Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae.

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by ß-carotene hydroxylase (crtZ) and ß-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.

Efficacy and Safety of Teriflunomide in Chinese Patients with Relapsing Forms of Multiple Sclerosis: A Subgroup Analysis of the Phase 3 TOWER Study.

BACKGROUND: Disease-modifying therapy is the standard treatment for patients with multiple sclerosis (MS) in remission. The primary objective of the current analysis was to assess the efficacy and safety of two teriflunomide doses (7 mg and 14 mg) in the subgroup of Chinese patients with relapsing MS included in the TOWER study. METHODS: TOWER was a multicenter, multinational, randomized, double-blind, parallel-group (three groups), placebo-controlled study. This subgroup analysis includes 148 Chinese patients randomized to receive either teriflunomide 7 mg (n = 51), teriflunomide 14 mg (n = 43), or placebo (n = 54). RESULTS: Of the 148 patients in the intent-to-treat population, adjusted annualized relapse rates were 0.63 (95% confidence interval [CI]: 0.44, 0.92) in the placebo group, 0.48 (95% CI: 0.33, 0.70) in the teriflunomide 7 mg group, and 0.18 (95% CI: 0.09, 0.36) in the teriflunomide 14 mg group; this corresponded to a significant relative risk reduction in the teriflunomide 14 mg group versus placebo (-71.2%, P = 0.0012). Teriflunomide 14 mg also tended to reduce 12-week confirmed disability worsening by 68.1% compared with placebo (hazard ratio: 0.319, P = 0.1194). There were no differences across all treatment groups in the proportion of patients with treatment-emergent adverse events (TEAEs; 72.2% in the placebo group, 74.5% in the teriflunomide 7 mg group, and 69.8% in the teriflunomide 14 mg group); corresponding proportions for serious adverse events were 11.1%, 3.9%, and 11.6%, respectively. The most frequently reported TEAEs with teriflunomide versus placebo were neutropenia, increased alanine aminotransferase, and hair thinning. CONCLUSIONS: Teriflunomide was as effective and safe in the Chinese subpopulation as it was in the overall population of patients in the TOWER trial. Teriflunomide has the potential to meet unmet medical needs for MS patients in China. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00751881; https://clinicaltrials.gov/ct2/show/NCT00751881?term=NCT00751881&rank=1.

Neuroprotection and CD131/GDNF/AKT Pathway of Carbamylated Erythropoietin in Hypoxic Neurons.

Carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to neuroprotective effects without erythropoiesis. However, little is known about molecular mechanisms behind CEPO-mediated neuroprotection. In primary neurons with oxygen-glucose deprivation (OGD) and mice with hypoxia-reoxygenation, the neuroprotection and possible molecular mechanism of CEPO were performed by immunohistochemistry and immunocytochemistry, Western blot, RT-PCR, and ELISA. The comparisons were analyzed by ANOVA followed by unpaired two-tailed Student's t test. Both CEPO and EPO showed the neuroprotective effects in OGD model and hypoxic brain. CEPO did not trigger JAK-2 but activated AKT through glial cell line-derived neurotrophic factor (GDNF). It has been shown that CEPO acts upon a heteroreceptor complex comprising both the EPO receptor and the common ß receptor subunit (ßcR, also known as CD131). The blockage of CD131 reduced CEPO-mediated GDNF production, while GFR receptor blockage and GDNF neutralization inhibited CEPO-induced neurogenesis. Addition of GDNF to cultured neurons increased phosphorylation of AKT. CEPO protects neurons possible through the CD131/GDNF/AKT pathway.

The lack of CD131 and the inhibition of Neuro-2a growth by carbamylated erythropoietin.

Recombinant human erythropoietin (EPO), a glycohormone, is one of the leading biopharmaceutical products, while carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to its neuroprotective effects without erythropoiesis in several cells and animal models. However, exogenous EPO promotes an angiogenic response from tumor cells and is associated with tumor growth, but knowledge of CEPO on tumor growth is lacking. Here we show that CEPO, but not EPO, inhibited Neuro-2a growth and viability. As expected, CEPO--unlike EPO--did not activate JAK-2 either in primary neurons or in Neuro-2a cells. Interestingly, CEPO did not induce GDNF expression and subsequent AKT activation in Neuro-2a cells. Before CEPO/EPO treatment, glial cell line-derived neurotrophic factor (GDNF) neutralization and GFR receptor blocking decreased the viability of EPO-treated Neuro-2a cells but did not influence CEPO-treated Neuro-2a cells. As compared to primary neurons, the expression of CD131, as a receptor complex binding to CEPO, is almost lacking in Neuro-2a cells. In BABL/C-nu mice, CEPO did not promote the growth of Neuro-2a cells nor extended the survival time compared to mice treated with EPO. The results indicate that CEPO did not promote tumor growth because of lower expression of CD131 and subsequent dysfunction of CD131/GDNF/AKT pathway in Neuro-2a cells, revealing its therapeutic potential in future clinical application.

Histone decacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection against MPP+.

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disease, characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra. Recent investigations have shown that mitochondrial fragmentation, an early event during apoptosis, is implicated in the degeneration of DA neurons in PD, and more importantly, preventing mitochondrial fragmentation could rescue cell death in several PD models. Therefore, mitochondrial dynamics may be a therapeutic target for early intervention in PD. However, much remains unknown about the mechanism underlying mitochondrial fragmentation in PD. METHODS: The alterations in mitochondrial morphology, cell apoptosis, and mitochondrial shaping protein levels were detected after SH-SY5Y cells were treated with various doses of MPP+ or rotenone. RESULTS: Mitochondrial fragmentation is an early event during apoptosis caused by MPP+ but not rotenone, and Trichostatin A (TSA), a commonly used histone deacetylase (HDAC) inhibitor, selectively rescues mitochondrial fragmentation and cell death induced by lower doses of MPP+. Mitochondrial fragmentation triggered by lower doses of MPP+ may be a result of Mfn2 down-regulation, which could be completely reversed by TSA. Further investigation suggests that TSA prevents MPP+-induced Mfn2 down-regulation via inhibiting histone deacetylation over Mfn2 promoter and alleviating its transcriptional dysfunction. CONCLUSIONS: Histone deacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection in PD cell model induced by MPP+. Hence, HDAC inhibitors may be a potential early treatment for PD.

Novel mutations m.3959G>A and m.3995A>G in mitochondrial gene MT-ND1 associated with MELAS.

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are progressive neurodegenerative disorder associated with polygenetic, maternally inherited mutations in mitochondrial DNA. Approximately 80% of MELAS cases are caused by the mutation m.3243A>G of the mitochondrial tRNA(Leu (UUR)) gene (MT-TL1). We reported two probands with MELAS features. Muscle biopsy identified ragged-red fibers (RRF) in Gomori Trichrome staining. A respiratory chain function study showed decreased activity of mitochondrial respiratory chain complex I in both probands. Sequencing of the mitochondrial DNA revealed two novel MT-ND1 gene missense mutations, m.3959G>A and m.3995A>G, which are highly conserved among species. Protein secondary structure predictions demonstrated that these mutations may alter the peptide structure and may lead to decreased ND1 gene stability. Our findings suggest that these two novel mutations may contribute to the MELAS phenotypes of the patients in our study.

Carbamylated erythropoietin ameliorates hypoxia-induced cognitive and behavioral defects with the generation of choline acetyltransferase-positive neurons.

Carbamylated erythropoietin (CEPO) is attracting widespread interest because of its neuroprotective effects without influencing erythropoiesis. Here we show that CEPO, unlike EPO, does not stimulate erythropoiesis. Both CEPO and EPO inhibit the death/apoptosis of neurons in the hypoxic model of primary neurons and induce neuron proliferation and differentiation in hypoxic mice. Hypoxic mice show apparent memory deficits at 3 and 30 days after hypoxia. The administration of CEPO/EPO significantly improves cognitive and behavioral defects after hypoxic insults. Further investigation shows that CEPO/EPO induces neuron proliferation and differentiation and promotes the generation of choline acetyltransferase (ChAT)(+) neurons in hypoxic mice. Phosphorylated AKT was colabeled with ChAT(+) neurons and coexpressed in bromodeoxyuridine-positive cells, suggesting that the PI3K/AKT pathway may play a pivotal role in CEPO/EPO-cholinergic neuron generation. These results reveal that CEPO/EPO ameliorates hypoxia-induced cognitive and behavioral defects possibly through the generation of ChAT-positive neurons.

HLA-DQA1*03:02/DQB1*03:03:02 is strongly associated with susceptibility to childhood-onset ocular myasthenia gravis in Southern Han Chinese.

OBJECTIVE: Our aim was to investigate the correlation between onset age, clinical features and HLA-DQA1/DQB1 genetic variability in myasthenia gravis (MG) patients in Southern Han Chinese. METHODS: 205 MG patients and 100 controls were genotyped for HLA-DQA1 and -DQB1 using sequence-based typing (SBT) and analyzed for haplotype frequencies. Anti-acetylcholine receptor (AChR) autoantibodies were measured in all, and muscle-specific tyrosine kinase (MuSK) antibodies were tested in AChR antibody negative patients. RESULTS: HLA-DQA1/DQB1 haplotypes showed association only with childhood-onset MG. Haplotype DQA1*03:02/DQB1*03:03:02 (DQ9) was positively associated with the childhood-onset MG, while haplotype DQA1*02:01/DQB1*02:02 and DQA1*05:01:01/DQB1*02:01:01 (DQ2) were negatively associated with this group. Childhood-onset ocular MG patients had an extremely high phenotype frequency of DQ9 haplotype (90.1% of patients, 34.0% of controls, p≤0.0001, OR=17.8). CONCLUSIONS: The childhood-onset ocular MG in Southern Han Chinese may present a particular subgroup of distinct genetic background. Its correlation to the HLA haplotype DQA1*03:02/DQB1*03:03:02 might explain the phenotypic difference of MG between Han Chinese and Caucasians.

The polymorphism of the ATP-binding cassette transporter 1 gene modulates Alzheimer disease risk in Chinese Han ethnic population.

BACKGROUND: Recent studies highlight a potential role of cholesterol metabolic disturbance in the pathophysiology of Alzheimer disease (AD). The adenosine triphosphate (ATP)-binding cassette transporter 1 (ABCA1) gene resides within proximity of linkage peaks on chromosome 9q influence AD and plays a key role in cellular cholesterol efflux in the brain. METHODS: We studied the role of R219K and V825I polymorphisms of ABCA1 in modulating the risk of AD in 321 AD patients and 349 comparisons of Chinese Han. Genotyping of R219K and V825I were performed by PCR-restriction fragment length polymorphism analysis. RESULTS: The genotype distribution of R219K was different with more RK in total AD group (χ(2) = 8.705, df = 2, p = 0.013), late-onset AD (LOAD) group (χ(2) = 10.636, df = 2, p = 0.005), APOE non-ε4ε4 group (χ(2) = 9.900, df = 2, p = 0.007), and female AD group (χ(2) = 8.369, df = 2, p = 0.015). Logistic regression manifested the risk of AD increased in RK carriers in total AD group (Wald = 6.102, df = 1, p = 0.014, odds ratio [OR]: 1.546, 95% confidence interval [95% CI]: 1.094-2.185), LOAD group (Wald = 7.746, df = 1, p = 0.005, OR: 1.921, 95% CI: 1.213-3.041), and APOE non-ε4ε4 group (Wald = 6.399, df = 1, p = 0.011, OR: 1.586, 95% CI: 1.109-2.266). K allele (RK + KK) also increased the risk of AD compared with RR allele in LOAD group (Wald = 4.750, df = 1, p = 0.029, OR: 1.619, 95% CI: 1.050-2.497). However, no discrepancy was found in V825I. In R219K, age at onset (AAO) was significantly lower by 4.9 years on average in patients of KK genotype than those of RK in APOE ε4 carrying group and higher by 5.5 years in patients of KK genotype than those of RR in APOE ε4 noncarrying group. In V825I, AAO was diseased by 4.3 years in II genotype compared with VV genotype in APOE ε4 noncarrying group and 3.4 years in APOE ε4ε4 noncarrying group. CONCLUSION: The results indicated that the RK genotype or K allele (RK + KK) of R219K may relate to the development of AD in the east of China.

TSPO-specific ligand vinpocetine exerts a neuroprotective effect by suppressing microglial inflammation.

Vinpocetine has long been used for cerebrovascular disorders and cognitive impairment. Based on the evidence that the translocator protein (TSPO, 18 kDa) was expressed in activated microglia, while Vinpocetine was able to bind TSPO, we explored the role of Vinpocetine on microglia treated with lipopolysaccharide (LPS) and oxygen-glucose deprivation (OGD) in vitro. Our results show that both LPS and OGD induced the up-regulation of TSPO expression on BV-2 microglia by RT-PCR, western blot and immunocytochemistry. Vinpocetine inhibited the production of nitrite oxide and inflammatory factors such as interleukin-1ß (IL-1ß), IL-6 and tumour necrosis factor-α (TNF-α) in BV-2 microglia, in which cells were treated with LPS or exposed to OGD, regardless of the time Vinpocetine was added. Next, we measured cell death-related molecules Akt, Junk and p38 as well as inflammation-related molecules nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Vinpocetine did not change cell death-related molecules, but inhibited the expression of NF-κB and AP-1 in LPS-stimulated microglia, indicating that Vinpocetine has an anti-inflammatory effect by partly targeting NF-κB/AP-1. Next, conditioned medium from Vinpocetine-treated microglia protected from primary neurons. As compared with in vitro, the administration of Vinpocetine in hypoxic mice also inhibited inflammatory molecules, indicating that Vinpocetine as a unique anti-inflammatory agent may be beneficial for the treatment of neuroinflammatory diseases.