Mild Hypoxia Enhances Proliferation and Multipotency of Human Neural Stem Cells

Background

Neural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it''s estimated to be 2.5 to 3%.

Methodology/Principal Findings

We investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of β-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.

Conclusions/Significance

These findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson''s disease, multiple sclerosis, and Alzheimer''s disease.     

PEGylated Nanoparticles based on a polyaspartamide. preparation, physico-chemical characterization, and intracellular uptake

Nanoparticles with different surface PEGylation degree were prepared by using as starting material alpha,beta-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA). PHEA was functionalized with a PEG amino-derivative for obtaining PHEA-PEG(2000) copolymer. Both PHEA and PHEA-PEG(2000) were derivatized with methacrylic anhydride (MA) for obtaining poly(hydroxyethylaspartamide methacrylated) (PHM) and poly(hydroxyethylaspartamide methacrylated)-PEGylated (PHM-PEG(2000)), respectively. Nanoparticles were obtained by UV irradiation of an inverse microemulsion, using as internal phase an aqueous solution of PHM alone or of the PHM/PHM-PEG(2000) mixture at different weight ratio and as external phase a mixture of propylene carbonate and ethyl acetate. Obtained nanoparticles were characterized by FT-IR analysis, dimensional analysis, and TEM micrography. XPS analysis and zeta potential measurements demonstrated the presence of PEG onto the nanoparticle surface. Moreover, the partial degradation of nanoparticles in the presence of esterase as a function of time was demonstrated. Finally, nanoparticles did not possess any cytotoxic activity against K-562 cells and were able to escape from phagocytosis depending on the surface PEGylation degree.     

Effects of radiofrequency field exposure on proteotoxic-induced and heat-induced HSF1 response in live cells using the bioluminescence resonance energy transfer technique

As of today, only acute effects of RF fields have been confirmed to represent a potential health hazard and they are attributed to non-specific heating (≥ 1 °C) under high-level exposure. Yet, the possibility that environmental RF impact living matter in the absence of temperature elevation needs further investigation. Since HSF1 is both a thermosensor and the master regulator of heat-shock stress response in eukaryotes, it remains to assess HSF1 activation in live cells under exposure to low-level RF signals. We thus measured basal, temperature-induced, and chemically induced HSF1 trimerization, a mandatory step on the cascade of HSF1 activation, under RF exposure to continuous wave (CW), Global System for Mobile (GSM), and Wi-Fi-modulated 1800 MHz signals, using a bioluminescence resonance energy transfer technique (BRET) probe. Our results show that, as expected, HSF1 is heat-activated by acute exposure of transiently transfected HEK293T cells to a CW RF field at a specific absorption rate of 24 W/kg for 30 min. However, we found no evidence of HSF1 activation under the same RF exposure condition when the cell culture medium temperature was fixed. We also found no experimental evidence that, at a fixed temperature, chronic RF exposure for 24 h at a SAR of 1.5 and 6 W/kg altered the potency or the maximal capability of the proteasome inhibitor MG132 to activate HSF1, whatever signal used. We only found that RF exposure to CW signals (1.5 and 6 W/kg) and GSM signals (1.5 W/kg) for 24 h marginally decreased basal HSF1 activity.Electronic supplementary materialThe online version of this article (10.1007/s12192-020-01172-3) contains supplementary material, which is available to authorized users.     

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.     

Immunoconjugate generation between the ribosome inactivating protein restrictocin and an anti-human breast carcinoma MAB

Summary Treatment of Wistar/AG rats with a single i.p. injection of 1 mg/kg of synthetic double-stranded polynucleotides, either polyadenylic-polyuridylic acid (rA_n.rU_n), or a mismatched analogue of polyinosinic-polycytidylic acid (rI_n.r(C₁₂U)_n), enhanced the cytotoxicity of natural killer (NK) cells among peripheral blood leukocytes and lung intracapillary leukocytes (LICL). The enhancement reached a peak 24 h after treatment and returned to control values after 4 days. In rats given repeated injections of double-stranded polynucleotides (2 per week), the NK cytotoxicity expressed by LICL reached more than ten times (in lytic units) the control levels between day 8, after 3 injections, and day 360, after 100 injections. No hypore-sponsiveness was observed. Moreover, NK activity was frequently and significantly higher in rats given multiple injections than in those given a single injection. In rats with experimentally induced P77 lung fibrohistiocytoma colonies, repeated injections of rI_n.r(C₁₂U)_n stimulated NK activity and reduced the number of metastatic nodules from 172 to 19. The same significant reduction (from 172 to 27) was also observed in animals given repeated injections of rA_n.rU_n. However, with two models of spontaneous metastases, significant reduction in lung metastases (M37 bronchioloalveolar carcinoma) or lack of effect (S₄T₁₉ rhabdomyosarcoma) were observed.     

Azorellane diterpenes from Azorella cryptantha

Azorella cryptantha yielded the diterpenes, azorellolide and the dihydroderivative, dihydroazorellolide, together with the known yaretol and 1alpha,10beta,4beta,5alpha-diepoxy-7alpha-germacran-6beta-ol. Both possess a carbon skeleton type that may originate from rearrangement of the mulinane skeleton.     

A Rapid Method for Screening Large Numbers of Environmental Microorganisms for Antiviral Activity

A new method for screening microbial colonies endowed with antiviral activity is described. It is based on close contact between microbial agar cultures and agar-covered virus-infected-cell monolayers and allows the screening of large numbers of colonies in just a few months.