ETS1 loss in mice impairs cardiac outflow tract septation via a cell migration defect autonomous to the neural crest.

Ets1 deletion in some mouse strains causes septal defects and has been implicated in human congenital heart defects in Jacobsen syndrome, in which one copy of the Ets1 gene is missing. Here, we demonstrate that loss of Ets1 in mice results in a decrease in neural crest (NC) cells migrating into the proximal outflow tract cushions during early heart development, with subsequent malalignment of the cushions relative to the muscular ventricular septum, resembling double outlet right ventricle (DORV) defects in humans. Consistent with this, we find that cultured cardiac NC cells from Ets1 mutant mice or derived from iPS cells from Jacobsen patients exhibit decreased migration speed and impaired cell-to-cell interactions. Together, our studies demonstrate a critical role for ETS1 for cell migration in cardiac NC cells that are required for proper formation of the proximal outflow tracts. These data provide further insights into the molecular and cellular basis for development of the outflow tracts, and how perturbation of NC cells can lead to DORV.

The Rise of Induced Pluripotent Stem Cell Approach to Hair Restoration.

SUMMARY: The advent of pluripotent stem cells following the discovery of Shinya Yamanaka (2012 Nobel prize in Medicine) brought about a regenerative medicine approach to virtually every human condition including hair loss. It is now possible to reprogram somatic cells (eg, blood or skin cells) from a person experiencing hair loss to generate autologous induced pluripotent stem cells (iPSCs), which could be amplified and cryopreserved. Subsequently, these iPSCs could be differentiated into various cell types such as dermal papilla cells, epithelial cells, melanocytes, and other cell types constituting functional hair follicle. Transplantation of human iPSC-derived folliculogenic cells into the nude mice has successfully generated xenografts with hair outgrowth. Because iPSCs provide a virtually unlimited source of folliculogenic cells for de novo formation of hair follicles, this approach has major advantages over current surgical hair restoration procedures, which merely redistribute existing hair follicles from one part of the sculp to another. Combined with robotics and automation of the transplantation process, this novel regenerative medicine approach is well poised to make hair restoration a routine procedure affordable for everybody who can benefit from it.

Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

The role of HSP27 in RACK1-mediated PKC activation in THP-1 cells.

Receptor for Activated C Kinase 1 (RACK1) pseudosubstrate is a commercially available peptide that directly activates protein kinase C-ß (PKCß). We have recently shown that RACK1 pseudosubstrate, alone or in combination with classical immune activators, results in increased cytokine production and CD86 upregulation in primary leukocytes. Furthermore, we demonstrated a role of PKCß and RACK1 in chemical allergen-induced CD86 expression and IL-8 production in both THP-1 cells and primary human dendritic cells. Aim of this study was to shed light on the mechanisms underlying RACK1 pseudosubstrate-induced immune activation and to compare it to lipopolysaccharide (LPS). The human promyelocytic cell line THP-1 was used throughout the study. RACK1 pseudosubstrate induced rapid (5 min) and dose-related PKCß activation as assessed by its membrane translocation. Among the proteins phosphorylated, we identified Hsp27. Both RACK1 pseudosubstrate and LPS induce its phosphorylation and release in culture medium. The release of Hsp27 induced by RACK1 pseudosubstrate was also confirmed in peripheral blood mononuclear cells. To evaluate the role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, we conducted Hsp27 silencing and neutralization experiments. Both strategies confirmed the central role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, as assessed by IL-8 production and upregulation of CD86.

Nature-Inspired Multifunctional Ligands: Focusing on Amyloid-Based Molecular Mechanisms of Alzheimer's Disease.

The amyloidogenic pathway is a prominent feature of Alzheimer's disease (AD). However, growing evidence suggests that a linear disease model based on ß-amyloid peptide (Aß) alone is not likely to be realistic, which therefore calls for further investigations on the other actors involved in the play. The pro-oxidant environment induced by Aß in AD pathology is well established, and a correlation among Aß, oxidative stress, and conformational changes in p53 has been suggested. In this study, we applied a multifunctional approach to identify allyl thioesters of variously substituted trans-cinnamic acids for which the pharmacological profile was strategically tuned by hydroxy substituents on the aromatic moiety. Indeed, only catechol derivative 3 [(S)-allyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enethioate] inhibited Aß fibrilization. Conversely, albeit to different extents, all compounds were able to decrease the formation of reactive oxygen species in SH-SY5Y neuroblastoma cells and to prevent alterations in the conformation of p53 and its activity mediated by soluble sub-lethal concentrations of Aß. This may support an involvement of oxidative stress in Aß function, with p53 emerging as a potential mediator of their functional interplay.

Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase ß, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors.

Immunostimulatory effects of RACK1 pseudosubstrate in human leukocytes obtained from young and old donors.

Aims of this study were to investigate the ability of RACK1 pseudosubstrate alone or in combination with classical immune stimuli to activate human leukocytes, and to restore age-associated immune defects.A total of 25 donors (17 old donors, 77-79 yrs; 8 young donors, 25-34 yrs) were enrolled. To evaluate the effect of RACK1 pseudosubstrate on cytokine production and CD86 expression the whole blood assay was used. Cultures were treated with RACK1 pseudosubstrate in the presence or absence of lipopolysaccharide (LPS) or phytohaemagglutinin (PHA) and incubated for 24 h or 48 h for LPS-induced CD86 expression, TNF-α, IL-6, IL-8, IL-10 production, and PHA-induced IL-4, IL-10, IFN-γ, respectively. RACK1 pseudosubstrate alone induced IL-6, IL-8, and CD86 expression in both young and old donors, and IFN-γ in old donors. In combination with LPS an increase in IL-8, IL-10 and TNF-α was observed, also resulting in restoration of age-associated defective production, while no changes in the other parameters investigated were found.Even if based on a small sample size, these results suggest the possibility to by-pass some of age-associated immune alterations, which may be beneficial in situations were natural immune stimulation is required, and highlight a different role of PKCß in immune cells activation.

DHEA modulates the effect of cortisol on RACK1 expression via interference with the splicing of the glucocorticoid receptor.

BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) is thought to be an anti-glucocorticoid hormone known to be fully functional in young people but deficient in aged humans. Our previous data suggest that DHEA not only counteracts the effect of cortisol on RACK1 expression, a protein required both for the correct functioning of immune cells and for PKC-dependent pathway activation, but also modulates the inhibitory effect of cortisol on LPS-induced cytokine production. The purpose of this study was to investigate the effect of DHEA on the splicing mechanism of the human glucocorticoid receptor (GR). EXPERIMENTAL APPROACH: The THP1 monocytic cell line was used as a cellular model. Cytokine production was measured by specific elisa. Western blot and real-time RT-PCR were used, where appropriate, to determine the effect of DHEA on GRs, serine/arginine-rich proteins (SRp), and RACK1 protein and mRNA. Small-interfering RNA was used to down-regulate GRß. KEY RESULTS: DHEA induced a dose-related up-regulation of GRß and GRß knockdown completely prevented DHEA-induced RACK1 expression and modulation of cytokine release. Moreover, we showed that DHEA influenced the expression of some components of the SRps found within the spliceosome, the main regulators of the alternative splicing of the GR gene. CONCLUSIONS AND IMPLICATIONS: These data contribute to our understanding of the mechanism of action of DHEA and its effect on the immune system and as an anti-glucocorticoid agent.

Corticosteroids modulate the expression of the PKC-anchoring protein RACK-1 and cytokine release in THP-1 cells.

We demonstrated that cortisol reduces the expression of RACK-1 (Receptor for Activated C Kinase-1), a protein required for immune cell activation. The aim of this study was to evaluate whether and to what extent other clinically relevant corticosteroids may modulate RACK-1 expression. We used the human promyelocytic cell line THP-1 to investigate the effects of cortisol, prednisone, prednisolone, budesonide, betamethasone and methylprednisolone on RACK-1 expression and cytokine production. As anticipated, all corticosteroids inhibited at non-cytotoxic concentrations in a dose and time related manner LPS-induced TNF-α and IL-8 release, with budesonide, betamethasone and methylprednisolone being the most active followed by prednisolone, cortisol and prednisone. To a similar extent, all corticosteroids also reduced RACK-1 mRNA expression and RACK-1 protein levels as assessed by Real Time PCR and Western blot, respectively. Prednisone was the least potent compound while betamethasone and methylprednisolone where the most active. A good correlation was observed between RACK-1 mRNA or protein levels and cytokine release (Pearson r=0.7376, p=0.0471 for RACK-1 mRNA and TNF-α release, and Pearson r=0.8108, p=0.0252 for RACK-1 protein and IL-8 release). Mifepristone, a potent glucocorticoid receptor (GR) antagonist, completely prevented the effect of cortisol, demonstrating that RACK-1 downregulation is via GR. Furthermore, to by-pass the defective PKC activation due to the decrease in RACK-1, we used a RACK-1 pseudosubstrate, that directly activates PKC-beta. RACK-1 pseudosubstrate was able to restore LPS-induced cytokine production affected by cortisol, supporting the role of RACK-1 in the anti-inflammatory effect of corticosteroids. These results confirm the involvement of RACK-1 in immune cell activation and identify this protein as a novel transcriptional target of corticosteroid-induced anti-inflammatory effects.

Role of PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in THP-1 cells.

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells' responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-ß. The purpose of this study was to investigate the role of RACK-1 and PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-ß and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-ε (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-ß, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-ß and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-ß in the initiation of chemical allergen-induced DC activation.

Zyxin is a novel target for ß-amyloid peptide: characterization of its role in Alzheimer's pathogenesis.

Zyxin is an adaptor protein recently identified as a novel regulator of the homeodomain-interacting protein kinase 2 (HIPK2)-p53 signaling in response to DNA damage. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer 's disease (AD), because of a deregulation of HIPK2 activity, leading to an impaired and dysfunctional response to stressors. Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 cells and SH-SY5Y neuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Aß) 1-40 and 1-42 induce zyxin deregulation, thus affecting the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A, which is in turn responsible for the induction of an altered conformational state of p53. We demonstrate for the first time that zyxin is a novel target of Aß activities in AD. These results may help the studies on the pathogenesis of AD, through the fine dissection of events related to beta-amyloid activities.

Rapid identification of Bartonella henselae by real-time polymerase chain reaction in a patient with cat scratch disease.

We report a localized submandibular lymph node infection in a patient with cat scratch disease. Directly performing real-time polymerase chain reaction assay on the biopsy sample, Bartonella henselae DNA was simultaneously detected and identified.