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Tungsten and its alloys have a high atomic number, high melting temperature, and high thermal conductivity, which make them fairly appropriate for use in nuclear applications in an extremely harsh radioactive environment. In recent years, there has been growing research interest in using additive manufacturing techniques to produce tungsten components with complex structures. However, the critical bottleneck for tungsten engineering manufacturing is the high melting temperature and high ductile-to-brittle transition temperature. In this study, laser powder bed fusion has been studied to produce bulk pure tungsten. And finite element analysis was used to simulate the temperature and stress field during laser irradiation. The as-printed surface as well as transverse sections were observed by optical microscopy and scanning electron microscopy to quantitatively study processing defects. The simulated temperature field suggests small-sized powder is beneficial for homogenous melting and provides guidelines for the selection of laser energy density. The experimental results show that ultra-dense tungsten bulk has been successfully obtained within a volumetric energy density of 200-391 J/mm3. The obtained relative density can be as high as 99.98%. By quantitative analysis of the pores and surface cracks, the relationships of cracks and pores with laser volumetric energy density have been phenomenologically established. The results are beneficial for controlling defects and surface quality in future engineering applications of tungsten components by additive manufacturing.
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PURPOSE: The incidence of pneumonia caused by multidrug-resistant gram-negative bacteria (MDR GNB) is increasing, which imposes significant burden on public health. Inhalation combined with intravenous polymyxins has emerged as a viable treatment option. However, pharmacokinetic studies focusing on intravenous and inhaled polymyxin B (PMB) are limited. METHODS: This study included seven patients with MDR GNB-induced pneumonia who were treated with intravenous plus inhaled PMB from March 1 to November 30, 2022, in the intensive care unit of the First Affiliated Hospital of Zhejiang University School of Medicine. Clinical outcomes and therapeutic drug monitoring data of PMB in both plasma and epithelial lining fluid (ELF) were retrospectively reviewed. RESULTS: Median PMB concentrations in the ELF were 7.83 (0.72-66.5), 116.72 (17.37-571.26), 41.1 (3.69-133.78) and 33.82 (0.83-126.68) mg/L at 0, 2, 6 and 12 h, respectively, and were much higher than those detected in the serum. ELF concentrations of PMB at 0, 2, 6 and 12 h were higher than the minimum inhibitory concentrations of pathogens isolated from the patients. Steady-state concentrations of PMB in the plasma were >2 mg/L in most patients. Of the patients, 57.14% were cured and 71.43% showed a favourable microbiological response. The incidence of side effects with PMB was low. CONCLUSIONS: Inhaled plus intravenous PMB can achieve high ELF concentrations and favourable clinical outcomes without an increased adverse effect profile. This treatment approach appears promising for the treatment of patients with pneumonia caused by MDR-GNB.
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Fungal phytochemicals derived from higher fungi, particularly those from the culinary-medicinal genus Hericium, have gained significant attention in drug discovery and healthcare. This review aims to provide a comprehensive analysis of the chemical structures, biosynthetic pathways, biological activities, and pharmacological properties of monomeric compounds isolated from Hericium species. Over the past 34 years, 253 metabolites have been identified from various Hericium species, including cyathane diterpenes, alkaloids, benzofurans, chromenes, phenols, pyrones, steroids, and other miscellaneous compounds. Detailed investigations into the biosynthesis of erinacines, a type of cyathane diterpene, have led to the discovery of novel cyathane diterpenes. Extensive research has highlighted the biological activities and pharmacological properties of Hericium-derived compounds, with particular emphasis on their neuroprotective and neurotrophic effects, immunomodulatory capabilities, anti-cancer activity, antioxidant properties, and antimicrobial actions. Erinacine A, in particular, has been extensively studied. Genomic, transcriptomic, and proteomic analyses of Hericium species have facilitated the discovery of new compounds and provided insights into enzymatic reactions through genome mining. The diverse chemical structures and biological activities of Hericium compounds underpin their potential applications in medicine and as dietary supplements. This review not only advances our understanding of Hericium compounds but also encourages further research into Hericium species within the realms of medicine, health, functional foods, and agricultural microbiology. The broad spectrum of compound types and their diverse biological activities present promising opportunities for the development of new pharmaceuticals and edible products.
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Hericium , Hericium/química , Humanos , Metabolismo Secundário , Estrutura Molecular , Animais , Alcaloides/farmacologia , Alcaloides/química , Alcaloides/biossíntese , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/químicaRESUMO
α-Synuclein accumulation and transmission are vital to the pathogenesis of Parkinson's disease, although the mechanisms underlying misfolded α-synuclein accumulation and propagation have not been conclusively determined. The expression of low-density lipoprotein receptor-related protein 1, which is abundantly expressed in neurons and considered to be a multifunctional endocytic receptor, is elevated in the neurons of patients with Parkinson's disease. However, whether there is a direct link between low-density lipoprotein receptor- related protein 1 and α-synuclein aggregation and propagation in Parkinson's disease remains unclear. Here, we established animal models of Parkinson's disease by inoculating monkeys and mice with α-synuclein pre-formed fibrils and observed elevated low-density lipoprotein receptor-related protein 1 levels in the striatum and substantia nigra, accompanied by dopaminergic neuron loss and increased α-synuclein levels. However, low-density lipoprotein receptor-related protein 1 knockdown efficiently rescued dopaminergic neurodegeneration and inhibited the increase in α-synuclein levels in the nigrostriatal system. In HEK293A cells overexpressing α-synuclein fragments, low-density lipoprotein receptor-related protein 1 levels were upregulated only when the N-terminus of α-synuclein was present, whereas an α-synuclein fragment lacking the N-terminus did not lead to low-density lipoprotein receptor-related protein 1 upregulation. Furthermore, the N-terminus of α-synuclein was found to be rich in lysine residues, and blocking lysine residues in PC12 cells treated with α-synuclein pre-formed fibrils effectively reduced the elevated low-density lipoprotein receptor-related protein 1 and α-synuclein levels. These findings indicate that low-density lipoprotein receptor-related protein 1 regulates pathological transmission of α-synuclein from the striatum to the substantia nigra in the nigrostriatal system via lysine residues in the α-synuclein N-terminus.
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The Chinese medicinal fungi "Sanghuang" have been long recognized for their significant and valued medicinal properties, as documented in ancient medical literature. However, in traditional folk medicine, various macrofungi sharing similar appearance, habitat, and therapeutic effects with Sanghuang were erroneously used. These Sanghuang-like fungi mainly belong to the Porodaedalea, Phellinus, and Inonotus genera within the Hymenochaetaceae family. Despite the establishment of the Sanghuangporus genus and the identification of multiple species, the emerging taxonomic references based on morphological, ITS, and mycelial structural features have been inadequate to differentiate Sanghuangporus and Sanghuang-like fungi. To address this limitation, this study presents the first comparative and phylogenetic analysis of Sanghuang-related fungi based on mitogenomes. Our results show that Sanghuangporus species show marked convergence in mitochondrial genomic features and form a distinct monophyletic group based on phylogenetic analyses of five datasets. These results not only deepen our understanding of Sanghuang-like fungi but also offer novel insights into their mitochondrial composition and phylogeny, thereby providing new research tools for distinguishing members of the Sanghuangporus genus. KEY POINTS: ⢠Sanghuangporus, Inonotus, and Porodaedalea are monophyly in sanghuang-like species. ⢠Mitogenome-based analysis exhibits high resolution in sanghuang-like genus. ⢠The mitogenomes provide strong evidence for reclassifying Phellinus gilvus S12 as Sanghuangporus vaninii.
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Filogenia , Genoma Mitocondrial , Basidiomycota/genética , Basidiomycota/classificação , DNA Fúngico/genética , Medicina Tradicional Chinesa , Análise de Sequência de DNARESUMO
Intelligent polymer nanocomposites are multicomponent and multifunctional materials that show immense potential across diverse applications. However, to exhibit intelligent traits such as adaptability, reconfigurability and dynamic properties, these materials often require a solvent or heating environment to facilitate the mobility of polymer chains and nanoparticles, rendering their applications in everyday settings impractical. Here intelligent azopolymer nanocomposites that function effectively in a solvent-free, room-temperature environment based on photocontrolled reversible solid-fluid transitions via switching flow temperatures (Tfs) are shown. A range of nanocomposites is synthesized through the grafting of Au nanoparticles, Au nanorods, quantum dots, or superparamagnetic nanoparticles with photoresponsive azopolymers. Leveraging the reversible cis-trans photoisomerization of azo groups, the azopolymer nanocomposites transition between solid (Tf above room temperature) and fluid (Tf below room temperature) states. Such photocontrolled reversible solid-fluid transitions empower the rewriting of nanopatterns, correction of nanoscale defects, reconfiguration of complex multiscale structures, and design of intelligent optical devices. These findings highlight Tf-switchable polymer nanocomposites as promising candidates for the development of intelligent nanomaterials operative in solvent-free, room-temperature conditions.
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The possible contamination routes, environmental adaptation, and genetic basis of Cronobacter spp. in infant and follow-up formula production factories and retailed products in mainland China have been determined by laboratory studies and whole-genome comparative analysis in a 7-year nationwide continuous surveillance spanning from 2012 to 2018. The 2-year continuous multicenter surveillance of the production process (conducted in 2013 and 2014) revealed that the source of Cronobacter spp. in the dry-blending process was the raw dry ingredients and manufacturing environment (particularly in the vibro sieve and vacuum cleaner), while in the combined process, the main contamination source was identified as the packing room. It is important to note that, according to the contamination control knowledge obtained from the production process surveillance, the contamination rate of retail powdered infant formula (PIF) and follow-up formula (FUF) products in China decreased significantly from 2016 onward, after improving the hygiene management practices in factories. The prevalence of Cronobacter spp. in retailed PIF and FUF in China in 2018 was dramatically reduced from 1.55 % (61/3925, in 2012) to an average as low as 0.17 % (13/7655 in 2018). Phenotype determination and genomic analysis were performed on a total of 90 Cronobacter spp. isolates obtained from the surveillance. Of the 90 isolates, only two showed resistance to either cefazolin or cefoxitin. The multilocus sequence typing results revealed that C. sakazakii sequence type 1 (ST1), ST37, and C. malonaticus ST7 were the dominant sequence types (STs) collected from the production factories, while C. sakazakii ST1, ST4, ST64, and ST8 were the main STs detected in the retailed PIF and FUF nationwide. One C. sakazakii ST4 isolate (1.1 %, 1/90) had strong biofilm-forming ability and 13 isolates (14.4 %, 13/90) had weak biofilm-forming ability. Genomic analysis revealed that Cronobacter spp. have a relatively stable core-genome and an increasing pan-genome size. Plasmid IncFIB (pCTU3) was prevalent in this genus and some contained 14 antibacterial biocide- and metal-resistance genes (BMRGs) including copper, silver, and arsenic resistant genes. Plasmid IncN_1 was predicted to contain 6 ARGs. This is the first time that a multi-drug resistance IncN_1 type plasmid has been reported in Cronobacter spp. Genomic variations with respect to BMRGs, virulence genes, antimicrobial resistance genes (ARGs), and genes involved in biofilm formation were observed among strains of this genus. There were apparent differences in copies of bcsG and flgJ between the biofilm-forming group and non-biofilm-forming group, indicating that these two genes play key roles in biofilm formation. The findings of this study have improved our understanding of the contamination characteristics and genetic basis of Cronobacter spp. in PIF and FUF and their production environment in China and provide important guidance to reduce contamination with this pathogen during the production of PIF and FUF.
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Cronobacter , Fórmulas Infantis , China , Cronobacter/genética , Microbiologia de Alimentos , Contaminação de Alimentos/análise , Humanos , LactenteRESUMO
A novel deoxygenative alkynylation of amides promoted by a synergistic action of a divalent rare-earth element and a transition metal has been developed. In this method, α-alkynyl substituted amines are synthesized from unactivated amides and alkynes in a single transformation. Broad substrate scope and excellent selectivity for CîO cleavage has been demonstrated. This approach represents a general method for the construction of versatile α-alkynyl substituted amines from unactivated amide bonds.
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Compared with the healthy patients, patients with osteoporosis had a lower Hounsfield unit (HU) value and a higher vertebral bone quality (VBQ) score. Both the HU value and VBQ score can simply distinguish patients with osteoporosis (OP), with a cutoff value of HU value < 97.06 and VBQ score > 3.08. INTRODUCTION: The purpose of this study is to determine whether the opportunistic use of computed tomography (CT) or magnetic resonance imaging (MRI) is effective for identifying spine surgical patients with OP. METHODS: We retrospectively evaluated 109 lumbar spine surgery patients who received lumbar quantitative CT (QCT) and MRI. Using the area under the curve, the CT-based HU value and MRI-based VBQ score were calculated. Then, based on the QCT results, receiver operating characteristic (ROC) curves were constructed to determine the diagnostic performance of the HU value and VBQ score. RESULTS: The HU value was significantly lower in the OP group, and the VBQ score was significantly higher in the OP group. Using the area under the curve, the diagnostic performance of the HU value and VBQ score for OP were 0.959 and 0.880, respectively. The diagnostic threshold values determined with optimal sensitivity and specificity were an HU value of 97.06 and a VBQ score of 3.08. CONCLUSION: Opportunistic use of CT and MRI can simply distinguish patients with OP, which are expected to be potential alternatives to T-score for the osteoporosis screening.
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Densidade Óssea , Vértebras Lombares , Imageamento por Ressonância Magnética , Osteoporose , Tomografia Computadorizada por Raios X , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/fisiopatologia , Feminino , Idoso , Imageamento por Ressonância Magnética/métodos , Estudos Retrospectivos , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X/métodos , Masculino , Osteoporose/diagnóstico por imagem , Densidade Óssea/fisiologia , Idoso de 80 Anos ou mais , Programas de Rastreamento/métodos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: To determine COVID-19 vaccine uptake among physicians in Ontario, Canada from 14 December 2020 to 13 February 2022. DESIGN: Population-based retrospective cohort study. SETTING: All registered physicians in Ontario, Canada using data from linked provincial administrative healthcare databases. PARTICIPANTS: 41 267 physicians (including postgraduate trainees) who were Ontario residents and registered with the College of Physicians and Surgeons of Ontario were included. Physicians who were out of province, had not accessed Ontario Health Insurance Plan-insured services for their own care for ≥5 years and those with missing identifiers were excluded. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcomes were the proportions of physicians who were recorded to have received at least one, at least two and three doses of a Health Canada-approved COVID-19 vaccine by study end date. Secondary outcomes were how uptake varied by physician characteristics (including age, sex, specialty and residential location) and time elapsed between doses. RESULTS: Of 41 267 physicians, (56% male, mean age 47 years), 39 359 (95.4%) received at least one dose, 39 148 (94.9%) received at least two doses and 35 834 (86.8%) received three doses of a COVID-19 vaccine. Of those who received three doses, the proportions were 90.4% among those aged ≥60 years and 81.2-89.5% among other age groups; 88.7% among family physicians and 89% among specialists. 1908 physicians (4.6%) had no record of vaccination, and this included 3.4% of family physicians and 4.1% of specialists; however, 28% of this group had missing specialty information. CONCLUSIONS: In Ontario, within 14 months of COVID-19 vaccine availability, 86.8% of physicians had three doses of a COVID-19 vaccine, compared with 45.6% of the general population. Findings may signify physicians' confidence in the safety and effectiveness of COVID-19 vaccines.
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Vacinas contra COVID-19 , COVID-19 , Médicos , Humanos , Ontário , Masculino , Feminino , Estudos Retrospectivos , Vacinas contra COVID-19/administração & dosagem , Pessoa de Meia-Idade , COVID-19/prevenção & controle , Adulto , Médicos/estatística & dados numéricos , SARS-CoV-2 , Vacinação/estatística & dados numéricos , Idoso , Padrões de Prática Médica/estatística & dados numéricosRESUMO
A robust palladium-catalyzed Suzuki-Miyaura reaction of carboxylic-phosphoric anhydrides via highly selective C(O)-O bond cleavage under inorganic base-free conditions has been reported. Carboxylic-phosphoric anhydrides, generated through activating carboxylic acids using phosphates by esterification or direct dehydrogenative reaction with phosphites, have been employed as highly reactive electrophiles for Suzuki-Miyaura cross-coupling reactions. Broad substrate scope and excellent functional group tolerance have been demonstrated to be a general and practical approach for the synthesis of highly valuable ketones.
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Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: ⢠Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. ⢠Three polyketides and one asterriquinone were isolated from HDAC deleted strain. ⢠Two different PKSs were reported in C. olivaceum SD-80A.
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Chaetomium , Histona Desacetilases , Família Multigênica , Policetídeos , Metabolismo Secundário , Chaetomium/genética , Chaetomium/enzimologia , Chaetomium/metabolismo , Metabolismo Secundário/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Policetídeos/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Vias Biossintéticas/genética , Epigênese GenéticaRESUMO
The genus Agrocybe, situated within the Strophariaceae family, class Agaricomycetes, and phylum Basidiomycota, encompasses a myriad of species exhibiting significant biological activities. This review presents an integrative overview of the secondary metabolites derived from Agrocybe species, elucidating their respective biological activities and potential pharmacological applications. The metabolites under scrutiny encompass a diverse array of biological macromolecules, specifically polysaccharides and lectins, as well as a diverse group of 80 documented small molecular chemical constituents, classified into sterols, sesquiterpenes, volatile compounds, polyenes, and other compounds, their manifesting anti-inflammatory, anticancer, antioxidant, hepatoprotective, antimicrobial, and antidiabetic activities, these metabolites, in which polysaccharides exhibit abundant activities, underscore the potential of the Agrocybe genus as a valuable source of biologically active natural products. The present review emphasises the need for escalated research into Agrocybe, including investigations into the biosynthetic pathways of these metabolites, which could foster the development of novel pharmaceutical therapies to address various health challenges.
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Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. During in vitro culture, many stressful conditions can affect embryo quality and viability, leading to adverse clinical outcomes such as abortion and congenital abnormalities. In this study, we found that valeric acid (VA) increased the mitochondrial membrane potential and ATP content, decreased the level of reactive oxygen species that the mitochondria generate, and thus improved mitochondrial function during early embryonic development in pigs. VA decreased expression of the autophagy-related factors LC3B and BECLIN1. Interestingly, VA inhibited expression of autophagy-associated phosphorylation-adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylation-UNC-51-like autophagy-activated kinase 1 (p-ULK1, Ser555), and ATG13, which reduced apoptosis. Short-chain fatty acids (SCFAs) can signal through G-protein-coupled receptors on the cell membrane or enter the cell directly through transporters. We further show that the monocarboxylate transporter 1 (MCT1) was necessary for the effects of VA on embryo quality, which provides a new molecular perspective of the pathway by which SCFAs affect embryos. Importantly, VA significantly inhibited the AMPK-ULK1 autophagic signaling pathway through MCT1, decreased apoptosis, increased expression of embryonic pluripotency genes, and improved embryo quality.
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Proteínas Quinases Ativadas por AMP , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Desenvolvimento Embrionário , Mitocôndrias , Transportadores de Ácidos Monocarboxílicos , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Suínos/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transdução de Sinais/efeitos dos fármacos , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Técnicas de Cultura Embrionária/veterinária , SimportadoresRESUMO
Eremophilanes exhibit diverse biological activities and chemical structures. This study reports the bioinformatics-guided reconstitution of the biosynthetic machinery of fungal eremophilanes, eremofortin C and sporogen-AO1, to elucidate their biosynthetic pathways. Their biosyntheses include P450-catalyzed multistep oxidation and enzyme-catalyzed isomerization by the DUF3237 family protein. Successful characterization of six P450s enabled us to discuss the functions of eremophilane P450s in putative eremophilane biosynthetic gene clusters, providing opportunities to understand the oxidative modification pathways of fungal eremophilanes.
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Sesquiterpenos , Oxirredução , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Fungos/química , Fungos/metabolismo , Vias Biossintéticas , Biologia Computacional/métodosRESUMO
Flammulina velutipes, a popular edible mushroom, contains sesquiterpenes with potential health benefits. We characterized 12 sesquiterpene synthases and one P450 enzyme in F. velutipes using Aspergillus oryzae as a heterologous expression system, culminating in the biosynthesis of 16 distinct sesquiterpene compounds. An enzyme encoded by the axeB gene responsible for the synthesis of the spiro [4.5] decane compound axenol was discovered, and the mechanism of spirocycle formation was elucidated through quantum mechanical calculations. Furthermore, we delineated the role of a P450 enzyme colocated with AxeB in producing the novel compound 3-oxo-axenol. Our findings highlight the diverse array of sesquiterpene skeletons and functional groups biosynthesized by these enzymes in F. velutipes and underscore the effectiveness of the A. oryzae system as a heterologous host for expressing genes in the Basidiomycota genome. These insights into the biosynthesis of bioactive compounds in F. velutipes have significant implications for functional food and drug development.
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Diarrheagenic Escherichia coli (DEC) is a kind of foodborne pathogen that poses a significant threat to both food safety and human health. To address the current challenges of high prevalence and difficult subtyping of DEC, this study developed a method that combined multiplex PCR with high-resolution melting (HRM) analysis for subtyping 5 kinds of DEC. The target genes are amplified by multiplex PCR in a single well, and HRM curve analysis was applied for distinct amplicons based on different melting temperature (Tm) values. The method enables discrimination of different DEC types based on characteristic peaks and distinct Tm values in the thermal melting curve. The assay exhibited 100% sensitivity and 100% specificity with a detection limit of 0.5 to 1 ng/µL. The results showed that different DNA concentrations did not influence the subtyping results, demonstrating this method owed high reliability and stability. In addition, the method was also used for the detection and subtyping of DEC in milk. This method streamlines operational procedures, shorts the detection time, and offers a novel tool for subtyping DEC.
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Escherichia coli , Leite , Reação em Cadeia da Polimerase em Tempo Real , Leite/microbiologia , Animais , Escherichia coli/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sensibilidade e Especificidade , Reprodutibilidade dos TestesRESUMO
As a conservation and breeding institution for birds, Taipei Zoo plays an important role in restoring endangered species. As approximately half of all bird species are monomorphic, precisely confirming the sex of individuals is critical for the management of ex-situ conservation breeding populations, as well as for understanding the sex ratio of those in the wild. Generally, PCR is used more reliably for sex determination versus traditional methods such as plumage, behavior or hormone levels. Nevertheless, the various primer sets and annealing temperatures vary between species, and so inaccurate sexing can occasionally happen due to inadequate PCR conditions. To reduce the probability of misidentification, and to establish a PCR condition database for sex determination across the diverse range of avian taxa, we tested multiple primer sets and annealing temperatures for amplification of the bird sex-specific gene fragments (CHD1) for each captive or rescued avian species held at Taipei Zoo since 2014. A total of 162 species across 22 orders were tested using one or two primer sets. One hundred and fifty-five species were successfully sexed by the primer set 2550F/2718R and the success rate of sex typing reached over 90% of species tested in each order. Most species have suitable PCR annealing temperatures between 45°C and 55°C, and the species in the same avian taxa showed similar results in temperature. This indicates that it is possible to select the annealing temperature of other species in the same family when the species had not been tested before. We expect this study will improve the success rate of identifying sex by using applicable PCR conditions and reduce the time for searching references every time before attempts to PCR sex birds.
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Animais de Zoológico , Aves , Análise para Determinação do Sexo , Animais , Aves/fisiologia , Aves/genética , Aves/classificação , Análise para Determinação do Sexo/métodos , Análise para Determinação do Sexo/veterinária , Taiwan , Feminino , Masculino , Reação em Cadeia da Polimerase/veterinária , Espécies em Perigo de ExtinçãoRESUMO
Menispermaceae species, as early-diverging eudicots, can synthesize valuable benzylisoquinoline alkaloids (BIAs) like bisbenzylisoquinoline alkaloids (bisBIAs) and sinomenines with a wide range of structural diversity. However, the evolutionary mechanisms responsible for their chemo-diversity are not well understood. Here, a chromosome-level genome assembly of Menispermum dauricum is presented and demonstrated the occurrence of two whole genome duplication (WGD) events that are shared by Ranunculales and specific to Menispermum, providing a model for understanding chromosomal evolution in early-diverging eudicots. The biosynthetic pathway for diverse BIAs in M. dauricum is reconstructed by analyzing the transcriptome and metabolome. Additionally, five catalytic enzymes - one norcoclaurine synthase (NCS) and four cytochrome P450 monooxygenases (CYP450s) - from M. dauricum are responsible for the formation of the skeleton, hydroxylated modification, and C-O/C-C phenol coupling of BIAs. Notably, a novel leaf-specific MdCYP80G10 enzyme that catalyzes C2'-C4a phenol coupling of (S)-reticuline into sinoacutine, the enantiomer of morphinan compounds, with predictable stereospecificity is discovered. Moreover, it is found that Menispermum-specific CYP80 gene expansion, as well as tissue-specific expression, has driven BIA diversity in Menispermaceae as compared to other Ranunculales species. This study sheds light on WGD occurrences in early-diverging eudicots and the evolution of diverse BIA biosynthesis.
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Benzilisoquinolinas , Sistema Enzimático do Citocromo P-450 , Menispermaceae , Benzilisoquinolinas/metabolismo , Benzilisoquinolinas/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Menispermaceae/genética , Menispermaceae/metabolismo , Menispermaceae/química , Alcaloides/metabolismo , Filogenia , Evolução Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: ⢠The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. ⢠TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. ⢠The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.