RÉSUMÉ
The mRNA 5'cap-binding eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in the control of mRNA translation in health and disease. One mechanism of regulation of eIF4E activity is via phosphorylation of eIF4E by MNK kinases, which promotes the translation of a subset of mRNAs encoding pro-tumorigenic proteins. Work on eIF4E phosphatases has been paltry. Here, we show that PPM1G is the phosphatase that dephosphorylates eIF4E. We describe the eIF4E-binding motif in PPM1G that is similar to 4E-binding proteins (4E-BPs). We demonstrate that PPM1G inhibits cell proliferation by targeting phospho-eIF4E-dependent mRNA translation.
Sujet(s)
Prolifération cellulaire , Facteur-4E d'initiation eucaryote , Biosynthèse des protéines , Protein phosphatase 2C , ARN messager , Facteur-4E d'initiation eucaryote/métabolisme , Facteur-4E d'initiation eucaryote/génétique , Humains , Prolifération cellulaire/génétique , Protein phosphatase 2C/métabolisme , Protein phosphatase 2C/génétique , Phosphorylation , ARN messager/génétique , ARN messager/métabolisme , Phosphoprotein Phosphatases/métabolisme , Phosphoprotein Phosphatases/génétique , Liaison aux protéines , Cellules HEK293 , AnimauxRÉSUMÉ
Bacterial cellulose (BC) has been found extensive applications in diverse domains for its exceptional attributes. However, the lack of antibacterial properties hampers its utilization in food and biomedical sectors. Leucocin, a bacteriocin belonging to class IIa, is synthesized by Leuconostoc that demonstrates potent efficacy against the foodborne pathogen, Listeria monocytogenes. In the current study, co-culturing strategy involving Kosakonia oryzendophytica FY-07 and Leuconostoc carnosum 4010 was used to confer anti-listerial activity to BC, which resulted in the generation of leucocin-containing BC (BC-L). The physical characteristics of BC-L, as determined by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA), were similar to the physical characteristics of BC. Notably, the experimental results of disc diffusion and growth curve indicated that the BC-L film exhibited a potent inhibitory effect against L. monocytogenes. Scanning electron microscopy (SEM) showed that BC-L exerts its bactericidal activity by forming pores on the bacterial cell wall. Despite the BC-L antibacterial mechanism, which involves pore formation, the mammalian cell viability remained unaffected by the BC-L film. The measurement results of zeta potential indicated that the properties of BC changed after being loaded with leucocin. Based on these findings, the anti-listerial BC-L generated through this co-culture system holds promise as a novel effective antimicrobial agent for applications in meat product preservation and packaging.
Sujet(s)
Antibactériens , Cellulose , Listeria monocytogenes , Listeria monocytogenes/effets des médicaments et des substances chimiques , Listeria monocytogenes/croissance et développement , Antibactériens/pharmacologie , Antibactériens/composition chimique , Cellulose/composition chimique , Cellulose/pharmacologie , Cellulose/biosynthèse , Techniques de coculture , Tests de sensibilité microbienne , Leuconostoc/métabolisme , Bactériocines/pharmacologie , Bactériocines/composition chimiqueRÉSUMÉ
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent â¼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.
RÉSUMÉ
In lactic acid bacteria, the global transcriptional regulator CcpA regulates carbon metabolism by repressing and activating the central carbon metabolism pathway, thus decreasing or increasing the yield of certain metabolites to maximize carbon flow. However, there are no reports on the deregulation of the inhibitory effects of CcpA on the metabolism of secondary metabolites. In this study, we identified a single-base mutant strain of Lactococcus lactis N8-2 that is capable of metabolizing 2,3-butanediol. It has been established that CcpA dissociates from the catabolite responsive element (cre) site due to a mutation, leading to the activation of derepression and expression of the 2,3-butanediol dehydrogenase gene cluster (butB and butA). Transcriptome analysis and quantitative polymerase chain reaction (Q-PCR) results showed significant upregulation of transcription of butB and butA compared to the unmutated strain. Furthermore, micro-scale thermophoresis experiments confirmed that CcpA did not bind to the mutated cre. Furthermore, in a bacterial two-plasmid fluorescent hybridization system, it was similarly confirmed that the dissociation of CcpA from cre eliminated the repressive effect of CcpA on downstream genes. Finally, we investigated the differing catalytic capacities of the 2,3-butanediol dehydrogenase gene cluster in L. lactis N8-1 and L. lactis N8-2 for 2,3-butanediol. This led to increased expression of butB and butA, which were deregulated by CcpA repression. This is the first report on the elimination of the deterrent effect of CcpA in lactic acid bacteria, which changes the direction of enzymatic catalysis and alters the direction of carbon metabolism. This provides new perspectives and strategies for metabolizing 2,3-butanediol using bacteria in synthetic biology.
RÉSUMÉ
Antigen-presenting cells (APCs) constantly express major histocompatibility complex II (MHC II), including macrophages and dendritic cells (DCs) which deliver antigens to CD4+ T cells and play an important role in adaptive immunity. The expression of MHC II is controlled by the transcriptional coactivator CIITA. Interleukin-27 (IL-27), a newly discovered IL-12 family cytokine, is composed of p28 and EBI3 subunits. In this study, we used IL-27p28 conditional knock-out mice to investigate the regulatory effects of IL-27p28 on macrophage polarization and the expression of MHC II in macrophages. We found that MHC II expression was upregulated in the bone marrow-derived and peritoneal exudate macrophages (BMDMs; PEMs) from IL-27p28-deficient mice, with their inflammation regulating function unaffected. We also demonstrated that in the APCs, IL-27p28 selectively regulated MHC II expression in macrophages but not in dendritic cells. During Pseudomonas aeruginosa (P. aeruginosa) reinfection, higher survival rate, bacterial clearance, and ratio of CD4+/CD8+ T cells in the spleen during the specific immune phase were observed in IL-27p28 defect mice, as well as an increased MHC II expression in alveolar macrophages (AMs). But these did not occur in the first infection. For the first time we discovered that IL-27p28 specifically regulates the expression of MHC II in macrophages by regulating CIITA, while its absence enhances antigen presentation and adaptive immunity against P. aeruginosa.
Sujet(s)
Lymphocytes T CD8+ , Antigènes d'histocompatibilité de classe II , Interleukines , Macrophages , Animaux , Souris , Présentation d'antigène , Antigènes d'histocompatibilité de classe II/métabolisme , Macrophages/métabolisme , Souris de lignée C57BL , Interleukines/génétique , Interleukines/métabolismeRÉSUMÉ
The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR posttranslational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their posttranslational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.
Sujet(s)
Protéine CFTR , Mucoviscidose , Humains , Protéine CFTR/génétique , Protéine CFTR/métabolisme , Pliage des protéines , Mucoviscidose/génétique , Mutation , Réticulum endoplasmique/métabolismeRÉSUMÉ
The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence Summary: Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.
RÉSUMÉ
Hydrophobins (HFBs) are a group of small, secreted amphipathic proteins of fungi with multiple physiological functions and potential commercial applications. In this study, HFB genes of the edible mushroom, Grifola frondosa, were systematically identified and characterized, and their transcriptional profiles during fungal development were determined. In total, 19 typical class I HFB genes were discovered and bioinformatically analyzed. Gene expression profile examination showed that Gf.hyd9954 was particularly highly upregulated during primordia formation, suggesting its major role as the predominant HFB in the lifecycle of G. frondosa. The wettability alteration profile and the surface modification ability of recombinant rGf.hyd9954 were greater than for the Grifola HFB HGFII-his. rGf.hyd9954 was also demonstrated to form the typical class I HFB characteristic-rodlet bundles. In addition, rGf.hyd9954 was shown to possess nanoparticle characteristics and emulsification activities. This research sheds light on the regulation of fungal development and its association with the expression of HFB genes.
RÉSUMÉ
Curcumin, a polyphenol derived from turmeric, has multiple biological functions, such as anti-inflammatory, antioxidant, antibacterial and, above all, antitumor activity. Colorectal cancer is a common malignancy of the gastrointestinal tract with an extremely high mortality rate. However, the low bioavailability and poor targeting properties of curcumin generally limit its clinical application. In the present study, we designed a fusion protein GE11-HGFI as a nanodrug delivery system. The protein was connected by flexible linkers, inheriting the self-assembly properties of hydrophobin HGFI and the targeting ability of GE11. The data show that the encapsulation of curcumin by fusion protein GE11-HGFI can form uniform and stable nanoparticles with a size of only 80 nm. In addition, the nanocarrier had high encapsulation efficiency for curcumin and made it to release sustainably. Notably, the drug-loaded nanosystem selectively targeted colorectal cancer cells with high epidermal growth factor receptor expression, resulting in high aggregated concentrations of curcumin at tumor sites, thus showing a significant anticancer effect. These results suggest that the nanocarrier fusion protein has the potential to be a novel strategy for enhancing molecular bioactivity and drug targeting in cancer therapy.
Sujet(s)
Antinéoplasiques , Tumeurs colorectales , Curcumine , Nanoparticules , Humains , Vecteurs de médicaments , Systèmes de délivrance de médicaments , Récepteurs ErbB/génétique , Tumeurs colorectales/traitement médicamenteux , Tumeurs colorectales/génétiqueRÉSUMÉ
Hydrophobins are small proteins from filamentous fungi, which have remarkable self-assembly properties of great potential, e.g., as drug carriers and as anti-bacterial agents, but different hydrophobins, with improved properties, are needed. HGFI (a hydrophobin from Grifola frondosa) is a class I hydrophobin, which can self-assemble into rodlet structures with a length range 100-150 nm. In this study, we identified a new hydrophobin gene (hgfII) from the mycelium of G. frondosa with a much higher transcriptional level than hgfI. Heterologous expression of hgfII was accomplished in the Pichia pastoris. X-ray photoelectron spectroscopy and water contact angle assay measurements revealed that HGFII can self-assemble into a protein film at the air-solid interface, with circular dichroism and thioflavin T fluorescence studies showing that this effect was accompanied by a decrease in α-helix content and an increase in ß-sheet content. Using atomic force microscopy, it was shown that HGFII self-assembled into rodlet-like structures with a diameter of 15-30 nm, showing that it was a class I hydrophobin, with self-assembly behavior different from HGFI. The surface hydrophobicity of HGFII was stronger than that of HGFI, meanwhile, in emulsification trials, HGFII displayed better dispersive capacity to the soybean oil than HGFI, producing a more stable and durable emulsion.
RÉSUMÉ
Seed viability depends upon the maintenance of functional lipids; however, how membrane lipid components dynamically change during the seed aging process remains obscure. Seed storage is accompanied by the oxidation of membrane lipids and loss of seed viability. Understanding membrane lipid changes and their effect on the cell membrane during seed aging can contribute to revealing the mechanism of seed longevity. In this study, the potential relationship between oxidative stress and membrane lipid metabolism was evaluated by using a non-targeted lipidomics approach during artificial aging of Glycine max L. Merr. Zhongdou No. 27 seeds. We determined changes in reactive oxygen species, malondialdehyde content, and membrane permeability and assessed antioxidant system activity. We found that decreased non-enzymatic antioxidant contents and catalase activity might lead to reactive oxygen species accumulation, resulting in higher electrolyte leakage and lipid peroxidation. The significantly decreased phospholipids and increased glycerolipids and lysophospholipids suggested that hydrolysis of phospholipids to form glycerolipids and lysophospholipids could be the primary pathway of membrane metabolism during seed aging. Moreover, the ratio of phosphatidylcholine to phosphatidylethanolamine, double bond index, and acyl chain length of phospholipids were found to jointly regulate membrane function. In addition, the observed changes in lipid metabolism suggest novel potential hallmarks of soybean seed aging, such as diacylglycerol 36:4; phosphatidylcholine 34:2, 36:2, and 36:4; and phosphatidylethanolamine 34:2. This knowledge can be of great significance for elucidating the molecular mechanism underlying seed aging and germplasm conservation.
RÉSUMÉ
A real-time label-free lateral offset spliced coreless fiber (CF) Mach-Zehnder interferometer (MZI) biosensor functionalized with hydrophobin Grifola frondosa I (HGFI) was proposed for the detection of cytokine tumour necrosis factor alpha (TNF-α). The nanolayer self-assembled on the optical fiber surfaces by HGFI rendered the immobilization of probe TNF-α antibody and recognition of antigen TNF-α. Trifluoroacetic acid was utilized to remove the HGFI layer from the glass surface, which was validated by field emission scanning electron microscopy (FESEM) and water contact angle (WCA). Results demonstrated that the processes of HGFI modification, antibody immobilization and specific antibody detection can be monitored in real time. The proposed biosensor exhibited good specificity, repeatability and low detection limit for TNF-α, extending its application in inflammation and disease monitoring.
RÉSUMÉ
BACKGROUND: Aromatic compounds, such as p-coumaric acid (p-CA) and caffeic acid, are secondary metabolites of various plants, and are widely used in diet and industry for their biological activities. In addition to expensive and unsustainable methods of plant extraction and chemical synthesis, the strategy for heterologous synthesis of aromatic compounds in microorganisms has received much attention. As the most abundant renewable resource in the world, lignocellulose is an economical and environmentally friendly alternative to edible, high-cost carbon sources such as glucose. RESULTS: In the present study, carboxymethyl-cellulose (CMC) was utilized as the sole carbon source, and a metabolically engineered Saccharomyces cerevisiae strain SK10-3 was co-cultured with other recombinant S. cerevisiae strains to achieve the bioconversion of value-added products from CMC. By optimizing the inoculation ratio, interval time, and carbon source content, the final titer of p-CA in 30 g/L CMC medium was increased to 71.71 mg/L, which was 155.9-fold higher than that achieved in mono-culture. The de novo biosynthesis of caffeic acid in the CMC medium was also achieved through a three-strain co-cultivation. Caffeic acid production was up to 16.91 mg/L after optimizing the inoculation ratio of these strains. CONCLUSION: De novo biosynthesis of p-CA and caffeic acid from lignocellulose through a co-cultivation strategy was achieved for the first time. This study provides favorable support for the biosynthesis of more high value-added products from economical substrates. In addition, the multi-strain co-culture strategy can effectively improve the final titer of the target products, which has high application potential in the field of industrial production.
Sujet(s)
Génie métabolique , Saccharomyces cerevisiae , Acides caféiques , Carbone/métabolisme , Carboxyméthylcellulose de sodium/métabolisme , Techniques de coculture , Acides coumariques , Milieux de culture/métabolisme , Escherichia coli/métabolisme , Génie métabolique/méthodes , Saccharomyces cerevisiae/métabolismeRÉSUMÉ
AIMS: To overcome the defective unstable production of p-coumaric acid (p-CA) using episomal plasmids and simultaneously achieve genetic stability and high-copy integration in Saccharomyces cerevisiae. METHODS AND RESULTS: Two-micron plasmids were used to obtain high titres of p-CA, but p-CA production was decreased significantly in a nonselective medium after 72 h. To overcome the defect of unstable p-CA production during fermentation, delta integration with the triosephosphate isomerase gene from Schizosaccharomyces pombe (POT1) was employed as a selection marker to integrate heterologous p-CA synthesis cassette, and the high-level p-CA-producing strain QT3-20 was identified. In shake flask fermentation, the final p-CA titre of QT3-20 reached 228.37 mg L-1 at 168 h, 11-fold higher than integrated strain QU3-20 using URA3 as the selective marker, and 9-fold higher than the best-performing episomal expression strain NKE1. Additionally, the p-CA titre and gene copy number remained stable after 100 generations of QT3-20 in a nonselective medium. CONCLUSION: We achieved high-copy genome integration and stable heterologous production of p-CA via a POT1-mediated strategy in S. cerevisiae. SIGNIFICANCE AND IMPACT OF STUDY: With superior genetic stability and production stability in a nonselective medium during fermentation, the high-level p-CA-producing strain constructed via POT1-mediated delta integration could serve as an efficient platform strain, to eliminate the threat of unstable and insufficient supply for future production of p-CA derivatives, make downstream processing and biosynthesis much simpler.
Sujet(s)
Protéines de Saccharomyces cerevisiae , Protéines de Schizosaccharomyces pombe , Schizosaccharomyces , Acides coumariques/métabolisme , Fermentation , Génie métabolique , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , Schizosaccharomyces/métabolisme , Protéines de Schizosaccharomyces pombe/génétique , Protéines de Schizosaccharomyces pombe/métabolisme , Complexe shelterine , Protéines télomériques/génétique , Protéines télomériques/métabolismeRÉSUMÉ
Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 µ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for de novo synthesis of caffeic acid in S. cerevisiae suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant S. cerevisiae.
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The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquitinated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorders.
Sujet(s)
Autophagie/physiologie , Calcium/métabolisme , Endosomes/métabolisme , Homéostasie protéique/physiologie , Ubiquitin-protein ligases/métabolisme , Animaux , Arginine/métabolisme , Cellules CHO , Lignée cellulaire tumorale , Cricetulus , Cellules HeLa , Humains , Lysosomes/métabolisme , Protéolyse , Transduction du signal/physiologie , Ubiquitine/métabolismeRÉSUMÉ
The intestinal flora plays an important role in the development of many human and animal diseases. Microbiome association studies revealed the potential regulatory function of intestinal bacteria in many liver diseases, such as autoimmune hepatitis, viral hepatitis and alcoholic hepatitis. However, the key intestinal bacterial strains that affect pathological liver injury and the underlying functional mechanisms remain unclear. We found that the gut microbiota from gentamycin (Gen)-treated mice significantly alleviated concanavalin A (ConA)-induced liver injury compared to vancomycin (Van)-treated mice by inhibiting CD95 expression on the surface of hepatocytes and reducing CD95/CD95L-mediated hepatocyte apoptosis. Through the combination of microbiota sequencing and correlation analysis, we isolated 5 strains with the highest relative abundance, Bacteroides acidifaciens (BA), Parabacteroides distasonis (PD), Bacteroides thetaiotaomicron (BT), Bacteroides dorei (BD) and Bacteroides uniformis (BU), from the feces of Gen-treated mice. Only BA played a protective role against ConA-induced liver injury. Further studies demonstrated that BA-reconstituted mice had reduced CD95/CD95L signaling, which was required for the decrease in the L-glutathione/glutathione (GSSG/GSH) ratio observed in the liver. BA-reconstituted mice were also more resistant to alcoholic liver injury. Our work showed that a specific murine intestinal bacterial strain, BA, ameliorated liver injury by reducing hepatocyte apoptosis in a CD95-dependent manner. Determination of the function of BA may provide an opportunity for its future use as a treatment for liver disease.
Sujet(s)
Bacteroides/physiologie , Microbiome gastro-intestinal , Maladies du foie/prévention et contrôle , Antigènes CD95/métabolisme , Animaux , Apoptose , Bactéries/classification , Bactéries/génétique , Bactéries/isolement et purification , Bacteroides/génétique , Bacteroides/isolement et purification , Fèces/microbiologie , Glutathion/métabolisme , Hépatocytes/cytologie , Hépatocytes/métabolisme , Humains , Maladies du foie/métabolisme , Maladies du foie/microbiologie , Maladies du foie/physiopathologie , Souris , Souris de lignée C57BL , Antigènes CD95/génétiqueRÉSUMÉ
In this study, a co-culturing Enterobacter sp. and Lactococcus lactis strategy was developed to alter bacterial cellulose (BC) properties and increase nisin yields. We generated high nisin yields (6260 IU/mL) by altering inoculum ratios and inoculation times in a novel co-culture system. Critically, these were 85% higher than L. lactis monocultures. By monitoring fermentation broth pH and lactic acid yields, the pH was higher and lactic acid yields lower during co-culture conditions when compared with L. lactis monocultures, suggesting that co-culturing was more suitable for L. lactis nisin production. We also determined BC film yields and properties (BC, BC-N, and BC-N after nisin release). BC yields produced by co-culturing were not very different from Enterobacter sp. monocultures, but crystallinity was significantly altered. Collectively, our co-culture system adequately and economically modified BC fibers by interfering with self-assembly and crystallization processes during BC synthesis, with significantly improved nisin yields.
Sujet(s)
Lactococcus lactis , Nisine , Cellulose , Techniques de coculture , Fermentation , Lactococcus lactis/métabolismeRÉSUMÉ
Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
Sujet(s)
Cryomicroscopie électronique/instrumentation , Protéines fongiques/composition chimique , Adsorption , Air , Interactions hydrophobes et hydrophiles , Protéines/composition chimique , Électricité statique , Eau/composition chimiqueRÉSUMÉ
Bacterial non-coding RNAs (ncRNAs) play important regulatory roles in various physiological metabolic pathways. In this study, a novel ncRNA CsiR (ciprofloxacin stress-induced ncRNA) involved in the regulation of ciprofloxacin resistance in the foodborne multidrug-resistant Proteus vulgaris (P. vulgaris) strain P3M was identified. The survival rate of the CsiR-deficient strain was higher than that of the wild-type strain P3M under the ciprofloxacin treatment condition, indicating that CsiR played a negative regulatory role, and its target gene emrB was identified through further target prediction, quantitative real-time PCR (qRT-PCR), and microscale thermophoresis (MST). Further studies showed that the interaction between CsiR and emrB mRNA affected the stability of the latter at the post-transcriptional level to a large degree, and ultimately affected the ciprofloxacin resistance of P3M. Notably, the base-pairing sites between CsiR and emrB mRNAs were highly conserved in other sequenced P. vulgaris strains, suggesting that this regulatory mechanism may be ubiquitous in this species. To the best of our knowledge, this is the first identification of a novel ncRNA involved in the regulation of ciprofloxacin resistance in P. vulgaris species, which lays a solid foundation for comprehensively expounding the antibiotic resistance mechanism of P. vulgaris.