miRNA expression analysis in the human heart: Undifferentiated progenitors vs. bioptic tissues-Implications for proliferation and ageing.

In developed countries, cardiovascular diseases are currently the first cause of death. Cardiospheres (CSs) and cardiosphere-derived cells (CDCs) have been found to have the ability to regenerate the myocardium after myocardial infarction (MI). In recent years, much effort has been made to gain insight into the human heart repair mechanisms, in which miRNAs have been shown to play an important role. In this regard, to elucidate the involvement of miRNAs, we evaluated the miRNA expression profile across human heart biopsy, CSs and CDCs using microarray and next-generation sequencing (NGS) technologies. We identified several miRNAs more represented in the progenitors, where some of them can be responsible for the proliferation or the maintenance of an undifferentiated state, while others have been found to be downregulated in the undifferentiated progenitors compared with the biopsies. Moreover, we also found a correlation between downregulated miRNAs in CSs/CDCs and patient age (eg miR-490) and an inverse correlation among miRNAs upregulated in CSs/CDCs (eg miR-31).

Two-Step Coimmunoprecipitation (TIP) Enables Efficient and Highly Selective Isolation of Native Protein Complexes.

Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.

Two-Step Co-Immunoprecipitation (TIP).

In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4+ T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley & Sons, Inc.

Zika virus infection induces MiR34c expression in glioblastoma stem cells: new perspectives for brain tumor treatments.

Zika virus (ZIKV) is a flavivirus with a marked effect on fetal nervous system development. ZIKV treatment has recently been found to also have a benefit against glioblastoma, a highly aggressive brain tumor with a poor prognosis. The reported data do not completely explain the mechanism beyond this effect. Nevertheless, in the majority of the cases no adverse effect has been found in healthy adult humans. In this study, we characterized the ZIKV infection mechanism on glioblastoma stem cells, which are considered responsible for the tumor progression and resistance to conventional therapies. Moreover, we explain why the action of this virus is directed to the stem cells in the nervous system counterpart. Our results confirm the effectiveness of ZIKV treatment against glioblastoma, indicating novel molecular targets that can be introduced for more powerful therapies.

MiR34 inhibition induces human heart progenitor proliferation.

MiR34 involvement in myocardial injury repair and ageing has been well documented in mouse model. Our aim was to establish whether the inhibition of miR34 expression through locked nucleic acid (LNA) could be used as a pharmacological intervention to enhance human heart repair. Cardiac progenitor cells were obtained by right atrial specimen collection during intraoperative procedures. Our analysis revealed a direct correlation between miR34 expression and patient age, and its silencing by LNA promoted the cardiac progenitor growth rate up to twofold ( ± 0.8). Our results confirmed the relevance of miR34a in human heart ageing, as previously demonstrated in mouse. Moreover, the decrease of miR34 expression in the cardiac progenitor cell population indicates its role in maintaining an undifferentiated status and consequently in a lower proliferation rate with the involvement of genes such as Notch-1, Numb, and p63.

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.

Numb Expression Contributes to the Maintenance of an Undifferentiated State in Human Epidermis.

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmental signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differentiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer.

Chk1/2 inhibition overcomes the cisplatin resistance of head and neck cancer cells secondary to the loss of functional p53.

Despite the use of multimodality therapy using cisplatin to treat patients with advanced stage squamous cell carcinoma of the head and neck (HNSCC), there is an unacceptably high rate of treatment failure. TP53 is the most commonly mutated gene in HNSCC, and the impact of p53 mutation on response to cisplatin treatment is poorly understood. Here, we show unambiguously that wild-type TP53 (wtp53) is associated with sensitivity of HNSCC cells to cisplatin treatment, whereas mutation or loss of TP53 is associated with cisplatin resistance. We also show that senescence is the major cellular response to cisplatin in wtp53 HNSCC cells and that cisplatin resistance in p53-null or -mutant TP53 cells is due to their lack of senescence. Given the dependence on checkpoint kinase (Chk)1/2 kinases to mediate the DNA damage response in p53-deficient cells, there is potential to exploit this to therapeutic advantage through targeted inhibition of the Chk1/2 kinases. Treatment of p53-deficient HNSCC cells with the Chk inhibitor AZD7762 sensitizes them to cisplatin through induction of mitotic cell death. This is the first report showing the ability of a Chk kinase inhibitor to sensitize TP53-deficient HNSCC to cisplatin in a synthetic lethal manner, which has significance given the frequency of TP53 mutations in this disease and because cisplatin has become part of standard therapy for aggressive HNSCC tumors. These preclinical data provide evidence that a personalized approach to the treatment of HNSCC based on Chk inhibition in p53-mutant tumors may be feasible.

CD200 expression in patients with Multiple Myeloma: another piece of the puzzle.

CD200 is a relatively ubiquitously expressed molecule that plays a role in cancer immune evasion through interaction with its receptors. High expression levels of CD200 have been described in different human malignancies. For example, CD200 has been shown to be targeted after RAS/RAF/MEK/ERK activation in melanoma. Here we present the analysis of CD200 expression in human Multiple Myeloma (MM) samples. We found that CD200-positive cells express ERK and p-ERK. Moreover, UO126, a MEK inhibitor, reduces CD200 expression. Furthermore, we observe that CD200-positive cells show reduced immunogenicity compared to normal lymphocytes and that such immunogenicity increases when UO126 is used. We therefore hypothesize that CD200 expression in MM could suppress antitumor response and that anti-CD200 treatment might be therapeutically beneficial in CD200-expressing tumors.

MARCH-I expression in cord blood CD34+KDR+ cells.

Hematopoietic stem cells transplantation has been successfully used in the treatment of patients with hematological malignances. A better knowledge of the mechanisms beyond their ability to completely repopulate the entire hematopoietic system would help in the treatment of hematological diseases. For this reason we focused our studies on a cell population that has been demonstrated to have some peculiar characteristics among the stem cells: CD34+KDR+ cells. These cells, an extremely rare population among the CD34 (0.1%-0.5%) cells, have been demonstrated from different groups to have the potential to give rise to the hematopoietic and endothelial lineage. By a subtraction library approach we found different sequences more expressed in CD34+KDR+ than their CD34+KDR- counterpart. In particular, we found an open reading frame correspondent to a newly characterized E3 ligase, MARCH-I. This gene is part of a recently described family involved in immune response modulation through the proteosomal mediated degradation. MARCH-I expression in stem cells could be important for their intrinsic immune properties.