Ion-Concentration-Hopping Heterolayer Gel for Ultrahigh Gradient Energy Conversion.

Conventional solid ion channel systems relying on single one- or two-dimensional confined nanochannels enabled selective and ultrafast convective ion transport. However, due to intrinsic solid channel stacking, these systems often face pore-pore polarization and ion concentration blockage, thereby restricting their efficiency in macroscale ion transport. Here, we constructed a soft heterolayer-gel system that integrated an ion-selective hydrogel layer with a water-barrier organogel layer, achieving ultrahigh cation selectivity and flux and effectively providing high-efficiency gradient energy conversion on a macroscale order of magnitude. Specifically, the hydrogel layer featured an unconfined 3D network, where the fluctuations of highly hydrated polyelectrolyte chains driven by thermal dynamics enhanced cation selectivity and mitigated transfer energy barriers. Such chain fluctuation mechanisms facilitated ion-cluster internal transmission, thereby enhancing ion concentration hopping for more efficient ion-selective transport. Compared to the existing rigid nanochannel-based gradient energy conversion systems, such a heterogel-based power generator exhibited a record power density of 192.90 and 1.07 W/m2 at the square micrometer scale and square centimeter scale, respectively (under a 500-fold artificial solution). We anticipate that such heterolayer gels would be a promising candidate for energy separation and storage applications.

Broadband and ascendant nonlinear optical properties of the wide bandgap material GaN nanowires.

Gallium nitride (GaN) nanowire, as a type of wide bandgap nanomaterial, has attracted considerable interest because of its outstanding physicochemical properties and applications in energy storage and photoelectric devices. In this study, we prepared GaN nanowires via a facile chemical vapor deposition method and investigated their nonlinear absorption responses ranging from ultraviolet to near-infrared in the z-scan technology under irradiation by picosecond laser pulses. The experiment revealed that GaN nanowires exhibit remarkable nonlinear absorption characteristics attributed to their wide bandgap and nanostructure, including saturable absorption and reverse saturable absorption. When compared to bulk GaN crystals, the nanowires provide a richer and more potent set of nonlinear optical effects. Furthermore, we conducted an analysis of the corresponding electronic transition processes associated with photon absorption. Under high peak power density laser excitation, two-photon absorption or three-photon absorption dominate, with maximum modulation depths of 73.6%, 74.9%, 63.1% and 64.3% at 266 nm, 355 nm, 532 nm, and 1064 nm, respectively, corresponding to absorption coefficients of 0.22 cm/GW, 0.28 cm/GW, 0.08 cm/GW, and 2.82 ×10-4 cm3/GW2. At lower peak energy densities, GaN nanowires demonstrate rare and excellent saturation absorption characteristics at wavelength of 355 nm due to interband transitions, while saturable absorption is also observed at 532 nm and 1064 nm due to band tail absorption. The modulation depths are 85.2%, 41.9%, and 13.7% for 355 nm, 532 nm, and 1064 nm, corresponding to saturation intensities of 3.39 GW/cm2, 5.58 GW/cm2 and 14.13 GW/cm2. This indicates that GaN nanowires can be utilized as broadband optical limiters and high-performance pulse laser modulating devices, particularly for scarce ultraviolet optical limiters, and saturable absorbers for ultraviolet and visible lasers. Furthermore, our study demonstrates the application potential of wide bandgap nanomaterials in nonlinear optical devices.

Hmga1-overexpressing lentivirus protects against osteoporosis by activating the Wnt/ß-catenin pathway in the osteogenic differentiation of BMSCs.

Postmenopausal osteoporosis is associated with bone formation inhibition mediated by the impaired osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs). However, identifying and confirming the essential genes in the osteogenic differentiation of BMSCs and osteoporosis remain challenging. The study aimed at revealing the key gene that regulated osteogenic differentiation of BMSCs and led to osteoporosis, thus exploring its therapeutic effect in osteoporosis. In the present study, six essential genes related to the osteogenic differentiation of BMSCs and osteoporosis were identified, namely, fibrillin 2 (Fbn2), leucine-rich repeat-containing 17 (Lrrc17), heat shock protein b7 (Hspb7), high mobility group AT-hook 1 (Hmga1), nexilin F-actin-binding protein (Nexn), and endothelial cell-specific molecule 1 (Esm1). Furthermore, the in vivo and in vitro experiments showed that Hmga1 expression was increased during the osteogenic differentiation of rat BMSCs, while Hmga1 expression was decreased in the bone tissue of ovariectomized (OVX) rats. Moreover, the expression of osteogenic differentiation-related genes, the activity of alkaline phosphatase (ALP), and the number of mineralized nodules were increased after Hmga1 overexpression, which was partially reversed by a Wnt signaling inhibitor (DKK1). In addition, after injecting Hmga1-overexpressing lentivirus into the bone marrow cavity of OVX rats, the bone loss, and osteogenic differentiation inhibition of BMSCs in OVX rats were partially reversed, while osteoclast differentiation promotion of BMSCs in OVX rats was unaffected. Taken together, the present study confirms that Hmga1 prevents OVX-induced bone loss by the Wnt signaling pathway and reveals that Hmga1 is a potential gene therapeutic target for postmenopausal osteoporosis.

Unveiling Small-Sized Plastic Particles Hidden behind Large-Sized Ones in Human Excretion and Their Potential Sources.

Health risks of microplastic exposure have drawn growing global concerns due to the widespread distribution of microplastics in the environment. However, more evidence is needed to understand the exposure characteristics of microplastics owing to the limitation of current spectrum technologies, especially the missing information on small-sized particles. In the present study, laser direct infrared spectroscopy and thermal desorption-gas chromatography-mass spectrometry combined pyrolysis using a tubular furnace (TD-GC/MS) were employed to comprehensively detect the presence of plastic particles down to 0.22 µm in human excreted samples. The results showed that polyethylene (PE), polyvinyl chloride, PE terephthalate (PET), and polypropylene dominated large-sized (>20 µm) and small-sized plastic plastics (0.22-20 µm) in feces and urine. Moreover, fragments accounted for 60.71 and 60.37% in feces and urine, respectively, representing the most pervasive shape in excretion. Surprisingly, the concentration of small-sized particles was significantly higher than that of large-sized microplastics, accounting for 56.54 and 50.07% in feces (345.58 µg/g) and urine (6.49 µg/mL). Significant positive correlations were observed between the level of plastic particles in feces and the use of plastic containers and the consumption of aquatic products (Spearman correlation analysis, p < 0.01), suggesting the potential sources for plastic particles in humans. Furthermore, it is estimated that feces was the primary excretory pathway, consisting of 94.0% of total excreted microplastics daily. This study provides novel evidence regarding small-sized plastic particles, which are predominant fractions in human excretion, increasing the knowledge of the potential hazards of omnipresent microplastics to human exposure.

Isoindigo-Thiophene D-A-D-Type Conjugated Polymers: Electrosynthesis and Electrochromic Performances.

Four novel isoindigo-thiophene D-A-D-type precursors are synthesized by Stille coupling and electrosynthesized to yield corresponding hybrid polymers with favorable electrochemical and electrochromic performances. Intrinsic structure-property relationships of precursors and corresponding polymers, including surface morphology, band gaps, electrochemical properties, and electrochromic behaviors, are systematically investigated. The resultant isoindigo-thiophene D-A-D-type polymer combines the merits of isoindigo and polythiophene, including the excellent stability of isoindigo-based polymers and the extraordinary electrochromic stability of polythiophene. The low onset oxidation potential of precursors ranges from 1.10 to 1.15 V vs. Ag/AgCl, contributing to the electrodeposition of high-quality polymer films. Further kinetic studies illustrate that isoindigo-thiophene D-A-D-type polymers possess favorable electrochromic performances, including high optical contrast (53%, 1000 nm), fast switching time (0.8 s), and high coloration efficiency (124 cm2 C-1). These features of isoindigo-thiophene D-A-D-type conjugated polymers could provide a possibility for rational design and application as electrochromic materials.

[Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism].

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aß_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aß_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aß_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1ß(IL-1ß), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aß_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1ß, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aß_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1ß, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1ß, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aß and expression of p-Tau, thereby restoring the hippocampal morphological structure.

The role of low-frequency oscillations in three-dimensional perception with depth cues in virtual reality.

Currently, vision-related neuroscience studies are undergoing a trend from simplified image stimuli toward more naturalistic stimuli. Virtual reality (VR), as an emerging technology for visual immersion, provides more depth cues for three-dimensional (3D) presentation than two-dimensional (2D) image. It is still unclear whether the depth cues used to create 3D visual perception modulate specific cortical activation. Here, we constructed two visual stimuli presented by stereoscopic vision in VR and graphical projection with 2D image, respectively, and used electroencephalography to examine neural oscillations and their functional connectivity during 3D perception. We find that neural oscillations are specific to delta and theta bands in stereoscopic vision and the functional connectivity in the two bands increase in cortical areas related to visual pathways. These findings indicate that low-frequency oscillations play an important role in 3D perception with depth cues.

11ß-Hydroxysteroid dehydrogenase 2: A key mediator of high susceptibility to osteoporosis in offspring after prenatal dexamethasone exposure.

Epidemiological investigations have shown that individuals treated with dexamethasone during pregnancy have an increased risk of osteoporosis after birth. Our studies reported that peak bone mass was decreased in the prenatal dexamethasone exposure (PDE) offspring before chronic stress, while further decrease was observed after chronic stress. Simultaneously, increase of bone local active corticosterone was observed in the PDE offspring, while further increase was also observed after chronic stress. Moreover, the histone H3 lysine 9 acetylation (H3K9ac) level of 11-beta hydroxysteroid dehydrogenase 2 (11ß-HSD2) and its expression in bone tissue of PDE offspring rats remained lower than the control before and after birth. Injection of 11ß-HSD2 overexpression lentivirus into the bone marrow cavity could partially alleviate the accumulation of bone local active corticosterone and bone loss induced by PDE. In vitro, dexamethasone inhibited the expression of 11ß-HSD2 and aggravated the inhibitory effect of corticosterone on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Overexpression of 11ß-HSD2 partially alleviated the inhibitory effect of corticosterone. Moreover, dexamethasone promoted the nuclear translocation of glucocorticoid receptor (GR), which resulted in the stimulation of 11ß-HSD2 expression due to the binding of GR to the 11ß-HSD2 promoter region directly, as well as increasing H3K9ac level in the 11ß-HSD2 promoter region by recruiting histone deacetylase 11 (HDAC11). Our results indicated that low expression of 11ß-HSD2 in bone tissue is an important mediator for the high susceptibility to osteoporosis in PDE adult offspring.

Intrauterine programming of cartilaginous 11ß-HSD2 induced by corticosterone and caffeine mediated susceptibility to adult osteoarthritis.

Our previous study reported that prenatal caffeine exposure (PCE) could induce chondrodysplasia and increase the susceptibility to osteoarthritis in offspring rats. However, the potential mechanisms and initiating factors remain unknown. This study aims to investigate whether 11ß-HSD2, a glucocorticoid-metabolizing enzyme, is involved in the susceptibility of osteoarthritis induced by PCE and to further explore its potential mechanisms and initiating factors. Firstly, we found that PCE reduced cartilage matrix synthesis (aggrecan/Col2a1 expression) in male adult offspring rats and exhibited an osteoarthritis phenotype following chronic stress, which was associated with persistently reduced H3K9ac and H3K27ac levels at the promoter of 11ß-HSD2 as well as its expression in the cartilage from fetus to adulthood. The expression of 11ß-HSD2, aggrecan and Col2a1 were all decreased by corticosterone in the fetal chondrocytes, while overexpression of 11ß-HSD2 could partially alleviate the decrease of matrix synthesis induced by corticosterone in vitro. Furthermore, the glucocorticoid receptor (GR) activated by glucocorticoids directly bonded to the promoter region of 11ß-HSD2 to inhibit its expression. Meanwhile, the activated GR reduced the H3K9ac and H3K27ac levels of 11ß-HSD2 by recruiting HDAC4 and promoting GR-HDAC4 protein interaction to inhibit the 11ß-HSD2 expression. Moreover, caffeine could reduce the expression of 11ß-HSD2 by inhibiting the cAMP/PKA signaling pathway but without reducing the H3K9ac and H3K27ac levels of 11ß-HSD2, thereby synergistically enhancing the corticosterone effect. In conclusion, the persistently reduced H3K9ac and H3K27ac levels of 11ß-HSD2 from fetus to adulthood mediated the inhibition of cartilage matrix synthesis and the increased susceptibility to osteoarthritis. This epigenetic programming change in utero was induced by glucocorticoids with synergistic effect of caffeine.

Combined-Acupoint Electroacupuncture Induces Better Analgesia via Activating the Endocannabinoid System in the Spinal Cord.

Electroacupuncture (EA) therapy has been widely reported to alleviate neuropathic pain with few side effects in both clinical practice and animal studies worldwide. However, little is known about the comparison of the therapeutic efficacy among the diverse EA schemes used for neuropathic pain. The present study is aimed at investigating the therapeutic efficacy discrepancy between the single and combined-acupoint EA and to reveal the difference of mechanisms behind them. Electroacupuncture was given at both Zusanli (ST36) and Huantiao (GB30) in the combined group or ST36 alone in the single group. Paw withdrawal mechanical threshold (PWMT) was measured to determine the pain level. Electrophysiology was performed to detect the effects of EA on synaptic transmission in the spinal dorsal horn of the vGlut2-tdTomato mice. Spinal contents of endogenous opioids, endocannabinoids, and their receptors were examined. Inhibitors of CBR (cannabinoid receptor) and opioid receptors were used to study the roles of opioid and endocannabinoid system (ECS) in EA analgesia. We found that combined-acupoint acupuncture provide stronger analgesia than the single group did, and the former inhibited the synaptic transmission at the spinal level to a greater extent than later. Besides, the high-intensity stimulation at ST36 or normal stimulation at two sham acupoints did not mimic the similar efficacy of analgesia in the combined group. Acupuncture stimulation in single and combined groups both activated the endogenous opioid system. The ECS was only activated in the combined group. Naloxone totally blocked the analgesic effect of single-acupoint EA; however, it did not attenuate that of combined-acupoint EA unless coadministered with CBR antagonists. Hence, in the CCI-induced neuropathic pain model, combined-acupoint EA at ST36 and GB30 is more effective in analgesia than the single-acupoint EA at ST36. EA stimulation at GB30 alone neither provided a superior analgesic effect to EA treatment at ST36 nor altered the content of AEA, 2-AG, CB1 receptor, or CB2 receptor compared with the CCI group. Activation of the ECS is the main contributor of the better analgesia by the combined acupoint stimulation than that induced by single acupoint stimulation.

Joint Estimation of Mass and Center of Gravity Position for Distributed Drive Electric Vehicles Using Dual Robust Embedded Cubature Kalman Filter.

The accurate estimation of the mass and center of gravity (CG) position is key to vehicle dynamics modeling. The perturbation of key parameters in vehicle dynamics models can result in a reduction of accurate vehicle control and may even cause serious traffic accidents. A dual robust embedded cubature Kalman filter (RECKF) algorithm, which takes into account unknown measurement noise, is proposed for the joint estimation of mass and CG position. First, the mass parameters are identified based on directly obtained longitudinal forces in the distributed drive electric vehicle tires using the whole vehicle longitudinal dynamics model and the RECKF. Then, the CG is estimated with the RECKF using the mass estimation results and the vertical vehicle model. Finally, different virtual tests show that, compared with the cubature Kalman algorithm, the RECKF reduces the root mean square error of mass and CG by at least 7.4%, and 2.9%, respectively.

Toward High-Performance Electrochromic Conjugated Polymers: Influence of Local Chemical Environment and Side-Chain Engineering.

Three homologous electrochromic conjugated polymers, each containing an asymmetric building block but decorated with distinct alkyl chains, were designed and synthesized using electrochemical polymerization in this study. The corresponding monomers, namely T610FBTT810, DT6FBT, and DT48FBT, comprise the same backbone structure, i.e., an asymmetric 5-fluorobenzo[c][1,2,5]thiadiazole unit substituted by two thiophene terminals, but were decorated with different types of alkyl chain (hexyl, 2-butyloctyl, 2-hexyldecyl, or 2-octyldecyl). The effects of the side-chain structure and asymmetric repeating unit on the optical absorption, electrochemistry, morphology, and electrochromic properties were investigated comparatively. It was found that the electrochromism conjugated polymer, originating from DT6FBT with the shortest and linear alkyl chain, exhibits the best electrochromic performance with a 25% optical contrast ratio and a 0.3 s response time. The flexible electrochromic device of PDT6FBT achieved reversible colors of navy and cyan between the neutral and oxidized states, consistent with the non-device phenomenon. These results demonstrate that subtle modification of the side chain is able to change the electrochromic properties of conjugated polymers.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

HEMGN and SLC2A1 might be potential diagnostic biomarkers of steroid-induced osteonecrosis of femoral head: study based on WGCNA and DEGs screening.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a chronic and crippling bone disease. This study aims to reveal novel diagnostic biomarkers of SONFH. METHODS: The GSE123568 dataset based on peripheral blood samples from 10 healthy individuals and 30 SONFH patients was used for weighted gene co-expression network analysis (WGCNA) and differentially expressed genes (DEGs) screening. The genes in the module related to SONFH and the DEGs were extracted for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Genes with |gene significance| > 0.7 and |module membership| > 0.8 were selected as hub genes in modules. The DEGs with the degree of connectivity ≥5 were chosen as hub genes in DEGs. Subsequently, the overlapping genes of hub genes in modules and hub genes in DEGs were selected as key genes for SONFH. And then, the key genes were verified in another dataset, and the diagnostic value of key genes was evaluated by receiver operating characteristic (ROC) curve. RESULTS: Nine gene co-expression modules were constructed via WGCNA. The brown module with 1258 genes was most significantly correlated with SONFH and was identified as the key module for SONFH. The results of functional enrichment analysis showed that the genes in the key module were mainly enriched in the inflammatory response, apoptotic process and osteoclast differentiation. A total of 91 genes were identified as hub genes in the key module. Besides, 145 DEGs were identified by DEGs screening and 26 genes were identified as hub genes of DEGs. Overlapping genes of hub genes in the key module and hub genes in DEGs, including RHAG, RNF14, HEMGN, and SLC2A1, were further selected as key genes for SONFH. The diagnostic value of these key genes for SONFH was confirmed by ROC curve. The validation results of these key genes in GSE26316 dataset showed that only HEMGN and SLC2A1 were downregulated in the SONFH group, suggesting that they were more likely to be diagnostic biomarkers of SOFNH than RHAG and RNF14. CONCLUSIONS: Our study identified that two key genes, HEMGN and SLC2A1, might be potential diagnostic biomarkers of SONFH.

Generation of a simultaneous orthogonally polarized dual-wavelength Raman laser with power ratio tunability by a single hexagonal crystal: Cs2TeMo3O12.

A new generation of orthogonally polarized dual-wavelength lasers was demonstrated using a dye mode-locked neodymium-doped yttrium aluminum garnet laser for the first time. With a hexagonal Cs2TeMo3O12 as the Raman medium, efficient dual-wavelength stimulated Raman scattering was obtained at 1175 and 1154 nm with similar output power, corresponding to the stretching vibration of Mo-O and the asymmetric stretching vibrations of Mo-O and Te-O groups, respectively. The power ratio of two Raman components can be flexibly adjusted by tuning the polarization of the incident laser, which can be tuned from 0% to 100%. Laser sources with such a small wavelength separation could prove interesting for the difference-frequency generation of terahertz waves in the 4.6 THz range. Our study provides a simple and flexible method to achieve a promising dual-wavelength laser source in orthogonal polarization by Raman-based nonlinear frequency conversions.

CCR7, CD80/86 and CD83 in yellow catfish (Pelteobagrus fulvidraco): Molecular characteristics and expression patterns with bacterial infection.

Dendritic cells (DCs) have a strong ability to stimulate naive T lymphocyte proliferation, so DCs play an important regulatory role in the initiation of the specific immune response. DCs cannot play the role of antigen presentation without the expression of surface molecules. The chemokine receptor CCR7 and the costimulatory molecules CD80/86 and CD83 are not only markers of DC maturation but also important functional molecules in the immune response of DC-T cells. In this study, partial cDNA sequences of CCR7, CD80/86 and CD83 were obtained by rapid amplification of cDNA ends (RACE) technology from yellow catfish. Bioinformatics analysis of deduced amino acid sequences of these three genes showed that CCR7, CD80/86 and CD83 genes in yellow catfish have similar functional domains to the homologs in other vertebrates, which indicated that the functions of these genes may be somewhat conserved during the evolution process. Afterward, the expression characteristics of these three genes in different tissues were detected by q-PCR. This result indicated that CCR7, CD80/86 and CD83 were expressed in all examined tissues, and the highest expression levels of CCR7 and CD80/86 and CD83 were detected in the trunk kidney, muscle and midgut, respectively. Meanwhile, the expression levels of CCR7 and CD80/86 were lowest in the gill, and the expression of CD83 was lowest in the stomach. Finally, healthy yellow catfish were infected with A.hydrophila (1.0 × 107 CFU/mL) or E.ictaluri (1.0 × 106 CFU/mL), q-PCR results indicated that both pathogenic bacteria can induce significant upregulation of CCR7, CD80/86 and CD83 in immune organs, and the expression levels of these genes in the intestine were higher than those in the skin and gill. Our results in this study provide a molecular basis for exploring the role of CCR7, CD80/86 and CD83 in the immune responses induced by bacteria, and can help us to understand the difference of immune responses induced by extracellular and intracellular bacteria.

ROS induced by spring viraemia of carp virus activate the inflammatory response via the MAPK/AP-1 and PI3K signaling pathways.

Spring viraemia of carp virus (SVCV) can cause a high mortality in common carp (Cyprinus carpio), and its main pathological processes include the inflammatory response. However, the detailed mechanism is still unclear. Reactive oxygen species (ROS) have been shown to play critical roles in the immune response, including inflammation, in different models. Our previous studies have demonstrated that SVCV infection results in the accumulation of ROS, including H2O2, in epithelioma papulosum cyprini (EPC) cells. In this study, we aimed to explore the relationship between H2O2 accumulation and inflammation during SVCV infection. After EPC cells were infected with SVCV, the expression levels of the inflammatory factors tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and interleukin (IL)-8 were up-regulated, while the expression of the anti-inflammatory factor interleukin (IL)-10 was down-regulated, compared with that in mock-infected EPC cells. The antioxidant N-acetyl-l-cysteine (NAC) could dampen the increased TNF-ɑ and COX-2 expression induced by SVCV and H2O2, suggesting a relationship between ROS accumulation and inflammation during SVCV infection. Dual luciferase reporter assays demonstrated that SVCV could not activate the NF-κB pathway. In addition, inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC) treatment had no effect on the expression of inflammatory factors. Furthermore, inhibition of the ERK, JNK, and p38MAPK signaling pathways by U0126, SP600125, and SB203580, respectively, reduced the expression of TNF-ɑ, COX-2, and IL-8, indicating that these three signaling pathways were all involved in the inflammatory response after SVCV infection. In addition, the PI3K signaling pathway was involved in the expression of the chemokine IL-8 in the SVCV-induced inflammatory response. We also showed that inhibition of the MAPK or PI3K signaling pathway facilitated the expression of SVCV-G as well as increased the SVCV viral titer. Altogether these results reveal the mechanism of the SVCV-mediated inflammatory response. Thus, targeting these signaling pathways may provide novel treatment strategies for SVCV-mediated diseases.

Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations.

Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. Escherichia coli primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.

Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing.

Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redß and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.

Pharmacokinetics of florfenicol in blunt-snout bream (Megalobrama amblycephala) at two water temperatures with single-dose oral administration.

The pharmacokinetics and residue elimination of florfenicol (FFC) and its metabolite florfenicol amine (FFA) were studied in healthy blunt-snout bream (Megalobrama amblycephala, 50 ± 10 g). The study was conducted with a single-dose (25 mg/kg) oral administration at a water temperature of 18 or 28°C, while in the residue elimination study, fish were administered at 25 mg/kg daily for three consecutive days by oral gavage to determine the withdrawal period (WDT) at 28°C. The FFC and FFA levels in plasma and tissues (liver, kidneys and muscle) were analysed using high-performance liquid chromatography (HPLC). A no-compartment model was used to analyse the concentration versus time data of M. amblycephala. In the two groups at 18 and 28°C, the maximum plasma concentration (Cmax ) of FFC was 5.89 and 6.21 µg/ml, while the time to reach Cmax (Tmax ) was 5.97 and 2.84 hr, respectively. These suggested that higher temperature absorbed more drug and more quickly at M. amblycephala. And the elimination half-life (T1/2 kß ) of FFC was calculated as 26.75 and 16.14 hr, while the total body clearance (CL) was 0.09 and 0.15 L kg-1  hr-1 , and the areas under the concentration-time curves (AUCs) were 265.87 and 163.31 µg hr/ml, respectively. The difference demonstrated that the elimination rate of FFC in M. amblycephala at 28°C was more quickly than that at 18°C. The results of FFA showed the same trend in tissues of M. amblycephala. After multiple oral doses (25 mg/kg daily for 3 days), the k (eliminate rate constant) of FFA in M. amblycephala muscle was 0.017, the C0 (initial concentration) was 3.07 mg/kg, and the WDT was 10 days (water temperature 28°C).