Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.

The interplay between lysosome, protein corona and biological effects of cationic carbon dots: Role of surface charge titratability.

Carbon dots (CDs) are nanoparticles (NPs) with potential applications in the biomedical field. When in contact with biological fluids, most NPs are covered by a protein corona. As well, upon cell entry, most NP are sequestered in the lysosome. However, the interplay between the lysosome, the protein corona and the biological effects of NPs is still poorly understood. In this context, we investigated the role of the lysosome in the toxicological responses evoked by four cationic CDs exhibiting protonatable or non-protonatable amine groups at their surface, and the associated changes in the CD protein corona. The four CDs accumulated in the lysosome and led to lysosomal swelling, loss lysosome integrity, cathepsin B activation, NLRP3 inflammasome activation, and cell death by pyroptosis in a human macrophage model, but with a stronger effect for CDs with titratable amino groups. The protein corona formed around CDs in contact with serum partially dissociated under lysosomal conditions with subsequent protein rearrangement, as assessed by quantitative proteomic analysis. The residual protein corona still contained binding proteins, catalytic proteins, and proteins involved in the proteasome, glycolysis, or PI3k-Akt KEGG pathways, but with again a more pronounced effect for CDs with titratable amino groups. These results demonstrate an interplay between lysosome, protein corona and biological effects of cationic NPs in link with the titratability of NP surface charges.

Surface charge influences protein corona, cell uptake and biological effects of carbon dots.

Carbon dots are emerging nanoparticles (NPs) with tremendous applications, especially in the biomedical field. Herein is reported the first quantitative proteomic analysis of the protein corona formed on CDs with different surface charge properties. Four CDs were synthesized from citric acid and various amine group-containing passivation reagents, resulting in cationic NPs with increasing zeta (ζ)-potential and density of positive charges. After CD contact with serum, we show that protein corona identity is influenced by CD surface charge properties, which in turn impacts CD uptake and viability loss in macrophages. In particular, CDs with high ζ-potential (>+30 mV) and charge density (>2 µmol mg-1) are the most highly internalized, and their cell uptake is strongly correlated with a corona enriched in vitronectin, fibulin, fetuin, adiponectin and alpha-glycoprotein. On the contrary, CDs with a lower ζ-potential (+11 mV) and charge density (0.01 µmol mg-1) are poorly internalized, while having a corona with a very different protein signature characterized by a high abundance of apolipoproteins (APOA1, APOB and APOC), albumin and hemoglobin. These data illustrate how corona characterization may contribute to a better understanding of CD cellular fate and biological effects, and provide useful information for the development of CDs for biomedical applications.

Combining label-free and label-based accurate quantifications with SWATH-MS: Comparison with SRM and PRM for the evaluation of bovine muscle type effects.

Mass spectrometry has proven to be a valuable tool for the accurate quantification of proteins. In this study, the performances of three targeted approaches, namely selected reaction monitoring (SRM), parallel reaction monitoring (PRM) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS), to accurately quantify ten potential biomarkers of beef meat tenderness or marbling in a cohort of 64 muscle samples were evaluated. So as to get the most benefit out of the complete MS2 maps that are acquired in SWATH-MS, an original label-free quantification method to estimate protein amounts using an I-spline regression model was developed. Overall, SWATH-MS outperformed SRM in terms of sensitivity and dynamic range, while PRM still performed the best, and all three strategies showed similar quantification accuracies and precisions for the absolute quantification of targets of interest. This targeted picture was extended by 585 additional proteins for which amounts were estimated using the label-free approach on SWATH-MS; thus, offering a more global profiling of muscle proteomes and further insights into muscle type effect on candidate biomarkers of beef meat qualities as well as muscle metabolism.

Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway.

The biosynthetic secretory pathway is particularly challenging to investigate as it is underrepresented compared to the abundance of the other intracellular trafficking routes. Here, we combined the retention using selective hook (RUSH) to a CRISPR-Cas9 gene editing approach (eRUSH) and identified Rab7-harboring vesicles as an important intermediate compartment of the Golgi-to-plasma membrane transport of neosynthesized transferrin receptor (TfR). These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles. Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before fusion with the plasma membrane. Together, our study reveals a role for Rab7 in the biosynthetic secretory pathway of the TfR, highlighting the diversity of the secretory vesicles' nature.

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.

Safety, Immunogenicity, and Glycemic Control of Insulin Aspart Biosimilar SAR341402 Versus Originator Insulin Aspart in People with Diabetes Also Using Insulin Glargine: 12-Month Results from the GEMELLI 1 Trial.

Background: SAR341402 (SAR-Asp) is a biosimilar/follow-on of the originator insulin aspart-NovoLog®/NovoRapid® (NN-Asp). This study investigated whether the efficacy, safety, and immunogenicity findings for SAR-Asp versus NN-Asp, observed over 6 months in people with type 1 (n = 497) or type 2 diabetes (n = 100) treated with multiple daily injections in combination with insulin glargine (Lantus®), are maintained after 12 months. Materials and Methods: GEMELLI 1 was a multicenter, randomized, open-label, phase 3 study. Participants completing the initial 6-month treatment period continued on SAR-Asp or NN-Asp, as randomized, for a 6-month safety extension. Results: Of the 597 participants randomized, 264 out of 301 (87.7%) and 263 out of 296 (88.9%) assigned to SAR-Asp and NN-Asp, respectively, completed 12 months of treatment. Improved glycemic control was sustained at 12 months in both treatment groups, with similar least-squares mean reductions in glycated hemoglobin (HbA1c) from baseline (SAR-Asp: -0.25%; NN-Asp: -0.26%). Fasting plasma glucose and seven-point self-monitored plasma glucose profile changes, including postprandial glucose excursions, and changes in mealtime and basal insulin dosages were similar between groups. Safety and tolerability, including anti-insulin aspart antibodies (AIAs; incidence, prevalence, titers, cross-reactivity to human insulin), neutralizing antibodies (incidence, prevalence), hypoglycemia, and treatment-emergent adverse events (including hypersensitivity events and injection site reactions), were similar between groups. No relationship was observed between maximum individual AIA titers and change in HbA1c or insulin dose, hypoglycemia, or hypersensitivity reactions or between efficacy/safety measures and subgroups by presence or absence of treatment-emergent AIA. Conclusions: SAR-Asp and NN-Asp demonstrated similar efficacy and safety (including immunogenicity) in people with diabetes over 12 months of treatment.

Efficacy and Safety of Insulin Aspart Biosimilar SAR341402 Versus Originator Insulin Aspart in People with Diabetes Treated for 26 Weeks with Multiple Daily Injections in Combination with Insulin Glargine: A Randomized Open-Label Trial (GEMELLI 1).

Background: This study compared the efficacy, safety, and immunogenicity of insulin aspart biosimilar/follow-on biologic product SAR341402 (SAR-Asp) with originator insulin aspart-NovoLog®/NovoRapid® (NN-Asp) in people with type 1 diabetes (T1D) or type 2 diabetes (T2D) treated with multiple daily injections in combination with insulin glargine (Lantus®; Gla-100). Materials and Methods: This 6-month, randomized, open-label, phase 3 study (NCT03211858) enrolled 597 people with T1D (n = 497) or T2D (n = 100). Participants were randomized 1:1 to mealtime SAR-Asp (n = 301) or NN-Asp (n = 296) in combination with Gla-100. The primary objective was to demonstrate noninferiority (by 0.3% margin in the intent-to-treat population) of SAR-Asp versus NN-Asp in HbA1c change from baseline to week 26. Immunogenicity was also assessed in terms of anti-insulin aspart antibody (AIA) status (positive/negative) and titers during the study. Results: HbA1c was similarly improved in both treatment groups (SAR-Asp -0.38%; NN-Asp -0.30%); the least squares mean difference at week 26 for SAR-Asp minus NN-Asp was -0.08% (95% confidence interval: -0.192 to 0.039), thus meeting the criteria for noninferiority between SAR-Asp and NN-Asp and inverse noninferiority of NN-Asp versus SAR-Asp. Changes in fasting plasma glucose and seven-point self-monitored plasma glucose profile, including postprandial glucose excursions, and insulin dosages were similar in both groups at week 26. Safety and tolerability, including AIA responses (incidence, prevalence), hypoglycemia, and adverse events (including hypersensitivity events and injection site reactions), were similar between groups. Conclusions: SAR-Asp demonstrated effective glycemic control with a similar safety and immunogenicity profile to NN-Asp in people with diabetes treated for 26 weeks.

Zika virus enhances monocyte adhesion and transmigration favoring viral dissemination to neural cells.

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.

Corrigendum: Morphine Binds Creatine Kinase B and Inhibits Its Activity.

[This corrects the article DOI: 10.3389/fncel.2018.00464.].

Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo.

Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs' hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo.

Morphine Binds Creatine Kinase B and Inhibits Its Activity.

Morphine is an analgesic alkaloid used to relieve severe pain, and irreversible binding of morphine to specific unknown proteins has been previously observed. In the brain, changes in the expression of energy metabolism enzymes contribute to behavioral abnormalities during chronic morphine treatment. Creatine kinase B (CK-B) is a key enzyme involved in brain energy metabolism. CK-B also corresponds to the imidazoline-binding protein I2 which binds dopamine (a precursor of morphine biosynthesis) irreversibly. Using biochemical approaches, we show that recombinant mouse CK-B possesses a µM affinity for morphine and binds to morphine in vitro. The complex formed by CK-B and morphine is resistant to detergents, reducing agents, heat treatment and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). CK-B-derived peptides CK-B1-75 and CK-B184-258 were identified as two specific morphine binding-peptides. In vitro, morphine (1-100 µM) significantly reduces recombinant CK-B enzymatic activity. Accordingly, in vivo morphine administration (7.5 mg/kg, i.p.) to mice significantly decreased brain extract CK-B activity compared to saline-treated animals. Together, these results show that morphine strongly binds CK-B and inhibits its activity in vitro and in vivo.

Stable isotope-labelled morphine to study in vivo central and peripheral morphine glucuronidation and brain transport in tolerant mice.

BACKGROUND AND PURPOSE: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels. EXPERIMENTAL APPROACH: Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms. KEY RESULTS: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study. CONCLUSIONS AND IMPLICATIONS: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine.

The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles.

Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.

Multi-OMICS analyses unveil STAT1 as a potential modifier gene in mevalonate kinase deficiency.

OBJECTIVES: The objective of the present study was to explain why two siblings carrying both the same homozygous pathogenic mutation for the autoinflammatory disease hyper IgD syndrome, show opposite phenotypes, that is, the first being asymptomatic, the second presenting all classical characteristics of the disease. METHODS: Where single omics (mainly exome) analysis fails to identify culprit genes/mutations in human complex diseases, multiomics analyses may provide solutions, although this has been seldom used in a clinical setting. Here we combine exome, transcriptome and proteome analyses to decipher at a molecular level, the phenotypic differences between the two siblings. RESULTS: This multiomics approach led to the identification of a single gene-STAT1-which harboured a rare missense variant and showed a significant overexpression of both mRNA and protein in the symptomatic versus the asymptomatic sister. This variant was shown to be of gain of function nature, involved in an increased activation of the Janus kinase/signal transducer and activator of transcription signalling (JAK/STAT) pathway, known to play a critical role in inflammatory diseases and for which specific biotherapies presently exist. Pathway analyses based on information from differentially expressed transcripts and proteins confirmed the central role of STAT1 in the proposed regulatory network leading to an increased inflammatory phenotype in the symptomatic sibling. CONCLUSIONS: This study demonstrates the power of a multiomics approach to uncover potential clinically actionable targets for a personalised therapy. In more general terms, we provide a proteogenomics analysis pipeline that takes advantage of subject-specific genomic and transcriptomic information to improve protein identification and hence advance individualised medicine.

Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods.

The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the 50 most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass-spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.

A ruthenium anticancer compound interacts with histones and impacts differently on epigenetic and death pathways compared to cisplatin.

Ruthenium complexes are considered as potential replacements for platinum compounds in oncotherapy. Their clinical development is handicapped by a lack of consensus on their mode of action. In this study, we identify three histones (H3.1, H2A, H2B) as possible targets for an anticancer redox organoruthenium compound (RDC11). Using purified histones, we confirmed an interaction between the ruthenium complex and histones that impacted on histone complex formation. A comparative study of the ruthenium complex versus cisplatin showed differential epigenetic modifications on histone H3 that correlated with differential expression of histone deacetylase (HDAC) genes. We then characterized the impact of these epigenetic modifications on signaling pathways employing a transcriptomic approach. Clustering analyses showed gene expression signatures specific for cisplatin (42%) and for the ruthenium complex (30%). Signaling pathway analyses pointed to specificities distinguishing the ruthenium complex from cisplatin. For instance, cisplatin triggered preferentially p53 and folate biosynthesis while the ruthenium complex induced endoplasmic reticulum stress and trans-sulfuration pathways. To further understand the role of HDACs in these regulations, we used suberanilohydroxamic acid (SAHA) and showed that it synergized with cisplatin cytotoxicity while antagonizing the ruthenium complex activity. This study provides critical information for the characterization of signaling pathways differentiating both compounds, in particular, by the identification of a non-DNA direct target for an organoruthenium complex.

Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP.

The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction.

The tapetum is a single layer of secretory cells which encloses the anther locule and sustains pollen development and maturation. Upon apoptosis, the remnants of the tapetal cells, consisting mostly of lipids and proteins, fill the pits of the sculpted exine to form the bulk of the pollen coat. This extracellular matrix forms an impermeable barrier that protects the male gametophyte from water loss and UV light. It also aids pollen adhesion and hydration and retains small signaling compounds involved in pollen-stigma communication. In this study, we have updated the list of the pollen coat's protein components and also discussed their functions in the context of sexual reproduction.