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From assisting division to molecular transport, cells depend on microtubules for several vital functions. Their fundamental nature is reflected in the extensive links between disease and mutations in their building blocks: heterodimers of tubulin-α and -β proteins. The first reported tubulin mutations were solely associated with neurodevelopment. Since then, disorders including bleeding disorders and infertility have also been attributed to tubulins, with several isotypes in the tubulin family now implicated in disease. However, the mechanisms governing how these mutations give rise to disease remain unclear. This thesis integrates computational and experimental strategies to address this question. First, I focused on comparing isotypes using both approaches. Bioinformatic tools allowed me to evaluate how tubulin isotypes differ in tissue expression, sequence divergence and evolutionary constraint. I then complemented this by utilising cell biology techniques to investigate the capability of tubulin isotypes to be incorporated into microtubules. Through this work, I could also interrogate strategies for tagging tubulin isotypes in vivo. Next, I systematically analysed mutations across the tubulin family to understand how they can result in disease with the view to improve predictions of their phenotypic effects. Variant effect predictors (VEPs) and structural analyses showed an inadequate ability to distinguish between pathogenic mutations and putatively benign variants. Moreover, their performance was markedly worse in genes linked to diseases outside of neurodevelopment. I then highlighted the mutations that had the poorest mutations so I could characterise them experimentally. Mutant tubulin incorporated into microtubules successfully, indicating that their pathogenic phenotype results from dominant-negative effects. Finally, I interrogated mutations in TUBβ4B associated with primary ciliary dyskinesia (PCD), a disease newly ascribed to tubulin. As these mutations varied in structural location and predicted effect, they also provided an excellent case study for identifying differences in pathogenic mechanisms. Indeed, experimental characterisation in vitro showed that mutations occurring close to the heterodimeric binding interface and predicted more likely to be pathogenic (P259S and P259L) disrupted heterodimer formation. In contrast, a mutation predicted more poorly (P358S) close to the microtubule lumen formed a stable heterodimer, suggesting it may exert dominant-negative effects. In vivo experiments confirmed these differences in terms of microtubule incorporation. Overall, this thesis highlights tubulin mutations as a blind spot for pathogenicity prediction, and this is potentially attributed to their ability to cause disease through dominant-negative effects.

Cilia are small microtubule-based structures found on the surface of most mammalian cells, which have key sensory and sometimes motile functions. Primary ciliary dyskinesia (PCD) is a type of ciliopathy caused by defects in motile cilia. The genetic basis of PCD is only partially understood. Studying a cohort of 11 human patients with PCD, we find thatde novomutations inTUBB4B, a beta tubulin isotype, cause three distinct classes of ciliopathic disease.In vivostudies in mice show thatTubb4bplays a specific role in cilia, building centrioles and axonemes in multiciliated cells. Examining the effects of specific TUBB4B variants in cells and in mice, we further demonstrate that distinctTUBB4Bmutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Finally, structure-function studies reveal that different TUBB4B mutations disrupt distinct tubulin interfaces. Importantly, these molecular differences correlate with disease features. We show that tubulin heterodimer-impairing TUBB4B variants underlie nonsyndromic PCD, whilst additional renal and sensorineural ciliopathic features in a syndromic PCD subtype arise from microtubule lumenal interface-impaired TUBB4B variants. These findings suggest that specific tubulin isotypes have distinct and non-redundant subcellular functions, and demonstrate that human tubulinopathies can be drivers of ciliopathic syndromes.

Additional file 1: Fig. S1 A. Construction of OTUs and effective reads data. ~1450 bacterial OTUs were detected in stool or tissue samples combined. The y1-axis represents the number of reads. Red and blue bars represent the number of effective and annotated reads, respectively. We also detected some "Unique Reads" (orange bars) with a frequency of 1 that only occurs in one sample. The y2-axis titled "OTUs Number" represents the number of OTUs displayed as purple bars to identify the numbers of OTUs in different samples. B-E. Relative abundance of bacterial kingdom. Agglomeration of bacteria at the class, order, family, and genus levels. The y1-axis represents the number of reads. Red and blue bars represent the number of effective and annotated reads, respectively. We also detected some "Unique Reads" (orange bars) with a frequency of 1 that only occurs in one sample. The y2-axis titled "OTUs Number" represents the number of OTUs displayed as purple bars to identify the numbers of OTUs in different samples.

Additional file 4. Accuracy metrics for the machine learning models.

Additional file 2: Fig. S2 Schematic of our major findings. The microbiome from fecal specimens with that obtained from normal mucosa in children with FAP and from fecal specimens in healthy controls was compared in addition to biopsies from an adenoma to synchronous normal mucosa. Observations including significant preferential expression of probiotic candidate bacteria in control stool vs. decreased potentially protective bacterial subtypes in polyp compared with normal mucosa were made along with a reduction in alpha- and beta-diversities in polyp compared with apparently normal mucosa. Machine learning further distinguished bacterial populations between patients with and without polyposis. Changes are indicated through up or down arrows, respectively. Partially created in BioRender.

GC chromatograms and photos of extracts

GC chromatograms and photos of extracts

To meet decarbonisation goals and implement a more sustainable circular economy model, the chemical industry needs to transition from fossil to renewable sources of carbon. Current chemical production is dominated by petroleum, where this broadly uniform feedstock is separated using a single, simple process to give a small range of heteroatom-free molecules that are the platform to a myriad of products. In a biorefinery, however, many feedstocks of widely varying composition using markedly different technologies are processed to give one or two of a wide range of bioderived platform molecules. Here, recent publications are used to highlight selection of the most suitable second or third generation feedstocks, converted using integrated, complementary processes to generate multiple products. This approach generates a range of chemicals, more fully using the carbon source of choice in a sustainable manner, generating more value, which together makes the realisation of the biorefinery concept draw ever closer.

International audience; The European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) Polyposis Working Group developed recommendations to assist clinicians and health care providers with appropriate management of patients with juvenile polyposis. This is the first juvenile polyposis Position Paper published by ESPGHAN with invited experts. Many of the published studies were descriptive and/or retrospective in nature, consequently after incorporating a modified version of the GRADE system many of the recommendationsare based on expert opinion. This ESPGHAN Position Paper provides a guide for diagnosis, assessment and management of Juvenile polyposis syndrome (JPS) in children and adolescents, and will be helpful in the appropriate management and timing of procedures in children and adolescents. The formation of international collaboration and consortia is proposed in order to monitor patients prospectively to advance our understanding of juvenile polyposis conditions.

Immense global generation of food waste calls for advanced technologies to maximize the use of such renewable carbon-based resources. In this study, corn, taro, lettuce, and bean sprout, were valorized for the production of value-added chemicals via sequential supercritical CO2 (scCO2) extraction and thermochemical conversion. The scCO2 extraction was performed at 350 bar and 50 °C for 60 min. The extracts of the lettuce contained sterols (764 μg g–1) that have potential anticancer properties. While bean sprout extracts had a higher content of saturated fatty acids (641 μg g–1), corn extracts comprised polyunsaturated fatty acids (405 μg g–1) as one of the major compounds, which are beneficial to cholesterol control. There were also notable amounts of wax esters (75–774 μg g–1) in these food waste extracts. Taro extracts were rich in both saturated (2313 μg g–1) and unsaturated fatty acids (1605 μg g–1) and, in particular, contained difatty acids that exhibit pharmaceutical activities. Moreover, the solid residues after scCO2 extraction served as the substrates for platform chemical production. The starch-rich substrates, i.e., taro and corn, resulted in 11–20% hydroxymethylfurfural (HMF) after microwave heating at 140 °C for 5–10 min using SnCl4 catalyst. In comparison, due to the high fiber content, lettuce and bean sprout required a higher temperature of 170–190 °C for chemical decomposition over H2SO4, generating a levulinic acid yield of ∼7%, in company with glucose and fructose as the coproducts. This study on the combined technologies suggested good compatibility between scCO2 extraction and subsequent thermochemical conversion, producing a wide spectrum of value-added chemicals from biomass waste. We herein highlight the vast potential of integrated technologies for food waste valorization in achieving sustainable and carbon-efficient biorefineries.

The decline in petrochemical wax supply coupled with the ever-growing demand for bio-products means that the development of a sustainable process to renewably sourced waxes is paramount. This review focuses on recent advances in supercritical extraction as a clean efficient process for extracting waxes from waste biomass as a part of a holistic biorefinery. The use of supercritical carbon dioxide leads to reductions in solvent waste and leaves no solvent residues meaning that the biomass can further processed without the need for energy-intensive solvent removal steps. This technology crucially improves the downstream conversion of residual cellulosic biomass for the sustainable production of sugars, consumer products and biofuels (up to a 40% increase in ethanol production) leading to higher energy efficiencies and higher economic returns. Supercritical carbon dioxide extraction is not only an important technology for the cleaner production of waxes, it is a sustainable pre-treatment of biomass as part of an integrated holistic biorefinery and significantly, it can improve the safety of products, e.g. less off gassing of wood pellets.

Up to 33% of hemp by mass can be lost in the form of dust during processing for fibre production. Heptane Soxhlet extractions and supercritical carbon dioxide extractions (scCO2) of hemp dust samples yielded significant quantities of high value lipophilic molecules including fatty acids, policosanols (fatty alcohols), fatty aldehydes, hydrocarbons, sterols, triterpenoids and cannabinoids (cannabidiol (CBD)). Dust collected from different stages of the mechanical processing of hemp fibres gave rise to lipophilic extractives with varying compositions, thus making the isolation and purification of these compounds easier. Of particular interest is CBD (5832.5 ± 118.9 μg/g of dust), which has attracted much attention for clinical-level studies due to its therapeutic efficacy in the treatment of a variety of central nervous system (CNS) disorders. Factorial experimental design was carried out to optimise the scCO2 extraction, with 350 bar and 50 °C yielding the selective extraction of higher value components.

Supercritical carbon dioxide was utilized as a sustainable alternative to solvent extraction of waxes from the waste needles of two spruce species, namely Norwegian and Sitka spruce. These extracts were rich in nonacosan-10-ol, an organic compound with hydrophobic properties that lends its use in the preparation of superhydrophobic coatings. The highest crude yields were 1.7% w/w of needles obtained at 400 bar and 60 °C, while nonacosan-10-ol was selectively extracted at 200 bar and 60 °C (8070 ± 91.1 μg/g of needles). Purification of nonacosan-10-ol from the wax extracts was conducted using a simple rapid green recrystallization technique. This yielded a recovery of 44.6% ± 2% and 48.4% ± 2% of the total nonacosan-10-ol from the original crude Sitka (3600 μg/g of needles) and Norwegian wax (1920 μg/g of needles) respectively. Application of nonacosan-10-ol to a filter paper led to the formation of highly hydrophobic surfaces, with preliminary contact angles of up to 149°. This sustainable production method may develop opportunities to valorize forestry waste within a holistic biorefinery.

Recurrent abdominal pain in childhood, is classically defined as three or more episodes of pain severe enough to interfere with daily activities over the span of more than three months. It is a common presenting complaint to both general practice and pediatricians with a prevalence of 10-15 % of school age children. However an organic underlying disorder is rare; most studies suggest in the order of 5-10%. The clinical characteristics that facilities the recognition of children who are more likely to harbor organic disease are therefore important to the practicing primary care provider and pediatricians in order to avoid unnecessary, costly and invasive investigations., peer-reviewed

Gastrointestinal symptoms are frequently attributes to food hypersensitivity both by patients and increasingly by the medical community. Indeed, up to 35 % of the general population in Western countries think they have food allergy, although this is objectively confirmed in only 1-2% of the population. The incident of food hypersensitivity appears to be on the rise, in parallel with the overall rise in atopic disorders over the last 30-40 years and concurrent with a decline in infectious disorders. This has lead to the emergence if the 'hygiene hypothesis' which raised awareness of the rose of microbes and their products in immune regulation; more recently this had prompted important observations on the impact of beneficial bacteria (probiotics) in the prevention of allergic processes. This article refers to clinical scenarios of food hypersensitivity in infancy, in the older child and young adult; Eosinophilic Esophagitis (EE), celiac disease, milk allergy and lactose intolerance, peer-reviewed

Chronic constipation is defined as a decrease in frequency or the painful passage of bowel movements present for several weeks. Retentive encopresis is the term reserved for the involuntary and uncontrollable soiling that occurs in children with chronic constipation. Constipation is a relatively common pediatric complaint accounting for up to 3% of general paediatirc outpatient visits and 25% of visits to a pediatric gastroenterologist. Encopresis is also common, occurring in at least 1.5% of all children, peer-reviewed

Clinicians are increasingly involved with probiotics in the wake of society’s increased concern with the deleterious effects of antibiotics along with a trust towards more healthful foods and supplements. Pre- and probiotic agents however are not created equal and a familiarity with the key concepts and published literature is desirable., peer-reviewed

With an approximate length of 15 feet, the small intestine presents a daunting challenge to the clinician faced with symptoms referable to this relatively inaccessible area. In the last decade, capsule endoscopy (CE) has established an increasingly broad niche in the diagnostic armamentarium of both paediatric and adult clinicians; it is now the established standard of care in the work up of obscure gastrointestinal hemorrhage, suspected Crohn's Disease, polyposis syndromes and arguably, chronic abdominal pain., peer-reviewed