Solid Phase Synthesis and Biological studies on metal conjugates of bioactive peptides for targeted delivery

Summary Peptide-based drug delivery systems and therapeutics have gained an enormous attention during the last ten years. Progress in this field will lead to site specific delivery, improved receptor affinities, efficient cellular uptake and/or nuclear targeting. Thus, in this research project, metal conjugates of some bioactive peptides were synthesized and biologically tested in order to evaluate the effects of these metal moieties on the biological activities such as the cellular uptake, nuclear targeting and binding affinity, of these selected peptides. In this study, the two redox active metallocenes, ferrocene and cobaltocenium and in addition the cytotoxic platinum were chosen as metal labels. The investigated peptides were the Simian Virus 40 nuclear localisation signal (NLS), the HIV transactivator of transcription (TAT) peptide and the small regulatory peptide, the neurotensin (NT). Metal conjugates of these bioactive peptides were successfully synthesized by solid phase peptide synthesis (SPPS) which was used not only for the covalent bonding of the metallocenes to the peptides but also for the complexation of platinum to the peptides and fluorescence labelling with FITC. Comprehensive characterisation of the synthesized bioconjugates was carried out by various techniques such as NMR, RP-HPLC, MS and electrochemistry. The cellular uptake and nuclear localisation of the metallocene-NLS and -TAT bioconjugates was monitored by fluorescence microscopy in living liver cancer cells (Hep G2). The metallocene-NLS conjugates were efficiently internalized by the cells and were localised in the nuclei of the Hep G2 cells. The metallocene moiety is responsible for the enhanced cellular uptake of these bioconjugates and the NLS transports the organometallic species into the nuclei. This is the first example of the directed nuclear delivery of ferrocene and the cobaltocenium cation, by conjugation to the NLS peptide. The use of the scrambled NLS sequence (NLSscr) abolishes the nuclear targeting property of the conjugates. All metallocene-NLS bioconjugates were found to be non-toxic in concentrations up to 1mM in the WST-1 proliferation assay. In case of the metallocene-TAT bioconjugates, the ferrocene moiety plays a role in the escape of the conjugate from the endosomes, which is an advantage because the utility of TAT peptide as a vector for cellular delivery is limited by its inability to escape from the endosomes. Moreover, these peptides are toxic in higher concentration due to cell membrane perturbation. This was also demonstrated in the present study using the WST-1 proliferation assay. In the last part of the project, metal conjugates of wild type NT(8-13) and the modified Pseudoneurotensin (pNT) were synthesized and tested for their binding affinity to the NTR1 receptors in the HT 29 adenocarcinoma cell line. Replacement of Arg8-Arg9 with Lysines in pNT led to a significant decrease in the binding affinity. The metal-NT bioconjugates showed good receptor affinity especially the cobaltocenium-NT conjugate (IC50=2.3nM). In this case, the lipophilicity of the metallocene bioconjugate may facilitate the crossing of the blood brain barrier which is a limiting factor in any centrally intended therapy. The Pt-NT bioconjugate also showed good affinity (IC50=6.8nM) for the receptors. Thus, such bioconjugates may be specifically and selectively delivered to the tumour tissues that overexpress the neurotensin receptors. In conclusion, the optimised synthesis procedures for the studied metals and the peptides were established. The biological studies demonstrate a great potential of these metals for the improvement of the biological functions of the tested peptides especially for use as vectors for cellular uptake and targeted nuclear delivery. This represents a novel application of bioorganometallic chemistry in biological systems

Deployment of social nets in multilayer model to identify key individuals using majority voting

Social web and social media are evidenced to be a rich source of user-generated social content. Social media includes multiple numbers of social dimensions represented by different social networks. The identification of important player in these real-world social networks has been in high emphasis due to its effectiveness in multiple disciplines, especially in law enforcement areas working on dark networks. Many algorithms have been proposed to identify key players according to the objective of interest using suitable network centrality measures. This paper proposes a new perspective of dealing with key player identification by redefining it as a problem of “Key Individual Identification,” across multiple social dimensions. Research deals with each social dimension as a layer in the multiple-layer social network model. The proposed technique extracts a number of features from each network based on social network analysis. The features are assembled to formulate a global feature set representing the behaviors of individuals in all networks individually. The technique then attempts to find key individuals using hybrid classifiers. The results from all classifiers are formulated, and the final decision of an individual to be part of the individual key set is based on majority voting. This novel technique gives good results on a number of known networks

Increased Circulating VAP-1 Levels Are Associated with Liver Fibrosis in Chronic Hepatitis C Infection

Vascular adhesion protein-1 (VAP-1) is a multifunction protein. While membrane-bound VAP-1 is an adhesion protein, soluble VAP-1 catalyzes the deamination of primary amines through its semicarbazide-sensitive amino oxidase (SSAO) activity. VAP-1 supports the transmigration of leukocytes and increases oxidative stress. In chronic liver diseases, it plays a role in leukocyte infiltration and fibrogenesis. Here, we measured VAP-1 plasma concentration and its SSAO activity in 322 patients with chronic hepatitis C infection and evaluated the association of VAP-1 with fibrosis stages. VAP-1 concentration strongly correlated with liver stiffness and was the second strongest influencing variable after gamma-glutamytransferase (GGT) for liver stiffness in regression analysis. The VAP-1 concentration increased with advancing fibrosis stages and the highest concentrations were found in patients with cirrhosis. According to the receiver operating characteristic (ROC) analysis, a VAP-1 cut-off value of 541 ng/mL predicted histologically confirmed cirrhosis (sensitivity 74%; specificity 72%). SSAO activity correlated only moderately with liver stiffness, showing a relatively small increase in advanced fibrosis. To our knowledge, this is the first study on VAP-1 in chronic hepatitis C infection showing its association with progressive fibrosis. In conclusion, VAP-1 plasma concentration, rather than its SSAO activity, may represent a non-invasive biomarker for monitoring fibrogenesis in patients with chronic hepatitis C infection

The Gut Microbiota and Hematopoietic Stem Cell Transplantation: Challenges and Potentials.

The human gut microbiota gained tremendous importance in the last decade as next-generation technologies of sequencing and multiomics analyses linked the role of the microbial communities to host physiology and pathophysiology. A growing number of human pathologies and diseases are linked to the gut microbiota. One of the main mechanisms by which the microbiota influences the host is through its interactions with the host immune system. These interactions with both innate and adaptive host intestinal and extraintestinal immunity, although usually commensalistic even mutualistic with the host, in some cases lead to serious health effects. In the case of allogenic hematopoietic stem cell transplantation (allo-HSCT), the disruption of the intestinal microbiota diversity is associated with acute graft-versus-host disease (GvHD). Causing inflammation of the liver, skin, lungs, and the intestine, GvHD occurs in 40-50% of patients undergoing allo-HSCT and results in significant posttransplantation mortality. In this review, we highlight the impact of the gut microbiota on the host immunity in GvHD and the potential of microbiota in alleviation or even prevention of GvHD

Gene expression profile of CD14+ blood monocytes following lifestyle-induced weight loss in individuals with metabolic syndrome.

Lifestyle-induced weight loss is regarded as an efficient therapy to reverse metabolic syndrome (MetS) and to prevent disease progression. The objective of this study was to investigate whether lifestyle-induced weight loss modulates gene expression in circulating monocytes. We analyzed and compared gene expression in monocytes (CD14+ cells) and subcutaneous adipose tissue biopsies by unbiased mRNA profiling. Samples were obtained before and after diet-induced weight loss in well-defined male individuals in a prospective controlled clinical trial (ICTRP Trial Number: U1111-1158-3672). The BMI declined significantly (- 12.6%) in the treatment arm (N = 39) during the 6-month weight loss intervention. This was associated with a significant reduction in hsCRP (- 45.84%) and circulating CD14+ cells (- 21.0%). Four genes were differentially expressed (DEG's) in CD14+ cells following weight loss (ZRANB1, RNF25, RB1CC1 and KMT2C). Comparative analyses of paired CD14+ monocytes and subcutaneous adipose tissue samples before and after weight loss did not identify common genes differentially regulated in both sample types. Lifestyle-induced weight loss is associated with specific changes in gene expression in circulating CD14+ monocytes, which may affect ubiquitination, histone methylation and autophag

Increased Circulating VAP-1 Levels Are Associated with Liver Fibrosis in Chronic Hepatitis C Infection.

Vascular adhesion protein-1 (VAP-1) is a multifunction protein. While membrane-bound VAP-1 is an adhesion protein, soluble VAP-1 catalyzes the deamination of primary amines through its semicarbazide-sensitive amino oxidase (SSAO) activity. VAP-1 supports the transmigration of leukocytes and increases oxidative stress. In chronic liver diseases, it plays a role in leukocyte infiltration and fibrogenesis. Here, we measured VAP-1 plasma concentration and its SSAO activity in 322 patients with chronic hepatitis C infection and evaluated the association of VAP-1 with fibrosis stages. VAP-1 concentration strongly correlated with liver stiffness and was the second strongest influencing variable after gamma-glutamytransferase (GGT) for liver stiffness in regression analysis. The VAP-1 concentration increased with advancing fibrosis stages and the highest concentrations were found in patients with cirrhosis. According to the receiver operating characteristic (ROC) analysis, a VAP-1 cut-off value of 541 ng/mL predicted histologically confirmed cirrhosis (sensitivity 74%; specificity 72%). SSAO activity correlated only moderately with liver stiffness, showing a relatively small increase in advanced fibrosis. To our knowledge, this is the first study on VAP-1 in chronic hepatitis C infection showing its association with progressive fibrosis. In conclusion, VAP-1 plasma concentration, rather than its SSAO activity, may represent a non-invasive biomarker for monitoring fibrogenesis in patients with chronic hepatitis C infection

Towards a 21st-century roadmap for biomedical research and drug discovery:consensus report and recommendations

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and nongovernmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathway-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this

Effect of Phytosynthesized Selenium and Cerium Oxide Nanoparticles on Wheat (Triticum aestivum L.) against Stripe Rust Disease

In this study, selenium nanoparticles (SeNPs) and cerium oxide nanoparticles (CeONPs) were synthesized by using the extract of Melia azedarach leaves, and Acorus calamusas rhizomes, respectively, and investigated for the biological and sustainable control of yellow, or stripe rust, disease in wheat. The green synthesized NPs were characterized by UV-Visible spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and X-ray diffraction (XRD). The SeNPs and CeONPs, with different concentrations (i.e., 10, 20, 30, and 40 mg/L), were exogenously applied to wheat infected with Puccinia striformis. SeNPs and CeONPs, at a concentration of 30 mg/L, were found to be the most suitable concentrations, which reduced the disease severity and enhanced the morphological (plant height, root length, shoot length, leaf length, and ear length), physiological (chlorophyll and membrane stability index), biochemical (proline, phenolics and flavonoids) and antioxidant (SOD and POD) parameters. The antioxidant activity of SeNPs and CeONPs was also measured. For this purpose, different concentrations (50, 100, 150, 200 and 400 ppm) of both SeNPs and CeONPs were used. The concentration of 400 ppm most promoted the DPPH, ABTS and reducing power activity of both SeNPs and CeONPs. This study is considered the first biocompatible approach to evaluate the potential of green synthesized SeNPs and CeONPs to improve the health of yellow, or stripe rust, infected wheat plants and to provide an effective management strategy to inhibit the growth of Puccinia striformis.This research was funded by projects APOGEO (Cooperation Program INTERREG-MAC 2014–2020, with European Funds for Regional Development-FEDER). “Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) del Gobierno de Canarias” (Project ProID2020010134), and Fundación CajaCanarias (Project 2019SP43).Peer reviewe

A shift in paradigm towards human biology-based systems for cholestatic-liver diseases.

Cholestatic-liver diseases (CLDs) arise from diverse causes ranging from genetic factors to drug-induced cholestasis. The so-called diseases of civilization (obesity, diabetes, metabolic disorders, non-alcoholic liver disease, cardiovascular diseases, etc.) are intricately implicated in liver and gall bladder diseases. Although CLDs have been extensively studied, there seem to be important gaps in the understanding of human disease. Despite the fact that many animal models exist and substantial clinical data are available, translation of this knowledge towards therapy has been disappointingly limited. Recent advances in liver cell culture such as in vivo-like 3D cultivation of human primary hepatic cells, human induced pluripotent stem cell-derived hepatocytes; and cutting-edge analytical techniques such as 'omics' technologies and high-content screenings could play a decisive role in deeper mechanistic understanding of CLDs. This Topical Review proposes a roadmap to human biology-based research using omics technologies providing quantitative information on mechanisms in an adverse outcome/disease pathway framework. With modern sensitive tools, a shift in paradigm in human disease research seems timely and even inevitable to overcome species barriers in translation

The Changing Paradigm in Preclinical Toxicology: in vitro and in silico Methods in Liver Toxicity Evaluations

In vitro methods, based on human primary cells, cell lines, and genetically modified reporter cell lines, have greatly expanded the scope of in vitro toxicology. Other significant progress in the area of human-induced pluripotent stem cells (hiPSCs) (Asgari et al., 2010; Schwartz et al., 2014; Shinde et al., 2016; Shtrichman, Germanguz and Itskovitz-Eldor, 2013) is allowing the application of patient and disease-specific hiPSCs (Ghodsizadeh et al., 2010; McCracken et al., 2014; Siller et al., 2013). Moreover, the tools of precise genome editing with engineered nucleases, such as the zinc finger nucleases (zfns), the transcription activator-like effecter nucleases (talens) and, more recently, the Clustered Regularly Interspaced Short Palindromic Repeats (crispr) associated Cas9 technology (Gaj, Gersbach and Barbas, 2013; Kim, 2016; Komor, Badran and Liu, 2017) have opened up tremendous opportunities for the development of cell lines, especially those of human origin (Tobita, Guzman-Lepe and de L’Hortet, 2015). crispr/Cas9 technology was reported for genome editing in hiPSCs (Flaherty and Brennand, 2015; Li et al., 2014; Seah et al., 2015; Suzuki et al., 2014). Another study reported on the simultaneous reprogramming and gene correction of patient fibroblasts (Howden et al., 2015). Since 2015, more than 3,000 articles were published on studies using crispr/Cas9 genome editing, including more than 900 articles using the technology in mammalian cells (PubMed, accessed June 11, 2017). With further technological developments, these human in vitro cellular models shall be highly useful in the screening of compounds for personalized medicine, allowing optimum therapy with minimum or no adverse effects, and in the study of adverse outcomes in different strata of population. In addition to high-content screening, where several parameters are measured as simultaneous readouts in single cells (Gasparri, 2009), high-content imaging will play an important complimentary role in systems biology approaches (van Vliet et al., 2014). High-content platforms have been already used for the screening of compounds (Bale et al., 2014; Sirenko et al., 2014; Tolosa et al., 2014)