Unlocking the treasure box: the role of HEPES buffer in disassembling an uncommon ferritin nanoparticle

Ferritins are ideal nanoparticles as drug delivery systems due to their hollow-sphere structure and the ability to target specific receptors on the cell surface. Here, we develop and characterize a new ferritin derived from the chimeric humanized A. fulgidus one, already designed to recognize the TfR1 receptor. Starting from the synthetic gene of this chimeric protein, we replaced two positively charged amino acids with two alanine residues to close the large triangular pores on its surface. These mutations make the protein nanoparticle suitable to incorporate even small therapeutics without leakage. Size-exclusion chromatography shows that the assembling/disassembling of this new protein cage can be easily fine-tuned by varying the HEPES buffer and MgCl2 concentration. The protein cage can be opened using 150 mM HEPES buffer without magnesium ions. Adding this divalent cation to the solution promotes the quick assembly of the ferritin as a 24-mer. The development of this new protein cage paves the way for encapsulation and delivery studies of small molecules for therapeutic and diagnostic purposes

Proteomic analysis of plasma exosomes from cystic echinococcosis patients provides in vivo support for distinct immune response profiles in active vs inactive infection and suggests potential biomarkers

The reference diagnostic method of human abdominal Cystic Echinococcosis (CE) is imaging, particularly ultrasound, supported by serology when imaging is inconclusive. However, current diagnostic tools are neither optimal nor widely available. The availability of a test detecting circulating biomarkers would considerably improve CE diagnosis and cyst staging (active vs inactive), as well as treatments and follow-up of patients. Exosomes are extracellular vesicles involved in intercellular communication, including immune system responses, and are a recognized source of biomarkers. With the aim of identifying potential biomarkers, plasma pools from patients infected by active or inactive CE, as well as from control subjects, were processed to isolate exosomes for proteomic label-free quantitative analysis. Results were statistically processed and subjected to bioinformatics analysis to define distinct features associated with parasite viability. First, a few parasite proteins were identified that were specifically associated with either active or inactive CE, which represent potential biomarkers to be validated in further studies. Second, numerous identified proteins of human origin were common to active and inactive CE, confirming an overlap of several immune response pathways. However, a subset of human proteins specific to either active or inactive CE, and central in the respective protein-protein interaction networks, were identified. These include the Src family kinases Src and Lyn, and the immune-suppressive cytokine TGF-β in active CE, and Cdc42 in inactive CE. The Src and Lyn Kinases were confirmed as potential markers of active CE in totally independent plasma pools. In addition, insights were obtained on immune response profiles: largely consistent with previous evidence, our observations hint to a Th1/Th2/regulatory immune environment in patients with active CE and a Th1/inflammatory environment with a component of the wound healing response in the presence of inactive CE. Of note, our results were obtained for the first time from the analysis of samples obtained in vivo from a well-characterized, large cohort of human subjects

Unregulated Custody Transfers: Why the Practice of Rehoming Should Be Considered a Form of Illegal Adoption and Human Trafficking

In this work, the authors prepared and characterized two different graphene oxides: one chemically synthesized (GO sample) and the other one electrochemically synthesized (GO(LiCl)). Both samples were fully characterized with atomic force microscopy (AFM), Raman and Fourier transform infrared (FTIR) spectroscopies, X-ray photo electron spectroscopy (XPS), thermal analysis (TG/DTA), and Z-potential. The antibacterial properties of both graphene oxides were studied using Gram-negative Escherichia coli ATCC 25922 and Gram-positive Staphylococcus aureus ATCC 25923 by spectrophotometer and viable cell count as indirect and direct methods, respectively. Results demonstrated that the GO(LiCl) exhibited a significant antibacterial activity compared to GO that showed a bacteriostatic effect on both pathogens. Electron microscopy analysis confirmed the antibacterial effects of both graphene oxides toward the pathogens, especially working at 80 μg/mL, for 24 h. Additional studies were also performed and both GO samples were not cytotoxic at 2 μg/mL toward neuroblastoma cells. Moreover, 2 μg of GO was suitable to carry the minimum effective dose (5.74 ng/mL) of kinase inhibitor S29 (1-(2-chloro-2-(4-chlorophenyl)ethyl)-N-(4-fluorobenzyl)-1H-pyrazolo[3,4-d] pyrimidin-4-amine), providing negligible side effects related to the S29 treatment (this latter being specifically active on the neuroblastoma cell lines (SK-N-BE(2)))

Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-β5, Survivin, TGF-β, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools

NECROTIC CELL DEATH IN HUMAN AMNIOTIC CELLS INFECTED BY LISTERIA MONOCYTOGENES

Listeria monocytogenes can cause a placental-foetal infection that results in spontaneous abortion, premature labour, stillbirth, or neonatal sepsis and meningitis. Bacteria cross the maternofoetal barrier at the villous syncytiotrophoblast level and subsequently spread from the placenta to the fetus. L. monocytogenes is able to induce different kinds of death in a variety of cells. Murine hepatocytes, murine T and human B lymphocytes, and murine dendritic cells die by apoptosis, whereas bacterial infection of murine and human macrophages leads mainly to necrotic cell death. As we previously described the efficient infection and growth of L. monocytogenes in a human amniotic cell line, we investigated the fate of these cells in order to analyse the mode of cell death. Our results provide biochemical and morphological evidence of necrotic death induced by L. monocytogenes infection

Erythrocyte Remodeling in Plasmodium berghei Infection: The Contribution of SEP Family Members

Host-parasite interactio

Survey for virulence determinants among Enterococcus faecalis isolated from different sources

A collection of Enterococcus faecalis strains from clinical isolates, healthy individuals and the environment was screened for the presence of virulence factor genes, such as those for collagen-binding protein (ace), endocarditis antigen (efaA), haemolysin activator (cylA), gelatinase (gelE), aggregation substances (asa1 and asa373), a surface protein (esp) and two novel putative surface antigens (EF0591 and EF3314). Apart from some genes that were present in all strains (ace, efaA and EF3314), the gelE gene was the most common factor, although its presence did not correlate with its expression. The genes that encode Esp and CylA were never detected in endocarditis isolates, whereas an association was noted between the esp gene and isolates from urinary tract infection (UTI) and bacteraemia. An aggregation substance gene was always present in commensal strains. As for gelatinase, the presence of the cylA and asa genes did not correlate completely with their phenotypic expression. Generally, isolates from endocarditis, biliary stents and the environment were equipped with fewer virulence factors than isolates from other sources. UTI strains possessed the highest number of factors

Receptor-mediated endocytosis of biofilm-forming Enterococcus faecalis by rat peritoneal macrophages

Background & objectives: Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics. Studies were taken up to identify virulence factors and to characterise pathogenic mechanisms of such infections to evaluate potential targets for treatments alternative to antibotic therapy. This study was carried out to evaluate the contribution of extracellular polysaccharide expressed by Enterococcus faecalis to resistance to phagocytosis and survival within rat peritoneal macrophages. Methods: Six E. faecalis clinical isolates were tested for their ability to survive within rat peritoneal macrophages. Cytochalasin D, colchicine and monodansylcadaverine were used to investigate the route of enterococcal entry inside macrophages. Results: Four of the isolates were able to produce extracellular polysaccharide and form biofilm after growth in glucose-supplemented medium, while no production could be detected in glucose deficient medium. Two isolates were polysaccharide-negative in both conditions. Isolates expressing extracellular polysaccharide were able to survive for more than 24 h compared to polysaccharide-negative bacterial cells of the same strain grown in glucose-deficient medium, which were readily cleared. Cytochalasin D virtually abolished the number of viable intracellular bacteria, after growth in either trypticase soy broth (TSB) or TSB supplemented with glucose; colchicine and monodansylcadaverine strongly affected survival of polysaccharide-positive bacteria, significantly more than that of polysaccharide-negative ones. Interpretation & conclusion: Biofilm-forming E. faecalis survived within rat peritoneal macrophages significantly better than polysaccharide-negative isolates. Perturbators of cytoskeleton and of surface receptors turnover, indicated receptors-mediated endocytosis as the most likely route for enterococcal entry into macrophages