Determining the effects of Candida utilis-fermented pea starch vs. unfermented pea starch, alone or in whole diets, on palatability and glycemic response in dogs and cats.

Current research suggests yeast fermentation has the potential to improve palatability of pea-based diets for both cats and dogs. However, to be useful, fermentation should not compromise other healthy attributes of peas such as a low glycemic response. Fermentation of uncooked pea starch with Candida utilis (ATCC 9950) appeared to increase crude protein, crude fiber content, inorganic compounds (phosphorus and iron) and phenols. Whole diets were designed with fermented and unfermented pea starch to assess palatability, food intake, and glycemic responses in unacclimated, mixed sex Beagle dogs and mixed breed cats (n = 8 and n = 7, respectively). For palatability testing, a control diet was formulated with 30% corn starch as well as test diets with 30% inclusion of fermented or unfermented pea starch (all lab-made), then compared to a commercial diet containing pea starch (Legacy/Horizon). Fermentation had little effect on rapidly digestible starch either in uncooked starch form or when incorporated into whole diets, but did decrease resistant starch by 15% and increase slowly digestible starch by 20%. Palatability tests using either two choices or four choices at a time revealed a significant preference for the fermented pea starch diet (p < 0.01) in both species. For the glycemic responses, a total of four different pea products were included: unfermented pea starch, fermented pea starch, and 30% inclusion of unfermented and fermented pea starch in whole formulated diets. There were no significant changes in glycemic responses with the fermented pea diet compared to the unfermented diet, demonstrating that healthful low glycemic properties of pea starch were retained after C. utilis fermentation. Overall, C. utilis-fermentation technique was successfully adapted to pea starch where it resulted in increased palatability and food intake in dogs and cats, with potential to positively contribute to overall health benefits for both species.

Review: A species comparison of the kinetic homogeneous and heterogeneous organization of sodium-dependent glucose transport systems along the intestine.

The targeted use of carbohydrates by feed and food industries to create balanced and cost-effective diets has generated a tremendous amount of research in carbohydrate digestion and absorption in different species. Specifically, this research has led us to a larger observation that identified different organizations of intestinal sodium-dependent glucose absorption across species, which has not been previously collated and reviewed. Thus, this review will compare the kinetic segregation of sodium-dependent glucose transport across the intestine of different species, which we have termed either homogeneous or heterogeneous systems. For instance, the pig follows a heterogeneous system of sodium-dependent glucose transport with a high-affinity, super-low-capacity (Ha/sLc) in the jejunum, and a high-affinity, super-high-capacity (Ha/sHc) in the ileum. This is achieved by multiple sodium-dependent glucose transporters contributing to each segment. In contrast, tilapia have a homogenous system characterized by high-affinity, high-capacity (Ha/Hc) throughout the intestine. Additionally, we are the first to report glucose transporter patterns across species presented from vertebrates to invertebrates. Finally, other kinetic transport systems are briefly covered to illustrate possible contributions/modulations to sodium-dependent glucose transporter organization. Overall, we present a new perspective on the organization of glucose absorption along the intestinal tract.

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase.

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry. Supplementary Information: The online version contains supplementary material available at 10.1186/s40643-023-00633-8.

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity.

BACKGROUND: TlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis. METHODS: TlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis. RESULTS: Purified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments. CONCLUSIONS: Oligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence. GENERAL SIGNIFICANCE: Our results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.

The Effects of Fermentation of Low or High Tannin Fava Bean-Based Diets on Glucose Response, Cardiovascular Function, and Fecal Bile Acid Excretion during a 28-Day Feeding Period in Dogs: Comparison with Commercial Diets with Normal vs. High Protein.

We have shown that feeding dogs fava bean (FB)-based diets for 7 days is safe and FB flour fermentation with Candida utilis has the potential to decrease FB anti-nutritional factors. In the present study, the effects of 28-day feeding of 4 different FB-based test dog foods containing moderate protein (~27% dry matter (DM)) were compared with two commercial diets with normal protein (NP, grain-containing, ~31% DM protein) or high protein (HP, grain-free, ~41% DM protein). Health parameters were investigated in beagles fed the NP or HP diets or using a randomized, crossover, 2 × 2 Latin square design of the FB diets: unfermented high-tannin (UF-HT), fermented high-tannin (FM-HT), unfermented low-tannin (UF-LT), and fermented low-tannin (FM-LT). The results showed that fermentation increased glucose tolerance, increased red blood cell numbers and increased systolic blood pressure, but decreased flow-mediated vasodilation. Taken together, the overall effect of fermentation appears to be beneficial and improved FB nutritional value. Most interesting, even though the HP diet was grain-free, the diet did contain added taurine, and no adverse effects on cardiac function were observed, while glucose tolerance was impaired compared to NP-fed dogs. In summary, this study did not find evidence of adverse cardiac effects of pulses in 'grain-free' diets, at least not in the relatively resistant beagle breed over a 28-day period. More importantly, fermentation with C. utilis shows promise to enhance health benefits of pulses such as FB in dog food.

Glycemic, insulinemic and methylglyoxal postprandial responses to starches alone or in whole diets in dogs versus cats: Relating the concept of glycemic index to metabolic responses and gene expression.

Species differences between domestic cats (Felis catus) and dogs (Canis familiaris) has led to differences in their ability to digest, absorb and metabolize carbohydrates through poorly characterized mechanisms. The current study aimed to first examine biopsied small intestine, pancreas, liver and skeletal muscle from laboratory beagles and domestic cats for mRNA expression of key enzymes involved in starch digestion (amylase), glucose transport (sodium-dependent SGLTs and -independent glucose transporters, GLUT) and glucose metabolism (hexokinase and glucokinase). Cats had lower mRNA expression of most genes examined in almost all tissues compared to dogs (p < 0.05). Next, postprandial glucose, insulin, methylglyoxal (a toxic glucose metabolite) and d-lactate (metabolite of methylglyoxal) after single feedings of different starch sources were tested in fasted dogs and cats. After feeding pure glucose, peak postprandial blood glucose and methylglyoxal were surprisingly similar between dogs and cats, except cats had a longer time to peak and a greater area under the curve consistent with lower glycolytic enzyme expression. After feeding starches or whole diets to dogs, postprandial glycemic response, glycemic index, insulin, methylglyoxal and d-lactate followed reported glycemic index trends in humans. In contrast, cats showed very low to negligible postprandial glycemic responses and low insulin after feeding different starch sources, but not whole diets, with no relationship to methylglyoxal or d-lactate. Thus, the concept of glycemic index appears valid in dogs, but not cats. Differences in amylase, glucose transporters, and glycolytic enzymes are consistent with species differences in starch and glucose handling between cats and dogs.

Critical transporters of methionine and methionine hydroxyl analogue supplements across the intestine: What we know so far and what can be learned to advance animal nutrition.

DL-methionine (DL-Met) and its analogue DL-2-hydroxy-4-(methylthio) butanoic acid (DL-methionine hydroxyl analogue or DL-MHA) have been used as nutritional supplements in the diets of farmed raised animals. Knowledge of the intestinal transport mechanisms involved in these products is important for developing dietary strategies. This review provides updated information of the expression, function, and transport kinetics in the intestine of known Met-linked transporters along with putative MHA-linked transporters. As a neutral amino acid (AA), the transport of DL-Met is facilitated by multiple apical sodium-dependent/-independent high-/low-affinity transporters such as ASCT2, B0AT1 and rBAT/b0,+AT. The basolateral transport largely relies on the rate-limiting uniporter LAT4, while the presence of the basolateral antiporter y+LAT1 is probably necessary for exchanging intracellular cationic AAs and Met in the blood. In contrast, the intestinal transport kinetics of DL-MHA have been scarcely studied. DL-MHA transport is generally accepted to be mediated simply by the proton-dependent monocarboxylate transporter MCT1. However, in-depth mechanistic studies have indicated that DL-MHA transport is also achieved through apical sodium monocarboxylate transporters (SMCTs). In any case, reliance on either a proton or sodium gradient would thus require energy input for both Met and MHA transport. This expanding knowledge of the specific transporters involved now allows us to assess the effect of dietary ingredients on the expression and function of these transporters. Potentially, the resulting information could be furthered with selective breeding to reduce overall feed costs.

Characterization of the segmental transport mechanisms of DL-methionine hydroxy analogue along the intestinal tract of rainbow trout with an additional comparison to DL-methionine.

The aim of this study was to identify the unknown transport mechanism of the extensively used monocarboxylate methionine feed supplement DL-methionine hydroxy analogue (DL-MHA) in rainbow trout intestine. Transport across the pyloric caeca (PC), midgut (MG), and hindgut (HG) regions were kinetically studied in Na+- and H+-dependent manners. Gene expression of monocarboxylate (MCTs) and sodium monocarboxylate transporters (SMCTs) were assessed. Results demonstrated that DL-MHA transport from 0.2-20 mM was Na+-dependent and obeyed Michaelis-Menten kinetics with low affinity in PC & MG in apical/basal pH of 7.7/7.7. Changes in apical/basal pH (6.0/6.0, 6.0/7.7, and 7.7/8.7) had insignificant effects on kinetics. In contrast, HG flux kinetics were only obtained in pH 7.7/8.7 or in the presence of lactate with medium affinity. Additionally, DL-MHA transport from 0-150 µM demonstrated the presence of a Na+-dependent high-affinity transporter in PC & MG. Conclusively, two distinct carrier-mediated DL-MHA transport mechanisms along the trout gut were found: 1) in PC & MG: apical transport was regulated by Na+-requiring systems that possibly contained low- and high-affinity transporters, and basolateral transport was primarily achieved through a H+-independent transporter; 2) in HG: uptake was apically mediated by a Na+-dependent transporter with medium affinity, and basolateral exit was largely controlled by an H+-dependent transporter. Finally, two major methionine feed supplements, DL-MHA and DL-methionine (DL-Met) were compared to understand the differences in their bioefficacy. Flux rates of DL-MHA were only about 42.2-66.0% in PC and MG compared to DL-Met, suggesting intestinal transport of DL-MHA was lower than DL-Met.

Impairment of electroneutral Na+ transport and associated downregulation of NHE3 contributes to the development of diarrhea following in vivo challenge with Brachyspira spp.

The effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na+ transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na+ absorption was assessed in Ussing chambers by radiolabeled 22Na flux and pharmacological inhibitory studies. Basal radiolabeled 22Na flux experiments revealed that mucosal-to-serosal flux (Jms) was significantly impaired in B. hyodysenteriae and B. hampsonii-diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current (Isc) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na+ absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na+/H+ exchanger (NHE1, NHE2, and NHE3) mRNA expression in the proximal, apex, and distal colonic segments paired with decreased protein expression of the critical NHE3 isoform. The decrease in NHE3 mRNA expression appears not to be attributed to the host's cytokine response as human IL-1α did not modify NHE3 mRNA expression in Caco-2 cells. However, a whole cell B. hampsonii lysate significantly downregulated NHE3 mRNA expression and significantly increased p38 phosphorylation in Caco-2 cells. Together these findings provide a likely mechanism for the spirochete-induced malabsorptive diarrhea, indicated by a decrease in electroneutral Na+ absorption in the porcine colon due to Brachyspira's ability to inhibit NHE3 transcription, resulting in diarrheal disease.NEW & NOTEWORTHY This research demonstrates that diarrheal disease caused by two infectious spirochete spp. is a result of impaired electroneutral Na+ absorption via Na+/H+ exchanger 3 (NHE3) in the porcine colon. Our findings suggest that the decrease in NHE3 mRNA and protein is not likely a result of the host's cytokine response. Rather, it appears that these two Brachyspira spp. directly inhibit the transcription and translation of NHE3, resulting in the development of diarrhea.

Comparison of intestinal glucose flux and electrogenic current demonstrates two absorptive pathways in pig and one in Nile tilapia and rainbow trout.

The mucosal-to-serosal flux of 14C 3-O-methyl-d-glucose was compared against the electrogenic transport of d-glucose across ex vivo intestinal segments of Nile tilapia, rainbow trout, and pig in Ussing chambers. The difference in affinities (Km "fingerprints") between pig flux and electrogenic transport of glucose, and the absence of this difference in tilapia and trout, suggest two absorptive pathways in the pig and one in the fish species examined. More specifically, the total mucosal-to-serosal flux revealed a super high-affinity, high-capacity (sHa/Hc) total glucose transport system in tilapia; a super high-affinity, low-capacity (sHa/Lc) total glucose transport system in trout and a low-affinity, low-capacity (La/Lc) total glucose transport system in pig. Comparatively, electrogenic glucose absorption revealed similar Km in both fish species, with a super high-affinity, high capacity (sHa/Hc) system in tilapia; a super high-affinity/super low-capacity (sHa/sLc) system in trout; but a different Km fingerprint in the pig, with a high-affinity, low-capacity (Ha/Lc) system. This was supported by different responses to inhibitors of sodium-dependent glucose transporters (SGLTs) and glucose transporter type 2 (GLUT2) administered on the apical side between species. More specifically, tilapia flux was inhibited by SGLT inhibitors, but not the GLUT2 inhibitor, whereas trout lacked response to inhibitors. In contrast, the pig responded to inhibition by both SGLT and GLUT2 inhibitors with a higher expression of GLUT2. Altogether, it would appear that two pathways are working together in the pig, allowing it to have continued absorption at high glucose concentrations, whereas this is not present in both tilapia and trout.

SLC transporters ASCT2, B0 AT1-like, y+ LAT1, and LAT4-like associate with methionine electrogenic and radio-isotope flux kinetics in rainbow trout intestine.

Methionine (Met) is an important building block and metabolite for protein biosynthesis. However, the mechanism behind its absorption in the fish gut has not been elucidated. Here, we describe the fundamental properties of Met transport along trout gut at µmol/L and mmol/L concentration. Both electrogenic and unidirectional DL-[14 C]Met flux were employed to characterize Met transporters in Ussing chambers. Exploiting the differences in gene expression between diploid (2N) and triploid (3N) and intestinal segment as tools, allowed the association between gene and methionine transport. Specifically, three intestinal segments including pyloric caeca (PC), midgut (MG), and hindgut (HG) were assessed. Results at 0-150 µmol/L concentration demonstrated that the DL-Met was most likely transported by apical transporter ASCT2 (SLC1A5) and recycled by basolateral transporter y+ LAT1 (SLC7A7) due to five lines of observation: (1) lack of Na+ -independent kinetics, (2) low expression of B0 AT2-like gene, (3) Na+ -dependent, high-affinity (Km , µmol/L ranges) kinetics in DL-[14 C]Met flux, (4) association mRNA expression with the high-affinity kinetics and (5) electrogenic currents induced by Met. Results at 0.2-20 mmol/L concentration suggested that the DL-Met transport is likely transported by B0 AT1-like (SLC6A19-like) based on gene expression, Na+ -dependence and low-affinity kinetics (Km , mmol/L ranges). Similarly, genomic and gene expression analysis suggest that the basolateral exit of methionine was primarily through LAT4-like transporter (SLC43A2-like). Conclusively, DL-Met uptake in trout gut was most likely governed by Na+ -dependent apical transporters ASCT2 and B0 AT1-like and released through basolateral LAT4-like, with some recycling through y+ LAT1. A comparatively simpler model than that previously described in mammals.

Sigmoidal kinetics define porcine intestinal segregation of electrogenic monosaccharide transport systems as having multiple transporter population involvement.

Kinetic characterization of electrogenic sodium-dependent transport in Ussing chambers of d-glucose and d-galactose demonstrated sigmoidal/Hill kinetics in the porcine jejunum and ileum, with the absence of transport in the distal colon. In the jejunum, a high-affinity, super-low-capacity (Ha/sLc) kinetic system accounted for glucose transport, and a low-affinity, low-capacity (La/Lc) kinetic system accounted for galactose transport. In contrast, the ileum demonstrated a high-affinity, super-high-capacity (Ha/sHc) glucose transport and a low-affinity, high-capacity (La/Hc) galactose transport systems. Jejunal glucose transport was not inhibited by dapagliflozin, but galactose transport was inhibited. Comparatively, ileal glucose and galactose transport were both sensitive to dapagliflozin. Genomic and gene expression analyses identified 10 of the 12 known SLC5A family members in the porcine jejunum, ileum, and distal colon. Dominant SGLT1 (SLC5A1) and SGLT3 (SLC5A4) expression was associated with the sigmoidal Ha/sLc glucose and La/Lc galactose transport systems in the jejunum. Comparatively, the dominant expression of SGLT1 (SLC5A1) in the ileum was only associated with Ha glucose and La galactose kinetic systems. However, the sigmoidal kinetics and overall high capacity (Hc) of transport is unlikely accounted for by SGLT1 (SLC5A1) alone. Finally, the absence of transport and lack of pharmacological inhibition in the colon was associated with the poor expression of SLC5A genes. Altogether, the results demonstrated intestinal segregation of monosaccharide transport fit different sigmoidal kinetic systems. This reveals multiple transporter populations in each system, supported by gene expression profiles and pharmacological inhibition. Overall, this work demonstrates a complexity to transporter involvement in intestinal electrogenic monosaccharide absorption systems not previously defined.

Von Willebrand Factor Type A domain of hCLCA1 is sufficient for U-937 macrophage activation.

The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1ß mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.

The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca2+/calmodulin.

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Intestinal electrogenic sodium-dependent glucose absorption in tilapia and trout reveal species differences in SLC5A-associated kinetic segmental segregation.

Electrogenic sodium-dependent glucose transport along the length of the intestine was compared between the omnivorous Nile tilapia ( Oreochromis niloticus) and the carnivorous rainbow trout ( Oncorhynchus mykiss) in Ussing chambers. In tilapia, a high-affinity, high-capacity kinetic system accounted for the transport throughout the proximal intestine, midintestine, and hindgut segments. Similar dapagliflozin and phloridzin dihydrate inhibition across all segments support this homogenous high-affinity, high-capacity system throughout the tilapia intestine. Genomic and gene expression analysis supported findings by identifying 10 of the known 12 SLC5A family members, with homogeneous expression throughout the segments with dominant expression of sodium-glucose cotransporter 1 (SGLT1; SLC5A1) and sodium-myoinositol cotransporter 2 (SMIT2; SLC5A11). In contrast, trout's electrogenic sodium-dependent glucose absorption was 20-35 times lower and segregated into three significantly different kinetic systems found in different anatomical segments: a high-affinity, low-capacity system in the pyloric ceca; a super-high-affinity, low-capacity system in the midgut; and a low-affinity, low-capacity system in the hindgut. Genomic and gene expression analysis found 5 of the known 12 SLC5A family members with dominant expression of SGLT1 ( SLC5A1), sodium-glucose cotransporter 2 (SGLT2; SLC5A2), and SMIT2 ( SLC5A11) in the pyloric ceca, and only SGLT1 ( SLC5A1) in the midgut, accounting for differences in kinetics between the two. The hindgut presented a low-affinity, low-capacity system partially attributed to a decrease in SGLT1 ( SLC5A1). Overall, the omnivorous tilapia had a higher electrogenic glucose absorption than the carnivorous trout, represented with different kinetic systems and a greater expression and number of SLC5A orthologs. Fish differ from mammals, having hindgut electrogenic glucose absorption and segment specific transport kinetics.

Decreased electrogenic anionic secretory response in the porcine colon following in vivo challenge with Brachyspira spp. supports an altered mucin environment.

Brachyspira spp. cause diarrheal disease in multiple animal species by colonization of the colon, resulting in colitis, mucus induction, and disrupted ion transport. Unique to spirochete pathogenesis is the immense production of mucus, resulting in a niche mucin environment likely favoring spirochete colonization. Mucin rheological properties are heavily influenced by anionic secretion, and loss of secretory function has been implicated in diseases such as cystic fibrosis. Here, the effects on the agonist-induced electrogenic anionic secretory response by infectious colonic spirochete bacteria Brachyspira hyodysenteriae and Brachyspira hampsonii were assessed in the proximal, apex, and distal sections of colon in Ussing chambers. Activation of secretion via isoproterenol, carbachol, and forskolin/3-isobutyl-1-methylxanthine demonstrated a significantly decreased change in short-circuit current ( Isc) in Brachyspira-infected pigs in all sections. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in anionic secretion as indicated by a decrease in bumetanide inhibitable Isc. Quantitative RT-PCR and Western blot analyses determined that the major anionic channels of the epithelium were downregulated in diarrheic pigs paired with altered mucin gene expression. The investigated cytokines were not responsible for the downregulation of anion channel gene transcripts. Although IL-1α was upregulated in all segments, it did not alter cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in Caco-2 monolayers. However, a whole cell Brachyspira hampsonii lysate significantly reduced CFTR mRNA expression in Caco-2 monolayers. Together, these findings indicate that these two Brachyspira spp. may directly cause a decreased anionic secretory response in the porcine colon, supporting an altered mucin environment likely favoring spirochete colonization. NEW & NOTEWORTHY This research demonstrates for the first time that the niche mucin environment produced by two infectious spirochete spp. is supported by a decrease in the electrogenic anionic secretory response throughout the porcine colon. Our findings suggest that the host's cytokine response is not likely responsible for the decrease in anionic secretory function. Rather, it appears that Brachyspira spp. directly impede ion channel transcription and translation, potentially altering colonic mucin rheological properties, which may favor spirochete colonization.

Redundant contribution of a Transient Receptor Potential cation channel Member 1 exon 11 single nucleotide polymorphism to equine congenital stationary night blindness.

BACKGROUND: Congenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts. RESULTS: Retinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression. CONCLUSIONS: These finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.

Evaluation of inhibition of F4ac positive Escherichia coli attachment with xanthine dehydrogenase, butyrophilin, lactadherin and fatty acid binding protein.

BACKGROUND: Neonatal and post-weaning colibacillosis caused by enterotoxigenic E. coli is responsible for substantial economic losses encountered by the pork industry. Intestinal colonization of young piglets by E. coli depends on the efficiency of bacterial attachment to host gastrointestinal epithelium that is mediated by fimbriae. We tested the effect of porcine individual milk fat globule membrane (MFGM) proteins on F4ac positive E. coli attachment to porcine enterocytes in vitro. RESULTS: Butyrophilin, lactadherin and fatty acid binding protein inhibited fimbriae-dependent adherence of E. coli to enterocytes in vitro, while xanthine dehydrogenase did not. The inhibiting activity was dose-dependent for all three proteins, but the inhibiting efficiency was different. CONCLUSIONS: The results indicate that MFGM proteins may interfere with attachment of E. coli to porcine neonatal intestinal mucosa.

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane.

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.

Les Escherichia coli entérotoxinogénique (ETEC) positif pour F4ac doivent s'attacher à la muqueuse intestinale pour causer la diarrhée chez les porcelets. L'empêchement de l'attachement bactérien à la muqueuse intestinale est le moyen de défense le plus efficace contre la diarrhée induite par les ETEC. Les membranes de globules de gras de lait porcin (MFGM) ont été montré comme étant capable d'inhiber l'attachement des ETEC à la bordure en brosse intestinale; toutefois, les composantes spécifiques des MFGM porcines qui inhibaient l'attachement des ETEC aux entérocytes ne furent pas identifiées. Ainsi, le but de la présente étude était d'identifier les protéines des MFGM liant F4ac par immunobuvardage par superposition et chromatographie d'affinité. Le protéome des MFGM porcine fut caractérisé et les protéines liant F4as suivantes furent détectées par immunobuvardage par superposition et chromatographie d'affinité : lactadhérine, butyrophiline, adipophiline, acyl-CoA synthétase 3, et la protéine 3 liant les acides gras. La fonction biologique de ces protéines ne fut pas étudiée mais il est possible que leur interaction avec les fimbriae F4ac interfère avec l'attachement bactérien et la colonisation.(Traduit par Docteur Serge Messier).

Systemic lipopolysaccharide-mediated alteration of cortical neuromodulation involves increases in monoamine oxidase-A and acetylcholinesterase activity.

BACKGROUND: Lipopolysaccharide (LPS)-mediated sickness behaviour is known to be a result of increased inflammatory cytokines in the brain. Inflammatory cytokines have been shown to mediate increases in brain excitation by loss of GABAA-mediated inhibition through receptor internalization or inactivation. Inflammatory pathways, reactive oxygen species and stress are also known to increase monoamine oxidase-A (MAO-A) and acetylcholinesterase (ACh-E) activity. Given that neuromodulator actions on neural circuits largely depend on inhibitory pathways and are sensitive to alteration in corresponding catalytic enzyme activities, we assessed the impact of systemic LPS on neuromodulator-mediated shaping of a simple cortical network. METHODS: Extracellular field recordings of evoked postsynaptic potentials in adult mouse somatosensory cortical slices were used to evaluate effects of a single systemic LPS challenge on neuromodulator function 1 week later. Neuromodulators were administered transiently as a bolus (100 µl) to the bath perfusate immediately upstream of the recording site to mimic phasic release of neuromodulators and enable assessment of response temporal dynamics. RESULTS: Systemic LPS administration resulted in loss of both spontaneous and evoked inhibition as well as alterations in the temporal dynamics of neuromodulator effects on a paired-pulse paradigm. The effects on neuromodulator temporal dynamics were sensitive to the Monoamine oxidase-A (MAO-A) antagonist clorgyline (for norepinephrine and serotonin) and the ACh-E inhibitor donepezil (for acetylcholine). This is consistent with significant increases in total MAO and ACh-E activity found in hemi-brain samples from the LPS-treated group, supporting the notion that systemic LPS administration may lead to longer-lasting changes in inhibitory network function and enzyme (MAO/ACh-E) activity responsible for reduced neuromodulator actions. CONCLUSIONS: Given the significant role of neuromodulators in behavioural state and cognitive processes, it is possible that an inflammatory-mediated change in neuromodulator action plays a role in LPS-induced cognitive effects and could help define the link between infection and neuropsychiatric/degenerative conditions.