The porous structure induced heterogeneous and localized failure of the biofilm in microfluidic channels.

Understanding the mechanism of biofilm distribution and detachment is very important to effectively improve water treatment and prevent blockage in porous media. The existing research is more related to the local biofilm evolving around one or few microposts and the lack of the integral biofilm evolution in a micropost array for a longer growth period. This study combines microfluidic experiments and mathematical simulations to study the distribution and detachment of biofilm in porous media. Microfluidic chips with an array of microposts with different sizes are designed to simulate the physical pore structure of soil. The research shows that the initial formation and distribution of biofilm are influenced by bacterial transport velocity gradients within the pore space. Bacteria prefer to aggregate areas with smaller microposts, leading to the development of biofilm in those regions. Consequently, impermeable blockage structures form in this area. By analyzing experimental images of biofilm structures at the later stages, as well as coupling fluid flow and porous medium, and the finite element simulation, we find that the biofilm detachment is correlated with the morphology and permeability (kb) (from 10-15 to 10-9 m2) of the biofilm. The simulations show that there are two modes of biofilm detachment, such as internal detachment and external erosion.

Single and joint toxic effects of waterborne exposure to copper and cadmium on Coregonus ussuriensis Berg.

Heavy metal contamination severely affects the aquatic environment and organisms. Copper (Cu) and cadmium (Cd) are two of the most common heavy metal contaminants that impair the survival, development, and reproduction of aquatic organisms. With the growth of agriculture and industry, there is a possibility of heavy metal pollution in Coregonus ussuriensis Berg's water source. However, there are no published studies on the toxicity to C. ussuriensis. Acute toxicity experiments in C. ussuriensis revealed the 96-h median lethal concentrations of copper and cadmium to be 0.492 mg·L-1 (95% confidence interval: 0.452-0.529) and 1.548 mg·L-1 (95% confidence interval: 1.434-1.657), respectively, and safe concentrations of 4.92 µg·L-1 and 15.48 µg·L-1, respectively. C. ussuriensis was then treated for 96 h with Cu (20% of 96 h LC50), Cd (20% of 96 h LC50), and a combination of Cu and Cd (20% of Cu 96 h LC50 + 20% of Cd 96 h LC50). The histological damage caused by the three different exposure modes to the liver and gills of C. ussuriensis was verified using hematoxylin and eosin staining. All three exposure modes caused different degrees of vacuolization, nuclear consolidation, and necrosis in the liver tissue of C. ussuriensis and edema, hyperplasia, laminar fusion, and epithelial elevation in the gill tissue compared with the reference group. The severity of the damage increased with increasing exposure time. Anti-oxidant activity in the gill and liver tissues were measured using enzyme activity assay kits to reflect oxidative stress induced by copper and cadmium exposure alone and in combination. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were substantially higher than those in the reference groups. However, the activities of the enzymes decreased with increasing exposure time. Malondialdehyde (MDA) activity significantly increased during exposure in relation to that in the reference group. Analysis of immune gene expression in C. ussuriensis gill and liver tissues was executed using real-time inverse transcript polymerase chain response (RT-PCR). The expression levels of the pro-inflammatory cytokines interleukin one beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) were positively correlated with exposure time and were significantly upregulated with increasing exposure time. Metallothionein (MT) gene expression levels were significantly upregulated in the short term after exposure compared to the reference group but decreased with increasing exposure time. Our results indicate that exposure to aqueous copper and cadmium solutions, either alone or in combination, causes histopathological damage, oxidative stress, and immunotoxicity in C. ussuriensis gill and liver tissue. This study investigated the toxic effects of copper and cadmium on C. ussuriensis to facilitate the monitoring of heavy metals in water sources for healthy aquaculture.

miR-301b-5p and its target gene nfatc2ip regulate inflammatory responses in the liver of rainbow trout (Oncorhynchus mykiss) under high temperature stress.

Rainbow trout (Oncorhynchus mykiss) is a typical cold-water aquaculture fish and a high-end aquatic product. When water temperature exceeds its optimal range of 12-18 °C, the immune system of rainbow trout becomes weakened and unbalanced. High temperature in summer and global warming severely impact rainbow trout industry. The focus of this study was to explore the mechanisms regulating the immune response of rainbow trout under high temperature stress and identify molecular elements that account for resistance to high temperature. In this study, individual fish were screened in a high temperature stress experiment and divided into resistant (R) and sensitive (S) groups. The hepatic transcriptome sequencing and analysis of mRNAs and microRNAs of the R, S, and control groups showed that the number of the differentially expressed genes (DEGs) in the S group (9259) was higher than that in the R group (5313). Furthermore, the 1233 genes differentially expressed between S and R groups were mainly enriched in immune-related pathways, including cytokine-cytokine receptor interaction, TNF signaling and IL-17 signaling. Among these DEGs were miR-301b-5p and its target gene that encodes nuclear factor of activated T cells two interacting protein (nfatc2ip). The dual-luciferase reporter system and immunofluorescence experiments verified the relationship between miR-301b-5p and nfatc2ip. We also showed that expression levels of miR-301b-5p and nfatc2ip significantly negatively correlated in the liver of rainbow trout under high temperature stress. By performing functional experiments, we showed that activation of miR-301b-5p expression or inhibition of nfatc2ip expression stimulated the phosphorylation of p65, p38, and JNK in the classical nuclear factor kappa-B and mitogen-activated protein kinase pathways under high temperature stress. These manipulations initially promoted the secretion of the pro-inflammatory factor IL-1ß and then increased the levels of IL-6, IL-12, and TNF-α. In addition, activation of miR-301b-5p expression or inhibition of nfatc2ip expression stimulated the repair of the hepatic ultrastructural damage caused by high temperature stress by activating the inflammatory response in rainbow trout liver.

Matrix-producing cells formed 'Van Gogh bundles' facilitate Bacillus subtilis biofilm self-healing.

Biofilms grow and expand through cell differentiation into various phenotypes, which have different functions and cooperate with each other. In our experiments, we find that biofilms can heal after damaged, and we also find there is a special structure near the cut, which is called the 'Van Gogh bundles' by Jordi et al. because of its resemblance to Van Gogh's famous painting 'The Starry Night'. Here, we study the 'Van Gogh bundles' structure evolution near the cut area, and how 'Van Gogh bundles' structure facilitates the cut healing by observing microscopic images of bacterial colonies growing from wild-type and mutant strains. We find that the amount of matrix-producing cells contributes to the 'Van Gogh bundles' structure, such as curvature. Through the comparison of curvatures of 'Van Gogh bundles' and the rate of the cut healing, we find that the smaller the curvature, the faster healing rate. To better explain the above experiment observations, we establish an individual-based model and simulate the formation and growth of 'Van Gogh bundles' along the cut by giving rules for an individual cell like cell growth, division and turning rules, and also 'Van Gogh bundles' fold division rule.

Screening and Validation of p38 MAPK Involved in Ovarian Development of Brachymystax lenok.

Brachymystax lenok (lenok) is a rare cold-water fish native to China that is of high meat quality. Its wild population has declined sharply in recent years, and therefore, exploring the molecular mechanisms underlying the development and reproduction of lenoks for the purposes of artificial breeding and genetic improvement is necessary. The lenok comparative transcriptome was analyzed by combining single molecule, real-time, and next generation sequencing (NGS) technology. Differentially expressed genes (DEGs) were identified in five tissues (head kidney, spleen, liver, muscle, and gonad) between immature [300 days post-hatching (dph)] and mature [three years post-hatching (ph)] lenoks. In total, 234,124 and 229,008 full-length non-chimeric reads were obtained from the immature and mature sequencing data, respectively. After NGS correction, 61,405 and 59,372 non-redundant transcripts were obtained for the expression level and pathway enrichment analyses, respectively. Compared with the mature group, 719 genes with significantly increased expression and 1,727 genes with significantly decreased expression in all five tissues were found in the immature group. Furthermore, DEGs and pathways involved in the endocrine system and gonadal development were identified, and p38 mitogen-activated protein kinases (MAPKs) were identified as potentially regulating gonadal development in lenok. Inhibiting the activity of p38 MAPKs resulted in abnormal levels of gonadotropin-releasing hormone, follicle-stimulating hormone, and estradiol, and affected follicular development. The full-length transcriptome data obtained in this study may provide a valuable reference for the study of gene function, gene expression, and evolutionary relationships in B. lenok and may illustrate the basic regulatory mechanism of ovarian development in teleosts.

Molecular characterization and antibacterial immunity functional analysis of the antimicrobial peptide hepcidin from Coregonus ussuriensis berg.

Antimicrobial peptides are immune system molecules existing in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine rich antimicrobial peptides, which plays an important role in fish response to a variety of pathogens. In this study, we cloned and identified Hepcidin from the Coregonus ussuriensis Berg, and its functions in vivo and in vitro was investigated. Our results showed that, CuHepc contains a 267 bp coding sequence (CDS) region that encodes 88 putative amino acids with a molecular weight of 9.77 kD. Hepcidin transcripts were most abundant in the liver of healthy C. ussuriensis Berg. The synthesized Hepcidin peptide exhibited a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria in vitro, and the results of in vivo bacterial attack assays showed that the CuHepc gene was differentially up-regulated in the six tissues investigated after infection with Aeromonas hydrophila. To analyze the changes in protein levels in C. ussuriensis, we generated Hepc polyclonal antibodies in rabbits and verified that the protein expression was increased after bacterial infection with Western blot assay. MIC assay results showed a geometric mean value of 5.513 µM for CuHepc peptide. In the in vivo experiment, immune-related genes IL-10, NF-κB, TLR3 were up-regulated post-infection CuHepc peptide in liver and intestine. Finally, CuHepc peptide reduced the tissues microbial load compared to infection with Aeromonas hydrophila. The above results indicate that Hepc plays a role in the immune response of C. ussuriensis to exogenous disturbances, indicate that CuHepc might act a candidate for modulation of the innate immune system in C. ussuriensis.

Biofilm streamer growth dynamics in various microfluidic channels.

Biofilms are microbial colonies that are encapsulated in extracellular polymers secreted by cells through their proliferation and differentiation. Biofilms exist on solid surfaces, liquid surfaces, or in liquid media, where the growth of the bacterial biofilm is closely related to the velocity of the secondary flow, main flow, and geometry of the channel, which are difficult to measure in a natural fluid environment, making the study of the biofilm streamer growth process difficult. In this study, we used microfluidic channels made of polydimethylsiloxane to study the growth dynamics of Bacillus subtilis biofilm streamers. We observed that the biofilm streamer growth undergoes three stages with different growth characteristics. First, we found that the initial growth of the streamer is located at the position with the maximum value of P = secondary flow velocity × main flow velocity. Second, the biofilm underwent floating growth around the microcolumn obstacle. After the transition stage, the last growth stage includes two types because of the different attachment strengths and mechanical properties of the biofilm. Our research provides new insights into the formation and shedding of biofilm streamers in natural and industrial environments and helps us to better understand biofilm growth in fluid flow.

Effect of microfluidic channel geometry on Bacillus subtilis biofilm formation.

Biofilms are microbial colonies encased in an extracellular polymer matrix self-secreted through bacterial proliferation and differentiation. Biofilms exist almost everywhere such as sewers, rivers and oceans. In the fluid environment, the formation of biofilms is closely related to the relevant parameters of the flow field, such as the shear stress, the secondary flow, and the Reynolds number. In this paper, we use microfluidic channels made of polydimethylsiloxane to study the channel-geometry effect on Bacillus subtilis biofilms formation, such as the biofilm adhesion and structure. Our study shows that both the shear stress and the secondary flow play roles in the biofilm adhesion at the initial stage, the shear stress decides whether the biofilm adheres, if yes, then the secondary flow determines the adhesion rate. Our study further shows that after the biofilm forms, its structure evolves from loose to dense, with a concomitant 20-times rise in adhesion. Our study provides new insights into the adhesion of biofilms in natural and industrial fluid environments and helps understand the growth of biofilms.

Osmotic pressure induced by extracellular matrix drives Bacillus subtilis biofilms' self-healing.

The formation of bacterial biofilms is due to the bacteria adhering to the contact surface, secreting exopolysaccharide (EPS) and proteins, which make a large number of bacteria aggregate to form communities. In our experiments, we find that biofilms can heal after being destroyed like cut. To understand how biofilms self-heal, we use a diffusion-reaction continuum model to simulate the biofilm self-healing process, by using the extended finite element and level set method through MATLAB. The extended finite element method is used to calculate the diffusion of nutrients and the pressure field in the biofilm during the self-healing process, and the level set method is used to track the biofilm edge expansion and the cutting edge healing. The result can well describe the experimental observation, we find that the cut in the young biofilm heals almost completely, while old biofilms heal only at the edge. According to the phenotype observation, we find that matrix producing cells contribute to the biofilm self-healing, matrix producing cells secrete exopolysaccharide causing the difference of macromolecular substances' concentration in the biofilm and the agar substrate, which results in osmotic pressure promoting the transport of nutrients and leads to cut healing. Our simulation demonstrates that the nutrient concentration and the osmotic pressure are confinements for the biofilm healing.

The self-healing of Bacillus subtilis biofilms.

Self-healing is an intrinsic ability that exists widely in every multicellular biological organism. Our recent experiments have shown that bacterial biofilms also have the ability to self-heal after man-make cuts, but the mechanism of biofilm self-healing have not been studied. We find that the healing process of cuts on the biofilm depends on cut geometries like its location or direction, the biofilm itself like the biofilm age, the growing substrate properties like its hardness, and also the environments such as the competitive growth of multiple biofilms. What is more, the healing rate along the cut is heterogeneous, and the maximum healing rate can reach 260 µm/h, which is three times the undestroyed biofilm expansion rate. The cut does not change the rounded shape growth of biofilms. Further study of phenotypic evolution shows that the cut delays bacterial differentiation; motile cells perceive the cut and move to the cut area, while the cut only heals when there are enough matrix-producing cells in the cut area. Our work suggests new ideas for developing self-healing materials.

Comprehensive analysis of miRNA-mRNA/lncRNA during gonadal development of triploid female rainbow trout (Oncorhynchus mykiss).

Chromosomal ploidy manipulation is one of the means to create excellent germplasm. Triploid fish could provide an ideal sterile model for searching of a underlying mechanism of abnormality in meiosis. The complete understanding of the coding and noncoding RNAs regulating sterility caused by meiosis abnormality is still not well understood. By high-throughput sequencing, we compared the expression profiles of gonadal mRNA, long non-coding RNA (lncRNA), and microRNA (miRNA) at three different developmental stages between the diploid (XX) and triploid (XXX) female rainbow trout. These stages were gonads before differentiation (65 days post fertilisation, dpf), at the beginning of morphological differences (180 dpf) and showing clear difference between diploids and triploids (600 dpf), respectively. A majority of differentially expressed (DE) RNAs were identified, and 22 DE mRNAs related to oocyte meiosis and homologous recombination were characterized. The predicted miRNA-mRNA/lncRNA networks of 3 developmental stages were constructed based on the target pairs of DE lncRNA-miRNA and DE mRNA-miRNA. According to the networks, meiosis-related gene of ccne1 was targeted by dre-miR-15a-5p_R + 1, and 6 targeted DE lncRNAs were identified. Also, qRT-PCR was performed to validate the credibility of the network. Overall, this study explored the potential interplay between coding and noncoding RNAs during the gonadal development of polyploid fish. The mRNA, lncRNA and miRNA screened in this study may be helpful to identify the functional elements regulating fertility of rainbow trout, which may provide reference for character improvement in aquaculture.

Cell differentiation and motion determine the Bacillus subtilis biofilm morphological evolution under the competitive growth.

The growth discrepancy of Bacillus subtilis biofilms along different directions under the competitive growth drive the formation of anisotropic biofilm morphology directly. Two biofilms growing from two inoculating positions with different distances exhibit promoting or inhibiting growth behavior. Here we develop an optical imaging technology to observe the cell differentiation and the growth dynamics when the biofilm grows. It shows that the spatiotemporal distribution of different phenotypes affects the biofilm morphological evolution. We develop a program to calculate the velocity of cell motion within the biofilm, which is based on the feature point matching approach. We find the cell differentiation ununiformity in the neighboring region and its opposite region leads to the cell velocity difference in the competitive environment, the different cell motion further influences the biofilm morphology evolution. When biofilms grow with a long inoculating distance, there is always a gap between the them; when biofilms grow with a short inoculating distance, two biofilms gradually merge into a whole. Our work establishes a relationship between microscopic cells and macroscopic biofilm morphological which enables us to study the competitive growth process of biofilms from multiple perspectives.