Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs.

Inhibitory effect of acarbose on tyrosinase: application of molecular dynamics integrating inhibition kinetics.

Due to its clinical and cosmetic applications, investigators have paid attention to tyrosinase (TYR) inhibitor development. In this study, a TYR inhibition study with acarbose was investigated to gain insights into the regulation of the catalytic function. Biochemical assay results indicated that acarbose was turned to be an inhibitor of TYR in a reversible binding manner and probed as a distinctive mixed-type inhibitor via measurement of double-reciprocal kinetic (Ki = 18.70 ± 4.12 mM). Time-interval kinetic measurement indicated that TYR catalytic function was gradually inactivated by acarbose in a time-dependent behavior displaying with a monophase process that was evaluated by semi-logarithmic plotting. Spectrofluorimetric measurement by integrating with a hydrophobic residue detector (1-anilinonaphthalene-8-sulfonate) showed that the high dose of acarbose derived a conspicuous local structural deformation of the TYR catalytic site pocket. Computational docking simulation showed that acarbose bound to key residues such as HIS61, TYR65, ASN81, HIS244, and HIS259. Our study extends an understanding of the functional application of acarbose and proposes that acarbose is an alternative candidate drug for a whitening agent via direct retardation of TYR catalytic function and it would be applicable for the relevant skin hyperpigmentation disorders concerning the dermatologic clinical purpose.Communicated by Ramaswamy H. Sarma.

Characterization and activity-folding relationship of serine protease from Antarctic krill (Euphausia superba).

Euphausia superba (Antarctic krill) serine protease (ESP) was investigated to gain insights into the activity-structural relationship, folding behavior, and regulation of the catalytic function. We purified ESP from the krill muscle and characterized biochemical distinctions via enzyme kinetics. Studies of inhibition kinetics and unfolding in the presence of a serine residue modifier, such as phenylmethanesulfonyl fluoride, were conducted. Structural characterizations were measured by spectrofluorimetry, including 1-anilinonaphthalene-8-sulfonate dye labeling for hydrophobic residues. The computational simulations such as docking and molecular dynamics were finally conducted to detect key residues and folding behaviors in a nano-second range. The kinetic parameters of ESP were measured as KmBANH = 0.97 ± 0.15 mM and kcat/KmBANH = 4.59 s-1/mM. The time-interval kinetics measurements indicated that ESP inactivation was transformed from a monophase to a biphase process to form a thermodynamically stable state. Spectrofluorimetry measurements showed that serine is directly connected to the regional folding of ESP. Several osmolytes such as proline and glycine only partially protected the inactive form of ESP by serine modification. Computational molecular dynamics and docking simulations showed that three serine residues (Ser183, Ser188, and Ser207) and Cys184, Val206, and Gly209 are key residues of catalytic functions. Our study revealed the functional roles of serine residues as key residues of catalytic function at the active site and of the structural conformation as key folding factors, where ESP displays a flexible property of active site pocket compared to the overall structure.Communicated by Ramaswamy H. Sarma.

Characterization and tissue expression analysis of mitochondrial creatine kinases (types I and II) from Pelodiscus sinensis.

The aim of this study was to characterize the functions of the mitochondrial creatine kinases in the Chinese soft-shelled turtle Pelodiscus sinensis (PSCK-MT1 and PSCK-MT2) to characterize function in relation to hibernation. Computational prediction via molecular dynamics simulations showed that PSCK-MT1 had stronger kinase- and creatine-binding affinity than PSCK-MT2. We measured PSCK-MT1 and PSCK-MT2 levels in the myocardium, liver, spleen, lung, kidney, and ovary of P. sinensis before and after hibernation and found that the expression of these enzymes was the most significantly upregulated in the ovary. We enumerated the ovarian follicles and evaluated the physiological indices of P. sinensis and discovered that fat was the main form of energy storage in P. sinensis. Moreover, both PSCK-MTs promoted follicular development during hibernation. Immunohistochemistry was used to study follicular development and revealed that both PSCK-MTs were expressed primarily in the follicular fluid and granulosa layer before and after hibernation. We found that PSCK-MT1 and PSCK-MT2 could play important roles in ovarian follicular development under hibernation. Hence, both PSCK-MTs probably function effectively under the conditions of low temperature and oxygen during hibernation. Communicated by Ramaswamy H. Sarma.

Study on the pattern of spermatogenesis during the breeding season of the Chinese soft-shelled turtle, Pelodiscus sinensis (Reptilia: Trionychidae).

The Chinese soft-shelled turtle, Pelodiscus sinensis (Reptilia: Trionychidae) is a typical seasonal breeding species and its spermatogenesis pattern is complex. In this study, the process of sperm cell development was studied using histology. The process of sperm cell development may be divided into six stages based on a combination of different cell types in the seminiferous epithelium. A close examination revealed two patterns of sperm cell development in the seminiferous tubules during the breeding season. The first is a normal sperm cell development pattern, in which the process of sperm cell development and maturation are completed in the seminiferous epithelium without round spermatozoa in the lumen. The second is rapid sperm cell development, in which the first batches of round spermatozoa fall off the seminiferous epithelium before they mature, thus beginning a second batch of sperm cell development. The round sperm cells are shed into the lumen and further mature in the seminiferous tubules and epididymis. This rapid sperm cell development process of the Chinese soft-shelled turtle is rare in other vertebrate species and may be an adaptation to cope with seasonal breeding. The results of this study provide insight into the theory of seasonal reproduction in reptiles.

Seasonal spermatogenesis, epididymal storage, and creatine kinase expression in Pelodiscus sinensis.

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.

Effects of isorhamnetin on tyrosinase: inhibition kinetics and computational simulation.

We studied the inhibitory effects of isorhamnetin on mushroom tyrosinase by inhibition kinetics and computational simulation. Isorhamnetin reversibly inhibited tyrosinase in a mixed-type manner at Ki=0.235±0.013 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate(ANS)-binding fluorescence showed that isorhamnetin did not induce significant changes in the tertiary structure of tyrosinase. To gain insight into the inactivation process, the kinetics were computed via time-interval measurements and continuous substrate reactions. The results indicated that inactivation induced by isorhamnetin was a first-order reaction with biphasic processes. To gain further insight, we simulated docking between tyrosinase and isorhamnetin. Simulation was successful (binding energies for Dock6.3: -32.58 kcal/mol, for AutoDock4.2: -5.66 kcal/mol, and for Fred2.2: -48.86 kcal/mol), suggesting that isorhamnetin interacts with several residues, such as HIS244 and MET280. This strategy of predicting tyrosinase interaction in combination with kinetics based on a flavanone compound might prove useful in screening for potential natural tyrosinase inhibitors.

Integrating Radiomics with Genomics for Non-Small Cell Lung Cancer Survival Analysis.

Purpose: The objectives of our study were to assess the association of radiological imaging and gene expression with patient outcomes in non-small cell lung cancer (NSCLC) and construct a nomogram by combining selected radiomic, genomic, and clinical risk factors to improve the performance of the risk model. Methods: A total of 116 cases of NSCLC with CT images, gene expression, and clinical factors were studied, wherein 87 patients were used as the training cohort, and 29 patients were used as an independent testing cohort. Handcrafted radiomic features and deep-learning genomic features were extracted and selected from CT images and gene expression analysis, respectively. Two risk scores were calculated through Cox regression models for each patient based on radiomic features and genomic features to predict overall survival (OS). Finally, a fusion survival model was constructed by incorporating these two risk scores and clinical factors. Results: The fusion model that combined CT images, gene expression data, and clinical factors effectively stratified patients into low- and high-risk groups. The C-indexes for OS prediction were 0.85 and 0.736 in the training and testing cohorts, respectively, which was better than that based on unimodal data. Conclusions: Combining radiomics and genomics can effectively improve OS prediction for NSCLC patients.

The geriatric nutritional risk index is an effective tool to detect GLIM-defined malnutrition in rectal cancer patients.

Background: This study aimed to investigate the value of the Geriatric Nutritional Risk Index (GNRI), prognostic nutritional index (PNI), and advanced lung cancer inflammation index (ALI) scores in detecting malnutrition in patients with rectal cancer; the Global Leadership Initiative on Malnutrition (GLIM) was used as the reference criterion. Materials and methods: This study included patients with rectal cancer who underwent proctectomy. GNRI, PNI, and ALI were calculated to detect the GLIM-defined malnutrition using the Receiver operating characteristic (ROC) curves. Univariate and multivariate logistic regression analyses were used to evaluate the association between the nutritional tools and postoperative complications. Kaplan-Meier survival curves, log-rank tests, and univariate and multivariate Cox regression analyses were used to clarify the relationship between nutritional tools and overall survival (OS). Results: This study enrolled 636 patients with rectal cancer. The GNRI demonstrated the highest sensitivity (77.8%), pretty specificity (69.0%), and the largest AUC (0.734). The GNRI showed good property in predicting major postoperative complications. All three nutritional tools were independent predictors of OS. Conclusion: The GNRI can be used as a promising alternative to the GLIM and is optimal in perioperative management of patients with rectal cancer.

A study of Pb2+ induced unfolding and aggregation of arginine kinase from Euphausia superba: kinetics and computational simulation integrating study.

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 Pb2+-induced inhibition and aggregation of Euphausia superba arginine kinase (ESAK) and found that significantly inactivated ESAK in a dose-dependent manner (IC50 = 0.058 ± 0.002 mM). Spectrofluorimetry results showed that Pb2+ 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 Pb2+ occurred with multi-phase kinetics. The addition of osmolytes did not show protective effect on Pb2+-induced inactivation of ESAK. The computational molecular dynamics (MD) simulation showed that three Pb2+ 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 Pb2+-induced misfolding of ESAK and the complete loss of activity irreversibly, which cannot be recovered by osmolytes.Communicated by Ramaswamy H. Sarma.

Seasonal expression of cytoplasmic creatine kinase in the epididymal epithelium of Pelodiscus sinensis.

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.

Detection of Melanogenesis and Anti-Apoptosis-Associated Melanoma Factors: Array CGH and PPI Mapping Integrating Study.

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.

Functional study of acetaldehyde dehydrogenase 1 (ALDH1) in keratinocytes: microarray integrating bioinformatics approaches.

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.

A Knock-Down Cell-Based Study for the Functional Analysis of Chloride Intracellular Channel 1 (CLIC1): Integrated Proteomics and Microarray Study.

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.

Biochemical Study of Fibrinolytic Protease from Euphausia superba Possessing Multifunctional Serine Protease Activity.

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 Km BApNA = 0.629 ± 0.02 mM and kcat/Km 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.

A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium.

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley ß-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-ß-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

Tyrosinase-mediated melanogenesis in melanoma cells: Array comparative genome hybridization integrating proteomics and bioinformatics studies.

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.

Screening and analysis of agouti signaling protein interaction partners in Pelodiscus sinensis suggests a role in lipid metabolism.

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.

Comparative studies of the expression of creatine kinase isoforms under immune stress in Pelodiscus sinensis.

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.

Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches.

Previously, we detected that 14-3-3 protein epsilon (YWHAE) was involved in the pathogenesis of atopic dermatitis (AD) and tyrosinase-mediated pigmentation. In this study, we aimed to identify critical factors associated with YWHAE in human keratinocytes using high-throughput screening (HTS) approaches to reveal its functions in skin. We overexpressed YWHAE in human HaCaT keratinocytes and then conducted serial HTS studies, including RNA sequencing integrated with antibody arrays and the implementation of bioinformatics algorithms. Cumulatively, these approaches identified several novel genes in keratinocytes associated with the function of YWHAE including KRT9, KRT1, KRT6C, BST2, CIB2, APH1B, ACTC1, IFI27, TUBA1A, CAPN6, UTY, MX2, and MAPK15, based on RNA sequencing data, and MAPK1, MMP2, TYK2, NOS3, and CASP3, based on antibody array data. In particular, CD37 is a unique gene that was detected and validated in all the methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of YWHAE in skin keratinocytes. The approach used here could contribute to the clinical understanding of YWHAE-associated applications in the treatment of AD disease. AbbreviationsDAVIDthe database for annotation, visualization and integrated discoveryHTSHigh-throughput screeningKEGGKyoto Encyclopedia of Genes and GenomesPPIprotein-protein interactionsCommunicated by Ramaswamy H. Sarma.