Lactobacillus Persisters Formation and Resuscitation.

Lactobacillus is a commonly used probiotic, and many researchers have focused on its stress response to improve its functionality and survival. However, studies on persister cells, dormant cells that aid bacteria in surviving general stress, have focused on pathogenic bacteria that cause infection, not Lactobacillus. Thus, understanding Lactobacillus persister cells will provide essential clues for understanding how Lactobacillus survives and maintains its function under various environmental conditions. We treated Lactobacillus strains with various antibiotics to determine the conditions required for persister formation using kill curves and transmission electron microscopy. In addition, we observed the resuscitation patterns of persister cells using single-cell analysis. Our results show that Lactobacillus creates a small population of persister cells (0.0001-1% of the bacterial population) in response to beta-lactam antibiotics such as ampicillin and amoxicillin. Moreover, only around 0.5-1% of persister cells are heterogeneously resuscitated by adding fresh media; the characteristics are typical of persister cells. This study provides a method for forming and verifying the persistence of Lactobacillus and demonstrates that antibiotic-induced Lactobacillus persister cells show characteristics of dormancy, sensitivity of antibiotics, same as exponential cells, multi-drug tolerance, and resuscitation, which are characteristics of general persister cells. This study suggests that the mechanisms of formation and resuscitation may vary depending on the characteristics, such as the membrane structure of the bacterial species.

Lactobacillus amylovorus KU4 induces adipose browning in obese mice by regulating PP4C.

We previously reported that Lactobacillus amylovorus KU4 (LKU4) promotes adipocyte browning in mice fed a high-fat diet (HFD mice) in part by remodeling the PPARγ transcription complex. However, the mechanism through which LKU4 enables PPARγ to drive adipocyte browning remains elusive. Here, we report that LKU4 inhibits the expression of PP4C in inguinal white adipose tissue of HFD mice and in insulin-resistant 3T3-L1 adipocytes, which promotes SIRT1-dependent PPARγ deacetylation by activating AMPK, leading to the browning of adipocytes. Consistently, the silencing of PP4C further enhances this pathway. Furthermore, we observed that lactate, a key LKU4 metabolite, reduces insulin-induced PP4C expression and suppresses PP4C inhibition of PPARγ deacetylation and transcriptional activity via AMPK-SIRT1, thereby facilitating the browning of adipocytes. Together, these data demonstrate that LKU4 promotes the AMPK-SIRT1-PPARγ pathway by inhibiting PP4C, thereby facilitating adipocyte browning in HFD mice.

Inhibitory Effects of Latilactobacillus curvatus BYB3 Cell-Free Extract on Human Melanoma B16F10 Cells and Tumorigenic Mice.

Latilactobacillus curvatus BYB3 (BYB3) is a species of lactic acid bacteria, formerly named Lactobacillus curvatus, which is isolated from kimchi. In this study, the effect of BYB3, Lactobacillus rhamnosus GG, and Lactobacillus acidophilus GP1B strain extracts at various concentrations was examined on B16F10, a mouse melanoma cell line. Cell viability was examined via MTT assay, and the results indicated that compared to the other two probiotics, BYB3 significantly decreased the total percentages of viable cells. The effects of BYB3 on cell migration and proliferation in B16F10 cells were evaluated using wound healing mobility and proliferation assays, respectively; the results indicated that BYB3 inhibits cell migration and proliferation in a concentration-dependent manner. Using human dermal fibroblast cells to investigate BYB3 extract in vivo had no effect on skin-related cells. Nonetheless, the BYB3 extract inhibited tumor growth in a mouse model, as demonstrated by liver slices. Therefore, this suggests that using BYB3 extract to inhibit melanoma may be a novel approach.

The PKA-SREBP1c Pathway Plays a Key Role in the Protective Effects of Lactobacillus johnsonii JNU3402 Against Diet-Induced Fatty Liver in Mice.

SCOPE: The present study aims to assess the protective effect of Lactobacillus johnsonii JNU3402 (LJ3402) against diet-induced non-alcoholic fatty liver disease (NAFLD) and determine the mechanism underlying its beneficial effect on the liver in mice. METHODS AND RESULTS: Seven-week-old male mice are fed a high-fat diet (HFD) with or without oral supplementation of LJ3402 for 14 weeks. In mice fed an HFD, LJ3402 administration alleviates liver steatosis, diet-induced obesity, and insulin resistance with a decreased hepatic expression of sterol-regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), and an increased phosphorylation of SREBP-1c. The mechanistic study shows that LJ3402 inhibits SREBP-1c transcriptional activity by enhancing protein kinase A (PKA)-mediated phosphorylation and reduces the expression of its lipogenic target genes in AML12 and HepG2 cells, thereby attenuating hepatic lipid accumulation. Moreover, silencing the PKA α catalytic subunit or the inhibition of PKA activity by H89 abolishes LJ3402 suppression of free fatty acid (FFA)-induced SREBP-1c activity in hepatocytes. In addition, LJ3402 administration elevates the plasma lactate levels in mice fed an HFD; this lactate increases PKA-mediated SREBP-1c phosphorylation in AML12 cells with a decreased expression of its target genes, reducing hepatic lipid accumulation. CONCLUSION: LJ3402 attenuates HFD-induced fatty liver in mice through the lactate-PKA-SREBP-1c pathway.

Probiotics that Ameliorate Cognitive Impairment through Anti-Inflammation and Anti-Oxidation in Mice.

The gut-brain axis encompasses a bidirectional communication pathway between the gastrointestinal microbiota and the central nervous system. There is some evidence to suggest that probiotics may have a positive effect on cognitive function, but more research is needed before any definitive conclusions can be drawn. Inflammation-induced by lipopolysaccharide (LPS) may affect cognitive function. To confirm the effect of probiotics on oxidative stress induced by LPS, the relative expression of antioxidant factors was confirmed, and it was revealed that the administration of probiotics had a positive effect on the expression of antioxidant-related factors. After oral administration of probiotics to mice, an intentional inflammatory response was induced through LPS i.p., and the effect on cognition was confirmed by the Morris water maze test, nitric oxide (NO) assay, and interleukin (IL)-1ß enzyme-linked immunosorbent assay performed. Experimental results, levels of NO and IL-1 ß in the blood of LPS i.p. mice were significantly decreased, and cognitive evaluation using the Morris water maze test showed significant values in the latency and target quadrant percentages in the group that received probiotics. This proves that intake of these probiotics improves cognitive impairment and memory loss through anti-inflammatory and antioxidant mechanisms.

Machine learning-based predictive model for prevention of metabolic syndrome.

Metabolic syndrome (MetS) is a chronic disease caused by obesity, high blood pressure, high blood sugar, and dyslipidemia and may lead to cardiovascular disease or type 2 diabetes. Therefore, the detection and prevention of MetS at an early stage are imperative. Individuals can detect MetS early and manage it effectively if they can easily monitor their health status in their daily lives. In this study, a predictive model for MetS was developed utilizing solely noninvasive information, thereby facilitating its practical application in real-world scenarios. The model's construction deliberately excluded three features requiring blood testing, specifically those for triglycerides, blood sugar, and HDL cholesterol. We used a large-scale Korean health examination dataset (n = 70, 370; the prevalence of MetS = 13.6%) to develop the predictive model. To obtain informative features, we developed three novel synthetic features from four basic information: waist circumference, systolic and diastolic blood pressure, and gender. We tested several classification algorithms and confirmed that the decision tree model is the most appropriate for the practical prediction of MetS. The proposed model achieved good performance, with an AUC of 0.889, a recall of 0.855, and a specificity of 0.773. It uses only four base features, which results in simplicity and easy interpretability of the model. In addition, we performed calibrations on the prediction probability and calibrated the model. Therefore, the proposed model can provide MetS diagnosis and risk prediction results. We also proposed a MetS risk map such that individuals could easily determine whether they had metabolic syndrome.

Complete genome sequence of Limosilactobacillus fermentum JNU532 as a probiotic candidate for the functional food and feed supplements.

Lactic acid bacteria (LAB) have been reported to possess various beneficial properties and are commonly used as probiotics. LAB play a crucial role in milk fermentation, industrial lactic acid fermentation, and health and medicine. Limosilactobacillus fermentum isolated from fermented dairy and food products is considered as 'Generally Recognized as Safe' by FDA. Limosilactobacillus fermentum plays an important role in modulation of the intestinal microbiota, enhancing the host immune system and improving feed digestibility. We isolated a probiotic candidate that was identified and named Limosilactobacillus fermentum JNU532. In a previous report, cell-free culture of L. fermentum JNU532 exhibited anti-melanogenic and antioxidant activities. In this study, we present the complete genome assembly of the bacterial strain JNU532. The final genome consists of one circular chromosome (2,077,416 base pairs) with a guanine + cytosine (GC) ratio of 51.5%.

Complete genome and two plasmids sequences of Lactiplantibacillus plantarum L55 for probiotic potentials.

In this study, we report the complete genome sequence of Lactiplantibacillus plantarum L55, a probiotic strain of lactic acid bacteria isolated from kimchi. The genome consists of one circular chromosome (2,077,416 base pair [bp]) with a guanine cytosine (GC) content of 44.5%, and two circular plasmid sequences (54,267 and 19,592 bp, respectively). We also conducted a comprehensive analysis of the genome, which identified the presence of functional genes, genomic islands, and antibiotic-resistance genes. The genome sequence data presented in this study provide insights into the genetic basis of L. plantarum L55, which could be beneficial for the future development of probiotic applications.

Metabolites of Latilactobacillus curvatus BYB3 and Indole Activate Aryl Hydrocarbon Receptor to Attenuate Lipopolysaccharide-Induced Intestinal Barrier Dysfunction.

This study aimed to investigate the effects of the metabolites of Latilactobacillus curvatus BYB3 and indole-activated aryl hydrocarbon receptor (AhR) to increase the tight junction (TJ) proteins in an in vitro model of intestinal inflammation. In a Western blot assay, the metabolites of L. curvatus BYB3 reduced the TJ demage in lipoploysaccharide (LPS) stimulated-Caco-2 cells. This reduction was a result of upregulating the expression of TJ-associated proteins and suppressing the nuclear factor-κB signaling. Immunofluorescence images consistently revealed that LPS disrupted and reduced the expression of TJ proteins, while the metabolites of L. curvatus BYB3 and indole reversed these alterations. The protective effects of L. curvatus BYB3 were observed on the intestinal barrier function when measuring transepithelial electrical resistance. Using high-performance liquid chromatography analysis the metabolites, the indole-3-latic acid and indole-3-acetamide concentrations were found to be 1.73±0.27 mg/L and 0.51±0.39 mg/L, respectively. These findings indicate that the metabolites of L. curvatus BYB3 have increasing mRNA expressions of cytochrome P450 1A1 (CYP1A1) and AhR, and may thus be applicable for therapy of various inflammatory gut diseases as postbiotics.

Culturomic-, metagenomic-, and transcriptomic-based characterization of commensal lactic acid bacteria isolated from domestic dogs using Caenorhabditis elegans as a model for aging.

In tandem with the fast expansion of the pet-economy industry, the present aging research has been noticing the function of probiotics in extending the healthy lifetime of domestic animals. In this study, we aimed to understand the bacterial compositions of canine feces and isolating lactic acid bacteria (LAB) as commensal LAB as novel potential probiotics for the use of antiaging using Caenorhabditis elegans surrogate animal model. Under an anaerobic, culturomic, and metagenomic analysis, a total of 305 commensal LAB were isolated from diverse domestic dogs, and four strains, Lactobacillus amylolyticus, L. salivarius, Enterococcus hirae, and E. faecium, made prominence as commensal LAB by enhancing C. elegans life span and restored neuronal degeneration induced by aging by upregulating skn-1, ser-7, and odr-3, 7, 10. Importantly, whole transcriptome results and integrative network analysis revealed extensive mRNA encoding protein domains and functional pathways of naturally aging C. elegans were examined and we built the gene informatics basis. Taken together, our findings proposed that a specific gene network corresponding to the pathways differentially expressed during the aging and selected commensal LAB as potential probiotic strains could be provided beneficial effects in the aging of domestic animals by modulating the dynamics of gut microbiota.

In tandem with the fast expansion of the pet-economy industry, the present aging research has been noticing the function of probiotics in extending the healthy lifetime of domestic animals. In this study, collaborating with understanding the characteristics of gut microbiome from canine feces by multiomics approaches including culturomics, metagenomics, and transcriptomics, we isolate and identify commensal lactic acid bacteria (LAB) as novel potential probiotics for the use of antiaging using Caenorhabditis elegans surrogate animal model and multiomics analysis. The selected commensal LAB could be provided beneficial effects in the aging of domestic animals by modulating the dynamics of gut microbiome and applied in the future companion animal market by clarifying their purpose and function.

Complete genome sequences of Lactococcus lactis JNU 534, a potential food and feed preservative.

A new bacteriocin-producing lactic acid bacteria isolated from kimchi was identified as Lactococcus lactis JNU 534, presenting preservative properties for foods of animal origin. In this study, we present the complete genome sequence of the bacterial strain JNU 534. The final complete genome assembly consists of one circular chromosome (2,443,687 bp [base pair]) with an overall GC (guanine-cytosine) content of 35.2%, one circular plasmid sequence (46,387 bp) with a GC content of 34.5%, and one circular contig sequence (7,666 bp) with a GC content of 36.2%.

Roles of Milk Fat Globule Membrane on Fat Digestion and Infant Nutrition.

Milk fats are present as globules emulsified in the aqueous phase of milk and stabilized by a delicate membrane architecture called milk fat globule membrane (MFGM). The unique structure and composition of the MFGM play an important role in fat digestion and the metabolic programming of neonates. The objective of this review is to compare the structure, composition, and physicochemical characteristics of fat globules in human milk, bovine milk, and infant formula. It provides an overview of the fat digestion process and enzymes in healthy infants, and describes the possible roles of the MFGM in association with factors affecting fat digestion. Lastly, the health benefits of the MFGM on infant nutrition and future perspectives are discussed with a focus on brain development, metabolic response, and gut health.

Development of the Integrated Glaucoma Risk Index.

Various machine-learning schemes have been proposed to diagnose glaucoma. They can classify subjects into 'normal' or 'glaucoma'-positive but cannot determine the severity of the latter. To complement this, researchers have proposed statistical indices for glaucoma risk. However, they are based on a single examination indicator and do not reflect the total severity of glaucoma progression. In this study, we propose an integrated glaucoma risk index (I-GRI) based on the visual field (VF) test, optical coherence tomography (OCT), and intraocular pressure (IOP) test. We extracted important features from the examination data using a machine learning scheme and integrated them into a single measure using a mathematical equation. The proposed index produces a value between 0 and 1; the higher the risk index value, the greater the risk/severity of glaucoma. In the sanity test using test cases, the I-GRI showed a balanced distribution in both glaucoma and normal cases. When we classified glaucoma and normal cases using the I-GRI, we obtained a misclassification rate of 0.07 (7%). The proposed index is useful for diagnosing glaucoma and for detecting its progression.

Latilactobacillus curvatus BYB3 Isolated from Kimchi Alleviates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice by Inhibiting IL-6 and TNF-R1 Production.

Recent studies have shown that probiotics have health-promoting effects, particularly intestinal immune modulation. In this study, we focused on the immunomodulatory properties of Latilactobacillus curvatus BYB3, formerly called Lactobacillus curvatus, isolated from kimchi. In a mouse model of 14-day dextran sulfate sodium (DSS)-induced colitis, treatment with L. curvatus BYB3 significantly decreased the disease activity index, colon length, and weight loss. Moreover, histological analyses showed that L. curvatus BYB3 protected the structural integrity of the intestinal epithelial layer and mucin-secreting goblet cells from DSS-induced damage, with only slight infiltration by immune cells. To evaluate the molecular mechanisms underlying L. curvatus BYB3-driven inhibition of interleukin 6 production, possible in vivo anti-inflammatory effects of L. curvatus BYB3 were examined in the same mouse model. In addition, significantly lower levels of IL-6 and tumor necrosis factor receptor 1 upregulation were seen in the DSS+BYB3 group (compared to that in the DSS group). These results indicate that L. curvatus BYB3 exhibits health-promoting effects via immune modulation; and therefore, it can be used to treat various inflammatory diseases.

Genome analysis of Limosilactobacillus fermentum JN2019 applied to tumeric fermentation for animal feed.

Limosilactobacillus fermentum JN2019, formerly named Lactobacillus fermentum JN2019, was isolated from kimchi. Its genome was completely sequenced using the PacBio RSII sequencing system to explore beneficial phenotypes. In a previous study, L. fermentum JN2019 was used to ferment the by-product of tumeric for use in livestock feed. The 2.3 Mb genome had a high guanine (G) + cytosine (C) content of 50.6% and a 30 kb plasmid. The data will inform the comprehensive understanding of JN2019 and provide insights for potential applications.

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

Optimizing the composition of the medium for the viable cells of Bifidobacterium animalis subsp. lactis JNU306 using response surface methodology.

This research improved the growth potential of Bifidobacterium animalis subsp lactis strain JNU306, a commercial medium that is appropriate for large-scale production, in yeast extract, soy peptone, glucose, L-cysteine, and ferrous sulfate. Response surface methodology (RSM) was used to optimize the components of this medium, using a central composite design and subsequent analyses. A second-order polynomial regression model, which was fitted to the data at first, significantly lacked fitness. Thus, through further analyses, the model with linear and quadratic terms plus two-way, three-way, and four-way interactions was selected as the final model. Through this model, the optimized medium composition was found as 2.8791% yeast extract, 2.8030% peptone soy, 0.6196% glucose, 0.2823% L-cysteine, and 0.0055% ferrous sulfate, w/v. This optimized medium ensured that the maximum biomass was no lower than the biomass from the commonly used blood-liver (BL) medium. The application of RSM improved the biomass production of this strain in a more cost-effective way by creating an optimum medium. This result shows that B. animalis subsp lactis JNU306 may be used as a commercial starter culture in manufacturing probiotics, including dairy products.

Antioxidant and Antimelanogenic Activities of Kimchi-Derived Limosilactobacillus fermentum JNU532 in B16F10 Melanoma Cells.

Melanin is a natural skin pigment produced by specialized cells called melanocytes via a multistage biochemical pathway known as melanogenesis, involving the oxidation and polymerization of tyrosine. Melanogenesis is initiated upon exposure to ultraviolet (UV) radiation, causing the skin to darken, which protects skin cells from UVB radiation damage. However, the abnormal accumulation of melanin may lead to the development of certain skin diseases, including skin cancer. In this study, the antioxidant and antimelanogenic activities of the cell-free supernatant (CFS) of twenty strains were evaluated. Based on the results of 60% 2,2-diphenyl-1-picrylhydrazyl scavenging activity, 21% 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) scavenging capacity, and a 50% ascorbic acid equivalent ferric reducing antioxidant power value, Limosilactobacillus fermentum JNU532 was selected as the strain with the highest antioxidant potential. No cytotoxicity was observed in cells treated with the CFS of L. fermentum JNU532. Tyrosinase activity was reduced by 16.7% in CFStreated B16F10 cells (but not in the cell-free system), with >23.2% reduction in melanin content upon treatment with the L. fermentum JNU532-derived CFS. The inhibitory effect of the L. fermentum JNU532-derived CFS on B16F10 cell melanogenesis pathways was investigated using quantitative reverse transcription polymerase chain reaction and western blotting. The inhibitory effects of the L. fermentum JNU532-derived CFS were mediated by inhibiting the transcription of TYR, TRP-1, TRP-2, and MITF and the protein expression of TYR, TRP-1, TRP-2, and MITF. Therefore, L. fermentum JNU532 may be considered a potentially useful, natural depigmentation agent.

Evaluation of the Effects of Developmental Trauma on Neurotransmitter Systems Using Functional Molecular Imaging.

Early life stress (ELS) is strongly associated with psychiatric disorders such as anxiety, depression, and schizophrenia in adulthood. To date, biological, behavioral, and structural aspects of ELS have been studied extensively, but their functional effects remain unclear. Here, we examined NeuroPET studies of dopaminergic, glutamatergic, and serotonergic systems in ELS animal models. Maternal separation and restraint stress were used to generate single or complex developmental trauma. Body weights of animals exposed to single trauma were similar to those of control animals; however, animals exposed to complex trauma exhibited loss of body weight when compared to controls. In behavioral tests, the complex developmental trauma group exhibited a decrease in time spent in the open arm of the elevated plus-maze and an increase in immobility time in the forced swim test when compared to control animals. In NeuroPET studies, the complex trauma group displayed a reduction in brain uptake values when compared to single trauma and control groups. Of neurotransmitter systems analyzed, the rate of decrease in brain uptake was the highest in the serotonergic group. Collectively, our results indicate that developmental trauma events induce behavioral deficits, including anxiety- and depressive-like phenotypes and dysfunction in neurotransmitter systems.

Explainable Machine Learning Model for Glaucoma Diagnosis and Its Interpretation.

The aim is to develop a machine learning prediction model for the diagnosis of glaucoma and an explanation system for a specific prediction. Clinical data of the patients based on a visual field test, a retinal nerve fiber layer optical coherence tomography (RNFL OCT) test, a general examination including an intraocular pressure (IOP) measurement, and fundus photography were provided for the feature selection process. Five selected features (variables) were used to develop a machine learning prediction model. The support vector machine, C5.0, random forest, and XGboost algorithms were tested for the prediction model. The performance of the prediction models was tested with 10-fold cross-validation. Statistical charts, such as gauge, radar, and Shapley Additive Explanations (SHAP), were used to explain the prediction case. All four models achieved similarly high diagnostic performance, with accuracy values ranging from 0.903 to 0.947. The XGboost model is the best model with an accuracy of 0.947, sensitivity of 0.941, specificity of 0.950, and AUC of 0.945. Three statistical charts were established to explain the prediction based on the characteristics of the XGboost model. Higher diagnostic performance was achieved with the XGboost model. These three statistical charts can help us understand why the machine learning model produces a specific prediction result. This may be the first attempt to apply "explainable artificial intelligence" to eye disease diagnosis.