Uncoupling of osteoblast–osteoclast regulation in a chemical murine model of Gaucher disease

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Type I GD is the mildest form and is characterized by the absence of neuronopathic affection. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood.     

Targeting COX-2/PGE2 Pathway in HIPK2 Knockdown Cancer Cells: Impact on Dendritic Cell Maturation

Background

Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE₂). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE₂ production after HIPK2 depletion and how to modulate it.

Methodology/Principal findings

We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE₂ generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE₂/VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation.

Conclusion/Significance

These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE₂/VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation.     

Divergent modulation of neuronal differentiation by caspase-2 and -9

Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ～100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.     

Turning text into research networks: information retrieval and computational ontologies in the creation of scientific databases

Background

Web-based, free-text documents on science and technology have been increasing growing on the web. However, most of these documents are not immediately processable by computers slowing down the acquisition of useful information. Computational ontologies might represent a possible solution by enabling semantically machine readable data sets. But, the process of ontology creation, instantiation and maintenance is still based on manual methodologies and thus time and cost intensive.

Method

We focused on a large corpus containing information on researchers, research fields, and institutions. We based our strategy on traditional entity recognition, social computing and correlation. We devised a semi automatic approach for the recognition, correlation and extraction of named entities and relations from textual documents which are then used to create, instantiate, and maintain an ontology.

Results

We present a prototype demonstrating the applicability of the proposed strategy, along with a case study describing how direct and indirect relations can be extracted from academic and professional activities registered in a database of curriculum vitae in free-text format. We present evidence that this system can identify entities to assist in the process of knowledge extraction and representation to support ontology maintenance. We also demonstrate the extraction of relationships among ontology classes and their instances.

Conclusion

We have demonstrated that our system can be used for the conversion of research information in free text format into database with a semantic structure. Future studies should test this system using the growing number of free-text information available at the institutional and national levels.     

Induction of osteoclastogenesis in an in vitro model of Gaucher disease is mediated by T cells via TNF-α

Gaucher disease is a lysosomal storage disorder caused by deficiency of glucocerebrosidase enzymatic activity leading to accumulation of its substrate glucocerebrosidase mainly in macrophages. Skeletal disorder of Gaucher disease is the major cause of morbidity and is highly refractory to enzyme replacement therapy. However, pathological mechanisms of bone alterations in Gaucher disease are still poorly understood. We hypothesized that cellular alteration in Gaucher disease produces a proinflammatory milieu leading to bone destruction through enhancement of monocyte differentiation to osteoclasts and osteoclasts resorption activity. Against this background we decided to investigate in an in vitro chemical model of Gaucher disease, the capacity of secreted soluble mediators to induce osteoclastogenesis, and the mechanism responsible for this phenomena. We demonstrated that soluble factors produced by CBE-treated PBMC induced differentiation of osteoclasts precursors into mature and active osteoclasts that express chitotriosidase and secrete proinflammatory cytokines. We also showed a role of TNF-α in promoting osteoclastogenesis in Gaucher disease chemical model. To analyze the biological relevance of T cells in osteoclastogenesis of Gaucher disease, we investigated this process in T cell-depleted PBMC cultures. The findings suggest that T cells play a role in osteoclast formation in Gaucher disease. In conclusion, our data suggests that in vitro GCASE deficiency, along with concomitant glucosylceramide accumulation, generates a state of osteoclastogenesis mediated in part by pro-resorptive cytokines, especially TNF-α. Moreover, T cells are involved in osteoclastogenesis in Gaucher disease chemical model.     

Bactericidal Activity and Synergy Studies of Peptide AP-CECT7121 Against Multi-resistant Bacteria Isolated from Human and Animal Soft Tissue Infections

AP-CECT7121 is an antimicrobial peptide, produced by Enterococcus faecalis CECT7121, with bactericidal activity against Gram-positive bacteria. The aim of this study was to evaluate the bactericidal activity of AP-CECT7121, alone and with gentamicin, against multi-resistant bacteria isolated from human and animals with soft tissue infections. During the period 2014–2015, bacterial strains producing human and animal soft tissue infections were studied. Samples from patients attended at a general hospital and cattle from four dairies in the Province of Buenos Aires (Argentina) were included. Twenty-two methicillin-resistant Staphylococcus aureus (11, human blood samples; 11, cow milk) and five vancomycin-resistant Ent. faecium strains isolated from four mastitic dairy cows were tested. AP-CECT7121 (12 mg/L) potency was assessed by time-kill curves alone or with sub-inhibitory concentrations of gentamicin. All staphylococcal strains were susceptible to gentamicin; enterococci did not show high-level gentamicin resistance. Colony counts were carried out at 0, 2, 4, 8, and 24 h of incubation. AP-CECT7121 showed bactericidal activity against all the enterococcal strains. In addition, AP-CECT7121 had a bactericidal effect on most staphylococci (16/22). Early AP-CECT7121/gentamicin synergy (4–8 h) for all staphylococci was detected. At 24 h, synergy (19/22) and indifference (3/22) were observed. Synergy with gentamicin was detected for staphylococci. AP-CECT7121 constitutes an attractive candidate for its use as a natural therapeutic tool for the treatment of infections produced by multi-resistant Staph. aureus and vancomycin-resistant Ent. faecium isolated from humans and animals.     

The mutation K30D disrupts the only salt bridge at the subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis and changes the mechanism of cooperativity.

The subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis, HbI, is stabilized by a network of interactions that involve several hydrogen-bonded structural water molecules, a hydrophobic patch, and a single, symmetrical salt bridge between residues Lys-30 and Asp-89. Upon mutation of Lys-30 to Asp, the interface is destabilized markedly. Sedimentation equilibrium and velocity experiments allowed the estimate of the dimerization constants for the unliganded (K(1,2D) = 8 x 10(4) M(-1)) and for the CO-bound (K(1,2L) = 1 x 10(3) m(-1)) and oxygenated (K(1,2L) = 70 m(-1)) derivatives. For the oxygenated derivative, the destabilization of the subunit interface with respect to native HbI corresponds to about 8 kcal/mol, an unexpectedly high figure. In the K30D mutant, at variance with the native protein, oxygen affinity and cooperativity are strongly dependent on protein concentration. At low protein concentrations (e.g. 1.2 x 10(-5) m heme), at which the monomeric species becomes significant also in the unliganded derivative, oxygen affinity increases and cooperativity decreases. At protein concentrations where both derivatives are dimeric (e.g. 3.3 x 10(-3) m heme), both cooperativity and oxygen affinity decrease. Taken together, the experimental data indicate that in the K30D mutant, the mechanism of cooperativity is drastically altered and is driven by a ligand-linked monomer-dimer equilibrium rather than being based on a direct heme-heme communication as in native HbI.     

Differences Between d-Fenfluramine and d-Norfenfluramine in Serotonin Presynaptic Mechanisms

Abstract: The abilities of d -fenfluramine ( d -F) and that of d -norfenfluramine ( d -NF) to inhibit [³H]serotonin ([³H]5-HT) accumulation in normal and reserpinized synaptosomes were compared to establish to what extent the serotonin-releasing activity of the two drugs might contribute to reduced accumulation of [₃H]5-HT. The results indicate that the inhibitory action of ( d -NF) on [³H]5-HT accumulation is due principally to its ability to release [³H]5-HT. In contrast, the interference of release in accumulation studies does not seem to play an important role for d -F, suggesting that release from the granular pool and true uptake inhibition are two different mechanisms by which d -F affects serotonin neurons in vitro .