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1.
Z Gastroenterol ; 57(9): 1067-1076, 2019 Sep.
Artigo em Alemão | MEDLINE | ID: mdl-31525799

RESUMO

Intestinal tuberculosis is an infectious disease of the extrapulmonary manifestation with the Mycobacteria tuberculosis complex. In developed countries, this disease is rarely seen. The clinical features are heterogeneous and unspecific. Furthermore, intestinal tuberculosis poses diagnostic challenges. Regarding intestinal tuberculosis the Ziehl-Neelsen staining for acid-fast bacillus, PCR examination and culture methods show only poor sensitivity and specificity. In this case series, we present three patients suffering from intestinal tuberculosis, who were diagnosed and treated successfully. Furthermore, we review the literature about the pitfalls of the diagnostic approaches and the treatment options of intestinal tuberculosis.


Assuntos
Tuberculose Gastrointestinal/diagnóstico , Biópsia , Colonoscopia , Corantes , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Coloração e Rotulagem , Tuberculose Gastrointestinal/patologia
2.
Adv Exp Med Biol ; 1148: 105-114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482496

RESUMO

Fluorescence spectroscopy is one of the most important techniques in the study of therapeutic enzymes. The fluorescence phenomenon has been discovered and exploited for centuries, while therapeutic enzymes have been used in treatment of disease for only decades. This chapter provides a brief summary of the current applications of fluorescence methods in studying therapeutic enzymes to provide some insights on the selection of proper method tailored to the goal. First a brief introduction about therapeutic enzymes and history of fluorescence were provided, followed by discussions on how fluorescence was applied in the studies. Four popular fluorescence methods are discussed: fluorescence tracing, fluorescence resonance energy transfer (FRET), fluorescence quenching and fluorescence polarization. Selected application of the fluorescence methods in studying therapeutic enzymes are listed, and discussed in details in the following paragraphs.


Assuntos
Enzimas/química , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Fluorescência , Enzimas/farmacologia , Coloração e Rotulagem
3.
Zhonghua Fu Chan Ke Za Zhi ; 54(8): 527-533, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31461809

RESUMO

Objective: To investigate the feasibility of a non-invasive sampling method by collecting menstrual blood and obtaining endometrium for further research in menstruation-related diseases. Methods: On the second day of menstruation, menstrual blood was collected with menstrual cups for 4 hours, and the menstrual endometrium was filtered through a metal screen for weighing, cryopreserved, immunohistochemical staining and cell culture. Results: The collection process was painless and non-invasive. In the control group, the menstrual volume was (9.1±0.7) ml, and the endometrial tissue weight was (91.0±14.7) g. In the endometriosis group, the menstrual volume was (9.6±1.9) ml (P=0.022), and the endometrial tissue weight was (134.7±43.9) g (P=0.057). Endometrial cell culture was successful in all patients and should not be contaminated. The growth curve was a finite cell line type. The expression of cytokeratin 19 and vimentin in menstrual endometrium and cells were positive. Conclusions: By collecting menstrual blood and filtering endometrial tissue, it is an ideal non-invasive sampling method. In combination with advanced experimental technology, menstrual endometrium make further researches of endometriosis, endometrial lesions or other menstruation-related diseases possible.


Assuntos
Endometriose/etiologia , Endométrio , Menstruação/fisiologia , Feminino , Humanos , Ciclo Menstrual , Coloração e Rotulagem
4.
Mol Biol (Mosk) ; 53(4): 685-691, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397442

RESUMO

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Humanos , Focalização Isoelétrica , Proteoma/química , Espectrometria de Massas em Tandem
5.
Arch Virol ; 164(10): 2585-2592, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377889

RESUMO

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.


Assuntos
Anguilla/virologia , Doenças dos Peixes/virologia , Infecções por Reoviridae/veterinária , Reoviridae/isolamento & purificação , Animais , Encéfalo/virologia , Linhagem Celular , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia Eletrônica de Transmissão , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Reoviridae/classificação , Reoviridae/genética , Infecções por Reoviridae/virologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Coloração e Rotulagem , Proteínas Virais/genética , Vírion/ultraestrutura , Cultura de Vírus
6.
Zhonghua Bing Li Xue Za Zhi ; 48(7): 531-536, 2019 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-31288308

RESUMO

Objective: To investigate the diagnostic role of H3F3A G34W immunohistochemistry in giant cell tumor of bone. Methods: A total of 275 tumors were collected from 2016 to 2018 at Shanghai Jiaotong University Affiliated Sixth People's Hospital, including 136 giant cell tumors of bone, 31 general osteosarcomas, 3 osteoclast-rich osteosarcomas, 3 brown tumors, 34 chondroblastomas, 29 giant cell tumors of tendon sheath, 20 primary arteromatoid bone cysts and 19 non-ossifying fibromas. Results: Among the 136 cases of giant cell tumor of bone,82 patients were male and 54 were female. The age ranged from 15 to 72 years (median age 38 years). Nuclear positivity for H3F3A G34W was seen in 119/136(87.5%) giant cell tumors of bone and 1/31(3.2%) general osteosarcoma,while all osteoclast-rich osteosarcomas, brown tumors, chondroblastomas, giant cell tumors of tendon sheath, primary arteromatoid bone cysts and non-ossifying fibroma were negative. Conclusions: The monoclonal antibody against the G34W mutated site of H3F3A is a specific biomarker for giant cell tumor of bone and useful for the diagnosis and differential diagnosis of giant cell tumor of bone. Meanwhile,for those cases in which giant cell tumor of bone are diagnosed basing on clinical,pathologic and radiographic features but are negative for H3F3A G34W,should be tested for rare mutations or H3F3A wild type.


Assuntos
Neoplasias Ósseas , Tumor de Células Gigantes do Osso , Adolescente , Adulto , Idoso , Biomarcadores Tumorais , China , Feminino , Histonas , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Coloração e Rotulagem , Adulto Jovem
7.
Virchows Arch ; 475(2): 191-199, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31264038

RESUMO

Pre-analytical factors, such as fixation time, influence morphology of diagnostic and predictive immunohistochemical staining, which are increasingly used in the evaluation of lung cancer. Our aim was to investigate if variations in fixation time influence the outcome of immunohistochemical staining in lung cancer. From lung resections, specimen with tumor size bigger than 4 cm, 10 samples were obtained: 2 were put through the standard fixation protocol, 5 through the delayed, and 3 through the prolonged fixation protocol. After paraffin embedding, tissue microarrays (TMAs) were made. They were stained with 20 antibodies and scored for quality and intensity of staining. Samples with delay in fixation showed loss of TMA cores on glass slides and deterioration of tissue quality leading to reduction in the expression of CK 7, Keratin MNF116, CAM 5.2, CK 5/6, TTF-1, C-MET, Napsin A, D2-40, and PD-L1. Prolonged fixation had no influence on the performance of immunohistochemical stains. Delay of fixation negatively affects the expression of different immunohistochemical markers, influencing diagnostic (cytokeratins) and predictive (PD-L1) testing. These results emphasize the need for adequate fixation of resection specimen.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/patologia , Fixação de Tecidos/métodos , Humanos , Coloração e Rotulagem/métodos
8.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1335-1347, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328490

RESUMO

Docosahexaenoic acid (DHA) has many unique physiological functions such as promoting the development of brain and retina in infants. Therefore, it is widely applied to food, pharmacy, breeding and other industries. To obtain engineered strains of Aurantiochytrium limacinum SR21 suitable for industrial application with increased lipid and DHA production, we designed a simple, fast, accurate and high-throughput screening method based on Nile red staining of oil droplets. First, ultraviolet C (UVC) mutagenesis was used to generate a random mutant library of A. limacinum. Second, screening conditions were optimized including staining conditions of Nile red and the sorting criterion. Thereby, three putative high-lipid mutants (D03432, D05106 and D01521) were selected from the mutant library containing 3 648 mutated clones. The three mutants grew faster and produced higher amounts of lipids and DHA compared to wild type (WT). In 100 mL cultures, the lipid content of D03432 and D05106 mutants reached 64.74% and 63.13% of dry cell weight respectively, whereas the wild strain exhibited only 43.19%. DHA yield in these two mutants were even 2.26-fold and 2.37-fold higher than that of the wild strain. Experiment with 5 L fermentor further confirmed that D03432 and D05106 mutants had better performance than the wild strain on DHA yield (45.51% and 66.46% more than that of the wild strain, respectively), and demonstrated their high potential for industrial application. This work not only generated several high-DHA content mutants with high potential for industrial use, but also provided vital guidance for high-throughput screening of lipid hyper-accumulating mutants in other oil-producing microorganisms.


Assuntos
Estramenópilas , Reatores Biológicos , Ácidos Docosa-Hexaenoicos , Mutagênese , Coloração e Rotulagem
9.
Nat Commun ; 10(1): 2947, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270320

RESUMO

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.


Assuntos
Epitopos/metabolismo , Sondas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/metabolismo , Coloração e Rotulagem , Peixe-Zebra/embriologia
10.
Int J Nanomedicine ; 14: 4849-4866, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308662

RESUMO

Stem cells possess a promising potential in the clinical field. The application and effective delivery of stem cells to the desired target organ or site of injury plays an important role. This review describes strategies on understanding the effective delivery of stem cells labeled with superparamagnetic iron oxide nanoparticles (SPION) using an external magnet to enhance stem cell migration in vivo and in vitro. Fourteen total publications among 174 articles were selected. Stem cell type, SPION characteristics, labeling time, and magnetic force in vivo are considered important factors affecting the effective delivery of stem cells to the homing site. Most papers reported that the efficiency was increased when magnet is applied compared to those without. Ten studies analyzed the homing competency of SPION-labeled MSCs in vitro by observing the migration of the cell toward the external magnet. In cell-based experiments, the mechanism of magnetic attraction, the kind of nanoparticles, and various stem cells were studied well. Meta-analysis has shown the mean size of nanoparticles and degree of recovery or regeneration of damaged target organs upon in vivo studies. This strategy may provide a guideline for designing studies involving stem cell homing and further expand stem cell.


Assuntos
Magnetismo/métodos , Nanopartículas de Magnetita/química , Coloração e Rotulagem , Células-Tronco/metabolismo , Animais , Movimento Celular , Humanos , Hidrodinâmica , Tamanho da Partícula , Células-Tronco/citologia
11.
Plant Dis ; 103(9): 2330-2336, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31298992

RESUMO

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.


Assuntos
Azul Evans , Plasmodioforídeos , Esporos de Protozoários , Coloração e Rotulagem , Azul Evans/metabolismo , Doenças das Plantas , Plasmodioforídeos/fisiologia , Esporos de Protozoários/fisiologia , Coloração e Rotulagem/métodos , Coloração e Rotulagem/normas
12.
Nat Commun ; 10(1): 2539, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182711

RESUMO

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.


Assuntos
Glicina/química , Insulina/química , Proteínas/química , Coloração e Rotulagem/métodos , Aldeídos/química , Escherichia coli , Corantes Fluorescentes/química , Flúor , Células HEK293 , Humanos , Receptor de Insulina/química
13.
Nat Commun ; 10(1): 2770, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235780

RESUMO

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.


Assuntos
Bactérias/isolamento & purificação , Citometria de Fluxo/métodos , Microbiota , Microbiologia do Solo , Aminoácidos/análise , Aminoácidos/química , Bactérias/genética , Bactérias/metabolismo , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Coloração e Rotulagem/métodos
14.
Forensic Sci Int ; 301: e44-e48, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31208773

RESUMO

Hanging can be suicidal, accidental, or homicidal, and these backgrounds must be discriminated by police and forensic pathologists. We herein report a case involving a 33-year-old man who was found dead on the floor behind the entrance door of an apartment house. The man's brother declared that he had found him hanging in the gap between the stairs on the top floor. When his brother tried to cut him down, the victim fell three floors down through the gap between the stairs. Autopsy was performed to confirm suicidal hanging and a postmortem fall into the narrow gap. In this case, however, a homicide was suspected, and the version of events told by the victim's brother was initially doubted. Homicidal hanging may be uncommon, but intensive scene investigation and thorough autopsy are necessary in hanging cases to rule out homicide.


Assuntos
Asfixia/patologia , Homicídio , Lesões do Pescoço/patologia , Suicídio , Adulto , Aquaporinas/metabolismo , Epiderme/metabolismo , Medicina Legal/métodos , Fraturas de Cartilagem/patologia , Humanos , Cartilagens Laríngeas/lesões , Cartilagens Laríngeas/patologia , Masculino , Coloração e Rotulagem
15.
Eur J Histochem ; 63(2)2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31232013

RESUMO

Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are "hard to hold onto" once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, the aim was the establishment of a reliable and reproducible protocol. Tissue samples were available from pig feeding experiments. In the present study, we focus on a fixation / staining procedure without making comparisons between differently fed pigs. Several fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section. Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin's solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with intact mucin containing vesicles within the cells, which was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was possible. In conclusion, reliable mucus quantification and assessment of mucus quality requires strictly tested procedures. According to our experience, the most important aim after cryosectioning is fast fixation of the mucosubstances, which requires a combination of different fixation steps.


Assuntos
Trato Gastrointestinal/química , Histocitoquímica/métodos , Mucinas/análise , Coloração e Rotulagem/métodos , Animais , Vesículas Citoplasmáticas/química , Trato Gastrointestinal/citologia , Células Caliciformes/química , Células Caliciformes/citologia , Mucinas/química , Mucinas/classificação , Suínos
16.
Eur J Histochem ; 63(2)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243942

RESUMO

The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.


Assuntos
Autofagia , Citometria de Fluxo/métodos , Proteínas Associadas aos Microtúbulos/análise , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Cloroquina/farmacologia , Fluorescência , Humanos , Leucócitos Mononucleares/microbiologia , Mycobacterium bovis/química , Coloração e Rotulagem , Células THP-1
17.
Forensic Sci Int ; 301: 284-288, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195249

RESUMO

Troponin I (TnI) is the inhibitory subunit of the troponin complex in the sarcomeric thin filament of striated muscle and plays a central role in the calcium regulation of contraction and relaxation. Vertebrate TnI has evolved into three isoforms encoded by three homologous genes: TNNI1 for slow skeletal muscle TnI, TNNI2 for fast skeletal muscle TnI and TNNI3 for cardiac TnI, which are expressed under muscle type-specific and developmental regulations in both the atrium and ventricle of the heart. Skeletal muscle TnI (both sTnI iso-forms) have been proposed as a sensitive and fast fiber-specific serum marker of skeletal muscle damage; fsTnI concentration in increased peripheral blood when fast twitch fibers were damaged. In our study we investigate if the 'Troponin I, fast skeletal muscle' can also be used as a reliable diagnostic tool in forensic practice, to perform differential diagnosis about vitality in suicide by hanging and simulated hanging (suspension of the victim after murder). We selected 8 women and 13 men, mean age 52.2 years, who died from suicidal hanging. The ligature material used for hanging was soft material in 11 cases and hard material in 10 cases. We chose cases as a control group of adults (n = 10; six women, four men, mean age 47.3 years) that died from opioid overdose (n = 2), car accident (n = 3) and sudden cardiac death (n = 5). Those deaths were characterized by their rapidity. To test the Anti-Troponin I fast skeletal muscle Antibody (Abcam clone-134,838), we used a case of a subject who died of myocardial infarction (timing infarct dated to 24-36 h prior to death). The reactions to Troponin I (namely the amount and extent of marker depletion) was scored for each section from 0 to -3: 0 = no loss of staining; -1 = minimal decrease in staining, compared to normally stained tissue; -2 = clear decrease in staining with some positivity (brown color) remaining; and -3 = no positive (brown) staining. The set of results obtained leads us to believe that the use of this antibody (Anti-Troponin I fast skeletal muscle antibody) is very promising to be able to make a certain differential diagnosis between antemortem and postmortem hangings. It should be emphasized that the present study seems to open new and promising horizons in the possibility to discriminate between suicidal hanging and simulated hanging (suspension of the victim after murder).


Assuntos
Asfixia/diagnóstico , Lesões do Pescoço/diagnóstico , Músculos do Pescoço/metabolismo , Suicídio , Troponina I/metabolismo , Anticorpos/análise , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Patologia Legal/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem , Troponina I/imunologia
18.
Vet Immunol Immunopathol ; 212: 23-26, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31213248

RESUMO

Renal α-enolase has variable expression in inflammatory and neoplastic diseases. Therefore, in order to define the distribution of α-enolase in renal tissues of cats, an immunohistochemistry assay was validated and described here. Tissues from 29 cats with IRIS Stage 2-4 CKD, 8 control cats < 2 years of age, and 4 control cats> 10 years of age were assessed. Interstitial nephritis was the predominant histopathological finding in the CKD group. The control cats < 2 years of age had moderate α-enolase immunoreactivity in tubular epithelium but staining was absent to mild in glomeruli. In contrast, α-enolase was moderate to high in tubular epithelium and glomeruli in control cats > 10 years of age. In cats with CKD, α-enolase was decreased in tubules that were degenerative or atrophic, similar to normal tubules in control groups, and moderate to high in glomeruli. When compared between the study groups, the results suggest that alpha-enolase decreases in damaged tubules and increases in the glomeruli of older cats prior to the development of detectable CKD. Further studies will be required to determine whether these findings relate to the pathogenesis or could be used in the diagnosis of feline CKD.


Assuntos
Rim/enzimologia , Rim/patologia , Fosfopiruvato Hidratase/análise , Insuficiência Renal Crônica/enzimologia , Insuficiência Renal Crônica/patologia , Animais , Doenças do Gato/enzimologia , Doenças do Gato/patologia , Gatos , Imuno-Histoquímica , Coloração e Rotulagem
19.
Anal Bioanal Chem ; 411(15): 3221-3227, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31037373

RESUMO

High-quality matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) of lipids in biological tissue relies on the fabrication of a homogeneous matrix coating featuring best possible analyte integration. This communication addresses a matrix vapor deposition/recrystallization process for the application of 1,5-diaminonaphthalene (1,5-DAN) onto slices of human aortic tissue. The matrix coating is compatible with both positive- as well as negative-ion-mode MALDI MSI facilitating a significantly enhanced detection of lipid-related signals in different cell layers of blood vessel walls. Graphical abstract.


Assuntos
Aorta/química , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Aorta/ultraestrutura , Temperatura Baixa , Humanos , Coloração e Rotulagem , Vácuo
20.
Nat Commun ; 10(1): 1930, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036827

RESUMO

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.


Assuntos
Cromatina/química , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Cromatina/metabolismo , Regulação da Expressão Gênica , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transposases/genética , Transposases/metabolismo
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