Maternal and/or post-weaning supplementation with Bacillus altitudinis spores modulates the microbial composition of colostrum, digesta and faeces in pigs.

This study examined the effects of maternal and/or post-weaning Bacillus altitudinis supplementation on the microbiota in sow colostrum and faeces, and offspring digesta and faeces. Sows (n = 12/group) were assigned to: (1) standard diet (CON), or (2) CON supplemented with probiotic B. altitudinis spores (PRO) from day (d)100 of gestation to weaning (d26 of lactation). At weaning, offspring were assigned to CON or PRO for 28d, resulting in: (1) CON/CON, (2) CON/PRO, (3) PRO/CON, and (4) PRO/PRO, after which all received CON. Samples were collected from sows and selected offspring (n = 10/group) for 16S rRNA gene sequencing. Rothia was more abundant in PRO sow colostrum. Sow faeces were not impacted but differences were identified in offspring faeces and digesta. Most were in the ileal digesta between PRO/CON and CON/CON on d8 post-weaning; i.e. Bacteroidota, Alloprevotella, Prevotella, Prevotellaceae, Turicibacter, Catenibacterium and Blautia were more abundant in PRO/CON, with Firmicutes and Blautia more abundant in PRO/PRO compared with CON/CON. Lactobacillus was more abundant in PRO/CON faeces on d118 post-weaning. This increased abundance of polysaccharide-fermenters (Prevotella, Alloprevotella, Prevotellaceae), butyrate-producers (Blautia) and Lactobacillus likely contributed to previously reported improvements in growth performance. Overall, maternal, rather than post-weaning, probiotic supplementation had the greatest impact on intestinal microbiota.

Maternal supplementation with Bacillus altitudinis spores improves porcine offspring growth performance and carcass weight.

The objective of this study was to evaluate the effect of feeding Bacillus altitudinis spores to sows and/or offspring on growth and health indicators. On day (D) 100 of gestation, twenty-four sows were selected and grouped as: control (CON), fed with a standard diet; and probiotic (PRO), fed the standard diet supplemented with B. altitudinis WIT588 spores from D100 of gestation until weaning. Offspring (n 144) from each of the two sow treatments were assigned to either a CON (no probiotic) or PRO (B. altitudinis-supplemented) treatment for 28 d post-weaning (pw), resulting in four treatment groups: (1) CON/CON, non-probiotic-supplemented sow/non-probiotic-supplemented piglet; (2) CON/PRO, non-probiotic-supplemented sow/probiotic-supplemented piglet; (3) PRO/CON, probiotic-supplemented sow/non-probiotic-supplemented piglet and (4) PRO/PRO, probiotic-supplemented sow/probiotic-supplemented piglet. B. altitudinis WIT588 was detected in the faeces of probiotic-supplemented sows and their piglets, and in the faeces and intestine of probiotic-supplemented piglets. Colostrum from PRO sows had higher total solids (P = 0·02), protein (P = 0·04) and true protein (P = 0·05), and lower lactose (P < 0·01) than colostrum from CON sows. Maternal treatment improved offspring feed conversion ratio at D0-14 pw (P < 0·001) and increased offspring body weight at D105 and D127 pw (P = 0·01), carcass weight (P = 0·05) and kill-out percentage (P < 0·01). It also increased small intestinal absorptive capacity and impacted the haematological profile of sows and progeny. There was little impact of pw treatment on any of the parameters measured. Overall, the lifetime growth benefits in the offspring of B. altitudinis-supplemented sows offer considerable economic advantages for pig producers in search of alternatives to in-feed antibiotics/zinc oxide.

Rethinking the bile acid/gut microbiome axis in cancer.

Dietary factors, probiotic agents, aging and antibiotics/medicines impact on gut microbiome composition leading to disturbances in localised microbial populations. The impact can be profound and underlies a plethora of human disorders, including the focus of this review; cancer. Compromised microbiome populations can alter bile acid signalling and produce distinct pathophysiological bile acid profiles. These in turn have been associated with cancer development and progression. Exposure to high levels of bile acids, combined with localised molecular/genome instability leads to the acquisition of bile mediated neoplastic alterations, generating apoptotic resistant proliferation phenotypes. However, in recent years, several studies have emerged advocating the therapeutic benefits of bile acid signalling in suppressing molecular and phenotypic hallmarks of cancer progression. These studies suggest that in some instances, bile acids may reduce cancer phenotypic effects, thereby limiting metastatic potential. In this review, we contextualise the current state of the art to propose that the bile acid/gut microbiome axis can influence cancer progression to the extent that classical in vitro cancer hallmarks of malignancy (cell invasion, cell migration, clonogenicity, and cell adhesion) are significantly reduced. We readily acknowledge the existence of a bile acid/gut microbiome axis in cancer initiation, however, in light of recent advances, we focus exclusively on the role of bile acids as potentially beneficial molecules in suppressing cancer progression. Finally, we theorise that suppressing aggressive malignant phenotypes through bile acid/gut microbiome axis modulation could uncover new and innovative disease management strategies for managing cancers in vulnerable cohorts.

Exploiting Interkingdom Interactions for Development of Small-Molecule Inhibitors of Candida albicans Biofilm Formation.

A rapid decline in the development of new antimicrobial therapeutics has coincided with the emergence of new and more aggressive multidrug-resistant pathogens. Pathogens are protected from antibiotic activity by their ability to enter an aggregative biofilm state. Therefore, disrupting this process in pathogens is a key strategy for the development of next-generation antimicrobials. Here, we present a suite of compounds, based on the Pseudomonas aeruginosa 2-heptyl-4(1H)-quinolone (HHQ) core quinolone interkingdom signal structure, that exhibit noncytotoxic antibiofilm activity toward the fungal pathogen Candida albicans In addition to providing new insights into what is a clinically important bacterium-fungus interaction, the capacity to modularize the functionality of the quinolone signals is an important advance in harnessing the therapeutic potential of signaling molecules in general. This provides a platform for the development of potent next-generation small-molecule therapeutics targeting clinically relevant fungal pathogens.

Influence of a low-carbohydrate diet on endothelial microvesicles in overweight women.

Low-carbohydrate diets (LCD) are increasing in popularity, but their effect on vascular health has been questioned. Endothelial microvesicles (EMV) are membrane-derived vesicles with the potential to act as a sensitive prognostic biomarker of vascular health and endothelial function. The aim of this study was to examine the influence of a LCD on EMV and other endothelial biomarkers of protein origin. Twenty-four overweight women (age, 48.4 ± 0.6 years; height, 1.60 ± 0.07 m; body mass, 76.5 ± 9.1 kg; body mass index, 28.1 ± 2.7 kg·m(-2); waist circumference, 84.1 ± 7.4 cm; mean ± standard deviation) were randomised to either 24 weeks on their normal diet (ND) or a LCD, after which they crossed over to 24 weeks on the alternative diet. Participants were assisted in reducing carbohydrate intake, but not below 40 g·day(-1). Body composition and endothelial biomarkers were assessed at the crossover point and at the end of the study. Daily carbohydrate intake (87 ± 7 versus 179 ± 11 g) and the percentage of energy derived from carbohydrate (29% versus 44%) were lower (p < 0.05) on the LCD compared to the ND, but absolute fat and saturated fat intake were unchanged. Body mass and waist circumference were 3.7 ± 0.8 kg and 3.5 ± 1.0 cm lower (p < 0.05), respectively, after the LCD compared with the ND phases. CD31(+)CD41(-)EMV, soluble (s) thrombomodulin, sE-selectin, sP-selectin, serum amyloid A and C-reactive protein were lower (p < 0.05) after the LCD compared to the ND, but serum lipids and apolipoproteins were not different. EMV along with a range of endothelial and inflammatory biomarkers are reduced by a LCD that involves modest weight loss.

Harnessing Bacterial Signals for Suppression of Biofilm Formation in the Nosocomial Fungal Pathogen Aspergillus fumigatus.

Faced with the continued emergence of antibiotic resistance to all known classes of antibiotics, a paradigm shift in approaches toward antifungal therapeutics is required. Well characterized in a broad spectrum of bacterial and fungal pathogens, biofilms are a key factor in limiting the effectiveness of conventional antibiotics. Therefore, therapeutics such as small molecules that prevent or disrupt biofilm formation would render pathogens susceptible to clearance by existing drugs. This is the first report describing the effect of the Pseudomonas aeruginosa alkylhydroxyquinolone interkingdom signal molecules 2-heptyl-3-hydroxy-4-quinolone and 2-heptyl-4-quinolone on biofilm formation in the important fungal pathogen Aspergillus fumigatus. Decoration of the anthranilate ring on the quinolone framework resulted in significant changes in the capacity of these chemical messages to suppress biofilm formation. Addition of methoxy or methyl groups at the C5-C7 positions led to retention of anti-biofilm activity, in some cases dependent on the alkyl chain length at position C2. In contrast, halogenation at either the C3 or C6 positions led to loss of activity, with one notable exception. Microscopic staining provided key insights into the structural impact of the parent and modified molecules, identifying lead compounds for further development.

Resistance exercise increases endothelial progenitor cells and angiogenic factors.

INTRODUCTION: Bone marrow-derived endothelial progenitor cells (EPC) are involved in vascular growth and repair. They increase in the circulation after a single bout of aerobic exercise, potentially related to muscle ischemia. Muscular endurance resistance exercise (MERE) bouts also have the potential to induce muscle ischemia if appropriately structured. PURPOSE: The objective of this study is to determine the influence of a single bout of MERE on circulating EPC and related angiogenic factors. METHODS: Thirteen trained men age 22.4 ± 0.5 yr (mean ± SEM) performed a bout of MERE consisting of three sets of six exercises at participants' 15-repetition maximum without resting between repetitions or exercises. The MERE bout duration was 12.1 ± 0.6 min. Blood lactate and HR were 11.9 ± 0.9 mmol·L and 142 ± 5 bpm, respectively, at the end of MERE. Blood was sampled preexercise and at 10 min, 2 h, and 24 h postexercise. RESULTS: Circulating EPC and serum concentrations of vascular endothelial growth factors (VEGF-A, VEGF-C, and VEGF-D), granulocyte colony stimulating factor, soluble Tie-2, soluble fms-like tyrosine kinase-1, and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, and MMP-9) were higher (P < 0.05) in the postexercise period. Circulating EPC levels were unchanged at 10 min postexercise but higher at 2 h postexercise (P < 0.05). The concentration of most angiogenic factors and metalloproteinases were higher at 10 min postexercise (VEGF-A, +38%; VEGF-C, +40%; VEGF-D, +9%; soluble Tie-2, +15%; soluble fms-like tyrosine kinase-1, +24%; MMP-1, +62%; MMP-2, +3%; MMP-3, +54%; and MMP-9, +45%; all P < 0.05). Soluble E-selectin was lower (P < 0.05) at 2 and 24 h postexercise, with endothelial microparticles and thrombomodulin unchanged. CONCLUSIONS: Short intense bouts of MERE can trigger increases in circulating EPC and related angiogenic factors, potentially contributing to vascular adaptation and vasculoprotection.

Essential role of hIST1 in cytokinesis.

The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, a new positive modulator of the ESCRT pathway, has been described recently, but an essential function of this highly conserved protein has not been identified. Here, we describe the previously uncharacterized KIAA0174 as the human homologue of IST1 (hIST1), and we report its conserved interaction with VPS4, CHMP1A/B, and LIP5. We also identify a microtubule interacting and transport (MIT) domain interacting motif (MIM) in hIST1 that is necessary for its interaction with VPS4, LIP5 and other MIT domain-containing proteins, namely, MITD1, AMSH, UBPY, and Spastin. Importantly, hIST1 is essential for cytokinesis in mammalian cells but not for HIV-1 budding, thus providing a novel mechanism of functional diversification of the ESCRT machinery. Last, we show that the hIST1 MIM activity is essential for cytokinesis, suggesting possible mechanisms to explain the role of hIST1 in the last step of mammalian cell division.

Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane.

Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including remodelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 interacts physically with the plasma-membrane-associated phosphoinositide 4-kinase, Stt4. In the present study, we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature-sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphoinositide 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependent events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals that there are substantially fewer plasma-membrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [(3)H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality under non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphoinositide 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of PtdIns4P in sac1Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have on levels of this phosphoinositide.

Fab1p and AP-1 are required for trafficking of endogenously ubiquitylated cargoes to the vacuole lumen in S. cerevisiae.

In S. cerevisiae synthesis of phosphatidylinositol (3,5)-bisphosphate [PtdIns(3,5)P2] by Fab1p is required for several cellular events, including an as yet undefined step in the ubiquitin-dependent trafficking of some integral membrane proteins from the trans-Golgi network to the vacuole lumen. AP-1 is a heterotetrameric clathrin adaptor protein complex that binds cargo proteins and clathrin coats, and regulates bi-directional protein trafficking between the trans-Golgi network and the endocytic/secretory pathway. Like fab1Delta cells, AP-1 complex component mutants have lost the ability to traffic ubiquitylated cargoes to the vacuole lumen - the first demonstration that AP-1 is required for this process. Deletion mutants of AP-1 complex components are compromised in their ability to synthesize PtdIns(3,5)P2, indicating that AP-1 is required for correct in vivo activation of Fab1p. Furthermore, wild-type protein sorting can be restored in AP-1 mutants by overexpression of Fab1p, implying that the protein-sorting defect in these cells is as a result of disruption of PtdIns(3,5)P2 synthesis. Finally, we show that Fab1p and Vac14p, an activator of Fab1p, are also required for another AP-1-dependent process: chitin-ring deposition in chs6Delta cells. Our data imply that AP-1 is required for some Fab1p and PtdIns(3,5)P2-dependent processes.

Caloric restriction increases longevity substantially only when the reaction norm is steep.

Evolutionary theory leads to the general expectation that dietary restriction will often result in increased survival probabilities, and thus increased lifespan. The reaction norm is a basic tool of evolutionary analysis that quantifies the relationship between environmental parameters and functional characters, including reproduction and longevity. In rodents, the reaction norm connecting adult longevity to caloric intake is fairly steep; small changes in intake lead to large changes in longevity. If this strong quantitative relationship were evolutionarily conserved among all mammals, then the prospects for a substantial increase in human lifespan from caloric restriction would be very good. In theory, however, reaction norms are expected to evolve for fitness related characters such as reproduction and survival. It has been shown experimentally in Drosophila that dietary reaction norms readily evolve in the laboratory, suggesting that they can do so among mammals as well, particularly over the millions of years since contemporary rodents and primates last shared a common ancestor. Our previous work crudely estimates that the dietary reaction norms of rodents and humans have diverged substantially, with a very flat dietary reaction norm for human longevity. These general principles and our specific results suggest that the benefits from human caloric restriction would be minor.

Weak acid and alkali stress regulate phosphatidylinositol bisphosphate synthesis in Saccharomyces cerevisiae.

Weak organic acids are used as food preservatives to inhibit the growth of spoilage yeasts, including Saccharomyces cerevisiae. Long-term adaptation to weak acids requires the increased expression of the ATP-binding cassette transporter Pdr12p, which catalyses the active efflux of the weak acids from the cytosol; however, very little is known about the signalling events immediately following application of weak acid stress. We have investigated the effects of weak acids on two stress-responsive signalling molecules, PtdIns(3,5)P2 and PtdIns(4,5)P2, which in S. cerevisiae are synthesized by Fab1p and Mss4p respectively. At low extracellular pH, benzoic acid, sorbic acid and acetic acid all cause a transient reduction in PtdIns(3,5)P2 accumulation and a more persistent rise in PtdIns(4,5)P2 levels. The increase in PtdIns(4,5)P2 levels is accompanied by a reorganization of the actin cytoskeleton. However, changes in PtdInsP2 levels are independent of weak acid-induced Pdr12p expression. In contrast, changing the extracellular medium to alkaline pH provokes a prolonged and substantial rise in PtdIns(3,5)P2 levels. As PtdIns(3,5)P2 synthesis is required for correct vacuole acidification, it is possible that levels of this molecule are modulated to maintain intracellular pH homoeostasis in response to weak acid and alkali stresses. In conclusion, we have expanded the repertoire of stress responses that affect PtdInsP2 levels to include weak acid and alkali stresses.

Why dietary restriction substantially increases longevity in animal models but won't in humans.

Caloric restriction (CR) extends maximum longevity and slows aging in mice, rats, and numerous non-mammalian taxa. The apparent generality of the longevity-increasing effects of CR has prompted speculation that similar results could be obtained in humans. Longevity, however, is not a trait that exists in a vacuum; it evolves as part of a life history and the physiological mechanisms that determine longevity are undoubtedly complex. Longevity is intertwined with reproduction and there is a cost to reproduction. The impact of this cost on longevity can be age-independent or age-dependent. Given the complexity of the physiology underlying reproductive costs and other mechanisms affecting life history, it is difficult to construct a simple model for the relationship between the particulars of the physiology involved and patterns of mortality. Consequently, we develop a hypothesis-neutral model describing the relationship between diet and longevity. Applying this general model to the special case of human longevity and diet indicates that the benefits of caloric restriction in humans would be quantitatively small.

Breakdown in correlations during laboratory evolution. I. Comparative analyses of Drosophila populations.

We provide evidence from comparisons of populations of Drosophila that evolutionary correlations between longevity and stress resistance break down over the course of laboratory evolution. Using 15 distinct evolutionary regimes, we created 75 populations that were differentiated for early fecundity, longevity, starvation resistance, desiccation resistance, and developmental time. In earlier experiments, selection for postponed aging produced increases in stress resistance, whereas selection for increased stress resistance produced increases in longevity. Direct estimates of correlations also indicated an antagonistic relationship between early fecundity on one hand and longevity or stress resistance on the other. Laboratory evolution of extreme values of stress resistance, however, led to a breakdown in these evolutionary relationships. There was no evidence that these significant changes in correlation resulted from genotype-by-environment interactions or inbreeding. These findings suggest that correlations between functional characters are not necessarily durable features of a species, and that short-term evolutionary responses cannot be extrapolated reliably to longer-term evolutionary patterns.

Breakdown in correlations during laboratory evolution. II. Selection on stress resistance in Drosophila populations.

We trace the evolutionary correlation between stress resistance and longevity in populations of Drosophila melanogaster selected for stress resistance over many generations. Females selected for desiccation resistance and both females and males selected for increasing starvation resistance initially show concurrent increases in longevity, but then begin to decrease in longevity, even as stress resistance continues to increase. We demonstrate that the correlation between two fitness traits can change and that this change is due to sustained selection rather than a genotype-by-environment interaction or inbreeding depression. The breakdown in evolutionary correlation we report underscores the difficulty of extrapolating the results from short-term selection experiments to predictions of long-term evolution.

Crystal structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein.

We report the 2.1 A crystal structure of the core G protein domain of the unusual Rho family member RhoE/Rnd3 in complex with endogenous GTP and magnesium. Unlike other small G proteins, RhoE, along with two other proteins Rnd1/Rho6 and Rnd2/RhoN, does not hydrolyze GTP. The main reason for this is the presence of serines in the positions equivalent to Ala59 and Gln61 in Ras. The structure shows that there are still water molecules in similar positions to the waters thought to be involved in the hydrolysis reaction in other G proteins. The structure suggests three not necessarily exclusive explanations for the lack of hydrolysis. The lack of the conserved glutamine raises the energy of the transition state inhibiting hydrolysis. The serines may restrain the waters from moving closer to the GTP, a step that is required to attain the transition state. They also stabilize the GTP-bound conformation of switch II and could prevent conformational changes required during hydrolysis. By superposition of the RhoE structure on structures of Rho family proteins in complex with binding partners, we make predictions on RhoE interactions with these partners.

The X-ray crystal structure and putative ligand-derived peptide binding properties of gamma-aminobutyric acid receptor type A receptor-associated protein.

The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABA(A) receptor and microtubules. We present the three-dimensional structure of GABARAP obtained by x-ray diffraction at 1.75 A resolution. The structure was determined by molecular replacement using the structure of the homologous protein GATE-16. NMR spectroscopy of isotope-labeled GABARAP showed the structure in solution to be compatible with the overall fold but showed evidence of conformation heterogeneity that is not apparent in the crystal structure. We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the gamma2 subunit of the GABA(A) receptor and the acidic carboxyl-terminal tails of human alpha- and beta-tubulin. There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides. These results are compatible with a more general role in membrane targeting and transportation for the GABARAP family of proteins.