RESUMO
A simple, reliable method for identifying ß-lactoglobulin (ß-LG) in dairy products is needed to protect those with ß-LG allergies. A common, practical strategy for target detection is designing simplified nucleic acid nanodevices by integrating functional components. This work presents a label-free modular ß-LG aptasensor consisting of an aptamer-loop G-quadruplex (G4), the working conformation of which is regulated by conformational antagonism to ensure respective module functionality and the related signal transduction. The polymorphic conformations of the module-fused sequence are systematically characterized, and the cause is revealed as shifting antagonistic equilibrium. Combined with conformational folding dynamics, this helped regulate functional conformations by fine-tuning the sequences. Furthermore, the principle of specific ß-LG detection by parallel G4 topology is examined as binding on the G4 aptamer loop by ß-LG to reinforce the G4 topology and fluorescence. Finally, a label-free, assembly-free, succinct, and turn-on fluorescent aptasensor is established, achieving excellent sensitivity across five orders of magnitude, rapidly detecting ß-LG within 22-min. This study provides a generalizable approach for the conformational regulation of module-fused G4 sequences and a reference model for creating simplified sensing devices for a variety of targets.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Quadruplex G , Lactoglobulinas , Lactoglobulinas/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodosRESUMO
Liposomes are widely used in the biological field due to their good biocompatibility and surface modification properties. With the development of biochemistry and material science, many liposome structures and their surface functional components have been modified and optimized one by one, pushing the liposome platform from traditional to functionalized and intelligent, which will better satisfy and expand the needs of scientific research. However, a main limiting factor effecting the efficiency of liposomes is the complicated environmental conditions in the living body. Currently, in order to overcome the above problem, functionalized liposomes have become a very promising strategy. In this paper, binding strategies of liposomes with four main functional elements, namely nucleic acids, antibodies, peptides, and stimuli-responsive motif have been summarized for the first time. In addition, based on the construction characteristics of functionalized liposomes, such as drug-carrying, targeting, long-circulating, and stimulus-responsive properties, a comprehensive overview of their features and respective research progress are presented. Finally, the paper critically presents the limitations of these functionalized liposomes in the current applications and also prospectively suggests the future development directions, aiming to accelerate realization of their industrialization.
Assuntos
Lipossomos , Lipossomos/química , Humanos , Sistemas de Liberação de Medicamentos/métodos , Peptídeos/químicaRESUMO
Sinapate esters, which are induced in plants under ultraviolet-B (UV-B) irradiation, have important roles not only in the protection against UV-B irradiation but also in the regulation of stomatal closure. Here, we speculated that sinapate esters would function in the stomatal closure of Arabidopsis thaliana in response to UV-B. We measured the stomatal aperture size of the wild-type (WT) and bright trichomes 1 (brt1) and sinapoylglucose accumulator 1 (sng1) mutants under UV-B irradiation; the latter two mutants are deficient in the conversion of sinapic acid to sinapoylglucose (SG) and SG to sinapoylmalate (SM), respectively. Both the brt1 and sng1 plants showed smaller stomatal apertures than the WT under normal light and UV-B irradiation conditions. The accumulation of SM and malate were induced by UV-B irradiation in WT and brt1 plants but not in sng1 plants. Consistently, exogenous malate application reduced UV-B-induced stomatal closure in WT, brt1 and sng1 plants. Nonetheless, levels of reactive oxygen species (ROS), nitric oxide (NO) and cytosolic Ca2+ were higher in guard cells of the sng1 mutant than in those of the WT under normal white light and UV-B irradiation, suggesting that disturbance of sinapate metabolism induced the accumulation of these signaling molecules that promote stomatal closure. Unexpectedly, exogenous sinapic acid application prevented stomatal closure of WT, brt1 and sng1 plants. In summary, we hypothesize that SG or other sinapate esters may promote the UV-B-induced malate accumulation and stomatal closure, whereas sinapic acid inhibits the ROS-NO pathway that regulates UV-B-induced cytosolic Ca2+ accumulation and stomatal closure.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Ésteres/metabolismo , Malatos/metabolismo , Cálcio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estômatos de Plantas/fisiologia , Ácido Abscísico/metabolismoRESUMO
Recent investigations in Arabidopsis thaliana suggest that SUPPRESSOR of MORE AXILLARY GROWTH 2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2) are negative regulators of karrikin (KAR) and strigolactone (SL) signaling during plant growth and development, but their functions in drought resistance and related mechanisms of action remain unclear. To understand the roles and mechanisms of SMAX1 and SMXL2 in drought resistance, we investigated the drought-resistance phenotypes and transcriptome profiles of smax1 smxl2 (s1,2) double-mutant plants in response to drought stress. The s1,2 mutant plants showed enhanced drought-resistance and lower leaf water loss when compared with wild-type (WT) plants. Transcriptome comparison of rosette leaves from the s1,2 mutant and the WT under normal and dehydration conditions suggested that the mechanism related to cuticle formation was involved in drought resistance. This possibility was supported by enhanced cuticle formation in the rosette leaves of the s1,2 mutant. We also found that the s1,2 mutant plants were more sensitive to abscisic acid in assays of stomatal closure, cotyledon opening, chlorophyll degradation and growth inhibition, and they showed a higher reactive oxygen species detoxification capacity than WT plants. In addition, the s1,2 mutant plants had longer root hairs and a higher root-to-shoot ratio than the WT plants, suggesting that the mutant had a greater capacity for water absorption than the WT. Taken together, our results indicate that SMAX1 and SMXL2 negatively regulate drought resistance, and disruption of these KAR- and SL-signaling-related genes may therefore provide a novel means for improving crop drought resistance.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Resistência à Seca , Germinação/genética , Ácido Abscísico/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Amino metabolites are essential for life activities and can be used clinically as biomarkers for disease diagnosis and treatment. Solid-phase-supported chemoselective probes can simplify sample handling and enhance detection sensitivity. However, the low efficiency and complicated preparation of traditional probes limit their further application. In this work, a novel solid-phase-supported probe Fe3O4-SiO2-polymers-phenyl isothiocyanate (FSP-PITC) was developed by immobilizing phenyl isothiocyanate on magnetic beads with disulfide as an orthogonal cleavage site, which can couple amino metabolites directly regardless of whether proteins and other matrixes were removed. After purification, the targeted metabolites were released by dithiothreitol and detected by high-resolution mass spectrometry. The simplified processing steps shorten the analysis time, and the introduction of polymers results in a 100-1000-fold increase in probe capacity. With high stability and specificity, FSP-PITC pretreatment allows accurate qualitative and quantitative (R2 > 0.99) analysis, facilitating the detection of metabolites in subfemtomole quantities. Using this strategy, 4158 metabolite signals were detected in negative ion mode. Among them, 352 amino metabolites including human cells (226), serum (227), and mouse samples (274) were searched from the Human Metabolome Database. These metabolites participate in metabolic pathways of amino acids, biogenic amine, and the urea cycle. All these results indicate that FSP-PITC is a promising probe for novel metabolite discovery and high-throughput screening.
Assuntos
Metaboloma , Dióxido de Silício , Humanos , Animais , Camundongos , Espectrometria de Massas/métodos , Aminas Biogênicas , Metabolômica/métodosRESUMO
Playing a great role in human physiologies and pathologies, carbonyl metabolites are intimately associated with a variety of diseases, though the effective analysis method of them remains a challenge. A hydrazide-terminated polyurea-modified magnetic particle (HPMP) with versatile probes is developed to address this issue. The capture ability of HPMPs for carbonyl metabolite is more than 1200 µmol g-1 , which is increased by 4 orders of magnitude via the introduction of polyurea. With a broad linear range of over 4 orders of magnitude, remarkably improved sensitivity, and limit of detection at attomole quantities, HPMPs are applied in relative quantification of more than 1500 carbonyl metabolites in 113 human serum samples with high throughput and high coverage. The combined indicators of these metabolites demonstrates a great diagnostic accuracy for distinguishing between health and disease subjects as well as differentiating the patients with benign lung disease and lung cancer. Combining powerful capture ability, low-cost preparation, and convenient operation, the HPMPs demonstrate extensive application in biomarker discovery and the detailed study of the biochemical landscape.
Assuntos
Neoplasias Pulmonares , Polímeros , Humanos , Neoplasias Pulmonares/metabolismo , Biomarcadores , Fenômenos MagnéticosRESUMO
The karrikin (KAR) receptor and several related signaling components have been identified by forward genetic screening, but only a few studies have reported on upstream and downstream KAR signaling components and their roles in drought tolerance. Here, we characterized the functions of KAR UPREGULATED F-BOX 1 (KUF1) in drought tolerance using a reverse genetics approach in Arabidopsis (Arabidopsis thaliana). We observed that kuf1 mutant plants were more tolerant to drought stress than wild-type (WT) plants. To clarify the mechanisms by which KUF1 negatively regulates drought tolerance, we performed physiological, transcriptome, and morphological analyses. We found that kuf1 plants limited leaf water loss by reducing stomatal aperture and cuticular permeability. In addition, kuf1 plants showed increased sensitivity of stomatal closure, seed germination, primary root growth, and leaf senescence to abscisic acid (ABA). Genome-wide transcriptome comparisons of kuf1 and WT rosette leaves before and after dehydration showed that the differences in various drought tolerance-related traits were accompanied by differences in the expression of genes associated with stomatal closure (e.g. OPEN STOMATA 1), lipid and fatty acid metabolism (e.g. WAX ESTER SYNTHASE), and ABA responsiveness (e.g. ABA-RESPONSIVE ELEMENT 3). The kuf1 mutant plants had higher root/shoot ratios and root hair densities than WT plants, suggesting that they could absorb more water than WT plants. Together, these results demonstrate that KUF1 negatively regulates drought tolerance by modulating various physiological traits, morphological adjustments, and ABA responses and that the genetic manipulation of KUF1 in crops is a potential means of enhancing their drought tolerance.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Secas , Proteínas de Arabidopsis/metabolismo , Estômatos de Plantas/fisiologia , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico/genética , Ácido Abscísico/farmacologia , Ácido Abscísico/metabolismo , Água/metabolismo , Plantas Geneticamente Modificadas/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system has been widely used in gene editing of various organisms. However, food-grade gene editing systems in lactic acid bacteria are still preliminary. Red/ET-dependent homologous recombination or CRISPR-based systems have been developed to gene editing in Lactococcus lactis, but these methods are overall inefficient. In the present study, a recombinant system based on CRISPR/Cas9 technology combined with Red/ET was developed using the plasmid pMG36e derived from Lactococcus lactis. Then, the developed recombinant system was applied to Lactococcus lactis. Knockout efficiency was significantly higher using the developed system (91%). In addition, this system showed the potential to be used as a high-throughput method for hierarchical screening. Finally, a gene-edited strain was obtained, and no antibiotics or exogenous genes were introduced using the developed gene editing system. Thus, the efficient system in lactic acid bacteria was constructed and optimized.
Assuntos
Edição de Genes , Lactococcus lactis , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Plasmídeos/genética , Recombinação HomólogaRESUMO
Yogurt and its related products are popular worldwide. During transportation and storage, Lactobacillus delbrueckii ssp. bulgaricus in yogurt continues to metabolize to form lactic acid, the postacidification phenomenon of yogurt. Postacidification of yogurt is a widespread phenomenon in the dairy industry. Many scholars have done research on controlling the postacidification process, but few report on the molecular mechanisms involved. In this study, we used a molecular-assisted approach to screen food additives that can inhibit postacidification and analyzed its effects on yogurt quality as well as its regulatory mechanism from multi-omics perspectives in combination. The copper ion was found to upregulate the expression of the LDB_RS05285 gene, and the copper transporter-related genes were regulated by copper. Based on the metabolic-level analysis, copper was found to promote lactose hydrolysis, accumulate a large amount of glucose and galactose, inhibit the conversion of glucose to lactic acid, and reduce the production of lactic acid. The significantly greater abundance of l-isoleucine and l-phenylalanine increased the abundance of 3-methylbutyraldehyde (â¼1.2 times) and benzaldehyde (â¼7.9 times) to different degrees, which contributed to the formation of the overall flavor of yogurt. Copper not only stabilizes the acidity of yogurt, but also it improves the flavor of yogurt. Through this established method involving quantitative and correlation analyses at the transcriptional and metabolic levels, this study provides guidance for the research and development of food additives that inhibit postacidification of yogurt and provide a reference for studying the changes of metabolites during storage of yogurt.
Assuntos
Cobre , Lactobacillus delbrueckii , Animais , Fermentação , Cobre/metabolismo , Iogurte/análise , Lactobacillus delbrueckii/metabolismo , Glucose/metabolismo , Óperon , Ácido Láctico/metabolismo , Streptococcus thermophilus/metabolismoRESUMO
PURPOSE: The study aimed to compare the perioperative complications, short-term clinical outcomes, patient-reported outcomes, and radiographic parameters of tibiofibular proximal osteotomy combined with absorbable spacer insertion (TPOASI) and open-wedge high tibial osteotomy (OWHTO) in a two year postoperative time period. METHODS: A total of 160 patients with Kellgren-Lawrence classification grade 3 medial compartmental knee OA were randomized to receive either TPOASI (n = 82) or OWHTO (n = 78). The primary and secondary outcomes were measured preoperatively, postoperatively, and at each follow-up examination. The primary outcomes were the between-group change in the Western Ontario and McMaster Universities Global score (WOMAC). Secondary measures included visual analog scale (VAS), radiographic parameters, American Knee Society Score (KSS), operation time, blood loss, length of incision, hospital stay, and relevant complications. Postoperative radiographic parameters, including the femorotibial angle (FTA), varus angle (VA), and joint line convergence angle (JLCA), were measured to evaluate the correction of varus deformity. RESULTS: No significant differences were found in the baseline data between the two groups. Both methods improved functional status and pain postoperatively. For primary outcomes of both groups, statistical difference was observed in WOMAC scores at the 6-month follow-up (P < 0.001). For secondary outcomes, no statistical difference was observed between the groups during the 2-year follow-up (P > 0.05). For TPOASI vs. OWHTO, the mean hospital stay (6.6 ± 1.3 days vs. 7.8 ± 2.1 days) was shorter (P < 0.001), and both blood loss (70.56 ± 35.58 vs. 174.00 ± 66.33 mL) and complication rate (3.7% vs. 12.8%) were significantly lower (P < 0.005 for both). CONCLUSIONS: Both approaches showed satisfactory functional outcomes and alleviated pain. However, TPOASI is a simple, feasible method with few complications, and it could be widely used.
Assuntos
Osteoartrite do Joelho , Humanos , Osteoartrite do Joelho/cirurgia , Tíbia/cirurgia , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/cirurgia , Osteotomia/efeitos adversos , Osteotomia/métodos , Dor , Estudos RetrospectivosRESUMO
A deepening understanding of the relationship between transient receptor potential canonical channel 5 (TRPC5) and chronic kidney disease (CKD), has led to the emergence of several types of TRPC5 inhibitors displaying clear therapeutic effect. Herein, we report the synthesis and biological evaluation of a series of pyrroledione TRPC5 inhibitors, culminating in the discovery of compound 16g with subtype selectivity. Compared with GFB-8438, a potent TRPC5 inhibitor (Goldfinch Bio), compound 16g showed improved inhibition of TRPC5 and enhanced protective effect against protamine sulfates (PS)-induced podocyte injury in vitro. In addition, compound 16g did not induce cell death in primary cultured hepatocytes and immortalized podocytes in a preliminary toxicity assessment, indicating its utility as a potent and safe inhibitor for studying the function of TRPC5.
Assuntos
Descoberta de Drogas , Pirróis/farmacologia , Canais de Cátion TRPC/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Protaminas , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Canais de Cátion TRPC/metabolismoRESUMO
Transient receptor potential canonical (TRPC) channels are a class of non-selective cation channels expressed in a variety of tissues and organ systems where they functionally regulate physiological and pathological processes. TRPC5 has been shown to be a promising target for focal segmental glomerulosclerosis treatment. In this study, we report the synthesis and biological evaluation of a novel series of benzimidazole-based TRPC5 inhibitors. One compound, 8b, is 100-fold more potent than the parent compound, AC1903, in the suppression of TRPC5 channel activity. Interestingly, both AC1903 and 8b also suppressed TRPC4 channel activity with similar potency. Compound 8b also significantly blunts protamine sulfate-induced reorganization of podocyte cytoskeleton, interleukin (IL)-17-induced cell proliferation, and the expression of proinflammatory mediators in human keratinocyte HaCaT cells.
Assuntos
Indazóis , Canais de Cátion TRPC , Humanos , Indazóis/farmacologia , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismoRESUMO
A simple, rapid and ultrasensitive visual sensing method for the detection of Cronobacter sakazakii (C. sakazakii) based on a biohybrid interface was established. During the entire sensing process, quadruple-cascade amplification showed its superior sensing performance. First, the prepared immunomagnetic beads (IMB) were used to isolate and enrich specific targets from the food matrix. After adding the fusion aptamer, the aptamer sequence specifically recognized the target and formed the immune sandwich structure of antibody-target-fusion aptamer. In addition, the fusion aptamer also included the template sequence of exponential amplification reaction (EXPAR), which contained the antisense sequence of the G-rich sequence. Therefore, a large number of G-rich sequences can be generated after EXPAR can be triggered in the presence of Bst. DNA polymerase, nicking endonuclease, cDNA, and dNTP. They were self-assembled into G-quadruplex structures and then combined with hemin to form G4/hemin DNAzyme, resulting in visible coloration and measuring absorbance at 450 nm for quantitative detection. The assay showed a limit of detection (LOD) of 2 CFU/mL in pure culture and 12 CFU/g in milk powder in optimal conditions. This method provides a promising strategy for rapid and point-of-care testing (POCT) since it does not require DNA extraction, medium culturing, and expensive instrumentation. KEY POINTS: â¢Single-cell level detection of C. sakazakii with ultrasensitive and rapidness â¢The fusion aptamer integrated recognition and amplification â¢Sensing analysis of C. sakazakii based on cascade amplification of biohybrid interface.
Assuntos
Cronobacter sakazakii , Cronobacter sakazakii/genética , DNA Polimerase Dirigida por DNA , Hemina/química , Limite de DetecçãoRESUMO
OBJECTIVES: To produce nattokinase in a food-grade expression system and evaluate its thrombolytic activity in vitro. RESULTS: No nattokinase activity from reconstituted strains was observed in simulated gastric juice, but the enzyme was stable in intestinal fluid, the relative activity of which was found to be 60% after 4 h. Due to the nattokinase being produced intracellularly by recombinant bacterial strains, the persistence of the bacteria in gastric juice ensured transmission of the nattokinase into intestinal juice. Because of subsequent disintegration of the bacteria, the highest nattokinase activity was observed after 3 h at approximately 32%, following its carriage within the recombinant strains to the intestinal fluid. CONCLUSIONS: This study demonstrated that nattokinase from recombinant strains exhibited good thrombolytic activity in vitro and may be used by the dairy fermentation industry for the development of novel thrombolytic functional foods.
Assuntos
Secreções Intestinais/enzimologia , Lactobacillus delbrueckii/crescimento & desenvolvimento , Subtilisinas/química , Subtilisinas/genética , Animais , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Indústria de Laticínios , Estabilidade Enzimática , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Microbiologia de Alimentos , Alimento Funcional/microbiologia , Expressão Gênica , Lactobacillus delbrueckii/genética , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Subtilisinas/farmacologia , Suínos , Transformação BacterianaRESUMO
Fermented milk is an effective carrier for probiotics, the consumption of which improves host health. The beneficial effects of probiotics, prebiotics, and synbiotics on gut dysbiosis have been reported previously. However, the way in which specific probiotics, prebiotics, and synbiotics regulate intestinal microbes remains unclear. Therefore, the probiotics Lactobacillus rhamnosus AS 1.2466 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 and the prebiotics xylooligosaccharide and red ginseng extracts were fed to mice to determine their effects on the intestinal microbiota. Then, mice were administered xylooligosaccharide and L. rhamnosus (synthesis) by gavage, and the number of L. rhamnosus was determined in the intestine at different times. The results show that probiotics and prebiotics can quickly reduce the Firmicutes/Bacteroidetes ratio, inhibit harmful bacteria (such as Klebsiella and Escherichia coli), and accelerate the recovery of beneficial intestinal microorganisms (such as Lactobacillus). In a complex intestinal microecology, different probiotics and prebiotics have different effects on specific intestinal microorganisms that cannot be recovered in the short term. In addition, after 20 d of intragastric xylooligosaccharide addition at 0.12 g/kg of body weight, L. rhamnosus colonization in the mouse ileum was 7.48 log cfu/mL, which was higher than in the low-dose group, prolonging colonization time and increasing the number of probiotics in the intestine. Therefore, this study demonstrated that probiotics and prebiotics can promote the balance of intestinal microbiota by regulating specific microbes in the intestine, and the effects of a suitable combination of synbiotics are beneficial, laying the foundation for the development of new dairy products rich in synbiotics.
Assuntos
Bactérias/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Prebióticos , Probióticos/farmacologia , Simbióticos , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Microbioma Gastrointestinal/fisiologia , Glucuronatos/administração & dosagem , Glucuronatos/farmacologia , Lactobacillus delbrueckii/química , Lacticaseibacillus rhamnosus/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligossacarídeos/administração & dosagem , Oligossacarídeos/farmacologia , Panax/química , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Prebióticos/administração & dosagem , Probióticos/administração & dosagem , Organismos Livres de Patógenos Específicos , Simbióticos/administração & dosagemRESUMO
The health-promoting effects of the probiotic strain Lactobacillus rhamnosus are based on its adherence and colonization ability. However, little is known about its adhesion and colonization rates. Lactobacillus rhamnosus in mouse intestinal mucosa a mutant of the red fluorescence protein (RFP) DSred2 was used to tag L. rhamnosus to observe the adhesion and distribution of L. rhamnosus in mouse intestinal mucosa. A mutant of the red fluorescence protein (RFP) Dsred2 was used to tag L. rhamnosus to allow us to observe and distinguish it in the mouse intestine. Seven-week-old female BALB/c mice were fed once (at day 0) with an oral administration of the labeled L. rhamnosus, and the number of labeled bacteria was detected in different regions of the intestinal tract at 3 h and at day 1, 2, 3, 4, 5, 6, 7, and 15 after administration. The labeling process changed the morphology of L. rhamnosus, as it appeared after observation under the microscope, but did not change its basic probiotic properties in vitro. In vivo, labeled L. rhamnosus reached the colonization peak at the fourth day after gavage. From the distribution point of view, the number of colonization strains increased from the proximal to the distal small intestine (duodenum < jejunum < ileum) and the number of strains in the colon was less than the distal small intestine (ileum). The labeling protocol actually allowed the detection of the distribution and adhesion of this bacterium to the intestine, thus demonstrating that the health-promoting effects of this probiotic are satisfied. This study provides a scientific basis in the use of probiotics such as L. rhamnosus in functional foods.
Assuntos
Aderência Bacteriana , Intestinos/microbiologia , Lacticaseibacillus rhamnosus/fisiologia , Proteínas Luminescentes/metabolismo , Probióticos , Animais , Contagem de Colônia Microbiana , Feminino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestinos/anatomia & histologia , Intestinos/química , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , Lacticaseibacillus rhamnosus/metabolismo , Proteínas Luminescentes/genética , Camundongos Endogâmicos BALB C , Probióticos/administração & dosagem , Probióticos/metabolismo , Proteína Vermelha FluorescenteRESUMO
Euglena gracilis, a model organism of the eukaryotic supergroup Discoba harbouring also clinically important parasitic species, possesses diverse metabolic strategies and an atypical electron transport chain. While structures of the electron transport chain complexes and supercomplexes of most other eukaryotic clades have been reported, no similar structure is currently available for Discoba, limiting the understandings of its core metabolism and leaving a gap in the evolutionary tree of eukaryotic bioenergetics. Here, we report high-resolution cryo-EM structures of Euglena's respirasome I + III2 + IV and supercomplex III2 + IV2. A previously unreported fatty acid synthesis domain locates on the tip of complex I's peripheral arm, providing a clear picture of its atypical subunit composition identified previously. Individual complexes are re-arranged in the respirasome to adapt to the non-uniform membrane curvature of the discoidal cristae. Furthermore, Euglena's conformationally rigid complex I is deactivated by restricting ubiquinone's access to its substrate tunnel. Our findings provide structural insights for therapeutic developments against euglenozoan parasite infections.
Assuntos
Euglena , Membranas Mitocondriais , Transporte de Elétrons , Membranas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo EnergéticoRESUMO
It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.