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As a metal additive manufacturing process, laser cladding (LC) is employed as a novel and beneficial repair technology for damaged steel structures. This study employed LC technology with 316 L stainless steel powder to repair locally corroded steel plates. The influences of interface slope and scanning pattern on the mechanical properties of repaired specimens were investigated through tensile tests and finite element analysis. By comparing the tensile properties of the repaired specimens with those of the intact and corroded specimens, the effectiveness of LC repair technology was assessed. An analysis of strain variations in the LC sheet and substrate during the load was carried out to obtain the cooperation mechanism between the LC sheet and substrate. The experimental results showed that the decrease in interface slope slightly improved the mechanical properties of repaired specimens. The repaired specimens have similar yield strength and ultimate strength to the intact specimens and better ductility as compared to the corroded specimen. The stress-strain curve of repaired specimens can be divided into four stages: elastic stage, substrate yield-LC sheet elastic stage, substrate hardening-LC sheet elastic stage, and plastic stage. These findings suggest that the LC technology with 316 L stainless steel powder is effective in repairing damaged steel plates in civil engineering structures and that an interface slope of 1:2.5 with the transverse scanning pattern is suitable for the repair process.
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OBJECTIVES: To investigate the effect of reactive oxygen species (ROS)/silent information regulator 1 (SIRT1) on hyperoxia-induced mitochondrial injury in BEAS-2B cells. METHODS: The experiment was divided into three parts. In the first part, cells were divided into H0, H6, H12, H24, and H48 groups. In the second part, cells were divided into control group, H48 group, H48 hyperoxia+SIRT1 inhibitor group (H48+EX 527 group), and H48 hyperoxia+SIRT1 agonist group (H48+SRT1720 group). In the third part, cells were divided into control group, 48-hour hyperoxia+N-acetylcysteine group (H48+NAC group), and H48 group. The ROS kit was used to measure the level of ROS. Western blot and immunofluorescent staining were used to measure the expression levels of SIRT1 and mitochondria-related proteins. Transmission electron microscopy was used to observe the morphology of mitochondria. RESULTS: Compared with the H0 group, the H6, H12, H24, and H48 groups had a significantly increased fluorescence intensity of ROS (P<0.05), the H48 group had significant reductions in the expression levels of SIRT1 protein and mitochondria-related proteins (P<0.05), and the H24 and H48 groups had a significant reduction in the fluorescence intensity of mitochondria-related proteins (P<0.05). Compared with the H48 group, the H48+SRT1720 group had significant increases in the expression levels of mitochondria-related proteins and the mitochondrial aspect ratio (P<0.05), and the H48+EX 527 group had a significant reduction in the mitochondrial area (P<0.05). Compared with the H48 group, the H48+NAC group had a significantly decreased fluorescence intensity of ROS (P<0.05) and significantly increased levels of SIRT1 protein, mitochondria-related proteins, mitochondrial area, and mitochondrial aspect ratio (P<0.05). CONCLUSIONS: The ROS/SIRT1 axis is involved in hyperoxia-induced mitochondrial injury in BEAS-2B cells.
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Bronquios , Células Epiteliales , Hiperoxia , Especies Reactivas de Oxígeno , Sirtuina 1 , Sirtuina 1/metabolismo , Sirtuina 1/fisiología , Sirtuina 1/genética , Humanos , Especies Reactivas de Oxígeno/metabolismo , Hiperoxia/complicaciones , Hiperoxia/metabolismo , Células Epiteliales/metabolismo , Bronquios/metabolismo , Mitocondrias/metabolismo , Células Cultivadas , Línea CelularRESUMEN
Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving Cupriavidus necator H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD600 value of 56.0 and a dry cell weight of 15.2 g L-1, compared to the wild type (WT) strain's final OD600 of 39.1 and dry cell weight of 8.4 g L-1. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD600 of 48.9 and a dry cell weight of 12.7 g L-1, in contrast to the WT strain's final OD600 of 9.0 and dry cell weight of 3.1 g L-1. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.
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Cupriavidus necator , Glicerol , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Glicerol/metabolismo , Glicerol/química , Cationes Bivalentes , Evolución Molecular Dirigida/métodosRESUMEN
OBJECTIVES: Harmonization has been recommended by the International Organization for Standard (ISO) to achieve equivalent results across in vitro diagnostic measurement devices (IVD-MDs). We aim to evaluate the effectiveness of Bland-Altman plot-based harmonization algorithm (BA-BHA) created in this study and compare it with weighted Deming regression-based harmonization algorithm (WD-BHA) proposed in ISO 21151:2020. METHODS: Eighty patient sera were used as the harmonization reference material (HRM) to develop IVD-MD-specific harmonization algorithms. Another panel of 40 patient sera was used to validate the effectiveness of harmonization algorithms. We compared regression slopes, intercepts, Bland-Altman plot layouts, percent differences, limits of agreement (LoAs), between-method coefficients of variation (CV) before and after harmonization. RESULTS: After harmonization by WD-BHA, acceptable slopes and intercepts between measured values and HRM targets were observed in weighted Deming regression, but not in Passing-Bablok analysis. Mean differences were -5.5 to 5.0â¯% and differences at specific levels were -33.9 to 23.9â¯%. LoAs were -64.6 to 74.6â¯%. Between-method CV was 22.9â¯% (±12.9â¯%). However, after harmonization by BA-BHA, both weighted Deming and Passing-Bablok regressions equations presented harmonized results. Mean differences were -0.3 to 0.2â¯% and differences at specific levels were -1.1 to 1.6â¯%. LoAs were -23.3 to 23.2â¯%. Between-method CV was 8.4â¯% (±4.0â¯%). The data points were evenly distributed at both sides of the mean in Bland-Altman plots. CONCLUSIONS: The inequivalence of test results between different methods can be improved but unacceptable analytical differences at specific levels may be hidden in terms of an acceptable slope and intercept on WD-BHA. The new protocol BA-BHA may be a viable alternative to optimize the harmonization for immunoassays.
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Vertebrate life begins with fertilization, and then the zygote genome is activated after transient silencing, a process termed zygotic genome activation (ZGA). Despite its fundamental role in totipotency and the initiation of life, the precise mechanism underlying ZGA initiation remains unclear. The existence of minor ZGA implies the possible critical role of noncoding RNAs in the initiation of ZGA. Here, we delineate the expression profile of long noncoding RNAs (lncRNAs) in early mouse embryonic development and elucidate their critical role in minor ZGA. Compared with protein-coding genes (PCGs), lncRNAs exhibit a stronger correlation with minor ZGA. Distinct H3K9me3 profiles can be observed between lncRNA genes and PCGs, and the enrichment of H3K9me3 before ZGA might explain the suspended expression of major ZGA-related PCGs despite possessing PolII pre-configuration. Furthermore, we identified the presence of PolII-enriched MuERV-L around the transcriptional start site of minor ZGA-related lncRNAs, and these repeats are responsible for the activation of minor ZGA-related lncRNAs and subsequent embryo development. Our study suggests that MuERV-L mediates minor ZGA lncRNA activation as a critical driver between epigenetic reprogramming triggered by fertilization and the embryo developmental program, thus providing clues for understanding the regulatory mechanism of totipotency and establishing bona fide totipotent stem cells.
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Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Genoma , ARN Largo no Codificante , Cigoto , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cigoto/metabolismo , Ratones , Desarrollo Embrionario/genética , Genoma/genética , Femenino , Histonas/metabolismo , Epigénesis Genética , Embrión de Mamíferos/metabolismoRESUMEN
BACKGROUND: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.
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Glicerol , Levaduras , Glicerol/química , Ácidos Grasos/química , Carbono , BiocombustiblesRESUMEN
Notch-RBP-J signaling plays an essential role in the maintenance of myeloid homeostasis. However, its role in monocyte cell fate decisions is not fully understood. Here, we showed that conditional deletion of transcription factor RBP-J in myeloid cells resulted in marked accumulation of blood Ly6Clo monocytes that highly expressed chemokine receptor CCR2. Bone marrow transplantation and parabiosis experiments revealed a cell-intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes. RBP-J-deficient Ly6Clo monocytes exhibited enhanced capacity competing with wildtype counterparts in blood circulation. In accordance with alterations of circulating monocytes, RBP-J deficiency led to markedly increased population of lung tissues with Ly6Clo monocytes and CD16.2+ interstitial macrophages. Furthermore, RBP-J deficiency-associated phenotypes could be genetically corrected by further deleting Ccr2 in myeloid cells. These results demonstrate that RBP-J functions as a crucial regulator of blood Ly6Clo monocytes and thus derived lung-resident myeloid populations, at least in part through regulation of CCR2.
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Monocitos , Células Mieloides , Macrófagos , Trasplante de Médula Ósea , Homeostasis , Receptores de QuimiocinaRESUMEN
BACKGROUND: The skin is the largest organ in the human body and serves as a barrier for protective, immune, and sensory functions. Continuous and permanent exposure to the external environment results in different levels of skin and extracellular matrix damage. During skin wound healing, the use of good dressings and addition of growth factors to the wound site can effectively modulate the rate of wound healing. A dressing containing bioactive substances can absorb wound exudates and reduce adhesion between the wound and dressing, whereas growth factors, cytokines, and signaling factors can promote cell motility and proliferation. AIM AND OBJECTIVES: We prepared a functional wound dressing by combining platelet-rich plasma (PRP) and zwitterionic hydrogels. Functional wound dressings are rich in various naturally occurring growth factors that can effectively promote the healing process in various types of tissues and absorb wound exudates to reduce adhesion between wounds and dressings. Furthermore, PRP-incorporated zwitterionic hydrogels have been used to repair full-thickness wounds in Sprague-Dawley rats with diabetes (DM SD). MATERIALS AND METHODS: Fibroblasts and keratinocytes were cultured with PRP, zwitterionic hydrogels, and PRP-incorporated zwitterionic hydrogels to assess cell proliferation and specific gene expression. Furthermore, PRP-incorporated zwitterionic hydrogels were used to repair full-thickness skin defects in DM SD rats. RESULTS: The swelling ratio of hydrogel, hydrogel + PRP1000 (108 platelets/mL), and hydrogel + PRP1000 (109 platelets/mL) groups were similar (~07.71% ± 1.396%, 700.17% ± 1.901%, 687.48% ± 4.661%, respectively) at 144 hours. The tensile strength and Young modulus of the hydrogel and hydrogel + PRP10000 groups were not significantly different. High concentrations of PRP (approximately 108 and 109 platelets/mL) effectively promoted the proliferation of fibroblasts and keratinocytes. The zwitterionic hydrogels were not cytotoxic to any cell type. High PRP concentration-incorporated zwitterionic hydrogels increased the rate of cell proliferation and significantly increased the expression of characteristic genes such as collagen, fibronectin, involucrin, and keratin. Subsequently, zwitterionic hydrogels with high PRP concentrations were used to repair full-thickness skin defects in DM SD rats, and a wound healing rate of more than 90% was recorded on day 12. CONCLUSIONS: PRP contains high concentrations of growth factors that promote cell viability, enhance specific gene expression, and have a high medical value in cell therapy. Zwitterionic hydrogels have a 3-dimensional interconnected microporous structure and can resist cell adhesion without causing cytotoxicity. Platelet-rich plasma-incorporated zwitterionic hydrogels further enhance the cellular properties and provide an effective therapeutic option for wound healing.
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Diabetes Mellitus , Plasma Rico en Plaquetas , Ratas , Humanos , Animales , Cicatrización de Heridas , Hidrogeles , Ratas Sprague-Dawley , Plasma Rico en Plaquetas/química , Plasma Rico en Plaquetas/metabolismo , Diabetes Mellitus/metabolismo , Adherencias TisularesRESUMEN
IMPORTANCE: Over the past decade, increasing evidence has shown that circular RNAs (circRNAs) play important regulatory roles in viral infection and host antiviral responses. However, reports on the role of circRNAs in Zika virus (ZIKV) infection are limited. In this study, we identified 45 differentially expressed circRNAs in ZIKV-infected A549 cells by RNA sequencing. We clarified that a downregulated circRNA, hsa_circ_0007321, regulates ZIKV replication through targeting of miR-492 and the downstream gene NFKBID. NFKBID is a negative regulator of nuclear factor-κB (NF-κB), and we found that inhibition of the NF-κB pathway promotes ZIKV replication. Therefore, this finding that hsa_circ_0007321 exerts its regulatory role on ZIKV replication through the miR-492/NFKBID/NF-κB signaling pathway has implications for the development of strategies to suppress ZIKV and possibly other viral infections.
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ARN Circular , Infección por el Virus Zika , Virus Zika , Humanos , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Circular/genética , Transducción de Señal , Virus Zika/genética , Virus Zika/metabolismo , Infección por el Virus Zika/genéticaRESUMEN
OBJECTIVE: Bronchopulmonary dysplasia is a chronic lung disease in premature infants with alveolar simplification and pulmonary vascular development disorder as the main pathological feature and hyperoxia as the main etiology. Autophagy is a highly conserved cytological behavior of self-degrading cellular components and is accompanied by oxidative stress. Studies have reported that autophagy is regulated by FOXO1 posttranslational modification. However, whether autophagy can be involved in the regulation of endothelial cell injury induced by hyperoxia and its mechanism are still unclear. STUDY DESIGN: We have activated and inhibited autophagy in human umbilical vein endothelial cells under hyperoxia and verified the role of autophagy in endothelial cell-related functions from both positive and negative aspects. RESULTS: Our research showed that the expression level of autophagy-related proteins decreased, accompanied by decreased cell migration ability and tube formation ability and increased cell reactive oxygen species level and cell permeability under hyperoxia conditions. Using an autophagy agonist alleviated hyperoxia-induced changes and played a protective role. However, inhibition of autophagy aggravated the cell damage induced by hyperoxia. Moreover, the decrease in autophagy proteins was accompanied by the upregulation of FOXO1 phosphorylation and acetylation. CONCLUSION: We concluded that autophagy was a protective mechanism against endothelial cell injury caused by hyperoxia. Autophagy might participate in this process by coregulating posttranslational modifications of FOXO1. KEY POINTS: · Hyperoxia induces vascular endothelial cell injury.. · Autophagy may has a protective role under hyperoxia conditions.. · FOXO1 posttranslational modification may be involved in the regulation of autophagy..
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Mitophagy is a selective degradation mechanism that maintains mitochondrial homeostasis by eliminating damaged mitochondria. Many viruses manipulate mitophagy to promote their infection, but its role in Zika virus (ZIKV) is unclear. In this study, we investigated the effect of mitophagy activation on ZIKV replication by the mitochondrial uncoupling agent niclosamide. Our results demonstrate that niclosamide-induced mitophagy inhibits ZIKV replication by eliminating fragmented mitochondria, both in vitro and in a mouse model of ZIKV-induced necrosis. Niclosamide induces autophosphorylation of PTEN-induced putative kinase 1 (PINK1), leading to the recruitment of PRKN/Parkin to the outer mitochondrial membrane and subsequent phosphorylation of ubiquitin. Knockdown of PINK1 promotes ZIKV infection and rescues the anti-ZIKV effect of mitophagy activation, confirming the role of ubiquitin-dependent mitophagy in limiting ZIKV replication. These findings demonstrate the role of mitophagy in the host response in limiting ZIKV replication and identify PINK1 as a potential therapeutic target in ZIKV infection.
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Infección por el Virus Zika , Virus Zika , Ratones , Animales , Mitofagia , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Infección por el Virus Zika/tratamiento farmacológico , Niclosamida/farmacología , Ubiquitina/metabolismoRESUMEN
Liver fibrosis is a wound-healing response following chronic liver injury caused by hepatitis virus infection, obesity, or excessive alcohol. It is a dynamic and reversible process characterized by the activation of hepatic stellate cells and excess accumulation of extracellular matrix. Advanced fibrosis could lead to cirrhosis and even liver cancer, which has become a significant health burden worldwide. Many studies have revealed that noncoding RNAs (ncRNAs), including microRNAs, long noncoding RNAs and circular RNAs, are involved in the pathogenesis and development of liver fibrosis by regulating signaling pathways including transforming growth factor-ß pathway, phosphatidylinositol 3-kinase/protein kinase B pathway, and Wnt/ß-catenin pathway. NcRNAs in serum or exosomes have been reported to tentatively applied in the diagnosis and staging of liver fibrosis and combined with elastography to improve the accuracy of diagnosis. NcRNAs mimics, ncRNAs in mesenchymal stem cell-derived exosomes, and lipid nanoparticles-encapsulated ncRNAs have become promising therapeutic approaches for the treatment of liver fibrosis. In this review, we update the latest knowledge on ncRNAs in the pathogenesis and progression of liver fibrosis, and discuss the potentials and challenges to use these ncRNAs for diagnosis, staging and treatment of liver fibrosis. All these will help us to develop a comprehensive understanding of the role of ncRNAs in liver fibrosis.
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MicroARNs , ARN Largo no Codificante , Humanos , ARN no Traducido/genética , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/genética , Cirrosis Hepática/terapia , Fibrosis , ARN Largo no Codificante/genética , Vía de Señalización WntRESUMEN
Epigenetic reprogramming of the parental genome is essential for zygotic genome activation and subsequent embryo development in mammals. Asymmetric incorporation of histone H3 variants into the parental genome has been observed previously, but the underlying mechanism remains elusive. In this study, we discover that RNA-binding protein LSM1-mediated major satellite RNA decay plays a central role in the preferential incorporation of histone variant H3.3 into the male pronucleus. Knockdown of Lsm1 disrupts nonequilibrium pronucleus histone incorporation and asymmetric H3K9me3 modification. Subsequently, we find that LSM1 mainly targets major satellite repeat RNA (MajSat RNA) for decay and that accumulated MajSat RNA in Lsm1-depleted oocytes leads to abnormal incorporation of H3.1 into the male pronucleus. Knockdown of MajSat RNA reverses the anomalous histone incorporation and modifications in Lsm1-knockdown zygotes. Our study therefore reveals that accurate histone variant incorporation and incidental modifications in parental pronuclei are specified by LSM1-dependent pericentromeric RNA decay.
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Núcleo Celular , Histonas , Animales , Masculino , Histonas/metabolismo , Núcleo Celular/metabolismo , Desarrollo Embrionario/genética , Cigoto/metabolismo , Estabilidad del ARN , ARN/metabolismo , Mamíferos/genéticaRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.
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Células Madre Hematopoyéticas , N-Metiltransferasa de Histona-Lisina , Pez Cebra , Animales , Humanos , Diferenciación Celular , Linaje de la Célula , Hematopoyesis , Células Madre Hematopoyéticas/patología , N-Metiltransferasa de Histona-Lisina/genética , Inflamación/patología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.
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Tolerancia Inmunológica , Intestinos , Linfocitos , Microbiota , Linfocitos T Reguladores , Animales , Inmunidad Innata , Integrina alfaV/metabolismo , Interleucina-2/inmunología , Intestinos/inmunología , Intestinos/microbiología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Microbiota/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Análisis de la Célula Individual , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Factores de Transcripción/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patologíaRESUMEN
PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.
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Clonación Molecular , ADN , Biblioteca de Genes , ADN/genética , Mutagénesis , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Chronic cutaneous wounds from tissue trauma or extensive burns can impair skin barrier function and cause severe infection. Fabrication of a customizable tissue-engineered skin is a promising strategy for regeneration of uneven wounds. Herein, a planar-/curvilinear-bioprintable hydrogel is developed to produce tissue-engineered skin and evaluated in rat models of chronic and irregular wounds. The hydrogel is composed of biodegradable polyurethane (PU) and gelatin. The hydrogel laden with cells displays good 3D printability and structure stability. The circular wounds of normal and diabetes mellitus (DM) rats treated with planar-printed tri-cell-laden (fibroblasts, keratinocytes, and endothelial progenitor cells (EPCs)) hydrogel demonstrate full reepithelization and dermal repair as well as large amounts of neovascularization and collagen production after 28 days. Furthermore, the curvilinear module is fabricated based on the corresponding wound topography for curvilinear-bioprinting of the irregular tissue-engineered skin. The large and irregular rat skin wounds treated with curvilinear-printed tri-cell-laden hydrogel demonstrate full repair after 28 days. This planar-/curvilinear-bioprintable tri-cell-laden hydrogel shows great potential for customized biofabrication in skin tissue engineering.
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Bioimpresión , Hidrogeles , Animales , Gelatina/química , Gelatina/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Impresión Tridimensional , Ratas , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
As a fast-growing, woody grass plant, Moso bamboo (Phyllostachys edulis) can supply edible shoots, building materials, fibrous raw material, raw materials for crafts and furniture and so on within a relatively short time. Rapid growth of Moso bamboo occurs after the young bamboo shoots are covered with a shell and emerge from the ground. However, the molecular reactions of bioenergetic processes essential for fast growth remain undefined. Herein, total and mitochondrial transcriptomes and proteomes were compared between spring and winter shoots. Numerous key genes and proteins responsible for energy metabolism were significantly upregulated in spring shoots, including those involved in starch and sucrose catabolism, glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, significant decreases in starch and soluble sugar, higher ATP content and higher rates of respiration and glycolysis were identified in spring shoots. Further, the upregulated genes and proteins related to mitochondrial fission significantly increased the number of mitochondria, indirectly promoting intracellular energy metabolism. Moreover, enhanced alternate-oxidase and uncoupled-protein pathways in winter shoots showed that an efficient energy-dissipating system was important for winter shoots to adapt to the low-temperature environment. Heterologous expression of PeAOX1b in Arabidopsis significantly affected seedling growth and enhanced cold-stress tolerance. Overall, this study highlights the power of comparing total and mitochondrial omics and integrating physiochemical data to understand how bamboo initiates fast growth through modulating bioenergetic processes.
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Arabidopsis , Transcriptoma , Arabidopsis/genética , Metabolismo Energético , Regulación de la Expresión Génica de las Plantas , Mitocondrias/metabolismo , Poaceae , Proteómica , Almidón/metabolismo , Transcriptoma/genéticaRESUMEN
Background: MicroRNAs (miRNAs) play critical roles in regulating virus infection and replication. However, the mechanism by which miRNA regulates Zika virus (ZIKV) replication remains elusive. We aim to explore how the differentially expressed miR-103a-3p regulates ZIKV replication and to clarify the underlying molecular mechanism. Methods: Small RNA sequencing (RNA-Seq) was performed to identify differentially expressed miRNAs in A549 cells with or without ZIKV infection and some of the dysregulated miRNAs were validated by quantitative real time PCR (qRT-PCR). The effect of miR-103a-3p on ZIKV replication was examined by transfecting miR-103a-3p mimic or negative control (NC) into A549 cells with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and expression levels of ZIKV NS5 mRNA and NS1 protein were detected by qRT-PCR and Western blot, respectively. The potential target genes for miR-103a-3p were predicted by four algorithms and further validated by mutation analysis through luciferase reporter assay. The predicated target gene OTU deubiquitinase (DUB) 4 (OTUD4) was over-expressed by plasmid transfection or silenced by siRNA transfection into cells prior to ZIKV infection. Activation status of p38 MAPK signaling pathway was revealed by looking at the phosphorylation levels of p38 (p-p38) and HSP27 (p-HSP27) by Western blot. Results: Thirty-five differentially expressed miRNAs in ZIKV-infected A549 cells were identified by RNA-Seq analysis. Five upregulated and five downregulated miRNAs were further validated by qRT-PCR. One of the validated upregulated miRNAs, miR-103a-3p significantly stimulated ZIKV replication both at mRNA (NS5) and protein (NS1) levels. We found p38 MAPK signaling was activated following ZIKV infection, as demonstrated by the increased expression of the phosphorylation of p38 MAPK and HSP27. Blocking p38 MAPK signaling pathway using SB203580 inhibited ZIKV replication and attenuated the stimulating effect of miR-103a-3p on ZIKV replication. We further identified OTUD4 as a direct target gene of miR-103a-3p. MiR-103a-3p over-expression or OTUD4 silencing activated p38 MAPK signaling and enhanced ZIKV replication. In contrast, OTUD4 over-expression inhibited p38 MAPK activation and decreased ZIKV replication. In addition, OTUD4 over-expression attenuated the stimulating effect of miR-103a-3p on ZIKV replication and activation of p38 MAPK signaling. Conclusion: Zika virus infection induced the expression of miR-103a-3p, which subsequently activated p38 MAPK signaling pathway by targeting OTUD4 to facilitate ZIKV replication.
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Shigella flexneri is a serious threat to global public health, and a rapid detection method is urgently needed. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and in vitro diagnosis. Here, we combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a to develop a novel diagnostic test (CRISPR/Cas12a-E-LAMP) for the diagnosis of S. flexneri. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for S. flexneri templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene hypothetical protein (accession: AE014073, region: 4170556-4171,068) of S. flexneri. After the LAMP reaction at 60°C for 20 min, the pre-loaded CRISPR/Cas12a regents were mixed with the LAMP products in one tube at 37°C for 20 min, and the final results can be viewed by naked eyes with a total time of 40 min. The sensitivity of CRISPR/Cas12a-E-LAMP to detect S. flexneri was 4 × 100 copies/µl plasmids and without cross-reaction with other six closely related non-S. flexneri. Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of S. flexneri and may be applied in other pathogen infection detection.