Comparative study of ultra-lightweight pulp foams obtained from various fibers and reinforced by MFC.

A range of cellulose-based, ultra-lightweight pulp foams with different morphologies were prepared and reinforced with microfibrillated cellulose (MFC). By careful design of the pulp foam forming process, free-standing ultra-lightweight pulp foams were obtained through high velocity mixing and air/oven drying from cellulose fiber in the presence of surfactant, MFC, and retention aid. The effects of different types of fibers and surfactants on the air uptake volumes and mechanical properties of the foam were systematically investigated. The structures characterized using an optical microscope and scanning electron microscope (SEM) showed that the foam was composed of wood fibers into two- or three-dimensional microstructures within random orientations surrounding gas bubbles. The results indicated that in spite of the strength of the foam could be manipulated by varying the surfactants and processing parameters, the addition of MFC indeed improved strength of pulp foams further. The process developed in this work provides a cost effective approach to fabricate the strong and ultra-lightweight pulp foam, with a density lower than 0.02g/cm3, using a standard handsheet former.

Association between IL-10 genetic variations and cervical cancer susceptibility in a Chinese population.

We conducted an investigation into the role of the IL-10 polymorphisms -592A/C (rs1800872), -819C/T (rs1800871), and -1082A/G (rs1800896) in cervical cancer risk in a Chinese population. A case-control study was carried out, including 165 newly diagnosed cervical cancer patients and 165 control subjects. The polymerase chain reaction-restriction fragment length polymorphism method was used to genotype the three IL-10 variant loci. Using conditional logistic regression analysis, we observed that homozygous IL-10 -819C/T TT carriers were at significantly increased risk of cervical cancer compared to homozygous CC individuals, with an adjusted odds ratio (OR) of 2.23 and a 95% confidence interval (CI) of 1.16-4.30. Moreover, the CT+TT genotype was significantly associated with cervical cancer in comparison to the wild-type variant (OR = 1.69, 95%CI = 1.04-2.76; P = 0.03). In conclusion, our study suggests that the IL-10 -819C/T genetic variation may contribute to cervical cancer risk in the Chinese population examined.

Isolation and characterization of polymorphic microsatellite markers for blue fox (Alopex lagopus).

The blue fox, belonging to the family Canidae, is a coat color variant of the native arctic fox (Alopex lagopus). To date, microsatellite loci in blue fox are typically amplified using canine simple sequence repeat primers. In the present study, we constructed an (AC)n enrichment library, and isolated and identified 17 polymorphic microsatellite markers for blue fox. The number of alleles per locus is from two to seven based on 24 examined individuals. The expected and observed heterozygosities were in the range of 0.3112 to 0.8236 and 0.2917 to 0.8750, respectively. The polymorphic information content per locus ranged from 0.2583 to 0.8022. These polymorphic markers can be useful for future population genetic studies of both farmed blue foxes and wild arctic foxes.

De novo assembly and characterization of farmed blue fox (Alopex lagopus) global transcriptome using Illumina paired-end sequencing.

The blue fox (Alopex lagopus), a coat-color variant of the Arctic fox, is a domesticated fur-bearing mammal. In the present study, transcriptome data generated from a pool of nine different tissues were obtained with Illumina HiSeq2500 paired-end sequencing technology. After filtering from raw reads, 32,358,290 clean reads were assembled into 161,269 transcripts and 97,252 unigenes by the Trinity fragment assembly software. Of the assembled unigenes, 37,967 were annotated in the National Center for Biotechnology Information (NCBI) Non-Redundant (NR) protein database and 26,264 in the Swiss-Prot database. Among the annotated unigenes, 24,839 and 24,267 were assigned using the Gene Ontology (GO) and euKaryotic Orthologous Groups (KOG) databases, respectively. Altogether, 17,057 unigenes were mapped onto 227 pathways using the Kyoto Encyclopedia of Genes and Genomes database. In addition, 6394 simple sequence repeats were identified by examining 12,965 unigenes (>1 kb), which could contribute to the development of molecular markers. This study generated transcriptome data for the blue fox that will promote further progress in expression profiling studies, and provide a good annotation basis for genomic studies.

The characterisation and functional ß-cell differentiation of duck pancreas-derived mesenchymal cells.

The generation of insulin-producing pancreatic ß-cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes research. The fractionation, expansion and conversion of primary duck pancreas-derived mesenchymal stem cells (PSCs) into functional ß-cells are described in this study. The cell surface antigens of PSCs, FOXA2, SOX9, NKX6.1 and INS were detected by immunofluorescent stain and flow cytometry for determining the biological characteristics of PSCs. The genes CD44, Ki67, Vimentin, C-myc, glucagon, PDX1 and insulin were detected by reverse-transcription polymerase chain reaction techniques. The growth curves of different passages were all typically sigmoidal. Karyotype analysis was conducted to estimate the stability of PSCs. A simple protocol was developed to assess functional differentiation by assessing the expression of pancreas ß-cell markers, the staining of dithizone and confirmation of insulin secretion. Insulin and PDX1 were all increased in differentiated cells compared to controls. Differentiated cells secreted insulin in a glucose-responsive manner.

Identification of cDNA sequences and alternative splicing patterns of canine AMEL genes (AMELX and AMELY).

Amelogenin is a major protein of the developing enamel matrix. There are two amelogenin genes (AMELX and AMELY) located on the X and Y chromosomes, respectively, in dogs. In the present study, we characterized full-length cDNAs and alternative splicing patterns of the AMEL genes in the tooth tissue of a dog by 5'- and 3'-rapid amplification of cDNA ends and AMEL-specific RT-PCR. Sequence analysis revealed that the coding regions of AMELX and AMELY were 579 and 576 bp (accession Nos. KP244310 and KP244311), respectively. The coding sequence of AMELX had 95.1% identity to that of AMELY. The AMEL genes on X and Y chromosomes were both expressed in developing tooth tissue. Eight different alternatively spliced transcripts were identified, five from AMELX and three from AMELY.

Cloning and identification of the ASIP gene in Chinese raccoon dog (Nyctereutes procyonoides procyonoides).

The quantity, quality, and distribution of eumelanin and pheomelanin determine a wide variety of coat colors in animals. Three coat color variants exist in farmed wild-type Chinese raccoon dog (Nyctereutes procyonoides procyonoides), which is an important fur-bearing animal species. The ASIP gene is an important candidate gene for coat color variation in some species. In this study, the complete cDNA sequences of ASIP were amplified from a wild-type Chinese raccoon dog. Sequence analysis revealed the coding region of ASIP in Chinese raccoon dog to be 396-bp in length and two transcripts (accession Nos. KT224450 and KT224451) were identified due to the alternative use of exon 1 (1A and 1C). However, the alternative splicing pattern and the coding sequence of ASIP in three types of coat color variants were the same as those identified in the wild-type individual. Based on the results obtained in this study, we can exclude a role for alternative splicing of exon 1 and the coding sequence of ASIP in coat color variation in Chinese raccoon dog.

Isolation and characterization of novel microsatellite markers from the sika deer (Cervus nippon) genome.

Microsatellite markers are widely and evenly distributed, and are highly polymorphic. Rapid and convenient detection through automated analysis means that microsatellite markers are widely used in the construction of plant and animal genetic maps, in quantitative trait loci localization, marker-assisted selection, identification of genetic relationships, and genetic diversity and phylogenetic tree construction. However, few microsatellite markers remain to be isolated. We used streptavidin magnetic beads to affinity-capture and construct a (CA)n microsatellite DNA-enriched library from sika deer. We selected sequences containing more than six repeats to design primers. Clear bands were selected, which were amplified using non-specific primers following PCR amplification to screen polymorphisms in a group of 65 unrelated sika deer. The positive clone rate reached 82.9% by constructing the enriched library, and we then selected positive clones for sequencing. There were 395 sequences with CA repeats, and the CA repeat number was 4-105. We selected sequences containing more than six repeats to design primers, of which 297 pairs were designed. We next selected clear bands and used non-specific primers to amplify following PCR amplification. In total, 245 pairs of primers were screened. We then selected 50 pairs of primers to randomly screen for polymorphisms. We detected 47 polymorphic and 3 monomorphic loci in 65 unrelated sika deer. These newly isolated and characterized microsatellite loci can be used to construct genetic maps and for lineage testing in deer. In addition, they can be used for comparative genomics between Cervidae species.

Development of novel polymorphic microsatellite markers for the silver fox (Vulpes vulpes).

The silver fox (Vulpes vulpes), a coat color variant of the red fox, is one of the most important fur-bearing animals. To date, development of microsatellite loci for the silver fox has been limited and mainly based on cross-amplification by using canine SSR primers. In this study, 28 polymorphic microsatellite markers were isolated and identified for silver fox through the construction and screening of an (AC)n-enriched library. The number of alleles per locus ranged from 2 to 8 based on 48 individuals tested. The expected and observed hetero- zygosity and polymorphism information content per locus ranged from 0.2544 to 0.859, 0.2083 to 0.7917, and 0.2181 to 0.821, respectively. The polymorphic markers presented in this study may be useful for future analysis of the genetic diversity and population structure of farmed silver fox and wild red fox.

Cloning and association analysis of KIT and EDNRB polymorphisms with dominant white coat color in the Chinese raccoon dog (Nyctereutes procyonoides procyonoides).

The Chinese raccoon dog (Nyctereutes procyonoides procyonoides) is one of the most important fur-bearing animal species. The dominant white individual, a coat color variant in farmed Chinese raccoon dog, shows a completely white phenotype over the entire body. The KIT and EDNRB genes have been reported to be associated with the dominant white coat color in some mammalian species. In the present study, the full-length coding sequences of KIT and EDNRB were amplified from a dominant white and a wild-type Chinese raccoon dog. Sequence analysis revealed that the coding region of KIT and EDNRB in Chinese raccoon dog was 2919 and 1332 base pairs in length (accession No. KM083121 and KM083122), respectively, and 2 single-nucleotide polymorphisms (SNPs; c.600C>T and c.967G>A) in KIT and 1 SNP (c.259A>C) in EDNRB was found only in the dominant white individual. An alternative splicing site at the boundary of 4 and 5 of the KIT gene was identified in both individuals. We further investigated the association between the 3 SNPs of KIT and EDNRB and dominant white coat color by genotyping 18 individuals. We found no association between these SNPs and dominant white coat color. Based on these results, we can exclude the coding regions of the KIT and EDNRB genes as determinants of the dominant white coat color in Chinese raccoon dog.

A base substitution in the donor site of intron 12 of KIT gene is responsible for the dominant white coat colour of blue fox (Alopex lagopus).

The dominant white coat colour of farmed blue fox is inherited as a monogenic autosomal dominant trait and is suggested to be embryonic lethal in the homozygous state. In this study, the transcripts of KIT were identified by RT-PCR for a dominant white fox and a normal blue fox. Sequence analysis showed that the KIT transcript in normal blue fox contained the full-length coding sequence of 2919 bp (GenBank Acc. No KF530833), but in the dominant white individual, a truncated isoform lacking the entire exon 12 specifically co-expressed with the normal transcript. Genomic DNA sequencing revealed that a single nucleotide polymorphism (c.1867+1G>T) in intron 12 appeared only in the dominant white individuals and a 1-bp ins/del polymorphism in the same intron showed in individuals representing two different coat colours. Genotyping results of the SNP with PCR-RFLP in 185 individuals showed all 90 normal blue foxes were homozygous for the G allele, and all dominant white individuals were heterozygous. Due to the truncated protein with a deletion of 35 amino acids and an amino acid replacement (p.Pro623Ala) located in the conserved ATP binding domain, we propose that the mutant receptor had absent tyrosine kinase activity. These findings reveal that the base substitution at the first nucleotide of intron 12 of KIT gene, resulting in skipping of exon 12, is a causative mutation responsible for the dominant white phenotype of blue fox.

Development and characterization of polymorphic microsatellite markers for Chinese raccoon dog (Nyctereutes procyonoides procyonoides).

Chinese raccoon dog (Nyctereutes procyonoides procyonoides) is one of the most important fur-bearing animal species. Information about the genetic background of farmed Chinese raccoon dogs is limited. In this study, 17 polymorphic microsatellite markers were isolated and identified from an (AC)n-microsatellite-enriched library of Chinese raccoon dogs. The number of alleles per locus ranged from 2 to 8 based on 48 individuals tested. The expected and observed heterozygosity and polymorphism information content per locus ranged from 0.383 to 0.8378, 0.3200 to 0.8696, and 0.3047 to 0.7947, respectively. Cross-species amplification of these loci in 2 other Canidae species indicated that 9 and 11 of these loci could also be amplified successfully in the arctic and silver fox, respectively. These microsatellite loci developed in the present report will provide useful tools for population genetic studies, individual identification, and phylogenetic analysis in the Chinese raccoon dog and other Canidae species.

Molecular characteristics and expression profiles of glycerol-3-phosphate dehydrogenase 1 (GPD1) gene in pig.

The cytosolic activity of glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays an important role in the synthesis of triacylglycerol and in the transport of reducing equivalents from the cytosol to mitochondria. Here we report the full-length genomic sequence of porcine GPD1 gene including promoter region. Porcine GPD1 gene contains eight exons and seven introns. Using the ImpRH, the GPD1 gene was mapped on chromosome 5. Sub-cellular localization of the pig GPD1 was localized in cytoplasm by GFP reporter gene. The full-length CDS of porcine GPD1 gene comprises 1050 nucleotides and it encodes 349 amino acids. Using the CDS sequences of 17 species, we built the phylogeny tree of GPD1 gene. We investigated the expression level of the gene in 13 different tissues and time course from birth to postnatal day 28 in longissinus doris muscle (LD) and in cerebrum. The result shows that porcine GPD1 gene is expressed in almost all tissues we tested but its levels of expression varies widely over 2 orders of magnitude. LD and the cerebrum have similar expression pattern that is at a low level at birth and increasing with aging to the highest level at postnatal day 8 in LD and postnatal day 14 in cerebrum. But weaning decreased the expression level of the GPD1 gene. This may partially explains the effects of weaning on energy metabolism.

Establishment and biological characteristics of Qingyuan partridge chicken fibroblast line.

The aim of this work was to seek a different approach to conserving important domestic animals in imminent danger. A Qingyuan partridge chicken embryonic fibroblast line, containing 336 cryovials with 8x10(6) cells each, was successfully established from 60 Qingyuan partridge chicken embryos using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was a typical "S" shape having a detention phase, a logarithmic phase, and a plateau phase. The population doubling time was approximately 72 h. Tests for bacteria, fungi, viruses, and Mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis suggested that the chromosome number of a normal cell was 2n=78 and 93.4% of the entire population was diploid. The transfection efficiencies of 6 fluorescent proteins (pEGFP-C1, pEGFP-N3, pDsRed-N1, pEYFP-N1, pECFP-N1, and pECFP-mito) optimal at 48 h were from 15.1 to 39.8%. The cell line met all of the criteria from the American Type Culture Collection. Not only has the germline of this important chicken breed been preserved at the cell level, but also valuable material has been provided for genome, postgenome, and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

Expression of fascin in oral and oropharyngeal squamous cell carcinomas has prognostic significance - a tissue microarray study of 129 cases.

AIMS: To elucidate the role of fascin in oral and oropharyngeal squamous cell carcinoma (OSCC) by correlation with clinical parameters. METHODS AND RESULTS: Paraffin sections using tissue microarrays of 129 patients with OSCC were investigated immunohistochemically. Fascin protein was overexpressed in OSCC cells compared with their non-neoplastic epithelial counterparts. For evaluating the intensity of fascin, 39 (30.2%) were classified as weakly immunoreactive, 76 (58.9%) as moderate reactive and 14 (10.9%) as intensely reactive. For evaluating the distribution of fascin, 64 (49.6%) were classified as < 55% and 65 (50.4%) were classified as >/= 55%. Fascin protein expression was correlated with size or extent of the tumour (P < 0.001), positive lymph node metastasis (P < 0.001), distant metastasis (P = 0.014) and clinical staging (P < 0.001). The immunoreactivity scores of fascin in OSCC were variable but showed significant correlation with histological grade, clinical TNM system and stage. CONCLUSION: Expression of fascin protein may play an important role in progression of OSCC. Overexpression of fascin contributes to a more aggressive clinical course and suggests the potential of fascin as a new molecular target for therapeutic intervention.