RESUMEN
OBJECTIVE: We investigated the anti-inflammatory and anti-pathogenic activities of Lacticaseibacillus rhamnosus IDCC 3201 isolated from the feces of breast-fed infants. METHODS: Cell viability, nitric oxide (NO) production, and expression of inflammatory markers by L. rhamnosus IDCC 3201 were quantitatively analyzed in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The antibacterial and antifungal activities of L. rhamnosus IDCC 3201 against various pathogens were also investigated. RESULTS: Treatment of LPS-induced macrophages with cell-free supernatant of L. rhamnosus IDCC 3201 significantly decreased the expression levels of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). Nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) levels also significantly decreased in LPS-induced macrophages. Phenotypically, the treatment of L. rhamnosus IDCC 3201 reduced the production of nitric oxide (NO) in LPS-induced macrophages. Furthermore, L. rhamnosus IDCC 3201 was proven to have potent inhibitory activities against various pathogens responsible for inflammatory responses in the gastrointestinal tract (i.e., Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, and Salmonella Typhimurium), respiratory system (i.e., Streptococcus pneumoniae), and vagina (i.e., Candida albicans). CONCLUSION: L. rhamnosus IDCC 3201 has anti-inflammatory activity in terms of decreased expression of cytokines, inflammation-inducible enzymes in LPS-induced macrophages, and anti-pathogenic activity.
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Lacticaseibacillus rhamnosus , Lipopolisacáridos , Lactante , Femenino , Humanos , Óxido Nítrico , Heces , Antiinflamatorios/farmacología , Factor de Necrosis Tumoral alfaRESUMEN
This study evaluated the safety of Lactobacillus johnsonii IDCC 9203 and investigated its anti-inflammatory activity in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Genomic analysis revealed that this strain has no virulence and antibiotic resistance gene except tetW, which is a tetracycline resistance gene. Minimum inhibitory concentration data showed that the strain is resistant to tetracycline and aminoglycosides. Further analysis indicated that the transferability of the tetW gene is extremely low, and resistance to aminoglycosides is due to the intrinsic resistance of L. johnsonii IDCC 9203. Phenotypic safety assessment showed that the strain has neither ß-hemolytic nor ß-glucuronidase activity, and no biogenic amine production. When LPS-induced RAW 264.7 cells were treated with L. johnsonii IDCC 9203, the level of nitric oxide and expression of pro-inflammatory cytokines significantly decreased (p < 0.05). Therefore, L. johnsonii IDCC 9203 strain is considered as safe and beneficial probiotic for human consumption.
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Lactobacillus johnsonii , Aminoglicósidos , Antibacterianos , Antiinflamatorios/farmacología , Heces , Femenino , Humanos , Lactante , Lactobacillus/genética , LipopolisacáridosRESUMEN
Probiotics are beneficial microorganisms, and the evaluation of their safety for human use in the food industry has become critical. This study examines the safety of Bacillus coagulans IDCC 1201 isolated from green malt by analyzing its genomic and phenotypic characteristics and determining its toxicity. The presence of antibiotic resistance and toxigenic genes and gene transferability were investigated using whole-genome analysis. The strain's hemolytic and enzyme activities, minimum inhibitory concentrations of antibiotics, and biogenic amine and D-lactate production were also examined. Furthermore, the principal properties of B. coagulans IDCC 1201 as probiotics, such as resistance to abiotic stress and intestinal adhesion, were studied. The whole-genome analysis demonstrated that B. coagulans IDCC 1201 had no antibiotic resistance or toxigenic genes; the strain was susceptible to the nine antibiotics proposed by the European Food Safety Authority. Moreover, this strain lacked hemolytic and ß-glucuronidase activities. Additionally, it was confirmed that B. coagulans IDCC 1201 produced undesirable metabolites, including biogenic amines or D-lactate, at a safe level. Finally, the strain exhibited functional potential as a probiotic in terms of abiotic tolerance, such as bile tolerance and intestinal adhesion in in vitro experiments. In conclusion, B. coagulans IDCC 1201 can be considered as a safe probiotic with regard to human health.
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Bacillus coagulans/efectos de los fármacos , Bacillus coagulans/genética , Probióticos , Células A549 , Animales , Antibacterianos/farmacología , Aminas Biogénicas/metabolismo , Línea Celular , Farmacorresistencia Microbiana , Femenino , Estudio de Asociación del Genoma Completo , Inestabilidad Genómica , Genómica , Células HaCaT , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácido Láctico/metabolismo , Metaboloma , Pruebas de Sensibilidad Microbiana , Modelos Animales , Filogenia , Probióticos/toxicidad , Ratas , Factores de Virulencia/genética , Secuenciación Completa del GenomaRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) has been implicated in both familial and sporadic Parkinson's disease (PD), yet its pathogenic role remains unclear. A previous screen in Drosophila identified Scar/WAVE (Wiskott-Aldrich syndrome protein-family verproline) proteins as potential genetic interactors of LRRK2 Here, we provide evidence that LRRK2 modulates the phagocytic response of myeloid cells via specific modulation of the actin-cytoskeletal regulator, WAVE2. We demonstrate that macrophages and microglia from LRRK2-G2019S PD patients and mice display a WAVE2-mediated increase in phagocytic response, respectively. Lrrk2 loss results in the opposite effect. LRRK2 binds and phosphorylates Wave2 at Thr470, stabilizing and preventing its proteasomal degradation. Finally, we show that Wave2 also mediates Lrrk2-G2019S-induced dopaminergic neuronal death in both macrophage-midbrain cocultures and in vivo. Taken together, a LRRK2-WAVE2 pathway, which modulates the phagocytic response in mice and human leukocytes, may define an important role for altered immune function in PD.
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Citofagocitosis/fisiología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Células Mieloides/citología , Enfermedad de Parkinson/fisiopatología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Línea Celular , Drosophila , Humanos , Ratones , Microglía , Células Mieloides/fisiología , Transducción de Señal/fisiologíaRESUMEN
Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, and Xenopus oocyte expression, we first identified fragment Asp-21-Ser-42 to be functionally important. We then found that deletion mutant Δ1-36 (TRPP3 missing fragment Met-1-Arg-36) has a similar function as wild-type TRPP3, whereas Δ1-38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1-Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3.
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Canales de Calcio/metabolismo , Lipoilación/fisiología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Canales de Calcio/genética , Células HEK293 , Humanos , Mutación Missense , Fosforilación/fisiología , Dominios Proteicos , Receptores de Superficie Celular/genética , Eliminación de Secuencia , Xenopus laevisRESUMEN
The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.
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Etanol/metabolismo , Expresión Génica , Ingeniería Metabólica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Aerobiosis , Anaerobiosis , Medios de Cultivo/química , Fermentación , Manihot/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nitrógeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Almidón/metabolismo , Proteína de Unión a TATA-Box/genética , Zea mays/metabolismoRESUMEN
Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 3' untranslated region (3'UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 3'UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pull-down assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 3'UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD.
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Regiones no Traducidas 3'/fisiología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Biosíntesis de Proteínas , Canales Catiónicos TRPP/genética , Animales , Células Cultivadas , Proteínas de Unión al ARN , Pez CebraRESUMEN
Present sterilization methods for biofilms in medical devices have limitations. Therefore, an alternative sterilization method using supercritical carbon dioxide (SC-CO2) was tested on Candida albicans biofilms. The effect of varying pressure, temperature, and treatment time on the inactivation of C. albicans spores in suspensions and in biofilms was examined. The parameters such as treatment time, pressure, and temperature that led to the complete inactivation of C. albicans biofilms ranged 5-20 min, 100-200 bar, and 35-45 °C, respectively. Notably, treatment of SC-CO2 at either 100 bar and 40 °C or 200 bar and 30 °C induced complete inactivation of spores within 5 min. Furthermore, it was found that wet biofilms (0.4 %, w/w) had higher sensitivity to SC-CO2 than dried biofilms. Finally, spore inactivation was confirmed by confocal laser scanning microscopy. In this study, the use of a low-temperature SC-CO2 sterilization method was proven to be effective in fungal biofilm inactivation, and the moisture content of biofilms was revealed to be the key factor for biofilm inactivation.
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Antibacterianos/administración & dosificación , Antibacterianos/química , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Ensayo de Materiales , Presión , Esterilización/métodosRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, and it affects over 10 million people worldwide. It is characterized by cyst formation in the kidney, liver and pancreas. Dosage changes in PKD1/PKD2 are important in ADPKD pathogenesis; therefore, their expression and function has to be strictly regulated. However, how they are regulated remain poorly understood. Recent studies have linked PKD2 regulation to endoplasmic reticulum (ER) stress that is implicated in neuronal, cardiac, and renal diseases. One major ER stress downstream is phosphorylation of eukaryotic initiation factor eIF2α by kinase PERK, which attenuates global protein translation and enhances translation of selected proteins. Here, we showed in several mammalian cell lines that PKD2 protein expression is up-regulated by different stresses that all increase phosphorylated eIF2α (P-eIF2α). Increasing P-eIF2α by overexpression or inhibiting the phosphatase activity resulted in increased PKD2. PCR and polysome-binding assays showed that ER stress does not affect the PKD2 mRNA level but increase its binding with ribosomes, indicating that P-eIF2α translationally up-regulates PKD2. By mutation analysis, we found that the upstream open reading frame (uORF) in the 5'-untranslated region of PKD2 mRNA represses PKD2 translation. Thus, ER stress and P-eIF2α translationally up-regulates PKD2 through bypassing the inhibitory uORF.
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Estrés del Retículo Endoplásmico , Biosíntesis de Proteínas , Canales Catiónicos TRPP/biosíntesis , Regulación hacia Arriba , Regiones no Traducidas 5' , Animales , Perros , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Mutación , Polirribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canales Catiónicos TRPP/genéticaRESUMEN
A CO2-added ammonia explosion pretreatment was performed for bioethanol production from rice straw. The pretreatment conditions, such as ammonia concentration, CO2 loading level, residence time, and temperature were optimized using response surface methodology. The response for optimization was defined as the glucose conversion rate. The optimized pretreatment conditions resulting in maximal glucose yield (93.6 %) were determined as 14.3 % of ammonia concentration, 2.2 MPa of CO2 loading level, 165.1 °C of temperature, and 69.8 min of residence time. Scanning electron microscopy analysis showed that pretreatment of rice straw strongly increased the surface area and pore size, thus increasing enzymatic accessibility for enzymatic saccharification. Finally, an ethanol yield of 97 % was achieved via simultaneous saccharification and fermentation. Thus, the present study suggests that CO2-added ammonia pretreatment is an appropriate process for bioethanol production from rice straw.
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Amoníaco/metabolismo , Dióxido de Carbono/metabolismo , Etanol/metabolismo , Oryza/metabolismo , Fermentación , Microscopía Electrónica de RastreoRESUMEN
Obesity is a common metabolic disease characterized by abnormal fat accumulation. It contributes to health issues, such as type 2 diabetes, cardiovascular disease, and dyslipidemia, necessitating continuous management through diet and physical activity. Probiotics, particularly Bifidobacterium lactis IDCC 4301 (B. lactis Fit™), have shown promise in positively regulating the gut microbiota. Therefore, this study aimed to evaluate the anti-obesity effect of B. lactis IDCC 4301 (B. lactis Fit™) in obese women. A randomized, double-blind, placebo-controlled, parallel-arm study was performed in 99 volunteers with a body mass index (BMI) of 25-30 kg m-2. The participants were randomly assigned to probiotics (n = 49, >5.0 × 109 CFU day-1) or placebo (n = 50) groups. Body fat, lipid profiles, and adipokine levels were assessed at baseline and at 12 weeks. After 12 weeks, changes in total fat (placebo -0.16 ± 0.83 kg; probiotics -0.45 ± 0.83 kg; p = 0.0407), trunk fat (placebo -0.03 ± 0.50 kg; probiotics -0.22 ± 0.51 kg; p = 0.0200), and serum triglyceride concentration (placebo 13 ± 60 mg dL-1; probiotics -15 ± 62 mg dL-1; p = 0.0088) were significantly different between the groups. The difference in total fat mass change between groups among postmenopausal women was greater than that of all women. A significant positive correlation was found between the change in total fat mass and log leptin/adiponectin ratio (R = 0.371, p = 0.0112) in the probiotics group. In addition, BMI (26.6 ± 1.9 kg m-2 to 26.4 ± 2.0 kg m-2, p = 0.0009) and leg fat (42 ± 5% to 41 ± 5%, p = 0.0006) significantly decreased in the probiotics group after 12 weeks, but there was no difference in the placebo group. In conclusion, B. lactis IDCC 4301 (B. lactis Fit™) may be associated with body fat loss through changes in metabolic health parameters, such as serum triglyceride and adipokine levels. The clinical trial registry number is KCT0007425 (https://cris.nih.go.kr).
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Adipoquinas , Tejido Adiposo , Bifidobacterium animalis , Obesidad , Probióticos , Triglicéridos , Humanos , Femenino , Obesidad/dietoterapia , Obesidad/terapia , Obesidad/sangre , Triglicéridos/sangre , Adulto , Método Doble Ciego , Persona de Mediana Edad , Adipoquinas/sangre , Tejido Adiposo/metabolismo , Índice de Masa Corporal , Suplementos Dietéticos , Microbioma GastrointestinalRESUMEN
As a renewable energy from biomass, isobutanol is considered as a promising alternative to fossil fuels. To biotechnologically produce isobutanol, strain development using industrial microbial hosts, such as Escherichia coli, has been conducted by introducing a heterologous isobutanol synthetic pathway. However, the toxicity of produced isobutanol inhibits cell growth, thereby restricting improvements in isobutanol titer, yield, and productivity. Therefore, the development of robust microbial strains tolerant to isobutanol is required. In this study, isobutanol-tolerant mutants were isolated from two E. coli parental strains, E. coli BL21(DE3) and MG1655(DE3), through adaptive laboratory evolution (ALE) under high isobutanol concentrations. Subsequently, 16 putative genes responsible for isobutanol tolerance were identified by transcriptomic analysis. When overexpressed in E. coli, four genes (fadB, dppC, acs, and csiD) conferred isobutanol tolerance. A fermentation study with a reverse engineered isobutanol-producing E. coli JK209 strain showed that fadB or dppC overexpression improved isobutanol titers by 1.5 times, compared to the control strain. Through coupling adaptive evolution with transcriptomic analysis, new genetic targets utilizable were identified as the basis for the development of an isobutanol-tolerant strain. Thus, these new findings will be helpful not only for a fundamental understanding of microbial isobutanol tolerance but also for facilitating industrially feasible isobutanol production.
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Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Butanoles/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión GénicaRESUMEN
Obesity and overweight among companion animals are significant concerns, paralleling the issues observed in human populations. Recent research has highlighted the potential benefits of various probiotics in addressing weight-related changes, obesity, and associated pathologies. In this study, we delved into the beneficial probiotic mechanisms in high-fat-induced obese canines, revealing that Enterococcus faecium IDCC 2102 (IDCC 2102) and Bifidobacterium lactis IDCC 4301 (IDCC 4301) have the capacity to mitigate the increase in body weight and lipid accumulation in obese canines subjected to a high-fat diet and hyperlipidemic Caenorhabditis elegans (C. elegans) strain VS29. Both IDCC 2102 and IDCC 4301 demonstrated the ability to reduce systemic inflammation and hormonal disruptions induced by obesity. Notably, these probiotics induced modifications in the microbiota by promoting lactic acid bacteria, including Lactobacillaceae, Ruminococcaceae, and S24-7, with concomitant activation of pyruvate metabolism. IDCC 4301, through the generation of bacterial short-chain fatty acids and carboxylic acids, facilitated glycolysis and contributed to ATP synthesis. Meanwhile, IDCC 2102 produced bacterial metabolites such as acetic acid and butyric acid, exhibiting a particular ability to stimulate dopamine synthesis in a canine model. This stimulation led to the restoration of eating behavior and improvements in glucose and insulin tolerance. In summary, we propose novel probiotics for the treatment of obese animals based on the modifications induced by IDCC 2102 and IDCC 4301. These probiotics enhanced systemic energy utilization in response to high caloric intake, thereby preventing lipid accumulation and restoring stability to the fecal microbiota. Consequently, this intervention resulted in a reduction in systemic inflammation caused by the high-fat diet.IMPORTANCEProbiotic supplementation affected commensal bacterial proliferation, and administering probiotics increased glycolysis and activated pyruvate metabolism in the body, which is related to propanate metabolism as a result of pyruvate metabolism activation boosting bacterial fatty acid production via dopamine and carboxylic acid specialized pathways, hence contributing to increased ATP synthesis and energy metabolism activity.
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Microbioma Gastrointestinal , Probióticos , Humanos , Perros , Animales , Caenorhabditis elegans/metabolismo , Dopamina , Obesidad/terapia , Obesidad/veterinaria , Obesidad/metabolismo , Pérdida de Peso , Ácidos Grasos Volátiles , Metabolismo Energético , Inflamación , Piruvatos , Adenosina Trifosfato/metabolismoRESUMEN
Probiotics can exert direct or indirect influences on various aspects of health claims by altering the composition of the gut microbiome and producing bioactive metabolites. The aim of this study was to examine the effect of Lacticaseibacillus rhamnosus IDCC3201 on skeletal muscle atrophy in dexamethasone-induced C2C12 cells and a mouse animal model. Dexamethasone treatment significantly reduced C2C12 muscle cell viability, myotube diameter, and levels of muscle atrophic markers (Atrogin-1 and MuRF-1). These effects were alleviated by conditioned media (CM) and cell extract (EX) derived from L. rhamnosus IDCC3201. In addition, we assessed the in vivo therapeutic effect of L. rhamnosus IDCC3201 in a mouse model of dexamethasone (DEX)-induced muscle atrophy. Supplementation with IDCC3201 resulted in significant enhancements in body composition, particularly in lean mass, muscle strength, and myofibril size, in DEX-induced muscle atrophy mice. In comparison to the DEX-treatment group, the normal and DEX + L. rhamnosus IDCC3201 groups showed a higher transcriptional level of myosin heavy chain family genes (MHC1, MHC1b, MHC2A, 2bB, and 2X) and a reduction in atrophic muscle makers. These analyses revealed that L. rhamnosus IDCC3201 supplementation led to increased production of branched-chain amino acids (BCAAs) and improved the Allobaculum genus within the gut microbiota of muscle atrophy-induced groups. Taken together, our findings suggest that L. rhamnosus IDCC3201 represents a promising dietary supplement with the potential to alleviate sarcopenia by modulating the gut microbiome and metabolites.
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Dexametasona , Suplementos Dietéticos , Microbioma Gastrointestinal , Lacticaseibacillus rhamnosus , Probióticos , Sarcopenia , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Sarcopenia/metabolismo , Probióticos/farmacología , Probióticos/administración & dosificación , Masculino , Atrofia Muscular/metabolismo , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/inducido químicamente , Modelos Animales de Enfermedad , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Ratones Endogámicos C57BL , Proteínas Musculares/metabolismoRESUMEN
Probiotics, specifically Lacticaseibacillus rhamnosus, have garnered attention for their potential health benefits. This study focuses on evaluating the probiotic properties of candidate probiotics L. rhamnosus IDCC 3201 (3201) using the Caenorhabditis elegans surrogate animal model, a well-established in vivo system for studying host-bacteria interactions. The adhesive ability to the host's gastrointestinal tract is a crucial criterion for selecting potential probiotic bacteria. Our findings demonstrated that 3201 exhibits significantly higher adhesive capabilities compared with Escherichia coli OP50 (OP50), a standard laboratory food source for C. elegans and is comparable with the widely recognized probiotic L. rhamnosus GG (LGG). In lifespan assay, 3201 significantly increased the longevity of C. elegans compared with OP50. In addition, preconditioning with 3201 enhanced C. elegans immune response against four different foodborne pathogenic bacteria. To uncover the molecular basis of these effects, transcriptome analysis elucidated that 3201 modulates specific gene expression related to the innate immune response in C. elegans. C-type lectin-related genes and lysozyme-related genes, crucial components of the immune system, showed significant upregulation after feeding 3201 compared with OP50. These results suggested that preconditioning with 3201 may enhance the immune response against pathogens. Metabolome analysis revealed increased levels of fumaric acid and succinic acid, metabolites of the citric acid cycle, in C. elegans fed with 3201 compared with OP50. Furthermore, there was an increase in the levels of lactic acid, a well-known antimicrobial compound. This rise in lactic acid levels may have contributed to the robust defense mechanisms against pathogens. In conclusion, this study demonstrated the probiotic properties of the candidate probiotic L. rhamnosus IDCC 3201 by using multi-omics analysis.
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Caenorhabditis elegans , Lacticaseibacillus rhamnosus , Longevidad , Probióticos , Animales , Caenorhabditis elegans/inmunología , Caenorhabditis elegans/microbiología , Perfilación de la Expresión Génica , Inmunidad Innata , MultiómicaRESUMEN
Pkd2L1 (also called TRPP3) is a non-selective cation channel permeable to Ca(2+), Na(+), and K(+) and is activated by Ca(2+). It is also part of an acid-triggered off-response cation channel complex. We previously reported roles of the Pkd2L1 C-terminal fragments in its channel function, but the role of the N terminus remains unclear. Using a yeast two-hybrid screening, we found that the Pkd2L1 N terminus interacts with the receptor for activated C kinase 1 (RACK1), a scaffolding/anchoring protein implicated in various cellular functions. This interaction requires the last two Trp-Asp (WD) motifs of RACK1 and fragment Ala(19)-Pro(45) of Pkd2L1. The interaction was confirmed by GST pulldown, blot overlay, and co-immunoprecipitation assays. By (45)Ca tracer uptake and two-microelectrode voltage clamp electrophysiology, we found that in Xenopus oocytes with RACK1 overexpression Pkd2L1 channel activity is abolished or substantially reduced. Combining with oocyte surface biotinylation experiments, we demonstrated that RACK1 inhibits the function of Pkd2L1 channel on the plasma membrane in addition to reducing its total and plasma membrane expression. Overexpressing Pkd2L1 N- or C-terminal fragments as potential blocking peptides for the Pkd2L1-RACK1 interaction, we found that Pkd2L1 N-terminal fragment Met(1)-Pro(45), but not Ile(40)-Ile(97) or C-terminal fragments, abolishes the inhibition of Pkd2L1 channel by overexpressed and oocyte-native RACK1 likely through disrupting the Pkd2L1-RACK1 association. Taken together, our study demonstrated that RACK1 inhibits Pkd2L1 channel function through binding to domain Met(1)-Pro(45) of Pkd2L1. Thus, Pkd2L1 is a novel target channel whose function is regulated by the versatile scaffolding protein RACK1.
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Canales de Calcio/química , Canales de Calcio/fisiología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/fisiología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Animales , Sitios de Unión/fisiología , Calcio/metabolismo , Canales de Calcio/genética , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Mutagénesis/fisiología , Proteínas de Neoplasias/genética , Oocitos/fisiología , Técnicas de Placa-Clamp , Dominios y Motivos de Interacción de Proteínas/fisiología , Estructura Terciaria de Proteína/fisiología , ARN Mensajero/farmacología , Receptores de Cinasa C Activada , Receptores de Superficie Celular/genética , Técnicas del Sistema de Dos Híbridos , XenopusRESUMEN
Previously, it was shown that overexpression of either of two SPT15 mutant alleles, SPT15-M2 and SPT15-M3, which encode mutant TATA-binding proteins, confer enhanced ethanol tolerance in Saccharomyces cerevisiae. In this study, we demonstrated that strains overexpressing SPT15-M2 or SPT15-M3 were tolerant to hyperosmotic stress caused by high concentrations of glucose, salt, and sorbitol. The enhanced tolerance to high glucose concentrations in particular improved ethanol production from very high gravity (VHG) ethanol fermentations. The strains displayed constitutive and sustained activation of Hog1, a central kinase in the high osmolarity glycerol (HOG) signal transduction pathway of S. cerevisiae. However, the cell growth defect known to be caused by constitutive and sustained activation of Hog1 was not observed. We also found that reactive oxygen species (ROS) were accumulated to a less extent upon exposure to high glucose concentration in our osmotolerant strains. We identified six new genes (GPH1, HSP12, AIM17, SSA4, USV1, and IGD1), the individual deletion of which renders cells sensitive to 50 % glucose. In spite of the presence of multiple copies of stress response element in their promoters, it was apparent that those genes were not controlled at the transcriptional level by the HOG pathway under the high glucose conditions. Combined with previously published results, overexpression of SPT15-M2 or SPT15-M3 clearly provides a basis for improved tolerance to ethanol and osmotic stress, which enables construction of strains of any genetic background that need enhanced tolerance to high concentrations of ethanol and glucose, promoting the feasibility for VHG ethanol fermentation.
Asunto(s)
Mutación , Presión Osmótica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Alelos , Etanol/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Glicerol/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The demand for plant-based proteins as alternative meat sources continues to increase because of environmental concerns, animal welfare, and religious reasons. However, plant-based proteins have low digestibility than real meat, which should be overcome. In the present study, the effect of co-administration of legumin protein mixture and the probiotic strain on plasma concentration of amino acids was investigated as a strategy of enhancement in protein digestion. First, the proteolytic activity of the four probiotic strains was compared. As a result, Lacticaseibacillus casei IDCC 3451 was identified as an optimal probiotic strain that efficiently digested the legumin protein mixture by forming the largest halo produced by proteolysis. Next, to investigate whether the co-administration of legumin protein mixture and L. casei IDCC 3451 could synergically improve digestibility, mice were fed either a high-protein diet or a high-protein diet with L. casei IDCC 3451 for 8 weeks. Compared to only in the high-protein diet only group, the concentrations of branched chain amino acids and essential amino acids were 1.36 and 1.41 times higher in the co-administered group, respectively. Therefore, co-supplementation of plant-based proteins with L. casei IDCC 3451 can be suggested to improve protein digestibility based on the this study.
RESUMEN
Melanogenesis is responsible for skin pigmentation and the enzymatic browning of foods. Tyrosinases play a major role in melanin synthesis, and many attempts have been made to identify new natural tyrosinase inhibitors, but few have sought to do in microbes. Postbiotics are bioactive compounds produced by the metabolism of probiotics and have been reported to be safe and effective. In this study, we evaluated the tyrosinase inhibitory effects of culture supernatants of probiotics and discovered novel bacterial metabolites that can be used as a potent tyrosinase inhibitor based on metabolomics. Cultures of Bifidobacterium bifidum IDCC 4201 and Lactiplantibacillus plantarum IDCC 3501 showed effective anti-tyrosinase, reduced melanin synthesis, and altered protein expression associated with the melanogenesis pathway. Comparative metabolomics analyses conducted by GC-MS identified metabolites commonly produced by B. bifidum and L. plantarum. Of eight selected metabolites, phenyllactic acid exhibited significant tyrosinase-inhibitory activity. Our findings suggest that applications of probiotic culture supernatants containing high amounts of phenyllactic acid have potential use as anti-melanogenesis agents in food and medicines.
RESUMEN
SCOPE: Chronic hypernutrition promotes lipid accumulation in the body and excessive lipid accumulation leads to obesity. An increase in the number and size of adipocytes, a characteristic of obesity is closely associated with adipose dysfunction. Recent in vitro and in vivo studies have shown that probiotics may prevent this dysfunction by regulating lipid metabolism. However, the mechanisms of action of probiotics in obesity are not fully understood and their usage for treating obesity remains limited. METHODS AND RESULTS: Bifidobacterium lactis IDCC 4301 is selected for its anti-obesity potential after evaluating inhibitory activity of pancreatic lipase and cholesterol reducing activity. Next, this study investigates the roles of B. lactis IDCC 4301 on lipid metabolism in 3T3-L1 preadipocytes and high-fat diet (HFD)-fed mice. B. lactis IDCC 4301 inhibits cell differentiation and lipid accumulation by suppressing the expression of adipogenic enzymes in 3T3-L1 cells. Moreover, the administration of B. lactis IDCC 4301 decreases body and adipose tissue weight, improves serum lipid levels, and downregulates adipogenic mRNA expression in HFD-fed mice. Additionally, metabolomic analysis suggests that 2-ketobutyrate should be a possible target compound against obesity. CONCLUSIONS: B. lactis IDCC 4301 may be used as an alternative treatment for obesity.