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1.
Methods Mol Biol ; 2350: 105-123, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331282

RESUMO

Early detection of malignant tumors, micrometastases, and disseminated tumor cells is one of the effective way of fighting cancer. Among the many existing imaging methods like computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), optical imaging with fluorescent probes is one of the most promising alternatives because it is fast, inexpensive, safe, sensitive, and specific. However, traditional fluorescent probes, based on organic fluorescent dyes, suffer from the low signal-to-noise ratio. Furthermore, conventional organic fluorescent dyes are unsuitable for deep tissue imaging because of the strong visible light absorption by biological tissues. The use of fluorescent semiconductor nanocrystals, or quantum dots (QDs), may overcome this limitation due to their large multiphoton cross section, which ensures efficient imaging of thick tissue sections inaccessible with conventional fluorescent probes. Moreover, the lower photobleaching and higher brightness of fluorescence signals from QDs ensures a much better discrimination of positive signals from the background. The use of fluorescent nanoprobes based on QDs conjugated to uniformly oriented high-affinity single-domain antibodies (sdAbs) may significantly increase the sensitivity and specificity due to better recognition of analytes and deeper penetration into tissues due to small size of such nanoprobes.Here, we describe a protocol for the fabrication of nanoprobes based on sdAbs and QDs, preparation of experimental xenograft mouse models for quality control, and multiphoton imaging of deep-tissue solid tumors, micrometastases, and disseminated tumor cells.


Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Pontos Quânticos , Anticorpos de Domínio Único , Linhagem Celular Tumoral , Imunofluorescência/normas , Humanos , Imunoconjugados/química , Imuno-Histoquímica/métodos , Sondas Moleculares , Imagem Multimodal/métodos , Nanopartículas , Micrometástase de Neoplasia , Imagem Óptica/métodos
2.
Methods Mol Biol ; 2135: 259-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32246341

RESUMO

Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.


Assuntos
Análise Serial de Proteínas/métodos , Pontos Quânticos/química , Anticorpos/imunologia , Biotina/química , Corantes Fluorescentes/química , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Imunoensaio/métodos , Análise em Microsséries/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estreptavidina/química
3.
Polymers (Basel) ; 11(12)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817649

RESUMO

The possibility of the application of acrylate compositions and Bayfol HX photopolymers in holographic technologies is considered. The holographic characteristics of materials, their advantages, and limitations in relation to the tasks of obtaining holographic elements based on periodic structures are given. The conditions for obtaining controlled two and multichannel diffraction beam splitters are determined with advantages in terms of the simplicity of the fabrication process. The diffraction and selective properties of volume and hybrid periodic structures by radiation incidence in a wide range of angles in three-dimensional space are investigated, and new properties are identified that are of interest for the development of elements of holographic solar concentrators with advantages in the material used and the range of incidence angles. A new application of polymer materials in a new method of holographic 3D printing for polymer objects with arbitrary shape fabrication based on the projection of a holographic image of the object into the volume of photopolymerizable material is proposed, the advantage of which, relative to additive 3D printing technologies, is the elimination of the sequential synthesis of a three-dimensional object. The factors determining the requirements for the material, fabrication conditions, and properties of three-dimensional objects are identified and investigated.

4.
Genes (Basel) ; 10(3)2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836702

RESUMO

Interspecific crossing is a promising approach for introgression of valuable traits to develop cultivars with improved characteristics. Allium fistulosum L. possesses numerous pest resistances that are lacking in the bulb onion (Allium cepa L.), including resistance to Stemphylium leaf blight (SLB). Advanced generations were produced by selfing and backcrossing to bulb onions of interspecific hybrids between A. cepa and A. fistulosum that showed resistance to SLB. Molecular classification of the cytoplasm established that all generations possessed normal (N) male-fertile cytoplasm of bulb onions. Genomic in situ hybridization (GISH) was used to study the chromosomal composition of the advanced generations and showed that most plants were allotetraploids possessing the complete diploid sets of both parental species. Because artificial doubling of chromosomes of the interspecific hybrids was not used, spontaneous polyploidization likely resulted from restitution gametes or somatic doubling. Recombinant chromosomes between A. cepa and A. fistulosum were identified, revealing that introgression of disease resistances to bulb onion should be possible.


Assuntos
Cromossomos de Plantas/genética , Resistência à Doença , Hibridização In Situ/métodos , Cebolas/microbiologia , Citoplasma , Introgressão Genética , Genômica , Cariótipo , Cebolas/genética , Melhoramento Vegetal , Saccharomycetales/patogenicidade , Tetraploidia
5.
Plants (Basel) ; 8(2)2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30720753

RESUMO

We exploited the advantages of genomic in situ hybridization (GISH) to monitor the introgression process at the chromosome level using a simple and robust molecular marker in the interspecific breeding of bulb onion (Allium cepa L.) that is resistant to downy mildew. Downy mildew (Peronospora destructor [Berk.] Casp.) is the most destructive fungal disease for bulb onions. With the application of genomic in situ hybridization (GISH) and previously developed DMR1 marker, homozygous introgression lines that are resistant to downy mildew were successfully produced in a rather short breeding time. Considering that the bulb onion is a biennial plant, it took seven years from the F1 hybrid production to the creation of S2BC2 homozygous lines that are resistant to downy mildew. Using GISH, it was shown that three progeny plants of S2BC2 possessed an A. roylei homozygous fragment in the distal region of the long arm of chromosomes 3 in an A. cepa genetic background. Previously, it was hypothesized that a lethal gene(s) was linked to the downy mildew resistance gene. With the molecular cytogenetic approach, we physically mapped more precisely the lethal gene(s) using the homozygous introgression lines that differed in the size of the A. roylei fragments on chromosome 3.

6.
Nanoscale Res Lett ; 13(1): 44, 2018 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-29417375

RESUMO

Studies on the methods of nanoparticle (NP) synthesis, analysis of their characteristics, and exploration of new fields of their applications are at the forefront of modern nanotechnology. The possibility of engineering water-soluble NPs has paved the way to their use in various basic and applied biomedical researches. At present, NPs are used in diagnosis for imaging of numerous molecular markers of genetic and autoimmune diseases, malignant tumors, and many other disorders. NPs are also used for targeted delivery of drugs to tissues and organs, with controllable parameters of drug release and accumulation. In addition, there are examples of the use of NPs as active components, e.g., photosensitizers in photodynamic therapy and in hyperthermic tumor destruction through NP incorporation and heating. However, a high toxicity of NPs for living organisms is a strong limiting factor that hinders their use in vivo. Current studies on toxic effects of NPs aimed at identifying the targets and mechanisms of their harmful effects are carried out in cell culture models; studies on the patterns of NP transport, accumulation, degradation, and elimination, in animal models. This review systematizes and summarizes available data on how the mechanisms of NP toxicity for living systems are related to their physical and chemical properties.

7.
Microbiologyopen ; 2(3): 471-81, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23554367

RESUMO

A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4'-isoleucine and l-4,4'-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4'-hydroxylating) (HilA) and l-4'-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4')-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4')-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Isoleucina/metabolismo , Redes e Vias Metabólicas/genética , Pantoea/enzimologia , Pantoea/metabolismo , Biotransformação , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica
8.
Appl Microbiol Biotechnol ; 97(6): 2467-72, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22584432

RESUMO

L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.


Assuntos
Dioxigenases/isolamento & purificação , Dioxigenases/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Leucina/análogos & derivados , Leucina/metabolismo , Nostoc/enzimologia , Etionina/metabolismo , Metionina/metabolismo , Norleucina/metabolismo , Safrol/análogos & derivados , Safrol/metabolismo
9.
Brain Topogr ; 26(3): 410-27, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23104186

RESUMO

The auditory magnetic event-related fields (ERF) qualitatively change through the child development, reflecting maturation of auditory cortical areas. Clicks presented with long inter-stimulus interval produce distinct ERF components, and may appear useful to characterize immature EFR morphology in children. The present study is aimed to investigate morphology of the auditory ERFs in school-age children, as well as lateralization and repetition suppression of ERF components evoked by the clicks. School-age children and adults passively listened to pairs of click presented to the right ear, left ear or binaurally, with 8-11 s intervals between the pairs and a 1 s interval within a pair. Adults demonstrated a typical P50m/N100m response. Unlike adults, children had two distinct components preceding the N100m-P50m (at ~65 ms) and P100m (at ~100 ms). The P100m dominated the child ERF, and was most prominent in response to binaural stimulation. The N100m in children was less developed than in adults and partly overlapped in time with the P100m, especially in response to monaural clicks. Strong repetition suppression was observed for P50m both in children and adults, P100m in children and N100m in adults. Both children and adults demonstrated ERF amplitude and/or latency right hemispheric advantage effects that may reflect right hemisphere dominance for preattentive arousal processes. Our results contribute to the knowledge concerning development of auditory processing and its lateralization in children and have implications for investigation of the auditory evoked fields in developmental disorders.


Assuntos
Córtex Auditivo/fisiologia , Potenciais Evocados Auditivos/fisiologia , Lateralidade Funcional/fisiologia , Inibição Psicológica , Priming de Repetição/fisiologia , Estimulação Acústica , Adolescente , Adulto , Fatores Etários , Análise de Variância , Mapeamento Encefálico , Criança , Feminino , Humanos , Imageamento por Ressonância Magnética , Magnetoencefalografia , Masculino , Pessoa de Meia-Idade , Tempo de Reação/fisiologia , Fatores de Tempo , Adulto Jovem
10.
PLoS One ; 7(6): e39906, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22768163

RESUMO

Auditory sensory modulation difficulties are common in autism spectrum disorders (ASD) and may stem from a faulty arousal system that compromises the ability to regulate an optimal response. To study neurophysiological correlates of the sensory modulation difficulties, we recorded magnetic field responses to clicks in 14 ASD and 15 typically developing (TD) children. We further analyzed the P100m, which is the most prominent component of the auditory magnetic field response in children and may reflect preattentive arousal processes. The P100m was rightward lateralized in the TD, but not in the ASD children, who showed a tendency toward P100m reduction in the right hemisphere (RH). The atypical P100m lateralization in the ASD subjects was associated with greater severity of sensory abnormalities assessed by Short Sensory Profile, as well as with auditory hypersensitivity during the first two years of life. The absence of right-hemispheric predominance of the P100m and a tendency for its right-hemispheric reduction in the ASD children suggests disturbance of the RH ascending reticular brainstem pathways and/or their thalamic and cortical projections, which in turn may contribute to abnormal arousal and attention. The correlation of sensory abnormalities with atypical, more leftward, P100m lateralization suggests that reduced preattentive processing in the right hemisphere and/or its shift to the left hemisphere may contribute to abnormal sensory behavior in ASD.


Assuntos
Estimulação Acústica , Córtex Auditivo/fisiopatologia , Transtornos Globais do Desenvolvimento Infantil/fisiopatologia , Sensação/fisiologia , Comportamento , Criança , Demografia , Feminino , Humanos , Masculino , Localização de Som , Fatores de Tempo
11.
FEMS Microbiol Lett ; 331(2): 97-104, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22448874

RESUMO

L-isoleucine-4-hydroxylase (IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. L-methionine, L-leucine, L-isoleucine and L-threonine were found to be catalysed by the investigated enzymes, producing L-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free L-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed.


Assuntos
Aminoácidos/metabolismo , Bactérias/enzimologia , Dioxigenases/metabolismo , Escherichia coli/enzimologia , Bactérias/classificação , Bactérias/genética , Clonagem Molecular , Dioxigenases/classificação , Dioxigenases/genética , Escherichia coli/genética , Hidroxilação , Isoleucina/metabolismo , Cinética , Leucina/metabolismo , Metionina/metabolismo , Especificidade por Substrato , Treonina/metabolismo
12.
Appl Environ Microbiol ; 77(19): 6926-30, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21821743

RESUMO

We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.


Assuntos
Aminoácidos/metabolismo , Bacillus thuringiensis/enzimologia , Bacillus thuringiensis/metabolismo , Dioxigenases/metabolismo , Hidroxilação , Ácidos Cetoglutáricos/metabolismo , Especificidade por Substrato
13.
Appl Microbiol Biotechnol ; 88(3): 719-26, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20665018

RESUMO

The stereo-specific L-isoleucine-4-hydroxylase (L-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to "shunt" destroyed TCA, thereby coupling L-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of L-isoleucine into 4-HIL with an 82% yield.


Assuntos
Escherichia coli/metabolismo , Isoleucina/análogos & derivados , Sequência de Aminoácidos , Sequência de Bases , Biotransformação , Clonagem Molecular , Dioxigenases/metabolismo , Escherichia coli/crescimento & desenvolvimento , Fermentação , Regulação Bacteriana da Expressão Gênica , Isoleucina/biossíntese , Complexo Cetoglutarato Desidrogenase/metabolismo , Dados de Sequência Molecular
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