Cedrin identified from <i>Cedrus deodara</i> (Roxb.) G. Don protects PC12 cells against neurotoxicity induced by Aβ_1–42

<p>Alzheimer’s disease is a severe neurodegenerative disease affecting elder worldwide and closely related to the neurotoxicity induced by amyloid β. To find efficient therapeutics, we have investigated the protective effects of cedrin from <i>Cedrus deodara</i> (Roxb.) G. Don on PC12 cells against the neurotoxicity induced by amyloid β_1–42. The results have shown the viability of PC12 cells injured by amyloid β_1–42 can be improved by cedrin. Cedrin can reduce reacrive oxygen species overproduction, increase the activity of superoxide dismutase and decrease malondialdehyde content. Meanwhile, the loss of mitochondrial membrane potential and mitochondrial permeability transition pore opening in PC12 cells, and elevated Caspase-3 activity, downregulated Bcl-2 and upregulated Bax are meliorated. These results demonstrate the protective effect of cedrin is related to the inhibition of oxidative stress, improvement of mitochondrial dysfunction and suppression of apoptosis. This investigation gives evidences for the application of cedrin in practice and further investigation <i>in vivo</i>.</p

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

<div><p>Background</p><p>Recent studies have linked certain single nucleotide polymorphisms in the <i>leucine-rich repeat kinase 2 (LRRK2)</i> gene with Parkinson’s disease (PD). Among the mutations, <i>LRRK2</i> c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.</p><p>Principle Findings</p><p>Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.</p><p>Conclusion</p><p>Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.</p></div

Neurons ectopically expressing R1628P displayed a higher sensitivity to MPP+ in a Cdk5-dependent manner.

<p><b>a.</b> Neurons with the R1628P mutation display a higher sensitivity to MPP+. Primary-cultured cortical neurons from wild-type mice were transfected with GFP-tagged wild-type (WT) or R1628P mutant LRRK2 plasmids for 24 h, and then exposed to MPP+ (30 μM) for 24 h. The dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. <b>b.</b> Cdk5 deletion protects the neurons with the R1628P mutation from MPP+ toxicity. The above procedures were performed in primary-cultured cortical neurons from the neuronal Cdk5 conditional knockout mice. Single cell survival assay was conducted as above. The results represent at least three independent experiments as the mean ± SD, **P<0.01 (ANOVA). <b>c.</b> The higher sensitivity of R1628P to MPP+ requires the phosphorylation of S1627 on LRRK2. Primary-cultured cortical neurons from wild-type mice were transfected with GFP vector, GFP-tagged wild-type (WT) LRRK2 or R1628P, S1627A:R1628P, S1627D:R1628P mutant for 24 h, and then exposed to MPP+ (30 μM) for 24 h. The dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. The results represent at least three independent experiments as the mean ± SD, *P<0.05, (ANOVA)</p

R1628P mutation do not alter the LRRK2 activity and cause neuronal toxicity directly.

<p><b>a.</b> Kinase activity of LRRK2 mutants tested by kinase assay using MBP as substrate. HEK293 cells were transfected with HA-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N as indicated. After 24 h, LRRK2 was immunoprecipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. The bottom panel shows equal expression of HA-LRRK2 and MBP used in the lysates. <b>b.</b> The graph is the quantification of LRRK2 kinase activities in Fig 1A. The numbers are relative values, with WT set to 1. <b>c.</b> Kinase activity of LRRK2 mutants tested by kinase assay using LRRKtide as substrate. HA-tagged WT LRRK2 or mutants were immunoprecipitated as above, then <i>in vitro</i> LRRK2 kinase assay was performed using LRRKtide as substrate. <b>d.</b> Overexpression of R1628P mutation do not cause neuronal cell death. Primary-cultured cortical neurons were transfected with GFP-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N. After 48 h transfection, the dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. All the above results represent at least three independent experiments as the mean ± SD, *P<0.05, **P<0.01 (ANOVA).</p

The Schematic diagram shows that the R1628P genetic mutation of LRRK2 provide a potential “two-hit” target of environment toxic MPP+-induced Cdk5 activation, and Cdk5 could phosphorylate the adjacent amino residue S1627 of R1628P mutation, thus activate LRRK2 kinase activity and cause neuronal death.

<p>The Schematic diagram shows that the R1628P genetic mutation of LRRK2 provide a potential “two-hit” target of environment toxic MPP+-induced Cdk5 activation, and Cdk5 could phosphorylate the adjacent amino residue S1627 of R1628P mutation, thus activate LRRK2 kinase activity and cause neuronal death.</p

Additional file 2: Figure S1. of The association between obstructive sleep apnea and metabolic syndrome: a systematic review and meta-analysis

Meta-analysis for all studies including two conference reports that were not subsequently published. Two conference reports referred by Velazquez, 2011 and Papatsimpas, 2011. OR: odds ratio; CI: confidence interval. (TIFF 156 kb

Additional file 3: Figure S2. of The association between obstructive sleep apnea and metabolic syndrome: a systematic review and meta-analysis

Funnel plots among all studies including two conference reports that were not subsequently published. Two conference reports referred by Velazquez, 2011 and Papatsimpas, 2011. (TIFF 45 kb

Phosphorylation of R1628P mutant by Cdk5 increases the LRRK2 activity and causes cell death.

<p><b>a.</b> Plasmids of HA-tagged wild-type (WT) or R1628P mutant LRRK2 were cotransfected with Cdk5/p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. HA-LRRK2, Cdk5, and p35 levels were determined by Western blotting as a loading control. <b>b.</b> The graph is the quantification of LRRK2 kinase activities in Fig 3A. The numbers are relative values, with WT w/o Cdk5/p35 set to 1. <b>c.</b> Plasmids of HA-tagged wild-type (WT), R1628P, and S1627A:R1628P mutant LRRK2 were cotransfected with Cdk5 or dominant-negative form of Cdk5 (dnCdk5) and p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase activity was measured for 30min using LRRKtide as substrate. The results represent as the mean ± SD, **P<0.01 (compared with WT:control group) ##P<0.01 (compared with WT:Cdk5/p35 group) (ANOVA). <b>d.</b> Phosphorylation mimic of S1627 (S1627D) increased the LRRK2 kinase activity. HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P double mutant. LRRK2 kinase activities were measured as above. <b>e.</b> The graph is the quantification of LRRK2 kinase activities in Fig 3D. The numbers are relative values, with WT set to 1. **P<0.01 (ANOVA) <b>f.</b> HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P. LRRK2 kinase activities were measured as above for indicated period of time using LRRKtide as substrate. **P<0.01 (compared with WT group, ANOVA)</p

Additional file 1: of Effectiveness and safety of bifidobacteria and berberine in people with hyperglycemia: study protocol for a randomized controlled trial

SPIRIT 2013 Checklist: recommended items to address in a clinical trial protocol and related documents*. (DOCX 591 kb

Gender Differences in the Prevalence and Development of Metabolic Syndrome in Chinese Population with Abdominal Obesity

<div><p>Background</p><p>Not all the people with metabolic syndrome (MS) have abdominal obesity (AO). The study aimed to investigate gender differences in the prevalence and development of MS in Chinese population with abdominal obesity, which has rarely been reported.</p> <p>Methods</p><p>Data were obtained from the 2007-08 China National Diabetes and Metabolic Disorders Study, and participants were divided into two samples for analysis. Sample 1 consisted of 19,046 people with abdominal obesity, while sample 2 included 2,124 people meeting pre-specified requirements. Survival analysis was used to analyze the development of MS.</p> <p>Results</p><p>The age-standardized prevalence of MS in Chinese population with AO was 49.5%. The prevalence in males (73.7%) was significantly higher than that in females (36.9%). Males had significantly higher proportions of combinations of three or four MS components than females (36.4% <i>vs</i>. 30.2% and 18.4% <i>vs</i>. 5%, respectively). MS developed quick at first and became slow down later. Half of the participants with AO developed to MS after 3.9 years (95% CI: 3.7–4.1) from the initial metabolic abnormal component, whereas 75% developed to MS after 7.7 years (95% CI: 7.5–7.9).</p> <p>Conclusion</p><p>Compared with females, Chinese males with AO should receive more attention because of their higher prevalence of MS and its components, more complex and risky combinations of abnormal components, and faster development of MS.</p> </div