Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Stimuli-Responsive Dual Cross-Linked N-Carboxyethylchitosan Hydrogels with Tunable Dissolution Rate

Here, we discuss the applicability of (methylenebis(salicylaldehyde)—MbSA) for the fabrication of the stimuli-responsive N-carboxyethylchitosan (CEC) hydrogels with a tunable dissolution rate under physiological conditions. In comparison with non-covalent salicylimine hydrogels, MbSA cross-linking via covalent bis(‘imine clip’) and non-covalent hydrophobic interactions allowed the fabrication of hydrogels with storage moduli > 1 kPa at ten-fold lower aldehyde/CEC molar ratio with the preservation of pH- and amino-acid responsive behavior. Although MbSA-cross-linked CEC hydrogels were stable at neutral and weakly alkaline pH, their disassembly in cell growth medium (Dulbecco’s modified Eagle’s medium, DMEM) under physiological conditions was feasible due to transimination reaction with amino acids contained in DMEM. Depending on the cross-linking density, the complete dissolution time of the fabricated hydrogels varied from 28 h to 11 days. The cytotoxicity of MbSA cross-linked CEC hydrogels toward a human colon carcinoma cell line (HCT 116) and primary human dermal fibroblasts (HDF) was remarkably lower in comparison with CEC-salicylimine hydrogels. Fast gelation, relatively low cytotoxicity, and tunable stimuli-induced disassembly under physiological conditions make MbSA cross-linked CEC hydrogels promising for drug encapsulation and release, 3D printing, cell culturing, and other biomedical applications.

Development of a Hierarchical Support Vector Regression-Based In Silico Model for Caco-2 Permeability

Drug absorption is one of the critical factors that should be taken into account in the process of drug discovery and development. The human colon carcinoma cell layer (Caco-2) model has been frequently used as a surrogate to preliminarily investigate the intestinal absorption. In this study, a quantitative structure–activity relationship (QSAR) model was generated using the innovative machine learning-based hierarchical support vector regression (HSVR) scheme to depict the exceedingly confounding passive diffusion and transporter-mediated active transport. The HSVR model displayed good agreement with the experimental values of the training samples, test samples, and outlier samples. The predictivity of HSVR was further validated by a mock test and verified by various stringent statistical criteria. Consequently, this HSVR model can be employed to forecast the Caco-2 permeability to assist drug discovery and development.

The Inhibition of Proliferation and Invasion of Human Colon Carcinoma Cell Line (Caco-2 cells) by Cell-free Supernatants from Lactobacillus Rhamnosus and Lactobacillus Acidophilus

Background: The epidemiological studies indicated that colorectal cancer is one of the most common types of cancer in the world and is considered a leading cause of cancer-related death. The present study aimed to investigate the inhibitory effect of lactobacillus acidophilus supernatant (LAS) and lactobacillus rhamnosus supernatant (LRS) on the growth and invasiveness of the human colon carcinoma cell line (Caco2) in-vitro. Methods: In this experimental study, the anti-proliferative activity and anti-invasion potential of LAS and LRS were determined by MTT and transwell chambers assays, respectively. The expression of mitochondrial membrane potential-9 (MMP-9) and matrix metalloproteinase-12 (MMP12) genes were analyzed by real-time PCR.Results: The results indicated that supernatants of these two lactobacilli had cytotoxic effects on Caco-2 cells at a concentration of 25% v/v and higher. Thus, the minimum concentrations (25% V/V) of supernatants were chosen for further experiments. LAS and LRS could significantly suppress the invasiveness of Caco-2 cells. Also, the expression of MMP12 was significantly increased in Caco-2 cells when treated with LAS, whereas LRS had no significant effect on the invasive capacity and the gene expression levels of MMP12. The expression of MMP-9 was statistically decreased in Caco2 cells treated with LAS and LRS (P<0.00001).Conclusion: In general, it was shown that LAS and LRS exert anti-cancer activity against the growth, invasion, and metastasis of Caco2 cells in-vitro. It seems that these two bacteria could be used as prophylactic and therapeutic agents for the prevention and treatment of colorectal cancer.

Abstract 532: Isolation of Human Placenta-derived Cdx2 Cells for Cardiac Regeneration

Coronary artery disease and heart failure are among the leading causes of mortality in United States and worldwide. The limited proliferation of the adult mammalian heart has prompted the need to recognize novel strategies that can restore contractile function in heart disease. We recently reported that cells expressing trophoblast marker Cdx2 isolated from murine end-gestation placentas are multipotent and immune-privileged, with homing ability to travel to sites of cardiac injury to regenerate injured myocardium. Since murine Cdx2 cells are immunologically naïve and trophoblast-mediated placentation is a conserved phenomenon, our findings are ripe for translational application. Here we demonstrate the expression of CDX2 and isolation of CDX2 cells from human term placentas highlighting a potentially critical cell source for allogeneic therapy for cardiac regeneration. We studied de-identified human term placenta tissues from three different anatomical sites from three different patients. Formalin-fixed paraffin embedded samples, second trimester chorionic villus samples and de-identified fresh chorion samples were utilized. Using a multiparametric approach including transcript analysis and Sanger sequencing, immunoblot and immunofluorescence analysis, with the subsequent screening of different anatomical sites, we observed that CDX2 is present in the chorionic (fetal cytotrophoblast) portion of human term placentas including the chorionic villous samples (CVS). Subsequent validation on positive control cells DLD1 (human colon carcinoma cell line), versus HeLa cells (low endogenous CDX2 expression) was performed. Visualization of CDX2 expression showed the nuclear localization in the villi region. We have further demonstrated using a CDX2 promoter-driven fluorescence-based lentiviral expression system, we can isolate viable CDX2 cells from fresh term placenta. We demonstrate for the first time that CDX2 cells are present and can be isolated from term human placentas for prospective cardiomyocgenic differentiation. A step closer to the translational approach, our results herein may represent a paradigmatic shift in the way we approach early embryonic lineages and cell fate choices towards the identification of an unexplored human cell-based approach for cardiac regeneration.

Novel Anticancer NHC*-Gold(I) Complexes Inspired by Lepidiline A

N-Heterocyclic carbene gold(I) complexes derived from 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*) represent a promising class of anticancer drugs. Complexes of the type NHC*-Au-L (L = Br−, I−, C≡C-R) and [NHC*-Au-L]+ (L = NHC*, PPh3) have been synthesised. The X-ray crystal structures of all gold(I) complexes are presented; aurophilic interactions were observed in five of the complexes. The anticancer activity was assessed via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based proliferation assays against the human colon carcinoma cell line HCT-116wt and the multidrug-resistant human breast carcinoma cell line MCF-7topo. Most complexes showed good cytotoxicity with IC50 values in the low micromolar range, while excellent sub-micromolar activity was observed for 2c, 3a and 3b. Generally, the activity of the ligands studied was as follows: carbene > phosphine > alkyne > halide, with an exception for the highly active iodido derivative 2c.

Highly Concentrated Multifunctional Silver Nanoparticle Fabrication through Green Reduction of Silver Ions in Terms of Mechanics and Therapeutic Potentials

Background: Green synthesis of silver nanoparticles (AgNPs) is limited to produce AgNPs with only relatively low concentrations, and is unsuitable for large-scale productions. The use of Myrtus communis (MC) leaf methanolic extract (rich in hydrolyzable tannins) has been recommended to resolve the issues related to the aggregation of nanoparticles at high concentrations of silver ions with added facet of antioxidant properties. Methods: The produced highly concentrated MC-AgNPs were characterized by using imaging and spectroscopic methods. Subsequently, antioxidant, anticancer and antifungal activities of the nanoparticles were evaluated. Results: The thermogravimetric analysis and energy dispersive spectroscopy quantitative results suggested that the nanoparticles are biphasic in nature (bio-molecule + Ag0) and layered in structure, suggesting the formation of nanoparticles through a different mechanism than those described in the literature. MC-AgNPs showed greater scavenging activity of nitric oxide and iron (II) chelating ability than the extract. It also showed good reducing power compared to the standard antioxidant. Remarkable anticancer activity of MC-AgNPs (IC50 = 5.99µg/mL) was found against HCT-116 (human colon carcinoma) cell lines after 24h exposure with a therapeutic index value 2-fold higher than the therapeutic index of standard doxorubicin. Furthermore, distinct antifungal activity (MIC = 4µg/mL) was found against Candida krusei. Conclusion: The current method outperforms the existing methods because it produces a large amount of multifunctional nanoscale hybrid materials more efficiently using natural sources; thus, it may be used for diverse biomedical applications.