Selection of Reference Genes for Gene Expression Studies in Porcine Whole Blood and Peripheral Blood Mononuclear Cells under Polyinosinic:Polycytidylic Acid Stimulation

Wang, Jiying,Wang, Yanping,Wang, Huaizhong,Hao, Xiaojing,Wu, Ying,Guo, Jianfeng Asian Australasian Association of Animal Productio 2014 Animal Bioscience Vol.27 No.4

Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation.

Direct Interaction between Ras Homolog Enriched in Brain and FK506 Binding Protein 38 in Cashmere Goat Fetal Fibroblast Cells

Wang, Xiaojing,Wang, Yanfeng,Zheng, Xu,Hao, Xiyan,Liang, Yan,Wu, Manlin,Wang, Xiao,Wang, Zhigang Asian Australasian Association of Animal Productio 2014 Animal Bioscience Vol.27 No.12

Ras homolog enriched in brain (Rheb) and FK506 binding protein 38 (FKBP38) are two important regulatory proteins in the mammalian target of rapamycin (mTOR) pathway. There are contradictory data on the interaction between Rheb and FKBP38 in human cells, but this association has not been examined in cashmere goat cells. To investigate the interaction between Rheb and FKBP38, we overexpressed goat Rheb and FKBP38 in goat fetal fibroblasts, extracted whole proteins, and performed coimmunoprecipitation to detect them by western blot. We found Rheb binds directly to FKBP38. Then, we constructed bait vectors (pGBKT7-Rheb/FKBP38) and prey vectors (pGADT7-Rheb/FKBP38), and examined their interaction by yeast two-hybrid assay. Their direct interaction was observed, regardless of which plasmid served as the prey or bait vector. These results indicate that the 2 proteins interact directly in vivo. Novel evidence is presented on the mTOR signal pathway in Cashmere goat cells.

Distinct growth phenomenon observed on L-Arg․ CF3COOH crystals

Xiaojing Liu,Zeyan Wang,Guanghui Zhang,Guangwei Yu,Aidong Duan,Xinqiang Wang,Dong Xu 한국물리학회 2009 Current Applied Physics Vol.9 No.1

Atomic force microscopy has been employed to study the growth behaviour on the {101} facets of L-Arg ․ CF3COOH crystals grown at 25℃ at a supersaturation of σ = 0.2. Nucleation on the wider step terraces and a large nucleus composed of two separate 2D nuclei were observed for the first time. For the region where blocking occurs convex pattern are found in the macrosteps, which correspond to the sites of the impurities that act as pinning points hindering step propagation. Finally, liquid inclusions will be generated because of disparate formation mechanisms. Atomic force microscopy has been employed to study the growth behaviour on the {101} facets of L-Arg ․ CF3COOH crystals grown at 25℃ at a supersaturation of σ = 0.2. Nucleation on the wider step terraces and a large nucleus composed of two separate 2D nuclei were observed for the first time. For the region where blocking occurs convex pattern are found in the macrosteps, which correspond to the sites of the impurities that act as pinning points hindering step propagation. Finally, liquid inclusions will be generated because of disparate formation mechanisms.

Molecular identification of DNA barcoding of Leguminous toxic species and quantitative analysis by ELISA kits

Wang Jie,Wang Shuangyu,Sun Fenglin,Liu Chang,Zhao Jinquan,Yu Hongwei,Lv Xiaojing,Liu Ze,Bu Shuhua,Yu Weisen 한국식물생명공학회 2024 Plant biotechnology reports Vol.18 No.2

Some edible Leguminous are toxic when raw, and the Chinese are particularly fond of beans, so Leguminous poisoning is very common in China. Rapid and accurate identification of poisoned species and determination of their toxic components would better assist physicians in treating patients. However, traditional morphology-based identification methods possess many limitations. DNA barcoding technique is a new species identification technique developed in recent years, which is expected to make up for the shortcomings of traditional morphological identification. In this study, a comprehensive evaluation system based on DNA barcoding and ELISA kits was attempted. A total of 30 Leguminous toxic plants were collected, involving 9 genera and 10 species. We used simulated gastric fluid (SGF) to simulate the human gastric environment. Three markers (rbcL, trnH-psbA, and ITS) were amplified and sequenced for all untreated and 15 mock-digested samples. The validity of DNA barcoding for species identification was assessed using the Basic Local Alignment Search Tool (BLAST) method and the tree construction method. The levels of three toxic components (saponin, phytoagglutin and trasylol) were determined in all samples using ELISA kits. The amplification success rate of all three regions was high (rbcL 96.67%, trnH-psbA 100%, and ITS 100%), but the sequencing of the trnH-psbA region was less satisfactory (66.67%), and SGF had a significant impact on the sequencing of the ITS region (After 40 min of SGF treatment, the sequencing success rate decreased by 46.67%). The samples from different species and origins contained different levels of toxic components, and the levels of all three sub- stances decreased significantly after undergoing SGF digestion. After 1 h of SGF treatment, the saponin content decreased to 0–8.60% in untreated content (PHA decreased to 8.62–36.88%, trasylol decreased to 4.70–47.06%). The current results suggest that DNA barcoding has great potential for rapid identification of Leguminous poisoning in clinical settings. Toxins are probably not detectable in the patient for longer periods of poisoning. We recommend DNA barcoding technology as a first step for rapid screening and combined with toxin analysis for clinical diagnosis.

Proteasome subunit beta type 1 interacts directly with Rheb and regulates the cell cycle in Cashmere goat fetal fibroblasts

Yanfeng Wang,Xudong Guo,Zhigang Wang,Zhixin Guo,Mingtao Liu,Keyu Zhao,Xue Feng,Xu Zheng,Xiaojing Wang,Huifang Hao 한국통합생물학회 2017 Animal cells and systems Vol.21 No.5

PSMB1 (proteasome subunit beta type 1) is a core component of the 20S proteasome, and based on its structure, it might have a crucial function in the transcription of certain genes. Rheb (Ras homolog enriched in brain) is a Ras-like small GTPase that acts as an upstream positive regulator of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. We cloned and characterized PSMB1 (KY310590.1) to determine its function in cell cycle progression and proliferation of goat fetal fibroblasts (GFbs). Further, by yeast two-hybrid screen and coimmunoprecipitation, we conformed that PSMB1 interacts directly with Rheb. An siRNA was designed and expressed targeting PSMB1 mRNA in GFbs and inducing cell cycle arrest. Rheb overexpression in GFbs significantly increased the number of S phase cells and growth efficiency compared with control cells. These data indicate that PSMB1 and Rheb have important functions in the cell cycle and proliferation of GFbs, indicating that their interaction governs many processes in GFbs.

Proteomic Analysis on Exosomes Derived from Patients’ Sera Infected with Echinococcus granulosus

Wen Wang,Xiaojing Zhou,Fang Cui,Chunli Shi,Yulan Wang,Yanfei Men,Wei Zhao,Jiaqing Zhao 대한기생충학ㆍ열대의학회 2019 The Korean Journal of Parasitology Vol.57 No.5

Cystic echinococcosis (CE), a zoonotic disease caused by Echinococcus granulosus at the larval stage, predominantly develops in the liver and lungs of intermediate hosts and eventually results in organ malfunction or even death. The interaction between E. granulosus and human body is incompletely understood. Exosomes are nanosized particles ubiquitously present in human body fluids. Exosomes carry biomolecules that facilitate communication between cells. To the best of our knowledge, the role of exosomes in patients with CE is not reported. Here, we isolated exosomes from the sera of patients with CE (CE-exo) and healthy donors and subjected them to liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis identified 49 proteins specifically expressed in CE-exo, including 4 proteins of parasitic origin. The most valuable parasitic proteins included tubulin alpha-1C chain and histone H4. And 8 proteins were differentially regulated in CE-exo (fold change>1.5), as analyzed with bioinformatic methods such as annotation and functional enrichment analyses. These findings may improve our understanding about the interaction between E. granulosus and human body, and may contribute to the diagnosis and prevention of CE.

A Pilot Study of Clinical Evaluation and Formation Mechanism of Irritable Bowel Syndrome-like Symptoms in Inflammatory Bowel Disease Patients in Remission

( Haiyang Wang ),( Xiaojing Zhao ),( Xiufang Cui ),( Meifeng Wang ),( Chunhua Jiao ),( Jiajia Li ),( Yan Yang ),( Yi Li ),( Hongjie Zhang ) 대한소화기기능성질환·운동학회 2021 Journal of Neurogastroenterology and Motility (JNM Vol.27 No.4

Background/Aims Some inflammatory bowel disease (IBD) patients in remission suffer from irritable bowel syndrome (IBS)-like symptoms (IBD-IBS). The pathogenesis has not yet been elucidated. The study aim is to evaluate relationships among quality of life (QOL), psychological status, and visceral sensitivity, and explore the formation mechanism of IBD-IBS. Methods Forty-seven patients with Crohn’s disease in remission, 24 ulcerative colitis in remission, 26 IBS, and 20 healthy controls were included in the study. The abdominal pain, QOL, anxiety, and depression were evaluated through questionnaires. Visceral sensitivity was measured by rectal balloon distension. The serum levels of 5-hydroxytryptamine (5-HT) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay. The expressions of tryptase, 5-HT, NGF, and related receptors in colonic tissues were detected by immunohistochemistry and western blot. Results Prevalence of IBS-like symptoms in Crohn’s disease and ulcerative colitis patients in clinical remission was 29.8% and 50.0%, respectively. The QOL was lower, the anxiety/depression scores were higher in IBD-IBS patients than those without IBS-like symptoms. Additionally, patients with IBD-IBS existed visceral hypersensitivity. Besides, abdominal pain was associated with poor QOL, visceral hypersensitivity, anxiety, and depression in IBD-IBS patients. The number of mast cells (MCs) and expressions of 5-HT, NGF, and related receptors were higher in IBD-IBS patients than those with no such symptoms. The serum levels of 5-HT and NGF positively correlated with abdominal pain and visceral hypersensitivity. Conclusion IBD-IBS patients may have low QOL and psychological abnormalities, as wells as visceral hypersensitivity which may be related to increased 5-HT and NGF levels released from activated mast cells. (J Neurogastroenterol Motil 2021;27:612-625)

Interfacial behaviors in Cu/molten Sn–58Bi/Cu solder joints under coupling with thermal and current stressing

Fengjiang Wang,Hong Chen,Dongyang Li,Zhijie Zhang,Xiaojing Wang 대한금속·재료학회 2019 ELECTRONIC MATERIALS LETTERS Vol.15 No.1

The interfacial behaviors of Cu/molten Sn–58Bi/Cu solder joints under the coupling eff ect of a temperature gradient and thecurrent stressing have been investigated. The most obvious change of the interfacial behaviors under the individual electromigration(EM) and thermomigration (TM) in molten solder was the asymmetrical growth of interfacial Cu–Sn intermetalliccompounds (IMCs), which grew rapidly as the stressing time prolonged. The growth rates of the interfacial IMCs inducedby TM under a temperature gradient of 40 °C/cm were slightly faster than EM under the current density of 0.5 × 10 4 A/cm 2 . However, the microstructure evolution and interfacial behaviors changed obviously when the thermal was distributedunevenly across the entire solder stripe under current stressing. It was found that there was a Bi-rich layer adhered to theanode side and a distributed Cu 6 Sn 5 phase existed in the solder matrix when the anode and the cold end were at the sameend. Additionally, a large number of Bi-blocks and Cu–Sn IMCs were dramatically observed in the solder matrix when theanode and the hot end were on the same side. The main reason for this result may be attributed to the signifi cant change ofthe diff usion of Bi atoms under the thermo-electric coupling conditions. In the initial melting stage of solder, the Bi atomsin molten Sn–58Bi solder rapidly diff used to the anode and then began to be reversely dissolved into the solder matrix. Theexperimental results proved the additional temperature gradient played a positive or negative role on the reverse dissolutionof Bi atoms in the heat preservation process.

Mycobacterium tuberculosis PE_PGRS45 (Rv2615c) Promotes Recombinant Mycobacteria Intracellular Survival via Regulation of Innate Immunity, and Inhibition of Cell Apoptosis

Xu Tao,Wang Chutong,Li Minying,Wei Jing,He Zixuan,Qian Zhongqing,Wang Xiaojing,Wang Hongtao 한국미생물학회 2024 The journal of microbiology Vol.62 No.1

Tuberculosis (TB), a bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is a significant global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study, we successfully constructed a recombinant Mycobacterium smegmatis (M. smegmatis) strain which heterologously expresses the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent factor actively involved in the interaction with the host macrophage.

Application of Biophysical Properties of Meridians in the Visualization of Pericardium Meridian

Xiong Feng,Xu Ruimin,Li Tongju,Wang Jinyu,Hu Qingchuan,Song Xiaojing,Wang Guangjun,Su Huanhuan,Jia Shuyong,Wang Shuyou,Zhu Zongxiang,Zhang Weibo 사단법인약침학회 2023 Journal of Acupuncture & Meridian Studies Vol.16 No.3

Background: The biophysical properties of the meridian system, an important concept of traditional Chinese medicine, include low impedance, resounding voice, and high acoustic conductance, all of which are helpful for elucidating the essence of meridians. Objectives: To visualize the human pericardium meridian (PC) based on the resounding voice property of meridians. Methods: Visualization of the PC was performed by injection of fluorescein sodium at the PC6 acupoint (Neiguan) on the PC. Before injection, percussion active points (PAPs) were identified by the virtue of their resounding voice properties. After injection, the trajectories of fluorescein migration throughout the body surface were recorded and analyzed. The distribution of fluorescein in the tissue was further studied using cross-sections of hind limbs of mini-pigs, in which fluorescein was injected into low impedance points. Results: The identified PAP lines were colocalized with PC. Following intradermal fluorescein injection, 1-3 fluorescent lines, which were unrelated to the arm veins, were observed in 7 of 10 participants; 85.4% of fluorescent signals were coincident with PAPs and their intensity had a negative correlation with the body mass index (r = –0.56, p = 0.045). Cross-sections showed a Y-shaped fluorescence pattern where the two migration lines on the surface were the two vertices of the “Y.” Conclusion: The trajectories of fluorescein in the body are suggestive of the anatomical structure of meridians. The PC is related to the deep horizontal interstitial channels that connect to the body surface through vertical interstitial spaces. These biophysical properties and techniques for meridian visualization are valuable for revealing the anatomical structure of meridians.