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1.
Proc Natl Acad Sci U S A ; 117(50): 31639-31647, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33262279

RESUMO

Hierarchical nanomaterials have received increasing interest for many applications. Here, we report a facile programmable strategy based on an embedded segmental crystallinity design to prepare unprecedented supramolecular planar nanobrush-like structures composed of two distinct molecular packing motifs, by the self-assembly of one particular diblock copolymer poly(ethylene glycol)-block-poly(N-octylglycine) in a one-pot preparation. We demonstrate that the superstructures result from the temperature-controlled hierarchical self-assembly of preformed spherical micelles by optimizing the crystallization-solvophobicity balance. Particularly remarkable is that these micelles first assemble into linear arrays at elevated temperatures, which, upon cooling, subsequently template further lateral, crystallization-driven assembly in a living manner. Addition of the diblock copolymer chains to the growing nanostructure occurs via a loosely organized micellar intermediate state, which undergoes an unfolding transition to the final crystalline state in the nanobrush. This assembly mechanism is distinct from previous crystallization-driven approaches which occur via unimer addition, and is more akin to protein crystallization. Interestingly, nanobrush formation is conserved over a variety of preparation pathways. The precise control ability over the superstructure, combined with the excellent biocompatibility of polypeptoids, offers great potential for nanomaterials inaccessible previously for a broad range of advanced applications.

2.
Psychosom Med ; 83(4): 322-327, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33284256

RESUMO

OBJECTIVE: This study aimed to compare the mental health and psychological responses in Wuhan, a severely affected area, and other areas of China during the coronavirus disease 2019 (COVID-19) epidemic. METHODS: This cross-sectional study was conducted on February 10-20, 2020. A set of online questionnaires was used to measure mental health and responses. A total of 1397 participants from Wuhan (age, 36.4 ± 10.7 years; male, 36.1%) and 2794 age- and sex-matched participants from other areas of China (age, 35.9 ± 9.9 years; male, 39.0%) were recruited. RESULTS: Compared with their counterparts, participants from Wuhan had a significantly higher prevalence of any mental health problems (46.6% versus 32.2%; adjusted odds ratio [OR] = 1.89, 95% confidence interval [CI] = 1.65-2.17), anxiety (15.2% versus 6.2%; adjusted OR = 2.65, 95% CI = 2.14-3.29), depression (18.3% versus 9.7%; adjusted OR = 2.11, 95% CI = 1.74-2.54), suicidal ideation (10.5% versus 7.1%; adjusted OR = 1.60, 95% CI = 1.28-2.02), and insomnia (38.6% versus 27.6%; adjusted OR = 1.70, 95% CI = 1.48-1.96). Participants from Wuhan had a slightly higher rate of help-seeking behavior (7.1% versus 4.2%; adjusted OR = 1.76, 95% CI = 1.12-2.77) but similar rate of treatment (3.5% versus 2.7%; adjusted OR = 1.23, 95% CI = 0.68-2.24) for mental problems than did their counterparts. In addition, compared with their counterparts, participants from Wuhan gave higher proportions of responses regarding "fearful" (52% versus 36%, p < .001), "discrimination against COVID-19 cases" (64% versus 58%, p = .006), "strictly comply with preventive behaviors" (98.7% versus 96%, p = .003), and "fewer living and medical supplies" (<2 weeks: 62% versus 57%, p = .015). CONCLUSIONS: The COVID-19 epidemic has raised enormous challenges regarding public mental health and psychological responses, especially in the highly affected Wuhan area. The present findings provide important information for developing appropriate strategies for the prevention and management of mental health problems during COVID-19 and other epidemics.


Assuntos
COVID-19/psicologia , Saúde Mental , Adulto , Ansiedade/epidemiologia , Ansiedade/etiologia , China/epidemiologia , Estudos Transversais , Depressão/epidemiologia , Depressão/etiologia , Epidemias , Feminino , Humanos , Masculino , Transtornos Mentais/epidemiologia , Transtornos Mentais/etiologia , Saúde Mental/estatística & dados numéricos , Escalas de Graduação Psiquiátrica , Ideação Suicida , Inquéritos e Questionários
3.
Fish Shellfish Immunol ; 111: 6-15, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33387658

RESUMO

Two interleukin (IL)-17 N genes (CcIL-17Na and b) present on different linkage groups were identified in the common carp (Cyprinus carpio) genome and confirmed by polymerase chain reaction (PCR) and real time (RT)-PCR in this experiment. Synteny analysis revealed that IL-17 N is transcribed by the complement sequence of TOP3B's intron 2. It is flanked by SDF2L and PPM1F in all fish studied to date, except fugu (Takifugu rubripes). The open reading frames of the two CcIL-17Ns are 411 base pairs long and encode 136 amino acids. The amino acid identity/similarity between CcIL-17Na and b is 91.2%/97.1%. The CcIL-17Ns share identity (46.8-90.4%) with their orthologs from other teleosts. Identities/similarities to other members of the IL-17 family in common carp were low at 21.4-30.2%/31.4-51.4%. In the phylogenetic tree, IL-17Ns from spotted gar (Lepisosteus oculatus, the ancestor of teleosts) and coelacanth (Latimeria chalumnae, the ancestor of tetrapods) were grouped within the same branch with a high bootstrap value of 97%, which indicates that IL-17 N is an ancient and conserved gene. Quantitative RT-PCR results showed that CcIL-17Ns were most highly expressed in the brain of healthy individuals. The expression in brain was significantly induced at 6 h post Aeromonas hydrophila infection; at 1 day post infection, expression in liver, muscle, skin, spleen, and head kidney was up-regulated. In addition, the upregulated expression of proinflammatory cytokines IL-1ß, IFN-γ, IL-6, chemokine CCL20, NF - κ B and TRAF6 in kidney tissue by ccIL-17 N recombinant protein also indicate that IL-17 N can promote inflammation through NF-κB pathway and induce the expression of chemokines and inflammatory factors.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucina-17/genética , Interleucina-17/imunologia , Perciformes/genética , Perciformes/imunologia , Imunidade Adaptativa/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Interleucina-17/química , Filogenia , Alinhamento de Sequência/veterinária
4.
Genomics ; 112(1): 55-64, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31404627

RESUMO

Coilia nasus (C. nasus) is an important anadromous fish species that resides in the Yangtze River in China. However, wild C. nasus have suffered serious damage as a result of overfishing and environmental pollution. We performed comparative liver and brain transcriptome analyses of C. nasus from the Jingjiang (JJ) and Dangtu (DT) sections of the Yangtze River. The results indicate that, during migration, most signal pathways in C. nasus livers were downregulated, indicating that the liver has a function in energy conservation. The brain assumes more of a regulatory role, and the signal transduction pathways and relevant genes were upregulated. This study provides genetic information for screening the key regulatory genes of gonad development of C. nasus, which can be applied in the artificial breeding of C. nasus, providing high-quality fish fry for proliferation and release and may also contribute to efforts towards the restoration of wild C. nasus.


Assuntos
Migração Animal , Peixes/genética , Transdução de Sinais/genética , Animais , Encéfalo/metabolismo , China , Metabolismo Energético/genética , Feminino , Peixes/metabolismo , Peixes/fisiologia , Perfilação da Expressão Gênica , Genoma , Fígado/metabolismo , Masculino
5.
Genomics ; 112(6): 4647-4656, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32798716

RESUMO

Eriocheir sinensis is an important euryhaline catadromous crustacean of the Yangtze River and an important commercial species for breeding in China. However, wild E. sinensis have suffered serious damage attributed to overfishing, climate change, etc. The Ministry of Agriculture of China issued a notice banning the commercial fishing of wild E. sinensis. E. sinensis megalopa migrates upriver into fresh water for growth and fattening, which creates optimal conditions to experimentally explore its hyposaline osmoregulation mechanism. We performed comparative transcriptome analyses of E. sinensis megalopae under hyposaline stress. The results suggest that KEGG pathways and genes related to genetic information processing, developmental regulation, immune and anti-stress responses were differentially expressed. The present study reveals the most significantly enriched pathways and functional gene groups, and explores the hyposaline osmoregulation mode of E. sinensis megalopae. This study lays a theoretical foundation for further studies on the osmoregulation and developmental mechanisms of E. sinensis.


Assuntos
Braquiúros/genética , Osmorregulação/genética , Animais , Braquiúros/crescimento & desenvolvimento , Braquiúros/imunologia , Braquiúros/metabolismo , Pressão Osmótica , RNA-Seq/estatística & dados numéricos , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
6.
Genomics ; 112(1): 71-81, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31759119

RESUMO

The Chinese mitten crab (Eriocheir sinensis), an economically valuable crustacean that is popular for its flavor, exhibits catadromous spawning migration. Overfishing and environmental pollution have inflicted serious damage on wild E. sinensis populations, and the Chinese government has banned the commercial fishing of this species in the Yangtze River. Studies have examined the sexual dimorphism in the body size and morphology of crabs, but there are few reports on the molecular regulatory mechanisms that occur during the reproduction of E. sinensis. In this study, we performed the first comparative transcriptome analyses of the cerebral ganglion and hepatopancreas of E. sinensis during reproduction. The results indicate that E. sinensis has significant sexual dimorphism in signal transduction, metabolism, substance transportation, and cellular protection. This study aims to provide information that can be used as a basis for further research on the molecular mechanisms that underlie sexual dimorphism in E. sinensis during reproduction. Furthermore, the results can be used to support the development of the E. sinensis breeding industry and the restoration of wild E. sinensis.


Assuntos
Braquiúros/genética , Gânglios/metabolismo , Hepatopâncreas/metabolismo , Caracteres Sexuais , Animais , Braquiúros/metabolismo , Braquiúros/fisiologia , Feminino , Masculino , RNA-Seq , Reprodução/genética , Transdução de Sinais
7.
J Fish Biol ; 99(1): 25-36, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33534139

RESUMO

Five group III secreted phospholipase (pla2g3s) homologous genes located on different linkage groups were identified from common carp (Cyprinus carpio), which we named Ccpla2g3a1, Ccpla2g3a2, Ccpla2g3b, Ccpla2g3c1 and Ccpla2g3c2. The five genes encode 530, 525, 461, 752 and 753 amino acids, respectively. Sequence analysis showed that the Ccpla2g3as contain seven exons and the others contain four exons. Synteny analysis of fish pla2g3s indicated that pla2g3a and pla2g3b were from the same ancestor gene, and Ccpla2g3a1, Ccpla2g3a2, Ccpla2g3c1 and Ccpla2g3c2 were from the specific genome duplication of common carp. Due to the significant variation of the pla2g3bs from common carp and zebrafish (Danio rerio), they formed a separate group in the phylogenetic tree. The tissue distributions of Ccpla2g3s coincided with their expression profiles during the embryo stages. The expression levels of Ccpla2g3as and Ccpla2g3cs were low at the embryo stages, and they were abundant in the liver and brain, respectively, whereas the expression of Ccpla2g3b was high at 0.5 h after fertilization and in the ovary. We obtained three soluble recombinant proteins of the bee venom-like PLA2 (BVLP) from Ccpla2g3 and evaluated their PLA2 enzyme properties. The optimum pHs of MBP-a1-BVLP, MBP-b-BVLP and MBP-c1-BVLP were 7.5, 7.0 and 8.0, respectively, and specific activities were 7.68 ± 0.66, 4.155 ± 0.158 and 1.93 ± 0.05 U µmol-1 , respectively. The Kd for Ca2+ of MBP-b-BVLP was the lowest (2.6 µM), whereas the values for both MBP-a1-BVLP and MBP-c1-BVLP were about 15 µM. The Km values of three proteins ranged from 31.9 to 41.91 µM.


Assuntos
Carpas , Fosfolipases A2 Secretórias , Animais , Carpas/genética , Feminino , Filogenia , Sintenia , Peixe-Zebra
8.
BMC Genomics ; 21(1): 410, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32552858

RESUMO

BACKGROUND: Coilia nasus (C. nasus) is an important anadromous fish species that resides in the Yangtze River of China, and has high ecological and economical value. However, wild resources have suffered from a serious reduction in population, attributed to the over-construction of water conservancy projects, overfishing, and environmental pollution. The Ministry of Agriculture and Rural Affairs of the People's Republic of China has issued a notice banning the commercial fishing of wild C. nasus in the Yangtze River. Wild C. nasus populations urgently need to recover. A better understanding of C. nasus migration patterns is necessary to maximize the efficiency of conservation efforts. Juvenile C. nasus experience a simultaneous effect of increasing salinity and cold stress during seaward migration, and the brain plays a comprehensive regulatory role during this process. Therefore, to explore the early seaward migration regulation mechanism of juvenile C. nasus, we performed a comparative transcriptome analysis on the brain of juvenile C. nasus under salinity and cold stress simultaneously. RESULTS: Relevant neurotransmitters, receptors, and regulatory proteins from three categories of regulatory pathway play synergistic regulatory roles during the migration process: neuronal signaling, the sensory system, and environmental adaptation. The significant differential expression of growth-related hormones, thyroid receptors, haptoglobin, and prolactin receptors was similar to the results of relevant research on salmonids and steelhead trout. CONCLUSIONS: This study revealed a regulatory network that the brain of juvenile C. nasus constructs during migration, thereby providing basic knowledge on further studies could build on. This study also revealed key regulatory genes similar to salmonids and steelhead trout, thus, this study will lay a theoretical foundation for further study on migration regulation mechanism of anadromous fish species.


Assuntos
Proteínas de Peixes/genética , Peixes/fisiologia , Perfilação da Expressão Gênica/veterinária , Migração Animal , Animais , Encéfalo/metabolismo , Resposta ao Choque Frio , Regulação da Expressão Gênica , Estresse Salino
9.
Neural Plast ; 2020: 1273198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32273888

RESUMO

Aging is a multifactorial process involving the cumulative effects of inflammation, oxidative stress, and mitochondrial dynamics, which can produce complex structural and biochemical alterations to the nervous system and lead to dysfunction of microcirculation, blood-brain barrier (BBB), and other problems in the brain. Long-term injection of D-galactose (D-gal) can induce chronic inflammation and oxidative stress, accelerating aging. The model of accelerated aging with long-term administration of D-gal have been widely used in anti-aging studies, due to the increase of chronic inflammation and decline of cognition that similarity with natural aging in animals. However, despite extensive researches in the D-gal-induced aging rats, studies on their microvasculature remain limited. Endothelial progenitor cells (EPCs), which are precursors to endothelial cells (ECs), play a significant role in the repair and regeneration process of endogenous blood vessel, and adiponectin (APN), a protein derived from adipocyte, has many effects on protective vascular endothelium and anti-inflammatory. Recently, many studies have shown that APN can promote improvements in cognitive function. Under these circumstances, we investigated the neuroprotective effect of the APN-transfected EPC (APN-EPC) treatment on rats after administration with D-gal and explored the likely underlying mechanisms. Compared to model group for D-gal administration, better cognitive function and denser microvessels were significantly found in the APN-EPC treatment group, and indicated APN-EPC treatment in aging rats could improve the cognitive dysfunction and microvessel density. The level of proinflammatory cytokines IL-1ß, IL-6, and TNF-α, activated astrocytes and apoptosis rate were significantly reduced in the APN-EPC group compared with the model group, showed that APN-EPCs alleviated the neuroinflammation in aging rats. In addition, the APN-EPC group inhibited the decrease of BBB-related proteins claudin-5, occludin, and Zo-1 in aging rats and attenuated BBB dysfunction significantly. These results of our study indicated that APN-EPC treatment in D-gal-induced aging rats have a positive effect on improving cognitive and BBB dysfunction, increasing angiogenesis, and reducing neuroinflammation and apoptosis rate. This research suggests that cell therapy via gene modification may provide a safe and effective approach for the treatment of age-related neurogenerative diseases.


Assuntos
Envelhecimento/fisiologia , Envelhecimento/psicologia , Células Endoteliais/fisiologia , Galactose/administração & dosagem , Aprendizagem em Labirinto/fisiologia , Células-Tronco/fisiologia , Envelhecimento/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Encefalite/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/transplante , Masculino , Ratos Sprague-Dawley , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos
10.
Neural Plast ; 2017: 6809745, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29201467

RESUMO

Endothelial progenitor cells (EPCs), as precursors to endothelial cells, play a significant part in the process of endogenous blood vessel repair and maintenance of endothelial integrity. Adiponectin (APN) is an adipocyte-specific adipocytokine. In this study, we aim to test whether we transplant a combined graft of EPCs transfected with the adiponectin gene into a rat model of cerebral ischemia could improve functional recovery after middle cerebral artery occlusion (MCAO). Sprague-Dawley (SD) rats were randomly divided into a MCAO control group, a MCAO EPC treatment group, and a MCAO LV-APN-EPC treatment group. A focal cerebral ischemia and reperfusion model was induced by the intraluminal suture method. After 2 h of reperfusion, EPCs were transplanted by injection through the tail vein. A rotarod test was conducted to assess behavioral function before MCAO and on days 1, 7, and 14 after MCAO. After 14 d, TTC staining, CD31 immunofluorescence, and TUNEL staining were used to evaluate infarct volume, microvessel density, and cell apoptosis. Results revealed that behavioral function, infarct area percentage, microvessel density, and cell apoptosis rates were more favorable in the LV-APN-EPC treatment group than in the EPC treatment group. These data suggested that gene-modified cell therapy may be a useful approach for the treatment of ischemic stroke.


Assuntos
Adiponectina/metabolismo , Isquemia Encefálica/metabolismo , Células Progenitoras Endoteliais/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Traumatismo por Reperfusão/metabolismo , Adiponectina/genética , Animais , Apoptose/fisiologia , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia
11.
Anal Chem ; 87(2): 1097-102, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25522095

RESUMO

Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven (15)N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide. Individual QconCATs were mixed with recombinant human clusterin at a 1:1 molar ratio and digested, and the actual ratios for each combination of peptide/flanking sequence were measured with a multiple reaction monitoring assay. Data analysis suggested that natural flanking sequences shorter than +6 residues can cause a quantitative error because the random appearance of other amino acid residues in close proximity to trypsin cleavage sites has unpredictable consequences for the digestion rates of QconCATs.


Assuntos
Cromatografia Líquida/métodos , Clusterina/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/normas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo , Sequência de Aminoácidos , Clusterina/química , Humanos , Marcação por Isótopo , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Proteínas Recombinantes/química , Padrões de Referência , Homologia de Sequência de Aminoácidos
12.
J Biol Chem ; 288(41): 29267-80, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-23970548

RESUMO

The benefits of antioxidant therapy for treating age-related macular degeneration, a devastating retinal disease, are limited. Perhaps species other than reactive oxygen intermediates should be considered as therapeutic targets. These could be lipid peroxidation products, including isolevuglandins (isoLGs), prototypical and extraordinarily reactive γ-ketoaldehydes that avidly bind to proteins, phospholipids, and DNA and modulate the properties of these biomolecules. We found isoLG adducts in aged human retina but not in the retina of mice kept under dim lighting. Hence, to test whether scavenging of isoLGs could complement or supplant antioxidant therapy, we exposed mice to bright light and found that this insult leads to retinal isoLG-adduct formation. We then pretreated mice with pyridoxamine, a B6 vitamer and efficient scavenger of γ-ketoaldehydes, and found that the levels of retinal isoLG adducts are decreased, and morphological changes in photoreceptor mitochondria are not as pronounced as in untreated animals. Our study demonstrates that preventing the damage to biomolecules by lipid peroxidation products, a novel concept in vision research, is a viable strategy to combat oxidative stress in the retina.


Assuntos
Ácidos Graxos Insaturados/antagonistas & inibidores , Luz , Piridoxamina/farmacologia , Retina/efeitos dos fármacos , Retina/efeitos da radiação , Idoso , Animais , Olho/metabolismo , Olho/ultraestrutura , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Degeneração Macular/metabolismo , Degeneração Macular/prevenção & controle , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Piridoxamina/sangue , Piridoxamina/metabolismo , Retina/metabolismo , Complexo Vitamínico B/sangue , Complexo Vitamínico B/metabolismo , Complexo Vitamínico B/farmacologia
13.
Clin Proteomics ; 11(1): 43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25593565

RESUMO

BACKGROUND: In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. METHOD: Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. RESULTS: The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. CONCLUSION: Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

14.
Anal Chem ; 85(1): 303-7, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23186391

RESUMO

It is likely that expression and/or post-translational generation of various protein isoforms can be indicative of initial pathological changes or pathology development. However, selective quantification of individual protein isoforms remains a challenge, because they simultaneously possess common and unique amino acid sequences. Quantification concatamer (QconCAT) internal standards were originally designed for a large-scale proteome quantification and are artificial proteins that are concatamers of tryptic peptides for several proteins. We developed a QconCAT for quantification of various isoforms of amyloid precursor protein (APP). APP-QconCAT includes tryptic peptides that are common for all isoforms of APP concatenated with those tryptic peptides that are unique for specific APP isoforms. Isotope-labeled APP-QconCAT was expressed, purified, characterized, and further used for quantification of total APP, APP695, and amyloid-ß (Aß) in the human frontal cortex from control and severe Alzheimer's disease donors. Potential biological implications of our quantitative measurements are discussed. It is also expected that using APP-QconCAT(s) will advance our understanding of biological mechanism by which various APP isoforms involved in the pathogenesis of Alzheimer's disease.


Assuntos
Precursor de Proteína beta-Amiloide/análise , Espectrometria de Massas em Tandem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/normas , Isótopos de Carbono/química , Córtex Cerebral/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Marcação por Isótopo , Dados de Sequência Molecular , Isótopos de Nitrogênio/química , Peptídeos/análise , Peptídeos/normas , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/normas , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Tripsina/metabolismo
15.
Anal Chem ; 85(22): 11014-9, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24131330

RESUMO

Carbapenems are broad spectrum antibiotics considered as a "last resort" medicine to treat bacterial infections. Carbapenem-hydrolyzing ß-lactamases (also called carbapenemases), however, can confer bacterial resistance and represent a serious health threat. Here, we report a novel approach using (18)O labeling and selected reaction monitoring to detect carbapenemase activity from pathogenic microorganisms in a rapid and quantitative manner. Four model bacterial strains bearing various classes of ß-lactamases were tested for their capability to hydrolyze Meropenem, an FDA-approved carbapenem drug. We were able to predict the Meropenem resistance of these bacteria on the basis of their carbapenemase activity, suggesting the great potential of our method in clinical diagnostics.


Assuntos
Bactérias/enzimologia , Infecções Bacterianas/diagnóstico , Proteínas de Bactérias/metabolismo , Radioisótopos de Oxigênio , Espectrometria de Massas em Tandem/métodos , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Bactérias/isolamento & purificação , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/classificação , Carbapenêmicos/química , Carbapenêmicos/farmacologia , Cromatografia Líquida , Resistencia a Medicamentos Antineoplásicos , Meropeném , Tienamicinas/química , Tienamicinas/farmacologia , beta-Lactamases/classificação
16.
Anal Chem ; 85(3): 1773-7, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23286534

RESUMO

Cluster of differentiation 4 (CD4) is an important glycoprotein containing four extracellular domains, a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs a critical role in multiple cellular functions such as signal amplification and activation of T cells. It is well-known as a clinical cell surface protein marker for study of HIV progression and for defining the T helper cell population in immunological applications. Moreover, CD4 protein has been used as a biological calibrator for quantification of other surface and intracellular proteins. However, flow cytometry, the conventional method of quantification of the CD4 density on the T cell surface depends on antibodies and has suffered from variables such as antibody clones, the fluorophore and conjugation chemistries, the fixation conditions, and the flow cytometric quantification methods used. In this study, we report the development of a highly reproducible nano liquid chromatography-multiple reaction monitoring mass spectrometry-based quantitative method to quantify the CD4 receptor density in units of copy number per cell on human CD4+ T cells. The method utilizes stable isotope-labeled full-length standard CD4 as an internal standard to measure endogenous CD4 directly, without the use of antibodies. The development of the mass spectrometry-based approach of CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method. It also provides new support for quantitative understanding and advanced characterization of CD4 on CD4+ T cells.


Assuntos
Antígenos CD4/análise , Linfócitos T CD4-Positivos/química , Citometria de Fluxo/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , Humanos
17.
Anal Chem ; 85(21): 10362-8, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24074274

RESUMO

Transferrin, an iron transport protein, is a clinically important biomarker in diseases such as iron-deficiency anemia. Current diagnostic methods for transferrin levels lack quantitative accuracy, suggesting the need for alternative approaches like LC-MS with isotope-labeled peptides as internal standards. Besides solid-phase synthesis, isotope-labeled peptides are also generated by a method called QconCAT where peptides are expressed from DNA in the presence of heavy isotope media. After evaluation of the expressed QconCAT, this study compares transferrin levels obtained by synthetic peptides versus QconCAT peptides as internal standards. Transferrin levels obtained by both internal standards give overlapping, or nearly overlapping, uncertainty values and are near ≈200 mg/dL of transferrin in human serum. Close agreement between the two methods suggests that the quantitative values are reasonable. Using QconCAT and synthetic peptides in parallel gives a refined focus on method development, and the resulting methods should be applicable to other clinically relevant proteins.


Assuntos
Transferrina/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Humanos , Dados de Sequência Molecular , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
18.
Front Oncol ; 13: 1161496, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37035181

RESUMO

Background: Reoperation may be beneficial for patients with recurrent gliomas. Minimizing the residual tumor volume (RTV) while ensuring the functionality of relevant structures is the goal of the reoperation of recurrent gliomas. Intraoperative ultrasound (IoUS) may be helpful for intraoperative tumor localization, intraoperative real-time imaging to guide surgical resection, and postoperative evaluation of the RTV in the reoperation for recurrent gliomas. Objective: To assess the effect of real-time ioUS on minimizing RTV in recurrent glioma surgery compared to Non-ioUS. Methods: We retrospectively analyzed the data from 92 patients who had recurrent glioma surgical resection: 45 were resected with ioUS guidance and 47 were resected without ioUS guidance. RTV, Karnofsky Performance Status (KPS) at 6 months after the operation, the number of recurrent patients, and the time to recurrence were evaluated. Results: The average RTV in the ioUS group was significantly less than the Non-ioUS group (0.27 cm3 vs. 1.33 cm3, p = 0.0004). Patients in the ioUS group tended to have higher KPS scores at 6 months of follow-up after the operation than those in the Non-ioUS group (70.00 vs. 60.00, p = 0.0185). More patients in the Non-ioUS group experienced a recurrence than in the ioUS group (43 (91.49%) vs. 32 (71.11%), p = 0.0118). The ioUS group had a longer mean time to recurrence than the Non-ioUS group (7.9 vs. 6.3 months, p = 0.0013). Conclusion: The use of ioUS-based real-time for resection of recurrent gliomas has been beneficial in terms of both RTV and postoperative outcomes, compared to the Non-ioUS group.

19.
Sci Data ; 10(1): 843, 2023 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-38036563

RESUMO

To gain a deeper understanding of the genetic factors influencing the growth and development of Eriocheir sinensis, a well-known species of hairy crab found in Yangcheng Lake, this study focused on the de novo genome and full-length transcriptome information of the selected subjects. Specifically, Yangcheng Lake hairy crabs were chosen as the experimental samples. Initially, a genome analysis was performed, resulting in the identification of gene fragments with a combined length of 1266,092,319 bp. Subsequently, a transcriptome analysis was conducted on a mixture of tissues from four different sites, namely muscle, brain, eye, and heart, to further investigate the genetic characteristics at the transcriptome level. The Pacific Biosciences (Pacio) single-molecule real-time sequencing system generated a total of 36.93 G sub-fragments and 175,90041 effective inserts. This research contributes to the indirect comprehension of genetic variations underlying individual traits. Furthermore, a comparison of the obtained data with relevant literature emphasizes the advantages of this study and establishes a basis for further investigations on the Chinese mitten crab.


Assuntos
Braquiúros , Perfilação da Expressão Gênica , Transcriptoma , Humanos , Genoma , Genômica , Braquiúros/genética
20.
Metabolites ; 13(1)2023 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-36677027

RESUMO

Aplodinotus grunniens, known as freshwater drum, is a kind of eurythermal freshwater fish that is widely distributed in North America. In 2019, our research group reached a milestone on its artificial breeding and cultivation and have investigated its physiological adaption to the environment, providing a breakthrough and prospects for aquaculture. However, its adaptability and metabolic homeostasis to hypothermia is not fully understood. In this experiment, cold stress was conducted at 18 °C (LT18) and 10 °C (LT10) with 25 °C as control (Con) for 8 days to explore the effects of short-term hypothermia on the physiology and metabolism of freshwater drum. From the results, the level of free essential amino acids in LT18 and LT10 decreased significantly after 2 days cold stress compared with Con. Furthermore, plasma total triglyceride (TG) content and lipase (LPS) activity were decreased at LT10 for 2d. With RNA-seq in the liver, metabolic-related signaling, especially amino acid synthesis and lipid metabolism, was inhibited by hypothermia. Specifically, the PPAR signaling pathway is correlated with the inhibition of lipid and amino acid metabolism induced by hypothermia. These data confirmed that PPAR signaling maintains lipid and amino acid metabolic homeostasis during cold stress. These results give a theoretical foundation for hypothermia resistance in the area of metabolic homeostasis for freshwater drum.

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