Search

Background: As a ferroptosis-inducing platform, based on its release of iron and ferrous ions to induce production of reactive oxygen species (ROS), it has attracted extensive attention in cancer treatment. Since iron-based nanomaterials such as prussian blue (PB), can only produce limited ROS. Bufotalin (CS-5), a promising ferroptosis inducer, was introduced to improve the efficiency of ferroptosis. However, the clinical application of CS-5 was limited by cardiotoxicity and high renal clearance rate. Therefore, we constructed a biomimetic nanosystem (PB@CS-5@M) with satisfactory tumor enrichment ability and long blood circulation time for the treatment of colorectal cancer induced by ferroptosis. Results: In this work, the PB@CS-5@M has efficient loading of CS-5, which significantly reduces the cardiotoxicity of CS-5. After photothermal therapy, the high temperature caused by laser stimulation triggers the release of CS-5, iron and ferrous ions to kill colorectal cancer cells. Furthermore, the ability of CS-5 to significantly down-regulate the expression of GPX4 and HIF-1a, induce ROS production and damage mitochondria to induce ferroptosis in colorectal cancer was amplified. In addition, the down-regulation of HSP70 expression under laser stimulation effectively improved the chemosensitivity of HCT116 cells to CS-5. Conclusions: The chemotherapeutic effect of CS-5 combined with the photothermal effect of PB can effectively treat colon cancer by inducing ferroptosis.

The dysbiosis of gut microbiota is closely related to the occurrence and development of inflammatory bowel disease (IBD). The manipulation of intestinal flora through prebiotics or probiotics is expected to induce and maintain the remission of IBD symptoms. 6-week-old C57BL/J mice were daily gavaged with fructooligosaccharides (FOS) or the synbiotic two weeks before the administration of dextran sulfate sodium (DSS). The supplementation of FOS or synbiotic could significantly ameliorate the body weight loss and colon histological damage in DSS-induced acute colitis mice. The altered composition of gut microbiota in acute colitis mice was reversed by FOS or Synbiotic supplementation, with a characteristic of decreased abundance of Mucispirillum. Both FOS and synbiotic mitigated DSS-induced loss of mucus protein (MUC2) and epithelium tight junction proteins (ZO-1, Occluding, Claudin1) in colon mucosa. The expression of pro-inflammatory cytokines (IL-6 and TNF-α) was decreased by FOS or synbiotic treatment, while the expression of Tbx21 and IL-10 was increased. The results suggested that the modulation of gut microbiota by FOS or synbiotic supplementation could decrease the inflammation potential of colonized commensals, which prevented the impairment of the intestinal barrier and induced a regulation of immune response in DSS-induced acute colitis mice.

Microtubule (MT) cytoskeleton of pepper male-sterile microsporocytes during meiosis was examined to characterise the defects during microsporogenesis by comparison with that of fertile ones. In ste...

Background: Some studies have demonstrated that Hapto and Gremlin1 play an important biological role in many neoplasms. However, the role of Hapto and Gremlin1 in extrahepatic cholangiocarcinoma (ECC) remains to be revealed. Thus, this study investigated the prognostic and clinicopathological significance of Hapto and Gremlin1 expression in ECC. Methods: We examined Hapto and Gremlin1 expression in 100 ECC, 30 peritumoral tissues, 10 adenoma and 15 normal biliary tract tissues using EnVision immunohistochemistry. The relationship between Hapto and Gremlin 1 expression and clinicopathological parameters was evaluated using the χ2 test or Fisher's exact test. The overall survival of patients was analyzed using Kaplan-Meier univariate survival analysis and log-rank tests. Results: Hapto and Gremlin1 proteins were overexpressed in ECC compared to peritumoral tissues, adenoma, and normal biliary tract (P

Aim EphB3 and dysadherin are involved in tumorigenesis and progression of many neoplasms. However, the roles of EphB3 and dysadherin in extrahepatic cholangiocarcinoma (ECC) remain to be revealed. In this study, we aimed to evaluate the expression of EphB3 and dysadherin, and investigate their clinicopathological significance in ECC. Methods We examined EphB3 and dysadherin expression in 100 ECC, 30 peritumoral tissues, 10 adenoma and 15 normal biliary tract tissues using EnVision immunohistochemistry. The relationship between EphB3 or dysadherin expression and clinicopathological features was evaluated using the χ2 test or Fisher's exact test. The overall survival of ECC patients was analyzed using Kaplan-Meier univariate survival analysis and Log rank tests. Results We found that EphB3 expression was significantly down-regulated and dysadherin expression was significantly up-regulated in ECC tissues compared with normal tissues (P < 0.01). EphB3 expression was negatively correlated with dysadherin expression in ECC (P < 0.01). The positive rate of EphB3 expression and negative rate of dysadherin expression was significantly higher in patients with well-differentiated type, no lymph node metastasis, no surrounding tissues and organs invasion, early TNM stages (I + II) and radical resection (P < 0.01). The survival of ECC patients with positive EphB3 or negative dysadherin expression was significantly longer than patients with negative EphB3 or positive dysadherin expression (P < 0.01). Cox multivariate analysis demonstrated that negative EphB3 or positive dysadherin expression were independent poor prognostic factors in ECC patients. The ROC curves suggested that EphB3 and dysadherin combined diagnostic efficacy (AUC=0.688, 95%CI: 0.603-0.772) was significantly higher EphB3 diagnostic efficacy (AUC=0.654, 95%CI: 0.564-0.743) or dysadherin diagnostic efficacy (AUC=0.648, 95%CI: 0.558-0.737) alone. Conclusion EphB3 and dysadherin are involved in the carcinogenesis and progression of ECC, and ECC patients with negative EphB3 or positive dysadherin expression have a poor prognosis.

Extrahepatic cholangiocarcinoma is a malignant tumor and poor prognosis with intrinsic resistance to cytotoxic agents. The molecular mechanism associated with high malignancy and resistance to chemotherapy and radiotherapy has not been fully elucidated. This study aims to investigate the clinicopathological significances of HMGA2 and Thy1 expression in extrahepatic cholangiocarcinoma.The expressions of HMGA2 and Thy1 in 100 extrahepatic cholangiocarcinoma, 30 peritumoral tissues, 10 adenoma and 15 normal biliary tract tissues were assayed using EnVision immunohistochemistry.The HMGA2 and Thy1 proteins were overexpression in extrahepatic cholangiocarcinoma compared to peritumoral tissues, adenoma, and normal biliary tract tissues (P 0.05 or P 0.01). Adenoma and pericancerous tissues with positive HMGA2 or/and Thy1 protein expression exhibited atypical hyperplasia. The positive correlation was found between the expression of HMGA2 and Thy1 in extrahepatic cholangiocarcinoma (P 0.01). The positive rates of HMGA2 and Thy1 expression were significantly higher in cases with poor differentiation, lymph node metastasis, invasion, and TNM stage III or IV and no resection (biopsy only) (P 0.05 or P 0.01). Kaplan-Meier survival analysis showed that the survival of extrahepatic cholangiocarcinoma patients with positive HMGA2 and/or Thy1 expression is significantly shorter than patients with negative HMGA2 and/or Thy1 expression (P = 0.000). Cox multivariate analysis revealed that positive HMGA2 and/or Thy1 expressions were independently poor prognosis factors in extrahepatic cholangiocarcinoma patients. We calculated the AUC for HMGA2 (AUC = 0.610, 95%CI: 0.519-0.702), or Thy1 (AUC = 0.675, 95%CI: 0.588-0.762), respectively.The present study indicated that positive HMGA2 and Thy1 expression are closely associated with the pathogenesis, clinical, pathological and biological behaviors, and poor prognosis in patients with extrahepatic cholangiocarcinoma.

Graphical abstract, Background Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. Methods The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. Results mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 μg/L, with a precision coefficient of variance below 10 %. Conclusion The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.

Bacterial infection, especially multidrug-resistant bacteria-induced infection, threatens human health seriously, which has posed great challenges for clinical therapy. The overuse of conventional antibiotics has given rise to bacterial resistance that severely restricts the clinical treatment options of conventional antibiotics. The development of highly effective antibacterial materials and therapeutic strategies to inhibit the multidrug-resistant bacteria-induced infections is of great urgency. Although silver nanoparticles (AgNPs) have exhibited certain effectiveness in killing multidrug-resistant bacteria, their antibacterial efficacy and biosafety are still unsatisfactory. In this work, we prepared graphene quantum dots (GQDs) by a green synthesis method with the natural polymer starch as a precursor for uniformly decorating AgNPs to form GQDs coated AgNPs (GQDs@Ag). The nanocomplex was comprehensively characterized, and its antibacterial activity and biosafety were systematically investigated. The characterization results revealed that the successfully constructed GQDs@Ag hybrids with improved dispersion and stability are composed of AgNPs closely and uniformly surrounded by the GQDs. Furthermore, in vitro and in vivo results demonstrated that GQDs@Ag hybrids with superior biosafety showed a markedly enhanced effect in killing MRSA and accelerating MRSA-infected wound healing as compared to AgNPs alone. Collectively, these results suggest that the biocompatible nanosystem of GQDs@Ag exhibits great potential in clinical application for MRSA infection.

Recently, accumulating evidence has suggested that gut microbiota is involved in modulating brain function and behavior through the gut-brain axis. Manipulation of gut microbiota by functional foods, such as prebiotics and probiotics, might improve mental health in stress-related psychiatric disorders. In present study, we found that supplementation of fructooligosaccharides could improve the depression-like behaviors and had a significant effect on the composition of gut flora in chronic stress mice. FOS treatment exhibited no effects on the elevated level of serum corticosterone in chronic stress mice, but significantly reversed the alterations of colonic tight junction proteins (Claudin1, Occludin, ZO-1) and reduced the pro-inflammatory cytokines (TNF-α, IL-6) in colon and brain. Thus, FOS might exert an anti-depressant effect via the prevention of intestinal barrier impairment and neuroinflammation induced by chronic stress.

Background: X-box-binding protein 1 (XBP1) and N-acetyltransferase 1 (NAT1) are involved in oncogenesis and progression of many human cancer types. However, the roles of XBP1 and NAT1 in gallbladder cancer (GBC) are never reported.Methods: We examined XBP1 and NAT1 expression in GBC and matched adjacent non-tumor tissues via Western blotting. Then, we assayed XBP1 and NAT1 expression in 215 GBCs, including 69 squamous cell/adenosquamous carcinomas (SC/ASCs) and 146 adenocarcinomas (ACs) with immunohistochemistry. Their prognostic and clinicopathological significance was further evaluated using the χ2 test or Fisher's exact test, Kaplan–Meier univariate survival analysis, and log-rank tests.Results: XBP1 expression was upregulated, and NAT1 expression was downregulated in GBC. Immunohistochemical results showed that XBP1 expression was negatively associated with NAT1 expression in GBC, including SC/ASC and AC. The rate of patients with an age of more than 45 years, positivity of lymph node metastasis, and invasion were significantly higher in SC/ASC than those in AC (all P < 0.05). The percentage of XBP1-positive and NAT1-negative expression was significantly higher in the cases with poor differentiation, advanced tumor, nodes, and metastases (TNM) stage, lymph node metastasis, invasion, and only receiving biopsy in GBC, SC/ASC, and AC (all P < 0.05). XBP1-positive and NAT1-negative expression was positively related to larger tumor size (>3 cm) in GBC and AC. There was a negative association between XBP1 and NAT1 expression in GBC, SC/ASC, and AC (all P < 0.05). Positive XBP1 and negative NAT1 expression was closely associated with decreased overall survival in GBC, SC/ASC, and AC patients (all P < 0.05). The multivariate Cox regression analysis showed that positive XBP1 or negative NAT1 expression was an independent factor for poor prognosis in gallbladder SC/ASC and AC patients.Conclusions: This study indicates that positive XBP1 and negative NAT1 expression are closely associated with the clinicopathological and biological behaviors and poor prognosis in GBC.

CMS, which refers to the inability to generate functional pollen grains while still producing a normal gynoecium, has been widely used for pepper hybrid seed production. Pepper line 8214A is an excellent CMS line exhibiting 100% male sterility and superior economic characteristics. A TUNEL assay revealed the nuclear DNA is damaged in 8214A PMCs during meiosis. TEM images indicated that the 8214A PMCs exhibited asynchronous meiosis after prophase I, and some PMCs degraded prematurely with morphological features typical of PCD. Additionally, at the end of meiosis, the 8214A PMCs formed abnormal non-tetrahedral tetrads that degraded in situ. To identify the genes involved in the pollen abortion of line 8214A, the transcriptional profiles of the 8214A and the 8214B anthers (i.e., from the fertile maintainer line) during meiosis were analyzed using an RNA-seq approach. A total of 1355 genes were determined to be differentially expressed, including 424 and 931 up- and down- regulated genes, respectively, in the 8214A anthers during meiosis relative to the expression levels in the 8214B. The expression levels of ubiquitin ligase and cell cycle-related genes were apparently down-regulated, while the expression of methyltransferase genes was up-regulated in the 8214A anthers during meiosis, which likely contributed to the PCD of these PMCs during meiosis. Thus, our results may be useful for revealing the molecular mechanism regulating the pollen abortion of CMS pepper.

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line...

Rac activation is precisely regulated temporally and spatially by intracellular signaling pathways in migrating cells to guarantee the formation of specific cell protrusions-lamellipodia at the leading edge. Integrins-mediated adhesions also control the signaling pathway for localized Rac activation in the cells, but very few studies have been addressed in this field. In the study, we aim to focus on how integrin-mediated signaling affects localized Rac activation by reducing the paxillin expression with shRNA targeting paxillin. The results revealed that reduction of the paxillin expression in the cells inhibited the formation of focal adhesions and Rac activation. By using Rac FRET biosensor, Rac activation was localized at the leading edge of the cell, within the lamellipodium. A ternary complex of paxillin-GIT1-PIX could establish the signaling pathway in front of the cells. Thus, we described a mechanism of integrin-mediated signaling for localized Rac activation that upon ligand binding, activated integrin via the signaling pathway paxillin-GIT1-PIX promotes localized Rac activation at the leading edge and cell migration.

Background Serum procalcitonin (PCT) has been recognized as a primary biomarker in bacterial infections, and monitoring its concentration could help to evaluate the prognosis of sepsis and guide the antibiotic administration. We aimed to establish a fast and accurate immunoassay for PCT quantitation. Methods Our newly developed monoclonal antibodies (mAbs) against human PCT were preliminarily evaluated by enzyme-linked immunosorbent assay and then used to develop a chemiluminescence enzyme immunoassay (CLEIA). The proposed CLEIA was assessed in analytical performance and applied to measurement of serum PCT. Results mAb 2D3 and mAb 8F6 were selected as capture and detection antibody respectively, due to the highest sensitivity for PCT detection with no cross reaction to calcitonin gene-related peptides. The proposed CLEIA based on mAb pair of 2D3/8F6-AP was characterized for a working range from 0.03 to 100 μg/L. An excellent correlation was observed between our proposed assay and the VIDAS BRAHMS PCT assay (r: 0.9825). Conclusion Our newly developed mAbs and CLEIA can serve as important diagnostic tools for measurement of human PCT in serum.

The rapid and sensitive detection of heart failure biomarker galectin-3 (Gal-3) is of great value in the prevention, diagnosis, treatment, and prognosis-evaluation of heart failure. Here, a high-performance dual-signal sandwich-type electrochemical immunosensor for Gal-3 was constructed by the covalent immobilization of the first antibody on electroreduced carboxyl graphene and the adsorption immobilization of the second antibody in aminated mesoporous silica nanoparticles labeled with methylene blue (MB) and gold nanoparticles (AuNPs). The relevant electrodes and materials were characterized by electrochemistry, quartz crystal microbalance, scanning electron microscopy and energy dispersive spectroscopy. Under optimized conditions, the MB-based in-situ microdroplet voltammetry showed a linear detection range (LDR) of Gal-3 from 50 fg mL−1 to 500 ng mL−1, with a limit of detection (LOD, S/N = 3) of 2.0 fg mL−1; and the AuNPs-based in-situ microdroplet anodic stripping voltammetry showed an LDR of Gal-3 from 0.5 fg mL−1 to 500 ng mL−1, with a LOD (S/N = 3) of 0.17 fg mL−1. The detection of clinical serum samples by our method returned satisfactory results. This research may help the high-sensitivity voltammetric detection of both noble metal nanomaterials and other electroactive biolabels for wider bioanalysis applications.

Summary: Periostin (POSTN) is a multifunctional extracellular component that regulates cell-matrix interactions and cell-cell crosstalk. POSTN deletion significantly decreases leukemia burden in mice; however, the underlying mechanisms by which POSTN promotes B cell acute lymphoblastic leukemia (B-ALL) progression remain largely unknown. Here, we demonstrate that bone marrow (BM)-derived mesenchymal stromal cells (BM-MSCs) express higher levels of POSTN when co-cultured with B-ALL cells in vitro and in vivo. POSTN deficiency in BM-MSCs significantly decreases CCL2 expression in co-cultured B-ALL cells in vitro and in vivo. Moreover, POSTN treatment increases expression of CCL2 in B-ALL cells by activating the integrin-ILK-NF-κB pathway. Conversely, CCL2 treatment upregulates expression of POSTN in BM-MSCs via STAT3 activation. Furthermore, there is a positive correlation between POSTN expression and CCL2 level in the BM of mice and patients with B-ALL. These findings suggest that B-ALL cell-derived CCL2 contributes to the increased leukemia burden promoted by BM-MSC-derived POSTN. : Ma et al. show that BM-MSC-derived periostin promotes leukemia progression by activating the integrin-ILK-NF-κB-CCL2 pathway in leukemia cells and that leukemia cell-derived CCL2 increases periostin expression in BM-MSCs by activating STAT3. This work identifies critical crosstalk between leukemia cells and stromal cells via periostin and CCL2 in B-ALL progression. Keywords: periostin, CCL2, MSC, B-ALL, leukemia, bone marrow, extracellular matrix, matricellular protein

Procalcitonin (PCT) is an important indicator for bacterial inflammatory diseases, and its sensitive, accurate and rapid detection has important clinical value. On the basis of sandwich immunoassay, glucose oxidase-catalyzed gold deposition and in-situ microliter-droplet anodic stripping voltammetry (ASV) of the enzyme-generated gold directly on the immunoelectrode, the ultrasensitive electrochemical detection of PCT is achieved. A new method of the chemical dissolution of gold by an appropriately diluted aqua regia and the simultaneous cathodic preconcentration of gold on the immunoelectrode is suggested, which gives the better performance for the ASV analysis of gold than the reported one. Under optimized conditions, the ASV peak current is linear with the common logarithm of PCT concentration from 0.05 fg mL−1 to 500 ng mL−1, with a limit of detection (LOD, S/N = 3) as low as 0.04 fg mL−1. Our method has also been used for detection of PCT in serum samples with satisfactory results.

It has been shown that CD40 is required for optimal B cell activation. Casitas-B-lineage lymphoma-b (Cbl-b), a RING finger E3 ubiquitin ligase, inhibits B cell activation. In this report, we demonstrate that CD40 stimulation markedly enhances IgM-induced B cell proliferation in wild-type (WT) mice, whereas this cell proliferation was reduced in CD40-deficient (Cd40−/−) mice. Interestingly, CD40 ligation strongly augments IgM-induced Cbl-b ubiquitination and degradation in primary mouse B cells, which closely correlates with their proliferation capacity. Cbl-b deficiency uncouples BCR-induced B cell proliferation from CD40 costimulation. Our results indicate that Cbl-b negatively regulates costimulation of BCR and CD40, possibly by setting the threshold for B cell activation via controlling Cbl-b expression., Highlights • CD40 is required for optimal B cell activation. • B cell activation threshold is controlled by Cbl-b. • CD40 costimulation promotes Cbl-b ubiquitination and degradation.

Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase delta, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2alpha could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2alpha indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2alpha to this region showed opposite effects.

Remarkable progress was made in the development of white-light-emitting diodes (LEDs). White LEDs provided a light element having a semiconductor InGaN light-emitting chip (blue or UV LEDs) and luminescent phosphors. Here we reported the sialon s-phase of (Sr, Eu) 2 Al 2 Si 10 N 14 O 4 . Eu 2+ activator ions that were substituted for the strontium site represented a new type of yellow-green phosphor that could be excited by blue LEDs used for application in the fabrication of white LEDs.

Focal adhesions are large multi-protein complexes that serve as the linkage between extracellular matrix (ECM) and actin cytoskeleton and control the network of signaling cascades underlying cell migration. Talin plays a key role in focal adhesion turnover, and calpain-mediated proteolysis of talin is central to focal adhesion disassembly, but its regulation is not well elucidated. Here we demonstrate that talin phosphorylation at three high stoichiometry sites on its head domain, T144 and T150, or S446, have contrasting effects on calpain-mediated cleavage of talin and cell migration by using site-directed mutagenesis to inhibit phosphorylation. Expression of talinT144A+T150A stimulated calpain-mediated cleavage of talin and accelerated focal adhesion disassembly, whereas expression of talinS446A fully inhibited talin cleavage by calpain, preventing focal adhesion disassembly. A large decrease in phospho-threonine or phospho-serine levels was seen with talinT144A+T150A or talinS446A respectively, while more active ERK was present in talinT144A+T150A than in talinS446A. Cell adhesion and transwell assays using uniformly expressing cells showed that expression of talinT144A+T150A or talinS446A have opposing effects on cell adhesion and migration. These findings define and highlight the integral role of site-specific high stoichiometry phosphorylation of talin in regulating calpain-mediated cleavage of talin and focal adhesion disassembly, thus controlling adhesion stability, cell adhesion and ultimately, cell migration.

A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.

The suboptimal features of the spectral properties of the leading fluorescence resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), limit the full promise of FRET imaging. To overcome the drawbacks, we developed FRET-based, intra-molecular biosensors consisting of CFP/discomona sp red fluorescent protein (DsRed) or green fluorescent protein (GFP)/DsRed as donor/acceptor fluorophores. The biosensors were expressed in NIH3T3 cells. In vitro fluorescence spectroscopy and Rho GTPase activation assays were used to confirm that Rac1 or Cdc42 was activated in serum-starved cells following stimulation with insulin or bradykinin. The transient changes of the amount, location, and translocation of activated Rac1 or Cdc42 in living cells were tracked with confocal imaging. The increase of FRET efficiency was achieved in the cells expressing the biosensors and was proportional to the levels of activated Rac1 or Cdc42. The localized, transitional, and transient FRET signals were directly and quantitatively imaged with high spatial and temporal resolution. The biosensors were used to analyze and judge the GEF or GAP activities of putative regulatory proteins for Rac1 or Cdc42. DsRed is a more suitable acceptor in FRET pair with CFP than with GFP in terms of the spectral overlap between the donor and acceptor. The approach can also be applied to many other types of protein behavior in living cells.

Hepatitis B virus (HBV) infection can cause the severe threat to the health of the people around the world. It depends upon the development of efficient diagnostic reagent and vaccine to prevent the prevalence of HB. In this study, we constructed the high expression recombinant Pichia pastoris and performed the screening tests in shake flasks to obtain the optimal values of several key fermentation parameters. Based on their effects on the growth and expression level of recombinant strains, FBS was the optimal industrial medium. The optimal values for the dissolve oxygen (DO), the final concentrations of methanol and the pH values were 50 mL, 1% (V/V) and 5.4-6.0, respectively. The optimal values of the parameters simulated in shake flasks were successfully scaled up to 10 L bioreactors to achieve high-throughput production: 310 OD600 in biomass and 27 mg/L in recombinant HBsAg. The expressed recombinant HBsAg in P. Pastoris was confirmed by SDS-PAGE and Western blotting. Electron microscopy examination showed that the purified protein could be self-assembled to 22 nm virus-like particles. The results provided a basis for industrial scale-up production of diagnostic reagent and vaccine of next generation against HB.

Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an immediate-early response gene of endothelium induced by TNF alpha. However, little is really known concerning the TNFAIP1 expression regulation. To better understand how TNFAIP1 expression is regulated, we functionally characterized the promoter region of human TNFAIP1 gene. Deletion mutation analysis, gel electrophoretic mobility shift, and site-directed mutagenesis assays allowed the identification of one functional Sp1-binding site within the human TNFAIP1 core promoter region. Moreover, chromatin immunoprecipitation analysis indicated that Sp1 was associated in vivo with the TNFAIP1 promoter. Further, Sp1 overexpression enhanced TNFAIP1 promoter activity. These findings suggest that Sp1 is implicated in the control of basal TNFAIP1 gene expression. Accordingly, Sp1 is supposed to be involved in the elevation of TNFAIP1 in response to TNF alpha induction, and thus participate in inflammation-associated angiogenesis.

Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase delta, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2alpha could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2alpha indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2alpha to this region showed opposite effects.

We identified potassium channel tetramerization domain-containing 1 (KCTD1) gene in a human brain cDNA library. Here, we report that the KCTD1 gene contains seven exons, encoding 257 amino acid residues with a predicted molecular mass of 29.4 kDa. Sequence alignments showed KCTD1 protein contains an N-terminal broad-complex, tramtrack, and bric-a-brac (BTB) domain. Northern blot analysis revealed that KCTD1 is expressed in the mammary gland, kidney, brain, and ovary compared to other tissues. Further, the subcellular localization results showed that KCTD1 is localized in the nuclei of HeLa and HBL100 cells. Reporter gene assays in HEK293FT and NIH3T3 cells further indicated that KCTD1 acts as a potent transcriptional repressor and inhibits the transcriptional activity via its BTB domain, though KCTD1 transcriptional repression is unaffected by the HDAC inhibitors, trichostatin A, and sodium butyrate. Finally, we found that the BTB domain of KCTD1 mediates homomeric protein-protein interactions by co-immunoprecipitation and GST pull-down assays. These data present the first characterization of human KCTD1 and suggest that KCTD1 is a nuclear protein that functions as a transcriptional repressor and mediates protein-protein interactions through a BTB domain.