Abstract :

Adult olfactory neurogenesis provides waves of new neurons involved in memory encoding. However, how the olfactory bulb deals with neuronal renewal to ensure the persistence of pertinent memories and the flexibility to integrate new events remains unanswered. To address this issue, mice performed two successive olfactory discrimination learning tasks with varying times between tasks. We show that with a short time between tasks, adult-born neurons supporting the first learning task appear to be highly sensitive to interference. Furthermore, targeting these neurons using selective light-induced inhibition altered memory of this first task without affecting that of the second, suggesting that neurons in their critical period of integration may only support one memory trace. A longer period between the two tasks allowed for an increased resilience to interference. Hence, newly formed adult-born neurons regulate the transience or persistence of a memory as a function of information relevance and retrograde interference.      

DEC 2019     

DOI : 10.1038/s41467-019-13521-7    

Pubmed ID : 31811134

Abstract :

Wheat-germ cell-free protein synthesis (WG-CFPS) is a potent platform for the high-yield production of proteins. It is especially of interest for difficult-to-express eukaryotic proteins, such as toxic and transmembrane proteins, and presents an important tool in high-throughput protein screening. Until recently, an assumed drawback of WG-CFPS was a reduced capacity for post-translational modifications. Meanwhile, phosphorylation has been observed in WG-CFPS; yet, authenticity of the respective phosphorylation sites remained unclear. Here we show that a viral membrane protein, the duck hepatitis B virus (DHBV) large envelope protein (DHBs L), produced by WG-CFPS, is phosphorylated upon translation at the same sites as DHBs L produced during DHBV infection of primary hepatocytes. Furthermore, we show that alternative translation initiation of the L protein, previously identified in virus-producing hepatic cells, occurs on WG-CFPS as well. Together, these findings further strengthen the high potential of WG-CFPS to include the reproduction of specific modifications proteins experience in vivo.     

DEC 2019     

DOI : 10.3389/fmolb.2019.00138

Pubmed ID : 31850370

Abstract :

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.        

NOV 2019     

DOI : 10.1038/s41598-019-53528-0    

Pubmed ID : 31784555

Abstract :

Pancreatic ductal adenocarcinoma (PDAC) is one of the solid tumors with the poorest prognosis. The stroma of this tumor is abundant and composed of extracellular matrix and stromal cells (including cancer-associated fibroblasts and immune cells). Nerve fibers invading this stroma represent a hallmark of PDAC, involved in neural remodeling, which participates in neuropathic pain, cancer cell dissemination and tumor relapse after surgery. Pancreatic cancer-associated neural remodeling is regulated through functional interplays mediated by physical and molecular interactions between cancer cells, nerve cells and surrounding Schwann cells, and other stromal cells. In the present study, we show that Schwann cells (glial cells supporting peripheral neurons) can enhance aggressiveness (migration, invasion, tumorigenicity) of pancreatic cancer cells in a transforming growth factor beta (TGFβ)-dependent manner. Indeed, we reveal that conditioned medium from Schwann cells contains high amounts of TGFβ able to activate the TGFβ-SMAD signaling pathway in cancer cells. We also observed in human PDAC samples that high levels of TGFβ signaling activation were positively correlated with perineural invasion. Secretome analyses by mass spectrometry of Schwann cells and pancreatic cancer cells cultured alone or in combination highlighted the central role of TGFβ in neuro-epithelial interactions, as illustrated by proteomic signatures related to cell adhesion and motility. Altogether, these results demonstrate that Schwann cells are a meaningful source of TGFβ in PDAC, which plays a crucial role in the acquisition of aggressive properties by pancreatic cancer cells.        

DEC 2019     

DOI : 10.1038/s41419-019-2116-x   

Pubmed ID : 31767842

Abstract :

Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of β2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.        

NOV 2019     

DOI : 10.1038/s41467-019-13113-5   

Pubmed ID : 31767837

Abstract :

Chronic kidney disease is associated with an increased cardiovascular risk, and altered biological properties of high-density lipoproteins (HDL) may play a role in these events. This study aimed to describe the HDL proteome from non-diabetic hemodialysis patients and identify potential pathways affected by the dysregulated expression of HDL proteins. HDL were sampled from nine non-diabetic hemodialysis (HD) and eight control patients. Samples were analyzed using a nano-RSLC coupled with a Q-Orbitrap. Data were processed by database searching using SequestHT against a human Swissprot database and quantified with a label-free quantification approach. Proteins that were in at least five of the eight control and six of the nine HD patients were analyzed. Analysis was based on pairwise ratios and the ANOVA hypothesis test. Among 522 potential proteins, 326 proteins were identified to be in the HDL proteome from HD and control patients, among which 10 were significantly upregulated and nine downregulated in HD patients compared to the control patients (p < 0.05). Up and downregulated proteins were involved in lipid metabolism, hemostasis, wound healing, oxidative stress, and apoptosis pathways. This difference in composition could partly explain HDL dysfunction in the chronic kidney disease (CKD) population and participate in the higher cardiovascular risk observed in this population.  

NOV 2019     

DOI : 10.3390/toxins11110671     

Pubmed ID : 31731787

Abstract :

The NF-κB pathway is constitutively activated in adult T cell leukemia, an aggressive malignancy caused by Human T Leukemia Virus type 1 (HTLV-1). The viral oncoprotein Tax triggers this constitutive activation by interacting with the ubiquitin-rich IKK complex. We previously demonstrated that Optineurin and TAX1BP1, two members of the ubiquitin-binding, Sequestosome-1 (SQSTM-1/p62)-like selective autophagy receptor family, are involved in Tax-mediated NF-κB signaling. Here, using a proximity-dependent biotinylation approach (BioID), we identify p62 as a new candidate partner of Tax and confirm the interaction in infected T cells. We then demonstrate that p62 knock-out in MEF cells as well as p62 knock-down in HEK293T cells significantly reduces Tax-mediated NF-κB activity. We further show that although p62 knock-down does not alter NF-κB activation in Jurkat T cells nor in infected T cells, p62 does potentiate Tax-mediated NF-κB activity upon over-expression in Jurkat T cells. We next show that p62 associates with the Tax/IKK signalosome in cells, and identify the 170–206 domain of p62 as sufficient for the direct, ubiquitin-independent interaction with Tax. However, we observe that this domain is dispensable for modulating Tax activity in cells, and functional analysis of p62 mutants indicates that p62 could potentiate Tax activity in cells by facilitating the association of ubiquitin chains with the Tax/IKK signalosome. Altogether, our results identify p62 as a new ubiquitin-dependent modulator of Tax activity on NF-κB, further highlighting the importance of ubiquitin in the signaling activity of the viral Tax oncoprotein..        

NOV 2019     

DOI : 10.1038/s41598-019-52408-x   

Pubmed ID : 31690813

Abstract :

Chromosome segregation in bacteria is poorly understood outside some prominent model strains and even less is known about how it is coordinated with other cellular processes. This is the case for the opportunistic human pathogen Streptococcus pneumoniae (the pneumococcus), which lacks the Min and the nucleoid occlusion systems, and possesses only an incomplete chromosome partitioning Par(A)BS system, in which ParA is absent8. The bacterial tyrosine kinase9 CpsD, which is required for capsule production, was previously found to interfere with chromosome segregation10. Here, we identify a protein of unknown function that interacts with CpsD and drives chromosome segregation. RocS (Regulator of Chromosome Segregation) is a membrane-bound protein that interacts with both DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the cell division protein FtsZ and hinders cell division. Altogether, this work reveals that RocS is the cornerstone of a nucleoid protection system ensuring proper chromosome segregation and cell division in coordination with the biogenesis of the protective capsular layer.        

OCT 2019     

DOI : 10.1038/s41564-019-0472-z     

Pubmed ID : 31182798

Abstract :

Our previous single-cell based gene expression analysis pointed out significant variations of LDHA level during erythroid differentiation. Deeper investigations highlighted that a metabolic switch occurred along differentiation of erythroid cells. More precisely we showed that self-renewing progenitors relied mostly upon lactate-productive glycolysis, and required LDHA activity, whereas differentiating cells, mainly involved mitochondrial oxidative phosphorylation (OXPHOS). These metabolic rearrangements were coming along with a particular temporary event, occurring within the first 24h of erythroid differentiation. The activity of glycolytic metabolism and OXPHOS rose jointly with oxgene consumption dedicated to ATP production at 12-24h of the differentiation process before lactate-productive glycolysis sharply fall down and energy needs decline. Finally, we demonstrated that the metabolic switch mediated through LDHA drop and OXPHOS upkeep might be necessary for erythroid differentiation. We also discuss the possibility that metabolism, gene expression and epigenetics could act together in a circular manner as a driving force for differentiation.  

SEP 2019     

DOI : 10.1371/journal.pone.0221472    

Pubmed ID : 31483850

Abstract :

N6-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t6A) is a universal modification essential for translational accuracy and efficiency. The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex.

SEP 2019     

DOI : 10.1038/s41467-019-11951-x   

Pubmed ID : 31481669

Abstract :

Caspase-4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non-canonical inflammasome, which drives inflammatory responses during Gram-negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome-wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS-mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase-4 levels in human monocytes and iPSC-derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non-canonical inflammasome. Upon IFN-γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase-4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram-negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non-canonical inflammasome.

SEP 2019     

DOI : 10.15252/embr.201948235 

Pubmed ID : 31353801

Abstract :

Studying skin aging to better understand age-related physiological changes or to find novel molecules that could act on aging, could be relatively long and costly. Fish animal models could be particularly appropriated since skin structure has been shown remarkably useful to investigate pigment biology for example, in normal or tumoral cells (Schartl et al.,2016). In particular, the Japanese Medaka, a small fish model with attractive experimental characteristics, may accumulate over lifespan some biomarkers currently associated with aging features in other vertebrates. However, very few are known about aging of the medaka fish skin, especially in its biomechanical properties and skin architecture. In our study, we first confirmed that classical molecular changes occurred in Medaka skin during aging, as well as SA-β-galactosidase accumulation. Moreover, we performed for the first time measurements of Medaka skin stiffness by Atomic Force Microscopy (AFM) which revelated modulation of the bio-mechanical properties of the Medaka skin throughout life. Since a recent study showed a remodelling of the dermis matrix in aged mouse (Kaur et al., 2019), we thus investigated the correlation between skin stiffness and dermis aging in human skin reconstructs. Finally, we observed a modulation of collagen density in both human reconstructed and Medaka skins by Second Harmonic Generation (SHG) microscopy. Overall, our study unveiled the potential of Medaka fish to study skin aging and the AFM as an innovative technology to characterize this biological phenomenon.

SEP 2019     

DOI : doi.org/10.1016/j.jid.2019.07.668

Abstract :

Background: Resistance to thyroid hormone alpha (RTHα) is a rare genetic disease due to mutations in the THRA gene, which encodes thyroid hormone receptor alpha 1 (TRα1). Since its first description in 2012, 46 cases of RTHα have been reported worldwide, corresponding to 26 different mutations of TRα1. RTHα patients share some common symptoms with hypothyroid patients, without significant reduction in thyroid hormone level. The high variability of clinical features and the absence of reliable biochemical markers make the diagnosis of this disease difficult. Some of these mutations have been recently modeled in mice. Methods: In our study, we used four different mouse models heterozygous for frameshift mutations in the Thra gene. Two of them are very close to human mutations, while the two others have not yet been found in patients. We characterized the metabolic phenotypes of urine and plasma samples collected from these four animal models using an untargeted nuclear magnetic resonance (NMR)-based metabolomic approach. Results: Multivariate statistical analysis of the metabolomic profiles shows that biofluids of mice that carry human-like mutations can be discriminated from controls. Metabolic signatures associated with Thra mutations in urine and plasma are stable over time and clearly differ from the metabolic fingerprint of hypothyroidism in the mouse. Conclusion: Our results provide a proof-of-principle that easily accessible NMR-based metabolic fingerprints of biofluids could be used to diagnose RTHα in humans.

SEP 2019     

DOI :  10.1089/thy.2018.0730

Pubmed ID : 31298651

Abstract :

Protein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop.        

SEP 2019     

DOI : 10.3389/fmicb.2019.01942         

Pubmed ID : 31551943

Abstract :

To assess evolutionary processes in deep time, it is essential to understand the roles of development and environment, both recorded through the morphological variability of fossil assemblages. Thanks to their great abundance and the high temporal resolution of their fossil record, conodont elements are ideal to address this issue. In this paper, we present the first quantitative study of a Carnian–Norian (Late Triassic) assemblage of closely related P1 conodont elements. Using geometric morphometrics (landmarks, sliding landmarks, and elliptic Fourier analysis), we explore the main axes of phenotypic variation and relate them to classically used taxonomic characters. We show that some important taxonomic features follow laws of covariation, hence highlighting developmental constraints. Furthermore, the intraspecific variation within all considered species, either Carnian or Norian forms, is similarly restricted, emphasizing, for the first time in conodont P1 elements, a common line of least resistance to evolution, which means that similar intrinsic (developmental) factors were acting on these taxa and likely biased the evolutionary trajectories of all these taxa in a similar way. Because the evolution between Carnian and Norian forms is known to have followed a trajectory that is significantly different from the line of least resistance, strong extrinsic pressures, such as environmental disturbances, were probably at play around the Carnian/Norian boundary to counteract the effects of these intrinsic, developmental constraints.       

AUG 2019     

DOI : doi.org/10.1017/pab.2019.14        

Abstract :

The Tar DNA-Binding Protein 43 (TDP-43) and its phosphorylated isoform (pTDP-43) are the major components associated with ubiquitin positive/Tau-negative inclusions found in neurons and glial cells of patients suffering of amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration-TDP-43 (FTLD-TDP). Many studies have revealed that TDP-43 is also in the protein inclusions associated with neurodegenerative conditions other than ALS and FTLD-TDP, thus suggesting that this protein may be involved in the pathogenesis of a variety of neurological disorders. In brains of Huntington-affected patients, pTDP-43 aggregates were shown to co-localize with mutant Huntingtin (mHtt) inclusions. Here, we show that expression of mHtt carrying 80-97 polyglutamines repeats in human cell cultures induces the aggregation and the phosphorylation of endogenous TDP-43, whereas non-pathological Htt with 25 polyglutamines repeats has no effect. Mutant Htt aggregation precedes accumulation of pTDP-43 and pTDP-43 co-localizes with mHtt inclusions reminding what it was previously described in brains of Huntington-affected patients. Detergent-insoluble fractions from cells expressing mHtt and containing mHtt-pTDP-43 co-aggregates can function as seeds for further TDP-43 aggregation in human cell culture. The human cellular prion protein PrPC was previously identified as a negative modulator of mHtt aggregation; here, we show that PrPC-mediated reduction of mHtt aggregation is tightly correlated with a decrease of TDP-43 aggregation and phosphorylation, thus confirming the close relationships between TDP-43 and mHtt.      

JUL 2019     

DOI : 10.1007/s00018-019-03059-8  

Pubmed ID : 30863908   

Abstract :

Fatal neurological syndromes can occur after measles virus (MeV) infection of the brain. The mechanisms controlling MeV spread within the central nervous system (CNS) remain poorly understood. We analyzed the role of type I interferon (IFN-I) receptor (IFNAR) signaling in the control of MeV infection in a murine model of brain infection. Using organotypic brain cultures (OBC) from wild-type and IFNAR-knockout (IFNARKO) transgenic mice ubiquitously expressing the human SLAM (CD150) receptor, the heterogeneity of the permissiveness of different CNS cell types to MeV infection was characterized. In the absence of IFNAR signaling, MeV propagated significantly better in explant slices. In OBC from IFNAR-competent mice, while astrocytes and microglia were infected on the day of explant preparation, they became refractory to infection with time, in contrast to neurons and oligodendrocytes, which remained permissive to infection. This selective loss of permissiveness to MeV infection was not observed in IFNARKO mouse OBC. Accordingly, the development of astrogliosis related to the OBC procedure was exacerbated in the presence of IFNAR signaling. In the hippocampus, this astrogliosis was characterized by a change in the astrocyte phenotype and by an increase of IFN-I transcripts. A proteome analysis showed the upregulation of 84 out of 111 secreted proteins. In the absence of IFNAR, only 27 secreted proteins were upregulated, and none of these were associated with antiviral activities. Our results highlight the essential role of the IFN-I response in astrogliosis and in the permissiveness of astrocytes and microglia that could control MeV propagation throughout the CNS.     

JUN 2019     

DOI : 10.1128/JVI.00618-19         

Pubmed ID : 31019048

Abstract :

Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.        

MAY 2019     

DOI : 10.1126/science.aav6390           

Pubmed ID : 31123134

Abstract :

Type I interferon (IFN-I) is critical for antiviral defense, and plasmacytoid dendritic cells (pDCs) are a predominant source of IFN-I during virus infection. pDC-mediated antiviral responses are stimulated upon physical contact with infected cells, during which immunostimulatory viral RNA is transferred to pDCs, leading to IFN production via the nucleic acid sensor TLR7. Using dengue, hepatitis C, and Zika viruses, we demonstrate that the contact site of pDCs with infected cells is a specialized platform we term the interferogenic synapse, which enables viral RNA transfer and antiviral responses. This synapse is formed via aLb2 integrin-ICAM-1 adhesion complexes and the recruitment of the actin network and endocytic machinery. TLR7 signaling in pDCs promotes interferogenic synapse establishment and provides feed-forward regulation, sustaining pDC contacts with infected cells. This interferogenic synapse may allow pDCs to scan infected cells and locally secrete IFN-I, thereby confining a potentially deleterious response.  

MAY 2019     

DOI :10.1016/j.chom.2019.03.005     

Pubmed ID : 31003939

Abstract :

Hepatitis D virus (HDV) doesn't encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.       

MAY 2019     

DOI :10.1038/s41467-019-10117-z    

Pubmed ID : 31068585

Abstract :

A stabilized cartilage construct without signs of hypertrophy in chondrocytes is still a challenge. Suspensions of adipose stem/stromal cells (ASCs) and cartilage progenitor cells (CPCs) were seeded into micromolded nonadhesive hydrogel to produce spheroids (scaffold- and serum-free method) characterized by size, immunohistochemistry, fusion, and biomechanical properties. After cell dissociation, they were characterized for mesenchymal cell surface markers, cell viability, and quantitative real-time polymerase chain reaction. Both targeted and nontargeted (shotgun mass spectrometry) analyses were conducted on the culture supernatants. Induced ASC spheroids (o = 350 mu m) showed high cell viability and CD73 downregulation contrasting to CD90. The transforming growth factor (TGF)-beta 3/TGF-beta 1 ratio and SOX9 increased (p < 0.05), whereas interleukin (IL)-6, IL-8, RUNX2, and ALPL decreased. Induced ASC spheroids were able to completely fuse and showed a higher force required to compression at day 14 (p < 0.0001). Strong collagen type II in situ was associated with gradual decrease of collagen type X and a lower COLXA1 gene expression at day 14 compared with day 7 (p = 0.0352). The comparison of the secretome content of induced and non-induced ASCs and CPCs identified 138 proteins directly relevant to chondrogenesis of 704 proteins in total. Although collagen X was absent, thrombospondin-1 (TSP-1), described as antiangiogenic and antihypertrophic, and cartilage oligomeric matrix protein (COMP), a biomarker of chondrogenesis, were upregulated in induced ASC spheroids. Our scaffold- and serum-free method mimics stable cartilage acting as a tool for biomarker discovery and for regenerative medicine protocols. Impact Statement Promising adult stem cell sources for cartilage regeneration include adipose stem/stromal cells (ASCs) from subcutaneous adipose tissue. Our main objective was the development of a reproducible and easy-to-handle scaffold- and serum-free method to obtain stable cartilage from induced ASC spheroids. In addition to targeted protein profiling and biomechanical analysis, we provide the first characterization of the secretome composition for ASC spheroids, providing a useful tool to monitor in vitro chondrogenesis and a noninvasive quality control of tissue-engineered constructs. Furthermore, our secretome analysis revealed a potential novel biomarker-thrombospondin-1 (TSP-1), known by its antiangiogenic properties and recently described as an antihypertrophic protein.                                        

MAY 2019

DOI :10.1089/ten.tea.2018.0311        

Pubmed ID : 30734654

Abstract :

To gain insight into the biology of NK cells, others and we previously identified the NK-cell signature, defined as the set of transcripts which expression is highly enriched in these cells compared to other immune subtypes. The transcript encoding the Serine/threonine/tyrosine kinase 1 (Styk1) is part of this signature. However, the role of Styk1 in the immune system is unknown. Here, we report the generation of a novel transgenic mouse model, in which Styk1 expression is invalidated and replaced by an EGFP reporter cassette. We demonstrated that Styk1 expression is a hallmark of NK cells and other NK1.1 expressing cells such as liver type 1 innate lymphoid cells (ILC1) and NK1.1(+) gamma delta T cells. Styk1 expression is maintained by IL-15 in NK cells and negatively correlates with the expression of educating NK-cell receptors. Analysis of phosphorylation levels of mTOR substrates suggested that Styk1 could moderately contribute to the activity of the PI3K/Akt/mTOR pathway. However, Styk1-deficient NK cells develop normally and have normal in vitro and in vivo effector functions. Thus Styk1 expression is a hallmark of NK cells, ILC1 and NK1.1(+) T cells but is dispensable for their development and immune functions.         

MAY 2019     

DOI :10.1002/eji.201847721 

Pubmed ID : 30690705

Abstract :

Collagens are the most abundant vertebrate extracellular matrix proteins. They form a superfamily of 28 members that show a remarkable diversity in molecular and supramolecular organization, tissue distribution and function and mutations in collagen genes result in a wide range of inherited connective tissue diseases. In the recent years, unexpected and very diverse regulatory and mechanical collagen functions have been reported. But the structural and functional landscape of the collagen superfamily is still far from being complete. Zebrafish has emerged over the last decades as a powerful model to interrogate gene function and there are numerous advantages of using zebrafish for collagen research, including recent advances in genome editing technologies and the characterization of the zebrafish matrisome. One can confidently predict that zebrafish will rapidly become a popular vertebrate model to investigate the role of collagens in development, disease and regeneration as discussed in this chapter.      

MAY 2019     

DOI : 10.1016/j.semcdb.2018.10.002 

Pubmed ID : 30312775

Abstract :

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumorsuppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylationmediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.         

MAY 2019     

DOI : 10.1038/s41418-018-0169-5      

Pubmed ID : 30042493

Abstract :

The development of cytosolic 5'-nucleotidase II (cN-II) inhibitors is essential to validate cN-II as a potential target for the reversion of resistance to cytotoxic nucleoside analogues. We previously reported a fragment-based approach combined with molecular modelling, herein, the selected hit-fragments were used again in another computational approach based on the Ilib-diverse (a software enabling to build virtual molecule libraries through fragment based de novo design) program to generate a focused library of potential inhibitors. A molecular scaffold related to a previously identified compound was selected and led to a novel series of compounds. Ten out of nineteen derivatives showed 50-75% inhibition on the purified recombinant protein at 200 AM and among them three derivatives (12,13 and 18) exhibited k(1) the sub-millimolar range (0.84, 2.4 and 0.58 mM, respectively). Despite their only modest potency, the cN-II inhibitors showed synergistic effects when used in combination with cytotoxic purine nucleoside analogues on cancer cells. Therefore, these derivatives represent a family of non-nucleos(t)idic cN-II inhibitors with potential usefulness to overcome cancer drug resistance especially in hematological malignancies in which cN-II activity has been described as an important parameter. (C) 2019 Elsevier Masson SAS. All rights reserved.              

APR 2019     

DOI : 10.1016/j.ejmech.2019.02.040  

Pubmed ID : 30798051

Abstract :

Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.           

APR 2019     

DOI : 10.1096/fj.201801860RR

Abstract :

Nasal reconstruction remains a challenge for every reconstructive surgeon. Alloplastic implants are proposed to repair nasal cartilaginous defects but they are often associated with high rates of extrusion and infection and poor biocompatibility. In this context, a porous polymeric scaffold filled with an autologous cartilage gel would be advantageous. In this study, we evaluated the capacity of IEIK13 self-assembling peptide (SAP) to serve as support to form such cartilage gel. Human nasal chondrocytes (HNC) were first amplified with FGF-2 and insulin, and then redifferentiated in IEIK13 with BMP-2, insulin, and T3 (BIT). Our results demonstrate that IEIK13 fosters HNC growth and survival. HNC phenotype was assessed by RT-PCR analysis and neo-synthesized extracellular matrix was characterized by western blotting and immunohistochemistry analysis. BIT-treated cells embedded in IEIK13 displayed round morphology and expressed cartilage-specific markers such as type II and type IX collagens and aggrecan. In addition, we did not detect significant production of type I and type X collagens and gene products of dedifferentiated and hypertrophic chondrocytes that are unwanted in hyaline cartilage. The whole of these results indicates that the SAP IEIK13 represents a suitable support for hydrogel-based tissue engineering of nasal cartilage.            

APR 2019     

DOI : 10.1002/jbm.a.36612    

Pubmed ID : 30650239

Abstract :

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV's low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV's intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV's in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8(+) T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.            

MAR 2019  

DOI : 10.1038/s41598-019-40071-1    

Pubmed ID : 30842485

Abstract :

ARHGEF1 is a RhoA-specific guanine nucleotide exchange factor expressed in hematopoietic cells. We used whole-exome sequencing to identify compound heterozygous mutations in ARHGEF1, resulting in the loss of ARHGEF1 protein expression in 2 primary antibody-deficient siblings presenting with recurrent severe respiratory tract infections and bronchiectasis. Both ARHGEF1-deficient patients showed an abnormal B cell immunophenotype, with a deficiency in marginal zone and memory B cells and an increased frequency of transitional B cells. Furthermore, the patients' blood contained immature myeloid cells. Analysis of a mediastinal lymph node from one patient highlighted the small size of the germinal centers and an abnormally high plasma cell content. On the molecular level, T and B lymphocytes from both patients displayed low RhoA activity and low steady-state actin polymerization (even after stimulation of lysophospholipid receptors). As a consequence of disturbed regulation of the RhoA downstream target Rho-associated kinase I/II (ROCK), the patients' lymphocytes failed to efficiently restrain AKT phosphorylation. Enforced ARHGEF1 expression or drug-induced activation of RhoA in the patients' cells corrected the impaired actin polymerization and AKT regulation. Our results indicate that ARHGEF1 activity in human lymphocytes is involved in controlling actin cytoskeleton dynamics, restraining PI3K/AKT signaling, and confining B lymphocytes and myelocytes within their dedicated functional environment.     

MAR 2019     

DOI : 10.1172/JCI120572        

Pubmed ID : 30521495

Abstract :

Background The protein kinase mechanistic target of rapamycin (mTOR) controls cellular growth and metabolism. Although balanced mTOR signalling is required for proper muscle homeostasis, partial mTOR inhibition by rapamycin has beneficial effects on various muscle disorders and age-related pathologies. Besides, more potent mTOR inhibitors targeting mTOR catalytic activity have been developed and are in clinical trials. However, the physiological impact of loss of mTOR catalytic activity in skeletal muscle is currently unknown. Methods We have generated the mTORmKOKI mouse model in which conditional loss of mTOR is concomitant with expression of kinase inactive mTOR in skeletal muscle. We performed a comparative phenotypic and biochemical analysis of mTORmKOKI mutant animals with muscle-specific mTOR knockout (mTORmKO) littermates. Results In striking contrast with mTORmKO littermates, mTORmKOKI mice developed an early onset rapidly progressive myopathy causing juvenile lethality. More than 50% mTORmKOKI mice died before 8 weeks of age, and none survived more than 12 weeks, while mTORmKO mice died around 7 months of age. The growth rate of mTORmKOKI mice declined beyond 1 week of age, and the animals showed profound alterations in body composition at 4 weeks of age. At this age, their body weight was 64% that of mTORmKO mice (P < 0.001) due to significant reduction in lean and fat mass. The mass of isolated muscles from mTORmKOKI mice was remarkably decreased by 38-56% (P < 0.001) as compared with that from mTORmKO mice. Histopathological analysis further revealed exacerbated dystrophic features and metabolic alterations in both slow/oxidative and fast/glycolytic muscles from mTORmKOKI mice. We show that the severity of the mTORmKOKI as compared with the mild mTORmKO phenotype is due to more robust suppression of muscle mTORC1 signalling leading to stronger alterations in protein synthesis, oxidative metabolism, and autophagy. This was accompanied with stronger feedback activation of PKB/Akt and dramatic down-regulation of glycogen phosphorylase expression (0.16-fold in tibialis anterior muscle, P < 0.01), thus causing features of glycogen storage disease type V. Conclusions Our study demonstrates a critical role for muscle mTOR catalytic activity in the regulation of whole-body growth and homeostasis. We suggest that skeletal muscle targeting with mTOR catalytic inhibitors may have detrimental effects. The mTORmKOKI mutant mouse provides an animal model for the pathophysiological understanding of muscle mTOR activity inhibition as well as for mechanistic investigation of the influence of skeletal muscle perturbations on whole-body homeostasis.              

FEB  2019     

DOI : 10.1002/jcsm.12336      

Pubmed ID : 30461220

Abstract :

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-, and modulation of FXR activity by ligands alters HBV replication. Mechanisms of HBV control by FXR remain to be unveiled. FXR silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXR agonist GW4064 inhibited FXR proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXR acts as a proviral factor by 2 different mechanisms, which are abolished by FXR stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXR expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXR can be envisioned as a target for HBV treatment.

FEB  2019     

DOI : 10.1096/fj.201801181R

Pubmed ID : 30307769

Abstract :

Inactivated influenza vaccines (IIVs) lack broad efficacy. Cellular immunity to a conserved internal antigen, the nucleoprotein (NP), has been correlated to protection against pandemic and seasonal influenza and thus could have the potential to broaden vaccine efficacy. We developed OVX836, a recombinant protein vaccine based on an oligomerized NP, which shows increased uptake by dendritic cells and immunogenicity compared with NP. Intramuscular immunization in mice with OVX836 induced strong NP-specific CD4+ and CD8+ T-cell systemic responses and established CD8+ tissue memory T cells in the lung parenchyma. Strikingly, OVX836 protected mice against viral challenge with three different influenza A subtypes, isolated several decades apart and induced a reduction in viral load. When co-administered with IIV, OVX836 was even more effective in reducing lung viral load.

JAN 2019     

DOI : 10.1038/s41541-019-0098-4      

Pubmed ID : 30701093

Abstract :

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.   

JAN 2019     

DOI : 10.1038/s41467-018-07845-z    

Pubmed ID : 30604748

Abstract :

How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a(-/-) regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.

JAN  2019     

DOI : 10.1016/j.matbio.2018.07.005  

Pubmed ID : 30031067

Abstract :

Nasal reconstruction remains a challenge for every reconstructive surgeon. Alloplastic implants are proposed to repair nasal cartilaginous defects but they are often associated with high rates of extrusion and infection and poor biocompatibility. In this context, a porous polymeric scaffold filled with an autologous cartilage gel would be advantageous. In this study, we evaluated the capacity of IEIK13 self-assembling peptide (SAP) to serve as support to form such cartilage gel. Human nasal chondrocytes (HNC) were first amplified with FGF-2 and insulin, and then redifferentiated in IEIK13 with BMP-2, insulin, and T3 (BIT). Our results demonstrate that IEIK13 fosters HNC growth and survival. HNC phenotype was assessed by RT-PCR analysis and neo-synthesized extracellular matrix was characterized by western blotting and immunohistochemistry analysis. BIT-treated cells embedded in IEIK13 displayed round morphology and expressed cartilage-specific markers such as type II and type IX collagens and aggrecan. In addition, we did not detect significant production of type I and type X collagens and gene products of dedifferentiated and hypertrophic chondrocytes that are unwanted in hyaline cartilage. The whole of these results indicates that the SAP IEIK13 represents a suitable support for hydrogel-based tissue engineering of nasal cartilage.            

APR 2019     

DOI : 10.1002/jbm.a.36612    

Pubmed ID : 30650239

Abstract : 

Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP's role in organ development and disorders associated with TCTP miss-expression.          

JAN 2019     

DOI : 10.1371/journal.pgen.1007899    

Pubmed ID : 30695029