The size-dependent effects of nanoplastics in mouse primary hepatocytes from cells to molecules.

Nanoplastics (NPs) are easily ingested by organisms and their major accumulation organ was determined to be liver. To date, the size-dependent cytotoxicity of NPs on mammalian hepatocytes remains unclear. This study utilized mouse primary hepatocytes and catalase (CAT) as specific receptors to investigate the toxicity of NPs from cells to molecules, focusing on size-dependent effects. Results showed that the larger the particle size of NP at low doses (≤ 50 mg/L), the most pronounced inhibitory effect on hepatocyte viability. 20 nm NPs significantly inhibit cell viability only at high doses (100 mg/L). Larger NP particles (500 nm and 1000 nm) resulted in a massive release of lactate dehydrogenase (LDH) from the cell (cell membrane damage). Reactive oxygen species (ROS), superoxide dismutase (SOD) and CAT tests suggest that NPs disturbed the cellular antioxidant system. 20 nm NPs show great strength in oxidizing lipids and disrupting mitochondrial function compared to NPs of other particle sizes. The degree of inhibition of CAT activity by different sized NPs was coherent at the cellular and molecular levels, and NP-500 had the most impact. This suggests that the structure and microenvironment of the polypeptide chain in the vicinity of the CAT active site is more susceptible to proximity and alteration by NP-500. In addition, the smaller NPs are capable of inducing relaxation of CAT backbone, disruption of H-bonding and reduction of α-helix content, whereas the larger NPs cause contraction of CAT backbone and increase in α-helix content. All NPs induce CAT fluorescence sensitization and make the chromophore microenvironment hydrophobic. This study provides new insights for NP risk assessment and applications.

The combined effects of polystyrene nanoplastics with nickel on oxidative stress and related toxic effects to earthworms from individual and cellular perspectives.

Nanoplastics may adsorb other pollutants in the environment due to their high specific surface area and small size. We used earthworms as experimental organisms to evaluate the ecotoxicity of NPs and Ni combined pollution at the individual and cellular levels. The results showed that when only 20 mg/L Ni2+ was added to the combined pollution system, the antioxidant system of earthworm coelomocytes was destroyed to a certain extent, the ROS level increased, the cell viability decreased significantly, and the redox balance was destroyed. With the introduction of PS-NPs and the increase of concentration, the oxidative damage in the coelomocytes of earthworms gradually increased, and finally tended to be stable when the maximum concentration of 50 mg/L PS-NPs and Ni were exposed together. At the animal level, the activities of CAT and SOD decreased within 28 days of exposure, and the combined pollution showed a synergistic effect. At the same time, it promoted the synthesis of GST in earthworms, improved their detoxification ability and reduced oxidative damage. The changes of T-AOC and MDA showed that the combined pollution caused the accumulation of ROS and caused more serious toxicological effects. With the increase of exposure time, the antioxidant system of earthworms was continuously destroyed, and the oxidative damage was serious, which induced more serious lipid peroxidation and caused the damage of earthworm body wall structure.

Compound effect and mechanism of oxidative damage induced by nanoplastics and benzo [a] pyrene.

Nanoplastics and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soil environments. In order to objectively evaluate the toxic interaction between polystyrene nanoplastics (PS NPs) and benzo [a] pyrene (BaP), oxidative damage at the level of earthworm cells and biomacromolecules was investigated by experiments combined with molecular dynamics simulation. Studies on cells reveal that PS NPs and BaP had synergistic toxicity when it came to causing oxidative stress. Cellular reactive oxygen species (ROS) levels under combined pollutant exposure were 24% and 19% higher, respectively than when PS NPs and BaP were exposed alone (compared to the blank group). In addition, BaP and PS NPs inhibited the ability of CAT to decompose H2O2 by affecting the structure of the proximal amino acid Tyr 357 in the active center of CAT, which exacerbated oxidative stress to a certain extent. Therefore, the synergistic toxic effect of BaP and PS NPs is due to the mutual complement of the two to the induction of protein structural looseness, and the strengthening of the stability of the conjugate (CAT-BaP-PS) under the weak interaction. This work provides a new perspective and approach on how to talk about the toxicity of combined pollutants.

Size-dependent effects of nanoplastics on structure and function of superoxide dismutase.

The ubiquitous existence of nano-plastics (NPs) has attracted widespread concern. Currently, the uptake of NPs by organisms and cells has been reported. However, knowledge about the interaction between NPs and protein is still limited, and there is a gap in research on the size-dependent toxicity of NPs toward protein. In this study, multi-spectroscopic techniques and enzyme activity determination were used to explore the structure and function changes of the main antioxidant enzyme superoxide dismutase (SOD), caused by the binding of NPs with different particle sizes. Results indicated NPs with different sizes can directly interact with SOD. NPs with smaller sizes result in looser skeletons of SOD, while the larger lead to tighter peptide chains. In addition, NPs can bind with SOD to form complexes, and the smaller the NPs are easier to be induced to coalesce by SOD. The surface curvature of 100 nm NPs was more conducive to varying the secondary structure of SOD. NPs of 100 nm and 500 nm can cause greater sensitization of SOD endogenous fluorescence, and increase the polarity around tyrosine residue. The enzyme activity assay further revealed the functional differences caused by the size-dependent effects of NPs. NPs of 100 nm and 20 nm induced a more significant change in SOD activity (increased by 20% and 8%, respectively), while NPs of 500 nm and 1000 nm had a little impact on it. Together, smaller NPs have a greater impact on the structure and function of SOD. This study revealed the size-dependent toxicity of NPs to protein, which provided a rationale for the necessary avoidance and substitution of NPs in engineering applications.

Comparison of Rumen Microbiota and Serum Biochemical Indices in White Cashmere Goats Fed Ensiled or Sun-Dried Mulberry Leaves.

Mulberry leaves, which have high nutritional value, have not been fully utilized. Few research systems have indicated whether mulberry leaves can replace traditional feed ingredients in goats. In this study, we investigated the effects of feeding white cashmere goats ensiled (Group E) or sun-dried mulberry leaves (Group S) on changes in ruminal microbial communities, rumen fermentation parameters and serum biochemical indices. The control group (Group C) received a typical total mixed ration (TMR). 16S rRNA gene sequencing revealed 209 genera belonging to 19 bacterial phyla dominated by Firmicutes and Bacteroidetes. Only the relative abundances of Erysipelotrichaceae_UCG-009 were significantly different among the three groups (p < 0.05). Physiological and biochemical findings revealed that only the serum leptin concentrations were significantly decreased when mulberry leaves were added to the diets (p < 0.05). Correlation analysis revealed that Ruminococcus_2 were significantly positively correlated with the butyrate concentration. These findings suggested that supplementation with mulberry leaves only induced minor changes in the abovementioned indicators, implying that the rumen fermentation status was still stable after adding mulberry leaves to the diets.

Alterations in Rumen Bacterial Community and Metabolome Characteristics of Cashmere Goats in Response to Dietary Nutrient Density.

This study was conducted to investigate the impacts of dietary energy and protein on rumen bacterial composition and ruminal metabolites. A total of 12 ruminal samples were collected from Shaanbei white cashmere goats which were divided into two groups, including high-energy and high-protein (Group H; crude protein, CP: 9.37% in dry matter; metabolic energy, ME: 9.24 MJ/kg) and control (Group C; CP: 8.73%; ME: 8.60 MJ/kg) groups. Thereby, 16S rRNA gene sequencing and a quantitative polymerase chain reaction were performed to identify the rumen bacterial community. Metabolomics analysis was done to investigate the rumen metabolites and the related metabolic pathways in Groups C and H. The high-energy and high-protein diets increased the relative abundance of phylum Bacteroidetes and genera Prevotella_1 and Succiniclasticum, while decreasing the number of Proteobacteria (p < 0.05). The dominant differential metabolites were amino acids, peptides, and analogs. Tyrosine metabolism played an important role among the nine main metabolic pathways. Correlation analysis revealed that both Prevotella_1 (r = 0.608, p < 0.05) and Ruminococcus_2 (r = 0.613, p < 0.05) showed a positive correlation with catechol. Our findings revealed that the diets with high energy and protein levels in Group H significantly altered the composition of ruminal bacteria and metabolites, which can help to improve the dietary energy and protein use efficiency in goats.

Transactivation of miR-202-5p by Steroidogenic Factor 1 (SF1) Induces Apoptosis in Goat Granulosa Cells by Targeting TGFßR2.

MicroRNAs play key roles during ovary development, with emerging evidence suggesting that miR-202-5p is specifically expressed in female animal gonads. Granulosa cells (GCs) are somatic cells that are closely related to the development of female gametes in mammalian ovaries. However, the biological roles of miR-202-5p in GCs remain unknown. Here, we show that miR-202-5p is specifically expressed in GCs and accumulates in extracellular vesicles (EVs) from large growth follicles in goat ovaries. In vitro assays showed that miR-202-5p induced apoptosis and suppressed the proliferation of goat GCs. We further revealed that miR-202-5p is a functional miRNA that targets the transforming growth factor-beta type II receptor (TGFßR2). MiR-202-5p attenuated TGF-ß/SMAD signaling through the degradation of TGFßR2 at both the mRNA and protein level, decreasing p-SMAD3 levels in GCs. Moreover, we verified that steroidogenic factor 1 (SF1) is a transcriptional factor that binds to the promoters of miR-202 and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) through luciferase reporter and chromatin immunoprecipitation (ChIP) assays. That contributed to positive correlation between miR-202-5p and CYP19A1 expression and estradiol (E2) release. Furthermore, SF1 repressed TGFßR2 and p-SMAD3 levels in GCs through the transactivation of miR-202-5p. Taken together, these results suggest a mechanism by which miR-202-5p regulates canonical TGF-ß/SMAD signaling through targeting TGFßR2 in GCs. This provides insight into the transcriptional regulation of miR-202 and CYP19A1 during goat ovarian follicular development.

Temperature and humidity index (THI)-induced rumen bacterial community changes in goats.

The rumen microbiome is thought to play an important role in maintaining normal gastrointestinal metabolism and nutrient absorption in ruminants. The present study was designed to investigate the effect of heat stress on the rumen microbiome of goats using 16S rRNA sequencing technology. Six female goats were randomly allocated into two control metabolic chambers: A and B (in which the temperature and humidity could be precisely controlled with a precision deviation of ± 0.5 °C and ± 5%, with three goats/chamber). Dynamic changes in the rumen bacterial community were detected under 16 gradually increasing temperature and humidity indexes (THIs). Heat stress had no significant effect on alpha diversity but affected the main phyla and genera of the goat rumen microbiota. With a deeper level of heat stress, the TH groups formed a distinct cluster that differed from that of the control check (CK) group. The dominant phylum transitioned from Firmicutes to Bacteroidetes, and co-exclusion occurred between these two phyla. With the increase in THI, the content of probiotics in the Lachnospiraceae_ND3007_group (P < 0.05) decreased, and the abundance of pathogenic bacteria, such as Erysipelotrichaceae_UCG-004 and Treponema_2, increased; however, the difference between the groups was not significant (P > 0.05). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict bacterial function, and we found that the ambient environment significantly affected the balance between carbohydrate and energy metabolism (P < 0.05). In conclusion, heat stress changed the composition of rumen microbes and affected metabolic function. This experiment provides a theoretical basis for exploring the effects of environmental factors on the rumen of goats.

Bacterial community diversity associated with different levels of dietary nutrition in the rumen of sheep.

The sheep rumen microbial community plays an important role in animal performance and the environment. Few studies have paid close attention to the impact of different levels of dietary nutrition on rumen microbial populations. A total of 112 healthy Tan sheep were selected and randomly allotted to one of four dietary treatments (groups I, II, III, and IV). Each treatment included four replicated pens with seven sheep each for a total of 28 sheep per treatment. The sheep were fed four diets with nutrient levels that were 84, 96, 108, or 120% of the recommendation. In this study, a next-generation sequencing strategy and quantitative real-time PCR analysis were applied to investigate changes in whole ruminal bacteria with increased dietary energy and protein levels. The study observed 133 genera belonging to 16 phyla distributed throughout the rumen samples, with Firmicutes and Bacteroidetes predominating. Additionally, the higher nutritional dietary level linearly increased (P < 0.05) the number of Bacteroidetes and Proteobacteria but linearly decreased (P < 0.05) the Firmicutes richness. At the species level, the abundance of Prevotella ruminicola, Ruminococcus flavefaciens, and Succinivibrio dextrinosolvens linearly increased (P < 0.05), whereas the abundance of Selenomonas ruminantium and Veillonella parvula did not (P > 0.05). Furthermore, we predicted the potential functions of rumen bacteria. In particular, the relative abundances of the genes related to carbohydrates were overrepresented, and the genes involved in amino acid metabolism linearly increased (P < 0.05). These findings provide the first deep insights into the rumen microbial composition and the targeted improvement of dietary protein and energy use efficiency in Tan sheep.

Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus.

This article describes a rapid, highly efficient and versatile method for seamlessly assembling multiple DNA fragments into a vector at any desired position. The inserted fragments and vector backbone were amplified by high-fidelity PCR containing 20 bp to 50 bp overlapping regions at 3' and/or 5' termini. These linearised fragments were equimolarly mixed, and then cyclised in a prolonged overlap extension PCR without adding primers. The resulting PCR products were DNA multimers that could be directly transformed into host strains, yielding the desired chimeric plasmid. The proposed method was illustrated by constructing an Escherichia coli co-expression vector. The feasibility of the method in Lactobacillus was further validated by assembling an E. coli-Lactobacillus shuttle vector. Results showed that three to four fragments could be simultaneously and precisely inserted in a vector in only 2-3 days using the proposed method. The acceptable transformation efficiency was determined through the tested host strains; more than 95% of the colonies were positive transformants. Therefore, the proposed method is sufficiently competent for high-efficiency insertion of multiple DNA fragments into a plasmid and has theoretically good application potential for gene cloning and protein expression because it is simple, easy to implement, flexible and yields highly positive clones.