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With the increased use of gene expression profiling for personalized oncology, optimized RNA sequencing (RNA-seq) protocols and algorithms are necessary to provide comparable expression measurements between exome capture (EC)-based and poly-A RNA-seq. Here, we developed and optimized an EC-based protocol for processing formalin-fixed, paraffin-embedded samples and a machine-learning algorithm, Procrustes, to overcome batch effects across RNA-seq data obtained using different sample preparation protocols like EC-based or poly-A RNA-seq protocols. Applying Procrustes to samples processed using EC and poly-A RNA-seq protocols showed the expression of 61% of genes (N = 20,062) to correlate across both protocols (concordance correlation coefficient > 0.8, versus 26% before transformation by Procrustes), including 84% of cancer-specific and cancer microenvironment-related genes (versus 36% before applying Procrustes; N = 1,438). Benchmarking analyses also showed Procrustes to outperform other batch correction methods. Finally, we showed that Procrustes can project RNA-seq data for a single sample to a larger cohort of RNA-seq data. Future application of Procrustes will enable direct gene expression analysis for single tumor samples to support gene expression-based treatment decisions., (© 2024. The Author(s).)

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and 5-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR'TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro , particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR'TCR-T, CAR-T, and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR'TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies., Competing Interests: K.T., I.E.Y.O., C.B., V.H., N.Winter, N.Werchau, S.K., C.W., N.J., K.B, B.E., T.S., and D.L. are employees of Miltenyi Biotec B.V. & Co. KG., (© 2024 The Author(s).)

Goal: Presentation of the first Russian computer program (www.carotidscore.ru) for risk stratification of postoperative complications of carotid endarterectomy (CEE)., Material and Methods: The present study is based on the analysis of a multicenter Russian database that includes 25,812 patients after CEE operated on from 01/01/2010 to 04/01/2022. The following types of CEE were implemented: 6814 classical CEE with plastic reconstruction of the reconstruction zone with a patch; 18,998 eversion CEE., Results: In the hospital postoperative period, 0.18% developed a lethal outcome, 0.14%-myocardial infarction, 0.35%-stroke. The combined endpoint was 0.68%. For each factor present in patients, a predictive coefficient was calculated. The prognostic coefficient was a numerical indicator reflecting the strength of the influence of each factor on the development of postoperative complications. Based on this formula, predictive coefficients were calculated for each factor present in patients in our study. The total contribution of these factors was reflected in "%" and denoted the risk of postoperative complications with a minimum value of 0% and a maximum of 100%. On the basis of the obtained calculations, a computer program CarotidSCORE was created. Its graphical interface is based on the QT framework (https://www.qt.io), which has established itself as one of the best solutions for desktop applications. It is possible not only to calculate the probability of developing a complication, but also to save all data about the patient in JSON format (for the patient's personal card and his anamnesis). The CarotidSCORE program contains 47 patient parameters, including clinical-demographic, anamnestic and angiographic characteristics. It allows you to choose one of the four types of CEE, which will provide an accurate stratification of the risk of complications for each of them in person., Conclusion: CarotidSCORE (www.carotidscore.ru) is able to determine the likelihood of postoperative complications in patients undergoing CEE., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum . So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 10 4 to 10 5 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.

Ex vivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of ex vivo expansion is strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O 2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O 2 . Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell-cell (ICAM1, HCAM/CD44) and cell-matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O 2 (20%). The number of ICAM-1 positive ASCs was increased under low O 2 as well. These alterations contributed into the ex vivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p < 0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration in vivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)