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ABSTRACT We assessed the effect of health sand dietary supplementation with methionine (Met) on White King pigeons. Paired pigeons (n = 180) were fed one of five diets; group T1 received no added Met, while T2, T3, T4 and T5 received 30, 60, 90 and 120 g of supplemental DL-Met/kg, respectively. Each treatment was replicated three times with 24 pairs in each replicate. The results showed that supplementary Met had a minor effect on the length of the fourth primary wing feather in 28-day-old squabs (p>0.05), but the length of 14-day-old squabs in T2 was significantly longer (p=0.010). Dietary Met had a minor effect on Wnt-7a and fibroblast growth factor receptors-2 (FGFR-2) mRNA levels in 28-day-old squabs (p>0.05). The IGF-1 concentration in plasma was highest in T4 and lowest in T2 (p=0.012), but there was no difference between T1, T2 and T5 (p>0.05). In the chest muscle, the expression of IGF-1 in T3 and T4 was higher than in T1 (p=0.172 and 0.015, respectively). In the leg muscle, IGF-1 mRNA level was higher in T4 and T3, and lower in T2 (p>0.05). The results indicate that the optimal Met supplement for increasing fourth primary wing feather length was 30 g/kg Met in health sand, and the feathers were the longest in 14-day-old squabs. Adding 90 g/kg Met to health sand can improve the concentration of IGF-1, which is important for growth performance of pigeon squabs.

Microalgae such as dinoflagellate and diatom are the major food source of bivalve species, and sufficient food intake contributes to the immunity and the growth of bivalves. In the present study, a monoamine oxidase gene (named as CgMAO), which is the rate-limiting enzyme of norepinephrine (NE) biosynthesis, was cloned from C. gigas. After the oysters were fed with a diet rich in diatom for 21 and 40 d, the NE contents in oyster serum, as well as the mRNA expression of CgMAO in oyster haemocytes, increased significantly compared with control group. Besides, the mRNA expression of cytokines CgTNF-1 and CgIL17-5 in haemocytes and the activities of immune-related enzymes (SOD and LYZ) in oyster serum also increased significantly after diatom feeding. These results collectively suggested that sufficient microalgae intake might significantly enhance the antibacterial capacity in oyster by prompting the biosynthesis of NE and triggering the subsequent antibacterial processes modulated by NE.

Recent years, invertebrate animals with clear genetic background and completed genome sequence have become very popular in biological research. Insulin and insulin-like peptides (ILPs) are a class of peptides having important physiological functions, including promotion of cell proliferation and differentiation, and body growth and development. To date, many invertebrates have been identified with multiple ILP families, whose structures and functions have become increasingly clear to scientists. This review summarizes the ILPs identified in the model Lepidopteran insect Bombyx mori, in the aspects of structures and classification, functions and pathways. A brief discussion on the ILPs from several other model insects, including Drosophila melanogaster, Aedes aegypti, and Apis mellifera, was also included.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most serious viral disease in silkworms. To investigate the mechanisms of the immune responses of B. mori to a BmNPV infection, a suitable reference gene (RG) is necessary for normalizing data when studying the expression of genes in BmNPV-infected silkworms or cells. Thus, quantitative real-time PCR polymerase chain reaction was used to compare the stability of expression of nine potential RGs, including the actin A3, translation initiation factor 3 (TIF-3), TIF-A4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S RNA, 28S RNA, TATA-binding protein (TBP), ribosomal protein L3 (Rpl3), and α-tubulin genes, in silkworms infected with BmNPV. The results were analyzed by BestKeeper, geNorm, and NormFinder software. Overall, α-tubulin exhibited the most stable gene expression in BmNPV-infected silkworms, and this was verified by western blotting of the α-tubulin protein. Moreover, we detected the expression of some genes involved in the immune signaling pathways of silkworms after BmNPV infection using α-tubulin as an internal RG.

Ste20-like kinases are critically multifuctional proteins which play important roles in varieties of celluar processes and physiological events. Here, We characterized a Ste20-like kinase gene (DjSlk) in planarian Dugesia japonica. Whole-mount in situ hybridizations revealed that DjSlk was expressed in the central nervous system (CNS) including cephalic ganglia and ventral nerve cords (VNCs) in intact and regenerating animals. After RNA interference (RNAi) of DjSlk, adult planarians became immobilized and wrinkled, then swelled and lysed eventually. DjSlk RNAi treated regenerating planarians could form the entire animals, and then displayed the similar phenotype transformation. These results suggest that loss of function of DjSlk leads to morphogenetic malformation of planarian D. japonica probably via regulating cell volume instead of disrupting the balance between cell proliferation and apoptosis.

Ectopic calcifications in intervertebral discs (IVDs) are known characteristics of IVD degeneration that are not commonly reported but may be implicated in structural failure and dysfunctional IVD cell metabolic responses. This study investigated the novel hypothesis that ectopic calcifications in the IVD are associated with advanced glycation end products (AGEs) via hypertrophy and osteogenic differentiation. Histological analyses of human IVDs from several degeneration stages revealed areas of ectopic calcification within the nucleus pulposus and at the cartilage endplate. These ectopic calcifications were associated with cells positive for the AGE methylglyoxal-hydroimidazolone-1 (MG-H1). MG-H1 was also co-localised with Collagen 10 (COL10) and Osteopontin (OPN) suggesting osteogenic differentiation. Bovine nucleus pulposus and cartilaginous endplate cells in cell culture demonstrated that 200 mg/mL AGEs in low-glucose media increased ectopic calcifications after 4 d in culture and significantly increased COL10 and OPN expression. The receptor for AGE (RAGE) was involved in this differentiation process since its inhibition reduced COL10 and OPN expression. We conclude that AGE accumulation is associated with endochondral ossification in IVDs and likely acts via the AGE/RAGE axis to induce hypertrophy and osteogenic differentiation in IVD cells. We postulate that this ectopic calcification may play an important role in accelerated IVD degeneration including the initiation of structural defects. Since orally administered AGE and RAGE inhibitors are available, future investigations on AGE/RAGE and endochondral ossification may be a promising direction for developing non-invasive treatment against progression of IVD degeneration.

X Chen* , F Zhu* , P Lü, S Ma, A Nouara, Y Yang, K Chen * These authors contributed equally to this work Institute of Life Sciences, Jiangsu University, Zhenjiang, 212013, Jiangsu, P.R. China Accepted March 10, 2016 Abstract Insulin/Insulin-like growth factors play important roles in promoting proliferation, differentiation, growth and development of organisms. In this article, bioinformatics analysis was performed to locate and identify the Bombyx mori insulin-like peptide (BmILP) gene, which had two introns, three exons, and a predicted signal peptide at the front, and it also determined that BmILP was the same to the gene Bombyxin Z1, a member of the bombyxin family. Both the full-length BmILP gene and the gene without the signal peptide expressed mainly existed in inclusion body forms at different induction conditions and it was noted that the signal peptide had significant influence on the expression level. In addition, protein-protein interaction experiments suggested that there was interaction between BmILP and the autophagy-related protein Atg8, indicating that BmILP may play a role in immunity. It was found was that BmILP predominantly produced by the ovary among different tissues in the silkworm. Particularly, the adult females had higher expression levels than the adult males, and the expression level appeared to be the highest in the mated female moths, suggesting that BmILP may be a key factor in the regulation of egg maturation in Bombyx mori.

Mercury (Hg), one of the most hazardous and persistent contaminants, is widespread in the aquatic environment. To establish an effective Hg monitoring strategy, the mRNA expression profiles of four antioxidant enzymes, as well as SOD enzymatic activities and MDA content were investigated at two sublethal HgCl2 exposure doses of 5 and 50 μg L-1 in Venerupis philippinarum gill tissue and hemocytes at 1, 2, 4 and 5 days. All parameters tested showed significant differences between the experimental and control groups at the various time points with tissue-specific manners. GST displayed a dose-dependent increase expression profiles in the two examined tissues. Also decrease expression patterns were detected in Trx and CYP414A1 in both gill and haemocytes with a significant positive relationship of 0.915 in the form tissue (p < 0.01). A positive relationship was found in those of SOD expression and the SOD enzymatic activities in hemocytes (0.683, p < 0.05). Taken together, we found that gill tissue is more suitable for biomarker identification compared to that of hemocytes because of lower variation. This study provides new evidences that mRNA expression of Trx, CYP414A1 and GST in gill tissue has a strong potential as a biomarker for marine mercury monitoring.

Di-(2-ethylhexyl) phthalate (DEHP) has many adverse effects on immunity and metabolic states. However, scarce information is available on its connection with toxicologically relevant proteomics response in marine invertebrates. In this study, GS-MS was employed to determine the bio-accumulated levels of DEHP in clam Venerupis philippinarum. After exposure to 0.4 mg L-1 and 4mg L-1 DEHP, the bio-accumulated DEHP in the clam foot was significantly increased in the first 24 h, and then sharply decreased from 0.203 ± 0.022 μg g-1 to 0.104 ± 0.011 μg g-1 , and from 1.689 ± 0.018 μg g-1 to 1.172 ± 0.012 μg g-1, respectively. Comparative proteomic was conducted to investigate the global protein expression changes towards this contaminant exposure. Twenty-eight proteins with significant differences in abundance were identified and characterized, among them six enzymes related to the glycolysis pathway were suppressed, and two members of TCA cycle were induced. The activity and mRNA expression level of malate dehydrogenase (MDH) were further assessed using qPCR and an enzymatic assay. The MDH activity and mRNA transcript levels were both elevated compared to those in the ethanol control group. Our findings indicated that a DEHP-treated clam modulated host toxicological effect through depressing glycogen synthesis and activating TCA cycle.

Ste20-like kinases are critically multifuctional proteins which play important roles in varieties of celluar processes and physiological events. Here, We characterized a Ste20-like kinase gene (DjSlk) in planarian Dugesia japonica. Whole-mount in situ hybridizations revealed that DjSlk was expressed in the central nervous system (CNS) including cephalic ganglia and ventral nerve cords (VNCs) in intact and regenerating animals. After RNA interference (RNAi) of DjSlk, adult planarians became immobilized and wrinkled, then swelled and lysed eventually. DjSlk RNAi treated regenerating planarians could form the entire animals, and then displayed the similar phenotype transformation. These results suggest that loss of function of DjSlk leads to morphogenetic malformation of planarian D. japonica probably via regulating cell volume instead of disrupting the balance between cell proliferation and apoptosis.

Bone marrow-derived, circulating endothelial progenitor cells (EPCs) contribute to neovascularization in various diseases, and represent a very interesting alternative cell source for enhancing vasculogenesis in regenerative medicine. In this study, we investigated the effects of Ginkgolide B (GB) on proliferation and differentiation of EPCs, and the involved signaling pathway in vitro. EPC proliferation, migration, adhesion and angiogenesis activities were assessed with the WST-8 assay, Transwell chamber assay, cell counting and angiogenesis kit, respectively. Apoptosis was detected with annexin V and propidium iodide staining. The protein expression of angiogenesis-related makers was detected by Western blot, and related gene expression was determined by real-time polymerase chain reaction (RT-PCR). The results showed that GB promoted the proliferation and endothelial gene expression, and markedly enhanced vascular endothelial growth factor-induced migration response and the capability to incorporate into the vascular networks in EPCs. GB protected EPCs from H2O2-induced cell death. GB induced the phosphorylation of eNOS, Akt and p38, which in turn promoted cell proliferation and function. In conclusion, the present study demonstrates that GB, at a near medical applied dose, increases the number and functional activities of EPCs with involvement of Akt/endothelial nitric oxide synthase and mitogen-activated protein kinase (MAPK)/p38 signal pathways. These findings raise the intriguing possibility that GB may play an important role in the protection and revascularization of blood vessels.

We present a study of the magnetic configuration due to step-induced magnetic frustration at ferromagnetic/antiferromagnetic (FM/AFM) interfaces. At a substrate monatomic step edge, a 180° domain wall emerges. A physically appealing form for the thickness dependence of the domain wall width is obtained. It follows a universal behaviour in the whole thickness range, from ultrathin film to bulk and in both cases of an AFM domain wall on top of the FM layer and a FM domain wall on top of an AFM substrate. In the ultrathin limit of the capping layer, the domain wall grows linearly with the slope depending only on the ratio of the inter-layer and intra-layer Heisenberg exchange constants, regardless of the presence of magneto-crystalline anisotropy. These findings are in good agreement with previous experimental observations. As the thickness grows beyond the ultrathin regime, the corresponding thickness dependence departs from linearity and tends to its bulk value. The analytical insights are supported by conclusive numerical simulations of two independent varieties, namely, the Monte Carlo method which also includes the growth kinetics and the object oriented micromagnetic framework based micromagnetic simulations. While the quantitative details of the study are naturally dependent on the specific material parameters of the complex magnetic system, the global features of the spin texture in the capping layer are dictated by the topological step-edge defect. The latter in itself is quantifiable by a winding number of $\pm \tfrac{1}{2}.$

Embryonic stem cells (ESC) are both a potential source of cells for tissue replacement therapies and an accessible tool to model early embryonic development. Chemical factors such as soluble growth factors and insoluble components of the extracellular matrix are known to affect the differentiation of murine ESCs. However, there is also evidence to suggest that undifferentiated cells can both sense the mechanical properties of their environment and differentiate accordingly. By growing ESCs on flexible polydimethylsiloxane substrates with varying stiffness, we tested the hypothesis that substrate stiffness can influence ESC differentiation. While cell attachment was unaffected by the stiffness of the growth substrate, cell spreading and cell growth were all increased as a function of substrate stiffness. Similarly, several genes expressed in the primitive streak during gastrulation and implicated in early mesendoderm differentiation, such as Brachyury, Mixl1 and Eomes, were upregulated in cell cultures on stiffer compared to softer substrates. Finally, we demonstrated that osteogenic differentiation of ESCs was enhanced on stiff substrates compared to soft substrates, illustrating that the mechanical environment can play a role in both early and terminal ESC differentiation. Our results suggest a fundamental role for mechanosensing in mammalian development and illustrate that the mechanical environment should be taken into consideration when engineering implantable scaffolds or when producing therapeutically relevant cell populations in vitro.

The system of a trapped ion translationally excited by a blue-detuned near-resonant laser, sometimes described as an instance of a phonon laser, has recently received attention as interesting in its own right and for its application to non-destructive readout of internal states of non-fluorescing ions. Previous theoretical work has been limited to cases of two-level ions. Here, we perform simulations to study the dynamics of a phonon laser involving the Λ -type $^{138}{\rm B}{{{\rm a}}^{+}}$ ion, in which coherent population trapping (CPT) effects lead to different behavior than in the previously studied cases. We explore optimization of the laser parameters to maximize amplification gain for initially seeded motion and consider the related signal-to-noise ratios for internal state readout. We find that good Doppler amplification and state readout performance can be obtained even when operating quite near the CPT dip.