Glucose-driven histone lactylation promotes the immunosuppressive activity of monocyte-derived macrophages in glioblastoma.

Immunosuppressive macrophages restrict anti-cancer immunity in glioblastoma (GBM). Here, we studied the contribution of microglia (MGs) and monocyte-derived macrophages (MDMs) to immunosuppression and mechanisms underlying their regulatory function. MDMs outnumbered MGs at late tumor stages and suppressed T cell activity. Molecular and functional analysis identified a population of glycolytic MDM expressing GLUT1 with potent immunosuppressive activity. GBM-derived factors promoted high glycolysis, lactate, and interleukin-10 (IL-10) production in MDMs. Inhibition of glycolysis or lactate production in MDMs impaired IL-10 expression and T cell suppression. Mechanistically, intracellular lactate-driven histone lactylation promoted IL-10 expression, which was required to suppress T cell activity. GLUT1 expression on MDMs was induced downstream of tumor-derived factors that activated the PERK-ATF4 axis. PERK deletion in MDM abrogated histone lactylation, led to the accumulation of intratumoral T cells and tumor growth delay, and, in combination with immunotherapy, blocked GBM progression. Thus, PERK-driven glucose metabolism promotes MDM immunosuppressive activity via histone lactylation.

Sequential nucleophilic aromatic substitutions on cyanuric chloride: synthesis of BODIPY derivatives and mechanistic insights.

Herein we report a study on the sequential substitution of different nucleophiles on cyanuric chloride to obtain potential candidates for metal sensors (5a-c). The set of nucleophiles on the 1,3,5-triazine ring includes a phenolic BODIPY, an aminoalkyl pyridine and aminoalkyl phosphoramidates, each one designed to play a specific role in the final fluoroionophore. Three new triazine triads were synthesized in similar yields: 5a (45%), 5b (43%) and 5c (52%) after a methodical sequential combination of the nucleophiles via thermodependent nucleophilic aromatic substitution of the three chlorine atoms of cyanuric chloride. To ratify the synthetic results we simulated the reaction mechanisms for the different nucleophiles, aiming to address the distinctive orthogonality and temperature control inherent in this process, identifying and providing a sound rationale for any preferential sequence of nucleophiles inserted into the triazine core. According to our experimental and computational analysis (thermo- and kinetic preferences), we have identified the following preferential order for the sequential substitution: p-hydroxybenzaldehyde > 2-(pyridin-2-yl)ethanamine > aminoalkyl phosphoramidate, indicating that all steps follow a single-step process (concerted) in two stages, where nucleophilic addition precedes leaving group dissociation. The Meisenheimer σ-complex was identified as a transition state structure, with insufficient stability to exist as an intermediate. We observed a consistent and progressive increase in barrier height: 2-8 kcal mol-1 for the first step, 9-15 kcal mol-1 for the second step, and >15 kcal mol-1 for the third substitution. These findings align with the experimental observation of thermodependency in the sequential substitution.

Exploring borderline SN1-SN2 mechanisms: the role of explicit solvation protocols in the DFT investigation of isopropyl chloride.

Nucleophilic substitution at saturated carbon is a crucial class of organic reactions, playing a pivotal role in various chemical transformations that yield valuable compounds for society. Despite the well-established SN1 and SN2 mechanisms, secondary substrates, particularly in solvolysis reactions, often exhibit a borderline pathway. A molecular-level understanding of these processes is fundamental for developing more efficient chemical transformations. Typically, quantum-chemical simulations of the solvent medium combine explicit and implicit solvation methods. The configuration of explicit molecules can be defined through top-down approaches, such as Monte Carlo (MC) calculations for generating initial configurations, and bottom-up methods that involve user-dependent protocols to add solvent molecules around the substrate. Herein, we investigated the borderline mechanism of the hydrolysis of a secondary substrate, isopropyl chloride (iPrCl), at DFT-M06-2X/aug-cc-pVDZ level, employing explicit and explicit + implicit protocols. Top-down and bottom-up approaches were employed to generate substrate-solvent complexes of varying number (n = 1, 3, 5, 7, 9, and 12) and configurations of H2O molecules. Our findings consistently reveal that regardless of the solvation approach, the hydrolysis of iPrCl follows a loose-SN2-like mechanism with nucleophilic solvent assistance. Increasing the water cluster around the substrate in most cases led to reaction barriers of ΔH‡ ≈ 21 kcal mol-1, with nine water molecules from MC configurations sufficient to describe the reaction. The More O'Ferrall-Jencks plot demonstrates an SN1-like character for all transition state structures, showing a clear merged profile. The fragmentation activation strain analyses indicate that energy barriers are predominantly controlled by solvent-substrate interactions, supported by the leaving group stabilization assessed through CHELPG atomic charges.

The Transcriptional Co-factor IRF2BP2: A New Player in Tumor Development and Microenvironment.

Interferon regulatory factor 2-binding protein 2 (IRF2BP2) encodes a member of the IRF2BP family of transcriptional regulators, which includes IRF2BP1, IRF2BP2, and IRF2BPL (EAP1). IRF2BP2 was initially identified as a transcriptional corepressor that was dependent on Interferon regulatory factor-2 (IRF-2). The IRF2BP2 protein is found in different organisms and has been described as ubiquitously expressed in normal and tumor cells and tissues, indicating a possible role for this transcriptional cofactor in different cell signaling pathways. Recent data suggest the involvement of IRF2BP2 in the regulation of several cellular functions, such as the cell cycle, cell death, angiogenesis, inflammation and immune response, thereby contributing to physiological cell homeostasis. However, an imbalance in IRF2BP2 function may be related to the pathophysiology of cancer. Some studies have shown the association of IRF2BP2 expression in hematopoietic and solid tumors through mechanisms based on gene fusion and point mutations in gene coding sequences, and although the biological functions of these types of hybrid and mutant proteins are not yet known, they are thought to be involved in an increase in the likelihood of tumor development. In this review, we address the possible involvement of IRF2BP2 in tumorigenesis through its regulation of important pathways involved in tumor development.

CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.

Roles of Non-Coding RNA in Sugarcane-Microbe Interaction.

Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408-a copper-microRNA-was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5'RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly.

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells.

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza's Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.