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BACKGROUND: Extracellular vesicles (EVs) are membrane-enclosed structures containing lipids, proteins, and RNAs that play a crucial role in cell-to-cell communication. However, the precise mechanism through which circulating EVs disrupt hepatic glucose homeostasis in gestational diabetes mellitus (GDM) remains unclear. RESULTS: Circulating EVs isolated from human plasma were co-cultured with mammalian liver cells to investigate the potential induction of hepatic insulin resistance by GDM-EVs using glucose output assays, Seahorse assays, metabolomics, fluxomics, qRT-PCR, bioinformatics analyses, and luciferase assays. Our findings demonstrated that hepatocytes exposed to GDM-EVs exhibited increased gluconeogenesis, attenuated energy metabolism, and upregulated oxidative stress. Particularly noteworthy was the discovery of miR-1299 as the predominant miRNA in GDM-EVs, which directly targeting the 3'-untranslated regions (UTR) of STAT3. Our experiments involving loss- and gain-of-function revealed that miR-1299 inhibits the insulin signaling pathway by regulating the STAT3/FAM3A axis, resulting in increased insulin resistance through the modulation of mitochondrial function and oxidative stress in hepatocytes. Moreover, experiments conducted in vivo on mice inoculated with GDM-EVs confirmed the development of glucose intolerance, insulin resistance, and downregulation of STAT3 and FAM3A. CONCLUSIONS: These results provide insights into the role of miR-1299 derived from circulating GDM-EVs in the progression of insulin resistance in hepatic cells via the STAT3/FAM3A axis and downstream metabolic reprogramming.
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Diabetes Gestacional , Vesículas Extracelulares , Glucosa , Hepatocitos , Homeostasis , Resistencia a la Insulina , Hígado , MicroARNs , Factor de Transcripción STAT3 , Animales , Femenino , Humanos , Ratones , Embarazo , Regiones no Traducidas 3' , Diabetes Gestacional/metabolismo , Diabetes Gestacional/genética , Vesículas Extracelulares/metabolismo , Glucosa/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones Endogámicos C57BL , MicroARNs/metabolismo , MicroARNs/genética , Estrés Oxidativo , Transducción de Señal , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
BACKGROUND: Society is burdened with stroke-associated pneumonia (SAP) after intracerebral haemorrhage (ICH). Cerebral small vessel disease (CSVD) complicates clinical manifestations of stroke. In this study, we redefined the CSVD burden score and incorporated it into a novel radiological-clinical prediction model for SAP. MATERIALS AND METHODS: A total of 1278 patients admitted to a tertiary hospital between 1 January 2010 and 31 December 2019 were included. The participants were divided into training and testing groups using fivefold cross-validation method. Four models, two traditional statistical models (logistic regression and ISAN) and two machine learning models (random forest and support vector machine), were established and evaluated. The outcomes and baseline characteristics were compared between the SAP and non-SAP groups. RESULTS: Among the of 1278 patients, 281(22.0%) developed SAP after their first ICH. Multivariate analysis revealed that the logistic regression (LR) model was superior in predicting SAP in both the training and testing groups. Independent predictors of SAP after ICH included total CSVD burden score (OR, 1.29; 95% CI, 1.03-1.54), haematoma extension into ventricle (OR, 2.28; 95% CI, 1.87-3.31), haematoma with multilobar involvement (OR, 2.14; 95% CI, 1.44-3.18), transpharyngeal intubation operation (OR, 3.89; 95% CI, 2.7-5.62), admission NIHSS score ≥ 10 (OR, 2.06; 95% CI, 1.42-3.01), male sex (OR, 1.69; 95% CI, 1.16-2.52), and age ≥ 67 (OR, 2.24; 95% CI, 1.56-3.22). The patients in the SAP group had worse outcomes than those in the non-SAP group. CONCLUSION: This study established a clinically combined imaging model for predicting stroke-associated pneumonia and demonstrated superior performance compared with the existing ISAN model. Given the poor outcomes observed in patients with SAP, the use of individualised predictive nomograms is vital in clinical practice.
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Hemorragia Cerebral , Aprendizaje Automático , Neumonía , Accidente Cerebrovascular , Humanos , Masculino , Femenino , Anciano , Persona de Mediana Edad , Hemorragia Cerebral/diagnóstico por imagen , Hemorragia Cerebral/complicaciones , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/diagnóstico por imagen , Neumonía/diagnóstico por imagen , Neumonía/complicaciones , Estudios Retrospectivos , Modelos Logísticos , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Enfermedades de los Pequeños Vasos Cerebrales/complicaciones , Tomografía Computarizada por Rayos X , Factores de Riesgo , Modelos Estadísticos , Anciano de 80 o más AñosRESUMEN
BACKGROUND: To evaluate the effect of perioperative esketamine administration on postpartum depression in pregnant women undergoing cesarean section. METHODS: Data sources was PubMed, Embase, Web of Science, and Cochrane Library from inception to February 1, 2024. Randomized controlled trials in pregnant women undergoing cesarean section were selected and compared to the use of esketamine in the perioperative period. The primary outcome measure was the incidence of postpartum maternal depression. Preferred reporting items for systematic reviews and meta-analyses were used. Data pooled by random-effects models are presented as risk ratios (RR) (95% confidence intervals, 95% CI) or mean differences (95% CI). This review was registered in PROSPERO (ID: CRD42023431197). RESULTS: We included 8 studies with a total of 1655 participants. The quality of the studies was rated high or unclear. Seven studies involving 1485 participants reported the incidence of postpartum depression. Compared with pregnant women undergoing cesarean section without the use of esketamine, those using esketamine in the perioperative period showed a 48% decreased risk of developing postpartum depression (RR: 0.52, 95% CI: 0.35-0.79) and a 1.43-point reduction in EPDS (Edinburgh Postnatal Depression Scale) (mean difference: -1.43, 95% CI: -2.32 to -0.54). For immediate intraoperative adverse reactions, the application of esketamine caused maternal nausea and vomiting (RR: 2.16, 95% CI: 1.22-3.81), dizziness (RR: 6.11, 95% CI: 1.49-24.98), and hallucinations (RR: 6.83, 95% CI: 1.57-29.68) compared to no esketamine use. CONCLUSIONS: Perioperative use of esketamine in pregnant women undergoing cesarean section may reduce postpartum depression and increase intraoperative adverse reactions, but has no significant effect on postoperative adverse reactions.
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Cesárea , Depresión Posparto , Ketamina , Humanos , Ketamina/administración & dosificación , Ketamina/uso terapéutico , Femenino , Cesárea/efectos adversos , Embarazo , Depresión Posparto/prevención & control , Depresión Posparto/epidemiología , Ensayos Clínicos Controlados Aleatorios como Asunto , Atención Perioperativa/métodosRESUMEN
The bacterium Natranaerobius thermophilus is an extremely halophilic alkalithermophile that can thrive under conditions of high salinity (3.3-3.9 M Na+), alkaline pH (9.5), and elevated temperature (53°C). To understand the molecular mechanisms of salt adaptation in N. thermophilus, it is essential to investigate the protein, mRNA, and key metabolite levels on a molecular basis. Based on proteome profiling of N. thermophilus under 3.1, 3.7, and 4.3 M Na+ conditions compared to 2.5 M Na+ condition, we discovered that a hybrid strategy, combining the "compatible solute" and "salt-in" mechanisms, was utilized for osmotic adjustment dur ing the long-term salinity adaptation of N. thermophilus. The mRNA level of key proteins and the intracellular content of compatible solutes and K+ support this conclusion. Specifically, N. thermophilus employs the glycine betaine ABC transporters (Opu and ProU families), Na+/solute symporters (SSS family), and glutamate and proline synthesis pathways to adapt to high salinity. The intracellular content of compatible solutes, including glycine betaine, glutamate, and proline, increases with rising salinity levels in N. thermophilus. Additionally, the upregulation of Na+/ K+/ H+ transporters facilitates the maintenance of intracellular K+ concentration, ensuring cellular ion homeostasis under varying salinities. Furthermore, N. thermophilus exhibits cytoplasmic acidification in response to high Na+ concentrations. The median isoelectric points of the upregulated proteins decrease with increasing salinity. Amino acid metabolism, carbohydrate and energy metabolism, membrane transport, and bacterial chemotaxis activities contribute to the adaptability of N. thermophilus under high salt stress. This study provides new data that support further elucidating the complex adaptation mechanisms of N. thermophilus under multiple extremes.IMPORTANCEThis study represents the first report of simultaneous utilization of two salt adaptation mechanisms within the Clostridia class in response to long-term salinity stress.
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Proteínas Bacterianas , Potasio , Estrés Salino , Potasio/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Adaptación Fisiológica , SalinidadRESUMEN
OBJECTIVE: Radiomics can reflect the heterogeneity within the focus. We aim to explore whether radiomics can predict recurrent intracerebral hemorrhage (RICH) and develop an online dynamic nomogram to predict it. METHODS: This retrospective study collected the clinical and radiomics features of patients with spontaneous intracerebral hemorrhage seen in our hospital from October 2013 to October 2016. We used the minimum redundancy maximum relevancy and the least absolute shrinkage and selection operator methods to screen radiomics features and calculate the Rad-score. We use the univariate and multivariate analyses to screen clinical predictors. Optimal clinical features and Rad-score were used to construct different logistics regression models called the clinical model, radiomics model, and combined-logistic regression model. DeLong testing was performed to compare performance among different models. The model with the best predictive performance was used to construct an online dynamic nomogram. RESULTS: Overall, 304 patients with intracerebral hemorrhage were enrolled in this study. Fourteen radiomics features were selected to calculate the Rad-score. The patients with RICH had a significantly higher Rad-score than those without (0.5 vs. -0.8; P< 0.001). The predictive performance of the combined-logistic regression model with Rad-score was better than that of the clinical model for both the training (area under the receiver operating curve, 0.81 vs. 0.71; P = 0.02) and testing (area under the receiver operating curve, 0.65 vs. 0.58; P = 0.04) cohorts statistically. CONCLUSIONS: Radiomics features were determined related to RICH. Adding Rad-score into conventional clinical models significantly improves the prediction efficiency. We developed an online dynamic nomogram to accurately and conveniently evaluate RICH.
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Nomogramas , Radiómica , Humanos , Estudios Retrospectivos , Hemorragia Cerebral/diagnóstico por imagen , HospitalesRESUMEN
Cladophora rupestris is ubiquitous in many kinds of waterbodies, and C. rupestris biomass can serve as a carrier for adsorbing and transferring heavy metals. Batch experiments and characterization were performed. Results showed that the organic frameworks of C. rupestris (CROF) had a specific surface area of 2.58 m2/g and an external surface area of 2.06 m2/g. Many mesopores were present in CROF, mainly distributed in 2.5-7.5 nm. The zeta potentials were within the range of - 4.46 to - 13.98 mV in the tested pH of 2.0-9.0. CROF could effectively adsorb Pb2+ in large pH range. The maximum adsorption capacity (qmax) of Pb2+ on CROF was 15.02 mg/g, and 97% of Pb2+ was adsorbed onto CROF after 25 min. CROF had a preferential adsorption of Pb2+. The protein secondary structures and carbon skeletons of CROF all worked in adsorption. The main Pb2+ adsorption mechanisms were pore filling, electrostatic attraction, Pb-π interaction, and surface complexation. Therefore, it is valuable as a biosorbent for the removal of Pb2+ from waterbodies.
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Chlorophyta , Metales Pesados , Contaminantes Químicos del Agua , Plomo , Metales Pesados/química , Física , Cinética , Adsorción , Concentración de Iones de Hidrógeno , Contaminantes Químicos del Agua/análisisRESUMEN
Objective: Post-stroke hyperglycemia as a common phenomenon is associated with unfavorable outcomes. Focusing on admission hyperglycemia, other markers of dysglycemia were overlooked. This study aimed to explore the contribution of acute phase blood glucose levels in combination with other radiological signs to the prognostication of functional outcomes in patients with spontaneous intracerebral hemorrhage (sICH). Methods: Consecutive patients with sICH with at least five random plasma glucose measurements and complete radiological data during hospitalization were included. We calculated the average, maximum, minimum, standard deviation, and coefficient of variation of blood glucose levels for each patient. Radiological data, including island, black hole, blend, and satellite signs were collected. Functional outcomes were evaluated using the Barthel index. Unfavorable outcomes were defined as a Barthel index score ≤ 60. Univariate and multivariate analyses were performed to identify independent predictors of unfavorable outcomes. Results: Two hundred and thirty-eight patients (mean age 58.5, 163 men and 75 women) were included, and 71 had a history of diabetes. Unfavorable outcomes occurred in 107 patients (45.0%) at 3 months. Multivariate logistic regression analysis demonstrated that maximum blood glucose levels (odds ratio, 1.256; 95% confidence interval, 1.124â1.404; p < 0.001) and island sign (odds ratio, 2.701; 95% confidence interval, 1.322â5.521; p = 0.006) were independent predictors of unfavorable outcomes in the nondiabetic group. Meanwhile, patients without diabetes who experienced hematoma expansion had higher average (p = 0.036) and maximum blood glucose levels (p = 0.014). Interpretation: Maximum blood glucose levels and island sign were independently associated with unfavorable outcomes in patients without diabetes, whereas no glycemic variability indices were associated with unfavorable outcomes. Glucose levels influenced hematoma expansion and functional outcomes, particularly in patients without diabetes with sICH. Thus, clinical management of blood glucose levels should be strengthened for patients with sICH with or without a history of diabetes.
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BACKGROUND: Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct. This is a major drawback of the engineered Mycolicibacteria for industrial application, which could be attributed to the co-existence of androstene-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) sub-pathways in the degradation of the sterol C17 side chain. Since the key mechanism underlying the HBC sub-pathway has not yet been clarified, the above shortcoming has not been resolved so far. RESULTS: The key gene involved in the putative HBC sub-pathway was excavated from the genome of M. neoaurum by comparative genomic analysis. Interestingly, an aldolase- encoding gene, atf1, was identified to be responsible for the first reaction of the HBC sub-pathway, and it exists as a conserved operon along with a DUF35-type gene chsH4, a reductase gene chsE6, and a transcriptional regulation gene kstR3 in the genome. Subsequently, atf1 and chsH4 were identified as the key genes involved in the HBC sub-pathway. Therefore, an updated strategy was proposed to develop engineered C-19 or C-22 steroid-producing strains by simultaneously modifying the AD and HBC sub-pathways. Taking the development of 4-HBC and 9-OHAD-producing strains as examples, the improved 4-HBC-producing strain achieved a 20.7 g/L production titer with a 92.5% molar yield and a 56.4% reduction in byproducts, and the improved 9-OHAD producing strain achieved a 19.87 g/L production titer with a 94.6% molar yield and a 43.7% reduction in byproduct production. CONCLUSIONS: The excellent performances of these strains demonstrated that the primary operon involved in the HBC sub-pathway improves the industrial strains in the conversion of phytosterols to steroid synthons.
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Endocrine-disrupting compounds (EDCs) are widely distributed in the environment. Here, we present a CRISPR/Cas12a (CAS) biosensor based on DNA aptamers for point-of-care detection of EDCs. Two typical EDCs, 17ß-estradiol (E2) and bisphenol A (BPA), were selected to be detected by the CAS biosensors via the plug-and-play of their DNA aptamers. The results indicated that the performance of the CAS biosensors can be well regulated by controlling the trans-cleavage activity of Cas12a on a single-stranded DNA reporter and optimizing the sequence and ratio of DNA aptamer and activator DNA. Ultimately, two reliable and specific biosensors were developed, with the linear range and limit of detection of 0.2-25 nM and 0.08 nM for E2 and of 0.1-250 nM and 0.06 nM for BPA, respectively. Compared to the existing detection methods, the CAS biosensors showed higher reliability and sensitivity with simple operation, short detection time, and no costly equipment.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Aptámeros de Nucleótidos/genética , Sistemas CRISPR-Cas , Reproducibilidad de los Resultados , Estradiol , Técnicas Biosensibles/métodosRESUMEN
Introduction: Spinocerebellar ataxia type 3 (SCA3) is a common autosomal dominant hereditary ataxia, which is caused by a cytosine-adenine-guanine (CAG) repeat expansion on the causative gene ATXN3, usually with lower extremity ataxia as the first symptom, and effective treatment is scarce. Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique that regulates the cerebellum and the neural network connected to it. Methods: Herein, we report familial cases of SCA3 in two nephews and their aunt, each of whom was treated with high-frequency (5 Hz) rTMS. The rTMS treatment lasted 2 weeks, once daily for 5 consecutive days a week, about 20 minutes each session. The Scale for the Assessment and Rating of Ataxia (SARA), the International Cooperative Ataxia Rating Scale (ICARS), and proton magnetic resonance spectroscopy (1H-MRS) examination were evaluated before and after rTMS treatment. Results: We found that the ICARS scores improved significantly (p = 0.04), and the NAA/Cr values were elevated in vermis and both cerebellar hemispheres after rTMS treatment. Conclusion: Our study suggested that high-frequency rTMS therapy can contribute to the improvement of cerebellar NAA/Cr value of SCA3 patients, and improve posture and gait as well as limb kinetic function in SCA3 patients.
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BACKGROUND: Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. RESULTS: In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and ß-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of ß-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). CONCLUSIONS: This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.
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Methotrexate (MTX) is a first-line treatment for rheumatoid arthritis (RA), but its clinical use is greatly limited by the adverse effects and poor patient compliance caused by traditional oral administration or injection. In recent years, some transdermal drug delivery systems have received considerable attention due to overcoming these shortcomings. In this study, we developed dissolving microneedle patch (DMNP) for transdermal delivery of MTX to treat RA safely and effectively. The morphology, mechanical strength, skin insertion, drug content, in vitro transdermal delivery, and other properties of DMNP were characterized. Meanwhile, the adjuvant-induced arthritis model of rats was established to investigate the therapeutic effect of MTX-loaded DMNP in vivo. The results showed that the microneedles had excellent morphology with neat array and complete needles, good puncture performance and mechanical strength, and rapid intradermal dissolution rate. In vitro transdermal delivery results indicated that microneedles could significantly increase drug transdermal permeation compared with the cream group. The pharmacological study showed that MTX-loaded DMNP significantly alleviated paw swelling, inhibit inflammatory response via downregulating the levels of TNF-α and IL-1ß, relieved synovium destruction with less cartilage erosion, and slowed the progression of RA in AIA rats. Besides, DMNP presented better therapeutic performance than cream or intragastric administration at the same dosage of MTX. In conclusion, the MTX-loaded dissolving microneedle patch has advantages of safety, convenience, and high efficacy over conventional administrations, laying a foundation for the transdermal drug delivery system treatment of rheumatoid arthritis.
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Artritis Reumatoide , Metotrexato , Ratas , Animales , Administración Cutánea , Sistemas de Liberación de Medicamentos , Artritis Reumatoide/tratamiento farmacológico , Agujas , Parche TransdérmicoRESUMEN
l-homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l-homoserine-producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l-homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l-homoserine production, including enhancement of precursors for l-homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l-homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid-losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l-homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5-L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.
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Antitoxinas , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Homoserina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Antibacterianos/metabolismo , Plásmidos/genética , Antitoxinas/genética , Antitoxinas/metabolismo , Ingeniería Metabólica/métodosRESUMEN
Macrophages work with monocytes and dendritic cells to form a monocyte immune system, which constitutes a powerful cornerstone of the immune system with their powerful antigen presentation and phagocytosis. Macrophages play an essential role in infection, inflammation, tumors and other pathological conditions, but these cells also have non-immune functions, such as regulating lipid metabolism and maintaining homeostasis. Propofol is a commonly used intravenous anesthetic in the clinic. Propofol has sedative, hypnotic, anti-inflammatory and anti-oxidation effects, and it participates in the body's immunity. The regulation of propofol on immune cells, especially macrophages, has a profound effect on the occurrence and development of human diseases. We summarized the effects of propofol on macrophage migration, recruitment, differentiation, polarization, and pyroptosis, and the regulation of these propofol-regulated macrophage functions in inflammation, infection, tumor, and organ reperfusion injury. The influence of propofol on pathology and prognosis via macrophage regulation is also discussed. A better understanding of the effects of propofol on macrophage activation and function in human diseases will provide a new strategy for the application of clinical narcotic drugs and the treatment of diseases.
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PURPOSE: Bladder carcinoma (BLCA) is the most common urinary tract malignancy and exhibits a poor response to chemotherapy. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in a wide variety of regulatory cellular processes, including apoptosis and the DNA-damage response (DDR). LB100, a small molecule inhibitor of PP2A, has been shown to act as a chemo-sensitizer in multiple types of cancer. However, the anti-tumor effect and mode of action of LB100 in BLCA have yet to be identified. METHODS: In vitro and in vivo experiments were performed to assess the anti-tumor effect of LB100 alone or in combination with gemcitabine. Mass spectrometry (MS)-based phosphoproteomics analysis was used to identify the downstream substrates of PP2A and to explore the mechanism underlying LB100-induced DNA damage and apoptosis. In addition, we established a chemo-resistant BLCA cell line (RT-112-R) by prolonged drug exposure and determined the effect of LB100 in enhancing genotoxicity in BLCA cell lines and xenograft mouse models. RESULTS: We found that LB100 is sufficient to induce an anti-tumor response in BLCA cells by inducing DNA damage and apoptosis both in vitro and in vivo. Furthermore, we found that PP2A potentially dephosphorylates p-p21-ser130 to stabilize p21. Inhibition of PP2A by LB100 increased the level of p-p21-ser130, subsequently leading to a reduction in p21 level in a dose-dependent manner. In addition, we found that treatment of LB100 abrogated the G1/S cell cycle checkpoint, resulting in increased phosphorylation of γH2AX in BLCA cells. Moreover, LB100 enhanced genotoxicity in chemo-resistant BLCA cells by inducing DNA damage and apoptosis in vitro and in vivo. CONCLUSION: Our findings indicate that PP2A may serve as a potential therapeutic target in BLCA through regulating p21 stability.
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Proteína Fosfatasa 2 , Neoplasias de la Vejiga Urinaria , Animales , Humanos , Ratones , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were â¼100% for no more than three-copy integration events and decreased sharply to â¼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.
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Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce ß-carotene. After seven rounds of iterative evolution over two months, a ß-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in ß-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.
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Sistemas CRISPR-Cas , Saccharomyces cerevisiae , Sistemas CRISPR-Cas/genética , Edición Génica , Ingeniería Genética , Mutagénesis , ARN Guía de Kinetoplastida/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta Caroteno/metabolismoRESUMEN
BACKGROUND: 7ß-hydroxylated steroids (7ß-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7ß-OHSt is still a challenge due to the lack of cheap 7ß-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7ß-hydroxy precursor, 7ß-hydroxyandrost-4-ene-3,17-dione (7ß-OH-AD) in Mycolicibacterium, for 7ß-OHSt synthesis. RESULTS: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7ß-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7ß-OH-AD was increased from 34.24 mg L- 1 to 66.25 mg L- 1. To further enhance the performance of 7ß-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L- 1 7ß-OH-AD in the cofactor recycling and regeneration system. CONCLUSIONS: This was the first report on the one-pot biosynthesis of 7ß-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.
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Fitosteroles , Biotransformación , Mycobacteriaceae , Fitosteroles/metabolismo , Regeneración , EsteroidesRESUMEN
TRIP13 is a member of the large superfamily of the AAA + ATPase proteins and is associated with a variety of activities. Emerging evidence has shown that TRIP13 may serve as an oncogene. However, the function of TRIP13 in breast cancer (BC) has not yet been elucidated. Here, a variety of bioinformatic tools and laboratory experiments were combined to analyse the expression patterns, prognostic value and functional network of TRIP13 in BC. Multiple databases and immunohistochemistry (IHC) indicated a higher TRIP13 expression in BC tissue compared with normal tissue. TRIP13 was highly expressed in lung metastatic lesions compared with primary tumours in a 4T1 cell implantation BALB/c mouse model of BC. Kaplan-Meier plots also revealed that high TRIP13 expression correlated with poor survival in patients with BC. Furthermore, gene set enrichment analysis revealed that TRIP13 was primarily enriched in the signalling pathway of PI3K-AKT-mTOR. Suppressing TRIP13 could inhibit the expression of related genes, as well as the proliferation and migration of BC cell. Finally, 10 hub genes with a high score of connectivity were filtered from the protein-protein interaction (PPI) network, including MAD2L1, CDC20, CDC5L, CDK1, CCNA2, BUB1B, RAD51, SPO11, KIF11 and AURKB. Thus, TRIP13 may be a promising prognostic biomarker and an effective therapeutic target for BC.
Asunto(s)
Neoplasias de la Mama , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Animales , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genéticaRESUMEN
Patchoulol is a natural sesquiterpene, which is widely used in perfumes and cosmetics. In the work, the mitochondria of S. cerevisiae were engineered for patchoulol production. The patchoulol titer of mitochondria-compartmentalized strain (1.79 mg/L) was 2.71-fold higher than that of control strain (0.66 mg/L) using genome-integrated patchoulol synthase, indicating that mitochondria compartmentation resulted in higher concentration of FPP (farnesyl pyrophosphate) precursor for patchoulol production. Moreover, when fused FPP synthase and patchoulol synthase was overexpressed in the strain with a mitochondria-localized DMAPP (dimethylallyl diphosphate) pathway, the production of patchoulol increased significantly to 19.24 mg/L, indicating more precursors were provided for patchoulol production. Nevertheless, the introduction of excess foreign proteins into mitochondria might cause a certain stress on mitochondria and showed a negative effect on the growth of yeast cells, which could hinder the expression of foreign pathways and reduce the patchoulol production. In conclusion, mitochondria-engineered yeast cells showed important potential for the enhanced biosynthesis of patchoulol, and further engineering could be considered based on the present work.