Pseudorabies Virus Infection Activates the TLR-NF-κB Axis and AIM2 Inflammasome To Enhance Inflammatory Responses in Mice.

Pseudorabies virus (PRV) infection activates inflammatory responses to release robust proinflammatory cytokines, which are critical for controlling viral infection and clearance of PRV. However, the innate sensors and inflammasomes involved in the production and secretion of proinflammatory cytokines during PRV infection remain poorly studied. In this study, we report that the transcription and expression levels of some proinflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α), are upregulated in primary peritoneal macrophages and in mice during PRV infection. Mechanistically, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR5 were induced by the PRV infection to enhance the transcription levels of pro-IL-1ß, pro-IL-18, and gasdermin D (GSDMD). Additionally, we found that PRV infection and transfection of its genomic DNA triggered AIM2 inflammasome activation, apoptosis-related speckle-like protein (ASC) oligomerization, and caspase-1 activation to enhance the secretion of IL-1ß and IL-18, which was mainly dependent on GSDMD, but not GSDME, in vitro and in vivo. Taken together, our findings reveal that the activation of the TLR2-TLR3-TRL4-TLR5-NF-κB axis and AIM2 inflammasome, as well as GSDMD, is required for proinflammatory cytokine release, which resists the PRV replication and plays a critical role in host defense against PRV infection. Our findings provide novel clues to prevent and control PRV infection. IMPORTANCE PRV can infect several mammals, including pigs, other livestock, rodents, and wild animals, causing huge economic losses. As an emerging and reemerging infectious disease, the emergence of PRV virulent isolates and increasing human PRV infection cases indicate that PRV is still a high risk to public health. It has been reported that PRV infection leads to robust release of proinflammatory cytokines through activating inflammatory responses. However, the innate sensor that activates IL-1ß expression and the inflammasome involved in the maturation and secretion of proinflammatory cytokines during PRV infection remain poorly studied. In this study, our findings reveal that, in mice, activation of the TLR2-TLR3-TRL4-TLR5-NF-κB axis and AIM2 inflammasome, as well as GSDMD, is required for proinflammatory cytokine release during PRV infection, and it resists PRV replication and plays a critical role in host defense against PRV infection. Our findings provide novel clues to prevent and control PRV infection.

GmMDE genes bridge the maturity gene E1 and florigens in photoperiodic regulation of flowering in soybean.

Soybean (Glycine max) is highly sensitive to photoperiod, which affects flowering time and plant architecture and thus limits the distribution range of elite soybean cultivars. The major maturity gene E1 confers the most prominent effect on photoperiod sensitivity, but its downstream signaling pathway remains largely unknown. Here, we confirm that the encoded E1 protein is a transcriptional repressor. The expression of seven GmMDE genes (Glycine max MADS-box genes downregulated by E1) was suppressed when E1 was overexpressed and promoted when E1 was knocked out through clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis. These GmMDEs exhibited similar tissue specificity and expression patterns, including in response to photoperiod, E1 expression, and E1 genotype. E1 repressed GmMDE promoter activity. Results for two GmMDEs showed that E1 epigenetically silences their expression by directly binding to their promoters to increase H3K27me3 levels. The overexpression of GmMDE06 promoted flowering and post-flowering termination of stem growth. The late flowering phenotype of E1-overexpressing soybean lines was reversed by the overexpression of GmMDE06, placing GmMDE06 downstream of E1. The overexpression of GmMDE06 increased the expression of the soybean FLOWERING LOCUS T orthologs GmFT2a and GmFT5a, leading to feedback upregulation of GmMDE, indicating that GmMDE and GmFT2a/GmFT5a form a positive regulatory feedback loop promoting flowering. GmMDE06 also promoted post-flowering termination of stem growth by repressing the expression of the shoot identity gene Dt1. The E1-GmMDEs-GmFT2a/5a-Dt1 signaling pathway illustrates how soybean responds to photoperiod by modulating flowering time and post-flowering stem termination.

Characterization of the lipidomic profile of clam Meretrix petechialis in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a devastating pathogen of clam Meretrix petechialis, which brings about huge economic losses in aquaculture breeding industry. In our previous study, we have found that Vibrio infection is closely associated with lipid metabolism of clams. In this study, an untargeted lipidomics approach was used to explore the lipid profiling changes upon Vibrio infection. The results demonstrated that the hepatopancreas of clams was composed of five lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids and sterol lipids. And the content of lipid classes altered during Vibrio infection, implying that Vibrio infection altered intracellular lipid homeostasis in clams. Meanwhile, a total of 200 lipid species including 82 up-regulated and 118 down-regulated significantly were identified in response to Vibrio infection, of which ceramide (Cer), phosphatidylcholine (PC) and triglyceride (TG) accounted for the largest proportion. Notably, all Cers showed a significantly decreased trend while nearly all TG species were increased significantly during Vibrio infection, which suggested that Cer and TG could be determined as effective biomarkers. Furthermore, these differentially expressed lipid species were enriched in 20 metabolic pathways and sphingolipid metabolism was one of the most enriched pathways. These results evidenced how the lipid metabolism altered in the process of Vibrio infection and opened a new perspective on the response of marine bivalves to pathogen infection.

Genome-Wide Identification and Characterization of Copper Chaperone for Superoxide Dismutase (CCS) Gene Family in Response to Abiotic Stress in Soybean.

Copper Chaperone For Superoxide Dismutase (CCS) genes encode copper chaperone for Superoxide dismutase (SOD) and dramatically affect the activity of SOD through regulating copper delivery from target to SOD. SOD is the effective component of the antioxidant defense system in plant cells to reduce oxidative damage by eliminating Reactive oxygen species (ROS), which are produced during abiotic stress. CCS might play an important role in abiotic stress to eliminate the damage caused by ROS, however, little is known about CCS in soybean in abiotic stress regulation. In this study, 31 GmCCS gene family members were identified from soybean genome. These genes were classified into 4 subfamilies in the phylogenetic tree. Characteristics of 31 GmCCS genes including gene structure, chromosomal location, collinearity, conserved domain, protein motif, cis-elements, and tissue expression profiling were systematically analyzed. RT-qPCR was used to analyze the expression of 31 GmCCS under abiotic stress, and the results showed that 5 GmCCS genes(GmCCS5, GmCCS7, GmCCS8, GmCCS11 and GmCCS24) were significantly induced by some kind of abiotic stress. The functions of these GmCCS genes in abiotic stress were tested using yeast expression system and soybean hairy roots. The results showed that GmCCS7/GmCCS24 participated in drought stress regulation. Soybean hairy roots expressing GmCCS7/GmCCS24 showed improved drought stress tolerance, with increased SOD and other antioxidant enzyme activities. The results of this study provide reference value in-depth study CCS gene family, and important gene resources for the genetic improvement of soybean drought stress tolerance.

Improvement of Lightweight Convolutional Neural Network Model Based on YOLO Algorithm and Its Research in Pavement Defect Detection.

To ensure the safe operation of highway traffic lines, given the imperfect feature extraction of existing road pit defect detection models and the practicability of detection equipment, this paper proposes a lightweight target detection algorithm with enhanced feature extraction based on the YOLO (You Only Look Once) algorithm. The BIFPN (Bidirectional Feature Pyramid Network) network structure is used for multi-scale feature fusion to enhance the feature extraction ability, and Varifocal Loss is used to optimize the sample imbalance problem, which improves the accuracy of road defect target detection. In the evaluation test of the model in the constructed PCD1 (Pavement Check Dataset) dataset, the mAP@.5 (mean Average Precision when IoU = 0.5) of the BV-YOLOv5S (BiFPN Varifocal Loss-YOLOv5S) model increased by 4.1%, 3%, and 0.9%, respectively, compared with the YOLOv3-tiny, YOLOv5S, and B-YOLOv5S (BiFPN-YOLOv5S; BV-YOLOv5S does not use the Improved Focal Loss function) models. Through the analysis and comparison of experimental results, it is proved that the proposed BV-YOLOv5S network model performs better and is more reliable in the detection of pavement defects and can meet the needs of road safety detection projects with high real-time and flexibility requirements.

Cationic P,O-Coordinated Nickel(II) Catalysts for Carbonylative Polymerization of Ethylene: Unexpected Productivity via Subtle Electronic Variation.

Transition-metal-catalyzed copolymerization of ethylene with carbon monoxide affords polyketones materials with excellent mechanical strength, photodegradability, surface and barrier properties. Unlike the widely used and rather expensive Pd catalysts, Ni-catalyzed carbonylative polymerization is very difficult since the strong binding affinity of CO to Ni deactivates the highly electrophilic metal center easily. In this study, various cationic P,O-coordinated Ni complexes were synthesized using the electronic modulation strategy, and the catalyst with strong electron-donating substituents exhibits an excellent productivity of 104  g polymer (g Ni)-1 , which represents a rare discovery that a Ni complex could operate with such exceptional efficiency in comparison with Pd catalysts. Notably, those Ni catalysts were also efficient for terpolymerization of ethylene, propylene with CO for producing commercial polyketone materials with low melting temperatures and easy processibility.

A Hybrid Deep Learning Model for Recognizing Actions of Distracted Drivers.

With the rapid spreading of in-vehicle information systems such as smartphones, navigation systems, and radios, the number of traffic accidents caused by driver distractions shows an increasing trend. Timely identification and warning are deemed to be crucial for distracted driving and the establishment of driver assistance systems is of great value. However, almost all research on the recognition of the driver's distracted actions using computer vision methods neglected the importance of temporal information for action recognition. This paper proposes a hybrid deep learning model for recognizing the actions of distracted drivers. Specifically, we used OpenPose to obtain skeleton information of the human body and then constructed the vector angle and modulus ratio of the human body structure as features to describe the driver's actions, thereby realizing the fusion of deep network features and artificial features, which improve the information density of spatial features. The K-means clustering algorithm was used to preselect the original frames, and the method of inter-frame comparison was used to obtain the final keyframe sequence by comparing the Euclidean distance between manually constructed vectors representing frames and the vector representing the cluster center. Finally, we constructed a two-layer long short-term memory neural network to obtain more effective spatiotemporal features, and one softmax layer to identify the distracted driver's action. The experimental results based on the collected dataset prove the effectiveness of this framework, and it can provide a theoretical basis for the establishment of vehicle distraction warning systems.

Phosphatase AtDBP1 negatively regulates drought and salt tolerance through altering leaf surface permeability in Arabidopsis.

In our previous study, AtDBP1 encoding a DBP factor was identified as a putative abiotic stress candidate gene. DBP factors are important regulators that participate in both transcriptional regulation and post-translational regulation, but their roles in abiotic stress are still not well-understood. So we conducted a detailed study on the function of AtDBP1 in abiotic stress. It is found that expression of AtDBP1 could be induced by drought and salt, and the induction by salt was inhibited in ABA-deficient mutant aba2-3, indicating the expression of AtDBP1 was ABA-inducible. Overexpression of AtDBP1 resulted in a rapid stomatal closure, and elevated expression of drought/salt-responsive genes, which should help Arabidopsis to enhance the drought and salt tolerance. Unexpectedly, overexpression of AtDBP1 decreased the drought and salt tolerance of Arabidopsis. Further analysis suggested that AtDBP1 is involved in cuticle wax and cuticle membrane regulation. Overexpression of AtDBP1 showed increased cuticular conductance due to a decreased cuticle wax accumulation and cuticle membrane thickness. The cuticular wax provides an essential barrier for decreasing nonstomatal water loss during drought stress, so overexpression of AtDBP1 showed decreased drought tolerance possibly ascribed to the change of cuticle membrane structure. Our previous study elucidated that AtDBP1 was also involved in flowering time regulation. Taken together, the results above indicated that AtDBP1 was involved in both plant development and stress regulation. The mechanism of AtDBP1 in this study indicates that genes involved in both plant development and stress regulation might be not suitable for production application in breeding. Collectively, our results provide some new ideas on purposefully increasing the abiotic stress without influence on plant growth and development.

Multilabel Image Classification Based Fresh Concrete Mix Proportion Monitoring Using Improved Convolutional Neural Network.

Proper and accurate mix proportion is deemed to be crucial for the concrete in service to implement its structural functions in a specific environment and structure. Neither existing testing methods nor previous studies have, to date, addressed the problem of real-time and full-scale monitoring of fresh concrete mix proportion during manufacturing. Green manufacturing and safety construction are hindered by such defects. In this study, a state-of-the-art method based on improved convolutional neural network multilabel image classification is presented for mix proportion monitoring. Elaborately planned, uniformly distributed, widely covered and high-quality images of concrete mixtures were collected as dataset during experiments. Four convolutional neural networks were improved or fine-tuned based on two solutions for multilabel image classification problems, since original networks are tailored for single-label multiclassification tasks, but mix proportions are determined by multiple parameters. Various metrices for effectiveness evaluation of training and testing all indicated that four improved network models showed outstanding learning and generalization ability during training and testing. The best-performing one was embedded into executable application and equipped with hardware facilities to establish fresh concrete mix proportion monitoring system. Such system was deployed to terminals and united with mechanical and weighing sensors to establish integrated intelligent sensing system. Fresh concrete mix proportion real-time and full-scale monitoring and inaccurate mix proportion sensing and warning could be achieved simply by taking pictures and feeding pictures into such sensing system instead of conducting experiments in laboratory after specimen retention.

Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus).

As an important maricultured fish, the olive flounder Paralichthys olivaceus shows sex-dimorphic growth. Thus, the molecular mechanisms involved in sex control in P. olivaceus have attracted researchers' attention. Among the sex-related genes, forkhead box protein L2 (foxl2) exhibits significant sex-dimorphic expression patterns and plays an important role in fish gonad differentiation and development. The present study first investigated the expression levels and promoter methylation dynamics of foxl2 during flounder gonad differentiation under treatments of high temperature and exogenous 17ß-oestradiol (E2). During high temperature treatment, the expression of flounder foxl2 may be repressed via maintenance of DNA methylation. Then, flounder with differentiated testis at Stages I-II were treated with exogenous 5ppm E2 or 5ppm E2+150ppm trilostane (TR) to investigate whether exogenous sex hormones could induce flounder sex reversal. The differentiated testis exhibited phenotypic variations of gonadal dysgenesis with upregulation of female-related genes (foxl2 and cytochrome P450 family 19 subfamily A (cyp19a)) and downregulation of male-related genes (cytochrome P450 family 11 subfamily B member 2 (cyp11b2), doublesex- and mab-3 related transcription factor 1 (dmrt1), anti-Mullerian hormone (amh) and SRY-box transcription factor 9 (sox9)). Furthermore, a cotransfection assay of the cells of the flounder Sertoli cell line indicated that Foxl2 was able alone or with nuclear receptor subfamily 5 group A member 2 (Nr5a2) jointly to upregulate expression of cyp19a. Moreover, Foxl2 and Nr5a2 repressed the expression of dmrt1. In summary, Foxl2 may play an important role in ovarian differentiation by maintaining cyp19a expression and antagonising the expression of dmrt1. However, upregulation of foxl2 is not sufficient to induce the sex reversal of differentiated testis.

Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus.

The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17ß-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 µM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 µM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.

Significant association of cyp19a promoter methylation with environmental factors and gonadal differentiation in olive flounder Paralichthys olivaceus.

The sex ratio of olive flounder Paralichthys olivaceus is sensitive to temperature or exogenous hormone exposures in the period of gonadal differentiation. Among sex-related genes, cyp19a, encoding cytochrome P450 aromatase, exhibits significant sex-dimorphic expression pattern and plays an important role in fish gonadal differentiation and development. The present study investigated the expression levels and promoter methylation dynamics of cyp19a and its regulators (nr5a2 and nr0b1), and sex-steroid hormone levels during flounder gonadal differentiation under the treatments of high temperature and estradiol-17ß (E2). The results showed that levels of flounder cyp19a expression and estradiol-17ß were repressed by high temperature treatment during this period. The up-regulation of nr5a2 by E2 treatment may be related to the all-female formation, and up-regulation of nr0b1 by high temperature treatment may be associated with masculinization. Co-transfection assay indicated that nr5a2 and nr0b1 were antagonist regulators of cyp19a. Furthermore, cyp19a promoter exhibited significant demethylation phenomenon at early stage of ovarian differentiation. While, high temperature could repress the demethylation process, resulting in hypermethylation maintenance in cyp19a promoter. The hypermethylation promoter was able to suppress cyp19a expression by blocking the nr5a2-mediated transactivation activity in vitro. The DNA methylation of epigenetic modification in cyp19a promoter might be the vital way linking environmental factors and gonadal differentiation in flounder.

The stimulatory effect of neuropeptide Y on growth hormone expression, food intake, and growth in olive flounder (Paralichthys olivaceus).

Neuropeptide Y (NPY) is a 36-amino acid peptide known to be a strong orexigenic (appetite-stimulating) factor in many species. In this study, we investigated the effect of NPY on food intake and growth in the olive flounder (Paralichthys olivaceus). Recombinant full-length NPY was injected intraperitoneally into olive flounder at the dose of 1 µg/g body weight; phosphate buffered saline was used as the negative control. In a long-term experiment, NPY and control groups were injected every fifth day over a period of 30 days. In a short-term experiment, NPY and control groups were given intraperitoneal injections and maintained for 24 h. Food intake and growth rates were significantly higher in fish injected with recombinant NPY than in the control fish (P < 0.05). Higher growth hormone (GH) and NPY mRNA transcript levels were observed in both experiments, indicating a stimulatory effect of NPY on GH release. These findings demonstrate that NPY is an effective appetite-stimulating factor in olive flounder with the potential to improve the growth of domestic fish species and enhance efficiency in aquaculture.

Characterization of pax3a and pax3b genes in artificially induced polyploid and gynogenetic olive flounder (Paralichthys olivaceus) during embryogenesis.

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.

p53-Induced inflammation exacerbates cardiac dysfunction during pressure overload.

The rates of death and disability caused by severe heart failure are still unacceptably high. There is evidence that the sterile inflammatory response has a critical role in the progression of cardiac remodeling in the failing heart. The p53 signaling pathway has been implicated in heart failure, but the pathological link between p53 and inflammation in the failing heart is largely unknown. Here we demonstrate a critical role of p53-induced inflammation in heart failure. Expression of p53 was increased in cardiac endothelial cells and bone marrow cells in response to pressure overload, leading to up-regulation of intercellular adhesion molecule-1 (ICAM1) expression by endothelial cells and integrin expression by bone marrow cells. Deletion of p53 from endothelial cells or bone marrow cells significantly reduced ICAM1 or integrin expression, respectively, as well as decreasing cardiac inflammation and ameliorating systolic dysfunction during pressure overload. Conversely, overexpression of p53 in bone marrow cells led to an increase of integrin expression and cardiac inflammation that reduced systolic function. Norepinephrine markedly increased p53 expression in endothelial cells and macrophages. Reducing ß2-adrenergic receptor expression in endothelial cells or bone marrow cells attenuated cardiac inflammation and improved systolic dysfunction during pressure overload. These results suggest that activation of the sympathetic nervous system promotes cardiac inflammation by up-regulating ICAM1 and integrin expression via p53 signaling to exacerbate cardiac dysfunction. Inhibition of p53-induced inflammation may be a novel therapeutic strategy for heart failure.

A new pattern of primordial germ cell migration in olive flounder (Paralichthys olivaceus) identified using nanos3.

The olive flounder (Paralichthys olivaceus) is an important cultured marine fish. However, little information is available on primordial germ cell (PGC) development and migration in this species; such information is vital for applications in artificial reproduction and for the preservation of genetic resources. Here, we sought to remedy this information deficit by isolating the germline-specific gene nanos3 and analyzing its expression in olive flounder. Sequencing analysis showed that olive flounder nanos3 contained a typical RNA-binding zinc finger domain. A phylogenetic analysis demonstrated that nanos3 of the olive flounder grouped with that of the barfin flounder (Verasper moseri). In the olive flounder, nanos3 was consistently expressed during embryogenesis. Whole-mount in situ hybridization showed that a new pattern of PGC migration was present in olive flounder, which combined elements of the PGC migration patterns of medaka, herring, and goby. In olive flounder, PGCs aligned along the lateral plate mesoderm in two loose, elongated lines at early embryogenesis, aggregated into a single loose cluster at mid-embryogenesis, then re-aligned into two tight clusters at late somitogenesis, and finally migrated to the genital ridge as two clusters. Furthermore, whole-mount in situ hybridization revealed that expression of stromal derived factor 1 (Sdf1) was important for guiding of PGC migration during somitogenesis. In particular, Sdf1 directed aggregation of PGCs into a single loose cluster from the two elongated lines during mid-embryogenesis. Additionally, PGCs in zebrafish were successfully visualized by injection of chimeric RNA containing the green fluorescent protein (GFP) and 3' untranslated region of olive flounder nanos3. These findings provide new insights into PGC migration and development in olive flounder and will also facilitate germ cell manipulation in this species.

Liver congestion in heart failure contributes to inappropriately increased serum hepcidin despite anemia.

Hepcidin is a key regulator of mammalian iron metabolism and mainly produced by the liver. Hepcidin excess causes iron deficiency and anemia by inhibiting iron absorption from the intestine and iron release from macrophage stores. Anemia is frequently complicated with heart failure. In heart failure patients, the most frequent histologic appearance of liver is congestion. However, it remains unclear whether liver congestion associated with heart failure influences hepcidin production, thereby contributing to anemia and functional iron deficiency. In this study, we investigated this relationship in clinical and basic studies. In clinical studies of consecutive heart failure patients (n = 320), anemia was a common comorbidity (41%). In heart failure patients without active infection and ongoing cancer (n = 30), log-serum hepcidin concentration of patients with liver congestion was higher than those without liver congestion (p = 0.0316). Moreover, in heart failure patients with liver congestion (n = 19), the anemia was associated with the higher serum hepcidin concentrations, which is a type of anemia characterized by induction of hepcidin. Subsequently, we produced a rat model of heart failure with liver congestion by injecting monocrotaline that causes pulmonary hypertension. The monocrotaline-treated rats displayed liver congestion with increase of hepcidin expression at 4 weeks after monocrotaline injection, followed by anemia and functional iron deficiency observed at 5 weeks. We conclude that liver congestion induces hepcidin production, which may result in anemia and functional iron deficiency in some patients with heart failure.

Inappropriate expression of hepcidin by liver congestion contributes to anemia and relative iron deficiency.

BACKGROUND: Anemia and relative iron deficiency (RID) are prevalent in patients with heart failure (HF). The etiology of anemia and RID in HF patients is unclear. Hepcidin expression may be closely related to anemia and RID in HF patients. Although hepcidin is produced mainly by the liver, and the most frequent histologic appearance of liver in HF patients is congestion, the influence of liver congestion (LC) on hepcidin production has not yet been investigated. We investigated whether hepcidin contributed to anemia and RID in rats with LC. METHODS AND RESULTS: LC was induced in rats by ligating the inferior vena cava and compared with bleeding anemia (BA) model induced by phlebotomy and hemolytic anemia (HA) model induced by injection of phenylhydrazine. BA and HA strongly suppressed expression of hepcidin in liver and so did not cause decrease in serum iron and transferrin saturation. However, hepcidin expression did not decrease in LC rats, which resulted in anemia and lower transferrin saturation. In addition, many cells with hemosiderin deposits were observed in the liver and spleen and not in the bone marrow, and this appeared to be related to suppression of hepcidin expression. Iron accumulated in hepatocytes, and bone morphogenetic protein 6, which induces hepcidin, increased. Inflammation was observed in the congestive liver, and there was an increase in interleukin-6, which also induced hepcidin and was induced by free heme and hemoglobin via Toll-like receptor 4. CONCLUSIONS: We conclude that LC contributes to RID and anemia, and it does so via inappropriate expression of hepcidin.

Differential regulation of IGF-I and IGF-II gene expression in skeletal muscle cells.

Insulin-like growth factor (IGF)-I and IGF-II play major roles in the regulation of skeletal muscle growth and differentiation, and both are locally expressed in muscle cells. Recent studies have demonstrated that IGF-II up-regulates its own gene expression during myogenesis and this auto-regulatory loop is critical for muscle differentiation. How local IGF-I is regulated in this process is unclear. Here, we report that while IGF-II up-regulated its own gene expression, it suppressed IGF-I gene expression during myogenesis. These opposite effects of IGF-II on IGF-I and IGF-II genes expression were time dependent and dose dependent. It has been shown that IGFs activate the PI3K-Akt-mTOR, p38 MAPK, and Erk1/2 MAPK pathways. In myoblasts, we examined their role(s) in mediating the opposite effects of IGF-II. Our results showed that both the PI3K-Akt-mTOR and p38 MAPK pathways played critical roles in increasing IGF-II mRNA expression. In contrast, mTOR was required for down-regulating the IGF-I gene expression by IGF-II. In addition, Akt, Erk1/2 MAPK, and p38 MAPK pathways were also involved in the regulation of basal levels of IGF-I and IGF-II genes during myogenesis. These findings reveal a previously unrecognized negative feedback mechanism and extend our knowledge of IGF-I and IGF-II gene expression and regulation during myogenesis.

Estimation of paracrine signaling using quantitative RT-PCR from multiple patchy lesion samples.

We investigated whether correlations between mRNA levels of cytokines versus other proteins from patchy lesion could estimate cytokine paracrine signaling in vivo. Experiments with rat experimental autoimmune myocarditis (EAM), a patchy myocarditis model, indicated IL-1 and other protein levels were correlated, indicating paracrine signaling pathways in vivo.