Efficacy of hydroethanolic extract of Randia aculeata seed against the southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on naturally infested cattle under field conditions.

The cattle tick Rhipicephalus (Boophilus) microplus is a major problem of concern for cattle industry in tropical and subtropical areas. Control of cattle tick is based mainly on the use of chemical acaricides, which has contributed to the emerging problem of selection of resistant tick lineages. Plants have been used as an alternative to conventional acaricidal drugs. On the other hand, the acaricidal activity of hydroethanolic extract of Randia aculeata seed (EHRA) has been demonstrated against R. microplus under laboratory conditions. However, the utility of EHRA seed as a potential acaricidal needs to be determined under field conditions. For this reason, the aim of this study was to evaluate the efficacy of the EHRA against R. microplus sprayed on naturally infested calves, determine the effect of the EHRA seed on acetylcholinesterase activity in R. microplus larval and identify the chemical composition of EHRA. Forty-five male calves were divided in three groups and treated with: G1 water; G2 EHRA 20% w/v and G3 coumaphos 0.2% v/v. Acetylcholinesterase (AChE) activity in R. microplus larvae was determined by a colorimetric assay. The chemical composition of EHRA was accessed through HPLC/MS. Significantly fewer ticks were observed after 24 h on the treated group compared to control group. EHRA significantly inhibited in vitro AChE activity in R. microplus at all tested concentrations. Chlorogenic acid, vanillinic acid, p-coumaric acid, caffeic acid. rutin, quercetin, (-)-epicatechin, 4-hydroxybenzoic acid, quercetin, vanillin, 2,4-dimethoxy-6-methylbenzoic acid, scopoletin and ferulic acid were identified in the extract. The results provided new data for the elucidation of the mechanisms of EHRA acaricide action and to further evaluate the use as a new alternative control agent against R. microplus under in vivo conditions.

Methodological advances and future directions of microalgal bioassays for evaluation of potential toxicity in environmental samples: A review.

Microalgal bioassays are widely applied to evaluate the potential toxicity of various persistent toxic substances in environmental samples due to multiple advantages, including high sensitivity, short test duration, and cost-effectiveness. Microalgal bioassay is gradually developing in method, and the scope of application to environmental samples is also expanding. Here, we reviewed the published literature on microalgal bioassays for environmental assessments, focusing on types of samples, sample preparation methods, and endpoints, and highlighted key scientific advancements. Bibliographic analysis was performed with the keywords 'microalgae' and 'toxicity' or 'bioassay', and 'microalgal toxicity'; 89 research articles were selected and reviewed. Traditionally, most studies implementing microalgal bioassays focused on water samples (44%) with passive samplers (38%). Studies using the direct exposure method (41%) of injecting microalgae into sampled water mainly evaluated toxic effects by growth inhibition (63%). Recently, various automated sampling techniques, in situ bioanalytical methods with multiple endpoints, and targeted and non-targeted chemical analyses have been applied. More research is needed to identify causative toxicants affecting microalgae and to quantify the cause-effect relationships. This study provides the first comprehensive overview of recent advances in microalgal bioassays performed with environmental samples, suggesting future research directions based on current understanding and limitations.

Cryostructuring of Polymeric Systems: 64. Preparation and Properties of Poly(vinyl alcohol)-Based Cryogels Loaded with Antimicrobial Drugs and Assessment of the Potential of Such Gel Materials to Perform as Gel Implants for the Treatment of Infected Wounds.

Physical macroporous poly(vinyl alcohol)-based cryogels formed by the freeze-thaw technique without the use of any foreign cross-linkers are of significant interests for biomedical applications. In the present study, such gel materials loaded with the antimicrobial substances were prepared and their physicochemical properties were evaluated followed by an assessment of their potential to serve as drug carriers that can be used as implants for the treatment of infected wounds. The antibiotic Ceftriaxone and the antimycotic Fluconazole were used as antimicrobial agents. It was shown that the Ceftriaxone additives caused the up-swelling effects with respect to the cryogel matrix and some decrease in its heat endurance but did not result in a substantial change in the gel strength. With that, the drug release from the cryogel vehicle occurred without any diffusion restrictions, which was demonstrated by both the spectrophotometric recording and the microbiological agar diffusion technique. In turn, the in vivo biotesting of such drug-loaded cryogels also showed that these materials were able to function as rather efficient antimicrobial implants injected in the artificially infected model wounds of laboratory rabbits. These results confirmed the promising biomedical potential of similar implants.

Cryostructuring of Polymeric Systems: 63. Synthesis of Two Chemically Tanned Gelatin-Based Cryostructurates and Evaluation of Their Potential as Scaffolds for Culturing of Mammalian Cells.

Various gelatin-containing gel materials are used as scaffolds for animal and human cell culturing within the fields of cell technologies and tissue engineering. Cryostructuring is a promising technique for the preparation of efficient macroporous scaffolds in biomedical applications. In the current study, two new gelatin-based cryostructurates were synthesized, their physicochemical properties and microstructure were evaluated, and their ability to serve as biocompatible scaffolds for mammalian cells culturing was tested. The preparation procedure included the dissolution of Type A gelatin in water, the addition of urea to inhibit self-gelation, the freezing of such a solution, ice sublimation in vacuo, and urea extraction with ethanol from the freeze-dried matter followed by its cross-linking in an ethanol medium with either carbodiimide or glyoxal. It was shown that in the former case, a denser cross-linked polymer phase was formed, while in the latter case, the macropores in the resultant biopolymer material were wider. The subsequent biotesting of these scaffolds demonstrated their biocompatibility for human mesenchymal stromal cells and HepG2 cells during subcutaneous implantation in rats. Albumin secretion and urea synthesis by HepG2 cells confirmed the possibility of using gelatin cryostructurates for liver tissue engineering.

Evaluation of ecotoxicological effects associated with coastal sediments of the Yellow Sea large marine ecosystem using the marine copepod Tigriopus japonicus.

A copepod bioassay with Tigriopus japonicus was applied to evaluate the relative ecotoxicity of sediments in the Yellow and Bohai seas, and contributions of individual PAHs to copepod toxicity were evaluated. Mean toxicity was greatest in the Yellow Sea of China, followed by the Bohai Sea and Yellow Sea of Korea. Elevated concentrations of sedimentary PAHs, alkylphenols, and styrene oligomers back-supported the significant toxicities observed in bioassay. Copepod toxicity in relation to PAHs indicated the greatest contribution by indeno[1,2,3-c,d]pyrene. However, lacked contribution by PAHs, viz., 2.4 and 3.0 % for the total immobilization and mortality, respectively, indicated a large proportion of unknown toxicants being widely distributed along the Yellow Sea Large Marine Ecosystem (YSLME) coastline. Overall, the present study provides useful baseline information for evaluating the potential sedimentary toxicants, with emphasizing further investigation to identify the unknown toxicants at an LME scale, and elsewhere.

Bioassay-based identification and removal of target and suspect toxicants in municipal wastewater: Impacts of chemical properties and transformation.

Municipal wastewater contains numerous chemicals and transformation products with highly diverse physiochemical properties and intrinsic toxicity; thus, it is imperative but challenging to identify major toxicants. Herein, toxicity identification evaluation (TIE) was applied to identify major toxicants in a typical municipal wastewater treatment plant (WWTP). Impacts of chemical properties on the removal of contaminants and toxicity at individual treatment stages were also examined. The WWTP influent caused 100% death of Daphnia magna and zebrafish embryos, and toxicity characterization suggested that organics, metals, and volatiles all contributed to the toxicity. Toxicity identification based on 189 target and approximately one-thousand suspect chemicals showed that toxicity contributions of organic contaminants, metals, and ammonia to D. magna were 77%, 4%, and 19%, respectively. Galaxolide, pyrene, phenanthrene, benzo[a]anthracene, fluoranthene, octinoxate, silver, and ammonia were identified as potential toxicants. Comparatively, the detected transformation products elicited lower toxicity than their respective parent contaminants. In contrast, the analyzed contaminants showed negligible contributions to the toxicity of zebrafish embryos. Removal efficiencies of these toxicants in WWTP were highly related to their hydrophobicity. Diverse transformation and removal efficiencies of contaminants in WWTPs may influence the chemical compositions in effluent and ultimately the risk to aquatic organisms in the receiving waterways.

Development of a robust reporter gene-based assay for the bioactivity determination of recombinant human follicle stimulating hormone (rhFSH) pharmaceutical products.

FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.

Time-Integrative Passive sampling combined with TOxicity Profiling (TIPTOP): an effect-based strategy for cost-effective chemical water quality assessment.

This study aimed at demonstrating that effect-based monitoring with passive sampling followed by toxicity profiling is more protective and cost-effective than the current chemical water quality assessment strategy consisting of compound-by-compound chemical analysis of selected substances in grab samples. Passive samplers were deployed in the Dutch river delta and in WWTP effluents. Their extracts were tested in a battery of bioassays and chemically analyzed to obtain toxicity and chemical profiles, respectively. Chemical concentrations in water were retrieved from publicly available databases. Seven different strategies were used to interpret the chemical and toxicity profiles in terms of ecological risk. They all indicated that the river sampling locations were relatively clean. Chemical-based monitoring resulted for many substances in measurements below detection limit and could only explain <20% of the observed in vitro toxicity. Effect-based monitoring yielded more informative conclusions as it allowed for ranking the sampling sites and for estimating a margin-of-exposure towards chronic effect ranges. Effect-based monitoring was also cheaper and more cost-effective (i.e. yielding more information per euro spent). Based on its identified strengths, weaknesses, opportunities, and threats (SWOT), a future strategy for effect-based monitoring has been proposed.

Ecotoxicological impacts of surface water and wastewater from conventional and advanced treatment technologies on brood size, larval length, and cytochrome P450 (35A3) expression in Caenorhabditis elegans.

Anthropogenic micropollutants and transformation products (TPs) negatively affect aquatic ecosystems and water resources. Wastewater treatment plants (WWTP) represent major point sources for (micro)pollutants and TPs in urban water cycles. The aim of the current study was to assess the removal of micropollutants and toxicity during conventional and advanced wastewater treatment. Using wild-type and transgenic Caenorhabditis elegans, the endpoint reproduction, growth, and cytochrome P450 (CYP) 35A3 induction (via cyp-35A3::GFP) were assessed. Samples were collected at four WWTPs and a receiving surface water. One WWTP included the advanced treatments: ozonation followed by granular activated carbon (GAC) or biological filtration (BF), respectively. Relevant micropollutants and WWTP parameters (n = 111) were included. Significant reproductive toxicity was detected for one WWTP effluent (31-83% reduced brood size). Three of four effluents significantly promoted the growth of C. elegans larvae (49-55% increased lengths). This effect was also observed for the GAC (34-41%) and BF (30%) post-treatments. Markedly, significant cyp-35A3::GFP induction was detected for one effluent before and after ozonation, being more pronounced for the ozonated samples (5- and 7.4-fold above controls). While the advanced treatments decreased the concentrations of most micropollutants, the observed effects may be attributed to effects of residual target compounds and/or compounds not included in the target chemical analysis. This highlights the need for an integrated assessment of (advanced) wastewater treatment covering both biological and chemical parameters.

Acute-Contact and Chronic-Systemic In Vivo Bioassays: Regional Monitoring of Susceptibility to Thiamethoxam in Soybean Aphid (Hemiptera: Aphididae) Populations From the North Central United States.

The risks associated with soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), in the North Central soybean systems has fostered the adoption of prophylactic chemical control practices, such as planting seeds treated with neonicotinoid insecticides, especially thiamethoxam. Consequently, there is a concern that increased selection pressure imposed on the arthropod-pest complex by this insecticide may lead to resistance. Therefore, in vivo bioassays were conducted to determine the susceptibility of soybean aphid to thiamethoxam among North Central U.S. POPULATIONS: Concentration-mortality data were collected using contact glass-vial and detached-leaf systemic bioassays. The results of these experiments indicate that both bioassays were reliable to detect shifts in susceptibility between different soybean aphid clones. The LC50s of field populations of soybean aphid were significantly different when mortality was recorded in contact and systemic exposure assays. Nevertheless, the magnitude of the resistance ratios was consistent in both methods. In addition, a significant increase in the LC50 and EC50 values was observed among field populations tested in detached-leaf systemic bioassays. These results represent the first extensive efforts to identify the variability in susceptibility of soybean aphid to thiamethoxam in the North Central United States Therefore, our results provide a baseline for future assessment and contribute to a better understanding of the applicability of in vivo bioassays for susceptibility monitoring and resistance detection of soybean aphid to thiamethoxam.

In vivo toxicity assessment of aflatoxin B1-contaminated corn after ozone degradation.

Corn is an important food and feedstuff in China and worldwide. The problems caused by aflatoxin B1-contaminated corn (ACC) are of great concern. Our previous studies have demonstrated that ozone can effectively degrade AFB1 in corn, prompting us to investigate the in vivo toxicity of treated ACC. In this study, 35 Kunming mice were used to assess the in vivo toxicity of ozone treated ACC. Results indicated that compared to mice fed with basal feedstuff (provided by the Shanghai SLAC Laboratory), those fed with ACC have significantly decreased mean weight as well as total protein (TP), albumin (ALB), and globulin (GLB) contents (p < 0.05). On the other hand, the liver and kidney/body weight ratio as well as the serum alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels significantly increased (p < 0.05). Obvious histopathological changes were found in the liver and kidney. When mice were fed with the ozone-treated ACC, no significant differences were observed in the mean weight, the liver and kidney/body weight ratio and in the major serum indexes ALT, TP, ALB, and GLB (p > 0.05). However, AST and ALP significantly increased (p < 0.05), and slight histopathological changes were found in liver tissues. This study indicated that ACC may lead to significant changes in various physiological characteristics and biochemical indexes in liver and kidney tissues, but ozone treatment of ACC could significantly reduce these changes.

Thyroid monitoring of adults and children after reactor accidents with a new dose rate measurement device.

A specialized dose rate measurement device that was designed for monitoring the thyroid dose of children and adults after reactor accidents was tested. In measurements with neck phantoms and a human patient, the device was found to be capable of measuring the required low dose rates, even within increased ambient radiation. It is suitable for the application in emergency care centers.

Factors influencing preclinical in vivo evaluation of mumps vaccine strain immunogenicity.

Immunogenicity testing in animals is a necessary preclinical assay for demonstration of vaccine efficacy the results of which are often the basis for the decision whether to proceed or withdraw the further development of the novel vaccine candidate. However, in vivo assays are rarely, if at all, optimized and validated. Here we clearly demonstrate the importance of in vivo assay (mumps virus immunogenicity testing in guinea pigs) optimization for gaining reliable results and the suitability of Fractional factorial design of experiments (DoE) for such a purpose. By the use of DoE with resolution IV (2IV((4-1))) we clearly revealed that the parameters significantly increasing assay sensitivity were interval between animal immunizations followed by the body weight of experimental animals. The quantity (0 versus 2%) of the stabilizer (fetal bovine serum, FBS) in the sample was shown as non-influencing parameter in DoE setup. However, the separate experiment investigating only the FBS influence, and performed under other parameters optimally set, showed that FBS also influences the results of immunogenicity assay. Such finding indicated that (a) factors with strong influence on the measured outcome can hide the effects of parameters with modest/low influence and (b) the matrix of mumps virus samples to be compared for immunogenicity must be identical for reliable virus immunogenicity comparison. Finally the 3 mumps vaccine strains widely used for decades in the licensed vaccines were for the first time compared in an animal model, and results obtained were in line with their reported immunogenicity in human population supporting the predictive power of the optimized in vivo assay.

Variation of prostaglandin E2 concentrations in ovaries and its effects on ovarian maturation and oocyte proliferation in the giant fresh water prawn, Macrobrachium rosenbergii.

Prostaglandins (PGs) are important bioactive mediators for many physiological functions. In some decapod crustaceans, prostaglandin E2 (PGE2) has been detected in reproductive organs, and may play a role in the control of ovarian maturation. However, in the freshwater prawn, Macrobrachium rosenbergii, the presences of PGE2 and key enzymes for PGE2 biosynthesis, as well as its effects on ovarian maturation have not yet been investigated. In this study we reported the presence of PGE2, cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) in the ovarian tissues of M. rosenbergii, using immunohistochemistry. Intense immunoreactivities of PGE2 (PGE2-ir), COX1 (Cox1-ir) and PGES (PGES-ir) were detected in previtellogenic oocytes (Oc1 and Oc2), while the immunoreactivities were absent in the late vitellogenic oocytes (Oc4). This finding supports the hypothesis that the PGE2 biosynthesis occurs in the ovary of this prawn. To ascertain this finding we used LC-MS/MS to quantitate PGE2 concentrations during ovarian developmental cycle. The levels of PGE2 were significantly higher in the early ovarian stages (St I and II) than in the late stages (St III and IV). Moreover, we found that administration of PGE2 stimulated the ovarian maturation in this species by shortening the length of the ovarian cycle, increasing ovarian-somatic index, oocyte proliferation, and vitellogenin (Vg) level in the hemolymph.

Collaborative study for the establishment of erythropoietin BRP batch 4.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin (EPO) is used as a working standard for potency determination of EPO preparations by in vivo bioassay as prescribed in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). The BRP batch 3 was calibrated in 2006 and its stocks are depleted. The European Directorate for the Quality of Medicines & HealthCare (EDQM) thus initiated a project to calibrate a replacement batch in International Units against the WHO 3(rd) International Standard (IS) for Erythropoietin, recombinant, for bioassay (11/170). A Ph. Eur. Chemical Reference Substance (CRS) was established recently for use as reference in some of the physicochemical tests prescribed in the monograph. Therefore, the EPO BRP batch 4 was only calibrated for the normocythaemic and polycythaemic mouse in vivo bioassays described in the Assay section of the Ph. Eur. monograph (1316). The collaborative study involved seven laboratories from Europe, the USA and South America. The results confirmed that the candidate BRP (cBRP) is suitable for use as a reference preparation in the potency determination of EPO medicinal products by bioassay (using the normocythaemic or polycythaemic mouse methods). The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as erythropoietin BRP batch 4 in November 2014 for use as a reference preparation solely for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 13 000 IU/vial. Furthermore, the potency of BRP3 was confirmed during the study, thus warranting a good continuity of the IU.

In vitro and in vivo toxicities of sediment and surface water in an area near a major steel industry of Korea: endocrine disruption, reproduction, or survival effects combined with instrumental analysis.

The influence of industrial and/or municipal contaminant inputs on the aquatic environment of Pohang, Korea was investigated, with a focus on bioassay combined with instrumental analysis. Pohang is the most heavily populated city in Gyeongsangbuk-do province of Korea, with more than half a million residents, and also hosts the nation's biggest steel manufacturer and related industries. Sediment (n=15) and surface water samples (n=17) were collected from Hyeongsan River which runs across the Pohang city, in two separate events, i.e., June 2010 and February 2011. Sediment samples were first Soxhlet-extracted (raw extract) and were measured for estrogenicity using H295R cell line, and also analyzed for alkylphenols (APs), bisphenol A (BPA), PAHs, and PCBs. For sediment samples which exhibited greatest effects in the cell line, further fractionation was performed into non-polar, mid-polar, and polar portions. In surface water samples, heavy metals were also analyzed. Among 15 sediment samples, station S2 near the steel industry complex and station M3 near the municipal area showed the greatest sex hormone changes, and these changes were generally explained by the fractions which contained APs and BPA. Principal component analysis (PCA) however suggests that chemicals that were not analyzed in the present study would better explain endocrine disruption capacity of sediments. In water samples, adverse effects on hatchability and growth of Japanese medaka fish, and on Daphnia reproduction were noted following exposure to six water samples collected from stations near industrial and municipal areas. Several heavy metals and nonylphenol (NP) concentrations exceeded surface water quality guidelines, suggesting adverse effects of contamination inputs from both industrial and municipal activities. Observed estrogenicities in stations such as S2 and M3 warrant further investigations on longer term ecosystem impacts near industrial and municipal areas. The levels of major organic chemicals in sediments are quite comparable to those reported in ~10 years ago, emphasizing a need for source control.