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Protein capsules are promising drug delivery vehicles for cancer research therapies. Apoferritin (AFt) is a self-assembling 12 nm diameter hollow nanocage with many desirable features for drug delivery, however, control of drug retention inside the protein cage remains challenging. Here we report the encapsulation of copper(ii)-1,10-phenanthroline (Cu(phen)) within the horse spleen AFt (HSAFt) nanocage, by diffusion of the metal through the pores between the protein subunits. Transmission electron microscopy revealed the formation of organised copper adducts inside HSAFt, without affecting protein integrity. These structures proved stable during storage (>4 months at -20 °C). Exposure to physiologically relevant conditions (37 °C) showed some selectivity in cargo release after 24 h at pH 5.5, relevant to the internalisation of AFt within the endosome (60% release), compared to pH 7.4, relevant to the bloodstream (40% release). Co-encapsulation of temozolomide, a prodrug used to treat glioblastoma multiforme, and Cu(phen) enabled entrapment of an average of 339 TMZ molecules per cage. In vitro results from MTT and clonogenic assays identified cytotoxic activity of the Cu(phen), HSAFt-Cu(phen) and HSAFt-Cu(phen)-TMZ adducts against colorectal cancer cells (HCT-116) and glioblastoma cells (U373V, U373M). However, the presence of the metal also contributed to more potent activity toward healthy MRC5 fibroblasts, a result that requires further investigation to assess the clinical viability of this system.
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[This corrects the article DOI: 10.1021/acsomega.2c00997.].
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Excipients are included within protein biotherapeutic solution formulations to improve colloidal and conformational stability but are generally not designed for the specific purpose of preventing aggregation and improving cryoprotection in solution. In this work, we have explored the relationship between the structure and antiaggregation activity of excipients by utilizing coarse-grained molecular dynamics modeling of protein-excipient interaction. We have studied human serum albumin as a model protein, and we report the interaction of 41 excipients (polysorbates, fatty alcohol ethoxylates, fatty acid ethoxylates, phospholipids, glucosides, amino acids, and others) in terms of the reduction of solvent accessible surface area of aggregation-prone regions, proposed as a mechanism of aggregation prevention. Polyoxyethylene sorbitan had the greatest degree of interaction with aggregation-prone regions, decreasing the solvent accessible surface area of APRs by 20.7 nm2 (40.1%). Physicochemical descriptors generated by Mordred are employed to probe the structure-property relationship using partial least-squares regression. A leave-one-out cross-validated model had a root-mean-square error of prediction of 4.1 nm2 and a mean relative error of prediction of 0.077. Generally, longer molecules with a large number of alcohol-terminated PEG units tended to interact more, with qualitatively different protein interactions, wrapping around the protein. Shorter or less ethoxylated compounds tend to form hemimicellar clusters at the protein surface. We propose that an improved design would feature many short chains of 5 to 10 PEG units in many distinct branches and at least some hydrophobic content in the form of medium-length or greater aliphatic chains (i.e., six or more carbon atoms). The combination of molecular dynamics simulation and quantitative modeling is an important first step in an all-purpose protein-independent model for the computer-aided design of stabilizing excipients.
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Produtos Biológicos , Excipientes , Humanos , Excipientes/química , Excipientes/metabolismo , Proteínas , Aminoácidos/química , SolventesRESUMO
We report a liquid chromatography-isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C-glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R 2 ≥ 0.99, limits of detection were ≤1.0 µM, limits of quantification were ≤10 µM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid "cycle" were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production.
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The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new 'direct' InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expanding on the triclosan structure. Low Minimum Inhibitory Concentrations (MICs) were obtained for benzylphenyl compounds (12, 43 and 44) and di-triclosan derivative (39), against Mycobacterium bovis BCG although these may also be inhibiting other enzymes. The ether linked di-triclosan derivative (38) displayed excellent in vitro isolated enzyme inhibition results comparable with triclosan, but at a higher MIC (125 µg mL-1). These compounds offer good opportunities as leads for further optimisation.
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Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. 'Direct inhibitors' of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC50 of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC99 of 12.9 µM against M. bovis.
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Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Triclosan/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Oxirredutases/metabolismo , Relação Estrutura-Atividade , Triclosan/síntese química , Triclosan/química , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/metabolismoRESUMO
Recombinant spider silk has the potential to provide a new generation of biomaterial scaffolds as a result of its degree of biocompatibility and lack of immunogenicity. These recombinant biomaterials are, however, reported to exhibit poor cellular adhesion which limits their potential for use in applications such as tissue engineering and regenerative medicine. In this study, a simple chemical functionalization approach is described that specifically addresses this issue and significantly improves the adhesion of human mesenchymal stem cells (CiMSCs) to a recombinant spider silk biomaterial. This utilizes copper-catalyzed or strain-promoted azide-alkyne cycloaddition (CuAAC/SPAAC) "click" chemistry to covalently attach cyclo(RGDfK) peptides to the azide group of l-azidohomoalanine, a methionine analogue previously site specifically incorporated into the primary sequence of a thioredoxin (TRX)-tagged silk fusion protein, TRX-4RepCT, to give TRX3Aha -4RepCT3Aha . This method is used to produce cyclo(RGDfK) functionalized films and macroscopic fibers. Over 24 h, cyclo(RGDfK) functionalized TRX3Aha -4RepCT3Aha films and 4RepCT3Aha fibers display significantly improved performance in CiMSC culture, yielding far greater cell numbers than the controls. This approach circumvents the previously observed lack of cell adhesion, thus allowing spider silk derived biomaterials to be used where such adhesion is critical, in tissue engineering, regenerative medicine and wound healing.
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Proteínas de Artrópodes/química , Materiais Biocompatíveis/farmacologia , Seda/farmacologia , Cicatrização/efeitos dos fármacos , Alcinos/química , Proteínas de Artrópodes/síntese química , Azidas/química , Materiais Biocompatíveis/química , Química Click , Cobre/química , Reação de Cicloadição/métodos , Fibroínas/química , Fibroínas/genética , Fibroínas/uso terapêutico , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Seda/químicaRESUMO
Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 µM for AFt-TMZ, compared to 35 and 376 µM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.
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Antineoplásicos Alquilantes , Apoferritinas/química , Neoplasias Encefálicas/metabolismo , Nanoestruturas/química , Temozolomida , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Glioblastoma/metabolismo , Humanos , Temozolomida/análogos & derivados , Temozolomida/química , Temozolomida/farmacocinética , Temozolomida/farmacologiaRESUMO
INTRODUCTION: Advancement of novel anticancer drugs into clinical use is frequently halted by their lack of solubility, reduced stability under physiological conditions, and non-specific uptake by normal tissues, causing systemic toxicity. Their progress to use in the clinic could be accelerated by the development of new formulations employing suitable and complementary drug delivery vehicles. METHODS: A robust method for apoferritin (AFt)-encapsulation of antitumour benzothiazoles has been developed for enhanced activity against and drug delivery to benzothiazole-sensitive cancers. RESULTS: More than 70 molecules of benzothiazole 5F 203 were encapsulated per AFt cage. Post-encapsulation, the size and integrity of the protein cages were retained as evidenced by dynamic light scattering. ToF-SIMS depth profiling using an argon cluster beam confirmed 5F 203 exclusively within the AFt cavity. Improved encapsulation of benzothiazole lysyl-amide prodrugs was achieved (~130 molecules of Phortress per AFt cage). Transferrin receptor 1, TfR1, was detected in lysates prepared from most cancer cell lines studied, contributing to enhanced anticancer potency of the AFt-encapsulated benzothiazoles (5F 203, Phortress, GW 610, GW 608-Lys). Nanomolar activity was demonstrated by AFt-formulations in breast, ovarian, renal and gastric carcinoma cell lines, whereas GI50 >50 µM was observed in non-tumourigenic MRC-5 fibroblasts. Intracellular 5F 203, a potent aryl hydrocarbon receptor (AhR) ligand, and inducible expression of cytochrome P450 (CYP) 1A1 were detected following exposure of sensitive cells to AFt-5F 203, confirming that the activity of benzothiazoles was not compromised following encapsulation. CONCLUSION: Our results show enhanced potency and selectivity of AFt-encapsulated 5F 203 against carcinomas derived from breast, ovarian, renal, colorectal as well as gastric cancer models, and offer realistic prospects for potential refinement of tumour-targeting and treatment, and merit further in vivo investigations.
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Antineoplásicos/farmacologia , Apoferritinas/metabolismo , Sistemas de Liberação de Medicamentos , Tiazóis/farmacologia , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Tiazóis/químicaRESUMO
We report on the synthesis of water-soluble gold nanoclusters capped with polyethylene glycol (PEG)-based ligands and further functionalized with folic acid for specific cellular uptake. The dihydrolipoic acid-PEG-based ligands terminated with -OMe, -NH2 and -COOH functional groups are produced and used for surface passivation of Au nanoclusters (NCs) with diameters <2 nm. The produced sub 2 nm Au NCs possess long-shelf life and are stable in physiologically relevant environments (temperature and pH), are paramagnetic and biocompatible. The paramagnetism of Au NCs in solution is also reported. The functional groups on the capping ligands are used for direct conjugation of targeting molecules onto Au NCs without the need for post synthesis modification. Folic acid (FA) is attached via an amide group and effectively target cells expressing the folate receptor. The combination of targeting ability, biocompatibility and paramagnetism in FA-functionalized Au NCs is of relevance for their exploitation in nanomedicine for targeted imaging.
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Receptores de Folato com Âncoras de GPI/análise , Ácido Fólico/química , Ouro/química , Nanopartículas Metálicas/química , Linhagem Celular Tumoral , Humanos , Nanotecnologia , Polietilenoglicóis/químicaRESUMO
Despite important advances in the synthesis of inorganic perovskite nanocrystals (NCs), the long-term instability and degradation of their quantum yield (QY) over time need to be addressed to enable the further development and exploitation of these nanomaterials. Here we report stable CsPbI3 perovskite NCs and their use in hybrid light emitting diodes (LEDs), which combine in one system the NCs and a blue GaN-based LED. Nanocrystals with improved morphological and optical properties are obtained by optimizing the post-synthesis replacement of oleic acid ligands with iminodibenzoic acid: the NCs have a long shelf-life (>2 months), stability under different environmental conditions, and a high QY, of up to 90%, in the visible spectral range. Ligand replacement enables the engineering of the morphological and optical properties of the NCs. Furthermore, the NCs can be used to coat the surface of a GaN-LED to realize a stable diode where they are excited by blue light from the LED under low current injection conditions, resulting in emissions at distinct wavelengths in the visible range. The high QY and fluorescence lifetime in the nanosecond range are key parameters for visible light communication, an emerging technology that requires high-performance visible light sources for secure, fast energy-efficient wireless transmission.
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Listeria innocua DNA binding protein from starved cells (LiDps) belongs to the ferritin family and provides a promising self-assembling spherical 12-mer protein scaffold for the generation of functional nanomaterials. We report the creation of a Gaussia princeps luciferase (Gluc)-LiDps fusion protein, with chemical conjugation of Zinc (II)-protoporphyrin IX (ZnPP) to lysine residues on the fusion protein (giving Gluc-LiDps-ZnPP). The Gluc-LiDps-ZnPP conjugate is shown to generate reactive oxygen species (ROS) via Bioluminescence Resonance Energy Transfer (BRET) between the Gluc (470-490â¯nm) and ZnPP. In vitro, Gluc-LiDps-ZnPP is efficiently taken up by tumorigenic cells (SKBR3 and MDA-MB-231 breast cancer cells). In the presence of coelenterazine, this construct inhibits the proliferation of SKBR3 due to elevated ROS levels. Following exposure to Gluc-LiDps-ZnPP, migration of surviving SKBR3 cells is significantly suppressed. These results demonstrate the potential of the Gluc-LiDps-ZnPP conjugate as a platform for future development of an anticancer photodynamic therapy agent.
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Copépodes/enzimologia , Listeria/metabolismo , Luciferases/metabolismo , Medições Luminescentes , Nanopartículas/química , Fotoquimioterapia , Protoporfirinas/uso terapêutico , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Protoporfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Paramagnetic gadolinium ions (GdIII), complexed within DOTA-based chelates, have become useful tools to increase the magnetic resonance imaging (MRI) contrast in tissues of interest. Recently, "on/off" probes serving as 19F·MRI biosensors for target enzymes have emerged that utilize the increase in transverse (T 2 ∗ or T 2) relaxation times upon cleavage of the paramagnetic GdIII centre. Molecular 19F·MRI has the advantage of high specificity due to the lack of background signal but suffers from low signal intensity that leads to low spatial resolution and long recording times. In this work, an "on/off" probe concept is introduced that utilizes responsive deactivation of paramagnetic relaxation enhancement (PRE) to generate 19F longitudinal (T 1) relaxation contrast for accelerated molecular MRI. The probe concept is applied to matrix metalloproteinases (MMPs), a class of enzymes linked with many inflammatory diseases and cancer that modify bioactive extracellular substrates. The presence of these biomarkers in extracellular space makes MMPs an accessible target for responsive PRE deactivation probes. Responsive PRE deactivation in a 19F biosensor probe, selective for MMP-2 and MMP-9, is shown to enable molecular MRI contrast at significantly reduced experimental times compared to previous methods. PRE deactivation was caused by MMP through cleavage of a protease substrate that served as a linker between the fluorine-containing moiety and a paramagnetic GdIII-bound DOTA complex. Ultrashort echo time (UTE) MRI and, alternatively, short echo times in standard gradient echo (GE) MRI were employed to cope with the fast 19F transverse relaxation of the PRE active probe in its "on-state." Upon responsive PRE deactivation, the 19F·MRI signal from the "off-state" probe diminished, thereby indicating the presence of the target enzyme through the associated negative MRI contrast. Null point 1H·MRI, obtainable within a short time course, was employed to identify false-positive 19F·MRI responses caused by dilution of the contrast agent.
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Imagem por Ressonância Magnética de Flúor-19/métodos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Estrutura MolecularRESUMO
Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6-methylguanine (O6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease.
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Spiders produce multiple silks with different physical properties that allow them to occupy a diverse range of ecological niches, including the underwater environment. Despite this functional diversity, past molecular analyses show a high degree of amino acid sequence similarity between C-terminal regions of silk genes that appear to be independent of the physical properties of the resulting silks; instead, this domain is crucial to the formation of silk fibers. Here, we present an analysis of the C-terminal domain of all known types of spider silk and include silk sequences from the spider Argyroneta aquatica, which spins the majority of its silk underwater. Our work indicates that spiders have retained a highly conserved mechanism of silk assembly, despite the extraordinary diversification of species, silk types and applications of silk over 350 million years. Sequence analysis of the silk C-terminal domain across the entire gene family shows the conservation of two uncommon amino acids that are implicated in the formation of a salt bridge, a functional bond essential to protein assembly. This conservation extends to the novel sequences isolated from A. aquatica. This finding is relevant to research regarding the artificial synthesis of spider silk, suggesting that synthesis of all silk types will be possible using a single process.
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Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Seda/química , Seda/genética , Aranhas/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Evolução Molecular , Perfilação da Expressão Gênica , Família Multigênica , Filogenia , Domínios Proteicos/genética , Aranhas/classificaçãoRESUMO
In a new, versatile approach to fun-ction-alizing recombinant spider silk, L-azidohomoalanine is introduced residue-specifically in the minispidroin protein 4RepCT through expression in an E. coli methionine auxotroph. Both fluorophores and the antibiotic levofloxacin are attached to this bio-orthogonal amino acid using copper-catalyzed click chemistry, either before or after the silk fibers are self-assembled.
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Due to low fluorine background signal in vivo, 19F is a good marker to study the fate of exogenous molecules by magnetic resonance imaging (MRI) using equilibrium nuclear spin polarization schemes. Since 19F MRI applications require high sensitivity, it can be important to assess experimental feasibility during the design stage already by estimating the minimum detectable fluorine concentration. Here we propose a simple method for the calibration of MRI hardware, providing sensitivity estimates for a given scanner and coil configuration. An experimental "calibration factor" to account for variations in coil configuration and hardware set-up is specified. Once it has been determined in a calibration experiment, the sensitivity of an experiment or, alternatively, the minimum number of required spins or the minimum marker concentration can be estimated without the need for a pilot experiment. The definition of this calibration factor is derived based on standard equations for the sensitivity in magnetic resonance, yet the method is not restricted by the limited validity of these equations, since additional instrument-dependent factors are implicitly included during calibration. The method is demonstrated using MR spectroscopy and imaging experiments with different 19F samples, both paramagnetically and susceptibility broadened, to approximate a range of realistic environments.
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Imageamento por Ressonância Magnética , Calibragem , Radioisótopos de Flúor/química , Gadolínio/química , Limite de Detecção , Imageamento por Ressonância Magnética/normas , Modelos Teóricos , Razão Sinal-RuídoRESUMO
Magnetic interactions of Mn2+ ions in lead sulfide (PbS) nanocrystals with protons in water are probed by NMR and MRI. A thin layer of capping molecules enables free solvent diffusion to the nanocrystal surface resulting in a decrease of proton relaxation times. Magnetic resonance imaging of neuronal cell pellets exposed to (PbMn)S at non-toxic concentrations demonstrates their prospects as MRI-labels.
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BACKGROUND: Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence. RESULTS: We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism's ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions. CONCLUSIONS: We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.
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Clostridium/genética , Genoma Bacteriano , Análise de Sequência de DNA/métodos , Curadoria de Dados/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Anticancer drug Gefitinib encapsulated within human heavy chain apoferritin by diffusion allows pH-controlled sustained release of cargo. The combination of increased cellular uptake, and potent and enhanced antitumor activity against the HER2 overexpressing SKBR3 cell line compared to Gefitinib alone, makes it a promising carrier for delivery of drugs to tumor sites.