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The porous materials (PM) were prepared by the Pickering high internal phase emulsion (PHIPE) template. Firstly, the nanoparticles named as ZHMNPs or MZHMNPs were fabricated based on zein, Hohenbuehelia serotina polysaccharides and Malus baccata (Linn.) Borkh polyphenols without or with Maillard reaction, the average particle sizes and zeta potentials of which were distributed in a range of 718.1-979.4 nm and -21.6-25.2 mV. ZHMNPs possessed the relatively uniform spherical morphology, while MZHMNPs were irregular in shape. With ZHMNPs or MZHMNPs serving as the stabilizers, the PHIPEs were prepared, and exhibited the good viscoelasticity and excellent storage and freeze-thaw stabilities. Based on above PHIPEs template, the constructed PM possessed the large specific surface area and uniform pore structure. Through the investigations of adsorption performances, PM showed the outstanding adsorption capacities on Pb2+ and Cu2+ ions regardless of dissolving in deionized water or simulated gastrointestinal digestive fluid. Furthermore, the results also showed that the pH, temperature and adsorbent dosage had certain impacts on the adsorption performances of PM on Pb2+ and Cu2+ ions.
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Plomo , Nanopartículas , Emulsiones/química , Porosidad , Adsorción , Polifenoles , Agua , Polisacáridos , Nanopartículas/química , Tamaño de la PartículaRESUMEN
Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.
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Antibacterianos , Infecciones por Pseudomonas , Humanos , Antibacterianos/farmacología , Pseudomonas aeruginosa/metabolismo , Biopelículas , Infecciones por Pseudomonas/microbiología , Fimbrias BacterianasRESUMEN
Nanomedicine has brought significant advancements to healthcare by utilizing nanotechnology in medicine. Despite much promise, the further development of nanocarriers for clinical use has been hindered by a lack of understanding and visualization of nano-bio interactions. Conventional imaging methods have limitations in resolution, sensitivity, and specificity. This study introduces a label-free optical approach using stimulated Raman scattering (SRS) microscopy to image poly(lactic-co-glycolic acid) (PLGA) nanocarriers, the most widely used polymeric nanocarrier for delivery therapeutic agents, with single-particle sensitivity and quantification capabilities. A unique Raman peak was identified for PLGA ester, enabling generalized bio-orthogonal bond imaging. We demonstrated quantitative SRS imaging of PLGA nanocarriers across different biological systems from cells to animal tissues. This label-free imaging method provides a powerful tool for studying this prevalent nanocarrier and quantitatively visualizing their distribution, interaction, and clearance in vivo.
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Microscopía , Nanopartículas , Animales , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Nanopartículas/químicaRESUMEN
Nafamostat mesylate, a synthetic serine protease inhibitor, has been shown to have antiviral activity against SARS-CoV-2 and anticoagulant properties that may be beneficial in the treatment of COVID-19. We conducted a meta-analysis to evaluate the effectiveness and safety of nafamostat mesylate for the treatment of COVID-19. PubMed, Embase, Cochrane Library, Scopus, Web of Science, medRxiv, and bioRxiv were searched up to July 2023 for studies comparing the outcomes of nafamostat mesylate treatment and no nafamostat mesylate treatment in patients with COVID-19. Mortality, disease progression, and adverse events were analyzed. Six studies involving 16,195 patients were included in the analysis. Meta-analysis revealed no significant difference in mortality (odds ratio [OR]: 0.88, 95% CI: 0.20-3.75, P = 0.86) or disease progression (OR: 2.76, 95% CI: 0.31-24.68, P = 0.36) between groups. However, nafamostat mesylate was associated with an increased risk of hyperkalemia (OR: 7.15, 95% CI: 2.66-19.24, P < 0.0001). Nafamostat mesylate did not improve mortality or morbidity in hospitalized patients with COVID-19. The risk of hyperkalemia is a serious concern that requires monitoring and preventive measures. Further research in different COVID-19 populations is required.
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Benzamidinas , Tratamiento Farmacológico de COVID-19 , COVID-19 , Guanidinas , SARS-CoV-2 , Humanos , Benzamidinas/uso terapéutico , Guanidinas/uso terapéutico , Guanidinas/efectos adversos , COVID-19/mortalidad , SARS-CoV-2/efectos de los fármacos , Antivirales/uso terapéutico , Antivirales/efectos adversos , Resultado del Tratamiento , Progresión de la Enfermedad , Hiperpotasemia/tratamiento farmacológicoRESUMEN
Nanomedicine has emerged as a revolutionary strategy of drug delivery. However, fundamentals of the nano-neuro interaction are elusive. In particular, whether nanocarriers can cross the blood-brain barrier (BBB) and release the drug cargo inside the brain, a basic process depicted in numerous books and reviews, remains controversial. Here, we develop an optical method, based on stimulated Raman scattering, for imaging nanocarriers in tissues. Our method achieves a suite of capabilities-single-particle sensitivity, chemical specificity, and particle counting capability. With this method, we visualize individual intact nanocarriers crossing the BBB of mouse brains and quantify the absolute number by particle counting. The fate of nanocarriers after crossing the BBB shows remarkable heterogeneity across multiple scales. With a mouse model of aging, we find that blood-brain transport of nanocarriers decreases with age substantially. This technology would facilitate development of effective therapeutics for brain diseases and clinical translation of nanocarrier-based treatment in general.
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Encefalopatías , Nanomedicina , Animales , Ratones , Encéfalo/diagnóstico por imagen , Barrera Hematoencefálica/diagnóstico por imagen , EnvejecimientoRESUMEN
Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. In this study, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that under specific conditions, biofilms lacking RpoS and/or Crc show increased sensitivity to phenazines indicating that the increased metabolic activity in these mutants comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.
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Fenazinas , ARN , Metilación , Fenazinas/farmacología , ARN/metabolismo , Biopelículas , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.
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Plants growing in open environments are frequently coinfected by multiple strains of the same pathogen. However, few investigations have been carried out to reveal the outcomes and underlying mechanisms of such infections. This study aimed to observe the behaviors of two different strains under coinfection and cocultivation. We constructed an experimental system to study such interactions directly by labeling Magnaporthe oryzae strains with the green fluorescent proteins and mushroom cherry fluorescent protein to observe mixed strain behavior in vivo and in vitro. Moreover, multiomics analyses were conducted to explore the underlying mechanisms at the genomic, transcriptomic, and metabolomic levels. Our results revealed that coinfection with two strains can affect disease severity and that the more weakly virulent strain benefits from the coinfection system. We also found that amino acid variation might negatively influence such interactions at transcriptomic and metabolomic levels. In addition, we showed that the overexpression of a glutamine-related gene improved strain competitiveness during mixture cultivation. Collectively, our results provided experimental methods to analyze the interaction between two strains of M. oryzae and preliminarily explored the interacted mechanism of two strains under cocultivation through multiomics analyses.
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Coinfección , Magnaporthe , Oryza , Oryza/metabolismo , Magnaporthe/genética , Multiómica , Enfermedades de las Plantas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
Three new segmented trapdoor spider species belonging to the family Heptathelidae Kishida, 1923, i.e., Luthelaasukasp. nov. (ââ, Sichuan), L.beijingsp. nov. (ââ, Beijing), and L.kagamisp. nov. (ââ, Sichuan), are described from China. Their phylogenetic position and relationships within Heptathelidae are tested and assessed using a combination available COI data downloaded from GenBank with new DNA sequences obtained in this study. The results show that the new species form a clade with eight known and one undescribed species of Luthela. High-definition illustrations of the male palps and female genitalia, diagnoses, and DNA barcodes are provided for these three new species, and their distributions are mapped.
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High motional resistance and incompatibility with post-CMOS fabrication due to thermal budget constraints are imperative issues associated with the back-end-of-line integration of lateral extensional vibrating micromechanical resonators. This paper presents piezoelectric ZnO-on-nickel resonators as a viable means for mitigating both of the issues. Lateral extensional mode resonators equipped with thin-film piezoelectric transducers can exhibit much lower motional impedances than their capacitive counterparts due to piezo-transducers' higher electromechanical coupling coefficients. Meanwhile, the employment of electroplated nickel as the structural material allows the process temperature to be kept lower than 300 °C, which is low enough for the post-CMOS resonator fabrication. In this work, various geometrical rectangular and square plates resonators are investigated. Moreover, parallel combination of several resonators into a mechanically coupled array was explored as a systematic approach to lower motional resistance from ~1 kΩs to 0.562 kΩs. Higher order modes were investigated for achieving higher resonance frequencies up to 1.57 GHz. Local annealing by Joule heating was also exploited for quality factor improvement after device fabrication by ~2× enhancement and breaking the record of MEMS electroplated nickel resonators in lowering insertion loss to ~10 dB.
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Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. Here, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that biofilms lacking Crc show increased sensitivity to an exogenously added methylated phenazine, indicating that the increased metabolic activity in this mutant comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.
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Flexible and functional perovskite oxide sheets with high orientation and crystallization are the next step in the development of next-generation devices. One promising synthesis method is the lift-off and transfer method using a water-soluble sacrificial layer. However, the suppression of cracks during lift-off is a crucial problem that remains unsolved. In this study, we demonstrated that this problem can be solved by depositing amorphous Al2O3 capping layers on oxide sheets. Using this simple method, over 20 mm2 of crack-free, deep-ultraviolet transparent electrode La:SrSnO3 and ferroelectric Ba0.75Sr0.25TiO3 flexible sheets were obtained. By contrast, the sheets without any capping layers broke. The obtained sheets showed considerable flexibility and high functionality. The La:SrSnO3 sheet simultaneously exhibited a wide bandgap (4.4 eV) and high electrical conductivity (>103 S/cm). The Ba0.75Sr0.25TiO3 sheet exhibited clear room-temperature ferroelectricity with a remnant polarization of 17 µC/cm2. Our findings provide a simple transfer method for obtaining large, crack-free, high-quality, single-crystalline sheets.
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Purpose: To explore the effect of Shenkang injection (SKI) combined with Jinshuibao for early diabetic nephropathy (DN) and its effect on the coagulation fibrinolysis system and urinary protein. Methods: 136 patients with early DN admitted to our hospital from March 2018 to October 2019 were divided into the observation group (n = 68) and the control group (n = 68) randomly. On the basis of the conventional treatment, the control group was treated with SKI, and the observation group was treated with SKI and Jinshuibao. Two weeks later, the therapeutic effects of the 2 groups were compared. The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), and D-dimer (D-D) were observed and compared before and after the treatment. 24 hour urine total protein (24 h-UTP), urine albumin excretion rate (UAER), and urine ß 2 microglobulin (ß 2-MG) were measured and compared before and after the treatment. Adverse reactions in the two groups were recorded during the treatment. Results: The effective rate of the observation group after treatment was 92.65% higher than the control group 79.41%. the difference was statistically significant (P < 0.05). The levels of PT, APTT, TT, FIB, PAI-1, and D-D in the two groups after treatment were lower, and t-PA levels after treatment were higher than those before, and all of the above indicators were significantly changed in the observation group than in the control group. The difference was statistically significant (P < 0.05). The 24 h-UTP, UAER, and ß 2-MG in the two groups after treatment were lower than those before, and all of the above indicators were significantly changed in the observation group than in the control group. The difference was statistically significant (P < 0.05). There was no statistically significant difference during the treatment for 2 groups in terms of adverse reactions. The difference was statistically significant (P > 0.05). Conclusion: SKI combined with Jinshuibao has a significant effect in the treatment of early DN, which can reduce the risk of hyperfunction of coagulation and fibrinolysis system, further reduce the content of urine protein, and delay the process of DN.
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Melatonin, as a dietary supplement, has a potent neuroprotective effect and exerts a certain antidepressant effect. This study explored the molecular mechanisms and targets of melatonin on chronic stress-induced hippocampal damage from the perspective of inhibiting inflammatory cytokines release. Our results indicated that melatonin alleviated chronic restraint stress (CRS)-induced inflammatory response and apoptosis, thus improving hippocampal structural damage and subsequent depression-like behaviors in rats. The radar map displayed that the change of TNF-α content was the most significant. Meanwhile, correlation analysis showed that TNF-α content was highly positively correlated with apoptosis. Molecular autodocking studies suggested that TNF-α converting enzyme ADAM17 as a potential target has a priority in docking with melatonin. Molecular mechanism studies indicated that melatonin inhibited CRS-induced activation of the ADAM17/TNF-α axis and its downstream proteins p38 and p53 phosphorylation in the hippocampus. Analogously, Both ADAM17 inhibitor TMI-1 and TNF-α inhibitor thalidomide relieved the effects of CRS on ADAM17/TNF-α axis and its downstream proteins phosphorylation, hippocampal apoptosis, hippocampal inflammatory response, and depression-like behaviors in rats. Altogether, these findings reveal that melatonin relieves CRS-induced inflammatory response and apoptosis, and subsequent depression-like behaviors by inhibiting ADAM17/TNF-α axis.
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Proteína ADAM17 , Apoptosis , Hipocampo , Melatonina , Enfermedades Neuroinflamatorias , Fármacos Neuroprotectores , Estrés Psicológico , Animales , Ratas , Proteína ADAM17/antagonistas & inhibidores , Citocinas/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/patología , Melatonina/farmacología , Melatonina/uso terapéutico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Talidomida/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/etnología , Estrés Psicológico/complicaciones , Enfermedad CrónicaRESUMEN
A rod-like melem with high fluorescence quantum yield of 71.3 % was prepared in this work to enhance the chemiluminescence (CL) intensity of Na2SO3-Ce (â £) system. The results showed that the CL intensity of Na2SO3-Ce (â £) system could be increased by 350 times based on chemiluminescence resonance energy transfer (CRET) mechanism. Furthermore, a CL sensor based on Na2SO3-Ce (â £)-melem system was designed to detect reduced glutathione (G-SH). It was indicated that the CL sensor exhibited excellent G-SH detection performance with a detection limit of 0.065 nM and a linear range from 0.32 to 650 µM. This study applied melem for CL detection and provided a new way for the detection of G-SH.
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Glutatión , Puntos Cuánticos , Transferencia de Energía , Fluorescencia , Compuestos Heterocíclicos con 3 Anillos , Luminiscencia , Mediciones Luminiscentes/métodos , TriazinasRESUMEN
Magnaporthe oryzae, the causal agent of rice blast disease, produces devastating damage to global rice production. It is urgent to explore novel strategies to overcome the losses caused by this disease. 9-phenanthrol is often used as a transient receptor potential melastatin 4 (TRPM4) channel inhibitor for animals, but we found its fungal toxicity to M. oryzae. Thus, we explored the antimicrobial mechanism through transcriptome and metabolome analyses. Moreover, we found that overexpression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase involved in the tyrosine degradative pathway enhanced the tolerance of 9-phenanthrol in M. oryzae. Thus, our results highlight the potential fungal toxicity mechanism of 9-phenanthrol at metabolic and transcriptomic levels and identify a gene involving 9-phenanthrol alleviation. Importantly, our results demonstrate the novel mechanism of 9-phenanthrol on fungal toxicity that will provide new insights of 9-phenanthrol for application on other organisms.
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4-Hidroxifenilpiruvato Dioxigenasa , Magnaporthe , Micotoxinas , Oryza , 4-Hidroxifenilpiruvato Dioxigenasa/genética , Ascomicetos , Proteínas Fúngicas/metabolismo , Magnaporthe/genética , Magnaporthe/metabolismo , Metaboloma , Micotoxinas/metabolismo , Oryza/metabolismo , Fenantrenos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , TranscriptomaRESUMEN
In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.
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Manganese is a crucial trace element that constitutes the cofactors of many enzymes. However, excessive Mn2+ can be toxic for both prokaryotes and eukaryotes. The mechanism of fungal genetics and metabolism in response to Mn2+ stress remains understudied, warranting further studies. Magnaporthe oryzae is well-established as the most destructive pathogen of rice. A field strain, YN2046, more sensitive to Mn2+ toxicity than other strains, was obtained from a previous study. Herein, we explored the genetic mechanisms of Mn2+ sensitivity in YN2046 through comparative transcriptomic analyses. We found that many genes previously reported to participate in Mn2+ stress were not regulated in YN2046. These non-responsive genes might cause Mn2+ sensitivity in YN2046. Weight gene correlation network analysis (WGCNA) was performed to characterize the expression profile in YN2046. Some overexpressed genes were only found in the Mn2+ tolerant isolate YN125. Among these, many single nucleotide polymorphism (SNP) were identified between YN125 and YN2046, which might disrupt the expression levels of Mn responsive genes. We cloned two uncharacterized genes, MGG_13347 and MGG_16609, from YN125 and transformed them to YN2046 with a strong promoter. Our results showed that the heterologous overexpression of two genes in YN2046 restored its sensitivity. Transcriptomic and biochemical analyses were performed to understand Mn tolerance mechanisms mediated by the two heterologous overexpressed genes. Our results showed that heterologous overexpression of these two genes activated downstream gene expression and metabolite production to restore M. oryzae sensitivity to Mn, implying that SNPs in responsive genes account for different phenotypes of the two strains under Mn stress. IMPORTANCE Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. The comparative transcriptome analysis showed that fewer genes were regulated in the Mn-sensitive strain. WGCNA and SNP analyses found that mutations in promoter and coding sequence regions might disrupt the expression of genes involved in Mn detoxification in the sensitive strain. We transferred two unannotated genes that were cloned from the Mn-tolerant strain into a sensitive strain with strong promoters, and the transformants exhibited an enhanced tolerance to Mn2+ toxicity. Transcriptome and biochemistry results indicated that heterologous overexpression of the two genes enhanced the tolerance to Mn toxicity by reactivation of downstream genes in M. oryzae.
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Magnaporthe , Oryza , Ascomicetos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Magnaporthe/genética , Magnaporthe/metabolismo , Manganeso/metabolismo , Manganeso/toxicidad , Oryza/genética , Oryza/metabolismo , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Polimorfismo de Nucleótido Simple , TranscriptomaRESUMEN
Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.
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Colorantes , Espectrometría Raman , Biomarcadores , Formaldehído , Microscopía , Adhesión en Parafina , Proteínas/metabolismo , Espectrometría Raman/métodos , Fijación del Tejido , VibraciónRESUMEN
PROBLEM: To investigate how asymptomatic bacterial imbalance affects the clinical pregnancy rate after artificial insemination with the husband's semen (AIH). METHODS: This study included married heterosexual couples who underwent AIH. According to the follow-up results, participants were divided into the pregnancy and non-pregnancy groups. Based on the first 10 pair participants in each group with vaginal flora bacterial 16S rRNA sequencing results, six semen samples received bacterial-sperm mixed test. Moreover, 34 cytokines were detected in the peripheral blood sera of the first three pairs by high-throughput Luminex, which were verified in vaginal secretions, cervical mucus, and blood sera from the first 200 pairs by ELISA. RESULTS: The results of the 16S sequencing of vaginal secretions showed that compared with the pregnant group, the non-pregnant group had a significantly increased bacterial species diversity, which was mainly manifested by a decrease in Lactobacillus crispatus and an increase in Prevotella bivia. When Prevotella bivia or Lactobacillus crispatus were mixed with sperms, the sperm motility was decreased (p < .05). The vaginal posterior fornix secretions, cervical mucus, and peripheral blood sera of the non-pregnant group showed decreased levels of MIP-1α and increased levels of IL-17A (p < .05). CONCLUSION: The imbalance of vaginal flora leading to the increase of Prevotella bivia and the decrease of Lactobacillus crispatus may cause an imbalance of immune regulation. Low expression of MIP-1α and high expression of IL-17A were associated with reduced clinical pregnancy rate in AIH.