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Spermatogenesis in eukaryotes is a process that occurs within a very narrow temperature threshold, typically not exceeding 36 °C. SPO11 was isolated from the temperature-sensitive mutant receptor of Saccharomyces cerevisiae and is thought to be the only protein that functions during meiosis. This suggested that SPO11 may be the key protein that influenced the temperature of spermatogenesis not exceeding 36 °C. Elevated temperatures typically damage the spermatogenic cells. Birds have a core body temperature of 41-42 °C, and their testis are located inside their bodies, providing an alternative perspective to investigate the potential impact of temperature threshold on spermatogenesis. The objective of this study was to ascertain whether elevated ambient temperatures affect spermatogenesis in birds and whether SPO11 is the key gene affecting the temperature threshold for spermatogenesis. STRA8, SCP3, SPO11, γ-H2AX, and RAD51 were all crucial components in the process of meiotic initiation, synapsis, DNA double-strand break (DSB) induction, homologous chromosome crossover recombination, and repair of DSB. In this study, 39-day-old Japanese quail were subjected to heat stress (HS) at 38 °C for 8 h per day for 3 (3d HS) and 13 (13d HS) consecutive days and analyzed the expression of meiotic signaling molecules (STRA8, SCP3, SPO11, γ-H2AX, and RAD51) using molecular biology techniques, including Immunohistochemistry (IHC), Western Blot (WB), and Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). We found that spermatogenesis was normal in both groups exposed to HS. Meiotic signaling molecules were expressed normally in the 3d HS group. All detected signaling molecules were normally expressed in the 13d HS group, except for SPO11, which showed a significant increase in expression, indicating that SPO11 was temperature-sensitive. We examined the localized expression of each meiotic signaling molecule in quail testis, explored the temperature sensitivity of SPO11, and determined that quail testis can undergo normal spermatogenesis at ambient temperatures exceeding 36 °C. This study concluded that SPO11 is not the key protein influencing spermatogenesis in birds. These findings enhance our understanding of avian spermatogenesis.
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Idiopathic pulmonary fibrosis (IPF) is considered to be associated with aging. Both ER stress and the unfolded protein response (UPR) have been associated with pulmonary fibrosis via key mechanisms including AEC apoptosis, EMT, altered myofibroblast differentiation, and M2 macrophage polarization. A relationship between ER stress and aging has also been demonstrated in vitro, with increased p16 and p21 levels seen in lung epithelial cells of older IPF patients. The mechanism underlying ER stress regulation of IPF fibroblasts is still unclear. In this study, we aimed to delineate ER stress regulation in IPF-derived fibroblasts. Here, we found that ER stress markers (p-eIF2α, p-IREα, ATF6) and fibrosis markers (α-SMA and Collagen-I) were significantly increased in lung tissues of IPF patients and bleomycin-induced mouse models. Notably, the expression of PGC-1α was decreased in fibroblasts. In vivo experiments were designed using an AAV-6 vector mediated conditional PGC-1α knockout driven by a specific α-SMA promoter. Ablation of PGC-1α expression in fibroblasts promoted ER stress and supported the development of pulmonary fibrosis in a bleomycin-induced mouse model. In another experimental group, mice with conditional knockout of PGC-1α in fibroblasts and injected intraperitoneally with 4-PBA (an endoplasmic reticulum stress inhibitor) were protected from lung fibrosis. We further constructed an AAV-6 vector mediated PGC-1α overexpression model driven by a specific Collagen-I promoter. Overexpression of PGC-1α in fibroblasts suppressed ER stress and attenuated development of pulmonary fibrosis in bleomycin-induced mouse models. Taken together, this study identified PGC-1α as a promising target for developing novel therapeutic options for the treatment of lung fibrosis.
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Bleomicina , Estresse do Retículo Endoplasmático , Fibroblastos , Fibrose Pulmonar Idiopática , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fenilbutiratos , Animais , Feminino , Humanos , Masculino , Camundongos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fenilbutiratos/farmacologiaRESUMO
BACKGROUND: Phospholipase C (PLC) γ1 is a critical enzyme regulating nuclear factor-κB (NF-κB), extracellular signal-related kinase, mitogen-activated protein kinase, and nuclear factor of activated T cells signaling pathways, yet germline PLCG1 mutation in human disease has not been reported. OBJECTIVE: We aimed to investigate the molecular pathogenesis of a PLCG1 activating variant in a patient with immune dysregulation. METHODS: Whole exome sequencing was used to identify the patient's pathogenic variants. Bulk RNA sequencing, single-cell RNA sequencing, quantitative PCR, cytometry by time of flight, immunoblotting, flow cytometry, luciferase assay, IP-One ELISA, calcium flux assay, and cytokine measurements in patient PBMCs and T cells and COS-7 and Jurkat cell lines were used to define inflammatory signatures and assess the impact of the PLCG1 variant on protein function and immune signaling. RESULTS: We identified a novel and de novo heterozygous PLCG1 variant, p.S1021F, in a patient presenting with early-onset immune dysregulation disease. We demonstrated that the S1021F variant is a gain-of-function variant, leading to increased inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and increased phosphorylation of extracellular signal-related kinase, p65, and p38. The transcriptome and protein expression at the single-cell level revealed exacerbated inflammatory responses in the patient's T cells and monocytes. The PLCG1 activating variant resulted in enhanced NF-κB and type II interferon pathways in T cells, and hyperactivated NF-κB and type I interferon pathways in monocytes. Treatment with either PLCγ1 inhibitor or Janus kinase inhibitor reversed the upregulated gene expression profile in vitro. CONCLUSIONS: Our study highlights the critical role of PLCγ1 in maintaining immune homeostasis. We illustrate immune dysregulation as a consequence of PLCγ1 activation and provide insight into therapeutic targeting of PLCγ1.
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Mutação com Ganho de Função , NF-kappa B , Humanos , NF-kappa B/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Fosfolipase C gama/genéticaRESUMO
Early bovine embryo sexing both increases the number of offspring of the desired sex, and reduces the subsequent costs of processing unwanted offspring of the opposite sex. The need for cattle of different sexes varies from industry to industry, and a range of tools have been set up to meet this need, but most are energy- and time-consuming, hence it is important to establish a fast and convenient method for bovine embryo determination. Herein, we established a recombinase polymerase amplification (RPA) method combined with CFI dye (RPA-CFI) for sexing of bovine embryos. The assay is highly sensitive, specific, rapid and simple; it can be carried out in only 5 min at 37 °C in a metal bath, and results are visualised using a fluorescent colorimeter. Highly specific male-female common and male-specific primers were designed based on the 1399 bp repeating unit of bovine 1.715 satellite DNA and the male-specific S4 repeating sequence, respectively. The limit of detection (LOD) of RPA-CFI with male-female common primers was 1 pg/µL, and the LOD with male-specific primers was 2 pg/µL. RPA-CFI could determine the sex of bovine embryos from only two cells. This is the first report using RPA-CFI for sex determination of bovine embryos. The assay could be applied to other economically important animals to improve efficiency in livestock industries. Additionally, the assay could relieve pressure on food demand due to human population growth, and contribute to economic development of global stockbreeding.
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OBJECTIVE: To explore the clinical characteristics and genetic variants in a patient with adult ceroid lipofuscinosis neuronal type 7 (ACLN7). METHODS: A female patient diagnosed with ACLN7 in Henan Provincial People's Hospital in June 2021 was selected as the study subject. Clinical data, auxiliary examination and result of genetic testing were retrospectively analyzed. RESULTS: The patient, a 39-year-old female, has mainly presented progressive visual loss, epilepsy, cerebellar ataxia and mild cognitive decline. Neuroimaging analysis has revealed generalized brain atrophy, prominently cerebellum. Fundus photography has revealed retinitis pigmentosa. Ultrastructural skin examination has revealed granular lipofuscin deposits in the periglandular interstitial cells. Whole exome sequencing revealed that she has harbored compound heterozygous variants of the MSFD8 gene, namely c.1444C>T (p.R482*) and c.104G>A (p.R35Q). Among these, c.1444C>T (p.R482*) was a well established pathogenic variant, while c.104G>A (p.R35Q) was a missense variant unreported previously. Sanger sequencing confirmed that the daughter, son and elder brother of the proband have respectively carried heterozygous c.1444C>T (p.R482*), c.104G>A (p.R35Q), and c.104G>A (p.R35Q) variants of the same gene. The family has therefore fit with the autosomal recessive inheritance pattern of the CLN7. CONCLUSION: Compared with previously reported cases, this patient has the latest onset of the disease with a non-lethal phenotype. Her clinical features have involved multiple systems. Cerebellar atrophy and fundus photography may be indicative of the diagnosis. The c.1444C>T (p.R482*) and c.104G>A (p.R35Q) compound heterozygous variants of the MFSD8 gene probably underlay the pathogenesis in this patient.
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Proteínas de Membrana Transportadoras , Lipofuscinoses Ceroides Neuronais , Masculino , Feminino , Humanos , Proteínas de Membrana Transportadoras/genética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/diagnóstico , Estudos Retrospectivos , Atrofia , MutaçãoRESUMO
Background: The deficiency of adenosine deaminase 2 (DADA2) is caused by an autosomal recessive bi-allelic loss-of-function mutation in the adenosine deaminase 2 (ADA2) gene. DADA2 is a monogenic inherited autoinflammatory disorder characterized by early-onset vasculopathy for which the symptoms range from skin lesions to very severe multiorgan involvement, including life-threatening ischemia and/or hemorrhagic strokes. Owing to the diversity of clinical presentation and the absence of suggestive features, differentiating DADA2 from other inflammatory disorders in the early stages of disease presentation is difficult. Here, we describe the case of a 3-year-old boy who had been misdiagnosed for nearly 2 years before he was definitively diagnosed with DADA2. Case Description: A previously healthy 3-year-old boy was initially diagnosed with systemic onset juvenile idiopathic arthritis (soJIA) owing to recurrent unprovoked fever and elevated acute phase reactants. He developed intractable hypertension during treatment, which his doctor considered an adverse drug reaction. Monogenic inherited autoinflammatory disorders were not suspected until the patient developed intestinal perforation and ensuing recurrent abdominal pain that coincided with fever. Gene sequence analysis revealed a novel compound heterozygous mutation in ADA2. The ADA2 enzyme activity was almost completely lost in the patient. Conclusions: The broad phenotypic spectrum of DADA2 makes early diagnosis challenging. DADA2 should be considered in case of early-onset vasculitis, which is the most common phenotype of DADA2. Early identification and treatment will result in significant improvement of the disease.
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BACKGROUND: Stimulator of interferon gene (STING)-associated vasculopathy with onset in infancy (SAVI), caused by gain-of-function mutations in human transmembrane protein 173 (TMEM173), is characterized by widespread chronic inflammation primarily affecting the skin and lungs. Although SAVI is an inflammatory disease, typical anti-inflammatory agents have limited or no effect. METHODS AND RESULTS: A 1-year-old boy presented with recurrent facial rashes since he was 8 months. Moreover, he suffered from recurrent oral ulcers, chronic cough, and failure to thrive. Laboratory parameters showed elevated erythrocyte sedimentation rate (ESR) and immunoglobulin levels. Chest high-resolution computed tomography (HRCT) showed interstitial lung disease (ILD). Whole-exome sequencing revealed a heterozygous mutation in the TMEM173 gene (c.463Gâ >â A, p.V155M). Ultimately, the patient was diagnosed with SAVI. Tofacitinib was initiated at the age of 19 months, resulting in the alleviation of facial rashes and improvement of ILD within 3 months. CONCLUSION: SAVI is a difficult-to-treat type I interferonopathy. We hope that JAKi treatment will prove valuable for SAVI patients.
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Mutação com Ganho de Função , Humanos , Lactente , ChinaRESUMO
Different studies on the effects of high-fat diet (HFD) on Alzheimer's disease (AD) pathology have reported conflicting findings. Our previous studies showed HFD could moderate neuroinflammation and had no significant effect on amyloid-ß levels or contextual memory on AD mice. To gain more insights into the involvement of HFD, we performed the whole-transcriptome sequencing and ribosome footprints profiling. Combined with competitive endogenous RNA analysis, the transcriptional regulation mechanism of HFD on AD mice was systematically revealed from RNA level. Mmu-miR-450b-3p and mmu-miR-6540-3p might be involved in regulating the expression of Th and Ddc expression. MiR-551b-5p regulated the expression of a variety of genes including Slc18a2 and Igfbp3. The upregulation of Pcsk9 expression in HFD intervention on AD mice might be closely related to the increase of cholesterol in brain tissues, while Huanglian Jiedu Decoction significantly downregulated the expression of Pcsk9. Our data showed the close connection between the alterations of transcriptome and translatome under the effect of HFD, which emphasized the roles of translational and transcriptional regulation were relatively independent. The profiled molecular responses in current study might be valuable resources for advanced understanding of the mechanisms underlying the effect of HFD on AD.
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PSTPIP1 (proline-serine-threonine phosphatase-interactive protein 1)-associated myeloid-related proteinemia inflammatory (PAMI) syndrome is a rare autoinflammatory disease caused by heterozygous gain-of-function mutation in PSTPIP1. As one of the PSTPIP1-associated inflammatory diseases (PAIDs), neutropenia is a distinct manifestation to separate PAMI syndrome from other PAIDs. This study aimed to investigate the potential role of neutrophils and inflammatory signatures in the pathogenesis of PAMI. PAMI neutrophils displayed markedly increased production of interleukin-1ß (IL-1ß) and IL-18 by enzyme linked immunosorbent assay (ELISA) assay and intracellular cytokine staining. ASC speck formation and lactic dehydrogenase (LDH) release are also increased in patient neutrophils suggesting elevated pyrin inflammasome activation followed by upregulated cell death in PAMI neutrophils. RNA sequencing result showed strong inflammatory signals in both nuclear-factor kappa B (NF-κB) pathway and interferon (IFN) pathway in patient neutrophils. This study highlighted that elevated proinflammatory cytokines IL-1ß and IL-18, increased pyrin inflammasome activation, and upregulation of NF-κB and IFN signaling pathways in neutrophils play important roles in pathogenicity of PAMI syndrome.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inflamassomos , Neutrófilos , Doenças Autoimunes , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Interferons , Interleucina-18 , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Oxirredutases , Fosfoproteínas Fosfatases , Prolina , Pirina , SíndromeRESUMO
The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.
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Proteínas Ferro-Enxofre , Ferro , Citosol/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Enxofre/metabolismoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by the complex interaction of multiple mechanisms. Recent studies examining the effect of high-fat diet (HFD) on the AD phenotype have demonstrated a significant influence on both inflammation and cognition. However, different studies on the effect of high-fat diet on AD pathology have reported conflicting conclusions. To explore the involvement of HFD in AD, we investigated phenotypic and metabolic changes in an AD mouse model in response to HFD. The results indicated there was no significant effect on Aß levels or contextual memory due to HFD treatment. Of note, HFD did moderate neuroinflammation, despite spurring inflammation and increasing cholesterol levels in the periphery. In addition, diet affected gut microbiota symbiosis, altering the production of bacterial metabolites. HFD created a favorable microenvironment for bile acid alteration and arachidonic acid metabolism in APP/PS1 mice, which may be related to the observed improvement in LXR/PPAR expression. Our previous research demonstrated that Huanglian Jiedu decoction (HLJDD) significantly ameliorated impaired learning and memory. Furthermore, HLJDD may globally suppress inflammation and lipid accumulation to relieve cognitive impairment after HFD intervention. It was difficult to define the effect of HFD on AD progression because the results were influenced by confounding factors and biases. Although there was still obvious damage in AD mice treated with HFD, there was no deterioration and there was even a slight remission of neuroinflammation. Moreover, HLJDD represents a potential AD drug based on its anti-inflammatory and lipid-lowering effects.
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Spontaneous unilateral cryptorchid boars have one testis in the abdomen or inguinal canal, causing its temperature to be at or near the body temperature, which impairs spermatogenesis, although the histomorphometry and molecular mechanisms underlying this process remain unclear. The aim of the present study was to determine the histomorphometry, proliferation, apoptosis, and autophagy alterations in spermatogonia and Sertoli cells in unilateral cryptorchid, scrotal (contrascrotal), and preweaning piglet (preweaning) testes. Histomorphometrical analysis of cryptorchid testes showed that the seminiferous tubules contained only Sertoli cells and a few spermatogonia, but did not contain post-meiotic germ cells. The number of spermatogonia markedly decreased, and the number of Sertoli cells did not change remarkably in cryptorchid testes. TUNEL assay results showed that apoptosis signals were predominantly observed in spermatogonia. In cryptorchid and contrascrotal testes, proliferating cell nuclear antigen (PCNA) and LC3 were located in spermatogonia. The number of PCNA-positive, TUNEL-positive, and LC3-positive germ cells was low, and the protein and mRNA levels of PCNA and LC3 were significantly decreased in cryptorchid testes. Taken together, the number of Sertoli cells did not change remarkably, whereas the number of germ cells decreased in the cryptorchid testes, compared with that in the contrascrotal testes. Insufficient proliferation, excessive apoptosis, and autophagy were involved in the regulation of the decrease in spermatogonia in cryptorchid boar testes.
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With recent availability of COVID-19 vaccine, post-vaccination neurological complications had been occasionally reported. Here, we reported for the first time a case of neuromyelitis optica spectrum disorder (NMOSD) that developed after the first dose of inactivated virus vaccine for COVID-19. The patient developed mild fever, vomiting, diarrhea, and cough after receiving the first dose of inactivated virus vaccine. Two months later, she experienced dizziness and unsteady walking. MRI scanning of the brain revealed lesions in area postrema and bilateral hypothalamus, typical for NMOSD. Serum antibodies for AQP4, ANA, SSA, SSB, Ro-52, and p-ANCA were positive. The patient was diagnosed as AQP4-positive NMOSD with coexisting systemic autoimmunity. After treatment with methylprednisolone (500 mg for 5 days), symptoms were greatly relieved. As NMOSD is seriously harmful and curative, it is important to be aware of the NMOSD symptoms after vaccination. Cautions should be given for those with preexisting systemic autoimmune abnormalities in vaccination for COVID-19.
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COVID-19 , Neuromielite Óptica , Aquaporina 4 , Autoanticorpos , Vacinas contra COVID-19 , Feminino , Humanos , SARS-CoV-2 , Vacinação/efeitos adversosRESUMO
"Shanghuo" ("excessive internal heat") is caused by exuberant endogenous fire, which does not have a comprehensive and systematic traditional Chinese medicine theory. In previous study, we had evaluated the therapeutic effect of Huanglian Jiedu Decoction (HLJDD) (granule) on patients with "Shanghuo", however, the specific mechanism was not clear, which need further exploration. To explain its intervention mechanism, we select 57 patients with oral diseases caused by "Shanghuo" and 20 health volunteers to divide into oral disease group, HLJDD intervention group and healthy control group. Firstly, biochemical indicators before and after HLJDD intervention are detected, such as inflammatory factors, oxidative stress factors and energy metabolism factors. The results exhibit that HLJDD significantly decreases indicators succinic acid (p < 0.001); tumor necrosis factor-alpha, adenosine triphosphate, citric acid (p < 0.01); interleukin-8 (IL-8), 4-hydroxynonenal, pyruvic acid, lactate dehydrogenase (p < 0.05). The levels of glucocorticoid, adrenocorticotropic hormone (p < 0.01); lactic acid, IL-4, IL-10 (p < 0.05) significantly increase after HLJDD intervention. In addition, we adopt multi-omics analysis approach to investigate the potential biomarkers. Nontargeted metabolomics demonstrate that the levels of 7 differential metabolites approach that in the healthy control group after HLJDD intervention, which are correlated with histidine metabolism, beta-alanine metabolism and sphingolipid metabolism through metabolic pathway analysis. Targeted lipidomics results and receiver operating characteristic curve analysis show that 13 differential lipids are identified in the three groups mainly focuse on lysophosphatidylcholines, lysophosphatidylethanolamines. Finally, the network associations of those differential biomarkers reveal the regulation of adenosine triphosphate and tricarboxylic acid cycle play essential role in the therapeutic effect mechanism of HLJDD in "Shanghuo". The study has laid the foundation for further revealing the mechanism and finding clinical biomarkers related to "Shanghuo".
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BACKGROUND: In recent years, excellent results have suggested an association between the "brain-gut" axis and Alzheimer's disease (AD) progression, yet the role of the "brain-gut" axis in AD pathogenesis still remains obscure. Herein, we provided a potential link between the central and peripheral neuroinflammatory disorders in AD progression. METHODS: The Morris water maze (MWM) test, immunohistochemistry, ELISA, ProcartaPlex Multiplex immunoassay, multiple LC-MS/MS methods, and the V3-V4 regions of 16S rRNA genes were applied to explore potential biomarkers. RESULTS: In Tg-APP/PS1 mice, gut dysbiosis and lipid metabolism were highly associated with AD-like neuroinflammation. The combination of inflammatory factors (IL-6 and INF-γ), phosphatidylcholines (PCs) and SCFA-producing bacteria were expected to be early diagnostic biomarkers for AD. Huanglian Jiedu decoction (HLJDD) suppressed gut dysbiosis and the associated Aß accumulation, harnessed neuroinflammation and reversed cognitive impairment. CONCLUSION: Together, our findings highlighted the roles of neuroinflammation induced by gut dysbiosis and lipid metabolism disorder in AD progression. This integrated metabolomics approach showed its potential to understand the complex mechanisms of HLJDD in the treatment of AD.
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Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Animais , Encéfalo , Cromatografia Líquida , Progressão da Doença , Medicamentos de Ervas Chinesas , Camundongos , RNA Ribossômico 16S , Espectrometria de Massas em TandemRESUMO
Barley yellow mosaic disease, caused mainly by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus, is a devastating disease of barley and is a threat to Eurasian barley production. Early detection is essential for effective management of the pathogens and to assure food security. In this study, a simple, rapid, specific, sensitive, and visual method was developed to detect BaYMV using loop-mediated isothermal amplification (LAMP). Two pairs of oligonucleotide primers (inner and outer primers) were designed to amplify the gene encoding the coat protein of BaYMV. The optimal conditions for the LAMP method were determined, and a one-step reverse transcription (RT)-LAMP method was also developed. Subsequently, the fastest processing time for RT-LAMP was determined. Among eight plant viruses examined using the LAMP method, only BaYMV was detectable, suggesting that the assay was highly specific. The RT-LAMP method was 10 times more sensitive than the RT-PCR method in the sensitivity test. To further shorten the virus detection process, a dye was added to the RT-LAMP products, and positive reactions were simply read by the naked eye via a color change (from orange to light green) under visible light. Barley samples from the middle and lower reaches of the Yangtze River basin, where BaYMV broke out very seriously in 1970s, were detected by the newly established RT-LAMP method. The results showed that all samples were positive for BaYMV, indicating the potential risk of the virus in these areas. This newly established LAMP/RT-LAMP method could be a promising tool for barley protection and food security control.
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Doenças das Plantas , Transcrição Reversa , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , PotyviridaeRESUMO
Nowadays, the root of Rose cymosa Tratt. (Rosaceae) is widely used in clinic as one of the sources of Chinese herb medicine Jinyinggen. However, only few studies have been done on its chemical composition and quality control. In this study, 27 monomeric compounds were obtained from the ethanol extract of the roots of R. cymosa Tratt., including two undescribed triterpenes, one of which contains a distinctive contracted five-membered A-ring ursane-type skeleton and the other is a common ursane-type tritepene. Then, triterpenoids, the main components of the R. cymosa root, were qualitatively and quantitatively analyzed by thin-layer chromatography and high-performance liquid chromatography methods. Thin-layer chromatography can identify seven triterpenoids in R. cymosa Tratt. spontaneously. For the high-performance liquid chromatography fingerprint, total of 16 chromatographic peaks were selected as the common peaks of 20 batches of samples, ten of which were identified by reference substances. At the same chromatographic condition, five abundant triterpenoids were quantitatively assayed. R. cymose, as one of the origins of Jinyinggen, was similar to R. laevigata in triterpenoids compounds, which demonstrated that both of them could be used in the clinical medication. These work also laid a foundation for the further research and development of triterpenoids in R. cymosa root.
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Medicamentos de Ervas Chinesas/análise , Extratos Vegetais/análise , Raízes de Plantas/química , Rosaceae/química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Conformação Molecular , Controle de Qualidade , EstereoisomerismoRESUMO
Glycyrrhiza uralensis Fisch., known as licorice, is one of the most famous traditional Chinese medicines. In this study, we perform a metabolome analysis using liquid chromatography-tandem mass spectrometry to assign bioactive components in different parts of licorice from different geographical origins in Gansu province of China. Sixteen potential biomarkers of taproots from different geographical origins were annotated, such as glycycoumarin, gancaonin Z, licoricone, and dihydroxy kanzonol H mainly exist in the sample of Jiuquan; neoliquiritin, 6'-acetylliquiritin, licochalcone B, isolicoflavonol, glycyrol, and methylated uralenin mainly exist in Glycyrrhiza uralensis from Lanzhou; gancaonin L, uralenin, and glycybridin I mainly exist in licorice from Wuwei for the first time.
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Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Glycyrrhiza uralensis/metabolismo , Metabolômica , Óxido Nítrico/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , China , Cromatografia Líquida , Glycyrrhiza uralensis/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Medicina Tradicional Chinesa , Camundongos , Óxido Nítrico/biossíntese , Células RAW 264.7 , Espectrometria de Massas em TandemRESUMO
All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.