show an increased need for scalable and closed manufacturing of cell therapies. Single-use bioreactor systems have proven suitable as a platform to meet the industry’s needs. Key process parameters for cell culture performance in these systems include pH, dissolved oxygen (DO) and agitation rates. Especially important is the understanding and application of appropriate solid-liquid mixing, which is essential for microcarrier-based cultures used for adherent stem cells. Agitation rates that are too high in microcarrier-based cultures can be correlated with smaller eddy lengths that impact cellular shear. Conversely, agitation rates that are low do not support the consistent microcarrier turnover required for cell access to nutrients, DO, and maintenance of pH. Moreover, suboptimal agitation rates may impact cell-to-microcarrier attachment and transfer. Here, we summarize a stepwise approach to optimizing pH, DO and agitation set-points in the Mobius® 3L single-use bioreactor for mesenchymal stem/stromal cells (MSCs). The theoretical agitation operating range best suited for microcarrier cultures was calculated based on the Zwietering equation for suspension of solids in stirred tanks, and verified experimentally with human bone marrow-derived MSCs. Upper agitation limits were defined by Kolmogorov\u27s theory of turbulent eddy lengths, and were substantially higher than the agitation rates required to keep microcarriers suspended. Identifying optimal pH, DO and agitation rates for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a robust platform for use in a controlled manufacturing environment

Environmental DNA metabarcoding for fish diversity assessment in a macrotidal estuary: A comparison with established fish survey methods

Fishes are a dominant component of the macrofauna in estuaries and are important for assessing the health of these threatened ecosystems. Several studies have applied environmental DNA (eDNA) metabarcoding to assess the biodiversity of fishes in estuaries. However, none have combined measurement of physicochemical variables with a spatially extensive sampling design across the full salinity gradient. This study aimed to compare spatial fish assemblage composition detected via eDNA metabarcoding of surface water samples with conventional fishing gear surveys in a macrotidal estuary (river Dee, North Wales, UK). In addition, eDNA assemblage composition across seasons was investigated. In autumn 2018, triplicate eDNA samples were taken at 13 stations in a spatially systematic design alongside seine, fyke and beam trawl sampling. In summer 2019, eDNA samples from eight of the 13 original stations were collected again in the upper and lower estuary. DNA was extracted from samples and subjected to metabarcoding analysis using an established assay targeting teleost fishes. The key findings were that in autumn, eDNA detected 17 of the 26 (71%) species caught by fishing gears, which included the most abundant species. Overall, eDNA detected a greater species richness, per 30 samples, than seine or fyke nets (but not beam trawling). Additionally, there was a clear correlation between salinity and assemblage composition, which was consistent across seasons. Overall, the study indicates that eDNA metabarcoding could enhance existing fish sampling methods, by generating a more comprehensive picture of estuarine fish biodiversity and providing additional information for ecological inference and management actions

Pump it Up workshop report

Workshop held 28-29 September 2017, Cape Cod, MAA two-day workshop was conducted to trade ideas and brainstorm about how to advance our understanding of the ocean’s biological pump. The goal was to identify the most important scientific issues that are unresolved but might be addressed with new and future technological advances

WormBase: better software, richer content

WormBase (), the public database for genomics and biology of Caenorhabditis elegans, has been restructured for stronger performance and expanded for richer biological content. Performance was improved by accelerating the loading of central data pages such as the omnibus Gene page, by rationalizing internal data structures and software for greater portability, and by making the Genome Browser highly customizable in how it views and exports genomic subsequences. Arbitrarily complex, user-specified queries are now possible through Textpresso (for all available literature) and through WormMart (for most genomic data). Biological content was enriched by reconciling all available cDNA and expressed sequence tag data with gene predictions, clarifying single nucleotide polymorphism and RNAi sites, and summarizing known functions for most genes studied in this organism

Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M−1cm−1in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives

Optimising species detection probability and sampling effort in lake fish eDNA surveys

Environmental DNA (eDNA) metabarcoding is transforming biodiversity monitoring in aquatic environments. Such an approach has been developed and deployed for monitoring lake fish communities in Great Britain, where the method has repeatedly shown a comparable or better performance than conventional approaches. Previous analyses indicated that 20 water samples per lake are sufficient to reliably estimate fish species richness, but it is unclear how reduced eDNA sampling effort affects richness, or other biodiversity estimates and metrics. As the number of samples strongly influences the cost of monitoring programmes, it is essential that sampling effort is optimised for a specific monitoring objective. The aim of this project was to explore the effect of reduced eDNA sampling effort on biodiversity metrics (namely species richness and community composition) using algorithmic and statistical resampling techniques of a data set from 101 lakes, covering a wide spectrum of lake types and ecological quality. The results showed that reliable estimation of lake fish species richness could, in fact, usually be achieved with a much lower number of samples. For example, in almost 90% of lakes, 95% of complete fish richness could be detected with only 10 water samples, regardless of lake area. Similarly, other measures of alpha and beta-diversity were not greatly affected by a reduction in sample size from 20 to 10 samples. We also found that there is no significant difference in detected species richness between shoreline and offshore sampling transects, allowing for simplified field logistics. This could potentially allow the effective sampling of a larger number of lakes within a given monitoring budget. However, rare species were more often missed with fewer samples, with potential implications for monitoring of invasive or endangered species. These results should inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals

The CCP4 suite: integrative software for macromolecular crystallography

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.Jon Agirre is a Royal Society University Research Fellow (UF160039 and URF\R\221006). Mihaela Atanasova is funded by the UK Engineering and Physical Sciences Research Council (EPSRC; EP/R513386/1). Haroldas Bagdonas is funded by The Royal Society (RGF/R1/181006). Jose´ Javier Burgos-Ma´rmol and Daniel J. Rigden are supported by the BBSRC (BB/S007105/1). Robbie P. Joosten is funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 871037 (iNEXTDiscovery) and by CCP4. This work was supported by the Medical Research Council as part of United Kingdom Research and Innovation, also known as UK Research and Innovation: MRC file reference No. MC_UP_A025_1012 to Garib N. Murshudov, which also funded Keitaro Yamashita, Paul Emsley and Fei Long. Robert A. Nicholls is funded by the BBSRC (BB/S007083/1). Soon Wen Hoh is funded by the BBSRC (BB/T012935/1). Kevin D. Cowtan and Paul S. Bond are funded in part by the BBSRC (BB/S005099/1). John Berrisford and Sameer Velankar thank the European Molecular Biology Laboratory–European Bioinformatics Institute, who supported this work. Andrea Thorn was supported in the development of AUSPEX by the German Federal Ministry of Education and Research (05K19WWA and 05K22GU5) and by Deutsche Forschungsgemeinschaft (TH2135/2-1). Petr Kolenko and Martin Maly´ are funded by the MEYS CR (CZ.02.1.01/0.0/0.0/16_019/0000778). Martin Maly´ is funded by the Czech Academy of Sciences (86652036) and CCP4/STFC (521862101). Anastassis Perrakis acknowledges funding from iNEXT (grant No. 653706), iNEXT-Discovery (grant No. 871037), West-Life (grant No. 675858) and EOSC-Life (grant No. 824087) funded by the Horizon 2020 program of the European Commission. Robbie P. Joosten has been the recipient of a Veni grant (722.011.011) and a Vidi grant (723.013.003) from the Netherlands Organization for Scientific Research (NWO). Maarten L. Hekkelman, Robbie P. Joosten and Anastassis Perrakis thank the Research High Performance Computing facility of the Netherlands Cancer Institute for providing and maintaining computation resources and acknowledge the institutional grant from the Dutch Cancer Society and the Dutch Ministry of Health, Welfare and Sport. Tarik R. Drevon is funded by the BBSRC (BB/S007040/1). Randy J. Read is supported by a Principal Research Fellowship from the Wellcome Trust (grant 209407/Z/17/Z). Atlanta G. Cook is supported by a Wellcome Trust SRF (200898) and a Wellcome Centre for Cell Biology core grant (203149). Isabel Uso´n acknowledges support from STFC-UK/CCP4: ‘Agreement for the integration of methods into the CCP4 software distribution, ARCIMBOLDO_LOW’ and Spanish MICINN/AEI/FEDER/UE (PID2021-128751NB-I00). Pavol Skubak and Navraj Pannu were funded by the NWO Applied Sciences and Engineering Domain and CCP4 (grant Nos. 13337 and 16219). Bernhard Lohkamp was supported by the Ro¨ntgen A˚ ngstro¨m Cluster (grant 349-2013-597). Nicholas Pearce is currently funded by the SciLifeLab and Wallenberg Data Driven Life Science Program (grant KAW 2020.0239) and has previously been funded by a Veni Fellowship (VI.Veni.192.143) from the Dutch Research Council (NWO), a Long-term EMBO fellowship (ALTF 609-2017) and EPSRC grant EP/G037280/1. David M. Lawson received funding from BBSRC Institute Strategic Programme Grants (BB/P012523/1 and BB/P012574/1). Lucrezia Catapano is the recipient of an STFC/CCP4-funded PhD studentship (Agreement No: 7920 S2 2020 007).Peer reviewe

GA4GH: International policies and standards for data sharing across genomic research and healthcare.

The Global Alliance for Genomics and Health (GA4GH) aims to accelerate biomedical advances by enabling the responsible sharing of clinical and genomic data through both harmonized data aggregation and federated approaches. The decreasing cost of genomic sequencing (along with other genome-wide molecular assays) and increasing evidence of its clinical utility will soon drive the generation of sequence data from tens of millions of humans, with increasing levels of diversity. In this perspective, we present the GA4GH strategies for addressing the major challenges of this data revolution. We describe the GA4GH organization, which is fueled by the development efforts of eight Work Streams and informed by the needs of 24 Driver Projects and other key stakeholders. We present the GA4GH suite of secure, interoperable technical standards and policy frameworks and review the current status of standards, their relevance to key domains of research and clinical care, and future plans of GA4GH. Broad international participation in building, adopting, and deploying GA4GH standards and frameworks will catalyze an unprecedented effort in data sharing that will be critical to advancing genomic medicine and ensuring that all populations can access its benefits