2,214 research outputs found
Bioactive Self-Assembled Protein Nanosheets for Stem Cell-Based Biotechnologies
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
A journey from molecule to physiology and in silico tools for drug discovery targeting the transient receptor potential vanilloid type 1 (TRPV1) channel
The heat and capsaicin receptor TRPV1 channel is widely expressed in nerve terminals of dorsal root ganglia (DRGs) and trigeminal ganglia innervating the body and face, respectively, as well as in other tissues and organs including central nervous system. The TRPV1 channel is a versatile receptor that detects harmful heat, pain, and various internal and external ligands. Hence, it operates as a polymodal sensory channel. Many pathological conditions including neuroinflammation, cancer, psychiatric disorders, and pathological pain, are linked to the abnormal functioning of the TRPV1 in peripheral tissues. Intense biomedical research is underway to discover compounds that can modulate the channel and provide pain relief. The molecular mechanisms underlying temperature sensing remain largely unknown, although they are closely linked to pain transduction. Prolonged exposure to capsaicin generates analgesia, hence numerous capsaicin analogs have been developed to discover efficient analgesics for pain relief. The emergence of in silico tools offered significant techniques for molecular modeling and machine learning algorithms to indentify druggable sites in the channel and for repositioning of current drugs aimed at TRPV1. Here we recapitulate the physiological and pathophysiological functions of the TRPV1 channel, including structural models obtained through cryo-EM, pharmacological compounds tested on TRPV1, and the in silico tools for drug discovery and repositioning
Investigating the effects of palmitoylation on the dopamine 1 receptor (D1)
The dopamine D1 receptor (D1) is a G protein-coupled receptor (GPCR) which regulates various key brain functions like attention, movement, reward, and memory. Understanding D1 signalling may open the horizon for novel treatments for neurological disorders. Upon agonist activation, the heterotrimeric G proteins Gαs activate adenylyl cyclase to increase cAMP/PKA signalling. D1 also engages β-arrestin proteins leading to β-arrestin dependent signalling. The D1 has two palmitoylation sites on cysteines 347&351 in its C-tail domain. However, the distinct roles and implications of palmitoylation on the D1 signalling, trafficking and β-arrestins recruitment are still largely unexplored. A palmitoylation D1 mutant was generated and luminescent based techniques such as BRET and split-Nanoluc complementation assay were employed, to delineate D1 palmitoylation effects on its pharmacology and signalling. The D1 agonists induced 50% less cAMP production in the mutant compared to wildtype (WT) and WT showed a more efficient dissociation of its Gαs. Moreover, the mutant receptor failed to recruit β-arrestin1&2, induced less ERK1/2 activation and internalises in an agonist-independent process while showing an altered intracellular Golgi trafficking. Also, in β-arrestin 1&2 KO HEK 293 cells similar cAMP production levels were reported for D1 WT and palmitoylation mutant. β-arrestin 1&2 KO blocked agonist-induced WT D1 plasma membrane trafficking, indicating that these β-arrestins are driving the differences between WT and the palmitoylation mutant D1. Taken together, our studies indicate that Gαs is the main transducer for D1 cAMP and ERK1/2 signalling and that palmitoylation is essential for its β-arrestin 1&2 interactions and modulating D1 signalling cascades in a drug-dependant process
Extrinsic Factors and RPE Regeneration
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that closely interacts with photoreceptor outer segments of the outer vertebrate retina to maintain visual function. Damage to the RPE, for instance in a disease such as Age-Related Macular Degeneration, results in photoreceptor degeneration and subsequently, vision loss. In contrast to mammals, zebrafish can intrinsically regenerate a functional RPE layer after injury. Specific molecular pathways are known to regulate RPE proliferation in culture, but the pathways that function in vivo to promote RPE regeneration remain largely unknown. My aim is to determine potential pathways that influence RPE regeneration in zebrafish. First, I examine the importance of the secreted ligand Semaphorin 3F (SEMA3F), expressed in the RPE of both mammals and zebrafish, in RPE regeneration. I use a sema3fa homozygous mutant zebrafish on a transgenic RPE injury background (Tg(rpe65a:NTR-EGFP)) where timed application of the drug metronidazole (MTZ) to the bath results in nitroreductase-mediated RPE-specific cell death. My data suggest Sema3fa has no effect on the extent of RPE injury in this model, though RPE apoptosis may be delayed and increased in the absence of Sema3fa. Further, loss of Sema3fa may induce an initial increase in proliferation in the RPE as well as increased proliferation in the photoreceptor outer nuclear layer. Second, I provide an initial assessment of the involvement of additional pathways in zebrafish RPE regeneration. These pathways impact proliferation and/or migration of cells in culture and are expressed within the RPE. I use in situ hybridization to visualize larval RPE expression of 10 candidate genes before and after RPE injury. Genes that may show changes in expression post-injury include bmp7b, caska, foxm1, her4.1, msnb, rpe65a, trpm7, and vrk1. Future work could include using loss-of-function approaches in the RPE injury model to determine potential roles of these genes in RPE regeneration. In the long-term, this work may impact gene therapies for patients suffering from retinal degenerative diseases
Analytical validation of innovative magneto-inertial outcomes: a controlled environment study.
peer reviewe
Funduse sinine ja lähi-infrapuna autofluorestsentsuuring autosoom-retsessiivse Stardgardti tõve, koroidereemia, PROM1-maakuli düstroofia ja okulaarse albinismi patsientidel
Väitekirja elektrooniline versioon ei sisalda publikatsiooneFunduse sinine ja lähi-infrapuna autofluorestsentsuuring autosoom-retsessiivse Stardgardti tõve, koroidereemia, PROM1-maakuli düstroofia ja okulaarse albinismi patsientidel
Pärilikud võrkkestahaigused on juhtivaks nägemiskaotuse põhjuseks tööealise elanikkonna seas arenenud riikides. Tegemist on kliiniliselt ja geneetiliselt väga heterogeense haiguste grupiga, mistõttu diagnostika ja haiguse patogeneesi uurimine on olnud vaevarikas. Võrkkesta piltdiagnostika on oluline mitte-invasiivne meetod haiguste diagnoosimiseks ja uurimiseks. Konfokaalne skanneeriv laseroftalmoskoop valgustab võrkkesta erineva lainepikkusega laserkiirega ning salvestab tagasikiirgavat valgust luues silmapõhjast pildi. Funduse autofluorestsents (AF) uuringul kasutatakse ära silmapõhja enda naturaalseid fluorofoore. Lipofustsiini ergastamiseks kasutatakse sinise spektri laserkiirt (sinine AF) ja melaniini jaoks lähipuna laserkiirt (lähipuna AF). Nende fluorofooride jaotus ja kogus silmapõhjas muutub erinevate haigusprotsesside mõjul ning need muutused on tuvastatavad AF uuringul.
Antud doktoritöös uurisime sinise ja lähipuna AF uuringu pilte autosoom-retsesiivse Stargardti tõve (STGD1), koroidereemia, PROM1-maakuli düstroofia ning okulaarse albinismi patsientidel. Töö eesmärgiks oli paremini mõista sinise ja lähipuna AF signaali allikaid erinevate haigusseisundite korral, kus võrkkesta fluorofooride jaotus ning kogused on muutunud. Lisaks kvalitatiivsele piltide hindamisele kasutamise kvantitatiivset AF signaali tugevuse mõõtmist hindamaks lipofustsiini ja melaniini taset.
Uurimustöös näitasime, et melaniin on lähipuna AF signaali peamiseks allikaks. Lisaks näitasime, et melanin võib kaudselt moduleerida lipofustsiinist tuleneva sinise AF signaali, sest okulaarse albinismi kandjate hüpopigmenteeritud võrkkesta alade sinise AF signal oli tavapärasest kõrgem. AF signaali tugevuse mõõtmisel leidsime, et lipofustsiini kuhjumine võrkkestas põhjustab lisaks sinise AF signaali tõusule ka lähipuna AF signaali tõusu STGD1 patsientidel. Kvantitatiivsel analüüsil näitasime ka, et PROM1-maakuli düstroofia patsientide sinise AF signaal oli võrreldav terve silmapõhja signaali tugevusega, eristades seda fenotüübiliselt sarnasest STGD1 haigusest ning viidates ka sellele, et lipofustsiini üleliigne kuhjumine ei ole antud haigusele omane mehhanism. Koroidereemia ja STGD1 haigete uurimisel leidsime, et pigmentepiteeli rakkude kärbumine on nähtav AF signaali hääbumisena, samas lähipuna AF uuringaitab tuvastada varasemaid muutusi kui sinine AF uuring. Lipofustsiin ja melanin on mõlemad olulised võrkkesta rakkude seisundi biomarkerid, mida on võimalik mitte-invasiivsel moel AF uuringu abil analüüsida ning hinnata haiguse progressiooni.Inherited retinal diseases are the leading cause of visual impairment among the working age-group in the developed countries. Because of genetic and phenotypical heterogeneity, diagnosis and understanding pathogenesis of inherited retinal disease has been challenging. Retinal imaging studies which are noninvasive, are an invaluable source of information. Fundus autofluorescence (FAF) utilizes natural fluorophores to create an image of the retina. Lipofuscin is the primary source for short-wavelength autofluorescence (SW-AF) and melanin for near-infrared autofluorescence (NIR-AF). The amount and distribution of these fluorophores changes in the different disease processes and is detectable in FAF images.
In this study we analyzed SW-AF and NIR-AF images in cases of genetically confirmed recessive Stargardt disease (STGD1), choroideremia, PROM1-macular disease and ocular albinism. The aim was to qualitatively describe FAF in conditions with varying levels of lipofuscin or melanin as well as to quantify FAF signal intensities. We also aimed at finding new clinical implications for autofluorescence imaging in evaluating inherited retinal disease.
We confirmed that melanin is the major source of NIR-AF signal by analyzing ocular albinism carriers and mice models with varying fundus pigmentation, but we also found that presence of melanin can modulate SW-AF signal strength. As a novel finding we confirmed that lipofuscin contributes to NIR-AF signal intensity in cases with excessive bisretinoid lipofuscin levels like seen in STGD1. The analysis of choroideremia and STGD1 patients showed that retinal pigment epithelium atrophy causes loss of signal in both SW-AF and NIR-AF, but NIR-AF could be more sensitive in detecting early cell degeneration. Quantifying the autofluorescence signal intensity helps to further understand disease processes as it is an indirect measure for levels of retinal fluorophores. We showed PROM1-macular dystrophy does not present with elevated levels of SW-AF indicating that excessive lipofuscin accumulation is likely not part of its disease mechanism. That knowledge is valuable in differentiating it from phenotypically similar STGD1 or when developing therapeutic approaches. Lipofuscin and melanin are both valuable retinal biomarkers for evaluating retinal health by using non-invasive autofluorescence imaging.https://www.ester.ee/record=b555738
Plant condensates: no longer membrane-less?
Cellular condensation is a reinvigorated area of study in biology, with scientific discussions focusing mainly on the forces that drive condensate formation, properties, and functions. Usually, condensates are called 'membrane-less' to highlight the absence of a surrounding membrane and the lack of associated contacts. In this opinion article we take a different direction, focusing on condensates that may be interfacing with membranes and their possible functions. We also highlight changes in condensate material properties brought about by condensate-membrane interactions, proposing how condensates-membrane interfaces could potentially affect interorganellar communication, development, and growth, but also adaptation in an evolutionary context. We would thus like to stimulate research in this area, which is much less understood in plants com-pared with the animal field
Development of Novel Therapeutic Strategies to Target Therapy Resistance and Cancer Stem Cells
This thesis focuses on the core issues of multidrug resistance (MDR) in cancer, a process that hinders the success of chemotherapeutic treatments. MDR involves various mechanisms, including the upregulation of ABC transporter pumps, like MRP1, and increased cancer stemness, which contributes to malignancy and recurrence.
The thesis comprises seven chapters: a literature review (Chapter 1), methodology (Chapter 2), results (Chapters 3-5), and discussions on findings and future studies (Chapters 6) and final discussion and overall summary (Chapter 7).
Chapter 3 delves into the novel roles of MRP1 in cellular iron metabolism and proliferation, its interaction with c-Myc, and the effects on cellular proliferation. Silencing and inhibition studies reveal MRP1's role in regulating iron regulatory proteins through c-Myc.
Chapter 4 investigates the role of ABC transporters in cancer stemness, revealing their connection with stemness states in different tumor types.
Chapter 5 explores strategies for targeting drug-resistant cancer cells, demonstrating how doxycycline reduces the stemness marker SOX2 across multiple tumor types through a unique pathway.
Chapter 6 examines the alteration of metabolism and stemness in drug-resistant cancer cells and strategies for targeting the cysteine metabolism pathway. The findings provide insights into cancer stemness regulation and potential therapeutic strategies, improving the efficacy of chemotherapeutics.
The work reported in this thesis reveals an underlying and unique mechanism in regulation of SOX2-mediated cancer stemness. Moreover, the use of DXC to remove stemness was demonstrated to be a promising therapeutic strategy in combination with other common chemotherapeutics agents. These findings presented in this thesis enables us to understand cancer stemness better and improve the efficacy of current chemotherapeutics, which ultimately improve overall quality of life
Toward the appropriate interpretation of Alphafold2
In life science, protein is an essential building block for life forms and a crucial catalyst for metabolic reactions in organisms. The structures of protein depend on an infinity of amino acid residues' complex combinations determined by gene expression. Predicting protein folding structures has been a tedious problem in the past seven decades but, due to robust development of artificial intelligence, astonishing progress has been made. Alphafold2, whose key component is Evoformer, is a typical and successful example of such progress. This article attempts to not only isolate and dissect every detail of Evoformer, but also raise some ideas for potential improvement
A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain
γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl−, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates
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