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The asexual sporulation of filamentous fungi is an important mechanism for their reproduction, survival, and pathogenicity. In Aspergillus and several filamentous fungi, BrlA, AbaA, and WetA are the key elements of a central regulatory pathway controlling conidiation, and MedA is a developmental modifier that regulates temporal expression of central regulatory genes; however, their roles are largely unknown in nematode-trapping (NT) fungi. Arthrobotrys oligospora is a representative NT fungus, which can capture nematodes by producing adhesive networks (traps). Here, we characterized the function of AoMedA and three central developmental regulators (AoBrlA, AoAbaA, and AoWetA) in A. oligospora by gene disruption, phenotypic comparison, and multi-omics analyses, as these regulators are required for conidiation and play divergent roles in mycelial development, trap formation, lipid droplet accumulation, vacuole assembly, and secondary metabolism. A combined analysis of phenotypic traits and transcriptome showed that AoMedA and AoWetA are involved in the regulation of peroxisome, endocytosis, and autophagy. Moreover, yeast one-hybrid analysis showed that AoBrlA can regulate AoMedA, AoAbaA, and AoWetA, whereas AoMedA and AoAbaA can regulate AoWetA. Our results highlight the important roles of AoMedA, AoBrlA, AoAbaA, and AoWetA in conidiation, mycelia development, trap formation, and pathogenicity of A. oligospora and provide a basis for elucidating the relationship between conidiation and trap formation of NT fungi. IMPORTANCE Conidiation is the most common reproductive mode for many filamentous fungi and plays an essential role in the pathogenicity of fungal pathogens. Nematode-trapping (NT) fungi are a special group of filamentous fungi owing to their innate abilities to capture and digest nematodes by producing traps (trapping devices). Sporulation plays an important role in the growth and reproduction of NT fungi, and conidia are the basic components of biocontrol reagents for controlling diseases caused by plant-parasitic nematodes. Arthrobotrys oligospora is a well-known NT fungus and is a routinely used model fungus for probing the interaction between fungi and nematodes. In this study, the functions of four key regulators (AoMedA, AoBrlA, AoAbaA, and AoWetA) involved in conidiation were characterized in A. oligospora. A complex interaction between AoMedA and three central regulators was noted; these regulators are required for conidiation and trap formation and play a pleiotropic role in multiple intracellular activities. Our study first revealed the role of AoMedA and three central regulators in conidiation, trap formation, and pathogenicity of A. oligospora, which contributed to elucidating the regulatory mechanism of conidiation in NT fungi and helped in developing effective reagents for biocontrol of nematodes.
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Ascomicetos , Nematodos , Animales , Metabolismo Secundario , Ascomicetos/fisiología , Saccharomyces cerevisiaeRESUMEN
Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.
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Vacunas Antirrábicas , Virus de la Rabia , Rabia , Chlorocebus aethiops , Humanos , Animales , Ratones , Porcinos , Virus de la Rabia/genética , Rabia/prevención & control , Células HEK293 , Células Vero , Anticuerpos Antivirales , Glicoproteínas/genética , Vacunas Antirrábicas/genética , Proteínas del Envoltorio Viral/genética , Proteínas RecombinantesRESUMEN
Mobile edge computing (MEC) is a promising technique to support the emerging delay-sensitive and compute-intensive applications for user equipment (UE) by means of computation offloading. However, designing a computation offloading algorithm for the MEC network to meet the restrictive requirements towards system latency and energy consumption remains challenging. In this paper, we propose a joint user-association, task-partition, and resource-allocation (JUTAR) algorithm to solve the computation offloading problem. In particular, we first build an optimization function for the computation offloading problem. Then, we utilize the user association and smooth approximation to simplify the objective function. Finally, we employ the particle swarm algorithm (PSA) to find the optimal solution. The proposed JUTAR algorithm achieves a better system performance compared with the state-of-the-art (SOA) computation offloading algorithm due to the joint optimization of the user association, task partition, and resource allocation for computation offloading. Numerical results show that, compared with the SOA algorithm, the proposed JUTAR achieves about 21% system performance gain in the MEC network with 100 pieces of UE.
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Currently, the gold standard and workhorse in mandibular reconstruction is the free vascularized fibula flap. Particularly for patients who have had mandibulectomy for a long time, it is still difficult to precisely reconstruct the mandibular contour and successfully restore the patient's chewing function and esthetics. For the restoration and rehabilitation of long-term mandibular abnormalities, three-dimensional (3D) virtual surgical planning (VSP) and 3D-printed surgical guides are essential. Digital design and manufacturing were used to improve the accuracy of prostheses and facilitate occlusal reconstruction. Therefore, equipped with the methods of 3D VSP, 3D-printed surgical guides, free vascularized fibular flap, and immediate dental implants, this clinical report provides a feasible solution for mandibular reconstruction.
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Implantes Dentales , Colgajos Tisulares Libres , Reconstrucción Mandibular , Humanos , Peroné/cirugía , Estética Dental , Mandíbula/cirugía , Colgajos Tisulares Libres/cirugía , Reconstrucción Mandibular/métodos , Trasplante Óseo/métodosRESUMEN
Resistance to inhibitors of cholinesterase 8 (Ric8) is a conserved guanine nucleotide exchange factor that is involved in the regulation of G-protein signalling in filamentous fungi. Here, we characterized an orthologous Ric8 (AoRic8) in Arthrobotrys oligospora by multi-omics analyses. The Aoric8 deletion (ΔAoric8) mutants lost an ability to produce traps essential for nematode predation, accompanied by a marked reduction in cAMP level. Yeast two-hybrid assay revealed that AoRic8 interacted with G-protein subunit Gα1. Moreover, the mutants were compromised in mycelia growth, conidiation, stress resistance, endocytosis, cellular components and intrahyphal hyphae. Revealed by transcriptomic analysis differentially upregulated genes in the absence of Aoric8 were involved in cell cycle, DNA replication and recombination during trap formation while downregulated genes were primarily involved in organelles, carbohydrate metabolism and amino acid metabolism. Metabolomic analysis showed that many compounds were markedly downregulated in ΔAoric8 mutants versus the wild-type strain. Our results demonstrated a crucial role for AoRic8 in the fungal growth, environmental adaption and nematode predation through control of cell cycle, organelle and secondary metabolism by G-protein signalling.
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Ascomicetos , Nematodos , Animales , Ascomicetos/genética , Colinesterasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Nematodos/genética , Nematodos/microbiologíaRESUMEN
AIMS: Phospholipase C (PLC) is a hydrolase involved in signal transduction in eukaryotic cells. This study aimed to understand the function of PLC in the nematode-trapping fungus Arthrobotrys oligospora. METHODS AND RESULTS: Orthologous PLC (AoPLC2) of A. oligospora was functionally analysed using gene disruption and multi-phenotypic analysis. Disrupting Aoplc2 caused a deformation of partial hyphal cells (about 10%) and conidia (about 50%), decreased the number of nuclei in both conidia and hyphal cells, and increased the accumulation of lipid droplets. Meanwhile, the sporulation-related genes fluG and abaA were downregulated in ΔAoplc2 mutants than in the wild-type strain. Moreover, ΔAoplc2 mutants were more sensitive to osmotic stressors. Importantly, the number of traps, electron-dense bodies in traps, and nematicidal activity of ΔAoplc2 mutants were reduced, and the shape of the traps was deformed. In addition, AoPLC2 was involved in the biosynthesis of secondary metabolites in A. oligospora. CONCLUSIONS: AoPLC2 plays an important role in the development of hyphae, spores, and cell nuclei, responses to stress, formation of traps, and predation of nematodes in A. oligospora. SIGNIFICANCE AND IMPACT OF STUDY: This study reveals the various functions of phospholipase C and elucidates the regulation of trap morphogenesis in nematode-trapping fungi.
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Ascomicetos , Nematodos , Fosfolipasas de Tipo C , Animales , Ascomicetos/enzimología , Ascomicetos/genética , Morfogénesis , Nematodos/microbiología , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , Virulencia/genéticaRESUMEN
The phenomenon of fusion is a type of tooth dysplasia, but few studies have systematically described the treatment of this kind of abnormality. This paper summarizes the treatment methods for fused teeth and classifies the management schemes according to whether the pulp is fused. Then, the treatment for a patient with bilateral anterior tooth fusion is reported. After orthodontic treatment, porcelain veneers were used to restore the normal shape and aesthetic appearance of the anterior teeth.
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Dientes Fusionados , Porcelana Dental , Coronas con Frente Estético , Estética Dental , HumanosRESUMEN
The 38 kDa protein is a major antigen of mycobacterium tuberculosis and has been widely used in TB serodiagnosis, due to its highly sensitivity and specificity. Here we attempt to establish a production platform of recombinant 38 kDa protein in mammalian cells and to evaluate the potential value of 38 kDa protein in TB serodiagnosis. The 38 kDa gene is synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-38 kDa-eGFP was packaged, titered, and then transduced into HEK 293 T cells. Recombinant cell lines were selected by limiting dilution. Supernatants were collected and purified by HisTrapTM HP column. Western blot showed a molecular weight of approximate 38 kDa in cell supernatants as expected. ELISA assay confirmed the immunological specificity of the obtained protein in the presence of MTB-infected human serum samples. In all, we have obtained a stable cell line with long-term and robust expression of secretory MTB 38 kDa protein, which may provide a promising candidate antigen for the development of TB serological diagnosis.
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Antígenos Bacterianos/genética , Expresión Génica , Lipoproteínas/genética , Mycobacterium tuberculosis/metabolismo , Antígenos Bacterianos/análisis , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/aislamiento & purificación , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Lipoproteínas/análisis , Lipoproteínas/biosíntesis , Lipoproteínas/aislamiento & purificación , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tuberculosis/microbiologíaRESUMEN
Small GTPases of the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development and pathogenicity in yeast and filamentous fungi; however, little is known about their roles in nematode-trapping (NT) fungi. In this study, an ortholog of Arf-GAP Glo3 (AoGlo3) in Saccharomyces cerevisiae was characterized in the NT fungus Arthrobotrys oligospora. Deletion of the Aoglo3 gene resulted in growth defects and an increase in hyphal septum. Meanwhile, the sporulation capacity of the ΔAoglo3 mutant was decreased by 98%, and 67.1-71.2% spores became gourd or claviform in shape (from obovoid), which was accompanied by a significant decrease in the spore germination rate. This reduced sporulation capacity correlated with the transcriptional repression of several sporulation-related genes including fluG, rodA, abaA, medA, and lreA. The ΔAoglo3 mutant was also sensitive to several chemical stressors such as Congo red, NaCl, and sorbitol. Additionally, AoGlo3 was found to be involved in endocytosis, and more myelin figures were observed in the ΔAoglo3 mutant than in the wild-type strain, which was consistent with the presence of more autophagosomes observed in the mutant. Importantly, AoGlo3 affected the production of mycelial traps and serine proteases for nematode predation. In summary, AoGlo3 is involved in the regulation of multiple cellular processes such as mycelial growth, conidiation, environmental adaption, endocytosis, and pathogenicity in A. oligospora.
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Factores de Ribosilacion-ADP/metabolismo , Aldehído Oxidorreductasas/metabolismo , Ascomicetos , Nematodos/microbiología , Animales , Ascomicetos/genética , Ascomicetos/patogenicidad , Ascomicetos/fisiología , Ascomicetos/ultraestructura , Autofagia , Endocitosis , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Micelio/crecimiento & desarrollo , Serina Proteasas/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , VirulenciaRESUMEN
Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic ß-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme's optimal reaction conditions and catalytic efficiency (Km /kcat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors.IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.
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Basidiomycota/metabolismo , Dimetilaliltranstransferasa/metabolismo , Proteínas Fúngicas/metabolismo , Lactonas/metabolismo , Basidiomycota/enzimología , Escherichia coli/metabolismo , Microorganismos Modificados Genéticamente/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AND OBJECTIVE: Peri-implantitis is a plaque-associated pathological condition occurring in tissues around dental implants, characterized by inflammation in the peri-implant mucosa and subsequent progressive loss of supporting bone. Wnt5a is the activating ligand of the non-canonical Wnt signaling pathways and plays important roles in leukocyte infiltration and cytokine/ chemokine production in inflammatory disorders. Previous studies showed that Wnt5a was significantly up-regulated in gingival tissues of chronic and aggressive periodontitis. However, the roles and the regulatory mechanisms of Wnt5a in peri-implantitis are not well known. METHODS: The expression of Wnt5a in gingival tissues collected from 8 healthy implant patients and 8 peri-implantitis patients was analyzed by Western blotting and immunofluorescence. Porphyromonas gingivalis infected macrophages isolated from the peripheral blood of healthy volunteers were used as an in vitro cellular model of peri-implantitis. Using neutralizing antibodies, inhibitors and siRNA, the production and roles of Wnt5a in peri-implantitis were assessed by immunofluorescence, quantitative polymerase chain reaction (RT-PCR) and Western blotting. Unpaired two-tailed Student's t test was used to compare qRT-PCR and Western blotting results. P ≤ .05 was considered statistically significant. RESULTS: Wnt5a was highly expressed in the gingival tissues of peri-implantitis patients. Compared to controls, Wnt5a increased in P gingivalis infected macrophages. Wnt5a production in response to P gingivalis infection was dependent on LOX-1 and TLR4. Compared to controls, Wnt5a knockdown impaired IL-1ß, MCP-1, and MMP2 production induced by P gingivalis infection. CONCLUSION: Our results indicate that Wnt5a is involved in LOX-1 and TLR4 induced inflammatory signature via inflammatory cytokines production in response to P gingivalis infection. These findings demonstrate that Wnt5a maybe an important component of the host immune response in peri-implantitis.
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Implantes Dentales , Periimplantitis/metabolismo , Receptores Depuradores de Clase E/metabolismo , Receptor Toll-Like 4/aislamiento & purificación , Proteína Wnt-5a/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Macrófagos/microbiología , Porphyromonas gingivalisRESUMEN
Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1-3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different ß-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.
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Cupriavidus/genética , Vectores Genéticos , Plásmidos/genética , Origen de Réplica , Secuencia de Bases , Clonación Molecular , Escherichia/genética , Dosificación de Gen , Ingeniería Genética , Ingeniería Metabólica , Operón , Éteres Fenílicos/metabolismo , Plásmidos/aislamiento & purificación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADNRESUMEN
Because cleft palate (CP) is one of the most common congenital deformities, surgeons have tried for longtime to achieve an ideal reconstruction of the palatal bone defects and restoration of muscle attachments. In this study, a new CP model on rhesus was established and corrected by an approach of distraction osteogenesis (DO), and then quantitative studies of regulation of osteogenesis genes and expression of alkaline phosphatase (ALP), insulin-like growth factor-I (IGF-I), osteopontin (OPN), and osteocalcin (OC) in different phases of new bone formation were preformed. The CP models (23 animals) were established surgically. In the experimental group (21 animals), the tissue defects were repaired by means of DO at the rhythm of 0.4 mm twice per day. The specimens were retrieved in 1, 2, 4, 6, 8, 12, and 24 weeks (3 animals each) after completion of distraction. The IGF-I, ALP, OPN, and OC messenger RNA (mRNA) were detected by real-time reverse transcription polymerase chain reaction, and their proteins were then analyzed by enzyme-linked immunosorbent assay tests. The results are compared with those of the experimental control and empty control groups (2 animals each). In the distraction gap, the mRNA and protein expressions levels of IGF-I and ALP were both highly upregulated and reaching apex in the early phase of new bone formation. Otherwise, the mRNA and protein expressions of OPN and OC demonstrated high level during intermediate and later remodeling stages. These results suggest that the reconstruction of CP bone defect by means of DO could get definitely intramembraneous new bone formation and eventually quite normal bone structure via consecutive remodeling in situ.
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Fisura del Paladar/cirugía , Osteogénesis por Distracción/métodos , Osteogénesis/genética , Procedimientos de Cirugía Plástica/métodos , Fosfatasa Alcalina/análisis , Animales , Remodelación Ósea/genética , Fisura del Paladar/genética , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Factor I del Crecimiento Similar a la Insulina/análisis , Macaca mulatta , Osteocalcina/análisis , Osteopontina/análisis , ARN Mensajero/análisis , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia ArribaRESUMEN
OBJECTIVE: To compare five selective media to isolate human Bifidobacterium. METHODS: Feces from six healthy human volunteers were diluted and cultivated on five Bifidobacterium selective media. After anaerobic cultivation, bacterial colonies were counted, selected and identified. Meanwhile, bacterial genomic DNA was extracted from the feces samples, and the Denaturing Gradient Gel Electrophoresis (DGGE) and Quantitative Polymerase Chain Reaction (q-PCR) were applied to reveal the diversity of Bifidobacterium. RESULTS: The amount of Bifidobacterium grown on BSM and BLM media was similar to the result detected by q-PCR and was significantly higher than that on three other media. Bifidobacterium isolated from BLM medium was similar to the identified result of DGGE profile. CONCLUSION: BLM medium is the best selective medium for Bifidobacterium isolation from human gastrointestinal tract.
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Bifidobacterium/aislamiento & purificación , Medios de Cultivo/química , Heces/microbiología , Adulto , Bifidobacterium/clasificación , Bifidobacterium/genética , Bifidobacterium/metabolismo , Medios de Cultivo/metabolismo , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , FilogeniaRESUMEN
Arthrobotrys oligospora, a widespread nematode-trapping fungus which can produce conidia for asexual reproduction and form trapping devices (traps) to catch nematodes. However, little is known about the sporulation mechanism of A. oligospora. This research characterized the functions and regulatory roles of the upstream spore-producing regulatory genes, AosfgA and AofluG, in A. oligospora. Our analysis showed that AosfgA and AofluG interacted with each other. Meanwhile, the AofluG gene was downregulated in the ΔAosfgA mutant strain, indicating that AosfgA positively regulates AofluG. Loss of the AosfgA and AofluG genes led to shorter hyphae and more septa, and the ΔAosfgA strain responded to heat and chemical stresses. Surprisingly, the number of nuclei was increased in the mycelia but reduced in the conidia of the ΔAosfgA and ΔAofluG mutants. In addition, after nematode induction, the number and volume of vacuoles were remarkably increased in the ΔAosfgA and ΔAofluG mutant strains. The abundance of metabolites was markedly decreased in the ΔAosfgA and ΔAofluG mutant strains. Collectively, the AosfgA and AofluG genes play critical roles in mycelial development, and they are also involved in vacuole assembly, the stress response, and secondary metabolism. Our study provides distinct insights into the regulatory mechanism of sporulation in nematode-trapping fungi.
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Introduction: Arbuscular mycorrhizal fungi (AMF) have been demonstrated their ability to enhance the arsenic (As) tolerance of host plants, and making the utilization of mycorrhizal plants a promising and practical approach for remediating As-contaminated soils. However, comprehensive transcriptome analysis to reveal the molecular mechanism of As tolerance in the symbiotic process between AMF and host plants is still limited. Methods: In this study, transcriptomic analysis of Gossypium seedlings was conducted with four treatments: non-inoculated Gossypium under non-As stress (CK0), non-inoculated Gossypium under As stress (CK100), F. mosseae-inoculated Gossypium under non-As stress (FM0), and F. mosseae-inoculated Gossypium under As stress (FM100). Results: Our results showed that inoculation with F. mosseae led to a reduction in net fluxes of Ca2+, while increasing Ca2+ contents in the roots and leaves of Gossypium under the same As level in soil. Notably, 199 and 3129 differentially expressed genes (DEGs) were specially regulated by F. mosseae inoculation under As stress and non-As stress, respectively. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analyses, we found that under As stress, F. mosseae inoculation up-regulated a significant number of genes related to the Ca2+ signaling pathway genes, involved in cellular process, membrane part, and signal transduction. This suggests a potential role in mitigating As tolerance in Gossypium seedlings. Furthermore, our analysis identified specific DEGs in transcription factor families, including ERF, MYB, NAC, and WRKY, that were upregulated by F. mosseae inoculation. Conversely, MYB and HB-other were down-regulated. The ERF and MYB families exhibited the highest number of up- and down-regulated DEGs, respectively, which were speculated to play an important role in alleviating the As toxicity of Gossypium. Discussion: Our findings provided valuable insights into the molecular theoretical basis of the Ca2+ signaling pathway in improving As tolerance of mycorrhizal plants in the future.
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Rab GTPases regulate vesicle trafficking in organisms and play crucial roles in growth and development. Arthrobotrys oligospora is a ubiquitous nematode-trapping (NT) fungus, it can form elaborate traps to capture nematodes. Our previous study found that deletion of Aorab7A abolished the trap formation and sporulation. Here, we investigated the regulatory mechanism of AoRab7A using transcriptomic, biochemical, and phenotypic comparisons. Transcriptome analysis, yeast library screening, and yeast two-hybrid assay identified two vacuolar protein sorting (Vps) proteins, AoVps41 and AoVps35, as putative targets of AoRab7A. The deletion of Aovps41 and Aovps35 caused considerable defects in multiple phenotypic traits, such as conidiation and trap formation. We further found a close connection between AoRab7A and Vps proteins in vesicle-vacuole fusion, which triggered vacuolar fragmentation. Further transcriptome analysis showed that AoRab7A and AoVps35 play essential roles in many cellular processes and components including proteasomes, autophagy, fatty acid degradation, and ribosomes in A. oligospora. Furthermore, we verified that AoRab7A, AoVps41, and AoVps35 are involved in ribosome and proteasome functions. The absence of these proteins inhibited the biosynthesis of nascent proteins and enhanced ubiquitination. Our findings suggest that AoRab7A interacts with AoVps41 and AoVps35 to mediate vacuolar fusion and influence lipid droplet accumulation, autophagy, and stress response. These proteins are especially required for the conidiation and trap development of A. oligospora.
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Ascomicetos , Nematodos , Complejo de la Endopetidasa Proteasomal , Animales , Vacuolas , Saccharomyces cerevisiae , RibosomasRESUMEN
Polyetheretherketone (PEEK) has been one of the most promising materials in bone tissue engineering in recent years, with characteristics such as biosafety, corrosion resistance, and wear resistance. However, the weak bioactivity of PEEK leads to its poor integration with bone tissues, restricting its application in biomedical fields. This research effectively fabricated composite porous scaffolds using a combination of PEEK, nano-hydroxyapatite (nHA), and carbon fiber (CF) by the process of fused deposition molding (FDM). The experimental study aimed to assess the impact of varying concentrations of nHA and CF on the biological performance of scaffolds. The incorporation of 10% CF has been shown to enhance the overall mechanical characteristics of composite PEEK scaffolds, including increased tensile strength and improved mechanical strength. Additionally, the addition of 20% nHA resulted in a significant increase in the surface roughness of the scaffolds. The high hydrophilicity of the PEEK composite scaffolds facilitated the in vitro inoculation of MC3T3-E1 cells. The findings of the study demonstrated that the inclusion of 20% nHA and 10% CF in the scaffolds resulted in improved cell attachment and proliferation compared to other scaffolds. This suggests that the incorporation of 20% nHA and 10% CF positively influenced the properties of the scaffolds, potentially facilitating bone regeneration. In vitro biocompatibility experiments showed that PEEK composite scaffolds have good biosafety. The investigation on osteoblast differentiation revealed that the intensity of calcium nodule staining intensified, along with an increase in the expression of osteoblast transcription factors and alkaline phosphatase activities. These findings suggest that scaffolds containing 20% nHA and 10% CF have favorable properties for bone induction. Hence, the integration of porous PEEK composite scaffolds with nHA and CF presents a promising avenue for the restoration of bone defects using materials in the field of bone tissue engineering.
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Fibula transplantation plays an irreplaceable role in restoring the function and morphology of the defected mandible. However, the complex load-bearing environment of the mandible makes it urgent to accurately reconstruct the mandible, ensure the position of the condyle after surgery, and restore the patient's occlusal function and contour. The intervention of digital design and three-dimensional (3D) printed titanium mesh provides a more efficient method and idea to solve this problem. Digital design guides the accurate positioning, osteotomy, and simultaneous implant placement during surgery, and 3D printed titanium mesh ensures stable condyle position after surgery, restoring good mandibular function. The double-layer folded fibula maintains the vertical height of the mandible and a good facial contour, and simultaneous implant placement can establish a good occlusal relationship. This study conducted a retrospective analysis of five patients with jaw defects who underwent digital fibula reconstruction over the past 3 years. It was found that the surgical protocol combining digital design, 3D printed intraoperative guides, 3D printed titanium mesh, free fibula flap, immediate implant, and occlusal reconstruction to repair jaw defects had more ideal facial appearance and biological function. It will provide a more reliable surgical protocol for clinical management of large mandibular defects.
RESUMEN
OBJECTIVES: This study aimed to use a carboxybetaine methacrylate (CBMA) copolymer solution to surface treat 3D printed clear aligners at different fabrication stages, to impart antifouling properties, and assess the surface treatment at various fabrication stages' impact on physico-mechanical characteristics. METHODS: Surface treatments using a blend of 2-hydroxyethyl methacrylate (HEMA) and CBMA, termed CCS, were performed at various stages of 3D printed clear aligner fabrication. Experimental groups, CB1, CB2, and CB3, were determined by the stage of surface treatment during post-processing. CB1, CB2, and CB3 received treatment before post-curing, after post-curing, and after post-processing, respectively. Untreated samples served as controls. Physical and mechanical properties were assessed through tensile testing, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and UV-Vis spectroscopy. The surface was further characterized through scanning electron microscopy and contact angle measurements. The cytotoxicity was assessed with 7-day elution and agar diffusion assays. Lastly, bacterial biofilm resistance was evaluated using confocal laser scanning microscopy. Crystal violet assay was performed using Streptococcus mutans. RESULTS: Surface treatment during CB1 stage exerted the most significantly unfavorable influence on properties of the 3D printed aligner resin. CB2 samples showed the maximum preservation of translucency even after 7-day aging. CB2 and CB3 phases showed enhanced hydrophilicity of sample surfaces with reduced adhesion of multispecies biofilm and S. mutans. SIGNIFICANCE: Application of CCS surface treatment immediately after post-curing (CB2) can enhance the biofilm resistance of 3D printed clear aligners while maintaining high fidelity to optical translucency and constituent mechanical properties.