Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

Structural plasticity and enzyme action: crystal structures of mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacterium tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme from Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule.     

Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome

BACKGROUND: The retinoic acid (RA)-catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1-null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS: Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by chi(2) analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS: Mutational screening allowed us to identify 6 SNPs, 4 of which (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS: Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects.     

Novel polymorphisms of the APOA2 gene and its promoter region affect body traits in cattle

Apolipoprotein A-II (APOA2) is one of the major constituents of high-density lipoprotein and plays a critical role in lipid metabolism and obesity. However, similar research for the bovine APOA2 gene is lacking. In this study, polymorphisms of the bovine APOA2 gene and its promoter region were detected in 1021 cows from four breeds by sequencing and PCR-RFLP methods. Totally, we detected six novel mutations which included one mutation in the promoter region, two mutations in the exons and three mutations in the introns. There were four polymorphisms within APOA2 gene were analyzed. The allele A, T, T and G frequencies of the four loci were predominant in the four breeds when in separate or combinations analysis which suggested cows with those alleles to be more adapted to the steppe environment. The association analysis indicated three SVs in Nangyang cows, two SVs in Qinchun cows and the 9 haplotypes in Nangyang cows were significantly associated with body traits (P < 0.05 or P < 0.01). The results of this study suggested the bovine APOA2 gene may be a strong candidate gene for body traits in the cattle breeding program.     

Association of cytochrome b translational activator proteins with the mitochondrial membrane: implications for cytochrome b expression in yeast.

Summary The products of the nuclear genes CBS1 and CBS2 are both required for translational activation of mitochondrial apocytochrome b in yeast. We report the intramitochondrial localization of both proteins by use of specific antisera. Based on its solubilization properties the CBS1 protein is presumed to be a component of the mitochondrial membrane; the detergent concentrations needed to release CBS1 from mitochondria are almost the same as for cytochrome c ₁. In contrast, CBS2 behaves like a soluble protein, with some characteristics of a membrane-associated protein. A model is presented for translational activation of cytochrome b, which might also be applicable to translational regulation of other mitochondrial genes.     

Shedding new light on lipid functions with CARS and SRS microscopy

Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. This article is part of a Special Issue entitled Tools to study lipid functions.     

Evolutionary history of a specialized p450 propane monooxygenase

The evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the ‘lineage’ from a naturally occurring C₁₂-C₂₀ fatty acid hydroxylase (P450_BM3) to a laboratory-evolved P450 propane monooxygenase (P450_PMO) having 20 heme domain substitutions compared to P450_BM3. Biochemical, crystallographic, and computational analyses show that a minimal perturbation of the P450_BM3 fold and substrate-binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (∼ 9000-fold increase in k_cat/K_m) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate-recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely respecialize the cytochrome P450 for function on a nonnative substrate.     

CD20-specific chimeric antigen receptor-expressing T cells as salvage therapy in rituximab-refractory/relapsed B-cell non-Hodgkin lymphoma

Background aimsThe infusion of chimeric antigen receptor (CAR) T cells that target specific tumor-associated antigens is a promising strategy that has exhibited encouraging results in clinical trials. However, few studies have focused on the effectiveness and safety of CD20 CAR T cells in rituximab-refractory/relapsed (R/R) B-cell non-Hodgkin lymphoma (B-NHL) patients, particularly those treated with rituximab for a short time. This prospective study aimed to assess the effectiveness and toxicity of CD20 CAR T cells in R/R B-NHL patients previously treated with rituximab.MethodsThe authors conducted a prospective, single-center phase I study on the effectiveness and toxicity of CD20 CAR T cells in rituximab-treated R/R B-NHL patients (no. ChiCTR2000036350). A total of 15 patients with R/R B-NHL were enrolled between November 21, 2017, and December 1, 2021.ResultsAn overall response rate of 100% was shown in enrolled patients, with 12 (80%) achieving complete remission and three (20%) achieving partial remission for the best response. The median follow-up time was 12.4 months. Progression-free survival and overall survival were not yet reached by the data cutoff day. No patient developed grade 4 cytokine release syndrome, and only one patient had immune effector cell-associated neurotoxicity syndrome.ConclusionsAll enrolled B-NHL patients who were previously R/R to rituximab achieved different degrees of clinical response with tolerable toxicities. Notably, patients who had received rituximab within 3 months had a poorer prognosis.