Herbal Preparation (Bromelain, Papain, Curcuma, Black Pepper) Enhances Mineralization and Reduces Glucocorticoid-Induced Osteoporosis in Zebrafish

Natural foods with antioxidant properties, such as curcuma, papain, bromelain and black pepper, have been indicated as a potential natural therapeutic approach against osteoporosis. Zebrafish are an excellent animal model to study the effects of herbal preparations on osteogenesis and bone metabolism, both in physiological and in pathological conditions. Our study was aimed at evaluating whether curcuma-bromelain-papain-pepper herbal preparation (CHP) administered in embryos and adult fish is capable of promoting bone wellness in physiological and osteoporotic conditions. The effect of CHP has been studied in embryonic osteogenesis and glucocorticoid-induced osteoporosis (GIOP) in an adult fish model in which drug treatment induces a bone-loss phenotype in adult scales very similar to that which characterizes the bones of human patients. CHP prevented the onset of the osteoporotic phenotype in the scales of GIOP in adult zebrafish, with the osteoblastic and osteoclastic metabolic activity maintaining unaltered. CHP is also able to attenuate an already established GIOP phenotype, even if the alteration is in an advanced phase, partially restoring the normal balance of the bone markers alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) and stimulating anabolic reparative processes. The results obtained indicated CHP as a potential integrative antioxidant therapy in human bone-loss diseases.

An optimized LC-HRMS untargeted metabolomics workflow for multi-matrices investigations in the three-spined stickleback

Environmental metabolomics has become a growing research field to understand biological and biochemical perturbations of organisms in response to various abiotic or biotic stresses. It focuses on the comprehensive and systematic analysis of a biologic system’s metabolome. This allows the recognition of biochemical pathways impacted by a stressor, and the identification of some metabolites as biomarkers of potential perturbations occurring in a body. In this work, we describe the development and optimization of a complete reliable methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) for untargeted metabolomics studies within a fish model species, the three-spined stickleback (Gasterosteus aculeatus). We evaluated the differences and also the complementarities between four different matrices (brain, gills, liver and whole fish) to obtain metabolome information. To this end, we optimized and compared sample preparation and the analytical method, since the type and number of metabolites detected in any matrix are closely related to these latter. For the sample preparation, a solid-liquid extraction was performed on a low quantity of whole fish, liver, brain, or gills tissues using combinations of methanol/water/heptane. Based on the numbers of features observed in LC-HRMS and on the responses of analytical standards representative of different metabolites groups (amino acids, sugars…), we discuss the influence of the nature, volume, and ratio of extraction solvents, the sample weight, and the reconstitution solvent. Moreover, the analytical conditions (LC columns, pH and additive of mobile phases and ionization modes) were also optimized so as to ensure the maximum metabolome coverages. Thus, two complementary chromatographic procedures were combined in order to cover a broader range of metabolites: a reversed phase separation (RPLC) on a C18 column followed by detection with positive ionization mode (ESI+) and a hydrophilic interaction chromatography (HILIC) on a zwitterionic column followed by detection with negative ionization mode (ESI-). This work provides information on brain, gills, liver, vs the whole body contribution to the stickleback metabolome. These information would help to guide ecotoxicological and biomonitoring studies.

Dynamic Analysis of a Phytoplankton-Fish Model with the Impulsive Feedback Control Depending on the Fish Density and Its Changing Rate

This paper proposes a comprehensive fishing strategy that takes into consideration the population density of fish and its current growth rate, which provides new ideas for fishing strategies. Firstly, we establish a phytoplankton-fish model with the impulsive feedback control depending on the density and rate of change of the fish. Secondly, the complex phase and impulse sets of this model are divided into three cases, then the Poincar´e map for the model is defined, and analyzed the properties of Poincar´e map. In addition, the sufficient and necessary conditions for the global asymptotic stability of the order-1 periodic solution and existence condition of order- k ( k ≥ 2) periodic solution are discussed. The action threshold depends on the density and rate of change of the fish, which is reasonable than earlier studies. The analysis method proposed in this paper also plays an important role in the analysis of impulse models with complex phase sets or impulse sets.

Determination of the Earliest time Point for Heat-shock Inducing Osteoporosis-like Phenotype in the rankl:HSE:CFP Medaka Fish Model for Osteoporosis

The transgenic medaka rankl:HSE:CFP expressing Rankl, a stimulator for osteoclastogenesis - the formation and activation of osteoclasts, bone “eating” cells, under control of a heat inducible promotor has been established as a model for osteoporosis to evaluate antiosteoporosis effects of substances. Transgenic larvae were usually heat-shocked for 90 minutes at 39oC when they were at 9 days post fertilization (dpf) and osteoporosis-like phenotype was analysed when they were at 11 dpf. In this study we investigated whether osteoporosis-like phenotype could be induced in the transgenic larvae when heat-shock was applied at earlier time points, when larvae were at 1, 2, 3, 4, 5, or 6 dpf. Results showed that heat-shocks from the time point of 3 dpf onwards resulted in osteoporosis-like phenotype while heat-shock at 1 or 2 dpf did not affect mineralized bone matrix in 11 dpf larvae. These provide important evidence for study on onset of Rankl induced osteoclasts in fish and help improving experimental procedures using this fish model for osteoporosis.

Measurement of Chromosomal Arms and FISH Reveal Complex Genome Architecture and Standardized Karyotype of Model Fish, Genus Carassius

The widely distributed ray-finned fish genus Carassius is very well known due to its unique biological characteristics such as polyploidy, clonality, and/or interspecies hybridization. These biological characteristics have enabled Carassius species to be successfully widespread over relatively short period of evolutionary time. Therefore, this fish model deserves to be the center of attention in the research field. Some studies have already described the Carassius karyotype, but results are inconsistent in the number of morphological categories for individual chromosomes. We investigated three focal species: Carassius auratus, C. carassius and C. gibelio with the aim to describe their standardized diploid karyotypes, and to study their evolutionary relationships using cytogenetic tools. We measured length (q+plength) of each chromosome and calculated centromeric index (i value). We found: (i) The relationship between q+plength and i value showed higher similarity of C. auratus and C. carassius. (ii) The variability of i value within each chromosome expressed by means of the first quartile (Q1) up to the third quartile (Q3) showed higher similarity of C. carassius and C. gibelio. (iii) The fluorescent in situ hybridization (FISH) analysis revealed higher similarity of C. auratus and C. gibelio. (iv) Standardized karyotype formula described using median value (Q2) showed differentiation among all investigated species: C. auratus had 24 metacentric (m), 40 submetacentric (sm), 2 subtelocentric (st), 2 acrocentric (a) and 32 telocentric (T) chromosomes (24m+40sm+2st+2a+32T) ; C. carassius: 16m+34sm+8st+42T; and C. gibelio: 16m+22sm+10st+2a+50T. (v) We developed R scripts applicable for the description of standardized karyotype for any other species. The diverse results indicated unprecedented complex genomic and chromosomal architecture in the genus Carassius probably influenced by its unique biological characteristics which make the study of evolutionary relationships more difficult than it has been originally postulated.

Do Single-Component and Mixtures Selected Organic UV Filters Induce Embryotoxic Effects in Zebrafish (Danio rerio)?

UVs are important ingredients in common cosmetic products (e.g., sunscreens, hairsprays, soap). After their use, they can enter the aquatic ecosystem and negatively affect non-target aquatic organisms. The aim of our study was to evaluate acute embryotoxicity of selected organic UVs 2-phenylbenzimidazole-5-sulfonic acid (PBSA), ethylhexyl methoxycinnamate (EHMC), octocrylene (OC), 4-methylbenzylidene camphor (4-MBC) and benzophenone-3 (BP-3). The chemicals were tested both as a single substance and their mixtures. The types of mixtures were chosen as follows: the combination of OC and 4-MBC; the combination of PBSA, EHMC and BP-3 and the combination of all five UV filters. The embryotoxicity was evaluated using a modified method of the Fish Embryo Acute Toxicity Test-OECD guideline 236 and zebrafish (Danio rerio) was selected as a suitable fish model organism. The toxic effects were studied by assessing mortality, hatching and the occurrence of malformations at 24, 48, 72 and 96 h post fertilization. The obtained results indicate that especially the mixture of OC and 4-MBC presents a potential risk of embryotoxicity for zebrafish due to a significant increase in mortality, which was 41.7% in the experimental group exposed to 10 μg/L at 96 h post fertilization. Based on our results, the most effected sub-lethal endpoints were hatching and malformation (e.g., edema of pericard, bent spine, yolk edema), but with no statistically significant effect. These results differ within groups with single UVs and with their mixtures, suggesting the interaction of these substances when they are exposed together.