Etablierung eines revers–genetischen Systems für feline Coronaviren

Im Rahmen dieser Arbeit erfolgte die Etablierung eines revers-genetischen Systems für ein Serotyp I felines Coronavirus (FCoV). Dabei wurden folgende Ergebnisse erzielt: 1) Als Grundlage für die Etablierung des Systems wurde die komplette Genomsequenz des FCoV Stammes Black bestimmt und anhand dieser Sequenz der Genomaufbau ermittelt. Es ließ sich der für Coronaviren typische Genomaufbau nachweisen. 2) Die Herstellung eines infektiösen Klons beinhaltete die Integration einer kompletten cDNA Kopie des FCoV Stammes Black in das Vaccinia Virus Genom, welches als Vektor diente. Die coronavirale cDNA wurde in vitro transkribiert und die synthetische RNA in Zellen elektroporiert. Rekombinante FCoV ließen sich nachweisen und somit war die synthetische RNA infektiös, also in der Lage, den coronaviralen Replikationszyklus zu initiieren und zu durchlaufen. Die Analyse der gewonnenen rekombinanten Viren erfolgte nach Infektion feliner Zellkulturen mit verschiedenen Ansätzen. Dabei konnte festgestellt werden, dass die rekombinanten Viren die gleichen Eigenschaften wie der ursprüngliche FCoV Stamm Black besitzen. 3) In nachfolgenden Versuchen erfolgte die Herstellung zweier Reportergenexprimierender FCoV mit dem etablierten revers-genetischen System. Dabei wurden im FCoV Stamm Black Genom die akzessorischen Gene 3abc durch das „green fluorescent protein“ (GFP) oder Renilla Luciferase ersetzt. Die stabile Expression der Reportergene konnte in felinen Zellkulturen nachgewiesen werden. 4) Feline Monozyten, Makrophagen und dendritische Zellen (DCs), die als Zielzellen sowohl von FCoV als auch von anderen Coronaviren gelten, wurden mit Wildtyp und den Reportergen-exprimierenden FCoV in vitro infiziert. Die Infektion von monozytären Zellen konnte ausschließlich mit Hilfe der GFP exprimierenden Mutanten nachgewiesen werden.In this study a reverse genetic system for a serotype I feline coronavirus was established. The following results were obtained: 1) As basis for the establishment of a reverse genetic system the complete genomic sequence of FCoV strain Black was determined. The sequence analysis revealed the typical genome organization for coronavirus. 2) For the generation of an infectious clone the full-length FCoV strain Black cDNA was introduced into vaccinia virus as cloning vector. The FCoV cDNA was used for in vitro transcription and the synthesized RNA electroporated into cells. Recombinant FCoV could be detected which showed that the in vitro transcribed RNA was infectious and could initiate the coronavirus replication cycle. The resulting recombinant virus was analyzed in feline cell culture using different approaches. It could be demonstrated that the properties of the recombinant virus are indistinguishable from those of FCoV wild type virus strain Black. 3) In subsequent experiments based on the newly established reverse genetic system, two reporter gene expressing recombinant FCoV were generated. The accessory genes 3abc were replaced by genes encoding the green fluorescent protein (GFP) and Renilla luciferase. The stable expression of these reporter genes was demonstrated in feline cell culture. 4) Feline monocytes, macrophages and dendritic cells (DCs) which are considered to be important target cells for FCoV and other coronaviruses were infected with the wildtype and the reporter gene expressing FCoV. The infection of the monocytic cells was exclusively shown with the GFP expressing mutant

Maternal prebiotic supplementation during pregnancy and lactation modifies the microbiome and short chain fatty acid profile of both mother and infant

Background & aims: Improving maternal gut health in pregnancy and lactation is a potential strategy to improve immune and metabolic health in offspring and curtail the rising rates of inflammatory diseases linked to alterations in gut microbiota. Here, we investigate the effects of a maternal prebiotic supplement (galacto-oligosaccharides and fructo-oligosaccharides), ingested daily from \u3c 21 weeks\u27 gestation to six months’ post-partum, in a double-blinded, randomised placebo-controlled trial. Methods: Stool samples were collected at multiple timepoints from 74 mother–infant pairs as part of a larger, double-blinded, randomised controlled allergy intervention trial. The participants were randomised to one of two groups; with one group receiving 14.2 g per day of prebiotic powder (galacto-oligosaccharides GOS and fructo-oligosaccharides FOS in ratio 9:1), and the other receiving a placebo powder consisting of 8.7 g per day of maltodextrin. The faecal microbiota of both mother and infants were assessed based on the analysis of bacterial 16S rRNA gene (V4 region) sequences, and short chain fatty acid (SCFA) concentrations in stool. Results: Significant differences in the maternal microbiota profiles between baseline and either 28-weeks’ or 36-weeks’ gestation were found in the prebiotic supplemented women. Infant microbial beta-diversity also significantly differed between prebiotic and placebo groups at 12-months of age. Supplementation was associated with increased abundance of commensal Bifidobacteria in the maternal microbiota, and a reduction in the abundance of Negativicutes in both maternal and infant microbiota. There were also changes in SCFA concentrations with maternal prebiotics supplementation, including significant differences in acetic acid concentration between intervention and control groups from 20 to 28-weeks’ gestation. Conclusion: Maternal prebiotic supplementation of 14.2 g per day GOS/FOS was found to favourably modify both the maternal and the developing infant gut microbiome. These results build on our understanding of the importance of maternal diet during pregnancy, and indicate that it is possible to intervene and modify the development of the infant microbiome by dietary modulation of the maternal gut microbiome

Stillbirth risk prediction using machine learning for a large cohort of births from Western Australia, 1980–2015

Quantification of stillbirth risk has potential to support clinical decision-making. Studies that have attempted to quantify stillbirth risk have been hampered by small event rates, a limited range of predictors that typically exclude obstetric history, lack of validation, and restriction to a single classifier (logistic regression). Consequently, predictive performance remains low, and risk quantification has not been adopted into antenatal practice. The study population consisted of all births to women in Western Australia from 1980 to 2015, excluding terminations. After all exclusions there were 947,025 livebirths and 5,788 stillbirths. Predictive models for stillbirth were developed using multiple machine learning classifiers: regularised logistic regression, decision trees based on classification and regression trees, random forest, extreme gradient boosting (XGBoost), and a multilayer perceptron neural network. We applied 10-fold cross-validation using independent data not used to develop the models. Predictors included maternal socio-demographic characteristics, chronic medical conditions, obstetric complications and family history in both the current and previous pregnancy. In this cohort, 66% of stillbirths were observed for multiparous women. The best performing classifier (XGBoost) predicted 45% (95% CI: 43%, 46%) of stillbirths for all women and 45% (95% CI: 43%, 47%) of stillbirths after the inclusion of previous pregnancy history. Almost half of stillbirths could be potentially identified antenatally based on a combination of current pregnancy complications, congenital anomalies, maternal characteristics, and medical history. Greatest sensitivity is achieved with addition of current pregnancy complications. Ensemble classifiers offered marginal improvement for prediction compared to logistic regression

Stillbirth risk prediction using machine learning for a large cohort of births from Western Australia, 1980–2015

Quantification of stillbirth risk has potential to support clinical decision-making. Studies that have attempted to quantify stillbirth risk have been hampered by small event rates, a limited range of predictors that typically exclude obstetric history, lack of validation, and restriction to a single classifier (logistic regression). Consequently, predictive performance remains low, and risk quantification has not been adopted into antenatal practice. The study population consisted of all births to women in Western Australia from 1980 to 2015, excluding terminations. After all exclusions there were 947,025 livebirths and 5,788 stillbirths. Predictive models for stillbirth were developed using multiple machine learning classifiers: regularised logistic regression, decision trees based on classification and regression trees, random forest, extreme gradient boosting (XGBoost), and a multilayer perceptron neural network. We applied 10-fold cross-validation using independent data not used to develop the models. Predictors included maternal socio-demographic characteristics, chronic medical conditions, obstetric complications and family history in both the current and previous pregnancy. In this cohort, 66% of stillbirths were observed for multiparous women. The best performing classifier (XGBoost) predicted 45% (95% CI: 43%, 46%) of stillbirths for all women and 45% (95% CI: 43%, 47%) of stillbirths after the inclusion of previous pregnancy history. Almost half of stillbirths could be potentially identified antenatally based on a combination of current pregnancy complications, congenital anomalies, maternal characteristics, and medical history. Greatest sensitivity is achieved with addition of current pregnancy complications. Ensemble classifiers offered marginal improvement for prediction compared to logistic regression

Maternal prebiotic supplementation during pregnancy and lactation modifies the microbiome and short chain fatty acid profile of both mother and infant

Background & aims: Improving maternal gut health in pregnancy and lactation is a potential strategy to improve immune and metabolic health in offspring and curtail the rising rates of inflammatory diseases linked to alterations in gut microbiota. Here, we investigate the effects of a maternal prebiotic supplement (galacto-oligosaccharides and fructo-oligosaccharides), ingested daily from <21 weeks' gestation to six months’ post-partum, in a double-blinded, randomised placebo-controlled trial. Methods: Stool samples were collected at multiple timepoints from 74 mother–infant pairs as part of a larger, double-blinded, randomised controlled allergy intervention trial. The participants were randomised to one of two groups; with one group receiving 14.2 g per day of prebiotic powder (galacto-oligosaccharides GOS and fructo-oligosaccharides FOS in ratio 9:1), and the other receiving a placebo powder consisting of 8.7 g per day of maltodextrin. The faecal microbiota of both mother and infants were assessed based on the analysis of bacterial 16S rRNA gene (V4 region) sequences, and short chain fatty acid (SCFA) concentrations in stool. Results: Significant differences in the maternal microbiota profiles between baseline and either 28-weeks’ or 36-weeks’ gestation were found in the prebiotic supplemented women. Infant microbial beta-diversity also significantly differed between prebiotic and placebo groups at 12-months of age. Supplementation was associated with increased abundance of commensal Bifidobacteria in the maternal microbiota, and a reduction in the abundance of Negativicutes in both maternal and infant microbiota. There were also changes in SCFA concentrations with maternal prebiotics supplementation, including significant differences in acetic acid concentration between intervention and control groups from 20 to 28-weeks’ gestation. Conclusion: Maternal prebiotic supplementation of 14.2 g per day GOS/FOS was found to favourably modify both the maternal and the developing infant gut microbiome. These results build on our understanding of the importance of maternal diet during pregnancy, and indicate that it is possible to intervene and modify the development of the infant microbiome by dietary modulation of the maternal gut microbiome

Factors influencing gastrointestinal tract and microbiota immune interaction in preterm infants

The role of microbial colonization is indispensable for keeping a balanced immune response in life. However, the events that regulate the establishment of the microbiota, their timing, and the way in which they interact with the host are not yet fully understood. Factors such as gestational age, mode of delivery, environment, hygienic measures, and diet influence the establishment of microbiota in the perinatal period. Environmental microbes constitute the most important group of exogenous stimuli in this critical time frame. However, the settlement of a stable gut microbiota in preterm infants is delayed compared to term infants. Preterm infants have an immature gastrointestinal tract and immune system which predisposes to infectious morbidity. Neonatal microbial dynamics and alterations in early gut microbiota may precede and/or predispose to diseases such as necrotizing enterocolitis (NEC), late-onset sepsis or others. During this critical period, nutrition is the principal contributor for immunological and metabolic development, and microbiological programming. Breast milk is a known source of molecules that act synergistically to protect the gut barrier and enhance the maturation of the gut-related immune response. Host-microbe interactions in preterm infants and the protective role of diet focused on breast milk impact are beginning to be unveiled.M.C. acknowledges a “Rio Hortega” Research Fellowship Grant (CM13/0017) and M.V. acknowledges grants PI11/0313 and RD12/0026/0012 (Red SAMID) from the Instituto Carlos III (Spanish Ministry of Economy and Competitivity). M.C.C. and G.P-M. were supported by the grant AGL2013-47420-R from the Spanish Ministry of Science and Innovation.Peer reviewe

Значение мотивации персонала на предприятии

Основная цель – систематизировать сведения о мотивации персонала и его значении на предприятии

Evidence-based guidelines for use of probiotics in preterm neonates

<p>Abstract</p> <p>Background</p> <p>Current evidence indicates that probiotic supplementation significantly reduces all-cause mortality and definite necrotising enterocolitis without significant adverse effects in preterm neonates. As the debate about the pros and cons of routine probiotic supplementation continues, many institutions are satisfied with the current evidence and wish to use probiotics routinely. Because of the lack of detail on many practical aspects of probiotic supplementation, clinician-friendly guidelines are urgently needed to optimise use of probiotics in preterm neonates.</p> <p>Aim</p> <p>To develop evidence-based guidelines for probiotic supplementation in preterm neonates.</p> <p>Methods</p> <p>To develop core guidelines on use of probiotics, including strain selection, dose and duration of supplementation, we primarily used the data from our recent updated systematic review of randomised controlled trials. For equally important issues including strain identification, monitoring for adverse effects, product format, storage and transport, and regulatory hurdles, a comprehensive literature search, covering the period 1966-2010 without restriction on the study design, was conducted, using the databases PubMed and EMBASE, and the proceedings of scientific conferences; these data were used in our updated systematic review.</p> <p>Results</p> <p>In this review, we present guidelines, including level of evidence, for the practical aspects (for example, strain selection, dose, duration, clinical and laboratory surveillance) of probiotic supplementation, and for dealing with non-clinical but important issues (for example, regulatory requirements, product format). Evidence was inadequate in some areas, and these should be a target for further research.</p> <p>Conclusion</p> <p>We hope that these evidence-based guidelines will help to optimise the use of probiotics in preterm neonates. Continued research is essential to provide answers to the current gaps in knowledge about probiotics.</p

They Are What You Eat: Can Nutritional Factors during Gestation and Early Infancy Modulate the Neonatal Immune Response?

The ontogeny of the human immune system is sensitive to nutrition even in the very early embryo, with both deficiency and excess of macro- and micronutrients being potentially detrimental. Neonates are particularly vulnerable to infectious disease due to the immaturity of the immune system and modulation of nutritional immunity may play a role in this sensitivity. This review examines whether nutrition around the time of conception, throughout pregnancy, and in early neonatal life may impact on the developing infant immune system