Comparative toxic effect of ammonia exposure on Mauremys sinensis and invasive species Trachemys scripta elegans.

As one of main pollutants, ammonia could cause adverse effects to aquatic animals. To explore the toxic effects of ammonia on Chinese striped-necked turtles (Mauremys sinensis) and invasive species red-eared slider (Trachemys scripta elegans), we compared the activities of antioxidant enzymes, the mRNA levels of genes involved in immune status, endoplasmic reticulum stress and apoptosis between T. s. elegans and M. sinensis under ammonia exposure for 30 days. The results showed that ammonia obviously increased the activities of SOD, CAT, GPX and T-AOC in both T. s. elegans and M. sinensis, especially CAT and GPX in T. s. elegans were higher than that in M. sinensis. The expression levels of JAK, RELA and Mcl-1 in T. s. elegans obviously increased, while IL-6 mRNA levels significantly increased in M. sinensis. In addition, Bip and IRE1 levels in M. sinensis showed a marked increase, and were significantly higher than that in T. s. elegans. Bcl-2 and Bcl-xL transcriptional levels in T. s. elegans showed an increase, especially Bcl-xL were significantly higher than that in M. sinensis. These results indicated that T. s. elegans exhibited more stronger antioxidant defense and immune function than M. sinensis under ammonia exposure. M. sinensis was more likely to occur endoplasmic reticulum stress and inflammation in ammonia environment. This research reveals the physiological response of turtles to ammonia, helps to understand adverse effects of environmental pressure on aquatic turtles, and further explains the tolerance of invasive species T. s. elegans to environmental pollution.

Ammonia stress influences intestinal histomorphology, immune status and microbiota of Chinese striped-neck turtle (Mauremys sinensis).

Ammonia is one of major pollutants in aquatic environment that induces severe stress and toxicity to organisms in aquatic system. The intestine acts a major defense line that protects living organisms from biotic and abiotic stresses. In the current study, we examined the effects of ammonia on intestinal histomorphology, transcriptional levels of intestinal barrier functioning genes and intestinal microbiota of Chinese striped-neck turtle (Mauremys sinensis). Thus, the turtles were placed in water with addition of ammonia at 0 (control), 100, 200 mg L-1 for 30 days. Our findings showed that ammonia reduced the villus length and induced the inflammatory cells appearance. In addition, the epithelial tight junction genes, claudin and zonola occludin significantly downregulated in ammonia exposed groups as compared to control group (P < 0.05). Similarly, the mRNA expression levels of MUC-2 gene also significantly decreased in ammonia treated groups (P < 0.05). However, the expression levels of intestinal immune related genes such as IL-10, IL-12, TGF-ß1, TNF-α and IFN-γ significantly increased (P < 0.05). Furthermore, ammonia changed gut microbial diversity variedly. At the phylum levels, Firmicutes increased, whereas Bacteroidota, Desulfobacterota and Synergistota decreased significantly. Likewise, Lachnospiraceae, Bacteroides, Eubacteriaceae, Desulfovibrio, Muribaculaceae, Bilophila, Cloacibacillus, Christensenellaceae, Ruminococcus and Parabacteroides decreased while, Romboutsia and Turicibacter increased in ammonia exposed groups. In conclusion, ammonia at 100 and 200 mg L-1 could alter the intestinal barrier function and change the composition of intestinal microbiota, leading to bad health status in M. sinensis.

Ammonia exposure induces endoplasmic reticulum stress and apoptosis in Chinese striped-necked turtle (Mauremys sinensis).

Ammonia is a major pollutant in the water environment, which could cause severe harm to aquatic organisms. To explore the pathological and physiological effects of ammonia in Chinese striped-necked turtles (Mauremys sinensis), the individuals (body mass: 218.26 ± 12.65 g) were divided into two groups: control group and ammonia exposed group (6.25 mM total ammonia), then the expression levels of signaling factors involved in the endoplasmic reticulum stress and apoptotic pathways were determined. The results showed that ammonia exposure up-regulated the transcriptional and protein levels of endoplasmic reticulum stress marker gene Bip. Meanwhile, the relative mRNA levels of key genes (PERK, ATF6, eIF2α, ATF4, IRE1α and XBP1) involved in unfolded protein response up-regulated, and the phosphorylation levels of PERK, eIF2α and IRE1α increased correspondingly. In addition, the protein and transcriptional levels of CHOP and JNK related to apoptotic pathway induced by unfolded protein reaction increased under ammonia exposure. Moreover, Bcl-2 mRNA expression levels and protein levels decreased, whereas BAX and caspase-3 showed an opposite trend, and the cleaved protein of caspase-3 appeared when the turtles in the elevated ammonia. Furthermore, the apoptotic cells in liver increased after ammonia exposure. These results suggested ammonia exposure induced endoplasmic reticulum stress, then activated unfolded protein response, followed by apoptosis in M. sinensis. The results will contribute to a better understanding of the toxicity mechanism of ammonia to aquatic turtles.

Endoplasmic reticulum unfolded protein response modulates the adaptation of Trachemys scripta elegans in salinity water.

Trachemys scripta elegans, as a freshwater invasive species, can survive and lay eggs in brackish water, which may lead to the expansion of its potential invasion range due to freshwater salinization. Our previous studies have shown that high salinity leads to the accumulation of serum lipid content, which may induce endoplasmic reticulum stress (ERS) in the turtle. To better understand whether ERS is triggered by salinity, and in turn whether the turtles promote the protection mechanism, we exposed the turtles to the freshwater (CK), 5‰ salinity water (S5) and 15‰ salinity water (S15), and sampled at 6 h, 24 h and 30 d. 13 differentially expressed genes (DEGs) related to ERS pathways were found in the comparison of CK vs. S15 by transcriptomics analysis. Then, the mRNA and protein expression of ERS and its related activation pathways were further investigated. ERS marker glucose regulated protein 78 kD (GRP78) increased significantly (p < 0.05) in both the transcript and protein levels after exposure to 15‰ salinity water, which clearly indicated that salinity could induce ERS in T. s. elegans. Meanwhile, the three unfolded protein response (UPR) including transducers protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α) and activating transcription factor-6 (ATF6) were promoted by salinity, suggesting that the turtle might promote physiological process to eliminate damaged cells and cope with unfolded proteins accumulation induced by ERS. Our results provide new insight into the mechanism of salinity adaptation in T. s. elegans and salt-tolerant biological invasion.

Toxic effects of ammonia on intestinal health and microbiota in red-eared slider (Trachemys scripta elegans).

Ammonia is an important environmental pollutant and can induce serious damages to the organs of aquatic animals, especially the intestine which is mostly exposed to external environment. As important species of aquatic ecosystems, turtles may be potential risk targets of ammonia. However, it is not clear whether ammonia shows toxic effects on the intestines of turtles. Therefore, the worldwide species red-eared slider (Trachemys scripta elegans) was selected, to investigate the effects of ammonia on intestinal health and the composition of microbiota. Results showed that ammonia significantly changed the structure of intestines by decreasing the thickness of intestinal wall, shortening the length of intestinal villus, extending lamina proprias, and inducing inflammatory cells appearance when the turtles were exposed to ammonia (1.418 mg NH3 L-1) for 30 d. In addition, the downregulation of epithelial tight junction genes indicated that ammonia increased selective paracellular permeability. Simultaneously, the upregulation of cytokines suggested that ammonia induced intestinal immune and inflammatory responses. Furthermore, ammonia altered the dominant bacterial composition, and decreased the abundance of beneficial intestinal bacteria in the host. Our results demonstrated that ammonia impaired the intestinal health and changed the composition of residential microbiota in T. s. elegans. This study provides a new insight to evaluate the toxic effects of ammonia on aquatic turtles and helps to build a framework for the effective conservation of turtles.

Modulation of the intestinal barrier adaptive functions in red-eared slider (Trachemys scripta elegans) invading brackish waters.

Globally, the increase in sea levels is leading to salinization of freshwater, which might influence the freshwater organisms such as red-eared slider, Trachemys scripta elegans. The turtle can invade brackish water environments, in which it must deal with elevated salinity in the gastrointestinal tract that could impact the intestinal function. The intestinal barrier provides a front-line of organismal defense against the chemical and biological environmental insults. In this study, the adaptive functions of the intestinal barrier including intestinal histomorphology, genes involved in intestinal barrier functions, and the intestinal micro-ecosystem were analyzed in the turtles exposed to freshwater (S0), 5‰ salinity (S5) and 15‰ salinity (S15) water for 30 days. The results showed that the intestine of T. s. elegans maintained normal histomorphological structure in the S5 group, whereas the villus height, crypt depth and the number of goblet cells in the S15 group were lower than that in the S5 and S0 groups. In addition, the relative expression levels of epithelial tight junction-related genes and intestinal immune-related genes in the gut were significantly upregulated in the S15 group, compared to the freshwater group. Mucin-2 gene expression was downregulated, but mucin-1 transcript levels were upregulated in salinity-treated groups. Furthermore, the abundances of phylum Proteobacteria, and genera Morganella and Aeromonas in the intestine were particularly enhanced in the S15 group than the S0 and S5 groups. Taken together, these results indicate that the intestinal barrier plays a protective role in T. s. elegans adaptation to brackish water environments. Our results provide a perspective on the evolution of salinity tolerance and help to evaluate the potential danger of the turtle to other species, and understand the challenges that other species must meet with rising sea levels.

Effects of ammonia exposure on antioxidant function, immune response and NF-κB pathway in Chinese Strip-necked Turtle (Mauremys sinensis).

As one of the main toxic substances in aquaculture water, ammonia causes seriously physiological harm to aquatic animals. In order to investigate the effects of ammonia exposure on the antioxidant defense, immune response, and NF-κB signaling pathway in Chinese Strip-necked Turtle (Mauremys sinensis), we designed two experimental groups (control and 6.45 mM ammonia), and sampled at 6 h, 24 h, 48 h, re 24 h (recover 24 h), and re 48 h. The results showed that the blood ammonia (BA) content was significantly increased when the turtles were subjected to ammonia, and the activities of cholinesterase (CHE) and aspartate aminotransferase (AST) in the serum also showed a significant upward trend. The malondialdehyde (MDA) content continuously increased during ammonia exposure, and more than doubled at 48 h compared with the control group. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and their corresponding relative mRNA expression levels in the liver during ammonia exposure were obviously increased when compared to the control group, but most decreased to the normal levels at re 48 h. In addition, the relative mRNA and protein expression levels of NF-E2 related factor 2 (Nrf2) showed similar up-regulation patterns to antioxidase during ammonia exposed periods; whereas kelch-like ECH-binding protein 1 (Keap1), as Nrf2 negative regulator, showed opposite patterns. Moreover, the relative mRNA expression levels of heat shock proteins (HSP70, HSP90) significantly elevated upon the exposure of ammonia. Furthermore, ammonia increased the relative mRNA and protein expression levels of p50 and p65 at different exposed times. The reative mRNA expression levels of immune cytokines (BAFF and IL-6) were upregulated during ammonia exposured time, while there was a decline but did not return to normal levels, in the recovery periods. Taken together, these results indicated that antioxidation, immunity, and NF-κB signaling played a certain protective role for Mauremys sinensis under ammonia exposure. Our results will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in turtles.

The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes.

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.

Elastic properties of induced pluripotent stem cells.

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

Direct extraction of photosynthetic electrons from single algal cells by nanoprobing system.

There are numerous sources of bioenergy that are generated by photosynthetic processes, for example, lipids, alcohols, hydrogen, and polysaccharides. However, generally only a small fraction of solar energy absorbed by photosynthetic organisms is converted to a form of energy that can be readily exploited. To more efficiently use the solar energy harvested by photosynthetic organisms, we evaluated the feasibility of generating bioelectricity by directly extracting electrons from the photosynthetic electron transport chain before they are used to fix CO(2) into sugars and polysaccharides. From a living algal cell, Chlamydomonas reinhardtii, photosynthetic electrons (1.2 pA at 6000 mA/m(2)) were directly extracted without a mediator electron carrier by inserting a nanoelectrode into the algal chloroplast and applying an overvoltage. This result may represent an initial step in generating "high efficiency" bioelectricity by directly harvesting high energy photosynthetic electrons.

Pulsed direct current electric fields enhance osteogenesis in adipose-derived stromal cells.

Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy-based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration.

Open micro-fluidic system for atomic force microscopy-guided in situ electrochemical probing of a single cell.

Ultra-sharp nano-probes and customized atomic force microscopy (AFM) have previously been developed in our laboratory for in situ sub-cellular probing of electrochemical phenomena in living plant cells during their photosynthesis. However, this AFM-based electrochemical probing still has numerous engineering challenges such as immobilization of the live cells, compatibility of the immobilization procedure with AFM manipulation of the probe, maintenance of biological activity of the cells for an extended time while performing the measurements, and minimization of electrochemical noise. Thus, we have developed an open micro-fluidic channel system (OMFC) in which individual cells can be immobilized in micro-traps by capillary flow. This system affords easy AFM access and allows for maintenance of the cells in a well-defined chemical environment, which sustains their biological activity. The use of micro-channels for making the electrochemical measurements significantly reduces parasitic electrical capacitances and allows for current detection in the sub-pico-ampere range at high signal bandwidths. The OMFC was further studied using simulation packages for optimal design conditions. This system was successfully used to measure light-dependent oxidation currents of a few pico-amperes from the green alga Chlamydomonas reinhardtii.