Novel half Salphen cobalt(III) complexes: synthesis, DNA binding and anticancer studies.

While platinum(II)-based drugs continue to be employed in cancer treatments, the escalating occurrence of severe side effects has spurred researchers to explore novel sources for potential therapeutic agents. Notably, cobalt(III) has emerged as a subject of considerable interest due to its ubiquitous role in human physiology. Several studies investigating the anticancer effects of Salphen complexes derived from cobalt(III) have unveiled intriguing antiproliferative properties. In a bid to enhance our understanding of this class of compounds, we synthesized and characterized two novel half Salphen cobalt(III) complexes. Both compounds exhibited notable stability, even in the presence of physiologically relevant concentrations of glutathione. The application of spectroscopic and computational methodologies unravelled their interactions with duplex and G4-DNAs, suggesting an external binding affinity for these structures, with preliminary indications of selectivity trends. Importantly, antiproliferative assays conducted on 3D cultured SW-1353 cancer cells unveiled a compelling anticancer activity at low micromolar concentrations, underscoring the potential therapeutic efficacy of this novel class of cobalt(III) complexes.

Modulating the release of bioactive molecules of human mesenchymal stromal cell secretome: Heparinization of hyaluronic acid-based hydrogels.

An amine derivative of hyaluronic acid (HA) was crosslinked to obtain a 3D dried sponge. The sponge was subsequently rehydrated using secretome from human mesenchymal stromal cells (MSCs), resulting in the formation of a hydrogel. The release kinetics analysis demonstrated that the hydrogel effectively sustained secretome release, with 70% of the initially loaded wound-healing-associated cytokines being released over a 12-day period. Tuning the hydrogel properties through heparin crosslinking resulted in a biomaterial with a distinct mechanism of action. Specifically, the presence of heparin enhanced water uptake capacity of the hydrogel and increased its sensitivity to enzymatic degradation. Notably, the heparin crosslinking also led to a significant retention of cytokines within the hydrogel matrix. Overall, the secretome-rehydrated HA hydrogel holds promise as a versatile device for regenerative medicine applications: the non-heparinized hydrogel may function as a biomaterial with low reabsorption rates, sustaining the release of bioactive molecules contained in MSC secretome. In contrast, the heparinized hydrogel may serve as a depot of bioactive molecules with faster reabsorption rates. Given its patch-like characteristic, the HA-based hydrogel appears suitable as topical treatment for external organs, such as the skin.

An In Vitro Model of Glioma Development.

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

Galectin-9 and Interferon-Gamma Are Released by Natural Killer Cells upon Activation with Interferon-Alpha and Orchestrate the Suppression of Hepatitis C Virus Infection.

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.

High-Resolution Secretome Analysis of Chemical Hypoxia Treated Cells Identifies Putative Biomarkers of Chondrosarcoma.

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.

Quantitative Proteomics Reveals That ADAM15 Can Have Proteolytic-Independent Functions in the Steady State.

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.

A retrospective molecular epidemiological scenario of carbapenemase-producing Klebsiella pneumoniae clinical isolates in a Sicilian transplantation hospital shows a swift polyclonal divergence among sequence types, resistome and virulome.

In this work, we assessed and characterized the epidemiological scenario of carbapenem-resistant Klebsiella pneumoniae strains (CR-Kp) at IRCCS-ISMETT, a transplantation hospital in Palermo, Italy, from 2008 to 2017. A total of 288 K. pneumoniae clinical isolates were selected based on their resistance to carbapenems. Molecular characterization was also done in terms of the presence of virulence and resistance genes. All patients were inpatients from our facility and clinical isolates were collected from several sources, either from infection or colonization cases. We observed that, in agreement with the Italian epidemiological scenario, initially only ST258 and ST512 clade II (but not from clade I) were identified from 2008 to 2011. From 2012 onwards, other STs have been observed, including the clinically relevant ST101 and ST307, but also others not previously observed in other Italian health settings, such as ST220 and ST753. The presence of genes involved in resistance and virulence was confirmed, and a heterogeneous genetic resistance profile throughout the years was observed. Our work highlights that resistance genes are rapidly disseminating between different and novel K. pneumoniae clones which, combined with resistance to multiple antibiotics, can derive into more aggressive and pathogenic multidrug-resistant strains of clinical importance. Our results stress the importance of continuous surveillance of CR Enterobacterales in health facilities so that novel STs carrying resistance and virulence genes that may become increasingly pathogenic can be identified and adequate therapies to adopted to avoid their dissemination and derived pathologies.

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.

Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates.

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.

Extracellular Vesicle-Derived microRNAs of Human Wharton's Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis.

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton's jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.

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.

Comparison of Immunosuppressive and Angiogenic Properties of Human Amnion-Derived Mesenchymal Stem Cells between 2D and 3D Culture Systems.

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies.

Unbiased and quantitative proteomics reveals highly increased angiogenesis induction by the secretome of mesenchymal stromal cells isolated from fetal rather than adult skin.

Scarless wound healing and functional regeneration are typical processes of the fetus, gradually lost during postnatal life, and maximally attributed to fetal skin tissue and induced by fetal skin fibroblasts. The latter have been successfully applied to postnatal wounds, with clear advantages compared with autologous dermis grafts or adult fibroblast applications. Our goal was to functionally identify and uncover key factors and mechanisms through the analysis of secretomes, the principal players in all cell therapies based on mesenchymal stromal cells (MSCs). Cell secretomes also putatively mediate skin regenerative effects achieved in clinical applications of fetal skin fibroblasts. An innovative and unbiased approach of comparative and quantitative proteomics of cell conditioned media enabled us to gain knowledge of key molecules and processes from a translational perspective. Using banks of fetal and adult skin fibroblasts that we previously characterized as being MSCs, we discovered secretome changes by identification and comparative quantification, distinguishing secretome signatures of fetal skin MSCs putatively relevant for therapeutic microenvironment modulation. The uncovered proteins can trigger, directly and by modulation of extracellular matrix, angiogenesis, thus highlighting its key role towards scarless wound healing. The angiogenic trigger was functionally validated and corroborated in vitro, with fetal skin MSC secretomes stabilizing and inducing the formation of capillary-like networks by endothelial cells and fetal liver MSCs, respectively. Our approach and our results may aid in the development of cell-based and cell-free products for skin regeneration in acute or chronic injury, and also for wound healing in the regeneration of other tissues. Copyright © 2017 John Wiley & Sons, Ltd.

Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice.

Exosomes are nano-sized vesicles of endolysosomal origin, released by several cytotypes in physiological and pathological conditions. Tumor derived exosomes, interacting with other cells of the tumor microenvironment, modulate tumor progression, angiogenic switch, metastasis, and immune escape. Recently, extracellular vesicles were proposed as excellent biomarkers for disease monitoring and prognosis in cancer patients. Non-small cell lung cancer (NSCLC) has a poor 5-year survival rate due to the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Exosomes might be promising beneficial tools as biomarker candidates in the scenario of NSCLC, since they contain both, proteins and miRNAs. The clinical case reported in this manuscript is a proof of concept revealing that NSCLC exosomes and sorted miRNAs might constitute, in a near future, novel biomarkers. This review summarizes the role of exosomes in NSCLC, focusing on the importance of exosomal microRNAs in lung cancer diagnosis and prognosis.