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1.
Small ; : e2103374, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34636153

RESUMO

Multilevel luminescence materials have aroused wide attention for their advanced anti-counterfeiting abilities. However, various complicated stimuli factors involved in multilevel luminescence anti-counterfeiting (MlLA) limit the practical applications of such materials. Herein, carbon dots (CDs) are in situ introduced into Eu-substituted AlPO4 -5 zeolite (named CDs@EuAPO-5) via a solvent-free thermal crystallization method, which exhibits triple emissions including pink fluorescence mainly associated with Eu3+ in the zeolite framework, blue fluorescence and green room temperature phosphorescence (RTP) associated with CDs. CDs are uniformly embedded in the EuAPO-5 zeolite matrix. Such composite displays excellent photo-, thermo-, and solvent resistance, as well as long-term storage-stability. Moreover, the triple emissions of the composite only need two kinds of common excitation lights to trigger, without involving other complicated stimuli. A triple-level luminescence anti-counterfeiting (TlLA) label has been built, realizing facile, quick, and advanced luminescence anti-counterfeiting that is hard to copy.

2.
Bioorg Chem ; 116: 105382, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34598087

RESUMO

The bone morphogenetic protein-2 (BMP-2) is an essential regulator of bone formation and remodeling, which has also been implicated in the pathogenesis of osteoarthritis and its closely related chondrocyte senescence. The BMP-2 uses a conformational wrist epitope and a linear knuckle epitope to interact with type-I (BMPR-I) and type-II (BMPR-II) receptors, respectively. Previously, the knuckle epitope has been intensely studied, but the wrist epitope still remains largely unexplored due to its discontinuous nature. In the present work, the intermolecular interaction of BMP-2 with BMPR-I was investigated systematically at structural, energetic and dynamic levels. Three discrete hotspots that represent the key BMPR-I recognition sites of BMP-2 were identified; they are spatially dispersed over the two monomers of BMP-2 dimer and totally account for 83.5 % binding potency of BMP-2 to BMPR-I (hotspot 1: residues 49-70 in monomer 1; hotspot 2: residues 24-31 in monomer 2; hotspot 3: residues 88-107 in monomer 2). Therefore, we defined the three discrete hotspot sites as the core region of wrist epitope; their contribution to the binding increases in the order: hotspot 2 < hotspot 3 < hotspot 1. We demonstrated that the primary hotspot 1 site has a native U-shaped conformation in the full-length BMP-2 protein context, but it cannot maintain in the native conformation when split from the context to obtain a free hotspot-1 peptide, thus largely impairing its binding potency to BMPR-I. We further employed disulfide-bonded cyclization and head-to-tail cyclization to constrain the peptide conformation, and found that only the former can effectively constrain the peptide into native conformation, thus considerably improving its binding affinity to BMPR-I, whereas the latter totally disorders the native conformation, thus rendering the peptide as a full nonbinder of BMPR-I.

3.
J Zhejiang Univ Sci B ; 22(9): 746-756, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34514754

RESUMO

Clinically, a large proportion of glaucoma patients undergo repeated intraocular pressure (IOP) spike (Spike IOP) attacks during their sleep, which may facilitate retinopathy. In this study, we established a mouse model of repeated transient Spike IOP to investigate the direct damage to the retina following Spike IOP attacks, and elucidated the underlying molecular mechanism. We analyzed the changes in the number of retinal ganglion cells (RGCs) via immunofluorescence. Thereafter, we detected retinal cell apoptosis via terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining, and performed RNA sequencing (RNA-seq) to reveal the underlying molecular mechanism. Finally, we validated the expression of key molecules in the endoplasmic reticulum (ER) stress pathway using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Results revealed a time-dependent RGC loss in Spike IOP, evidenced by a reduction in the number of Brn3a-positive RGCs in experimental eyes following a 7-d continuous treatment with Spike IOP. In addition, TUNEL staining indicated that apoptosis of retinal cells started in the outer nuclear layer (ONL), and then spread to the ganglion cell layer (GCL) with time. RNA-seq analysis revealed that ER stress might be involved in Spike IOP-induced retinal injury. This result was corroborated by western blot, which revealed upregulation of ER stress-related proteins including binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78), phosphorylated inositol-requiring enzyme 1 (p-IRE1), unspliced X-box-binding protein 1 (XBP1-u), spliced X-box-binding protein 1 (XBP1-s), phosphorylated c-Jun N-terminal kinase (p-JNK), C/EBP-homologous protein (CHOP), and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax). These findings indicate that repeated IOP transients are detrimental to the retina, while ER stress plays an important role in retinal cell apoptosis in this situation. Notably, repeated Spike IOP among glaucoma patients is a crucial factor for progressive retinopathy.

4.
J Mol Endocrinol ; 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34582356

RESUMO

Increased inflammatory responses is one of the major characteristics of osteonecrosis of the femoral head (ONFH). We aimed to investigate the function of bone morphogenetic protein 2 (BMP-2)/interleukin (IL)-34 axis in the inflammatory responses of ONFH. The systemic and local expression of BMPs in ONFH patients were detected by qRT-PCR and ELISA. In vitro osteoclast differentiation and ONFH mouse models, induced by 20 mg/kg methylprednisolone through intramuscular injection, were established using wild type and BMP-2-/- mice to explore the regulatory role of BMP-2 in pro-inflammatory responses and bone defects of ONFH. IL-34 expression and function were examined in vitro and in vivo through qRT-PCR, TRAP staining, and gene knockout. The systemic and local expression of BMPs were elevated in ONFH patients. BMP-2 reduced the production of pro-inflammatory cytokines and inhibited the differentiation of osteoclasts. Mechanistically, BMP-2 inhibited osteoclasts formation through suppressing IL-34 expression, and then promoted bone repair and alleviated ONFH. In conclusion, our study reveals that BMP-2 inhibits inflammatory responses and osteoclast formation through down-regulating IL-34.

5.
ACS Infect Dis ; 7(8): 2565-2582, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34346692

RESUMO

Biofilms, structured communities of bacterial cells embedded in a self-produced extracellular matrix (ECM) which consists of proteins, polysaccharide intercellular adhesins (PIAs), and extracellular DNA (eDNA), play a key role in clinical infections and are associated with an increased morbidity and mortality by protecting the embedded bacteria against drug and immune response. The high levels of antibiotic tolerance render classical antibiotic therapies impractical for biofilm-related infections. Thus, novel drugs and strategies are required to reduce biofilm tolerance and eliminate biofilm-protected bacteria. Here, we showed that gallium, an iron mimetic metal, can lead to nutritional iron starvation and act as dispersal agent triggering the reconstruction and dispersion of mature methicillin-resistant Staphylococcus aureus (MRSA) biofilms in an eDNA-dependent manner. The extracellular matrix, along with the integral bacteria themselves, establishes the integrated three-dimensional structure of the mature biofilm. The structures and compositions of gallium-treated mature biofilms differed from those of natural or antibiotic-survived mature biofilms but were similar to those of immature biofilms. Similar to immature biofilms, gallium-treated biofilms had lower levels of antibiotic tolerance, and our in vitro tests showed that treatment with gallium agents reduced the antibiotic tolerance of mature MRSA biofilms. Thus, the sequential administration of gallium agents (gallium porphyrin and gallium nitrate) and relatively low concentrations of vancomycin (16 mg/L) effectively eliminated mature MRSA biofilms and eradicated biofilm-enclosed bacteria within 1 week. Our results suggested that gallium agents may represent a potential treatment for refractory biofilm-related infections, such as prosthetic joint infections (PJI) and osteomyelitis, and provide a novel basis for future biofilm treatments based on the disruption of normal biofilm-development processes.


Assuntos
Gálio , Staphylococcus aureus Resistente à Meticilina , Porfirinas , Biofilmes , DNA , Gálio/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Vancomicina/farmacologia
6.
Food Res Int ; 147: 110493, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399489

RESUMO

Pungency is increasingly being recognized as an important factor of overall sensory quality, palatability, and consumer preference of distilled spirits. The characterization of pungency is necessary to evaluate the potential sensory quality of distilled spirits. In this study, the temporal profiles of pungency of Baijiu with different aging times were evaluated using time-intensity (TI) and temporal dominance of sensations (TDS) methods, considering both pungency intensity and pungency sub-qualities. TI results indicated significant differences in release rate of pungency during Baijiu consumption. Compared to young Baijiu, old Baijiu tend to show higher release rate of pungency, the areas under the curve and duration of pungency were significantly decreased in old Baijiu. The TDS results showed significant differences in the combination of dominant sub-qualities, as well as in the maximum dominance rates and the dominant duration of sub-qualities among Baijiu. The young Baijiu were mainly characterized by the dominant sub-qualities of "burning" and "numbing", whereas for old Baijiu, "burning", "prickle", and "drying" were dominant. The application of TI and TDS provided dynamic and temporal profiles of pungency to fully characterize pungency differences of distilled beverages.


Assuntos
Sensação , Paladar , Bebidas , Comportamento do Consumidor
7.
J Cell Physiol ; 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34378805

RESUMO

Merlin is known as a tumor suppressor, while its role in osteomyelitis remains unclear. This study aimed to investigate the role of Merlin in Staphylococcus aureus-induced osteomyelitis and its underlying mechanisms. S. aureus-induced osteomyelitis mouse model was established in Merlinfl/fl Lyz2cre/+ and Merlinfl/fl Lyz2+/+ mice. Bone marrow-derived macrophages (BMDMs) were isolated and stimulated by lipopolysaccharide (LPS). Bioassays, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot analysis, and enzyme-linked immunosorbent assays, were conducted to determine the levels of target genes or proteins. Immunoprecipitation was applied to determine the interactions between proteins. DCAF1fl/fl mice were further crossed with Lyz2-Cre mice to establish myeloid cell conditional knockout mice (DCAF1fl/fl Lyz2cre/+ ). It was found that the level of Merlin was elevated in patients with osteomyelitis and S. aureus-infected BMDMs. Merlin deficiency in macrophages suppressed the production of inflammatory cytokines and ameliorated the symptoms of osteomyelitis induced by S. aureus. Merlin deficiency in macrophages also suppressed the production of proinflammatory cytokines in BMDMs induced by LPS. The inhibitory effects of Merlin deficiency on the inflammatory response were associated with DDB1-Cul4-associated factor 1 (DCAF1). In summary, Merlin deficiency ameliorates S. aureus-induced osteomyelitis through the regulation of DCAF1.

8.
Artigo em Inglês | MEDLINE | ID: mdl-34432433

RESUMO

Currently, light-based three-dimensional (3D) printing with submicron features is mainly developed based on photosensitive polymers or inorganic-polymer composite materials. To eliminate polymer/organic additives, a strategy for direct 3D assembly and printing of metallic nanocrystals without additives is presented. Ultrafast laser with intensity in the range of 1 × 1010 to 1 × 1012 W/cm2 is used to nonequilibrium heat nanocrystals and induce ligand transformation, which triggers the spontaneous fusion and localized assembly of nanocrystals. The process is due to the operation of hot electrons as confirmed by a strong dependence of the printing rate on laser pulse duration varied in the range of electron-phonon relaxation time. Using the developed laser-induced ligand transformation (LILT) process, direct printing of 3D metallic structures at micro and submicron scales is demonstrated. Facile integration with other microscale additive manufacturing for printing 3D devices containing multiscale features is also demonstrated.

9.
Sex Med ; 9(4): 100403, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34298329

RESUMO

INTRODUCTION: Testosterone can improve glucose metabolism through multiple cellular mechanisms. However, it remains unclear as to whether hypogonadal men with type 2 diabetes mellitus (T2DM) can benefit from testosterone replacement therapy (TRT). AIMS: To assess the relative effect of TRT on glycolipid metabolism among hypogonadal men with T2DM. METHODS: Electronic literature searches of the Cochrane Library, PubMed, MEDLINE, and EMBASE databases were conducted, up to the end of October 2020. Only studies that used randomized controlled trials (RCTs) were included in our systematic review. Main outcome measures From these studies, we extracted certain outcomes including changes in insulin resistance, glucose metabolism, and lipid parameters. RESULTS: There were a total of 8 studies that met our criteria. Four of these studies either did not have a consistent treatment strategy, or the control groups used untreated patients rather than patients that had been given a placebo. Thus, results from these four studies contributed to the variability in treatment outcomes. In four of the examined RCTs, there was no change in either the dose or the type of antidiabetic medication prescribed. Based on the homeostatic model assessment of insulin resistance, the pooled WMD was -0.34, 95% confidence interval (CI; -1.02, 0.34), P = .33; For fasting plasma glucose, the pooled WMD was -0.27, 95% CI (-1.02, 0.48), P = .48, the pooled WMD for HbA1c% was -0.00, 95% CI (-1.08, 1.08), P = 1.00. CONCLUSIONS: Although certain RCTs showed that TRT improved insulin resistance and glycolipid metabolism when compared with the placebo or untreated control groups, these findings may partly be due to changes in antidiabetic therapy during the course of the study. In the current meta-analysis, analyses showed that TRT did not significantly improve insulin resistance or glycolipid metabolism. Future studies need to be rigorous in design and delivery, and comprehensive descriptions of all aspects of their methods should be included to further enable a more accurate appraisal and interpretation of the results. Yu X, Wei Z, Liu Y, et al. Effects of Testosterone Replacement Therapy on Glycolipid Metabolism Among Hypogonadal Men with T2DM: A Meta-Analysis And System Review Of Randomized Controlled Trials. Sex Med 2021;XX:XX-XXX.

10.
Microb Cell Fact ; 20(1): 131, 2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34247591

RESUMO

BACKGROUND: Thermotolerant yeast has outstanding potential in industrial applications. Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins. RESULTS: Herein, we obtained a thermotolerant K. phaffii mutant G14 by mutagenesis and adaptive evolution. G14 exhibited oxidative and thermal stress cross-tolerance and high heterologous protein production efficiency. The reactive oxygen species (ROS) level and lipid peroxidation in G14 were reduced compared to the parent. Oxidative stress response (OSR) and heat shock response (HSR) are two major responses to thermal stress, but the activation of them was different in G14 and its parent. Compared with the parent, G14 acquired the better performance owing to its stronger OSR. Peroxisomes, as the main cellular site for cellular ROS generation and detoxification, had larger volume in G14 than the parent. And, the peroxisomal catalase activity and expression level in G14 was also higher than that of the parent. Excitingly, the gene knockdown of CAT encoding peroxisomal catalase by dCas9 severely reduced the oxidative and thermal stress cross-tolerance of G14. These results suggested that the augmented OSR was responsible for the oxidative and thermal stress cross-tolerance of G14. Nevertheless, OSR was not strong enough to protect the parent from thermal stress, even when HSR was initiated. Therefore, the parent cannot recover, thereby inducing the autophagy pathway and resulting in severe cell death. CONCLUSIONS: Our findings indicate the importance of peroxisome and the significance of redox balance in thermotolerance of yeasts.

11.
Biomolecules ; 11(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200257

RESUMO

Water content is an important factor in lipase-catalyzed reactions in organic media but is frequently ignored in the study of lipases by molecular dynamics (MD) simulation. In this study, Candida antarctica lipase B, Candida rugosa lipase and Rhizopus chinensis lipase were used as research models to explore the mechanisms of lipase in micro-aqueous organic solvent (MAOS) media. MD simulations indicated that lipases in MAOS systems showed unique conformations distinguished from those seen in non-aqueous organic solvent systems. The position of water molecules aggregated on the protein surface in MAOS media is the major determinant of the unique conformations of lipases and particularly impacts the distribution of hydrophilic and hydrophobic amino acids on the lipase surface. Additionally, two maxima were observed in the water-lipase radial distribution function in MAOS systems, implying the formation of two water shells around lipase in these systems. The energy landscapes of lipases along solvent accessible areas of catalytic residues and the minimum energy path indicated the dynamic open states of lipases in MAOS systems differ from those in other solvent environments. This study confirmed the necessity of considering the influence of the microenvironment on MD simulations of lipase-catalyzed reactions in organic media.


Assuntos
Microambiente Celular/fisiologia , Biologia Computacional/métodos , Lipase/química , Simulação de Dinâmica Molecular , Água/química , Basidiomycota/química , Basidiomycota/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipase/análise , Lipase/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomycetales/química , Saccharomycetales/metabolismo , Água/metabolismo
12.
Vis Neurosci ; 38: E005, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33934732

RESUMO

Previously, we reported the myelin regulatory factor (MYRF) as a candidate gene for nanophthalmos. We have also produced Myrf knockdown (Myrf+/-) mouse strain to investigate the cellular and molecular phenotypes of reduced MYRF expression in the retina. Myrf+/- mouse strain was generated using the CRISPR/Cas9 system. Optomotor response system, electroretinogram (ERG), spectral-domain optical coherence tomography (SD-OCT), histology, and immunohistochemistry were performed to evaluate retinal spatial vision, electrophysiological function, retinal thickness, and pathological changes in cone or rod photoreceptors, respectively. RNA sequencing (RNA-seq) was performed to investigate the underlying molecular mechanism linking Myrf deficiency with photoreceptor defects. The genotype and phenotype of CRISPR/Cas9-induced Myrf+/- mice and their offspring were comprehensively investigated. Photoreceptor defects were detected in the retinas of Myrf+/- mice. Visual acuity and ERG responses were decreased in Myrf+/- mice compared with the control mice (Myrf+/+). The loss of cone and rod neurons was proportional to the decreased outer nuclear layer (ONL) thickness. Moreover, RNA-seq revealed that phototransduction and estrogen signaling pathways played important roles in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Myrf+/- mouse strain provides a good model to investigate the function of the MYRF gene. Photoreceptor defects with impaired functions of spatial vision and retinal electrophysiology indicate an important role played by MYRF in retinal development. Alterations in phototransduction and estrogen signaling pathways play important roles in linking Myrf deficiency with retinal photoreceptor defects.

13.
Int Ophthalmol ; 41(9): 3087-3097, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33905050

RESUMO

PURPOSE: To describe a modified surgical approach with anterior vitrectomy, phacoemulsification (phaco) cataract extraction and irido-zonulo-hyaloid-vitrectomy (IZHV) in protracted acute angle closure crisis (AACC). PATIENTS AND METHODS: Non-comparative, retrospective case series including 21 eyes in 19 consecutive cases of protracted AACC, which persists for at least 7 days despite maximal medical and laser therapies, were included in this study. All patients underwent a modified surgical procedure with anterior vitrectomy, phaco cataract extraction, IOL implantation, goniosynechialysis (GSL) and IZHV, using modest phaco dynamic parameters with intraocular pressure (IOP) set at 30 mmHg through the procedure using Centurion® Vision System equipped with active fluidics while the anterior vitrectomy was set at 4000 or 5000 rpm. IOP and anterior chamber space were maintained through the procedure using ophthalmic viscosurgical device (OVD) injected through paracentesis whenever the Phaco or I/A probe was withdrawn from within the anterior chamber. Medical history, visual acuity (VA), IOP and anterior and posterior segment findings were recorded and compared before and after surgical treatment. RESULTS: The average age of all patients was 60.05 years old, while the average period of persistent AACC was 20.05 days. Preoperatively, the average IOP of all included eyes was 44.40 ± 8.42 mmHg despite maximal topical and systemic anti-glaucoma medications and/or laser surgeries, while the average VA was 1.46 ± 0.88 (log MAR). Postoperatively, IOP was well controlled in all patients with an average IOP at 12.06 ± 3.07 mmHg without any anti-glaucoma medications at follow-ups, which was decreased significantly from that in preoperative measurements (P < 0.001). Visual acuity was improved significantly at final follow-up with an average postoperative VA at 0.74 ± 0.77 (log MAR, P < 0.001). Anterior segment inflammation was surprisingly mild with no or minimal inflammatory cells or exudates. Anterior segment configuration was resolved in all the cases. There was no recurrent IOP spike, anterior chamber shallowing or severe complications during an average follow-up of 5.38 months (ranging from 3 to 6 months). CONCLUSIONS: Protracted AACC is a complex situation while a modified surgical strategy of anterior vitrectomy, phaco cataract extraction and IZHV provides a safe and efficient solution.


Assuntos
Extração de Catarata , Catarata , Glaucoma de Ângulo Fechado , Facoemulsificação , Catarata/complicações , Glaucoma de Ângulo Fechado/cirurgia , Humanos , Pressão Intraocular , Pessoa de Meia-Idade , Estudos Retrospectivos , Vitrectomia
14.
Int J Biol Sci ; 17(5): 1353-1360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33867851

RESUMO

Previous studies have described that NF-κB signaling mediated by NFκB-inducing kinase (NIK) plays a critical role of the differentiation of osteoclasts. We aim to explore the role of IKKe in methylprednisolone -induced osteonecrosis of the femoral head (ONFH). Methylprednisolone-induced ONFH mice model was successfully established, and subjected to micro computed tomography to detect the femoral head image of the mice. Bone marrow cells from experimental mice were collected and cultured. qPCR and immunoblot were performed to examine the possible signal pathways of IKKe involvement, and osteoclast-related gene expressions in IKKe+/+ and IKKe-/- cells in vitro and in vivo were examined. It was found that the levels of IKKe decreased in ONFH patients, and IKKe interacted with NIK in the NF-κB signal pathway to suppress osteoclasts via inhibiting the transcription of NIK. Furthermore, IKKe knockout promoted the osteoclastogenesis in mice model. Finally, IKKe knockout suppressed methylprednisolone-induced ONFH and pro-inflammatory responses in mice model. Our findings show a mechanism of IKKe inhibition of the progression of methylprednisolone-induced ONFH via the NIK/NF-κB pathway.

15.
J Agric Food Chem ; 69(14): 4263-4275, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33797235

RESUMO

Most fungal lipases contain a propeptide, which is very important for their function and substrate selectivity. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of the propeptide of the lipase. Conventional molecular dynamics (MD) and metadynamics simulations were used to explore the mechanism by which the propeptide affects the activity of the lipase, which was subsequently verified by mutation experiments. MD simulations indicated that the propeptide had an inhibitory effect on the lid movement of RCL and found a key region (Val5-Thr10) on the propeptide. Subsequently, site-directed mutations were created in this region. The mutations enhanced the lipase catalytic efficiency to 700% and showed the potential for the propeptide to shift the substrate specificity of RCL. The specificity and activity of RCL mutants also had similar trends to wild-type RCL toward triglycerides with varying chain lengths. The mutual corroboration of simulation and site-directed mutagenesis results revealed the vital role of the key propeptide region in the catalytic activity and substrate specificity of the lipase.


Assuntos
Lipase , Rhizopus , Lipase/genética , Lipase/metabolismo , Simulação de Dinâmica Molecular , Rhizopus/genética , Rhizopus/metabolismo , Especificidade por Substrato
16.
Invest Ophthalmol Vis Sci ; 62(3): 1, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33646289

RESUMO

Purpose: Patients with nanophthalmos who undergo intraocular surgery often present with abnormal ciliary zonules. In a previous study, we reported mutation in MYRF that is implicated in the pathogenesis of nanophthalmos. The aim of this study was to model the mutation in mice to explore the role of MYRF on zonule structure and its major molecular composition, including FBN1 and FBN2. Methods: Human MYRF nanophthalmos frameshift mutation was generated in mouse using the CRISPR-Cas9 system. PCR and Sanger sequencing were used for genotype analysis of the mice model. Anterior chamber depth (ACD) was measured using hematoxylin and eosin-stained histology samples. Morphologic analysis of ciliary zonules was carried out using silver staining and immunofluorescence. Transcript and protein expression levels of MYRF, FBN1, and FBN2 in ciliary bodies were quantified using quantitative real-time PCR (qRT-PCR) and Western blot. Results: A nanophthalmos frameshift mutation (c.789delC, p.N264fs) of MYRF in mice showed ocular phenotypes similar to those reported in patients with nanophthalmos. ACD was reduced in MYRF mutant mice (MYRFmut/+) compared with that in littermate control mice (MYRF+/+). In addition, the morphology of ciliary zonules showed reduced zonular fiber density and detectable structural dehiscence of zonular fibers. Furthermore, qRT-PCR analysis and Western blot showed a significant decrease in mRNA expression levels of MYRF, FBN1, and FBN2 in MYRFmut/+ mice. Conclusions: Changes in the structure and major molecular composition of ciliary zonules accompanied with shallowing anterior chamber were detected in MYRFmut/+ mice. Therefore, MYRF mutant mice strain is a useful model for exploring pathogenesis of zonulopathy, which is almost elusive for basic researches due to lack of appropriate animal models.


Assuntos
Corpo Ciliar/patologia , Mutação da Fase de Leitura , Glaucoma de Ângulo Fechado/genética , Hiperopia/genética , Ligamentos/patologia , Microftalmia/genética , Fatores de Transcrição/genética , Doenças da Úvea/genética , Animais , Câmara Anterior/patologia , Western Blotting , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Feminino , Fibrilina-1/genética , Fibrilina-2/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Genotipagem , Humanos , Imuno-Histoquímica , Ligamentos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Doenças da Úvea/metabolismo , Doenças da Úvea/patologia
17.
BMC Geriatr ; 21(1): 175, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33691628

RESUMO

BACKGROUND: Osteoarthritis (OA) is a commonly known prevalent joint disease, with limited therapeutic methods. This study aimed to investigate the functions of miRNA-132 (miR-132) in the modulation of PTEN/PI3K/AKT signaling pathway in the development and progression of osteoarthritis. METHODS: Eight male osteoarthritic patients and eight healthy males were recruited. Male Sprague Dawley (SD) rats were used for cellular experiments. QRT-PCR was performed to detect the expression levels of miR-132, PTEN, PI3K and AKT. MTT assay and apoptosis assay were carried out to measure the cell proliferation rate and cell apoptosis rate, respectively. Western blotting was employed to detect the protein expression of related RNAs and inflammatory factors. RESULTS: In osteoarthritic patients, the expression level of miR-132 was decreased, compared with that in the normal group. Over-expression of miR-132 elevated cell proliferation and decreased apoptosis of chondrocytes. Down-regulation of miR-132 decreased cell proliferation and induced apoptosis in chondrocytes. In addition, down-regulation of miR-132 promoted the expression of Bax protein and activated caspase-3/9, increased inflammation divisors. PTEN inhibitor antagonized the destructive effect of the miR-132 inhibitor on cell proliferation of chondrocytes. PI3K inhibitor increased the destructive effect of the miR-132 inhibitor on osteoarthritis. CONCLUSION: In conclusion, miR-132 is an important regulator of osteoarthritis in chondrocytes through the PTEN/PI3K/AKT signaling pathway.


Assuntos
MicroRNAs , Osteoartrite , Animais , Proliferação de Células , Humanos , Masculino , MicroRNAs/genética , Osteoartrite/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
18.
Int Immunopharmacol ; 93: 107345, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33563553

RESUMO

Osteonecrosis of the femoral head (ON-FH) is a common complication of steroid use. Pro-inflammatory macrophages play a crucial role in the apoptosis of osteocytes. The objective of the study was to evaluate a plant extract astragaloside IV (AS-IV) in treating ON-FN. Bone-marrow-derived macrophages (BMDMs) were treated with lipopolysaccharides (LPS), IFN-γ or IL-4 to induce M1 and M2-like phenotypes. Quantitative real-time PCR and Western blot were used to examine M1 and M2 phenotypic markers. Flow cytometry was used to analyze MHC II, CD206, F4/80, and CD11b levels and cell apoptosis. Glucocorticoid was used to induce ON-FN in mice. TNF-α and IL-1ß levels in femoral head were determined using enzyme-linked immunosorbent assay. AS-IV repolarized macrophages from M1 to M2 phenotypes. Culture medium from AS-IV treated M1 macrophages induced less cell apoptosis osteocytes compared to that from untreated M1 macrophages. In ON-FH mice, the ratio of M1 macrophages was decreased in the femoral head by AS-IV, concomitant with a decrease in TNF-α and IL-1ß levels. AS-IV is effective in alleviating ON-FH through its effects in repolarizing macrophages from M1-like phenotype to M2-like phenotype, promoting survival of osteocytes, reducing arthritic symptoms, and decreasing inflammatory cytokines.


Assuntos
Necrose da Cabeça do Fêmur/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Saponinas/uso terapêutico , Triterpenos/uso terapêutico , Animais , Células Cultivadas , Feminino , Cabeça do Fêmur/efeitos dos fármacos , Cabeça do Fêmur/imunologia , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/imunologia , Glucocorticoides , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Fenótipo , Saponinas/farmacologia , Triterpenos/farmacologia
19.
J Int Med Res ; 49(1): 300060520986680, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33472486

RESUMO

Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial tumor. The lesions may be single or multiple and usually behave in a benign or indolent fashion, sometimes persisting for many years. In the present case, a 37-year-old woman had experienced primary infertility for 12 years, and a diagnostic laparoscopy was performed. Approximately 200 mL of dark red, free fluid in the pelvis and more than 10 yellow-white nodules on the surface of the right round ligament, sacrum ligament, right fallopian tube, and both sides of the uterus were found. A lesionectomy was performed and immunohistochemical markers indicated WDPM with adenomatoid tumor. The patient was monitored by computed tomography and serum CA125 (cancer antigen 125) levels for 49 months with no recurrence. WDPM and adenomatoid tumor are both benign tumors of mesothelial origin. Because of the lack of effective radical treatment, regular follow-up is sufficient. However, the effects of estrogen and progesterone on WDPM and adenomatoid tumors during ovulation or pregnancy remains unclear. Although WDPM is not life threatening, a strategy to fulfill the fertility requirements of women with this condition is a new challenge for infertility doctors.


Assuntos
Infertilidade , Mesotelioma , Neoplasias Peritoneais , Adulto , Feminino , Humanos , Mesotelioma/complicações , Mesotelioma/diagnóstico por imagem , Mesotelioma/cirurgia , Recidiva Local de Neoplasia , Neoplasias Peritoneais/complicações , Neoplasias Peritoneais/diagnóstico por imagem , Neoplasias Peritoneais/cirurgia , Tomografia Computadorizada por Raios X
20.
Int J Biol Macromol ; 173: 1-12, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33476612

RESUMO

The catalytic mechanism of most lipases involves a step called "interfacial activation" which significantly increases lipases activity beyond the critical micellar concentration (CMC) of substrate. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of lipase interfacial activation beyond the CMC. Molecular dynamic (MD) simulations indicated the open- and closed-lid transitions and revealed that Phe113 was the critical site for RCL activation by its dynamic flipping. Such putative switch affecting interfacial activation has not been reported in lipase so far. The function of Phe113 was subsequently verified by mutation experiments. The F113W mutant increases the lipase catalytic efficiency (1.9 s-1·µM-1) to 280% at the optimum temperature (40 °C) and pH 8.5 with the addition of 0.12 µg protein in the 200 µL reaction system. MD simulations indicated that the fast flipping rate from the closed to the open state, the high open state proportion, and the exposure of the catalytic triad are the main reasons for the lipase activation. The mutual corroboration of simulations and site-directed mutagenesis results revealed the vital role of Phe113 in the lipase activation.


Assuntos
Butiratos/química , Proteínas Fúngicas/química , Lipase/química , Fenilalanina/química , Rhizopus/química , Sítios de Ligação , Biocatálise , Butiratos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/genética , Lipase/metabolismo , Simulação de Dinâmica Molecular , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizopus/enzimologia , Especificidade por Substrato , Temperatura , Termodinâmica , Água/química
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