RESUMEN
PURPOSE: Ulcerative colitis (UC) is a serious health problem with increasing morbidity and prevalence worldwide. The pathogenesis of UC is complex, currently believed to be influenced by genetic factors, dysregulation of the host immune system, imbalance in the intestinal microbiota, and environmental factors. Currently, UC is typically managed using aminosalicylates, immunosuppressants, and biologics as adjunctive therapies, with the risk of relapse and development of drug resistance upon discontinuation. Therefore, further research into the pathogenesis of UC and exploration of potential treatment strategies are necessary to improve the quality of life for affected patients. According to previous studies, Lactobacillus paracasei Jlus66 (Jlus66) reduced inflammation and may help prevent or treat UC. METHODS: We used dextran sulfate sodium (DSS) to induce a mouse model of UC to assess the effect of Jlus66 on the progression of colitis. During the experiment, we monitored mouse body weight, food and water consumption, as well as rectal bleeding. Hematoxylin-eosin staining was performed to assess intestinal pathological damage. Protein imprinting and immunohistochemical methods were used to evaluate the protein levels of nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and tight junction (TJ) proteins in intestinal tissues. Fecal microbiota was analyzed based on partial 16S rRNA gene sequencing. RESULTS: Jlus66 supplementation reduced the degree of colon tissue damage, such as colon shortening, fecal occult blood, colon epithelial damage, and weight loss. Supplementation with Jlus66 reduced DSS-induced upregulation of cytokine levels such as TNF-α, IL-1ß, and IL-6 (p < 0.05). The NF-κB pathway and MAPK pathway were inhibited, and the expression of TJ proteins (ZO-1, Occludin, and Claudin-3) was upregulated. 16S rRNA sequencing of mouse cecal contents showed that Jlus66 effectively regulated the structure of the intestinal biota. CONCLUSION: In conclusion, these data indicate that Jlus66 can alter the intestinal biota and slow the progression of UC, providing new insights into potential therapeutic strategies for UC.
RESUMEN
N-glycolylneuraminic acid (Neu5Gc), a sialic acid predominantly found in the non-neurohumoral fluids of hind-mouthed animals, is incapable of synthesizing Neu5Gc due to a deletion in the CMAH exon of the gene encoding human CMP-Neu5Gc hydroxylase. But consumption of animal-derived foods that contain Neu5Gc, such as red meat, can instigate an immune response in humans, as Neu5Gc is recognized as a foreign substance by the human immune system. This recognition leads to the production of anti-Neu5Gc antibodies, subsequently resulting in chronic inflammation. When Neu5Gc is consumed excessively or frequently, it may contribute to the development of heart disease and cancer. This makes Neu5Gc, an endogenous pathogenic factor derived from red meat, a new hot topic in red meat safety research. In this study, aptamers obtained by the magnetic bead SELEX technique were subjected to homology and secondary structure prediction analysis as well as affinity determination. The result indicated that the aptamer 2B.N2A9 exhibited a robust binding affinity, with an affinity constant (Ka) of 1.87 × 108 L/mol. This aptamer demonstrated optimal binding specificity within a pH range of 5.4 to 7.4. Molecular docking analysis further revealed that aptamer 2B.N2A9 formed stable binding interactions with the target Neu5Gc at specific sites, namely G-14, C-15, G-13, G-58, G-60, and C-59. An Enzyme-Linked Oligonucleotide Sorbent Assay (ELOSA) methodology was established to detect the endogenous pathogenic factor Neu5Gc present in red meat. This method demonstrated a limit of detection (LOD) of 0.71 ng/mL, along with an average recovery rate of 92.23%. The aptamer obtained in this study exhibited favorable binding properties to Neu5Gc. The assay was relatively convenient and demonstrated good sensitivity. Further investigation into the distribution of Neu5Gc in various red meats is of public health significance and scientific potential. A practical detection method should be provided to guide red meat diets and ensure the nutrition and safety of meat products.
Asunto(s)
Ácido N-Acetilneuramínico , Carne Roja , Animales , Humanos , Simulación del Acoplamiento Molecular , Inflamación , OligonucleótidosRESUMEN
Tyrosol is one of the main polyphenol compounds in white wine and extra virgin olive oil (EVOO), which plays an antioxidant and anti-inflammatory role in vitro. In the present study, we investigated the possible anti-inflammatory mechanism of tyrosol in Escherichia coli (ETEC)-induced diarrhea in mice. ICR mice were randomly divided into control group, ETEC group, and ETEC + Tyrosol group with 10 mice in each group. In addition to the control group, a bacterial diarrhea model was induced in mice by continuous administration of 0.2 ml × 109 CFU/ml ETEC. After 7 days, the ETEC + Tyrosol group was given tyrosol (20 mg/kg) once a day by gavage, during which the body weight of mice and the degree of diarrhea were measured daily. On the 15th day, all animals in this experiment were sacrificed, colon tissue was collected, and colon length was recorded. Our results indicate that tyrosol significantly attenuated the extent of ETEC-induced diarrhea, including inhibition of pro-inflammatory cytokine, repair of the intestinal epithelial mechanical barrier, and significant inhibition of NF-κB activation. This finding is helpful for the development and further application of tyrosol in the treatment of diarrhea.
Asunto(s)
Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Animales , Ratones , FN-kappa B , Infecciones por Escherichia coli/microbiología , Ratones Endogámicos ICR , Diarrea/microbiologíaRESUMEN
Due to their susceptibility to several human viruses, the mink has been proposed as potential animal models for the study of human viral infections. However, there are no specific monoclonal antibody (mAbs) currently available for the detection of mink-specific interferon-gamma (miIFN-γ). The BALB/c mice were immunized intraperitoneally with purified recombinant miIFN-γ protein. The splenocytes were obtained and fused with murine myeloma cells. Five of 24 hybridoma clones were obtained to produce mAbs steadily with the strongest affinity to recombinant miIFN-γ protein. The isotype of the 31A, 31B and 31G were lgG 2b. The isotype of 44 and 46 were lgG 2a and 1. All five mAbs were κ light chains. Western blotting and indirect ELISA method showed that 5 mAbs were positive to miIFN-γ. Immunofluorescence showed that 2 mAbs (44 and 46) had a positive reaction to miIFN-γ. The hybridoma clone 46 had the highest sensitivity for the detection of miIFN-γ. Most importantly, our primary sandwich ELISA system (mAbs 46 and polyclonal antiserum) detected endogenous IFN-γ in mink lymphocytes infected with canine distemper virus (CDV). We have thus developed a novel mAbs could recognize miIFN-γ, and have demonstrated the first ELISA-based measurement of IFN-γ in lymphocyte of the mink.
Asunto(s)
Anticuerpos Monoclonales , Visón , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Hibridomas/metabolismo , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Visón/metabolismoRESUMEN
Melanocortin 1 receptor (MC1R) is thought to be a marker of poor prognosis and a potential target for the treatment of melanoma. Studies have found that MC1R promotes several tumor behaviors, including cell proliferation and differentiation, pigment formation, and genome damage repair. Some single-nucleotide polymorphisms (SNPs) of MC1R are involved in the occurrence and development of melanoma. A few studies have reported a relationship between MC1R and colorectal cancer (CRC). In this research, our objective was to examine MC1R expression and MC1R SNPs and investigate their correlation with the clinicopathological features of human CRC tissues. We evaluated MC1R mRNA expression by performing bioinformatic analyses on human CRC expression datasets. We used Western blotting and RT-qPCR to compare MC1R expression in CRC tissues with that in normal tissues, and MC1R SNPs in CRC tissues were detected by PCR-direct sequencing (DS). The expression of MC1R was significantly decreased in CRC tissues compared with normal tissue, and its expression was negatively associated with P53 expression, MLH1 expression, and PMS2 expression, and high MC1R expression was significantly associated with microsatellite instability (MSI). MC1R SNPs were also associated with the clinicopathological characteristics of CRC; for example, the rs2228479 locus genotype was correlated with Ki67 status, and the rs885479 locus genotype was correlated with age and T stage. In conclusion, MC1R plays a crucial role in the progression of CRC and may be a marker of poor prognosis in CRC.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Regulación Neoplásica de la Expresión Génica , Inestabilidad de Microsatélites , Receptor de Melanocortina Tipo 1/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Biología Computacional/métodos , Perfilación de la Expresión Génica , Humanos , Proteínas de Punto de Control Inmunitario/genética , Proteínas de Punto de Control Inmunitario/metabolismo , Estimación de Kaplan-Meier , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Estadificación de Neoplasias , Polimorfismo de Nucleótido Simple , Pronóstico , Mapeo de Interacción de Proteínas , Receptor de Melanocortina Tipo 1/metabolismo , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Flujo de TrabajoRESUMEN
Given the great harm of pesticide residues to the environment and public health, exploring ultrasensitive and low-cost methods for their quantitative analysis becomes intensely necessary. Herein, we proposed a double-functionalized gold nanoparticle (AuNP) probe as a signal amplification immunoassay for the detection of acetochlor (ATC), metolachlor, and propisochlor. The AuNP was modified with IgG and fluorophore-labeled duplex DNA by a polyadenine-based freezing method. The quenched fluorescence can be effectively recovered via duplex-specific nuclease (DSN) with excellent cleaving activity. This approach provided limits of detection (LODs) down to 0.03 ng/mL for ATC, 0.10 ng/mL for metolachlor, 0.14 ng/mL for propisochlor, and 0.08 ng/mL for their mixture. The average recoveries of ATC, metolachlor, and propisochlor were 93.0-106.6% from a corn sample, which are in good agreement with the commercial kit (R2 = 0.9995). This "turn-off" fluorescence immunoassay presents considerable potential in the analysis of chloroacetamide herbicide due to its simple process of probe preparing and ultrahigh sensitivity.
Asunto(s)
Oro , Nanopartículas del Metal , Acetamidas , Inmunoensayo , ToluidinasRESUMEN
Pathogenic Yersinia (Y.) enterocolitica is the primary causative agent of Yersiniosis, with outbreaks in numerous countries around the world, and causes diarrhea and vomiting in animals and humans. Therefore, an instrument-free and convenient nucleic acid visualization method, RPA-SYBR Green I, was established, which combines recombinase polymerase amplification (RPA) with the fluorescent dye SYBR Green I for the detection of the adhesion gene ail in pathogenic Y. enterocolitica. After optimization of a series of conditions such as primer concentration, the detection of pathogenic Y. enterocolitica could be finally completed within about 20 min (from DNA extraction to observation of results) at an isothermal temperature of 39°C. RPA-SYBR Green I had no cross-reactivity with other bacteria and the detection limit was 101 CFU/µL, with sensitivity equal to that of conventional PCR. The method established in this paper and conventional PCR identified a total of 5 spiked samples and 15 meat samples stored in refrigerated, and it was concluded that there was 100% consistency between the two methods. Overall, RPA-SYBR Green I is a visual and facilitate detection assay that can accurately discover pathogenic Y. enterocolitica.
Asunto(s)
Benzotiazoles/química , Diaminas/química , Fluorometría/métodos , Microbiología de Alimentos/métodos , Carne/microbiología , Técnicas de Amplificación de Ácido Nucleico/métodos , Quinolinas/química , Yersinia enterocolitica/genética , Proteínas de la Membrana Bacteriana Externa/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Carne/análisis , Recombinasas/metabolismo , Reproducibilidad de los Resultados , Temperatura , Yersinia enterocolitica/aislamiento & purificaciónRESUMEN
Cholecystokinin-2 receptor (CCK2R) has been proven to be a specific biomarker for colorectal malignancies. Immunotoxins are a valuable class of immunotherapy agents consisting of a targeting element and a bacterial or plant toxin. Previous work demonstrated that targeting CCK2R is a good therapeutic strategy for the treatment of colorectal cancer (CRC). In the present study, we developed a new version of CCK2R-targeting immunotoxin GD9P using a targeted peptide, GD9, as the binding motif and a truncated Pseudomonas exotoxin A (PE38) as the cytokiller. BALB/c nude mice were treated with different doses of GD9P, and pharmacodynamics, pharmacokinetic, and toxicological data were obtained throughout this study. Compared to the parental immunotoxin rCCK8PE38, GD9P exhibited about 1.5-fold yield, higher fluorescence intensity, and increased antitumor activity against human CRC in vitro and in vivo. The IC50 values of GD9P in vitro ranged from 1.61 to 4.55 nM. Pharmacokinetic studies were conducted in mice with a T1/2 of 69.315 min. When tumor-bearing nude mice were treated with GD9P at doses ≥2 mg/kg for five doses, a rapid shrinkage in tumor volume and, in some cases, complete remission was observed. A preliminary safety evaluation demonstrated a good safety profile of GD9P as a Pseudomonas exotoxin A-based immunotherapy. The therapy in combination with oxaliplatin can increase the antitumor efficacy and reduce the toxic side effects caused by chemotherapy. In conclusion, the data support the use of GD9P as a promising immunotherapy targeting CCK2R-expressing colorectal malignancies.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Antineoplásicos/farmacología , Toxinas Bacterianas/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Exotoxinas/farmacología , Receptor de Colecistoquinina B/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Factores de Virulencia/farmacología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Toxinas Bacterianas/genética , Toxinas Bacterianas/uso terapéutico , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Exotoxinas/genética , Exotoxinas/uso terapéutico , Humanos , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Distribución Tisular , Pruebas de Toxicidad Aguda , Factores de Virulencia/genética , Factores de Virulencia/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosaRESUMEN
N- glycolylneuraminic acid (Neu5Gc) is a type of sialic acid, it can be synthesized by a range of mammals except chickens and healthy human. After entering human body, Neu5Gc in foods such as red meat and milk can cause chronic inflammation, thus promoting the development of cancer and related diseases. In this study, we identified a gene sequence of Neu5Gc-specific single-chain variable fragment (ScFv) by phage display from a primary chicken antibodies library. Then the gene sequence was used to express a 29 kDa anti-Neu5Gc ScFv protein as detection probe in competitive inhibition ELISA (IC-ELISA). The linear regression equation of the IC-ELISA was y = 23.12x+33.19 (R = 0.980), and the half-maximal inhibitory concentration (IC50) and the limit of detection (LOD) was 5.333 and 0.66 µg/mL. The mean recovery of the spiked samples was 83.04%, and the intra-assay and inter-assay coefficients of variation (CVs) were both 5.59%. The results suggested that the specific anti-Neu5Gc ScFv is a promising probe for the development of IC-ELISA and test strip in order to detect the presence of Neu5Gc in red meat, milk, and tumor tissues.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Ácidos Neuramínicos/química , Biblioteca de Péptidos , Anticuerpos de Cadena Única , Animales , Pollos , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
Ceftiofur (CEF) is a third-generation and the first animal-specific cephalosporin that is widely used in animal husbandry. As a heat-labile antibiotic, the cytotoxicity of CEF after thermal treatment has been reported. This study seeks to investigate the potential toxicity of thermally treated CEF (TTC) in vivo based on acute oral toxicity studies and acute intraperitoneal studies in mice. Our data indicated that TTC exhibited significant increased toxicity in mice compared with CEF. TTC resulted in weight gain, hypercholesterolemia, hepatocyte steatosis and hepatocyte mitochondrial damage, and downregulated ß-oxidation-related genes in mice in acute oral toxicity studies. In addition, TTC caused acute pulmonary congestion, increased levels of reactive oxygen species (ROS), prolonged coagulation time, and even death in mice in acute intraperitoneal toxicity studies. Our data showed that thermal treatment enhanced the toxicity of CEF in vivo. Lung and liver are the main target organs in the pathological damage process mediated by TTC. These findings suggested that residual CEF in animal-derived food may represent a potential food safety risk and pose a potential threat to human health.
Asunto(s)
Antibacterianos/toxicidad , Cefalosporinas/toxicidad , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Temperatura , Administración Oral , Animales , Antibacterianos/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Cefalosporinas/administración & dosificación , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Hígado/metabolismo , Hígado/patología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos ICR , Tamaño de los Órganos/efectos de los fármacos , Especies Reactivas de Oxígeno/análisisRESUMEN
Based on the etching of gold nanorods (GNRs) and enzyme-linked immunosorbent assay (ELISA), a multicolor immunosensor for visual detection of enterovirus 71 infection is proposed. Once the immunocomplex is formed, the horseradish peroxidase bound to the ELISA plate oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) into TMB2+ in the presence of hydrogen peroxide. Subsequently, TMB2+ quantitatively etches GNRs to the short GNRs, leading to a blue shift of longitudinal localized surface plasmon resonance and corresponding color responses. This change is used to develop two types of cut-off standards, which respond to the human anti-enterovirus at a concentration of 71 IgM antibody. The method has been validated with clinical serum samples and showed high sensitivity and specificity . This visual immunosensor has an important application value for point-of-care detection of EV71, especially in areas lacking detection equipment. Graphical abstract.
Asunto(s)
Técnicas Biosensibles/métodos , Enterovirus Humano A/patogenicidad , Oro/química , HumanosRESUMEN
High levels of glutamate dehydrogenase (GDH) activity are associated with hypoglycemia, cancer, and Parkinson's disease. Propylselen was synthesized to investigate its mechanism of GDH inhibition in comparison with Ebselen and Epigallocatechin gallate (EGCG). Because Ebselen was found to crosslink with the peptide (AA299-341) at the active site of E.coli GDH, the Cys, Pro, and Lys residues of the corresponding peptide were mutagenized to Ala residues. Using enzyme kinetics and biomolecular interaction assays, we found that the conserved GDH P320 residue is important for propylselen binding, C321 for Ebselen binding, and K341 for EGCG binding. In addition, these 3 mutations abolished NADP+ binding to E. coli GDH in the absence of glutamate substrate, but in the presence of glutamate, the catalytic activity of the mutants was reduced only by 2-4 fold, indicating that a substrate-induced fit mechanism exists in E. coli GDH. Furthermore, biochemical analysis showed that NADP+ had high affinity (Kd of 77â¯nM) for GDH; by targeting the NADP binding site, propylselen effectively inhibited both E. coli and human GDH activity and improved anticancer activity.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Azoles/química , Azoles/farmacología , Proliferación Celular/efectos de los fármacos , Glutamato Deshidrogenasa/antagonistas & inhibidores , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Escherichia coli/enzimología , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/metabolismo , Humanos , Isoindoles , Simulación del Acoplamiento Molecular , NADP/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimologíaRESUMEN
BACKGROUND: Brucellosis is a worldwide zoonotic infectious disease that is transmitted in various ways and causes great harm to humans and animals. The brucellosis pathogen is Brucella, which mainly resides in macrophage cells and survives and replicates in host cells. However, the mechanisms underlying Brucella survival in macrophage cells have not been thoroughly elucidated to date. Peroxiredoxin 6 (Prdx6) is a bifunctional protein that shows not only GSH peroxidase activity but also phospholipase A2 activity and plays important roles in combating oxidative damage and regulating apoptosis. RESULTS: Recombinant mouse (Mus musculus) Prdx6 (MmPrdx6) was expressed and purified, and monoclonal antibodies against MmPrdx6 were prepared. Using the Brucella suis S2 strain to infect RAW264.7 murine macrophages, the level of intracellular Prdx6 expression first decreased and later increased following infection. Overexpressing Prdx6 in macrophages resulted in an increase in B. suis S2 strain levels in RAW264.7 cells, while knocking down Prdx6 reduced the S2 levels in cells. CONCLUSIONS: Host Prdx6 can increase the intracellular survival of B. suis S2 strain and plays a role in Brucella infection.
Asunto(s)
Brucella suis/fisiología , Brucelosis/microbiología , Peroxiredoxina VI/metabolismo , Animales , Femenino , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7RESUMEN
The interleukin-1 family is an important component of the innate immune system and plays an important role in regulating immune responses on the invasion of intracellular parasites in the acquired immune system. Interleukin 1ß (IL-1ß) is one of the members of the IL-1 family that predominantly activates downstream signaling pathways to play immunological functions of stimulating T and B lymphocyte activation and promoting the various syntheses of inflammatory substances in conjunction with other cytokines. Here, a full-length IL-1ß cDNA (OaIL-1ß) of sheep (Ovis aries) was cloned using rapid amplification of cDNA ends (RACE), which consists of 1494 bp and contains a 5'-UTR region with a length of 83 bp, a complete ORF of 801 bp in length, and a 3'-UTR region with a length of 642 bp. Recombinant protein OaIL-1ß was expressed and purified, and the monoclonal antibody against IL-1ß of sheep is prepared. Western blotting results showed that the sheep IL-1ß protein was detected in the heart, liver, lung, kidney, stomach, intestine, muscle, lymph nodes and leukocytes with the highest expression in the muscle and the lowest expression in the lung. Different bacteria treating sheep white blood cells induced differential expression of OaIL-1ß. Compared with the normal sheep, OaIL-1ß in the buffy coat was differentially expressed in the Brucella melitensis-challenged group and the B. suis S2 strain-inoculated group. However, whether IL-1ß may be considered as a molecular biomarker for differing Brucella-infected animals from brucellosis-vaccinated animals or not need to be further studied.
Asunto(s)
Brucelosis/veterinaria , Perfilación de la Expresión Génica , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Enfermedades de las Ovejas/patología , Oveja Doméstica , Estructuras Animales/patología , Animales , Brucella melitensis/inmunología , Brucella suis/inmunología , Brucelosis/patología , Clonación Molecular , Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , OvinosRESUMEN
N-glycolylneuraminic acid (Neu5Gc) is a type of sialic acid that is not typically produced in healthy humans but detective in some visceral cancer cells. As a new carcinoma biomarker, the level change in the serum and urine from the patient could potentially have the relation to the disease progression. So the measurement of the Neu5Gc will help to take a better response to therapeutic schedule for the sufferers. A sensitive and rapid aptamer-nanoparticle immunochromatographic strip for the visual detection of Neu5Gc was developed. The assay is based on the competitive reaction of binding the DNA aptamer targeting the candidate molecule selected by SELEX between Neu5Gc and complementary DNA. The sensing results indicated that the aptamer-based strip was sufficiently sensitive to detect Neu5Gc. The visual limit of detection (LOD) for semi-quantitative detection was 30â¯ng/mL under the optimal conditions and a quantitative detection limit of 5.38â¯ng/mL could be obtained using a scanning strip reader. The average recovery of the spiked cancer cell samples was 88.86%, with a coefficient of variation (CV) of 5.27%. The detection could be performed in less than 15â¯min using a simple procedure without any complicated equipment, demonstrating that this aptamer-nanoparticle biosensor strip has great potential for use to Neu5Gc-related cancer diagnosis.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Cromatografía de Afinidad , Oro/química , Nanopartículas del Metal/química , Ácidos Neuramínicos/análisis , Humanos , Células Tumorales CultivadasRESUMEN
A competitive fluorescence-linked immunosorbent assay (FLISA) was developed using rhodamine B isothiocyanate (RBITC) as the model fluorescent dye conjugate monoclonal antibody (McAb) for detection of Phe and its homolog (acenaphthene, fluorene, fluoranthene, pyrene and indeno [1,2,3-cd] pyrene) in water samples. The detection range of the assay for Phe was from 2.10 to 91.95â¯ng/mL. The limit of detection was 1.05â¯ng/mL, which was approximately 2-fold lower than that of traditional ic-ELISA. Compared with traditional ic-ELISA, more than 70â¯min was saved because of only one immunoreaction step was needed to accomplish the assay. The average recoveries of Phe and its homolog from domestic water, contaminated water and natural water were 100.7%, 100.8% and 101.2% respectively. The accuracy and precision of the developed FLISA were validated with GC-MS/MS. There were good correlation between the two methods from tap water, contaminated water and river water samples were 0.9994, 0.9935 and 0.9967, respectively. The results suggested that the proposed FLISA could be a potential alternative format for rapid, sensitive, and quantitative detection of Phe and its homolog in environmental water.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Técnica del Anticuerpo Fluorescente/métodos , Fenantrenos/análisis , Contaminantes del Agua/análisis , Agua/análisisRESUMEN
rCCK8PE38 is a novel immunotoxin that targets choleystokinin B receptor, which is over-expressed in some tumor tissues. Although we constructed a prokaryotic expression vector to express rCCK8PE38 in our laboratory, thorough purification was necessary to quantitatively assess its anti-tumor effect. In this study, we established a purification protocol to obtain rCCK8PE38 with high purity from E. coli. Three different types of chromatography, hydrophobic chromatography, ion exchange chromatography and size exclusion chromatography, were used in combination. The purification technological parameters of each chromatography type were optimized. The whole process of purification was arranged to minimize the purification steps and achieve purity and bioactivity. Finally, through this optimized scheme, we obtained a recombinant protein with a purity of >95%; then, the protein was stored at -80°C after lyophilization. The purified protein was used in a tumor inhibition experiment and was effective in killing tumor cells that over-expressed choleystokinin B receptor. The results of this study may provide some valuable information about protein purification and lay the foundation for further clinical experiments with rCCK8PE38.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Inmunotoxinas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Sulfato de Amonio/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Precipitación Química , Cromatografía Liquida , Escherichia coli/genética , Humanos , Inmunotoxinas/química , Inmunotoxinas/genética , Inmunotoxinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Poultry husbandry is a very important aspect of the agricultural economy in China. However, chicks are often susceptible to infectious disease microorganisms, such as bacteria, viruses and parasites, causing large economic losses in recent years. In the present study, we isolated an Acinetobacter baumannii strain, CCGGD201101, from diseased chicks in the Jilin Province of China. Regression analyses of virulence and LD50 tests conducted using healthy chicks confirmed that A. baumannii CCGGD201101, with an LD50 of 1.81 (±0.11) × 10(4) CFU, was more virulent than A. baumannii ATCC17978, with an LD50 of 1.73 (±0.13) × 10(7) CFU. Moreover, TEM examination showed that the pili of A. baumannii CCGGD201101 were different from those of ATCC17978. Antibiotic sensitivity analyses showed that A. baumannii CCGGD201101 was sensitive to rifampicin but resistant to most other antibiotics. These results imply that A. baumannii strain CCGGD201101 had both virulence enhancement and antibiotic resistance characteristics, which are beneficial for A. baumannii survival under adverse conditions and enhance fitness and invasiveness in the host. A. baumannii CCGGD20101, with its high virulence and antimicrobial resistance, may be one of the pathogens causing death of diseased chicks.
Asunto(s)
Infecciones por Acinetobacter/veterinaria , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Enfermedades de las Aves de Corral/microbiología , Acinetobacter baumannii/genética , Acinetobacter baumannii/aislamiento & purificación , Acinetobacter baumannii/ultraestructura , Animales , China , Pruebas de Sensibilidad Microbiana , Fenotipo , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/mortalidad , VirulenciaRESUMEN
Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.
Asunto(s)
Brucella melitensis/genética , ADN Complementario/genética , Lisofosfolipasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Brucella melitensis/enzimología , Brucelosis/inmunología , Brucelosis/veterinaria , Clonación Molecular , Femenino , Ratones Endogámicos BALB C , Filogenia , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , OvinosRESUMEN
Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 108 M(-1), 1.47 × 108 M(-1), 1.23 × 108 M(-1) and 1.05 × 108 M(-1), respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X - 1.78. The IC50 of O31 was 3.39 ng·mL(-1), which was close to the value predicted by the OA ELISA (IC50 = 4.4 ng·mL(-1)); the IC10 was 0.33 ng·mL(-1). The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.