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

The PU.1 transcription factor induces cyclin D2 expression in U937 cells [16]

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Klebsiella pneumoniae Lipopolysaccharides Serotype O2afg Induce Poor Inflammatory Immune Responses Ex Vivo

Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium

Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients

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 developmen- tal 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 differ- entiation. 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

BRIEF REPORT Proteasome Inhibitors Synergize with Tumor Necrosis Factor-Related Apoptosis-Induced Ligand to Induce Anaplastic Thyroid Carcinoma Cell Death

Context: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC. Objective and Methods: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways. Results: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1␤ converting enzyme inhibitory protein, Bcl-2, Bcl-X L , and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway. Conclusions: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma. (J Clin Endocrinol Metab 92: 1938 -1942, 2007

Hedgehog Signaling Regulates the Survival of Gastric Cancer Cells by Regulating the Expression of Bcl-2

Gastric cancer is the second most common cause of cancer deaths worldwide. The underlying molecular mechanisms of its carcinogenesis are relatively poorly characterized. Hedgehog (Hh) signaling, which is critical for development of various organs including the gastrointestinal tract, has been associated with gastric cancer. The present study was undertaken to reveal the underlying mechanism by which Hh signaling controls gastric cancer cell proliferation. Treatment of gastric cancer cells with cyclopamine, a specific inhibitor of Hh signaling pathway, reduced proliferation and induced apoptosis of gastric cancer cells. Cyclopamine treatment induced cytochrome c release from mitochondria and cleavage of caspase 9. Moreover, Bcl-2 expression was significantly reduced by cyclopamine treatment. These results suggest that Hh signaling regulates the survival of gastric cancer cells by regulating the expression of Bcl-2

BRIEF REPORT Proteasome Inhibitors Synergize with Tumor Necrosis Factor-Related Apoptosis-Induced Ligand to Induce Anaplastic Thyroid Carcinoma Cell Death

Context: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC. Objective and Methods: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways. Results: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1␤ converting enzyme inhibitory protein, Bcl-2, Bcl-X L , and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway. Conclusions: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma. (J Clin Endocrinol Metab 92: 1938 -1942, 2007

Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits.

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH(2)-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922-20929). Here we used deletion and site- directed mutagenesis to determine that alpha residues 21-80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55-57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated alpha subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871-881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits

Prospective identification of parasitic sequences in phage display screens

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 10[superscript 9] random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ~10[superscript 6] diverse clones prevents the biased selection of parasitic clones