Gut Microbiota and Derived Short-Chain Fatty Acids Are Linked to Evolution of Heart Failure Patients.

There is a lack of direct evidence regarding gut microbiota dysbiosis and changes in short-chain fatty acids (SCFAs) in heart failure (HF) patients. We sought to assess any association between gut microbiota composition, SCFA production, clinical parameters, and the inflammatory profile in a cohort of newly diagnosed HF patients. In this longitudinal prospective study, we enrolled eighteen newly diagnosed HF patients. At admission and after 12 months, blood samples were collected for the assessment of proinflammatory cytokines, monocyte populations, and endothelial dysfunction, and stool samples were collected for analysis of gut microbiota composition and quantification of SCFAs. Twelve months after the initial HF episode, patients demonstrated improved clinical parameters and reduced inflammatory state and endothelial dysfunction. This favorable evolution was associated with a reversal of microbiota dysbiosis, consisting of the increment of health-related bacteria, such as genus Bifidobacterium, and levels of SCFAs, mainly butyrate. Furthermore, there was a decrease in the abundance of pathogenic bacteria. In vitro, fecal samples collected after 12 months of follow-up exhibited lower inflammation than samples collected at admission. In conclusion, the favorable progression of HF patients after the initial episode was linked to the reversal of gut microbiota dysbiosis and increased SCFA production, particularly butyrate. Whether restoring butyrate levels or promoting the growth of butyrate-producing bacteria could serve as a complementary treatment for these patients deserves further studies.

Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1ß (IL1-ß), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

Ultrasonically-Assisted and Conventional Extraction from Erodium Glaucophyllum Roots Using Ethanol:Water Mixtures: Phenolic Characterization, Antioxidant, and Anti-Inflammatory Activities.

The paper presents experimental results concerning the ultrasonically-assisted extraction of bioactive compounds from Erodium glaucophyllum roots. A comparison with conventional methodology is presented, and thereby the phytochemical composition and the antioxidant and anti-inflammatory activities of extracts are evaluated. The phenolic profile of Erodium extracts was analyzed by TOF-LC-MS-MS. The identification of phenolic compounds revealed that the major component was (+)-gallocatechin in the aqueous extracts obtained for the different extraction methodologies. The highest quantity of phenolic compounds and antioxidant capacity was found in the hydroethanolic extract obtained by conventional extraction (29.22-25.50 mg GAE/g DM; 21.174 mM Trolox equivalent). The highest content of carotenoids, varying from 0.035 to 0.114 mg/g dry matter, was reached by ultrasonic-assisted extraction. Furthermore, Erodium extracts showed a potent inhibition of the inflammatory reaction by means of the inhibition of tumor necrosis factor-alpha (TNF-α). The extracts obtained when ultrasound extraction was combined with ethanol:water (50:50, v/v) presented the greatest inhibition (92%).

Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei.

A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups.

The impact of liquid-pressurized extracts of Spirulina, Chlorella and Phaedactylum tricornutum on in vitro antioxidant, antiinflammatory and bacterial growth effects and gut microbiota modulation.

The impact of Spirulina, Chlorella and Phaeodactylum tricornutum (P. tricornutum) microalgal extracts obtained by pressurized liquid extraction (PLE) on antioxidant and anti-inflammatory activities, microbial growth and in vitro gut microbiota composition was evaluated. PLE, compared to conventional extraction, led to a significant (p < 0.05) increase in proteins, carbohydrates, polyphenols, and antioxidant capacities of the three microalgal extracts. Moreover, Spirulina and P. tricornutum extracts significantly (p < 0.05) reduced the in vitro activation of the inflammatory NF-κB pathway. The microalgal extracts had also an inhibitory effect on the pathogenic bacteria while potential beneficial Lactobacillus and Bifidobacterium strains increased growth. The effects of microalgal extracts on specific bacterial groups were analyzed by quantitative PCR technology, and bacterial gene copy numbers were affected by in vitro digestion process and colonic fermentation time. GC-MS results showed that microalgal biomolecules' digestion promoted the release of short-chain fatty acids (SCFAs) during in vitro colonic microbiota fermentation, particularly acetic, butanoic and propanoic, indicating that the biomolecules in microalgae extracts have potential health benefits for human gut.

Composition, structural properties and immunomodulatory activity of several aqueous Pleurotus ß-glucan-rich extracts.

In this work, aqueous extracts from six different Pleurotus species were obtained and their yield, gross composition, ß-glucan content, monosaccharide profile, thermal stability, molecular weight distribution, and FT-IR were analyzed before and after purification through ethanol precipitation of the carbohydrate-rich fractions. The bioactivity (anti-inflammatory and immunomodulatory activity) of the various fractions obtained was also analyzed in three different cell cultures and compared with a lentinan control. The trend observed after purification of the aqueous fractions was an increase in the concentration of polysaccharides (especially ß-glucans), a decrease in ash, glucosamine and protein content and the elimination of low molecular weight (Mw) compounds, thus leaving in the purified samples high Mw populations with increased thermal stability. Interestingly, all these purified fractions displayed immunomodulatory capacity when tested in THP-1 macrophages and most of them also showed significant activity in HEK-hTLR4 cells, highlighting the bioactivity observed for Pleurotus ostreatus (both the extracts obtained from the whole mushroom and from the stipes). This specific species was richer in heteropolysaccharides, having moderate ß-glucan content and being enriched upon purification in a high Mw fraction with good thermal stability.

Distinct breast milk microbiota, cytokine, and adipokine profiles are associated with infant growth at 12 months: an in vitro host-microbe interaction mechanistic approach.

Breast milk (BM) is important for adequate infant development, and it contains bioactive compounds, such as bacteria, cytokines and some adipokines which play a role in infant microbial, metabolic, and immunological maturation. However, little is known about its impact on growth and development in early life. The objective of this study was to evaluate the impact of milk microbiota, cytokine, and adipokine profiles on the risk of overweight at 12 months of life to find the possible mechanisms of host-microbe interactions. In this study, BM samples from 100 healthy women collected during 15 d after birth were included. BM microbiota was analysed by 16S rRNA gene sequencing, and cytokine and adipokine levels were measured by the Luminex approach. In addition, infant weight and length were recorded during the first 12 months and z-scores were obtained according to the WHO databases. Infants were classified as risk of overweight (ROW) and no-risk of overweight (NOROW) based on their body mass index z-score (BMIZ) and infant weight-for-length z-score (WLZ) at 12 months. In order to study host-microbe interactions, epithelial intestinal and mammary cell lines were exposed to milk microbes to assess the host response by interleukin (IL)-6 production as a potential inflammatory marker. BM was dominated by Staphylococcus and Streptococcus genera, and the most abundant cytokines were IL-6 and IL-18. Leptin levels were positively correlated with the pregestational body mass index (BMI). Higher relative abundance of the Streptococcus genus was associated with higher IL-10 and higher relative abundance of the Bifidobacterium genus was associated with lower IL-6 concentrations in milk. Infant WLZ at 12 months could be partially predicted by Streptococcus genus proportions and IL-10 and IL-18 levels in BM. BM microbiota significantly induced cytokine responses in mammary epithelial cells. Higher levels of IL-6 production were observed in mammary cells exposed to BM microbiota from mothers with ROW offspring compared to mothers with NOROW offspring. In conclusion, BM microbiota is related to the cytokine profile. IL-10 and IL-18 levels and the abundance of the Streptococcus genus could affect early infant development. Further research is needed to clarify the specific impact of BM microbiota and cytokines on infant growth and the risk of overweight.

Assessment of SARS-CoV-2 neutralizing antibody titers in breastmilk from convalescent and vaccinated mothers.

Breastmilk contains antibodies that could protect breastfed infants from infections. In this work, we examined if antibodies in breastmilk could neutralize SARS-CoV-2 in 84 breastmilk samples from women that were either vaccinated (Comirnaty, mRNA-1273, or ChAdOx1), infected with SARS-CoV-2, or both infected and vaccinated. The neutralization capacity of these sera was tested using pseudotyped vesicular stomatitis virus carrying either the Wuhan-Hu-1, Delta, or BA.1 Omicron spike proteins. We found that natural infection resulted in higher neutralizing titers and that neutralization correlated positively with levels of immunoglobulin A in breastmilk. In addition, significant differences in the capacity to produce neutralizing antibodies were observed between both mRNA-based vaccines and the adenovirus-vectored ChAdOx1 COVID-19 vaccine. Overall, our results indicate that breastmilk from naturally infected women or those vaccinated with mRNA-based vaccines contains SARS-CoV-2 neutralizing antibodies that could potentially provide protection to breastfed infants from infection.

M6P/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues.

BACKGROUND & AIMS: The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), a multifunctional protein, plays a central role in intracellular targeting of lysosomal enzymes and control of insulin-like growth factor II (IGF-II) bioactivity. Importantly, the gene encoding this receptor is frequently inactivated in a wide range of malignant tumors including hepatocellular carcinomas. Thus, M6P/IGF2R is considered a putative liver tumor suppressor. The aim of this study was to establish the impact of the receptor on the invasive properties of liver cells. METHODS: Reconstitution experiments were performed by expression of wild type and mutant M6P/IGF2R in receptor-deficient FRL14 fetal rat liver cells. RNA interference was used to induce M6P/IGF2R downregulation in receptor-positive MIM-1-4 mouse hepatocytes. RESULTS: We show that the M6P/IGF2R status exerts a strong impact on the invasiveness of tumorigenic rodent liver cells. M6P/IGF2R-deficient fetal rat liver cells hypersecrete lysosomal cathepsins and penetrate extracellular matrix barriers in a cathepsin-dependent manner. Forced expression of M6P/IGF2R restores intracellular transport of cathepsins to lysosomes and concomitantly reduces the tumorigenicity and invasive potential of these cells. Conversely, M6P/IGF2R knock-down in receptor-positive mouse hepatocytes causes increased cathepsin secretion as well as enhanced cell motility and invasiveness. We also demonstrate that functional M6P-binding sites are important for the anti-invasive properties of M6P/IGF2R, whereas the capacity to bind IGF-II is dispensable for the anti-invasive activity of the receptor in liver cells. CONCLUSIONS: M6P/IGF2R restricts liver cell invasion by preventing the pericellular action of M6P-modified proteins.

Defining microbiota for developing new probiotics.

The human body harbors complex communities of microbes that play a prominent role in human health. Detailed characterization of the microbiota in the target population forms the basis of probiotic use. Probiotics are defined as live bacterial preparations with clinically documented health effects in humans, and independent of their genus and species, probiotic strains are unique and their beneficial properties on human health have to be assessed in a case-by-case manner. Understanding the mechanisms by which probiotics influence microbiota would facilitate the use of probiotics for both dietary management and reduction in risk of specific diseases. The development of high throughput sequencing methods has allowed metagenomic approaches to study the human microbiome. These efforts are starting to generate an inventory of bacterial taxons and functional features bound to particular health or disease status that allow inferring aspects of the microbiome's function. In the future, this information will allow the rational design of dietary interventions aimed to improve consumer's health via modulation of the microbiota.

Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells.

Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains-Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG-in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.

Human milk microbiota: what did we learn in the last 20 years?

Human milk (HM) is the gold standard for infant nutrition during the first months of life. Beyond its nutritional components, its complex bioactive composition includes microorganisms, their metabolites, and oligosaccharides, which also contribute to gut colonization and immune system maturation. There is growing evidence of the beneficial effects of bacteria present in HM. However, current research presents limited data on the presence and functions of other organisms. The potential biological impacts on maternal and infant health outcomes, the factors contributing to milk microbes' variations, and the potential functions in the infant's gut remain unclear. This review provides a global overview of milk microbiota, what the actual knowledge is, and what the gaps and challenges are for the next years.

Persistence of Anti SARS-CoV-2 Antibodies in Breast Milk from Infected and Vaccinated Women after In Vitro-Simulated Gastrointestinal Digestion.

Breastfeeding is key for infant development and growth. Breast milk contains different bioactive compounds including antibodies. Recent studies have demonstrated the presence of breast milk SARS-CoV-2 antibodies after maternal infection and vaccination. However, the potential impact on the infant has not been explored yet. As a first step, we aimed at assessing the potential persistence of SARS-CoV-2 IgA and IgG antibodies from infected and vaccinated women in the gastrointestinal tract of the infants by means of an in vitro-simulated gastrointestinal digestion approach. Breast milk samples from 10 lactating women receiving mRNA vaccination against SARS-CoV-2 (n = 5 with BNT162b2 mRNA and n = 5 with mRNA-1273) and also, COVID-19 infected (n = 5) were included. A control group with women with no exposure to the virus (n = 10 pre-pandemic) were also studied. The presence of IgA and IgG SARS-CoV-2 antibody levels was determined by ELISA after the gastric and intestinal stages. The impact of digested antibodies on infant gut microbiota was tested by simulating colonic fermentation with two different fecal inoculums: infants from vaccinated and non-vaccinated mothers. Specific gut microbial groups were tested by targeted qPCR. In vitro infant gastrointestinal digestion significantly decreased the levels of both anti-SARS-CoV-2 IgA and IgG. However, both remained resistant in all the study groups except in that evaluating breast milk samples from infected women, in which IgG was degraded below the cut-off values in the intestinal phase. No effect of the antibodies on microbiota were identified after digestion. In conclusion, antibody levels against SARS-CoV-2 are reduced after in vitro-simulated gastrointestinal tract but remain present, so a positive biological effect could be expected from this infant immunization pathway.

Metallomic and Untargeted Metabolomic Signatures of Human Milk from SARS-CoV-2 Positive Mothers.

SCOPE: Lack of information about the impact of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the elemental and metabolomic profile of human milk (HM). METHODS AND RESULTS: An observational study on HM from mothers with COVID-19 is conducted including a prepandemic control group. Maternal-infant clinical records and symptomatology are recorded. The absolute quantification of elements and untargeted relative metabolomic profiles are determined by inductively coupled plasma mass spectrometry and gas chromatography coupled to mass spectrometry, respectively. Associations of HM SARS-CoV-2 antibodies with elemental and metabolomic profiles are studied. COVID-19 has a significant impact on HM composition. COVID-19 reduces the concentrations of Fe, Cu, Se, Ni, V, and Aluminium (Al) and increases Zn compared to prepandemic control samples. A total of 18 individual metabolites including amino acids, peptides, fatty acids and conjugates, purines and derivatives, alcohols, and polyols are significantly different in HM from SARS-CoV-2 positive mothers. Aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine, and linoleic acid pathways are significantly altered. Differences are obtained depending on COVID-19 symptomatic and asymptomatic status. CONCLUSIONS: This study provides unique insights about the impact of maternal SARS-CoV-2 infection on the elemental and metabolomic profiles of HM that warrants further research due the potential implications for infant health.

Anti-SARS-CoV-2 IgA and IgG in human milk after vaccination is dependent on vaccine type and previous SARS-CoV-2 exposure: a longitudinal study.

BACKGROUND: Breast milk is a vehicle to transfer protective antibodies from the lactating mother to the neonate. After SARS-CoV-2 infection, virus-specific IgA and IgG have been identified in breast milk, however, there are limited data on the impact of different COVID-19 vaccine types in lactating women. This study is aimed to evaluate the time course of induction of SARS-CoV-2-specific IgA and IgG in breast milk after vaccination. METHODS: In this prospective observational study in Spain, 86 lactating women from priority groups receiving the vaccination against SARS-CoV-2 were included. Breast milk samples were collected longitudinally at seven or eight-time points (depending on vaccine type). A group with confirmed SARS-CoV-2 infection (n=19) and a group of women from pre-pandemic time (n=20) were included for comparison. RESULTS: Eighty-six vaccinated lactating women [mean age, 34.6 ± 3.7 years] of whom 96% were Caucasian and 92% were healthcare workers. A total number of 582 milk samples were included, and vaccine distribution was BioNTech/Pfizer (BNT162b2, n=34), Moderna (mRNA-1273, n=20), and AstraZeneca (ChAdOx1 nCoV-19, n=32). For each vaccine, 7 and 8 longitudinal time points were collected from baseline up to 30 days after the second dose for mRNA vaccines and adenovirus-vectored vaccines, respectively. A strong reactivity was observed for IgG and IgA after vaccination mainly after the 2nd dose. The presence and persistence of specific SARS-CoV-2 antibodies in breast milk were dependent on the vaccine type, with higher IgG and IgA levels in mRNA-based vaccines when compared to AstraZeneca, and on previous virus exposure. High intra- and inter-variability were observed, being relevant for IgA antibodies. In milk from vaccinated women, anti-SARS-CoV-2 IgG was significantly higher while IgA levels were lower than in milk from COVID-19-infected women. Women with previous COVID-19 increased their IgG antibodies levels after the first dose to a similar level observed in vaccinated women after the second dose. CONCLUSIONS: COVID-19 vaccination induced anti-SARS-CoV-2 IgA and IgG in breast milk with higher levels after the 2nd dose. Levels of anti-SARS-CoV-2 IgA and IgG are dependent on the vaccine type. Further studies are warranted to demonstrate the protective antibody effect against COVID-19 in infants from vaccinated and infected mothers. TRIAL REGISTRATION: NCT04751734 (date of registration is on February 12, 2021).

Mycotoxin Interactions along the Gastrointestinal Tract: In Vitro Semi-Dynamic Digestion and Static Colonic Fermentation of a Contaminated Meal.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) naturally co-occur in several foods, but no studies have followed the fate of mycotoxins' interactions along the gastrointestinal tract using in vitro digestion models. This study used a novel semi-dynamic model that mimics gradual acidification and gastric emptying, coupled with a static colonic fermentation phase, in order to monitor mycotoxins' bioaccessibility by the oral route. AFB1 and OTA bioaccessibility patterns differed in single or co-exposed scenarios. When co-exposed (MIX meal), AFB1 bioaccessibility at the intestinal level increased by ~16%, while OTA bioaccessibility decreased by ~20%. Additionally, a significant increase was observed in both intestinal cell viability and NO production. With regard to mycotoxin-probiotic interactions, the MIX meal showed a null effect on Lactobacillus and Bifidobacterium strain growth, while isolated AFB1 reduced bacterial growth parameters. These results were confirmed at phylum and family levels using a gut microbiota approach. After colonic fermentation, the fecal supernatant did not trigger the NF-kB activation pathway, indicating reduced toxicity of mycotoxins. In conclusion, if single exposed, AFB1 will have a significant impact on intestinal viability and probiotic growth, while OTA will mostly trigger NO production; in a co-exposure situation, both intestinal viability and inflammation will be affected, but the impact on probiotic growth will be neglected.

SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain.

OBJECTIVES: To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies. DESIGN AND PATIENTS: This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included. SETTING: Seven health centres from different provinces in Spain. MAIN OUTCOME MEASURES: Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)-IgA, IgG and IgM-in breast milk samples from patients with COVID-19. RESULTS: All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation). CONCLUSIONS: Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244.

Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli.

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3'UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3'UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3'UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

Lactobacillus casei extracellular vesicles stimulate EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface.

In the complex interplay of beneficial bacteria with the host, there are few examples of bacterial metabolites and effector molecules that have been consistently identified. Protective effects on the intestinal epithelium have been ascribed to P40 and P75, two well characterized cell wall muramidases, present in the culture supernatant of strains belonging to the taxon Lactobacillus casei/paracasei/rhamnosus. This work reports that Lactobacillus casei BL23 extracellular vesicles (BL23 EVs) have a small size (17-20 nm or 24-32 nm, depending on the method used) and contain lipoteichoic acid (LTA). Interestingly, all detected P40 and most of P75 were associated to EVs and possibly located at their external surface, as shown by proteinase K digestion. Biosensor assays showed that both proteins bind LTA and vesicles, suggesting that they could bind to ligands like LTA present on BL23 EVs. Native BL23 EVs have a moderate proinflammatory effect and they were able to induce phosphorylation of the epidermal growth factor receptor (EGFR), showing an effect similar to purified P40 and P75 and leading to the conclusion that the activity described in the supernatant (postbiotic) of these bacteria would be mainly due to P40 and P75 bound to EVs.

Differences in the expression of cell envelope proteinases (CEP) in two Lactobacillus paracasei probiotic strains.

Proteinase PrtP (EC:3.4.21.96) is a cell envelope proteinase (CEP) highly expressed in the probiotic strain Lactobacillus paracasei BL312(VSL#3) that accounts for its anti-inflammatory properties. The main aim of this work is to understand differences in CEP expression between this strain and L. paracasei BL23. Hence, differences in the regulation by amino acid sources of four proteinase related genes (prtP, prsA, prtR1 and prtR2) were determined by RT-qPCR in BL312(VSL#3) and BL23 using as a reference BL368, a BL23 derepressed mutant lacking the response regulator (RR) PrcR. BL312(VSL#3) showed greater expression of prtP (2- to 3-fold) than BL23, and prtP was highly repressed by peptone in both strains. Two other putative CEP genes, prtR1 and prtR2, showed a low expression profile. Interestingly, when the prsA-prtP promoter region from both strains, and deleted mutants, were cloned in vector pT1GR, expression of the gfp and mrfp fluorescent reporters was always repressed in BL23 (high or low peptone) and derepressed in BL368, revealing an interesting mechanism of regulation affecting specifically to this promoter. In conclusion, BL312(VSL#3) has higher expression of prtP and other CEP related genes than BL23, that could respond to a natural deregulation in this strain, possibly independent from the RR PrcR.