Sesamin and sesamolin potentially inhibit adipogenesis through downregulating the peroxisome proliferator-activated receptor γ protein expression and activity in 3T3-L1 cells.

Sesamin and sesamolin are major sesame lignans that have demonstrated anti-inflammatory, anticancer, and neuroprotective properties and potential benefits in the liver, cardiovascular diseases, and metabolic syndrome. However, despite previous research on their antiobesity effects and underlying mechanisms, a comprehensive investigation of these aspects is still lacking. In this study, we evaluated the regulatory effects of 20 to 80 µM sesamin and sesamolin on adipogenesis in vitro using 3T3-L1 cells as a model cell line. We hypothesized that the lignans would inhibit adipogenic differentiation in 3T3-L1 cells through the regulation of peroxisome proliferator-activated receptor γ (PPARγ). Our data indicate that sesamin and sesamolin inhibited the adipogenic differentiation of 3T3-L1 cells by dose-dependently decreasing lipid accumulation and triglyceride formation. Sesamin and sesamolin reduced the mRNA and protein expression of the adipogenesis-related transcription factors, PPARγ and CCAAT/enhancer-binding protein α, leading to the dose-dependent downregulations of their downstream targets, fatty acid binding protein 4, hormone-sensitive lipase, lipoprotein lipase, and glucose transporter 4. In addition, glucose uptake was dose-dependently attenuated by sesamin and sesamolin in both differentiated 3T3-L1 cells and HepG2 cells. Interestingly, our results suggested that sesamin and sesamolin might directly bind to PPARγ to inhibit its transcriptional activity. Finally, sesamin and sesamolin decreased the phosphorylation of 3 mitogen-activated protein kinase signaling components in differentiated 3T3-L1 cells. Taken together, our findings suggest that sesamin and sesamolin may exhibit antiobesity effects by potentially downregulating PPARγ and its downstream genes through the mitogen-activated protein kinase signaling pathway, offering important insights into the molecular mechanisms underlying the potential antiobesity effects of sesamin and sesamolin.

Differentially expressed genes in orbital adipose/connective tissue of thyroid-associated orbitopathy.

Background: Thyroid-associated orbitopathy (TAO) is a disease associated with autoimmune thyroid disorders and it can lead to proptosis, diplopia, and vision-threatening compressive optic neuropathy. To comprehensively understand the molecular mechanisms underlying orbital adipogenesis in TAO, we characterize the intrinsic molecular properties of orbital adipose/connective tissue from patients with TAO and control individuals. Methods: RNA sequencing analysis (RNA-seq) was performed to measure the gene expression of orbital adipose/connective tissues of TAO patients. Differentially expressed genes (DEGs) were detected and analyzed through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA). The protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified by the Cytoscape plug-in, cytoHubba. We validated several top DEGs through quantitative real-time polymerase chain reaction (qRT-PCR). Results: We identified 183 DEGs in adipose tissue between TAO patients (n = 3) and control patients (n = 3) through RNA sequencing, including 114 upregulated genes and 69 downregulated genes. The PPI network of these DEGs had 202 nodes and 743 edges. PCR-based validation results of orbital adipose tissue showed multiple top-ranked genes in TAO patients (n = 4) are immune and inflammatory response genes compared with the control individual (n = 4). They include ceruloplasmin isoform x3 (CP), alkaline tissue-nonspecific isozyme isoform x1 (ALPL), and angiotensinogen (AGT), which were overrepresented by 2.27- to 6.40-fold. Meanwhile, protein mab-21-like 1 (MAB21L1), phosphoinositide 3-kinase gamma-subunit (PIK3C2G), and clavesin-2 (CLVS2) decreased by 2.6% to 32.8%. R-spondin 1 (RSPO1), which is related to oogonia differentiation and developmental angiogenesis, was significantly downregulated in the orbital muscle tissues of patients with TAO compared with the control groups (P = 0.024). Conclusions: Our results suggest that there are genetic differences in orbital adipose-connective tissues derived from TAO patients. The upregulation of the inflammatory response in orbital fat of TAO may be consistent with the clinical phenotype like eyelid edema, exophthalmos, and excess tearing. Downregulation of MAB21L1, PIK3C2G, and CLVS2 in TAO tissue demonstrates dysregulation of differentiation, oxidative stress, and developmental pathways.

MiR-423-5p promotes Müller cell activation via targeting NGF signaling in diabetic retinopathy.

AIMS: Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus and one of the major causes of visual impairment and blindness in industrialized countries. The early neuro-glial perturbations, especially retinal Müller cells (rMC) activation, intimately associated with the vascular alterations. MicroRNAs (miRNAs) have been reported to play critical roles in the progression of DR. Here, we aimed to further explore the role and underlying mechanism of miR-423-5p in Müller cell activation in streptozotocin (STZ)-induced diabetic mice and oxygen-induced retinopathy (OIR) model. MATERIALS AND METHODS: Retinal histology, optical coherence tomography (OCT) and biochemical markers were assessed. KEY FINDINGS: Our data revealed that the expression of miR-423-5p was significantly increased under high-glucose environment. We also demonstrated that miR-423-5p overexpression markedly accelerated retinal vascular leakage, leukocytosis, and rMC activation. This response was ameliorated in animals pre-treated with the inhibition of miR-423-5p. Specifically, miR-423-5p bound to the nerve growth factor (NGF) 3' UTR region to induce its silencing. NGF inhibition significantly promoted retinal microvascular dysfunction. SIGNIFICANCE: These findings demonstrate that miR-423-5p is a critical miRNA that promotes microvascular dysfunction in DR.

Discovery and Characterization of a Dual-Function Peptide Derived from Bitter Gourd Seed Protein Using Two Orthogonal Bioassay-Guided Fractionations Coupled with In Silico Analysis.

The hydrolysate of bitter gourd seed protein, digested by the combined gastrointestinal proteases (BGSP-GPs), exhibited the most potent inhibition on angiotensin-I-converting enzyme (ACE) with an IC50 value of 48.1 ± 2.0 µg/mL. Using two independent bioassay-guided fractionations, fraction F5 from reversed-phase chromatography and fraction S1 from strong cation exchange chromatography exhibited the highest ACE inhibitory (ACEI) activity. Three identical peptides were simultaneously detected from both fractions and, based on the in silico appraisal, APLVSW (AW6) was predicted as a promising ACEI peptide. Their dipeptidyl peptidase-IV (DPP4) inhibitory (DPP4I) activity was also explored. The IC50 values of AW6 against ACE and DPP4 were calculated to be 9.6 ± 0.3 and 145.4 ± 4.4 µM, respectively. The inhibitory kinetics and intermolecular interaction studies suggested that AW6 is an ACE competitive inhibitor and a DPP4 non-competitive inhibitor. The quantities of AW6 in BGSP-GP hydrolysate, fractions F5 and S1, were also analyzed using liquid chromatography-tandem mass spectrometry. Notably, AW6 could resist hydrolysis in the human gastrointestinal tract according to the result of the simulated gastrointestinal digestion. To the best of our knowledge, this is the first discovery and characterization of a dual-function (ACEI and DPP4I activities) peptide derived from bitter gourd seed protein.

The circRNA MKLN1 regulates autophagy in the development of diabetic retinopathy.

Diabetic retinopathy (DR) is a common complication in patients with diabetes and has become an important cause of blindness in working-age people. However, the mechanisms involved have not been fully elucidated. Circular RNAs (circRNAs) can play an important role in DR, and they can accurately regulate the expression of target genes through a new regulatory model: the competing endogenous RNA (ceRNA) model. We isolated total RNA from extracellular vesicles in the serum of healthy individuals (Con) and individuals with diabetes mellitus without DR (DM), nonproliferative DR (NPDR), or proliferative DR (PDR) and subjected them to deep sequencing. We found aberrantly high expression of circMKLN1. In a streptozotocin (STZ)-induced mice model of diabetes, the inhibition of circMKLN1 with AAV2 transduction markedly ameliorated retinal acellular vessels and vascular leakage, which was reversed by intravitreal injection of rapamycin, a potent autophagy inducer. In addition, circMKLN1 adsorbs miR-26a-5p as a molecular sponge and mediates high glucose (HG)/methylglyoxal (MG)-induced autophagy in hRMECs. CircMKLN1-silencing treatment reduces HG/MG-related reactive autophagy and inflammation. In addition, miR-26a-5p targeting by circMKLN1 plays an important role in the regulation of Rab11a expression. Thus, either new biomarkers or new therapeutic targets may be identified with the translation of these findings.

The effects of using Tempeh as a supplement for type 2 diabetes.

Studies suggest that the consumption of Tempeh can improve abnormal blood glucose and lipid parameters, although it remains still unclear as to whether Tempeh can improve tissue damage. In our study, db/db obese diabetic mice were given Tempeh 1 (300 mg/kg) and Tempeh 2 (600 mg/kg) for 3 months. The tissue samples collected were stained using different tissue-staining methodologies and were compared with the diabetic control group that was not given any Tempeh. Our results demonstrated that consuming high-dose Tempeh for 1 month could significantly reduce serum glucose and body weight in mice whereas the tissue section of our result could validate that consuming high-dose Tempeh for 3 months effectively improves lipid droplet size and lipid accumulation in the liver, aorta, and kidney of the mice. Moreover, an indication of the recovery of the damaged tissue could be observed in the heart and pancreatic tissue when high dosage of Tempeh was given as a treatment. Thus, it can be concluded that continuous consumption of Tempeh as a treatment could improve both blood glucose and body weight of diabetic mice while also improving lipid accumulation and tissue damage.

Long Noncoding RNA PPT2-EGFL8 Regulates Pathological Retinal Neovascularization in PDR by Functioning as a Competing Endogenous RNA.

Diabetic retinopathy (DR) is a common complication in patients with diabetes, and proliferative DR (PDR) has become an important cause of blindness; however, the mechanisms involved have not been fully elucidated. miRNAs and long noncoding RNAs can play an important role in DR, and they can accurately regulate the expression of target genes through a new regulatory model: competing endogenous RNAs. We isolated total RNA of extracellular vesicles (EVs) in the serum of healthy individuals and individuals with diabetes without DR, non-PDR, or PDR, and performed deep sequencing. We found aberrantly low expression of PPT2-EGFL8 and significantly increased level of miR-423-5p. PPT2-EGFL8 adsorbs miR-423-5p as a molecular sponge and inhibits hypoxia-induced human retinal microvascular endothelial cells proliferation. In an oxygen-induced retinopathy (OIR) model and a streptozotocin-induced diabetes model, Egfl8-overexpression treatment reduces diabetes-related reactive gliosis, inflammation, and acellular capillaries and attenuates the development of pathological neovascularization. In addition, PPT2-EGFL8 targeting miR-423-5p plays an important role in hypoxia-induced peroxisome proliferator-activated receptor-ß/δ (PPARD)/angiopoietin-like 4 (ANGPTL4) signaling activation, especially the expression of the C-terminal ANGPTL4 fragment. Finally, ANGPTL4 significantly induces retinal vessel breakage in the inner limiting membrane and facilitates retinal vessel sprouting into the vitreous in the OIR mice. Thus, either new biomarkers or new therapeutic targets may be identified with translation of these findings.

Toll-like Receptor-Mediated Immunomodulation of Th1-Type Response Stimulated by Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2).

PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4+ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1ß and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

PGE2 promotes macrophage recruitment and neovascularization in murine wet-type AMD models.

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is a leading cause of blindness worldwide. Activated macrophages recruited to the injured eyes greatly contribute to the pathogenesis of choroidal neovascularization (CNV) in exudative AMD (wet AMD). This study describes the effects of cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) signalling on the macrophage activation and CNV formation of wet AMD. In a mouse model of laser-induced wet AMD, the mice received an intravitreal injection of celecoxib (a selective COX2 inhibitor). Optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal histology of the CNV lesions, and biochemical markers were assessed. The level of PGE2 expression was high in the laser-induced CNV lesions. Macrophage recruitment and CNV development were significantly less after celecoxib treatment. E-prostanoid1 receptor (EP1R)/protein kinase C (PKC) signalling was involved in M2 macrophage activation and interleukin-10 (IL-10) production of bone marrow-derived macrophages (BMDMs) in vitro. In addition, IL-10 was found to induce the proliferation and migration of human choroidal microvascular endothelial cells (HCECs). Thus, the PGE2/EP1R signalling network serves as a potential therapeutic target for CNV of the wet-type AMD. Video abstract.

Role of arginine supplementation on muscular metabolism and flesh quality of Pacific white shrimp (Litopenaeus vannamei) reared in freshwater.

It is no doubt that the improvement of flesh quality of Pacific white shrimp (Litopenaeus vannamei) reared in freshwater contributes to its development potential in aquaculture. In this study, we aimed to explore the effect of arginine supplementation on the flesh quality of L. vannamei reared in freshwater and its mechanism. L. vannamei were randomly fed with three diets for 56 days, of which arginine level was 10.15 g kg-1 (arginine-deficient diet), 21.82 g kg-1 (arginine-optimal diet), and 32.46 g kg-1 (arginine-excessive diet), respectively. Each diet was randomly assigned to triplicate tanks, and each tank was stocked with 35 shrimps (initial weight: 1.70 ± 0.02 g). Results showed the arginine-optimal diet increased the weight gain, flesh percentage, crude protein and flavor amino acid contents in muscle, and improved the flesh hardness by conversing fast myofibers to slow myofibers, increasing myofiber density and myofibrillar length, and promoting ornithine and collagen synthesis. The arginine-optimal diet influenced the purine metabolic pathway by reducing hypoxanthine, xanthine, and inosine contents. Ornithine, citrulline, and glutamate were identified as the key metabolites affecting flesh quality traits after arginine treatments. Only increasing the levels of dietary arginine did not result in an increase in endogenous creatine synthesis in muscle and hepatopancreas. Overall, the arginine-optimal diet improved the flesh quality traits of L. vannamei reared in freshwater due to the enhanced muscular hardness, protein deposition, and flavor, which may be contributing to the transformation of muscle fiber type and increase in protein synthesis by the metabolites of arginine (ornithine, citrulline, and glutamate).

Novel insights for PI3KC3 in mediating lipid accumulation in yellow catfish Pelteobagrus fulvidraco.

In this study, the transcriptional regulation of PI3KC3 by three transcription factors (PPARγ, PPARα, and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5'-deletion assay, overexpression assay, site-mutation assay, and electrophoretic mobility shift assay suggested that PPARα, PPARγ, and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG) content, non-esterified fatty acid (NEFA) content, and lipid drops (LDs) content, the mRNA level of pparα, pparγ, stat3, and dnmt3b, the protein level of PPARα, PPARγ, and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.

Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response.

The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163+ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.

LXA4 protects against blue-light induced retinal degeneration in human A2E-laden RPE cells and Balb-c mice.

BACKGROUND: Age-related macular degeneration (AMD) is one of the leading causes of permanent visual impairment in the elderly. Blue light (BL) has been reported to cause retinal damage and contribute to the onset and development of severe AMD. N-retinylidene-N-retinylethanolamine (A2E), a lipofuscin fluorophore, accumulates with ageing in the retinal pigment epithelium (RPE) cells. Once exposed to BL, A2E easily oxidizes to A2E-epoxides, causing oxidative-stress injury to the retina. Lipoxin A4 (LXA4), an endogenous anti-antioxidant lipid, plays a key role in multiple organs by binding to the formyl-peptide receptor-like 1 (FPRL1). This study examined the protective effects of LXA4 on oxidative-stress injury induced by BL exposure, and clarified the underlying mechanisms in cultured RPE cells and Balb-c mice. METHODS: LXA4 diluent was orally administered to mice before retinal degeneration was established. Optical coherence tomography, retinal histology, and RPE cell injury were assessed. RESULTS: LXA4 administration significantly ameliorated retinal damage as evidenced by the thicknesses of the retinal layers and the tight junctions of RPE cells in vivo. LXA4 inhibited BL-induced reactive oxygen species (ROS) production, reduced tight junctions, and the death of A2E-laden RPE cells. LXA4 also potently increased the expression of haem oxygenase-1 (HO1) and NAD(P)H quinone oxidoreductase 1 (NQO1), probably by decreasing the association between nuclear factor erythroid 2-related factor 2 (NRF2) and Kelch-like ECH (Epichlorohydrin) -associated protein 1 (Keap1), and ameliorating NRF2 nuclear translocation and the antioxidant response element (ARE) deoxyribonucleic acid (DNA) binding activity. CONCLUSIONS: Our results showed that LXA4 ameliorated retinal degeneration, and should be considered in the prevention and treatment of AMD.

Data on effect of Tempeh Fermentation on patients with type II diabetes.

Type II diabetes (T2D) arises through insulin resistance and a progressive decrease in insulin secretion, which may be partly related to pancreatic beta-cell function decline, obesity, and eventual hyperglycemia [1]. The first line for managing hyperglycemia in patients with T2D includes lifestyle modifications and metformin monotherapy. However, many patients still showed poor glycemic control due to progressive deterioration during the course of T2D [2, 3]. On streptozotocin-induced T2D rats, tempeh fermentation has been shown to be a potentially beneficial dietary supplement for abnormal carbohydrate metabolism [4]. This study was a prospective open-label clinical trial. The data were collected from Kaohsiung Veterans General Hospital Pingtung Branch, Taiwan from August 2018 to July 2019. 35 eligible T2D participants with a mean age of 57.91 ± 10.17 years were enrolled. After taking 2 g tempeh capsules daily for a period of 3 months, the levels of HbA1C and triglyceride were noticeably decreased in the participants. A regression analysis revealed that cholesterol concentration had a significant positive correlation with the concentrations of LDL, but triglyceride concentration had a significant negative correlation with the concentrations of HDL in the pre- and the post-tempeh treatment.

Condition-Specific Molecular Network Analysis Revealed That Flagellar Proteins Are Involved in Electron Transfer Processes of Shewanella piezotolerans WP3.

Because of the ability to metabolize a large number of electron acceptors such as nitrate, nitrite, fumarate, and metal oxides, Shewanella species have attracted much attention in recent years. Generally, the use of these electron acceptors is mainly achieved through electron transfer proteins and their interactions which will dynamically change across different environmental conditions in cells. Therefore, functional analysis of condition-specific molecular networks can reveal biological information on electron transfer processes. By integrating expression data and molecular networks, we constructed condition-specific molecular networks for Shewanella piezotolerans WP3. We then identified condition-specific key genes and studied their potential functions with an emphasis on their roles in electron transfer processes. Functional module analysis showed that different flagellar assembly modules appeared under these conditions and suggested that flagellar proteins are important for these conditions. We also identified the electron transfer modules underlying these various environmental conditions. The present results could help with screening electron transfer genes and understanding electron transfer processes under various environmental conditions for the Shewanella species.

Genome-scale mutant fitness reveals versatile c-type cytochromes in Shewanella oneidensis MR-1.

Shewanella has been widely investigated for its metabolic versatility and use of a large number of extracellular electron acceptors. Many c-type cytochromes are responsible for this diversity, mainly in condition-specific fashions. By using genome-scale mutant fitness data, we studied which genes (particularly c-type cytochromes) were used to coordinate various electron transfer processes in the present work. First, by integrating fitness profiles with protein-protein interaction (PPI) networks, we showed that the genes with a high total fitness value were generally more important in PPI networks than those with low fitness values. Then, we identified genes that are important across many experiments, and further fitness analysis confirmed five versatile c-type cytochromes: ScyA (SO0264), PetC (SO0610), CcoP (SO2361), CcoO (SO2363) and CytcB (SO4666), which are considered to be crucial in most experimental conditions. Finally, we demonstrated a mediating role in the periplasm for the less-reported CytcB by combining protein structure, subcellular localization and disordered region analysis. Comparative genome analysis further revealed that it is distinctive in Shewanella species. Collectively, these results suggest that periplasmic electron transfer processes are more diverse and flexible than previously reported, giving insight for further experimental studies of Shewanella oneidensis MR-1.

Expression, epitope prediction and IgE-binding of the Tyrophagus putrescentiae group 13 allergen.

Storage mites, such as Tyrophagus putrescentiae, are an important source of allergens that cause allergic diseases in humans. It has previously been indicated that T. putrescentiae has a high sensitization rate as an allergen in some Asian and European countries. Identifying and cloning the allergens in this species may enable improved diagnostic and therapeutic approaches. The aim of the present study was to clone and sequence the T. putrescentiae group 13 allergen (Tyr p 13) isolated from storage mites in China, to use bioinformatics tools to model its biophysical characteristics and to induce protein expression to test its IgE-binding activity. The full-length cDNA comprised 486 bp and was predicted to include a signal peptide of 22 amino acids. Its secondary structure was shown to comprise an α-helix (10.79%), extended strand (33.81%) and random coils (55.40%). Using homology modeling, the present study constructed a reasonable tertiary structure of Tyr p 13. Linear Bcell epitopes at amino acids 47-53, 70-76, 81-86, 101-105 and 112120 were predicted. Three discontinuous B-cell epitopes were also predicted: i) 47, 48, 49, 50, 51, 52, 53, 70, 71, 72 and 73; ii) 91, 92, 93, 94, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 and 138; and iii) 74, 76, 79, 81, 82, 83, 84, 86, 101, 102, 103, 104 and 105. SDS-PAGE identified a specific band at the predicted molecular weight of the recombinant Tyr p 13 (rTyr p 13), demonstrating its successful expression. The rTyr p 13 bound to IgE in the serum of 13.2% (5/38) of patients allergic to T. putrescentiae, according to ELISA. The successful cloning of Tyr p 13 and basic bioinformatics analysis of the protein provided a foundation for the further study of this allergen with regards to the diagnosis and treatment of patients allergic to storage mites. These results provided a theoretical basis for the design of rTyr p 13 with modified B-cell epitopes.

Molecular Dynamics Simulations of Mite Aquaporin DerfAQP1 from the Dust Mite Dermatophagoides farinae (Acariformes: Pyroglyphidae).

Aquaporins are a large family of transmembrane channel proteins that facilitate the passive but highly selective transport of water and other small solutes across biological membranes. House dust mite (Dermatophagoides farinae) is the major source of household immunogens, and we have recently reported six cDNA sequence encoding aquaporins from this mite species. To better understand the structure and role of mite aquaporin, we constructed a tertiary structure for DerfAQP1 by homology modeling from the X-ray structure of malaria aquaporin PfAQP (Protein Data Bank code No. 3C02) and conducted molecular dynamics simulation. The simulation arranged seven water molecules in a single file through the pores of the DerfAQP1. Further, two conserved Asn-Pro-Ala motifs were located on Asn203 and Asn77; residues Arg206, Trp57, Met190, Gly200, and Asp207 constituted an extracellular vestibule of the pore; and residues His75, Val80, Ile65, and Ile182 constituted the cytoplasmic portions. The overall free energy profile for water transport through DerfAQP1 revealed an energy barrier of ~2.5 kcal/mol. These results contribute to the understanding of mite physiology and pathology.

Identification of LincRNA from Dermatophagoides farinae (Acari: Pyroglyphidae) for Potential Allergen-Related Targets.

Long noncoding RNAs (lncRNAs), especially their important subclass of long intergenic noncoding RNAs (lincRNAs), have been identified in some insects. They play important roles in the regulation of biological processes, such as immune response or cell differentiation and as possible evolutionary precursors for protein coding genes. House dust mites (HDMs) are recognized as allergenic mites because allergens are found in their feces and bodies. Dermatophagoides farinae is one of the most important pyroglyphid mites because of its abundance in the household. To determine if lincRNAs can regulate allergen presentation in HDMs, we analyzed RNA-seq data for HDMs. We identified 11 lincRNAs that are related to mRNAs coding for allergens in HDMs. Using qRT-PCR, we amplified 10 lincRNAs and their putative target allergen-encoding mRNAs, confirming expression of these lincRNAs and allergen genes. The results suggest that lincRNAs might be involved in the regulation of allergen production in HDMs and might represent potential acaricidal candidates to inhibit mite allergen production.

Anti-hypertensive effects of Radix Rehmanniae and its active ingredients.

Radix Rehmanniae (RR) is the root tuber of Rehmannia glutinosa Libosch. Herein, the methanol extracts of dried RR (DRR) and processed RR (PRR) were partitioned to obtain ethyl acetate, aqueous, and n-butanol layers. The angiotensin-I converting enzyme (ACE) inhibition test indicated that the ethyl acetate extracts of DRR (DRRE) and PRR (PRRE) show better inhibitory activity. Therefore, changes in blood pressure were tested over 24 h in spontaneously hypertensive rats, with DRR showing good anti-hypertensive activity. DRRE was further subjected to column chromatography; 28 fractions were separated and tested for ACE inhibition. Ultimately, six compounds were identified by spectral analysis, and literature comparison. Specifically, ursolic acid and oleanolic acid showed better ACE inhibition than the other compounds. This study confirmed that DRR has anti-hypertensive activity. In future, DRR's potential as a health food should be further assessed.