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Synthetic cells, expressing proteins using cell-free transcription-translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a "silencing oligo": a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence-dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells.
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Células Artificiais , Biossíntese de Proteínas , Escherichia coli/genética , Sistema Livre de Células/metabolismo , Transfecção , Inativação Gênica , RNA Interferente Pequeno/metabolismoRESUMO
The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.
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Proteínas de Bactérias/metabolismo , Mananas/metabolismo , Manose/metabolismo , Streptomyces/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Carboximetilcelulose Sódica/metabolismo , Galactanos/metabolismo , Galactose/análogos & derivados , Insetos/microbiologia , Manosidases/genética , Manosidases/metabolismo , Gomas Vegetais/metabolismo , Streptomyces/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
Several studies have suggested that non-stenotic carotid plaque was a risk factor for embolic stroke of undetermined source in some patients. However, individual backgrounds of these patients is unclear. We encountered a 64-years-old female with cerebral emboli, from an apparently stable non-stenotic carotid plaque (only 1.42mm thick) at the distal left common carotid artery, caused by violent tic movement of thyroid cartilage under well controlled dyslipidemia. Even though the plaque appeared thin and stable, mechanical stimulation could cause multiple, unnaturally localized emboli by stimulation-induced atherogenesis and plaque rupture, resulting in a misdiagnose of embolic stroke of undetermined source with non-stenotic carotid plaque.
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Doenças das Artérias Carótidas/complicações , Embolia Intracraniana/etiologia , Placa Aterosclerótica , Acidente Vascular Cerebral/etiologia , Cartilagem Tireóidea/inervação , Tiques/complicações , Doenças das Artérias Carótidas/diagnóstico por imagem , Diagnóstico Diferencial , Erros de Diagnóstico , Feminino , Humanos , Embolia Intracraniana/diagnóstico por imagem , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Acidente Vascular Cerebral/diagnóstico por imagem , Cartilagem Tireóidea/diagnóstico por imagem , Tiques/diagnóstico por imagem , Tiques/fisiopatologiaRESUMO
Objectives: Using a murine model of systemic Kawasaki disease (KD)-like vasculitis induced by Candida albicans cell-wall-derived mannan · ß-glucan · protein complexes, the objective was to elucidate the relationships of ß-glucan receptor dectin-1 (D1) and α-mannan receptor dectin-2 (D2) to the onset of that vasculitis.Methods: The incidence and histological severity of vasculitis were compared among mice lacking the genes for D1 or D2 (i.e. D1-/- and D2-/-) and wild-type (WT) mice.Results: The incidences of vasculitis in the three animal groups were 100% (18/18) in the WT group, 100% (18/18) in the D1-/- group, and 0% (0/18) in the D2-/- group. In the WT and D1-/- mice, severe inflammatory cell infiltration, consisting mainly of neutrophils and macrophages, was seen in the aortic root and the coronary arteries. On the other hand, in the D2-/- mice, not even mild vascular lesions such as endoarteritis were seen.Conclusion: Recognition of α-mannan by D2 played an important role in the onset of vasculitis in the studied murine model.
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Lectinas Tipo C/metabolismo , Mananas/farmacologia , Síndrome de Linfonodos Mucocutâneos/metabolismo , Vasculite/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Candida albicans/química , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Lectinas Tipo C/genética , Macrófagos/metabolismo , Mananas/toxicidade , Camundongos , Síndrome de Linfonodos Mucocutâneos/etiologia , Síndrome de Linfonodos Mucocutâneos/patologia , Vasculite/etiologia , Vasculite/patologiaRESUMO
Coronary subclavian steal syndrome (CSSS) is a coronary steal phenomenon secondary to subclavian artery stenosis in patients who have undergone coronary bypass surgery with the internal thoracic artery. Most commonly, CSSS is diagnosed angiographically. Our case emphasizes that stress ultrasound assessment using reactive hyperemia in the ipsilateral arm elicits a functional diagnosis of CSSS.
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Ponte de Artéria Coronária , Síndrome do Roubo Coronário-Subclávio/complicações , Síndrome do Roubo Coronário-Subclávio/diagnóstico por imagem , Hiperemia/etiologia , Complicações Pós-Operatórias/diagnóstico por imagem , Ultrassonografia/métodos , Idoso , Síndrome do Roubo Coronário-Subclávio/fisiopatologia , Antebraço/diagnóstico por imagem , Antebraço/fisiopatologia , Humanos , Hiperemia/fisiopatologia , Masculino , Complicações Pós-Operatórias/fisiopatologiaRESUMO
The morphological determinants of left ventricular outflow tract (LVOT) obstruction in hypertrophic cardiomyopathy (HCM) are not completely understood. We aimed to identify the anatomical risks of the obstruction using echocardiography.Fifty patients with untreated HCM were classified into two groups: those with LVOT pressure gradient (LVOTPG) ≥ 30 mmHg (obstructive HCM [HOCM] group) and those with LVOTPG < 30 mmHg (HNCM group). The echocardiographic morphological variables were analyzed to determine whether they were predictive of LVOT obstruction. Systolic anterior motions of the mitral valve were observed in 100% of patients in the HOCM group but only in 58% in the HNCM group. There were no significant differences in wall thickness, end-systolic LV dimension (LVDs), or LVOT diameter between the two groups. However, HOCM subjects had a shorter distance from papillary muscles to the inter-ventricular septum (5.97 ± 2.3 versus 9.20 ± 1.9 mm, respectively, P < 0.0001) and a longer anterior mitral leaflet (AML) length (24.7 ± 5.8 versus 20.1 ± 5.4 mm, respectively, P < 0.01) compared to the HNCM group. The AML length/LVDs ratio was significantly higher in the HOCM group compared to the HNCM group (1.02 ± 0.34 versus 0.78 ± 0.26, P < 0.01), and an LVOT obstruction was predicted with an area under the curve of 0.71 (P < 0.05). Multiple linear regression revealed that only the AML length/LVDs ratio was independently associated with LVOTPG (P < 0.01).The AML length/LVDs ratio has a significant predictive value for LVOT obstruction and a strong relationship with LVOTPGs. The AML length/LVDs ratio determines the anatomical risk of LVOT obstruction in HCM.
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Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/patologia , Ecocardiografia , Obstrução do Fluxo Ventricular Externo/etiologia , Idoso , Idoso de 80 Anos ou mais , Cardiomiopatia Hipertrófica/complicações , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Curva ROC , Estudos Retrospectivos , Obstrução do Fluxo Ventricular Externo/diagnóstico por imagemRESUMO
We herein report a 61-year-old woman with a history of mitral valve replacement for rheumatic fever who presented with crural edema and ascites. Computed tomography showed massive left atrial (LA) calcification involving the interatrial septum, termed "coconut atrium." Catheterization revealed not only pulmonary artery hypertension but also a large V-wave in the pulmonary artery wedge pressure waveform and a dip-and-plateau pattern of right ventricular pressure. Three-dimensional transthoracic echocardiography confirmed the early attainment of peak LA volume and a decreased LA expansion index. Stiff LA syndrome due to coconut LA results in the development of restrictive right ventricular physiology.
Assuntos
Átrios do Coração , Humanos , Feminino , Pessoa de Meia-Idade , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/fisiopatologia , Disfunção Ventricular Direita/fisiopatologia , Disfunção Ventricular Direita/diagnóstico por imagem , Disfunção Ventricular Direita/etiologia , Calcinose/diagnóstico por imagem , Calcinose/fisiopatologia , Tomografia Computadorizada por Raios X , Ecocardiografia TridimensionalRESUMO
We herein report a case of coarctation of the aorta (CoA) in an asymptomatic adult who had a preserved exercise capacity. A 56-year-old man with mild hypertension exhibited left ventricular hypertrophy on an electrocardiogram during an annual medical checkup. Echocardiography showed a bicuspid aortic valve and cor triatriatum sinister, and subsequent computed tomography revealed CoA with developed collaterals. Cardiopulmonary exercise testing showed a good exercise capacity. He refused surgical repair and has been asymptomatic for five years. This case is of special interest, as CoA is usually rarely noticed during adulthood if there are no severe congenital anomalies, and in addition, this patient had good exercise capacity.
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Coartação Aórtica , Masculino , Humanos , Adulto , Pessoa de Meia-Idade , Coartação Aórtica/diagnóstico por imagem , Coartação Aórtica/cirurgia , Tolerância ao Exercício , Aorta , Ecocardiografia , EletrocardiografiaRESUMO
Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms.
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Bioengenharia , Corantes , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema Livre de CélulasRESUMO
BACKGROUND: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. RESULTS: Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. CONCLUSIONS: The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.
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AIMS: Few studies have investigated the subclinical atherosclerotic changes in the brain and carotid artery, and in East Asian populations. We sought to investigate whether gravidity, delivery, the age at menarche and menopause and estrogen exposure period are associated with subclinical atherosclerosis of the brain and carotid arteriopathy. METHODS: This cross-sectional study formed part of a cohort study of Ohasama residents initiated in 1986. Brain atherosclerosis and carotid arteriopathy were diagnosed as white matter hyperintensity (WMH) and lacunae evident on brain magnetic resonance imaging (MRI) and carotid intimal media thickness (IMT) or plaque revealed by ultrasound, respectively. The effect of the reproductive events on brain atherosclerosis and carotid arteriopathy was investigated using logistic regression and general linear regression models after adjusting for covariates. RESULTS: Among 966 women aged ≥ 55 years in 1998, we identified 622 and 711 women (mean age: 69.2 and 69.7 years, respectively) who underwent either MRI or carotid ultrasound between 1992-2008 or 1993-2018, respectively. The highest quartile of gravidity (≥ 5 vs. 3) and delivery (≥ 4 vs. 2), and the highest and second highest (3 vs. 2) quartiles of delivery were associated with an increased risk of WMH and carotid artery plaque, respectively. Neither of age at menarche, menopause, and estrogen exposure period estimated by subtracting age at menarche from age at menopause was associated with atherosclerotic changes of brain and carotid arteries. CONCLUSIONS: Higher gravidity and delivery are associated with subclinical atherosclerosis of the brain and carotid plaque.
Assuntos
Aterosclerose , Doenças das Artérias Carótidas , Placa Aterosclerótica , Idoso , Feminino , Humanos , Aterosclerose/diagnóstico , Aterosclerose/epidemiologia , Aterosclerose/complicações , Encéfalo , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/epidemiologia , Doenças das Artérias Carótidas/complicações , Espessura Intima-Media Carotídea , Estudos de Coortes , Estudos Transversais , Estrogênios , Placa Aterosclerótica/patologia , Fatores de Risco , Pessoa de Meia-IdadeAssuntos
Aneurisma/diagnóstico por imagem , Colo/irrigação sanguínea , Fluordesoxiglucose F18 , Íleo/irrigação sanguínea , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos , Idoso , Aneurisma/complicações , Artérias , Humanos , Inflamação/complicações , Inflamação/microbiologia , Masculino , Infecções Estreptocócicas/complicaçõesRESUMO
Synthetic cells are engineered vesicles that can mimic one or more salient features of life. These features include directed localization, sense-and-respond behavior, gene expression, metabolism, and high stability. In nanomedicine, many of these features are desirable capabilities of drug delivery vehicles but are difficult to engineer. In this focus article, we discuss where synthetic cells offer unique advantages over nanoparticle and living cell therapies. We review progress in the engineering of the above life-like behaviors and how they are deployed in nanomedicine. Finally, we assess key challenges synthetic cells face before being deployed as drugs and suggest ways to overcome these challenges. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures.
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Células Artificiais , Nanopartículas , Nanoestruturas , Sistemas de Liberação de Medicamentos , NanomedicinaRESUMO
Background: The carotid sinus baroreceptor reflex controls the neural regulation of blood pressure. Baroreceptor disorders due to carotid sinus atherosclerosis have detrimental cardiovascular effects. This study investigated the medium-term effects of carotid artery revascularization (CAR) on sympathetic and cardiac function and systemic blood pressure variability in patients with carotid artery stenosis. Methods: This study included 21 consecutive patients (median age 70 years, 18 men) with carotid artery stenosis scheduled for CAR. 123I metaiodobenzylguanidine (MIBG) scintigraphy, echocardiography, brain natriuretic peptide levels, 24-h Holter electrocardiography (ECG), and ambulatory blood pressure monitoring assessed approximately 3 months postoperatively were compared to preoperative data. Results: All 21 enrolled patients underwent CAR. Carotid artery stenting was done in three patients with cardiovascular risk or anatomical difficult for carotid endarterectomy. The mean common carotid artery end-diastolic velocity improved significantly (P < 0.01) by 1.6-fold, from 10.8 ± 3.2 to 16.1 ± 7.1 cm/s. In 123I-MIBG scintigraphy, the heart-to-mediastinum (H/M) count ratio was significantly higher than preoperatively (from 2.66 ± 0.48 to 2.86 ± 0.56, P = 0.03). Holter ECG analysis revealed a significant decrease in the low-frequency/high-frequency (LF/HF) ratio compared to preoperatively (from 2.17 ± 1.20 to 1.62 ± 0.68, P = 0.04). These findings suggest decreased myocardial sympathetic activation. In echocardiography, the tissue Doppler-derived e' increased, and E/e' decreased significantly (P < 0.05) from 11.7 ± 5.1 to 10.1 ± 4.0, suggesting an improved left ventricular diastolic capacity. The mean 24-h and nighttime blood pressures were unchanged. Conclusions: CAR in patients with carotid stenosis may provide medium-term improvement in cardiac sympathetic nerve activity and left ventricular diastolic dysfunction.
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Liposomal encapsulation serves as the basis for the engineering of biomimetic and novel synthetic cells. Liposomes are normally formed using such methods as thin film rehydration (TFH), density-mediated reverse emulsion encapsulation (REE), or one of many microfluidics-based approaches-with the latter of these two methods being used mainly for the encapsulation of various lumen constituents such as cell-free protein expression reactions. Here, we describe the simultaneous formation and encapsulation of liposomes and various cell-mimetic lumen chemistries, respectively, using a 3D-printable microcapillary-based microfluidics device based off of the droplet-shooting and size-filtration (DSSF) liposome preparation method.
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Lipossomos , Microfluídica , Biomimética , Emulsões , Microfluídica/métodos , Impressão TridimensionalRESUMO
(1) Background: Although the ankle-brachial index (ABI) and skin perfusion pressure (SPP) are commonly used to evaluate the peripheral circulation in critical limb ischemia (CLI), they often cannot be performed on sore areas. We investigated the utility of superb microvascular imaging (SMI) for assessing foot perfusion in CLI patients. (2) Methods: We measured the SMI-based vascular index (SMI-VI) at six sites in the foot before and after endovascular treatment (EVT) in 50 patients with CLI who underwent EVT of the superficial femoral artery and compared the results with SPP values and the ABI. (3) Results: SMI visualized foot perfusion in all subjects in accordance with the angiosome, including the toe areas, while the ABI was unmeasurable in three patients on hemodialysis and SPP failed in four patients. SMI-VI values were significantly lower in the CLI group than in controls, and the plantar SMI-VI had the highest diagnostic performance for CLI (sensitivity 88.6%, specificity 95.6%). After EVT, the increase in the SMI-VI was positively correlated with the increase in SPP but not that in the ABI, implying that the SMI-VI reflects foot microcirculation. (4) Conclusions: SMI enables the visualization and quantification of foot microcirculation based on the angiosome. SMI has high utility as a tool for assessing foot perfusion in CLI.
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BACKGROUND: Our aim was to analyze the incidence of P-wave abnormalities in embolic and non-embolic strokes, and evaluate its clinical usefulness for predicting stroke etiology. METHODS: We included 376 consecutive patients hospitalized for acute ischemic stroke from January 2015 to September 2021. Among the patients in sinus rhythm at admission, 31 had ischemic stroke due to atrial fibrillation (AF)-related embolism, 59 had embolic stroke of unknown source (ESUS), and 143 had non-embolic stroke. P-wave abnormalities were defined as 1. P-wave axis abnormality (PWAA); 2. P-wave terminal force in V1 (PTFV1) ≤ -4000 µV*ms; 3. advanced inter-atrial block (A-IAB). RESULTS: The prevalence of each type of abnormality was consistently lower in patients with non-embolic stroke than in those with AF-related embolism (AF-related vs. ESUS vs. non-embolic; PWAA, 45% vs. 20% vs. 14%; PTFV1, 36% vs. 37% vs. 15%; and A-IAB, 55% vs. 31% vs. 13%, respectively). The identification of at least one type of P-wave abnormality improved the sensitivity compared to using a single abnormality parameter (sensitivity 72%, specificity 62%), while at least two types of abnormality had low sensitivity, but high specificity (sensitivity 29%, specificity 95%). Multivariate regression analysis revealed that identification of at least one type of P-wave abnormality was independently associated with embolic stroke (odds ratio 3.11, 95%CI 1.46-6.63). CONCLUSIONS: The incidence of each type of P-wave abnormality was significantly lower in patients with non-embolic stroke. A combination of PWAA, PTFV1, and A-IAB parameters could be useful for distinguishing embolic from non-embolic stroke.
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Fibrilação Atrial , Embolia , Embolia Intracraniana , AVC Isquêmico , Acidente Vascular Cerebral , Fibrilação Atrial/complicações , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/epidemiologia , Eletrocardiografia/efeitos adversos , Embolia/diagnóstico , Embolia/epidemiologia , Humanos , Embolia Intracraniana/diagnóstico , Embolia Intracraniana/epidemiologia , Embolia Intracraniana/etiologia , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologiaRESUMO
Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.
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Vaga-Lumes , Luminescência , Animais , Vaga-Lumes/genética , Vaga-Lumes/metabolismo , Genes Reporter , Indicadores e Reagentes , Luciferases/genética , Luciferases/metabolismo , Luciferases de Vaga-Lume/genéticaRESUMO
Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.
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Proteínas de Escherichia coli , Escherichia coli , Sistema Livre de Células/metabolismo , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismoRESUMO
Recently, the emergence and rapid dissemination of extended-spectrum beta-lactamase (ESBL)-producing bacteria, particularly of the family Enterobacteriaceae, has posed serious healthcare challenges. Here, we determined the antimicrobial susceptibility and genetic characteristics of 164 Escherichia coli strains isolated from infected patients in two hospitals in Ghana. In total, 102 cefotaxime-resistant isolates (62.2%) were identified as ESBL-producers. Multilocus sequence typing of the ESBL-producers identified 20 different sequence types (STs) with ST131 (n = 25, 24.5%) as the dominant group. Other detected STs included ST410 (n = 21, 20.6%) and ST617 (n = 19, 18.6%). All identified ESBL-producers harbored blaCTX-M-14, blaCTX-M-15, or blaCTX-M-27, with blaCTX-M-15 (n = 96, 94.1%) being the most predominant ESBL allele. Further analysis showed that the immediate genetic environment around blaCTX-M-15 is conserved within blaCTX-M-15 containing strains. Five of the 25 ST131 isolates were clustered with clade A, one with sub-clade C1, and 19 with the dominant sub-clade C2. The results show that fluoroquinolone-resistant, blaCTX-M-14- and blaCTX- M-15-producing ESBL E. coli ST131 strains belonging to clade A and sub-clades C1 and C2 are disseminating in Ghanaian hospitals. To the best of our knowledge, this is the first report of the ST131 phylogeny in Ghana.