Uptake, translocation, and metabolism of organophosphate esters (OPEs) in plants and health perspective for human: A review.

Plant uptake, accumulation, and transformation of organophosphate esters (OPEs) play vital roles in their geochemical cycles and exposure risks. Here we reviewed the recent research advances in OPEs in plants. The mean OPE concentrations based on dry/wet/lipid weight varied in 4.80-3,620/0.287-26.8/12,000-315,000 ng g-1 in field plants, and generally showed positive correlations with those in plant habitats. OPEs with short-chain substituents and high hydrophilicity, particularly the commonly used chlorinated OPEs, showed dominance in most plant samples, whereas some tree barks, fruits, seeds, and roots demonstrated dominance of hydrophobic OPEs. Both hydrophilic and hydrophobic OPEs can enter plants via root and foliar uptake, and the former pathway is mainly passively mediated by various membrane proteins. After entry, different OPEs undergo diverse subcellular distributions and acropetal/basipetal/intergenerational translocations, depending on their physicochemical properties. Hydrophilic OPEs mainly exist in cell sap and show strong transferability, hydrophobic OPEs demonstrate dominant distributions in cell wall and limited migrations owing to the interception of Casparian strips and cell wall. Additionally, plant species, transpiration capacity, growth stages, commensal microorganisms, and habitats also affect OPE uptake and transfer in plants. OPE metabolites derived from various Phase I transformations and Phase II conjugations are increasingly identified in plants, and hydrolysis and hydroxylation are the most common metabolic processes. The metabolisms and products of OPEs are closely associated with their structures and degradation resistance and plant species. In contrast, plant-derived food consumption contributes considerably to the total dietary intakes of OPEs by human, particularly the cereals, and merits specifical attention. Based on the current research limitations, we proposed the research perspectives regarding OPEs in plants, with the emphases on their behavior and fate in field plants, interactions with plant-related microorganisms, multiple uptake pathways and mechanisms, and comprehensive screening analysis and risk evaluation.

Uptake mechanism, translocation, and transformation of organophosphate esters in water hyacinth (Eichhornia crassipes): A hydroponic study.

Owing to their dominant wastewater origin, bioavailability, and toxicity, the occurrence and behavior of organophosphate esters (OPEs) in aquatic systems have attracted considerable attention over the past two decades. Aquatic plants can accumulate and metabolize OPEs in water, thereby playing an important role in their behavior and fate in waterbodies. However, their uptake, translocation and transformation mechanisms in plants remain incompletely characterized. We investigated the accumulation and transformation of OPEs in water hyacinth (Eichhornia crassipes) through a series of hydroponic experiments using three representative OPEs, tris(2-chloroethyl) phosphate (TCEP), tris(2-butoxyethyl) phosphate (TBEP), and triphenyl phosphate (TPP). These OPEs can not only be adsorbed onto and enter plant roots via passive diffusion pathways, which are facilitated by anion channels and/or aquaporins, but also can return to the solution when concentration gradients exist. After entry, hydrophilic TCEP showed a dominant distribution in the cell sap, strong acropetal transportability, and rapid translocation rate, whereas hydrophobic TPP was mostly retained in the root cell wall and therefore demonstrated weak acropetal transportability; TBEP with moderate hydrophilicity remained in the middle. All these OPEs can be transformed into diesters, which presented higher proportions in the cell sap and therefore have stronger acropetal transferability than their parent OPEs. TCEP exhibits the lowest biodegradability, followed by TPP and TBEP. These OPEs exerted apparent effects on plant growth, photosynthesis, and the diversity and composition of the rhizosphere microbial community.

The value of vestibular graviceptive pathway evaluation in the diagnosis of unilateral peripheral vestibular dysfunction.

BACKGROUND: Evaluation of vestibular graviceptive pathway (VGP) in patients with unilateral peripheral vestibular dysfunction (UPVD) has received increasing attention from researchers. The study aimed to investigate the value of VGP evaluation in the diagnosis of UPVD. METHODS: Ninety-five UPVD patients were divided into attack and remission phase groups. VGP evaluation-related indicators, including subjective visual vertical (SVV), subjective visual horizontal (SVH), head tilt, ocular torsion (OT), and skew deviation (SD), were measured, and their correlations with cochleovestibular function test results were analyzed. The possible etiologies of contralesional VGP (c-VGP) were analyzed. RESULTS: Positive rates of SVV, SVH, OT, and SD were significantly higher, and the degrees of SVV, SVH, and OT were significantly greater in the attack phase group than the remission phase group. The sides with abnormal VGP evaluation results were correlated with the sides with hearing loss, abnormal caloric, and video head impulse test (vHIT) results. A total of 14 patients showed c-VGP, and possible etiologies included contralateral benign paroxysmal positional vertigo (n = 4), bilateral hearing loss (n = 8), bilateral vHIT gain reduction (n = 1), autoimmune diseases (n = 6), vascular risk factors (n = 6), lacunar infarction (n = 3), and endolymphatic hydrops (n = 3). CONCLUSIONS: Alterations in SVV, SVH, OT, and SD were noted in UPVD patients in different phases, which are presumed to be related to dynamic vestibular compensation; correlations between VGP evaluation results and cochleovestibular function test results indicate that VGP evaluation may be helpful for the diagnosis of the side affected in UPVD; the presence of c-VGP may be related to bilateral labyrinth lesions or endolymphatic hydrops on the affected side; and the involvement of autoimmune mechanisms also deserves attention.

Safety of butylphthalide and edaravone in patients with ischemic stroke: a multicenter real-world study.

BACKGROUND: Butylphthalide (NBP) and edaravone (EDV) injection are common acute ischemic stroke medications in China, but there is a lack of large real-world safety studies on them. This study aimed to determine the incidence of adverse events, detect relevant safety signals, and assess the risk factors associated with these medications in real-world populations. METHODS: In this study, data of acute ischemic stroke patients were extracted from the electronic medical record database of six tertiary hospitals between January 2019 and August 2021. Baseline confounders were eliminated using propensity score matching. The drugs' safety was estimated by comparing the results of 24 laboratory tests standards on liver function, kidney function, lipid level, and coagulation function. The drugs' relative risk was estimated by logistic regression. A third group with patients who did not receive NBP or EDV was constructed as a reference. Prescription sequence symmetry analysis was used to evaluate the associations between adverse events and NBP and EDV, respectively. RESULTS: 81,292 patients were included in this study. After propensity score matching, the NBP, EDV, and third groups with 727 patients in each group. Among the 15 test items, the incidence of adverse events was lower in the NBP group than in the EDV group, and the differences were statistically significant. The multivariate logistic regression equation revealed that NBP injection was not a promoting factor for abnormal laboratory test results, whereas EDV had statistically significant effects on aspartate transaminase, low-density lipoprotein cholesterol and total cholesterol. Prescription sequence symmetry analysis showed that NBP had a weak correlation with abnormal platelet count. EDV had a positive signal associated with abnormal results in gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase, prothrombin time, and platelet count. CONCLUSIONS: In a large real-world population, NBP has a lower incidence of adverse events and a better safety profile than EDV or other usual medications.

Spontaneous nystagmus with an upbeat component: Central or peripheral vestibular disorders?

Objective: To determine the topical diagnosis and etiologies of spontaneous nystagmus (SN) with an upbeat component. Methods: We retrospectively recruited 43 patients with SN with an upbeat component at a university hospital in China from 2020 to 2022. SN with an upbeat component was divided into pure upbeat nystagmus (UBN), SN with a predominant upbeat component, and SN with a non-predominant upbeat component. We analyzed their clinical and neurotologic findings and the final diagnosis. Results: Fourteen (32.6%) of them showed pure UBN, while 29 (67.4%) exhibited SN mixed with an upbeat component, mixed upbeat-horizontal in 15, mixed upbeat-horizontal-torsional in 13, and upbeat-torsional in the remaining one. Pure UBN and SN with a predominant upbeat component were more common in central than in peripheral vestibular disorders [16 (80.0%) vs. 0 (0%), Chi-Square test, p < 0.001]. Central vestibular disorders were diagnosed in 20 (46.5%) patients, peripheral in 14 (32.6%), and undetermined in nine (20.9%) patients. The underlying causes mainly included acute unilateral peripheral vestibulopathy (n = 11), posterior circulation infarction (n = 9), benign recurrent vertigo (n = 4), vestibular migraine (VM, n = 3), and VM of childhood (n = 2). Conclusion: SN with an upbeat component can be seen in both central and peripheral vestibular disorders. Pure UBN was a characteristic sign of central vestibular dysfunction. Central vestibular disorders should be highly suspected when patients show pure UBN or SN with a predominant upbeat component.

Potential drug-drug interactions in drug therapy for older adults with chronic coronary syndrome at hospital discharge: A real-world study.

Introduction: Polypharmacy are commonly observed among older adults with cardiovascular disease. However, multiple medications lead to increased risk of drug-drug interactions (DDIs). Therefore, identification and prevention actions related to harmful DDIs are expected in older adults. The study aimed to describe the prevalence of potential DDIs (pDDIs) in discharge prescriptions among older adults with chronic coronary syndrome (CCS). Methods: A single-center cross-sectional study was performed in a tertiary public hospital in Beijing, China. CCS patients aged 65 years and above who were admitted to cardiology wards over a 3-month period and alive at discharge were included. Electronic medical records and discharge prescriptions were reviewed. pDDIs were evaluated through the Lexi-Interact online. Results: pDDIs were identified in 72.9% of the 402 individuals (n = 293). A total of 864 pDDIs were obtained. 72.1% of patients were found with C DDIs (n = 290) and 20.3% were categorized in D and X DDIs (n = 82). The only X DDI was between cyclosporine and atorvastatin. Under category D, glycemia alterations within antidiabetics and increased chances of bleeding with antithrombotic were the most common. Concomitant use of clopidogrel and calcium channel blockers was a frequent situation within category C, followed by synergic blood pressure lowering agents and increased rosuvastatin concentration induced by clopidogrel. Conclusion: DDIs exposure was common in older CCS. DDIs screening tools should be introduced to alert potential adverse effects. Prescribers need to rigorously review or modulate therapies to prevent DDI-related adverse outcomes. Clinical pharmacists should be more involved in complex drug regimen management.

NMR Assignments of Six Asymmetrical N-Nitrosamine Isomers Determined in an Active Pharmaceutical Ingredient by DFT Calculations.

N-nitrosamines, which are well-known pro-mutagens, are found in drugs, pickled food and tobacco. Therefore, controlling their concentrations is very important. When an HPLC, GC or NMR analysis is conducted to investigate certain asymmetrical N-nitrosamines, two sets of signals attributed to the asymmetric N-nitrosamine isomers are usually observed. However, few reports on the NMR assignment of asymmetrical N-nitrosamine isomers have been published. In this study, we investigated the NMR assignments of the Z/E isomers of six asymmetrical N-nitrosamines by means of density functional theory (DFT) calculations. The configuration of the major isomer of asymmetrical N-nitrosamine 3 was the Z-configuration. The configuration of the major isomers of asymmetrical N-nitrosamines 4-7 was the E-configuration. Then, we determined the Z/E ratios of these asymmetrical N-nitrosamines by means of variable temperature (VT) and room temperature (RT) 1H-NMR experiments. The ratios of the Z/E isomer 3 quickly increased beyond 100% in the VT 1H NMR experiments. The ratios of Z/E isomers 4-7 were increased in the range of 10-60% in the VT 1H NMR experiments. The results of this study indicate that identifying the isomers of asymmetrical N-nitrosamine is necessary to control the quality of N-nitrosamines for active pharmaceutical ingredients (APIs).

The Protective Effect of Dexmedetomidine Against Ischemia-Reperfusion Injury after Hepatectomy: A Meta-Analysis of Randomized Controlled Trials.

Background: Hepatic inflow occlusion proceeded to reduce blood loss during hepatectomy induces ischemia-reperfusion (IR) injury in the remnant liver. Dexmedetomidine, a selective α2-adrenoceptor agonist used as an anesthetic adjuvant, has been shown to attenuate IR injury in preclinical and clinical studies. However, a meta-analysis is needed to systematically evaluate the protective effect of perioperative dexmedetomidine use on IR injury induced by hepatectomy. Methods: A prospectively registered meta-analysis following Cochrane and PRISMA guidelines concerning perioperative dexmedetomidine use on IR injury after hepatectomy was performed via searching Cochrane Library, PubMed, EMBASE, ClinicalTrials.gov, Web of Science, CNKI, WanFang, and Sinomed for eligible randomized controlled trials up to 2021.3.31. The main outcome is postoperative liver function. Risk of bias was assessed by the Cochrane Risk of Bias tool. Review Manager 5.3 and Stata12.0 were applied to perform data analyses. Results: Eight RCTs enrolling 468 participants were included. Compared with 0.9% sodium chloride, dexmedetomidine decreased serum concentration of ALT (WMD = -66.54, 95% CI: -92.10--40.98), AST (WMD= -82.96, 95% CI: -106.74--59.17), TBIL (WMD = -4.51, 95% CI: -7.32--1.71), MDA (WMD = -3.09, 95% CI: -5.17--1.01), TNF-α (WMD = -36.54, 95% CI: -61.33--11.95) and IL-6 (WMD = -165.05, 95% CI: -225.76--104.34), increased SOD activity (WMD = 24.70, 95% CI: 18.09-31.30) within postoperative one day. There was no significant difference in intraoperative or postoperative recovery parameters between groups. Conclusions: Perioperative administration of dexmedetomidine can exert a protective effect on liver IR injury after hepatectomy. Additional studies are needed to further evaluate postoperative recovery outcomes of dexmedetomidine with different dosing regimens.

Metal-organic framework IRMOFs coated with a temperature-sensitive gel delivering norcantharidin to treat liver cancer.

BACKGROUND: Norcantharidin (NCTD) is suitable for the treatment of primary liver cancer, especially early and middle primary liver cancer. This compound can reduce tumors and improve immune function. However, the side effects of NCTD have limited its application. There is a marked need to reduce the side effects and increase the efficacy of NCTD. AIM: To develop a nanomaterial carrier, NCTD-loaded metal-organic framework IRMOF-3 coated with a temperature-sensitive gel (NCTD-IRMOF-3-Gel), aiming to improve the anticancer activity of NCTD and reduce the drug dose. METHODS: NCTD-IRMOF-3-Gel was obtained by a coordination reaction. The apparent characteristics and in vitro release of NCTD-IRMOF-3-Gel were investigated. Cell cytotoxicity assays, flow cytometry, and apoptosis experiments in mouse hepatoma (Hepa1-6) cells were used to determine the anti-liver cancer activity of NCTD-IRMOF-3-Gel in in vitro models. RESULTS: The particle size of NCTD-IRMOF-3-Gel was 50-100 nm, and the particle size distribution was uniform. The release curve showed that NCTD-IRMOF-3-Gel had an obvious sustained-release effect. The cytotoxicity assays showed that the free drug NCTD and NCTD-IRMOF-3-Gel treatments markedly inhibited Hepa1-6 cell proliferation, and the inhibition rate increased with increasing drug concentration. By flow cytometry, NCTD-IRMOF-3-Gel was observed to block the Hepa1-6 cell cycle in the S and G2/M phases, and the thermosensitive gel nanoparticles may inhibit cell proliferation by inducing cell cycle arrest. Apoptosis experiments showed that NCTD-IRMOF-3-Gel induced the apoptosis of Hepa1-6 cells. CONCLUSION: Our results indicated that the NCTD-IRMOF-3-Gel may be beneficial for liver cancer disease treatment.

Potentially Inappropriate Medication and Associated Factors Among Older Patients with Chronic Coronary Syndrome at Hospital Discharge in Beijing, China.

PURPOSE: Medication therapy is crucial in the management of chronic coronary syndrome (CCS). The use of potentially inappropriate medications (PIMs) contributes to poor outcomes in older patients, making it a major public health concern. However, few studies are available on PIMs use in older Chinese CCS patients. To investigate the frequency of prescribed PIMs at discharge and explore risk factors in older adults with CCS. PATIENTS AND METHODS: The cross-sectional study was conducted in a tertiary hospital in China over three months, from 1st October to 31st December, 2019. CCS patients aged over 60 years who were discharged alive were recruited. Information on demographics and medications at discharge was collected. Clinical data including diagnoses, frailty status, New York Heart Association (NYHA) class and age-adjusted Charlson Comorbidity Index (ACCI) were evaluated in each patient. PIMs were identified using the 2019 Beers criteria. Binary logistic regression was performed to recognize variables related to PIMs. RESULTS: A total of 447 eligible patients with 2947 medications were included. The prevalence of PIMs use was 38%. Medications to be avoided, to be used with caution, and with drug-drug interactions were 38.4%, 48.9% and 12.7% of the PIMs, respectively. Medications with drug-disease/syndrome interactions and those adjusted for kidney function were not identified. The common PIMs were diuretics (37.1%), benzodiazepines and benzodiazepine receptor agonist hypnotics (15.2%), glimepiride (13.1%), and co-prescription of potassium-sparing diuretics and renin-angiotensin system (RAS) inhibitors (9.7%). Individuals with frailty syndrome, polypharmacy, multiple comorbidities, atrial fibrillation, psychiatric disorders and greater NYHA class severity were more likely to receive PIMs. CONCLUSION: Prescription of PIMs was a common burden in older adults. A CCS multidisciplinary team is needed to control PIMs, especially in vulnerable older patients.

Anaphylaxis effect and substance basis of honeysuckle extract.

Objective: To explore the anaphylaxis effect and anaphylaxis substances of honeysuckle. Methods: Rat peritoneal mast cells (PMC) were separated and purified, the cells were incubated with compound 48/80 (0.02 g/L), physiological saline and honeysuckle extract (120 g/L) at 37 °C for 0, 15, 30, 45 and 60 min. Degranulation were observed by optical microscope and transmission electron microscope. Annexin V positive cell rate was detected by flow cytometry to reflect the degranulation rate of PMC. SD rats were supplied with honeysuckle extract by intravenous injection at a dose of 2.25 g/L. After administration, different parameters were analyzed, including the symptoms, histamine (HIS) and tryptase (MCT) levels, which were determined to explore the effect of anaphylaxis. Regression analysis was used to calculate the relationships between the peaks and the pharmacological effects to explore potentially anaphylactoid components. Results: The percentage of Annxin V positive cells and the degranulation ratio were markedly elevated in PMC treated with honeysuckle extract for more than 15 min (P < 0.05). HIS and MCT level were significantly elevated after injection of honeysuckle extract for more than 15 min. Morphology of PMC and systemic symptoms were also changed compared with the controlled group (P < 0.05). Regression analysis was used to calculate the relationship between peaks and pharmacological effects, and to determine peaks 7, 10 and 13 as possible anaphylactoid ingredients. Conclusion: This study established a prospective method to clarify the anaphylactoid components of honeysuckle extract, which would provide guidance for screening anaphylactoid components in traditional Chinese medicine injections containing honeysuckle in the prescription.

Hippocampus and serum metabolomic studies to explore the regulation of Chaihu-Shu-Gan-San on metabolic network disturbances of rats exposed to chronic variable stress.

Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine formula, has been effectively used for the treatment of depression. However, studies of its anti-depressive mechanism are challenging, due to the complex pathophysiology of depression, and complexity of CSGS with multiple constituents acting on different receptors. In the present work, metabolomic studies of biochemical changes in the hippocampus and serum of chronic variable stress (CVS)-induced depression rats after treatment with CSGS were performed using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Partial least squares-discriminate analysis indicated that the metabolic perturbation induced by CVS was reduced by treatment with CSGS. A total of twenty-six metabolites (16 from the hippocampus and 10 from serum) were considered as potential biomarkers involved in the development of depression. Among them, 11 were first reported to have potential relevance in the pathogenesis of depression, and 25 may correlate to the regulation of CSGS treatment on depression. The results combined with a previous study indicated that CSGS mediated synergistically abnormalities of the metabolic network, composed of energy metabolism, synthesis of neurotransmitters, tryptophan, phospholipids, fatty acid and bile acid metabolism, bone loss and liver detoxification, which may be helpful for understanding its mechanism of action. Furthermore, the extracellular signal-regulated kinase (ERK) signal pathway, involved in the neuronal protective mechanism of depression related to energy metabolism, was investigated by western blot analysis. The results showed that CSGS reversed disruptions of BDNF, ERK1/2 and pERK1/2 in CVS rats, which provides the first evidence that the ERK signal system may be one of the targets related to the antidepressant action of CSGS.

Identification of a linear B-cell epitope within the Bluetongue virus serotype 8 NS2 protein using a phage-displayed random peptide library.

The NS2 protein of Bluetongue virus (BTV) is an important non-structural protein and plays important roles in viral replication and assembly. In this study, one monoclonal antibody (mAb), 4D4, was raised against BTV8 NS2. Phage display technology was used and identified the consensus binding motif SNYD recognized by mAb 4D4. To define the minimal region required for antibody binding, a panel of synthetic peptides encompassing SNYD derived from the BTV8 NS2 was then used to more specifically define the 4D4 epitope as (149)RSNYDV(154). Furthermore, amino acid sequence alignments of different BTV serotypes and other orbiviruses suggested that this epitope is highly conserved among the BTV serotypes. The mAb reagent generated in this study may be applied to the development of BTV diagnosis and surveillance programs and the epitope defined here can lead to important insights into how BTV might interact with the sheep's immune system.

Integration of ¹H NMR and UPLC-Q-TOF/MS for a comprehensive urinary metabonomics study on a rat model of depression induced by chronic unpredictable mild stress.

Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing (1)H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21-25, and 27-36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1-2, 4-6, 15, 18, 20-23, 27, 32, 35-36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of (1)H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways.

[Application and advance of artificial antigens in studies on allergic reaction of traditional Chinese medicine injections].

There are some small molecules with potential allergenicity in traditional Chinese medicine injections. They are lack of immunogenicity due to their small molecular weight, but they can lead to allergic reactions when they were coupled with appropriate vectors. Therefore, how to couple small molecule semi-antigens with vectors to prepare complete antigens with immunogenicity and reactogenicity is the key for screening small molecular allergenic substances out of traditional Chinese medicine injections. In terms of semi-antigen characteristics of traditional Chinese medicine injections, vector selection and application, coupling method and complete antigen purification and identification, the author introduces the latest research situations of artificial antigen and antibody preparation technology, the advance in experimental studies on screening of allergenic substances in traditional Chinese medicine injections, as well as the application prospect of immuno-chip technology in studies on allergenic substances in traditional Chinese medicine injections, with the aim of providing new experimental thoughts and methods for safety control of traditional Chinese medicine injections.

Identification of two novel BTV16-specific B cell epitopes using monoclonal antibodies against the VP2 protein.

The VP2 protein of bluetongue virus (BTV) is an important structural protein and is the principal antigen responsible for BTV serotype specificity. In this study, we mapped the reactivity of two BTV16-specific monoclonal antibodies (MAbs) and identified two novel serotype-specific linear B cell epitopes on the BTV16 VP2 protein. By screening a series of peptides derived from the BTV16 VP2 protein and expressed as mannose-binding protein fusions, we determined that the linear epitopes recognized by the VP2-specific MAbs 3 G10 and 2B4 were located within the peptides 34EWSGHDVTEIPNRRMF49 and 540KNEDPYVKRTVKPIRA555, respectively. To define the minimal region required for antibody binding within these peptide regions, a series of progressively shorter peptides were synthesized and evaluated for 3 G10 and 2B4 binding. This work defined the motifs 34EWSGHDVTEIPNRRMF49 and 543DPYVKRTVK555 as the minimal linear peptides required for 3 G10 and 2B4 binding, respectively. Alignment of amino acid sequences from a number of BTV16 strains isolated from different regions indicated that these two epitopes are highly conserved among BTV16 strains. Furthermore, these two epitopes are not conserved among other BTV serotypes or prototype members of the genus Orbivirus in the family Reoviridae, as shown by sequence alignments. The MAb reagents and linear epitopes defined here provide the basis for the development of epitope-based serotype-specific differential diagnostic tools and may be useful in the design of epitope-based vaccines.

Identification of three novel linear B-cell epitopes on the VP5 protein of BTV16.

Bluetongue virus (BTV) VP5 protein is an important antigenic protein which is centrally involved in serotype determination and the virus entry process. Very little is known about the B-cell epitopes on the BTV VP5 protein recognized by humoral immune responses. In this study, we generated five BTV16 VP5 protein-specific monoclonal antibodies (MAbs), named 3B11, 2B10, 1H7, 4A6 and 3G9, and defined the linear epitopes recognized by MAbs using a series of peptides expressed as maltose-binding protein (MBP)-fusion polypeptides. Three novel linear B-cell epitopes were identified: 3B11 and 3G9 recognized the motif ITANTREIQHIKEE; 2B10 recognized the motif LSGID; and 4A6 recognized the motif STMVKEYRQKIDALKA. Exact sequences corresponding to the three motifs identified were found in the BTV16 VP5 protein ((310)ITANTREIQHIKEE(323), (265)LSGID(269) and (188)STMVKEYRQKIDALKA(203)). These motifs represent the minimal linear peptide sequence required for MAb reactivity, as binding of each MAb was abolished when additional amino acids were removed from the amino and carboxy termini of the peptide. Amino acid sequence alignment indicated that three epitopes were totally conserved among different BTV16 strains. The MAbs generated along with identified epitopes will be useful for examining VP5 protein function and the development of epitope-based marker vaccines against BTV.

Monoclonal antibodies against VP7 of bluetongue virus.

VP7 is a major group-specific protein of the bluetongue virus (BTV), and is therefore a candidate for use as a diagnostic reagent. In this study, BALB/c mice were immunized with BTV16, and the lymphocyte hybridoma technique and indirect ELISA screening method were employed to obtain two strains of hybridoma cells secreting specific monoclonal antibodies (MAbs) to BTV16. Eukaryotic recombinant plasmids coding for 10 segments of BTV16 separately were transfected into BHK-21 cells, respectively, followed by immunofluorescence, showing that two MAbs only reacted with BTV-VP7. Western blot analysis showed the same result. Indirect immunofluorescence results indicated that two of the MAbs present different response spectrums with BTV1~24 serotypes. These results indicate that these MAbs may be good candidates for a specific diagnostic method and functional exploration of the VP7 protein.