Abstract :

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG(+) counterparts do not. Antigen boost modifies the gene expression profile of IgM(+) plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge.

DEC  2016  

DOI :  10.1038/ncomms13600       

Pubmed : 27924814

Abstract :

Background: B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective: Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods: We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCID gamma c(-/-)(NSG) mouse. Results: Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTRLVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEVLVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/gamma c(-/-) (NSG) [non-obese diabetic (NOD), severe combined immunodeficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions: The BaEV-LVs might represent a valuable tool for therapeutic protein secretion from autologous B cells in vivo in the treatment of hemophilia and other acquired or inherited diseases.

DEC  2016

DOI : 10.1111/jth.13520

Pubmed : 27685947

Abstract :

Memory CD8 T lymphocyte populations are remarkably heterogeneous and differ in their ability to protect the host. In order to identify the whole range of qualities uniquely associated with protective memory cells we compared the gene expression signatures of two qualities of memory CD8 T cells sharing the same antigenic-specificity: protective (Influenza-induced, Flu-TM) and non-protective (peptide-induced, TIM) spleen memory CD8 T cells. Although Flu-TM and TIM express classical phenotypic memory markers and are polyfunctional, only Flu-TM protects against a lethal viral challenge. Protective memory CD8 T cells express a unique set of genes involved in migration and survival that correlate with their unique capacity to rapidly migrate within the infected lung parenchyma in response to influenza infection. We also enlighten a new set of poised genes expressed by protective cells that is strongly enriched in cytokines and chemokines such as Ccl1, Ccl9 and Gm-csf. CCL1 and GM-CSF genes are also poised in human memory CD8 T cells. These immune signatures are also induced by two other pathogens (vaccinia virus and Listeria monocytogenes). The immune signatures associated with immune protection were identified on circulating cells, i.e. those that are easily accessible for immuno-monitoring and could help predict vaccines efficacy.                    

NOV  2016

DOI: 10.1038/srep37651           

Pubmed : 27883012

Abstract :

Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31(-) cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31(-) DP cell population contained 1.4% of CD56(+), 1.5% of CD146(+), 2.4% of CD271(+) and 6.3% of MSCA-1(+) cells but very few Stro-1(+) cells (<= 1%). CD56(+), CD146(+), CD271(+), and MSCA-1(+) cell subpopulations expressed various levels of these markers. CD146(+)MSCA-1(+), CD271(+)MSCA-1(+), and CD146(+)CD271(+) cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56(+), CD271(+), MSCA-1(+), and Stro-1(+) cells, and MSCA-1 by 15-30% of CD56(+), CD146(+), CD271(+), and Stro-1(+) cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by,,1% of the expanded cell populations. Interestingly, the percentage of CD56 (+) cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146(+)CD56(+), MSCA-1(+)CD56(+), and CD146(+)MSCA-1(+) cells were the most abundant DP MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products.

nov-08  2016     

DOI : 10.3339/fphys.2016.00512          

Pubmed : 27877132

Abstract :

The actin cable is a supracellular structure that embryonic epithelia produce to close gaps. However, the action of the cable remains debated. Here, we address the function of the cable using Drosophila dorsal closure, a paradigm to understand wound healing. First, we show that the actin cytoskeleton protein Zasp52 is specifically required for actin cable formation. Next, we used Zasp52 loss of function to dissect the mechanism of action of the cable. Surprisingly, closure dynamics are perfect in Zasp52 mutants: the cable is therefore dispensable for closure, even in the absence of the amnioserosa. Conversely, we observed that the cable protects cellular geometries from robust morphogenetic forces that otherwise interfere with closure: the absence of cable results in defects in epithelial organization that lead to morphogenetic scarring. We propose that the cable prevents morphogenetic scarring by stabilizing cellular interactions rather than by acting on closure dynamics.

NOV  2016  

DOI :  10.1038/ncb3421

Pubmed : 27749820

Abstract :

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons. However, given that GAPDH does not produce ATP, it is unclear whether glycolysis per se is sufficient to propel vesicles. Although many proteins regulating transport have been identified, the molecular composition of transported vesicles in neurons has yet to be fully elucidated. Here we selectively enrich motile vesicles and perform quantitative proteomic analysis. In addition to the expected molecular motors and vesicular proteins, we find an enrichment of all the glycolytic enzymes. Using biochemical approaches and super-resolution microscopy, we observe that most glycolytic enzymes are selectively associated with vesicles and facilitate transport of vesicles in neurons. Finally, we provide evidence that mouse brain vesicles produce ATP from ADP and glucose, and display movement in a reconstituted in vitro transport assay of native vesicles. We conclude that transport of vesicles along microtubules can be autonomous.             

OCT   24-2016   

DOI :   10.1038/ncomms13233         

Pubmed : 27775035

Abstract :

Glucagon and thyroid hormone (T-3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T-3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T-3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T-3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T-3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease

OCT  20-2016   

DOI :  10.1016/j.cell.2016.09.014   

Pubmed : 27720451

Abstract :

Activation of TGF-β by dendritic cells (DCs) expressing αvβ8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal Ags. We have recently shown that αvβ8 integrin is preferentially expressed by CD103(+) DCs and confers their ability to activate TGF-β and generate Tregs. However, how these DCs become specialized for this vital function is unknown. In this study, we show that β8 expression is controlled by a combination of factors that include DC lineage and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-β itself, along with retinoic acid and TLR signaling, drives expression of αvβ8 in DCs. However, these signals only result in high levels of β8 expression in cells of the cDC1 lineage, CD8α(+), or CD103(+)CD11b(-) DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvβ8-expressing DCs specialized for activation of TGF-β to facilitate Treg generation.

SEP 2016     

DOI :  10.4049/jimmunol.1600244           

Pubmed :  27481847

Abstract :

Development of vaccines able to induce mucosal immunity in the genital and gastrointestinal tracts is a major challenge to counter sexually transmitted pathogens such as HIV-1 and HSV-2. Herein, we showed that intradermal (ID) immunisation with sub-unit vaccine antigens (i.e., HIV-1 gp140 and HSV-2 gD) delivered with Poly(I:C) or CpG1668 as adjuvant induces long-lasting virus-specific immunoglobulin (Ig)-G and IgA antibodies in the vagina and feces. Poly(I: C)-supplemented sub-unit viral vaccines caused minimal skin reactogenicity at variance to those containing CpG1668, promoted a delayed-type hypersensitivity (DTH) to the vaccine and protected mice from genital and neurological symptoms after a lethal vaginal HSV-2 challenge. Interestingly, Poly(I:C-12U) (Ampligen), a Poly(I:C) structural analogue that binds to TLR3 but not MDA-5, promoted robust mucosal and systemic IgG antibodies, a weak skin DTH to the vaccine but not IgA responses and failed to confer protection against HSV-2 infection. Moreover, Poly(I:C) was far superior to Poly(I:C-12U) at inducing prompt and robust upregulation of IFN beta transcripts in lymph nodes draining the injection site. These data illustrate that ID vaccination with glycoproteins and Poly(I:C) as adjuvant promotes long-lasting mucosal immunity and protection from genital HSV-2 infection, with an acceptable skin reactogenicity profile. The ID route thus appears to be an unexpected inductive site for mucosal immunity and anti-viral protection suitable for sub-unit vaccines. This works further highlights that TLR3/MDA5 agonists such as Poly(I:C) may be valuable adjuvants for ID vaccination against sexually transmitted diseases.  

AUG 2016     

DOI :  10.1038/npjvaccines.2016.10            

Pubmed : 29263853

Abstract :

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.

AUG 2016         

DOI :  10.1002/adhm.201600091                   

Pubmed : 27113918

Abstract :

Cancer Stem Cells (CSCs) in Head and Neck Squamous Cell Carcinoma (HNSCC) have extremely aggressive profile (high migratory and invasive potential). These characteristics can explain their resistance to conventional treatment. Efficacy of photon and carbon ion irradiation with addition of cetuximab (5 nM) is studied on clonogenic death, migration and invasion of two HNSCC populations: SQ20B and SQ20B/CSCs. SQ20B express E-cadherin and overexpress EGFR while SQ20B/CSCs express N-cadherin and low EGFR. Cetuximab strongly inhibits SQ20B proliferation but has no effect on SQ20B/CSCs. 2 Gy photon irradiation enhances migration and invasiveness in both populations (p < 0.05), while cetuximab only stops SQ20B migration (p < 0.005). Carbon irradiation significantly inhibits invasion in both populations (p < 0.05), and the association with cetuximab significantly inhibits invasion in both populations (p < 0.005). These results highlight CSCs characteristics: EGFRLow, cetuximab-resistant, and highly migratory. Carbon ion irradiation appears to be a very promising therapeutic modality counteracting migration/invasion process in both parental cells and CSCs in contrast to photon irradiation. 

JUL 2016

DOI : 10.18632/oncotarget.10281       

Pubmed : 27374096

Abstract :

Hedonic value is a dominant aspect of olfactory perception. Using optogenetic manipulation in freely behaving mice paired with immediate early gene mapping, we demonstrate that hedonic information is represented along the antero-posterior axis of the ventral olfactory bulb. Using this representation, we show that the degree of attractiveness of odors can be bidirectionally modulated by local manipulation of the olfactory bulb's neural networks in freely behaving mice.         

JUL 2016  

DOI :  10.1038/nn.4317              

Pubmed : 27273767

Abstract :

African mole-rats are fossorial rodents that consist of five chisel-tooth digging genera (Heterocephalus, Heliophobius, Georychus, Fukomys, and Cryptomys) and one scratch digger (Bathyergus). They are characterized by striking physiological, morphological, and behavioral adaptations intimately related to their subterranean life. The influence of their mode of life in shaping the cranial morphology has yet to be evaluated in comparison to other Ctenohystrica, especially fossorial genera, which include the subterranean genera Spalacopus and Ctenomys. In our study, we seek to determine to what extent subterranean life affects the morpho-functional properties of the skull among fossorial ctenohystricans. 3D geometric morphometric analyses were performed on 277 skulls, encompassing 63 genera of Ctenohystrica, and complemented by biomechanical studies. African mole-rats and other subterranean Ctenohystrica, especially chisel-tooth diggers, have a short snout, a wide cranium with enlarged zygomatic arches, and a strongly hystricognathous mandible. Even if convergences are also manifest between most fossorial Ctenohystrica, subterranean rodents departed from the main ctenohystrican allometric trends in having a skull shape less size-dependent, but under stronger directional selection with intense digging activity as a major constraint. African mole-rats, notably chisel-tooth diggers, show important mechanical advantage for the temporalis muscles favoring higher forces at the bite point, while mechanical advantage of the superficial masseter muscles is lower compared to other Ctenohystrica. If subterranean species can be clearly discriminated based on their skull morphology, the intrinsic mosaic of anatomical characters of each genus (e.g., skull, teeth, and muscles) can be understood only in the light of their ecology and evolutionary history.               

JUN  2016     

DOI :    10.1007/s10914-015-9305-x       

Pubmed :

Abstract :

Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

JUN 2016       

DOI :  10.1038/ncomms12071                

Pubmed : 27346279

Abstract :

The stage V sporulation protein G (SpoVG) homolog of Staphylococcus aureus is a modulator of virulence factor synthesis and antibiotic resistance in this clinically important gram-positive pathogen. Here we demonstrate that SpoVG can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation positively affects its DNA-binding properties. Mass spectrometric analyses and site directed mutagenesis identified Thr4, Thr13, Thr24 and Ser41 as phospho-acceptors. Stk1-mediated phosphorylation markedly enhanced the DNA binding activity of SpoVG towards the promoter regions of target genes such as capA, lip, and nuc1. Similarly, trans-complementation of the S. aureus Delta yabJ-spoVG mutant SM148 with a SpoVG derivative that mimics constitutive phosphorylation, SpoVG_Asp, exhibited capA, lip, and nuc1 transcript levels that were comparable to the levels seen with the wild-type, whereas trans-complementation with a phosphoablative variant of SpoVG (SpoVG_A1a) produced transcript levels similar to the ones seen in SM148. Our data suggest that the expression activity of this transcription factor is tightly controlled in S. aureus by transcriptional, post-transcriptional and post-translational mechanisms. (C) 2016 Elsevier Inc. All rights reserved.            

MAY 13 2016   

DOI : 10.1016/j.bbrc.2016.04.044                      

Pubmed : 27091430

Abstract :

Nucleolin (NCL) is a major component of the cell nucleolus, which has the ability to rapidly shuttle to several other cells' compartments. NCL plays important roles in a variety of essential functions, among which are ribosome biogenesis, gene expression, and cell growth. However, the precise mechanisms underlying NCL functions are still unclear. Our study aimed to provide new information on NCL functions via the identification of its nuclear interacting partners. Using an interactomics approach, we identified 140 proteins co-purified with NCL, among which 100 of them were specifically found to be associated with NCL after RNase digestion. The functional classification of these proteins confirmed the prominent role of NCL in ribosome biogenesis and additionally revealed the possible involvement of nuclear NCL in several pre-mRNA processing pathways through its interaction with RNA helicases and proteins participating in pre-mRNA splicing, transport, or stability. NCL knockdown experiments revealed that NCL regulates the localization of EXOSC10 and the amount of ZC3HAV1, two components of the RNA exosome, further suggesting its involvement in the control of mRNA stability. Altogether, this study describes the first nuclear interactome of human NCL and provides the basis for further understanding the mechanisms underlying the essential functions of this nucleolar protein.

MAY  2016     

DOI : 10.1021/acs.jproteome.6b00126                 

Pubmed : 27049334

Abstract :

Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans.

DOI : 10.1084/jem.20150794                 

Pubmed : 27069116

Abstract :

The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an alpha-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.   

MAY  2016     

DOI : 10.1007/s00018-015-2118-5           

Pubmed : 26708291

Abstract :

Head and neck squamous cell carcinomas (HNSCC) are common human malignancies with poor clinical outcomes. The 5-year survival rates for patients with advanced stage HNSCC have not changed appreciably in the past few decades, underscoring a dire need for improved therapeutic options. HNSCC is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio-and chemoresistance and poor clinical outcome. The aim of this study was to evaluate combined effects of radiation and ABT-737, a BH3-mimetic molecule, in HNSCC. Although ABT-737, as a single agent, was largely ineffective at promoting HNSCC cell death, we found that combining ABT-737 and radiation induced strong synergistic apoptosis in HNSCC cell lines and delayed tumoral growth in vivo. Moreover, we demonstrated for the first time that ABT-737, alone or in combination with radiation, can efficiently eliminate cancer stem cells (CSCs). Altogether, our results indicate that therapy targeting anti-apoptotic Bcl-2 family members could be a highly effective potential adjuvant to radiotherapy capable of targeting CSCs in HNSCC and therefore overcoming cancer recurrence and metastasis.             

Mars-29 2016       

DOI :10.18632/oncotarget.7744           

Pubmed : 26934442

Abstract :

Nowadays, head and neck squamous cell carcinoma (HNSCC) treatment failure is mostly explained by locoregional progression or intrinsic radioresistance. Radiotherapy (RT) has recently evolved with the emergence of heavy ion radiations or new fractionation schemes of photon therapy, which modify the dose rate of treatment delivery. The aim of the present study was then to evaluate the in vitro influence of a dose rate variation during conventional RT or carbon ion hadrontherapy treatment in order to improve the therapeutic care of patient. In this regard, two HNSCC cell lines were irradiated with photons or 72 MeV/n carbon ions at a dose rate of 0.5, 2, or 10 Gy/min. For both radiosensitive and radioresistant cells, the change in dose rate significantly affected cell survival in response to photon exposure. This variation of radiosensitivity was associated with the number of initial and residual DNA double-strand breaks (DSBs). By contrast, the dose rate change did not affect neither cell survival nor the residual DNA DSBs after carbon ion irradiation. As a result, the relative biological efficiency at 10% survival increased when the dose rate decreased. In conclusion, in the RT treatment of HNSCC, it is advised to remain very careful when modifying the classical schemes toward altered fractionation. At the opposite, as the dose rate does not seem to have any effects after carbon ion exposure, there is less need to adapt hadrontherapy treatment planning during active system irradiation.          

MARCH 2016    

DOI : 10.3389/fonc.2016.00058             

Pubmed : 27014633

Abstract :

The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss-and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development.  

MARCH 2016    

DOI : 10.1523/JNEUROSCI.2847-15.2016               

Pubmed : 26937007

Abstract :

The sea urchin endoskeleton consists of a magnesium-rich biocalcite comprising a small amount of occluded organic macromolecules. This structure constitutes a key-model for understanding the mineral--organics interplay, and for conceiving in vitro bio-inspired materials with tailored properties. Here we employed a deep-clean technique to purify the occluded proteins from adult Paracentrotus lividus tests. We characterized them by 1- and 2D-electrophoreses, ELISA and immunoblotting, and using liquid chromatography coupled with Mass Spectrometry (nanoLC-MS/MS), we identified two metalloenzymes (carbonic anhydrase and MMP), a set of MSP130 family members, several C-type lectins (SM29, SM41, PM27) and cytoskeletal proteins. We demonstrate the effect of the protein extract on the crystals, with an in vitro crystallization assay. We suggest that this small set of biomineralization proteins may represent a 'minimal molecular crystallization toolkit'.

MARCH 2016    

DOI : 10.1016/j.jprot.2016.01.001             

Pubmed : 26778142

Abstract :

In most animal species, juvenile growth is marked by an exponential gain in body weight and size. Here we show that the microbiota of infant mice sustains both weight gain and longitudinal growth when mice are fed a standard laboratory mouse diet or a nutritionally depleted diet. We found that the intestinal microbiota interacts with the somatotropic hormone axis to drive systemic growth. Using monocolonized mouse models, we showed that selected lactobacilli promoted juvenile growth in a strain-dependent manner that recapitulated the microbiota's effect on growth and the somatotropic axis. These findings show that the host's microbiota supports juvenile growth. Moreover, we discovered that lactobacilli strains buffered the adverse effects of chronic undernutrition on the postnatal growth of germ-free mice.

FEB 19   2016     

DOI : 10.1126/science.aad8588                

Pubmed : 26912894   

Abstract :

Intestinal DCs orchestrate gut immune homeostasis by dampening proinflammatory T-cell responses and inducing anti-inflammatory IgA responses. Although no specific DC subset has been strictly assigned so far to govern IgA response, some candidate subsets emerge. In particular, plasmacytoid DCs (pDCs), which notoriously promote anti-viral immunity and T-cell tolerance to innocuous antigens (Ags), contribute to IgA induction in response to intestinal viral infection and promote T-cell-independent IgA responses in vitro. Here, using two transgenic mouse models, we show that neither short-term nor long-term pDC depletion alters IgA class switch recombination in Peyer's patches and frequency of IgA plasma cells in intestinal mucosa at steady state, even in the absence of T-cell help. In addition, pDCs are dispensable for induction of intestinal IgA plasma cells in response to oral immunization with T-cell-dependent or T-cell-independent Ags, and are not required for proliferation and IgA switch of Ag-specific B cells in GALT. These results show that pDCs are dispensable for noninfectious IgA responses, and suggest that various DC subsets may play redundant roles in the control of intestinal IgA responses.

FEB   2016     

DOI : 10.1002/eji.201545977   26518732           

Pubmed :

Abstract :

Macromolecule crystal formation can be divided in two major steps: 1. the formation of a nucleus and 2. the growth of this nucleus into a full mature crystal. The latter is well described and understood, while the former remains elusive due to the difficulty to study it and is described by nucleation theories. Here we report the structure of the Escherichia coli outer membrane porin OmpF in two centered monoclinic space groups. Strikingly, the two crystals originate from the same building block, made of two trimers of OmpF interacting via their rough side. The different crystallization conditions trigger the formation of distinct arrangement of these building blocks, leading to the formation of translational non-crystallographic symmetry (tNCS) in one case, made possible by the loose lateral packing mediated by detergents. In light of nucleation theories, these results allow us to speculate that these two crystals originate from nuclei made of either clusters of building blocks, or already forming columns that later associate laterally using detergents as glue. (C) 2015 Elsevier B.V. All rights reserved.

FEB   2016     

DOI : 10.1016/j.bbamem.2015.11.021              

Pubmed : 26620074

Abstract :

Due to the emergence of methicillin-resistant strains, Staphylococcus aureus has become as major public health threat. Studies aimed at deciphering the molecular mechanism of virulence are thus required to identify new targets and develop efficient therapeutic agents. Protein phosphorylations are known to play key regulatory functions and their roles in pathogenesis are under intense scrutiny. Here we analyzed the protein tyrosine phosphatase PtpA of S. aureus, a member of the family of low molecular weight protein tyrosine phosphatases that are often secreted by pathogenic bacteria. We report for the first time that PtpA is phosphorylated in vitro by the S. aureus tyrosine kinase CapA1B2. A mass spectrometry approach allowed determining that Tyr122 and Tyr123 were the only two residues phosphorylated by this kinase. This result was confirmed by analysis of a double PtpA_Y122A/Y123A mutant that showed no phosphorylation by CapA1B2. Interestingly, PtpA phosphatase activity was abrogated in this mutant, suggesting a key regulatory function for these two tyrosine residues. This was further reinforced by the observation that CapA1B2-mediated phosphorylation significantly increased PtpA phosphatase activity. Moreover, we provide evidence that PtpA is secreted during growth of S. aureus. Together our results suggest that PtpA is an exported S. aureus signaling molecule controlled by tyrosine phosphorylation which may interfere with host cell signaling. (C) 2015 Elsevier Inc. All rights reserved.

Janv 15 2016     

DOI :  10.1016/j.bbrc.2015.11.123

Pubmed : 26679607

Abstract :

Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.

Janv 2016     

DOI: 10.1111/tpj.13099.

Pubmed : 26662936

Abstract :

Adoptive cell therapy represents a promising approach for several chronic diseases. This study describes an innovative strategy for biofunctionalization of nanoparticles, allowing the generation of synthetic particulate antigens (SPAg). SPAg activate polyclonal B cells and vectorize noncognate proteins into their endosomes, generating highly efficient stimulators for ex vivo expansion of antigen-specific CD4+ T cells. This method also allows harnessing the ability of B cells to polarize CD4+ T cells into effectors or regulators.

Janv 2016     

DOI: 110.1021/acs.nanolett.5b03801

Pubmed : 26650819