RESUMEN
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for â¼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a â¼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.
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Nicotiana , Ribulosa-Bifosfato Carboxilasa , Nicotiana/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Fotosíntesis/genética , Cloroplastos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Dióxido de Carbono/metabolismoRESUMEN
SignificanceWe report the development of peptidomimetic antibiotics derived from a natural antimicrobial peptide, human α-defensin 5. By engaging multiple bacterial targets, the lead compound is efficacious in vitro and in vivo against bacteria with highly inducible antibiotic resistance, promising a useful therapeutic agent for the treatment of infections caused by antibiotic-resistant bacteria.
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Antibacterianos/química , Defensinas/química , Descubrimiento de Drogas/métodos , Peptidomiméticos/química , Antibacterianos/farmacología , Defensinas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Peptidomiméticos/farmacología , Relación Estructura-ActividadRESUMEN
Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.
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Orgánulos/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Synechococcus/metabolismo , Carbono/metabolismo , Ciclo del Carbono , Microscopía por Crioelectrón , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fotosíntesis , Subunidades de Proteína/metabolismo , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/genética , Synechococcus/genética , TranscriptomaRESUMEN
The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single ß-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.
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Ambiente , Orgánulos/metabolismo , Orgánulos/ultraestructura , Synechococcus/metabolismo , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Luz , Modelos Biológicos , Orgánulos/efectos de la radiación , Synechococcus/efectos de la radiación , Synechococcus/ultraestructuraRESUMEN
BACKGROUND: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. RESULTS: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. CONCLUSIONS: We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.
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Proteínas , Proteómica , Biblioteca de Genes , Técnicas Genéticas , Péptidos , Proteínas/análisisRESUMEN
In 2019, a new strain of coronavirus pneumonia spread quickly worldwide. Viral propagation may be simulated using the Susceptible Infectious Removed (SIR) model. However, the SIR model fails to consider that separation of patients in the COVID-19 incubation stage entails difficulty and that these patients have high transmission potential. The model also ignores the positive effect of quarantine measures on the spread of the epidemic. To address the two flaws in the SIR model, this study proposes a new infectious disease model referred to as the Susceptible Quarantined Exposed Infective Removed (SQEIR) model. The proposed model uses the weighted least squares for the optimal estimation of important parameters in the infectious disease model. Based on these parameters, new differential equations were developed to describe the spread of the epidemic. The experimental results show that this model exhibits an accuracy 6.7% higher than that of traditional infectious disease models.
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Rubisco is the essential enzyme mediating the fixation of atmospheric CO2 during photosynthesis. In cyanobacteria, Rubisco enzymes are densely packed and encapsulated in a specialized organelle known as the carboxysome. Well-defined Rubisco assembly and carboxysome formation are pivotal for efficient CO2 fixation. Numerous chaperone proteins, including RbcX, are essential for proper protein folding and Rubisco assembly. In this study, we investigated the in vivo function of RbcX in the cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) using molecular, biochemical, and live-cell fluorescence imaging approaches. Our results show that genetic deletion of the rbcX gene affects Rubisco abundance, as well as carboxysome formation and spatial distribution. Moreover, RbcX appears as one component of the carboxysome and shows a dynamic interaction with Rubisco enzymes. These in vivo observations provide insight into the role of RbcX from Syn7942 in mediating carboxysome assembly. Understanding the molecular mechanism underlying Rubisco assembly and carboxysome biogenesis will provide essential information required for engineering functional CO2-fixing complexes in heterogeneous organisms, especially plants, with the aim of boosting photosynthesis and agricultural productivity.
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Proteínas Bacterianas/fisiología , Chaperonas Moleculares/fisiología , Synechococcus/metabolismo , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Biología Computacional , Chaperonas Moleculares/metabolismo , Orgánulos/metabolismo , Fotosíntesis , FilogeniaRESUMEN
BACKGROUND: Although Mitochondrial DNA depletion syndrome (MDS) can be classified into three forms: myopathic, encephalomyopathic and hepatocerebral form, it is difficult to identify its form due to its clinical heterogeneity. Therefore, it is very important to conduct molecular genetic analysis on suspected patients. This study presented a male 38 weeks and 5 days infant with liver cytolysis and leukodystrophy. CASE PRESENTATION: A male infant proband was admitted to the department of NICU for feeding intolerance, irregular rhythm of respiration, hypoglycemia, lactic acidosis, liver cytolysis and neurological abnormalities. He was onset of mild jaundice with leukodystrophy and high lactate and phenylderivatives for urine organic acids on the 7th day. Whole exome sequencing (WES) and Sanger sequencing were performed to screen and confirm the suspicious pathogenic mutations. The results revealed this proband carried two compound heterozygous mutations in TWNK: c.1186 C > T / p.Pro396Ser and c.1844 G > C / p.Gly615Ala inherited by an autosomal recessive form from his parents, of which protein conservative analysis and structural modeling supported the pathogenicity of the two mutations. Unfortunately, the conditions described above were not improved until he was discharged from the hospital on the 23rd day and died at 4 months of age. CONCLUSIONS: In this study, we investigated a Chinese family with the hepatocerebral form of MDS and conducted WES and Sanger sequencing to explore the causative mutations for this proband born from non-consanguineous and healthy parents. We identified two novel TWNK c.1186 C > T/ c.1844 G > C compound heterozygous mutations which were probably the disease-causing mutations of hepatocerebral form of MDS and described the clinical manifestations of the proband, which expanded the phenotypic spectrum of MDS caused by variants in TWNK. This study also emphasized WES technology can provide the genetic diagnosis of Mendelian genetic disease.
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ADN Helicasas/genética , ADN Mitocondrial/genética , Secuenciación del Exoma , Heterocigoto , Seudoobstrucción Intestinal/genética , Proteínas Mitocondriales/genética , Distrofia Muscular Oculofaríngea/genética , Mutación , Pueblo Asiatico , Secuencia de Bases , Predisposición Genética a la Enfermedad/genética , Humanos , Lactante , Masculino , Modelos Moleculares , Oftalmoplejía/congénito , Linaje , Análisis de Secuencia de ProteínaRESUMEN
Deltamethrin is widely used worldwide due to its valuable insecticidal activity against pests and parasites. Increasing evidence has shown that deltamethrin causes varying degrees of toxicity. Moreover, oxidative stress and metabolism are highly correlated with toxicity. For the first time, this review systematically summarizes the deltamethrin toxicity mechanism from the perspective of oxidative stress, including deltamethrin-mediated oxidative damage, antioxidant status, oxidative signaling pathways and modulatory effects of antagonists, synergists and placebos on oxidative stress. Further, deltamethrin metabolism, including metabolites, metabolic enzymes and pathways and deltamethrin metabolite toxicity are discussed. This review will shed new light on deltamethrin toxicity mechanisms and provide effective strategies to ensure pest control and prevention of human and animal poisoning.
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Insecticidas/toxicidad , Nitrilos/toxicidad , Piretrinas/toxicidad , Animales , Estrés OxidativoRESUMEN
WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Pyroptosis, a type of proinflammatory programmed cell death, drives cytokine storm. Caspase-11-dependent macrophage pyroptosis contributes to mortality during sepsis. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling can amplify interleukin-1ß secretion in endotoxin-induced inflammation. Here, we hypothesized that S1PR2 signaling increases caspase-11-dependent macrophage pyroptosis and worsens Gram-negative sepsis outcome. METHODS: A Gram-negative sepsis model was induced through intraperitoneal injection of Escherichia coli. Primary peritoneal macrophages isolated from wild-type, S1pr2-deficient (S1pr2), or nucleotide-binding oligomerization domain-like receptor protein-3-deficient mice were treated with E. coli. Caspase-11 activation, macrophage pyroptosis, and Ras homolog gene family, member A-guanosine triphosphate levels were assessed in those cells. Additionally, monocyte caspase-4 (an analog of caspase-11) expression and its correlation with S1PR2 expression were determined in patients with Gram-negative sepsis (n = 11). RESULTS: Genetic deficiency of S1PR2 significantly improved survival rate (2/10 [20%] in wild-type vs. 7/10 [70%] in S1pr2, P = 0.004) and decreased peritoneal macrophage pyroptosis (pyroptosis rate: 35 ± 3% in wild-type vs. 10 ± 3% in S1pr2, P < 0.001). Decreased caspase-11 activation in S1PR2 deficiency cells contributed to the reduced macrophage pyroptosis. In addition, RhoA inhibitor abrogated the amplified caspase-11 activation in wild-type or S1PR2-overexpressing cells. In patients with Gram-negative sepsis, caspase-4 increased significantly in monocytes compared to nonseptic controls and was positively correlated with S1PR2 (r = 0.636, P = 0.035). CONCLUSIONS: S1PR2 deficiency decreased macrophage pyroptosis and improved survival in E. coli sepsis. These beneficial effects were attributed to the decreased caspase-11 activation of S1PR2-deficient macrophages. S1PR2 and caspase-11 may be promising new targets for treatment of sepsis.
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Bacteriemia/metabolismo , Caspasas/metabolismo , Escherichia coli , Macrófagos/metabolismo , Piroptosis/fisiología , Receptores de Lisoesfingolípidos/deficiencia , Animales , Bacteriemia/patología , Caspasas Iniciadoras , Células Cultivadas , Humanos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sepsis/metabolismo , Sepsis/patología , Transducción de Señal/fisiología , Receptores de Esfingosina-1-FosfatoRESUMEN
BACKGROUND: Pheromone binding proteins (PBPs) of male Lepidoptera function in chemical communication, mate attraction and recognition. Directional selection was previously predicted between PBP3 orthologs of Ostrinia furnacalis and Ostrinia nubilalis were interpreted as being involved in sexual isolation. RESULTS: In vitro assays show that recombinant male OfurPBP3 bound O. furnacalis sex pheromones, Z-12-tetradecenyl acetate (Z12-14:OAc) and E-12-tetradecenyl acetate (E12-14:OAc), as well as to ECB pheromones Z11- and E11-14:OAc. Recombinant OfurPBP4 and OfurPBP5 bound E11- and Z11-14:OAc with greater affinity compared to Z12- and E12-14:OAc, and OfurPBP4 incapable of binding with E12-14:OAc. In silico molecular docking predicted OfurPBP3 residues Phe12, Ile52, Leu94, Ile113 within a hydrophobic ligand-binding pocket and may participate in E12- and Z12-14:OAc binding. Independent site-directed mutagenesis experiments demonstrated that Ser12, Asn52, Arg94, and Asn113 residues variants caused an approximately 1.7- to 4.6-fold reduction in OfurPBP3 affinity for Z12- and E12-14:OAc, and a 2.7- to 8.4-fold decrease in affinity towards E11- and Z11-14:OAc. CONCLUSIONS: Five PBPs of O. furnacalis play important functions in Ostrinia pheromones binding. These four amino acids may play a role in binding of sex pheromone, but this study does not address questions regarding specific response between males of O. furnacalis and O. nubilalis. Additional studies are required determine the role, if any, PBPs play in the evolution of sex pheromone communication.
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Proteínas Portadoras/metabolismo , Proteínas de Insectos/metabolismo , Lepidópteros/metabolismo , Atractivos Sexuales/metabolismo , Animales , Proteínas Portadoras/genética , Femenino , Proteínas de Insectos/genética , Lepidópteros/genética , Masculino , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conducta Sexual AnimalRESUMEN
Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO2 fixation. The central CO2-fixing machinery is the carboxysome, which is composed of an icosahedral proteinaceous shell encapsulating the key carbon fixation enzyme, Rubisco, in the interior. Controlled biosynthesis and ordered organization of carboxysomes are vital to the CO2-fixing activity of cyanobacterial cells. However, little is known about how carboxysome biosynthesis and spatial positioning are physiologically regulated to adjust to dynamic changes in the environment. Here, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of ß-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that ß-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between ß-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for us to modulate the ß-carboxysome biosynthesis and function in cyanobacteria. In translational terms, the knowledge is instrumental for design and synthetic engineering of functional carboxysomes into higher plants to improve photosynthesis performance and CO2 fixation.
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Ciclo del Carbono/fisiología , Fotosíntesis/fisiología , Synechococcus/fisiología , Diurona/farmacología , Transporte de Electrón , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luz , Microscopía Fluorescente/métodos , Organismos Modificados Genéticamente , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Synechococcus/efectos de los fármacosRESUMEN
BACKGROUND: Phytohormones are chemical messengers that have a positive effect on biodiesel production of microalgae at low concentrations. However, the effect of phytohormone 6-benzylaminopurine on lipid and docosahexaenoic acid (DHA) production in marine DHA-producer Aurantiochytrium has never been reported. In this study, a GC-MS-based metabolomics method combined with a multivariate analysis is applied to reveal the metabolic mechanism of 6-benzylaminopurine enhancing production of lipid and DHA in Aurantiochytrium sp.YLH70. RESULTS: In total, 71 metabolites were identified by GC-MS. The PCA model revealed that 76.9% of metabolite variation was related to 6-benzylaminopurine treatment, and overall metabolomics profiles between the 6-benzylaminopurine and control groups were clearly discriminated. Forty-six metabolites identified by the PLS-DA model were responsible for responding to 6-benzylaminopurine. Metabolic analysis showed that 6-benzylaminopurine could accelerate the rate of utilization of glucose in Aurantiochytrium sp. YLH70, and the metabolic flux from glycolysis, TCA cycle and mevalonate pathway to fatty acids biosynthesis was promoted. Moreover, the anti-stress mechanism in Aurantiochytrium sp.YLH70 might be induced by 6-benzylaminopurine. CONCLUSION: Metabolomics is a suitable tool to discover the metabolic mechanism for improving lipid and DHA accumulation in a microorganism. 6-benzylaminopurine has the potential to stimulate lipid and DHA production of Aurantiochytrium sp.YLH70 for industrial purposes. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Copy number variation (CNV), referring to a genome structure variation, has attracted researchers' great interests. Thirty-two CNV regions (CNV region, CNVR) have been detected on chromosome 13 in our previous work. In order to detect the genes located in these CNVRs, we first obtained the annotated information from Ensembl database, and searched gene functional enrichments using DAVID online tools. In the 32 CNVRs, a total of 236 genes were identified, in which 169 genes were annotated. Gene Ontology (GO) analysis revealed that these genes mainly participate in proteolysis, cell adhesion, and macromolecular catabolic process. To study the genetic law of these CNVs, we chose the RCAN1 (regulators of calcineurin 1) gene as the candidate. We quantified the copy number of RCAN1 gene in 38 Laiwu pigs by using QPCR method, and analyzed the genetic laws in three Laiwu families including 15 pigs. QPCR results showed that both duplication and deletion occurred in RCAN1 gene among Laiwu pigs and the heredity mode corresponds with Mendelian genetic law.
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Cromosomas de los Mamíferos/genética , Variaciones en el Número de Copia de ADN/genética , Porcinos , Animales , Femenino , Patrón de Herencia/genética , Masculino , Anotación de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Objective: Using meta-analysis to comprehensively and quantitatively evaluate the impact of acute sleep deprivation on different sports performance of athletes, this study aims to provide scientific guidance for coaches in optimizing and adjusting training and competition arrangements. Methods: Establishing literature inclusion and exclusion criteria, we conducted searches in both Chinese and English databases. Using stata 14.0, we analyzed 75 indicators from 27 included literature, focusing on three aspects: the impact of acute sleep deprivation on overall athletic performance, the impact on sporting performance across various athletic abilities, and the disparities in athletic performance between morning and afternoon following acute sleep deprivation. Results: The effect size of acute sleep deprivation on overall athletic performance was -0.56 (P<0.05). Sub-analyses revealed effect sizes of -0.23 (P<0.05) for whole night sleep deprivation, -1.17 (P<0.05) for partial sleep deprivation at the end of the night, and -0.25 (P>0.05) for partial sleep deprivation in the beginning of the night. The effect sizes of acute sleep deprivation on high intensity intermittent exercise, skill control, speed, aerobic endurance, and explosive power indicators were -1.57, -1.06, -0.67, -0.54, and -0.39 respectively (P<0.05). The effect sizes of acute sleep deprivation on the overall athletic performance in the morning and afternoon were -0.30, and -1.11, respectively (P<0.05). Conclusion: Acute sleep deprivation significantly impairs the overall athletic performance of athletes, with a more pronounced negative impact observed with partial sleep deprivation at the end of the night. Various types of exercise performance are adversely affected by acute sleep deprivation, with magnitude of impact ranking high intensity intermittent, skill control, speed, aerobic endurance, and explosive power. Following acute sleep deprivation, athletes' overall sporting performance in the afternoon is inferior to that in the morning.
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Because it is safe and has a simple genome, recombinant adeno-associated virus (rAAV) is an extremely appealing vector for delivery in in vivo gene therapy. However, its low transduction efficiency for some cells, limits its further application in the field of gene therapy. Bleomycin is a chemotherapeutic agent approved by the FDA whose effect on rAAV transduction has not been studied. In this study, we systematically investigated the effect of Bleomycin on the second-strand synthesis and used CRISPR/CAS9 and RNAi methods to understand the effects of Bleomycin on rAAV vector transduction, particularly the effect of DNA repair enzymes. The results showed that Bleomycin could promote rAAV2 transduction both in vivo and in vitro. Increased transduction was discovered to be a direct result of decreased cytoplasmic rAAV particle degradation and increased second-strand synthesis. TDP1, PNKP, and SETMAR are required to repair the DNA damage gap caused by Bleomycin, TDP1, PNKP, and SETMAR promote rAAV second-strand synthesis. Bleomycin induced DNA-PKcs phosphorylation and phosphorylated DNA-PKcs and Artemis promoted second-strand synthesis. The current study identifies an effective method for increasing the capability and scope of in-vivo and in-vitro rAAV applications, which can amplify cell transduction at Bleomycin concentrations. It also supplies information on combining tumor gene therapy with chemotherapy.
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Daño del ADN , Terapia Genética , Transducción Genética , ADN , Roturas del ADN , Dependovirus/genética , Vectores Genéticos , Reparación del ADNRESUMEN
Gram-negative sepsis has become a substantial and escalating global healthcare challenge due to the growing antibiotic resistance crisis and the sluggish development of new antibiotics. LL-37, a unique Cathelicidin species found in humans, exhibits a wide range of bioactive properties, including direct bactericidal effects, inflammation regulation, and LPS neutralization. KR-12, the smallest yet potent peptide fragment of LL-37, has been modified to create more effective antimicrobials. In this study, we designed two myristoylated derivatives of KR-12, referred to as Myr-KR-12N and Myr-KR-12C. These derivatives displayed remarkable ability to spontaneously assemble into nanoparticles when mixed with deionized water. Myristoylated KR-12 derivatives exhibited broad-spectrum and intensified bactericidal activity by disrupting bacterial cell membranes. In particular, Myr-KR-12N showed superior capability to rescue mice from lethal E. coli-induced sepsis in comparison with the conventional antibiotic meropenem. We also confirmed that the myristoylated KR-12 nanobiotic possesses significant LPS binding capacity and effectively reduces inflammation in vitro. In an in vivo context, Myr-KR-12N outperformed polymyxin B in rescuing mice from LPS-induced sepsis. Crucially, toxicological assessments revealed that neither Myr-KR-12N nor Myr-KR-12C nanobiotics induced meaningful hemolysis or caused damage to the liver and kidneys. Collectively, our study has yielded an innovative nanobiotic with dual capabilities of bactericidal action and LPS-neutralization, offering substantial promise for advancing the clinical translation of antimicrobial peptides and the development of novel antibiotics. This addresses the critical need for effective solutions to combat Gram-negative sepsis, a pressing global medical challenge.
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Infecciones por Escherichia coli , Sepsis , Humanos , Animales , Ratones , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Lipopolisacáridos/química , Escherichia coli/metabolismo , Catelicidinas/química , Catelicidinas/metabolismo , Catelicidinas/farmacología , Bacterias , Sepsis/tratamiento farmacológico , Antibacterianos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: Streptococcus pneumoniae (S. pneumoniae) is a common pathogen that causes bacterial pneumonia. However, with increasing bacterial resistance, there is an urgent need to develop new drugs to treat S. pneumoniae infections. Nanodefensin with a 14-carbon saturated fatty acid (ND-C14) is a novel nanoantimicrobial peptide designed by modifying myristic acid at the C-terminus of human α-defensin 5 (HD5) via an amide bond. However, it is unclear whether ND-C14 is effective against lung infections caused by S. pneumoniae. METHODS: In vitro, three groups were established, including the control group, and the HD5 and ND-C14 treatment groups. A virtual colony-count assay was used to evaluate the antibacterial activity of HD5 and ND-C14 against S. pneumoniae. The morphological changes of S. pneumoniae treated with HD5 or ND-C14 were observed by scanning electron microscopy. In vivo, mice were divided into sham, vehicle, and ND-C14 treatment groups. Mice in the sham group were treated with 25 µL of phosphate-buffered saline (PBS). Mice in the vehicle and ND-C14 treatment groups were treated with intratracheal instillation of 25 µL of bacterial suspension with 2×108 CFU/mL (total bacterial count: 5×106 CFU), and then the mice were given 25 µL PBS or intratracheally injected with 25 µL of ND-C14 (including 20 µg or 50 µg), respectively. Survival rates were evaluated in the vehicle and ND-C14 treatment groups. Bacterial burden in the blood and bronchoalveolar lavage fluid were counted. The lung histology of the mice was assessed. A propidium iodide uptake assay was used to clarify the destructive effect of ND-C14 against S. pneumoniae. RESULTS: Compared with HD5, ND-C14 had a better bactericidal effect against S. pneumoniae because of its stronger ability to destroy the membrane structure of S. pneumoniae in vitro. In vivo, ND-C14 significantly delayed the death time and improved the survival rate of mice infected with S. pneumoniae. ND-C14 reduced bacterial burden and lung tissue injury. Moreover, ND-C14 had a membrane permeation effect on S. pneumoniae, and its destructive ability increased with increasing ND-C14 concentration. CONCLUSION: The ND-C14 may improve bactericidal effects on S. pneumoniae both in vitro and in vivo.
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Biohybrid photocatalysts are composite materials that combine the efficient light-absorbing properties of synthetic materials with the highly evolved metabolic pathways and self-repair mechanisms of biological systems. Here, we show the potential of conjugated polymers as photosensitizers in biohybrid systems by combining a series of polymer nanoparticles with engineered Escherichia coli cells. Under simulated solar light irradiation, the biohybrid system consisting of fluorene/dibenzo [b,d]thiophene sulfone copolymer (LP41) and recombinant E. coli (i.e., a LP41/HydA BL21 biohybrid) shows a sacrificial hydrogen evolution rate of 3.442 mmol g-1 h-1 (normalized to polymer amount). It is over 30 times higher than the polymer photocatalyst alone (0.105 mmol g-1 h-1), while no detectable hydrogen was generated from the E. coli cells alone, demonstrating the strong synergy between the polymer nanoparticles and bacterial cells. The differences in the physical interactions between synthetic materials and microorganisms, as well as redox energy level alignment, elucidate the trends in photochemical activity. Our results suggest that organic semiconductors may offer advantages, such as solution processability, low toxicity, and more tunable surface interactions with the biological components over inorganic materials.