ABSTRACT
There are currently no standard methods for diagnosing cardiac diseases in dolphins. These diseases may consequently be overlooked and go undiagnosed. The presence and severity of cardiac diseases in humans can be determined using blood tests. Human atrial natriuretic peptide (hANP) used in human cardiac examinations has low species specificity. There have already been reports of homology between dolphin ANP and hANP; however, its potential for clinical application in dolphins has not been tested. This study was conducted to establish a reference for plasma hANP levels in healthy bottlenose dolphins. Healthy bottlenose dolphins (seven females; estimated to be 7-30 years of age) at an aquarium in Japan were sampled. Each animal was tested for hANP at least three times, and the mean value and standard deviation were calculated to be 43.4 ± 19.2 pg/mL. In humans, patients with high plasma hANP levels have a poor prognosis. In veterinary medicine, cutoff values for the diagnosis of mitral regurgitation and heart failure in dogs have been established and used to predict prognosis. The results of the present study may contribute to the health management of bottlenose dolphins, particularly in the early detection and treatment of cardiac disease.
ABSTRACT
Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of γ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11's nature in colorectal cancers and others.
ABSTRACT
Background: Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-ß1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-ß1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods: LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results: Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-κB (NF-κB). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-ß activated kinase 1 (TAK1) were required for the activation of NF-κB through Iκß kinase kinase (IKKα/ß) phosphorylation. Conclusion: Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-ß1-TRAF4-TAK1-IKKα/ß-Iκßα-NF-κB-MMP9, is crucial for TNBC cell invasiveness.
ABSTRACT
Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-ß1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.
ABSTRACT
Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson's disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration.
Subject(s)
Parkinson Disease , Rotenone , Humans , Animals , Mice , Rotenone/pharmacology , Rotenone/metabolism , Neurons/metabolism , Parkinson Disease/metabolism , Cell Death , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolismABSTRACT
Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-γ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-γ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-γ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death.
Subject(s)
Axons , NAD , NAD/metabolism , Vincristine/metabolism , Axons/metabolism , Neurons/metabolism , Cell Death , Armadillo Domain Proteins/metabolismABSTRACT
Background: LOX family members are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer. Methods: We established the MDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells' activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach. Results: Our in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin ß-1, resulting in the locking of integrin ß-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth. Conclusions: LOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin ß-1 on the cell surface.
ABSTRACT
Background: EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. Methods: We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (ΔZn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. Results: MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (ΔZn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. Conclusions: These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells.
ABSTRACT
The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3-namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. KEY MESSAGES: ⢠REIC/Dkk-3 protein effectively suppresses breast cancer progression through an acceleration of PD-L1 degradation. ⢠PD-L1 stability on the cancer cell membrane is kept high by binding with mainly CMTM6. ⢠Competitive binding of REIC/Dkk-3 protein with CMTM6 liberates PD-L1, leading to PD-L1 degradation.
Subject(s)
B7-H1 Antigen , Breast Neoplasms , Female , Humans , Intercellular Signaling Peptides and Proteins , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
Previous studies on dolphin electrocardiograms have shown that they are mainly composed of increased negative waves, similar to ungulates. The electrocardiogram waveform was determined by the distribution of the Purkinje fibers. Based on the waveform of the dolphin electrocardiogram, Hamlin predicted that the Purkinje fibers would be distributed within the ventricular muscle, as in ungulates. The purpose of this study was to confirm the histological distribution of Purkinje fibers in dolphins. In the present study, bottlenose dolphin hearts were observed both grossly and histologically, and the effects of Purkinje fiber distribution and cardiac morphology on electrocardiogram waveforms were examined. This study showed that the Purkinje fibers of dolphins run just below the endocardium, as in humans, dogs, and cats, whose electrocardiograms mainly show positive waves. When the cardiac morphology of dolphins was observed carefully, the right ventricle was found to be extremely dilated compared to that of terrestrial mammals. In human recreational divers, right ventricular dilatation is induced by diving. We hypothesized that the dolphin's heart is in a state similar to that of the right heart dilatation in terrestrial animals. The dolphin electrocardiogram waveform was considered to be due to right axis deviation. Based on the above, we concluded that the dolphin electrocardiogram waveform was due to its ability to live in water. We found that the dolphins are genetically related to ungulates, particularly the hippopotamus, but that their hearts have evolved differently.
Subject(s)
Bottle-Nosed Dolphin , Animals , Humans , Dogs , Bottle-Nosed Dolphin/physiology , Thorax , Mammals , Electrocardiography , Heart VentriclesABSTRACT
(1) Background: Lung ischemia-reperfusion (IR) injury increases the mortality and morbidity of patients undergoing lung transplantation. The objective of this study was to identify the key initiator of lung IR injury and to evaluate pharmacological therapeutic approaches using a functional inhibitor against the identified molecule. (2) Methods: Using a mouse hilar clamp model, the combination of RNA sequencing and histological investigations revealed that neutrophil-derived S100A8/A9 plays a central role in inflammatory reactions during lung IR injury. Mice were assigned to sham and IR groups with or without the injection of anti-S100A8/A9 neutralizing monoclonal antibody (mAb). (3) Results: Anti-S100A8/A9 mAb treatment significantly attenuated plasma S100A8/A9 levels compared with control IgG. As evaluated by oxygenation capacity and neutrophil infiltration, the antibody treatment dramatically ameliorated the IR injury. The gene expression levels of cytokines and chemokines induced by IR injury were significantly reduced by the neutralizing antibody. Furthermore, the antibody treatment significantly reduced TUNEL-positive cells, indicating the presence of apoptotic cells. (4) Conclusions: We identified S100A8/A9 as a novel therapeutic target against lung IR injury.
ABSTRACT
Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.
Subject(s)
Chemotaxis , Pancreatic Neoplasms , Mice , Animals , Humans , Mice, Nude , Cell Line, Tumor , Cell Movement , Pancreatic Neoplasms/pathology , Pancreas/pathology , Cell Transformation, Neoplastic , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , RNA , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Pancreatic NeoplasmsABSTRACT
Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.
Subject(s)
Toll-Like Receptor 4 , Urinary Bladder Neoplasms , Humans , Calgranulin A/metabolism , Calgranulin B/metabolism , Membrane Glycoproteins/metabolism , Receptors, Interleukin-1 , Toll-Like Receptor 4/metabolism , Urinary Bladder/metabolismABSTRACT
The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.
Subject(s)
Calgranulin A/metabolism , Calgranulin B/metabolism , Lung Neoplasms , Melanoma, Experimental , Proteins/metabolism , Animals , Calgranulin A/blood , Calgranulin A/genetics , Calgranulin B/blood , Chemotactic Factors , Ligands , Lung Neoplasms/metabolism , MiceABSTRACT
Although bronchiolitis obliterans syndrome (BOS) is a major cause of death after lung transplantation, an effective drug therapy for BOS has not yet developed. Here, we assessed the effectiveness of a neutralizing anti-S100 calcium binding protein (S100) A8/A9 antibody against BOS. A murine model of heterotopic tracheal transplantation was used. Mice were intraperitoneally administered control IgG or the S100A8/A9 antibody on day 0 and twice per week until they were sacrificed. Tissue sections were used to evaluate the obstruction ratio, epithelium-preservation ratio, α-smooth muscle actin (SMA)-positive myofibroblast infiltration, and luminal cell death. Quantitative reverse transcriptase-polymerase chain reaction analysis was performed to analyze the mRNA-expression levels of collagen, inflammatory cytokines, and chemokines on days 7, 14, and 21. The anti-S100A8/A9 antibody significantly improved the obstruction ratio and epithelium-preservation ratio, with less α-SMA-positive myofibroblast infiltration compared to the control group. Antibody treatment reduced the type-III collagen: type-I collagen gene-expression ratio. The antibody also significantly suppressed the number of dead cells in the graft lumen. The expression levels of tumor growth factor ß1 and C-C motif chemokine 2 on day 21, but not those of interleukin-1ß, interleukin-6, and tumor necrosis factor α, were significantly suppressed by S100A8/A9 antibody treatment. These findings suggest that S100A8/A9 may be a potential therapeutic target for BOS after lung transplantation.
Subject(s)
Airway Obstruction , Interleukin-6 , Actins/metabolism , Animals , Calgranulin A/genetics , Calgranulin A/metabolism , Calgranulin B/metabolism , Cytokines/metabolism , Disease Models, Animal , Immunoglobulin G/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Mice , RNA, Messenger , RNA-Directed DNA Polymerase/metabolism , S100 Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy.
ABSTRACT
The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.
Subject(s)
Sperm Motility , Spermatozoa , Animals , Male , Mice , Mice, Knockout , Sperm Motility/genetics , Testis , Wnt Signaling Pathway/geneticsABSTRACT
OBJECTIVES: The aim of this study was to verify the safety and efficacy of transvaginal mesh by analyzing the 2-year follow-up data of patients performed by a surgeon with a high volume of procedures. METHODS: A total of 617 patients with pelvic organ prolapse underwent transvaginal mesh by a single surgeon. Complications and anatomical status of each patient were examined up to 24 months after surgery. Risk factors for the recurrence were also analyzed. RESULTS: Regarding complications, we experienced 10 patients (3.8%) of bladder injuries in anterior transvaginal mesh and eight (3.4%) in anterior and posterior transvaginal mesh. Massive blood loss was observed in four patients, but there was no case of blood transfusion. Mesh exposures were seen in seven patients (1.2%). A total of 100 patients (16.2%) had prolapse recurrence, defined as the Pelvic Organ Prolapse Quantification System stage ≥II. As to recurrences on the operated compartments, we observed five patients (2.0%) for anterior transvaginal mesh, three (6.5%) for posterior transvaginal mesh, five (7.4%) for combined transvaginal mesh, and 31 (14.2%) in anterior and posterior transvaginal mesh. Regarding Point C before operation in the anterior and posterior transvaginal mesh, the recurrence rates were more than 23% in patients with a Point C of 4 or more. Binominal regression analyses showed that higher body mass index, younger age, and higher stage of uterine prolapse were significant risk factors. CONCLUSIONS: The transvaginal mesh surgery is safe when conducted by experts. However, the recurrence rate may exceed 20% for high-stage uterine prolapse even when conducted by experts.
Subject(s)
Pelvic Organ Prolapse , Surgeons , Uterine Prolapse , Body Mass Index , Female , Humans , Pelvic Organ Prolapse/surgery , Surgical Mesh/adverse effectsABSTRACT
Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2-) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2-, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2- generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.