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PURPOSE: We investigated the aqueous humor proteome and associated plasma proteome in patients with infectious or noninfectious uveitis. METHODS: AH and plasma were obtained from 28 patients with infectious uveitis (IU), 29 patients with noninfectious uveitis (NIU) and 35 healthy controls undergoing cataract surgery. The proteins profile was analyzed by SomaScan technology. RESULTS: We found 1844 and 2484 proteins up-regulated and 124 and 161 proteins down-regulated in the AH from IU and NIU groups, respectively. In the plasma, three proteins were up-regulated in NIU patients, and one and five proteins were down-regulated in the IU and NIU patients, respectively. The results of pathway enrichment analysis for both IU and NIU groups were related mostly to inflammatory and regulatory processes. CONCLUSION: SomaScan was able to detect novel AH and plasma protein biomarkers in IU and NIU patients. Also, the unique proteins found in both AH and plasma suggest a protein signature that could distinguish between infectious and noninfectious uveitis.
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Extração de Catarata , Uveíte , Humanos , Proteoma , Uveíte/diagnóstico , BiomarcadoresRESUMO
OBJECTIVE: Abnormal complement activation is associated with periodontitis. W54011 is a novel non-peptide C5aR antagonist (C5aRA) that exhibits favorable anti-inflammatory effects in various inflammatory models. However, whether W54011 inhibits periodontitis has not yet been fully elucidated. To address this, we have investigated the probable anti-inflammatory mechanism of W54011 in LPS-treated inflammation in human gingival fibroblasts (HGFs). METHODOLOGY: HGFs were isolated from healthy gingival tissue samples using the tissue block method and were identified with immunofluorescence staining. The CCK8 assay and reverse transcription-PCR (RT-PCR) were used to select the optimal induction conditions for Lipopolysaccharide (LPS) and C5aRA (according to supplementary data S1, S2 and S3). The levels of inflammatory cytokines, C5aR, and the activation of NF-κB/MAPK signaling pathways were determined by RT-quantitative PCR (RT-qPCR) and Western blotting. RESULTS: Immunofluorescence results showed that vimentin and FSP-1 were positive in HGFs and Keratin was negative in HGFs. Immunofluorescence staining demonstrated that C5aRA inhibited LPS-stimulated nuclear translocation of p-p65. RT-qPCR and Western blotting showed that C5aRA reduced the expression of IL-1ß, IL-6, TNF-α, C5aR, p-p65, p-IκBα, p-JNK, p-c-JUN, and TLR4 in LPS-induced HGFs. CONCLUSION: These findings suggested that C5aRA attenuated the release of inflammatory cytokines in LPS-induced HGFs by blocking the activation of the NF-κB and MAPK signaling pathways.
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NF-kappa B , Periodontite , Humanos , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Transdução de Sinais , Inflamação , Citocinas/metabolismo , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Periodontite/tratamento farmacológico , Periodontite/metabolismo , FibroblastosRESUMO
BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.
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Testículo , Ubiquitina-Proteína Ligases , Masculino , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Testículo/metabolismo , Espermatogênese/fisiologia , Linhagem Celular , Proliferação de Células , Apoptose , Ubiquitinas/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Abstract Objective Abnormal complement activation is associated with periodontitis. W54011 is a novel non-peptide C5aR antagonist (C5aRA) that exhibits favorable anti-inflammatory effects in various inflammatory models. However, whether W54011 inhibits periodontitis has not yet been fully elucidated. To address this, we have investigated the probable anti-inflammatory mechanism of W54011 in LPS-treated inflammation in human gingival fibroblasts (HGFs). Methodology HGFs were isolated from healthy gingival tissue samples using the tissue block method and were identified with immunofluorescence staining. The CCK8 assay and reverse transcription-PCR (RT-PCR) were used to select the optimal induction conditions for Lipopolysaccharide (LPS) and C5aRA (according to supplementary data S1, S2 and S3). The levels of inflammatory cytokines, C5aR, and the activation of NF-κB/MAPK signaling pathways were determined by RT-quantitative PCR (RT-qPCR) and Western blotting. Results Immunofluorescence results showed that vimentin and FSP-1 were positive in HGFs and Keratin was negative in HGFs. Immunofluorescence staining demonstrated that C5aRA inhibited LPS-stimulated nuclear translocation of p-p65. RT-qPCR and Western blotting showed that C5aRA reduced the expression of IL-1β, IL-6, TNF-α, C5aR, p-p65, p-IκBα, p-JNK, p-c-JUN, and TLR4 in LPS-induced HGFs. Conclusion These findings suggested that C5aRA attenuated the release of inflammatory cytokines in LPS-induced HGFs by blocking the activation of the NF-κB and MAPK signaling pathways.
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BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.
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Humanos , Masculino , Testículo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Repressoras/metabolismo , Espermatogênese/fisiologia , Ubiquitinas/metabolismo , Linhagem Celular , Apoptose , Proliferação de Células , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Oxigenases de Função Mista/metabolismoRESUMO
Atrial fibrillation (AF) represents the most common type of sustained cardiac arrhythmia in humans and confers a significantly increased risk for thromboembolic stroke, congestive heart failure and premature death. Aggregating evidence emphasizes the predominant genetic defects underpinning AF and an increasing number of deleterious variations in more than 50 genes have been involved in the pathogenesis of AF. Nevertheless, the genetic basis underlying AF remains incompletely understood. In the current research, by whole-exome sequencing and Sanger sequencing analysis in a family with autosomal-dominant AF and congenital patent ductus arteriosus (PDA), a novel heterozygous variation in the PRRX1 gene encoding a homeobox transcription factor critical for cardiovascular development, NM_022716.4:c.373G>T;p.(Glu125*), was identified to be in co-segregation with AF and PDA in the whole family. The truncating variation was not detected in 306 unrelated healthy individuals employed as controls. Quantitative biological measurements with a reporter gene analysis system revealed that the Glu125*-mutant PRRX1 protein failed to transactivate its downstream target genes SHOX2 and ISL1, two genes that have been causally linked to AF. Conclusively, the present study firstly links PRRX1 loss-of-function variation to AF and PDA, suggesting that AF and PDA share a common abnormal developmental basis in a proportion of cases.
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Sand flies are hematophagous insects responsible for the transmission of vector-borne diseases to humans. Prominent among these diseases is Leishmaniasis that affects the skin and mucous surfaces and organs such as liver and spleen. Importantly, the function of blood-sucking arthropods goes beyond merely transporting pathogens. The saliva of vectors of disease contains pharmacologically active components that facilitate blood feeding and often pathogen establishment. Transcriptomic and proteomic studies have enumerated the repertoire of sand fly salivary proteins and their potential use for the control of Leishmaniasis, either as biomarkers of vector exposure or as anti-Leishmania vaccines. However, a group of specific sand fly salivary proteins triggers formation of cross-reactive antibodies that bind the ectodomain of human desmoglein 1, a member of the epidermal desmosomal cadherins. These cross-reactive antibodies are associated with skin autoimmune blistering diseases, such as pemphigus, in certain immunogenetically predisposed individuals. In this review, we focus on two different aspects of sand fly salivary proteins in the context of human disease: The good, which refers to salivary proteins functioning as biomarkers of exposure or as anti-Leishmania vaccines, and the bad, which refers to salivary proteins as environmental triggers of autoimmune skin diseases.
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Leishmaniose , Psychodidae , Animais , Autoimunidade , Leishmaniose/prevenção & controle , Proteômica , Proteínas e Peptídeos SalivaresRESUMO
BACKGROUND: Emerging evidence indicates the important role of herbal medicine for neuroinflammation, which is closely associated with neurodegenerative diseases. OBJECTIVE: To clarify the characteristics and primary mechanisms of action of the traditional herbal medicine Daphne kiusiana var. atrocaulis (Rehd.) F. Maekawa in neuroinflammation by phytochemistry and bioassays using both in vitro and in vivo assays. METHODS: The chemical composition of D. kiusiana var. atrocaulis was clarified using multiple chromatography technologies and spectroscopic analysis. The anti-neuroinflammatory effects of the identified components were evaluated in LPS-induced BV-2 cells by monitoring the production of nitric oxide. C57BL/6 mice were used to construct a neuroinflammatory model by injecting LPS into the lateral ventricle of the brain. The most promising component was evaluated in vivo by measuring the number of Iba-1 cells and expression of inflammatory factors. Furthermore, the anti-neuroinflammatory mechanism involved in the activation of the NF-κB pathway was investigated using western blot and immunofluorescence. RESULTS: Thirty-two constituents (1-32), including five new compounds, were successfully identified from D. kiusiana var. atrocaulis. Compounds 3, 5, 12-15, and 20 (IC50 values from 5.41 to 57.27 µM) could considerably inhibit the LPS-induced production of NO in BV-2 cells, displaying stronger anti-neuroinflammatory activities than that of minocycline (IC50 = 67.08 µM). The concentration of the most potential compound 13 (IC50 5.41 µM) was 5.4% of the ethyl acetate fraction. Acutissimalignan B (13) could reduce the mRNA expression of iNOs, TNF-α, IL-1ß, and IL-6, inhibit the phosphorylation of IκBα, and inhibit the nuclear translocation of NK-κB p65 in BV-2 cells induced by LPS. Moreover, in the LPS-induced mouse model, compound 13 was found to exert anti-neuroinflammatory activity by attenuating the activation of microglia in the cortex and hippocampus, repressing the phosphorylation of IκBα, inhibiting the nuclear translocation of NK-κB p65, and decreasing the mRNA expression of iNOs, TNF-α, IL-1ß, and IL-6 in the cortex. CONCLUSION: We found that D. kiusiana var. atrocaulis had an inhibitory activity on neuroinflammation. In addition, the main active component (-)-acutissimalignan B (13) showed anti-neuroinflammatory effects in both in vivo and in vitro assays. Its mechanism of action may be associated with the inhibition of the NF-κB signaling pathway. Our current findings provide new information on D. kiusiana var. atrocaulis in the treatment of neuroinflammation.
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Anti-Inflamatórios não Esteroides/farmacologia , Daphne/química , Inflamação/tratamento farmacológico , Lignanas/farmacologia , NF-kappa B/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Avaliação Pré-Clínica de Medicamentos , Inflamação/metabolismo , Inflamação/patologia , Lignanas/química , Lipopolissacarídeos/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Preparações de Plantas/química , Preparações de Plantas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
As the main signal for the maternal recognition in ruminants, interferon-tau (IFNT) stimulates expression of interferon-stimulated genes (ISGs) in uterus and many extrauterine tissues. However, it is unclear that early pregnancy induces expression of signal transducer and activator of transcription 1 (STAT1), myxovirusresistance 1 (Mx1), interferon-gamma-inducible protein 10 (IP-10) and ubiquitin activating enzyme E1-like protein (UBE1L) in maternal thymus. In this study, ovine thymuses were sampled on day 16 of the estrous cycle and on days 13, 16 and 25 of gestation, and the expression of STAT1, Mx1, IP-10 and UBE1L was detected by real-time quantitative PCR, Western blot and immunohistochemistry. The results revealed that the expression of STAT1 and IP-10 reached peaks on day 16 of pregnancy, and expression of Mx1 was enhanced on day 25 of pregnancy, and STAT1 protein was located in the epithelial reticular cells, capillaries and thymic corpuscles. However, expression of UBE1L was declined during early pregnancy. In conclusion, early pregnancy influences expression of STAT1, Mx1, IP-10 and UBE1L in maternal thymus, which may participate in regulation of maternal immune tolerance during early pregnancy in sheep.
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Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.