Sec22b is a critical and nonredundant regulator of plasma cell maintenance.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.

When human cord blood-derived CD34+ cells are induced to differentiate, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. In particular, the cells pass through a transitory phase known as "multilineage-primed" state. These cells are characterized by a mixed gene expression profile, different in each cell, with the coexpression of many genes characteristic for concurrent cell lineages. The aim of our study is to understand the mechanisms of the establishment and the exit from this transitory state. We investigated this issue using single-cell RNA sequencing and ATAC-seq. Two phases were detected. The first phase is a rapid and global chromatin decompaction that makes most of the gene promoters in the genome accessible for transcription. It results 24 h later in enhanced and pervasive transcription of the genome leading to the concomitant increase in the cell-to-cell variability of transcriptional profiles. The second phase is the exit from the multilineage-primed phase marked by a slow chromatin closure and a subsequent overall down-regulation of gene transcription. This process is selective and results in the emergence of coherent expression profiles corresponding to distinct cell subpopulations. The typical time scale of these events spans 48 to 72 h. These observations suggest that the nonspecificity of genome decompaction is the condition for the generation of a highly variable multilineage expression profile. The nonspecific phase is followed by specific regulatory actions that stabilize and maintain the activity of key genes, while the rest of the genome becomes repressed again by the chromatin recompaction. Thus, the initiation of differentiation is reminiscent of a constrained optimization process that associates the spontaneous generation of gene expression diversity to subsequent regulatory actions that maintain the activity of some genes, while the rest of the genome sinks back to the repressive closed chromatin state.

Differentiation is accompanied by a progressive loss in transcriptional memory.

BACKGROUND: Cell differentiation requires the integration of two opposite processes, a stabilizing cellular memory, especially at the transcriptional scale, and a burst of gene expression variability which follows the differentiation induction. Therefore, the actual capacity of a cell to undergo phenotypic change during a differentiation process relies upon a modification in this balance which favors change-inducing gene expression variability. However, there are no experimental data providing insight on how fast the transcriptomes of identical cells would diverge on the scale of the very first two cell divisions during the differentiation process. RESULTS: In order to quantitatively address this question, we developed different experimental methods to recover the transcriptomes of related cells, after one and two divisions, while preserving the information about their lineage at the scale of a single cell division. We analyzed the transcriptomes of related cells from two differentiation biological systems (human CD34+ cells and T2EC chicken primary erythrocytic progenitors) using two different single-cell transcriptomics technologies (scRT-qPCR and scRNA-seq). CONCLUSIONS: We identified that the gene transcription profiles of differentiating sister cells are more similar to each other than to those of non-related cells of the same type, sharing the same environment and undergoing similar biological processes. More importantly, we observed greater discrepancies between differentiating sister cells than between self-renewing sister cells. Furthermore, a progressive increase in this divergence from first generation to second generation was observed when comparing differentiating cousin cells to self renewing cousin cells. Our results are in favor of a gradual erasure of transcriptional memory during the differentiation process.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment.

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned).

Combination of imaging flow cytometry and time-lapse microscopy for the study of label-free morphology dynamics of hematopoietic cells.

Cell differentiation is a longitudinal and dynamic process. Studying and quantifying such a process require tools combining precise time resolution and statistical power. Imaging flow cytometry (IFC) provides statistically significant number of microscopy images of individual cells in a sample at a given time point. Time-lapse microscopy (TLM) is the method of choice for studying the dynamics of cell processes at a high temporal, but low statistical resolution. In this work, we show that the dynamic changes of cord-blood derived CD34+ cells in response to cytokine stimulation can be successfully studied, in a label-free way, by the combination of the IFCs statistical power and the TLM's high time resolution. Cell morphology phenotypes were quantified through roundness and surface area, measured both in IFC and with a homemade segmentation algorithm in TLM. Two distinct morphologies-polarized and round-were observed in cord-blood derived CD34+. We show that some cells have the ability to fluctuate between these morphologies, suggesting that the apparent stable composition of round and polarized cells may actually represent a dynamic equilibrium. This example demonstrates that the different resolutions and modalities of IFC and TLM are complementary and allow the study of complex dynamic biological processes. © 2017 International Society for Advancement of Cytometry.

Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family.

Improving Interpretability of Leucocyte Classification with Multimodal Network.

White blood cell classification plays a key role in the diagnosis of hematologic diseases. Models can perform classification either from images or based on morphological features. Image-based classification generally yields higher performance, but feature-based classification is more interpretable for clinicians. In this study, we employed a Multimodal neural network to classify white blood cells, utilizing a combination of images and morphological features. We compared this approach with image-only and feature-only training. While the highest performance was achieved with image-only training, the Multimodal model provided enhanced interpretability by the computation of SHAP values, and revealed crucial morphological features for biological characterization of the cells.

Artificial intelligence workflow quantifying muscle features on Hematoxylin-Eosin stained sections reveals dystrophic phenotype amelioration upon treatment.

Cell segmentation is a key step for a wide variety of biological investigations, especially in the context of muscle science. Currently, automated methods still struggle to perform skeletal muscle fiber quantification on Hematoxylin-Eosin (HE) stained histopathological whole slide images due to low contrast. On the other hand, the Deep Learning algorithm Cellpose offers new perspectives considering its increasing adoption for segmentation of a wide range of cells. Combining two open-source tools, Cellpose and QuPath, we developed MyoSOTHES, an automated Myofibers Segmentation wOrkflow Tuned for HE Staining. MyoSOTHES enables solving segmentation inconsistencies encountered by default Cellpose model in presence of large range size cells and provides information related to muscle Feret's diameter distribution and Centrally Nucleated Fibers, thus depicting muscle health and treatment effects. MyoSOTHES achieves high quality segmentation compared to baseline workflow with a detection F1-score increasing from 0.801 to 0.919 and a Root Mean Square Error (RMSE) on diameter improved by 31%. MyoSOTHES was validated on an animal study featuring gene transfer in [Formula: see text]-Sarcoglycanopathy, for which dose-response effect is visible and conclusions drawn are consistent with those previously published. MyoSOTHES thus paves the way for wide quantification of HE stained muscle sections and retrospective analysis of HE labeled slices used in laboratories for decades.

Glutamate-Induced Deregulation of Krebs Cycle in Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-Like Episodes (MELAS) Syndrome Is Alleviated by Ketone Body Exposure.

(1) Background: The development of mitochondrial medicine has been severely impeded by a lack of effective therapies. (2) Methods: To better understand Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-like episodes (MELAS) syndrome, neuronal cybrid cells carrying different mutation loads of the m.3243A > G mitochondrial DNA variant were analysed using a multi-omic approach. (3) Results: Specific metabolomic signatures revealed that the glutamate pathway was significantly increased in MELAS cells with a direct correlation between glutamate concentration and the m.3243A > G heteroplasmy level. Transcriptomic analysis in mutant cells further revealed alterations in specific gene clusters, including those of the glutamate, gamma-aminobutyric acid pathways, and tricarboxylic acid (TCA) cycle. These results were supported by post-mortem brain tissue analysis from a MELAS patient, confirming the glutamate dysregulation. Exposure of MELAS cells to ketone bodies significantly reduced the glutamate level and improved mitochondrial functions, reducing the accumulation of several intermediate metabolites of the TCA cycle and alleviating the NADH-redox imbalance. (4) Conclusions: Thus, a multi-omic integrated approach to MELAS cells revealed glutamate as a promising disease biomarker, while also indicating that a ketogenic diet should be tested in MELAS patients.

DNAJB2 expression in normal and diseased human and mouse skeletal muscle.

DNAJB2, a co-chaperone regulator of Hsp70 that is expressed principally in the nervous system, has been recently reported to be up-regulated in human skeletal muscle during its recovery from damage. Here we identified DNAJB2 expression in regenerating fibers in skeletal muscles of the dystrophic mdx mouse and patients with Duchenne muscular dystrophy. Surprisingly, in both dystrophic and control mice and patients, DNAJB2 was also expressed in non-regenerating fibers at the postsynaptic side of the neuromuscular junction. DNAJB2 functions as an adaptor molecule for the evacuation and degradation of proteins through the ubiquitin-proteasome system, and overexpression of DNAJB2 in models of the neurodegenerative disease spinobulbar muscular atrophy was shown to result in the reduction of protein inclusions. We therefore studied the possible relation of DNAJB2 expression to protein inclusion formation in skeletal muscle in biopsies of several muscle pathologies associated with protein aggregation and found in all of them a strong immunoreactivity with anti-DNAJB2 in aggregates and vacuoles. We conclude that DNAJB2 is expressed in mouse and human skeletal muscle at the neuromuscular junction of normal fibers, in the cytoplasm and membrane of regenerating fibers, and in protein aggregates and vacuoles in protein aggregate myopathies. Therefore, we propose a role for DNAJB2 in protein turnover processes in skeletal muscle.

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

Exposure of Human Skin Organoids to Low Genotoxic Stress Can Promote Epithelial-to-Mesenchymal Transition in Regenerating Keratinocyte Precursor Cells.

For the general population, medical diagnosis is a major cause of exposure to low genotoxic stress, as various imaging techniques deliver low doses of ionizing radiation. Our study investigated the consequences of low genotoxic stress on a keratinocyte precursor fraction that includes stem and progenitor cells, which are at risk for carcinoma development. Human skin organoids were bioengineered according to a clinically-relevant model, exposed to a single 50 mGy dose of γ rays, and then xeno-transplanted in nude mice to follow full epidermis generation in an in vivo context. Twenty days post-xenografting, mature skin grafts were sampled and analyzed by semi-quantitative immuno-histochemical methods. Pre-transplantation exposure to 50 mGy of immature human skin organoids did not compromise engraftment, but half of xenografts generated from irradiated precursors exhibited areas displaying focal dysplasia, originating from the basal layer of the epidermis. Characteristics of epithelial-to-mesenchymal transition (EMT) were documented in these dysplastic areas, including loss of basal cell polarity and cohesiveness, epithelial marker decreases, ectopic expression of the mesenchymal marker α-SMA and expression of the EMT promoter ZEB1. Taken together, these data show that a very low level of radiative stress in regenerating keratinocyte stem and precursor cells can induce a micro-environment that may constitute a favorable context for long-term carcinogenesis.

The intestinal quorum sensing 3-oxo-C12:2 Acyl homoserine lactone limits cytokine-induced tight junction disruption.

The intestine is home to the largest microbiota community of the human body and strictly regulates its barrier function. Tight junctions (TJ) are major actors of the intestinal barrier, which is impaired in inflammatory bowel disease (IBD), along with an unbalanced microbiota composition. With the aim to identify new actors involved in host-microbiota interplay in IBD, we studied N-acyl homoserine lactones (AHL), molecules of the bacterial quorum sensing, which also impact the host. We previously identified in the gut a new and prominent AHL, 3-oxo-C12:2, which is lost in IBD. We investigated how 3-oxo-C12:2 impacts the intestinal barrier function, in comparison to 3-oxo-C12, a structurally close AHL produced by the opportunistic pathogen P. aeruginosa. Using Caco-2/TC7 cells as a model of polarized enterocytes, we compared the effects on paracellular permeability and TJ integrity of these two AHL, separately or combined with pro-inflammatory cytokines, Interferon-γ and Tumor Necrosis Factor-α, known to disrupt the barrier function during IBD. While 3-oxo-C12 increased paracellular permeability and decreased occludin and tricellulin signal at bicellular and tricellular TJ, respectively, 3-oxo-C12:2 modified neither permeability nor TJ integrity. Whereas 3-oxo-C12 potentiated the hyperpermeability induced by cytokines, 3-oxo-C12:2 attenuated their deleterious effects on occludin and tricellulin, and maintained their interaction with their partner ZO-1. In addition, 3-oxo-C12:2 limited the cytokine-induced ubiquitination of occludin and tricellulin, suggesting that this AHL prevented their endocytosis. In conclusion, the role of 3-oxo-C12:2 in maintaining TJ integrity under inflammatory conditions identifies this new AHL as a potential beneficial actor of host-microbiota interactions in IBD.

Robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta.

BACKGROUND: The monogenic disease osteogenesis imperfecta (OI) is due to single mutations in either of the collagen genes ColA1 or ColA2, but within the same family a given mutation is accompanied by a wide range of disease severity. Although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of DNA from OI patients has not been reported. Promising genome wide marker-independent physical methods for identifying disease-related loci have lacked robustness for widespread applicability. Therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to OI. RESULTS: We have improved methods for enriching regions of identity-by-descent (IBD) shared between related, afflicted individuals. The extent of enrichment exceeds 10- to 50-fold for some loci. The efficiency of the new process is shown by confirmation of the identification of the Col1A2 locus in osteogenesis imperfecta patients from Amish families. Moreover the analysis revealed additional candidate linkage loci that may harbour modifier genes for OI; a locus on chromosome 1q includes COX-2, a gene implicated in osteogenesis. CONCLUSION: Technology for physical enrichment of IBD loci is now robust and applicable for finding genes for monogenic diseases and genes for complex diseases. The data support the further investigation of genetic loci other than collagen gene loci to identify genes affecting the clinical expression of osteogenesis imperfecta. The discrimination of IBD mapping will be enhanced when the IBD enrichment procedure is coupled with deep resequencing.

Stable and functional lymphoid reconstitution in artemis-deficient mice following lentiviral artemis gene transfer into hematopoietic stem cells.

Patients with mutations in the Artemis gene display a complete absence of T- and B lymphocytes, together with increased cellular radiosensitivity; this leads to a radiosensitive severe combined immunodeficiency (RS-SCID). Allogenic hematopoietic stem-cell (HSC) transplantation is only partially successful in the absence of an human leukocyte antigen-genoidentical donor, and this has prompted a search for alternative therapeutic approaches such as gene therapy. In this study, a self-inactivated lentiviral vector expressing Artemis was used to complement the Artemis knockout mouse (Art(-/-)). Transplantation of Artemis-transduced HSCs into irradiated Art(-/-) mice restored a stable (over a 15-month period of follow-up) and functional T- and cell repertoire that was comparable to that of control mice. The success of secondary transplantations demonstrated that the HSCs had been transduced. One of thirteen mice developed a thymoma 6 months after gene therapy. Although thymic cells were seen to be carrying two lentiviral integration sites, there was no evidence of lentivirus-driven oncogene activation. The Art(-/-) mice were found to be prone to develop T-cell lymphomas, either spontaneously or after irradiation. These data indicate that the observed lymphoproliferation was probably the consequence of the chromosomal instability associated with the Artemis-deficient background. As a whole, our work provides a basis for supporting the gene therapy approach in Artemis-deficient SCID.

Epigenetic gene expression noise and phenotypic diversification of clonal cell populations.

Spontaneous emergence of phenotypic heterogeneity in cultures of genetically identical cells is a frequently observed phenomenon that provides a simple in vitro experimental system to model the problems of in vivo differentiation. In the present study, we have investigated whether stochastic variation of gene expression levels could contribute to phenotypic change in human cells. We have applied the two fluorescence-coding gene method and the expression variability of the two reporter genes to human cells in culture. We have quantified the portion of gene expression variation determined by global, promoter-specific, or by epigenetic sources. These two types of variation appear to contribute, in different ways, to the phenotypic diversification of clonal cell populations. Global, or promoter-specific, gene expression noise increases with cellular stress and contributes to the emergence of cellular diversity by diversifying the gene-expression levels. Epigenetic mechanisms act to increase the robustness of the cellular state by stabilizing gene transcription levels or by reinforcing the silenced state.

In Vivo Monitoring of Calpain Activity by Forster Resonance Energy Transfer.

Calpains are a 15-member class of calcium-activated nonlysosomal neutral proteases. They are involved in many cellular processes and are highly upregulated in pathological conditions. Some are ubiquitously expressed (CAPN1, CAPN2, CAPN4, CAPN5, CAPN7, and CAPN10), but others are thought to be localized in specific tissues. The monitoring of in vivo calpain activity is required for physiological, pathological, and therapeutic evaluations. This past decade, a tool for monitoring calpain activity in such conditions was developed using Forster resonance energy transfer (FRET). Studies showed that the level of calpain activity correlates with a decrease in FRET between the two fluorescent proteins. This chapter describes the methodologies from the design of the construct to the imaging procedure and analysis to evaluate ubiquitous calpain activity in vivo.

Constraints on Human CD34+ Cell Fate due to Lentiviral Vectors Can Be Relieved by Valproic Acid.

The initial stages following the in vitro cytokine stimulation of human cord blood CD34+ cells overlap with the period when lentiviral gene transfer is typically performed. Single-cell transcriptional profiling and time-lapse microscopy were used to investigate how the vector-cell crosstalk impacts on the fate decision process. The single-cell transcription profiles were analyzed using a new algorithm, and it is shown that lentiviral transduction during the early stages of stimulation modifies the dynamics of the fate choice process of the CD34+ cells. The cells transduced with a lentiviral vector are biased toward the common myeloid progenitor lineage. Valproic acid, a histone deacetylase inhibitor known to increase the grafting potential of the CD34+ cells, improves the transduction efficiency to almost 100%. The cells transduced in the presence of valproic acid can subsequently undergo normal fate commitment. The higher gene transfer efficiency did not alter the genomic integration profile of the vector. These observations open the way to substantially improving lentiviral gene transfer protocols.

An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative treatment for any of the sarcoglycanopathies. A first clinical trial to evaluate the safety of a recombinant AAV2/1 vector expressing γ-sarcoglycan using an intramuscular route of administration showed limited expression of the transgene and good tolerance of the approach. In this report, we undertook a dose-effect study in mice to evaluate the efficiency of an AAV2/8-expressing γ-sarcoglycan controlled by a muscle-specific promoter with a systemic mode of administration. We observed a dose-related efficiency with a nearly complete restoration of gamma sarcoglycan (SGCG) expression, histological appearance, biomarker level, and whole-body strength at the highest dose tested. In addition, our data suggest that a high expression threshold level must be achieved for effective protection of the transduced muscle, while a suboptimal transgene expression level might be less protective in the context of mechanical stress.

Vectofusin-1, a potent peptidic enhancer of viral gene transfer forms pH-dependent α-helical nanofibrils, concentrating viral particles.

Gene transfer using lentiviral vectors has therapeutic applications spanning from monogenic and infectious diseases to cancer. Such gene therapy has to be improved by enhancing the levels of viral infection of target cells and/or reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1, a recently developed cationic amphipathic peptide with a pronounced capacity to enhance such viral transduction, strongly promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium. To clarify the molecular basis of its action the peptide was investigated on a molecular and a supramolecular level by a variety of biophysical approaches. We show that in culture medium vectofusin-1 rapidly forms complexes in the 10 nm range that further assemble into annular and extended nanofibrils. These associate with viral particles allowing them to be easily pelleted for optimal virus-cell interaction. Thioflavin T fluorescence, circular dichroism and infrared spectroscopies indicate that these fibrils have a unique α-helical structure whereas most other viral transduction enhancers form ß-amyloid fibrils. A vectofusin-1 derivative (LAH2-A4) is inefficient in biological assays and does not form nanofibrils, suggesting that supramolecular assembly is essential for transduction enhancement. Our observations define vectofusin-1 as a member of a new class of α-helical enhancers of lentiviral infection. Its fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols.