Alterations in the gut microbiome and its metabolites are associated with the immune response to mucosal immunization with Lactiplantibacillus plantarum-displaying recombinant SARS-CoV-2 spike epitopes in mice.

Lactic acid bacteria (LAB) expressing foreign antigens have great potential as mucosal vaccines. Our previous study reported that recombinant Lactiplantibacillus plantarum SK156 displaying SARS-CoV-2 spike S1 epitopes elicited humoral and cell-mediated immune responses in mice. Here, we further examined the effect of the LAB-based mucosal vaccine on gut microbiome composition and function, and gut microbiota-derived metabolites. Forty-nine (49) female BALB/c mice were orally administered L. plantarum SK156-displaying SARS-CoV-2 spike S1 epitopes thrice (at 14-day intervals). Mucosal immunization considerably altered the gut microbiome of mice by enriching the abundance of beneficial gut bacteria, such as Muribaculaceae, Mucispirillum, Ruminococcaceae, Alistipes, Roseburia, and Clostridia vadinBB60. Moreover, the predicted function of the gut microbiome showed increased metabolic pathways for amino acids, energy, carbohydrates, cofactors, and vitamins. The fecal concentration of short-chain fatty acids, especially butyrate, was also altered by mucosal immunization. Notably, alterations in gut microbiome composition, function, and butyrate levels were positively associated with the immune response to the vaccine. Our results suggest that the gut microbiome and its metabolites may have influenced the immunogenicity of the LAB-based SARS-CoV-2 vaccine.

Multispecies probiotic supplementation in diet with reduced crude protein levels altered the composition and function of gut microbiome and restored microbiome-derived metabolites in growing pigs.

Both crude protein (CP) and probiotics can modulate the gut microbiome of the host, thus conferring beneficial effects. However, the benefits of low CP diet supplemented with multispecies probiotics on gut microbiome and its metabolites have not been investigated in pigs. Thus, we investigated the combinatory effects of low CP diet supplemented with multispecies probiotics on gut microbiome composition, function, and microbial metabolites in growing pigs. In total, 140 6 week-old piglets (Landrace × Yorkshire × Duroc) were used in this study. The pigs were divided into four groups with a 2 × 2 factorial design based on their diets: normal-level protein diet (16% CP; NP), low-level protein diet (14% CP; LP), NP with multispecies probiotics (NP-P), and LP with multispecies probiotics (LP-P). After the feeding trial, the fecal samples of the pigs were analyzed. The fecal scores were improved by the probiotic supplementation, especially in LP-P group. We also observed a probiotic-mediated alteration in the gut microbiome of pigs. In addition, LP-P group showed higher species richness and diversity compared with other groups. The addition of multispecies probiotics in low CP diet also enhanced gut microbiota metabolites production, such as short-chain fatty acids (SCFAs) and polyamines. Correlation analysis revealed that Oscillospiraceae UCG-002, Eubacterium coprostanoligenes, Lachnospiraceae NK4A136 group, and Muribaculaceae were positively associated with SCFAs; and Prevotella, Eubacterium ruminantium, Catenibacterium, Alloprevotella, Prevotellaceae NK3B31 group, Roseburia, Butyrivibrio, and Dialister were positively correlated with polyamines. Supplementation with multispecies probiotics modulated the function of the gut microbiome by upregulating the pathways for protein digestion and utilization, potentially contributing to enriched metabolite production in the gut. The results of this study demonstrate that supplementation with multispecies probiotics may complement the beneficial effects of low CP levels in pig feed. These findings may help formulate sustainable feeding strategies for swine production.

Characterization of Novel Amylase-Sensitive, Anti-Listerial Class IId Bacteriocin, Agilicin C7 Produced by Ligilactobacillus agilis C7.

Among various biological agents, bacteriocins are important candidates to control Listeria monocytogenes which is a foodborne pathogen. In this study, a novel bacteriocin, named agilicin C7, was isolated from Ligilactobacillus agilis C7 showing inhibitory activity against L. monocytogenes. Agilicin C7 biosynthesis gene was characterized by bioinformatics analyses and heterologously expressed in Escherichia coli for further study. The anti-listeria activity of recombinant agilicin C7 (r-agilicin C7) was lost by proteases and α-amylase, suggesting that agilicin C7 is a glycoprotein. r-Agilicin C7 has wide pH and thermal stability and is also stable in various organic solvents. It destroyed L. monocytogenes by damaging the integrity of the cell envelope. These properties of r-agilicin C7 indicate that agilicin C7 is a novel amylase-sensitive anti-listerial Class IId bacteriocin. Physicochemical stability and inhibitory activity against L. monocytogenes of r-agilicin C7 suggest that it can be applied to control L. monocytogenes in the food industry, including dairy and meat products.

Mucosal immunization with lactiplantibacillus plantarum-displaying recombinant SARS-CoV-2 epitopes on the surface induces humoral and mucosal immune responses in mice.

BACKGROUND: The use of probiotic lactic acid bacteria as a mucosal vaccine vector is considered a promising alternative compared to the use of other microorganisms because of its "Generally Regarded as Safe" status, its potential adjuvant properties, and its tolerogenicity to the host. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), is highly transmissible and pathogenic. This study aimed to determine the potential of Lactiplantibacillus plantarum expressing SARS-CoV-2 epitopes as a mucosal vaccine against SARS-CoV-2. RESULTS: In this study, the possible antigenic determinants of the spike (S1-1, S1-2, S1-3, and S1-4), membrane (ME1 and ME2), and envelope (E) proteins of SARS-CoV-2 were predicted, and recombinant L. plantarum strains surface-displaying these epitopes were constructed. Subsequently, the immune responses induced by these recombinant strains were compared in vitro and in vivo. Most surface-displayed epitopes induced pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α and interleukin (IL)-6] and anti-inflammatory cytokines (IL-10) in lipopolysaccharide-induced RAW 264.7, with the highest anti-inflammatory to pro-inflammatory cytokine ratio in the S1-1 and S1-2 groups, followed by that in the S1-3 group. When orally administered of recombinant L. plantarum expressing SARS-CoV-2 epitopes in mice, all epitopes most increased the expression of IL-4, along with induced levels of TNF-α, interferon-gamma, and IL-10, specifically in spike protein groups. Thus, the surface expression of epitopes from the spike S1 protein in L. plantarum showed potential immunoregulatory effects, suggesting its ability to potentially circumvent hyperinflammatory states relevant to monocyte/macrophage cell activation. At 35 days post immunization (dpi), serum IgG levels showed a marked increase in the S1-1, S1-2, and S1-3 groups. Fecal IgA levels increased significantly from 21 dpi in all the antigen groups, but the boosting effect after 35 dpi was explicitly observed in the S1-1, S1-2, and S1-3 groups. Thus, the oral administration of SARS-CoV-2 antigens into mice induced significant humoral and mucosal immune responses. CONCLUSION: This study suggests that L. plantarum is a potential vector that can effectively deliver SARS-CoV-2 epitopes to intestinal mucosal sites and could serve as a novel approach for SARS-CoV-2 mucosal vaccine development.

Bioinformatics and its role in the study of the evolution and probiotic potential of lactic acid bacteria.

Due to their numerous well-established applications in the food industry, there have been many studies regarding the adaptation and evolution of lactic acid bacteria (LAB) in a wide variety of hosts and environments. Progress in sequencing technology and continual decreases in its costs have led to the availability of LAB genome sequence data. Bioinformatics has been central to the extraction of valuable information from these raw genome sequence data. This paper presents the roles of bioinformatics tools and databases in understanding the adaptation and evolution of LAB, as well as the bioinformatics methods used in the initial screening of LAB for probiotic potential. Moreover, the advantages, challenges, and limitations of employing bioinformatics for these purposes are discussed.

Predictive values of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and other prognostic factors in pediatric vestibular neuritis patients.

OBJECTIVE: To analyze the relationship between severity and hemodynamic factor in pediatric vestibular neuritis patients and find out their effectiveness as a prognostic factor. STUDY DESIGN: Retrospective review. SETTING: Tertiary medical center. PATIENTS: Pediatric patients diagnosed between January 2010 and December 2019 at a tertiary medical institution who were hospitalized with dizziness. MAIN OUTCOME MEASURE: A study of the duration of spontaneous nystagmus loss and the severity of the disease using questionnaires (PVSQ, DHI) in pediatric patients admitted to Vestibular neuritis (VN) and correlation with NLR, PLR, MPV and RDW conducted at the time of admission. RESULT: This study was conducted on 22 children with VN and 30 children in control group. For the evaluate of the severity of the symptoms of VN patients, two group were classified based on the period from the onset of the symptom to the loss of the spontaneous nystagmus.: mild VN and severe VN, and the PVSQ and DHI questionnaires were used to assess the degree of improvement. PVSQ and DHI score generally improved during spontaneous nystagmus loss, and comparing the severity of the symptoms with the values of NLR, PLR, MPV and RDW showed a positive correlation between the severity of the symptoms and the value of the NLR and PLR value. CONCLUSION: NLR, PLR value is thought to be an efficient indicator of pediatric VN patents and could be a clue to the current unknown cause of childhood VN disease.

Whole genome sequence analysis of Ligilactobacillus agilis C7 isolated from pig feces revealed three bacteriocin gene clusters.

We here report the whole genome sequence of Ligilactobacillus agilis C7 with anti-listerial activity, which was isolated from pig feces. The genome size of L. agilis C7 (~ 3.0 Mb) is relatively larger compared with other L. agilis strains. L. agilis C7 carries three bacteriocin gene clusters encoding garvicin Q, salivaricin A, and Blp family class II bacteriocin. Garvicin Q and salivaricin A are reported to be active against Listeria monocytogenes and Micrococcus luteus, respectively, as well as against other Gram-positive bacteria. Meanwhile, the bacteriocin encoded in the blp cassette was shown to be active against pneumococci, mediating intraspecies competition. This report highlights the potential of L. agilis C7 for the production of bacteriocins inhibiting pathogenic bacteria.

Multispecies probiotics alter fecal short-chain fatty acids and lactate levels in weaned pigs by modulating gut microbiota.

Short-chain fatty acids (SCFAs) are metabolic products produced during the microbial fermentation of non-digestible fibers and play an important role in metabolic homeostasis and overall gut health. In this study, we investigated the effects of supplementation with multispecies probiotics (MSPs) containing Bacillus amyloliquefaciens, Limosilactobacillus reuteri, and Levilactobacillus brevis on the gut microbiota, and fecal SCFAs and lactate levels of weaned pigs. A total of 38 pigs weaned at 4 weeks of age were fed either a basal diet or a diet supplemented with MSPs for 6 weeks. MSP administration significantly increased the fecal concentrations of lactate (2.3-fold; p < 0.01), acetate (1.8-fold; p < 0.05), and formate (1.4-fold; p < 0.05). Moreover, MSP supplementation altered the gut microbiota of the pigs by significantly increasing the population of potentially beneficial bacteria such as Olsenella, Catonella, Catenibacterium, Acidaminococcus, and Ruminococcaceae. MSP supplementation also decreased the abundance of pathogenic bacteria such as Escherichia and Chlamydia. The modulation of the gut microbiota was observed to be strongly correlated with the changes in fecal SCFAs and lactate levels. Furthermore, we found changes in the functional pathways present within the gut, which supports our findings that MSP modulates the gut microbiota and SCFAs levels in pigs. The results support the potential use of MSPs to improve the gut health of animals by modulating SCFAs production.

Cudrania tricuspidata Combined with Lacticaseibacillus rhamnosus Modulate Gut Microbiota and Alleviate Obesity-Associated Metabolic Parameters in Obese Mice.

The aim of the presented study was to investigate the synbiotic effects of L. rhamnosus 4B15 and C. tricuspidata extract administration on the gut microbiota and obesity-associated metabolic parameters in diet-induced obese mice. Thirty-one 6-week-old male C57BL/N6 mice were divided into five diet groups: normal diet (ND, n = 7) group; high-fat diet (HFD, n = 6) group; probiotic (PRO, n = 5) group; prebiotic (PRE, n = 7) group; and synbiotic (SYN, n = 6) group. After 10 weeks, the percent of fat mass, serum triglyceride, and ALT levels were significantly reduced in SYN-fed obese mice, compared with other treatments. SYN treatment also modulated the abundance of Desulfovibrio, Dorea, Adlercreutzia, Allobaculum, Coprococcus, unclassified Clostridiaceae, Lactobacillus, Helicobacter, Flexispira, Odoribacter, Ruminococcus, unclassified Erysipelotrichaceae, and unclassified Desulfovibrionaceae. These taxa showed a strong correlation with obesity-associated indices. Lastly, the SYN-supplemented diet upregulated metabolic pathways known to improve metabolic health. Further investigations are needed to understand the mechanisms driving the synbiotic effect of C. tricuspidata and L. rhamnosus 4B15.

Complete genome sequence of Lactobacillus plantarum SK156, a candidate vehicle for mucosal vaccine delivery.

Lactobacillus plantarum SK156 was isolated from traditional Korean food. The genome of SK156 strain consists of a circular chromosome (3,231,383 bp) with guanine (G) + cytosine (C) content of 44.56%. Among the predicted 2,991 protein-coding genes, the genome included genes encoding for α-amylase, which hydrolyzes α-bonds of polysaccharides. Genomic sequencing of L. plantarum SK156 will give information on the mechanism involved in the enzymatic degradation of polysaccharides and its application for improving feed efficiency.

Construction of a Bile-responsive Expression System in Lactobacillus plantarum.

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the ß-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, pULP3-PLDH, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.

Isolation and Characterization of an Anti-listerial Bacteriocin from Leuconostoc lactis SD501.

Although bacteriocins with anti-listerial activity have been isolated from a wide variety of lactic acid bacteria, little is known about those from Leuconostoc lactis, a heterofermentative bacterium that produces diacetyl and exopolysaccharides in dairy foods. In this study, an anti-listerial bacteriocin was isolated from Leuc. lactis SD501 and characterized. It was particularly potent against Listeria monocytogenes and also inhibited Enterococcus faecalis. Anti-listerial activity reached a maximum during the early stationary phase and then decreased gradually. The anti-listerial substance was sensitive to proteinase K and ɑ-chymotrypsin, confirming its proteinaceous nature. Its activity remained stable at pH values ranging from 1 to 10. In addition, it was strongly resistant to high temperatures, retaining its activity even after incubation for 15 min at 121℃. The apparent molecular mass of the partially purified anti-listerial bacteriocin was approximately 7 kDa. The characteristics of the SD501 bacteriocin, including its small molecular size (<10 kDa), strong anti-listerial activity, wide pH stability and good thermostability, indicate its classification as a Class IIa bacteriocin.

Enhanced device performance of germanium nanowire junctionless (GeNW-JL) MOSFETs by germanide contact formation with Ar plasma treatment.

In this study, germanium nanowire junctionless (GeNW-JL) metal-oxide-semiconductor-field-effect-transistors (MOSFETs) exhibited enhanced electrical performance with low source/drain (S/D) contact resistance under the influence of Ar plasma treatment on the contact regions. We found that the transformation of the surface oxide states by Ar plasma treatment affected the S/D contact resistance. With Ar plasma treatment, the germanium dioxide on the GeNW surface was effectively removed and increased oxygen vacancies were formed in the suboxide on the GeNW, whose germanium-enrichment surface was obtained to form a germanide contact at low temperature. After a rapid thermal annealing process, Ni-germanide contacts were formed on the Ar-plasma-treated GeNW surface. Ni-germanide contact resistance was improved by more than an order of magnitude compared to that of the other devices without Ni-germanide contact. Moreover, the peak field effect mobility value of the GeNW-JL MOSFETs was dramatically improved from 15 cm(2)/(V s) to 550 cm(2)/(V s), and the Ion/off ratio was enhanced from 1 × 10 to 3 × 10(3) due to Ar plasma treatment. The Ar plasma treatment process is essential for forming uniform Ni-germanide-contacts with reduced time and low temperature. It is also crucial for increasing mass productivity and lowering the thermal budget without sacrificing the performance of GeNW-JL MOSFETs.

A synergistic interaction between transcription factors nuclear factor-κB and signal transducers and activators of transcription 3 promotes gastric cancer cell migration and invasion.

BACKGROUND: The transcription factor nuclear factor-κB (NF-κB) has been implicated in gastric cancer metastasis, but the underlying molecular mechanisms remain unclear. We investigated the role of the interaction between NF-κB and signal transducers and activators of transcription 3 (STAT3) in controlling metastatic potential of gastric cancer cells. METHODS: Immunohistochemistry for NF-κB p65 (RelA), phospho-Tyr705-STAT3 (pSTAT3), or matrix metalloproteinase 9 (MMP9) was performed on tissue array slides containing 255 gastric carcinoma specimens. NF-κB inhibition in SNU-638 and MKN1 gastric cancer cell lines were performed by transduction with a retroviral vector containing NF-κB repressor mutant of IκBα, and STAT3 was silenced by RNA interference. We also did luciferase reporter assay, double immunofluorescence staining and immunoblotting. Cell migration and invasion were determined by wound-healing assay and invasion assay, respectively. RESULTS: NF-κB and STAT3 were constitutively activated and were positively correlated (P=0.038) in gastric cancer tissue specimens. In cell culture experiments, NF-κB inhibition reduced STAT3 expression and activation, whereas STAT3 silencing did not affect NF-κB activation. Moreover, both NF-κB inhibition and STAT3 silencing decreased gastric cancer cell migration and invasion in a synergistic manner. In addition, both NF-κB activation and STAT3 activation were positively correlated with MMP9 in gastric cancer tissues (P=0.001 and P=0.022, respectively), decreased E-cadherin expression and increased Snail and MMP9 expressions in cultured cells. CONCLUSION: NF-κB and STAT3 are positively associated and synergistically contribute to the metastatic potential of gastric cancer cells. Thus, dual use of NF-κB and STAT3 inhibitors may enhance the efficacy of the anti-metastatic treatment of gastric cancer.