Domain 4 of pneumolysin from Streptococcus pneumoniae is a multifunctional domain contributing TLR4 activating and hemolytic activity.

The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae.

Toll-like receptor 9 agonist enhances anti-tumor immunity and inhibits tumor-associated immunosuppressive cells numbers in a mouse cervical cancer model following recombinant lipoprotein therapy.

BACKGROUND: Although cytotoxic T lymphocytes (CTLs) play a major role in eradicating cancer cells during immunotherapy, the cancer-associated immunosuppressive microenvironment often limits the success of such therapies. Therefore, the simultaneous induction of cancer-specific CTLs and reversal of the immunosuppressive tumor microenvironment may be more effectively achieved through a single therapeutic vaccine. A recombinant lipoprotein with intrinsic Toll-like receptor 2 (TLR2) agonist activity containing a mutant form of E7 (E7m) and a bacterial lipid moiety (rlipo-E7m) has been demonstrated to induce robust CTL responses against small tumors. This treatment in combination with other TLR agonists is able to eliminate large tumors. METHODS: Mouse bone marrow-derived dendritic cells (DCs) were employed to determine the synergistic production of pro-inflammatory cytokines upon combination of rlipo-E7m and other TLR agonists. Antigen-specific CTL responses were investigated using immunospots or in vivo cytolytic assays after immunization in mice. Mice bearing various tumor sizes were used to evaluate the anti-tumor effects of the formulation. Specific subpopulations of immunosuppressive cells in the tumor infiltrate were quantitatively determined by flow cytometry. RESULTS: We demonstrate that a TLR9 agonist (unmethylated CpG oligodeoxynucleotide, CpG ODN) enhances CTL responses and eradicates large tumors when combined with rlipo-E7m. Moreover, combined treatment with rlipo-E7m and CpG ODN effectively increases tumor infiltration by CTLs and reduces the numbers of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. CONCLUSION: These findings suggest that the dramatic anti-tumor effects of the recombinant lipoprotein together with CpG ODN may reflect the amplification of CTL responses and the repression of the immunosuppressive environment. This promising approach could be applied for the development of additional therapeutic cancer vaccines.

Development and integration of LensHooke® R10 for automatic and standardized diagnosis for sperm DNA fragmentation.

BACKGROUND: The sperm chromatin dispersion assay is commonly used to assess sperm DNA integrity. This approach is time-consuming, demonstrates poor chromatin preservation, and provides an ambiguous and unstandardized evaluation of fragmented chromatin. OBJECTIVES: We aimed to (i) develop an optimized sperm chromatin dispersion assay with reduced operation time, (ii) validate R10 test accuracy by comparing it to a conventional sperm chromatin dispersion assay, and (iii) standardize the sperm DNA fragmentation analysis procedure by integrating artificial intelligence optical microscopic technology. MATERIALS AND METHODS: This cross-section study included 620 semen samples. Aliquots were analyzed by a conventional Halosperm® G2 assay (G2) and LensHooke® R10 assay (R10). The DNA fragmentation index was scored manually, and R10 slides were automatically determined by a LensHooke® X12 PRO semen analysis system (X12). RESULTS: We demonstrated significant improvements in total assay time (40 vs. 72 min, p < 0.001) and in the halo-cytological resolution using R10 compared to G2. Comparing the G2 and R10, DNA fragmentation index results demonstrated good agreement between the two methods (Spearman's rank correlation, rho = 0.8517, p < 0.0001). We introduced the integration of an auto-calculation system to diagnose sperm DNA fragmentation. X12 interpretation showed excellent agreement with manual interpretation (Spearman's rank correlation, rho = 0.9323, p < 0.0001), but had a low coefficient of variation compared to manual interpretation (4% for R10 by X12 vs. 19% for R10 by manual scoring vs. 25% for G2 by manual scoring). DNA fragmentation index was more correlated with total motility (coefficients = -0.3607, p < 0.0001) than sperm morphology and was positively associated with asthenozoospermic semen samples (p = 0.0001). CONCLUSION: The R10 sperm chromatin dispersion assay combined with the X12 semen analysis system is faster, more objective, and provides standardization for sperm DNA fragmentation.

Integrating Network Pharmacology and In vivo Experimental Validation to Reveal the Mechanism of FuZheng YiLiu Formula on Estrogen Receptor Positive Breast Cancer.

BACKGROUND AND PURPOSE: FuZheng YiLiu Formula (FZYL) is a commonly used formula for postoperative estrogen receptor-positive (ER+) breast cancer and post-radiotherapy deficiency of both Qi and Yin. FZYL has been used in clinical practice for decades because of its ability to effectively improve the symptoms of deficiency in cancer patients. However, its mechanism needs to be further clarified. In this paper, we will observe the effect of FZYL on mice with ER+ breast cancer and explore the mechanism by which it improves the symptoms of ER+ breast cancer. MATERIALS AND METHODS: A tumor xenograft mouse model was established to detect tumor growth in vivo in order to evaluate the pharmacological effects of FZYL on ER+ breast cancer. The main targets of FZYL were identified by extracting the FZYL components and the corresponding potential target genes of breast cancer from the established database and constructing a protein-protein interaction network of shared genes using the string database. GO functional annotation and KEGG pathway enrichment analysis were performed, and molecular docking, molecular dynamics simulations, western blotting analysis, and RT-qPCR were performed to confirm the validity of targets in the relevant pathways. RESULTS: FZYL was able to significantly reduce the size of tumors in vivo and had a significant therapeutic effect on tumor xenograft mice. GO and KEGG pathway enrichment analyses indicated that the effects of FZYL may be mediated by oxidative stress levels, apoptotic signaling pathways, and cell cycle proliferation. By RT-qPCR and protein blotting assays, FZYL targeted the key targets of TP53, JUN, ESR1, RELA, MYC, and MAPK1 to exert its effects. The key active components of FZYL are quercetin, luteolin, stigmasterol, and glycitein. Molecular docking and molecular dynamics simulation results further demonstrated that the key active components of FZYL are stably bound to the core targets. CONCLUSION: In this study, the potential active ingredients, potential core targets, key biological pathways, and signaling pathways involved in the treatment of breast cancer with FZYL were identified, providing a theoretical basis for further anti ER+ breast cancer research.

Innovative technology for evaluation of sperm DNA double-strand breaks diagnoses male factor infertility and prevents reproductive failures.

Neutral comet assay has been available for two decades to evaluate sperm double-strand breaks (DSBs). However, its clinical usability is limited due to its complex and time-consuming procedure, as well as the lack of a standardized scoring system. The aim of this study was to: develop a rapid diagnostic method for DSBs, Sperm DNA Fragmentation Releasing Assay (SDFR), and explore the association between DSBs and reproductive outcomes. We pioneered the use of polyacrylamide (PA) for embedding sperm chromatin and optimized the porosity of PA to be between 10 and 13%. The refined PA network allowed the trapping of DSBs, which dispersed halo on an immunological slide; in contrast, intact chromatin failed to develop a halo. A strong correlation was showed between reproducible values obtained from SDFR and neutral comet assay. SDFR were responsive to dose-/time-dependent simulated DSBs, indicating high sensitivity and specificity. Furthermore, we conducted a retrospective study of couples with embryonic aneuploidy screening, and recording DSB profiles of the male partners. Our findings revealed that DSB enabled to predict embryonic aneuploidy whereas basic semen parameters did not. In conclusion, SDFR offers a rapid and user-friendly approach for evaluating DSBs, with potential implications for predictive healthcare in reproductive medicine.

Virtual and real-world implementation of deep-learning-based image denoising model on projection domain in digital tomosynthesis and cone-beam computed tomography data.

Reducing the radiation dose will cause severe image noise and artifacts, and degradation of image quality will also affect the accuracy of diagnosis. To find a solution, we comprise a 2D and 3D concatenating convolutional encoder-decoder (CCE-3D) and the structural sensitive loss (SSL), via transfer learning (TL) denoising in the projection domain for low-dose computed tomography (LDCT), radiography, and tomosynthesis. The simulation and real-world practicing results show that many of the figures-of-merit (FOMs) increase in both projections (2-3 times) and CT imaging (1.5-2 times). From the PSNR and structural similarity index of measurement (SSIM), the CCE-3D model is effective in denoising but keeps the shape of the structure. Hence, we have developed a denoising model that can be served as a promising tool to be implemented in the next generation of x-ray radiography, tomosynthesis, and LDCT systems.

Fuzheng Yiliu Formula Regulates Tumor Invasion and Metastasis through Inhibition of WAVE3 Expression.

OBJECTIVE: To explore the mechanism of action of Fuzheng Yiliu formula (FZYLF) in regulation of the invasion and metastasis of MDA-MB-231/Adr human breast cancer cells through WAVE3. METHODS: The MDA-MB-231/Adr cells with high invasive ability were screened by Transwell, and the plasmid with high WAVE3 expression was made for transfection. Plasmid transfection efficiency and protein expression level were verified by polymerase chain reaction (PCR) and western blotting (WB). The effect of FZYLF on cell proliferation and invasion was investigated before and after WAVE3 silencing by flow cytometry. A nude mouse model of tumor metastasis was established to study the antitumor activity of FZYLF. RESULTS: The expression levels of mRNA and proteins of intracellular WAVE3 increased significantly after plasmid transfection, mRNA from 1.37± 0.41 to 9.88 ± 1.31 and protein from 1 ± 0.08 to 5.09 ± 0.03 (P < 0.01). Intervention with FZYLF could significantly affect the activity of MDA-MB-231/Adr cells and inhibit invasion and metastasis, IC50 from 71.04 to 46.41 mg/mL and from 162 ± 14.82 to 81.4 ± 12.05 (P < 0.05 or P < 0.01), and significantly reduce the expression levels of WAVE3 (from 1 ± 0.02 to 0.63 ± 0.04), MMP-9 (from 1 ± 0.05 to 0.63 ± 0.03), NF-κB (p65) (from 1 ± 0.02 to 0.62 ± 0.02), and p-IκBα (from 1 ± 0.03 to 0.68 ± 0.02) (P < 0.05 or P < 0.01). The T/C (%) of FZYLF (13 g crude drug/kg) was 62.06% for MDA-MB-231/Adr tumor xenografted in nude mice, with a tumor inhibition rate of 39.64%. CONCLUSION: FZYLF can inhibit the invasion and proliferation of the MDA-MB-231/Adr human breast cancer cells, and the mechanism of action may be related to the regulation of WAVE3 expression.

Asiatic Acid Interferes with Invasion and Proliferation of Breast Cancer Cells by Inhibiting WAVE3 Activation through PI3K/AKT Signaling Pathway.

OBJECTIVE: To explore the ability of asiatic acid to interfere with the invasion and proliferation of breast cancer cells by inhibiting WAVE3 expression and activation through the PI3K/AKT signaling pathway. METHODS: The MDA-MB-231 cells with strong invasiveness were screened by transwell assay, and plasmids with high expression of WAVE3 were constructed for transfection. The transfection effect and protein expression level of plasmids were verified by PCR and WB. The effects of asiatic acid on cell proliferation and invasion were investigated by flow cytometry. The xenografted tumor models in nude mice were established to study the antitumor activity of asiatic acid. RESULTS: Asiatic acid significantly inhibited the activity of MDA-MB-231 cells, and the expression level of WAVE3 increased significantly in the tissue of ductal carcinoma in situ and was lower than that in the metastasis group. After plasmid transfection, the mRNA and protein expression of WAVE3 increased significantly in the cells. Asiatic acid at different concentrations had an impact on cell apoptosis and invasion and could significantly inhibit the expression of WAVE3, P53, p-PI3K, p-AKT, and other proteins. The T/C(%) of asiatic acid (50 mg/kg) for MDA-MB-231(F10) xenografted tumor in nude mice was 46.33%, with a tumor inhibition rate of 59.55%. Asiatic acid could significantly inhibit the growth of MDA-MB-231 (F10) xenografted tumors in nude mice (p < 0.05). CONCLUSIONS: Asiatic acid interferes with the ability of breast cancer cells to invade and proliferate by inhibiting WAVE3 expression and activation and the mechanism of action may be related to the PI3K/AKT signaling pathway.

Morin inhibits the growth of human leukemia HL-60 cells via cell cycle arrest and induction of apoptosis through mitochondria dependent pathway.

The effects of morin (3,5,7,20,40-pentahydroxyflavone) on human leukemia HL-60 cells in vitro were investigated and the molecular mechanisms of morin-induced G2/M arrest and apoptosis in HL-60 cells were examined. Morin induced morphological changes and decreased the percentage of viable cells via induction of G2/M-phase arrest and apoptosis. Morin-induced G2/M-phase arrest was accompanied by the promotion of p21 and Wee1, and decreased levels of Cdc25c and cyclins A and B1 complex. Morin-induced apoptosis in HL-60 cells was also confirmed by flow cytometric assay, DNA gel electrophoresis for DNA fragmentation and DAPI staining. Morin induced apoptosis in time- and dose-dependent manners. Morin-induced apoptosis was associated with elevated intracellular reactive oxygen species (ROS) increased and Ca2+ production; decreased the levels of mitochondria membrane potential (deltapsi(m)) and increased caspase-3 activation. Collectively, these results suggest that the morin-induced apoptosis in HL-60 cells may result from the activation of caspase-3 and intracellular Ca2+ release, as well as the mitochondria membrane potential pathway.

Gemcitabine enhances antitumor efficacy of recombinant lipoimmunogen-based immunotherapy.

Although immunotherapy is an attractive approach for cancer treatment, increasing evidence has shown that the combination of immunotherapy with other treatment modalities may improve the outcome of advanced malignancy. We combined the anticancer drug gemcitabine (Gem) with recombinant lipoprotein-based immunotherapy (rlipo-E7m/CpG) to treat advanced cancer. Mice bearing huge solid tumors (≧ 12 mm in diameter) or orthotopic cervical cancer were treated with a therapeutic regimen consisting of rlipo-E7m/CpG and Gem. In addition, tumor-infiltrating immune cells were quantified by flow cytometry following the chemotherapy and/or immunotherapy. We observed the eradication of huge tumors following the administration of Gem on days 21, 24, and 27 or following rlipo-E7m/CpG therapy on day 30 post-tumor implantation. The combination therapy substantially reduced the number of immunosuppressive cells (CD11b+Gr-1+, CD11b+F4/80+, and CD4+CD25+FOXP3+) and increased the number of tumor-infiltrating antigen-specific CD8+ T cells compared to Gem or rlipo-E7m/CpG monotherapy. Interestingly, the administration of Gem and rlipo-E7m/CpG reduced the quantity of programmed cell death protein 1 (PD-1)-expressing antigen-specific cytotoxic T lymphocytes (CTLs) in the regressing tumors. These findings demonstrated that Gem enhances the eradication of huge tumors by inhibiting a broad range of immunosuppressive cells when combined with immunotherapy. Based on the promising results from this animal study, Gem chemotherapy combined with recombinant lipoimmunogen-based immunotherapy represents a feasible approach for cancer therapy.

Carboxyl-terminal fusion of E7 into Flagellin shifts TLR5 activation to NLRC4/NAIP5 activation and induces TLR5-independent anti-tumor immunity.

Flagellin has the capacity to activate both Toll-like receptor 5 (TLR5) and Nod-like receptor C4 (NLRC4)/neuronal apoptosis inhibitory protein 5 (NAIP5) inflammasome signaling. We fused E7m (the inactivated E7 of human papillomavirus) to either end of the flagellin protein, and the resulting recombinant flagellin-E7m proteins (rFliCE7m and rE7mFliC) were used as immunogens. Both fusion proteins activated receptor signaling to different degrees. rE7mFliC-induced TLR5 activity was 10-fold higher than that of rFliCE7m, whereas rFliCE7m activated the NLRC4/NAIP5 pathway more strongly. Therefore, these recombinant proteins provided a tool to investigate which signaling pathway is critical for the induction of antigen-specific T cell responses and anti-tumor immunity. We demonstrated that rFliCE7m induced higher levels of E7-specific IFN-gamma-secreting cells and cytotoxic T lymphocytes (CTLs) than rE7mFliC, and a single injection with rFliCE7m but not rE7mFliC inhibited E7-expressing tumor growth in vivo. Furthermore, we confirmed that CD8(+) T cells played a major role in the anti-tumor immunity induced by rFliCE7m. These findings suggested that the NLRC4/NAIP5 intracellular signaling pathway was critical for the induction of anti-tumor immunity. These observations provide important information for the rational design of flagellin-based immunotherapy.

Beneficial Effect of Astragaloside on Alzheimer's Disease Condition Using Cultured Primary Cortical Cells Under ß-amyloid Exposure.

ß-amyloid (Aß)-mediated neuronal apoptosis contributes to the pathogenesis of Alzheimer's disease (AD). This study aimed to investigate whether astragalosides (AST) could inhibit Aß-induced apoptosis in vivo and in vitro and to explore the underlying mechanisms. Amyloid ß-protein fragment 25-35 (Aß25-35) was administered to cerebral lateral ventricle of rats to make the AD models in vivo. AST was able to attenuate both cortical cell degeneration and memory deficits in the AD rats. AST also inhibited Aß25-35-induced cytotoxicity (e.g., decreased cell viability); apoptosis (e.g., increased caspase-3 expression, increased DNA fragmentation, and Tau hyperphosphorylation); synaptotoxicity (e.g., increased loss of both a dendritic marker, microtubule-associated protein 2 (MAP-2) and synaptic proteins, synaptophysins); and mitochondrial dysfunction (e.g., increased mitochondrial membrane potential) in cultured primary rat cortical cells. The beneficial effect of AST in reducing Aß-induced cytotoxicity, apoptosis, and mitochondrial dysfunction in cortical cells were blocked by inhibition of phosphoinositide 3-kinase (PI3K)-dependent protein kinase B (PKB, as known as AKT) activation with LY294002. In addition, inhibition of extracellular protein kinase (ERK) with U0126 shared with the AST the same beneficial effects in reducing Aß-induced apoptosis. Our data suggest that the cortical PI3K/AKT and MAPK (or ERK) pathways as appealing therapeutic targets in treating AD, and AST may have a positive impact on AD treatment via modulation of both PI3K/AKT and ERK pathways.

Self-adjuvanting lipoimmunogens for therapeutic HPV vaccine development: potential clinical impact.

The goal of therapeutic HPV vaccines is the induction of cytotoxic T lymphocyte immunity against HPV-associated cancers. Recombinant proteins and synthetic peptides have high safety profiles but low immunogenicity, which limits their efficacy when used in a vaccine. Self-adjuvanting lipid moieties have been conjugated to synthetic peptides or expressed as lipoproteins to enhance the immunogenicity of vaccine candidates. Mono-, di- and tri-palmitoylated peptides have been demonstrated to activate dendritic cells and induce robust cellular immunity against infectious diseases and cancer. Recently, a platform technology using the high-yield production of recombinant lipoproteins with Toll-like receptor 2 agonist activity was established for the development of novel subunit vaccines. This technology represents a novel strategy for the development of therapeutic HPV vaccines. In this review, we describe recent progress in the design of therapeutic HPV vaccines using lipoimmunogens.

Recombinant lipidated HPV E7 induces a Th-1-biased immune response and protective immunity against cervical cancer in a mouse model.

The E7 oncoprotein of human papillomavirus (HPV) is an ideal target for developing immunotherapeutic strategies against HPV-associated tumors. However, because protein-based immunogens alone are poor elicitors of the cytotoxic T-lymphocyte (CTL) responses, they have been difficult to exploit for therapeutic purposes. In this study, we report that a recombinant lipoprotein consisting of inactive E7 (E7m) biologically linked to a bacterial lipid moiety (rlipo-E7m) induces the maturation of mouse bone marrow-derived dendritic cells through toll-like receptor 2 (TLR2), skews the immune responses toward the Th1 responses and induces E7-specific CTL responses. We further studied the ability of rlipo-E7m to provide protection against a TC-1 tumor cell challenge in an animal model. Mice prophylactically immunized with two 10-µg doses of rlipo-E7m were found to be free of TC-1 tumor growth. Experiments in a therapeutic immunization model showed that the tumor volume in mice receiving a single dose of rlipo-E7m was less than 0.01 cm(3) on day 40, whereas the tumor volume in mice treated with rE7m was 2.28±1.21 cm(3). The tumor volume of the entire control group was over 3 cm(3). In addition, we demonstrated that the CD8+ T cells play a major role in anti-tumor immunity when administration of rlipo-E7m. These results demonstrate that rlipo-E7m could be a promising candidate for treating HPV-associated tumors.

A recombinant lipoprotein containing an unsaturated fatty acid activates NF-kappaB through the TLR2 signaling pathway and induces a differential gene profile from a synthetic lipopeptide.

The lipid moiety of a novel recombinant lipoprotein, which contains a dengue virus envelope protein domain 3, rlipo-D1E3, has been shown to activate antigen-presenting cells (APCs) as an intrinsic adjuvant. Because the lipid moiety of rlipo-D1E3 contains an unsaturated fatty acid, it is unclear if the receptor usage by bacterially derived lipoproteins is the same as that of the synthetic lipopeptide palmitoyl-3-Cys-Ser-(Lys)(4) (Pam3). In the present study, we show that the rlipo-D1E3 lipoprotein can induce the activation of spleen cells and bone marrow-derived dendritic cells (BM-DCs) in wild-type and TLR4-deficient mice, but not in TLR2(-/-) mice. After analyzing the co-receptor usage of TLR2 using TLR1(-/-) or TLR6(-/-) mice, the TLR2 signaling triggered by rlipo-D1E3 and Pam3 could use either TLR1 or TLR6 as a co-receptor. Analysis of the MAPK signaling pathway revealed that rlipo-D1E3 could initiate the phosphorylation of p38, ERK1/2 and JNK1/2 earlier than the synthetic lipopeptide. In addition, the expression levels of IL-23, IL-27 and MIP-1 alpha in BM-DCs stimulated by rlipo-D1E3 were higher than the expression levels in BM-DCs stimulated by Pam3. Taken together, these results demonstrate that different TLR2 ligands can promote various immune responses by inducing different levels of biological cytokines and chemokines.

A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design.

We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.