MiR-124 is significantly upregulated during neurogenic transdifferentiation of ADMSCs.

<p>(A) A protocol outlined describing the strategy to induce neurogenic transdifferentiation of ADMSCs. (B) Representative morphological images of ADMSCs before induction (up) and on day 1 post-induction (down). (C) Representative immunofluorescent images of neuronal markers (NSE and Tuj-1) and glial cell marker (GFAP) expressed in differentiated ADMSCs on day 1 post-induction. Red: neurogenic markers. Blue: DAPI. (D) A total of 12 differential expressed miRNAs, including 6 up-regulated and 6 down-regulated, were identified in the ADMSCs on day 0, 5 and 10 of the induction. The criteria was fold change ≥3 from day 0 to day 10, <i>p</i><0.05. Columns represent samples and rows represent miRNAs (black, green, and red correspond to unchanged, down-regulated and upregulated, respectively). D0: day 0, D5: day 5, D10: day 10. (E) QRT-PCR analysis of miR-124 expression of the ADMSCs on day 0, day 5 and day 10 of induction. Transdiff. = transdifferentiated. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

The schematic diagram of miR-124’s regulation over neurogenic transdifferentiation of ADMSCs.

<p>The schematic diagram of miR-124’s regulation over neurogenic transdifferentiation of ADMSCs.</p

MiR-124 deficiency can affect neurogenic transdifferentiation of ADMSCs.

<p>(A) QRT-PCR analysis of miR-124 expression in ADMSCs infected with the pLV-miR-124 locker lentiviral particles or the vector negative control (Vector NC). (B) QRT-PCR analysis of NSE, Tuj-1 and GFAP mRNA expression in ADMSCs (with or without miR-124 knockdown) on day 1 post-induction. (C) Representative images of flow cytometry analysis of ADMSCs (with or without miR-124 knockdown) with positive neurogenic markers on day 1 post-induction. (D) Quantification of the proportion of ADMSCs with positive neurogenic markers showed in (C). * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

MiR-124 knockdown hampers the acquired electrophysiological properties due to transdifferentiation.

<p>(A) Representative traces of intracellular calcium dynamics (ratiometric acquisition) showing lack of response of untransdifferentiated ADMSCs at biochemical depolarization with 50 nM KCI while still preserving their functional response following exposure to 1 mM ATP. (B) Representative traces of intracellular calcium dynamics (ratiometric acquisition) showing functional response of transdiff. ADMSCs (without miR-124 knockdown: up; with miR-124 knockdown: down) both at biochemical depolarization with KCI as well as the electrical field potential stimulation. However, the responses of the transdiff. ADMSCs/miR-124(-) were significantly weaker. (C) Representative traces of intracellular calcium dynamics (ratiometric acquisition) of mature hippocampal neurons (HN) at biochemical depolarization with 50 nM KCI. (D) Quantification of the peak of intensity of response of transdiff. ADMSC and neurons following exposure to 50 mM KCI (Δ340/380; ADMSCs naive = 0.002±0.001, n = 6; transdiff. ADMSCs = 0.052±0.006, n = 6; transdiff. ADMSCs/miR-124(-) = 0.023±0.008, n = 6; HN = 0.133±0.021, n = 6). (E) Quantitative evaluation of membrane potential values in ADMSCs, transdiff. ADMSCs (with or without miR-124 knockdown) and in hippocampal neurons (ADMSCs naive: 10.13±4.22 mV, n = 6; transdiff. ADMSCs = -42.52±5.31 mV, n = 6; transdiff. ADMSCs/miR-124(-) = -16.37±4.42 mV, n = 6; HN = -52.75±7.48, n = 6). *: comparison with ADMSCs; #: comparison with transdiff. ADMSCs. * and # <i>p</i><0.05, ** and ## <i>p</i><0.01, *** and ### <i>p</i><0.001.</p

MiR-124 enhances neuronal transdifferentiation of ADMSCs partly through RhoA/ROCK1 signaling pathway.

<p>(A) QRT-PCR analysis of RhoA, ROCK1 and ROCK2 mRNA expression in ADMSCs infected with the RhoA shRNA lentiviral particles, ROCK1 shRNA lentiviral particles, ROCK2 shRNA lentiviral particles or the negative control. (B) QRT-PCR analysis of NSE, Tuj-1 and GFAP mRNA expression in ADMSCs infected with the pLV-miR-124 locker lentiviral particles, co-infected with the pLV-miR-124 locker lentiviral particles and the RhoA shRNA lentiviral particles, ROCK1 shRNA lentiviral particles or ROCK2 shRNA lentiviral particles on day 1 after transdifferentiation induction. (C, E and G) Representative images of flow cytometry analysis of neurogenic markers (NSE, Tuj-1 and GFAP) positive ADMSCs infected with the pLV-miR-124 locker lentiviral particles and co-infected with the pLV-miR-124 locker lentiviral particles and the RhoA shRNA lentiviral particles (C), ROCK1 shRNA lentiviral particles (E) or ROCK2 shRNA lentiviral particles (G) on day 1 after transdifferentiation induction. (D, F and H) Quantification of the proportion of ADMSCs with positive neurogenic markers showed in C, E and G. *: comparison with shRNA NC or vector NC; #: comparison with transdiff. ADMSCs/miR-124(-). * and # <i>p</i><0.05, ** and ## <i>p</i><0.01, *** and ### <i>p</i><0.001.</p

MiR-124 can directly target RHOA mRNA and regulate its expression.

<p>(A) QRT-PCR (up) and western blot (down) analysis of RhoA expression in ADMSCs on day 0, 5 and 10 during the transdifferentiation induction. (B) Predicted binding site between 3’UTR of RhoA mRNA and miR-124 and the designed mutant sequence. (C) Dual luciferase assay of relative luciferase activity of pGL3-RhoA-WT (RhoA-WT) and pGL3-RhoA-MUT (RhoA-WT) transfected with 50 nM miR-124 mimics or the negative control in HEK 293T cells. Firefly luciferase activity was normalized to that of Renilla luciferase. (D) QRT-PCR (up) and western blot (down) analysis of RhoA expression in ADMSCs infected with the pLV-miR-124 locker lentiviral particles or the negative control. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Data and Code.zip

The data and code of the paper titled "A prototype-based sampling method for spatial data sampling and its application in landslide susceptibility mapping".</p

Image_1_Lead exposure dose-dependently affects oxidative stress, AsA-GSH, photosynthesis, and mineral content in pakchoi (Brassica chinensis L.).jpeg

Lead (Pb) is a heavy metal pollutant and negatively affects agriculture and ecosystems. Pb can cause oxidative stress and abnormal plant growth. The ascorbic acid-glutathione (AsA-GSH) cycle mainly exists in chloroplasts and resists oxidative stress, scavenges reactive oxygen radicals, and maintains normal photosynthesis. However, the dosage related effects of Pb on pakchoi photosynthesis, via oxidative stress and the AsA-GSH system, remains unclear. In this study, various Pb dosage stress models were tested (low: 300 mg/kg; medium: 600 mg/kg; high: 900 mg/kg). Pb stress induced a dose-dependent increase in Pb content in pakchoi leaves (P < 0.05). Principal component analysis showed that Se, B, and Pb were significantly and negatively correlated. Pb stress also increased MDA content and decreased antioxidant enzymes SOD, GSH-Px, and T-AOC activities (P < 0.05). We also found that Vc content, as well as the GSH/GSSG ratio, decreased. Additionally, Pb stress destroyed chloroplast structure, decreased photosynthesis indicators Pn, Tr, Gs, Ci and VPD, and attenuated Fv/Fm and Fv/Fo (P < 0.05). In the high-dose group, the contents of chlorophyll a, chlorophyll b, and carotenoids decreased significantly, while the expression of chloroplast development genes (GLK, GLN2) decreased (P < 0.05). Our data suggest that Pb stress leads to dosage-dependent, aberrant photosynthesis by inhibiting the AsA-GSH system in pakchoi. This study expands the Pb toxicology research field and provides indications for screening antagonists.</p

Table_1_Lead exposure dose-dependently affects oxidative stress, AsA-GSH, photosynthesis, and mineral content in pakchoi (Brassica chinensis L.).docx

Lead (Pb) is a heavy metal pollutant and negatively affects agriculture and ecosystems. Pb can cause oxidative stress and abnormal plant growth. The ascorbic acid-glutathione (AsA-GSH) cycle mainly exists in chloroplasts and resists oxidative stress, scavenges reactive oxygen radicals, and maintains normal photosynthesis. However, the dosage related effects of Pb on pakchoi photosynthesis, via oxidative stress and the AsA-GSH system, remains unclear. In this study, various Pb dosage stress models were tested (low: 300 mg/kg; medium: 600 mg/kg; high: 900 mg/kg). Pb stress induced a dose-dependent increase in Pb content in pakchoi leaves (P < 0.05). Principal component analysis showed that Se, B, and Pb were significantly and negatively correlated. Pb stress also increased MDA content and decreased antioxidant enzymes SOD, GSH-Px, and T-AOC activities (P < 0.05). We also found that Vc content, as well as the GSH/GSSG ratio, decreased. Additionally, Pb stress destroyed chloroplast structure, decreased photosynthesis indicators Pn, Tr, Gs, Ci and VPD, and attenuated Fv/Fm and Fv/Fo (P < 0.05). In the high-dose group, the contents of chlorophyll a, chlorophyll b, and carotenoids decreased significantly, while the expression of chloroplast development genes (GLK, GLN2) decreased (P < 0.05). Our data suggest that Pb stress leads to dosage-dependent, aberrant photosynthesis by inhibiting the AsA-GSH system in pakchoi. This study expands the Pb toxicology research field and provides indications for screening antagonists.</p

Table_2_Lead exposure dose-dependently affects oxidative stress, AsA-GSH, photosynthesis, and mineral content in pakchoi (Brassica chinensis L.).xlsx

Lead (Pb) is a heavy metal pollutant and negatively affects agriculture and ecosystems. Pb can cause oxidative stress and abnormal plant growth. The ascorbic acid-glutathione (AsA-GSH) cycle mainly exists in chloroplasts and resists oxidative stress, scavenges reactive oxygen radicals, and maintains normal photosynthesis. However, the dosage related effects of Pb on pakchoi photosynthesis, via oxidative stress and the AsA-GSH system, remains unclear. In this study, various Pb dosage stress models were tested (low: 300 mg/kg; medium: 600 mg/kg; high: 900 mg/kg). Pb stress induced a dose-dependent increase in Pb content in pakchoi leaves (P < 0.05). Principal component analysis showed that Se, B, and Pb were significantly and negatively correlated. Pb stress also increased MDA content and decreased antioxidant enzymes SOD, GSH-Px, and T-AOC activities (P < 0.05). We also found that Vc content, as well as the GSH/GSSG ratio, decreased. Additionally, Pb stress destroyed chloroplast structure, decreased photosynthesis indicators Pn, Tr, Gs, Ci and VPD, and attenuated Fv/Fm and Fv/Fo (P < 0.05). In the high-dose group, the contents of chlorophyll a, chlorophyll b, and carotenoids decreased significantly, while the expression of chloroplast development genes (GLK, GLN2) decreased (P < 0.05). Our data suggest that Pb stress leads to dosage-dependent, aberrant photosynthesis by inhibiting the AsA-GSH system in pakchoi. This study expands the Pb toxicology research field and provides indications for screening antagonists.</p