Search

This study aimed to evaluate the expression level of caseins during mastitis of goats. Whole goat caseins were used as primary antigens for mouse immunization. A monoclonal antibody (mAb) named 5B with high specificity to goat αs1-casein was developed. Further results showed that mAb 5B can successfully be applied to western blot and ELISA. In addition, immunofluorescence analysis using this mAb showed increased milk αs1-casein level in mastitis-affected goats. In conclusion, this study has established an effective tool to evaluate the expression level of αs1-casein during mastitis development. [ABSTRACT FROM AUTHOR]

Functional constipation (FC) is a common gastrointestinal disorder, the incidence of which is increasing each year with the changes in life style and diet, and the age of patients is becoming younger. Correct categorization will guide the treatment of FC and in the present study colonic transit time (CTT) and pelvic floor electromyography (PFE) were evaluated for their clinical significance in categorizing FC.Thirty-two cases of FC diagnosed by Rome II criteria with a duration of 6 months to 30 years were enrolled. Radio-opaque markers were used to detect CTT, the transit index (TI) and colonic evacuation rate; PFE was used to observe whether there was any paradoxical movement between the pelvic floor and abdominal muscles.Colonic transit time was recorded in 32 patients: 20 had outlet obstruction constipation and 12 had slow transit type. In the outlet obstruction type, PFE showed varying degrees of paradoxical movement between the muscles, whereas in the slow transit type, only 4 cases showed it.Functional constipation can be categorized by CTT and PFE, which are simple, painless and feasible to perform. [ABSTRACT FROM AUTHOR]

• The differential expression of proteins and miRNAs in plasma EVs of profound SSNHL patients and the related biological function was identified. • Complement C3 is one of the most significant proteins with differentially expressed in plasma EVs of profound SSNHL patients. • Plasma EVs-derived complement C3 is associated with the severity and early efficacy of profound SSNHL patients. Sudden sensorineural hearing loss (SSNHL) is characterized by rapid, unexplained loss of hearing within a 72-hour period and exhibits a high incidence globally. Despite this, the outcomes of therapeutic interventions remain largely unpredictable, especially for those with profound hearing loss. Extracellular vesicles (EVs), nano-sized entities containing biological materials, are implicated in the development of numerous diseases. The specific relationship between EVs and both the severity and treatment effectiveness of SSNHL, however, is not well understood. This study involved the analysis of medical records from the Department of Otolaryngology (September 1, 2020 − December 31, 2022) of patients diagnosed with SSNHL according to the 2015 Guidelines for Diagnosis and Treatment of Sudden Deafness in China. Peripheral blood samples from patients with various types of SSNHL before and after treatment were collected, alongside samples from healthy volunteers serving as controls. Plasma EVs were isolated using gel rejection chromatography and analyzed for concentration, marker presence, and morphology using Nanosight, Western blot, and transmission electron microscopy (TEM), respectively. Proteomics and miRNA assessments were conducted to identify differentially expressed proteins and miRNAs in the plasma EVs of SSNHL patients and healthy volunteers. Key proteins were further validated through Western blot analysis. Enzyme-linked immunosorbent assay (ELISA) was utilized to determine the levels of complement C3 in plasma EVs, and correlation analyses were performed with audiological data pre- and post-treatment. Plasma from SSNHL patients of varying types was collected and their EVs were successfully isolated and characterized. Proteomic analysis revealed that complement C3 levels in the plasma EVs of patients with profound SSNHL were significantly higher compared to healthy controls. Differential expression of miRNAs in plasma EVs and their related functions were also identified. The study found that the level of complement C3 in plasma EVs, but not the total plasma complement C3, positively correlated with the severity of SSNHL in patients exhibiting positive therapeutic responses, particularly in those with initially lower levels of EV-associated complement C3. After treatment, complement C3 level was decreased in patients with initially higher levels of EV-associated complement C3. No significant correlation was observed between changes in plasma EV-derived complement C3 levels and the degree of hearing loss in either responders or non-responders among patients with profound SSNHL. Differential profiles of proteins and miRNAs were identified in patients with profound SSNHL. Notably, plasma EV-derived complement C3 was linked to both the severity and early treatment effectiveness of patients with profound SSNHL. [ABSTRACT FROM AUTHOR]

Four field sources of citrus tristeza virus (CTV) (Y3, Y6, Y7 and Y23) collected from grapefruit trees at groves in Fort Pierce, Florida, and isolate T36were used to evaluate the transmission and separation of different virus genotypes by single brown citrus aphids (BrCA). Analysis of the field sources of CTV by inoculation to indicator plants, ELISA and RT-PCR showed that Y6 was a decline-inducing isolate and Y23 a nondecline-inducing isolate. Assays of genotype by RT-PCR indicated that Y6 contained the T36 genotype while Y23 contained the T30 genotype. Both Y3 and Y7 were a mixture of decline-inducing and nondecline-inducing CTV isolates and were a mixture of T36 and T30 genotypes. When Y6 and Y23 were the acquisition host for single BrCA, only the T36 or T30 genotypes, respectively, were detected by RT-PCR in 'Mexican' Lime receptor plants. Only the T36 genotype was transmitted to receptor plants from infected Y3 and Y7 plants although these acquisition plants contained more than one genotype. No T3 or VT genotypes were detected in any acquisition or receptor plants. CTV genotype mixtures in the various field sources were separated by single BrCA transmission and that the T36 genotype in T36/T30 mixtures was more easily transmitted than the T30 genotype when the acquisition plant was 'Duncan' grapefruit and the receptor plant was 'Mexican' lime. [ABSTRACT FROM AUTHOR]

Reverse transcription—polymerase chain reaction (RT-PCR) was compared with enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) for detection of non-decline-inducing and decline-inducing isolates of Citrus tristeza virus (CTV) in 21 field sweet orange and grapefruit plants on sour orange rootstock in Fort Pierce, FL. Among these samples, seven, six, and eight were infected with decline-inducing, non-decline-inducing, and both decline-inducing and non-decline-inducing isolates of CTV, respectively. However, there was not a good correlation between field symptoms and detection of the decline-inducing isolate. The results confirmed that RT-PCR is not only able to detect and differentiate decline-inducing and non-decline-inducing isolates of CTV in Florida, but also can detect both isolate types in a single field sweet orange or grapefruit tree. For most samples, results from RT-PCR, ELISA, and DTBIA were the same. However, the 320-bp fragments produced only from decline-inducing isolates were amplified from two sweet orange and two grapefruit samples that did not react with decline-inducing CTV-specific monoclonal antibody MCA13 in ELISA or DTBIA, indicating that RT-PCR has a higher sensitivity than these immunological tests for field sweet orange or grapefruit samples. Thus, RT-PCR is a simple, rapid, and specific procedure for CTV identification applicable to both research and diagnostic needs. [ABSTRACT FROM AUTHOR]

Abstract Concentrations, composition and seasonal variations of organochlorine pesticides (OCPs) in the atmosphere (particulate phase and gaseous phase) at coastal cities in northern China were determined. OCP transport from emission source areas and lifetime excess cancer risks by inhalation exposure to specific OCPs were also investigated. The annual average concentration of total OCPs in gaseous phase ranged from 1.0 ng/m3 to 6.3 ng/m3, with the peak observed in summer at most sites. Particulate phase concentrations ranged from 29.9 pg/m3 to 103.3 pg/m3, with the maximum found in the local heating period at most locations. The detection rates of gaseous samples were considerably higher than those of particulate ones. The dominant components included endosulfan (I and II), (α- and γ-) chlordane, pentachlorobiphenyl (PeCB), hexachlorobenzene (HCB), heptachlor, (α-, β- and γ-) hexachlorocyclohexane (HCH), dichloro-diphenyl-trichloroethane (DDT) and their metabolic products. The specific ratios indicated different applications of DDT, technical HCH and endosulfan at most sites. Large differences in compositional profiles occurred in January (typical heating period) and July (representative non-heating period), and diurnal changes in component concentrations may have been influenced by local emission pattern. The potential source contribution function (PSCF) manifested seasonal concentrations of airborne OCPs affected by the input of potential sources in different regions. The emission sources with higher contribution probabilities to the sites were primarily distributed in the surrounding areas. The lifetime excess cancer risks for the local residents by inhalation exposure to specific components were not high, though the potential threat of α-HCH and HCB should be concerned. Capsule Gaseous OCPs reached peak values in summer and dominated relative to particulate (PM 10) values; meanwhile, surrounding sources affected air OCP concentrations, and cancer risks of OCPs by inhalation exposure were not high. Graphical abstract Unlabelled Image Highlights • Gaseous phase OCPs reached the highest levels in summer at most sampling sites. • Endosulfan (I and II), α- and γ‑chlordane, PeCB and HCB were dominant components. • Potential sources of OCPs may exist in the surrounding areas. • Lifetime excess cancer risks by inhalation exposure to specific OCPs were not high. [ABSTRACT FROM AUTHOR]

The nematicidal activity of 92 Bacillus thuringiensis strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world's top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bacillus thuringiensis BRC-XQ12 were the most toxic to Bursaphelenchus xylophilus, with a lethal concentration 50 (LC50) of 32.13 pg/ml. Because the ICPs expressed by Bacillus thuringiensis BRC-XQ12 were closest to CrylEa6 and B. thuringiensis BRC-XQ12 contained four kinds of cry I subgenes (crylAa, crylCh, crylEa, and cry l hi), CrylEa was most likely to be the key active component against the nematode. The 3,516-bp crylEall gene front BRC-XQ12, as designated by the B. thuringiensis 8-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified CrylEal 1 showed an LC50 of 32.53 and 23.23 pg/ml at 24 and 48 h, with coiresponding virulence equations of Y = 32.15X + 1.38 (R2 = 0.9951) and Y = 34.29X + 3.16 (R2 = 0.9792), respectively. In order to detect the pathway of B. thuringiensis CrylEal 1 into Bursaphelenchus xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-CrylEall. The results of confocal laser-scanning microscopy showed that the 159-kDa GST-CrylEall could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-CrylEal 1 could enter into the nematode through the stylet. As far as we know, no Cry 1 proteins have been shown to have activity against plant-parasitic nematodes before. These results demonstrate that CrylEal I is a promising nematicidal protein for controlling pine wilt disease rendered by B. .xylophilus, further dramatically broadening the spectrum of Bacillus thuringiensis ICPs. [ABSTRACT FROM AUTHOR]

The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests in the rice fields of Asia. Like other hemipteran insects, BPH is not susceptible to Cry toxins of Bacillus thuringiensis (Bt) or transgenic rice carrying Bt cry genes. Lack of Cry receptors in the midgut is one of the main reasons that BPH is not susceptible to the Cry toxins. The main Cry-binding proteins (CBPs) of the susceptible insects are cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP). In this study, we analyzed and validated de novo assembled transcripts from transcriptome sequencing data of BPH to identify and characterize homologs of cadherin, APN, and ALP. We then compared the cadherin-, APN-, and ALP-like proteins of BPH to previously reported CBPs to identify their homologs in BPH. The sequence analysis revealed that at least one cadherin, one APN, and two ALPs of BPH contained homologous functional domains identified from the Cry-binding cadherin, APN, and ALP, respectively. Quantitative real-time polymerase chain reaction used to verify the expression level of each putative Cry receptor homolog in the BPH midgut indicated that the CBPs homologous APN and ALP were expressed at high or medium-high levels while the cadherin was expressed at a low level. These results suggest that homologs of CBPs exist in the midgut of BPH. However, differences in key motifs of CBPs, which are functional in interacting with Cry toxins, may be responsible for insusceptibility of BPH to Cry toxins. [ABSTRACT FROM AUTHOR]

In our previous study, several Bacillus thuringiensis (Bt) strains were isolated from bryophyte samples, indicating that bryophytes could serve as Bt reservoirs in the wild. SFR13 is a wild strain isolated from the bryophyta Physcomitrium japonicum. In order to understand its ecological properties, green fluorescent protein (GFP)-labelled SFR13 (SFR13GFP) was generated to evaluate the colonization capability in bryophytes, using dynamic tracing and cell counting to observe the process and patterns of colonization. Our results showed that genetic stability, growth curve dynamics, and insecticidal crystal production were not affected by GFP expression in Bt. Fluorescence microscopy was used to track the dynamic distribution of SFR13GFP. Distribution patterns showed that SFR13GFP can establish stable and long-term colonization in leaves and stems by the 26th day after inoculation. A better understanding of how Bt colonizes plants in the wild will not only result in increased knowledge of plant-microbe interactions but will also lead to a more successful and reliable use of bacterial inoculants. [ABSTRACT FROM AUTHOR]

Gene expression profiles are important data to reveal the functions of genes putatively involved in crucial biological processes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and specifically primed reverse transcription polymerase chain reaction (RT-PCR) were combined to screen differentially expressed genes following development of a commercial Bacillus thuringiensis subsp. kurstaki strain 8010 (serotype 3a3b). Six differentially expressed transcripts (RAP1 to RAP6) were obtained. RAP1 encoded a putative triple helix repeat-containing collagen or an exosporium protein H related to spore pathogenicity. RAP2 was homologous to a ClpX protease and an ATP-dependent protease La (LonB), which likely acted as virulence factors. RAP3 was homologous to a beta subunit of propionyl-CoA carboxylase required for the development of Myxococcus xanthus. RAP4 had homology to a quinone oxidoreductase involved in electron transport and ATP formation. RAP5 showed significant homology to a uridine kinase that mediates phosphorylation of uridine and azauridine. RAP6 shared high sequence identity with 3-methyl-2-oxobutanoate-hydroxymethyltransferase (also known as ketopantoate hydroxymethyltransferase or PanB) involved in the operation of the tricarboxylic acid cycle. The findings described here would help to elucidate the molecular mechanisms underlying the differentiation process of B. thuringiensis and unravel novel pathogenic genes. Les profils d'expression génique fournissent d'importants éléments révélant les fonctions de gènes potentiellement impliqués dans des processus biologiques déterminants. On a combiné des réactions de polymérisation en chaîne de l'ARN utilisant des amorces arbitraires (RAR-PCR) et de PCR á transcription inverse (RT-PCR) á amorces spécifiques afin de rechercher des gènes exprimés de manière différentielle lors du développement de Bacillus thuringiensis subsp. kurstaki souche 8010 (sérotype 3a3b). On a obtenu 6 transcrits exprimés différentiellement (RAP1 á RAP6). RAP1 codait un collagène putatif á répétition trihélicoïdale ou une protéine H d'exine liée á la pathogénicité de la spore. RAP2 était homologue á une protéase ClpX et á une protéase La dépendante de l'ATP (LonB) dont la fonction était probablement liée á la virulence. RAP3 était homologue á une sous-unité bêta de la propionyl-CoA carboxylase nécessaire au développement de Myxococcus xanthus. RAP4 était homologue á une quinone oxydoréductase participant au transport d'électrons et á la formation d'ATP. RAP5 présentait une forte homologie á une uridine kinase responsable de la phosphorylation de l'uridine et de l'azauridine. RAP6 partageait une identité de séquence élevée avec la 3-méthyl-2-oxobutanoate-hydroxyméthyltransférase (aussi connue comme la kétopantoate hydroxyméthyltransférase ou PanB) impliquée dans le cycle de Krebs. Ces trouvailles permettraient d'élucider les mécanismes moléculaires sous-tendant le processus de différenciation de B. thuringiensis et de mettre au jour de nouveaux gènes pathogènes. [ABSTRACT FROM AUTHOR]

Widespread fecal pollution of surface waters in developing countries is a threat to public health and may represent a significant pathway for the global dissemination of antibiotic resistance. The Minjiang River drainage basin in Fujian Province is one of China's most intensive livestock and poultry production areas and is home to several million people. In the study reported here, Escherichia coli isolates (n = 2,788) were sampled (2007 and 2008) from seven surface water locations in the basin and evaluated by PCR for carriage of selected genes encoding virulence factors, primarily for swine disease. A subset of isolates (n = 500) were evaluated by PCR for the distribution and characteristics of class 1 integrons, and a subset of these (n = 200) were evaluated phenotypically for resistance to a range of antibiotics. A total of 666 (24%) E. coli isolates carried at least one of the virulence genes elt, fedA, astA, fasA, estA, stx2e, paa, and sepA. Forty-one percent of the isolates harbored class 1 integrons, and these isolates had a significantly higher probability of resistance to tobramycin, cefoperazone, cefazolin, ciprofloxacin, norfloxacin, azitromycin, and rifampin than isolates with no class 1 integron detected. Frequencies of resistance to selected antibiotics were as high as or higher than those in fecal, wastewater, and clinical isolates in published surveys undertaken in China, North America, and Europe. Overall, E. coli in the Minjiang River drainage basin carry attributes with public health significance at very high frequency, and these data provide a powerful rationale for investment in source water protection strategies in this important agricultural and urban setting in China. [ABSTRACT FROM AUTHOR]

Abstract  InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects. [ABSTRACT FROM AUTHOR]