EMBO/sd-character-level-ner huggingface dataset:
Intracellular trafficking by kinesins UNC‐116 and UNC‐104 is required for necrotic cell death.Figure 5(A) Depletion of the kinesin 1 heavy chain UNC‐116 suppresses necrosis induced by both mec‐4(d) and deg‐3(d) toxic alleles. (B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, unc‐116 and unc‐104 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test). (C) Percentage of unc‐116 animals that survive after treatment with sodium azide. (D) Depletion of the monomeric kinesin UNC‐104 protects neurons against MEC‐4(d)‐induced degeneration. (E) unc‐104 mutant animals are more resistant to hypoxic death induced by sodium azide. (F) Combined inactivation of endocytosis and kinesin‐mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC‐4(d)‐induced degeneration. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).
Figure 8Nuclear dsDNA released into cytoplasm by several cell death pathways during brain ischemia is a damage-associated molecular pattern, which can drive cGAS signaling and AIM2 inflammasome activation in microglia. Once activated, microglia can release both non-inflammasome-dependent cytokines (e.g., TNFα, IL-6) and inflammasome-dependent cytokines (IL-1β and IL-18). Furthermore, microglia also undergo pyroptosis that could subsequently amplify the inflammation through triggering the release of neurotoxic and inflammatory mediators. Moreover, cytokines secreted could further drive a persistent inflammatory cascade and activate infiltrating neutrophils, causing neuronal death. cGAS antagonizing synthetic oligonucleotide A151 attenuates the overall neuroinflammatory response caused by cytoplasmic dsDNA through reducing microglia activation, preventing microglial pyroptosis, and inhibiting the peripheral neutrophils infiltration. Inhibiton of dsDAN sensing cGAS and AIM2 may thus exert neuroprotection and improve stroke outcomes by interrupting this inflammatory positive feedback loop.
Novel G-insertion mutation identified in male with FXS symptoms.Figure 1Predicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus. Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability. FMR1 mRNA levels were analyzed via RT-qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD. Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.
Antibiotics remove NOD2−/−-associated dysbiosis contributing to insulin resistance.Figure 6A Principal coordinates analysis (PCoA) performed on pairwise unweighted UniFrac distances in HFD-fed mice shows a strong antibiotic effect (PCoA1: 28%) and a strong similarity within donors and recipients (R) from the microbiota transfer experiment into germ-free mice (PCoA2: 16%). All mice were fed a HFD, each small dot is a mouse, and the most abundant bacterial genus are labeled and superimposed on the same PCoA plot. The size of the sphere representing a taxon is proportional to the mean relative abundance of the taxon across all samples. B Alpha diversity represented by the Shannon index (n ≥ 5/group), *P = 0.03 and **P = 0.0001. C Average relative abundance of the major genus in the cecum of each group of mice (n ≥ 5/group). D, E Impact of antibiotics (ATB) on relative population of Lactococcus genus and Lachnospiraceae family in all mice (E) (n ≥ 10/group), *P = 0.0001. F, Body mass (F) in 16 week HFD-fed WT and NOD2−/−mice without (CON) or with ATB in the drinking water for the final 4 weeks of the HFD (n ≥ 5/group), *P = 0.04, **P = 0.0008, and #P = 0.004. G Fasting bloodglucose (G) in 16 week HFD-fed WT and NOD2−/−mice without (CON) or with ATB in the drinking water for the final 4 weeks of the HFD (n ≥ 5/group), *P = 0.04, **P = 0.0008, and #P = 0.004. H, I Percentage change in fasting blood glucose (H) and AUC during GTTs (I) that was induced by ATB in HFD-fed WT and NOD2−/− mice (n ≥ 5/group), *P = 0.04 and **P = 0.005.
Stimulation of ATP synthase activity is related to decreased mobility.Figure 4A) Localizations and trajectory map of CV SU γ-HaloTag/TMRHTL particles in a single mitochondrion. Scale bar: 1 µm. Cumulative images of 2000 frames (recording 30 Hz). Each trajectory in a different color. Trajectories of the F1FO ATP synthase were generated by using the Multi-Target-Tracing-Tool (MTT) Sergé et al., 2008(). B) Diffusion coefficients for ATP synthase under control conditions (5.6 mM glucose), and under OXPHOS conditions. The probability density function (PDF) and cumulative density function (CDF) of -log D is plotted. Number of trajectories analyzed: 8622 (Glc) and 10333 (Gal) from mitochondria in at least 5 different cells. C) Mean diffusion coefficients D calculated from all individual trajectories. Mann-Whitney test. ***, p<0.001; **, p<0.01; *, p<0.5. Cells analyzed >5 per condition; number of trajectories: 4011 (Glc), 10333 (Gal); Q1: 0.00387 (Glc), 0.0031 (Gal); median DGlc: 0.0142 µm2/s, DGal: 0.00898 µm2/s; Q3: 0.0504 (Glc); 0.02969 Gal; p<10-31.
Figure 3Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.
Figure 5(A) Volcano curve analysis of dysregulated genes in miR-574-/- versus WT heart. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure. The dots above the dashed line show Padj<0.05. (B) Heatmap of significantly upregulated genes in miR-574-/- mice at baseline analyzed by RNA-Seq. P60 male mice, n=3 per group, Padj<0.05. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure. (C) Gene Ontology analysis of enriched pathways of upregulated genes in RNA-Seq. The top three pathways are listed with enriched gene sets. (D) Identification of Fam210a as a target of miR-574-5p and miR-574-3p. Seed sequence-bearing target genes were predicted by TargetScan. (E) Expression of Fam210a mRNA in the heart of miR-574-/- and WT mice from 3 biological replicates. (F) Expression of FAM210A protein in the heart of miR-574-/- and WT mice. Protein intensity was quantified in the right panel (n=6 per group). (G) Seed sequences of miR-574-5p and miR-574-3p in Fam210a mRNA 3'UTR (TargetScan) and their mutants. (H) Dual luciferase reporter assays with co-transfection of miR-574-5p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates. (I) Dual luciferase reporter assays with co-transfection of miR-574-3p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.
Genome-wide annotation of chromatin states highlights putative primary transcription factor targets.Figure 5A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8-FL cells. B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment. Enrichment over a random value and the p-value (based on the hypergeometric test) are shown (bars corresponding to non-significant enrichment values are semi-transparent). Analysis was performed separately for genes up- and down-regulated. C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.
Fig.Figure 2A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm. B) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. basic medium organoids 48 h after infection and normalized to GAPDH (n=6). C) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. full medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=9). D) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in basic medium vs. basic medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). E) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium + IFN-γ vs. basic medium+ IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). (B-E) Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by Student's t-test F) qPCR data comparing the relative virus load of SARS-CoV-2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium (left) and basic medium (right) untreated or treated with IFN-γ, indicating increase in virus load in differentiated and IFN-γ treated conditions (n=8). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test G) Electron microscopy imaging of organoids grown in basic medium, pretreated with IFN-γ for 3 days and infected for 48 h with SARS-CoV-2. Upper: Overview showing large virus-loaded vesicles in an epithelial cell. Scale bar: 500nm. Lower: Disintegrated organoid cell containing high virus load, scale bar: 2.5µm, higher magnification to visualize virus particles, scale bar: 100nm. H) Immunofluorescence for cleaved-caspase3 (red) and E-cadherin (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Scale bar: 50 µm. (below) Quantification of apoptotic cells, indicating increased cell death in the differentiated and IFN-γ treated condition after SARS-CoV-2 infection (n=7). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test
Figure 3A and B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice, respectively, for PSD95 (green, to label presynaptic photoreceptor terminals) and Cav1.4 (red, to label presynaptic Ca2+-channel). Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (A1 to A4) Magnification of Cav1.4 staining in the outer plexiform layer (OPL) in the retina of RBEWT/WT and (B1 to B4) RBEKO/KO mice. White arrow heads indicate Ca2+-channel distribution at selected synapses. Images represent 2 µm z-stack projections. Scale bar: 2 µm.
Figure 1Gating strategy for sorting of EpCAMhighCD24lowSca‑1+ cells and rMC from lung homogenate of adult mice. Time-course of epithelial stem/progenitor cell proliferation and differentiation within BALO at days 8, 11 and 21 of co-culture with rMC. Clonal expansion of EpCAMhighCD24lowSca‑1+ cells derived from either GFP-expressing mice or tdTomato-expressing ROSAmT/mG mice at day 21 of co‑culture. Percentages of SCGB1A1+SFTPC+ (BALO), SCGB1A1+SFTPC- (bronchiolospheres) and SCGB1A1-SFTPC+ (alveolospheres) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca-1+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice (n=4 biological replicates). Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca-1+SCGB1A1+SFTPC+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice. Heat map (left) and tSNE plot (right) (F) of digested day 21 BALO cultures depicting four distinct clusters (C1, airway, blue; C2, alveolar, purple; C3, MYO, orange, and C4, LIF, green). Violin plots of selected genes representing airway associated-genes (G) (Itgb4, Trp63, Krt7, and Sox2) and alveoli associated‑genes (H) (Cxcl15, Lyz2, Sftpc, and Hopx). Each violin plot shows the frequency distribution of the mean transcript level (log2).
Figure 6 A) Larval brains at 16 h ALH from wild-type (yw) and E-cadR69 were analyzed for EdU incorporation, and larval brains were labeled with EdU, Dpn and Mira  B) Quantification graph of the percentage of qNSCs with primary protrusion in wild-type (yw), E-cadR69 and E-cadR69.Ubi-p63E-E-cad cells. GFP at 16 h ALH. Larval brains were labeled with Dpn and Mira (not shown). n=19 BL for control; n=23 BL for E-cadR69; n=11 BL for E-cadR69.Ubi-p63E-E-cad BL. ****p<0.0001; p=0.2690 (ns)  C) Quantification graph of NSC diameter at 16 h ALH from wild-type (yw), E-cadR69 and E-cadR69.Ubi-p63E-E-cad.GFP. NSCs were marked by Dpn and Mira. n=110 NSCs for control; n=131 NSCs for E-cadR69; n=114 NSCs for E-cadR69.Ubi-p63E-E-cad. ****p<0.0001; p=0.8151 (ns)  D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=10 BL for control; n=20 BL for per E-cadR69. ****p<0.0001  E) Larval brains at 24 h ALH from the wild-type control (yw) and klp64Dk5h/Df, kap3V6 and kap3 V6; g-kap3#11 were stained with EdU, Dpn, and Mira  F) Quantification graph of NSCs with primary protrusion per brain lobe for genotypes in (E) as well as klp64Dk5h, kap3V5, and kap3V6; g-kap3#31. n=26 BL for control; n=16 BL for klp64Dk5h; n=7 BL for klp64Dk5h/Df; n=20 BL for kap3V5; n=7 BL for kap3V6; n=6 BL for kap3V6; g-kap3#11; n=7 BL for kap3V6; g-kap3#31. ****p<0.0001; control vs. kap3V6; g-kap3#11, p=0.9998 (ns); control vs. kap3V6; g-kap3#31, p>0.9999 (ns)  G) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (E) as well as klp64Dk5h, kap3V5, and kap3V6; g-kap3#31, which were not shown in (E). n=33 BL for control; n=15 BL for klp64Dk5h; n=11 BL for klp64Dk5h/Df; n=20 BL for kap3V5; n=10 BL for kap3V6; n=10 BL for kap3V6; g-kap3#11; n=10 BL for kap3V6; g-kap3#31. ****p<0.0001; control vs. kap3V6; g-kap3#11, p=0.9713 (ns); control vs. kap3V6; g-kap3#31, p>0.9999 (ns)  H) Quantification graph of PH3-positive NSCs (Dpn+, Mira+) per brain lobe for wild-type control (yw), klp64Dk5h klp64Dk5h/Df, kap3V5, kap3V6 and kap3V6; g-kap3#11, and kap3V6; g-kap3#31 larval brains. n=26 BL for control; n=16 BL for klp64Dk5h; n=7 BL for klp64Dk5h/Df; n=20 BL for kap3V5; n=7 BL for kap3V6; n=6 BL for kap3V6; g-kap3#11; n=6 BL for kap3V6; g-kap3#31. ****p<0.0001; control vs. kap3V6; g-kap3#11, p=0.0977 (ns); control vs. kap3V6; g-kap3#31, p=0.1658 (ns)  I) Larval brains at 24h ALH from wild-type control (yw), klp64Dk5/Df, and kap3V5 were analyzed E-cad at the NSC-neuropil contact sites, and NSCs were labeled with Dpn and Msps  J) Quantification graph of the fold change of basal E-cad intensity in qNSCs with primary protrusion for genotypes in (I). n=85 NSCs for control; n=40 NSCs for klp64Dk5/Df; n=48 NSCs for kap3V5. ****p<0.0001.
Figure 4(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**). (D) High-magnification confocal images of the upper layer show that mH2A1.2 ko results in abnormally branched processes compared to WT. The GFP plasmid was electroporated into the WT and KO embryonic brains at E13.5 and harvested at P0. Scale bar represents 10 μm. (E) Quantification of dendritic numbers in WT and KO mouse cortices. n = 30 for all samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**). (F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H) Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(
a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h).figf4(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain. (b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin. (d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels. (e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli. (f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers. (g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.
deficits result in abnormal cell proliferation (a) A diagram of the BrdU labeling time point in electroporation experiments.Figure 2(a) A diagram of the BrdU labeling time point in electroporation experiments. (b) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm. (c) Statistical analysis of 2-hour BrdU labeling experiments. n=6 brains Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). (d) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs. (e) Immunostaining with pax6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. (f) Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n=3 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). (g) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains. White arrows represent GFP, sox2 double positive cells; Red arrows represent GFP, TBR2 double positive cells. area. Scale bar: 50 μm. (h) Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n=8 brains for each group. (i) Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n=5 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).