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Inflammation and fibrosis play important roles in diabetic kidney disease (DKD). Previous studies have shown that glucagon-like peptide-1 receptor (GLP-1R) agonists had renal protective effects. However, the mechanisms are not clear. The present study explored the effect of liraglutide, a GLP-1R agonist, on the downregulation of glomerular inflammation and fibrosis in DKD by regulating the TLR4/MyD88/NF-kappaB signaling pathway in mesangial cells (MCs). In vitro, rat MCs were cultured in high glucose (HG). We found that liraglutide treatment significantly reduced the HG mediated activation of the TLR4/MYD88/NF-κB signaling pathway, extracellular matrix (ECM)-related proteins and inflammatory factors. A combination of TLR4 inhibitor (TAK242) and liraglutide did not synergistically inhibited inflammatory factors and ECM proteins. Furthermore, in the presence of TLR4 siRNA, liraglutide significantly blunted HG-induced expression of fibronectin protein and inflammatory factors. Importantly, TLR4 selective agonist - LPS or TLR4 overexpression eliminated the improvement effects of liraglutide on the HG-induced response. In vivo, administration of liraglutide for 8 weeks significantly improved the glomerular damage in streptozotocin-induced diabetic mice, and reduced the expression of TLR4/MYD88/NF-κB signaling proteins, ECM protein, and inflammatory factors in renal cortex. TLR4-/- diabetic mice showed significant amelioration in urine protein excretion rate, glomerular pathological damage, inflammation and fibrosis. Liraglutide attenuated glomerular hypertrophy, renal fibrosis and inflammatory response in TLR4-/- diabetic mice. Taken together, our findings suggest that TLR4/MYD88/NF-κB signaling is involved in the regulation of inflammatory response and ECM protein proliferation in DKD. Liraglutide alleviates inflammation and fibrosis by downregulating the TLR4/MYD88/NF-κB signaling pathway in MCs.
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STUDY QUESTION: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER: A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY: The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.
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Acrossomo , Infertilidade Masculina , Proteínas dos Microfilamentos , Adulto , Humanos , Masculino , Acrossomo/patologia , Acrossomo/ultraestrutura , Actinas/metabolismo , Actinas/genética , Sequenciamento do Exoma , Fertilização/genética , Deleção de Genes , Infertilidade Masculina/genética , Cabeça do Espermatozoide/ultraestrutura , Cabeça do Espermatozoide/patologia , Injeções de Esperma Intracitoplásmicas , Espermatozoides/ultraestrutura , Espermatozoides/anormalidades , Proteínas dos Microfilamentos/genéticaRESUMO
PURPOSE: To compare the potential efficiency of [68Ga]Ga-LNC1007 with 2-[18F]FDG/[68Ga]Ga-PSMA PET/CT for detecting renal cell carcinoma (RCC) and to explore parameters derived from [68Ga]Ga-LNC1007 PET/CT for discriminating pathological characteristics in RCC. METHODS: Twenty-five RCC patients confirmed by pathology were enrolled in this prospective study. The maximum standardized uptake value (SUVmax), mean SUV (SUVmean), gross tumor volume (GTV) and total lesion-tracer (TL-tracer) of lesions were calculated from the corresponding PET/CT images. Pathological characteristics included World Health Organization/International Society of Urological Pathology (WHO/ISUP) grade and adverse pathological features (tumor necrosis or sarcomatoid or rhabdoid feature). RESULTS: [68Ga]Ga-LNC1007 PET/CT showed a higher detection rate for primary lesions than 2-[18F]FDG and [68Ga]Ga-PSMA (LNC1007 vs. FDG: 13/17 vs. 4/17, P = 0.005; LNC1007 vs. PSMA: 9/11 vs. 6/11, P = 0.361). [68Ga]Ga-LNC1007 PET/CT showed higher SUVmax (6.6 vs. 3.7, P = 0.005), SUVmean (4.1 vs. 2.3, P = 0.001) and TBR (2.6 vs. 1.7, P = 0.011) compared with 2-[18F]FDG PET/CT, and it also showed higher TBR (2.9 vs. 0.5, P = 0.003), TBR-delay (2.8 vs. 0.3, P = 0.003), GTV (84.1 vs. 42.9, P = 0.003) and TL-tracer (442.7 vs. 235.8, P = 0.008) compared with [68Ga]Ga-PSMA PET/CT. SUVmax and TBR derived from [68Ga]Ga-LNC1007 PET/CT could effectively differentiate WHO/ISUP grade (3-4 vs. 1-2) and adverse pathological features (positive vs. negative) (SUVmax: AUC 0.81, P = 0.04; AUC 0.80, P = 0.033; TBR: AUC 0.84, P = 0.026; AUC 0.85, P = 0.014). The SUVmax was positively correlated with the FAP expression, integrin αvß3 expression and the total expression of FAP and integrin αvß3 (r = 0.577, P = 0.006, r = 0.701, P < 0.001, and r = 0.702, P < 0.001, respectively). CONCLUSION: [68Ga]Ga-LNC1007 is a promising tracer for RCC imaging and can effectively identify aggressive pathological characteristics of RCC.
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Carcinoma de Células Renais , Neoplasias Renais , Humanos , Radioisótopos de Gálio , Fluordesoxiglucose F18 , Carcinoma de Células Renais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Estudos Prospectivos , Oligopeptídeos , Neoplasias Renais/diagnóstico por imagem , IntegrinasRESUMO
Diaminopropionate ammonia-lyase transforms D and L isomers of 2,3-diaminopropionate to pyruvate and ammonia. It catalyzes D- and l-serine less effectively. L-2,3-diaminopropionate is a precursor in the biosynthesis of oxalyl diaminopropionate as a neurotoxin in certain legume species. In this work, we cyclized the diaminopropionate ammonia-lyase from Salmonella typhimurium in vitro using the redox-responsive split intein, and identified that backbone cyclization afforded the enzyme with the improved activity, thermal stability and resistance to the exopeptidase proteolysis, different from effects of the incorporated sequence recognized by tobacco vein mottling virus protease at C-terminus. Using analyses of three fluorescent dyes including 8-anilino-1-naphthalenesulfonic acid, N-phenyl-1-naphthylamine, and thioflavin T, the same amounts of the cyclic protein displayed less fluorescence than those of the linear protein upon the heat treatment. The cyclic enzyme displayed the enhanced activity in Escherichia coli cells using the designed novel reporter. In this system, d-serine was added to the culture and transported into the cytoplasm. It was transformed by pre-overexpression of the diaminopropionate ammonia-lyase, and untransformed d-serine was oxidized by the coproduced human d-amino acid oxidase to generate hydrogen peroxide. This oxidant is monitored by the HyPer indicator. The current results presented that the cyclized enzyme could be applied as a better candidate to block the neurotoxin biosynthesis in certain plant species.
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Amônia-Liases , Neurotoxinas , Salmonella typhimurium , Humanos , Ciclização , Escherichia coli/genética , SerinaRESUMO
BACKGROUND: Cerebral small vessel injury, including loss of endothelial tight junctions, endothelial dysfunction, and blood-brain barrier breakdown, is an early and typical pathology for Alzheimer's disease, cerebral amyloid angiopathy, and hypertension-related cerebral small vessel disease. Whether there is a common mechanism contributing to these cerebrovascular alterations remains unclear. Studies have shown an elevation of BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) in cerebral vessels from cerebral amyloid angiopathy or Alzheimer's disease patients, suggesting that vascular BACE1 may involve in cerebral small vessel injury. METHODS: To understand the contribution of vascular BACE1 to cerebrovascular impairments, we combined cellular and molecular techniques, mass spectrometry, immunostaining approaches, and functional testing to elucidate the potential pathological mechanisms. RESULTS: We observe a 3.71-fold increase in BACE1 expression in the cerebral microvessels from patients with hypertension. Importantly, we discover that an endothelial tight junction protein, occludin, is a completely new substrate for endothelial BACE1. BACE1 cleaves occludin with full-length occludin reductions and occludin fragment productions. An excessive cleavage by elevated BACE1 induces membranal accumulation of caveolin-1 and subsequent caveolin-1-mediated endocytosis, resulting in lysosomal degradation of other tight junction proteins. Meanwhile, membranal caveolin-1 increases the binding to eNOS (endothelial nitric oxide synthase), together with raised circulating Aß (ß-amyloid peptides) produced by elevated BACE1, leading to an attenuation of eNOS activity and resultant endothelial dysfunction. Furthermore, the initial endothelial damage provokes chronic reduction of cerebral blood flow, blood-brain barrier leakage, microbleeds, tau hyperphosphorylation, synaptic loss, and cognitive impairment in endothelial-specific BACE1 transgenic mice. Conversely, inhibition of aberrant BACE1 activity ameliorates tight junction loss, endothelial dysfunction, and memory deficits. CONCLUSIONS: Our findings establish a novel and direct relationship between endothelial BACE1 and cerebral small vessel damage, indicating that abnormal elevation of endothelial BACE1 is a new mechanism for cerebral small vessel disease pathogenesis.
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Doença de Alzheimer , Angiopatia Amiloide Cerebral , Doenças de Pequenos Vasos Cerebrais , Hipertensão , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Angiopatia Amiloide Cerebral/complicações , Angiopatia Amiloide Cerebral/metabolismo , Humanos , Hipertensão/complicações , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/metabolismo , Ocludina/metabolismo , Proteínas de Junções Íntimas , Junções Íntimas/metabolismoRESUMO
Osimertinib (Osi) is widely used as a first-line treatment for non-small cell lung cancer (NSCLC) with EGFR mutations. However, the majority of patients treated with Osi eventually relapse within a year. The mechanisms of Osi resistance remain largely unexplored, and efficient strategies to reverse the resistance are urgently needed. Here, we developed a lactoferrin-modified liposomal codelivery system for the combination therapy of Osi and panobinostat (Pan), an epigenetic regulator of histone acetylation. We demonstrated that the codelivery liposomes could efficiently repolarize tumor-associated macrophages (TAM) from the M2 to M1 phenotype and reverse the epithelial-mesenchymal transition (EMT)-associated drug resistance in the tumor cells, as well as suppress glycolysis, lactic acid production, and angiogenesis. Our results suggested that the combination therapy of Osi and Pan mediated by liposomal codelivery is a promising strategy for overcoming Osi resistance in NSCLC.
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Acrilamidas , Compostos de Anilina , Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Indóis , Neoplasias Pulmonares , Panobinostat , Inibidores de Proteínas Quinases , Pirimidinas , Humanos , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Lipossomos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologiaRESUMO
OBJECTIVE: To determine the accuracy of salivary active matrix metalloproteinase (aMMP)-8 point-of-care test (POCT) for detecting periodontitis in adults, through meta-analysis. MATERIALS AND METHODS: Diagnostic studies evaluating the accuracy of salivary/oral rinse aMMP-8 POCT for detecting periodontitis in adults, when compared with clinical examination, were considered eligible. A comprehensive search was performed up to 31 August 2023 through five databases. Quality Assessment of Diagnostic Accuracy Studies 2 was utilized to evaluate the methodological quality of the included articles. Meta-analysis was performed using Bayesian bivariate hierarchical model and subgroup analysis. RESULTS: From 368 screened studies, 6 studies (4 cross-sectional and 2 longitudinal studies) were included in the meta-analysis. Overall, the pooled sensitivity and specificity of salivary aMMP-8-POCT for detecting periodontitis were 0.63 (95% CI: 0.41-0.82) and 0.84 (95% CI: 0.65-0.95), respectively. Subgroup analyses revealed that the 95% CI for oral fluid types, predefined diagnostic thresholds and the POCT systems largely overlapped, indicating that the differences between them may not be significant. CONCLUSION: Salivary aMMP-8 POCT shows fair accuracy for detecting periodontitis. The diagnostic accuracy cannot be significantly influenced by the types of oral fluids, predefined diagnostic thresholds or the specific POCT systems used. More research is needed to confirm the clinical utility and implementation of aMMP-8 POCT in the diagnosis of periodontitis.
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Metaloproteinase 8 da Matriz , Periodontite , Saliva , Adulto , Humanos , Metaloproteinase 8 da Matriz/análise , Periodontite/diagnóstico , Testes Imediatos , Saliva/enzimologia , Saliva/química , Sensibilidade e EspecificidadeRESUMO
AIMS: To explore the effect of post-stroke fatigue (PSF) on post-stroke depression (PSD) and examine the mediating effects of fear of disease progression (FOP) and resilience between PSF and PSD. DESIGN: A cross-sectional study. METHODS: A total of 315 stroke patients participated in the questionnaire survey between November 2022 and June 2023. Data were collected using the General Information Questionnaire, Fatigue Severity Scale, Fear of Disease Progression Questionnaire-Short Form, Connor-Davidson Resilience Scale-10 Item and Hospital Anxiety and Depression Scale-Depression Subscale. Data were analysed by descriptive analysis, Mann-Whitney U-test, Kruskal-Wallis H-test, Pearson or Spearman correlation, hierarchical regression analysis and mediation analysis. RESULTS: PSF had a significant positive total effect on PSD (ß = .354, 95% CI: .251, .454). Additionally, FOP and resilience played a partial parallel-mediating role in the relationship between PSF and PSD (ß = .202, 95% CI: .140, .265), and the total indirect effect accounted for 57.06% of the total effect. CONCLUSIONS: FOP and resilience parallelly mediated the effect of PSF on PSD, which may provide a novel perspective for healthcare professionals in preventing PSD. Targeted interventions aiming at reducing PSF, lowering FOP levels and enhancing resilience may be possible ways to alleviate PSD. IMPLICATIONS FOR THE PROFESSION AND PATIENT CARE: Interventions that tail to reducing PSF, lowering FOP levels and enhancing resilience may be considered as possible ways to alleviate PSD. IMPACT: This study enriched the literature by exploring the effect of PSF on PSD and further examining the mediating effects of FOP and resilience between PSF and PSD. Findings emphasized the important effects of PSF, FOP and resilience on PSD. REPORTING METHOD: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist for cross-sectional studies was used to guide reporting. PATIENT OR PUBLIC CONTRIBUTION: One tertiary hospital assisted participants recruitment.
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Inspired by transformation optics, we propose a new concept for plasmonic photocatalysis by creating a novel hybrid nanostructure with a plasmonic singularity. Our geometry enables broad and strong spectral light harvesting at the active site of a nearby semiconductor where the chemical reaction occurs. A proof-of-concept nanostructure comprising Cu2ZnSnS4 (CZTS) and Au-Au dimer (t-CZTS@Au-Au) is fabricated via a colloidal strategy combining templating and seeded growth. On the basis of numerical and experimental results of different related hybrid nanostructures, we show that both the sharpness of the singular feature and the relative position to the reactive site play a pivotal role in optimizing photocatalytic activity. Compared with bare CZTS, the hybrid nanostructure (t-CZTS@Au-Au) exhibits an enhancement of the photocatalytic hydrogen evolution rate by up to â¼9 times. The insights gained from this work might be beneficial for designing efficient composite plasmonic photocatalysts for diverse photocatalytic reactions.
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BACKGROUND: Nursing students encounter various stressors during their clinical practicum; however, the stressors are not the same during different periods. At present, studies on the stressors and coping styles of nursing students in the middle period of their clinical practicum are rare. AIMS: The current study aimed to explore the stressors and coping styles of nursing students in the middle period of their clinical practicum. METHODS: A qualitative study with a descriptive phenomenological method was conducted to collect data from 10 nursing students undergoing the middle period of their clinical practicum from December 2020 to February 2021. The data were collected by semistructured interviews using interview outlines prepared in advance. The data were analyzed by Colaizzi's analysis method. RESULTS: The stressors experienced by nursing students in the middle period of their clinical practicum mainly included personal reasons, teaching arrangements, interpersonal relationships, occupational particularity and career planning. Additionally, nursing students coped with the stressors that they face in the clinical practicum by eliminating stressors and regulating emotions. CONCLUSIONS: Nursing students experienced various stressors and used a variety of coping styles in the middle period of their clinical practicum, which was different from what occurred in the early and late periods. Targeted interventions should be formulated and implemented to relieve nursing students' stress and guide them to adopt effective coping styles.
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OBJECTIVE: To investigate the therapeutic utility of psychological nursing interventions for prostatic hyperplasia clients while they are receiving therapy. METHODS: Clinical data of 110 patients with prostate group hyperplasia who underwent treatment in our hospital were collected and analysed retrospectively, and the selected period was from October 2021 to October 2023. The 110 cases of prostate group hyperplasia patients were divided into a research group and a control group according to the different methods of care, and each group had 55 cases each. The research group received psychological nursing intervention based on the conventional nursing care given to the control group. The total treatment compliance rate and contentment with nursing were contrasted between the research and control groups, and changes in the Self Rating Anxiety Scale (SAS) score, Self Rating Depression Scale (SDS) score, Health Survey Short Form score, and sleep problems were observed between the research group and the control group. RESULTS: The research group's overall compliance rate was 94.55% (52/53), a substantial increase over the control group's rate, 69.09% (38/55), P < 0.01. Following nursing, the research group's SAS and SDS scores were considerably more reduced than those of the control group, and both groups' scores were substantially lower than they were prior to nursing (P < 0.05). CONCLUSION: This retrospective study found that psychological nursing intervention applied to patients with prostatic hyperplasia can effectively improve the patient's compliance with treatment, effectively reduce the occurrence of negative emotions, improve the patient's quality of life, and improve sleep problems. In addition, psychological nursing intervention can effectively alleviate the tension between nurses and patients, and is worthy of clinical application.
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Sepsis is a life-threatening disease caused by the dis-functioning of the immune response to pathogenic infections. Despite, the discovery of modern therapeutics and treatments of sepsis are lacking due to the resistance of pathogens. Metronidazole is an antibiotic commonly used to treat bacterial infections, but usage is limited and challenging by a short half-life period. In this research work, fabricate a pH-responsive drug delivery system for controlled release of metronidazole targeted molecules. We exemplified that, the encapsulation of hydrophilic metronidazole drug within a hydrophobic ZIF-90 framework can be enhanced the pH-responsive drug release under acidic conditions. The ZIF-90 frameworks only decompose in under acidic solutions, they are highly stable in physiological conditions. The pH-responsive protonation mechanism of ZIF-90 frameworks promotes the quick release of metronidazole within cells. The antimicrobial proficiency of zinc and metronidazole will expose a synergistic effect in ROS-mediated bacterial inhibition and auto-immunity boosting of normal cells. In vitro, antibacterial activity results revealed that the MI@ZIF-90 nano drug delivery system effectively eradicated human infectious pathogens at the lowest concentrations. In anti-fungal activity, studies show excellent growth inhibition against human pathogenic fungi Aspergillus fumigatus and Candida albicans. Finally, the PBMC cytocompatibility study concludes, that the fabricated MI@ZIF-90 drug delivery system is non-toxic to biomedical applications. The overall research findings highlight the design of a smart drug delivery system for sepsis treatment. In future it will be an efficient, low-cost, and biocompatible pharmaceutics for pediatric sepsis management processes.
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Estruturas Metalorgânicas , Nanopartículas , Sepse , Humanos , Criança , Metronidazol , Preparações Farmacêuticas , Leucócitos Mononucleares , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Anti-Inflamatórios , Concentração de Íons de Hidrogênio , Liberação Controlada de FármacosRESUMO
To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.
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Inteínas , Processamento de Proteína , Inteínas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , OxirreduçãoRESUMO
RESEARCH QUESTION: More than 100 variants have been identified in the TUBB8 gene, which account for approximately 30% of infertile women with oocyte maturation defects. But what is the correlation between the highly phenotypic diversity and genetic variability? Are there other variants in TUBB8 related to female infertility? DESIGN: TUBB8 resequencing was performed in 80 female subjects who were experiencing infertility and were seeking treatment with assisted reproductive technologies (ART), or had ever experienced ART failure due to oocyte maturation defects. All variants were evaluated with pedigree analysis, population frequency, in-silico analysis and molecular modelling. The effects of the variants on oocytes/arrested embryos were assessed by morphological observations, immunostaining, embryo biopsies and chromosome euploidy analysis. RESULTS: Nine missense variants and two frameshift variants from an additional 15 families were identified, including four novel variants and seven previously reported recurrent variants. These TUBB8 variants were related to highly variable phenotypes, including abnormalities in oocyte maturation or morphology, fertilization failure, embryonic development abnormalities and implantation failure. Also further clarified were the incomplete penetrance of heterozygous p.E108K, the likely benign significance of heterozygous p.A313V and the clinical effect of a novel variant of p.R380C. CONCLUSIONS: This study significantly expands the variant spectrum of the TUBB8 gene and, together with the available findings on TUBB8 variants and female infertility, will potentially facilitate the genetic counselling of infertile women in future.
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Infertilidade Feminina , Humanos , Gravidez , Feminino , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Mutação , Tubulina (Proteína)/genética , Oócitos , Técnicas de Reprodução Assistida , Testes GenéticosRESUMO
OBJECTIVE AND DESIGN: Post-traumatic urethral stricture is a clinical challenge for both patients and clinicians. Targeting glutamine metabolism to suppress excessive activation of urethral fibroblasts (UFBs) is assumed to be a potent and attractive strategy for preventing urethral scarring and stricture. MATERIAL OR SUBJECTS: In cellular experiments, we explored whether glutaminolysis meets the bioenergetic and biosynthetic demands of quiescent UFBs converted into myofibroblasts. At the same time, we examined the specific effects of M2-polarized macrophages on glutaminolysis and activation of UFBs, as well as the mechanism of intercellular signaling. In addition, findings were further verified in vivo in New Zealand rabbits. RESULTS: It revealed that glutamine deprivation or knockdown of glutaminase 1 (GLS1) significantly inhibited UFB activation, proliferation, biosynthesis, and energy metabolism; however, these effects were rescued by cell-permeable dimethyl α-ketoglutarate. Moreover, we found that exosomal miR-381 derived from M2-polarized macrophages could be ingested by UFBs and inhibited GLS1-dependent glutaminolysis, thereby preventing excessive activation of UFBs. Mechanistically, miR-381 directly targets the 3'UTR of Yes-associated protein (YAP) mRNA to reduce its stability at the transcriptional level, ultimately downregulating expression of YAP, and GLS1. In vivo experiments revealed that treatment with either verteporfin or exosomes derived from M2-polarized macrophages significantly reduced urethral stricture in New Zealand rabbits after urethral trauma. CONCLUSION: Collectively, this study demonstrates that exosomal miR-381 from M2-polarized macrophages reduces myofibroblast formation of UFBs and urethral scarring and stricture by inhibiting YAP/GLS1-dependent glutaminolysis.
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MicroRNAs , Estreitamento Uretral , Animais , Coelhos , Glutamina/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Cicatriz , Constrição Patológica , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibroblastos/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
BACKGROUND: Several fusion tags for separation handle have been developed, but the fused tag for simply and cheaply separating the target protein is still lacking. RESULTS: Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli were optimized via precipitation with calcium chloride (CaCl2) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na2). The HanA1-EmGFP absorbing with other three affinity matrix was detected, only it was strongly bound to heparin Sepharose. The separation efficiency of the HanA1-EmGFP was comparable with purification efficiency of the His6-tagged HanA1-EmGFP via metal ion affinity chromatography. Three fluorescent proteins for the EmGFP, mCherry red fluorescent protein and flavin-binding cyan-green fluorescent protein LOV from Chlamydomonas reinhardtii were used for naked-eye detection of the separation and purification processes, and two colored proteins including a red protein for a Vitreoscilla hemoglobin (Vhb), and a brown protein for maize sirohydrochlorin ferrochelatase (mSF) were used for visualizing the separation process. The added EDTA-Na2 disrupted the Fe-S cluster in the mSF, but it showed little impact on heme in Vhb. CONCLUSIONS: The selected five colored proteins were efficient for detecting the applicability of the highly selective hanA1 for fusion separation and purification handle. The fused hanA1 tag will be potentially used for simple and cheap affinity separation of the target proteins in industry and diagnosis.
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Anexina A1 , Humanos , Proteínas de Fluorescência Verde/metabolismo , Anexina A1/metabolismo , Ácido Edético/metabolismo , Cromatografia de Afinidade/métodos , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) have the potential to become malignant. Few studies have focused on the prevalence of the diabetes mellitus (DM) in IPMNs and its association with malignancy. We evaluated the association between DM and malignant IPMNs in this study. METHODS: A total of 226 patients with pathologically confirmed IPMNs were included. Demographic data, serum biochemical data, and imaging findings were collected. The malignant IPMNs were defined as those with high-grade dysplasia and associated invasive carcinoma. The association between DM and malignant IPMNs was studied using logistic regression analysis. RESULTS: The prevalence of DM was 17.7% for all type IPMNs and 31.7% for malignant IPMNs. Compared to patients without DM, those with DM had a 3.6-fold (odds ratio [OR]: 3.62; 95% confidence interval [CI]: 1.26-10.44) higher risk of invasive carcinoma and 2.5-fold (OR: 2.48; 95% CI: 1.06-5.77) higher risk of malignant IPMNs. Similar results were observed in main pancreatic duct (MPD) involved IPMNs. New-onset DM was associated with a 4.13-fold (OR: 4.13, 95% CI: 1.27-13.36) higher risk of invasive carcinoma. CONCLUSION: DM is associated with a higher risk of malignant IPMNs and invasive carcinoma, especially in patients with MPD-involved IPMNs.
Assuntos
Carcinoma Ductal Pancreático , Diabetes Mellitus , Neoplasias Intraductais Pancreáticas , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/patologia , Diabetes Mellitus/epidemiologia , Ductos Pancreáticos , Neoplasias Pancreáticas/patologia , Estudos Retrospectivos , Neoplasias Intraductais Pancreáticas/patologiaRESUMO
BACKGROUND: Lacrimal adenoid cystic carcinoma (LACC) is the most common orbital malignant epithelial neoplasm. LACC with high-grade transformation (LACC-HGT) has higher rates of recurrence, metastasis, and mortality than LACC without HGT. This study investigated the effects of microRNA-29a-3p (miR-29a-3p) in the pathogenesis of LACC-HGT. METHODS: An Agilent human miRNA microarray was used to screen the differentially expressed miRNAs (DEMs) in LACC and LACC-HGT tumor tissues. Then, the primary cells obtained in previous studies were used to determine the effect of miR-29a-3p. RESULTS: The expression of miR-29a-3p was abnormally lower in LACC-HGT than in LACC. miR-29a-3p can specifically target the 3' UTR of Quaking mRNA and down-regulate Quaking expression, thereby inhibiting the proliferation, migration, and epithelial-mesenchymal transition of LACC cells. CONCLUSIONS: This study illustrated that miR-29a-3p functions as a tumor suppressor by down-regulating the expression of Quaking to inhibit the tumorigenesis of LACC cells. This study may also reveal the pathogenesis of HGT in LACC cells and provide a reference for LACC-HGT targeted diagnosis.
Assuntos
Carcinoma Adenoide Cístico , Neoplasias de Cabeça e Pescoço , Aparelho Lacrimal , MicroRNAs , Humanos , Transição Epitelial-Mesenquimal/genética , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genéticaRESUMO
Carboxymethyl-ß-cyclodextrins (CM-ß-CDs) with five kinds of degrees of substitution were synthesized and characterized. Analytical enantioseparation of six basic drugs containing N-alkyl groups, including pheniramine, chlorpheniramine, labetalol, propranolol, venlafaxine, and trans-paroxol, was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) using the synthesized CM-ß-CD as chiral mobile phase additives. Key influence factors were optimized, including organic modifier, pH value, CM-ß-CD with different degrees of substitution, and concentration of CM-ß-CD. The mobile phase was composed of methanol and 10 mmol L-1 of phosphate buffer pH 4.0 containing 10 mmol L-1 of CM-ß-CD. Peak resolution for six racemic drugs was gradually increased with an increasing degree of substitution of the synthesized CM-ß-CD. The stoichiometric ratio and binding constants for the inclusion complex formed by CM-ß-CD and enantiomer were determined, which showed that the stoichiometric ratio for each inclusion complex was 1:1.
Assuntos
Cromatografia de Fase Reversa , beta-Ciclodextrinas , Cromatografia de Fase Reversa/métodos , Estereoisomerismo , Cromatografia Líquida de Alta Pressão/métodos , beta-Ciclodextrinas/química , Indicadores e ReagentesRESUMO
In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.