6,197 research outputs found

    Phosphate-based glass microspheres for bone repair and localised chemotherapy and radiotherapy treatment of bone cancers

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    Phosphate-based glasses (PBGs) are hugely promising materials for bone repair and regeneration as they can be formulated to be compositionally similar to the inorganic component of bone. Alterations to PBG formulations can be made to tailor their degradation rates and subsequent release of biotherapeutic ions to induce cellular responses, such as osteogenesis. In this work, novel invert-PBGs in the series xP2O5·(56-x)CaO·24MgO·20Na2O (mol%), where x is 40, 35, 32.5 and 30, were formulated to contain pyro (Q1) and orthophosphate (Q0) species. These PBGs were then processed into highly porous microspheres (PMS) via a flame spheroidisation process developed within the research group. Compositional and structural analysis using EDX and 31P-MAS NMR analysis revealed significant depolymerisation had occurred with reducing phosphate content, which increased further when PBGs were processed into PMS. A decrease from 50% to 0% of Q2 species and increase from 6% to 35% of Q0 species was observed for the PMS when the phosphate content decreased from 40 to 30 mol%. Ion release studies also revealed up to a 4-fold decrease in cations and an 8-fold decrease in phosphate anions released with decreasing phosphate content. In vitro bioactivity studies revealed that the orthophosphate rich PMS had favourable bioactivity responses after 28 days of immersion in SBF. Indirect and direct cell culture studies confirmed that the PMS were cytocompatible and supported cell growth and proliferation over 7 days of culture. The P30 PMS with ~65% pyro and ~35% ortho phosphate content revealed the most favourable properties and was proposed to be highly suitable for bone repair and regeneration, especially for orthobiologic applications owing to their highly porous morphology. Doxorubicin (DOX) was used as a model drug to assess its loading and release kinetics from porous phosphate-based glass microspheres to ascertain their suitability for localised drug delivery for the treatment of bone cancers. P40 PMS revealed a DOX loading efficiency of 55%, which was significantly greater than P30 PMS at 29.1%. Both P40 and P30 PMS released more DOX in phosphate buffered saline (PBS) at pH 5 as compared to release at pH 7.4. P40 PMS released 57% of DOX at pH 5 over a 48-hour period, whereas P30 PMS only released 15% of DOX. A pH-responsive DOX release in a more acidic environment suggests that the chemotherapeutic delivery and efficacy properties may lead to increased drug release within tumour tissues. Internal radiotherapy has been shown to be an effective treatment modality to destroy cancerous tissues and is usually achieved by the placement of radioactive sources at the tumour site. In this work, a novel processing method was established to combine yttrium oxide (Y2O3) with P40 phosphate glass particles to form uniform, solid microspheres containing very high yttrium levels via our flame spheroidisation process. The 30Y (~15 mol% Y2O3) and 50Y microspheres (~39 mol% Y2O3) had equivalent and superior yttrium content in comparison to clinically available microspheres used for internal radiotherapy (i.e., Therasphere®). The yttrium-containing microspheres formed were shown to be glass-ceramics, with crystalline phases present but with all elements homogenously distributed throughout the microspheres. Increasing yttrium addition resulted in increased durability of the microspheres, with 50Y microspheres revealing a 10-fold decrease in the release rate of some ions compared to P40 solid microspheres. Indirect and direct cell culture studies confirmed that the 30Y and 50Y microspheres were cytocompatible and supported cell growth and proliferation over 7 days of culture. No significant difference was observed in the metabolic and ALP activity for MG63s for both 30Y and 50Y microspheres from both indirect and direct cell culture studies. Yttrium was incorporated into the phosphate-based microspheres at a level that had not previously been achieved or observed from the literature studies and were shown to support bone cell attachment and growth. A high yttrium content could enable more radiation to be delivered per dose of microspheres, resulting in shorter neutron activation times which could prove beneficial for logistical issues associated with transportation of the biomaterials following nuclear activation. The radionuclide holmium-166 (166Ho) which is comparable to yttrium-90 (90Y) in that it emits β-radiation with a similar tissue penetration range and a significantly reduced half-life of 26.8 hours, was also investigated. The beneficial paramagnetic properties and density of 166Ho indicates that 166Ho-doped materials could be visualised through clinical imaging techniques, whilst simultaneously delivering a therapeutic dose of radiation. In this work, solid holmium-containing microspheres were similarly produced via the flame spheroidisation process using holmium oxide (Ho2O3) and P40 phosphate glass particles. The glass-ceramic microspheres produced had equivalent (30H: ~17mol% Ho2O3) and superior (50H: ~30mol% Ho2O3) holmium content in comparison to clinically used yttrium-doped microspheres (i.e. Therasphere®). Analogous to yttrium containing microspheres, elevated holmium content resulted in topographically unique features on the surface of some 50H microspheres. This increased holmium content resulted in significantly reduced ion release rates for all the ions and the holmium-microspheres did not show evidence of bioactivity. However, in vitro indirect and direct cell culture studies demonstrated their cytocompatibility. No significant difference was observed in the metabolic and ALP activity of MG63 cells for 30H and 50H microspheres in both the indirect and direct cell culture methods. This study appears to be the first to demonstrate microspheres containing high levels of holmium content that can also facilitate direct cell growth and proliferation of human osteoblast-like cells. The microspheres developed are therefore hugely promising biomaterials for both drug delivery and internal radiotherapy applications, as well as for promoting bone repair and regeneration at damaged sites. High holmium content could also result in higher specific activity per microsphere to increase radiotherapy delivery whilst also promoting higher visibility via imaging modalities

    Out-of-Distribution Generalization of Deep Learning to Illuminate Dark Protein Functional Space

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    Dark protein illumination is a fundamental challenge in drug discovery where majority human proteins are understudied, i.e. with only known protein sequence but no known small molecule binder. It\u27s a major road block to enable drug discovery paradigm shift from single-targeted which looks to identify a single target and design drug to regulate the single target to multi-targeted in a Systems Pharmacology perspective. Diseases such as Alzheimer\u27s and Opioid-Use-Disorder plaguing millions of patients call for effective multi-targeted approach involving dark proteins. Using limited protein data to predict dark protein property requires deep learning systems with OOD generalization capacity. Out-of-Distribution (OOD) generalization is a problem hindering the application and adoption of deep learning in real world problems. Classic deep learning setting in contrast is assuming training and testing data are independent identically distributed (iid). A well trained model under iid setting with reported 98% accuracy could deteriorate to worse than random guess in deployment to OOD data significantly different from training data. Numerous techniques in the research field emerged but are only addressing some specific OOD scenario instead of a general one. Dark protein illumination has unique complexity comparing to common deep learning tasks. There are three OOD axes, protein-OOD, compound-OOD, interaction-OOD. Previous research have only focused on compound-OOD, where new compound design algorithms are developed but still for 500 common proteins, instead of whole human genome 20,000 proteins, and only for single-targeted paradigm instead of multi-targeted. Focusing on an instrumental problem in drug discovery, dark protein function illumination problem is introduced from the OOD perspective. A series of dark protein OOD algorithms are developed to predict dark protein ligand interaction where multiple instrumental deep learning techniques are adapted to the biology context. By proposing the dark protein illumination problem, highlighting the neglected axes, demonstrating possibilities, numerous diseases now embrace new hopes

    Computational design of dynamic receptor-peptide signaling complexes applied to chemotaxis.

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    Engineering protein biosensors that sensitively respond to specific biomolecules by triggering precise cellular responses is a major goal of diagnostics and synthetic cell biology. Previous biosensor designs have largely relied on binding structurally well-defined molecules. In contrast, approaches that couple the sensing of flexible compounds to intended cellular responses would greatly expand potential biosensor applications. Here, to address these challenges, we develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides. To demonstrate the power of the approach, we create ultrasensitive chemotactic receptor-peptide pairs capable of eliciting potent signaling responses and strong chemotaxis in primary human T cells. Unlike traditional approaches that engineer static binding complexes, our dynamic structure design strategy optimizes contacts with multiple binding and allosteric sites accessible through dynamic conformational ensembles to achieve strongly enhanced signaling efficacy and potency. Our study suggests that a conformationally adaptable binding interface coupled to a robust allosteric transmission region is a key evolutionary determinant of peptidergic GPCR signaling systems. The approach lays a foundation for designing peptide-sensing receptors and signaling peptide ligands for basic and therapeutic applications

    Studium funkčních a farmakologických vlastností glutamátových receptorů

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    N-methyl-D-aspartátové receptory (NMDAR) jsou ionotropní glutamátové receptory, které se podílejí na regulaci téměř všech procesů v mozku. Proto i nepatrná porucha funkce NMDAR může vést k závažným patologickým důsledkům. Mutace v genech kódujících NMDAR, které vedou ke snížení jejich funkce, se podílejí na mnoha neuropsychiatrických poruchách, jako jsou například mentální retardace, schizofrenie, poruchy autistického spektra, epilepsie a poruchy pohybu. Nedostatečnou funkci NMDAR lze korigovat pozitivními alosterickými modulátory, včetně neurosteroidů. Mechanismus který je základem potenciačního účinku steroidů však není dosud dostatečně objasněn. Pomocí elektrofyziologické metody terčíkového zámku jsme změřili účinek nově syntetizovaných analogů pregnanů, které jsou podobné neurosteroidům, na rekombinantní NMDAR s podjednotkovým složením GluN1/GluN2B. Prokázali jsme, že sloučeniny s krátkými residui na uhlíku C3, jako je pregnanolon acetát (PA-Ace) a pregnanolon karboxylát (PA-Car), jsou negativními modulátory NMDAR, zatímco sloučeniny s delšími residui na C3, jako je pregnanolon butyrát (PA-But) a epipregnanolon butyrát (EPA-But), jsou pozitivními modulátory NMDAR. Dále jsme odhalili, že EPA-But má "disuse- dependentní" pozitivní alosterický účinek, přičemž je v tomto ohledu podobný endogennímu...N-methyl-D-Aspartate receptors (NMDAR) are ionotropic glutamate receptors that are involved in the regulation of nearly every process in the brain. Therefore, even a subtle disturbance in NMDAR function may result in severe pathological consequences. Loss-of- function mutations in the NMDAR-encoding genes have been implicated in numerous neuropsychiatric disorders, including intellectual disability, developmental delay, schizophrenia, autism spectrum disorders, epilepsy, and movement disorders. Insufficient NMDAR function can be rectified by positive allosteric modulators, including neurosteroids; however, the mechanism underlying the potentiating effect of steroids is not well understood. By employing patch-clamp electrophysiology we assessed the effect of newly synthesized neurosteroid-like pregnane analogues on recombinant GluN1/GluN2B receptors. We demonstrated that compounds with short C3 residues, such as pregnanolone acetate (PA- Ace) and pregnanolone carboxylate (PA-Car), are negative modulators of NMDAR, whereas compounds with longer C3 residues, such as pregnanolone butyrate (PA-But) and epipregnanolone butyrate (EPA-But), are positive modulators of NMDARs. Furthermore, we revealed that EPA-But has a disuse-dependent positive allosteric effect, being similar in that regard to endogenous...Mimofakultní pracoviště3. lékařská fakultaThird Faculty of Medicin

    Markov field models of molecular kinetics

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    Computer simulations such as molecular dynamics (MD) provide a possible means to understand protein dynamics and mechanisms on an atomistic scale. The resulting simulation data can be analyzed with Markov state models (MSMs), yielding a quantitative kinetic model that, e.g., encodes state populations and transition rates. However, the larger an investigated system, the more data is required to estimate a valid kinetic model. In this work, we show that this scaling problem can be escaped when decomposing a system into smaller ones, leveraging weak couplings between local domains. Our approach, termed independent Markov decomposition (IMD), is a first-order approximation neglecting couplings, i.e., it represents a decomposition of the underlying global dynamics into a set of independent local ones. We demonstrate that for truly independent systems, IMD can reduce the sampling by three orders of magnitude. IMD is applied to two biomolecular systems. First, synaptotagmin-1 is analyzed, a rapid calcium switch from the neurotransmitter release machinery. Within its C2A domain, local conformational switches are identified and modeled with independent MSMs, shedding light on the mechanism of its calcium-mediated activation. Second, the catalytic site of the serine protease TMPRSS2 is analyzed with a local drug-binding model. Equilibrium populations of different drug-binding modes are derived for three inhibitors, mirroring experimentally determined drug efficiencies. IMD is subsequently extended to an end-to-end deep learning framework called iVAMPnets, which learns a domain decomposition from simulation data and simultaneously models the kinetics in the local domains. We finally classify IMD and iVAMPnets as Markov field models (MFM), which we define as a class of models that describe dynamics by decomposing systems into local domains. Overall, this thesis introduces a local approach to Markov modeling that enables to quantitatively assess the kinetics of large macromolecular complexes, opening up possibilities to tackle current and future computational molecular biology questions

    Digital agriculture: research, development and innovation in production chains.

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    Digital transformation in the field towards sustainable and smart agriculture. Digital agriculture: definitions and technologies. Agroenvironmental modeling and the digital transformation of agriculture. Geotechnologies in digital agriculture. Scientific computing in agriculture. Computer vision applied to agriculture. Technologies developed in precision agriculture. Information engineering: contributions to digital agriculture. DIPN: a dictionary of the internal proteins nanoenvironments and their potential for transformation into agricultural assets. Applications of bioinformatics in agriculture. Genomics applied to climate change: biotechnology for digital agriculture. Innovation ecosystem in agriculture: Embrapa?s evolution and contributions. The law related to the digitization of agriculture. Innovating communication in the age of digital agriculture. Driving forces for Brazilian agriculture in the next decade: implications for digital agriculture. Challenges, trends and opportunities in digital agriculture in Brazil

    Understanding the Molecular Mechanism of Single-Strand Annealing Homologous DNA Recombination in Viruses, by Cryo-Electron Microscopy

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    The single-strand annealing homologous recombination (SSA) is one of the dsDNA break repair pathways, and albeit its importance from bacteria to bacteriophages, its molecular function is still unknown. The SSA reaction is catalysed by the enzyme complexes known as Exonuclease Annealase Two-component Recombinase (EATRs). The RecT and ORF6 proteins are single-stranded DNA-binding and annealing proteins expressed in E. coli and Kaposi’s sarcoma-associated herpesvirus (KSHV), respectively. RecT has already been shown to catalyse the SSA reaction. Although ORF6 has been shown to bind to ssDNA, further experimental evidence is needed to solidify its annealase activity. Since structure can dictate the function, this thesis aimed to determine the structure of the annealases RecT and ORF6 using a state-in-art cryo-electron microscopy technique. Furthermore, the shadow-casting EM technique has been established by optimising it for the equipment available at UOW, which is helpful for imaging the substrate DNA intermediates and the nucleoprotein complexes formed during SSA to better understand the molecular mechanistic details of this reaction. This thesis includes the details about RecT and ORF6 proteins’ cloning, expression, and purification, which were further optimised for purity and homogeneity for cryo-electron microscopy with the help of negative staining electron microscopy (NSEM). Additionally, based on several NSEM analyses, the C-terminal His-tag containing RecT (RecTCH) oligomerisation on ssDNA was studied, and a general mechanism of its oligomerisation is described. Unfortunately, during the RecTCH protein’s cryo-EM sample optimisation, the LiRecT structure was published by another group. Therefore, work on that project was ceased at that point. Several novel findings on ORF6 are reported in this thesis. Primarily, the concentration of the purified protein was increased 3 times more than the reports in the literature. Based on the NSEM and preliminary cryo-EM map of ORF6, it is shown that the ORF6 structure overall resembles the HSV1-ICP8 protein. Further, based on the steady-state and time-resolved fluorescence resonance energy transfer (FRET) experiments, a model for the ORF6 annealing mechanism is suggested. Towards generating a high-resolution structure, ORF6 monomers and filaments were optimised and imaged by using cryo-EM. Processing a data set obtained from a monomeric ORF6 sample showed the presence of conformational heterogeneity in the particles, which was expected as the ORF6 AlphaFold model shows that the N-terminal and C-terminal domains are connected by an 18 amino acids long loop, allowing C-terminal domain to be relatively flexible to move around. Processing of another data set obtained from a sample containing ORF6 filaments generated 2-dimensional averages that look promising for generating a high-resolution structure. This thesis also shows the details related to the installation and optimisation of the shadowing technique using a modern material, graphene oxide (GO), as a support film. This technique involves optimising both sample preparation and instrumentation for metal evaporation and deposition. For sample preparation, GO was deposited on cryo-EM holey grids, on which the sample was mounted. For instrumentation optimisation, a DENTON brand evaporator was used. The grid stage was re-engineered using AutoCAD to achieve the finest metal evaporation, and parameters such as amperage, vacuum, metal thickness, and angles were optimised. The optimised parameters were used to shadow-cast different lengths of DNA and their complexes with proteins, and good contrast images were acquired for qualitative and quantitative analyses. Overall, this thesis presents two main novel findings. First, RecTCH monomers oligomerise into an open ring-shaped structure, which stacks together to generate short filaments. Second, to anneal two complementary ssDNA strands, ORF6 first forms filaments with both ssDNA, which then come in contact with each other rapidly to anneal the complementary strands. Once the annealing finishes, the annealed dsDNA is released from the filaments as the filaments fall apart into monomers. We also found that ORF6 monomers oligomerise to form the helical and non-helical filaments in the presence of DTT+Mg2+ and DTT-containing buffer, respectively

    Glycomimetics for the inhibition and modulation of lectins

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    Carbohydrates are essential mediators of many processes in health and disease. They regulate self-/ non-self- discrimination, are key elements of cellular communication, cancer, infection and inflammation, and determine protein folding, function and life-times. Moreover, they are integral to the cellular envelope for microorganisms and participate in biofilm formation. These diverse functions of carbohydrates are mediated by carbohydrate-binding proteins, lectins, and the more the knowledge about the biology of these proteins is advancing, the more interfering with carbohydrate recognition becomes a viable option for the development of novel therapeutics. In this respect, small molecules mimicking this recognition process become more and more available either as tools for fostering our basic understanding of glycobiology or as therapeutics. In this review, we outline the general design principles of glycomimetic inhibitors (Section 2). This section is then followed by highlighting three approaches to interfere with lectin function, i.e. with carbohydrate-derived glycomimetics (Section 3.1), novel glycomimetic scaffolds (Section 3.2) and allosteric modulators (Section 3.3). We summarize recent advances in design and application of glycomimetics for various classes of lectins of mammalian, viral and bacterial origin. Besides highlighting design principles in general, we showcase defined cases in which glycomimetics have been advanced to clinical trials or marketed. Additionally, emerging applications of glycomimetics for targeted protein degradation and targeted delivery purposes are reviewed in Section 4

    End-to-End Full-Atom Antibody Design

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    Antibody design is an essential yet challenging task in various domains like therapeutics and biology. There are two major defects in current learning-based methods: 1) tackling only a certain subtask of the whole antibody design pipeline, making them suboptimal or resource-intensive. 2) omitting either the framework regions or side chains, thus incapable of capturing the full-atom geometry. To address these pitfalls, we propose dynamic Multi-channel Equivariant grAph Network (dyMEAN), an end-to-end full-atom model for E(3)-equivariant antibody design given the epitope and the incomplete sequence of the antibody. Specifically, we first explore structural initialization as a knowledgeable guess of the antibody structure and then propose shadow paratope to bridge the epitope-antibody connections. Both 1D sequences and 3D structures are updated via an adaptive multi-channel equivariant encoder that is able to process protein residues of variable sizes when considering full atoms. Finally, the updated antibody is docked to the epitope via the alignment of the shadow paratope. Experiments on epitope-binding CDR-H3 design, complex structure prediction, and affinity optimization demonstrate the superiority of our end-to-end framework and full-atom modeling.Comment: preprin
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