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1.
Plant J ; 118(2): 584-600, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38141174

RESUMO

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.


Assuntos
Germinação , Plântula , Fenótipo , Germinação/fisiologia , Sementes , Processamento de Imagem Assistida por Computador
2.
Nat Commun ; 14(1): 8222, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38086825

RESUMO

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules emerging as a powerful modality in drug discovery, with the potential to address outstanding medical challenges. However, the synthetic feasibility of PROTACs, and the empiric and complex nature of their structure-activity relationships continue to present formidable limitations. As such, modular and reliable approaches to streamline the synthesis of these derivatives are highly desirable. Here, we describe a robust ruthenium-catalysed late-stage C‒H amidation strategy, to access fully elaborated heterobifunctional compounds. Using readily available dioxazolone reagents, a broad range of inherently present functional groups can guide the C-H amidation on complex bioactive molecules. High selectivity and functional group tolerance enable the late-stage installation of linkers bearing orthogonal functional handles for downstream elaboration. Finally, the single-step synthesis of both CRBN and biotin conjugates is demonstrated, showcasing the potential of this methodology to provide efficient and sustainable access to advanced therapeutics and chemical biology tools.

3.
J Med Chem ; 66(19): 13400-13415, 2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37738648

RESUMO

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

4.
Org Biomol Chem ; 18(10): 1957-1967, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32101244

RESUMO

1,4- and 1,5-Disubstituted triazole amino acid monomers have gained increasing interest among peptidic foldamers, as they are easily prepared via Cu- and Ru-catalyzed click reactions, with the potential for side chain variation. While the latter is key to their applicability, the synthesis and structural properties of the chiral mono- or disubstituted triazole amino acids have only been partially addressed. We here present the synthesis of all eight possible chiral derivatives of a triazole monomer prepared via a ruthenium-catalyzed azide alkyne cycloaddition (RuAAC). To evaluate the conformational properties of the individual building units, a systematic quantum chemical study was performed on all monomers, indicating their capacity to form several low energy conformers. This feature may be used to effect structural diversity when the monomers are inserted into various peptide sequences. We envisage that these results will facilitate new applications for these artificial oligomeric compounds in diverse areas, ranging from pharmaceutics to biotechnology.


Assuntos
Peptidomiméticos/síntese química , Triazóis/síntese química , Alcinos/química , Azidas/química , Química Click , Reação de Cicloadição , Modelos Moleculares , Polimerização , Polímeros/síntese química , Teoria Quântica , Estereoisomerismo , Termodinâmica
5.
Lakartidningen ; 1162019 Feb 19.
Artigo em Sueco | MEDLINE | ID: mdl-31192391

RESUMO

Frequency of cancer in the urinary organs was significantly lower in patients with macroscopic hematuria associated with bacteriuria compared to those without bacteriuria. The predictive value of macroscopic hematuria was <1% in patients ≤ 75 years of age with concomitant bacteriuria. CT-urography added no diagnostic value over and above cystoscopy in patients with macroscopic hematuria with bacteriuria.Bacteriuria with other bacteria than E. coli or S. saprophyticus was associated with findings of bladder tumors.


Assuntos
Bacteriúria/diagnóstico , Hematúria/diagnóstico , Neoplasias Urogenitais/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Cistoscopia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pacotes de Assistência ao Paciente , Valor Preditivo dos Testes , Garantia da Qualidade dos Cuidados de Saúde , Fatores de Risco , Fumar , Tomografia Computadorizada por Raios X , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/microbiologia , Neoplasias Urogenitais/microbiologia , Urografia
6.
Clin Hemorheol Microcirc ; 71(2): 183-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30584128

RESUMO

Circulating blood cells are prone to varying flow conditions when contacting cardiovascular devices. For a profound understanding of the complex interplay between the blood components/cells and cardiovascular implant surfaces, testing under varying shear conditions is required. Here, we study the influence of arterial and venous shear conditions on the in vitro evaluation of the thrombogenicity of polymer-based implant materials.Medical grade poly(dimethyl siloxane) (PDMS), polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) films were included as reference materials. The polymers were exposed to whole blood from healthy humans. Blood was agitated orbitally at low (venous shear stress: 2.8 dyne · cm-2) and high (arterial shear stress: 22.2 dyne · cm-2) agitation speeds in a well-plate based test system. Numbers of non-adherent platelets, platelet activation (P-Selectin positive platelets), platelet function (PFA100 closure times) and platelet adhesion (laser scanning microscopy (LSM)) were determined.Microscopic data and counting of the circulating cells revealed increasing numbers of material-surface adherent platelets with increasing agitation speed. Also, activation of the platelets was substantially increased when tested under the high shear conditions (P-Selectin levels, PFA-100 closure times). At low agitation speed, the platelet densities did not differ between the three materials. Tested at the high agitation speed, lowest platelet densities were observed on PDMS, intermediate levels on PET and highest on PTFE. While activation of the circulating platelets was affected by the implant surfaces in a similar manner, PFA closure times did not reflect this trend.Differences in the thrombogenicity of the studied polymers were more pronounced when tested at high agitation speed due to the induced shear stresses. Testing under varying shear stresses, thus, led to a different evaluation of the implant thrombogenicity, which emphasizes the need for testing under various flow conditions. Our data further confirmed earlier findings where the same reference implants were tested under static (and not dynamic) conditions and with fresh human platelet rich plasma instead of whole blood. This supports that the application of common reference materials may improve inter-study comparisons, even under varying test conditions.


Assuntos
Materiais Biocompatíveis/uso terapêutico , Ativação Plaquetária/fisiologia , Adesividade Plaquetária/fisiologia , Humanos , Estresse Mecânico
7.
Am Heart J ; 201: 95-102, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29910060

RESUMO

BACKGROUND: Transcatheter foramen ovale closure (TPC) has emerged as a potential treatment option for patients with cryptogenic strokes and persistent foramen ovale (PFO). However, previous randomized controlled trials could hardly demonstrate any benefit compared to medical treatment (Med-Tx). Recently new data have become available which may change current practice of transcatheter PFO closure. METHODS: A systematic review and meta-analysis comparing TPC and Med-Tx based on all available multicentric randomized controlled trials was performed. The primary outcome of interest was the recurrence of stroke in both groups. RESULTS: Five studies met the inclusion criteria with 1829 patients in the TPC and 1622 in the Med-Tx group. The median follow-up was 4 years. In the intention-to-treat analysis we found a statistically significant relative risk reduction in recurrence of strokes in the TPC group compared to the Med-Tx group (pooled hazard ratio (HR): 0.32; 95% CI: 0.13-0.8; P = .018). Excluding one study due to potential publication bias resulted in a pooled HR of 0.48 (95% CI: 0.25-0.91, P = .024). Patients younger than 45 years of age (pooled HR: 0.35; 95% CI: 0.16-0.75; P = .007) and those with moderate to severe shunt (pooled HR: 0.28; 95% CI: 0.14-0.55; P < .001) were more likely to benefit from closure. CONCLUSION: According to our meta-analysis TPC plus antiplatelets was superior in terms of stroke prevention when compared to Med-Tx. Furthermore, patients with moderate to severe shunts and those younger than 45 years of age were found to benefit most from TPC.


Assuntos
Cateterismo Cardíaco/métodos , Forame Oval Patente/cirurgia , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Prevenção Secundária/métodos , Dispositivo para Oclusão Septal , Acidente Vascular Cerebral/prevenção & controle , Forame Oval Patente/complicações , Humanos , Análise de Intenção de Tratamento , Reoperação , Acidente Vascular Cerebral/etiologia
8.
ACS Pharmacol Transl Sci ; 1(2): 119-133, 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-32219208

RESUMO

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by proteolytic cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle, leading to activation of intracellular signaling pathways associated with inflammation and cancer. Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was probed by iterative combinations of receptor mutagenesis, agonist ligand modifications, and data-driven structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel peptide (1-benzyl-1H[1,2,3]triazole-4-yl-LIGKV) with functional potency higher than that of SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel agonists and competitive antagonists. The combined experimental and computational approach to characterize agonist binding to PAR2 can be extended to study the many other G protein-coupled receptors that recognize peptides or proteins.

9.
J Interv Cardiol ; 31(1): 83-90, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29193333

RESUMO

OBJECTIVES: The present study aimed to evaluate the outcome and potential limitations of a repeated MitraClip procedure (ReClip). BACKGROUND: The MitraClip procedure has emerged as a treatment option in high surgical risk patients suffering from severe mitral regurgitation (MR). However, despite successful initial repair a significant number of patients develops severe recurrent MR. METHODS: Patients undergoing a ReClip procedure in our institution were retrospectively identified. Baseline data and the procedural outcome were assessed to identify potential limitations of such procedures. RESULTS: Fifteen out of 234 patients undergoing a mitral-valve repair with the MitraClip device (Abbott Vascular) underwent a ReClip due to recurrent MR. In 11 patients, a MR reduction of at least one degree without causing mitral valve stenosis (trans-mitral mean gradient ≥5 mmHg) was achieved by performing a ReClip. After 1 year, two patients developed severe recurrent MR again. Pulmonary artery pressures significantly decreased after the procedure in individuals with successful repair (MR reduction of at least one degree and mitral valve mean gradient <5 mmHg). CONCLUSION: A ReClip procedure may be feasible in patients with recurrent MR but the risk benefit ratio should be carefully balanced against other treatment options.


Assuntos
Insuficiência da Valva Mitral , Complicações Pós-Operatórias , Reoperação/métodos , Idoso , Feminino , Implante de Prótese de Valva Cardíaca/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Valva Mitral/diagnóstico por imagem , Valva Mitral/cirurgia , Insuficiência da Valva Mitral/diagnóstico , Insuficiência da Valva Mitral/cirurgia , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Recidiva , Estudos Retrospectivos , Medição de Risco/métodos , Índice de Gravidade de Doença , Resultado do Tratamento
10.
Transgenic Res ; 26(6): 791-798, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28819706

RESUMO

Humanized mouse models are important tools in many areas of biological drug development including, within oncology research, the development of antagonistic antibodies that have the potential to block tumor growth by controlling vascularization and are key to the generation of in vivo proof-of-concept efficacy data. However, due to cross reactivity between human antibodies and mouse target such studies regularly require mouse models expressing only the human version of the target molecule. Such humanized knock-in/knock-out, KIKO, models are dependent upon the generation of homozygous mice expressing only the human molecule, compensating for loss of the mouse form. However, KIKO strategies can fail to generate homozygous mice, even though the human form is expressed and the endogenous mouse locus is correctly targeted. A typical strategy for generating KIKO mice is by ATG fusion where the human cDNA is inserted downstream of the endogenous mouse promoter elements. However, when adopting this strategy it is possible that the mouse promoter fails to express the human form in a manner compensating for loss of the mouse form or alternatively the human protein is incompatible in the context of the mouse pathway being investigated. So to understand more around the biology of KIKO models, and to overcome our failure with a number of ATG fusion strategies, we developed a range of humanized models focused on Delta-like 4 (Dll4), a target where we initially failed to generate a humanized model. By adopting a broader biologic strategy, we successfully generated a humanized DLL4 KIKO which led to a greater understanding of critical biological aspects for consideration when developing humanized models.


Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos Transgênicos/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/farmacologia , Proteínas de Ligação ao Cálcio , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos Knockout , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Chem Rev ; 116(23): 14726-14768, 2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27960271

RESUMO

The ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) affords 1,5-disubstituted 1,2,3-triazoles in one step and complements the more established copper-catalyzed reaction providing the 1,4-isomer. The RuAAC reaction has quickly found its way into the organic chemistry toolbox and found applications in many different areas, such as medicinal chemistry, polymer synthesis, organocatalysis, supramolecular chemistry, and the construction of electronic devices. This Review discusses the mechanism, scope, and applications of the RuAAC reaction, covering the literature from the last 10 years.

12.
Anal Biochem ; 484: 51-7, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25957128

RESUMO

A pre-labeling protocol based on Cy5 N-hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range-more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250pmol in 20µl using a Tris-HCl labeling buffer at pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of sub-nanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS-PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. Two-dimensional electrophoresis was used to examine the labeling of proteins in a cell lysate using both sulfonated and non-sulfonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points.


Assuntos
Carbocianinas/química , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas/análise , Proteínas/química , Animais , Células CHO , Cricetinae , Cricetulus , Cinética , Limite de Detecção , Proteínas/isolamento & purificação
13.
Org Biomol Chem ; 13(9): 2776-85, 2015 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-25605623

RESUMO

Peptidic foldamers have recently emerged as a novel class of artificial oligomers with properties and structural diversity similar to that of natural peptides, but possessing additional interesting features granting them great potential for applications in fields from nanotechnology to pharmaceuticals. Among these, foldamers containing 1,4- and 1,5-substitued triazole amino acids are easily prepared via the Cu- and Ru-catalyzed click reactions and may offer increased side chain variation, but their structural capabilities have not yet been widely explored. We here describe a systematic analysis of the conformational space of the two most important basic units, the 1,4-substitued (4Tzl) and the 1,5-substitued (5Tzl) 1,2,3-triazole amino acids, using quantum chemical calculations and NMR spectroscopy. Possible conformations of the two triazoles were scanned and their potential minima were located using several theoretical approaches (B3LYP/6-311++G(2d,2p), ωB97X-D/6-311++G(2d,2p), M06-2X/6-311++G(2d,2p) and MP2/6-311++G(2d,2p)) in different solvents. BOC-protected versions of 4Tzl and 5Tzl were also prepared via one step transformations and analyzed by 2D NOESY NMR. Theoretical results show 9 conformers for 5Tzl derivatives with relative energies lying close to each other, which may lead to a great structural diversity. NMR analysis also indicates that conformers preferring turn, helix and zig-zag secondary structures may coexist in solution. In contrast, 4Tzl has a much lower number of conformers, only 4, and these lack strong intraresidual interactions. This is again supported by NMR suggesting the presence of both extended and bent conformers. The structural information provided on these building units could be employed in future design of triazole foldamers.


Assuntos
Aminoácidos/química , Peptídeos/química , Triazóis/química , Conformação Molecular , Peptídeos/síntese química , Dobramento de Proteína , Teoria Quântica
14.
Bioorg Med Chem Lett ; 24(13): 2963-8, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24835983

RESUMO

Modification of a series of P2Y12 receptor antagonists by replacement of the ester functionality was aimed at minimizing the risk of in vivo metabolic instability and pharmacokinetic variability. The resulting ketones were then optimized for their P2Y12 antagonistic and anticoagulation effects in combination with their physicochemical and absorption profiles. The most promising compound showed very potent antiplatelet action in vivo. However, pharmacodynamic-pharmacokinetic analysis did not reveal a significant separation between its anti-platelet and bleeding effects. The relevance of receptor binding kinetics to the in vivo profile is described.


Assuntos
Plaquetas/efeitos dos fármacos , Fibrinolíticos/farmacologia , Cetonas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y12/metabolismo , Animais , Células CHO , Células CACO-2 , Cricetulus , Cães , Relação Dose-Resposta a Droga , Fibrinolíticos/administração & dosagem , Fibrinolíticos/química , Humanos , Cetonas/administração & dosagem , Cetonas/química , Cinética , Estrutura Molecular , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/química , Relação Estrutura-Atividade
15.
Chemistry ; 19(20): 6246-56, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23576496

RESUMO

The slow dissociation of DNA threading intercalators makes them interesting as model compounds in the search for new DNA targeting drugs, as there appears to be a correlation between slow dissociation and biological activity. Thus, it would be of great value to understand the mechanisms controlling threading intercalation, and for this purpose we have investigated how the length of the bridging ligand of binuclear ruthenium threading intercalators affects their DNA binding properties. We have synthesised a new binuclear ruthenium threading intercalator with slower dissociation kinetics from ct-DNA than has ever been observed for any ruthenium complex with any type of DNA, a property that we attribute to the increased distance between the ruthenium centres of the new complex. By comparison with previously studied ruthenium complexes, we further conclude that elongation of the bridging ligand reduces the sensitivity of the threading interaction to DNA flexibility, resulting in a decreased AT selectivity for the new complex. We also find that the length of the bridging ligand affects the enantioselectivity with increasing preference for the ΔΔ enantiomer as the bridging ligand becomes longer.


Assuntos
Substâncias Intercalantes/química , Rutênio/química , DNA/química , DNA/metabolismo , Cinética , Ligantes , Estereoisomerismo
16.
Nat Commun ; 3: 976, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22864569

RESUMO

Fbw7 is a ubiquitin-ligase that targets several oncoproteins for proteolysis, but the full range of Fbw7 substrates is not known. Here we show that by performing quantitative proteomics combined with degron motif searches, we effectively screened for a more complete set of Fbw7 targets. We identify 89 putative Fbw7 substrates, including several disease-associated proteins. The transcription factor NF-κB2 (p100/p52) is one of the candidate Fbw7 substrates. We show that Fbw7 interacts with p100 via a conserved degron and that it promotes degradation of p100 in a GSK3ß phosphorylation-dependent manner. Fbw7 inactivation increases p100 levels, which in the presence of NF-κB pathway stimuli, leads to increased p52 levels and activity. Accordingly, the apoptotic threshold can be increased by loss of Fbw7 in a p100-dependent manner. In conclusion, Fbw7-mediated destruction of p100 is a regulatory component restricting the response to NF-κB2 pathway stimulation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Biologia Computacional , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Subunidade p52 de NF-kappa B/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteômica , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem , Ubiquitina-Proteína Ligases/genética
17.
Proc Natl Acad Sci U S A ; 109(7): 2186-7, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22331872
18.
Mol Cell Proteomics ; 10(11): O111.011031, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21813417

RESUMO

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor ß by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.


Assuntos
Western Blotting/métodos , Anticorpos/química , Células Cultivadas , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Oligonucleotídeos/química , Fosforilação , Processamento de Proteína Pós-Traducional , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Razão Sinal-Ruído , Transferrina/metabolismo , Tubulina (Proteína)/metabolismo
19.
J Org Chem ; 76(7): 2355-9, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21388208

RESUMO

An experimentally simple sequential one-pot RuAAC reaction, affording 1,5-disubstituted 1H-1,2,3-triazoles in good to excellent yields starting from an alkyl halide, sodium azide, and an alkyne, is reported. The organic azide is formed in situ by treating the primary alkyl halide with sodium azide in DMA under microwave heating. Subsequent addition of [RuClCp*(PPh(3))(2)] and the alkyne yielded the desired cycloaddition product after further microwave irradiation.


Assuntos
Alcinos/química , Alcinos/síntese química , Azidas/química , Azidas/síntese química , Hidrocarbonetos Halogenados/química , Azida Sódica/química , Catálise , Ciclização , Micro-Ondas , Estrutura Molecular
20.
Bioresour Technol ; 101(2): 836-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19765984

RESUMO

The effect of a well-defined trace element solution and the elements nickel, cobalt and molybdenum on anaerobic digestion of a synthetic model substrate for maize silage was studied in batch reactor experiments at 35 degrees C. The defined substrate (dS) consisted of xylan and starch as the main carbon source, urea as nitrogen source and phosphorus from a 0.1M potassium phosphate buffer. Batch reactors were operated for 30 days with 1.5% organic dry matter (ODM) of inoculum sludge from a mono-maize biogas plant and 1% ODM of the defined substrate. Results showed an increase of methane yield of up to 30% upon addition of the trace element solution. With an addition of nickel at 10.6 microM, a final yield of 407 l kg(-1) ODM was reached and an enhanced methane production by 25% at day 25 of operation was observed. Total elimination of nickel from the trace element solution highly decreased methane formation and process stability. Cobalt in a concentration range of 0.4 up to 2.0 microM increased the methane production by 10% approximately. Interestingly, addition of molybdenum did not significantly effect methane production.


Assuntos
Metano/biossíntese , Oligoelementos/farmacologia , Zea mays/metabolismo , Reatores Biológicos , Fermentação , Esgotos
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