1,922 research outputs found
Detection of ice core particles via deep neural networks
Insoluble particles in ice cores record signatures of past climate parameters like vegetation dynamics, volcanic activity, and aridity. For some of them, the analytical detection relies on intensive bench microscopy investigation and requires dedicated sample preparation steps. Both are laborious, require in-depth knowledge, and often restrict sampling strategies. To help overcome these limitations, we present a framework based on flow imaging microscopy coupled to a deep neural network for autonomous image classification of ice core particles. We train the network to classify seven commonly found classes, namely mineral dust, felsic and mafic (basaltic) volcanic ash grains (tephra), three species of pollen (Corylus avellana, Quercus robur, Quercus suber), and contamination particles that may be introduced onto the ice core surface during core handling operations. The trained network achieves 96.8 % classification accuracy at test time. We present the system's potential and its limitations with respect to the detection of mineral dust, pollen grains, and tephra shards, using both controlled materials and real ice core samples. The methodology requires little sample material, is non-destructive, fully reproducible, and does not require any sample preparation procedures. The presented framework can bolster research in the field by cutting down processing time, supporting human-operated microscopy, and further unlocking the paleoclimate potential of ice core records by providing the opportunity to identify an array of ice core particles. Suggestions for an improved system to be deployed within a continuous flow analysis workflow are also presented
Marine Toxins from Harmful Algae and Seafood Safety
The rapid expansion of aquaculture around the world is increasingly being impacted by toxins produced by harmful marine microalgae, which threaten the safety of seafood. In addition, ocean climate change is leading to changing patterns in the distribution of toxic dinoflagellates and diatoms which produce these toxins. New approaches are being developed to monitor for harmful species and the toxins they produce. This Special Issue covers pioneering research on harmful marine microalgae and their toxins, including the identification of species and toxins; the development of new chemical and biological techniques to identify and monitor species and toxins; the uptake of marine biotoxins in seafood and marine ecosystems; and the distribution and abundance of toxins, particularly in relation to climate change
Single-molecule detection and characterisation of alpha-synuclein aggregates
Aberrant protein aggregation is a predominant feature of many neurodegenerative disorders.
It has long been recognised that aggregates of alpha-synuclein (α-syn) drive pathogenesis in
Parkinson’s Disease (PD), and it is widely accepted that small α-syn oligomers are the key
cytotoxic species in PD. Notably, however, these oligomeric species are difficult to characterise
using traditional biochemical ensemble methods due to their high level of heterogeneity
and low abundance. Single-molecule fluorescence microscopy techniques have emerged
as a suitable approach to circumventing this problem, enabling the detection of individual
aggregates amongst monomeric protein and thus facilitating the identification, quantification,
and characterisation of rare oligomeric species. However, cellular mechanisms of α-syn aggregation
are poorly understood. Furthermore, there remains some limitations to the singlemolecule
techniques currently available. This thesis describes the work completed to address
some of these issues.
Chapter 1 provides the contextual background for the work presented in this thesis, detailing
the biological aspects of α-syn, its aggregation, and its implications in PD, as well as outlining
the single-molecule techniques used to investigate aggregate species. Chapter 2 describes
the methodologies undertaken in this thesis, and chapters 3 to 5 describe the findings made
using the single-molecule techniques which were utilised and developed in this work.
One primary approach for studying species in single-molecule experiments involves directly
labelling biomolecules of interest with a suitable fluorophore. Early steps in α-syn aggregation
have previously been identified using fluorescently tagged α-syn and single-molecule Förster
resonance energy transfer (smFRET) in vitro; however, the characterisation of early aggregate
formation in cells has thus far been difficult to achieve. Chapter 3 describes the use of duallabelled
α-syn to detect and characterise aggregates formed both intracellularly and in vitro
via smFRET, using both single-molecule confocal microscopy coupled with microfluidics and
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total internal reflection fluorescence microscopy (TIRFM) to determine both the sizes and
structures of the oligomers formed. This work reveals the presence of distinct oligomeric
species in vitro and in neurons resulting from structural conversion during early aggregate
formation.
The approach taken in Chapter 3 is highly suitable for investigating aggregate formation
resulting from the addition of exogenous α-syn to samples of interest. However, such an
approach is not ideal for the detection and characterisation of endogenous aggregates due to
issues with the covalent labelling of cellular protein. Extrinsic amyloid dyes are typically used
as an alternative approach to labelled protein; however, such dyes are non-protein-specific
and bind to the common amyloid beta-sheet motif. As an alternative, the work presented in
Chapter 4 describes a novel single-molecule method to specifically detect and characterise
α-syn aggregates with high sensitivity, making use of a high-affinity antibody labelled with
orthogonal fluorophores which is combined with fast-flow microfluidics and single-molecule
confocal microscopy. This enables the quantification and size approximation of α-syn aggregates
at picomolar concentrations, both in vitro and in biological samples.
Although the kinetics of α-syn aggregation have been studied extensively, much of our current
knowledge stems from ensemble averaging techniques which are associated with high levels
of variability and are not conducive to detecting the earliest steps in aggregate formation.
In addition, there remains uncertainty surrounding the effect of familial variants and posttranslational
modifications (PTM) on aggregation. Chapter 5 encompasses the study of the effects
of the ubiquitous N-terminal acetylation PTM, in addition to the familial, rapid-onset G51D
mutation, on α-syn aggregation, using the novel detection method developed in Chapter 4.
This is used in conjunction with single-molecule detection with thioflavin-T (ThT) to reveal new
insights into the aggregation of α-syn variants.
Overall, the work presented here provides new insights into the aggregation of α-syn via the
use and development of single-molecule techniques. The advancements made have added
to the current understanding of the molecular mechanisms of α-syn aggregation, both in
vitro and in neurons, and have also been used to develop a novel single-molecule detection
method for α-syn aggregates. The work presented in this thesis has resulted in two published
papers, ’Pathological structural conversion of alpha-synuclein at the mitochondria induces
neuronal toxicity’ in Nature Neuroscience, and ’Single-molecule two-color coincidence detection
of unlabeled alpha-synuclein aggregates’ in Angewandte Chemie International Edition.
Furthermore, the novel detection method presented here holds promise for measuring α-syn
oligomeric load in clinical samples due to its high sensitivity and specificity for α-syn aggregates.
This may therefore be used in future studies for identifying, detecting, and studying
potential biomarkers in PD, with potential use in disease diagnosis. It is therefore expected
that the work from this thesis will be used to aid researchers towards better understanding the
mechanisms of α-syn aggregation, both in vitro and in clinical samples
Recommended from our members
Sonic heritage: listening to the past
History is so often told through objects, images and photographs, but the potential of sounds to reveal place and space is often neglected. Our research project ‘Sonic Palimpsest’1 explores the potential of sound to evoke impressions and new understandings of the past, to embrace the sonic as a tool to understand what was, in a way that can complement and add to our predominant visual understandings. Our work includes the expansion of the Oral History archives held at Chatham Dockyard to include women’s voices and experiences, and the creation of sonic works to engage the public with their heritage. Our research highlights the social and cultural value of oral history and field recordings in the transmission of knowledge to both researchers and the public. Together these recordings document how buildings and spaces within the dockyard were used and experienced by those who worked there. We can begin to understand the social and cultural roles of these buildings within the community, both past and present
Analysis of Polyphenols from Laminaria hyperborea for application as Life Science ingredients
Makroalger, også kjent som tang og tare, er anerkjent som en verdifull kilde til bioaktive forbindelser. Av disse har polyfenoler fått særlig oppmerksomhet. Polyfenoler har blitt assosiert med en rekke helsefordeler, og er mest kjent for sine ulike biologiske egenskaper, så som antioksidant-, antiinflammatorisk-, antidiabetisk-, og antikreftegenskaper. og. Dette prosjektet undersøkte to typer makroalger; den brune algen Laminaria hyperborea (nor.: stortare) og grønnalgen Ulva intestinalis (nor.: tarmgrønske). De første ekstraksjonene av L. hyperborea blader var utfordrerne grunnet dere høye polysakkarid innhold. Derfor ble det utført en innledende studie med grønn algen U. intestinalis for å utforske de ulike analyseteknikkene som brukes for kvantifisering og identifisering av polyfenoler. Teknikkene som ble benyttet i denne studien inkluderte tradisjonell DAD-HPLC, en totalt-fenolinnhold (TPC) test med Folin-Ciocalteu reagens, en totalt-flavonoidinnhold (TFC) test med en AlCl3 reagens, samt 1H kvantitativ NMR (qNMR) – inkludert 2D NMR. Resultatene viste at det polyfenoliske innholdet var komplekst, og med lav konsentrasjon. For råprøven (crude) av U. intestinalis ble det totale fenolinnholdet målt til 11.3 ± 1.4 mg GAE/g ved bruk av HPLC, 5.0 ± 1.0 mg GAE/g med TPC, og 27.3 ± 2.7 mg GAE/g med qNMR. TFC-testen oppdaget imidlertid ingen flavonoider i råprøven. Denne innledende studien avdekket et sterkt behov for å optimalisere de analytiske metodene som ble benyttet. I tillegg ble det utført LC-MS karakterisering på opprensede prøver av U. intestinalis. Disse identifiserte tentativt tilstedeværelsen av flere fenolsyrer og flavonoider. Noen av disse ble også bekreftet av referansestandarder.
Etter optimalisering av TPC-testen og 1H qNMR-metoden, ble kvantifisering og sammenlikning av L. hyperborea med fire andre brunalger gjennomført. Det totale polyfenolinnholdet i L. hyperborea ble funnet å være 5.51 ± 0.00 mg GAE/g (qNMR) og 5.72 ± 0.07 mg GAE/g (TPC). For tang som tilhører Laminariaceae-familien, som vokser i sublitoralsone, viste TPC-testen og selektiv qNMR liknende, men lave polyfenolkonsentrasjoner. Samtidig ble det for tang som vokser i epilittoralsone, som Ascophyllum nodosum (nor.: grisetang) og Fucus vesiculosus (no.: blæretang), observert en større forskjell mellom TPC og qNMR resultatet. Tang som vokser i strand sonen vil bli utsatt for mer lys, noe som kan føre til at det polyfenolske innholdet består av flere polyfenoler som inneholder flere reagerende grupper enn referanse standarden (gallesyre eller floroglucinol) som benyttes, noe som kan produsere overestimering av polyfenol innholdet ved bruk at TPC testen. Det er også mulig at disse artene som vokser nærmere havoverflaten inneholder en økt mengden TPC-forstyrrende forbindelser enn artene som vokser dypere i den sublitorale sonen. For å karakterisere det polyfenoliske innholdet i L. hyperborea, ble flash-kromatografi og preparativ HPLC benyttet til å rense opp tangprøven, for å opparbeide rensede fraksjoner til bruk i analysen. Resultatene viste at prøver som var mer opprenset korresponderte med høyere polyfenolinnhold både i TPC-testen og ved selektiv qNMR. I tillegg viste ORAC-testresultatene økende antioksidantaktivitet med økende renhet.
For å identifisere den molekylære strukturen til polyfenolene i L. hyperborea, ble både lavt- og høytoppløselig LC-MS benyttet. Analysene avdekket at polyfenolinnholdet hovedsakelig var sammensatt av lavmolekylære forbindelser, der 96% av de tentativt identifiserte forbindelsene hadde masser under 800 Da. 11 polyfenoliske forbindelser ble identifisert, inkludert fenolsyrer og florotanniner, og flere ble funnet å være sulfaterte, hvilket antas å skyldes en økologisk tilpasning til det marine miljøet.
Dette prosjektet gir den først omfattende beskrivelsen av stortares polyfenoliske innhold. Resultatene gir en grundig identifisering av polyfenolinnholdet i bladene av L. hyperborea og antyder potensialet for en «total utnyttelses»-strategi innen kommersiell alginatproduksjon. Videre gir prosjektet verdifull molekylær innsikt i det fenoliske innholdet i tang, noe som gir viktig informasjon til videre forskning på tang- bioraffineriet, kjemisk økologi, og havovervåkning.Macroalgae, or seaweed, has been recognized as a valuable source of bioactive compounds. Among these, polyphenols have gained particular attention. Polyphenols are well-known for their various bioactivities, such as antioxidant, anti-inflammatory, antidiabetic, and anticancer activities, and have been associated with a range of health benefits. This project focused on the investigation of two types of macroalgae, namely the leaf biomass of the brown algae Laminaria hyperborea (eng.: tangle/cuvie) and the green algae Ulva intestinalis (eng.: gut weed). The initial extractions of L. hyperborea leaves were challenging due to its high polysaccharide content. Therefore, an initial case study was conducted on U. intestinalis to explore the combination of analytical techniques employed for polyphenol quantification and identification in seaweeds. The techniques utilized in this investigation included DAD-HPLC, the total phenolic content (TPC) assay with the Folin–Ciocalteu (FC) reagent, total flavonoid content (TFC) assay with an AlCl3 reagent, and 1H qNMR – including 2D NMR. The analyses showed a relatively composed polyphenolic nature with lower individual concentrations. For the crude sample of U. intestinalis, the total phenolic content was found to be 11.3 ± 1.4 mg GAE/g using HPLC, 5.0 ± 1.0 mg GAE/g with TPC, and 27.3 ± 2.7 mg GAE/g with qNMR. However, the TFC assay detected no flavonoids in the crude sample. The case study strongly indicated the need for optimization of the analytical methods. In addition, characterization using LC-MS was performed on purified samples of U. intestinalis, which tentatively identified the presence of several phenolic acids and flavonoid aglycones. Some of these were confirmed using reference standards.
Following optimization of the TPC assay and the 1H qNMR method, the quantification and comparison of Laminaria hyperborea to four other brown algae was conducted. The total polyphenolic content of L. hyperborea was found to be 5.51 ± 0.00 mg GAE/g (qNMR) and 5.72 ± 0.07 mg GAE/g (TPC). For seaweeds belonging to the Laminariaceae family, which grow in the sublittoral zone, the TPC assay and selective qNMR showed similar, lower polyphenol yields. Still, a larger difference was observed between the TPC and qNMR results for seaweed growing in the eulittoral zone, such as Ascophyllum nodosum (eng.: knotted kelp) and Fucus vesiculosus (eng.: bladder wrack). Seaweed growing in more shallow waters will have higher light accessibility, possibly producing a polyphenolic pool with an increased portion of polyphenols containing an increased number of reacting groups compared to the reference standard (gallic acid or phloroglucinol) used, thus resulting in an overestimation when quantifying with the TPC assay. It is also possible that these species in more shallow waters have a higher occurrence of TPC-interfering compounds than seaweed species growing in the sublittoral zone. To characterize the polyphenolic content of L. hyperborea, flash chromatography and preparative HPLC were used to purify the seaweed sample, obtaining purified fractions for analysis. Results showed that increasing purification corresponded to higher polyphenolic content, as determined by both the TPC assay and selective qNMR. In addition, ORAC assay results revealed increasing antioxidant activity with increasing purification.
To identify the molecular structure of polyphenols in L. hyperborea, both low- and high-resolution LC-MS were utilized. Analysis showed that the polyphenolic matrix was mostly composed of low-molecular weight polyphenols, with 96% of the tentatively identified compounds having masses below 800 Da. Eleven polyphenolic compounds were confirmed, including phenolic acids and phlorotannins, and several were found to be sulfated, which is believed to be an ecological adaptation to the marine environment.
This project provides the first comprehensive characterization of the polyphenolic content of L. hyperborea leaves. These results provide a characterization of the polyphenolic content of L. hyperborea’s leaf biomass, important for the implementation of a “total utilization” strategy in commercial alginate production. Moreover, the project provides valuable molecular-level insights into the phenolic content of seaweed, yielding valuable implications for research across disciplines, such as in the seaweed biorefinery, chemical ecology, and ocean monitoring.Doktorgradsavhandlin
Non-energy Circular Economy Potential of Rice Husks: A Techno-eco-environmental Assessment
The non-energy circular bioeconomy potential of rice husks was examined via sustainability assessments, namely life cycle assessment (LCA), life cycle impact cost assessment (LCICA), and techno-economic assessment (TEA). The study was conducted with three objectives. The first objective was to review previous studies on the non-energy utilization potential of rice husks by the method of meta-analysis. This review followed a systematic approach where research papers were collected following a defined set of criteria. The study revealed 16 key utilization pathways, all of which showed promising results. However, a comprehensive sustainability assessment was lacking in all of the pathways. The second objective was to examine the circular bioeconomy potential of rice husks as a resource for bioplastic production. This study evaluated the techno-environmental assessment of three bioplastics, namely carboxymethyl cellulose, cellulose acetate, and cellulose nitrate relative to rice husks combustion. This provided information on the environmental impacts and the environmental impact costs of all three bioplastics. The result suggested that carboxymethylcellulose would be the most sustainable pathway, reducing the impact on human health and the cost of open-air combustion by 82% and 74%, respectively. The third objective was to examine the sustainable production of xylo-oligosaccharide from rice husk via a techno-economic and an environmental performance assessment. The study examined two production methods: autohydrolysis and enzymatic hydrolysis, considering a pilot and a large production scale for each. The results revealed that autohydrolysis is the best method to produce xylo-oligosaccharides, considering the damage to the environment and human health, and profitability (net profits of 42.4M for pilot and large-scale setups) hence, it is viable to thrive in the market
Analysis, Design and Fabrication of Micromixers, Volume II
Micromixers are an important component in micrototal analysis systems and lab-on-a-chip platforms which are widely used for sample preparation and analysis, drug delivery, and biological and chemical synthesis. The Special Issue "Analysis, Design and Fabrication of Micromixers II" published in Micromachines covers new mechanisms, numerical and/or experimental mixing analysis, design, and fabrication of various micromixers. This reprint includes an editorial, two review papers, and eleven research papers reporting on five active and six passive micromixers. Three of the active micromixers have electrokinetic driving force, but the other two are activated by mechanical mechanism and acoustic streaming. Three studies employs non-Newtonian working fluids, one of which deals with nano-non-Newtonian fluids. Most of the cases investigated micromixer design
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