The enduring effects of early-life stress on the microbiota-gut-brain axis are buffered by dietary supplementation with milk fat globule membrane and a prebiotic blend.

Nutritional interventions targeting the microbiota-gut-brain axis are proposed to modulate stress-induced dysfunction of physiological processes and brain development. Maternal separation (MS) in rats induces long-term alterations to behaviour, pain responses, gut microbiome and brain neurochemistry. In this study, the effects of dietary interventions (milk fat globule membrane [MFGM] and a polydextrose/galacto-oligosaccharide prebiotic blend) were evaluated. Diets were provided from postnatal day 21 to both non-separated and MS offspring. Spatial memory, visceral sensitivity and stress reactivity were assessed in adulthood. Gene transcripts associated with cognition and stress and the caecal microbiota composition were analysed. MS-induced visceral hypersensitivity was ameliorated by MFGM and to greater extent with the combination of MFGM and prebiotic blend. Furthermore, spatial learning and memory were improved by prebiotics and MFGM alone and with the combination. The prebiotic blend and the combination of the prebiotics and MFGM appeared to facilitate return to baseline with regard to HPA axis response to the restraint stress, which can be beneficial in times where coping mechanisms to stressful events are required. Interestingly, the combination of MFGM and prebiotic reduced the long-term impact of MS on a marker of myelination in the prefrontal cortex. MS affected the microbiota at family level only, while MFGM, the prebiotic blend and the combination influenced abundance at family and genus level as well as influencing beta-diversity levels. In conclusion, intervention with MFGM and prebiotic blend significantly impacted the composition of the microbiota as well as ameliorating some of the long-term effects of early-life stress.

Dietary polydextrose and galactooligosaccharide increase exploratory behavior, improve recognition memory, and alter neurochemistry in the young pig.

OBJECTIVES: Previous studies have shown that dietary prebiotics have the potential to improve memory, alter social behavior, and reduce anxiety-like behaviors in rodents. The present research sought to expand upon such results and describe the effects of feeding prebiotics early in life on cognition and neurochemistry using a translational piglet model. METHODS: Pigs were provided customized milk replacer containing 2 g/L each of polydextrose (PDX) and galactooligosaccharide (PDX/GOS) or 0 g/L (Control) from postnatal day (PND) 2-33. Beginning on PND 25, pigs were tested on the novel object recognition (NOR), novel location recognition (NLR), and backtest tasks to measure recognition memory and response to restraint stress. At study conclusion pigs were euthanized and intestine, blood, and brain tissues were collected and analyzed. RESULTS: PDX/GOS-fed pigs demonstrated recognition memory on the NOR task (P < 0.001) whereas Control pigs did not (P = 0.184). Additionally, PDX/GOS-fed pigs visited the novel and sample objects more frequently (all P < 0.05) while spending less time per visit exploring the sample object (P = 0.028) than Control pigs. Volatile fatty acids (VFAs) were decreased in the ascending colon (P < 0.012), whereas butyrate tended to be higher in blood (P = 0.080) and lower in the hippocampus (P = 0.061) of PDX/GOS-fed pigs. PDX/GOS-fed pigs exhibited lower serotonin (P = 0.016) in the hippocampus. CONCLUSION: These findings suggest that early life consumption of PDX/GOS supports recognition memory as measured by NOR while modulating the concentrations of VFAs in the colon, blood, and brain, as well as hippocampal serotonin.

Neurobehavioural effects of Lactobacillus rhamnosus GG alone and in combination with prebiotics polydextrose and galactooligosaccharide in male rats exposed to early-life stress.

Early life is a period of significant brain development when the brain is at its most plastic and vulnerable. Stressful episodes during this window of development have long-lasting effects on the central nervous system. Rodent maternal separation (MS) is a reliable model of early-life stress and induces alterations in both physiology and behaviour. Intriguingly, the gut microbiota of MS offspring differ from that of non-separated offspring, suggesting a mechanistic role for the microbiota-gut-brain axis. Hence, we tested whether dietary factors known to affect the gut microbiota alter the neurobehavioural effects of MS. The impact of consuming diet containing prebiotics polydextrose (PDX) and galactooligosaccharide (GOS) alone or in combination with live bacteria Lactobacillus rhamnosus GG (LGG) from weaning onwards in rats subjected to early-life MS was assessed. Adult offspring were assessed for anxiety-like behaviour in the open field test, spatial memory using the Morris water maze, and reactivity to restraint stress. Brains were examined via PCR for changes in mRNA gene expression. Here, we demonstrate that diets containing a combination of PDX/GOS and LGG attenuates the effects of early-life MS on anxiety-like behaviour and hippocampal-dependent learning with changes to hippocampal mRNA expression of genes related to stress circuitry, anxiety and learning.

Dermoscopy findings of a one mm lentigo maligna.

A 66-year-old man presented with a grayish 1mm macule on the left cheek. Dermatoscopic examination revealed annular-granular structures partially surrounding a single follicular ostium. Histopathologic examination demonstrated atypical, confluent single melanocytes at the basal layer with nesting, crowding, and upward migration descending along the hair follicle, consistent with the diagnosis of lentigo maligna (LM). This case presents evidence in support of the Schiffner progression model for LMs, starting with asymmetric pigmented follicular openings composed of annular-granular structures, which later coalesce into gray to dark black/brown streaks, and then form pseudo-networks or rhomboidal structures. The finding of annular-granular structures partially surrounding a single hair follicle may be the earliest recognizable dermatoscopic feature of an LM and may help clinicians diagnose facial LMs earlier.

Early life diets with prebiotics and bioactive milk fractions attenuate the impact of stress on learned helplessness behaviours and alter gene expression within neural circuits important for stress resistance.

Manipulating gut microbes may improve mental health. Prebiotics are indigestible compounds that increase the growth and activity of health-promoting microorganisms, yet few studies have examined how prebiotics affect CNS function. Using an acute inescapable stressor known to produce learned helplessness behaviours such as failure to escape and exaggerated fear, we tested whether early life supplementation of a blend of two prebiotics, galactooligosaccharide (GOS) and polydextrose (PDX), and the glycoprotein lactoferrin (LAC) would attenuate behavioural and biological responses to stress later in life. Juvenile, male F344 rats were fed diets containing either GOS and PDX alone, LAC alone, or GOS, PDX and LAC. All diets altered gut bacteria, while diets containing GOS and PDX increased Lactobacillus spp. After 4 weeks, rats were exposed to inescapable stress, and either immediately killed for blood and tissues, or assessed for learned helplessness 24 h later. Diets did not attenuate stress effects on spleen weight, corticosterone and blood glucose; however, all diets differentially attenuated stress-induced learned helplessness. Notably, in situ hybridization revealed that all diets reduced stress-evoked cfos mRNA in the dorsal raphe nucleus (DRN), a structure important for learned helplessness behaviours. In addition, GOS, PDX and LAC diet attenuated stress-evoked decreases in mRNA for the 5-HT1A autoreceptor in the DRN and increased basal BDNF mRNA within the prefrontal cortex. These data suggest early life diets containing prebiotics and/or LAC promote behavioural stress resistance and uniquely modulate gene expression in corresponding circuits.

Discovery of a novel insecticidal protein from Chromobacterium piscinae, with activity against Western Corn Rootworm, Diabrotica virgifera virgifera.

Western corn rootworm (WCR), Diabrotica virgifera virgifera, is one of the most significant pests of corn in the United States. Although transgenic solutions exist, increasing resistance concerns make the discovery of novel solutions essential. In order to find a novel protein with high activity and a new mode of action, a large microbial collection was surveyed for toxicity to WCR using in vitro bioassays. Cultures of strain ATX2024, identified as Chromobacterium piscinae, had very high activity against WCR larvae. The biological activity from the strain was purified using chromatographic techniques and fractions were tested against WCR larvae. Proteins in the final active fraction were identified by mass spectrometry and N-terminal sequencing and matched to the genome of ATX2024. A novel 58.9kDa protein, identified by this approach, was expressed in a recombinant expression system and found to have specific activity against WCR. Transgenic corn events containing this gene showed good protection against root damage by WCR, with average scores ranging between 0.01 and 0.04 on the Iowa State node injury scale. Sequence analysis did not reveal homology to any known insecticidal toxin, suggesting that this protein may act in a novel way to control WCR. The new WCR active protein is named GNIP1Aa, for Gram Negative Insecticidal Protein.

Dietary Isomers of Sialyllactose Increase Ganglioside Sialic Acid Concentrations in the Corpus Callosum and Cerebellum and Modulate the Colonic Microbiota of Formula-Fed Piglets.

BACKGROUND: Sialyllactose is a key human milk oligosaccharide and consists of sialic acid (SA) bound to a lactose molecule. Breastfed infants have increased accumulation of ganglioside-bound SA compared with formula-fed infants. OBJECTIVE: This study aimed to determine whether different isomers of sialyllactose enrich brain SA and modulate the microbiome of developing neonatal piglets. METHODS: Day-old pigs were randomly allocated to 6 diets (control, 2 or 4 g 3'-sialyllactose/L, 2 or 4 g 6'-sialyllactose/L, or 2 g polydextrose/L + 2 g galacto-oligosaccharides/L; n = 9) and fed 3 times/d for 21 d. Pigs were killed, and the left hemisphere of the brain was dissected into cerebrum, cerebellum, corpus callosum, and hippocampus regions. SA was determined by using a modified periodic acid-resorcinol reaction. Microbial composition of the intestinal digesta was analyzed with the use of 16S ribosomal DNA Illumina sequencing. RESULTS: Dietary sialyllactose did not affect feed intake, growth, or fecal consistency. Ganglioside-bound SA in the corpus callosum of pigs fed 2 g 3'-sialyllactose or 6'-sialyllactose/L increased by 15% in comparison with control pigs. Similarly, ganglioside-bound SA in the cerebellum of pigs fed 4 g 3'-sialyllactose/L increased by 10% in comparison with control pigs. Significant (P < 0.05, Adonis Test) microbiome differences were observed in the proximal and distal colons of piglets fed control compared with 4-g 6'-sialyllactose/L formulas. Differences were attributed to an increase in bacterial taxa belonging to species Collinsella aerofaciens (phylum Actinobacteria), genera Ruminococcus and Faecalibacterium (phylum Firmicutes), and genus Prevotella (phylum Bacteroidetes) (Wald test, P < 0.05, DeSeq2) compared with piglets fed the control diet. Taxa belonging to families Enterobacteriaceae and Enterococcaceae (phylum Proteobacteria), as well as taxa belonging to family Lachnospiraceae and order Lactobacillales (phylum Firmicutes), were 2.3- and 4-fold lower, respectively, in 6'-sialyllactose-fed piglets than in controls. CONCLUSIONS: Supplementation of formula with 3'- or 6'-sialyllactose can enrich ganglioside SA in the brain and modulate gut-associated microbiota in neonatal pigs. We propose 2 potential routes by which sialyllactose may positively affect the neonate: serving as a source of SA for neurologic development and promoting beneficial microbiota.

Prebiotics and Bioactive Milk Fractions Affect Gut Development, Microbiota, and Neurotransmitter Expression in Piglets.

OBJECTIVE: This study tested the hypothesis that the addition of prebiotics and 2 functional milk ingredients to infant formula would maintain normal growth and gut development, and modify microbiota composition and neurotransmitter gene expression in neonatal piglets. METHODS: Two-day-old male piglets (n = 24) were fed formula (CONT) or formula with polydextrose (1.2 g/100 g diet), galactooligosaccharides (3.5 g/100 g diet), bovine lactoferrin (0.3 g/100 g diet), and milk fat globule membrane-10 (2.5 g/100 g diet) (TEST) for 30 days. On study day 31, intestinal samples, ileal and colonic contents, and feces were collected. Intestinal histomorphology, disaccharidase activity, serotonin (5'HT), vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH) were measured. Gut microbiota composition was assessed by pyrosequencing of the V3-V5 regions of 16S rRNA and quantitative polymerase chain reaction. RESULTS: Body weight of piglets on TEST was greater (P ≤ 0.05) than CONT on days 17 to 30. Both groups displayed growth patterns within the range observed for sow-reared pigs. TEST piglets had greater jejunal lactase (P = 0.03) and higher (P = 0.003) ileal VIP expression. TEST piglets tended to have greater (P = 0.09) sucrase activity, longer (P = 0.08) ileal villi, and greater (P = 0.06) duodenal TH expression. Microbial communities of TEST piglets differed from CONT in ascending colon (AC, P = 0.001) and feces (P ≤ 0.05). CONT piglets had greater relative abundances of Mogibacterium, Collinsella, Klebsiella, Escherichia/Shigella, Eubacterium, and Roseburia compared with TEST piglets in AC. In feces, CONT piglets harbored lower (P ≤ 0.05) proportions of Parabacteroides, Clostridium IV, Lutispora, and Sutterella than TEST piglets. CONCLUSIONS: A mixture of bioactive ingredients improved weight gain and gut maturation, modulated colonic and fecal microbial composition, and reduced the proportions of opportunistic pathogens.

The prebiotics 3'Sialyllactose and 6'Sialyllactose diminish stressor-induced anxiety-like behavior and colonic microbiota alterations: Evidence for effects on the gut-brain axis.

There are extensive bidirectional interactions between the gut microbiota and the central nervous system (CNS), and studies demonstrate that stressor exposure significantly alters gut microbiota community structure. We tested whether oligosaccharides naturally found in high levels in human milk, which have been reported to impact brain development and enhance the growth of beneficial commensal microbes, would prevent stressor-induced alterations in gut microbial community composition and attenuate stressor-induced anxiety-like behavior. Mice were fed standard laboratory diet, or laboratory diet containing the human milk oligosaccharides 3'Sialyllactose (3'SL) or 6'Sialyllactose (6'SL) for 2 weeks prior to being exposed to either a social disruption stressor or a non-stressed control condition. Stressor exposure significantly changed the structure of the colonic mucosa-associated microbiota in control mice, as indicated by changes in beta diversity. The stressor resulted in anxiety-like behavior in both the light/dark preference and open field tests in control mice. This effect was associated with a reduction in immature neurons in the dentate gyrus as indicated by doublecortin (DCX) immunostaining. These effects were not evident in mice fed milk oligosaccharides; stressor exposure did not significantly change microbial community structure in mice fed 3'SL or 6'SL. In addition, 3'SL and 6'SL helped maintain normal behavior on tests of anxiety-like behavior and normal numbers of DCX+ immature neurons. These studies indicate that milk oligosaccharides support normal microbial communities and behavioral responses during stressor exposure, potentially through effects on the gut microbiota-brain axis.

EpicTope: narrating protein sequence features to identify non-disruptive epitope tagging sites.

Epitope tagging is an invaluable technique enabling the identification, tracking, and purification of proteins in vivo. We developed a tool, EpicTope, to facilitate this method by identifying amino acid positions suitable for epitope insertion. Our method uses a scoring function that considers multiple protein sequence and structural features to determine locations least disruptive to the protein's function. We validated our approach on the zebrafish Smad5 protein, showing that multiple predicted internally tagged Smad5 proteins rescue zebrafish smad5 mutant embryos, while the N- and C-terminal tagged variants do not, also as predicted. We further show that the internally tagged Smad5 proteins are accessible to antibodies in wholemount zebrafish embryo immunohistochemistry and by western blot. Our work demonstrates that EpicTope is an accessible and effective tool for designing epitope tag insertion sites. EpicTope is available under a GPL-3 license from: https://github.com/FriedbergLab/Epictope.

Functional characterization of QT interval associated SCN5A enhancer variants identify combined additive effects.

Several empirical and theoretical studies suggest presence of multiple enhancers per gene that collectively regulate gene expression, and that common sequence variation impacting on the activities of these enhancers is a major source of inter-individual variability in gene expression. However, for vast majority of genes, enhancers and the underlying regulatory variation remains unknown. Even for the genes with well-characterized enhancers, the nature of the combined effects from multiple enhancers and their variants, when known, on gene expression regulation remains unexplored. Here, we have evaluated the combined effects from five SCN5A enhancers and their regulatory variants that are known to collectively correlate with SCN5A cardiac expression and underlie QT interval association in the general population. Using small deletions centered at the regulatory variants in episomal reporter assays in a mouse cardiomyocyte cell line we demonstrate that the variants and their flanking sequences play critical role in individual enhancer activities, likely being a transcription factor (TF) binding site. By performing oligonucleotide-based pulldown assays on predicted TFs we identify the TFs likely driving allele-specific enhancer activities. Using all 32 possible allelic synthetic constructs in reporter assays, representing the five biallelic enhancers in tandem in their genomic order, we demonstrate combined additive effects on overall enhancer activities. Using transient enhancer assays in developing zebrafish embryos we demonstrate the four out the five enhancer elements act as enhancers in vivo . Together, these studies extend the previous findings to uncover the TFs driving the enhancer activities of QT interval associated SCN5A regulatory variants, reveal the additive effects from allelic combinations of these regulatory variants, and prove their potential to act as enhancers in vivo .

Computational Modeling of Ophthalmic Procedures: Computational Modeling of Ophthalmic Procedures.

PURPOSE: To explore how finite-element calculations can continue to contribute to diverse problems in ophthalmology and vision science, we describe our recent work on modeling the force on the peripheral retina in intravitreal injections and how that force increases with shorter, smaller gauge needles. We also present a calculation that determines the location and stress on a retinal pigment epithelial detachment during an intravitreal injection, the possibility that stress induced by the injection can lead to a tear of the retinal pigment epithelium. BACKGROUND: Advanced computational models can provide a critical insight into the underlying physics in many surgical procedures, which may not be intuitive. METHODS: The simulations were implemented using COMSOL Multiphysics. We compared the monkey retinal adhesive force of 18 Pa with the results of this study to quantify the maximum retinal stress that occurs during intravitreal injections. CONCLUSIONS: Currently used 30-gauge needles produce stress on the retina during intravitreal injections that is only slightly below the limit that can create retinal tears. As retina specialists attempt to use smaller needles, the risk of complications may increase. In addition, we find that during an intravitreal injection, the stress on the retina in a pigment epithelial detachment occurs at the edge of the detachment (found clinically), and the stress is sufficient to tear the retina. These findings may guide physicians in future clinical research. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Trends in Waste Production at a Community Hospital during the COVID-19 Pandemic.

INTRODUCTION: As of September 2021, the COVID-19 pandemic has led to 42,500,000 cases and 680,000 deaths in the United States. In Rhode Island, there have been 170,000 cases and 2,820 deaths. Investigating resource utilization and waste production during disease outbreaks can inform efforts at disaster preparedness. The purpose of this study was to examine trends in waste production during the COVID-19 pandemic. METHODS: This is a descriptive study examining trends in waste production during the COVID-19 pandemic. The study was conducted at a suburban community hospital in Rhode Island. Data was collected on regulated medical waste (RMW) and linen use from October 2019-July 2021. Adjusted patient days (APD) values were calculated using hospital census and revenue data. Total weight and weight/APD were calculated for each month of the study period. Data was then compared with overall COVID-19 cases and hospitalizations in Rhode Island. This data was gathered from the Rhode Island Department of Health (RIDOH) COVID Response Data Hub. RESULTS: Regulated Medical Waste (RMW) by total weight was lowest in April 2020, when the hospital census and adjusted patient days (APD) were at their lowest. In contrast, linen use remained largely consistent with pre-pandemic levels during the initial months of the pandemic despite a decrease in hospital census. The highest linen weight/APD value (23.32 lbs/APD) was in April 2020. Both RMW and linen use (weight/APD) decreased during the study period. Linen use was highest during months with increased COVID-19 cases and hospitalizations. CONCLUSIONS: This study examined trends in waste production at a community hospital during the COVID-19 pandemic. Linen use was highest during months of increased COVID-19 cases and hospitalizations, while RMW production decreased. There was a particular increase in linen use in April 2020, when the pandemic was in its initial phases.

DHA diet reduces AD pathology in young APPswe/PS1 Delta E9 transgenic mice: possible gender effects.

Epidemiological and clinical trial findings suggest that consumption of docosahexaenoic acid (DHA) lowers the risk of Alzheimer's disease (AD). We examined the effects of short-term (3 months) DHA enriched diet on plaque deposition and synaptic defects in forebrain of young APPswe/PS1 Delta E9 transgenic (tg) and non-transgenic (ntg) mice. Gas chromatography revealed a significant increase in DHA concomitant with a decrease of arachidonic acid in both brain and liver in mice fed with DHA. Female tg mice consumed relatively more food daily than ntg female mice, independent of diet. Plaque load was significantly reduced in the cortex, ventral hippocampus and striatum of female APPswe/PS1 Delta E9 tg mice on DHA diet compared to female tg mice on control diet. Immunoblot quantitation of the APOE receptor, LR11, which is involved in APP trafficking and A beta production, were unchanged in mice on DHA or control diets. Moreover drebrin levels were significantly increased in the hippocampus of tg mice on the DHA diet. Finally, in vitro DHA treatment prevented amyloid toxicity in cell cultures. Our findings support the concept that increased DHA consumption may play and important role in reducing brain insults in female AD patients.

It is all in the bag: collodion bag versus HistoGel cell block method.

INTRODUCTION: We performed a comparison of cell blocks prepared with the collodion bag and HistoGel to evaluate the ease of embedding and cutting, performance with low cellularity specimens, time and cost per specimen, and value to support immunohistochemistry and molecular diagnostics. MATERIALS AND METHODS: We processed 11 fresh, unfixed effusions using both the collodion bag and the HistoGel cell block preparation methods. Six immunohistochemistry stains were tested on 2 of the body fluids. DNA was extracted and quantified, and polymerase chain reaction cycle thresholds were evaluated from cell blocks prepared from 5 of the body fluids. The comparison parameters included embedding difficulty, cutting resistance, adequacy, cell yield, cell preservation, immunohistochemistry stain quality, DNA quantity, integrity, and purity. The time and cost to prepare each specimen was compared using normalized values for preparation of specimen, cost per year, and cost per specimen. RESULTS: Each parameter was assessed for both cell block preparation methods. All 3 of the samples with moderate or poor cell yield were low-volume (5-mL) samples prepared with the HistoGel method. In contrast, the collodion bag method produced a good yield with all three 5-mL samples. DNA recovery was greater in the collodion bag method. Similar crossing threshold values in purity reactions indicated equally high-quality matrix properties for the collodion bag and HistoGel preparations. Preparation of the specimen was 10 minutes faster with the collodion bag method, and the cost for the collodion bag method was $0.24 more expensive per cell block than using the HistoGel. CONCLUSIONS: The collodion bag method produced superior cell blocks for both morphologic and molecular studies more consistently, with lower volume specimens and with less time per specimen.

Dietary prebiotics alter novel microbial dependent fecal metabolites that improve sleep.

Dietary prebiotics produce favorable changes in the commensal gut microbiome and reduce host vulnerability to stress-induced disruptions in complex behaviors such as sleep. The mechanisms for how prebiotics modulate stress physiology remain unclear; however, emerging evidence suggests that gut microbes and their metabolites may play a role. This study tested if stress and/or dietary prebiotics (Test diet) alter the fecal metabolome; and explored if these changes were related to sleep and/or gut microbial alpha diversity. Male F344 rats on either Test or Control diet were instrumented for electroencephalography biotelemetry measures of sleep/wake. After 5 weeks on diet, rats were either stressed or remained in home cages. Based on untargeted mass spectrometry and 16S rRNA gene sequencing, both stress and Test diet altered the fecal metabolome/microbiome. In addition, Test diet prevented the stress-induced reduction in microbial alpha diversity based on PD_Whole_Tree, which has been previously published. Network propagation analysis revealed that stress increased members of the neuroactive steroidal pregnane molecular family; and that Test diet reduced this effect. We also discovered links between sleep, alpha diversity, and pyrimidine, secondary bile acid, and neuroactive glucocorticoid/pregnanolone-type steroidal metabolites. These results reveal novel microbial-dependent metabolites that may modulate stress physiology and sleep.

Evaluation of Dietary Bovine Milk Fat Globule Membrane Supplementation on Growth, Serum Cholesterol and Lipoproteins, and Neurodevelopment in the Young Pig.

Introduction: Milk fat globule membrane (MFGM) is a protein- and phospholipid-rich membrane that surrounds the lipid droplet in milk. We have previously reported that a diet composed of a combination of prebiotics, bovine MFGM (bMFGM), and lactoferrin (bLf) supported brain development in young pigs. Due to the growing interest of its potential benefits in neurodevelopment, the present study focused on the effects of dietary bMFGM alone using the pig as a translational model. Methods: Male pigs were provided ad libitum access to milk replacer with added whey protein-lipid concentrate (source of bMFGM) at 0 (CONT), 2.5 (MFGM-2.5), or 5 (MFGM-5.0) g/L from postnatal day (PND) 2 to 31. Blood was collected from pigs at PND 15 and 31, and pigs underwent behavioral testing using the novel object recognition task starting at PND 25. At PND 31, magnetic resonance imaging was conducted and animals were subsequently euthanized for tissue collection. Results: No group differences in body weight gain or milk intake were observed. At PND 31, few group differences were detected in absolute and relative brain volumes, brain water diffusivity outcomes, or behavioral parameters using the novel object recognition task. Serum lipoprotein was higher in pigs receiving diets with added dietary bMFGM compared with the CONT group. Serum cholesterol and high-density lipoprotein significantly higher (all P < 0.05) in the MFGM-2.5 compared with the CONT group. However, cholesterol concentrations within the brain prefrontal cortex and hippocampus did not differ among dietary groups. Conclusion: In this pig model, dietary supplementation with bMFGM was well-tolerated and supported growth and dietary intake similar to the control formula. Added dietary bMFGM was associated with increased serum lipoprotein, but no group differences in early brain cholesterol concentrations, macrostructure, microstructure, or recognition memory pigs at 31 days of age. Further examination of longitudinal brain development and myelination in the pig, particularly at later ages/maturation, is warranted.

Dietary Oligosaccharides Attenuate Stress-Induced Disruptions in Immune Reactivity and Microbial B-Vitamin Metabolism.

Background: Exposure to stressful stimuli dysregulates inflammatory processes and alters the gut microbiota. Prebiotics, including long-chain fermentable fibers and milk oligosaccharides, have the potential to limit inflammation through modulation of the gut microbiota. To determine whether prebiotics attenuate stress-induced inflammation and microbiota perturbations, mice were fed either a control diet or a diet supplemented with galactooligosaccharides, polydextrose and sialyllactose (GOS+PDX+SL) or sialyllactose (SL) for 2 weeks prior to and during a 6-day exposure to a social disruption stressor. Spleens were collected for immunoreactivity assays. Colon contents were examined for stressor- and diet- induced changes in the gut microbiome and metabolome through 16S rRNA gene sequencing, shotgun metagenomic sequencing and UPLC-MS/MS. Results: Stress increased circulating IL-6 and enhanced splenocyte immunoreactivity to an ex vivo LPS challenge. Diets containing GOS+PDX+SL or SL alone attenuated these responses. Stress exposure resulted in large changes to the gut metabolome, including robust shifts in amino acids, peptides, nucleotides/nucleosides, tryptophan metabolites, and B vitamins. Multiple B vitamins were inversely associated with IL-6 and were augmented in mice fed either GOS+PDX+SL or SL diets. Stressed mice exhibited distinct microbial communities with lower abundances of Lactobacillus spp. and higher abundances of Bacteroides spp. Diet supplementation with GOS+PDX+SL, but not SL alone, orthogonally altered the microbiome and enhanced the growth of Bifidobacterium spp. Metagenome-assembled genomes (MAGs) from mice fed the GOS+PDX+SL diet unveiled genes in a Bifidobacterium MAG for de novo B vitamin synthesis. B vitamers directly attenuated the stressor-induced exacerbation of cytokine production in LPS-stimulated splenocytes. Conclusions: Overall, these data indicate that colonic metabolites, including B vitamins, are responsive to psychosocial stress. Dietary prebiotics reestablish colonic B vitamins and limit stress-induced inflammation.

Characterization and plant expression of a glyphosate-tolerant enolpyruvylshikimate phosphate synthase.

BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials. CONCLUSION: These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.

Dietary Sialyllactose Does Not Influence Measures of Recognition Memory or Diurnal Activity in the Young Pig.

Sialic acid (SA) is an integral component of gangliosides and signaling molecules in the brain and its dietary intake may support cognitive development. We previously reported that feeding sialyllactose, a milk oligosaccharide that contains SA, alters SA content and diffusivity in the pig brain. The present research sought to expand upon such results and describe the effects of feeding sialyllactose on recognition memory and sleep/wake activity using a translational pig model. Pigs were provided ad libitum access to a customized milk replacer containing 0 g/L or 380 g/L of sialyllactose from postnatal day (PND) 2-22. Beginning on PND 15, pigs were fitted with accelerometers to track home-cage activity and testing on the novel object recognition task began at PND 17. There were no significant effects of diet on average daily body weight gain, average daily milk intake, or the gain-to-feed ratio during the study (all p ≥ 0.11). Pigs on both diets were able to display recognition memory on the novel object recognition task (p < 0.01), but performance and exploratory behavior did not differ between groups (all p ≥ 0.11). Total activity and percent time spent sleeping were equivalent between groups during both day and night cycles (all p ≥ 0.56). Dietary sialyllactose did not alter growth performance of young pigs, and there was no evidence that providing SA via sialyllactose benefits the development of recognition memory or gross sleep-related behaviors.