Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Introduction

Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods

Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results

We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion

We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.     

Delipidated retroviruses as potential autologous therapeutic vaccines--a pilot experiment

This pilot experiment in a simian immunodeficiency virus (SIV) chronic infection model aimed at extending our previous findings that vaccination with delipidated SIV resulted in more potent and diversified antiviral responses (1). Macaques chronically infected with SIVmac239 treated with antiretroviral therapy (ART) were vaccinated with autologous delipidated virus via consecutive lymph node targeted immunizations-1, 1 and 10 mug of virus spaced monthly. Results showed all animals had lasting viral load reduction approaching 1 log compared to set-point, and disease delay. Delipidation may enhance processing/ presentation of viral antigen eliciting potent antiviral control even at such late infection stage.     

Effects of a Longitudinal Training Program on Responses to Exercise in Overweight Men

Objective: The aim of this study was to determine how training modifies metabolic responses and lipid oxidation in overweight young male subjects. Research Methods and Procedures: Eleven overweight subjects were selected for a 4‐month endurance training program. Before and after the training period, they cycled for 60 minutes at 50% of their Vo ₂max after an overnight fast or 3 hours after eating a standardized meal. Various metabolic and endocrine parameters, and respiratory exchange ratio values were evaluated. Results: Exercise‐induced plasma norepinephrine concentration increases were similar before and after training in fasted or fed conditions. After food intake, exercise promoted a decrease in plasma glucose and a higher increase in epinephrine than in fasting conditions. The increase in epinephrine after the meal was more marked after training (264 ± 32 vs. 195 ± 35 pg/mL). Training lowered the resting plasma nonesterified fatty acids. During exercise, changes in glycerol were similar to those found before training. Lipid oxidation during exercise was higher in fasting than in fed conditions (15.5 ± 1.4 vs. 22.3 ± 1.7 g/h). Training did not significantly increase fat oxidation when exercise was performed in fed conditions, but it did in fasting conditions (18.6 ± 1.4 vs. 27.2 ± 1.8 g/h). Discussion: Endurance training decreased plasma nonesterified fatty acids, cholesterol, and insulin concentrations. Training increased lipid oxidation during exercise, in fasting conditions, and not when exercise was performed after the meal. During exercise in overweight subjects, the fasting condition seems more suited to oxidizing fat and maintaining glucose homeostasis than a 3‐hour wait after a standard meal.     

Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as β-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2–4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases. G. Podrygajlo is a Marie Curie Actions Fellow and M.A. Tegenge received a Georg Christoph Lichtenberg Fellowship from the Federal State of Lower Saxony. M. Stern and G. Bicker were supported by the Federal Ministry for Education and Research (BMBF). F. Paquet-Durand was supported by grants from the Kerstan Foundation, Tübingen, Germany and the German Research Council (DFG; PA 1751/1-1).     

Compatibility and thigmotropism in the lichen symbiosis: A reappraisal

The development of many complex stratified lichen thalli is made through stages of complex phenotypic interactions between a filamentous fungus (the mycobiont), and a trebouxioid alga (the photobiont). Typically, the second stage of this symbiotic development is marked by the envelopment of the photobiont by the mycobiont through increased lateral hyphal branching and the formation of appressoria. Previously, the mycobiont’s envelopment of photobiont cells was considered thigmotropic (a growth response due to shape) as a mycobiont can envelop algal sized objects in its environment. However, after growing the mycobiontCladonia grayi with various phototrophs and glass beads, we conclude that the mycobiont does not show this characteristic second stage morphological response when grown in non-compatible pairings. Instead,C. grayi displays a distinctive morphological growth response only in compatible symbiotic pairings, such as with its natural photobiontAsterochlor’is sp.     

Parasites, condition, immune responsiveness and carotenoid-based ornamentation in male red-legged partridge Alectoris rufa

Sexual ornaments might indicate better condition, fewer parasites or a greater immune responsiveness. Carotenoid-based ornaments are common sexual signals of birds and often influence mate choice. Skin or beaks pigmented by carotenoids can change colour rapidly, and could be particularly useful as honest indicators of an individual's current condition and/or health. This is because carotenoids must be acquired through diet and/or allocation for ornamental coloration might be to the detriment of self-maintenance needs. Here, we investigated whether the carotenoid-based coloration of eye rings and beak of male red-legged partridges Alectoris rufa predicted condition (mass corrected for size), parasite load (more specifically infection by coccidia, a main avian intestinal parasite) or a greater immune responsiveness (swelling response to a plant lectin, phytohaemagluttinin, or PHA). Redness of beak and eye rings positively correlated with plasma carotenoid levels. Also, males in better condition had fewer coccidia, more circulating carotenoids and a greater swelling response to PHA. Carotenoid-based ornamentation predicted coccidia abundance and immune responsiveness (redder males had fewer coccidia and greater swelling response to PHA), but was only weakly positively related to condition. Thus, the carotenoid pigmentation of beak and eye rings reflected the current health status of individuals. Our results are consistent with the hypothesis that allocation trade-offs (carotenoid use for ornamentation versus parasite defence needs) might ensure reliable carotenoid-based signalling.     

Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation

Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.Hepatitis C virus (HCV) is a highly variable enveloped virus. It is the sole member of the genus Hepacivirus within the family Flaviviridae (36). Based on sequence homology, patient isolates are classified into seven genotypes and more than 100 subtypes (17, 52).The genome of HCV is a single-stranded RNA molecule of positive polarity with a size of ∼9.6 kb. It encodes a polyprotein of ca. 3,000 amino acids and contains nontranslated regions (NTRs) at both the 5′ and 3′ termini that are required for translation and RNA replication (33). Cellular and two viral proteases, NS2-3 and NS3-4A, liberate the individual viral proteins. The N-terminal portion of the polyprotein contains the structural proteins core and envelope glycoproteins 1 and 2 (E1, E2), which constitute the virus particle. These proteins are cleaved from the polyprotein by the host cell signal peptidase (18, 24). In the case of the core protein, an additional cleavage step mediated by the signal peptide peptidase liberates its mature C terminus (41). Further downstream of the structural proteins the polyprotein harbors p7, a short membrane-associated polypeptide required for virus assembly and release (27, 55), and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Proteins NS3 to NS5B are the minimal components of the membrane-bound replication complexes that catalyze RNA replication (16, 38).Using the novel JFH1-based HCV infection model (35, 61, 65), it has been demonstrated recently that besides the canonical structural proteins core, E1, and E2, NS5A, p7, NS3, and NS2 also are crucial for the production of infectious HCV particles (1, 26, 27, 39, 40, 55, 57). These data highlight that HCV assembly and release is a coordinated process involving both structural and nonstructural proteins. However, how the aforementioned proteins contribute to the production of infectious virus particles remains poorly understood.HCV p7 comprises two helical domains connected by a polar loop. Studies with epitope-tagged p7 variants indicate that both termini of the protein are resident in the lumen of the endoplasmic reticulum (ER) (4) or that, in addition, a second alternative topology with the C terminus exposed to the cytoplasm can be adopted (25). Using such constructs for fluorescent microscopy, a complex localization of p7 was revealed. While most prominent staining generally was observed at the ER (4, 19, 23), pools of p7 also were detected at mitochondria (19) and at the plasma membrane (4). These data suggest that p7 influences virus replication at various sites within infected cells, and that the function and/or localization of p7 is regulated by different trafficking signals that could be exposed in a topology-dependent manner. However, caution is warranted since, due to the lack of antibodies, epitope-tagged p7 variants had to be employed for most analyses, and since localization studies of virus-producing cells with functional p7 still are lacking.One hallmark of p7 is its ability to form cation-selective channels in artificial membranes (20, 46, 49), a property that likely depends on the oligomerization of the protein (7, 21). There are intriguing correlations that link p7''s function as an ion channel protein in vitro to its role in the assembly and release of infectious HCV particles in tissue culture. First, the mutation of the conserved dibasic motif in the polar loop of p7 abrogates ion channel activity and interferes with virus production in tissue culture (20, 27, 55). Second, iminosugars coupled to long alkyl chains like N-nonyl deoxygalactonojirimycin (NN-DGJ) not only interfere with ion channel activity but also repress the release of infectious particles from transfected Huh-7 cells (46, 56). Taken together, these data suggest that the ion channel activity of p7 is crucial for its role in the late steps of the HCV replication cycle, and that this function is amenable to the development of selective inhibitors for antiviral therapy. However, presently it is unknown how mechanistically p7, as an ion channel protein, facilitates HCV assembly and release or if p7 also is a component of virus particles and participates in entry.Besides its function as an ion channel, p7 harbors a signal-like sequence in its C-terminal domain that directs the insertion of the N terminus of NS2 into the lumen of the ER (4). Strikingly, due to structural determinants within the C terminus of E2, p7, and the N terminus of NS2, signalase cleavages at the E2/p7 and the p7/NS2 sites are incomplete, thus yielding E2-p7-NS2 and E2-p7 precursor proteins (3, 18, 34, 42). Although these precursors are not absolutely essential for the production of infectious HCV particles (26, 27), a defined ratio between mature and precursor proteins might play a role to orchestrate optimal virus assembly. Given these circumstances, genetic studies of p7 function are complicated, since mutations may, on the one hand, affect ion channel activity, and on the other hand influence processing at the E2-p7 and p7-NS2 junctions.To circumvent this problem, in this study we developed a complementation system that permits the rescue of genomes with defects in p7 by the ectopic expression of p7 in trans. This enabled us to directly assess the function of p7 in the absence of secondary effects caused by aberrant polyprotein cleavage. Using this approach, we analyzed the role of the native signal sequence of p7 and p7-containing precursor proteins. In addition, we investigated key determinants that are essential for the optimal function of p7 in the course of HCV infectious particle production.