Additional file 1 of Multilayer factors associated with excess all-cause mortality during the omicron and non-omicron waves of the COVID-19 pandemic: time series analysis in 29 countries

Supplementary Material

Estrogen Receptor (ER)-α36 Is Involved in Estrogen- and Tamoxifen-Induced Neuroprotective Effects in Ischemic Stroke Models

<div><p>The neuroprotection by estrogen (E2) and tamoxifen is well documented in experimental stroke models; however, the exact mechanism is unclear. A membrane-based estrogen receptor, ER-α36, has been identified. Postmenopausal-levels of E2 act through ER-α36 to induce osteoclast apoptosis due to a prolonged activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) signaling. We hypothesized that ER-α36 may play a role in the neuroprotective activities of estrogen and tamoxifen. Here, we studied ER-α36 expression in the brain, as well as its neuroprotective effects against oxygen and glucose deprivation (OGD) in PC12 cells. We found that ER-α36 was expressed in both rat and human brain. In addition, OGD-induced cell death was prevented by l nmol/L 17β-estradiol (E2β). E2β activates the MAPK/ERK signaling pathway in PC12 cells under basal and OGD conditions by interacting with ER-α36 and also induces ER-α36 expression. Low-dose of tamoxifen up-regulated ER-α36 expression and enhanced neuronal survival in an ovariectomized ischemic stroke model. Furthermore, low-dose of tamoxifen enhanced neuroprotective effects by modulating activates or suppress ER-α36. Our results thus demonstrated that ER-α36 is involved in neuroprotective activities mediated by both estrogen and tamoxifen.</p></div

Expression of ER-α36 and caveolin-1 protein in the human cerebral cortex.

<p>Immunocytochemical staining of ER-α36 (green), caveolin-1 (red), and nuclei (blue) in brain slices at different magnifications. Scale bars = 75 μm/10 μm.</p

The ischemic brain of the MCAO model.

<p>Ischemia-reperfusion produced infarcts in the rat hippocampus, cortex and striatum. Red arrows show that ATP activity was decreased in the injured hippocampal CA1 region, as revealed by ATPase staining.</p

ER-α36 is involved in mitogen-activated protein kinase (MAPK) signaling pathway activation.

<p>(<b>A</b>) Low-dose E2β-induced activation of MAPK in PC12 cells under non-injury conditions. (B) Low-dose E2β induced activation of the ER-α36 promoter. The results represent a percentage of the control values (no exposure to OGD), expressed as mean ± standard deviation (<i>s</i>.<i>d</i>.; n = 3). *<i>p</i> < 0.05 vs. control.</p

ER-α36 mediates the ability of estradiol 17β (E2β) to protect against oxygen and glucose deprivation (OGD).

<p>(<b>A</b>) OGD reduced PC12 cell viability after 6 h. (<b>B</b>) Different IC162 concentrations increased the protection against OGD in PC12 cells. (<b>C</b>) Decreased expression of ER-α36 after knockdown. ER-α36 knockdown promoted apoptosis in PC12 cells exposed to 6-h OGD, and weakened the neuroprotective effect of E2β. The results represent a percentage of the control values (no exposure to OGD), expressed as mean ± standard deviation (<i>s</i>.<i>d</i>.; <i>n</i> = 3). *<i>p</i> < 0.05, ** <i>p</i> < 0.01 vs. control; ^#<i>p</i> < 0.05, ^##<i>p</i> < 0.01 vs. vehicle-treated cells.</p

Estrogen receptor (ER)-α36 expression in the rat hippocampus.

<p>(<b>A</b>) Hyperfluorescence of ER-α36 expressed in the CA1 and CA3 regions of the rat hippocampus, and co-staining of ER-α36 with the neuronal marker, NeuN. (<b>B</b>) Nissl staining of the hippocampus. (<b>C</b>) Statistical analysis of ER-α36 expression. Scale bar = 200 μm (200×), <i>n</i> = 3. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Diagram of the neuroprotective effects of estrogen and TAM.

<p>Diagram of the neuroprotective effects of estrogen and TAM.</p

ER-α36 expression and cell injury in ischemic stroke models.

<p>(<b>A</b>) Immunofluorescence staining revealed that ER-α36 expression decreased in the hippocampal CA1 region of rats subjected to ovariectomy (OVX) and/or middle cerebral artery occlusion (MCAO). (<b>B</b>) The results of Nissl staining and (<b>C</b>) western blots of caspase-3 protein showed obvious neural injury in the OVX/MCAO group. Scale bar = 200 μm (200×), <i>n</i> = 3. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Immunogenicity, antibody persistence, and safety of the 60 μg hepatitis B vaccine in hemodialysis patients: a multicenter, randomized, double-blind, parallel-controlled trial

<p><b>Background:</b> To evaluate the immunogenicity, antibody persistence, and safety of the 60 µg hepatitis B vaccine in hemodialysis patients in China.</p> <p><b>Methods:</b> We conducted a multicenter, randomized, double-blind, parallel-controlled trial including 352 hemodialysis patients who were centrally randomized in a ratio of 1:1 to receive a 20 µg (IM20 group) or 60 µg (IM60 group) recombinant hepatitis B vaccine at months 0, 1, and 6.</p> <p><b>Results:</b> The vaccine-elicited antibody responses peaked at month 7, and declined at month 12. At month 7, the IM60 group had stronger GMC of anti-HBs, and a higher proportion of seroconversion and high-level response than the IM20 group did (<i>P</i> < 0.05). Better immune responses were observed in the IM60 group, especially for those aged or in the high-frequency hemodialysis population.</p> <p><b>Conclusion:</b> The high dose 60 µg recombinant hepatitis B vaccines elicited stronger immune responses than the 20 µg hepatitis B vaccine did among hemodialysis patients.</p> <p><b>Clinical trial registration</b>: ClinicalTrials.gov, number NCT02963714.</p