bnct and nanoparticles a long way to a routine clinical method

is influenced by several parameters, including: the intrinsic properties of the nanomaterial (size, porosity, surface polarity), the characteristics of the medium (viscosity, pH, ionic strength, ionic composi tion, presence of molecules and/or macromolecules), and nanoparticle concentration. The addition of various molecules can partially stabilize some types of nanoparticles in suspension, but often these additives are not compatible for medical use, because toxic (e.g. alcohols, surfactants) or anyway contraindicated for intravenous injection (2,3). We have recently had the chance to read the editorial "Boron Neutron Capture Therapy of cancer as a part of modern nanomedicine" (1), by Alexander V. Safronov, in which the potential of nanomaterials as boron-carriers for the treatment of many types of tumors by BNCT is discusses. The author argues that "Most of the modern papers on BNCT report 'potential' BNCT agents (…) and don't even include cell studies", and "In some cases the abbreviation BNCT may become a 'golden ticket' for authors who just want to public their current study without intent to continue". We agree with the first assertion, which is unequivocally verifiable by reading the recent literature about BNCT, but not with the second, that is somewhat speculative. The study for a new drug is complex and may last ten years or more. It includes the preparation of the molecule in a pure form, the experimentation on cell lines, the animal testing, and the three phases of clinical trials on humans. In the case in which in vitro results already point to a possible cytotoxicity, additional in vivo tests on animals become inopportune on the basis of ethical principles that have been even reinforced by novel laws promulgated in some countries, such as the new European regulation (2010/63/UE), that strongly restrict inessential in vivo experiments. Therefore, a scientific work can be interrupted at an early stage and, unfortunately, the obtained data remain generally unpublished, staying hidden to the rest of the world. In a field such as nanotechnology, when little differences in size, shape and chemical-physical properties of nanoparticles induce great alterations in the interaction with biological systems, the failures and the frustrating difficulties in the interpretation of the results are widespread. A dramatic obstacle that is encountered in the application of nanomaterials for biological purposes (especially for BNCT, in which the compound must be administered as a homogeneous suspension by intravenous infusion), is their strong tendency to aggregate/agglomerate, in aqueous solvents, into large particles of micrometric size, by reason of their thermodynamic properties. These micrometric particles, besides having an elevated sedimentation rate, induce several adverse reactions to blood components (e.g. thrombi, inflammation, and hemolysis) and, most importantly, will never be internalized by target cells. The stability of nanoparticle suspension

Survival and senescence of human young red cells in vitro.

Background: A number of experimental investigations in vivo suggest that in humans a decrease of circulating erythrocyte number ensues whenever erythropoietin (EPO) plasma level decreases. Since the process seems to selectively eliminate young red cells (neocytes), it has been named neocytolysis. The experimental models in vivo have revealed and documented multiple forms of neocytolysis but have not fully elucidated the specificity of the target red cells and the relation with EPO level changes. In an attempt to better characterize the neocytolytic process, we have undertaken an in vitro investigation on age-ranked human red cells. Methods: By centrifugation on Percoll density gradient we separated the red cells population into three subsets, neocytes, middle-aged and old. Then we comparatively investigated the kinetics of survival of the subsets cultured under different conditions: with medium alone, with 10% autologous plasma, with EPO, alone or in combination with autologous monocytes. Results: Neocytes showed a viability and a survival rate lower than the other red cells when cultured in medium or with 10% plasma. EPO at physiological doses increased their survival rate, but not that of the other subsets. This effect was enhanced by co-culture with monocytes. Conclusion: Likely neocytes are more sensitive than the other RBCs subsets to presence or absence of survival signals, such as EPO or plasma or monocytes derived factors. These observations could provide an insight into the link between the decrease in EPO plasma level and the reduction of circulating red cells mass and account for the specificity of neocytes clearance

Membrane Rearrangements in the Maturation of Circulating Human Reticulocytes

Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a “per cell” basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC

Immortalized HEK 293 Kidney Cell Lines as Models of Renal Cells: Friends or Foes?

The immortalized cell lines derived from human embryonic kidney, named HEK 293, are extensively used as models of human renal cells in in vitro studies. Nevertheless, ample evidence in the literature shows that HEK 293 cells display genotypic and phenotypic characteristics that differ substantially from primary kidney cells, with potential detrimental effects on the quality of the experimental results. Among the differences documented between HEK 293 and renal cells, there is an altered pattern of expression of many proteins involved in the development and physiological functions of the kidney. Methionine sulfoxide reductase (Msr) enzymes are ubiquitous components of the cellular machinery, evolved to counteract the damages inflicted to methionine residues by oxidative stress, particularly intense in kidney tissues. In this article, we have compared the levels of expression of several different Msr enzymes in human kidney and in a HEK 293 strain and have observed significant differences between the two cell types

Continuous Change in Membrane and Membrane-Skeleton Organization During Development From Proerythroblast to Senescent Red Blood Cell

Within the context of erythropoiesis and the possibility of producing artificial red blood cells (RBCs) in vitro, a most critical step is the final differentiation of enucleated erythroblasts, or reticulocytes, to a fully mature biconcave discocyte, the RBC. Reviewed here is the current knowledge about this fundamental maturational process. By combining literature data with our own experimental evidence we propose that the early phase in the maturation of reticulocytes to RBCs is driven by a membrane raft-based mechanism for the sorting of disposable membrane proteins, mostly the no longer needed transferrin receptor (TfR), to the multivesicular endosome (MVE) as cargo of intraluminal vesicles that are subsequently exocytosed as exosomes, consistently with the seminal and original observation of Johnstone and collaborators of more than 30 years ago (Pan BT, Johnstone RM. Cell. 1983;33:967-978). According to a strikingly selective sorting process, the TfR becomes cargo destined to exocytosis while other molecules, including the most abundant RBC transmembrane protein, band 3, are completely retained in the cell membrane. It is also proposed that while this process could be operating in the early maturational steps in the bone marrow, additional mechanism(s) must be at play for the final removal of the excess reticulocyte membrane that is observed to occur in the circulation. This processing will most likely require the intervention of the spleen, whose function is also necessary for the continuous remodeling of the RBC membrane all along this cell's circulatory life

On the association of lipid rafts to the spectrin skeleton in human erythrocytes

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated-signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4 °C (Detergent Resistant Membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane-skeleton in human erythrocytes, and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils, that lead to proteolytic disruption of the supramolecular assembly of the membrane-skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions, that most likely involve the spectrin molecul

Kinetic resolution of phenyl methyl sulfoxides by mammalian methionine sulfoxide reductase A

Chiral sulfoxides are widely used in organic synthesis as chiral auxiliaries. There are numerous strategies for the preparation of enantiomerically pure sulfoxides, based either on the enantioselective oxidation of sulphides or the enantiospecific reduction of sulfoxides. For both cases, bioconversion techniques have been developed and proposed for large-scale synthesis. Methionine sulfoxide reductase enzymes (MsrA and MsrB) catalyse the stereoselective conversion of methionine sulfoxide to methionine. MsrA can also catalyse the reduction of other exogenous sulfoxides, including p-tolyl methyl sulfoxide. However, the stereoselectivity towards this type of substrate is not yet well characterized. The activity and enantioselectivity of MsrA toward several aryl methyl sulfoxides is presented in this paper