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Cystic fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel that impair airway salt and fluid secretion. Excessive release of pro-inflammatory cytokines and chemokines by CF bronchial epithelium during airway infection leads to chronic inflammation and a slow decline in lung function, thus there is much interest in finding safe and effective treatments that reduce inflammation in CF. We showed previously that the cyclic nucleotide phosphodiesterase (PDE) inhibitor ensifentrine (RPL554; Verona Pharma) stimulates the channel function of CFTR mutants with abnormal gating and also those with defective trafficking that are partially rescued using a clinically approved corrector drug. PDE inhibitors also have known anti-inflammatory effects, therefore we examined whether ensifentrine alters the production of pro-inflammatory cytokines in CF bronchial epithelial cells. Ensifentrine reduced the production of monocyte chemoattractant protein-1 (MCP-1) and granulocyte monocyte colony stimulating factor (GM-CSF) during challenge with Interleukin-1β. Comparing the effect of ensifentrine with milrinone and roflumilast, selective PDE3 and PDE4 inhibitors respectively, demonstrated that the anti-inflammatory effect of ensifentrine was mainly due to inhibition of PDE4. Beneficial modulation of GM-CSF was further enhanced when ensifentrine was combined with low concentrations of the β2-adrenergic agonist isoproterenol or the corticosteroid dexamethasone. The results indicate ensifentrine may have beneficial anti-inflammatory effects in CF airways particularly when used in combination with β2-adrenergic agonists or corticosteroids. Significance Statement Airways inflammation that is disproportionate to the burden of chronic airway infection causes much of the pathology in the Cystic Fibrosis (CF) lung. We show here that ensifentrine beneficially modulates the release of pro-inflammatory factors in well-differentiated CF bronchial epithelial cells that is further enhanced when combined with β2-adrenergic agonists or low-concentration corticosteroids. The results encourage further clinical testing of ensifentrine, alone and in combination with β2-adrenergic agonists or low-concentration corticosteroids, as a novel anti-inflammatory therapy for CF.

Treatment of individual trees in hardwood stands typically is conducted with herbicides that have no soil activity, such as triclopyr. However, triclopyr is not effective on some tree species. Applying a mixture of triclopyr and imazapyr would broaden the spectrum of species controlled, but nontarget mortality may be problematic as imazapyr may affect other trees through soil activity. We applied herbicide via girdle-and-spray as part of a forest stand improvement treatment in four upland hardwood stands in the Upper Coastal Plain of Alabama. We compared effects of using triclopyr alone with a mixture of triclopyr and imazapyr 18 months posttreatment. Only one untreated sweetgum (Liquidambar styraciflua) out of 440 trees was killed in the stands treated with the herbicide mixture (0.5 percent nontarget mortality rate). Nontarget mortality did not differ between treatments. However, the herbicide mixture controlled hickory (Carya spp.) and sourwood (Oxydendrum arboreum) better than triclopyr alone, with 56 percent of hickory treated with triclopyr still alive 18 months later, compared with 0 percent of hickory treated with the mixture. Our results indicate a mixture of triclopyr and imazapyr provides better control than triclopyr alone, and there is minimal risk to nontarget tree species in hardwood stands when used according to label recommendations.

Background Probiotic bacteria can provide health benefits when delivered in functional foods. This study involved isolation of lactic acid bacteria (LAB) from traditionally dried and salted anchovy fish and characterization of their survival in simulated gastrointestinal digestion. Promising strains were used to prepare fermented fish sausages which were then evaluated for cytotoxicity activity against two cancer cell-lines, antidiabetic activity as determined by α-amylase and α-glucosidase inhibition, and antioxidant and proteolytic activities in vitro, as compared to non-fermented control sausages. Results Out of 85 LAB obtained, 13 isolates with high tolerance to simulated gastrointestinal digestion were obtained, which were identified as Enterococcus spp. Four E. faecium strains, one E. faecalis, and one E. durans were used separately to make fermented fish sausages. The α-amylase and α-glucosidase inhibition from fish sausages fermented by Enterococcus spp. ranged from 29.2 to 68.7% and 23.9 to 41.4%, respectively, during 21 days of storage. The cytotoxicity activities against Caco2 and MCF-7 cells of fish sausages fermented with Enterococcus spp. ranged from 18.0 to 24% and 13.9 to 27.9%, respectively. Cytotoxicity activities correlated positively with proteolysis and antioxidant activities, α-amylase and α-glucosidase inhibition activities, but negatively with the pH in fermented fish sausages. Strains also exhibited antimicrobial activity against foodborne pathogens and presented no significant concerns with regards to antibiotic resistance or virulence gene content. Conclusions Fish sausages fermented by potential probiotic isolates of Enterococcus spp. from dried fish had valuable health-promoting benefits compared with non-fermented control sausages.

The selection of potential probiotic strains that possess the physiological capacity of performing successfully in the gastrointestinal tract (GIT) is a critical challenge. Probiotic microorganisms must tolerate the deleterious effects of various stresses to survive passage and function in the human GIT. Adhesion to the intestinal mucosa is also an important aspect. Recently, numerous studies have been performed concerning the selection and evaluation of novel probiotic microorganisms, mainly probiotic bacteria isolated from dairy and nondairy products. Therefore, it would be crucial to critically review the assessment methods employed to select the potential probiotics. This article aims to review and discuss the recent approaches, methods used for the selection, and outcomes of the evaluation of novel probiotic strains with the main purpose of supporting future probiotic microbial assessment studies. The findings and approaches used for assessing acid tolerance, bile metabolism and tolerance, and adhesion capability are the focus of this review. In addition, probiotic bile deconjugation and bile salt hydrolysis are explored. The selection of a new probiotic strain has mainly been based on the in vitro tolerance of physiologically related stresses including low pH and bile, to ensure that the potential probiotic microorganism can survive the harsh conditions of the GIT. However, the varied experimental conditions used in these studies (different types of media, bile, pH, and incubation time) hamper the comparison of the results of these investigations. Therefore, standardization of experimental conditions for characterizing and selecting probiotics is warranted.

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.

The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in β-lactam resistance, however, is unclear despite being first identified a decade ago., The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic β-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis. Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity.

Identifying risk factors for impaired oral rotavirus vaccine (ORV) efficacy in low-income countries may lead to improvements in vaccine design and delivery. We measured maternal rotavirus antibodies, environmental enteric dysfunction (EED), and bacterial gut microbiota development among infants receiving two doses of Rotarix in India (n = 307), Malawi (n = 119), and the UK (n = 60), using standardised methods across cohorts. ORV shedding and seroconversion rates were significantly lower in Malawi and India than the UK. Maternal rotavirus-specific antibodies in serum and breastmilk were negatively correlated with ORV response in India and Malawi, and this was mediated partly by a reduction in ORV replication. In the UK, ORV replication was not inhibited despite comparable maternal antibody levels. In both India and Malawi, pre-vaccination microbiota diversity was negatively correlated with ORV immunogenicity, suggesting that high early-life microbial exposure may contribute to impaired vaccine efficacy.

IntroductionChronic lung disease of prematurity (CLD), also known as bronchopulmonary dysplasia (BPD), is a cause of significant respiratory morbidity in childhood and beyond. Coupled with lung immaturity, infections (especially by Ureaplasma spp) are implicated in the pathogenesis of CLD through promotion of pulmonary inflammation. Azithromycin, which is a highly effective against Ureaplasma spp also has potent anti-inflammatory properties. Thus, azithromycin therapy may improve respiratory outcomes by targeting infective and inflammatory pathways. Previous trials using macrolides have not been sufficiently powered to definitively assess CLD rates. To address this, the azithromycin therapy for chronic lung disease of prematurity (AZTEC) trial aims to determine if a 10-day early course of intravenous azithromycin improves rates of survival without CLD when compared with placebo with an appropriately powered study.Methods and analysis796 infants born at less than 30 weeks’ gestational age who require at least 2 hours of continuous respiratory support within the first 72 hours following birth are being enrolled by neonatal units in the UK. They are being randomised to receive a double-blind, once daily dose of intravenous azithromycin (20 mg/kg for 3 days, followed by 10 mg/kg for a further 7 days), or placebo. CLD is being assessed at 36 weeks’ PMA. Whether colonisation with Ureaplasma spp prior to randomisation modifies the treatment effect of azithromycin compared with placebo will also be investigated. Secondary outcomes include necrotising enterocolitis, intraventricular/cerebral haemorrhage, retinopathy of prematurity and nosocomial infections, development of antibiotic resistance and adverse reactions will be monitored.Ethics and disseminationEthics permission has been granted by Wales Research Ethics Committee 2 (Ref 18/WA/0199), and regulatory permission by the Medicines and Healthcare Products Regulatory Agency (Clinical Trials Authorisation reference 21323/0050/001–0001). The study is registered on ISRCTN (ISRCTN11650227). The study is overseen by an independent Data Monitoring Committee and an independent Trial Steering Committee. We shall disseminate our findings via national and international peer-reviewed journals, and conferences. A summary of the findings will also be posted on the trial website.

This chapter describes methods used to isolate, identify, and partially characterize lactic acid bacteria (LAB) which exhibit inhibitory activity against Listeria monocytogenes from foods. Vegetal (plant based) sources are rich in naturally occurring LAB and therefore provide an easily accessible source of strains with potential antimicrobial activity for use in food-processing applications. From our previous work, the majority of LAB with inhibitory activity against L. monocytogenes were identified as generally recognized as safe (GRAS) Lactococcus lactis. Although these bacteria are most commonly known for their role in industrial dairy fermentations, they are believed to have originally derived from natural plant-based habitats. These isolates with anti-Listeria activity were all found to carry the genes involved in the production of nisin, which is an approved food-grade preservative (E234). These isolates may find various applications for in situ production of nisin allowing control of L. monocytogenes in various fermented and non-fermented foods and other environments.

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator ( cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.

The S100 family of proteins contains 25 known members that share a high degree of sequence and structural similarity. However, only a limited number of family members have been characterized in depth, and the roles of other members are likely undervalued. Their importance should not be underestimated however, as S100 family members function to regulate a diverse array of cellular processes including proliferation, differentiation, inflammation, migration and/or invasion, apoptosis, Ca2+ homeostasis, and energy metabolism. Here we detail S100 target protein interactions that underpin the mechanistic basis to their function, and discuss potential intervention strategies targeting S100 proteins in both preclinical and clinical situations.

Food processing treatments can alter the microbial biodiversity which can significantly affect shelf-life and safety. These changes in biodiversity can be analysed using culture-independent community profiling methods to provide a better understanding of which bacteria are resistant to the process. In this study, we used rice as a model food that contains a rich bacterial biodiversity to determine what effect variable high pressure processing (HPP) parameters and/or cooking has on the bacterial population. Two samples of milled rice each from two harvest years were pressured at 0, 200, 400 or 600 MPa for 10 min at 25 °C, then either cooked at 100 °C or left uncooked. Samples were stored at 25 °C for up to 8 weeks or until spoilage was detected by total aerobic plate count. Bacterial DNA was extracted and the community composition was analysed by Illumina sequencing of 250 bp of 16S rDNA. Pressured, uncooked samples spoiled after one day of storage at 25 °C and it was found that pressures at and above 400 MPa selected for aerobic sporeformers namely Bacillaceae being the dominant group. Of these, Bacillus cereus, Bacillus subtilis or Paenibacillus amylolyticus groups were the most abundant in samples. The untreated rehydrated rice control and rice treated at 200 MPa contained spoilage populations consisting of a mix of primarily Enterobacteriaceae and Bacillaceae suggesting that 200 MPa treatment was not severe enough to eliminate Gram-negative bacteria. Cooked rice samples spoiled after three days storage at 25 °C and contained almost exclusively Bacillus spp. with B. subtilis and B. cereus groups dominating the populations. With pressures of 400 and 600 MPa, cooked samples remained unspoilt after eight weeks of storage at 25 °C with sequences representing DNA from a highly heterogeneous mix of non-viable bacterial cells. The lower pressure treatment (200 MPa) combined with cooking resulted in one out of four samples spoiling due to growth of Bacillus flexus group bacteria. In summary, the culture-independent method used here demonstrates that higher HPP (400 and 600 MPa) treatment or cooking alone selected for sporeforming bacterial spoilage profiles. A combination of HPP (400 and 600 MPa) with cooking resulted in ambient temperature stable rice, with no growth of potentially hazardous sporeformers observed.

Hyperinsulinaemia potentially contributes to insulin resistance in metabolic tissues, such as skeletal muscle. The purpose of these experiments was to characterise glucose uptake, insulin signalling and relevant gene expression in primary human skeletal muscle-derived cells (HMDCs), in response to prolonged insulin exposure (PIE) as a model of hyperinsulinaemia-induced insulin resistance. Differentiated HMDCs from healthy human donors were cultured with or without insulin (100 nM) for 3 days followed by an acute insulin stimulation. HMDCs exposed to PIE were characterised by impaired insulin-stimulated glucose uptake, blunted IRS-1 phosphorylation (Tyr612) and Akt (Ser473) phosphorylation in response to an acute insulin stimulation. Glucose transporter 1 (GLUT1), but not GLUT4, mRNA and protein increased following PIE. The mRNA expression of metabolic (PDK4) and inflammatory markers (TNF-α) was reduced by PIE but did not change lipid (SREBP1 and CD36) or mitochondrial (UCP3) markers. These experiments provide further characterisation of the effects of PIE as a model of hyperinsulinaemia-induced insulin resistance in HMDCs.

The control of Listeria monocytogenes in soft cheese manufacture remains a challenge due to its widespread occurrence, ability to grow at refrigerated temperatures and salt resistance. The application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives for improving cheese quality and safety has attracted significant research interest. The aim of this study was to select non-dairy origin LAB strains with anti-Listeria activity and to characterise their industrially relevant properties for potential roles as biopreservative adjunct cultures for use in cheese production. A total of 897 LAB isolated from herbs, fruits and vegetables were screened for their anti-Listeria activity using an MRS agar overlay assay. Fourteen isolates with strong activity were identified by 16S rRNA gene sequencing as Lactococcus lactis and all contained the nisin gene cluster. Of these fourteen isolates, four strains also showed detectable anti-Listeria activity in a milk medium. These wild Lc. lactis strains possessed slow lactose utilisation and acidification capacity in milk despite exhibiting high growth rates. This was likely due to the adoption of the mixed acid fermentation pathway since ethanol was a major metabolic end product. Analysis of volatiles in milk and laboratory scale cheese fermented with wild and industrial Lc. lactis strain combination using headspace solid phase microextraction (SPME) GC-MS identified up to a 300–400 fold increase in the production of 3-methylbutanal, an important cheese odorant with a malty flavour. Whole genome sequencing of four wild strains provided some genetic insights into their phenotypes. The wild Lc. lactis lack the lactose utilisation operon (lacABCDFEG) commonly found in industrial strains, but contain genes encoding a putative β-galactosidase and a previously characterised alternative lactose assimilation pathway with slow lactose uptake. Wild strains also contain genes encoding α-keto-acid decarboxylase (kdcA) required for 3-methylbutanal production. These results provide insight into technological properties of wild non-dairy Lc. lactis strains which could be potentially used as biopreservative adjunct cultures to control the growth of L. monocytogenes in cheese.

High-moisture snacks, such as steamed buns and rice cakes, are traditional and popular in Asian countries. However, their shelf life is short, primarily due to microbial spoilage. Current manufacturing methods address this shortcoming through the use of chemical preservatives. To satisfy consumers’ demand for preservative-free food, thermal sterilization of a model high-moisture snack (steamed rice cakes) is investigated in this work. Bacillus cereus spores are heat-resistant pathogens typically found in rice products; hence, they constitute a suitable candidate to assess the effectiveness of thermal sterilization. A validated combination of predicted temperature profile of rice cakes based on thermal properties extracted experimentally with thermal inactivation kinetics of B. cereus spores allows us to assess the sensitivity of processing conditions to sterilization efficiency. Using both experimentation and modelling, it is shown that enhancement of heat transfer by improving convection from the heating medium (either water or steam) has a limited effect on inactivation due to the intrinsic kinetics of spore inactivation.

Diacetyl and the closely related compound acetoin impart desirable buttery flavour and odour to many foods including cheese and are generated through the metabolism of citrate by lactic acid bacteria (LAB). To increase the levels of these compounds, adjunct cultures capable of producing them can be added to cheese fermentations. In this study, we compared the diacetyl and acetoin producing abilities of 13 Lactobacillus rhamnosus strains from cheese sources. Diacetyl and acetoin production was found to be a common feature of Lb. rhamnosus grown in milk, with 12 strains producing these compounds. Whole genome sequencing of four strains revealed that genes encoding the citrate metabolising pathway present in other LAB are conserved in Lb. rhamnosus. One strain was, however, totally defective in diacetyl and acetoin production. This was likely due to an inability to produce the diacetyl/acetoin precursor compound acetolactate resulting from a frameshift mutation in the acetolactate synthase (als) gene. Complementation of this defective strain with a complete als gene from a diacetyl producing strain restored production of diacetyl and acetoin to levels equivalent to naturally high producing strains. Introduction of the same als-containing plasmid into the probiotic Lb. rhamnosus strain GG also increased diacetyl and acetoin levels. In model cheesemaking experiments, the als-complemented strain produced very high levels of diacetyl and acetoin over 35days of ripening. These findings identify the genetic basis for natural variation in production of a key cheese flavour compound in Lb. rhamnosus strains.

Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O2 ; 21-1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O2 ; with culture at 5% and 1% O2 causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O2 , myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O2 levels. At the molecular level we observed increases in mRNA expression of MuRF-1 only at 1% O2 whereas MAFbx expression was elevated at 10%, 5%, and 1% O2 . In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O2 , with no significant changes seen above this O2 level. Overall, these data suggest that engineered muscle exposed to O2 levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599-2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Transepithelial bicarbonate secretion by human airway submucosal glands and surface epithelial cells is crucial to maintain the pH-sensitive innate defence mechanisms of the lung. cAMP agonists stimulate HCO3 (-) secretion via coordinated increases in basolateral HCO3 (-) influx and accumulation, as well as CFTR-dependent HCO3 (-) efflux at the luminal membrane of airway epithelial cells. Here, we investigated the regulation of a basolateral located, DIDS-sensitive, Cl-/HCO3 (-) exchanger, anion exchanger 2 (AE2; SLC4A2) which is postulated to act as an acid loader, and therefore potential regulator of HCO3 (-) secretion, in human airway epithelial cells. Using intracellular pH measurements performed on Calu-3 cells, we demonstrate that the activity of the basolateral Cl-/HCO3 (-) exchanger was significantly downregulated by cAMP agonists, via a PKA-independent mechanism and also required Ca2+ and calmodulin under resting conditions. AE2 contains potential phosphorylation sites by a calmodulin substrate, protein kinase CK2, and we demonstrated that AE2 activity was reduced in the presence of CK2 inhibition. Moreover, CK2 inhibition abolished the activity of AE2 in primary human nasal epithelia. Studies performed on mouse AE2 transfected into HEK-293T cells confirmed almost identical Ca2+/calmodulin and CK2 regulation to that observed in Calu-3 and primary human nasal cells. Furthermore, mouse AE2 activity was reduced by genetic knockout of CK2, an effect which was rescued by exogenous CK2 expression. Together, these findings are the first to demonstrate that CK2 is a key regulator of Cl--dependent HCO3 (-) export at the serosal membrane of human airway epithelial cells.

Lactobacillus plantarum is a lactic acid bacterium commonly found on fruits and vegetables and also used in a variety of food fermentations. Strains from this species are also regularly reported as having antifungal or probiotic activity. Genotyping methods can be used to differentiate strains of the same species thus determining if strains are related or not. However for L. plantarum, the currently used methods have limitations including DNA band profile interpretation difficulty and cost. In this study, a new genotyping method based on multi-locus variable number tandem repeat analysis (MLVA) was developed and compared to a previously reported randomly amplified polymorphic DNA-PCR (RAPD-PCR) method for L. plantarum. With a selection of 13 antifungal strains of L. plantarum isolated from heterogeneous sources (cheese, silage, sauerkraut, vegetables and a probiotic product), RAPD-PCR revealed 9 different profiles resulting in a Hunter-Gaston discrimination index (D-value) of 0.94. The new MLVA method which compares the lengths of 4 repetitive regions within LPXTG motif-containing surface protein genes differentiated the 13 L. plantarum strains into 10 different subtypes leading to a D-value of 0.95. Interestingly 11 additional L. plantarum isolates obtained in a previous study during a screen for antifungal activity against the common cheese spoilage mould Penicillium commune all possessed the same RAPD-PCR and MLVA profile as each other and the commercial probiotic strain L. plantarum 299v. This study demonstrates that the new MLVA method can be used to simply and inexpensively differentiate L. plantarum strains and provide information regarding strain relatedness and thus potential insight into strain properties.

This study investigated the survival of probiotic Lactobacillus plantarum 299v microencapsulated in native maize starch or partially hydrolyzed maize starches after acid, bile and heat treatments. Scanning electron microscopy and confocal scanning laser microscopy confirmed that naturally present cavities and channels in native maize starch were enlarged by enzymatic hydrolysis allowing them to be filled with probiotics. The formulations using the modified starches had significantly higher initial viable cells compared to native starch after freeze-drying. Compared to free cells, the microencapsulated probiotic bacteria showed a significant improvement in acid tolerance. When comparing unmodified and modified starches, the enzymatic treatments did not significantly improve relative survival, but did result in significantly higher total probiotic numbers after exposure to acid (pH = 2.0, 1 h), bile salt (3% w/v, 4 h) and heat (60 °C, 15min). These results demonstrate that porous maize starch granules allow for a high probiotic loading efficiency and provide enhanced protection to various stressful conditions compared to free cells.

Cyclic di-AMP (c-di-AMP) is a second messenger which plays a major role in osmotic homeostasis in bacteria. In work by Quintana et al. (I. M. Quintana, J. Gibhardt, A. Turdiev, E. Hammer, et al., J Bacteriol 201:e00028-19, 2019, https://doi.org/10.1128/jb.00028-19), two Kup homologs from Lactococcus lactis were identified as high-affinity K+ importers whose activities are inhibited by direct binding of c-di-AMP. The results broaden the scope of K+ level regulation by c-di-AMP, with Kup homologs found in a number of pathogenic, commensal, and industrial bacteria.

Context The mechanisms responsible for dietary fat-induced insulin resistance of skeletal muscle and its microvasculature are only partially understood. Objective To determine the impact of high-fat overfeeding on postprandial glucose fluxes, muscle insulin signaling, and muscle microvascular endothelial nitric oxide synthase (eNOS) content and activation. Design Fifteen non-obese volunteers consumed a high-fat (64%) high-energy (+47%) diet for 7 days. Experiments were performed before and after the diet. Stable isotope tracers were used to determine glucose fluxes in response to carbohydrate plus protein ingestion. Muscle insulin signaling was determined as well as the content and activation state of muscle microvascular eNOS. Results High-fat overfeeding impaired postprandial glycemic control as demonstrated by higher concentrations of glucose (+11%; P = 0.004) and insulin (+19%; P = 0.035). Carbohydrate plus protein ingestion suppressed endogenous glucose production to a similar extent before and after the diet. Conversely, high-fat overfeeding reduced whole-body glucose clearance (–16%; P = 0.021) and peripheral insulin sensitivity (–26%; P = 0.006). This occurred despite only minor alterations in skeletal muscle insulin signaling. High-fat overfeeding reduced eNOS content in terminal arterioles (P = 0.017) and abolished the increase in eNOS Ser1177 phosphorylation that was seen after carbohydrate plus protein ingestion. Conclusion High-fat overfeeding impaired whole-body glycemic control due to reduced glucose clearance, not elevated endogenous glucose production. The finding that high-fat overfeeding abolished insulin-mediated eNOS Ser1177 phosphorylation in the terminal arterioles suggests that impairments in the vasodilatory capacity of the skeletal muscle microvasculature may contribute to early dietary fat-induced impairments in glycemic control.

The European Food Safety Authority and World Health Organization have been screening foods for acrylamide contamination and have published various guidelines to educate industries about mitigation strategies. There are several approaches to reduce the levels of acrylamide in food. Some aim to reduce the levels of asparagine and reduced sugars, which are involved in the formation of acrylamide, whereas others aim to change environmental factors such as temperature or pH. Meanwhile, biological methods to reduce acrylamide content have produced yielded results and, in some cases, have improved food quality. This study provides an updated and comprehensive review on biological acrylamide mitigation with a special focus on lactic acid bacteria (LAB), yeast, and microbial cell-free extracts. Nonetheless, we shed light on the current findings of acrylamide-related research on non-microbial approaches of mitigation and the updated dietary intake. Studies have revealed that the microbial approach to mitigate acrylamide is comparable to technological approaches. Several LAB species, yeast, and cell-free extracts displayed promising acrylamide removal capabilities. The literature has gaps in knowledge regarding acrylamide reduction using LAB, and thus, further efforts are warranted to maximize the role of microorganisms and understand their mechanisms of acrylamide removal.

In this work we investigated the potential of a new dairy-based fermented food which contains a nisin producing Lactococcus lactis strain with biopreservative function and a prebiotic. Lc lactis 537 was incubated in ultrahigh temperature (UHT) skim milk in the presence of 8 different commercially available prebiotics (inulin, fructooligosaccharide, xylooligosaccharide, lactulose, gum arabic, maltodextrin, isomaltooligosaccharides, galactooligosaccharides) at 3% concentration. The growth of Lc. lactis 537 was found to be enhanced in the presence of inulin only, as compared to in milk without prebiotic. Acidification was minimal, with all milks with or without prebiotic remaining above pH 6. Survival during storage for 14 days at 4 °C was high with or without prebiotic, with less than a 1-log CFU/mL reduction in viability seen. Antimicrobial production by Lc. lactis 537 was evaluated and found to be significantly higher when cells were grown in the presence of inulin. A nisB mutant of Lc. lactis 537 did not have any antimicrobial activity with or without prebiotic, thus demonstrating the effect was due to nisin. The findings of this study suggest that inulin is the most promising prebiotic inclusion for the development of a milk-based symbiotic product containing Lc. lactis 537.

Listeria monocytogenes is a potentially deadly psychrotrophic pathogen that can cause illness through contamination of ready-to-eat (RTE) foods. In this work we evaluated the use of a fermentate consisting of cells and supernatant of a nisin producing Lactococcus lactis strain to control L. monocytogenes in foods. In addition, to determine the role of nisin production in this activity, a transposon screen was carried out to isolate a nisin-negative mutant. Several independent mutants containing transposon insertions in the nisin gene cluster were obtained which exhibited a complete loss of anti-L. monocytogenes activity in an agar-based assay. A simple media using food grade components was found to support strong growth and nisin production of Lc. lactis 537 following incubation at between 20 and 35 °C. The ability of the fermentate to control L. monocytogenes in foods was evaluated in cut iceberg lettuce and RTE sliced ham stored at 4 °C for 10 days. Initial levels of 103–104 CFU/g L. monocytogenes were reduced to below detection limit (

Bacteria can sense environmental cues and alter their physiology accordingly through the use of signal transduction pathways involving second messenger nucleotides. One broadly conserved second messenger is cyclic-di-AMP (c-di-AMP) which regulates a range of processes including cell wall homeostasis, potassium uptake, DNA repair, fatty acid synthesis, biofilm formation and central metabolism in bacteria. The intracellular pool of c-di-AMP is maintained by the activities of diadenylate cyclase (DAC) and phosphodiesterase (PDE) enzymes, as well as possibly via c-di-AMP export. Whilst extracellular stimuli regulating c-di-AMP levels in bacteria are poorly understood, recent work has identified effector proteins which directly interact and alter the activity of DACs. These include the membrane bound CdaR and the phosphoglucosamine mutase GlmM which both bind directly to the membrane bound CdaA DAC and the recombination protein RadA which binds directly to the DNA binding DisA DAC. The genes encoding these multiprotein complexes are co-localised in many bacteria providing further support for their functional connection. The roles of GlmM in peptidoglycan synthesis and RadA in Holliday junction intermediate processing suggest that c-di-AMP synthesis by DACs will be responsive to these cellular activities. In addition to these modulatory interactions, permanent dysregulation of DAC activity due to suppressor mutations can occur during selection to overcome growth defects, rapid cell lysis and osmosensitivity. DACs have also been investigated as targets for the development of new antibiotics and several small compound inhibitors have recently been identified. This review aims to provide an overview of how c-di-AMP synthesis by DACs can be regulated.

OBJECTIVES CoNS are the most common cause of neonatal late-onset sepsis. Information on the vancomycin pharmacokinetics/pharmacodynamics against CoNS is limited. The aim of this study was to characterize vancomycin pharmacokinetic/pharmacodynamic relationships for CoNS and investigate neonatal optimal dosage regimens. METHODS A hollow fibre and a novel rabbit model of neonatal central line-associated bloodstream CoNS infections were developed. The results were then bridged to neonates by use of population pharmacokinetic techniques and Monte Carlo simulations. RESULTS There was a dose-dependent reduction in the total bacterial population and C-reactive protein levels. The AUC/MIC and Cmax/MIC ratios were strongly linked with total and mutant resistant cell kill. Maximal amplification of resistance was observed in vitro at an fAUC/MIC of 200 mg · h/L. Simulations predicted that neonates

Whey is a valuable co-product from cheese making that serves as a raw material for a wide range of products. Its rich nutritional content lends itself to rapid spoilage, thus it typically needs to be pasteurised and refrigerated promptly. Despite the extensive literature on milk spoilage bacteria, little is known about the spoilage bacteria of whey. The utility of carbon dioxide (CO2) to extend the shelf-life of raw milk and cottage cheese has been well established, but its application in whey preservation has not yet been explored. This study aims to characterise the microbial populations of fresh and spoiled sweet whey by culture-independent community profiling using 454 pyrosequencing of 16S rRNA gene amplicons and to determine whether carbonation is effective in inhibiting bacterial growth in sweet whey. The microbiota of raw Cheddar and Mozzarella whey was dominated by cheese starter bacteria. After pasteurisation, two out of the three samples studied became dominated by diverse environmental bacteria from various phyla, with Proteobacteria being the most dominant. Diverse microbial profiles were maintained until spoilage occurred, when the entire population was dominated by just one or two genera. Whey spoilage bacteria were found to be similar to those of milk. Pasteurised Cheddar and Mozzarella whey was spoiled by Bacillus sp. or Pseudomonas sp., and raw Mozzarella whey was spoiled by Pseudomonas sp., Serratia sp., and other members of the Enterobacteriaceae family. CO2 was effective in inhibiting bacterial growth of pasteurised Cheddar and Mozzarella whey stored at 15°C and raw Mozzarella whey stored at 4°C. The spoilage bacteria of the carbonated samples were similar to those of the non-carbonated controls.

Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist-stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP-regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin-stimulated elevations in intracellular cAMP as well as both adenosine- and forskolin-stimulated increases in CFTR-dependent transepithelial short-circuit current, in polarised cultures of Calu-3 human airway cells. This CO2 -induced reduction in anion secretion was not due to a decrease in HCO3 (-) transport given that neither a change in CFTR-dependent HCO3 (-) efflux nor Na(+) /HCO3 (-) cotransporter-dependent HCO3 (-) influx were CO2 -sensitive. Hypercapnia also reduced the volume of forskolin-stimulated fluid secretion over 24 h, yet had no effect on the HCO3 (-) content of the secreted fluid. Our data reveal that hypercapnia reduces CFTR-dependent, electrogenic Cl(-) and fluid secretion, but not CFTR-dependent HCO3 (-) secretion, which highlights a differential sensitivity of Cl(-) and HCO3 (-) transporters to raised CO2 in Calu-3 cells. Hypercapnia also reduced forskolin-stimulated CFTR-dependent anion secretion in primary human airway epithelia. Based on current models of airways biology, a reduction in fluid secretion, associated with hypercapnia, would be predicted to have important consequences for airways hydration and the innate defence mechanisms of the lungs.

The second messenger cyclic-di-adenosine monophosphate (c-di-AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c-di-AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c-di-AMP levels. In this study, a genetic screen was employed to identify components which affect the c-di-AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c-di-AMP phosphodiesterase gdpP mutants, which contain high c-di-AMP levels. Loss-of-function and gain-of-function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c-di-AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c-di-AMP level and a reduction in the key peptidoglycan precursor UDP-N-acetylglucosamine in L. lactis. C-di-AMP synthesis by CdaA was shown to be inhibited by GlmM(I154F) more than GlmM and GlmM(I154F) was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c-di-AMP level modulating protein and provide a direct connection between c-di-AMP synthesis and peptidoglycan biosynthesis.

Microbial exopolysaccharide (EPS) is of interest due to their applications in food processing and potential health benefits following consumption. The objectives of this study were to: 1) isolate, purify and characterize EPS produced by Enterococcus faecium MS79 (EPS-MS79); 2) investigate bioactive properties of EPS-MS79; and 3) investigate the rheological properties of the EPS-MS79 as influenced by salt type and pH level. The average molecular weight of the EPS-MS79 was 8.3 × 105 Da and it is composed of the three monosaccharides arabinose, mannose, and glucose in a molar ratio of 0.8:1.7:11.3. Scavenging activities of EPS-MS79 were 76% and 85% as measured by DPPH and 26% and 44% as measured by ABTS. The antiproliferative activities against colon (72% and 77%) and breast (43% and 56%) cancer cell lines. EPS-MS79 inhibited the growth of bacterial pathogens by up to 3 logs compared to the control. All EPS-MS79 solutions displayed shear thinning behavior. The storage and loss moduli of all EPS-MS79 solutions increased alongside with the frequency increase. Salt type and pH level impacted on the rheological properties of EPS-MS79. The EPS-MS79 could be a potent prebiotic promoting health and rheological properties of foods.

Foods containing edible probiotic bacteria, most commonly Lactobacillus and Bifidobacterium species, form a multi-billion-dollar industry worldwide. Currently marketed foods containing probiotics are mostly dairy based with yoghurts and fermented milks dominating the industry. Alternative foods as carriers of probiotics are being examined to reduce or eliminate lactose intolerance issues. Food categories including fruit juices, cheese, chocolate and even beer have been shown to be suitable for probiotic delivery. In addition, technologies such as encapsulation in food-grade alginate gels have allowed for improved probiotic survival in certain foodstuffs. We have explored the use of ready-to-eat vegetables such as baby spinach as carriers for commercial probiotics and found that high dose (>8 log CFU/g) can be achieved without having negative effects on appearance, taste or aroma. Leafy greens as well as other foods and beverages may be suitable probiotic containing new food products in the future.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress., Author summary Second messengers relay signals received from the environment to intracellular targets that adjust cellular physiology. One widespread bacterial cyclic-dinucleotide signalling molecule, cyclic-di-AMP (c-di-AMP) has been shown to regulate a range of cellular processes via binding to protein and riboswitch targets, with most identified thus far being linked to osmoregulation functions. C-di-AMP levels need to be carefully tuned under different environmental conditions to allow optimal growth. Here we show that a Lactococcus lactis GdpP phosphodiesterase mutant with a high intracellular pool of c-di-AMP is able to grow under hyperosmotic conditions after acquiring mutations which increase osmolyte (potassium [K+] or compatible solute) uptake or by actively exporting c-di-AMP. Interestingly, elevated K+ or glycine betaine uptake triggered accumulation of c-di-AMP and environmental osmolarity changes were also found to significantly impact c-di-AMP levels in various bacteria. These results support a model in which c-di-AMP negatively impacts osmoresistance through inhibition of the import of osmoprotectants and this system can sense both cellular and environmental changes causing water stress.

The Salmonella cytolethal distending toxin (S-CDT), first described as the “typhoid toxin” in Salmonella enterica subsp. enterica serotype Typhi, induces DNA damage in eukaryotic cells. Recent studies have shown that more than 40 nontyphoidal Salmonella (NTS) serotypes carry genes that encode S-CDT, yet very little is known about the activity, function, and role of S-CDT in NTS. Here we show that deletion of genes encoding the binding subunit (pltB) and a bacteriophage muramidase predicted to play a role in toxin export (ttsA) does not abolish toxin activity in the S-CDT-positive NTS Salmonella enterica subsp. enterica serotype Javiana. However, S. Javiana strains harboring deletions of both pltB and its homolog artB, had a complete loss of S-CDT activity, suggesting that S. Javiana carries genes encoding two variants of the binding subunit. S-CDT-mediated DNA damage, as determined by phosphorylation of histone 2AX (H2AX), producing phosphorylated H2AX (γH2AX), was restricted to epithelial cells in S and G2/M phases of the cell cycle and did not result in apoptosis or cell death. Compared to mice infected with a ΔcdtB strain, mice infected with wild-type S. Javiana had significantly higher levels of S. Javiana in the liver, but not in the spleen, ileum, or cecum. Overall, we show that production of active S-CDT by NTS serotype S. Javiana requires different genes (cdtB, pltA, and either pltB or artB) for expression of biologically active toxin than those reported for S-CDT production by S. Typhi (cdtB, pltA, pltB, and ttsA). However, as in S. Typhi, NTS S-CDT influences the outcome of infection both in vitro and in vivo., IMPORTANCE Nontyphoidal Salmonella (NTS) are a major cause of bacterial food-borne illness worldwide; however, our understanding of virulence mechanisms that determine the outcome and severity of nontyphoidal salmonellosis is incompletely understood. Here we show that S-CDT produced by NTS plays a significant role in the outcome of infection both in vitro and in vivo, highlighting S-CDT as an important virulence factor for nontyphoidal Salmonella serotypes. Our data also contribute novel information about the function of S-CDT, as S-CDT-mediated DNA damage occurs only during certain phases of the cell cycle, and the resulting damage does not induce cell death as assessed using a propidium iodide exclusion assay. Importantly, our data support that, despite having genetically similar S-CDT operons, NTS serotype S. Javiana has different genetic requirements than S. Typhi, for the production and export of active S-CDT.

This study investigated protein kinase activation and gene expression of angiogenic factors in response to low-load resistance exercise with or without blood flow restriction (BFR). In a repeated measures cross-over design, six males performed four sets of bilateral knee extension exercise at 20% 1RM (reps per set = 30:15:15:continued to fatigue) with BFR (110 mmHg) and without (CON). Muscle biopsies were obtained from the vastus lateralis before, 2 and 4 h post-exercise. mRNA expression was determined using real-time RT–PCR. Protein phosphorylation/expression was determined using Western blot. p38MAPK phosphorylation was greater (p = 0.05) at 2 h following BFR (1.3 ± 0.8) compared to CON (0.4 ± 0.3). AMPK phosphorylation remained unchanged. PGC-1α mRNA expression increased at 2 h (5.9 ± 1.3 vs. 2.1 ± 0.8; p = 0.03) and 4 h (3.2 ± 0.8 vs. 1.5 ± 0.4; p = 0.03) following BFR exercise with no change in CON. PGC-1α protein expression did not change following either exercise. BFR exercise enhanced mRNA expression of vascular endothelial growth factor (VEGF) at 2 h (5.2 ± 2.8 vs 1.7 ± 1.1; p = .02) and 4 h (6.8 ± 4.9 vs. 2.5 ± 2.7; p = .01) compared to CON. mRNA expression of VEGF-R2 and hypoxia-inducible factor 1α increased following BFR exercise but only eNOS were enhanced relative to CON. Matrix metalloproteinase-9 mRNA expression was not altered in response to either exercise. Acute low-load resistance exercise with BFR provides a targeted angiogenic response potentially mediated through enhanced ischaemic and shear stress stimuli.

Background: The role of Ureaplasma Urealyticum (UU) colonisation of the lungs remains controversial in the occurrence of bronchopulmonarydysplasia (BPD) of prematurity. The present European survey was conducted between April and July 2012, as part of the FP7-TINN2-project (Treat Infections in Neonates 2, www.tinn2-project.org). Aims and methods: It aimed to evaluate the position of neonatologists regarding the role of UU as a risk factor for BPD, use of azithromycin (AZY) for the prevention of BPD and factors that influence this practice in European neonatal intensive care units (NICUs). It was conducted using an online questionnaire of 64 questions. Results: 167 questionnaires from twenty-eight European countries were analyzed. All responders confirmed that the two major risk factors for BPD were prematurity ≤28weeks and high oxygen requirements. Various macrolides are used in Europe to treat or prevent Ureaplasma colonisation. Among them, AZY has antibacterial, anti-inflammatory and immuno-modulating properties, is employed throughout Europe (27% NICUs in 12countries), usually administered at a starting dose of 10mg/kg (60% of NICUs), followed by a maintenance dose of 5mg/kg/day (43% of NICUs). For 10 days. 78% of the NICUs were interested in the TINN2-project to evaluate the efficacy and safety of AZY versus placebo in reducing the risk of BPD in premature babies. For most neonatologists, newborns between 23 and 28weeks gestational age would be the best group of premature babies to be included in the trial.

Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on cystic fibrosis transmembrane conductance regulator (CFTR) function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by CFTR, and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways: autocrine activation of EP4 prostanoid receptors and stimulation of Ca2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs.

Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galβ] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates., IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.

The worldwide prevalence of diabetes has risen to 8.5% among adults, which represents a staggering rise in prevalence from 4.7% in 1980. Whilst some treatments work by increasing insulin secretion, over time their effectiveness decreases. We aim to increase insulin secretion by developing strategies that work through mechanisms independent of current treatment options. Isolated CD1 mouse islets, INS-1 pancreatic β-cells, or C2C12 mouse myotubes were incubated in standard tissue culture media, or media supplemented with 28 mM glucose, 200 μM palmitic acid, and 200 μM oleic acid as a cellular model of diabetic glucolipotoxicity. Intracellular reactive species content was assayed using 2′,7′-dichlorofluorescein diacetate dye, inducible nitric oxide synthase levels determined by Western blot, 3-nitrotyrosine and 4-hydrpxnonenal both assayed by ELISA, insulin secretion quantified using ELISA or radioimmunoassay, and glucose uptake determined through 2-deoxy glucose 6 phosphate luminescence. Our data indicate that carnosine, a histidine containing dipeptide available through the diet, is an effective scavenger of each of the aforementioned reactive species. This results in doubling of insulin secretion from isolated mouse islets or INS-1 β-cells. Crucially, carnosine also reverses glucolipotoxic inhibition of insulin secretion and enhances glucose uptake into skeletal muscle cells. Thus, carnosine, or non-hydrolysable carnosine analogs, may represent a new class of therapeutic agent to fight type 2 diabetes.

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan., IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis, a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

Skeletal muscle is an insulin sensitive tissue and accounts for approximately 80% of post-prandial glucose disposal. This study describes the effects of insulin, delivered for 72 hours, to skeletal muscle myoblasts during differentiation or to skeletal muscle myotubes. After chronic treatment, cultures were acutely stimulated with insulin and analysed for total and phosphorylated Akt (Ser473), mRNA expression of metabolic and myogenic markers and insulin-stimulated glucose uptake. Skeletal muscle cells differentiated in the presence of insulin chronically, reduced acute insulin stimulated phosphorylation of Akt Ser473. In addition, there was a reduction in mRNA expression of Hexokinase II (HKII), GLUT4 and PGC-1α. Insulin-stimulated glucose uptake was attenuated when cells were differentiated in the presence of insulin. In contrast, myotubes exposed to chronic insulin showed no alterations in phosphorylation of Akt Ser473. Both HKII and GLUT4 mRNA expression were reduced by chronic exposure to insulin; while PGC-1α was not different between culture conditions and was increased by acute insulin stimulation. These data suggest that there are differential responses in insulin signalling, transcription and glucose uptake of skeletal muscle cells when cultured in either the presence of insulin during differentiation or in myotube cultures.

Background: In the neonatal period, cortisol concentrations are expected to increase during significant stress and are important for survival. Extreme preterm infants may develop adrenal insufficiency in the early neonatal period. Cortisol is 90% bound to cortisol binding globulins (CBG) in the circulation; therefore measurements of plasma cortisol can be compromised by conditions that alter CBG levels. Measurement of free cortisol is the best indicator of adrenal glucocorticoid secretion and can be determined in the saliva. Few studies have been reported on salivary cortisol determination in the neonatal period and particularly, extremely premature infants. Methods: There were 65 infants (36 males). Mean gestation 25.3 ± 1.3 weeks. We obtained early morning plasma and salivary cortisol sampling before day 5 of postnatal age from extremely premature babies less than 28 weeks gestation. Saliva was obtained using 4 standard universal swabs by placing one swab at a time in the infant's mouth for 1-2 min. No salivary stimulants were used. Salivary cortisol was measured by competitive ELISA using a commercially available kit SLV-2930 (DRG, Germany) according to the manufacturer's instructions. Plasma cortisol was measured using DPC Immulite 2000 using a solid phase 2 site chemiluminescent immunometric assay. Results: Mean plasma cortisol levels were 400 nmol/L ± 42.8 SEM, and mean salivary cortisol levels were 127.5 nmol/L ± 66.5 SEM. Plasma cortisol was positively correlated with salivary cortisol (r=0.41, p

Inflammation occurs as a result of exposure of tissues and organs to harmful stimuli such as microbial pathogens, irritants, or toxic cellular components. The primary physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function to the affected area. These processes involve the major cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, T-cells, and B-cells. However, examination of a range of inflammatory lesions demonstrates the presence of specific leukocytes in any given lesion. That is, the inflammatory process is regulated in such a way as to ensure that the appropriate leukocytes are recruited. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signalling control mechanisms that characterise inflammation. This review will focus on the role of the main cytokines, chemokines, and their receptors in the pathophysiology of auto-inflammatory disorders, pro-inflammatory disorders, and neurological disorders involving inflammation. © 2014 Elsevier B.V.

In early pregnancy, maternal transfer of thyroxine (T4) significantly contributes to the foetal T4 requirements. Interruption of the maternal transfer of T4 may lead to inadequate T4 exposure, potentially leading to neurodevelopmental deficits.To determine if maternal factors are associated with the thyroid hormone status of extremely premature infants during the first five days of life.This prospective study looked at 117 mothers and their extremely premature babies (born before 28 weeks' gestation). The relationship between neonatal thyroid hormone status and maternal factors (gestation, route of delivery, exogenous maternal glucocorticoid administration, maternal free T4 (FT4), presence or absence of maternal chorioamnionitis, maternal smoking status, maternal body mass index (BMI) index, maternal thyroid peroxidase antibody status (TPO) and maternal haemoglobin levels) were evaluated. Multiple linear regression was used to study independent factors affecting neonatal thyroid function.Mean gestational age was 25(+5) ± 1.3 weeks (range 22(+0) to 27(+6)). Neonatal FT4 strongly correlated with gestation, with a greater severity of hypothyroxinaemia associated with lower gestation (r = 0.6, p 0.0001). Multiple regression found gestation to be the only independent factors affecting thyroid status (beta coefficient = 0.08, p = 0.01), and no maternal factors were found to be associated with neonatal thyroid status.Neonatal thyroid status in extreme preterm infants is independently affected by gestation and not maternal factors such as route of delivery, exogenous maternal glucocorticoid administration, third trimester maternal FT4, presence or absence of chorioamnionitis, smoking status, BMI, TPO status or haemoglobin levels. The severity of neonatal hypothyroxinaemia increases with lower gestational age.

Oral administration of dry vaccine formulations is acknowledged to offer major clinical and logistical benefits by eliminating the cold chain required for liquid preparations. A model antigen, bovine serum albumin (BSA) was encapsulated in alginate microspheres using aerosolisation. Hydrated microspheres 25 to 65 μm in size with protein loading of 3.3 % w/w were obtained. Environmental scanning electron microscopy indicated a stabilizing effect of encapsulated protein on alginate hydrogels revealed by an increase in dehydration resistance. Freeze drying of alginate microspheres without use of a cryoprotectant resulted in fragmentation and subsequent rapid loss of the majority of the protein load in simulated intestinal fluid in 2 h, whereas intact microspheres were observed following freeze-drying of BSA-loaded microspheres in the presence of maltodextrin. BSA release from freeze-dried preparations was limited to less than 7 % in simulated gastric fluid over 2 h, while 90 % of the protein load was gradually released in simulated intestinal fluid over 10 h. SDS-PAGE analysis indicated that released BSA largely preserved its molecular weight. These findings demonstrate the potential for manufacturing freeze-dried oral vaccines using alginate microspheres.

Standardised, concentrated neonatal parenteral nutrition (PN) regimens can overcome early nutritional deficits in very preterm infants. A PN regimen with increased macronutrient content (standardised, concentrated, added macronutrients parenteral (SCAMP)) has been shown to improve early head growth in a randomised controlled trial. Line complications including late onset sepsis were secondary outcomes of this study. Infants were started on standardised, concentrated PN at birth and randomised at 2–5 days to either switch to SCAMP or remain on control PN. Central venous catheter (CVC), blood culture (BC) and inflammatory marker data were collected for the 28-day intervention period. 150 infants were randomised with mean (SD) birth weight (g) of 900 (158) versus 884 (183) in SCAMP (n=74) and control (n=76) groups, respectively. There were no differences in CVC use/type or duration or in positive/negative BC with/without associated C reactive protein rise in SCAMP versus control groups. Increasing the macronutrient content of a standardised, concentrated neonatal PN regimen does not increase CVC complication rates. Trial registration number ISRCTN 76597892.