A defective Krab-domain zinc-finger transcription factor contributes to altered myogenesis in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder caused by a non-coding CTG repeat expansion that, in particular, provokes functional alteration of CUG-binding proteins. As a consequence, several genes with misregulated alternative splicing have been linked to clinical symptoms. In our search for additional molecular mechanisms that would trigger functional defects in DM1, we took advantage of mutant gene-carrying human embryonic stem cell lines to identify differentially expressed genes. Among the different genes found to be misregulated by DM1 mutation, one strongly downregulated gene encodes a transcription factor, ZNF37A. In this paper, we show that this defect in expression, which derives from a loss of RNA stability, is controlled by the RNA-binding protein, CUGBP1, and is associated with impaired myogenesis—a functional defect reminiscent of that observed in DM1. Loss of the ZNF37A protein results in changes in the expression of the subunit α1 of the receptor for the interleukin 13. This suggests that the pathological molecular mechanisms linking ZNF37A and myogenesis may involve the signaling pathway that is known to promote myoblast recruitment during development and regeneratio

Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice

A Quantitative ELISA Protocol for Detection of Specific Human IgG against the SARS-CoV-2 Spike Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with at least 3.8 million deaths to date. For that reason, finding an efficient vaccine for this virus quickly became a global priority. The majority of vaccines now marketed are based on the SARS-CoV-2 spike protein that has been described as the keystone for optimal immunization. In order to monitor SARS-CoV-2 spike-specific humoral responses generated by immunization or infection, we have developed a robust and reproducible enzyme-linked immunosorbent assay (ELISA) protocol. This protocol describes a method for quantitative detection of IgG antibodies against the SARS-CoV-2 spike protein using antigen-coated microtiter plates. Results showed that antibodies could be quantified between the range of 1.953 ng/mL to 500 ng/mL with limited inter- and intra-assay variability

A Functional GM-CSF Receptor on Dendritic Cells Is Required for Efficient Protective Anti-Tumor Immunity

Dendritic cells (DC) play a major role during the priming phase of anti-tumor immunization, as they are required for an efficient tumor-associated antigens presentation. At least one dendritic cell-based therapy has already been successfully approved by regulators for clinical application in prostate cancer patients. Moreover, DC development is dependent on the granulocyte macrophage colony stimulating factor (GM-CSF), a cytokine that has been successfully used as a potent inducer of anti-tumoral immunity. To better understand the relation between DC and GM-CSF in anti-tumor immunity, we studied the DC function in mice lacking the cytokine receptor common subunit beta (βc-/-) for GM-CSF, IL-3 and IL-5 and immunized with irradiated tumor cells. Such immunization induces a protective, specific tumor immunization in wild-type mice, while βc-/- mice failed to mount an immune response. Upon in vitro stimulation, DC from βc-/- mice (DCβc-/-) are unable to undergo a full maturation level. In vivo experiments show that they lack the ability to prevent tumor growth, in contrast to DCWT. Moreover, matured DCWT rescued immunization in βc-/- mice. DC maturation is dependent on a functional pathway involving GM-CSF signaling through a biologically functional receptor. These findings may contribute to new strategies for efficient anti-tumor immunotherapies

Liver X receptors and epididymal epithelium physiology

International audienc

Deciphering key residues involved in the virulence-promoting interactions between Streptococcus pneumoniae and human plasminogen

International audienceBacterial pathogens recruit circulating proteins to their own surfaces, coopting the host protein functions as a mechanism of virulence. Particular attention has focused on the binding of plasminogen (Plg) to bacterial surfaces, as it has been shown that this interaction contributes to bacterial adhesion to host cells, invasion of host tissues and evasion of the immune system. Several bacterial proteins are known to serve as receptors for Plg including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytoplasmic enzyme that appears on the cell surface in this moonlighting role. Although Plg typically binds to these receptors via several lysine-binding domains, the specific interactions that occur have not been documented in all cases. However, identification of the relevant residues could help define strategies for mitigating the virulence of important human pathogens, like Streptococcus pneumoniae (Sp). To shed light on this question, we have described a combination of peptide-spot array screening, competition and SPR assays, high-resolution crystallography and mutational analyses to characterize the interaction between SpGAPDH and Plg. We identified three SpGAPDH lysire residues that were instrumental in defining the kinetic and thermodynamic parameters of the interaction. Altogether, the integration of the data presented in this work allows us to propose a structural model for the molecular interaction of the SpGAPDH-Plg complex

Local sustained GM-CSF delivery by genetically engineered encapsulated cells enhanced both cellular and humoral SARS-CoV-2 spike-specific immune response in an experimental murine spike DNA vaccination model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with recurrences. Therefore, finding a vaccine for this virus became a priority for the scientific community. The SARS-CoV-2 spike protein has been described as the keystone for viral entry into cells and effective immune protection against SARS-CoV-2 is elicited by this protein. Consequently, many commercialized vaccines focus on the spike protein and require the use of an optimal adjuvant during vaccination. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated a powerful enhancement of acquired immunity against many pathogens when delivered in a sustained and local manner. In this context, we developed an encapsulated cell-based technology consisting of a biocompatible, semipermeable capsule for secretion of GM-CSF. In this study, we investigated whether murine GM-CSF (muGM-CSF) represents a suitable adjuvant for SARS-CoV-2 immunization, and which delivery strategy for muGM-CSF could be most beneficial. To test this, different groups of mice were immunized with intra-dermal (i.d.) electroporated spike DNA in the absence or presence of recombinant or secreted muGM-CSF. Results demonstrated that adjuvanting a spike DNA vaccine with secreted muGM-CSF resulted in enhancement of specific cellular and humoral immune responses against SARS-CoV-2. Our data also highlighted the importance of delivery strategies to the induction of cellular and humoral-mediated responses

Pneumococcal lysis induced by LytA promotes GAPDH surface localization.

<p>(A) Growth profiles of the R6 strain in CY and CY + 1% Cho and of the R6 Δ<i>lytA</i> mutant in CY medium. (B) Bacterial suspensions of the R6 strain grown in CY and in CY + 1% Cho and the R6 Δ<i>lytA</i> mutant grown in CY were treated by alkaline buffer to release surface-associated proteins. Proteins present in the pellet (P) fraction corresponding to the cytoplasmic extract and in the alkaline supernatant fraction, which contains proteins detached from the cell surface, were analyzed by Western blot using appropriate polyclonal antibodies. Samples were analyzed on the same polyacrylamide gel. Left panel: detection of FtsZ (44.4 kDa) used to monitor the non-lytic effect of the alkaline treatment. Right panel: detection of GAPDH (38 kDa). Equivalent amount of loading material was determined based on OD_600nm values and gel scanning quantification and not on CFU measurements since the chaining morphology of the R6 Δ<i>lytA</i> mutant and the one induced by the presence of 1% Cho alters colony counting [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125377#pone.0125377.ref065" target="_blank">65</a>]. This procedure was also applied in experiments showed in Figs 1C, 1D and 1E. (C) Quantification of pneumococcal GAPDH associated to the bacterial surface in the R6 strain grown in CY and in CY + 1% Cho and in the R6 Δ<i>lytA</i> mutant grown in CY. GAPDH was detected by Western blot and quantification of the signal was performed. The average of three independent experiments is shown. (D) Same protocol as 1C. Amount of GAPDH associated to the cell wall fraction at different stages of growth. (E) Same protocol as 1C. Amount of GAPDH associated to the cell wall fraction analyzed by subcellular fractionation.</p

GAPDH increases C1q deposition on pneumococcal cell wall.

<p>Pneumococcal sacculi containing only peptidoglycan were deposited on 96-wells plate and incubated with or without purified GAPDH. Normal human serum (NHS) was added and subsequent C1q and C4 deposition was measured using anti-C1q and anti-C4 antibodies. A representative experiment of 2 independent experiments is shown including the standard deviation of triplicate points.</p

Surface GAPDH promotes binding to C1q.

<p>Bacterial culture was withdrawn at mid-exponential growth phase (OD_600nm 0.3). FITC-labeled bacteria were incubated for 1 h at 4°C on 1 μg of C1q coated on 96-wells plate. After five washes, the fluorescence of FITC was measured. A representative experiment of 3 independent experiments is shown including the standard deviation of triplicate points. Significance was determined by t-test analysis on 3 independent experiments.</p