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Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies., (© 2024. The Author(s).)

Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and pathogenicity. A few phospholipases (PL) have been characterised so far at the gene and protein level in unicellular parasites including African trypanosomes (AT). They could play a role in different processes such as host-pathogen interaction, antigenic variation, intermediary metabolism. By mining the genome database of AT we found putative new phospholipase candidate genes and here we provided biochemical evidence that one of these has lipolytic activity. This protein has a unique non-canonical glycosome targeting signal responsible for its dual localisation in the cytosol and the peroxisomes-related organelles named glycosomes. We also show that this new phospholipase is excreted by these pathogens and that antibodies directed against this protein are generated during an experimental infection with T. brucei gambiense, a subspecies responsible for infection in humans. This feature makes this protein a possible tool for diagnosis., (© 2022. The Author(s).)

Trypanosome parasites are infecting mammals in Sub-Saharan Africa and are transmitted between hosts through bites of the tsetse fly. The transmission from the insect vector to the mammal host causes a number of metabolic and physiological changes. A fraction of the population continuously adapt to the immune system of the host, indicating heterogeneity at the population level. Yet, the cell to cell variability in populations is mostly unknown. We develop here an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of single-cell Trypanosoma brucei in emulsion droplets. We first show that mammalian stage trypanosomes survive for several hours to days in droplets, with an influence of droplet size on both survival and growth. We unravel various growth patterns within a population and find that droplet cultivation of trypanosomes results in 10-fold higher cell densities of the highest dividing cell variants compared to standard cultivation techniques. Some variants reach final cell titers in droplets closer to what is observed in nature than standard culture, of practical interest for cell production. Droplet microfluidics is therefore a promising tool for trypanosome cultivation and analysis with further potential for high-throughput single cell trypanosome analysis., (© 2021. The Author(s).)

Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology.

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.