Molecular Medicine

Correction to: Critical role of interleukin-23 in development of asthma promoted by cigarette smoke The original publication of this paper contains a mistake.

Pathological cardiac hypertrophy: the synergy of adenylyl cyclases inhibition in cardiac and immune cells during chronic catecholamine stress Abstract Response to stressors in our environment and daily lives is an adaptation conserved through evolution as it is beneficial in enhancing the survival and continuity of humans. Although stressors have evolved, the drastic physiological response they elicit still remains unchanged. The chronic secretion and circulation of catecholamines to produce physical responses when they are not required may result in pathological consequences which affect cardiac function drastically. This review seeks to point out the probable implication of chronic stress in inducing an inflammation disorder in the heart. We discussed the likely synergy of a G protein-independent stimuli signaling via β2-adrenergic receptors in both cardiomyocytes and immune cells during chronic catecholamine stress. To explain this synergy, we hypothesized the possibility of adenylyl cyclases having a regulatory effect on G protein-coupled receptor kinases. This was based on the negative correlations they exhibit during normal cardiac function and heart failures. As such, the downregulation of adenylyl cyclases in cardiomyocytes and immune cells during chronic catecholamine stress enhances the expressions of G protein-coupled receptor kinases. In addition, we explain the maladaptive roles played by G protein-coupled receptor kinase and extracellular signal-regulated kinase in the synergistic cascade that pathologically remodels the heart. Finally, we highlighted the therapeutic potentials of an adenylyl cyclases stimulator to attenuate pathological cardiac hypertrophy (PCH) and improve cardiac function in patients developing cardiac disorders due to chronic catecholamine stress.

Bone marrow chimeras—a vital tool in basic and translational research Abstract Bone marrow chimeras are used routinely in immunology research as well as in other fields of biology. Here, we provide a concise state-of-the-art review about the types of chimerisms that can be achieved and the type of information that each model generates. We include separate sections for caveats and future developments. We provide examples from the literature in which different types of chimerism were employed to answer specific questions. While simple bone marrow chimeras allow to dissect the role of genes in distinct cell populations such as the hematopoietic cells versus non-hematopoietic cells, mixed bone marrow chimeras can provide detailed information about hematopoietic cell types and the intrinsic and extrinsic roles of individual genes. The advantages and caveats of bone marrow chimerism for the study of microglia are addressed, as well as alternatives to irradiation that minimize blood-brain-barrier disruption. Elementary principles are introduced and their potential is exemplified through summarizing recent studies.

A rapid, safe, and quantitative in vitro assay for measurement of uracil-DNA glycosylase activity Abstract Base excision repair (BER) is a frontline repair mechanism that operates through the G1 phase of the cell cycle, which ensures the genome integrity by repairing thousands of DNA lesions due to endogenous and exogenous agents. Its correct functioning is fundamental for cell viability and the health of the organism. Uracil is one of the most prevalent lesions that appears in DNA arising by spontaneous or enzymatic deamination of cytosine or misincorporation of the deoxyuridine 5′-triphosphate nucleotide (dUTP) in place of deoxythymidine 5′-triphosphate (dTTP) during DNA replication. In the first pathway, the uracil will preferentially pair with adenine, leading to C:G → T:A transition. When uracil in DNA arises from misincorporation of dUTP instead of dTTP, this process will result in A:U pairs. Organisms counteract the mutagenic effects of uracil in DNA using the BER repair system, which is mediated by a member of the uracil-DNA glycosylase (UDG) superfamily. Several assays evaluating the in vitro BER enzyme activity have been described so far. Some of these measure the BER activity by an oligonucleotide incision assay using radiolabeled duplex oligo. Others use circular double-stranded DNA substrates containing a defined lesion. The novelty of our method resides in its rapidity and safety (radioactive free detection) as well as in the possibility of having a reliable quantitative determination of UDG activity in both cell and tissue extracts. We also demonstrated the effectiveness of our method in assessing UDG activity in cell lines with a reduced DNA repair capacity and in different kinds of tissues. Key messages • Base excision repair is a fundamental repair mechanism ensuring the genome integrity. • Uracil is one of the most prevalent lesions that appears in DNA. • The mutagenic effects of uracil in DNA are mitigated by the uracil-DNA glycosylase. • Several assays evaluating the in vitro BER activity have been described so far. • A safe and quantitative assay evaluating the in vitro UDG activity is required.

The protean world of non-coding RNAs in glioblastoma Abstract Non-coding ribonucleic acids (ncRNAs) are a diverse group of RNA molecules that are mostly not translated into proteins following transcription. We review the role of ncRNAs in the pathobiology of glioblastoma (GBM), and their potential applications for GBM therapy. Significant advances in our understanding of the protean manifestations of ncRNAs have been made, allowing us to better decipher the molecular complexity of GBM. A large number of regulatory ncRNAs appear to have a greater influence on the molecular pathology of GBM than thought previously. Importantly, also, a range of therapeutic approaches are emerging whereby ncRNA-based systems may be used to molecularly target GBM. The most successful of these is RNA interference, and some of these strategies are being evaluated in ongoing clinical trials. However, a number of limitations exist in the clinical translation of ncRNA-based therapeutic systems, such as delivery mechanisms and cytotoxicity; concerted research endeavors are currently underway in an attempt to overcome these. Ongoing and future studies will determine the potential practical role for ncRNA-based therapeutic systems in the clinical management of GBM. These applications may be especially promising, given that current treatment options are limited and prognosis remains poor for this challenging malignancy.

Mevalonate promotes differentiation of regulatory T cells Abstract Mevalonate is a precursor in a biosynthetic pathway that is important for the coordination of regulatory T cell (Treg) proliferation and upregulation of the suppressive function that establishes the functional competency of Tregs. The extensive role of mevalonate and its underlying effect on Treg differentiation are still unclear. We found that mevalonate increases in vitro differentiation of induced Tregs (iTregs) without broadly affecting Th1 and Th17 cell differentiation. Furthermore, an adoptive transfer study showed that mevalonate enhanced peripherally induced Treg cells (pTregs) in mesenteric lymphocytes in vivo. Mevalonate-treated iTregs exhibited greater suppressive activity against effector cells than untreated Tregs. Mechanistically, mevalonate enhanced transforming growth factor (TGF)-β signaling by increasing the phosphorylation of Smad3, but not Smad2, and by promoting Foxp3 expression. Furthermore, we demonstrated that mevalonate treatment ameliorated dextran sulfate sodium (DSS)-induced colitis and resulted in an increased percentage of Tregs in vivo. Our results suggest that mevalonate enhanced Treg differentiation and ameliorated DSS colitis, indicating its potential for treatment of inflammatory diseases.

RPSAP52 lncRNA is overexpressed in pituitary tumors and promotes cell proliferation by acting as miRNA sponge for HMGA proteins Abstract Long non-coding RNAs (lncRNAs) are emerging as fundamental players in cancer biology. Indeed, they are deregulated in several neoplasias and have been associated with cancer progression, tumor recurrence, and resistance to treatment, thus representing potential biomarkers for cancer diagnosis, prognosis, and therapy. In this study, we aimed to identify lncRNAs associated with pituitary tumorigenesis. We have analyzed the lncRNA expression profile of a panel of gonadotroph pituitary adenomas in comparison with normal pituitaries. Then, we focused on RPSAP52, a novel lncRNA antisense for the HMGA2 gene, whose overexpression plays a critical role in the development of pituitary adenomas. We report that RPSAP52 expression is highly upregulated in gonadotroph and prolactin-secreting pituitary adenomas, where it correlates with that of HMGA2, compared with normal pituitary tissues. Conversely, its expression showed a variable behavior in somatotroph adenomas. We also demonstrate that RPSAP52 enhances HMGA2 protein expression in a ceRNA-dependent way acting as sponge for miR-15a, miR-15b, and miR-16, which have been already described to be able to target HMGA2. Interestingly, RPSAP52 also positively modulates HMGA1, the other member of the High-Mobility Group A family. Moreover, functional studies indicate that RPSAP52 promotes cell growth by enhancing the G1-S transition of the cell cycle. The results reported here reveal a novel mechanism, based on the overexpression of the lncRNA RPSAP52, which contributes to pituitary tumorigenesis, and propose this lncRNA as a novel player in the development of these tumors. Key Messages RPSAP52 is overexpressed in pituitary adenomas. RPSAP52 increases HMGA protein levels. A ceRNA mechanism is proposed for the increased HMGA1/2 expression.

Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM Abstract Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray, and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on aspartic acid-to-valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in non-phosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-associated virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with serine-to-aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV- vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC mutations-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation. Key messages HCM-D166V mice show decreased RLC phosphorylation and decompensated function. AAV9-S15D-RLC gene therapy in HCM-D166V mice, but not in WT-RLC, results in improved heart performance. Global longitudinal strain analysis shows enhanced contractility in AAV vs controls. Increased systolic and diastolic function is paralleled by higher contractile force. Phosphomimic S15D-RLC has a therapeutic potential for HCM.

Targeted inhibition of histone deacetylase leads to suppression of Ewing sarcoma tumor growth through an unappreciated EWS-FLI1/HDAC3/HSP90 signaling axis Abstract Ewing sarcoma (ES) are aggressive pediatric bone and soft tissue tumors driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. Treatment of ES patients consists of up to 9 months of alternating courses of 2 chemotherapeutic regimens. Furthermore, EWS-ETS-targeted therapies have yet to demonstrate clinical benefit, thereby emphasizing a clinical responsibility to search for new therapeutic approaches. Our previous in silico drug screening identified entinostat as a drug hit that was predicted to reverse the ES disease signatures and EWS-FLI1-mediated gene signatures. Here, we establish preclinical proof of principle by investigating the in vitro and in vivo efficacy of entinostat in preclinical ES models, as well as characterizing the mechanisms of action and in vivo pharmacokinetics of entinostat. ES cells are preferentially sensitive to entinostat in an EWS-FLI1 or EWS-ERG-dependent manner. Entinostat induces apoptosis of ES cells through G0/G1 cell cycle arrest, intracellular reactive oxygen species (ROS) elevation, DNA damage, homologous recombination (HR) repair impairment, and caspase activation. Mechanistically, we demonstrate for the first time that HDAC3 is a transcriptional target of EWS-FLI1 and that entinostat inhibits growth of ES cells through suppressing a previously unexplored EWS-FLI1/HDAC3/HSP90 signaling axis. Importantly, entinostat significantly reduces tumor burden by 97.4% (89.5 vs. 3397.3 mm3 of vehicle, p < 0.001) and prolongs the median survival of mice (15.5 vs. 8.5 days of vehicle, p < 0.001), in two independent ES xenograft mouse models, respectively. Overall, our studies demonstrate promising activity of entinostat against ES, and support the clinical development of the entinostat-based therapies for children and young adults with metastatic/relapsed ES. Key messages • Entinostat potently inhibits ES both in vitro and in vivo. • EWS-FLI1 and EWS-ERG confer sensitivity to entinostat treatment. • Entinostat suppresses the EWS-FLI1/HDAC3/HSP90 signaling. • HDAC3 is a transcriptional target of EWS-FLI1. • HDAC3 is essential for ES cell viability and genomic stability maintenance.

Critical role of interleukin-23 in development of asthma promoted by cigarette smoke Abstract It has been recently reported that cigarette smoke exposure during allergen sensitization facilitates the development of allergic asthma; however, the underlying mechanisms remain elusive. We evaluated the role of interleukin (IL-23) in a cigarette smoke extract (CSE)-induced Dermatophagoides pteronyssinus (Dp)-allergic asthma mouse model. BALB/c mice were exposed to CSE during allergen sensitization period. Anti-IL-23p19 or IL-23R antibody was administered during the sensitization period. And we evaluated several immunological responses. The expression of IL-23 and IL-23 receptor (IL-23R) was examined in lung tissue. IL-23 and IL-23R expression was increased in the airway epithelium of Dp/CSE co-administered mice. CSE administration during the sensitization promoted Dp-allergic sensitization and the development of asthma phenotypes. Additionally, the proportion of innate lymphoid type 2 cells (ILC2) was also increased by CSE and Dp co-instillation. Anti-IL-23 or IL-23R antibody treatment during allergen sensitization significantly diminished phenotypes of allergic asthma and the ILC2 population. The levels of IL-33 and thymic stromal lymphopoietin (TSLP) were also significantly reduced by anti-IL-23 or IL-23R antibody treatment. IL-23 may thus play a significant role in cigarette smoke-induced allergic sensitization and asthma development. Clinically, the increase in allergen sensitization due to cigarette exposure causes onset of asthma, and IL-23 may be important in this mechanism. Key messages IL-23 and IL-23R expression was increased in the lung epithelium of Dp and CSE co-exposed mice during sensitization period. The population of ILC2s was increased in Dp and CSE co-exposed mice during sensitization period. Anti-IL23 or IL-23R antibody treatment with co-administration of CSE and HDM during sensitization period significantly suppresses ILC2. In vitro, IL-23 blockade in Dp and CSE-stimulated epithelial cells suppressed IL-13 expression in ILC2.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com