Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

Identification of Biomarkers for Systemic Distribution of Nanovesicles From Lactobacillus johnsonii N6.2.

The ability of bacterial extracellular vesicles (EV) to transport biological molecules has increased the research to determine their potential as therapeutic agents. In this study, Lactobacillus johnsonii N6.2-derived nanovesicles (NV) were characterized to identify components that may serve as biomarkers in host-microbe interactions. Comparative proteomic and lipidomic analyses of L. johnsonii N6.2 NV and cell membrane (CM) were performed. The lipidomic profiles indicated that both fractions contained similar lipids, however, significant differences were observed in several classes. LC-MS/MS proteomic analysis indicated that NV contained several unique and differentially expressed proteins when compared to the CM. Analysis of Gene Ontology (GO) terms, based on cellular component, showed significant enrichment of proteins in the cytoplasm/intracellular space category for the NV fraction. Based on these results, the proteins T285_RS00825 (named Sdp), Eno3 and LexA were selected for studies of localization and as potential biomarkers for host-microbe interactions. Immunogold staining, followed by scanning and transmission electron microscopy (SEM and TEM, respectively), revealed that Sdp was preferentially localized along the cell wall/membrane, and on NV-like structures surrounding the bacteria. These results were confirmed using immunofluorescence staining in Caco-2 cells incubated with NV. Consequently, we evaluated the potential for NV surface-exposed proteins to generate an immune response in the host. Plasma from individuals administered L. johnsonii N6.2 showed that IgA and IgG antibodies were generated against NV and Sdp domains in vivo. Altogether, these results show that L. johnsonii N6.2 NV have the potential to mediate host interactions through immune modulation.

Frozen Mother's Own Milk Can Be Used Effectively to Personalize Donor Human Milk.

Feeding preterm infants mother's own milk (MOM) lowers rates of sepsis, decreases necrotizing enterocolitis, and shortens hospital stay. In the absence of freshly expressed MOM, frozen MOM (FMOM) is provided. When MOM is unavailable, preterm infants are often fed pasteurized donor human milk (DHM), rendering it devoid of beneficial bacteria. We have previously reported that when MOM is inoculated into DHM to restore the live microbiota [restored milk (RM)], a similar microbial diversity to MOM can be achieved. Yet, it is unknown if a similar diversity to MOM can be obtained when FMOM is inoculated into DHM. The goal of this study was to determine whether a similar microbial composition to MOM could be obtained when FMOM is used to personalize DHM. To this end, a fresh sample of MOM was obtained and divided into fresh and frozen fractions. MOM and FMOM were inoculated into DHM at different dilutions: MOM/FMOM 10% (RM/FRM10) and MOM/FMOM 30% (RM/FRM30) and incubated at 37°C. At different timepoints, culture-dependent and culture-independent techniques were performed. Similar microbiota expansion and alpha diversity were observed in MOM, RM10, and RM30 whether fresh or frozen milk was used as the inoculum. To evaluate if microbial expansion would result in an abnormal activation on the innate immune system, Caco-2 epithelial cells were exposed to RM/FRM to compare interleukin 8 levels with Caco-2 cells exposed to MOM or DHM. It was found that RM samples did not elicit a significant increase in IL-8 levels when compared to MOM or FMOM. These results suggest that FMOM can be used to inoculate DHM if fresh MOM is unavailable or limited in supply, allowing both fresh MOM and FMOM to be viable options in a microbial restoration strategy.

Reconsidering the role of sex hormones in psychopathy development: Estrogen and psychopathy among male justice-involved youth.

Prominent theory suggests that factor one psychopathic traits may develop from increased input from hormones in the hypothalamic pituitary gonadal axis (HPG; i.e., testosterone) and decreased input from the hypothalamic pituitary adrenal axis (HPA; i.e., cortisol). Although there are extensive findings connecting low cortisol to psychopathy, less support has emerged for high levels of testosterone. This study examined whether incorporating the HPG hormone, estradiol, into this model would reveal relationships in line with theory: high levels of estradiol and testosterone in combination with low levels of cortisol would inform psychopathic traits. Baseline and reactive hormone levels were measured and compared to Psychopathy Checklist-Youth Version (PCL-YV) interviews among 66 male justice-involved youth (M age = 15.73) in a Southeastern juvenile detention center. The primary findings of this study were relationships between interacting HPA and HPG axis hormones with facet one and facet two psychopathic traits. Specifically, psychopathy total scores, interpersonal traits, and affective traits related to estradiol and testosterone reactivity, in that psychopathy scores were more likely with decreases in hormone reactivity (i.e., change in hormone level) following a stressor. Moreover, affective traits related to reactivity in all three hormones. These findings support inclusion of estradiol in neurobiological models of psychopathy and consideration of the individual components of psychopathy. This study adds to the growing body of research supporting interactions between variations in functioning of the HPA and HPG axes in relation to psychopathy.

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus.

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.

The Ferredoxin-Like Protein FerR Regulates PrbP Activity in Liberibacter asiaticus.

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase ß-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbPLas), mRNA levels of ferR of L. asiaticus (ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas The induction of ferRLas expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticusIMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas We show that the presence of FerR stabilizes and augments the activity of PrbPLas In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.

Personalization of the Microbiota of Donor Human Milk with Mother's Own Milk.

The American Academy of Pediatrics recommends that extremely preterm infants receive mother's own milk (MOM) when available or pasteurized donor breast milk (DBM) when MOM is unavailable. The goal of this study was to determine whether DBM could be inoculated with MOM from mothers of preterm infants to restore the live microbiota (RM). Culture dependent and culture independent methods were used to analyze the fluctuations in the overall population and microbiome, respectively, of DBM, MOM, and RM samples over time. Using MOM at time = 0 (T0) as the target for the restoration process, this level was reached in the 10% (RM-10) and 30% (RM-30) mixtures after 4 h of incubation at 37°C, whereas, the larger dilutions of 1% (RM-1) and 5% (RM-5) after 8 h. The diversity indexes were similar between MOM and DBM samples, however, different genera were prevalent in each group. Interestingly, 40% of the bacterial families were able to expand in DBM after 4 h of incubation indicating that a large percentage of the bacterial load present in MOM can grow when transferred to DBM, however, no core microbiome was identified. In summary, the microbiome analyses indicated that each mother has a unique microbiota and that live microbial reestablishment of DBM may provide these microbes to individual mothers' infants. The agreement between the results obtained from the viable bacterial counts and the microbiome analyses indicate that DBM incubated with 10-30% v/v of the MOM for 4 h is a reasonable restoration strategy.

Lactobacillus johnsonii N6.2 Modulates the Host Immune Responses: A Double-Blind, Randomized Trial in Healthy Adults.

Lactobacillus johnsonii N6.2 mitigates the onset of type 1 diabetes (T1D) in biobreeding diabetes-prone rats, in part, through changes in kynurenine:tryptophan (K:T) ratios. The goal of this pilot study was to determine the safety, tolerance, and general immunological response of L. johnsonii N6.2 in healthy subjects. A double-blind, randomized clinical trial in 42 healthy individuals with no known risk factors for T1D was undertaken to evaluate subject responses to the consumption of L. johnsonii N6.2. Participants received 1 capsule/day containing 108 colony-forming units of L. johnsonii N6.2 or placebo for 8 weeks. Comprehensive metabolic panel (CMP), leukocyte subpopulations by complete blood count (CBC) and flow cytometry, serum cytokines, and relevant metabolites in the indoleamine-2,3-dioxygenase pathway were assessed. L. johnsonii N6.2 survival and intestinal microbiota was analyzed. Daily and weekly questionnaires were assessed for potential effects of probiotic treatment on general wellness. The administration of L. johnsonii N6.2 did not modify the CMP or CBC of participants suggesting general safety. In fact, L. johnsonii N6.2 administration significantly decreased the occurrence of abdominal pain, indigestion, and cephalic syndromes. As predicted, increased serum tryptophan levels increased resulting in a decreased K:T ratio was observed in the L. johnsonii N6.2 group. Interestingly, immunophenotyping assays revealed that monocytes and natural killer cell numbers were increased significantly after washout (12 weeks). Moreover, an increase of circulating effector Th1 cells (CD45RO+CD183+CD196-) and cytotoxic CD8+ T cells subset was observed in the L. johnsonii N6.2 group. Consumption of L. johnsonii N6.2 is well tolerated in adult control subjects, demonstrates systemic impacts on innate and adaptive immune populations, and results in a decreased K:T ratio. These data provide support for the safety and feasibility of using L. johnsonii N6.2 in prevention trials in subjects at risk for T1D. TRIAL REGISTRATION: This trial was registered at http://clinicaltrials.gov as NCT02349360.