RESUMO
Bacterial infections pose a significant risk to human health. Magnolol, derived from Magnolia officinalis, exhibits potent antibacterial properties. Synthetic biology offers a promising approach to manufacture such natural compounds. However, the plant-based biosynthesis of magnolol remains obscure, and the lack of identification of critical genes hampers its synthetic production. In this study, we have proposed a one-step conversion of magnolol from chavicol using laccase. After leveraging 20 transcriptomes from diverse parts of M. officinalis, transcripts were assembled, enriching genome annotation. Upon integrating this dataset with current genomic information, we could identify 30 laccase enzymes. From two potential gene clusters associated with magnolol production, highly expressed genes were subjected to functional analysis. In vitro experiments confirmed MoLAC14 as a pivotal enzyme in magnolol synthesis. Improvements in the thermal stability of MoLAC14 were achieved through selective mutations, where E345P, G377P, H347F, E346C, and E346F notably enhanced stability. By conducting alanine scanning, the essential residues in MoLAC14 were identified, and the L532A mutation further boosted magnolol production to an unprecedented level of 148.83 mg/L. Our findings not only elucidated the key enzymes for chavicol to magnolol conversion, but also laid the groundwork for synthetic biology-driven magnolol production, thereby providing valuable insights into M. officinalis biology and comparative plant science.
Assuntos
Compostos Alílicos , Lignanas , Magnolia , Fenóis , Humanos , Magnolia/genética , Magnolia/química , Lacase , Lignanas/química , Compostos de Bifenilo/químicaRESUMO
Cognitive impairment in Alzheimer's disease (AD) is characterized by being deficient at learning and memory. Aß1-42 oligomers have been shown to impair rodent cognitive function. We previously demonstrated that activation of α7nAChR, inhibition of p38 or JNK could alleviate Aß-induced memory deficits in Y maze test. In this study, we investigated whether the effects of α7nAChR and MAPKs on Y maze test is reproducible with a hippocampus-dependent spatial memory test such as Morris water maze. We also assessed the possible co-existence of hippocampus-independent recognition memory dysfunction using a novel object recognition test and an alternative and stress free hippocampus-dependent recognition memory test such as the novel place recognition. Besides, previous research from our lab has shown that MAPKs pathways regulate Aß internalization through mediating α7nAChR. In our study, whether MAPKs pathways exert their functions in cognition by modulating α7nAChR through regulating glutamate receptors and synaptic protein, remain little known. Our results showed that activation of α7nAChR restored spatial memory, novel place recognition memory, and short-term and long-term memory in novel object recognition. Inhibition of p38 restored spatial memory and short-term and long-term memory in novel object recognition. Inhibition of ERK restored short-term memory in novel object recognition and novel place recognition memory. Inhibition of JNK restored spatial memory, short-term memory in novel object recognition and novel place recognition memory. Beside this, the activation of α7nAChR, inhibition of p38 or JNK restored Aß-induced levels of NMDAR1, NMDAR2A, NMDAR2B, GluR1, GluR2 and PSD95 in Aß-injected mice without influencing synapsin 1. In addition, these treatments also recovered the expression of acetylcholinesterase (AChE). Finally, we found that the inhibition of p38 or JNK resulted in the upregulation of α7nAChR mRNA levels in the hippocampus. Our results indicated that inhibition of p38 or JNK MAPKs could alleviate Aß-induced spatial memory deficits through regulating activation of α7nAChR via recovering memory-related proteins. Moreover, p38, ERK and JNK MAPKs exert different functions in spatial and recognition memory.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cognição/fisiologia , Sistema de Sinalização das MAP Quinases , Aprendizagem em Labirinto/fisiologia , Fragmentos de Peptídeos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/administração & dosagem , Animais , Modelos Animais de Doenças , MAP Quinase Quinase 4/metabolismo , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/administração & dosagem , Reconhecimento Psicológico/fisiologiaRESUMO
This study investigated the effect of sevoflurane postconditioning on post-ischaemic cardiac function, infarct size, myocardial mitochondrial ATP-sensitive potassium channel (mitoKATP) function and apoptosis in ageing rats to determine the possible mechanism underlying the cardioprotective property of sevoflurane. Ageing rat hearts were isolated and attached to a Langendorff apparatus. The hearts were then exposed or not to sevoflurane postconditioning in the presence or absence of 100 µmol/L 5-hydroxydecanoate (5-HD), a selective mitoKATP inhibitor. The infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Mitochondrial morphology was observed by electron microscopy and scored using FlaMeng semiquantitative analysis. In addition, the expression levels of Bax, Bcl-2, and cytochrome-C (Cyt-C) were determined by Western blot analysis at the end of reperfusion. Sevoflurane postconditioning increased coronary flow, improved functional recovery, reduced Bax/Bcl-2 and Cyt-C phosphorylation levels, and decreased mitochondrial lesion severity and the extent of apoptosis. The protective effects of sevoflurane postconditioning were prevented by the mitoKATP inhibitor 5-HD. Sevoflurane postconditioning significantly protected the function of ageing hearts that were subjected to ischaemia and reperfusion, and these protective effects were mediated by mitoKATP opening.
Assuntos
Envelhecimento , Apoptose/efeitos dos fármacos , Pós-Condicionamento Isquêmico/métodos , Canais KATP/metabolismo , Éteres Metílicos/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Isquemia Miocárdica/fisiopatologia , Animais , Circulação Coronária/efeitos dos fármacos , Ácidos Decanoicos/farmacologia , Hemodinâmica/efeitos dos fármacos , Hidroxiácidos/farmacologia , Canais KATP/antagonistas & inibidores , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Isquemia Miocárdica/complicações , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Sevoflurano , Proteína X Associada a bcl-2/metabolismoRESUMO
The widely used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is thought to have evolved from IS200/IS605 transposons. TnpB proteins, encoded by one type of IS200/IS605 transposon, are considered to be the evolutionary ancestors of Cas12 nucleases, which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells. TnpB nucleases, which are smaller than Cas nucleases, have been engineered for use in genome editing in animal systems, but the feasibility of this approach in plants remained unknown. Here, we obtained stably transformed genome-edited mutants in rice (Oryza sativa) by adapting three recently identified TnpB genome editing vectors, encoding distinct TnpB nucleases (ISAam1, ISDra2, and ISYmu1), for use in plants, demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes. ISDra2 and ISYmu1 precisely edited their target sequences, with no off-target mutations detected, showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants. Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00172-6.
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Piezo1 is a mechanically-gated calcium channel. Recent studies have shown that Piezo1, a mechanically-gated calcium channel, can attenuate both psychosine- and lipopolysaccharide-induced demyelination. Because oligodendrocyte damage and demyelination occur in intracerebral hemorrhage, in this study, we investigated the role of Piezo1 in intracerebral hemorrhage. We established a mouse model of cerebral hemorrhage by injecting autologous blood into the right basal ganglia and found that Piezo1 was largely expressed soon (within 48 hours) after intracerebral hemorrhage, primarily in oligodendrocytes. Intraperitoneal injection of Dooku1 to inhibit Piezo1 resulted in marked alleviation of brain edema, myelin sheath loss, and degeneration in injured tissue, a substantial reduction in oligodendrocyte apoptosis, and a significant improvement in neurological function. In addition, we found that Dooku1-mediated Piezo1 suppression reduced intracellular endoplasmic reticulum stress and cell apoptosis through the PERK-ATF4-CHOP and inositol-requiring enzyme 1 signaling pathway. These findings suggest that Piezo1 is a potential therapeutic target for intracerebral hemorrhage, as its suppression reduces intracellular endoplasmic reticulum stress and cell apoptosis and protects the myelin sheath, thereby improving neuronal function after intracerebral hemorrhage.
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Allium crop breeding remains severely hindered due to the lack of high-quality reference genomes. Here we report high-quality chromosome-level genome assemblies for three key Allium crops (Welsh onion, garlic and onion), which are 11.17 Gb, 15.52 Gb and 15.78 Gb in size with the highest recorded contig N50 of 507.27 Mb, 109.82 Mb and 81.66 Mb, respectively. Beyond revealing the genome evolutionary process of Allium species, our pathogen infection experiments and comparative metabolomic and genomic analyses showed that genes encoding enzymes involved in the metabolic pathway of Allium-specific flavor compounds may have evolved from an ancient uncharacterized plant defense system widely existing in many plant lineages but extensively boosted in alliums. Using in situ hybridization and spatial RNA sequencing, we obtained an overview of cell-type categorization and gene expression changes associated with spongy mesophyll cell expansion during onion bulb formation, thus indicating the functional roles of bulb formation genes.
Assuntos
Allium , Allium/genética , Melhoramento Vegetal , Cebolas/genética , Genoma , CromossomosRESUMO
Dipterocarpoideae, the largest subfamily of the Dipterocarpaceae, is a dominant component of Southeast Asian rainforests and is widely used as a source of wood, damar resin, medicine, and essential oil. However, many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance. Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes, providing clues to the function and evolution of these species. Here, we report on 13 high-quality Dipterocarpoideae genome assemblies, ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae. Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae. Based on evolutionary analysis, we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes. We discovered an expansion of genes encoding cellulose synthase (CesA), which is essential for cellulose biosynthesis and secondary cell-wall formation. We functionally identified five bornyl diphosphate synthase (BPPS) genes, which specifically catalyze the biosynthesis of borneol, a natural medicinal compound extracted from damar resin and oils, thus providing a basis for large-scale production of natural borneol in vitro.
Assuntos
Dipterocarpaceae , Humanos , Dipterocarpaceae/genética , Filogenia , Genoma , CanfanosRESUMO
BACKGROUND: Gemcitabine plus nab-paclitaxel (GA) is a commonly used first-line treatment regimen for metastatic pancreatic cancer, and many studies will add a novel targeted agent to this regimen for improving patient survival rate. However, the clinical effectiveness of GA is the most controversial issue. AIM: To compare the efficacy and safety of GA regimen with a targeted agent and GA regimen. METHODS: Up to 1 December 2021, the eligible randomized controlled trials (RCTs) relating to GA and GA with a targeted agent were searched on PubMed, EMBASE and Cochrane Library for eligible data. We screened out appropriate studies for overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and toxicity, which had been pooled and finally analyzed by using Stata version 15.1. In addition, we use Reference Citation Analysis (https://www.referencecitationanalysis.com/) to collect the latest related literature to improve the latest cutting-edge research results. RESULTS: Seven RCTs involving 1544 patients (848 men and 696 women) were included. There were no significant differences between GA with a targeted agent and GA in PFS [hazard ratio (HR): 1.18 95% confidence interval (CI): 0.91-1.53], OS (HR: 1.12 95%CI: 0.99-1.27), and ORR (HR: 0.96 95%CI: 0.71-1.29). There was no notable difference in the two groups in grade 3/4 toxicity (fatigue, anemia, vomiting and neutropenia), whereas the incidence of grade 3/4 diarrhea considerably increased in GA with a targeted drug. CONCLUSION: Adding a novel targeted agent to the GA regimen did not improve survival rate of patients with metastatic pancreatic cancer.
RESUMO
BACKGROUND: In South-east Asia, Dipterocarpoideae is predominant in most mature forest communities, comprising around 20% of all trees. As large quantity and high quality wood are produced in many species, Dipterocarpoideae plants are the most important and valuable source in the timber market. The d-borneol is one of the essential oil components from Dipterocarpoideae (for example, Dryobalanops aromatica or Dipterocarpus turbinatus) and it is also an important traditional Chinese medicine (TCM) formulation known as "Bingpian" in Chinese, with antibacterial, analgesic and anti-inflammatory effects and can enhance anticancer efficiency. METHODS: In this study, we analyzed 20 chloroplast (cp) genomes characteristics of Dipterocarpoideae, including eleven newly reported genomes and nine cp genomes previously published elsewhere, then we explored the chloroplast genomic features, inverted repeats contraction and expansion, codon usage, amino acid frequency, the repeat sequences and selective pressure analyses. At last, we constructed phylogenetic relationships of Dipterocarpoideae and found the potential barcoding loci. RESULTS: The cp genome of this subfamily has a typical quadripartite structure and maintains a high degree of consistency among species. There were slightly more tandem repeats in cp genomes of Dipterocarpus and Vatica, and the psbH gene was subjected to positive selection in the common ancestor of all the 20 species of Dipterocarpoideae compared with three outgroups. Phylogenetic tree showed that genus Shorea was not a monophyletic group, some Shorea species and genus Parashorea are placed in one clade. In addition, the rpoC2 gene can be used as a potential marker to achieve accurate and rapid species identification in subfamily Dipterocarpoideae. CONCLUSIONS: Dipterocarpoideae had similar cp genomic features and psbM, rbcL, psbH may function in the growth of Dipterocarpoideae. Phylogenetic analysis suggested new taxon treatment is needed for this subfamily indentification. In addition, rpoC2 is potential to be a barcoding gene to TCM distinguish.
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The 5-hydroxytryptamine (5-HT) receptor is significant for the regulation of mood and memory. However, the role of 5-HT1AR in ß-Amyloid protein (Aß)-induced cognitive decline, neuroinflammation and the possible mechanism remains elusive. Thus, we aimed to evaluate the effects of 5-HT1AR on Aß-induced learning and memory decline and neuroinflammation in mice. Novel object recognition and Morris water maze tests were performed to observe learning and memory behavior in mice. Protein levels of Iba1, GFAP, MAP2, TNF-α, Tß4, C-fos, IKK-ß, IKB-α, NF-κBp65, phospho-NF-κBp65 in the hippocampus were examined by immunostaining or western blotting. Aß1-42-treatment inducing learning and memory decline was shown in novel object recognition and Morris water maze tests; neuroinflammation shown in immunostaining. Our study found out that 5-HT1AR inhibitor WAY100635 showed significant improvement in Aß-induced learning and memory decline. Moreover, WAY100635 decreases levels of Iba1, GFAP, and TNF-α in the hippocampus, which were related to neuroinflammation. While treatment with 5-HT1AR agonist 8-OH-DPAT or ERK inhibitor U0126 exerted no effects or even aggravated Aß-induced learning and memory decline. In addition, WAY100635 could downregulate phospho-NF-κB in the hippocampus of Aß1-42-injected mice. These results provide new insight into the mechanism, for 5-HT1AR in Aß-induced cognitive impairments through crosstalk with the NF-κB signaling pathway. Our data indicated that WAY100635 was involved in the protective effects against neuroinflammation and improvement of learning and memory in Alzheimer's disease.
RESUMO
Patients with Alzheimer's disease often undergo anxiety and depression. Our previous studies have shown that α7nAChR protects against Aß-induced neurotoxicity via downregulation of p38 and JNK MAPKs, but the role of α7nAChR on Aß-induced anxiety and depressive-like behaviors and the effect of α7nAChR on the regulation of MAPKs pathways remain unknown. To examine the effects of α7nAChR and MAPKs pathways on Aß-induced anxiety and depression-like behaviors and to explore their relationships between them, elevated plus maze, open field and forced swim tests were performed. Protein levels of 5-HT1A receptor, 5-HT2C receptor, α7nAChR, t-ERK1/2 and p-ERK1/2 in the amygdala were analyzed by western blotting and immunostaining. Our study found out that Aß oligomers induced anxiety and depression-like behaviors in C56BL/6 mice with open field, elevated plus maze and forced swim tests. However, activation of α7nAChR or inhibition of ERK pathways showed significant antidepressant and anxiolytic-like effects on Aß-injected mice. Moreover, Aß significantly decreased the level of 5-HT1A receptor but increased the level of 5-HT2C receptor in the basolateral amygdala. Treatment with α7nAChR agonist PNU282987 or ERK inhibitor U0126 reversed Aß-induced 5-HT1A and 5-HT2C receptor changes. Moreover, activation of α7nAChR inhibited ERK pathway in the amygdala of Aß1-42-injected mice. Our study provides a new insight into the mechanism of α7nAChR in Aß-induced depression and anxiety-related symptoms through the regulation of ERK1/2 pathway and the potential association with serotonin receptors. Together, our data suggests that α7nAChR is protective against Aß-induced anxiety and depression-like behaviors in mice.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Ansiedade/metabolismo , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacos , Animais , Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/metabolismo , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Regulação para Cima/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular ß-amyloid protein (Aß) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aß caused cognition. It has high affinity with Aß and could mediate Aß internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aß internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aß is internalized by cholinergic and GABAergic neurons. The internalized Aß were found deposits in lysosomes/endosomes and mitochondria. Aß could form Aß-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aß internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aß-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aß. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Compostos Bicíclicos com Pontes/farmacologia , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/patologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/patologia , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Agonistas Nicotínicos/farmacologia , Fosforilação , Distribuição Aleatória , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidoresRESUMO
Amyloid ß peptide 1-42 (Aß1-42) could induce cognitive deficits through oxidative stress, inflammation, and neuron death in Alzheimer's disease (AD). MAPK pathways have been thought to mediate Aß1-42-induced neuroinflammation responses, neuron death and cognitive decline in AD. The α7 nicotinic acetylcholine receptor (α7nAChR) exerts a neuroprotective effect. However, whether α7nAChR alleviates Aß1-42-induced neurotoxicity through MAPKs (p38, ERK, JNK) in vivo remains unclear. In our study, memory was assessed in C57BL/6 mice using a Y-maze test. Cell death was assessed by Nissl and Hoechst staining and Bax, Bcl-2, Caspase 3, and Cytochrome C levels using Western blotting. Oxidative stress was assayed by superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels. Inflammation was examined with GFAP and Iba1 using immunohistochemistry. The Aß degrading enzymes insulin degrading enzyme (IDE) and neprilysin (NEP) were tested using Western blotting. We found that activating α7nAChR or inhibiting p38 or JNK pathway alleviated Aß1-42-induced cognitive deficits and neuron loss and death by reducing oxidative stress. In addition, activating α7nAChR or inhibiting p38 or JNK pathway also reduced inflammation, which was observed as reduced GFAP and Iba1 levels with different effects on Aß degrading enzymes. Finally, we found that the activation of α7nAChR led to the downregulation of pp38 and pJNK levels. Conversely, the inhibition of p38 or JNK resulted in the upregulation of α7nAChR levels in the hippocampus and cortex. Our data indicate that the activation of α7nAChR alleviates Aß1-42-induced neurotoxicity, and this protective effect might act through the downregulation of p38 and JNK MAPKs.