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Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cyclic nucleotides are critical secondary messengers in the neurohormonal regulation in the cardiovascular system. PDEs precisely control spatiotemporal subcellular distribution of cyclic nucleotides in a cell- and tissue-specific manner, playing critical roles in physiological responses to hormone stimulation in the heart and vessels. Dysregulation of PDEs has been linked to the development of several cardiovascular diseases, such as hypertension, aneurysm, atherosclerosis, arrhythmia, and heart failure. Targeting these enzymes has been proven effective in treating cardiovascular diseases and is an attractive and promising strategy for the development of new drugs. In this review, we discuss the current understanding of the complex regulation of PDE isoforms in cardiovascular function, highlighting the divergent and even opposing roles of PDE isoforms in different pathogenesis.
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Doenças Cardiovasculares , Dietilestilbestrol/análogos & derivados , Diester Fosfórico Hidrolases , Humanos , Inibidores de Fosfodiesterase/uso terapêutico , AMP Cíclico , GMP Cíclico , Isoformas de ProteínasRESUMO
The complement system is the first line of innate immune defense against microbial infections. To survive in humans and cause infections, bacterial pathogens have developed sophisticated mechanisms to subvert the complement-mediated bactericidal activity. There are reports that sialidases, also known as neuraminidases, are implicated in bacterial complement resistance; however, its underlying molecular mechanism remains elusive. Several complement proteins (e.g., C1q, C4, and C5) and regulators (e.g., factor H and C4bp) are modified by various sialoglycans (glycans with terminal sialic acids), which are essential for their functions. This report provides both functional and structural evidence that bacterial sialidases can disarm the complement system via desialylating key complement proteins and regulators. The oral bacterium Porphyromonas gingivalis, a "keystone" pathogen of periodontitis, produces a dual domain sialidase (PG0352). Biochemical analyses reveal that PG0352 can desialylate human serum and complement factors and thus protect bacteria from serum killing. Structural analyses show that PG0352 contains a N-terminal carbohydrate-binding module (CBM) and a C-terminal sialidase domain that exhibits a canonical six-bladed ß-propeller sialidase fold with each blade composed of 3-4 antiparallel ß-strands. Follow-up functional studies show that PG0352 forms monomers and is active in a broad range of pH. While PG0352 can remove both N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc), it has a higher affinity to Neu5Ac, the most abundant sialic acid in humans. Structural and functional analyses further demonstrate that the CBM binds to carbohydrates and serum glycoproteins. The results shown in this report provide new insights into understanding the role of sialidases in bacterial virulence and open a new avenue to investigate the molecular mechanisms of bacterial complement resistance.
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Neuraminidase , Ácidos Siálicos , Humanos , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Sistema Complemento , Fatores Imunológicos , Porphyromonas gingivalisRESUMO
Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine-meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome.
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Produtos Biológicos , Neoplasias da Mama , Humanos , Feminino , Proteoma/química , Lisina/química , Proteômica/métodos , Floroglucinol/farmacologia , Biomimética , Produtos Biológicos/farmacologiaRESUMO
The achievement of rapid multiplexed protein imaging is limited by the use of stimulating reagents, extensive incubating and washing steps, and the low fluorescence intensity of targets. In this study, sequentially-activated DNA tags are developed and combined them with primary antibodies using signal enhancement strategies to create sequentially-activated antibodies (SAAs). These SAAs enable rapid, wash-free sequential imaging of different protein targets. The samples are pre-processed to label all targets of interest with SAAs simultaneously, and the signal is turned ON for only one target in each stage. The sequential imaging of multiple targets is achieved through wash-free strand displacement reactions that exhibit rapid kinetics with t1/2 < 10 s in a cellular context. Remarkably, this method successfully demonstrates sequential imaging of nine different protein targets within just a few minutes. This all-in-one platform for multiplexed protein imaging holds great promise for diverse applications in immunofluorescence imaging.
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Anticorpos , Proteínas , Anticorpos/metabolismo , DNA , Diagnóstico por ImagemRESUMO
Sample preparation for mass spectrometry-based proteomics has many tedious and time-consuming steps that can introduce analytical errors. In particular, the steps around the proteolytic digestion of protein samples are prone to inconsistency. One route for reliable sample processing is the development and optimization of a workflow utilizing an automated liquid handling workstation. Diligent assessment of the sample type, protocol design, reagents, and incubation conditions can significantly improve the speed and consistency of preparation. When combining robust liquid chromatography-mass spectrometry with either discovery or targeted methods, automated sample preparation facilitates increased throughput and reproducible quantitation of biomarker candidates. These improvements in analysis are also essential to process the large patient cohorts necessary to validate a candidate biomarker for potential clinical use. This article reviews the steps in the workflow, optimization strategies, and known applications in clinical, pharmaceutical, and research fields that demonstrate the broad utility for improved automation of sample preparation in the proteomic field.
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Proteínas , Proteômica , Humanos , Proteômica/métodos , Espectrometria de Massas/métodos , Biomarcadores , Manejo de EspécimesRESUMO
BACKGROUND: The association between earlobe crease (ELC) and cerebral small vessel disease (CSVD), including white matter hyperintensities (WMHs) and brain atrophy, is unclear, especially in the setting of acute ischemic stroke (AIS). Here, we aimed to investigate the association between ELC and WMHs as well as brain atrophy among AIS patients. METHODS: A total of 730 AIS patients from China were enrolled. Patients were divided into groups without and with ELC, unilateral and bilateral ELC according to pictures of bilateral ears. Logistic regression models were employed to assess the impact of ELC, bilateral ELC on WMHs, periventricular hyperintensities (PVH), deep white matter hyperintensities (DWMH), and brain atrophy, as measured by the Fazekas scale and global cortical atrophy scale, in brain magnetic resonance imaging (MRI). RESULTS: There were 520 (71.2%) AIS patients with WMHs, 445 (61.0%) with PVH, 462 (63.3%) with DWMH and 586 (80.3%) with brain atrophy. Compared to those without ELC, patients with ELC were significant associated with an increased risk of PVH (odds ratio [OR] 1.79; 95% confidence interval [CI], 1.15-2.77) and brain atrophy (OR 6.18; 95% CI, 3.60-10.63), but not WMHs and DWMH. The presence of bilateral ELC significantly increased the odds of WMHs (OR 1.60; 95% CI, 1.00-2.56), PVH (OR 1.87; 95% CI, 1.18-2.96), and brain atrophy (OR 8.50; 95% CI, 4.62-15.66) when compared to individuals without ELC. Furthermore, we discovered that the association between bilateral ELC and WMHs, PVH, and DWMH was significant only among individuals aged ≤68 (median age) years (all P trend ≤0.041). However, this association was not observed in patients older than 68 years. CONCLUSIONS: In Chinese AIS patients, the presence of the visible aging sign, ELC, especially bilateral ELC, showed independent associations with both white matter hyperintensities and brain atrophy, particularly among those younger than 68 years old.
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Evidence presented that osteoporosis is closely related to the dysfunction of bone mesenchymal stem cells (BMSCs). But most studies are insufficient to reveal what actually happens to the osteoporotic BMSCs. In this study, BMSCs were harvested from ovariectomized and sham-operated rats. After checking the characteristics of rat models and stem cells, the BMSCs were carried out for RNA sequencing. Part of the findings were verified that seven mRNAs (Abi3bp, Aifm3, Ccl11, Cdkn1c, Chst10, Id2, Vcam1) were significantly up-regulated in osteoporotic BMSCs while seven mRNAs (Cep63, Fgfr3, Myc, Omd, Pou2f1, Smarcal1, Timm10b) were down-regulated. In addition, potential miRNA-mRNA and lncRNA-mRNA regulatory networks were illustrated. The changes in osteoporotic BMSCs covered a large set of biological processes, including cell viability, differentiation, immunoreaction, bone repairment and estrogen defect. This study enriched the pathophysiological mechanisms of BMSCs and osteporosis, as well as provided dozens of attractive RNA targets for further treatment.
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Células-Tronco Mesenquimais , Osteoporose , Ratos , Animais , Osteoporose/genética , Osteoporose/terapia , Diferenciação Celular/genética , Análise de Sequência de RNA , RNA Mensageiro , Osteogênese/genética , Células CultivadasRESUMO
As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.
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Asparagina/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribonuclease Pancreático/metabolismo , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Bovinos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Polissacarídeos/química , Polissacarídeos/genética , Conformação Proteica , Engenharia de Proteínas , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genéticaRESUMO
BACKGROUND: Heart failure is a syndrome with complex clinical manifestations. Due to increasing population aging, heart failure has become a major medical problem worldwide. In this study, we used the MIMIC-III public database to extract the temporal and spatial characteristics of electrocardiogram (ECG) signals from patients with heart failure. METHODS: We developed a NYHA functional classification model for heart failure based on a deep learning method. We introduced an integrating attention mechanism based on the CNN-LSTM-SE model, segmenting the ECG signal into 2 to 20 s long segments. Ablation experiments showed that the 12 s ECG signal segments could be used with the proposed deep learning model for superior classification of heart failure. RESULTS: The accuracy, positive predictive value, sensitivity, and specificity of the NYHA functional classification method were 99.09, 98.9855, 99.033, and 99.649%, respectively. CONCLUSIONS: The comprehensive performance of this model exceeds similar methods and can be used to assist in clinical medical diagnoses.
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Aprendizado Profundo , Insuficiência Cardíaca , Humanos , Arritmias Cardíacas/diagnóstico , Eletrocardiografia/métodos , Insuficiência Cardíaca/diagnóstico , Bases de Dados Factuais , AlgoritmosRESUMO
Telehealth, accessing healthcare and wellness remotely, should be a cost-effective and efficient way for individuals to receive care. The convenience of having a reliable remote collection device for blood tests will facilitate access to precision medicine and healthcare. Herein, we tested a 60-biomarker health surveillance panel (HSP), containing 35 FDA/LDT assays and covering at least 14 pathological states, on 8 healthy individuals' ability to collect their own capillary blood from a lancet finger prick and directly compared it to the traditional phlebotomist venous blood and plasma collection methods. All samples were spiked with 114 stable-isotope-labeled (SIL) HSP peptides and quantitatively analyzed by liquid chromatography-multiple reaction monitoring-mass spectrometry (LC/MRM-MS) scheduled method targeting 466 transitions from 114 HSP peptides and by a discovery data-independent acquisition mass spectrometry (DIA-MS) method. The average peak area ratio (PAR) of the HSP quantifier peptide transitions from all 8 volunteers' capillary blood (n = 48), venous blood (n = 48), and matched plasma (n = 24) was <20% coefficients of variation (CV). Heat map analysis of all 8 volunteers demonstrated that each individual had a unique biosignature. Biological replicates from capillary blood and venous blood clustered within each volunteer in k-means clustering analysis. Pearson statistical analysis of the three biofluids indicated that there was >90% similarity. Discovery DIA-MS analysis of the same samples using a plasma spectral library and a pan-human spectral library identified 1121 and 4661 total proteins, respectively. In addition, at least 122 FDA-approved biomarkers were identified. DIA-MS analysis reproducibly quantitated (<30% CV) â¼600-700 proteins in capillary blood, â¼800 proteins in venous blood, and â¼300-400 proteins in plasma, demonstrating that an expansive biomarker panel is possible with current mass spectrometry technology. Both targeted LC/MRM-MS and discovery DIA-MS analysis of whole blood collected on remote sampling devices are viable options for personal proteome biosignature stratification in precision medicine and precision health.
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Coleta de Amostras Sanguíneas , Peptídeos , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/química , BiomarcadoresRESUMO
OBJECTIVE AND DESIGN: The purpose of this study was to explore pathological processes during the first 4 weeks after anterior cruciate ligament reconstruction (ACLR). SUBJECTS: Sixteen ACL-injured patients (8 females/8 males, mean age = 19.1, mean BMI = 28.6). METHODS: Arthrocentesis was performed 1 and 4 weeks after ACLR. Proteins in the synovial fluid were identified using nanoLC-ESI-MS/MS. Differentially up- or down-regulated proteins were identified and quantified, and a pathway analysis was performed. All identified proteins were mapped into a protein-protein interaction (PPI) network, and networks of PPIs with a combined score > 0.9 were then visualized. RESULTS: Seven pathways were upregulated after ACLR: PI3K-AKT signaling pathway, extracellular matrix (ECM)-receptor interaction, focal adhesion, protein digestion and absorption, ameobiasis, and platelet activation. Network analyses identified 8 proteins that were differentially upregulated with strong PPI interactions (periostin and 7 collagen-related proteins). Increases in periostin moderately correlated with increases in a synovial fluid biomarker of type II cartilage degradation (ρ = 0.51, p = 0.06). CONCLUSION: Pro-inflammatory pathways and periostin were upregulated after ACLR. Periostin demonstrated strong network connections with markers of collagen breakdown, and future work is needed to determine whether periostin may offer a biomarker of early cartilage degradation after ACLR and/or play an active role in early post-traumatic osteoarthritis (PTOA) progression.
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Lesões do Ligamento Cruzado Anterior , Reconstrução do Ligamento Cruzado Anterior , Cartilagem Articular , Adulto , Feminino , Humanos , Masculino , Adulto Jovem , Lesões do Ligamento Cruzado Anterior/cirurgia , Lesões do Ligamento Cruzado Anterior/metabolismo , Lesões do Ligamento Cruzado Anterior/patologia , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Colágeno/metabolismo , Articulação do Joelho/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espectrometria de Massas em TandemRESUMO
Understanding the molecular mechanisms underlying osteoclast differentiation provides insights into bone loss and even osteoporosis. The specific mechanistic actions of cullin 4A (CUL4A) in osteoclast differentiation and resultant osteoporosis is poorly explored. We developed a mouse model of osteoporosis using bilateral ovariectomy (OVX) and examined CUL4A expression. It was noted that CUL4A expression was increased in the bone marrow of OVX mice. Overexpression of CUL4A promoted osteoclast differentiation, and knockdown of CUL4A alleviated osteoporosis symptoms of OVX mice. Bioinformatic analyses were applied to identify the downstream target genes of microRNA-340-5p (miR-340-5p), followed by interaction analysis. The bone marrow macrophages (BMMs) were isolated from femur of OVX mice, which were transfected with different plasmids to alter the expression of CUL4A, Zinc finer E-box binding homeobox 1 (ZEB1), miR-340-5p, and Toll-like receptor 4 (TLR4). ChIP assay was performed to detect enrichment of ZEB1 promoter by H3K4me3 antibody in BMMs. ZEB1 was overexpressed in the bone marrow of OVX mice. Overexpression of CUL4A mediated H3K4me3 methylation to increase ZEB1 expression, thus promoting osteoclast differentiation. Meanwhile, ZEB1 could inhibit miR-340-5p expression and upregulate HMGB1 to induce osteoclast differentiation. Overexpressed ZEB1 activated the TLR4 pathway by regulating the miR-340-5p/HMGB1 axis to induce osteoclast differentiation, thus promoting the development of osteoporosis. Overall, E3 ubiquitin ligase CUL4A can upregulate ZEB1 to repress miR-340-5p expression, leading to HMGB1 upregulation and the TLR4 pathway activation, which promotes osteoclast differentiation and the development of osteoporosis.
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Proteína HMGB1 , MicroRNAs , Osteoporose , Feminino , Camundongos , Animais , Receptor 4 Toll-Like/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Regulação para Cima , Osteoporose/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular , Osteogênese/genéticaRESUMO
Atmospheric turbulence reduces the detection accuracy of orbital angular momentum (OAM) modes, which affects the performance of OAM optical communication. In this paper, we propose a method based on interferometry and a residual network (ResNet) to detect the OAM modes of ring Airy Gaussian vortex beams (RAGVBs) disturbed by atmospheric turbulence. The RAGVBs first interfere with spherical waves to obtain the sign features of the OAM modes, and then ResNet is employed to recognize OAM modes from the interferograms. The results demonstrate that the detection accuracy is higher than that of the OAM spectrum method under different turbulence strengths. The detection accuracy can even reach over 99% under strong fluctuations. Our research provides a reference for improving the performance of OAM optical communication through atmospheric turbulence.
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OBJECTIVE: To analyze the incidence, indications, risk factors and pregnancy outcomes of postpartum hemorrhage resulting in peripartum hysterectomy (PH). METHODS: We retrospectively reviewed patients with postpartum hemorrhage requiring surgical procedures at ≥ 28 weeks of gestation from January 1, 2013 to December 31, 2022 at a tertiary hospital in Shanghai, China. The patients were divided into a PH group and a non-PH group. Maternal clinical characteristics, the management of postpartum hemorrhage, pregnancy outcomes were compared between groups. Logistic regression was used to analyze the correlations between risk factors and PH. RESULTS: The incidence of hysterectomy was 0.2/1000 deliveries (31/150194). The variables significantly associated with PH were placenta previa with placenta increta/percreta (OR36.26), uterine rupture (OR266.16) and an estimated blood loss ≥ 3513 mL (OR431.11). The proportion of cases involving hemorrhagic shock, disseminated intravascular coagulation, bladder injury, neonatal severe asphyxia, neonatal death and hypoxic-ischemic encephalopathy were significantly higher in the PH group (P < 0.05). CONCLUSION: The most common indications of PH were placental pathology. Efforts should be made to reduce the rate of cesarean deliveries and uterine curettage to lower the probability of abnormal placental invasion and appropriate medical indications for trial of labor after cesarean should be strictly followed to avoid the risk of uterine rupture.
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Inflammation plays a central role in the development of heart failure. Prostaglandin E2 (PGE2) is a key mediator of the inflammatory process in the cardiovascular system. However, the role of PGE2 in heart failure is complex and controversial. A recent report suggested that PGE2 inhibits acute ß adrenergic receptor (ß-AR) stimulation-enhanced cardiac contractility. The aim of this study was to characterize the influence of PGE2 on chronic ß-AR stimulation-induced heart failure. Male C57BL/6 J mice received isoproterenol (ISO) or vehicle for 4 weeks. PGE2 significantly reversed ISO-induced cardiac contractile dysfunction and remodeling. Mechanically, ventricular myocytes were found to be an important source of TGF-ß1 in ISO-model and PGE2 ablated TGF-ß1 synthesis in cardiomyocytes through inhibition of ß-AR activated PKA-CREB signaling. Furthermore, PGE2 significantly suppressed TGF-ß1-GRK2 crosstalk-induced pro-hypertrophy and pro-fibrotic signaling in cardiomyocytes and cardiac fibroblasts, respectively. Pharmacological inhibition of GRK2 also attenuated contractile dysfunction and cardiac hypertrophy and fibrosis in ISO-model. These studies elucidate a novel mechanism by which PGE2 reduces TGF-ß1 synthesis and its downstream signaling in heart failure and identify PGE2 or TGF-ß1-GRK2 crosstalk as plausible therapeutic targets for preventing or treating heart failure induced by chronic ß-AR stimulation.
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Cardiopatias , Insuficiência Cardíaca , Camundongos , Animais , Masculino , Miócitos Cardíacos/patologia , Dinoprostona , Fator de Crescimento Transformador beta1 , Camundongos Endogâmicos C57BL , Isoproterenol/farmacologia , Fibrose , Cardiopatias/patologiaRESUMO
Protein post-translational modifications serve to regulate a broad range of cellular functions including signal transduction, transcription, and metabolism. Protein lysine residues undergo many post-translational acylations and are regulated by a range of enzymes, such as histone acetyl transferases (HATs) and histone deacetylases (HDACs). KAT2A, well characterized as a lysine acetyltransferase for both histone and nonhistone substrates, has been reported to tolerate additional acyl-CoA substrates, such as succinyl-CoA, and shows nonacetyl transferase activity in specific biological contexts. In this work, we investigate the acyl-CoA substrate preference of KAT2A and attempt to determine whether and to what extent additional acyl-CoA substrates may be utilized by KAT2A in a cellular context. We show that while KAT2A can bind and utilize malonyl-CoA, its activity with succinyl-CoA or glutaryl-CoA is very weak, and acetylation is still the most efficient activity for KAT2A in vitro and in cells.
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Histonas , Lisina Acetiltransferases , Acetilação , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Lisina Acetiltransferases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.
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Sangue , Proteômica , Fluxo de Trabalho , Biomarcadores/sangue , Humanos , Peptídeos , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
In an imaging system, resolution and signal-to-noise ratio (SNR) are two important indexes to characterize imaging quality. Ghost imaging is a novel imaging method whose imaging resolution and SNR are affected by the speckle size. In this paper, the relation between speckle size and resolution as well as that between speckle size and SNR in the GI system is analyzed in detail. It is shown that the critical resolution, resolvable minimum-separation between two adjacent objects, is approximately equal to the speckle size (speckle diameter). There exists an optimum SNR when the speckle size is larger than the object size. Based on our conclusion, we propose a scheme to enhance the critical resolution of the GI system by using a vortex beam, and the enhancement ability under different topological charges is clearly presented, which can be quantized by a simple formula.
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Emerging evidence has shown that nonalcoholic fatty liver disease (NAFLD) may be both a consequence and a cause of hypertension. Recent studies have demonstrated that phosphodiesterase 4 (PDE4)-cAMP signaling represents a pathway relevant to the pathophysiology of metabolic disorders. This study aims to investigate the impact and the underlying mechanism of PDE4 in the pathogenesis of NAFLD and its associated hypertension. Here we demonstrated that high-fat-diet (HFD) fed mice developed NAFLD and hypertension, with an associated increase in hepatic PDE4D expression, which can be prevented and even reversed by PDE4 inhibitor roflumilast. Furthermore, we demonstrated that hepatic overexpression of PDE4D drove significant hepatic steatosis and elevation of blood pressure. Mechanistically, PDE4D activated fatty acid translocase CD36 signaling which facilitates hepatic lipid deposition, resulting in TGF-ß1 production by hepatocytes and excessive TGF-ß1 signaling in vessels and consequent hypertension. Specific silencing of TGF-ß1 in hepatocytes by siRNA using poly (ß-amino ester) nanoparticles significantly normalized hepatic PDE4D overexpression-activated TGF-ß1 signaling in vessels and hypertension. Together, the conclusions indicated that PDE4D plays an important role in the pathogenesis of NAFLD and associated hypertension via activation of CD36-TGF-ß1 signaling in the liver. PDE4 inhibitor such as roflumilast, which is clinically approved for chronic obstructive pulmonary disease (COPD) treatment, has the potential to be used as a preventive or therapeutic drug against NAFLD and associated hypertension in the future.
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Aminopiridinas/uso terapêutico , Benzamidas/uso terapêutico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Hipertensão/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Inibidores da Fosfodiesterase 4/uso terapêutico , Aminopiridinas/farmacologia , Animais , Aorta/citologia , Becaplermina/farmacologia , Benzamidas/farmacologia , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Ciclopropanos/farmacologia , Ciclopropanos/uso terapêutico , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/fisiologia , Nanopartículas/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Polímeros/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Sirtuína 1/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Idiopathic basal ganglia calcification (IBGC) is a genetic disorder of the nervous system commonly known as Fahr disease. IBGC patients with a genetic background are considered to have primary familial brain calcification (PFBC), also known as familial basal ganglia calcification (FBGC), or familial Fahr disease. It is a rare degenerative neurological disorder characterized by extensive bilateral basal ganglia calcification that can lead to a range of extrapyramidal symptoms and neuropsychiatric manifestations. Studies have suggested that more than 50 variants of SLC20A2 gene mutations account for approximately 50% of IBGC cases. There is a wide spectrum of mutation types, including frameshift, nonsense, and splice site mutations in addition to deletion and missense mutations. Here we report a case of familial basal ganglia calcification caused by a frameshift mutation in the SLC20A2 gene. We identified a heterozygous mutation in the SLC20A2 gene, c.1097delG (p.G366fs*89). To our knowledge, this mutation site has not been reported before. CASE PRESENTATION: A 57-year-old male patient was admitted to the hospital with "unstable walking and involuntary movements between the eyes and eyebrows for 6 months". Based on the patient's family history, symmetrical calcification foci in the bilateral caudate nucleus head, thalamus, cerebellum and parietal lobe indicated by head CT, and gene test results, the diagnosis of familial Fahr disease caused by mutations in the SLC20A2 gene, c.1097delG p.G366fs*89) was confirmed. CONCLUSION: For the first time, we identified c.1097delG (p.G366fs*89) as a frameshift mutation in the IBGC family. This frameshift mutation caused the condition in this family of patients. This mutation not only broadens the range of known SLC20A2 mutations but also aids in the genetic diagnosis of IBGC.