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Targeting the PI3K/mTOR pathway and modulating mitochondrial adaptation is expected to be a critical approach for cancer therapy. Although the regulation of mitochondria by the PI3K/mTOR pathway has been investigated, it is not well understood due to the complexity of its regulatory mechanisms. RNA-binding proteins (RBPs) selectively regulate gene expression through post-transcriptional modulation, playing a key role in cancer progression. LARP1, a downstream RBP of the mTOR pathway, is involved in mitochondria-mediated BCL-2 cell survival. Therefore, exploring the involvement of LARP1 in PI3K/mTOR-mediated translational regulation of mitochondria-associated proteins in ovarian cancer cells could help elucidate the role of mitochondria in the PI3K/mTOR pathway. We found that, unlike SKOV3 cells, the mitochondrial function of A2780 cells was not affected, which were insensitive to the dual PI3K/mTOR inhibitor PKI-402, suggesting that cell survival may be related to mitochondrial function. Knockdown of the LARP1 gene after PKI-402 treatment resulted in impaired mitochondrial function in A2780 cells, possibly due to decreased mRNA stability and reduced protein translation of the mitochondrial transcription initiation factor, TFB2M, and the respiratory chain complex II subunit, SDHB. LARP1 affects protein translation by binding to TFB2M mRNA, regulating mitochondrial DNA-encoded genes, or indirectly regulating the nuclear DNA-encoded SDHB gene, ultimately interfering with mitochondrial oxidative phosphorylation and leading to apoptosis. Therefore, LARP1 may be an important mediator in the PI3K/mTOR pathway for regulating mRNA translation and mitochondrial function. Targeting RBPs such as LARP1 downstream of the mTOR pathway may provide new insights and potential therapeutic approaches for ovarian cancer treatment.
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Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
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DNA de Cadeia Simples , Edição de Genes , Rad51 Recombinase , Reparo de DNA por Recombinação , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Humanos , Edição de Genes/métodos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Sistemas CRISPR-Cas , Células HEK293 , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
People often change their evaluations upon learning about their peers' evaluations, i.e., social learning. Given sleep's vital role in consolidating daytime experiences, sleep may facilitate social learning, thereby further changing people's evaluations. Combining a social learning task and the sleep-based targeted memory reactivation technique, we asked whether social learning-induced evaluation updating can be modulated during sleep. After participants had indicated their initial evaluation of snacks, they learned about their peers' evaluations while hearing the snacks' spoken names. During the post-learning non-rapid-eye-movement sleep, we re-played half of the snack names (i.e., cued snack) to reactivate the associated peers' evaluations. Upon waking up, we found that the social learning-induced evaluation updating further enlarged for both cued and uncued snacks. Examining sleep electroencephalogram (EEG) activity revealed that cue-elicited delta-theta EEG power and the overnight N2 sleep spindle density predicted post-sleep evaluation updating for cued but not for uncued snacks. These findings underscore the role of sleep-mediated memory reactivation and the associated neural activity in supporting social learning-induced evaluation updating.
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People preferentially endorse positive personality traits as more self-descriptive than negative ones, a positivity self-referential bias. Here, we investigated how to enhance positive self-referential processing, integrating wakeful cue-approach training task (CAT) and sleep-based targeted memory reactivation (TMR). In the CAT, participants gave speeded motor responses to cued positive personality traits. In a subsequent nap, we unobtrusively re-played half of the trained positive traits during slow-wave sleep (TMR). Upon awakening, CAT+TMR facilitated participants' speed in endorsing positive traits in immediate tests, and rendered participants endorse more positive traits as self-descriptive after one week. Notably, these enhancements were associated with the directionality of cue-related 1-4 Hz slow traveling waves (STW) that propagate across brain regions. Specifically, anterior-to-posterior backward STW was positively associated with these benefits, whereas forward STW showed negative associations. These findings demonstrate the potential benefits of integrated wakeful cue-approach training and sleep-based memory reactivation in strengthening positive self-referential processing.
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Colanic acid (CA) is exopolysaccharide that presents growing potential in the food and healthcare industry as a versatile polymer. Previously, we have constructed the Escherichia coli strain WWM16 which can efficiently produce CA. In this study, WWM16 has been further engineered to produce a higher yield of CA with low molecular mass and viscosity. The gene mcbR encoding a transcriptional factor, and the genes opgD, opgG, and opgH related to the biosynthesis of osmoregulated periplasmic glucans were deleted in E. coli WWM16, and the resulting strain WWM166 produced 18.1 g/L CA. The expression level of wcaD encoding the polymerase in WWM166 was downregulated using CRISPRi. As a result, the strain WWM166/pWpD1 could produce 49.9 g/L CA with lower molecular mass. CA products were purified from both WWM166 and WWM166/pWpD1, and their molecular mass, viscosity, fluidity, hygroscopicity, and antioxidant activity were determined and compared. These findings demonstrate the potential application of CA with different molecular masses to prolong life and protect skin in the food and cosmetic industries.
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Escherichia coli , Peso Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Viscosidade , Engenharia Metabólica , Polissacarídeos/metabolismo , Polissacarídeos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/químicaRESUMO
Prior studies have investigated the immediate impacts of substances on temporal perception, the impact of temporal outlook, and the consequences of modified temporal perception on addictive behaviors. These inquiries have provided valuable perspectives on the intricate associations between addiction and time perception, enriching the groundwork for forthcoming research and therapeutic strategies. This comprehensive review aims to further explore intricate correlation among diverse addictive substances-namely alcohol, cannabis, nicotine, opioids-and non-substance addictions such as internet gaming, elucidating their influence on temporal perception. Adhering to the PICOS method and adhering to PRISMA guidelines, we systematically reviewed and critically evaluated all existing research concerning temporal perception in individuals with substance and non-substance use disorders. Specifically, our analyses involved 31 pertinent articles encompassing six unique groups-alcohol, nicotine, cannabis, stimulants, opioids, and internet-related addictions-sourced from a pool of 551 papers. The findings revealed differences in time perception between addicts and control groups, as indicated by medium to large effect sizes (Hedge's g = 0.8, p < 0.001). However, the nature of these differences-whether they predominantly involve time overestimation or underestimation-is not yet definitively clear. This variability underscores the complexity of the relationship between addiction and temporal perception, paving the way for further research to unravel these intricate dynamics.
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Recollecting painful or traumatic experiences can be deeply troubling. Sleep may offer an opportunity to reduce such suffering. We developed a procedure to weaken older aversive memories by reactivating newer positive memories during sleep. Participants viewed 48 nonsense words each paired with a unique aversive image, followed by an overnight sleep. In the next evening, participants learned associations between half of the words and additional positive images, creating interference. During the following non-rapid-eye-movement sleep, auditory memory cues were unobtrusively delivered. Upon waking, presenting cues associated with both aversive and positive images during sleep, as opposed to not presenting cues, weakened aversive memory recall while increasing positive memory intrusions. Substantiating these memory benefits, computational modeling revealed that cueing facilitated evidence accumulation toward positive affect judgments. Moreover, cue-elicited theta brain rhythms during sleep predominantly predicted the recall of positive memories. A noninvasive sleep intervention can thus modify aversive recollection and affective responses.
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Sinais (Psicologia) , Rememoração Mental , Sono , Humanos , Feminino , Sono/fisiologia , Masculino , Rememoração Mental/fisiologia , Adulto , Adulto Jovem , Memória/fisiologiaRESUMO
In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification (HO), and we have developed a glucocorticoid-induced ectopic mineralization (EM) mouse model by systemic administration of a high dose of dexamethasone (DEX) to animals with muscle injury induced by cardiotoxin injection. In this model, dystrophic calcification (DC) developed into HO in a cell autonomous manner. However, it is not clear how DC is formed after DEX treatment. Therefore, in this study, we aimed to explore how glucocorticoids initiate muscle EM at a cellular and molecular level. We showed that DEX treatment inhibited inflammatory cell infiltration into injured muscle but inflammatory cytokine production in the muscle was significantly increased, suggesting that other non-inflammatory muscle cell types may regulate the inflammatory response and the muscle repair process. Accompanying this phenotype, transforming growth factor ß1 (TGF-ß1) expression in fibro-adipogenic progenitors (FAPs) was greatly down-regulated. Since TGF-ß1 is a strong immune suppressor and FAP's regulatory role has a large impact on muscle repair, we hypothesized that down-regulation of TGF-ß1 in FAPs after DEX treatment resulted in this hyperinflammatory state and subsequent failed muscle repair and EM formation. To test our hypothesis, we utilized a transgenic mouse model to specifically knock out Tgfb1 gene in PDGFRα positive FAPs to investigate if the transgenic mice could recapitulate the phenotype that was induced by DEX treatment. Our results showed that the transgenic mice completely phenocopied this hyperinflammatory state and spontaneously developed EM following muscle injury. On the contrary, therapeutics that enhanced TGF-ß1 signaling in FAPs inhibited the inflammatory response and attenuated muscle EM. In summary, these results indicate that FAPs-derived TGF-ß1 is a key molecule in regulating muscle inflammatory response and subsequent EM, and that glucocorticoids exert their effect via down-regulating TGF-ß1 in FAPs.
Heterotopic ossification (HO) is abnormal bone formation in soft tissue. Glucocorticoids, which have strong anti-inflammatory properties, have usually been used as HO therapeutics. However, our findings suggest that glucocorticoids can also promote HO formation. In this study, we tried to explain the underlying reason for these seemingly contradictory observations. We showed that glucocorticoids, in addition to exerting an anti-inflammatory effect on inflammatory cells, can also target another type of muscle cell to exert a pro-inflammatory effect. These cells are called fibro-adipogenic progenitors (FAPs), and we demonstrated that FAPs played a master regulatory role in the muscle inflammatory response by modulating the expression of transforming growth factor ß1 (TGF-ß1), a well-known immune suppressor. In summary, our findings highlighted the importance of FAP TGF-ß1 levels in affecting the progression and regression of muscle HO, and provided new treatment options for HO based on their ability to elevate TGF-ß1 levels in FAPs.
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When reminded of an unpleasant experience, people often try to exclude the unwanted memory from awareness, a process known as retrieval suppression. Here we used multivariate decoding (MVPA) and representational similarity analyses on EEG data to track how suppression unfolds in time and to reveal its impact on item-specific cortical patterns. We presented reminders to aversive scenes and asked people to either suppress or to retrieve the scene. During suppression, mid-frontal theta power within the first 500 ms distinguished suppression from passive viewing of the reminder, indicating that suppression rapidly recruited control. During retrieval, we could discern EEG cortical patterns relating to individual memories-initially, based on theta-driven visual perception of the reminders (0 to 500 ms) and later, based on alpha-driven reinstatement of the aversive scene (500 to 3000 ms). Critically, suppressing retrieval weakened (during 360 to 600 ms) and eventually abolished item-specific cortical patterns, a robust effect that persisted until the reminder disappeared (780 to 3000 ms). Representational similarity analyses provided converging evidence that retrieval suppression weakened the representation of target scenes during the 500 to 3000 ms reinstatement window. Together, rapid top-down control during retrieval suppression abolished cortical patterns of individual memories, and precipitated later forgetting. These findings reveal a precise chronometry on the voluntary suppression of individual memories.
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Conscientização , Eletroencefalografia , Rememoração Mental , Humanos , Masculino , Feminino , Adulto Jovem , Adulto , Conscientização/fisiologia , Rememoração Mental/fisiologia , Estado de Consciência/fisiologia , Memória/fisiologia , Percepção Visual/fisiologia , Encéfalo/fisiologiaRESUMO
Background and purpose: The associations of fundamental motor skills (FMS), health-related physical fitness (e.g., cardiorespiratory fitness, CRF), and moderate-vigorous physical activity (MVPA) have been demonstrated in Western children, but these associations have not yet been validated in a sample of Chinese children. The aims of this study, therefore, were to examine the association between FMS subdomains and MVPA in a sample of Chinese children and to evaluate whether this association is mediated by CRF. Methods: A cross-sectional study consisting of 311 children aged 8-12 years (49.2% girls; mean age = 9.9 years) from Shanghai was conducted. FMS, CRF and MVPA were assessed using the Test of Gross Motor Development-3rd Edition, Progressive Aerobic Cardiovascular Endurance Run and ActiGraph GT3X accelerometers. Preacher & Hayes's bootstrap method was used to test the mediating effects of CRF on the association between FMS and MVPA. Results: CRF fully mediated the association between total FMS and MVPA in girls (indirect effects, b = 0.21, 95% CI [0.07-0.37]), while the mediation was only partial in boys (indirect effects, b = 0.12, 95% CI [0.01-0.26]). CRF fully mediated the association between locomotor skills and MVPA in girls (indirect effects, b = 0.27, 95% CI [0.09- 0.51]), whereas CRF partially mediated the association between object control skills and MVPA in boys (indirect effects, b = 0.15, 95% CI [0.18-0.35]). Conclusion: In order to better design and implement sex-specific interventions aiming to increase MVPA, it is essential to consider FMS subdomains and CRF alongside the sex differences in the association between them.
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Acelerometria , Aptidão Cardiorrespiratória , Exercício Físico , Destreza Motora , Humanos , Feminino , Masculino , Criança , Aptidão Cardiorrespiratória/fisiologia , Estudos Transversais , Exercício Físico/fisiologia , China , Destreza Motora/fisiologia , Fatores Sexuais , População do Leste AsiáticoRESUMO
Nucleic acids are major structures detected by the innate immune system. Although intracellular single-stranded DNA (ssDNA) accumulates during pathogen infection or disease, it remains unclear whether and how intracellular ssDNA stimulates the innate immune system. Here, we report that intracellular ssDNA triggers cytokine expression and cell death in a CGT motif-dependent manner. We identified Schlafen 11 (SLFN11) as an ssDNA-activated RNase, which is essential for the innate immune responses induced by intracellular ssDNA and adeno-associated virus infection. We found that SLFN11 directly binds ssDNA containing CGT motifs through its carboxyl-terminal domain, translocates to the cytoplasm upon ssDNA recognition, and triggers innate immune responses through its amino-terminal ribonuclease activity that cleaves transfer RNA (tRNA). Mice deficient in Slfn9, a mouse homolog of SLFN11, exhibited resistance to CGT ssDNA-induced inflammation, acute hepatitis, and septic shock. This study identifies CGT ssDNA and SLFN11/9 as a class of immunostimulatory nucleic acids and pattern recognition receptors, respectively, and conceptually couples DNA immune sensing to controlled RNase activation and tRNA cleavage.
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DNA de Cadeia Simples , Imunidade Inata , Camundongos Endogâmicos C57BL , Animais , Feminino , Humanos , Masculino , Camundongos , DNA de Cadeia Simples/imunologia , Células HEK293 , Imunidade Inata/imunologia , Camundongos Knockout , Proteínas Nucleares/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ribonucleases/imunologia , Ribonucleases/metabolismoRESUMO
We present a case study detailing the occurrence of splenic lymphangioma in a 9-year-old girl. The multimodal imaging revealed a significant mass, a finding subsequently validated through histopathological examination. This case underscores the importance of considering splenic lymphangioma as a rare but notable differential diagnosis for solid masses observed in imaging studies.
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We conducted a phase I, randomized, double-blind, placebo-controlled trial including healthy adults in Sui County, Henan Province, China. Ninety-six adults were randomly assigned to one of three groups (high-dose, medium-dose, and low-dose) at a 3:1 ratio to receive one vaccine dose or placebo. Adverse events up to 28 days after each dose and serious adverse events up to 6 months after all doses were reported. Geometric mean titers and seroconversion rates were measured for anti-rotavirus neutralizing antibodies using microneutralization tests. The rates of total adverse events in the placebo group, low-dose group, medium-dose group, and high-dose group were 29.17 % (12.62 %-51.09 %), 12.50 % (2.66 %-32.36 %), 50.00 % (29.12 %-70.88 %), and 41.67 % (22.11 %-63.36 %), respectively, with no significant difference in the experimental groups compared with the placebo group. The results of the neutralizing antibody assay showed that in the adult group, the neutralizing antibody geometric mean titer at 28 days after full immunization in the low-dose group was 583.01 (95 % confidence interval [CI]: 447.12-760.20), that in the medium-dose group was 899.34 (95 % CI: 601.73-1344.14), and that in the high-dose group was 1055.24 (95 % CI: 876.28-1270.75). The GMT of serum-specific IgG at 28 days after full immunization in the low-dose group was 3444.26 (95 % CI: 2292.35-5175.02), that in the medium-dose group was 6888.55 (95 % CI: 4426.67-10719.6), and that in the high-dose group was 7511.99 (95 % CI: 3988.27-14149.0). The GMT of serum-specific IgA at 28 days after full immunization in the low-dose group was 2332.14 (95 % CI: 1538.82-3534.45), that in the medium-dose group was 4800.98 (95 % CI: 2986.64-7717.50), and that in the high-dose group was 3204.30 (95 % CI: 2175.66-4719.27). In terms of safety, adverse events were mainly Grades 1 and 2, indicating that the safety of the vaccine is within the acceptable range in the healthy adult population. Considering the GMT and positive transfer rate of neutralizing antibodies for the main immunogenicity endpoints in the experimental groups, it was initially observed that the high-dose group had higher levels of neutralizing antibodies than the medium- and low-dose groups in adults aged 18-49 years. This novel inactivated rotavirus vaccine was generally well-tolerated in adults, and the vaccine was immunogenic in adults (ClinicalTrials.gov number, NCT04626856).
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Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra Rotavirus , Vacinas de Produtos Inativados , Humanos , Adulto , Método Duplo-Cego , Masculino , Feminino , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Pessoa de Meia-Idade , Adulto Jovem , Adolescente , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas contra Rotavirus/imunologia , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/efeitos adversos , China , Imunogenicidade da Vacina , Infecções por Rotavirus/prevenção & controle , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Voluntários Saudáveis , Testes de NeutralizaçãoRESUMO
Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.
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Antivirais , Infecções por Rotavirus , Rotavirus , Replicação Viral , Rotavirus/efeitos dos fármacos , Rotavirus/fisiologia , Animais , Camundongos , Infecções por Rotavirus/tratamento farmacológico , Infecções por Rotavirus/virologia , Replicação Viral/efeitos dos fármacos , Humanos , Antivirais/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação , Camundongos Endogâmicos BALB C , Linhagem Celular , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Metabolic associated fatty liver disease (MAFLD) is a novel concept proposed in 2020, which is more practical for identifying patients with fatty liver disease with high risk of disease progression. Fatty liver is a driver for extrahepatic complications, particularly cardiovascular diseases (CVD). Although the risk of CVD in MAFLD could be predicted by carotid ultrasound test, a very early stage prediction method before the formation of pathological damage is still lacking. METHODS: Stool microbiomes and plasma metabolites were compared across 196 well-characterized participants encompassing normal controls, simple MAFLD patients, MAFLD patients with carotid artery pathological changes, and MAFLD patients with diagnosed coronary artery disease (CAD). 16S rDNA sequencing data and untargeted metabolomic profiles were interrogatively analyzed using differential abundance analysis and random forest (RF) machine learning algorithm to identify discriminatory gut microbiomes and metabolomic. RESULTS: Characteristic microbial changes in MAFLD patients with CVD risk were represented by the increase of Clostridia and Firmicutes-to-Bacteroidetes ratios. Faecalibacterium was negatively correlated with mean-intima-media thickness (IMT), TC, and TG. Megamonas, Bacteroides, Parabacteroides, and Escherichia were positively correlated with the exacerbation of pathological indexes. MAFLD patients with CVD risk were characterized by the decrease of lithocholic acid taurine conjugate, and the increase of ethylvanillin propylene glycol acetal, both of which had close relationship with Ruminococcus and Gemmiger. Biotin l-sulfoxide had positive correlation with mean-IMT, TG, and weight. The general auxin pesticide beta-naphthoxyacetic acid and the food additive glucosyl steviol were both positively correlated with the increase of mean-IMT. The model combining the metabolite signatures with 9 clinical parameters accurately distinguished MAFLD with CVD risk in the proband and validation cohort. It was found that citral was the most important discriminative metabolite marker, which was validated by both in vitro and in vivo experiments. CONCLUSIONS: Simple MAFLD patients and MAFLD patients with CVD risk had divergent gut microbes and plasma metabolites. The predictive model based on metabolites and 9 clinical parameters could effectively discriminate MAFLD patients with CVD risk at a very early stage.
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Fezes , Microbioma Gastrointestinal , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Fezes/microbiologia , Metabolômica/métodos , Doenças Cardiovasculares/sangue , Biomarcadores/sangue , Medição de Risco , Estudos de Casos e Controles , Idoso , Valor Preditivo dos Testes , Bactérias , Fatores de Risco de Doenças Cardíacas , Adulto , Hepatopatia Gordurosa não Alcoólica/sangue , Aprendizado de Máquina , Espessura Intima-Media CarotídeaRESUMO
Imbalance in carbon flux distribution is one of the most important factors affecting the further increase in the yield of high value-added natural products in microbial metabolic engineering. Meanwhile, the most common inducible expression systems are difficult to achieve industrial-scale production due to the addition of high-cost or toxic inducers during the fermentation process. Quorum sensing system, as a typical model for density-dependent induction of gene expression, has been widely applied in synthetic biology. However, there are currently few reports for efficient production of microbial natural products by using quorum sensing system to self-regulate carbon flux distribution. Here, we designed an artificial quorum sensing system to achieve efficient production of L-threonine in engineered Escherichia coli by altering the carbon flux distribution of the central metabolic pathways at specific periods. Under the combination of switch module and production module, the system was applied to divide the microbial fermentation process into two stages including growth and production, and improve the production of L-threonine by self-inducing the expression of pyruvate carboxylase and threonine extracellular transporter protease after a sufficient amount of cell growth. The final strain TWF106/pST1011, pST1042pr could produce 118.2â¯g/L L-threonine with a yield of 0.57â¯g/g glucose and a productivity of 2.46â¯g/(L· h). The establishment of this system has important guidance and application value for the production of other high value-added chemicals in microorganisms by self-regulation.
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Escherichia coli , Fermentação , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Percepção de Quorum , Treonina , Percepção de Quorum/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Treonina/metabolismo , Treonina/biossíntese , Redes e Vias Metabólicas/genética , Glucose/metabolismoRESUMO
BACKGROUND: Rotator cuff tears have been repaired using the transosseous method for decades. The direct suture (DS) technique has been widely used for rotator cuff tears; however, the retear rate is relatively high. Suture anchors are now used frequently for rotator cuff repair (RCR) in accordance with recent developments in materials. However, polyether ether ketone (PEEK) may still cause complications such as the formation of cysts and osteophytes. Some studies have developed the inlay suture (IS) technique for RCR. PURPOSE/HYPOTHESIS: To compare how 3 different surgical techniques-namely, the DS, IS, and PEEK suture anchor (PSA)-affect tendon-bone healing after RCR. We hypothesized that the IS technique would lead to better tendon-to-bone healing and that the repaired structure would be similar to the normal enthesis. STUDY DESIGN: Controlled laboratory study. METHODS: Acute infraspinatus tendon tears were created in 36 six-month-old male rabbits, which were divided into 3 groups based on the technique used for RCR: DS, IS, and PSA. Animals were euthanized at 6 and 12 weeks postoperatively and underwent a histological assessment and imaging. The expression of related proteins was demonstrated by immunohistochemistry and immunofluorescence staining. Mechanical properties were evaluated by biomechanical testing. RESULTS: At 12 weeks, regeneration of the enthesis was observed in the 3 groups. However, the DS group showed a lower type I collagen content than the PSA and IS groups, which was similar to the results for scleraxis. The DS group displayed a significantly inferior type II collagen expression and proteoglycan deposition after safranin O/fast green and sirius red staining. With regard to runt-related transcription factor 2 and alkaline phosphatase, the IS group showed upregulated expression levels compared with the other 2 groups. CONCLUSION: Compared with the DS technique, the PSA and IS techniques contributed to the improved maturation of tendons and fibrocartilage regeneration, while the IS technique particularly promoted osteogenesis at the enthesis. CLINICAL RELEVANCE: The IS and PSA techniques may be more beneficial for tendon-bone healing after RCR.
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Benzofenonas , Cetonas , Polietilenoglicóis , Polímeros , Lesões do Manguito Rotador , Manguito Rotador , Âncoras de Sutura , Técnicas de Sutura , Animais , Coelhos , Masculino , Lesões do Manguito Rotador/cirurgia , Manguito Rotador/cirurgia , Cicatrização , Modelos Animais de DoençasRESUMO
Muscle tissue engineering is a promising therapeutic strategy for volumetric muscle loss (VML). Among them, decellularized extracellular matrix (dECM) biological scaffolds have shown certain effects in restoring muscle function. However, researchers have inconsistent or even contradictory results on whether dECM biological scaffolds can efficiently regenerate muscle fibers and restore muscle function. This suggests that therapeutic strategies based on dECM biological scaffolds need to be further optimized and developed. In this study, we used a recellularization method of perfusing adipose-derived stem cells (ASCs) and L6 into adipose dECM (adECM) through vascular pedicles. On one hand, this strategy ensures sufficient quantity and uniform distribution of seeded cells inside scaffold. On the other hand, auxiliary L6 cells addresses the issue of low myogenic differentiation efficiency of ASCs. Subsequently, the treatment of VML animal experiments showed that the combined recellularization strategy can improve muscle regeneration and angiogenesis than the single ASCs recellularization strategy, and the TA of former had greater muscle contraction strength. Further single-nucleus RNA sequencing (snRNA-seq) analysis found that L6 cells induced ASCs transform into a new subpopulation of cells highly expressing Mki67, CD34 and CDK1 genes, which had stronger ability of oriented myogenic differentiation. This study demonstrates that co-seeding ASCs and L6 cells through vascular pedicles is a promising recellularization strategy for adECM biological scaffolds, and the engineered muscle tissue constructed based on this has significant therapeutic effects on VML. Overall, this study provides a new paradigm for optimizing and developing dECM-based therapeutic strategies.
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Matriz Extracelular Descelularizada , Doenças Musculares , Animais , Matriz Extracelular , Regeneração , Engenharia Tecidual/métodos , Células-Tronco , Obesidade , Músculo Esquelético/fisiologia , Alicerces TeciduaisRESUMO
Bioceramics are widely used in bone tissue engineering, yet the inherent high brittleness and low ductility of the ceramics lead to poor machinability, which restricts their clinical applications. Here, a flexible and processable 3D printed bioceramic scaffold with high ceramic content (66.7 %) and shape fidelity (volume shrinkage rate < 5 %) was developed by freeze-thaw cycles, which was assisted by polyvinyl alcohol (PVA) and silk fibroin (SF). The hydrogen bonding between PVA imparted printability to the ceramic ink and enabled the subsequent formation of flexible scaffolds, which can be twisted, bend and cut to match bone defects. After adding SF, the printability of the inks and hydrophilicity of the scaffolds were enhanced, owing to the interactions between PVA and SF. Further, combined with the formation of ß-sheet in SF, the scaffolds exhibited superior mechanical strength and excellent thermal stability, and can fully recover at 35 % compressive strain, which was breaking through the brittleness bottleneck of conventional ceramic scaffolds. Moreover, in vitro experiments showed excellent mineralization ability, osteogenic and angiogenic activities of the scaffolds, demonstrating its potential in bone regeneration. This initial study offers a promising personalized material for bone repair that can be used rapidly during surgery.
Assuntos
Fibroínas , Alicerces Teciduais , Osso e Ossos , Osteogênese , Engenharia Tecidual , Álcool de Polivinil , Impressão TridimensionalRESUMO
BACKGROUND: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. RESULTS: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in ß-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. CONCLUSIONS: Two novel P. pastoris chassis hosts with impaired ß-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.