Faecal lipid profile as a new marker of fat maldigestion, malabsorption and microbiota.

BACKGROUND: Fat malabsorption in children with cystic fibrosis (CF) leads to poor nutritional status and altered colonic microbiota. This study aimed at establishing the faecal lipid profile in children with CF, and exploring associations between the faecal lipidome and microbiota. METHODS: Cross-sectional observational study with children with CF and an age-matched control group. Faecal lipidome was analysed by UHLC-HRMS and microbiota profiling by 16S rRNA amplicon sequencing. RESULTS: Among 234 identified lipid species, five lipidome clusters (LC) were obtained with significant differences in triacylglycerols (TG), diacylglycerols (DG), monoacylglycerols (MG) and fatty-acids (FA): LC1 subjects with good digestion and absorption: low TG and low MG and FA; LC2 good digestion and poor absorption: low TG and high MG and FA; LC3 Mild digestion and poor absorption: intermediate TG and high MG and FA; LC4 poor digestion and absorption: high TG and high MG and FA; LC5 outliers. Bacteroidota and Verrucomicrobiota decreased over LC1-LC4, while Proteobacteria increased. Nutritional status indicators were significantly higher in LC1 and decreased over LC2-LC4. CONCLUSION: Assessing faecal lipidome may be relevant to determine how dietary lipids are digested and absorbed. This new evidence might be a method to support targeted nutritional interventions towards reverting fat maldigestion or malabsorption. IMPACT: Lipidomic analysis enabled the identification of the lipid species related to maldigestion (triglycerides) or malabsorption (monoglycerides and fatty acids). Children with cystic fibrosis can be grouped depending on the faecal lipidome profile related to dietary fat maldigestion or malabsorption. The lipidome profile in faeces is related to the composition of microbiota and nutritional status indicators.

Role of germinal center and CD39highCD73+ B cells in the age-related tonsillar involution.

BACKGROUND: The tonsils operate as a protection ring of mucosa at the gates of the upper aero-digestive tract. They show similarities with lymph nodes and participate as inductive organs of systemic and mucosal immunity. Based on the reduction of their size since puberty, they are thought to experience involution in adulthood. In this context, we have used tonsillar mononuclear cells (TMC) isolated from patients at different stages of life, to study the effect of ageing and the concomitant persistent inflammation on these immune cells. RESULTS: We found an age-dependent reduction in the proportion of germinal center B cell population (BGC) and its T cell counterpart (T follicular helper germinal center cells, TfhGC). Also, we demonstrated an increment in the percentage of local memory B cells and mantle zone T follicular helper cells (mTfh). Furthermore, younger tonsils rendered higher proportion of proliferative immune cells within the freshly isolated TMC fraction than those from older ones. We demonstrated the accumulation of a B cell subset (CD20+CD39highCD73+ cells) metabolically adapted to catabolize adenosine triphosphate (ATP) as patients get older. To finish, tonsillar B cells from patients at different ages did not show differences in their proliferative response to stimulation ex vivo, in bulk TMC cultures. CONCLUSIONS: This paper sheds light on the changing aspects of the immune cellular landscape, over the course of time and constant exposure, at the entrance of the respiratory and digestive systems. Our findings support the notion that there is a re-modelling of the immune functionality of the excised tonsils over time. They are indicative of a transition from an effector type of immune response, typically oriented to reduce pathogen burden early in life, to the development of an immunosuppressive microenvironment at later stages, when tissue damage control gets critical provided the time passed under immune attack. Noteworthy, when isolated from such histologic microenvironment, older tonsillar B cells seem to level their proliferation capacity with the younger ones. Understanding these features will not only contribute to comprehend the differences in susceptibility to pathogens among children and adults but would also impact on vaccine developments intended to target these relevant mucosal sites.

Predictors of surgical difficulty in upper third molar removal: a prospective cohort study.

BACKGROUND: Upper third molar (U3M) removal is a common surgical procedure. The aims of this study were to assess the patient-specific, radiological and surgical factors related to the difficulty of U3M removal, and to determine the incidence of intraoperative and postoperative complications. MATERIAL AND METHODS: A prospective cohort study was carried out in adult patients undergoing U3M removal. Operative time, surgeon-reported difficulty and the Parant classification were used to assess extraction difficulty. Clinical, radiological and surgical factors were recorded to determine their relationship with surgical difficulty. A descriptive, bivariate and multivariate statistical analysis was carried out. RESULTS: A total of 250 patients were included. The mean operative time was 10.4 (±12.3) minutes, mean surgeon-reported difficulty was 3.2/10 (±2.3). The multivariate analysis showed greater impaction against the second molar and greater soft tissue and bony impaction to significantly increase operative time and surgeon-perceived difficulty. Additionally, surgeon experience was related to perceived difficulty. The overall incidence of intraoperative complications was 0.8%, and no postoperative events were recorded. CONCLUSIONS: Upper third molars in close relation with the roots of the adjacent second molar and with soft tissue and bony impaction are significantly more difficult to extract. Perceived difficulty was related to surgeon experience. This procedure appears to produce few intra- and postoperative complications.

Noninvasive prediction models of intra-amniotic infection in women with preterm labor.

BACKGROUND: Among women with preterm labor, those with intra-amniotic infection present the highest risk of early delivery and the most adverse outcomes. The identification of intra-amniotic infection requires amniocentesis, perceived as too invasive by women and physicians. Noninvasive methods for identifying intra-amniotic infection and/or early delivery are crucial to focus early efforts on high-risk preterm labor women while avoiding unnecessary interventions in low-risk preterm labor women. OBJECTIVE: This study modeled the best performing models, integrating biochemical data with clinical and ultrasound information to predict a composite outcome of intra-amniotic infection and/or spontaneous delivery within 7 days. STUDY DESIGN: From 2015 to 2020, data from a cohort of women, who underwent amniocentesis to rule in or rule out intra-amniotic infection or inflammation, admitted with a diagnosis of preterm labor at <34 weeks of gestation at the Hospital Clinic and Hospital Sant Joan de Déu, Barcelona, Spain, were used. At admission, transvaginal ultrasound was performed, and maternal blood and vaginal samples were collected. Using high-dimensional biology, vaginal proteins (using multiplex immunoassay), amino acids (using high-performance liquid chromatography), and bacteria (using 16S ribosomal RNA gene amplicon sequencing) were explored to predict the composite outcome. We selected ultrasound, maternal blood, and vaginal predictors that could be tested with rapid diagnostic techniques and developed prediction models employing machine learning that was applied in a validation cohort. RESULTS: A cohort of 288 women with preterm labor at <34 weeks of gestation, of which 103 (35%) had a composite outcome of intra-amniotic infection and/or spontaneous delivery within 7 days, were included in this study. The sample was divided into derivation (n=116) and validation (n=172) cohorts. Of note, 4 prediction models were proposed, including ultrasound transvaginal cervical length, maternal C-reactive protein, vaginal interleukin 6 (using an automated immunoanalyzer), vaginal pH (using a pH meter), vaginal lactic acid (using a reflectometer), and vaginal Lactobacillus genus (using quantitative polymerase chain reaction), with areas under the receiving operating characteristic curve ranging from 82.2% (95% confidence interval, ±3.1%) to 85.2% (95% confidence interval, ±3.1%), sensitivities ranging from 76.1% to 85.9%, and specificities ranging from 75.2% to 85.1%. CONCLUSION: The study results have provided proof of principle of how noninvasive methods suitable for point-of-care systems can select high-risk cases among women with preterm labor and might substantially aid in clinical management and outcomes while improving the use of resources and patient experience.

SNAI1-expressing fibroblasts and derived-extracellular matrix as mediators of drug resistance in colorectal cancer patients.

Resistance to antitumor treatments is one of the most important problems faced by clinicians in the management of colorectal cancer (CRC) patients. Cancer-Associated Fibroblasts (CAFs) are the main producers and remodelers of the extracellular matrix (ECM), which is directly involved in drug resistance mechanisms. Primary Normal Fibroblasts (NFs) and CAFs and cell lines (fibroblasts and tumor cells), were used to generate ECM and to identify its role in the oxaliplatin and cetuximab chemoresistance processes of CRC cells mediated by SNAI1-expressing fibroblasts. Matrices generated by Snai1 KO MEFs (Knockout Mouse Embryonic Fibroblasts) confer less resistance on oxaliplatin and cetuximab than wild-type MEF-derived matrices. Similarly, matrices derived from CAFs cause greater survival of colorectal cancer cells than NF-derived matrices, in a similar way to Snai1 expression levels. In addition, Snail1 expression in fibroblasts regulates drug resistance and metabolism gene expression in tumor cells mediated by ECM. Finally, a series of 531 patients (TCGA) with CRC was used to assess the role of SNAI1 expression in patients' prognosis indicating an association between tumor SNAI1 expression and overall survival in colon cancer patients but not in rectal cancer patients. SNAI1 expression in CRC cancer patients, together with in vitro experimentation, suggests the possible use of SNAI1 expression in tumor-associated fibroblasts as a predictive biomarker of response to oxaliplatin and cetuximab treatments in patients with CRC.

Genomic and biochemical analysis of repeatedly observed variants in DBT in individuals with maple syrup urine disease of Central American ancestry.

Maple syrup urine disease (MSUD) is an intoxication-type inherited metabolic disorder in which hyperleucinemia leads to brain swelling and death without treatment. MSUD is caused by branched-chain alpha-ketoacid dehydrogenase deficiency due to biallelic loss of the protein products from the genes BCKDHA, BCKDHB, or DBT, while a distinct but related condition is caused by loss of DLD. In this case series, eleven individuals with MSUD caused by two pathogenic variants in DBT are presented. All eleven individuals have a deletion of exon 2 (delEx2, NM_001918.3:c.48_171del); six individuals are homozygous and five individuals are compound heterozygous with a novel missense variant (NM_001918.5:c.916 T > C [p.Ser306Pro]) confirmed to be in trans. Western Blot indicates decreased amount of protein product in delEx2;c.916 T > C liver cells and absence of protein product in delEx2 homozygous hepatocytes. Ultrahigh performance liquid chromatography-tandem mass spectrometry demonstrates an accumulation of branched-chain amino acids and alpha-ketoacids in explanted hepatocytes. Individuals with these variants have a neonatal-onset, non-thiamine-responsive, classical form of MSUD. Strikingly, the entire cohort is derived from families who immigrated to the Washington, DC, metro area from Honduras or El Salvador suggesting the possibility of a founder effect.

Maternal diet during pregnancy and intestinal markers are associated with early gut microbiota.

BACKGROUND: Diet has an important role in host-microbiome interplay, which may result in intestinal permeability changes and physiopathological effects at a systemic level. Despite the importance of maternal microbiota as the main contributor to the initial microbial seeding, little is known about the effects of maternal diet during pregnancy on maternal-neonatal microbiota. OBJECTIVES: This study aimed at ascertaining the possible associations between maternal dietary intake during pregnancy and neonatal microbiota at birth and to evaluate the relationship with maternal intestinal markers. METHODS: In a nested cross-sectional study in the longitudinal MAMI cohort, maternal-neonatal microbiota profiling at birth (n = 73) was assessed by 16S rRNA gene sequencing. Maternal intestinal markers as zonulin, intestinal alkaline phosphatase (IAP) activity and faecal calprotectin were measured in faeces. Furthermore, maternal-neonatal clinical and anthropometric data, as well as maternal nutrient intake during pregnancy obtained by FFQ questionnaires, were collected. RESULTS: Maternal diet is associated with both maternal and neonatal microbiota at the time of birth, in a delivery mode-dependent manner. The existing link between maternal diet, intestinal makers and neonatal gut microbiota would be mainly influenced by the intake of saturated (SFA) and monounsaturated fatty acids (MUFA). Members of Firmicutes in the neonatal microbiota were positively associated with maternal fat intake, especially SFA and MUFA, and negatively correlated to fibre, proteins from vegetable sources and vitamins. CONCLUSIONS: Maternal diet during pregnancy, mainly fat intake (SFA and MUFA), was related to intestinal markers, thus likely shifting the microbial transmission to the neonate and priming the neonatal microbial profile with potential health outcomes. CLINICAL TRIAL REGISTRY: NCT03552939.

Biochemical and anaplerotic applications of in vitro models of propionic acidemia and methylmalonic acidemia using patient-derived primary hepatocytes.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, which are caused by a deficiency in the enzyme propionyl-CoA carboxylase or the enzyme methylmalonyl-CoA (MM-CoA) mutase, respectively. The functional consequence of PA or MMA is the inability to catabolize P-CoA to MM-CoA or MM-CoA to succinyl-CoA, resulting in the accumulation of P-CoA and other metabolic intermediates, such as propionylcarnitine (C3), 3-hydroxypropionic acid, methylcitric acid (MCA), and methylmalonic acid (only in MMA). P-CoA and its metabolic intermediates, at high concentrations found in PA and MMA, inhibit enzymes in the first steps of the urea cycle as well as enzymes in the tricarboxylic acid (TCA) cycle, causing a reduction in mitochondrial energy production. We previously showed that metabolic defects of PA could be recapitulated using PA patient-derived primary hepatocytes in a novel organotypic system. Here, we sought to investigate whether treatment of normal human primary hepatocytes with propionate would recapitulate some of the biochemical features of PA and MMA in the same platform. We found that high levels of propionate resulted in high levels of intracellular P-CoA in normal hepatocytes. Analysis of TCA cycle intermediates by GC-MS/MS indicated that propionate may inhibit enzymes of the TCA cycle as shown in PA, but is also incorporated in the TCA cycle, which does not occur in PA. To better recapitulate the disease phenotype, we obtained hepatocytes derived from livers of PA and MMA patients. We characterized the PA and MMA donors by measuring key proximal biomarkers, including P-CoA, MM-CoA, as well as clinical biomarkers propionylcarnitine-to-acetylcarnitine ratios (C3/C2), MCA, and methylmalonic acid. Additionally, we used isotopically-labeled amino acids to investigate the contribution of relevant amino acids to production of P-CoA in models of metabolic stability or acute metabolic crisis. As observed clinically, we demonstrated that the isoleucine and valine catabolism pathways are the greatest sources of P-CoA in PA and MMA donor cells and that each donor showed differential sensitivity to isoleucine and valine. We also studied the effects of disodium citrate, an anaplerotic therapy, which resulted in a significant increase in the absolute concentration of TCA cycle intermediates, which is in agreement with the benefit observed clinically. Our human cell-based PA and MMA disease models can inform preclinical drug discovery and development where mouse models of these diseases are inaccurate, particularly in well-described species differences in branched-chain amino acid catabolism.

The Microbiota and Malnutrition: Impact of Nutritional Status During Early Life.

According to the developmental origins of health and disease hypothesis, our health is determined by events experienced in utero and during early infancy. Indeed, both our prenatal and postnatal nutrition conditions have an impact on the initial architecture and activity of our microbiota. Recent evidence has underlined the importance of the composition of the early gut microbiota in relation to malnutrition, whether it be undernutrition or overnutrition, that is, in terms of both stunted and overweight development. It remains unclear how early microbial contact is linked to the risk of disease, as well as whether alterations in the microbiome underlie the pathogenesis of malnutrition or are merely the end result of it, which indicates that thequestion of causality must urgently be answered. This review provides information on the complex interaction between the microbiota and nutrition during the first 1,000 days of life, taking into account the impact of both undernutrition and overnutrition on the microbiota and on infants' health outcomes in the short- and long-term.

Association of Maternal Secretor Status and Human Milk Oligosaccharides With Milk Microbiota: An Observational Pilot Study.

BACKGROUND AND OBJECTIVES: Breast milk contains several bioactive factors including human milk oligosaccharides (HMOs) and microbes that shape the infant gut microbiota. HMO profile is determined by secretor status; however, their influence on milk microbiota is still uncovered. This study is aimed to determine the impact of the FUT2 genotype on the milk microbiota during the first month of lactation and the association with HMO. METHODS: Milk microbiota from 25 healthy lactating women was determined by quantitative polymerase chain reaction and 16S gene pyrosequencing. Secretor genotype was obtained by polymerase chain reaction-random fragment length polymorphisms and by HMO identification and quantification. RESULTS: The most abundant bacteria were Staphylococcus and Streptococcus, followed by Enterobacteriaceae-related bacteria. The predominant HMO in secretor milk samples were 2'FL and lacto-N-fucopentaose I, whereas non-secretor milk was characterized by lacto-N-fucopentaose II and lacto-N-difucohexaose II. Differences in microbiota composition and quantity were found depending on secretor/non-secretor status. Lactobacillus spp, Enterococcus spp, and Streptococcus spp were lower in non-secretor than in secretor samples. Bifidobacterium genus and species were less prevalent in non-secretor samples. Despite no differences on diversity and richness, non-secretor samples had lower Actinobacteria and higher relative abundance of Enterobacteriaceae, Lactobacillaceae, and Staphylococcaceae. CONCLUSIONS: Maternal secretor status is associated with the human milk microbiota composition and is maintained during the first 4 weeks. Specific associations between milk microbiota, HMO, and secretor status were observed, although the potential biological impact on the neonate remains elusive. Future studies are needed to reveal the early nutrition influence on the reduction of risk of disease.

Oral microbiota maturation during the first 7 years of life in relation to allergy development.

BACKGROUND: Allergic diseases have become a major public health problem in affluent societies. Microbial colonization early in life seems to be critical for instructing regulation on immune system maturation and allergy development in children. Even though the oral cavity is the first site of encounter between a majority of foreign antigens and the immune system, the influence of oral bacteria on allergy development has not yet been reported. OBJECTIVE: We sought to determine the bacterial composition in longitudinally collected saliva samples during childhood in relation to allergy development. METHODS: Illumina sequencing of the 16S rDNA gene was used to characterize the oral bacterial composition in saliva samples collected at 3, 6, 12, 24 months, and 7 years of age from children developing allergic symptoms and sensitization (n = 47) and children staying healthy (n = 33) up to 7 years of age. RESULTS: Children developing allergic disease, particularly asthma, had lower diversity of salivary bacteria together with highly divergent bacterial composition at 7 years of age, showing a clearly altered oral microbiota in these individuals, likely as a consequence of an impaired immune system during infancy. Moreover, the relative amounts of several bacterial species, including increased abundance of Gemella haemolysans in children developing allergies and Lactobacillus gasseri and L. crispatus in healthy children, were distinctive during early infancy, likely influencing early immune maturation. CONCLUSION: Early changes in oral microbial composition seem to influence immune maturation and allergy development. Future experiments should test the probiotic potential of L. gasseri and L. crispatus isolates.

Shifts in gut microbiota composition in an APP/PSS1 transgenic mouse model of Alzheimer's disease during lifespan.

Alzheimer's disease (AD) is the most common form of dementia and one of the major causes of disability and dependency in older people. Accumulating evidences link gut microbiota with different diseases and its relationship with neurodegenerative diseases is becoming most intriguing. This study was aimed to compare the gut microbiota of transgenic APP/PS1 (TG) mice, a well-established deterministic mouse model of AD, with their C57BL/6 wild-type (WT) littermates. Faecal samples were collected from 3-, 6- and 24-month-old mice and analysed by pyrosequencing of the V1-V3 region of the bacterial 16S rRNA genes. Bacterial profiles were similar in all young mice (3 months old), and started to diverge so that 6-month-old WT and TG mice had different and more diverse microbiota. During ageing, Turicibacteriaceae (typical mice bacterial group) and Rikenellaceae increased in all groups, although total Bacteroidetes remained stable. TG mice were characterized by an increase in Proteobacteria after 6 months, particularly the genus Sutterella (Betaproteobacteria), interestingly also increased in autism disorder. Also, the inflammation related family Erysipelotrichaceae was more abundant in TG mice at 24 months compared to wild-type control. In summary, AD pathology in mice shifts the gut microbiota towards profiles that share features with autism and inflammatory disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: Alzheimer's disease is a neurodegenerative disease and neuroinflammation in the central nervous system appears to have a pivotal role. Using the transgenic APP/PS1 (TG) mouse model, we successfully characterized how AD pathology shifted gut microbiota composition during ageing towards an inflammation related bacterial profile related to Proteobacteria and Erysipelotrichaceae and suggest that these changes could contribute to disease progression and severity. Microbiota-targeted interventions could therefore represent a strategy to postpone disease symptoms.

Breast Milk Polyamines and Microbiota Interactions: Impact of Mode of Delivery and Geographical Location.

BACKGROUND/AIMS: The aim of the present study was to identify and quantify the polyamine levels in human milk obtained from different countries and through different modes of delivery, and to investigate their association with breast milk microbes. METHODS: Mature breast milk samples were obtained from 78 healthy mothers after 1 month of lactation from 4 different geographical locations: Finland, Spain (Europe); South Africa (Africa); and China (Asia). Polyamines were determined using HPLC after dansyl derivatization and milk microbiota was obtained by 16S rRNA gene sequencing. RESULTS: The mean values of polyamines in breast milk were 70.0, 424.2, and 610.0 nmol/dL for putrescine, spermidine and spermine, respectively, and 1,170.9 nmol/dL of total polyamines. The levels of putrescine were significantly higher in Spain (p < 0.05) and spermidine levels were significantly higher in Finland (p < 0.05) compared with other countries. Cesarean delivery had an impact on polyamine levels and it was related to an increase in the putrescine concentration being significant in Spanish samples (p < 0.01). Furthermore, putrescine levels were correlated positively with Gammaproteobacteria (r = 0.46, p < 0.001), especially with Pseudomonas fragi (r = 0.40, p < 0.001). CONCLUSIONS: The results demonstrate significant effect of geographical variations in human milk polyamine concentrations, being correlated with human milk microbiota composition. These differences may have an impact on infant development during lactation.

Validation of an Argentine version of Lupus Quality of Life questionnaire.

OBJECTIVE: To determine reproducibility and validity of an Argentine version of the Lupus Quality of Life questionnaire (LupusQoL) and to determine cut-off values in the questionnaire. MATERIALS AND METHODS: One hundred and forty-seven systemic lupus erythematosus patients (American College of Rheumatology 1982/1997) were assessed from April 2014 to July 2014. Demographic and socioeconomic variables were collected, as well as SELENA/SLEDAI, Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index Score, comorbidities and treatment data. Patients completed LupusQoL-Argentine version and European Quality of Life Questionnaire (EuroQoL-5D). Internal consistency and reliability were examined. Convergent validity with EuroQoL-5D was assessed through analysis of latent classes, which established homogeneous categories from the responses of each domain of LupusQoL and for the total. RESULTS: Out of 147 patients, 93.2% were female, mean age 36.4 ± 11.1 years, mean disease duration 2.7 ± 9 years, mean SELENA/SLEDAI 2.7 ± 3 points. The cut-off point that defined good or bad quality of life was 0.739 for EuroQoL 5D and 63 for LupusQoL. Cut-off values for each LupusQoL domain were also defined, creating two classes in each of them. There was moderate to high concordance to classify quality of life (Kappa = 0.74, 95% confidence interval = 0.54, 0.95). CONCLUSION: The Argentine version of LupusQoL is a valid, reliable and reproducible instrument to assess quality of life. In this study, cut-off points that allow the classification of patients regarding whether they have good or bad quality of life are established for the first time.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation.

OBJECTIVES: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. APPROACH AND RESULTS: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. CONCLUSIONS: The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

End-product diacylglycerol enhances the activity of PI-PLC through changes in membrane domain structure.

Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties.

Sphingosine induces the aggregation of imine-containing peroxidized vesicles.

Lipid peroxidation plays a central role in the pathogenesis of many diseases like atherosclerosis and multiple sclerosis. We have analyzed the interaction of sphingosine with peroxidized bilayers in model membranes. Cu(2+) induced peroxidation was checked following UV absorbance at 245nm, and also using the novel Avanti snoopers®. Mass spectrometry confirms the oxidation of phospholipid unsaturated chains. Our results show that sphingosine causes aggregation of Cu(2+)-peroxidized vesicles. We observed that aggregation is facilitated by the presence of negatively-charged phospholipids in the membrane, and inhibited by anti-oxidants e.g. BHT. Interestingly, long-chain alkylamines (C18, C16) but not their short-chain analogues (C10, C6, C1) can substitute sphingosine as promoters of vesicle aggregation. Furthermore, sphinganine but not sphingosine-1-phosphate can mimic this effect. Formation of imines in the membrane upon peroxidation was detected by (1)H-NMR and it appeared to be necessary for the aggregation effect. (31)P-NMR spectroscopy reveals that sphingosine facilitates formation of non-lamellar phase in parallel with vesicle aggregation. The data might suggest a role for sphingosine in the pathogenesis of atherosclerosis.

Revealing microbial recognition by specific antibodies.

BACKGROUND: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system. RESULTS: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin. CONCLUSIONS: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases.

Role of probiotics in reducing the risk of gestational diabetes.

Overweight and obesity currently constitute a major threat to human well-being. Almost half of the female population are currently overweight. Pregnant overweight women are at risk of gestational diabetes affecting the health of the mother and the child, in both the short and long term. Notwithstanding the extensive scientific interest centred on the problem, research efforts have thus far been unable to devise preventive strategies. Recent scientific advances point to a gut microbiota dysbiosis, with ensuing low-grade inflammation as a contributing element, in obesity and its comorbidities. Such findings would suggest a role for specific probiotics in the search for preventive and therapeutic adjunct applications in gestational diabetes. The aim of the present paper was to critically review recent demonstrations of the role of intestinal microbes in immune and metabolic regulation, which could be exploited in nutritional management of pregnant women by probiotic bacteria. By modulating specific target functions, probiotic dietary intervention may exert clinical effects beyond the nutritional impact of food. As this approach in pregnancy is new, an overview of the role of gut microbiota in shaping host metabolism, together with the definition of probiotics are presented, and finally, specific targets and potential mechanisms for probiotics in pregnancy are discussed. Pregnancy appears to be the most critical stage for interventions aiming to reduce the risk of non-communicable disease in future generations, beyond the immediate dangers attributable to the health of the mother, labour and the neonate. Specific probiotic interventions during pregnancy provide an opportunity, therefore, to promote the health not only of the mother but also of the child.

Membrane permeabilization induced by sphingosine: effect of negatively charged lipids.

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.