Fig 2: DOK7 gene therapy shows no obvious effects on motor neuron survival and motor axon atrophy in ALS miceWT or ALS mice treated or not with 1.2 × 1012 vg of AAV‐D7 at P90 were analyzed at P120. Nissl staining of the L4‐L5 spinal segment. Scale bars, 200 μm. Insets show higher magnification of the ventral horn in the boxed regions (scale bars, 100 μm). NT, not treated.Quantification of surviving motor neurons (n = 5 mice). Values are means ± SEM. ***P < 0.0001 by one‐way ANOVA with Bonferroni's post hoc test. n.s., not significant.Anti‐neurofilament‐H staining of the L4 ventral roots. Insets show higher magnification of the boxed regions. Scale bars, 50 μm.Size distribution of motor axons (n = 5 mice). Values are means ± SEM.

Fig 3: Overall endothelial-dependent mechanisms by which blueberry polyphenols may attenuate hypertension. Upregulated renin–angiotensin–aldosterone system results in the increased synthesis of angiotensin (Ang) II, which causes vasoconstriction due to reduced endothelial-derived nitric oxide (NO). (A) Ang II binds to Ang II type-1 receptor (AT1R), which results in the activation of NADPH-oxidase (NOX)1. NOX1 synthesizes superoxide anions (O2•−), which can react with NO, reducing NO bioavailability. (B) Blueberries may reverse this effect by increasing the synthesis of NO via increased endothelial NO synthase (eNOS) activity. (C) Mitogen-activated protein kinases (MAPKs), such as p38MAPK and stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK), are redox-sensitive kinases activated by upstream MAPK kinase kinase (MAPKKK). The phosphorylation of p38MAPK and SAPK/JNK expression was attenuated by blueberry polyphenols, suggesting reduced cellular oxidative stress. (D) Inhibitor of nuclear factor kappa B (IκB) kinase (IKK) is also a redox-sensitive kinase, which can undergo auto-phosphorylation upon interaction with reactive oxygen species (ROS) and is upstream of NF-κB. Following IKK phosphorylation, IκB is phosphorylated and the p65 subunit of NF-κB is then also phosphorylated. IκB then undergoes ubiquitin-dependent degradation, liberating NF-κB to translocate to the nucleus where it is involved in inflammatory transcription. Blueberries, however, reduced p65 phosphorylation, suggesting reduced IKK phosphorylation via reduced ROS. (E) These cytoprotective effects of blueberry polyphenols may be mediated by nuclear factor-erythroid factor 2-related factor 2 (NRF2), a master regulator of antioxidant enzymes. Kelch-like ECH-associated protein (KEAP)1 sequesters NRF2 in the cytosol and the entire complex undergoes ubiquitin-dependent degradation. However, conformational changes in KEAP1 prevent NRF2 binding, allowing NRF2 to translocate to the nucleus where it is involved in the transcription of antioxidant enzymes, NADPH dehydrogenase quinone (NQO)1 and superoxide dismutase (SOD)1, both of which can neutralize O2•−. Blueberries increase cellular NRF2 concentration possibly via interaction with KEAP1, resulting in increased NQO1 and SOD1, overall reducing NOX1-mediated oxidative stress preventing NO neutralization via O2•−.

Fig 4: TorsinA overexpression restores ER homeostasis to animals expressing G85R SOD1. (A) Bar chart representing normalized GFP pixel intensity values measured consistently in the boxed region shown in B. These worms express the hsp-4::GFP reporter alone or with other transgenic constructs as displayed in the figure. Values are the mean ± s.d. of three experiments where 30 animals were analyzed per replicate (*P<0.01; Student’s t-test). Animals expressing G85R SOD1 elicit an ER stress response, whereas WT SOD1 animals display an ER stress response that is comparable to hsp-4::GFP animals. (B) Representative fluorescent micrographs of animals expressing the ER stress reporter hsp-4::GFP alone (upper panel) and with an intestinally expressed G85R SOD1 transgene (lower panel). The image depicts the anterior region of C. elegans and the boxed region indicates the 100×100 μm region just below the pharynx where measurements of fluorescence were taken in all animals. (C) The stress response of animals expressing G85R SOD1 was temperature dependent. The relative stress level of G85R SOD1 worms increased significantly at 20°C and 25°C compared with the response at 15°C (**P<0.001; Student’s t-test). Furthermore, worms expressing G85R SOD1 also displayed significantly more ER stress compared with those expressing either WT SOD1 or the hsp-4::GFP control at 20°C and 25°C, confirming a physiological temperature-sensitive characteristic of the mutant G85R SOD1 protein (*P<0.01; **P<0.001; Student’s t-test). (D) Bar chart representing normalized GFP pixel intensity values. These worms express the hsp-4::GFP reporter alone or with other transgenic constructs as displayed in the figure. Values are the mean ± s.d. of three experiments where 30 animals were analyzed per replicate (*P<0.05; one-way ANOVA analysis with Tukey post-hoc test). Animals expressing G85R SOD1 elicit an ER stress response that is not restored by torsinA variants K108A and Δ1–40 torsinA, as seen with WT torsinA, where there is a significant reduction in ER stress response. All normalized values are to G85R SOD1 fluorescent intensities.

Fig 5: Effect of CHS on the activities of oxidative stress-related markers in rat pancreas after 4 weeks of intervention: (a) ROS level; (b) MDA level; (c) SOD activity; (d) GSH activity; (e) GPx activity; (f) the Nrf2 transcriptional activity; (g) effects of CHS on the expression levels of Nrf2 in cytoplasm and nuclear protein; (h) the ratio of nuclear/cytoplasm Nrf2 levels. The columns and error bars are presented as means ± SEM (n = 10). ##P < 0.01 compared with the CON group. ∗∗P < 0.01 and ∗P < 0.05 compared with the MOD group.