Stem cell decoupling underlies impaired lymphoid development during aging.

Mammalian aging is associated with multiple defects of hematopoiesis, most prominently with the impaired development of T and B lymphocytes. This defect is thought to originate in hematopoietic stem cells (HSCs) of the bone marrow, specifically due to the age-dependent accumulation of HSCs with preferential megakaryocytic and/or myeloid potential ("myeloid bias"). Here, we tested this notion using inducible genetic labeling and tracing of HSCs in unmanipulated animals. We found that the endogenous HSC population in old mice shows reduced differentiation into all lineages including lymphoid, myeloid, and megakaryocytic. Single-cell RNA sequencing and immunophenotyping (CITE-Seq) showed that HSC progeny in old animals comprised balanced lineage spectrum including lymphoid progenitors. Lineage tracing using the aging-induced HSC marker Aldh1a1 confirmed the low contribution of old HSCs across all lineages. Competitive transplantations of total bone marrow cells with genetically marked HSCs revealed that the contribution of old HSCs was reduced, but compensated by other donor cells in myeloid cells but not in lymphocytes. Thus, the HSC population in old animals becomes globally decoupled from hematopoiesis, which cannot be compensated in lymphoid lineages. We propose that this partially compensated decoupling, rather than myeloid bias, is the primary cause of the selective impairment of lymphopoiesis in older mice.

Morphological, behavioral and cellular analyses revealed different phenotypes in Wolfram syndrome wfs1a and wfs1b zebrafish mutant lines.

Wolfram syndrome (WS) is a rare genetic disease characterized by diabetes, optic atrophy and deafness. Patients die at 35 years of age, mainly from respiratory failure or dysphagia. Unfortunately, there is no treatment to block the progression of symptoms and there is an urgent need for adequate research models. Here, we report on the phenotypical characterization of two loss-of-function zebrafish mutant lines: wfs1aC825X and wfs1bW493X. We observed that wfs1a deficiency altered the size of the ear and the retina of the fish. We also documented a decrease in the expression level of unfolded protein response (UPR) genes in basal condition and in stress condition, i.e. after tunicamycin treatment. Interestingly, both mutants lead to a decrease in their visual function measured behaviorally. These deficits were associated with a decrease in the expression level of UPR genes in basal and stress conditions. Interestingly, basal, ATP-linked and maximal mitochondrial respirations were transiently decreased in the wfs1b mutant. Taken together, these zebrafish lines highlight the critical role of wfs1a and wfs1b in UPR, mitochondrial function and visual physiology. These models will be useful tools to better understand the cellular function of Wfs1 and to develop novel therapeutic approaches for WS.

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Factors associated with vaccine adherence among an underserved population: the adult Travellers in Nouvelle-Aquitaine, France.

BACKGROUND: A measles epidemic affected the Nouvelle-Aquitaine region from November 2017 to May 2018 with clusters among Travellers. This indicates that measles vaccination rates among Travellers remain lower than in the general population. The objective of this study was to estimate the 'declarative vaccination' against measles, mumps and rubella (MMR) and to propose a conceptual framework to help identify determinants of MMR vaccination uptake among adult Travellers in Nouvelle-Aquitaine in 2019-20. METHODS: A cross-sectional study using random sampling was performed and included 612 adult Travellers from 1 November 2019 to 31 March 2020. A conceptual framework to model vaccination adherence was tested among this underserved population by using structural equation modelling. This model included five latent variables: health literacy, attitudes toward preventive measures, stigma, accessibility to care and perceived needs and five measured variables: information received on vaccination, perception of barriers, support for administrative documents, social support and housing conditions. RESULTS: Individuals who did not answer all the questions linked to the variables included in the model were excluded, thus 347 adults were included in the final sample. The declared vaccination rate against MMR was 74.0%, and 72.4% of the participants were favorable to vaccination. Vaccination adherence was significantly correlated with favorable attitudes toward preventive measures such as having a history of MMR vaccination and not having already refused a recommended vaccine and finally satisfactory information received on vaccination. DISCUSSION: To improve vaccination adherence, health authorities should lean on personal history with vaccination and on transmitting information on vaccination.

RapaCaspase-9-based suicide gene applied to the safety of IL-1RAP CAR-T cells.

Even if adoptive cell transfer (ACT) has already shown great clinical efficiency in different types of disease, such as cancer, some adverse events consistently occur, and suicide genes are an interesting system to manage these events. Our team developed a new medical drug candidate, a chimeric antigen receptor (CAR) targeting interleukin-1 receptor accessory protein (IL-1RAP), which needs to be evaluated in clinical trials with a clinically applicable suicide gene system. To prevent side effects and ensure the safety of our candidate, we devised two constructs carrying an inducible suicide gene, RapaCasp9-G or RapaCasp9-A, containing a single-nucleotide polymorphism (rs1052576) affecting the efficiency of endogenous caspase 9. These suicide genes are activated by rapamycin and based on the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization. RapaCasp9-G- and RapaCasp9-A-expressing gene-modified T cells (GMTCs) were produced from healthy donors (HDs) and acute myeloid leukemia (AML) donors. The RapaCasp9-G suicide gene demonstrated better efficiency, and we showed its in vitro functionality in different clinically relevant culture conditions. Moreover, as rapamycin is not pharmacologically inert, we also demonstrated its safe use as part of our therapy.

Is breastfeeding duration related to the health of migrant mother-child dyads experiencing homelessness? The ENFAMS cross-sectional survey.

BACKGROUND: Literature from the general population shows a consensus about the health benefits associated with breastfeeding for both mothers and children. However, studies investigating these issues in the context of homelessness and migration are rare. This research aimed to examine the relations of any breastfeeding duration with health outcomes among migrant mother-child dyads experiencing homelessness. METHODS: Data were collected among sheltered and mainly foreign-born mothers experiencing homelessness, and their children aged 6 months to 5 years, from the ENFAMS cross-sectional survey (n = 481, 2013-Great Paris area). Any breastfeeding duration, along with various health outcomes of both the mother and her child, was ascertained by face-to-face questionnaires administered by trained interviewers to mothers (perceived physical and emotional health and maternal depression) or by trained psychologists to children (adaptive behaviours). Nurses measured weight and height [thus allowing them to calculate body mass index (BMI)] and haemoglobin concentration (mother-child dyad) and maternal blood pressure. Multivariable linear and modified Poisson regression analyses were performed to examine outcome-wide associations between any breastfeeding duration ≥6 months and the various mother-child outcomes. RESULTS: Any breastfeeding ≥6 months was associated with lower systolic blood pressure in mothers (B = -0.40, 95% confidence interval = -0.68 to -0.12). No association was observed with the other outcomes. CONCLUSIONS: The relevance of supporting breastfeeding to improve mothers' physical health holds true in the context of migration and homelessness. It is therefore important to support breastfeeding in these settings. Moreover, given the documented social complexity of breastfeeding practices, interventions should take mothers' socio-cultural heritage and the structural barriers they face into account.

Bi-allelic Variants in METTL5 Cause Autosomal-Recessive Intellectual Disability and Microcephaly.

Intellectual disability (ID) is a genetically and clinically heterogeneous disorder, characterized by limited cognitive abilities and impaired adaptive behaviors. In recent years, exome sequencing (ES) has been instrumental in deciphering the genetic etiology of ID. Here, through ES of a large cohort of individuals with ID, we identified two bi-allelic frameshift variants in METTL5, c.344_345delGA (p.Arg115Asnfs∗19) and c.571_572delAA (p.Lys191Valfs∗10), in families of Pakistani and Yemenite origin. Both of these variants were segregating with moderate to severe ID, microcephaly, and various facial dysmorphisms, in an autosomal-recessive fashion. METTL5 is a member of the methyltransferase-like protein family, which encompasses proteins with a seven-beta-strand methyltransferase domain. We found METTL5 expression in various substructures of rodent and human brains and METTL5 protein to be enriched in the nucleus and synapses of the hippocampal neurons. Functional studies of these truncating variants in transiently transfected orthologous cells and cultured hippocampal rat neurons revealed no effect on the localization of METTL5 but alter its level of expression. Our in silico analysis and 3D modeling simulation predict disruption of METTL5 function by both variants. Finally, mettl5 knockdown in zebrafish resulted in microcephaly, recapitulating the human phenotype. This study provides evidence that biallelic variants in METTL5 cause ID and microcephaly in humans and highlights the essential role of METTL5 in brain development and neuronal function.

Modulation of the ATM/autophagy pathway by a G-quadruplex ligand tips the balance between senescence and apoptosis in cancer cells.

G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound.

Use of Zebrafish Models to Boost Research in Rare Genetic Diseases.

Rare genetic diseases are a group of pathologies with often unmet clinical needs. Even if rare by a single genetic disease (from 1/2000 to 1/more than 1,000,000), the total number of patients concerned account for approximatively 400 million peoples worldwide. Finding treatments remains challenging due to the complexity of these diseases, the small number of patients and the challenge in conducting clinical trials. Therefore, innovative preclinical research strategies are required. The zebrafish has emerged as a powerful animal model for investigating rare diseases. Zebrafish combines conserved vertebrate characteristics with high rate of breeding, limited housing requirements and low costs. More than 84% of human genes responsible for diseases present an orthologue, suggesting that the majority of genetic diseases could be modelized in zebrafish. In this review, we emphasize the unique advantages of zebrafish models over other in vivo models, particularly underlining the high throughput phenotypic capacity for therapeutic screening. We briefly introduce how the generation of zebrafish transgenic lines by gene-modulating technologies can be used to model rare genetic diseases. Then, we describe how zebrafish could be phenotyped using state-of-the-art technologies. Two prototypic examples of rare diseases illustrate how zebrafish models could play a critical role in deciphering the underlying mechanisms of rare genetic diseases and their use to identify innovative therapeutic solutions.

Sigma-1 Receptor Is Critical for Mitochondrial Activity and Unfolded Protein Response in Larval Zebrafish.

The sigma-1 receptor (S1R) is a highly conserved transmembrane protein highly enriched in mitochondria-associated endoplasmic reticulum (ER) membranes, where it interacts with several partners involved in ER-mitochondria Ca2+ transfer, activation of the ER stress pathways, and mitochondria function. We characterized a new S1R deficient zebrafish line and analyzed the impact of S1R deficiency on visual, auditory and locomotor functions. The s1r+25/+25 mutant line showed impairments in visual and locomotor functions compared to s1rWT. The locomotion of the s1r+25/+25 larvae, at 5 days post fertilization, was increased in the light and dark phases of the visual motor response. No deficit was observed in acoustic startle response. A critical role of S1R was shown in ER stress pathways and mitochondrial activity. Using qPCR to analyze the unfolded protein response genes, we observed that loss of S1R led to decreased levels of IRE1 and PERK-related effectors and increased over-expression of most of the effectors after a tunicamycin challenge. Finally, S1R deficiency led to alterations in mitochondria bioenergetics with decreased in basal, ATP-linked and non-mitochondrial respiration and following tunicamycin challenge. In conclusion, this new zebrafish model confirmed the importance of S1R activity on ER-mitochondria communication. It will be a useful tool to further analyze the physiopathological roles of S1R.

In-vivo differentiation of adult hematopoietic stem cells from a single-cell point of view.

PURPOSE OF REVIEW: Although hematopoietic stem cell (HSC) function has long been studied by transplantation assays, this does not reflect what HSCs actually do in their native context. Here, we review recent technologic advances that facilitate the study of HSCs in their native context focusing on inducible HSC-specific lineage tracing and inference of hematopoietic trajectories through single-cell RNA sequencing (scRNA-Seq). RECENT FINDINGS: Lineage tracing of HSCs at the population level using multiple systems has suggested that HSCs make a major contribution to steady-state hematopoiesis. Although several genetic systems and novel methods for lineage tracing individual hematopoietic clones have been described, the technology for tracking these cellular barcodes (in particular mutations or insertion sites) is still in its infancy. Thus, lineage tracing of HSC clones in the adult bone marrow remains elusive. Static snapshots of scRNA-Seq of hematopoietic populations have captured the heterogeneity of transcriptional profiles of HSCs and progenitors, with some cells displaying a unilineage signature as well as others with bi or multipotent lineage profiles. Kinetic analysis using HSC-specific lineage tracing combined with scRNA-Seq confirmed this heterogeneity of progenitor populations and revealed a rapid and early emergence of megakaryocytic progeny, followed by erythroid and myeloid lineages, whereas lymphoid differentiation emerged last. SUMMARY: New approaches to study HSCs both in vivo through lineage tracing and at a high-resolution molecular level through scRNA-Seq are providing key insight into HSC differentiation in the absence of transplantation. Recent studies using these approaches are discussed here. These studies pave the way for integration of in-vivo clonal analysis of HSC behavior over time with single-cell sequencing data, including but not limited to transcriptomic, proteomic, and epigenomic, to establish a comprehensive molecular and cellular map of hematopoiesis.

Cytochrome P450 1B1 polymorphism drives cancer cell stemness and patient outcome in head-and-neck carcinoma.

BACKGROUND: Cytochrome P450 1B1 (CYP1B1) is mostly expressed in tumours and displays unusual properties. Its two polymorphic forms were differently associated with anticancer drug sensitivity. We decipher here the role of this polymorphism in anticancer drug efficacy in vitro, in vivo and in the clinical setting. METHODS: From head-and-neck squamous cell carcinoma cell lines not expressing CYP1B1, we generated isogenic derivatives expressing the two forms. Proliferation, invasiveness, stem cell characteristics, sensitivity to anticancer agents and transcriptome were analysed. Tumour growth and chemosensitivity were studied in vivo. A prospective clinical trial on 121 patients with advanced head-and-neck cancers was conducted, and a validation-retrospective study was conducted. RESULTS: Cell lines expressing the variant form displayed high rates of in vitro proliferation and invasiveness, stemness features and resistance to DNA-damaging agents. In vivo, tumours expressing the variant CYP1B1 had higher growth rates and were markedly drug-resistant. In the clinical study, overall survival was significantly associated with the genotypes, wild-type patients presenting a longer median survival (13.5 months) than the variant patients (6.3 months) (p = 0.0166). CONCLUSIONS: This frequent CYP1B1 polymorphism is crucial for cancer cell proliferation, migration, resistance to chemotherapy and stemness properties, and strongly influences head-and-neck cancer patients' survival.

Residual cancer burden index and tumor-infiltrating lymphocyte subtypes in triple-negative breast cancer after neoadjuvant chemotherapy.

PURPOSE: There is a need to refine the prognosis of triple-negative breast cancer (TNBC) patients after neoadjuvant chemotherapy (NAC) and to study the influence of the tumor microenvironment. We evaluated the prognostic value of pathological and immune markers in TNBC with residual disease (RD) after NAC. METHODS: In a series of 186 TNBC patients treated by NAC, we assessed the prognostic value of the Residual Cancer Burden (RCB) index. In 109 patients with RD, we studied the impact of clinicopathological features and tumor immune response in the residual tumor on overall survival (OS) and distant recurrence-free interval (DRFI). RESULTS: In the whole group, the OS and DRFI, at 3 years, were statistically different between the different classes of RCB (P = 0.0004 and P < 0.0001, respectively). In univariate analysis of the RD group, low RCB index and high ratios of stromal tumor-infiltrating lymphocytes (TILs), CD3 + TILs, CD4 + TILs, CD8 + TILs, and IDO1-positive cells were significant favorable prognostic factors for DRFI at 3 years. In the final multivariate model, CD4 + TILs and RCB index showed a statistically independent prognostic significance for DRFI [Hazard Ratio (HR) 2.88 (95%CI 1.34-6.17), P = 0.007 and HR 12.04 (95%CI 2.78-52.23, P < 0.0001), respectively]. The CD4 + TIL levels influenced survival in the different RCB classes with a significant effect observed in RCB-II and RCB-III classes (P = 0.05 and P = 0.05, respectively). CONCLUSIONS: These results suggest that the combination of pathological (RCB index) and tumor micro-environmental features (CD4 + TILs) help refining the prognosis of TNBC patients with RD following NAC.

Hydrophobicity of abiotic surfaces governs droplets deposition and evaporation patterns.

Surface contamination with droplets containing bacteria is of concern in the food industry and other environments where hygiene control is essential. Deposition patterns after the drying of contaminated droplets is affected by numerous parameters. The present study evaluated the rate of evaporation and the shape of deposition patterns after the drying of water droplets on a panel of materials with different surface properties (topography, hydrophobicity). The influence of the particle properties (in this study 1 µm-microspheres and two bacterial spores) was also investigated. Polystyrene microspheres were hydrophobic, while Bacillus spores were hydrophilic or hydrophobic, and surrounded by different surface features. In contrast to material topography, hydrophobicity was shown to deeply affect droplet evaporation, with the formation of small, thick deposits with microspheres or hydrophilic spores. Among the particle properties, the spore morphology (size and round/ovoid shape) did not clearly affect the deposition pattern. Conversely, hydrophobic spores aggregated to form clusters, which quickly settled on the materials and either failed to migrate, or only migrated to a slight extent on the surface, resulting in a steady distribution of spores or spore clusters over the whole contaminated area. Adherent bacteria or spores are known to be highly resistant to many stressful environmental conditions. In view of all the quite different patterns obtained following drying of spore-containing droplets, it seems likely that some of these would entail enhanced resistance to hygienic processes.

Global genetic insight contributed by consanguineous Pakistani families segregating hearing loss.

Consanguineous Pakistani pedigrees segregating deafness have contributed decisively to the discovery of 31 of the 68 genes associated with nonsyndromic autosomal recessive hearing loss (HL) worldwide. In this study, we utilized genome-wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported HL genes segregating in 321 Pakistani families. Of these, 70 (42.9%) variants identified in 29 genes are novel. As expected from genetic studies of disorders segregating in consanguineous families, the majority of affected individuals (94.4%) are homozygous for HL-associated variants, with the other variants being compound heterozygotes. The five most common HL genes in the Pakistani population are SLC26A4, MYO7A, GJB2, CIB2 and HGF, respectively. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling, and guide application of future gene-based therapies. These findings are also valuable in interpreting pathogenicity of variants that are potentially associated with HL in individuals of all ancestries. The Pakistani population, and its infrastructure for studying human genetics, will continue to be valuable to gene discovery for HL and other inherited disorders.

ELMOD3, a novel causative gene, associated with human autosomal dominant nonsyndromic and progressive hearing loss.

Autosomal dominant nonsyndromic hearing loss (ADNSHL) is a highly genetically heterogeneous disorder. Up to date only approximately 37 ADNSHL-causing genes have been identified. The goal of this study was to determine the causative gene in a five-generation Chinese family with ADNSHL. A Chinese family was ascertained. Simultaneously, two affected individuals and one normal hearing control from the family were analyzed by whole exome capture sequencing. To assess the functional effect of the identified variant, in-vitro studies were performed. novel missense variant, c.512A>G (p.His171Arg) in exon 8 of the ELMO domain-containing 3 (ELMOD3) gene, was identified as a causative variant in this family affected by late-onset and progressive ADNSHL. The variant was validated by Sanger sequencing and found to co-segregate with the phenotype within the pedigree and was absent in 500 ethnically matched unrelated normal hearing control subjects. To our knowledge, this is the first report of a family with ADNSHL caused by ELMOD3 mutation. Western blots and immunofluorescence staining demonstrated that p.His171Arg resulted in abnormal expression levels of ELMOD3 and abnormal subcellular localization. Furthermore, the analysis of the stability of the wild-type (WT) and mutant ELMOD3 protein shows that the decay of p.His171Arg is faster than that of the WT, suggesting a shorter halflife of the c.512A > G variant. A novel variant in the ELMOD3 gene, encoding a member of the engulfment and cell motility (ELMO) family of GTPase-activating proteins, was identified for the first time as responsible for ADNSHL.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

BACKGROUND: The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. METHODS: C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. RESULTS: Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. CONCLUSIONS: For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. GENERAL SIGNIFICANCE: In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation.

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals' survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged.