4,731 research outputs found

    Bioactive Self-Assembled Protein Nanosheets for Stem Cell-Based Biotechnologies

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    Tissue and stem cell culture methods have been dominated by glass and plastic substrates such as Tissue culture plastic. These solid substrates, although widely used, are associated with poor scalability for adherent stem cell expansion in systems such as 3D bioreactors and the design of parallel culture systems. Therefore, investigating strategies to bypass these obstacles in stem cell expansion is essential to enable the wider translation of stem cell technologies. An alternative strategy recently proposed consists in using a liquid surface instead, such as an oil, and associated oil droplets. Indeed, emulsions can be formed using protein nanosheets to stabilise oil/water interfaces to promote the adhesion of stem cells and enable their proliferation. These nanosheets exhibit enhanced interfacial mechanics and allow the introduction of bioactive components via recombinant protein expression to promote bioactivity. Beyond the application of resulting bioemulsions for the expansion of Mesenchymal stem cells, the impact of these bioactive interfaces on the differentiation of iPSCs and the development of cerebral organoids will be presented. The Bovine serum albumin protein was recombinantly modified to attach an N-terminal Avi-Tag, this was expressed and purified from the yeast P. pastoris expression system. The Avi-tag was then biotinylated in vitro by recombinantly expressed BirA. Emulsions of a specific size were formed using the newly biotinylated Bt-BSA protein and functionalized with a cascade of components to mimic cell-cell ligands, this resulted in bioemulsions with a bioactive surface that can interact with surrounding cells. These functionalised droplets were integrated into developing cerebral organoids and their impact on phenotype was studied. The droplets were found not to deform sufficiently to allow mechanical forces to be measured, yet the many of these droplets were retained within the organoids which led to an interesting phenotype within the organoids. The developing rosettes were found to develop enlarged lumens shown by an increase in area, this phenotype did not impact the differentiation into the cerebral lineage depicted by immunohistochemistry of hallmark marker of neuronal differentiation within organoids retaining droplets. The interfacial mechanics of fibrinogen nanosheets treated with varying concentrations of thrombin was studied using interfacial shear rheology. The effect of thrombin significantly altered the interfacial mechanics with the lower concentration of thrombin significantly increasing the toughness multiple folds and decreasing the elasticity of the nanosheets. Additionally, the nanostructure of nanosheets was studied using SEM and TEM and traditional fibrin fibres were found to not form at these interfaces, but local rearrangements and retractions in the thrombin treated nanosheets were observed. Finally, these enhanced mechanical properties promoted the proliferation and expansion of Mesenchymal stem cells on quasi-2D and 3D interfaces

    Urinary microbiota signatures associated with different types of urinary diversion: a comparative study

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    BackgroundRadical cystectomy and urinary diversion (UD) are gold standards for non-metastatic muscle-invasive bladder cancer. Orthotopic neobladder (or Studer), ileal conduit (or Bricker) and cutaneous ureterostomy (CU) are mainstream UD types. Little is known about urinary microbiological changes after UD. MethodsIn this study, urine samples were collected from healthy volunteers and patients with bladder cancer who had received aforementioned UD procedures. Microbiomes of samples were analyzed using 16S ribosomal RNA gene sequencing, and microbial diversities, distributions and functions were investigated and compared across groups. ResultsHighest urine microbial richness and diversity were observed in healthy controls, followed by Studer patients, especially those without hydronephrosis or residual urine, α-diversity indices of whom were remarkably higher than those of Bricker and CU groups. Studer UD type was the only independent factor favoring urine microbial diversity. The urine microflora structure of the Studer group was most similar to that of the healthy individuals while that of the CU group was least similar. Studer patients and healthy volunteers shared many similar urine microbial functions, while Bricker and CU groups exhibited opposite characteristics. ConclusionOur study first presented urinary microbial landscapes of UD patients and demonstrated the microbiological advantage of orthotopic neobladder. Microbiota might be a potential tool for optimization of UD management

    Structural and spectroscopic characterisation of Cytochrome c’ and Cytochrome P460 from Methylococcus capsulatus (Bath)

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    Many Ammonia-oxidising nonlithotrophic bacterium (ANB) and Ammonia oxidising bacteria (AOB) have been shown to contain two phylogenetically related cytochromes: a cytochrome P460 and a cytochrome c’-β. Cytochrome P460s (so named due to their 460 nm peak in the ferrous state) are enzymes known to convert hydroxylamine to nitrous oxide, a key step in the metabolism of ammonia in bacteria which is considered to be one of the largest sources of nitrous oxide in the environment. Cytochromes c’-β are so called as they have spectral properties similar to the better studied c’-α but they are predicted to all have a highly beta sheet structure instead of the alpha helixes normally associated with a cytochrome c’. Whilst the role of Cytochrome c’s has not been definitively proved it has been proposed that they are involved in NO scavenging and protecting cells against nitrosoative stress. P460s have been well studied in AOB but less so in ANB, whilst very few members of the cytochrome c’-β have been characterised at all. This thesis focusses on the cytochrome P460 and c’ from Methylococcus capsulatus (Bath) characterising their structural and spectroscopic properties through the use of cryogenic single crystal X-ray crystallography and UV-visible and EPR spectra, along with kinetic studies and activity assays, on both the wt proteins and single point mutants, to investigate how structural differences in the distal heme pockets for two proteins with very similar overall protein folds can give rise to two very different functions

    Clarifying the murk: unveiling bacterial dynamics in response to crude oil pollution, Corexit-dispersant, and natural sunlight in the Gulf of Mexico

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    The 2010 Deepwater Horizon (DwH) Oil spill released an enormous volume of oil into the Gulf of Mexico (GoM), prompting the widespread use of chemical dispersants like Corexit® EC9500A. The ecological consequences of this treatment, especially when combined with natural factors such as sunlight, remain unexplored in the context of marine bacterial communities’ dynamics. To address this knowledge gap, our study employed a unique metaproteomic approach, investigating the combined effects of sunlight, crude Macondo surrogate oil, and Corexit on GoM microbiome across different mesocosms. Exposure to oil and/or Corexit caused a marked change in community composition, with a decrease in taxonomic diversity relative to controls in only 24 hours. Hydrocarbon (HC) degraders, particularly those more tolerant to Corexit and phototoxic properties of crude oil and/or Corexit, proliferated at the expense of more sensitive taxa. Solar radiation exacerbated these effects in most taxa. We demonstrated that sunlight increased the dispersant’s toxicity, impacting on community structure and functioning. These functional changes were primarily directed by oxidative stress with upregulated proteins and enzymes involved in protein turnover, general stress response, DNA replication and repair, chromosome condensation, and cell division. These factors were more abundant in chemically treated conditions, especially in the presence of Corexit compared to controls. Oil treatment significantly enhanced the relative abundance of Alteromonas, an oil-degrading Gammaproteobacteria. In combined oil-Corexit treatments, the majority of identified protein functions were assigned to Alteromonas, with strongly expressed proteins involved in membrane transport, motility, carbon and amino acid metabolism and cellular defense mechanisms. Marinomonas, one of the most active genera in dark conditions, was absent from the light treatment. Numerous metabolic pathways and HC-degrading genes provided insights into bacterial community adaptation to oil spills. Key enzymes of the glyoxylate bypass, enriched in contaminant-containing treatments, were predominantly associated with Rhodobacterales and Alteromonadales. Several proteins related to outer membrane transport, photosynthesis, and nutrient metabolisms were characterized, allowing predictions of the various treatments on biogeochemical cycles. The study also presents novel perspectives for future oil spill clean-up processes

    Characterisation of peroxisomes in the fission Yeast Schizosaccharomyces pombe and slime mold Dictyostelium discoideum

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    Peroxisome is a compartment that is found in most eukaryotic organisms' cells. It has several crucial roles, such as fatty acid beta (β) oxidation and hydrogen peroxide (H2O2) detoxification. It contains many essential enzymes, including oxidase and catalase, and has several metabolic and non-metabolic pathways, depending on the environment and the organisms within its cells. This study investigates the role of peroxisomes in two organisms, S. pombe and D. discoideum. Although S. pombe is a well-studied yeast, there is only one study of this yeast that has focused on peroxisomes. This study offers a few crucial observations, including that S. pombe contains peroxisomes, that GFP containing a well-characterized PTS1 (SKL) is efficiently imported, and that peroxisome numbers increase in cells grown on a fatty acid as the sole carbon source, suggesting a role for peroxisomes in fatty acid degradation. The starting point in my research was initially a bioinformatics screen. This screening recognized the enzymes imported into peroxisomes based on the presence of a potential peroxisomal targeting signal. A few proteins were found. However, the low number of proteins with a classical PTS might be the result of different targeting signals that are not recognized by our bioinformatics parameters. Indeed, in other organisms, there are proteins without PTS1 that still use Pex5 for import. The first example is S. cerevisiae Acyl-CoA oxidase. In a global yeast two-hybrid screen, S. pombe Pex5 was found to bind S. pombe Str3 and Lys3. Consequently, we think that there is conserved targeting of a peroxisomal protein lacking a PTS1 and PTS2 imported into the peroxisome by Pex5. One of these is the Str3 case. Interestingly, proteins involved in peroxisomal fatty acid β -oxidation are absent from the S. pombe genome, casting doubt on the conclusions from the previous study and explaining the low number of potential peroxisomal enzymes. In D. discoideum, this study investigates the dynamic regulation of peroxisome numbers in response to growth conditions and identifies peroxisomal import and contents through a proximity labeling approach (BioID). Overall, this study sheds light on the roles and regulation of peroxisomes in these two organisms

    Emerging Evidence for Association of Transsulfuration Pathway with Hypoxia Responses

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    When people ascend to a high altitude (HA), the body’s oxygen (O2) sensing mechanisms can sense perturbation in partial pressure and trigger adaptive responses. Rapid ascending to HA without ample time for acclimatization culminates in high-altitude illnesses, which can derail the body functioning of lowlanders moving to HA. High-altitude native populations have undergone positive natural selection to efficiently overcome the challenges of chronic hypobaric hypoxia (HH) and thus offer a unique model to understand physiological and genetic adaptations at high altitudes. In addition, evolutionary shreds of evidence propose that sulfur belonging to the same periodic table family can mimic oxygen to bypass its metabolic oxygen demand and modulate energy production.Intriguingly, our group has identified a strong association between diminished hydrogen sulfide (H2S)levels and HH-induced pathological responses. We have recently presented experimental evidence of cysteine deficit, which functionally regulates both lowered levels of endogenous H2S and HH-induced neuropathological responses. In this review, we sought to understand the role of H2S and the transsulfuration pathway at HA

    Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast

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    Although many prokaryotes have glycolysis alternatives, it\u27s considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism

    Ecology of methanotrophs in a landfill methane biofilter

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    Decomposing landfill waste is a significant anthropogenic source of the potent climate-active gas methane (CH₄). To mitigate fugitive methane emissions Norfolk County Council are trialling a landfill biofilter, designed to harness the methane oxidizing potential of methanotrophic bacteria. These methanotrophs can convert CH₄ to CO₂ or biomass and act as CH₄ sinks. The most active CH₄ oxidising regions of the Strumpshaw biofilter were identified from in-situ temperature, CH₄, O₂ and CO₂ profiles. While soil CH₄ oxidation potential was estimated and used to confirm methanotroph activity and determine optimal soil moisture conditions for CH₄ oxidation. It was observed that most CH₄ oxidation occurs in the top 60cm of the biofilter (up to 50% of CH4 input) at temperatures around 50ºC, optimal soil moisture was 10-27.5%. A decrease in in-situ temperature following CH₄ supply interruption suggested the high biofilter temperatures were driven by CH₄ oxidation. The biofilter soil bacterial community was profiled by 16S rRNA gene analysis, with methanotrophs accounting for ~5-10% of bacteria. Active methanotrophs at a range of different incubation temperatures were identified by ¹³CH₄ DNA stable-isotope probing coupled with 16S rRNA gene amplicon and metagenome analysis. These methods identified Methylocella, Methylobacter, Methylocystis and Crenothrix as potential CH₄ oxidisers at the lower temperatures (30ºC/37ºC) observed following system start-up or gas-feed interruption. At higher temperatures typical of established biofilter operation (45ºC/50ºC), Methylocaldum and an unassigned Methylococcaceae species were the dominant active methanotrophs. Finally, novel methanotrophs Methylococcus capsulatus (Norfolk) and Methylocaldum szegediense (Norfolk) were isolated from biofilter soil enrichments. Methylocaldum szegediense (Norfolk) may be very closely related to or the same species as one of the most abundant active methanotrophs in a metagenome from a 50ºC biofilter soil incubation, based on genome-to-MAG similarity. This isolate was capable of growth over a broad temperature range (37-62ºC) including the higher (in-situ) biofilter temperatures (>50ºC)

    Investigating Heterologous Transgene Expression in N. Crassa

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    First adopted as a model organism by Shear and Dodge in 1927, the filamentous fungus Neurospora crassa is among the most well studied eukaryotic systems today, facilitating major discoveries in genetics, biochemistry, and molecular biology. The sequencing of the Neurospora genome was completed in 2003, which combined with the organism’s fast growth, simple culturing requirements, and haploid life cycle, make it a particularly attractive model system. Despite these advantages, Neurospora crassa’s research value is diminished by its recalcitrance to expressing ectopic sequences incorporated into its genome. As a result, molecular techniques requiring heterologous expression (such as CRISPR) are largely incompatible with Neurospora research, despite successful implementation in other model systems. Neurospora possesses multiple well-documented processes of neutralizing sequences deemed a threat to genomic integrity, such as transposable elements or mycoviruses. Even in the absence of characterized genome defense mechanisms in N. crassa, such as quelling, the expression of transgenic Cas9 endonuclease remains inhibited, suggesting the possibility of an additional genome defense mechanism operating within this model system. Here I propose that inhibition of Cas9 expression in N. crassa results from regulatory elements, encoded within its genome, that target transgenic sequences for silencing. I further propose that these regulatory elements are, themselves, vulnerable to inactivation through mutation. Utilizing an experimental transgenic strain containing inexpressible Cas9 fragment, mutagenesis assays have been performed and a potential heterologous expression positive (hep) mutant has been isolated. Sequencing of the transgenic construct in the mutant revealed no alterations, indicating that this mutation likely acts in trans. Furthermore, the results of sexual crosses and recombinant screening indicate that a single mutation located in the vicinity of the native leu-1 locus on chromosome three is likely responsible for the observed mutant phenotype
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