RESUMO
BACKGROUND: There has not been a consensus on the prothesis sizing strategy in type 0 bicuspid aortic stenosis (AS) patients undergoing transcatheter aortic valve replacement (TAVR). Modifications to standard annular sizing strategies might be required due to the distinct anatomical characteristics. We have devised a downsizing strategy for TAVR using a self-expanding valve specifically for patients with type 0 bicuspid AS. The primary aim of this study is to compare the safety and efficacy of downsizing strategy with the Standard Annulus Sizing Strategy in TAVR for patients with type 0 bicuspid AS. TRIAL DESIGN: It is a prospective, multi-center, superiority, single-blinded, randomized controlled trial comparing the Down Sizing and Standard Annulus Sizing Strategy in patients with type 0 bicuspid aortic stenosis undergoing transcatheter aortic valve replacement. Eligible participants will include patients with severe type 0 bicuspid AS, as defined by criteria such as mean gradient across aortic valve ≥40 mmHg, peak aortic jet velocity ≥4.0 m/s, aortic valve area (AVA) ≤1.0 cm², or AVA index ≤0.6 cm2/m2. These patients will be randomly assigned, in a 1:1 ratio, to either the Down Sizing Strategy group or the Standard Sizing Strategy group. In the Down Sizing Strategy group, a valve one size smaller will be implanted if the "waist sign" manifests along with less than mild regurgitation during balloon pre-dilatation. The primary end point of the study is a composite of VARC-3 defined device success, absence of both permanent pacemaker implantation due to high-degree atrioventricular block and new-onset complete left bundle branch block. CONCLUSION: This study will compare the safety and efficacy of Down Sizing Strategy with the Standard Annulus Sizing Strategy and provide valuable insights into the optimal approach for sizing in TAVR patients with type 0 bicuspid AS. We hypothesize that the Down Sizing Strategy will demonstrate superiority when compared to the Standard Annulus Sizing Strategy. (Down Sizing Strategy (HANGZHOU Solution) vs Standard Sizing Strategy TAVR in Bicuspid Aortic Stenosis (Type 0) (TAILOR-TAVR), NCT05511792).
Assuntos
Estenose da Valva Aórtica , Doença da Válvula Aórtica Bicúspide , Próteses Valvulares Cardíacas , Desenho de Prótese , Substituição da Valva Aórtica Transcateter , Humanos , Substituição da Valva Aórtica Transcateter/métodos , Estenose da Valva Aórtica/cirurgia , Doença da Válvula Aórtica Bicúspide/cirurgia , Doença da Válvula Aórtica Bicúspide/complicações , Estudos Prospectivos , Método Simples-Cego , Valva Aórtica/cirurgia , Valva Aórtica/anormalidades , Valva Aórtica/diagnóstico por imagem , Masculino , FemininoRESUMO
Mitral regurgitation (MR) is the most common heart valve disease, and transcatheter edge-to-edge repair (TEER) has been recommended as a therapy for severe MR patients by guidelines. The classic Carpentier classification used to guide surgical mitral valve repair but is inadequate for mitral TEER (M-TEER). We herein proposed a new modified Carpentier classification named after "type + segment," which is suitable for M-TEER. We shared our strategies in M-TEER procedure for screening and performing the M-TEER according to the new modified Carpentier classification.
RESUMO
Yarrowia lipolytica N12 and A13 with high lipase activity obtained by mutagenesis were inoculated into sour meat, and their effects on physicochemical properties, microbial community succession, free amino acids, and volatile compounds of sour meat were investigated. Inoculation fermentation increased the contents of free amino acids observably, rapidly reduced pH, promoted the accumulation of total acids, decreased 2-thiobarbituric acid reactive substances (TBARS) values. In addition, the addition of Y. lipolytica might contribute to the growth of lactic acid bacteria, Candida spp., and Debaryomyces udenii, which play an important role in production of volatile compounds. It was shown that inoculation promoted the production of esters, aldehydes, and alcohols, especially ethyl esters, giving sour meat a better meat flavor. Besides, it was found that Y. lipolytica A13 had better fermenting property. Sample of A13 group had higher contents of ethyl esters, free amino acids and dominant microorganisms. The results may help to provide new strains for sour meat fermentation.
Assuntos
Lactobacillales , Saccharomycetales , Yarrowia , Yarrowia/genética , Ésteres/metabolismo , Lactobacillales/genética , Lactobacillales/metabolismo , Fermentação , Aminoácidos/metabolismo , CarneRESUMO
BACKGROUND: There are limited data available on the development of arrhythmias in patients at risk of high-degree atrioventricular block (HAVB) or complete heart block (CHB) following transcatheter aortic valve replacement (TAVR). OBJECTIVE: This study aimed to explore the incidence and evolution of arrhythmias by monitoring patients at risk of HAVB or CHB after TAVR using smartwatches. METHODS: We analyzed 188 consecutive patients in the prospective SMART TAVR (smartwatch-facilitated early discharge in patients undergoing TAVR) trial. Patients were divided into 2 groups according to the risk of HAVB or CHB. Patients were required to trigger a single-lead electrocardiogram (ECG) recording and send it to the Heart Health App via their smartphone. Physicians in the central ECG core lab would then analyze the ECG. The incidence and timing of arrhythmias and pacemaker implantation within a 30-day follow-up were compared. All arrhythmic events were adjudicated in a central ECG core lab. RESULTS: The mean age of the patients was 73.1 (SD 7.3) years, of whom 105 (55.9%) were men. The mean discharge day after TAVR was 2.0 (SD 1.8) days. There were no statistically significant changes in the evolution of atrial fibrillation or atrial flutter, Mobitz I, Mobitz II, and third-degree atrial ventricular block over time in the first month after TAVR. The incidence of the left bundle branch block (LBBB) increased in the first week and decreased in the subsequent 3 weeks significantly (P<.001). Patients at higher risk of HAVB or CHB received more pacemaker implantation after discharge (n=8, 9.6% vs n=2, 1.9%; P=.04). The incidence of LBBB was higher in the group with higher HAVB or CHB risk (n=47, 56.6% vs n=34, 32.4%; P=.001). The independent predictors for pacemaker implantation were age, baseline atrial fibrillation, baseline right bundle branch block, Mobitz II, and third-degree atrioventricular block detected by the smartwatch. CONCLUSIONS: Except for LBBB, no change in arrhythmias was observed over time in the first month after TAVR. A higher incidence of pacemaker implantation after discharge was observed in patients at risk of HAVB or CHB. However, Mobitz II and third-degree atrioventricular block detected by the smartwatch during follow-ups were more valuable indicators to predict pacemaker implantation after discharge from the index TAVR. TRIAL REGISTRATION: ClinicalTrials.gov NCT04454177; https://clinicaltrials.gov/study/NCT04454177.
Assuntos
Arritmias Cardíacas , Substituição da Valva Aórtica Transcateter , Humanos , Substituição da Valva Aórtica Transcateter/efeitos adversos , Masculino , Feminino , Idoso , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Estudos Prospectivos , Idoso de 80 Anos ou mais , Eletrocardiografia , Bloqueio Atrioventricular/etiologia , Bloqueio Atrioventricular/terapiaRESUMO
BACKGROUND: Ergothioneine (EGT) is a high-value food functional factor that cannot be synthesized by humans and other vertebrates, and the low yield limits its application. RESULTS: In this study, the optimal fermentation temperature, fermentation time, initial pH, inoculum age, and inoculation ratio on EGT biosynthesis of Rhodotorula mucilaginosa DL-X01 were optimized. In addition, the effects of three key precursor substances - histidine, methionine, and cysteine - on fungal EGT synthesis were verified. The optimal conditions were further obtained by response surface optimization. The EGT yield of R. mucilaginosa DL-X01 under optimal fermentation conditions reached 64.48 ± 2.30 mg L-1 at shake flask fermentation level. Finally, the yield was increased to 339.08 ± 3.31 mg L-1 (intracellular) by fed-batch fermentation in a 5 L bioreactor. CONCLUSION: To the best of our knowledge, this is the highest EGT yield ever reported in non-recombinant strains. The fermentation strategy described in this study will promote the efficient biosynthesis of EGT in red yeast and its sustainable production in the food industry. © 2024 Society of Chemical Industry.
Assuntos
Ergotioneína , Monascus , Rhodotorula , Humanos , Animais , Rhodotorula/genética , Rhodotorula/metabolismo , Antioxidantes/metabolismo , Histidina , Fermentação , Monascus/metabolismoRESUMO
BACKGROUND: Studies have shown that either the addition of starter culture or enzyme can improve fermentation in fish or other products. However, little research has been carried out on the effects of coupling starter cultures with lipase on the microbial community and product quality. Suanzhayu is a Chinese fermented fish product that mainly relies on spontaneous fermentation, resulting in an unstable flavor and quality. The present study investigated the impact of lipase and Lactiplantibacillus plantarum 1-24-LJ on the quality of Suanzhayu. RESULTS: Inoculation decreased pH and 2-thiobarbituric acid reactive substances (TBARS) values, and also helped the dominance of the strain in the ecosystem, whereas lipase addition raised TBARS values and had little effect on pH, water activity (aw ) and microbiota. The addition of lipase and/or Lpb. plantarum increased the content of alcohols, aldehydes, ketones, esters and umami amino acids. The co-additions with the most significant effect and the total contents of volatile compounds (VCs) and free amino acids (FAAs) were 1801.92 g per 100 g and 21 357.05 mg per 100 g, respectively. Former-Lactobacillus was negatively correlated with pH, aw and Prevotella, but positively with VCs (ethyl ester of heptanoic acid, ethyl ester of octanoic acid) and FAAs (Tyr, Phe). Furthermore, adding Lpb. plantarum 1-24-LJ alone or in combination with lipase shortened the fermentation process. CONCLUSION: The present study provides a recommended Suanzhayu process approach for improving product quality and flavor, as well as shortening fermentation time, by adding Lpb. plantarum 1-24-LJ with or without lipase. © 2023 Society of Chemical Industry.
Assuntos
Lactobacillus plantarum , Animais , Lactobacillus plantarum/metabolismo , Lipase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ecossistema , Microbiologia de Alimentos , Fermentação , Aminoácidos/metabolismoRESUMO
BACKGROUND: Unlike N-terminal pro-B-type natriuretic peptide (NT-proBNP), which have been extensively studied, little is known about the role of N-terminal pro-C-type natriuretic peptide (NT-proCNP) for predicting survival post transcatheter aortic valve replacement (TAVR). METHODS: A total of 309 patients were included in the analysis. Patients were grouped into quartiles (Q1-4) according to the baseline NT-proCNP value. Blood for NT-proCNP analysis was obtained prior to TAVR procedure. The primary endpoint was mortality after a median follow-up of 32 months. Multivariable Cox proportional hazards regression models analyzed prognostic factors. The predictive capability was compared between NT-proBNP and NT-proCNP using receiver operator curve (ROC) analysis. RESULTS: A total of 309 subjects with the mean age of 76.8 ± 6.3 years, among whom 58.6% were male, were included in the analysis. A total of 58 (18.8%) patients died during follow-up. Cox multivariable analyses indicated society of thoracic surgeons (STS)-score was a strong independent predictor for mortality (hazard ratio (HR) 1.08, 95% confidential interval (CI) 1.05-1.12, P < 0.001). Elevated NT-proCNP was associated with a higher risk of cardiovascular mortality (HR 1.02, 95% CI 1.00-1.03, P = 0.025) and All-cause mortality (HR 1.01, 95% CI 1.00-1.03, P = 0.027), whereas NT-proBNP showed a small effect size on mortality. ROC analysis indicated that NT-proCNP was superior to NT-proBNP for TAVR risk evaluation in patients with left ventricular ejection fraction (LVEF) < 50% [(Area under the curve (AUC)-values of 0.79 (0.69; 0.87) vs. 0.59 (0.48; 0.69), P = 0.0453]. CONCLUSIONS: NT-proCNP and STS-Score were the independent prognostic factors of mortality among TAVR patients. Furthermore, NT-proCNP was superior to NT-proBNP for TAVR risk evaluation in patients with LVEF < 50%. Trial registration NCT02803294, 16/06/2016.
Assuntos
Substituição da Valva Aórtica Transcateter , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Diuréticos , Humanos , Masculino , Peptídeo Natriurético Encefálico , Peptídeo Natriurético Tipo C , Fragmentos de Peptídeos , Prognóstico , Volume Sistólico , Substituição da Valva Aórtica Transcateter/efeitos adversos , Vasodilatadores , Função Ventricular EsquerdaRESUMO
Intracranial aneurysms (IAs) remain a major public health concern and endovascular treatment (EVT) has become a major tool for managing IAs. However, the recurrence rate of IAs after EVT is relatively high, which may lead to the risk for aneurysm re-rupture and re-bleed. Thus, we aimed to develop and assess prediction models based on machine learning (ML) algorithms to predict recurrence risk among patients with IAs after EVT in 6 months. Patient population included patients with IAs after EVT between January 2016 and August 2019 in Hunan Provincial People's Hospital, and an adaptive synthetic (ADASYN) sampling approach was applied for the entire imbalanced dataset. We developed five ML models and assessed the models. In addition, we used SHapley Additive exPlanations (SHAP) and local interpretable model-agnostic explanation (LIME) algorithms to determine the importance of the selected features and interpret the ML models. A total of 425 IAs were enrolled into this study, and 66 (15.5%) of which recurred in 6 months. Among the five ML models, gradient boosting decision tree (GBDT) model performed best. The area under curve (AUC) of the GBDT model on the testing set was 0.842 (sensitivity: 81.2%; specificity: 70.4%). Our study firstly demonstrated that ML-based models can serve as a reliable tool for predicting recurrence risk in patients with IAs after EVT in 6 months, and the GBDT model showed the optimal prediction performance.
Assuntos
Aneurisma Roto , Aneurisma Intracraniano , Algoritmos , Aneurisma Roto/epidemiologia , Aneurisma Roto/cirurgia , Área Sob a Curva , Humanos , Aneurisma Intracraniano/cirurgia , Aprendizado de MáquinaRESUMO
ABSTRACT: This study aims to introduce a new sagittal cephalometric measurement, the sagittal G-triangle analysis, to accurately and reproducibly assess the sagittal jaw relationship. Sagittal G-triangle analysis, which consists of angles AXK and BXK, is based on an equilateral triangle (Bo-X-K) constructed using 5 cephalometric landmarks (Ba, Bo, Po, Or, and G). To test the diagnostic efficiency of this analysis, pretreatment cephalometric radiographs of 120 female and 120 male Chinese patients were randomly selected. For each enlisted subject, angles SNA and SNB as well as angles AXK and BXK were measured and recorded. On the basis of the SNA and SNB results, subjects were categorized into 6 groups: maxillary retrognathism, normal maxilla, maxillary prognathism, mandibular retrognathism, normal mandible, and mandibular prognathism. The diagnostic efficiency of angles AXK and BXK were evaluated using various statistical tests. A high correlation was detected between angles SNA and AXK as well as between angles SNB and BXK. Female patients with angle AXK between -2.255° and 2.860° and male patients with angle AXK between -2.615° and 2.120° were considered to have a normal maxilla position. Female patients with angle BXK between -2.61° and 2.93° and male patients with angle BXK between -2.275° and 0.610° were considered to have a normal mandible position. In conclusion, sagittal G-triangle analysis could be used as an alternative method for the evaluation of the sagittal position of the maxilla and mandible in cephalometric analysis.
Assuntos
Má Oclusão Classe III de Angle , Má Oclusão , Prognatismo , Retrognatismo , Cefalometria/métodos , Feminino , Humanos , Masculino , Mandíbula/diagnóstico por imagem , Maxila/diagnóstico por imagem , Retrognatismo/diagnóstico por imagemRESUMO
Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.
Assuntos
Agrobacterium tumefaciens/genética , Regulação Fúngica da Expressão Gênica , Mutação , Rhodotorula , Transcrição Gênica , beta Caroteno , Acetofenonas/metabolismo , Rhodotorula/genética , Rhodotorula/metabolismo , beta Caroteno/biossíntese , beta Caroteno/genéticaRESUMO
BACKGROUND: Paocai is a traditional Chinese fermented vegetable food. As the most important ingredient, salt has crucial effects on the bacterial community and volatile compounds of paocai. To demonstrate the effects of salt on the fermentation of paocai, the bacterial composition and volatile compounds were investigated using high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS). RESULTS: The salt had no significant effects on the bacterial community at the phylum level. Proteobacteria and Bacteroidetes gradually decreased during the fermentation, and Firmicutes gradually increased as the dominant bacteria in the late stage of fermentation. At the genus level, Lactobacillus and Lactococcus gradually increased in relative abundance during the fermentation and became the dominant bacteria in paocai. High salt levels can contribute to the growth of Lactobacillus, which became the dominant genus in paocai. The salt concentration affected the profiles of volatile compounds in paocai after fermentation. A total of 42 volatile components were detected by GC-MS, among which phenols, aldehydes, and nitriles were the main ones. A high salt concentration will increase the volatile compound content, mainly aldehydes and alcohols, and improve the flavor of paocai. At the same time, the electronic tongue analysis also showed that a high salt concentration made a major contribution to the flavor of paocai. CONCLUSIONS: These data are helpful to elucidate the effects of salt on the quality of paocai and contribute to improving the quality and reducing the use of salt. © 2021 Society of Chemical Industry.
Assuntos
Bactérias/metabolismo , Alimentos Fermentados/análise , Cloreto de Sódio/análise , Produtos Vegetais/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , China , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Microbiota , Cloreto de Sódio/metabolismo , Produtos Vegetais/análise , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
Tooth transposition is an uncommon disorder related to ectopic eruption; it can be classified as complete or incomplete on the basis of the position of the crowns and roots of transposed teeth. Aligning the transposed teeth to a normal sequence is always complex and challenging, especially in patients with complete transposition. The segmented archwire technique with cantilever or loops has been used in many transposition patients; however, it requires considerable laboratory work and is sometimes uncomfortable for the patient. In this case report, we present a novel orthodontic treatment for an 8-year-old boy with unilateral complete transposition of the maxillary central incisor and lateral incisor (Mx.I2.I1). During the alignment stage, the lateral incisor was moved palatally to bypass the central incisor, using a 0.012-in nickel-titanium wire continuously. Active orthodontic treatment was conducted for 44 months, and the final outcome was esthetically and functionally effective. Stable and satisfactory results were achieved within 4 years of follow-up.
Assuntos
Anormalidades Dentárias , Erupção Ectópica de Dente , Criança , Dente Canino , Humanos , Incisivo , Masculino , Maxila/diagnóstico por imagem , Maxila/cirurgiaRESUMO
Suancai is a popular fermented product of Brassica vegetable in China. As important additive, salt concentration has crucial effects on the quality of suancai. To investigate the effects of salt concentration on suancai fermentation, the microbial diversity and volatile compounds (VCs) during fermentation were investigated by using Illumina HiSeq sequencing and GC-MS. Firmicutes, Proteobacteria and Ascomycota were detected as the main phylum during the fermentation with different salt concentrations. Lactobacillus, Lactococcus, Klebsiella, Weissella, Pediococcus, Candida, Cladosporium, Gibberella, Aspergillus, etc., were detected were observed during the fermentation with different concentrations. After fermentation, Lactobacillus predominated the fermentation of suancai and was not affected by salt concentration. Pediococcus, Leuconostoc, Weissella, Sporobolomyces, Azospirillum, Klebsiella, Acinetobacter and Cladosporium were significant affected by salt concentration. Salt addition could affect the VCs profiles and reduce the isothiocyanates after fermentation. Seventy-nine VCs were detected and strongly correlated with the dominant genus Lactobacillus during suancai fermentation. The inoculated fermentation of Lactobacillus could improve the VCs during fermentation. In conclusion, 6% salt addition could acquire a higher Lactobacillus abundance and a better taste quality. These results may facilitate the understanding of the effect of salt concentration on the fermentation ecology to improve suancai characteristics.
Assuntos
Alimentos Fermentados/análise , Alimentos Fermentados/microbiologia , Microbiota/genética , Cloreto de Sódio/análise , Compostos Orgânicos Voláteis/análise , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , Brassica/microbiologia , China , Fermentação , Microbiologia de Alimentos , Qualidade dos Alimentos , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Compostos Orgânicos Voláteis/químicaRESUMO
The red yeast Rhodosporidium toruloides is a known lipid producer capable of accumulating large amounts of triacylglycerols and carotenoids. However, it remains challenging to study its carotenoid production profiles owing to limited biochemical information and inefficient genetic tools. Here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) to change its carotenoid production and profiles. We constructed R. toruloides NP11 mutant libraries with ATMT, selected three mutants with different colours, characterized their carotenoid products by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis and assured differences among those strains in terms of carotenoid production and its composition profiles. We then located T-DNA insertion sites using the genome walking technology and provided discussions in terms of the new phenotypes. This study is the first of its kind to change the carotenoid production profiles in R. toruloides. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Carotenoides/biossíntese , Carotenoides/química , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Rhodotorula/genética , Rhodotorula/metabolismo , Transformação Genética , Agrobacterium tumefaciens/genética , Cromatografia Líquida de Alta Pressão , Mapeamento Cromossômico , Passeio de Cromossomo , Genótipo , Espectrometria de Massas , Mutagênese Insercional , FenótipoRESUMO
OBJECTIVES: To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. RESULTS: The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. CONCLUSIONS: Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.
Assuntos
Agrobacterium/genética , Vias Biossintéticas/genética , Carotenoides/biossíntese , Marcação de Genes , Rhodotorula/genética , Rhodotorula/metabolismo , Transformação Genética , Técnicas de Inativação de Genes , Recombinação Homóloga , Análise de Sequência de DNARESUMO
The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to the basidiomycetous group of fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled a reporter gene expression cassette, consisting of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted it into the binary vector pZPK. Hygromycin-resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harbouring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than five-fold and followed a decreasing sequence of PPGI, PPGK, PFBA, PTPI, and PGPD. This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals.
Assuntos
Basidiomycota/genética , Engenharia Genética , Vetores Genéticos/genética , Genoma Fúngico/genética , Regiões Promotoras Genéticas/genética , Basidiomycota/efeitos dos fármacos , Cinamatos/farmacologia , Clonagem Molecular , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Fenótipo , Transformação GenéticaRESUMO
Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.
Assuntos
Gotículas Lipídicas/metabolismo , Proteoma/metabolismo , Ustilaginales/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Proteoma/genética , Ustilaginales/genéticaRESUMO
Monodisperse metal clusters provide a unique platform for investigating magnetic exchange within molecular magnets. Herein, the core-shell structure of the monodisperse molecule magnet of [Gd52 Ni56 (IDA)48 (OH)154 (H2 O)38 ]@SiO2 (1 a@SiO2 ) was prepared by encapsulating one high-nuclearity lanthanide-transition-metal compound of [Gd52 Ni56 (IDA)48 (OH)154 (H2 O)38 ]â (NO3 )18 â 164 H2 O (1) (IDA=iminodiacetate) into one silica nanosphere through a facile one-pot microemulsion method. 1 a@SiO2 was characterized using transmission electron microscopy, N2 adsorption-desorption isotherms, and inductively coupled plasma-atomic emission spectrometry. Magnetic investigation of 1 and 1 a revealed J1 =0.25â cm(-1) , J2 =-0.060â cm(-1) , J3 =-0.22â cm(-1) , J4 =-8.63â cm(-1) , g=1.95, and z J=-2.0×10(-3) â cm(-1) for 1, and J1 =0.26â cm(-1) , J2 =-0.065â cm(-1) , J3 =-0.23â cm(-1) , J4 =-8.40â cm(-1) g=1.99, and z J=0.000â cm(-1) for 1 a@SiO2 . The z J=0 in 1 a@SiO2 suggests that weak antiferromagnetic coupling between the compounds is shielded by silica nanospheres.
RESUMO
The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation (ATMT) protocol for effective gene integration into the R. toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R. toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals.
Assuntos
Basidiomycota/genética , Técnicas de Transferência de Genes , Genética Microbiana/métodos , Genoma Fúngico , Engenharia Metabólica/métodos , Biologia Molecular/métodos , Recombinação Genética , Agrobacterium/genética , Vetores GenéticosRESUMO
High thermal conductivity aluminium-based silicon carbide (Al/SiC) composites were successfully fabricated through post-oxidization of reaction-bonded silicon carbide preforms (RS preforms), utilizing vacuum pressure infiltration technology. The study investigated the regulation of interfacial reactions in conventional sintering and reaction sintering. Conventional sintering introduced a large amount of SiO2, which negatively impacted the thermal conductivity of the Al/SiC composite, but the reaction sintering not. The proposed post-oxidization treatment of RS preforms effectively removed residual carbon from the SiC particle surfaces, thereby forestalling the formation of Al4C3. Furthermore, the post-oxidization treatment effectively formed lightweight SiO2 deposits onto the surface of SiC particles, improving Al-SiC interfacial wettability and reducing thermal resistance, thereby enhancing composite thermal conductivity. Notably, the thermal conductivity of the post-oxidized sample exhibited an increase of 6.5% compared to the untreated sample. The study also evaluated the impact of particle size distribution on volume fraction and thermal properties. The optimized Al/SiC composites yielded thermal conductivity, coefficient of thermal expansion, bending strength, and Young's modulus values of 237.3 W/m K, 8.5 × 10-6/°C, 325 MPa, and 75.9 GPa, respectively.