Biological interactions of biocompatible and water-dispersed MoS2 nanosheets with bacteria and human cells

Two dimensional materials beyond graphene such as MoS2 and WS2 are novel and interesting class of materials whose unique physico-chemical properties can be exploited in applications ranging from leading edge nanoelectronics to the frontiers between biomedicine and biotechnology. To unravel the potential of TMD crystals in biomedicine, control over their production through green and scalable routes in biocompatible solvents is critically important. Furthermore, considering multiple applications of eco-friendly 2D dispersions and their potential impact onto live matter, their toxicity and antimicrobial activity still remain an open issue. Herein, we focus on the current demands of 2D TMDs and produce high-quality, few-layered and defect-free MoS2 nanosheets, exfoliated and dispersed in pure water, stabilized up to three weeks. Hence, we studied the impact of this material on human cells by investigating its interactions with three cell lines: two tumoral, MCF7 (breast cancer) and U937 (leukemia), and one normal, HaCaT (epithelium). We observed novel and intriguing results, exhibiting evident cytotoxic effect induced in the tumor cell lines, absent in the normal cells in the tested conditions. The antibacterial action of MoS2 nanosheets is then investigated against a very dangerous gram negative bacterium, such as two types of Salmonellas: ATCC 14028 and wild-type Salmonella typhimurium. Additionally, concentration and layer-dependent modulation of cytotoxic effect is found both on human cells and Salmonellas

CNF1 Improves Astrocytic Ability to Support Neuronal Growth and Differentiation In vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes

A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells

Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications

Biological interactions of biocompatible and water-dispersed MoS2 nanosheets with bacteria and human cells

Two dimensional materials beyond graphene such as MoS2 and WS2 are novel and interesting class of materials whose unique physico-chemical properties can be exploited in applications ranging from leading edge nanoelectronics to the frontiers between biomedicine and biotechnology. To unravel the potential of TMD crystals in biomedicine, control over their production through green and scalable routes in biocompatible solvents is critically important. Furthermore, considering multiple applications of eco-friendly 2D dispersions and their potential impact onto live matter, their toxicity and antimicrobial activity still remain an open issue. Herein, we focus on the current demands of 2D TMDs and produce high-quality, few-layered and defect-free MoS2 nanosheets, exfoliated and dispersed in pure water, stabilized up to three weeks. Hence, we studied the impact of this material on human cells by investigating its interactions with three cell lines: two tumoral, MCF7 (breast cancer) and U937 (leukemia), and one normal, HaCaT (epithelium). We observed novel and intriguing results, exhibiting evident cytotoxic effect induced in the tumor cell lines, absent in the normal cells in the tested conditions. The antibacterial action of MoS2 nanosheets is then investigated against a very dangerous gram negative bacterium, such as two types of Salmonellas: ATCC 14028 and wild-type Salmonella typhimurium. Additionally, concentration and layer-dependent modulation of cytotoxic effect is found both on human cells and Salmonellas

CNF1-induced changes in differentiating neurons are reversible.

<p>Neurons were either continuously exposed to CNF1 until DIV 21, or exposed to CNF1 until DIV 9 and then switched to CNF1-free medium until DIV 21. Control neurons received no treatment. Neurons were immunolabeled for MAP2 (red), actin (green) and synaptophisin (green). At DIV 21, neurons that had been exposed to CNF1 only until DIV 9 show less impressive changes than neurons exposed to CNF1 up to DIV 21. Dendrites are more numerous, although they are still thicker and fewer, compared to untreated cultures. Cell bodies also remain larger, with a veil-like appearance, but the actin cytoskeleton show the formation of thinner and longer projections. The number of synapses is increased. In micrograph showing F-actin and synaptophysin, nuclei are stained with DAPI (blue) (bar  = 50 μm).</p

CNF1-treated astrocytes provide a more efficient substrate to neuritogenesis and synaptogenesis.

<p>Pure astrocytic cell cultures, at confluence, were treated with CNF1 for 48 h. After change of the medium, hippocampal neurons were seeded on the astrocytic monolayer, fixed at DIV 14, and immunolabeled for MAP2 (red) and GFAP (green) or synaptophysin (green). The exposure of astrocytes to CNF1 causes a decrease in GFAP staining (A) whereas hippocampal neurons, growing on CNF1-treated astrocytes but in absence of direct CNF1 influence, produce a much more abundant dendritic network, as shown by MAP2 immunolabeling (A,B, bars  = 200 μm). In C, a detail of a black and white image used for morphometric analysis emphasizes the change in the dendritic tree, which, growing on control astrocytes, appear smooth and poorly branching, while on CNF1-pretreated astrocytes, show much wider ramifications (C). D. Morphometric analysis of MAP2-positive area. In hippocampal neurons co-cultured with astrocytes, after background subtraction, MAP2-positive area was measured as percentage of the total field area (0.15 mm²). Values obtained for each field were pooled to obtain a single mean value for each neuronal culture (n = 5). Bars represent mean values ± S.E.M (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g005" target="_blank">Figure 5C</a>, * = p<0.05, Wilcoxon Matched Pairs test). The richer dendritic tree is accompanied by an increased formation of synapses, as shown by synaptophysin immunolabeling, particularly enriched around cell bodies (E, bar  = 50 μm). Synaptophysin-positive puncta are also visible along dendrite bundles (F). G. Morphometric analysis of synaptic density. Synaptophysin-positive area was measured in 41.500 μm²-large fields obtained from 3 different experiments, conducted in duplicate from 2 different cultures. At least 16 images were analyzed for each condition and the results pooled. Histogram represents the values + S.E.M. Statistical analysis was conducted by the nonparametric Wilcoxon Matched Pairs tests.</p

CNF1 treatment reduces GFAP and IL-1β levels and glutamate-dependent intracellular Ca²⁺ rise in pure astrocytic cultures.

<p>The CNF1-induced decrease in GFAP level, observed by immunofluorescence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g005" target="_blank">Figure 5</a>, panel A), is confirmed by Western blot analysis (panel A). In the supernatants from control and CNF1-treated astrocytes, the expressions of TNF-α and IL-1β were measured by ELISA. While the levels of TNF-α are unaffected (panel B), IL-1β is significantly decreased in astrocytes challenged with the toxin (panel C). Intracellular Ca²⁺ levels were analyzed in astrocytes, following administration of glutamate (after a 3 min baseline), using fluorimetric recordings with Fura 2 AM (panels D,E). In D, the time course of a representative experiment is shown. The values of at least 6 cells were recorded and averaged. In E, Ca²⁺ levels were compared at the baseline and in the peak region. Data for three different experiments were pooled and analyzed. At least 6 cells were evaluated in each experiment. Bars represent mean values ± S.E.M. Glutamate induces significantly lower Ca²⁺ peaks in astrocytes treated with CNF1 than in control cells (panels C and E *p<0.05 unpaired Student's t-test).</p

CNF1 effects in mixed astrocytic/neuronal cultures and in differentiated cultures.

<p>Cortical mixed astrocytic/neuronal cultures were treated with CNF1 at DIV2 and fixed at DIV 14. Neurons were immunolabeled for MAP2 (red) and synaptophysin (green) (A, bar  = 50 μm). In the presence of astrocytes, CNF1 induces less evident cytoskeletal changes than in pure neuronal cultures, with a minor dendritic tree remodeling, while a few synapses persist (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g004" target="_blank">Figure 4B</a>). Western blot analysis, in both pure neuronal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g004" target="_blank">Figure 4C</a>, left) and mixed astrocytic/neuronal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g004" target="_blank">Figure 4C</a>, right) cultures, show that the levels of synaptic proteins, such as synaptophysin and SNAP23, or of components of the dendritic tree, such as spinophilin, are similar in CNF1-treated and control cultures. Differentiated neurons at DIV 12 were exposed to CNF1, fixed at DIV 14 and immunolabeled for actin (red) and synaptophysin (green) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g004" target="_blank">Figure 4D</a>, bar  = 20 μm). Under the influence of CNF1, actin cytoskeleton shows a marked remodeling, with increase in cell body size and development of fine and short neuritic branches. Synaptic density decreases, when compared to control cultures. Again, no changes in the expression of the synaptic proteins above mentioned was measured by Western blotting (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034115#pone-0034115-g004" target="_blank">Figure 4E</a>).</p