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Pedro Henrique Dias Moura Prazeres,1,2 Heloísa Ferreira,2 Pedro Augusto Carvalho Costa,2 Walison da Silva,2 Marco Túllio Alves,2 Marshall Padilla,3 Ajay Thatte,3 Anderson Kenedy Santos,4,5 Anderson Oliveira Lobo,6 Adriano Sabino,7 Helen Lima Del Puerto,1 Michael J Mitchell,3 Pedro Pires Goulart Guimaraes1,2 1Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 2Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 3Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; 4Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, CT, USA; 5Department of Genetics, Yale School of Medicine, New Haven, CT, USA; 6Department of Materials Engineering, Federal University of Piauí, Teresina, PI, Brazil; 7Department of Clinical and Toxicological Analysis, Federal University of Minas Gerais, Belo Horizonte, MG, BrazilCorrespondence: Pedro Pires Goulart Guimaraes, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, Email ppiresgo@reitoria.ufmg.brIntroduction: Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR.Methods: Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells.Results: LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro.Conclusion: Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.Keywords: lipid nanoparticles, pDNA delivery, CAR T cells, cell engineering, cancer immunotherapy

In the last 500 years, there have been an estimated 21 mammal extinctions in the Pernambuco Endemism Center. We critically reviewed the published historical and recent literature records and concluded that the actual number of mammal species extinction was seven, indicating that the previous figure of 21 species lost is an overestimation of approximately 30 %. Our checklist differs from previous publications by including species that are still extant (n = 5), and removing species that have never been recorded in the Pernambuco Endemism Center (n = 8). We point out that a more rigorous approach towards historical and recent records is needed when producing lists of regionally extinct fauna, given that the implications of misidentifications and false assumptions can potentially lead to loss of credibility by stakeholders and ultimately have a negative effect on species conservation.

This article addresses an urgent ecological crisis of this century. It explores the transformative potential of ecological hermeneutics as a current trend in Old Testament research in the context of the Book of Psalms. As we face the unprecedented ecological threat of global devastation, this article identifies a new kind of speech emerging from current ecological trends in biblical research. The article examines the historical implications of biblical texts in the discussion about ecological degradation by providing an overview of the development of ecological hermeneutics. It then presents methodologies such as dark green religion and the ecobosadi approach and advocates for ways in which they reshape culture and have the potential to play a crucial role in reshaping it. [ABSTRACT FROM AUTHOR]