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Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1'carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SIGNIFICANCE: This article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.
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Complejo Antígeno-Anticuerpo , Proteínas , Humanos , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Reactivos de Enlaces Cruzados/químicaRESUMEN
Verifying the geographical origin of soybeans (Glycine max [Linn.] Merr.) is a major challenge as there is little available information regarding non-parametric statistical origin approaches for Chinese domestic and imported soybeans. Commercially procured soybean samples from China (n = 33) and soybeans imported from Brazil (n = 90), the United States of America (n = 6), and Argentina (n = 27) were collected to characterize different producing origins using stable isotopes (δ2H, δ18O, δ15N, δ13C, and δ34S), non-metallic element content (% N, % C, and % S), and 23 mineral elements. Chemometric techniques such as principal component analysis (PCA), linear discriminant analysis (LDA), and BP-artificial neural network (BP-ANN) were applied to classify each origin profile. The feasibility of stable isotopes and elemental analysis combined with chemometrics as a discrimination tool to determine the geographical origin of soybeans was evaluated, and origin traceability models were developed. A PCA model indicated that origin discriminant separation was possible between the four soybean origins. Soybean mineral element content was found to be more indicative of origin than stable isotopes or non-metallic element contents. A comparison of two chemometric discriminant models, LDA and BP-ANN, showed both achieved an overall accuracy of 100% for testing and training sets when using a combined isotope and elemental approach. Our findings elucidate the importance of a combined approach in developing a reliable origin labeling method for domestic and imported soybeans in China.
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Not-from-concentrate (NFC) juice has better nutrition, flavor and higher price than reconstituted juice. Accordingly, NFC juice is prone to adulteration and is an ongoing industry problem that has not yet been resolved. Undeclared addition of water and sugar are the main forms of NFC juice adulteration. This paper investigates the carbon and oxygen stable isotope ratios (δ13C and δ18O values) of the bulk juice and different juice components from 21 fruit and vegetable juices, and qualitatively and quantitatively analyzes the addition of water and sugar in NFC juices. The results show that the use of fruit pulp can help to qualitatively and quantitatively indicate the presence of C4 plant sugars in NFC juice, and can reliably detect added C4 plant sugars above 7 %. Sugar-specific isotope analysis (SSIA) technology was used to determine the δ13C values of different sugars (sucrose, glucose and fructose) and carbon content to qualitatively infer C3 plant sugar addition. Pulp extracted from juice had a good linear relationship with the juice water δ18O values (R2 >0.90). The addition of water to NFC juice can also be determined by comparing δ18O values of extraneous water, pulp and filtered juice. Stable isotope technology confirmed NFC juice adulteration of in-market samples using the pulp as an internal reference and was found to be a useful tool to detect adulteration of in-market NFC juice.
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Jugos de Frutas y Vegetales , Frutas , Bebidas/análisis , Isótopos de Carbono/análisis , Frutas/química , Isótopos de Oxígeno/análisis , AzúcaresRESUMEN
Origin verification of 240 French wines from four regions of France was undertaken using isotope and elemental analyses. Our aim was to identify and differentiate the geographical origin of these red wines, and more importantly, to build a classification tool that can be used to verify geographic origin of French red wines using machine learning models. Multivariate analyses of the isotopic and elemental data revealed that it is possible to determine the geographical origin of French wines with a high level of confidence for most regions analyzed in this study. The wine verification accuracy of four French wine producing regions of Bordeaux, Burgundy, Languedoc-Roussillon and Rhone using an Artificial Neural Network (ANN) method was 98.2%. The results also show that ANN is more suitable than Discriminant Analysis for this verification purpose. The most important variables for French wine regional traceability were Mg, Mn, Na, Sr, Ti and Rb.
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Análisis de los Alimentos/métodos , Análisis de los Alimentos/estadística & datos numéricos , Metales/análisis , Vino/análisis , Isótopos de Carbono/análisis , Análisis Discriminante , Contaminación de Alimentos/análisis , Francia , Espectrometría de Masas/métodos , Análisis Multivariante , Redes Neurales de la Computación , Oligoelementos/análisisRESUMEN
PURPOSE: Cardiac resynchronization therapy (CRT) is well acknowledged as an effective treatment for dyssynchronous heart failure. However, the molecular mechanism is unclear to date. Mitochondrial dysfunction and impaired energetic metabolism are two important mechanisms that lead to heart failure. Therefore, we aim to screen the changes of mitochondria-associated proteins and signaling pathways involved in heart failure and CRT treatment. METHODS: A total of 24 beagle dogs were randomly assigned into control (CON), heart failure (HF), or CRT group. Myocardial mitochondria from the free wall of left ventricle was extracted for isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with two-dimensional liquid chromatography tandem mass spectrometry analysis (2DLC-MS/MS). RESULTS: A total of 2190 proteins were identified, among which 234 proteins were differentially expressed in HF compared with CON group, 151 proteins were differentially expressed in CRT compared with HF group. A total of 192 of the 234 differentially expressed proteins in HF group were changed oppositely by CRT treatment, and 128 of the 151 CRT-induced differentially expressed proteins showed opposite trend of expression to HF/CON. Gene Ontology analysis of the 128 proteins revealed that 16 were localized in mitochondria, 17 were associated with calcium signaling, and 7 could be secreted extracellularly for cell-to-cell signaling. Calpain-1 (CAPN1), which is localized to mitochondria and related to calcium signaling, was upregulated in HF and downregulated after CRT treatment. CRT treatment also improved mitochondrial morphology and function and reduced collagen areas of both interstitial and perivascular fibrosis. CONCLUSIONS: CRT treatment significantly improved cardiac function, reduced myocardial fibrosis, and enhanced mitochondrial function in the failing heart through CAPN1 downregulation.
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Terapia de Resincronización Cardíaca , Insuficiencia Cardíaca , Animales , Perros , Insuficiencia Cardíaca/terapia , Mitocondrias , Proteómica , Espectrometría de Masas en Tándem , Resultado del TratamientoRESUMEN
Quality by design (QbD) is an efficient but challenging approach for the development of biosimilar due to the complex relationship among process, quality, and efficacy. Here, the analytical similarity of adalimumab biosimilar HLX03 to Humira® was successfully established following a QbD quality study. Quality target product profile (QTPP) of HLX03 was first generated according to the public available information and initial characterization of 3 batches of Humira®. The critical quality attributes (CQAs) were then identified through risk assessment according to impact of each quality attribute on efficacy and safety. The anticipated range for each CQA was derived from similarity acceptance range and/or the corresponding regulatory guidelines. Finally, a panel of advanced and orthogonal physicochemical and functional tests and comparison of 6 batches of HLX03 and 10 batches of the reference standard demonstrated high similarity of HLX03 to Humira®, except for slightly lower percentage of high mannosylated glycans (%HM) in HLX03 which had no effect on FcγRIII binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity in human peripheral blood mononuclear cell (PBMC). All above demonstrated the feasibility and efficiency of QbD-based similarity assessment of a biosimilar monoclonal antibody (mAb).
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Adalimumab/análisis , Antiinflamatorios/análisis , Biosimilares Farmacéuticos/análisis , Investigación Cualitativa , Adalimumab/química , Animales , Antiinflamatorios/química , Biosimilares Farmacéuticos/química , Células CHO , Cricetinae , Cricetulus , Humanos , Células Jurkat , Células U937RESUMEN
BACKGROUND: A biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes. OBJECTIVES: The aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin® (EU-Herceptin®) and China-sourced Herceptin® (CN-Herceptin®) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation. METHODS: A panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021. RESULTS: The extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group. CONCLUSION: Besides having an overall high analytical similarity to both EU-Herceptin® and CN-Herceptin®, HLX02 is more similar to Herceptin® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.
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Anticuerpos Monoclonales Humanizados/química , Biosimilares Farmacéuticos/química , Trastuzumab/química , Anticuerpos Monoclonales Humanizados/análisis , Biosimilares Farmacéuticos/análisis , Cromatografía de Afinidad , Humanos , Receptores de IgG/inmunología , Trastuzumab/análisisRESUMEN
Multi-isotope and multi-elemental analyses were performed on 600 red wine samples imported into China from 7 different countries and compared with Chinese wine. Carbon and oxygen isotopes and 16 elements were used to determine origin traceability. Our goal was to build a classification tool using data modeling that can verify the geographic origin of wines imported into China. Multivariate analyses of the isotopic and elemental data revealed that it is possible to determine the geographical origin for most imported wines with a high level of confidence (>90%). The results show that Artificial Neural Network method had a high discrimination accuracy and is more suitable than Discrimination Analysis and Random Forest methods when it comes to classifying wine origin on a global scale. In conclusion, stable isotope and trace element analyses followed by multivariate processing of the data is a fast and efficient technique suitable for global wine traceability.
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Análisis de los Alimentos/métodos , Vino/análisis , Isótopos de Carbono/análisis , China , Análisis Discriminante , Espectrometría de Masas/métodos , Modelos Estadísticos , Análisis Multivariante , Isótopos de Oxígeno/análisis , Oligoelementos/análisisRESUMEN
Development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EU-rituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China's Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN- and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, efï¬cacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.
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Secuencia de Aminoácidos , Biosimilares Farmacéuticos/química , Rituximab/química , Rituximab/genética , Análisis de Secuencia de Proteína , Humanos , Rituximab/uso terapéuticoRESUMEN
Absolute quantification of clinical biomarkers by mass spectrometry (MS) has been challenged due to low sample-throughput of current multiple reaction monitoring (MRM) methods. For this problem to be overcome, in this work, a novel high-sample-throughput multiple reaction monitoring mass spectrometric (HST-MRM-MS) quantification approach is developed to achieve simultaneous quantification of 24 samples. Briefly, triplex dimethyl reagents (L, M, and H) and eight-plex iTRAQ reagents were used to label the N- and C-termini of the Lys C-digested peptides, respectively. The triplex dimethyl labeling produces three coelute peaks in MRM traces, and the iTRAQ labeling produces eight peaks in MS2, resulting in 24 (3×8) channels in a single experiment. HST-MRM-MS has shown good accuracy ( R2 > 0.98 for absolute quantification), reproducibility (RSD < 15%), and linearity (2-3 orders of magnitude). Moreover, the novel method has been successfully applied in quantifying serum biomarkers in hepatocellular carcinoma (HCC)-related serum samples. In conclusion, HST-MRM-MS is an accurate, high-sample-throughput, and broadly applicable MS-based absolute quantification method.
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Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Espectrometría de Masas/métodos , Humanos , Indicadores y Reactivos , Marcaje Isotópico , Reproducibilidad de los ResultadosRESUMEN
Activation of hepatic stellate cells reportedly contributes to progression of hepatocellular carcinoma (HCC). Herein, we use quantitative proteomics and ingenuity pathway analysis to show that transglutaminase 2 (TGM2) is upregulated in the course of activated hepatic stellate cells promoting epithelial-mesenchymal transition (EMT) in HCC-derived cells both in vivo and in vitro. Mechanistically, activated hepatic stellate cells promote TGM2 upregulation in HCC cells through inflammatory signalling; and TGM2-induced depletion of von Hippel-Lindau (VHL) protein, a key molecule in the degradation of hypoxia inducible factor-1a (HIF-1a) under normoxia, then causes HIF-1a to accumulate, thereby producing a pseudohypoxic state that promotes EMT in HCC cells. These findings suggest that the promotion of EMT in HCC cells by activated hepatic stellate cells is mediated by pseudohypoxia induced via TGM2/VHL/HIF-1a pathway.
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Due to the critical role glycation plays in many serious pathological conditions, such as diabetes, it is of great significance to discover protein glycation at an early stage for precaution and prediction of the disease. Here, a method of reductive amination combining dimethylation (RAD) was developed for the quantification of early-stage glycated proteins. The quantitative analysis was first carried out by reducing the samples using NaBH3CN or NaBD3CN, resulting in a 1 Da mass shift and the stabilization of early-stage protein glycation. The two samples were then digested and isotopically dimethylated to achieve the mass shift of 4 m + 3 n ( m represents the number of N-termini and Lys residues, and n represents the number of glycated sites) between light- and heavy-labeled glycated peptides for quantification. Consequently, the false positive result can be removed according to the different mass shifts of glycated peptides and non-glycated peptides. In quantification of glycated myoglobin, RAD showed good linearity ( R2 > 0.99) and reproducibility (CVs ≤ 1.6%) in 2 orders of magnitude (1:10-10:1). RAD was then applied to quantify the endogenous glycated proteins in the serum of diabetic patients, revealing significant differences in the glycation level between the patients with complicated retinal detachment and those without. In conclusion, RAD is an effective method for quantifying endogenous glycated proteins.
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Glicopéptidos/análisis , Glicoproteínas/análisis , Espectrometría de Masas en Tándem/métodos , Aminación , Diabetes Mellitus/sangre , Glicopéptidos/sangre , Glicoproteínas/sangre , Glicosilación , Humanos , Metilación , Oxidación-ReducciónRESUMEN
Benefiting from high sensitivity and great ability to measure multiple samples simultaneously, isobaric tandem Mass spectrometry (MS2) quantification has been widely applied for protein biomarker screening. Here, a newly developed isobaric MS2 quantification method named triplex quantification by isobaric termini labeling (Triplex-QITL) was established. This method enables the accurate comparison of various fragment ions (reporter ions, amino acid fragments and N-/C-terminal fragments) based quantification to be operated in a single run. To our knowledge, this is the first time that this kind of comparison is achieved. In Triplex-QITL, proteins were first digested with Lys-C to produce peptides with lysine (K) at the C-termini, then dimethylation reagents and mTRAQ reagents were used to label the N-termini and C-termini of the peptides respectively. N- and C-terminal fragment ion pairs, reporter ions from mTRAQ (113,117,121) and a1 ion pairs were simultaneously generated in MS2 spectra. In simple sample experiment, not much difference in using various fragment ions for quantification was observed. When analyzing SW480 cell lysate, comparing with a1 ions, about two times of reproducible quantification results were achieved by reporter ions and N- and C-terminal ions. Meanwhile the measured quantification results were much closer to the expected results even in large ratios (1:10:10) using N- and C-terminal ions. Finally, Triplex-QITL was successfully applied to profile metastatic differences of three hepatocellular carcinoma (HCC) cell lines. In all, Triplex-QITL shows a promising future in quantitative proteomics.
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Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Humanos , Marcaje Isotópico/métodos , Neoplasias/química , Neoplasias/patología , Péptidos/análisis , Proteínas/análisis , Proteoma/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The von Hippel-Lindau (VHL) is deficient in â¼70% of clear-cell renal cell carcinomas (ccRCC), which contributes to the carcinogenesis and drug resistance of ccRCC. Here we show that VHL-deficient ccRCC cells present enhanced cytotoxicity of anthracyclines in a hypoxia-inducible factor-independent manner. By subtractive proteomic analysis coupling with RNAi or overexpression verification, aldehyde dehydrogenase 2 (ALDH2) is found to be transcriptionally regulated by VHL and contributes to enhanced anthracyclines cytotoxicity in ccRCC cells. Furthermore, VHL regulates ALDH2 expression by directly binding the promoter of -130 bp to -160 bp to activate the transcription of hepatocyte nuclear factor 4 alpha (HNF-4α). In addition, a positive correlation is found among the protein expressions of VHL, HNF-4α and ALDH2 in ccRCC samples. These findings will deepen our understanding of VHL function and shed light on precise treatment for ccRCC patients.
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Aldehído Deshidrogenasa Mitocondrial/genética , Antraciclinas/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Regulación hacia Abajo/genética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Animales , Antraciclinas/farmacología , Antraciclinas/toxicidad , Carcinoma de Células Renales/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor Nuclear 4 del Hepatocito/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/patología , Masculino , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Proteómica , Transcripción Genética/efectos de los fármacosRESUMEN
In this study, we measured the concentrations of trace metals (Cr, Cu, Zn, As, Cd, Pb and Hg) in typical cultured animals (crabs, clams, and shrimps) and sediments from aquaculture ponds nearby mangrove wetlands in Zhangjiang estuary, China. The contents of Cr, Cu, Cd, and Pb in mangrove sediments were significantly higher than those in pond sediments, while an inverse distribution was observed for Zn, As, and Hg. Significantly higher concentrations of trace metals were found in clams from the mangrove mudflats compared to those from the aquaculture ponds. The sources of trace metals in the clams were primarily from organic fertilizer, whereas those in the shrimp were from contaminated sediment. The results of geo-accumulation index and the ecological risk assessment indicated that the aquaculture ponds near the mangrove wetlands in this subtropical estuary posed a special risk of endogenous and exogenous trace metal pollution to nearby systems.
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Acuicultura , Sedimentos Geológicos/análisis , Metales Pesados/análisis , Estanques , Contaminantes Químicos del Agua/análisis , Humedales , Animales , Bivalvos , Braquiuros , China , Decápodos , Monitoreo del Ambiente , RhizophoraceaeRESUMEN
This study aimed to evaluate the T2 relaxation time of the brain in severely scalded rats using a magnetic resonance (MR) T2 mapping sequence, and to investigate the correlation between T2 relaxation time and plasma glucose level. Twenty-eight Wistar rats were randomly divided into the scalded group (n=21) and control group (n=7). Magnetic resonance scans were performed with T1WI, T2WI, and T2-mapping sequences in the scalded group; the scans were performed 1 day prior to scalding and 1, 3, 5, and 7 days post-scalding; in addition, identical MR scans were performed in the control group at the same time points. T2-maps were generated and T2 relaxation times were acquired from the following brain regions: the hippocampus, thalamus, caudate-putamen, and cerebrum. Pathological changes of the hippocampus were observed. The plasma glucose level of each rat was measured before each MR scan, and a correlation analysis was performed between T2 relaxation time and plasma glucose level. We found that conventional T1WI and T2WI did not reveal any abnormal signals or morphological changes in the hippocampus, thalamus, caudate-putamen, or cerebrum post-scalding. Both the T2 relaxation times of the selected brain regions and plasma glucose levels increased 1, 3, and 5 days post-scalding, and returned to normal levels 7 days post-scalding. The most marked increase of T2 relaxation time was found in the hippocampus; similar changes were also revealed in the thalamus, caudate-putamen, and cerebrum. No correlation was found between T2 relaxation time and plasma glucose level in scalded rats. Pathological observation of the hippocampus showed edema 1, 3, and 5 days post-scalding, with recovery to normal findings at 7 days post-scalding. Thus, we concluded that T2 mapping is a sensitive method for detecting and monitoring scald injury in the rat brain. As the hippocampus is the main region for modulating a stress reaction, it showed significantly increased water content along with an increased plasma glucose level post-scalding.
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Glucemia/metabolismo , Encéfalo/diagnóstico por imagen , Quemaduras/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Animales , Encéfalo/patología , Quemaduras/sangre , Núcleo Caudado/diagnóstico por imagen , Núcleo Caudado/patología , Cerebro/diagnóstico por imagen , Cerebro/patología , Ayuno/sangre , Hipocampo/diagnóstico por imagen , Hipocampo/patología , Masculino , Putamen/diagnóstico por imagen , Putamen/patología , Distribución Aleatoria , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tálamo/diagnóstico por imagen , Tálamo/patología , Factores de TiempoRESUMEN
To elucidate the toxic mechanism of snake venom at the protein level, proteomics technology was applied to investigate the effect of venom on circulation in the mammalian body. Temporal proteomic analysis was performed to profile the dynamic changes in the sera of Sprague-Dawley rats administered with Chinese cobra venom or saline. Using 8-plex iTRAQ analysis, 392 and 636 serum proteins were identified to be linearly upregulated or downregulated over time in the low-dose group and high-dose group, respectively. These proteins were mainly associated with the acute phase response pathway, complement system, and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/RXR activation pathways. Compared with the low-dose group, the immune response and integrin pathways were inhibited in the high-dose group, although no obvious effect was observed. With consistently higher or lower expression in the high-dose group compared to the low-dose group throughout the whole process of venom poisoning, two proteins, Kininogen-1 (KNG1) and orosomucoid 1 (ORM1), which are involved in metabolism and immune response, occupied a core position in the pathway network and are considered venom dose-dependent biomarker candidates.
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This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an Escherichia coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55, and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.
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This study reported and analyzed the complete sequences of two oqxAB-bearing IncHI2 plasmids harbored by a clinical Salmonella enterica serovar Typhimurium strain and an S Indiana strain of animal origin, respectively. In particular, pA3T recovered from S Indiana comprised the resistance determinants oqxAB, aac(6')Ib-cr, fosA3, and blaCTX-M-14 Further genetic screening of 63 oqxAB-positive Salmonella isolates revealed that the majority carried IncHI2 plasmids, confirming that such plasmids play a pivotal role in dissemination of oqxAB in Salmonella spp.
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Plásmidos/genética , Salmonella/genética , Animales , Pollos/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Transferencia de Gen Horizontal , Humanos , Quinoxalinas/farmacología , Salmonella/efectos de los fármacos , Salmonella/aislamiento & purificación , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/aislamiento & purificaciónRESUMEN
Radical S-adenosyl-l-methionine (SAM) enzymes utilize a [4Fe-4S] cluster to bind SAM and reductively cleave its carbon-sulfur bond to produce a highly reactive 5'-deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical-based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical-based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside-containing compounds by using radical SAM-dependent reactions.