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 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

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 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

Table_2_Case report: A safeguard in the sea of variants of uncertain significance: a case study on child with high risk neuroblastoma and acute myeloid leukemia.xlsx

The increased availability of genetic technologies has significantly improved the detection of novel germline variants conferring a predisposition to tumor development in patients with malignant disease. The identification of variants of uncertain significance (VUS) represents a challenge for the clinician, leading to difficulties in decision-making regarding medical management, the surveillance program, and genetic counseling. Moreover, it can generate confusion and anxiety for patients and their family members. Herein, we report a 5-year-old girl carrying a VUS in the Succinate Dehydrogenase Complex Subunit C (SHDC) gene who had been previously treated for high-risk neuroblastoma and subsequently followed by the development of secondary acute myeloid leukemia. In this context, we describe how functional studies can provide additional insight on gene function determining whether the variant interferes with normal protein function or stability.</p

Image_1_Case report: A safeguard in the sea of variants of uncertain significance: a case study on child with high risk neuroblastoma and acute myeloid leukemia.tif

The increased availability of genetic technologies has significantly improved the detection of novel germline variants conferring a predisposition to tumor development in patients with malignant disease. The identification of variants of uncertain significance (VUS) represents a challenge for the clinician, leading to difficulties in decision-making regarding medical management, the surveillance program, and genetic counseling. Moreover, it can generate confusion and anxiety for patients and their family members. Herein, we report a 5-year-old girl carrying a VUS in the Succinate Dehydrogenase Complex Subunit C (SHDC) gene who had been previously treated for high-risk neuroblastoma and subsequently followed by the development of secondary acute myeloid leukemia. In this context, we describe how functional studies can provide additional insight on gene function determining whether the variant interferes with normal protein function or stability.</p

Table_3_Case report: A safeguard in the sea of variants of uncertain significance: a case study on child with high risk neuroblastoma and acute myeloid leukemia.docx

The increased availability of genetic technologies has significantly improved the detection of novel germline variants conferring a predisposition to tumor development in patients with malignant disease. The identification of variants of uncertain significance (VUS) represents a challenge for the clinician, leading to difficulties in decision-making regarding medical management, the surveillance program, and genetic counseling. Moreover, it can generate confusion and anxiety for patients and their family members. Herein, we report a 5-year-old girl carrying a VUS in the Succinate Dehydrogenase Complex Subunit C (SHDC) gene who had been previously treated for high-risk neuroblastoma and subsequently followed by the development of secondary acute myeloid leukemia. In this context, we describe how functional studies can provide additional insight on gene function determining whether the variant interferes with normal protein function or stability.</p

Table_1_Case report: A safeguard in the sea of variants of uncertain significance: a case study on child with high risk neuroblastoma and acute myeloid leukemia.docx

The increased availability of genetic technologies has significantly improved the detection of novel germline variants conferring a predisposition to tumor development in patients with malignant disease. The identification of variants of uncertain significance (VUS) represents a challenge for the clinician, leading to difficulties in decision-making regarding medical management, the surveillance program, and genetic counseling. Moreover, it can generate confusion and anxiety for patients and their family members. Herein, we report a 5-year-old girl carrying a VUS in the Succinate Dehydrogenase Complex Subunit C (SHDC) gene who had been previously treated for high-risk neuroblastoma and subsequently followed by the development of secondary acute myeloid leukemia. In this context, we describe how functional studies can provide additional insight on gene function determining whether the variant interferes with normal protein function or stability.</p

Table_4_Case report: A safeguard in the sea of variants of uncertain significance: a case study on child with high risk neuroblastoma and acute myeloid leukemia.docx

The increased availability of genetic technologies has significantly improved the detection of novel germline variants conferring a predisposition to tumor development in patients with malignant disease. The identification of variants of uncertain significance (VUS) represents a challenge for the clinician, leading to difficulties in decision-making regarding medical management, the surveillance program, and genetic counseling. Moreover, it can generate confusion and anxiety for patients and their family members. Herein, we report a 5-year-old girl carrying a VUS in the Succinate Dehydrogenase Complex Subunit C (SHDC) gene who had been previously treated for high-risk neuroblastoma and subsequently followed by the development of secondary acute myeloid leukemia. In this context, we describe how functional studies can provide additional insight on gene function determining whether the variant interferes with normal protein function or stability.</p

Metody pro hodnocení přesnosti digitálních měřících systémů

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta elektrotechniky a informatiky