Structural and functional insights into the self-sufficient flavin-dependent halogenase.

Flavin-dependent halogenases (FDHs) have tremendous applications in synthetic chemistry. A single-component FDH, AetF, exhibits both halogenase and reductase activities in a continuous polypeptide chain. AetF exhibits broad substrate promiscuity and catalyzes the two-step bromination of l-tryptophan (l-Trp) to produce 5-bromotryptophan (5-Br-Trp) and 5,7-dibromo-l-tryptophan (5,7-di-Br-Trp). To elucidate the mechanism of action of AetF, we solved its crystal structure in complex with FAD, FAD/NADP+, FAD/l-Trp, and FAD/5-Br-Trp at resolutions of 1.92-2.23 Å. The obtained crystal structures depict the unprecedented topology of single-component FDH. Structural analysis revealed that the substrate flexibility and dibromination capability of AetF could be attributed to its spacious substrate-binding pocket. In addition, highly-regulated interaction networks between the substrate-recognizing residues and 5-Br-Trp are crucial for the dibromination activity of AetF. Several Ala variants underwent monobromination with >98 % C5-regioselectivity toward l-Trp. These results reveal the catalytic mechanism of single-component FDH for the first time and contribute to efficient FDH protein engineering for biocatalytic halogenation.

The structural and functional investigation into an unusual nitrile synthase.

The biosynthesis of neurotoxin aetokthonotoxin (AETX) that features a unique structure of pentabrominated biindole nitrile involves a first-of-its-kind nitrile synthase termed AetD, an enzyme that shares very low sequence identity to known structures and catalyzes an unprecedented mechanism. In this study, we resolve the crystal structure of AetD in complex with the substrate 5,7-di-Br-L-Trp. AetD adopts the heme oxygenase like fold and forms a hydrophobic cavity within a helical bundle to accommodate the indole moiety. A diiron cluster comprising two irons that serves as a catalytic center binds to the carboxyl O and the amino N of the substrate. Notably, we demonstrate that the AetD-catalyzed reaction is independent of the bromination of the substrate and also solved crystal structures of AetD in complex with 5-Br-L-Trp and L-Trp. Altogether, the present study reveals the substrate-binding pattern and validates the diiron cluster-comprising active center of AetD, which should provide important basis to support the mechanistic investigations into this class of nitrile synthase.

Development and psychometric evaluation of public stigma of stroke scale (PSSS).

Stroke patients suffer from public stigma because strokes cause visible disability and heavy social burden. However, existing tools measuring stroke-related stigma do not consider public stigma. The aim of this study was to develop and evaluate a public stigma of stroke scale (PSSS). This cross-sectional study recruited 730 participants, aged above 18 years, with no diagnosis of stroke before. Scale items were generated after reviewing relevant literature and conducting interviews. An expert panel evaluated the validity and reliability of a preliminary scale. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA), bifactor CFA (B-CFA), Exploratory structural equation modelling (ESEM), bifactor-ESEM (B-ESEM) were performed to extract factors and evaluate fit on the factor structures. The Omega coefficient was 0.93, and the test-retest reliability coefficient was 0.721. The EFA extracted four factors: inherent ideology, aesthetic feelings, avoidance behaviour, and policy attitudes. These explained 61.57% of the total variance in the data. The four-factor model was confirmed by B-CFA, and met the fitness criteria. The PSSS yields satisfactory psychometric properties and can be used to assess stroke-related public stigma.

Stress/Resource Complex, Sense of Coherence and Professional Identity Among Nursing Students: A Latent Profile and Mediation Analysis.

Purpose: Sense of coherence is significant to mental health and professional development in nursing students. However, the association among stress/resource complex, sense of coherence, and professional identity is less explored in nursing students. This study was designed to identify latent subtypes of stress/resource complex and to evaluate the mediating role of sense of coherence between stress/resource complex types and professional identity in nursing students. Participants and Methods: A total of 595 nursing students were recruited from Be Resilient to Nursing Career (BRNC) between October and December 2021 and administered with 10-item Connor-Davidson Resilience Scale, General Self-efficacy Scale, 10-item Chinese Perceived Stress Scale, 13-item Sense of Coherence Scale, and Professional Identity Questionnaire for Undergraduate Students. Latent profile analysis and mediation analysis were performed. Results: Three latent subtypes of stress/resource complex were identified: Flexibility (14.8%), Ordinary (44.2%), and Maladjustment (41.0%). Nursing students with role model were prone to Ordinary (OR = 1.48, 95% CI 1.03-2.13, p = 0.035) and Flexibility (OR = 1.92, 95% CI 1.17-3.16, p = 0.011). The association between stress/resource complex types and professional identity was mediated by sense of coherence (P < 0.05). Conclusion: There exists heterogeneity in nursing students' stress/resource complex. The association between stress/resource complex subtypes and professional identity was mediated by sense of coherence.

Identifying Influential Nodes in Complex Networks Based on Multiple Local Attributes and Information Entropy.

Identifying influential nodes in complex networks has attracted the attention of many researchers in recent years. However, due to the high time complexity, methods based on global attributes have become unsuitable for large-scale complex networks. In addition, compared with methods considering only a single attribute, considering multiple attributes can enhance the performance of the method used. Therefore, this paper proposes a new multiple local attributes-weighted centrality (LWC) based on information entropy, combining degree and clustering coefficient; both one-step and two-step neighborhood information are considered for evaluating the influence of nodes and identifying influential nodes in complex networks. Firstly, the influence of a node in a complex network is divided into direct influence and indirect influence. The degree and clustering coefficient are selected as direct influence measures. Secondly, based on the two direct influence measures, we define two indirect influence measures: two-hop degree and two-hop clustering coefficient. Then, the information entropy is used to weight the above four influence measures, and the LWC of each node is obtained by calculating the weighted sum of these measures. Finally, all the nodes are ranked based on the value of the LWC, and the influential nodes can be identified. The proposed LWC method is applied to identify influential nodes in four real-world networks and is compared with five well-known methods. The experimental results demonstrate the good performance of the proposed method on discrimination capability and accuracy.

[CADASIL with clinical manifestations of lumbago, hunchback and Parkinson's syndrome].

OBJECTIVE: To report a patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) manifesting as lumbago, hunchback and Parkinson's syndrome. METHODS: A 49-years-old male CADASIL patient was reported. Results of clinical examination, neuroimaging and genetic testing were analyzed. His family members were also subjected to genetic testing. Related literature was reviewed. RESULTS: The patient had no typical symptoms of CADASIL such as headache, repeated stroke, dementia and emotional disorders, but progressive Parkinson's syndrome, late onset lumbago, hunchback, dysphagia, and diplopia. Brain MRI showed left basal ganglia and external capsule lacunar infarction. Genetic testing revealed a point mutation c.1630C>T (p.R544C) in exon 11 of the NOTCH3 gene. A heterozygous mutation was detected in the same gene in his mother, elder sister and younger brother, all of whom showed different clinical phenotypes. CONCLUSION: The clinical features of CADASIL are heterogeneous. Lumbago, humpback, and Parkinson's syndrome may be a rare clinical phenotype of CADASIL.

MicroRNA-135b alleviates MPP+-mediated Parkinson's disease in in vitro model through suppressing FoxO1-induced NLRP3 inflammasome and pyroptosis.

The present study focused on the novel roles and the underlying mechanisms of miR-135b in pyroptosis of MPP+-induced Parkinson's disease (PD). We established an in vitro PD model induced by MPP+. Our results demonstrated miR-135b was lower while FoxO1 was inversely higher in MPP+-treated SH-SY5Y and PC-12 cells. Luciferase reporter assay showed FoxO1 was a downstream target of miR-135b. MiR-135b mimics suppressed MPP+-induced pyroptosis and the upregulation of TXNIP, NLRP3, Caspase-1, ASC, GSDMDNterm and IL-1ß. Moreover, FoxO1 overexpression had no effect on miR-135b but reversed its own downregulation caused by miR-135b mimics. Meanwhile, overexpression of FoxO1 abolished the inhibitory effects of miR-135b on pyroptosis and reversed the downregulation of pyroptotic genes and LDH release. In summary, miR-135b played a protective role in Parkinson's disease via inhibiting pyroptosis by targeting FoxO1. MiR-135b might serve as a potential therapeutic target in the treatment of Parkinson's disease.

Predicting Urban Medical Services Demand in China: An Improved Grey Markov Chain Model by Taylor Approximation.

The sharp increase of the aging population has raised the pressure on the current limited medical resources in China. To better allocate resources, a more accurate prediction on medical service demand is very urgently needed. This study aims to improve the prediction on medical services demand in China. To achieve this aim, the study combines Taylor Approximation into the Grey Markov Chain model, and develops a new model named Taylor-Markov Chain GM (1,1) (T-MCGM (1,1)). The new model has been tested by adopting the historical data, which includes the medical service on treatment of diabetes, heart disease, and cerebrovascular disease from 1997 to 2015 in China. The model provides a predication on medical service demand of these three types of disease up to 2022. The results reveal an enormous growth of urban medical service demand in the future. The findings provide practical implications for the Health Administrative Department to allocate medical resources, and help hospitals to manage investments on medical facilities.

Development and Validation of a Grading Scale for Primary Pontine Hemorrhage.

BACKGROUND AND PURPOSE: We aimed to develop and validate a grading scale for predicting 30-day mortality and 90-day functional outcome in patients with primary pontine hemorrhage (PPH). METHODS: We retrospectively reviewed records of consecutive patients with first-ever pontine hemorrhage from 3 teaching hospitals between 2005 and 2012. Independent factors associated with 30-day mortality were identified by logistic regression to establish a risk stratification scale, named the new PPH score. For validation of the new PPH score, we prospectively recruited subjects from 10 units between December 2014 and November 2015. The performance of the new PPH score was presented as discrimination and calibration, measured by area under the curve of the receiver operating characteristic and Hosmer-Lemeshow goodness-of-fit, respectively. RESULTS: Data of 171 patients were available for scale development. The new PPH score consisted of 2 independent factors with individual points assigned as follows: Glasgow Coma Scale score 3 to 4 (=2 points), 5 to 7 (=1 point), and 8 to 15 (=0 point); PPH volume >10 mL (=2 points), 5 to 10 mL (=1 point), and <5 mL (=0 point). An independent cohort of 98 patients was applied as an external validation of the new PPH score. Results showed that the new PPH score was discriminative in predicting both 30-day mortality (area under the curve, 0.902) and 90-day good outcome (area under the curve, 0.927). Furthermore, the new PPH score revealed a good calibration (χ2=1.387; P=0.846) in 30-day mortality prediction. CONCLUSIONS: The new PPH score is simple and reliable in predicting short-term and long-term outcome for PPH patients. CLINICAL TRIAL REGISTRATION: URL: http://www.chictr.org.cn. Unique identifier: ChiCTR-OOC-14005533.

TFP5 prevents 1-methyl-4-phenyl pyridine ion-induced neurotoxicity in mouse cortical neurons.

The present study aimed to investigate the protective effect of a modified p5 peptide, TFP5, on 1-methyl-4-phenyl pyridine ion (MPP+)-induced neurotoxicity in cortical neurons and explore the therapeutic effect of TFP5 on Parkinson's disease (PD). MPP+ was applied to a primary culture of mouse cortical neurons to establish the cell model of PD. Neurons were divided into four groups: Control, model (MPP+), scrambled peptide (Scb) (Scb + MPP+) and TFP5 (TFP5 + MPP+) groups. Pretreatment with Scb or TFP5 was applied to the latter two groups, respectively, for 3 h, while phosphate-buffered saline was applied to the control and model groups. MPP+ was then applied to all groups, with the exception of the control group, and neurons were cultured for an additional 24 h. Neuron viability was evaluated using a Cell Counting kit-8 (CCK8) assay. To explore the mechanism underlying the protective effects of TFP5, the expression levels of p35, p25 and phosphorylated myocyte enhancer factor 2 (p-MEF2D) were determined by western blotting. Fluorescence microscopy showed that TFP5 was able to pass through cell membranes and distribute around the nucleus. CCK8 assay showed that neuronal apoptosis was dependent on MPP+ concentration and exposure time. Cell viability decreased significantly in the model group compared with the control group (55±7 vs. 100±0%; P<0.01), and increased significantly in the TFP5 group compared with the model group (98±2 vs. 55±5%; P<0.01) and Scb group (98±2 vs. 54±4%; P<0.01). Scb exhibited no protective effect. Western blotting results showed that MPP+ induced p25 and p-MEF2D expression, TFP5 and Scb did not affect MPP+-induced p25 expression, but TFP5 reduced MPP+-induced p-MEF2D expression. In summary, TFP5 protects against MPP+-induced neurotoxicity in mouse cortical neurons, possibly through inhibiting the MPP+-induced formation and elevated kinase activity of a cyclin-dependent kinase 5/p25 complex.

Cdk5/p25 specific inhibitory peptide TFP5 rescues the loss of dopaminergic neurons in a sub-acute MPTP induced PD mouse model.

Parkinson's disease (PD) is pathologically characterized by progressively loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the formation of Lewy bodies. In 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced PD mice models, the calpain- cyclin-dependent kinase 5 (Cdk5)-myocyte enhancer factor 2 (MEF2) signaling has been proven in governing dopaminergic neuronal death. Under MPTP insult, p35 is cleaved by calpain into p25, which binds to Cdk5 and exhibits hyperactivity of Cdk5/p25. Cdk5/p25 inactivates MEF2, a survivor factor, which is critical for DA neuronal death. In this study, neuroprotective effect of the Cdk5/p25 specific peptide, TFP5, was evaluated in sub-acute MPTP induced PD mouse model by intraperitoneal (i.p.) injection of MPTP for five consecutive days. The results indicated that the levels of p35 and p25, and p25/p35 ratio increased in the sub-acute MPTP mice. TFP5 broadly reached cortex neuron, hippocampus and SNpc areas after i.p. injections. Pretreatment with 45mg/kg/day TFP5, as well as 10mgkg/day Cdk5 inhibitor roscovitine, for three days significantly rescued DA neuronal loss up to 9.8% or 9.7% respectively compared to the saline treated group. Treatment of TFP5 and roscovitine reduced the levels of inactive form of MEF2 and cleaved caspase 3, thus protected apoptosis of DA neurons against MPTP insult. Our results propose that TFP5 might be a potential therapeutic candidate for PD.

[Effect of AKR1A1 knock-down on H2;O2; and 4-hydroxynonenal-induced cytotoxicity in human 1321N1 astrocytoma cells].

OBJECTIVE: To investigate the role of human aldo-keto reductase 1A1 (AKR1A1) in the resistance to oxidative stress and the metabolism of toxic aldehyde in astrocytoma cells. METHODS: The siRNA was transfected into 1321N1 astrocytoma cells using Lipofectamine(TM); RNAiMax. Western blotting and qRT-PCR were applied to evaluate the knock-down efficiency of AKR1A1. MTT assay was used to examine the cell viability after H2;O2; and 4-hydroxynonenal treatment in AKR1A1 knock-down cells. In addition, the effect of knocking down AKR1A1 on cellular reactive oxygen species (ROS) level in the presence of H2;O2; was measured using 2', 7'-dichlorofluorescein (DCFH-DA). RESULTS: Western blotting and qRT-PCR showed that the AKR1A1-specific siRNA inhibited AKR1A1 gene expression by about 70% in 1321N1 cells. Cells with knock-down of AKR1A1 were more sensitive to H2;O2; and 4-hydroxynonenal-induced cytotoxicity. Furthermore, cellular ROS level in the cells with knock-down of AKR1A1 was much higher than that in the control cells in the presence of H2;O2;. CONCLUSION: The specific siRNA could efficiently inhibit AKR1A1 expression in 1321N1 cells. AKR1A1 could be involved in the metabolism of 4-hydroxynonenal and play a role in the resistance to oxidative stress.

[Effect of AKR7A1 overexpression on crotonaldehyde-induced mutagenicity in V79-4 cells].

AIM: To investigate the effect of overexpressing rat aldo-keto reductases (AKR7A1) in V79-4 cells on crotonaldehyde-induced mutagenicity. METHODS: The expression level and enzyme activity of AKR7A1 protein expressed in V79-4 cells were measured by Western blotting and AKR enzyme assay, respectively. HGPRT assay was applied to evaluate the effect of AKR7A1 overexpression on crotonaldehyde-induced mutagenicity in V79-4 cells. RESULTS: Western blotting confirmed a high expression level of AKR7A1 protein in V79-4 cells. AKR enzyme assay showed that the overexpressed AKR7A1 protein was functionally active. HGPRT assay demonstrated that cells overexpressing AKR7A1 exhibited a significant increase in resistance to crotonaldehyde-induced mutagenicity compared to control cells. CONCLUSION: Overexpression of AKR7A1 can protect V79-4 cells against crotonaldehyde-induced mutagenicity.

[Purification of recombinant AKR7A5 protein and measurement of substrate specificity of AKR7A5 towards naphthoquinone and its derivatives].

AIM: To investigate the substrate specificity of mouse aldo-keto reductase AKR7A5 protein towards naphthoquinone and its derivatives. METHODS: The recombinant His-tagged AKR7A5 fusion protein in E.coli BL21pLysS cell strain was induced by IPTG and purified using FPLC system through HiTrap affinity column. The purified recombinant AKR7A5 protein was confirmed by SDS-PAGE and Western blotting. AKR enzyme assay was applied to measure the substrate specificity of recombinant AKR7A5 protein towards naphthoquinone and its derivatives. RESULTS: Recombinant His-AKR7A5 was successfully purified as confirmed by SDS-PAGE and Western blotting. AKR enzyme assay indicated that the recombinant AKR7A5 protein exhibited mild substrate specificity towards lawsone and low specificity towards juglone and vitamine K3, but no activity towards 1, 4-naphthoquinone. CONCLUSION: AKR7A5 has selective substrate specificity towards naphthoquinone derivatives, suggesting that the aldo-keto reductase could play an important role in metabolism of certain naphthoquinone derivatives.

Insulin-like growth factor-1 induces the phosphorylation of PRAS40 via the PI3K/Akt signaling pathway in PC12 cells.

Insulin-like growth factor-1 (IGF-1) is a polypeptide tropic factor that plays an important role in the survival and differentiation of both neuronal and non-neuronal cells. Numerous studies have demonstrated that IGF-1 promotes neuronal cell survival via the PI3K/Akt signaling pathway. Proline-rich Akt substrate of 40kDa (PRAS40) is a recently discovered downstream target of Akt. However, the relationship between IGF-1 and PRAS40 is not known. In this study, we characterized the phosphorylation of PRAS40 induced by IGF-1 in PC12 cells and explored the signaling pathway responsible for the effect of IGF-1. IGF-1 induced the phosphorylation of Akt at Thr473 and PRAS40 at Thr246 in PC12 cells. The phosphorylation of Akt and PRAS40 induced by IGF-1 (100ng/ml) was inhibited by the phosphatidylinositide 3-kinase (PI3K) specific inhibitor LY294002 (50µM), while no inhibitory effect was observed for a MAPK kinase pathway specific inhibitor PD98059 nor a p38 MAPK inhibitor PD169316, suggesting that the phosphorylation of PRAS40 induced by IGF-1 is mediated by the PI3K pathway in PC12 cells and primary cultured neurons. In further support this hypothesis, an Akt kinase specific inhibitor, Akt inhibitor VIII, attenuated IGF-1-induced phosphorylation of PRAS40 at the concentration that blocked the phosphorylation of Akt induced by IGF-1. Taken together, these data demonstrate that IGF-1 stimulates the phosphorylation of PRAS40 at Thr246 in neuronal cells and the effect of IGF-1 is mediated, at least in part, by the PI3K/Akt signaling pathway.

Endoplasmic reticulum stress induces tau pathology and forms a vicious cycle: implication in Alzheimer's disease pathogenesis.

Accumulation of unfolded proteins can disturb the functions of the endoplasmic reticulum (ER), leading to ER-stress or unfolded protein response (UPR). Recent data have shown that activation of UPR can be found in postmortem brains of Alzheimer's disease (AD) patients; and biological markers for activation of UPR are abundant in neurons with diffuse phosphorylated tau. Although these observations suggest a linkage between ER-stress and tau pathology, little is known of their relationship. In this study, we found that high levels of phosphorylated PKR-like ER-resident kinase (p-PERK) and phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) as markers for activation of UPR in the hippocampus of aged P301L mutant tau transgenic mice. The immunoreactivity of p-PERK was found to co-localize with that of phosphorylated tau. We then hypothesized that phosphorylation of tau could induce ER-stress and vice versa in promoting AD-like pathogenesis. By using the protein phosphatase 2A inhibitor okadaic acid (OA) as an inducer for phosphorylation of tau, we found that primary cultures of rat cortical neurons treated with OA triggered UPR as indicated by increased levels of p-PERK and p-eIF2α, splicing of mRNA for xbp-1 and elevated levels of mRNA for GADD153. On the other hand, thapsigargin as an ER-stress inducer stimulated phosphorylation of tau at Thr231, Ser262 and Ser396. Thapsigargin also induced activation of caspase-3 and cleavage of tau. These findings suggested that ER-stress and hyperphosphorylation of tau could be induced by each other to form a vicious cycle to propagate AD-like neurodegeneration.

Proline-rich Akt substrate of 40kDa (PRAS40): a novel downstream target of PI3k/Akt signaling pathway.

Modifications in signaling of the proline-rich Akt substrate of 40-kDa (PRAS40) pathway is implicated in type 2 diabetes and melanoma. PRAS40 is known for its ability to regulate the mammalian target of rapamycin complex 1 (mTORC1) kinase activity, possessing a key regulatory role at the cross point of signal transduction pathways activated by growth factor receptors. Recently it has been found that PRAS40 is regulated by its upstream phosphatidylinositol 3-kinase/Akt (PI3K/Akt) which is activated by many tyrosine kinase receptors growth factors including insulin-like growth factor 1. Also, PRAS40 functions downstream of mTORC1 and upstream from its effectors ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Phosphorylation of PRAS40 by Akt and mTORC1 disrupts the binding between mTORC1 and PRAS40, and relieves the inhibitory constraint of PRAS40 on mTORC1 activity. This review summarizes the signaling regulating PRAS40 phosphorylation, as well as the dual function of PRAS40 as substrate and inhibitor of mTORC1 upon growth factor stimulation and under pathophysiological conditions.

The association between high-density lipoprotein cholesterol level and cholesteryl ester transfer protein TaqIB gene polymorphism is influenced by alcohol drinking in a population-based sample.

Cholesteryl ester transfer protein (CETP) is a key enzyme in high-density lipoprotein (HDL) cholesterol metabolism. We studied the association between CETP TaqIB polymorphism and the HDL cholesterol levels considering environmental factors in a population-based sample consisting of 1729 participants who did not use lipid-lowering agents (659 men and 1070 women). The CETP TaqIB genotypes were determined by PCR-RFLP analysis. The serum HDL cholesterol levels of female participants with the B2B2 genotype were significantly higher than those with other genotypes (p<0.001). Multiple regression analysis with covariates such as age, waist to hip (W/H) ratio, alcohol drinking, current smoking, non-HDL cholesterol, and logarithm of triglyceride revealed that the CETP TaqIB genotype was an independent determinant of HDL cholesterol levels in men (p=0.049) and women (p<0.001). Subgroup analysis revealed that an interaction was observed between the CETP TaqIB polymorphism and alcohol consumption in the regulation of HDL cholesterol levels in men (p=0.049) and women (p=0.022). No interactions were observed between the CETP TaqIB polymorphism and current smoking status, body mass index, or W/H ratio in the regulation of HDL cholesterol levels. The association between the CETP TaqIB polymorphism and HDL cholesterol levels was more evident in alcohol consumers than in non-drinkers.