MiGut: A scalable in vitro platform for simulating the human gut microbiome—Development,validation and simulation of antibiotic-induced dysbiosis

In vitro models of the human colon have been used extensively in understanding the human gut microbiome (GM) and evaluating how internal and external factors affect the residing bacterial populations. Such models have been shown to be highly predictive of in vivo outcomes and have a number of advantages over animal models. The complexity required by in vitro models to closely mimic the physiology of the colon poses practical limits on their scalability. The scalable Mini Gut (MiGut) platform presented in this paper allows considerable expansion of model replicates and enables complex study design, without compromising on in vivo reflectiveness as is often the case with other model systems. MiGut has been benchmarked against a validated gut model in a demanding 9-week study. MiGut showed excellent repeatability between model replicates and results were consistent with those of the benchmark system. The novel technology presented in this paper makes it conceivable that tens of models could be run simultaneously, allowing complex microbiome-xenobiotic interactions to be explored in far greater detail, with minimal added resources or complexity. This platform expands the capacity to generate clinically relevant data to support our understanding of the cause-effect relationships that govern the GM.     

Characterisation of Aft1 a Fot1/Pogo type transposon of Aspergillus fumigatus

In recent years the filamentous fungus Aspergillus fumigatus has become a significant cause of infection in man and as such has become the focus of much study. It is thought to be the leading mould pathogen in leukaemia and transplant patients and is responsible for mortality in a large number of individuals with immunological disorders. In an attempt to develop molecular mutagenesis tools for assessment of this organism, the genome of A. fumigatus was analysed to identify possible functional transposable elements. An apparently intact Fot1/Pogo type transposon with 65% identity to the active Tan1 element of Aspergillus niger was identified and designated Aft1. Aft1 is a 1.9kb element present in multiple (>20) highly conserved copies. It encodes a 332 amino acid transposase which contains all the functional motifs required for transposition. In addition, the transposase was expressed in cultures grown at 37 degrees C in all three strains assessed and excision analysis suggests Aft1 may be active and of use in transposon tagging experiments. Southern hybridisation patterns indicate that Aft1 is widely distributed amongst clinical isolates of A. fumigatus with considerable variation in genomic localisation. A comprehensive analysis of the genomic localisation of Aft1 in the sequenced strain AF293 show that one insertion is 30 bases upstream of a predicted gene encoding a G-protein coupled receptor. Expression analysis indicates that this gene has been inactivated by the insertion.     

Oomycete RXLR effectors: delivery, functional redundancy and durable disease resistance

To manipulate host defences, plant pathogenic oomycetes secrete and translocate RXLR effectors into plant cells. Recent reports have indicated that RXLR effectors are translocated from the extrahaustorial matrix during the biotrophic phase of infection and that they are able to suppress PAMP-triggered immunity. Oomycete genomes contain potentially hundreds of highly diverse RXLR effector genes, providing the potential for considerable functional redundancy and the consequent ability to readily shed effectors that are recognised by plant surveillance systems without compromising pathogenic fitness. Understanding how these effectors are translocated, their precise roles in virulence, and the extent to which functional redundancy exists in oomycete RXLR effector complements, are major challenges for the coming years.     

Therapeutic dendritic cell vaccine preparation using tumor RNA transfection: A promising approach for the treatment of prostate cancer

Background

Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

Methods

Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

Results

Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery.

Conclusion

Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired.     

Parental Weight Status and Girls’ Television Viewing,Snacking, and Body Mass Indexes

Objective: The purpose of this study was to examine whether television viewing (TVV) provides a context for patterns of snacking fostering overweight in young girls from overweight and non‐overweight families. Research Methods and Procedures: Participants were 173 non‐Hispanic white girls and their parents from central Pennsylvania, assessed longitudinally when girls were 5, 7, and 9 years old. Path analysis was used to test patterns of relationships among girls’ TVV, snacking while watching television, snacking frequency, fat intake from energy‐dense snack food, and girls’ increase in body mass index (BMI) from age 5 to 9. Results: In both overweight and non‐overweight families, girls who watched more television consumed more snacks in front of the television. In families where neither parent was overweight, television viewing was the only significant predictor of girls’ increase in BMI. In families where one or both parents were overweight, girls who watched more television snacked more frequently, and girls who snacked more frequently had higher intakes of fat from energy‐dense snacks, which predicted their increase in BMI from age 5 to 9. TVV did not directly predict girls’ increase in BMI in girls from overweight families. Discussion: The results of this study support and extend previous findings that have shown that excessive television viewing and snacking patterns are risk factors for the development of overweight in children; however, patterns of relationships may differ based on parental weight status. For overweight families, TVV may provide a context for excessive snack consumption, in addition to inactivity.     

Developmental Trajectories of Girls' BMI Across Childhood and Adolescence

This study describes qualitatively distinct trajectories of BMI change among girls participating in a longitudinal study of non‐Hispanic, white girls (n = 182) and their parents, assessed at daughters' ages 5, 7, 9, 11, 13, and 15 years. Height, weight, body fat, fasting blood glucose and lipids, blood pressure, waist circumference, and pubertal status were measured, and participants self‐reported dietary, physical activity, and television (TV) viewing patterns. Growth mixture models were used to model heterogeneity in girls' BMI trajectories over 10 years. Statistical support was strongest for four distinct BMI trajectories: (i) upward percentile crossing (UPC; n = 25, 14%); (ii) delayed downward percentile crossing (DDPC; n = 37, 20%); (iii) 60th percentile tracking (60PT; n = 52, 29%); and (iv) 50th percentile tracking (50PT; n = 68, 37%). Girls in the UPC group had more metabolic risk factors at age 15 years, even after adjusting for concurrent weight status. Girls in the UPC group had mothers with the highest BMIs at study entry and were breast‐fed for a shorter duration. This novel approach for examining differences in growth trajectories revealed four distinct BMI trajectories that predicted adolescent metabolic health outcomes in girls. The present study provides support for BMI monitoring in girls and for the potential utility of combining data on BMI tracking with data on familial characteristics for the early identification of girls at elevated risk for obesity and metabolic syndrome.