The potential of organoids in toxicologic pathology: role of toxicologic pathologists in in vitro chemical hepatotoxicity assessment

The development of in vitro toxicity assessment methods using cultured cells has gained popularity for promoting animal welfare in animal experiments. Herein, we briefly discuss the current status of hepatoxicity assessment using human- and rat-derived hepatocytes; we focus on the liver organoid method, which has been extensively studied in recent years, and discuss how toxicologic pathologists can use their knowledge and experience to contribute to the development of in vitro chemical hepatotoxicity assessment methods for drugs, pesticides, and chemicals. We also propose how toxicological pathologists should assess toxicity regarding the putative distribution of undifferentiated and differentiated cells in the organoid when liver organoids are observed in hematoxylin and eosin–stained specimens. This was done while considering the usefulness and limitations of in vitro studies for toxicologic pathology assessment.     

Asynchrony in shoot and root phenological relationships in hybrid walnut

New Forests - Understanding the processes driving plant phenology is crucial for assessing how shifts in climate affect plant productivity and species’ distribution. Despite the key role of...     

Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.     

Identification of a mannose‐binding lectin‐like protein recognized by the anti‐CD25 antibody in haemocytes from Cherax quadricarinatus

The crustacean haemolymph contains three main cell populations; however, it is not clear which mechanisms participate in the regulation of cells related to innate immunity. This work aimed to identify potential interleukin‐like receptors that could regulate cellular responses in Cherax quadricarinatus. By histochemical analysis with murine anti‐CD25 staining (targeting the α‐chain of the IL‐2 receptor), we identified that this antibody recognizes cytoplasmic granules in semigranular and granular haemocytes. In haemocytes stimulated with phorbol myristate acetate (PMA), increased fluorescence was observed in these cytoplasmic granules, whereas staining with a human IL‐2 antibody after stimulation with 1–10 ng/ml PMA revealed no overexpression of the receptor or oxidative burst in haemocytes. Two‐dimensional Western blot analysis of haemocyte lysates showed that anti‐CD25 identified a 27.4‐kDa protein with an isoelectric point (pI) of 7.7 and a 46‐kDa protein with a pI of 6.9. De novo sequencing of these proteins identified that they had 32% homology with a mannose‐binding lectin (MBL) from Pacifastacus leniusculus. Our results indicate that a mannose‐binding lectin‐like protein could exert a protective effect that prevents damage from other activated immune responses.     

Genetic parameters of survival in four generations of Indian white shrimp (Fenneropeneaus indicus)

This study evaluated the genetic variance for survival at harvest of Indian white shrimp (Fenneropenaeus indicus) using a pedigreed synthetic population generated upon collecting broodstock from different fishery grounds in Egypt. The estimated heritabilities (h² ± SE) of survival from generations G₀ to G₃ ranged from 0.144 ± 0.026 to 0.008 ± 0.030 using a model that included the common environmental effect (c²). Low heritability (0.014 ± 0.015) was estimated across generations with the inclusion of common environmental effect (c²). However, the h² of survival from G₀ to G₁ ranged from 0.569 ± 0.071 to 0.410 ± 0.011 when c² was not included. The genetic correlation for survival between different ponds from G₀ to G₃ was moderate to high (0.79 ± 0.042 to 0.83 ± 0.012) but significantly different (P < 0.05). There were low correlation coefficients (?0.237 ± 0.089 to 0.316 ± 0.073) between body weight estimated breeding value (EBV) and survival EBV of families in four generations.     

Effect of dietary protein-energy levels and fish initial sizes on growth rate, development and production of Nile tilapia, Oreochromis niloticus L.

The effects of dietary protein‐energy levels on the growth rate, proximate composition and production were examined in Nile tilapia, Oreochromis niloticus, at two starting weights (22.9 and 39.8 g) reared in concrete ponds for 180 days. The highest weight gain (183.1 g) was obtained with fish fed a 30% protein and 10.5 kJ g^?1 diet for the small initial size and 180.2 g for a diet containing 25% protein and 12.6 kJ g^?1 for the large initial size. Dressed yields (edible mass) and fillets increased to 56.9% and 52.5% in fish fed diet with 25% protein and 10.5 kJ g^?1 at the initial size of 22.9 g, while fish started at 39.8 g exhibited the best values (56.5% and 52.1%) when fed the 30% protein and 10.5 kJ g^?1 diet. Proximate composition of soft tissue (wet weight basis) in small fish was significantly influenced by dietary protein‐energy levels. Protein was 26.1±0.3% in fish fed the high protein (30%) and low energy (10.5 kJ g^?1 diet), while lipid content was 6.4±0.3% at diet containing 20% protein and 14.7 kJ g^?1 diet. Large initial size fish fed the diet with 25% protein and 14.7 kJ g^?1 had the highest body protein (32.0±0.4%) and lowest lipid content (2.2±0.3%). Feed conversion ratio (FCR) and protein efficiency ratio varied with different dietary protein‐energy levels and initial fish sizes. Feed conversion ratio increased with increasing protein and decreasing energy level in the diet, and values in small fish were higher than values in large fish. Protein efficiency ratio decreased with increasing dietary protein level and decreasing energy level. The maximum total production (7.6 tons feddan^?1) was with dietary high protein (30%) and low energy (10.5 kJ g^?1) for small‐sized fish, while large initial fish had the highest production (3.7 tons feddan^?1) when fed the 25% protein and 12.6 kJ g^?1 diet energy. Starting with 22.9 g fish was more advantageous than the initial size of 39.8 g for rearing Nile tilapia. Small fish required a high‐protein and low‐energy diet, whereas large fish required a low‐protein and high‐energy diet to achieve highest production.     

Impact of wheat-Leymus racemosus added chromosomes on wheat adaptation and tolerance to heat stress

Adaptation of wheat (Triticum aestivum L.) to high temperatures could be improved by introducing alien genes from wild relatives. We evaluated the responses of wheat-Leymus racemosus chromosome introgression lines to high temperature to determine their potentiality for developing improved wheat cultivars. Introgression lines and their parent Chinese Spring were evaluated in a growth chamber at the seedling stage and in the field at the reproductive stage in two heat-stressed environments in Sudan. Optimum and late planting were used to ensure exposure of the plants to heat stress at the reproductive stage. The results revealed the impact of several Leymus chromosomes in improving wheat adaptation and tolerance to heat. Three lines possessed enhanced adaptation, whereas two showed high heat tolerance. Two addition lines showed a large number of kernels per spike, while one possessed high yield potential. Grain yield was correlated negatively with the heat susceptibility index, days to heading and maturity and positively with kernel number per spike and triphenyl tetrazolium chloride assay under late planting. The findings suggest that these genetic stocks could be used as a bridge to introduce the valuable Leymus traits into a superior wheat genetic background, thus helping maximize wheat yield in heat-stressed environments.     

Physiological and biochemical responses for two cultivars of Pisum sativum (“Merveille de Kelvedon” and “Lincoln”) to iron deficiency conditions

The aim of this work was to compare the tolerance and the physiological responses to Fe deficiency of two Pisum sativum cultivars (“Merveille de kelvedon” and “Lincoln”) commonly cultivated in Tunisia. We studied the effects of Fe deficiency on: (i) chlorophyll content, relative growth rate (RGR) and Fe status, (ii) rhizosphere acidification, (iii) changes under Fe-deficient conditions in the activities of two root enzymes, the first related to the proton extrusion (H⁺-ATPase) and the second to iron reduction mechanism (Fe(III)-chelate-reductase: FCR). Three treatments were used: C, control, complete nutrient solution (CNS) containing 30 μM Fe; DD, direct deficiency, CNS without iron; ID, indirect deficiency, CNS containing 30 μM Fe + lime. Fe deficiency led to a significant decrease of chlorophyll content in both cultivars. The below reduction was observed in Fe-deficient plants of Merveille de Kelvedon. In addition, relative growth of shoots and whole plant was not affected by Fe deficiency. H⁺-ATPase and FCR activities were more stimulated in Merveille de Kelvedon than in Lincoln, under DD and ID Fe deficiency. The capacity of this cultivar to maintain plant growth and to preserve adequate chlorophyll synthesis under iron-limiting conditions is related to its better Fe-use efficiency, in addition to its high acidification and root reducing capacities. This allows us to suggest that Merveille de Kelvedon is more effective in overcoming Fe deficiency than Lincoln.