Transcriptome analysis of genes related to gonad differentiation and development in Muscovy ducks.

BACKGROUND: Sex-related genes play a crucial role in gonadal differentiation into testes or ovaries. However, the genetic control of gonadal differentiation in Muscovy ducks remains unknown. Therefore, the objective of our study was to screen new candidate genes associated with ovarian and testicular development. RESULTS: In this study, 24 males before gonadal differentiation (MB), 24 females before gonadal differentiation (FB), 24 males after gonadal differentiation (MA) and 24 females after gonadal differentiation (FA) were selected from Putian Muscovy ducks, forming 4 groups. RNA-Seq revealed 101.76 Gb of clean reads and 2800 differentially expressed genes (DEGs), including 46 in MB vs FB, 609 in MA vs FA, 1027 in FA vs FB, and 1118 in MA vs MB. A total of 146 signalling pathways were enriched by KEGG analysis, among which 20, 108, 108 and 116 signalling pathways were obtained in MB vs FB, MA vs MB, MA vs FA and FA vs FB, respectively. In further GO and KEGG analyses, a total of 21 candidate genes related to gonad differentiation and development in Muscovy ducks were screened. Among these, 9 genes were involved in the differentiation and development of the testes, and 12 genes were involved in the differentiation and development of the ovaries. In addition, RNA-Seq data revealed 2744 novel genes. CONCLUSIONS: RNA-Seq data revealed 21 genes related to gonadal differentiation and development in Muscovy ducks. We further identified 12 genes, namely, WNT5B, HTRA3, RSPO3, BMP3, HNRNPK, NIPBL, CREB3L4, DKK3, UBE2R2, UBPL3KCMF1, ANXA2, and OSR1, involved in the differentiation and development of ovaries. Moreover, 9 genes, namely, TTN, ATP5A1, DMRT1, DMRT3, AMH, MAP3K1, PIK3R1, AGT and ADAMTSL1, were related to the differentiation and development of testes. Moreover, after gonadal differentiation, DMRT3, AMH, PIK3R1, ADAMTSL1, AGT and TTN were specifically highly expressed in males. WNT5B, ANXA2 and OSR1 were specifically highly expressed in females. These results provide valuable information for studies on the sex control of Muscovy ducks and reveal novel candidate genes for the differentiation and development of testes and ovaries.

Isolation of blue-green eggshell pigmentation-related genes from Putian duck through RNA-seq.

BACKGROUND: The diversity of avian eggshell colour plays important biological roles in ensuring successful reproduction. Eggshell colour is also an important trait in poultry, but the mechanisms underlying it are poorly understood in ducks. This study aimed to provide insights into the mechanism of blue-green eggshell colour generation. RESULTS: Here, white-shelled ducks (HBR) and blue-green-shelled ducks (HQR) were selected from Putian black ducks, and white-shelled ducks (BBR) were selected from Putian white ducks. Transcriptional changes in the shell gland were analysed using RNA-sequencing on the Illumina HiSeq 2500. Twenty-seven individual cDNA libraries were sequenced and generated an average of 7.35 million reads per library; 70.6% were mapped to the duck reference genome, yielding an average of 13,794 genes detected, which accounted for approximately 86.39% of all 15,967 annotated duck genes. A total of 899 differentially expressed genes (DEGs) were detected between the HQR and BBR groups, and 373 DEGs were detected between the HQR and HBR groups. We analysed the DEGs in the HQR-vs-BBR and HQR-vs-HBR comparisons. None of these DEGs were directly involved in the eggshell pigmentation process in HQR-vs-HBR, while UDP-glucuronosyltransferase 2A2 (UGT2A2) and UDP-glucuronosyltransferase 1-1-like (UGT1-1-like), which participate in biliverdin breakdown, were two of the DEGs in HQR-vs-BBR. In the RT-qPCR results, delta-aminolevulinic acid synthase 1 (ALAS1) and EPRS glutamyl-prolyl-tRNA synthetase were significantly upregulated in the HBR group compared with the HQR and BBR groups (P < 0.05). Haem oxygenase (HMOX1) was significantly downregulated in BBR compared with HQR and HBR (P < 0.05). Biliverdin reductase A (BLVRA), GUSB glucuronidase beta, cytochrome c-type haem lyase, protohaem IX farnesyltransferase and UGT2A2 were significantly upregulated in HBR and BBR compared with HQR (P < 0.05). CONCLUSIONS: We conducted a comparative transcriptome analysis of the shell glands of Putian white ducks and Putian black ducks. None of the differentially regulated pathways were directly involved in the eggshell pigmentation process in the HQR-vs-HBR comparison, while 2 DEGs related to biliverdin breakdown were found in HQR-vs-BBR. Based on the RT-qPCR results, we can speculate that both HQR and HBR can produce biliverdin, but HBR cannot accumulate it. Compared with HQR, BBR produced less biliverdin and did not accumulate it.

Theranostics Aspects of Various Nanoparticles in Veterinary Medicine.

Nanoscience and nanotechnology shows immense interest in various areas of research and applications, including biotechnology, biomedical sciences, nanomedicine, and veterinary medicine. Studies and application of nanotechnology was explored very extensively in the human medical field and also studies undertaken in rodents extensively, still either studies or applications in veterinary medicine is not up to the level when compared to applications to human beings. The application in veterinary medicine and animal production is still relatively innovative. Recently, in the era of health care technologies, Veterinary Medicine also entered into a new phase and incredible transformations. Nanotechnology has tremendous and potential influence not only the way we live, but also on the way that we practice veterinary medicine and increase the safety of domestic animals, production, and income to the farmers through use of nanomaterials. The current status and advancements of nanotechnology is being used to enhance the animal growth promotion, and production. To achieve these, nanoparticles are used as alternative antimicrobial agents to overcome the usage alarming rate of antibiotics, detection of pathogenic bacteria, and also nanoparticles being used as drug delivery agents as new drug and vaccine candidates with improved characteristics and performance, diagnostic, therapeutic, feed additive, nutrient delivery, biocidal agents, reproductive aids, and finally to increase the quality of food using various kinds of functionalized nanoparticles, such as liposomes, polymeric nanoparticles, dendrimers, micellar nanoparticles, and metal nanoparticles. It seems that nanotechnology is ideal for veterinary applications in terms of cost and the availability of resources. The main focus of this review is describes some of the important current and future principal aspects of involvement of nanotechnology in Veterinary Medicine. However, we are not intended to cover the entire scenario of Veterinary Medicine, despite this review is to provide a glimpse at potential important targets of nanotechnology in the field of Veterinary Medicine. Considering the strong potential of the interaction between the nanotechnology and Veterinary Medicine, the aim of this review is to provide a concise description of the advances of nanotechnology in Veterinary Medicine, in terms of their potential application of various kinds of nanoparticles, secondly we discussed role of nanomaterials in animal health and production, and finally we discussed conclusion and future perspectives of nanotechnology in veterinary medicine.

Effects of PB1-F2 on the pathogenicity of H1N1 swine influenza virus in mice and pigs.

Although several studies have exploited the effects of PB1-F2 in swine influenza viruses, its contribution to the pathogenicity of swine influenza viruses remains unclear. Herein, we investigated the effects of PB1-F2 on the pathogenicity of influenza virus using a virulent H1N1 A/swine/Kansas/77778/2007 (KS07) virus, which expresses a full-length PB1-F2, in mice and pigs. Using reverse genetics, we generated the wild-type KS07 (KS07_WT), a PB1-F2 knockout mutant (KS07_K/O) and its N66S variant (KS07_N66S). KS07_K/O showed similar pathogenicity in mice to the KS07_WT, whereas KS07_N66S displayed enhanced virulence when compared to the other two viruses. KS07_WT exhibited more efficient replication in lungs and nasal shedding in infected pigs than the other two viruses. Pigs infected with the KS07_WT had higher pulmonary levels of granulocyte-macrophage colony-stimulating factor, IFN-γ, IL-6 and IL-8 at 3 and 5 days post-infection, as well as lower levels of IL-2, IL-4 and IL-12 at 1 day post-infection compared to those infected with the KS07_K/O. These results indicate that PB1-F2 modulates KS07 H1N1 virus replication, pathogenicity and innate immune responses in pigs and the single substitution at position 66 (N/S) in the PB1-F2 plays a critical role in virulence in mice. Taken together, our results provide new insights into the effects of PB1-F2 on the virulence of influenza virus in swine and support PB1-F2 as a virulence factor of influenza A virus in a strain- and host-dependent manner.

Inactivation kinetics of formaldehyde on N-acetyl-ß-D-glucosaminidase from Nile tilapia (Oreochromis niloticus).

Formaldehyde is a widely used sanitizer in aquaculture in China, while the appropriate concentration is not available to be used effectively and without damage to tilapia much less to its reproductive function. N-acetyl-ß-D-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzing the oligomers of N-acetyl-ß-D-glucosamine into monomer, is proved to be correlated with reproduction of male animals. In this paper, NAGase from spermary of tilapia was chosen as the material to study the effects of formaldehyde on its activity in order to further investigate the effects of formaldehyde use on tilapia reproduction. The results showed the relationship between the residual enzyme activity and the concentration of formaldehyde was concentration dependent, and the IC50 value was estimated to be 3.2 ± 0.1 %. Appropriate concentration of formaldehyde leaded to competitive reversible inhibition on tilapia NAGase. Moreover, formaldehyde could reduce the thermal and pH stability of the enzyme. The inactivation kinetics of formaldehyde on the enzyme was studied using the kinetic method of substrate reaction. The inactivation model was setup, and the rate constants were determined. The results showed that the inactivation of formaldehyde on tilapia NAGase was a slow, reversible reaction with partially residual activity. The results will give some basis to determine the concentration of formaldehyde used in tilapia culture.

Mechanism of fatty acid transposase (CD36) promoting fat accumulation in mule ducks.

Mule ducks accumulate a large amount of fat in their livers when fed high-energy feed, which is predominantly used for producing fatty livers. Nevertheless, there is limited research on the molecular mechanisms underlying the formation of fatty liver in mule ducks. Fatty acid translocase (CD36) is a sensor for fatty acids and lipid metabolism regulator, which may play a crucial role in the accumulation of fat in the liver of mule ducks. In this study, Overexpression and CD36 gene interference for 24 h was followed by induction of liver cells with 400 µmol/L palmitic acid (PA) for 24 h. The results demonstrated that CD36 overexpression increased hepatic triglyceride content, lipid droplet deposition, oxidative stress, and cell apoptosis. However, interference with CD36 had the opposite effect. CD36 overexpression suppressed the expression of AMPK and CPT-1A genes but enhanced the expression of ACC1 and LKB1 genes, with interference yielding contrasting results. Additionally, the expression of CD36 inhibited the AMPK pathway, reduced AMPK phosphorylation, downregulated AMPK protein expression, and upregulated SREBP1 protein expression. This promoted palmitic acid-induced hepatocyte fat accumulation. In summary, CD36 promotes palmitic acid-induced fat accumulation in primary mule duck liver cells through the AMPK signaling pathway.

Mechanism of fibroblast growth factor 1 regulating fatty liver disorder in mule ducks.

Mule ducks tend to accumulate abundant fat in their livers via feeding, which leads to the formation of a fatty liver that is several times larger than a normal liver. However, the mechanism underlying fatty liver formation has not yet been elucidated. Fibroblast growth factor 1 (FGF1), a member of the FGF superfamily, is involved in cellular lipid metabolism and mitosis. This study aims to investigate the regulatory effect of FGF1 on lipid metabolism disorders induced by complex fatty acids in primary mule duck liver cells and elucidate the underlying molecular mechanism. Hepatocytes were induced by adding 1,500:750 µmol/L oleic and palmitic acid concentrations for 36 h, which were stimulated with FGF1 concentrations of 0, 10, 100, and 1000 ng/mL for 12 h. The results showed that FGF1 significantly reduced the hepatic lipid droplet deposition and triglyceride content induced by complex fatty acids; it also reduced oxidative stress; decreased reactive oxygen species fluorescence intensity and malondialdehyde content; upregulated the expression of antioxidant factors nuclear factor erythroid 2 related factor 2 (Nrf2), HO-1, and NQO-1; significantly enhanced liver cell activity; promoted cell cycle progression; inhibited cell apoptosis; upregulated cyclin-dependent kinase 1 (CDK1) and BCL-2 mRNA expression; and downregulated Bax and Caspase-3 expression. In addition, FGF1 promoted AMPK phosphorylation, activated the AMPK pathway, upregulated AMPK gene expression, and downregulated the expression of SREBP1 and ACC1 genes, thereby alleviating excessive fat accumulation in liver cells induced by complex fatty acids. In summary, FGF1 may alleviate lipid metabolism disorders induced by complex fatty acids in primary mule duck liver cells by activating the AMPK signaling pathway.

PiggyBac transposon-mediated gene transfer in Cashmere goat fetal fibroblast cells.

PiggyBac (PB) has recently been found to be functional in various organisms. To verify and exploit its application in the cashmere goat, a PB transposon system including donor and helper vector of was developed, in which the EGFP gene in donor of vector was used as reporter. Cashmere goat fetal fibroblasts cells (GFFs) were transfected with the PB transposon system and the efficiency of gene transfer was determined. Compared with random integration, PB-mediated EGFP expression levels increased 7.78-fold in the GFFs, confirming that the PB transposon system constructed successfully mediated efficient foreign gene integration in the GFFs. To further investigate the characteristics of PB-mediated integration instance, PB integration site distribution in the goat genome was examined. The results showed that PB had a preference for AT rich regions of the goat genome. Thus this study confirms the function of PB transposon in GFFs and provides a potential genetic tool for producing transgenic goats.

Differential expression of MSTN, IGF2BP1, and FABP2 across different embryonic ages and sexes in white Muscovy ducks.

To explore the effects of growth-related genes in both sexes and at different growth and development stages, male and female white Muscovy ducks at embryonic day E13, E17, E21, E25 and E29 were assessed in this study. RT-qPCR was used to determine the mRNA transcription levels of selected growth-related genes in the leg muscles of Muscovy ducks of both sexes and at different growth and developmental stages. MSTN, IGF2BP1 and FABP2 mRNAs were expressed in the leg muscles of male and female Muscovy ducks, but with different expression patterns. The MSTN and IGF2BP1 mRNA expression patterns were wavelike. MSTN mRNA expression was elevated at E13, increased at E17, decreased rapidly to the lowest level at E21, increased again at E25, and then decreased. IGF2BP1 mRNA expression was elevated at E13, increased at E17, decreased rapidly at E21, decreased rapidly to the lowest level at E25, and increased at E29. The expression trend of FABP2 mRNA was approximately "⊥" shape; the expression was the lowest at E13, increased slowly from E17 to E25, and increased extremely significantly at E29. In addition, the expression of MSTN in male Muscovy ducks was significantly higher than that in female ducks at E25 (P < 0.05). The expression of IGF2BP1 in male Muscovy ducks was extremely significantly higher than that in female ducks at E17 (P < 0.01). However, the expression of FABP2 in female Muscovy ducks was extremely significantly higher than that in male Muscovy ducks at E21 and E29 (P < 0.01). In conclusion, the mRNA expression of MSTN, IGF2BP1 and FABP2 in white Muscovy ducks is gestational age specific and sex specific. The differential gene expression patterns observed in this study provide a basis for understanding the physiological changes in white Muscovy ducks at different embryonic ages and in both sexes, supplementing the existing research on duck embryo muscle development. In addition, the findings provide a new framework for further discussion of poultry breeding.

SNP discovery and functional annotation in the Panax japonicus var. major transcriptome.

Due to the lack of a Panax japonicus var. major reference genome, we assembled a reference transcriptome from P. japonicus C. A. Mey transcriptome sequencing data, and 203 283 unigenes were obtained. In this study, with the assistance from the Trinity, Bowtie2 and SAMtools softwares, 218 465 single nucleotide polymorphisms (SNPs) were identified by mapping the Illumina sequences to the reference transcriptome. The SNP forms included 126 262 transformations and 92 203 transversions. A large number of SNP loci were associated with triterpenoid saponin synthesis: 54 SNPs were associated with cytochrome P450, one with glycosyl transferase and 94 with the biosynthesis of the triterpenoid saponin backbone.

Curcumin attenuates oxidative stress in RAW264.7 cells by increasing the activity of antioxidant enzymes and activating the Nrf2-Keap1 pathway.

Large-scale breeding environments often lead to oxidative stress. Macrophages play an important role in the immune system and are vulnerable to reactive oxygen species (ROS), which result in macrophage death. Curcumin is the main active component of turmeric and exerts antioxidant effects. Here, we measured the activity of some antioxidant enzymes and chose the Nrf2-Keap1 signaling pathway to study the protective effects of curcumin on macrophages under oxidative stress in vitro. We used RAW264.7 cells as a research model, and oxidative damage was induced by hydrogen peroxide (H2O2). Cell viability was measured by an MTT assay. Flow cytometry was used to measure cellular ROS and apoptosis. The effect of curcumin on Nrf2-Keap1 signaling pathway-related genes was analyzed by qRT-PCR. Furthermore, the translocation of Nrf2 protein was also investigated by Western blot analysis of total and nuclear proteins. All curcumin-treated groups exhibited increased activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX). Low- and middle-dose curcumin decreased malondialdehyde (MDA) and ROS levels, but high-dose curcumin increased MDA and ROS production. We found that low-dose curcumin protected cells from apoptosis, while apoptosis in the middle- and high-dose curcumin-treated groups were stagnant in the early stage. Furthermore, middle-dose curcumin upregulated Nrf2 expression after H2O2 treatment for 4 h. Low- and middle-dose curcumin could activate Nrf2 and promote it to migrate into nuclei. The translocation of Nrf2 to the nucleus to upregulate the expression of haemoxygenase-1 (HO-1) was promoted in the low- and middle-dose curcumin-treated groups. The middle-dose curcumin-treated group also exhibited enhanced expression of glutamate-cysteine ligase, a modifier subunit (GLCM), but inhibited transcription of glutamate-cysteine ligase, a catalytic subunit (GCLC). Curcumin resisted oxidants by increasing the activity of antioxidant enzymes and activating the Nrf2-Keap1 pathway, which could potentially promote cell survival.

The Degree of Sex Reversal in Muscovy Ducks (Cairina moschata domestica) Induced by an Aromatase Inhibitor.

Under the same feeding conditions, the growth and development of male Muscovy ducks is significantly greater than that of females. Thus, controlling their sex expression can have economic benefits. However, reports on the degree of sex reversal in Muscovy ducks are scarce. In this study, we obtained sex-reversed Muscovy ducks by injecting letrozole before sex differentiation. We analyzed the degree of sex reversal in Muscovy ducks in terms of hormone levels, gonadal tissue development, and growth and found that the estradiol levels of AI-females (letrozole-induced female-to-male sex reversal) were not significantly different from those of normal males (p > 0.05), but the testosterone levels were significantly lower than those in normal males (p < 0.05). AI-female gonad tissue had changed, and the right gonad presented ovotestis tissue. The growth and development of AI-females was significantly less than that of normal males (p < 0.05) but was not significantly different from that of normal females (p > 0.05). Letrozole can induce female Muscovy ducks to convert into males, but the reversal cannot be completed. Thus, further studies are needed to elucidate how to entirely attain the change.

Identification and evaluation of antivirals for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever (RVF) that affects both livestock and humans. There are neither fully licensed RVF vaccines available for human or animal use, nor effective antiviral drugs approved for human use in the U.S. To identify antiviral compounds effective for RVF, we developed and employed a cell-based high-throughput assay using a recombinant RVFV MP-12 strain, which expresses Renilla luciferase in place of the NSs protein, to screen 727 small compounds purchased from the National Institutes of Health. Twenty-three compounds were initially identified using the screening assay. Two compounds, 6-azauridine and mitoxantrone, also inhibited the replication of the parental MP-12 strain encoding the NSs gene, with limited cytotoxic effects. The respective 50% inhibitory concentrations were 29.07 µM and 79.85 µM when tested with the parental MP-12 strain at a multiplicity of infection of 2. The compounds were further evaluated using the STAT-1 KO mouse model. At one hour post intranasal inoculation of MP-12 strain, mice were intranasally treated with each indicated compound twice daily. Mice treated with either placebo or 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurologic symptoms. Onset of disease was, however, delayed in mice treated with either ribavirin or mitoxantrone. The results indicated that mitoxantrone can reduce the severity of diseases in RVFV-infected mice. Our studies build the foundation for the initial screening and efficacy studies of RVF antivirals in a BSL-2 environment, avoiding the higher risks of BSL-3 exposure with wild-type virus.

Generation of a transgenic cashmere goat using the piggyBac transposition system.

The development of transgenic technologies in the Cashmere goat (Capra hircus) has the potential to improve the quality of the meat and wool. The piggyBac (PB) transposon system is highly efficient and can be used to transpose specific target genes into the genome. Here, we developed a PB transposon system to produce transgenic Cashmere goat fetal fibroblasts (GFFs) with the enhanced green fluorescent protein (EGFP). We then used the genetically modified GFFs as nuclear donors to generate transgenic embryos by somatic cell nuclear transfer (SCNT). The embryos (n = 40) were implanted into female goats (n = 20). One transgenic kid that expressed EGFP throughout the surface features of its body was born. This result demonstrated the usefulness of PB transposon system in generating transgenic Cashmere goats.

Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells.

BACKGROUND: Zinc oxide nanoparticles (ZnO NPs) are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3). METHODS: ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. RESULTS: Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H2AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy. Finally, the Western blot analysis revealed upregulation of Bax, caspase-9, Rad51, γ-H2AX, p53, and LC3 and downregulation of Bcl-2. CONCLUSION: The study findings demonstrated that the ZnO NPs are able to induce significant cytotoxicity, apoptosis, and autophagy in human ovarian cells through reactive oxygen species generation and oxidative stress. Therefore, this study suggests that ZnO NPs are suitable and inherent anticancer agents due to their several favorable characteristic features including favorable band gap, electrostatic charge, surface chemistry, and potentiation of redox cycling cascades.

Novel biomolecule lycopene-reduced graphene oxide-silver nanoparticle enhances apoptotic potential of trichostatin A in human ovarian cancer cells (SKOV3).

BACKGROUND: Recently, there has been much interest in the field of nanomedicine to improve prevention, diagnosis, and treatment. Combination therapy seems to be most effective when two different molecules that work by different mechanisms are combined at low dose, thereby decreasing the possibility of drug resistance and occurrence of unbearable side effects. Based on this consideration, the study was designed to investigate the combination effect of reduced graphene oxide-silver nanoparticles (rGO-AgNPs) and trichostatin A (TSA) in human ovarian cancer cells (SKOV3). METHODS: The rGO-AgNPs were synthesized using a biomolecule called lycopene, and the resultant product was characterized by various analytical techniques. The combination effect of rGO-Ag and TSA was investigated in SKOV3 cells using various cellular assays such as cell viability, cytotoxicity, and immunofluorescence analysis. RESULTS: AgNPs were uniformly distributed on the surface of graphene sheet with an average size between 10 and 50 nm. rGO-Ag and TSA were found to inhibit cell viability in a dose-dependent manner. The combination of rGO-Ag and TSA at low concentration showed a significant effect on cell viability, and increased cytotoxicity by increasing the level of malondialdehyde and decreasing the level of glutathione, and also causing mitochondrial dysfunction. Furthermore, the combination of rGO-Ag and TSA had a more pronounced effect on DNA fragmentation and double-strand breaks, and eventually induced apoptosis. CONCLUSION: This study is the first to report that the combination of rGO-Ag and TSA can cause potential cytotoxicity and also induce significantly greater cell death compared to either rGO-Ag alone or TSA alone in SKOV3 cells by various mechanisms including reactive oxygen species generation, mitochondrial dysfunction, and DNA damage. Therefore, this combination chemotherapy could be possibly used in advanced cancers that are not suitable for radiation therapy or surgical treatment and facilitate overcoming tumor resistance and disease progression.

The crystal structure of the endoglucanase Cel10, a family 8 glycosyl hydrolase from Klebsiella pneumoniae.

Cellulases are produced by microorganisms that grow on cellulose biomass. Here, a cellulase, Cel10, was identified in a strain of Klebsiella pneumoniae isolated from Chinese bamboo rat gut. Analysis of substrate specificity showed that Cel10 is able to hydrolyze amorphous carboxymethyl cellulose (CMC) and crystalline forms of cellulose (Avicel and xylan) but is unable to hydrolyze p-nitrophenol ß-D-glucopyranoside (p-NPG), proving that Cel10 is an endoglucanase. A phylogenetic tree analysis indicates that Cel10 belongs to the glycoside hydrolase 8 (GH8) subfamily. In order to further understanding of its substrate specificity, the structure of Cel10 was solved by molecular replacement and refined to 1.76 Šresolution. The overall fold is distinct from those of most other enzymes belonging to the GH8 subfamily. Although it forms the typical (α/α)6-barrel motif fold, like Acetobacterxylinum CMCax, one helix is missing. Structural comparisons with Clostridium thermocellum CelA (CtCelA), the best characterized GH8 endoglucanase, revealed that sugar-recognition subsite -3 is completely missing in Cel10. The absence of this subsite correlates to a more open substrate-binding cleft on the cellooligosaccharide reducing-end side.

Metagenomics approach to identify lignocellulose-degrading enzymes in the gut microbiota of the Chinese bamboo rat cecum

BACKGROUND: Lignocellulose is considered a renewable organic material, but the industrial production of biofuel from lignocellulose is challenging because of the lack of highly active hydrolytic enzymes. The guts of herbivores contain many symbiotic microorganisms that have evolved to hydrolyze plant lignocellulose. Chinese bamboo rats mainly consume high-fiber foods, indicating that some members of the intestinal tract microbiota digest lignocellulose, providing these rats with the energy required for growth. RESULTS: Here, we used metagenomics to analyze the diversity and functions of the gut microbiota in Chinese bamboo rats. We identified abundant populations of lignocellulose-degrading bacteria, whose main functions involved carbohydrate, amino acid, and nucleic acid metabolism. We also found 587 carbohydrate-active enzyme genes belonging to different families, including 7 carbohydrate esterase families and 21 glycoside hydrolase families. The glycoside hydrolase 3, glycoside hydrolase 1, glycoside hydrolase 43, carbohydrate esterase 4, carbohydrate esterase 1, and carbohydrate esterase 3 families demonstrated outstanding performance. CONCLUSIONS: The microbes and enzymes identified in our study expand the existing arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. This study also describes a powerful approach for targeting gut microbes and enzymes in numerous industries.

[Genetic structure and phylogeny status of Chaidamu goat population].

Genetic structure and character of Chaidamu goats were studied through simple random sampling. Genetic structure was analysed from five aspects, and phylogeny status was also investigated. The results indicated that: (1) the average phenotypic heterogeneity degree of coat color and morphological character were 0.3419 and 0.5207, respectively; (2) polymorphous blood albumen existed in 6 loci and the average loci heterozygosity was 0.2584; and (3) polymorphism existed in marked genes by DND-RAPD with diversity of 0.4085 approximately 0.5318. Phylogeny status was investigated through clustering by Ward's method on Chaidamu Goats and other domestic goats. All these indicated that Chaidamu Goats was a domestic goat with less intensively selected breed.

Enzymatic characterizations and activity regulations of N-acetyl-ß-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).

N-Acetyl-ß-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 µM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme.