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In the present study, we conducted an integrative approach of network pharmacology and experimental validation study to elucidate the underlying mechanisms of Chelerythrine (CLT), in treating fibrotic diseases (FD), which are disorders characterised by excessive accumulation of extracellular matrix. 27 common targets of CLT against FD were analysed, and these common targets were used to construct the PPI network. The results of GO and KEGG enrichment analyses suggested that CLT exerted pharmacological effects on FD by regulating mTOR signalling pathway, AKT-PI3K pathway and apoptosis signalling pathway. Finally, molecular docking confirmed a strong binding affinity between CLT and the core target proteins. CLT has inhibitory effects on the proliferation and migration of L929 cells, CLT could promote cell apoptosis. CLT decreased levels of the Bcl-2, p-AKT/AKT, p-mTOR/mTOR and p-PI3K/PI3K, meanwhile increased levels of the Bax. Taken together, these results indicate that CLT may be a potential drug for anti-fibrotic diseases therapy.

Human pharyngeal squamous cell carcinoma (HPSCC) is the most common malignancy in the head and neck region, characterized by high mortality and a propensity for metastasis. Fucoxanthin, a carotenoid isolated from brown algae, exhibits pharmacological properties associated with the suppression of tumor proliferation and metastasis. Nevertheless, its potential to inhibit HPSCC proliferation and metastasis has not been fully elucidated. This study represents the first exploration of the inhibitory effects of fucoxanthin on two human pharyngeal squamous carcinoma cell lines (FaDu and Detroit 562), as well as the mechanisms underlying those effects. The results showed dose-dependent decreases in the proliferation, migration, and invasion of HPSCC cells after fucoxanthin treatment. Further studies indicated that fucoxanthin caused a significant reduction in the expression levels of proteins in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway, as well as the downstream proteins matrix metalloproteinase (MMP)-2 and MMP-9. Specific activators of PI3K/AKT reversed the effects of fucoxanthin on these proteins, as well as on cell proliferation and metastasis, in FaDu and Detroit 562 cells. Molecular docking assays confirmed that fucoxanthin strongly interacted with PI3K, AKT, mTOR, MMP-2, and MMP-9. Overall, fucoxanthin, a functional food component, is a potential therapeutic agent for HPSCC.

This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory effects of fucoxanthin on SCC-25 human tongue squamous carcinoma cells. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and total iron were measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to assess glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (Nrf2), Keap1, solute carrier family 7 member 11 (SLC7A11), transferrin receptor protein 1 (TFR1), p53, and heme oxygenase 1 (HO-1) expression. Molecular docking was performed to validate interactions. Compared with the control group, the activity of fucoxanthin-treated SCC-25 cells significantly decreased in a dose- and time-dependent manner. The levels of MMP, GSH, and SOD significantly decreased in fucoxanthin-treated SCC-25 cells; the levels of ROS, MDA, and total iron significantly increased. mRNA and protein expression levels of Keap1, GPX4, Nrf2, and HO-1 in fucoxanthin-treated cells were significantly decreased, whereas levels of TFR1 and p53 were significantly increased, in a concentration-dependent manner. Molecular docking analysis revealed that binding free energies of fucoxanthin with p53, SLC7A11, GPX4, Nrf2, Keap1, HO-1, and TFR1 were below -5 kcal/mol, primarily based on active site hydrogen bonding. Our findings suggest that fucoxanthin can induce ferroptosis in SCC-25 cells, highlighting its potential as a treatment for tongue cancer.

Background: Marburg virus disease is an acute haemorrhagic fever caused by Marburg virus. Marburg virus is zoonotic, maintained in nature in Egyptian fruit bats, with occasional spillover infections into humans and nonhuman primates. Although rare, sporadic cases and outbreaks occur in Africa, usually associated with exposure to bats in mines or caves, and sometimes with secondary human-to-human transmission. Outbreaks outside of Africa have also occurred due to importation of infected monkeys. Although all previous Marburg virus disease outbreaks have been brought under control without vaccination, there is nevertheless the potential for large outbreaks when implementation of public health measures is not possible or breaks down. Vaccines could thus be an important additional tool, and development of several candidate vaccines is under way., Methods: We developed a branching process model of Marburg virus transmission and investigated the potential effects of several prophylactic and reactive vaccination strategies in settings driven primarily by multiple spillover events as well as human-to-human transmission. Linelist data from the 15 outbreaks up until 2022, as well as an Approximate Bayesian Computational framework, were used to inform the model parameters., Results: Our results show a low basic reproduction number which varied across outbreaks, from 0.5 [95% CI 0.05-1.8] to 1.2 [95% CI 1.0-1.9] but a high case fatality ratio. Of six vaccination strategies explored, the two prophylactic strategies (mass and targeted vaccination of high-risk groups), as well as a combination of ring and targeted vaccination, were generally most effective, with a probability of potential outbreaks being terminated within 1 year of 0.90 (95% CI 0.90-0.91), 0.89 (95% CI 0.88-0.90), and 0.88 (95% CI 0.87-0.89) compared with 0.68 (0.67-0.69) for no vaccination, especially if the outbreak is driven by zoonotic spillovers and the vaccination campaign initiated as soon as possible after onset of the first case., Conclusions: Our study shows that various vaccination strategies can be effective in helping to control outbreaks of MVD, with the best approach varying with the particular epidemiologic circumstances of each outbreak., (© 2023. The Author(s).)

Background: Outbreaks of Marburg virus disease (MVD) are rare and small in size, with only 18 recorded outbreaks since 1967, only two of which involved more than 100 cases. It has been proposed, therefore, that Phase 3 trials for MVD vaccines should be held open over multiple outbreaks until sufficient end points accrue to enable vaccine efficacy (VE) to be calculated. Here we estimate how many outbreaks might be needed for VE to be estimated., Methods: We adapt a mathematical model of MVD transmission to simulate a Phase 3 individually randomised placebo controlled vaccine trial. We assume in the base case that vaccine efficacy is 70% and that 50% of individuals in affected areas are enrolled into the trial (1:1 randomisation). We further assume that the vaccine trial starts two weeks after public health interventions are put in place and that cases occurring within 10 days of vaccination are not included in VE calculations., Results: The median size of simulated outbreaks was 2 cases. Only 0.3% of simulated outbreaks were predicted to have more than 100 MVD cases. 95% of simulated outbreaks terminated before cases accrued in the placebo and vaccine arms. Therefore the number of outbreaks required to estimate VE was large: after 100 outbreaks, the estimated VE was 69% but with considerable uncertainty (95% CIs: 0%-100%) while the estimated VE after 200 outbreaks was 67% (95% CIs: 42%-85%). Altering base-case assumptions made little difference to the findings. In a sensitivity analysis, increasing R 0 by 25% and 50% led to an estimated VE after 200 outbreaks of 69% (95% CIs: 53-85%) and 70% (95% CIs: 59-82%), respectively., Conclusions: It is unlikely that the efficacy of any candidate vaccine can be calculated before more MVD outbreaks have occurred than have been recorded to date. This is because MVD outbreaks tend to be small, public health interventions have been historically effective at reducing transmission, and vaccine trials are only likely to start after these interventions are already in place. Hence, it is expected that outbreaks will terminate before, or shortly after, cases start to accrue in the vaccine and placebo arms., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: W John Edmunds reports financial support was provided by Department of Health and Social Care. Thibaut Jombart reports financial support was provided by MRC Centre for Global Infectious Disease Analysis. W John Edmunds reports financial support was provided by Japan Agency for Medical Research and Development. George Qian reports a relationship with Pfizer that includes: funding grants and non-financial support. GQ works on a separate project that is funded by Pfizer (please see ’Other Support’ section)., (© 2023 The Author(s).)

The soft-shelled turtle, Pelodiscus sinensis, is an important economic aquaculture species. Its reproduction exhibits seasonality; however, there is a lack of systematic studies focused on sperm maturation and epididymal storage. The testes and epididymides of P. sinensis were sampled from March to December. The seasonal reproduction and maturation of the spermatozoa were examined by anatomy, hematoxylin and eosin staining, AB-PAS staining, and immunohistochemistry. Spermatogenesis exhibited obvious seasonality in P. sinensis. It was found that the spermatogenic epithelium was most active during June to September, whereas the diameter of the epididymal tubules was smallest during June to October. As key enzymes of ATP metabolism, creatine kinases were highly expressed in the epididymal tubule epithelium during the breeding season, which may be important for the regulation of sperm maturation. In addition, the epididymal tubule epithelium changed with the season in June to September, the epididymal tubule epithelium proliferated to form villous structures, and secreted a large number of glycoproteins, which may be related to the rapid maturation of sperm during the breeding season. In conclusion, this study provided insights into the spermatogenesis of P. sinensis through histological analysis and enriched our understanding of reproduction in reptiles., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be defined as a potential conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Arginine kinase is a crucial phosphagen kinase in invertebrates, which is associated to the environmental stress response, plays a key role in cellular energy metabolism. In this study, we investigated the Pb 2+ -induced inhibition and aggregation of Euphausia superba arginine kinase (ESAK) and found that significantly inactivated ESAK in a dose-dependent manner ( IC 50 = 0.058 ± 0.002 mM). Spectrofluorimetry results showed that Pb 2+ induced tertiary structural changes via the internal polarity increased and the non-polarity decreased in ESAK and directly induced ESAK aggregation. The ESAK aggregation process induced by Pb 2+ occurred with multi-phase kinetics. The addition of osmolytes did not show protective effect on Pb 2+ -induced inactivation of ESAK. The computational molecular dynamics (MD) simulation showed that three Pb 2+ interrupt the entrance of the active site of ESAK and it could be the reason on the loss of activity of ESAK. Several important residues of ESAK were detected that were importantly contributed the conformation and catalytic function of ESAK. Our study showed that Pb 2+ -induced misfolding of ESAK and the complete loss of activity irreversibly, which cannot be recovered by osmolytes.Communicated by Ramaswamy H. Sarma.

Background: Cardiogenic shock continues to be a highly morbid complication that affects around 7%-10% of patients with acute myocardial infarction or heart failure. Similarly, obesity has become a worldwide epidemic., Aim: To analyze the impact of higher body mass index (BMI) on outcomes of patients with cardiogenic shock., Methods: A systematic and comprehensive search was undertaken on the electronic databases of PubMed, Embase, ScienceDirect, CENTRAL, and Google Scholar for all types of studies comparing mortality outcomes of patients with cardiogenic shock based on BMI. All studies defined overweight or obese patients based on the World Health Organization BMI criteria. The data were then extracted and assessed on the basis of the Reference Citation Analysis (https://www.referencecitationanalysis.com/)., Results: Five studies were included. On pooled analysis of multivariable-adjusted ratios, we noted a statistically significantly reduced risk of mortality in overweight/obese vs normal patients (three studies; odds ratio [OR] = 0.92, 95% confidence interval [CI]: 0.85-0.98, I 2 = 85%). On meta-analysis, we noted that crude mortality rates did not significantly differ between overweight/obese and normal patients after cardiogenic shock (OR = 0.95, 95%CI: 0.79-1.15, I 2 = 99%). The results were not stable on sensitivity analysis and were associated with substantial heterogeneity., Conclusion: Current evidence on the association between overweight/obesity and mortality after cardiogenic shock is scarce and conflicting. The obesity paradox might exist in patients with cardiogenic shock but could be confounded by the use of mechanical circulatory support. There is a need for further studies to clarify this relationship., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

By assessing and comparing the phenotypic changes on the stepwise acquisition of fluconazole resistant Candida albicans isolates, we could find and describe the relationship between drug resistance and biofilm formation ability in a series of clonal strains., Methods: We performed antifungal susceptibility of five drugs (fluconazole, itraconazole, voriconazole, caspofungin and amphotericin B) to further verify the antifungal activity of the six isolates in vitro . Then we combined hyphal formation assay, cell surface hydrophobicity test positively related to adherence ability, and biofilm assays in vitro to observe and compare the phenotypic characteristics of our six clonal strains., Results: Biofilm capability is enhanced for four drug- intermediate strains, whereas the initial susceptible strain and the final resistant strain are both poor in adherence, hyphal growth and biofilm formation., Conclusions: It was suggested that the biofilm formation ability were not absolutely related to the degree of fluconazole resistance., Competing Interests: Conflicts of interest: The authors reported no conflicts of interest., (Copyright © 2022 Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences, and Chinese Medical Association, published by Wolters Kluwer, Inc.)

During hibernation of Pelodiscus sinensis , sperm mature and are stored in the epididymis. We investigated seasonal changes in the morphology of epithelial cells of the epididymis of P. sinensis and changes in expression of cytoplasmic creatine kinase (CK). We found that the epididymal epithelium proliferates rapidly to form multiple layers from June to September, while the epididymal epithelial cells are arranged in a single layer from October to May. From the March before the mating period to the end of the mating period in September, a large amount of neutral glycoprotein is secreted in the epididymal epithelium and in the sperm aggregation area; after October, the glycoprotein in the epididymis decreases. At sperm maturation, cytoplasmic CK is expressed abundantly in the villous epithelium, which is formed by proliferation of epididymal epithelial cells. During hibernation and reproduction, the epididymal epithelium of P. sinensis exhibits different proliferation and secretion patterns as the animal adapts to two types of sperm storage. Cytoplasmic CK may participate in regulating the energy metabolism of the epididymal epithelium; it is an important enzyme for regulating sperm maturation.

Sargassum fusiforme (S. fusiforme) has been used as an ingredient in Chinese herbal medicine for thousands of years. However, there are a limited number of studies concerning its therapeutic mechanism. High performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight of the S. fusiforme polysaccharide, SFPS 191212, is 43 kDa. SFPS 191212 is composed of mannose, rhamnose, galactose, xylose, glucose, and fucose (at a molar ratio: 2.1 : 2.9 : 1.8 : 15.5 : 4.6 : 62.5) with α- and β-configurations. The present research evaluated the anti-tumor potential of the S. fusiforme polysaccharide in human erythroleukemia (HEL) cells in vitro. To explore the SFPS 191212's apoptosis mechanism in HEL cells, transcriptome analysis was performed on HEL cells that were incubated with SFPS 191212. The inhibitory effect of SFPS 191212 on HEL cell growth was also analyzed. It was found that SFPS 191212 inhibited HEL cell proliferation, reduced cell viability in a concentration-dependent manner, and induced an insignificant toxic effect on normal human embryonic lung (MRC-5) cells. Compared with the control group, transcriptome analysis identified a total of 598 differentially expressed genes (DEGs), including 243 up-regulated genes and 355 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on all DEGs, and 900 GO terms and 52 pathways were found to be significantly enriched. Finally, 23 DEGs were randomly selected and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, SFPS 191212 down-regulated the PI3K/Akt signal transduction pathway. Our results provide a framework for understanding the effect of SFPS 191212 on cancer cells and can serve as a resource for delineating the anti-tumor mechanisms of S. fusiforme., (Copyright © 2021 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.Communicated by Ramaswamy H. Sarma.

We investigated the tyrosinase-associated melanogenesis in melanoma cells by using OMICS techniques. We characterized the chromosome copy numbers, including Chr 11q21 where the tyrosinase gene is located, from several melanoma cell lines (TXM13, G361, and SK-MEL-28) by using array CGH. We revealed that 11q21 is stable in TXM13 cells, which is directly related to a spontaneous high melanin pigment production. Meanwhile, significant loss of copy number of 11q21 was found in G361 and SK-MEL-28. We further profiled the proteome of TXM13 cells by LC-ESI-MSMS and detected more than 900 proteins, then predicted 11 hub proteins (YWHAZ; HSP90AA1; HSPA5; HSPA1L; HSPA9; HSP90B1; HSPA1A; HSPA8; FKSG30; ACTB; DKFZp686DQ972) by using an interactomic algorithm. YWHAZ (25% interaction in the network) is thought to be a most important protein as a linking factor between tyrosinase-triggered melanogenesis and melanoma growth. Bioinformatic tools were further applied for revealing various physiologic mechanisms and functional classification. The results revealed clues for the spontaneous pigmentation capability of TXM13 cells, contrary to G361 and SK-MEL-28 cells, which commonly have depigmentation properties during subculture. Our study comparatively conducted the genome-wide screening and proteomic profiling integrated interactomics prediction for TXM13 cells and suggests new insights for studying both melanogenesis and melanoma., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Laparoscopic cholecystectomy (LC) is a common surgery accompanied by some unpleasant adverse effects. Clinical trials indicated that acupuncture therapy may help reduce complications in LC. However, no systematic reviews have been conducted on the topic. Therefore, we will evaluate the current evidence and provide a rank for the efficacy of acupuncture therapy in LC by performing Bayesian network meta-analysis., Methods: A total of 9 databases will be searched from inception to 10 December 2020. Randomized control trails met the criterion will be included. Quality evaluation of included studies will be performed using Cochrane risk-of-bias tool. STATA 14.0, Addis 1.16.8, R 3.6.3, and OpenBUGS 3.2.3 will be used to conduct pairwise meta-analysis and network meta-analysis. The evidence will be assessed by the Grades of Recommendations Assessment Development and Evaluation., Results: This review will be based on clinical evidence to choose the best choice of acupuncture treatment for LC. And the results will be submitted to a peer-reviewed journal for publication., Conclusion: Through this systematic review, we will summarize the best available evidence of acupuncture therapy in LC and help to improve the clinical decision-making ability in LC domain., Systematic Review Registration: The protocol has been registered on INPLASY2020120056., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Previously, we detected that chloride intracellular channel 1 (CLIC1) was involved in the pathogenesis of atopic dermatitis (AD)., Objective: In this study, we aimed to use high-throughput screening (HTS) approaches to identify critical factors associated with the function of CLIC1 in knock-down cells., Methods: We down-regulated CLIC1 in human A549 cells via siRNA and then conducted serial HTS studies, including proteomics integrated with a microarray and the implementation of bioinformatics algorithms., Results: Together, these approaches identified several important proteins and genes associated with the function of CLIC1. These proteins and genes included tumor rejection antigen (gp96) 1, nucleophosmin, annexin I, keratin 1 and 10, FLNA protein, enolase 1, and metalloprotease 1, which were found using two-dimensional electrophoresis (2-DE) proteomics. Separately, NTNG1, SEMA5A, CLEC3A, GRPR, GNGT2, GRM5, GRM7, DNMT3B, CXCR5, CCL11, CD86, IL2, MNDA, TLR5, IL23R, DPP6, DLGAP1, CAT, GSTA1, GSTA2, GSTA5, CYP2E1, ADH1A, ESR1, ARRDC3, A1F1, CCL5, CASP8, DNTT, SQSTM1, PCYT1A, and SLCO4C1 were found using a DNA microarray integrated with PPI mapping., Conclusion: CCL11 is thought to be a particularly critical gene among the candidate genes detected in this study. By integrating the datasets and utilizing the strengths of HTS, we obtained new insights into the functional role of CLIC1, including the use of CLIC1-associated applications in the treatment of human diseases such as AD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated., Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated., Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study., Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with K m BApNA = 0.629 ± 0.02 mM and k cat /K m BApNA = 7.08 s-1/mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data., Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill's body was suggested., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: We investigated melanogenesis- and anti-apoptosis-related melanoma factors in melanoma cells (TXM1, TXM18, A375P, and A375SM)., Objective: To find melanoma associated hub factor, high-throughput screening-based techniques integrating with bioinformatics were investigated., Methods: Array CGH analysis was conducted with a commercial system. Total genomic DNAs prepared individually from each cell line with control DNA were properly labeled with Cy3-dCTP and Cy5-dCTP and hybridizations and subsequently performed data treatment by the log2 green (G; test) to red (R; reference) fluorescence ratios (G/R). Gain or loss of copy number was judged by spots with log2-transformed ratios. PPI mapping analysis of detected candidate genes based on the array CGH results was conducted using the human interactome in the STRING database. Energy minimization and a short Molecular Dynamics (MD) simulation using the implicit solvation model in CHARMM were performed to analyze the interacting residues between YWHAZ and YWHAB., Results: Three genes (BMP-4, BFGF, LEF-1) known to be involved in melanogenesis were found to lose chromosomal copy numbers, and Chr. 6q23.3 was lost in all tested cell lines. Ten hub genes (CTNNB1, PEX13, PEX14, PEX5, IFNG, EXOSC3, EXOSC1, EXOSC8, UBC, and PEX10) were predicted to be functional interaction factors in the network of the 6q23.3 locus. The apoptosis-associated genes E2F1, p50, BCL2L1, and BIRC7 gained, and FGF2 lost chromosomal copy numbers in the tested melanoma cell lines. YWHAB, which gained chromosomal copy numbers, was predicted to be the most important hub protein in melanoma cells. Molecular dynamics simulations for binding YWHAB and YWHAZ were conducted, and the complex was predicted to be energetically and structurally stable through its 3 hydrogen-bond patterns. The number of interacting residues is 27., Conclusion: Our study compares genome-wide screening interactomics predictions for melanoma factors and offers new information for understanding melanogenesis- and anti-apoptosis-associated mechanisms in melanoma. Especially, YWHAB was newly detected as a core factor in melanoma cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The carapace from the Chinese soft-shelled turtle (Pelodiscus sinensis) is used as a traditional Chinese medicine. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from turtle carapace were isolated and characterized to screen novel collagen material in this study. Yields of 1.0% and 2.8% were obtained for ASC and PSC which contained glycine as the major amino acid and had high imino acid content. Both collagens had maximum ultraviolet absorption peaks of 220 nm. SDS-PAGE revealed that the structure of both collagens was similar, belonging to type I collagen. Relative viscosities of collagens were decreased as the temperature increased. Collagens showed minimum solubility at pH 8 and maximum solubility at a salt concentration of 3%. The denaturation temperature (Td) of PSC was higher whereas the melting temperature was lower than that of ASC. Both ASC and PSC appeared to be spongy like microstructure with fibrillar pores shown by scanning electron microscopy. The results suggest that collagens isolated from turtle carapace has high thermal stability with potential uses as new substitute for mammalian collagen in medicinal, food or biomaterial fields. However, their biological or pharmacological activities are needed to be further studied., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to investigate the effects of Sargassum fusiforme polysaccharide (SFPS I, II, and III) on the apoptosis and regulation of human erythroleukemia (HEL) cells. The effect of different doses of SFPS on HEL cell growth was detected using the Cell Counting Kit-8 method, and apoptosis was detected by Hoechst staining. Cell cycle distribution and apoptosis were detected using flow cytometry. Expression of the cell cycle gene, p53, antiapoptotic genes, Bcl-xL and Bcl-2, and pro-apoptotic genes, Bax, Bad, and Caspase-3, as well as the expression of the corresponding proteins, were detected using real-time quantitative polymerase chain reaction (qPCR) and Western blot. The results showed that SFPS II and III decreased HEL cell viability and induced HEL cell apoptosis. Different concentrations of SFPS (I, II, and III) were detected that induced much less toxic effect in normal human embryonic lung (MRC-5) cells, and SFPS I increased cell proliferation, indicating its favorable selectivity towards cancer cells. The mechanism by which SFPS induced apoptosis was also found to be related to the induction of cell cycle arrest in the G 0 /G 1 phase and the increased expression of apoptosis-related genes and proteins. We concluded that SFPS induces HEL cell apoptosis, possibly via activation of the Caspase pathway, providing the theoretical basis for the development of SFPS-based anti-tumor drug products., (Copyright © 2020 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Agouti signaling protein (ASP) is a secreted paracrine protein that has been widely reported to function in melanogenesis and obesity and could potentially be a core protein that regulates the color and fatty phenotype of P. sinensis. In this study, we screened out interacting proteins of ASP by combined co-immunoprecipitation mass spectrometry (CoIP-MS), yeast two hybrid (Y2H) analysis, and computational predictions. We performed docking of ASP with its well-known receptor melanocortin receptor 4 (MC4R) to predict the binding capacity and to screen out actual ASP interacting proteins, CoIP-MS was performed where identified 32 proteins that could bind with ASP and Y2H confirmed seven proteins binding with ASP directly. CoIP-MS and Y2H screening results including PPI prediction revealed that vitronectin (VTN), apolipoprotein A1 (APOA1), apolipoprotein B (APOB), and filamin B (FLNB) were the key interacting proteins of ASP. VTN, APOA1, and APOB are functional proteins in lipid metabolism and various skin disorders, suggesting ASP may function in lipid metabolism through these partners. This study provided protein-protein interaction information of ASP, and the results will promote further research into the diverse roles of ASP, as well as its binding partners, and their function in different strains of P. sinensis., (Copyright © 2019. Published by Elsevier B.V.)

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein-ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC 50 was measured (IC 50 = 1.738 ± 0.041 mM), and the dissociation constant was determined ( K i Slope = 0.46 ± 0.04 mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. AbbreviationsAKGalpha-ketoglutaric acidpNPG4-nitrophenyl-α-d-glucopyranosideANS1-anilinonaphthalene-8-sulfonateMDmolecular dynamics.Communicated by Ramaswamy H. Sarma.

Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (k m and V max ). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze-thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.

The capacities for thermal and inhibitor tolerance are critical for industrial enzymes and loss of activity is a major challenge in deploying natural enzymes for commercial applications. Protein engineering approaches, such as site-directed mutagenesis and directed evolution, have been devoted to modifying natural enzymes. Recently, a post-translation protein engineering strategy, the SpyTag/SpyCatcher system, was introduced. Here, we have generated a thermo- and ion-tolerant cyclized xylanase (C-TFX) by fusing the SpyTag and SpyCatcher peptides to its N- and C- terminus respectively. Compared with the linear enzyme, C-TFX retained greater residual activity after heating or metal ion exposure. Intrinsic fluorescence and circular dichroism analysis revealed that the isopeptide bond mediated by SpyTag/SpyCatcher cyclization contributed to enhanced thermo- and ion-stability, probably by stabilizing its secondary and conformational structure. In addition, the heat-challenged C-TFX was observed to degrade natural lignocellulosic substrates efficiently. The cyclized xylanase was more stable and resistent to denaturation and aggregation than the linear enzyme. The "superglue" SpyTag/SpyCatcher cyclization system enables the enzyme to maintain its structural conformation, which will be of particular interest in engineering of enzymes for industrial application such as feed additives and functional oligosaccharides production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Regulation of α-glucosidase (EC 3.2.1.20) and its inhibitors is of great interest to researchers due to its clinical relevance as a target enzyme for the treatment of α-glucosidase-mediated diseases, such as type 2 diabetes mellitus and Pompe disease. In this study, we conducted a phloroglucinol-induced inhibition kinetics assay and performed computational molecular dynamics (MD) simulations to assess binding manner in α-glucosidase. The results showed that phloroglucinol reversibly inhibited α-glucosidase in a dose-dependent but non-competitive manner (K i =2.07±0.16mM). Interestingly, the maximum peak wavelength and the hydrophobic surface remained unchanged during the inhibition reaction, with computational MD simulations further revealing that phloroglucinol bound in front of the active site pocket rather than in the α-glucosidase active site. Therefore, we speculate that phloroglucinol-specific inhibition is mild and the inhibitor likely binds to a single binding site near but not in the active site. Our study provided insight into the effects and mechanisms associated with a mild inhibitor of α-glucosidase activity and promotes fundamental research and potential applications of inhibitors for treatment of α-glucosidase-mediated clinical disease., (Copyright © 2018. Published by Elsevier B.V.)

Pyrogallol is naturally found in aquatic plant and has been proposed as a substrate of tyrosinase. In this study, we evaluated the dual effect of pyrogallol on tyrosinase as an inhibitor in the presence of L‑DOPA simultaneously via integrating methods of enzyme kinetics and computational molecular dynamics (MD) simulations. Pyrogallol was found to be a reversible inhibitor of tyrosinase in the presence of L‑DOPA and its induced mechanism was the parabolic non-competitive inhibition type (IC 50  = 0.772 ± 0.003 mM and K i  = 0.529 ± 0.022 mM). Kinetic measurements by real-time interval assay showed that pyrogallol induced rapid inactivation process composing with slight activations at the low dose. Spectrofluorimetry studies showed that pyrogallol mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational MD simulations further revealed that pyrogallol could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS85, HIS259, ASN260, HIS263, VAL283, and ALA296. Our study provides insight into the mechanism by which hydroxyl group composing pyrogallol inhibit tyrosinase and pyrogallol is a potential natural anti-pigmentation agent., (Copyright © 2018. Published by Elsevier B.V.)

The metal-insulator transition temperature T c in VO 2 is experimentally shown to be almost the same as a magnetic transition temperature T m characterized by an abrupt decrease in susceptibility, suggesting the evidence of the same underlying origin for both transitions. The measurement of susceptibility shows that it weakly increases on cooling for temperature range of T > T m , sharply decreases near T m and then unusually increases on further cooling. A theoretical approach for such unusual observations in susceptibility near T m or below is performed by modeling electrons from each two adjacent V 4+ ions distributed along V-chains as a two-electron system, which indicates that the spin exchange between electrons could cause a level splitting into a singlet (S = 0) level of lower energy and a triplet (S = 1) level of higher energy. The observed abrupt decrease in susceptibility near T m is explained to be due to that the sample enters the singlet state in which two electrons from adjacent V 4+ ions are paired into dimers in spin antiparallel. By considering paramagnetic contribution of unpaired electrons created by the thermal activation from singlet to triplet levels, an expression for susceptibility is proposed to quantitatively explain the unusual temperature-dependent susceptibility observed at low temperatures. Based on the approach to magnetic features, the observed metal-insulator transition is explained to be due to a transition from high-temperature Pauli paramagnetic metallic state of V 4+ ions to low-temperature dimerized state of strong electronic localization.

Creatine kinase (EC 2.7.3.2, CK) plays an important role in cellular energy metabolism and homeostasis by catalysing the transfer of phosphate between ATP and creatine phosphate. In this study, we investigated the effects of H 2 O 2 on PSCKM (muscle type creatine kinase from Pelodiscus sinensis) by the integrating method between enzyme kinetics and docking simulations. We found that H 2 O 2 strongly inactivated PSCKM (IC 50 =0.25mM) in a first-order kinetic process, and targeted the active site cysteine directly. A conformational study showed that H 2 O 2 did not induce the tertiary structural changes in PSCKM with no extensive exposure of hydrophobic surfaces. Sequential docking simulations between PSCKM and H 2 O 2 indicated that H 2 O 2 interacts with the ADP binding region of the active site, consistent with experimental results that demonstrated H 2 O 2 -induced inactivation. Our study demonstrates the effect of H 2 O 2 on PSCKM enzymatic function and unfolding, and provides important insight into the changes undergone by this central metabolic enzyme in ectothermic animals in response to the environment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Cu2+ is well known to play important roles in living organisms having bifacial distinction: essential microelement that is necessary for a wide range of metabolic processes but hyper-accumulation of Cu2+ can be toxic. The physiological function of Cu2+ in ectothermic animals such as Pelodiscus sinensis (Chinese soft-shelled turtle) has not been elucidated., Objective: In this study, we elucidated effect of Cu2+ on the energy producing metabolic enzyme creatine kinase (CK), which might directly affect energy metabolism and homeostasis of P. sinensis., Method: We first conducted molecular dynamics (MD) simulations between P-CK and Cu2+ and conducted the inactivation kinetics including spectrofluorimetry study., Results: MD simulation showed that Cu2+ blocked the binding site of the ATP cofactor, indicating that Cu2+ could directly inactivate P-CK. We prepared the muscle type of CK (P-CK) and confirmed that Cu2+ conspicuously inactivated the activity of P-CK (IC50 = 24.3 μM) and exhibited non-competitive inhibition manner with creatine and ATP in a first-order kinetic process. This result was well matched to the MD simulation results that Cu2+-induced non-competitive inactivation of P-CK. The spectrofluorimetry study revealed that Cu2+ induced tertiary structure changes in PCK accompanying with the exposure of hydrophobic surfaces. Interestingly, the addition of osmolytes (glycine, proline, and liquaemin) effectively restored activity of the Cu2+-inactivated P-CK., Conclusion: Our study illustrates the Cu2+-mediated unfolding of P-CK with disruption of the enzymatic function and the protective restoration role of osmolytes on P-CK inactivation. This study provides information of interest on P-CK as a metabolic enzyme of ectothermic animal in response to Cu2+ binding., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.

Thyrotropin receptor (TSHR) is a G-protein-coupled receptor that regulates the synthesis, storage, and secretion of thyroid hormones in the thyroid tissue. The aims of the present study were to characterize the full-length TSHR cDNA in largemouth bass (Micropterus salmoides), and to determine the TSHR gene transcription levels in different tissues. In addition, the response of TSHR transcription levels to daily feeding in thyroid tissue was investigated. The results showed that the full-length cDNA sequence was 2743 bp with an open reading frame of 2340 bp encoding a 779-amino acid peptide. BLAST analysis indicated that the amino acid sequence displayed 58.4-90.2% identity and 5.6-125.8 divergence, compared with other known fish species. The most abundant TSHR transcription levels were found in the spleen, head kidney, and kidney. Feeding did not affect the transcription level of TSHR in thyroid tissue over the course of the day. Thus, the current study suggests that there was no relationship between daily nutritional status and TSHR transcription level in the thyroid tissue of largemouth bass. The spleen, head kidney, and kidney exhibited the most abundant TSHR transcription levels., Competing Interests: The authors declare no conflict of interest.

Creatine kinase (EC 2.7.3.2, CK) plays an important role in cellular energy metabolism and homeostasis by catalyzing the transfer of phosphate between ATP and creatine phosphate. We investigated the effects of Cd2+ on muscle type of creatine kinase from Pelodiscus sinensis (PSCKM). Cd2+ conspicuously inactivated the activity of PSCKM (IC50=0.062 mM) in a first-order kinetic process and exhibited non-competitive inhibition with creatine and ATP. A conformational study showed that Cd2+ induced tertiary structure changes in PSCKM with exposure of hydrophobic surfaces. The addition of osmolytes, such as glycine and proline, partially reactivated the Cd2+-mediated inactive PSCKM. Additionally, molecular dynamics and docking simulations between PSCKM and Cd2+ were conducted to show that Cd2+ blocked the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd2+-induced inactivation of PSCKM. Our study demonstrates the effect of Cd2+ on PSCKM enzymatic function and unfolding, including the protective effects of osmolytes on PSCKM inactivation. This study provides important insights into the changes in the PSCKM metabolic enzyme of ectothermic animals in response to the environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Arginine kinase is an essential enzyme which is closely related to energy metabolism in marine invertebrates. Arginine kinase provides a significant role in quick response to environmental change and stress. In this study, we simulated a tertiary structure of Sepia pharaonis arginine kinase (SPAK) based on the gene sequence and conducted the molecular dynamics simulations between SPAK and Zn(2+). Using these results, the Zn(2+) binding sites were predicted and the initial effect of Zn(2+) on the SPAK structure was elucidated. Subsequently, the experimental kinetic results were compared with the simulation results. Zn(2+) markedly inhibited the activity of SPAK in a manner of non-competitive inhibitions for both arginine and ATP. We also found that Zn(2+) binding to SPAK resulted in tertiary conformational change accompanying with the hydrophobic residues exposure. These changes caused SPAK aggregation directly. We screened two protectants, glycine and proline, which effectively prevented SPAK aggregation and recovered the structure and activity. Overall, our study suggested the inhibitory effect of Zn(2+) on SPAK and Zn(2+) can trigger SPAK aggregation after exposing large extent of hydrophobic surface. The protective effects of glycine and proline against Zn(2+) on SPAK folding were also demonstrated.

We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 μM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.

To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH4)2SO4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography. The purified enzyme (62.6 kDa; 2.62 unit/mg) was unstable at temperatures exceeding 30°C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300 mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5 M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The regulation of enzymatic activity and unfolding studies of arginine kinase (AK) from various invertebrates have been the focus of investigation. To gain insight into the structural and folding mechanisms of AK from Euphausia superba (ESAK), we purified ESAK from muscle properly. The enzyme behaved as a monomeric protein with a molecular mass of about 40kDa and had pH and temperature optima of 8.0 and 30°C, respectively. The Km(Arg) and Km(ATP) for the synthesis of phosphoarginine were 0.30 and 0.47mM, respectively, and kcat/Km(Arg) was 282.7s(-1)/mM. A study of the inhibition kinetics of structural unfolding in the denaturant sodium dodecyl sulfate (SDS) was conducted. The results showed that ESAK was almost completely inactivated by 1.0mM SDS. The kinetics analyzed via time-interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to biphase as SDS concentrations increased. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence showed that SDS concentrations lower than 5mM did not induce conspicuous changes in tertiary structures, while higher concentrations of SDS exposed hydrophobic surfaces and induced conformational changes. These results confirmed that the active region of AK is more flexible than the overall enzyme molecule., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Arginine kinase (AK) is a key metabolic enzyme for maintaining energy balance in invertebrates and studies on AK from Euphausia superba might provide important insights into the metabolic enzymes in extreme climatic marine environments. A folding study of the AK from E. superba (ESAK) has not yet been reported. To gain insights into the structural and folding mechanisms of ESAK, the denaturants guanidine HCl and urea were applied in this study. We purified ESAK from the muscle of E. superba and evaluated the inhibition kinetics with structural unfolding studies under various conditions. The results revealed that ESAK was almost completely inactivated when using 1.0 M guanidine HCl and 8.25 M urea. The kinetics, characterized via time-interval measurements, showed that the inactivations by guanidine HCl and urea were first-order reactions, with the kinetic processes shifting from monophases to biphases as concentrations increased. Measurements of intrinsic and ANS (anilinonaphthalene-8-sulfonate)-binding fluorescences showed that guanidine HCl and urea induced conspicuous changes in tertiary structures and followed the regular unfolding mechanisms. Our study provides information regarding the folding of this muscle-derived metabolic enzyme and expands our knowledge and understanding of invertebrate metabolisms.

The protective effect of osmolytes on the thermal denaturation and aggregation of Pelodiscus sinensis muscle creatine kinase (PSCK) was investigated by a combination of spectroscopic methods and thermodynamic analysis. Our results demonstrated that the addition of osmolytes, such as glycine and proline, could prevent thermal denaturation and aggregation of PSCK in a concentration-dependent manner. When the concentration of glycine and proline increased in the denatured system, the relative activation was significantly enhanced; meanwhile, the aggregation of PSCK during thermal denaturation was decreased. Spectrofluorometer results showed that glycine and proline significantly decreased the tertiary structural changes of PSCK and that thermal denaturation directly induced PSCK aggregation. In addition, we also built the 3D structure of PSCK and osmolytes by homology models. The results of computational docking simulations showed that the docking energy was relatively low and that the clustering groups were spread to the surface of PSCK, indicating that osmolytes directly protect the surface of the protein. P. sinensis are poikilothermic and quite sensitive to the change of ambient temperature; however, there were few studies on the thermal denaturation of reptile CK. Our study provides important insight into the protective effects of osmolytes on thermal denaturation and aggregation of PSCK., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sargassum fusiforme is a kind of brown algae that has been widely consumed not only as food, but also as herbal medicine for thousands of years. The purpose of this study was to investigate the antioxidant activities and intestinal functions of polysaccharides extracted from S. fusiforme (SFP) in normal and cyclophosphamide-induced immunosuppressed mice. The experiment was performed on six groups of ICR mice, which treated with cyclophosphamide (CY, 200 mg/kg) or different dosages of SFP for 14 days. The results showed that administration of SFP was able to overcome the immunosuppression, and significantly increased the spleen index and antioxidant activities in mice (P<0.05). It also remarkably improved the numbers of jejunal intraepithelial lymphocytes (IELs) and goblet cells in immunosuppressed mice (P<0.05). For normal mice, SFP increased both thymus index and intestinal function parameters such as villus length/crypt depth ratio and intestinal IELs and goblet cells (P<0.05). The results suggested that SFP, possessing pronounced antioxidant activities, may play an important role in the improvement of intestinal function in mice. This might be one of the possible mechanisms of SFP for the immunomodulatory effects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, we investigated validamycin A as a tyrosinase inhibitor based on its structural properties. We found that the reversible inhibition of tyrosinase by validamycin A occurred in a mixed-type manner with Ki=5.893±0.038mM, as determined by integrating kinetics studies and computational simulations. Time-interval tyrosinase studies showed that the inhibition followed first-order kinetics with two phases. Fluorescence measurements of ANS binding showed that validamycin A induced changes in the tertiary protein structure of tyrosinase. To obtain further insight, computational docking and molecular dynamics were applied, and the results indicated that HIS85, HIS244, GLU256, HIS259, and ASN260 of tyrosinase interacted with validamycin A. This strategy of predicting tyrosinase inhibition based on hydroxyl group numbers might be useful in the design and screening of potential tyrosinase inhibitors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We studied the effects of Zn(2+) on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn(2+) inactivated the activity of PSCK (IC(50) = .079 ± .004 mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn(2+) induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn(2+) directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn(2+) bind to PSCK and one Zn(2+) is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn(2+) with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn(2+) on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.

The soft-shelled turtle Pelodiscus sinensis is a high-profile turtle species because of its nutritional and medicinal value in Asian countries. However, little is known about the genes that are involved in formation of their nutritional quality traits, especially the molecular mechanisms responsible for unsaturated fatty acid and collagen biosynthesis. In the present study, the transcriptomes from six tissues from Pelodiscus sinensis were sequenced using an Illumina paired-end sequencing platform. We obtained more than 47 million sequencing reads and 73954 unigenes with an average size of 754 bp by de-novo assembly. In total, 55.19% of the unigenes (40814) had significant similarity with proteins in the National Center of Biotechnology Information (NCBI) non-redundant protein database and Swiss-Prot database (E-value <10(-5)). Of these annotated unigenes, 9156 and 11947 unigenes were assigned to 52 gene ontology categories (GO) and 25 clusters of orthologous groups (COG), respectively. In total, 26496 (35.83%) unigenes were assigned to 242 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, we found a number of highly expressed genes involved in the regulation of P. sinensis unsaturated fatty acid biosynthesis and collagen formation, including desaturases, growth factors, transcription factors, and extracellular matrix components. Our data represent the most comprehensive sequence resource available for the Chinese soft-shelled turtle and could provide a basis for new research on this turtle as well as the molecular genetics and functional genomics of other terrapins. To our knowledge, we report for the first time, the large-scale RNA sequencing (RNA-Seq) of terrapin animals and would enrich the knowledge of turtles for future research.

Recovery of valuable materials from waste printed circuit boards (WPCBs) is quite difficult because WPCBs is a heterogeneous mixture of polymer materials, glass fibers, and metals. In this study, WPCBs was treated using ionic liquid (1-ethyl-3-methylimizadolium tetrafluoroborate [EMIM+][BF4-]). Experimental results showed that the separation of the solders went to completion, and electronic components (ECs) were removed in WPCBs when [EMIM+][BF4-] solution containing WPCBs was heated to 240 °C. Meanwhile, metallographic observations verified that the WPCBs had an initial delamination. When the temperature increased to 260 °C, the separation of the WPCBs went to completion, and coppers and glass fibers were obtained. The used [EMIM+][BF4-] was treated by water to generate a solid-liquid suspension, which was separated completely to obtain solid residues by filtration. Thermal analyses combined with infrared ray spectra (IR) observed that the solid residues were bromine epoxy resins. NMR (nuclear magnetic resonance) showed that hydrogen bond played an important role for [EMIM+][BF4-] dissolving bromine epoxy resins. This clean and non-polluting technology offers a new way to recycle valuable materials from WPCBs and prevent environmental pollution from WPCBs effectively., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

A folding study of creatine kinase from Pelodiscus sinensis has not yet been reported. To gain more insight into structural and folding mechanisms of P. sinensis CK (PSCK), denaturants such as SDS, guanidine HCl, and urea were applied in this study. We purified PSCK from the muscle of P. sinensis and conducted inhibition kinetics with structural unfolding studies under various conditions. The results revealed that PSCK was completely inactivated at 1.8 mM SDS, 1.05 M guanidine HCl, and 7.5 M urea. The kinetics via time-interval measurements showed that the inactivation by SDS, guanidine HCl, and urea were all first-order reactions with kinetic processes shifting from monophase to biphase at increasing concentrations. With respect to tertiary structural changes, PSCK was unfolded in different ways; SDS increased the hydrophobicity but retained the most tertiary structural conformation, while guanidine HCl and urea induced conspicuous changes in tertiary structures and initiated kinetic unfolding mechanisms. Our study provides information regarding PSCK and enhances our knowledge of the reptile-derived enzyme folding., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tyrosinase plays a central role in biological pigment formation, and hence knowledge of tyrosinase catalytic mechanisms and regulation may have medical, cosmetic, and agricultural applications. We found in this study that arabinose significantly inhibited tyrosinase, and this was accompanied by conformational changes in enzyme structure. Kinetic analysis showed that arabinose-mediated inactivation followed first-order kinetics, and single and multiple classes of rate constants were measured. Arabinose displayed a mixed-type inhibitory mechanism with K(i)=0.22±0.07 mM. Measurements of intrinsic and ANS-binding fluorescence showed that arabinose induced tyrosinase to unfold and expose inner hydrophobic regions. We simulated the docking between tyrosinase and arabinose (binding energies were -26.28 kcal/mol for Dock6.3 and -2.02 kcal/mol for AutoDock4.2) and results suggested that arabinose interacts mostly with His61, Asn260, and Met280. The present strategy of predicting tyrosinase inhibition by simulation of docking by hydroxyl groups may prove useful in screening for potential tyrosinase inhibitors, as shown here for arabinose., (Copyright © 2012 Elsevier B.V. All rights reserved.)

As the human brain is the most complex living organ, constructing its detailed model with exploration capabilities in a form of an atlas is a challenge. Our overall goal is to construct an advanced, detailed, parcellated, labeled, accurate, interactive, three-dimensional (3D), and scalable whole human brain atlas of structure, vasculature, tracts and systems. The objectives of this work are three-fold; to present: (1) method of atlas design and development including design principles, accuracy requirements, atlas content, architecture, functionality, user interface, and customized tools; (2) creation of an atlas of structure and systems including its modeling method and validation; and (3) integration of this atlas with the cerebrovasculature and tracts created earlier. The atlas is created from multiple in vivo 3/7 T scans. Its design based on "pyramidal principle" enables scalability while preserving design principles and exploits interaction paradigm "from blocks to brain". The atlas contains (1) navigator with modules for system/object/object state management, interaction, user interfacing, and rendering; and (2) brain model with cerebrum, cerebellum, brainstem, spinal cord, white matter, deep structures, systems, ventricles, arteries, veins, sinuses, and tracts. The brain model is parcellated, labeled, consistent, realistic, of high resolution, polygonal/volumetric, dissectible, extendable, and deformable. It has over 1700 3D components. The atlas has sub-voxel accuracy of 0.1mm and the smallest vessels of 80 μm. Brain exploration includes dynamic scene composition, manipulation-independent 3D labeling, interaction combined with animation, meta-labeling, and quantification. This atlas is useful in education, research, and clinical applications. It can potentially be foundation for a multi-level molecular-cellular-anatomical-physiological-behavioral platform., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important role in antioxidant defense in organisms exposed to oxygen. However, there is a lack of research into the regulation of SOD activity and structural changes during folding, especially for SOD originating from extremophiles. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of manganese-containing SOD (Mn-SOD) from Thermus thermophilus. TFE decreased the degree of secondary structure of Mn-SOD, which directly resulted in enzyme inactivation and disrupted the tertiary structure of Mn-SOD. The kinetic studies showed that TFE-induced inactivation of Mn-SOD is a first-order reaction and that the regional Mn-contained active site is very stable compared to the overall structure. We further simulated the docking between Mn-SOD and TFE (binding energy for Dock 6.3, -9.68 kcal/mol) and predicted that the LEU9, TYR13, and HIS29 residues outside of the active site interact with TFE. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of TFE and allow us to describe ligand binding via inhibition kinetics combined with computational predictions.

Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (-1.89 kcal/mol) and the hexamer (-1.97 kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.

Tyrosinase inhibition studies have recently gained the attention of researchers due to their potential application values. We simulated docking (binding energies for AutoDock Vina: -9.1 kcal/mol) and performed a molecular dynamics simulation to verify docking results between tyrosinase and rutin. The docking results suggest that rutin mostly interacts with histidine residues located in the active site. A 10 ns molecular dynamics simulation showed that one copper ion at the tyrosinase active site was responsible for the interaction with rutin. Kinetic analyses showed that rutin-mediated inactivation followed a first-order reaction and mono- and biphasic rate constants occurred with rutin. The inhibition was a typical competitive type with K(i) = 1.10±0.25 mM. Measurements of intrinsic and ANS-binding fluorescences showed that rutin showed a relatively strong binding affinity for tyrosinase and one possible binding site that could be a copper was detected accompanying with a hydrophobic exposure of tyrosinase. Cell viability testing with rutin in HaCaT keratinocytes showed that no toxic effects were produced. Taken together, rutin has the potential to be a potent anti-pigment agent. The strategy of predicting tyrosinase inhibition based on hydroxyl group number and computational simulation may prove useful for the screening of potential tyrosinase inhibitors.

Tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture to prevent hyperpigmentation or browning effects. Some of the flavonoids mostly found in herbal plants and fruits are revealed as tyrosinase inhibitors. We studied the inhibitory effects of one such flavonoid, hesperetin, on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin reversibly inhibited tyrosinase in a competitive manner with K(i)=4.03±0.26 mM. Measurements of ANS-binding fluorescence showed that hesperetin induced the hydrophobic disruption of tyrosinase. For further insight, we used the docking algorithms to simulate binding between tyrosinase and hesperetin. Simulation was successful (binding energies for Dock6.3: -34.41 kcal/mol and for AutoDock4.2: -5.67 kcal/mol) and showed that a copper ion coordinating with 3 histidine residues (HIS61, HIS85, and HIS259) within the active site pocket was chelated via hesperetin binding. Our study provides insight into the inhibition of tyrosinase in response to flavonoids. A combination of inhibition kinetics and computational prediction may facilitate the identification of potential natural tyrosinase inhibitors such as flavonoids and the prediction of their inhibitory mechanisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)