APC/C-regulated CPT1C promotes tumor progression by upregulating the energy supply and accelerating the G1/S transition.

BACKGROUND: In addition to functioning as a precise monitoring mechanism in cell cycle, the anaphase-promoting complex/cyclosome (APC/C) is reported to be involved in regulating multiple metabolic processes by facilitating the ubiquitin-mediated degradation of key enzymes. Fatty acid oxidation is a metabolic pathway utilized by tumor cells that is crucial for malignant progression; however, its association with APC/C remains to be explored. METHODS: Cell cycle synchronization, immunoblotting, and propidium iodide staining were performed to investigate the carnitine palmitoyltransferase 1 C (CPT1C) expression manner. Proximity ligation assay and co-immunoprecipitation were performed to detect interactions between CPT1C and APC/C. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) assays, cell-scratch assays, and transwell assays and xenograft transplantation assays were performed to investigate the role of CPT1C in tumor progression in vitro and in vivo. Immunohistochemistry was performed on tumor tissue microarray to evaluate the expression levels of CPT1C and explore its potential clinical value. RESULTS: We identified CPT1C as a novel APC/C substrate. CPT1C protein levels exhibited cell cycle-dependent fluctuations, peaking at the G1/S boundary. Elevated CPT1C accelerated the G1/S transition, facilitating tumor cell proliferation in vitro and in vivo. Furthermore, CPT1C enhanced fatty acid utilization, upregulated ATP levels, and decreased reactive oxygen species levels, thereby favoring cell survival in a harsh metabolic environment. Clinically, high CPT1C expression correlated with poor survival in patients with esophageal squamous cell carcinoma. CONCLUSIONS: Overall, our results revealed a novel interplay between fatty acid utilization and cell cycle machinery in tumor cells. Additionally, CPT1C promoted tumor cell proliferation and survival by augmenting cellular ATP levels and preserving redox homeostasis, particularly under metabolic stress. Therefore, CPT1C could be an independent prognostic indicator in esophageal squamous cell carcinoma.

Combining multisite functionalized magnetic nanomaterials with interference-free SERS nanotags for multi-target sepsis biomarker detection.

Surface-enhanced Raman scattering (SERS) is an ultra-sensitive vibration spectroscopy technology, with the advantages of multi-index and non-destructive quantitative detection, has attracted much attention in the joint detection of biomarkers. A novel SERS biosensor with multisite capture and interference-free quantification was designed for the joint detection of the sepsis biomarker interleukin-6 (IL-6) and procalcitonin (PCT). This biosensor had two interference-free core-shell SERS probes with highly efficient electromagnetic enhancement and a multisite functionalized magnetic nanomaterial with high adsorption capacity. They formed sandwich structure with the targets through boronic affinity and immunoreaction, and the multi-target quantitative analysis of biomarkers in serum was performed using a portable Raman spectrometer in the Raman-silent region. The SERS biosensor was exhibited highly sensitive with detection limits of 0.584 and 2.99 pg/mL for IL-6 and PCT, respectively. In addition, it exhibited excellent selectivity and specificity even with the interference of other proteins. As this SERS method showed excellent performance in the detection of sepsis, it has great potential for multi-index detection in clinical diagnosis of major diseases.

High-sensitivity biosensor based on SERS integrated with dendrimer-assisted boronic acid-functionalized magnetic nanoparticles for IL-6 detection in human serum.

High-sensitivity quantitative analysis of sepsis disease markers in circulating blood is essential for sepsis early diagnosis, rapid stratification, and interventional treatment. Herein, a high-sensitivity biosensor combining surface-enhanced Raman spectroscopy (SERS) and functionalized magnetic materials was developed to quantitatively detect interleukin-6 (IL-6), a glycoprotein disease marker closely related to sepsis. First, boronic acid-functionalized magnetic nanomaterials with high adsorption performance were synthesized by utilizing the branched polyethyleneimine to provide many binding sites for boronic acid. Under antibody-free conditions, dendrimer-assisted boronic acid-functionalized magnetic nanomaterials selectively capture glycoproteins in complex biological samples as bio-capture element. Then, a core-shell bimetallic material with plenty of 'hot spots' was designed and synthesized as the enhancement substrate. The 4-Mercaptobenzonitrile (4-MP) with a characteristic peak at 2224 cm-1(Raman-silent region) was embedded as the Raman reporter to form a SERS immune probe with highly efficient electromagnetic enhancement effect, achieving specific recognition and high-sensitivity detection of IL-6 on bio-capture elements. Using this strategy for quantitative analysis of IL-6, a wide detection range (0.5-5000 pg ml-1) and a low detection limit (0.453 pg ml-1) were obtained. Moreover, this method exhibited excellent detection performance for IL-6 in human serum samples, demonstrating its potential promise in screening clinically relevant diseases. The biosensor presented here not only provides a novel and universally applicable sensing strategy for the enrichment and detection of trace glycoprotein disease markers, but also the application of a portable Raman spectrometer provides a more reliable experimental basis for the diagnosis and treatment of major diseases in the clinic or remote and deprived areas.

Branch retinal artery occlusion in a young patient after radiotherapy for nasopharyngeal carcinoma.

PURPOSE: To report a case of branch retinal artery occlusion (BRAO) in a young patient who received previous neck radiotherapy for nasopharyngeal carcinoma. METHODS: We describe an interesting case of a branch retinal artery occlusion in a patient with previous neck radiotherapy for nasopharyngeal carcinoma 14 years ago. PATIENT: The patient was a 49 year old male, who presented to the retina service in Tan Tock Seng Hospital. RESULTS: Ultrasound of the carotid arteries revealed more than 50% bilateral common carotid arteries stenosis, and 80-99% bilateral internal carotid artery (ICA) stenosis. Magnetic resonance imaging of the brain showed presence of chronic infarcts. Screening for hypercoaguable states and cardioembolic causes were unremarkable. CONCLUSION: Head and neck irradiation is a significant risk factor for developing carotid stenosis and its consequent complications such as retinal artery occlusions and cerebrovascular events.

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.

[Research progress on the expression and function of erythropoietin-producing hepatomocellular receptors and their receptor-interacting proteins in oral-related diseases].

Erythropoietin-producing hepatomocellular receptors and their receptor-interacting proteins (Eph/ephrin) can participate in the regulation of growth and development and promote the development of diseases through short-distance signal transduction between cells. To study the mechanism of Eph/ephrin and oral-related diseases, we provided a new theoretical basis and a strategy for the treatment of oral diseases. The Eph/ephrin pathway has been used to regulate oral diseases, especially in periodontal disease prevention, orthodontic bone reconstruction, and biological treatment of oral tumors. This paper reviews the research progress of Eph/ephrin pathway in oral-related diseases.

Toll-Like Receptor 4-Myeloid Differentiation Primary Response Gene 88 Pathway Is Involved in the Inflammatory Development of Polymyositis by Mediating Interferon-γ and Interleukin-17A in Humans and Experimental Autoimmune Myositis Mouse Model.

OBJECTIVE: Toll-like receptor 4 (TLR4) is one of the key players in the development of many autoimmune diseases. To determine the possible role of TLR4 in polymyositis (PM) development, we collected muscle samples from PM patients and mice subjected to an experimental autoimmune myositis (EAM) model. METHODS: We measured TLR4-MyD88 pathway-related factors, interferon-γ (IFN-γ), and interleukin-17A (IL-17A) in EAM mice and PM patients. Then, we observed the changes of above factors and the inflammatory development of EAM mice with TLR4 antagonist TAK-242, IFN-γ, or IL-17A antibody treatment. RESULTS: The expression of TLR4, MyD88, and NF-κB was significantly upregulated in the muscle tissues both in 22 patients with PM and in the EAM model. As expected, increased levels of various cytokines, such as IL-1ß, IL-6, IL-10, IL-12, tumor necrosis factor-α, TGF-ß, IFN-γ, and IL-17A, were evident in the serum of EAM mice. Moreover, mRNA expression levels of IFN-γ and IL-17A were significantly increased in both PM patients and EAM mice. Consistently, the levels of these factors were positively correlated with the degree of muscle inflammation in EAM mice. However, when EAM mice were treated with TLR4 antagonist TAK-242, the expression of IFN-γ and IL-17A was decreased. When the cytokines were neutralized by anti-IFN-γ or anti-IL-17A antibody, the inflammatory development of EAM exacerbated or mitigated. CONCLUSION: The present study provided the important evidence that the TLR4-MyD88 pathway may be involved in the immune mechanisms of PM by mediating IFN-γ and IL-17A.

Bio-inspired virus imprinted polymer for prevention of viral infections.

A novel virus-imprinted polymer for prevention of viral infection was prepared by anchoring molecularly imprinted polymer (MIP) on the surface of poly-dopamine (PDA)-coated silica particles. The imprinting reaction was carried out via self-polymerization of dopamine in the presence of a virus template. Plaque forming assay indicated that the MIP exhibited selective anti-viral infection properties for the template virus in complex media containing different interfering substances, and even other types of viruses. Remarkable dose-dependent and time-dependent inhibition of virus infection was observed due to the MIP's selective binding to the template virus. When the MIP was incubated with the virus and host cells together, rapid and selective adsorption of template viruses by the MIP prevented the viruses to infect the host cells in a period of 12h. The MIP was biocompatible and non-toxic, and had excellent stability and reusability. Furthermore, the MIPs prepared using different viruses as templates showed similar anti-viral infection properties. The MIP synthesized using dopamine as monomer and crude virus as template provided an attractive possibility for clinical applications in the field of antiviral therapy. STATEMENT OF SIGNIFICANCE: This is the first report to prepare artificial antibody (molecularly imprinted polymer, MIP) that can selectively prevent virus infection using dopamine self-polymerization system. Only MIP anchoring on the surface of poly-dopamine coated silica particles and polymerized using ammonium persulfate as radical initiator showed dose-dependent and time-dependent inhibition of template virus infection in complex media containing interferences and even other viruses. Viruses bond to MIP lost infectious capability. When incubated with virus and host cells, MIP rebond viruses rapidly and selectively to prevent viruses infecting host cells for 12h. The achieved MIPs were biocompatibility, non-toxicity with excellent stability and reusability, and can be used to different viruses. The bio-mimic MIPs provided an attractive prospect for clinical applications in antiviral therapy.

[Subcellular localization of severe fever with thrombocytopenia syndrome virus in macrophages].

OBJECTIVE: To study the subcellular localization of severe fever with thrombocytopenia syndrome virus (SFTSV) in macrophages and understand the replication and assembly mechanism of SFTSV in host cells. METHODS: Using two types of human macrophage cell lines THP-1 and U937, the study analyzed the intracellular colocalization of SFTSV with Golgi apparatus and endoplasmic reticulum by immunefluorescence staining and confocal microscopy. RESULTS: SFTSV infected macrophage cell lines THP-1 and U937. Immunofluorescence staining showed that the SFTSV nuclear protein colocalized with Golgi apparatus and closely surrounded by endoplasmic reticulum in the perinuclear region. CONCLUSION: The results suggested that Golgi complex and endoplasmic reticulum are probably the sites for formation and maturation of SFTSV viral particles.

[Secreted expression of dengue virus type 2 envelope glycoprotein in eukaryotic cells].

OBJECTIVE: To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly. METHODS: The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot. RESULTS: After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis. CONCLUSION: Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.

[Expression of structural and non-structural proteins of severe fever with thrombocytopenia syndrome bunyavirus].

Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) is a novel phlebovirus, causing a life-threatening illness associated with the symptoms of severe fever and thrombocytopenia syndrome. The sequence and structure of the genome have already been illustrated in previous study. However, the characteristics and function of the structure and non-structure proteins is still unclear. In this study, we identified the density of the purified SFTSV virions as 1.135 g/mL in sucrose solution. Using RT-PCR method, we amplified the full coding sequence of RNA dependent RNA polymerase(RdRp), glycoprotein precursor (M), glycoprotein n (Gn), glycoprotein c (Gc), nuclear protein (NP) and non structural protein (NSs) of SFTSV (strain HB29). Respectively inserted the target genes into eukaryotic expression vector pcDNA5/FRT or VR1012, the target protein in 293T cell were successfully expressed. By analyzing the SFTSV virions in SDS-PAGE and using recombinant viral proteins with SFTS patients sera in Western blotting and Immunofluorescent assay, the molecule weight of structure and non-structure proteins of SFTSV were defined. The study provides the first step to understand the molecular characteristics of SFTSV.

[Expression and function analysis of dengue virus type 1 to 4 envelope domain III recombinant fusion protein].

OBJECTIVE: To observe the ability of dengue virus type 1-4 envelope domain III fusion protein to inhibit virus infection and analyze the neutralizing ability of polyclonal antibodies against rE III. METHODS: After being connected by linker peptide, E III protein of Dengue virus serotypes 1-4 were expressed in E coli BL21 (DE3) then purified. Fusion proteins were verified by Western Blot and ELISA. Rabbits were immunized with fusion proteins to produce anti-rE III serum. The activity of anti-rE III serum were detected through indirect immunofluorescence assay test. Inhibition of dengue virus type 1 to 4 infection in BHK-21 cells by rE III fusion protein were tested. Neutralizing activity of anti-rE III serum was analyzed. RESULTS: Dengue virus type 1 to 4 envelope domain III recombinant fusion protein was expressed in E coli BL21 and purified successfully. Then rE III fusion protein and anti-rE III serum were analyzed respectively and rE III fusion protein can effectively inhibit dengue virus type 1 to 4 from infecting BHK cells. The anti-rE III serums can neutralize dengue virus type 1 to 4 but with different neutralizing titer. CONCLUSION: Dengue virus type 1-4 envelope domain III fusion protein can directly inhibit DV infection. Antibodies induced by rE III fusion proteins can neutralize dengue virus type 1-4.

[Secreted expression of dengue virus type I envelope glycoprotein in 293T cells].

OBJECTIVE: To expression prM/E gene of dengue virus type I in mammalia cells. METHODS: The full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot. RESULTS: In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA. CONCLUSION: The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.

[Recombinant envelope glycoprotein domain III of dengue virus inhibit virus infection].

OBJECTIVE: To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody. METHODS: E III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay. RESULTS: The recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different. CONCLUSION: proteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.

[The differential expression of the human lung carcinoma cells infected with high pathogenic avian influenza virus A/Anhui/1/2005 (H5N1)].

OBJECTIVE: To identify genes in human cells infected with high pathogenic avian influenza viruses H5N1. METHODS: The lung carcinoma cells line A549 was infected with H5N1 and H1N1, respectively. We harvested the infected cells at the different time points after infection and screened the genes with differential expression via microarray technology. The candidate genes were selected and confirmed by quantitative real-time PCR. RESULTS: The spectrum of genes with the differential expression in the cells infected with H5N1 was obtained and 16 candidate genes were identified in the cellular apoptosis pathway, mTOR pathway, and the cellular immunity as well. CONCLUSIONS: Our results suggest that H5N1 exert a stronger impact on eliciting apoptosis of infected cells than the common influenza virus H1N1.

Phage display mediated immuno-PCR.

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10,000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity.

OBJECTIVE: To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. METHODS: By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. RESULTS: After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus. CONCLUSION: The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.

[Construction and stable expression of intracellular antibodies to glycoprotein of hantavirus].

OBJECTIVE: To understand the molecular mechanisms of hantavirus assembly and maturation by stably expressing the intracellular antibodies to hantavirus glycoprotein G1 and G2 in endoplasmic reticulum (ER) and cytoplasm (Cyto) of Vero E6 cell. METHODS: The genes of VH and VL of antibodies against glycoprotein of hantavirus were amplified by PCR and cloned into pOPE 101-215 (Yol) vector. The G1 and G2 proteins specific ScFv genes were first expressed in E.coli and the function and binding properties were identified. The gene of ScFv were further inserted into intracellular expression vectyrs pEF/ myc/ ER and pEF/ myc/ CYTO vector and transfected Vero E6 cell. The clonal cell line which stabl expresses ScFv were isolated under the pressure of G418. RESULTS: The ScFv genes of hantavirus G1 and G2 specific antibodies were successfully expressed in subcellular compartment ER and Cyto of Vero E6 cells and specifically targeted G1 and G2 protein after virus infection of the cells. CONCLUSIONS: The recombination of intrabody to Hantann virus glycoprotein was constructed successfully, and it may provide basic material for the studying antiviral gene therapy and the molecular mechanism of viral replication and infection.

[Generation of human recombinant antibody Fab fragment and its IgG to adeno-associated virus type II from phage display library].

BACKGROUND: To acquire the recombinant human monoclonal antibodies and IgG to adeno-associated virus type 2 (AAVs-2). METHODS: Construct and pan human Fab antibody library to AAVs-2 was established from normal volunteer donors by using phage display technology and secreted expression in E.Coli system. The positive Fab clones were selected and characterized through ELISA and immunofluorescent assay, and then the heavy and light chain were sequenced. The gene of light chain and heavy chain Fd fragment of recombinant mAb were inserted into baculovirus expression vector pAC-L-Fc and construct expression vectors of intact IgG, then transfected insert sf-9 cell secreted expression in Baculovirus/Insert system. Immunoprecipitation test was used to detect its recognizing region. RESULTS: One clone named AAVs-31 showed positive responses in ELISA and IFA, the Fab was composed of gamma chain and kappa chain IgG was positive in ELISA and IFA. The IgG failed to detect nonassembled or denatured capsid proteins, but recognized the AAVs-2 stock from immunoprecipitation test. CONCLUSION: The authors isolated a clone of Fab and IgG to adeno-associated virus type 2 by phage display technology, they perhaps recognize an epitope which is formed during capsid assembly.