Effects of plant-derived protein and rapeseed oil on growth performance and gut microbiomes in rainbow trout.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is becoming popular with the increased demand for fish protein. However, the limited resources and expense of fish meal and oil have become restrictive factors for the development of the rainbow trout related industry. To solve this problem, plant-derived proteins and vegetable oils have been developed as alternative resources. The present study focuses on evaluating the effects of two experimental diets, FMR (fish meal replaced with plant-derived protein) and FOR (fish oil replaced with rapeseed oil), through the alteration of the gut microbiota in triploid rainbow trout. The commercial diet was used in the control group (FOM). RESULTS: Amplicon sequencing of the 16S and 18S rRNA genes was used to assess the changes in gut bacteria and fungi. Our analysis suggested that the α-diversity of both bacteria and fungi decreased significantly in the FMR and FOR groups, and ß-diversity was distinct between FOM/FMR and FOM/FOR based on principal coordinate analysis (PCoA). The abundance of the Planctomycetota phylum increased significantly in the FMR group, while that of Firmicutes and Bacteroidetes decreased. We also found that the fungal phylum Ascomycota was significantly increased in the FMR and FOR groups. At the genus level, we found that the abundance of Citrobacter was the lowest and that of pathogenic Schlesneria, Brevundimonas, and Mycoplasma was highest in the FMR and FOR groups. Meanwhile, the pathogenic fungal genera Verticillium and Aspergillus were highest in the FMR and FOR groups. Furthermore, canonical correspondence analysis (CCA) and network analysis suggested that the relatively low-abundance genera, including the beneficial bacteria Methylobacterium, Enterococcus, Clostridium, Exiguobacterium, Sphingomonas and Bacteroides and the fungi Papiliotrema, Preussia, and Stachybotrys, were positively correlated with plant protein or rapeseed oil. There were more modules that had the above beneficial genera as the hub nodes in the FMR and FOR groups. CONCLUSIONS: Our study suggested that the FMR and FOR diets could affect the gut microbiome in rainbow trout, which might offset the effects of the dominant and pathogenic microbial genera. This could be the underlying mechanism of explaining why no significant difference was observed in body weight between the different groups.

Multiple convergent events created a nominal widespread species: Triplophysa stoliczkae (Steindachner, 1866) (Cobitoidea: Nemacheilidae).

BACKGROUND: Triplophysa stoliczkae is the most widespread species in the genus Triplophysa and may have originated from morphological convergence. To understand the evolutionary history of T. stoliczkae, we employed a multilocus approach to investigate the phylogenetics and the morphological evolution of T. stoliczkae on the Qinghai-Tibetan Plateau. RESULTS: All phylogenetic analyses (two mitochondrial and five nuclear loci), a genealogical sorting index and species tree inferences suggested that T. stoliczkae consists of distinct lineages that were not closest relatives. The time estimation indicated that the divergence events between "T. stoliczkae" and other Triplophysa species occurred from approximately 0.10 to 4.51 Ma. The ancestral state analyses supported the independent evolution of T. stoliczkae morphology in distinct lineages. The morphometric analysis and convergence estimates demonstrated significant phenotypic convergence among "T. stoliczkae" lineages. CONCLUSIONS: Triplophysa stoliczkae includes 4 different lineages with similar morphologies. The increasingly harsh environments that have occurred since the Pliocene have driven the occurrences of scrape-feeding fish in the genus Triplophysa. Morphological adaptations associated with scrape-feeding behavior resulted in convergences and the artificial lumping of four different species in the nominal taxon T. stoliczkae. A taxonomic revision for T. stoliczkae is needed.

Duplication of toll-like receptor 22 in teleost fishes.

The TLRs of teleost fishes have distinct features and are highly diverse, but the duplication characteristics and expression patterns of the tlr22 gene remain unclear. Here, we identified paralogous tlr22 genes in 13 teleost fishes by screening available fish genomic resources and using molecular cloning. We then conducted comprehensive bioinformatics analyses and investigated spatiotemporal differences in the expression patterns of the tlr22 genes in G. eckloni. The results indicated that more than three paralogous tlr22 genes were possessed by some teleost fishes. Of these, tlr22c is specific to some subfamilies of the Cyprinidae (e.g., Barbinae, Cyprininae, Schizothoracinae, and Leuciscinae). Phylogenetic and syntenic analyses showed that the paralogous tlr22 genes originated from two single-gene duplication events. Molecular clock calculations dated the two gene duplication events at 49.5 and 39.3 MYA, which is before the common carp-specific genome duplication event and well after the fish-specific genome duplication. Gene duplication of tlr22 was followed by gene loss or pseudogene events in certain lineages. Spatiotemporal expression differences between the three duplicated tlr22 genes from G. eckloni suggested that these genes diverged functionally after gene duplication.

Adaptive Evolution of the Eda Gene and Scales Loss in Schizothoracine Fishes in Response to Uplift of the Tibetan Plateau.

Schizothoracine is the predominant wild fish subfamily of the Tibetan plateau (TP). Their scales, pharyngeal teeth and barbels have gradually regressed with increasing altitude. Schizothoracine have been divided into three groups: primitive, specialized and highly specialized. Ectodysplasin-A (Eda) has been considered as a major gene that contributes to the development of skin appendages. The present study cloned the Eda genes of 51 Schizothoracine fish species which represent the three groups and five Barbinae species. Phylogenetic analyses indicated that Eda may have acted as the genetic trigger for scale loss in the Schizothoracine. Furthermore, 14 single nucleotide polymorphisms (SNPs) and two deletions (18 bp and 6 bp in size), were also detected in the Eda coding sequence of the highly specialized group compared to the primitive group. The same SNPs and two indels result in four non-synonymous and two G-X-Y and 1 XY motif indels, which possibly contribute to significant structure changes in the Eda gene. The domain including (G-X-Y)n motif in the Eda gene is relatively conserved amongst teleosts. Based on the above results, we hypothesize that the evolution of Eda gene might be associated with the scale loss in Schizothoracine fishes in response to the phased uplift of the TP.

Comprehensive transcriptomic analysis of Tibetan Schizothoracinae fish Gymnocypris przewalskii reveals how it adapts to a high altitude aquatic life.

BACKGROUND: Understanding the genetic basis of adaptation to high altitude life is of paramount importance for preserving and managing genetic diversity in highland animals. This objective has been addressed mainly in terrestrial fauna but rarely in aquatic animals. Tibetan Schizothoracinae fish is the ideal model system in evolutionary biology, carrying key insights into evolutionary genetics of speciation and adaptation at high altitude. Gymnocypris przewalskii is the newly formed Schizothoracinae fish species in the Tibetan Plateau, inhabits chronic cold, extreme saline and alkaline aquatic environment in Lake Qinghai, thus evolving the unique genomic signatures to adapt extremely severe environments. RESULTS: To characterize its genomic features, we assembled de novo transcriptome of G. przewalskii from Lake Qinghai. Intriguingly, by comparative genomic analyses of G. przewalskii and 8 other fish species, we identified potential expansions in gene families related to energy metabolism, transport and developmental functions, possibly underlying the adaptation to these environmental stresses. Through comprehensive molecular evolution analyses, we found that sets of genes controlling mitochondrion, ion homoeostasis, acid-base balance and innate immunity show significant signals of positive selection. Compared to previous studies on highland fishes, we failed to identify any positively selected genes related to hypoxia response. CONCLUSIONS: Our findings provide comprehensive insights into the genetic basis of teleost fish that underlie their adaptation to extreme high altitude aquatic life on the Tibetan Plateau.

Positive Darwinian selection within interferon regulatory factor genes of Gymnocypris przewalskii (Cyprinidae) on the Tibetan Plateau.

Tibetan Plateau (TP) had experienced phased uplift, resulting in inhospitable environment of low temperature, hypoxia and high ultraviolet radiation for Tibetan wildlife. Many organisms can well adapt to TP, it is of ecological and evolutionary interest to untangle how organisms adapt to extreme environment on TP through evolution. Previous studies mainly focused on hypoxia and metabolism related genes, but we know little about the evolutionary history of immune genes in Tibetan wildlife. In this study, we first identified 10 interferon regulatory factor (IRF) genes from Tibetan naked carp Gymnocypris przewalskii. Within this gene family, IRF3, IRF5, IRF7 and IRF8 contained positive selection sites. Evidences indicated that positive selection may lead to IRF genes functional alternations, presumably driving genes towards adaptation to the environmental changes. Taken together, our results suggested 4 candidate genes as interesting targets for further experimental confirmation of their functional variations and contributions to high altitude adaptation in Tibet fish.

Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting.

In addition to the advantages of scalable, affordable, and easy to engineer, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is superior for multiplex targeting, which is laborious and inconvenient when achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate multiplex targeting usage. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers.

Transcriptome profiling analysis of naked carp (Gymnocypris przewalskii) provides insights into the immune-related genes in highland fish.

The naked carp, Gymnocypris przewalskii, is one of the dominant aquaculture fish species in Qinghai Province, China. Its wild stocks have severely suffered from overfishing, and the farming species are vulnerable to various pathogens infections. Here we report the first immune-related tissues transcriptome of a wild naked carp using a deep sequencing approach. A total of 158,087 unigenes are generated, 2687 gill-specific gene and 3215 kidney-specific genes are identified, respectively. Gene ontology analysis shows that 51,671 unigenes are involved in three major functional categories: biological process, cellular component, and molecular function. Further analysis shows that numerous consensus sequences are homologous to known immune-related genes. Pathways mapping annotate 56,270 unigenes and identify a large number of immune-related pathways. In addition, we focus on the immune-related genes and gene family in Toll-like receptor signaling pathway involved in innate immunity, including toll-like receptors (TLRs), interferon regulatory factors (IRFs), interleukins (ILs) and tumor necrosis factors (TNFs). Eventually, we identify 5 TLRs, 4 IRFs, 3 ILs and 2 TNFs with a completed coding sequence though mining the transcriptome data. Phylogeny analysis shows these genes of naked carp are mostly close to zebrafish. Protein domain and selection pressure analyses together show that all these genes are highly conserved in gene sequence and protein domain structure with other species, and purifying selection underwent in these genes, implied functionally important features are conserved in the genes above. Intriguingly, we detect positive selection signals in naked carp TLR4, and significant divergence occurred among tested species TLR4, suggested that naked carp TLR4 function may be affected. Finally, we identify 23,867 simple sequence repeat (SSR) marks in this transcriptome. Taken together, this study not only contributes a large number of candidate genes in naked carp immunity, and also helps improve current understanding of immunogenetics basis and evolutionary history of immune related genes and gene family in highland fish species.

Transcriptome-wide identification, molecular evolution and expression analysis of Toll-like receptor family in a Tibet fish, Gymnocypris przewalskii.

Toll-like receptors (TLR) are key components of innate immunity that play significant roles in immune defense against pathogens invasion. Recent frequent outbreaks of the "white spot disease" caused by parasitic infection in farmed Tibetan fishes had resulted in great economic losses. However, to our knowledge, the roles of TLRs in mediating immune response to parasitic infection in Tibetan fishes remain to be determined. Here, we performed data-mining on a widely-farmed Tibetan fish (Gymnocypris przewalskii or Gp) transcriptome to determine the genetic variation and expression pattern of TLRs. We totally obtained 14 GpTLRs and identified 5 with a complete coding sequence. Phylogenetic analysis verified their identities and supported the classification of TLRs into six families as in other vertebrates. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in GpTLR1-5. Selective pressure test demonstrated that all known GpTLRs are under purifying selection, except GpTLR4 underwent positive selection. Further, site model analysis suggested that 11 positively selected sites are found in LRR domain of GpTLR4. Three positively selected sites are located on outside surface of TLR4 3D structure, indicating that function of GpTLR4 may be affected. Tissue specific expression analysis showed all GpTLRs are present in gill, head-kidney and spleen but the relative abundance varied among tissues. In response to parasite Ichthyophthirius multifiliis infection, 5 GpTLR (GpTLR1, -2, -4, -9 and -20) expressions were induced. Intriguingly, GpTLR4 was significantly up-regulated in gills, while GpTLR19 and GpTLR21 unexpectedly showed no any change. In summary, these results revealed the first genomic resources of TLR family and several parasitic infection responsive TLRs in Tibetan fish. These findings provide key information for future studies aiming to understand the molecular mechanisms underlying the immune response to pathogen invasion in Tibetan fishes.

A suicidal zinc finger nuclease expression coupled with a surrogate reporter for efficient genome engineering.

Genome editing with engineered nucleases, such as zinc-finger nucleases (ZFNs) and TALE nucleases, remains confronted with a high risk of cellular toxicity induced by off-targeting. Here we describe the construction of a suicidal nuclease expression vector in which a pair of ZFNs genes were flanked of its target sites. To further enrich the targeted cells, the suicidal ZFN expression cassette was also inserted within an eGFP reporter, to disrupt the ORF of eGFP gene. ZFN-associated toxicity was reduced by ~40 % with this new system, and the activities of ZFNs were ~4.5 % lower. We conclude that using this new suicidal ZFN expression and surrogate reporter system represents an improvement for genomic editing by reducing toxicity and allowing easy detection of edited cells by eGFP analysis.

Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases.

Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probability of identifying ZFN-mediated positive targeted disruption events is small. In this paper, we used the context-dependent assembly method to synthesize two pairs of ZFNs targeted to the sheep MSTN gene. We verified the activity of these ZFNs using an mRFP-MBS-eGFP dual-fluorescence reporter system in HEK293T cells and, according to the expression level of eGFP, we obtained a pair of ZFNs that can recognize and cut the targeted MSTN site in the reporter vector. The activity of ZFN was increased by cold stimulation at 30 °C and by mutant the wildtype FokI in ZFN with its counterpart Sharkeys. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two MSTN mutant cell lines, identified by flow cytometry and sequencing, were obtained.

Response of phytoplankton composition to environmental stressors under humidification in three alpine lakes on the Qinghai-Tibet Plateau, China.

Owning to their extreme environmental conditions, lakes on the Qinghai-Tibet Plateau have typically displayed a simplistic food web structure, rendering them more vulnerable to climate change compared to lakes in plains. Phytoplankton, undergoing a changing aquatic environment, play a crucial role in the material cycle and energy flow of the food chain, particularly important for the unique fish species of the Tibetan Plateau. To identify the changing environment indexes and determine the response of phytoplankton composition to the environment change in alpine lakes, three lakes-Lake Qinghai, Lake Keluke and Lake Tuosu-were selected as study areas. Seasonal sampling surveys were conducted in spring and summer annually from 2018 to 2020. Our findings revealed there were significant changes in physicochemical parameters and phytoplankton in the three lakes. Bacillariophyta was the predominant phytoplankton in Lake Qinghai from 2018 to 2020, with the genera Synedra sp., Navicula sp., Cymbella sp. and Achnanthidium sp. predominated alternately. Lake Keluke alternated between being dominated by Bacillariophyta and cyanobacteria during the same period. Dolichospermum sp., a cyanobacteria, was prevalent in the summer of 2018 and 2019 and in the spring of 2020. In Lake Tuosu, Bacillariophyta was the predominant phytoplankton from 2018 to 2020, except in the summer of 2019, which was dominated by cyanobacteria. Synedra sp., Oscillatoria sp., Pseudoanabaena sp., Chromulina sp. and Achnanthidium sp. appeared successively as the dominant genera. Analysis revealed that all three lakes exhibited higher phytoplankton abundance in 2018 that in 2019 and 2020. Concurrently, they experienced higher average temperatures in 2018 than in the subsequent years. The cyanobacteria, Bacillariophyta, Chlorophyta and overall phytoplankton increased with temperature and decreased with salinity and NH4-N. Besides, the ratios of cyanobacteria, and the ratios of Bacillariophyta accounted in total phytoplankton increased with temperature. These findings suggest that cyanobacteria and phytoplankton abundance, especially Bacillariophyta, may have an increase tendency in the three alpine lakes under warm and wet climate.

A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin.

BACKGROUND: Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. RESULTS: We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. CONCLUSIONS: Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

Effects of ghrelin gene genotypes on the growth traits in Chinese cattle.

Ghrelin is an important peptide that stimulates food intake and regulates energy balance of animals. Single nucleotide polymorphisms of ghrelin gene in three Chinese cattle populations were investigated through PCR-SSCP and DNA sequencing. Five over-lapped DNA fragments were analyzed and a total of three ones exhibited different genotypes. Three genotypes and four SNPs (-415 A > G, -414 T > C, -321 C > A, and -172 A > G) were found on the -544 to +35 bp region (G-1) of ghrelin gene. On the locus of -1037 to -509 bp (G-2), two genotypes and one SNP (-726 A > T) were discovered. And in the exon1, exon2, and intron1 (G-4 locus, (+4 to +427)), two genotypes and one SNP were detected (+205 C > T, located in intron1). Positions of the five SNPs in the 5' regulatory region might be the transcription factor binding sites. The SNPs at -415 and -414 in the core binding sequence were found to cause the change of the site. Though the SNP at -172 did not change the binding site, it generated one new site at the same time. The frequencies of the genotypes varied differently in the three breeds. Results of ANOVA showed that G-1 was correlative to the ischium width (IW) of Nanyang cattle aged 18 months (p = 0.043). The least square analysis between genotypes at G-1 locus and growth traits in Nanyang cattle showed that the individuals (aged 18 months) with C genotype had greater IW than that of the other two genotypes. The C genotype might serve as one potential candidate genetic marker for cattle growth and development.

Chromosome-level genome of Tibetan naked carp (Gymnocypris przewalskii) provides insights into Tibetan highland adaptation.

Gymnocypris przewalskii, a cyprinid fish endemic to the Qinghai-Tibetan Plateau, has evolved unique morphological, physiological and genetic characteristics to adapt to the highland environment. Herein, we assembled a high-quality G. przewalskii tetraploid genome with a size of 2.03 Gb and scaffold N50 of 44.93 Mb, which was anchored onto 46 chromosomes. The comparative analysis suggested that gene families related to highland adaptation were significantly expanded in G. przewalskii. According to the G. przewalskii genome, we evaluated the phylogenetic relationship of 13 schizothoracine fishes, and inferred that the demographic history of G. przewalskii was strongly associated with geographic and eco-environmental alterations. We noticed that G. przewalskii experienced whole-genome duplication, and genes preserved post duplication were functionally associated with adaptation to high salinity and alkalinity. In conclusion, a chromosome-scale G. przewalskii genome provides an important genomic resource for teleost fish, and will particularly promote our understanding of the molecular evolution and speciation of fish in the highland environment.

Insights Into miRNA-mRNA Regulatory Mechanisms of Cold Adaptation in Gymnocypris eckloni: Ubiquitin-Mediated Proteolysis Is Pivotal for Adaptive Energy Metabolism.

This study aimed to understand cold stress adaptations mechanism in fish. Thus, the transcriptional response to cold conditions in Gymnocypris eckloni was evaluated using RNA-seq and microRNA (miRNA)-seq analyses. Low-temperature (LT) group G. eckloni was cultivated outdoors in waters cooled to 2-4°C for 3 weeks, while individuals in the control temperature (CT) group were exposed to 14-16°C. Significantly different responses were observed in both mRNA and miRNA expression profiles, with more mRNAs (1,833 and 1,869 mRNAs were up- and downregulated, respectively) and fewer miRNAs (15 and 6 were up- and downregulated, respectively) observed in the LT group individuals relative to the CT group individuals. A miRNA-mRNA network involved in the regulation of G. eckloni responses to cold stress was constructed; this network included ubiquitin-mediated proteolysis, protein processing, and oxidative phosphorylation. These results provided new insights into mechanisms of cold tolerance by fish, including decreased metabolic activity in addition to proteolysis.

Genome-wide analysis of CNVs in three populations of Tibetan sheep using whole-genome resequencing.

Copy number variation (CNV), an important source of genomic structural variation, can disturb genetic structure, dosage, regulation and expression, and is associated with phenotypic diversity and adaptation to local environments in mammals. In the present study, 24 resequencing datasets were used to characterize CNVs in three ecotypic populations of Tibetan sheep and assess CNVs related to domestication and adaptation in Qinghai-Tibetan Plateau. A total of 87,832 CNV events accounting for 0.3% of the sheep genome were detected. After merging the overlapping CNVs, 2777 CNV regions (CNVRs) were obtained, among which 1098 CNVRs were shared by the three populations. The average length of these CNVRs was more than 3 kb, and duplication events were more frequent than deletions. Functional analysis showed that the shared CNVRs were significantly enriched in 56 GO terms and 18 KEGG pathways that were mainly concerned with ABC transporters, olfactory transduction and oxygen transport. Moreover, 188 CNVRs overlapped with 97 quantitative trait loci (QTLs), such as growth and carcass QTLs, immunoglobulin QTLs, milk yield QTLs and fecal egg counts QTLs. PCDH15, APP and GRID2 overlapped with body weight QTLs. Furthermore, Vst analysis showed that RUNX1, LOC101104348, LOC105604082 and PAG11 were highly divergent between Highland-type Tibetan Sheep (HTS) and Valley-type Tibetan sheep (VTS), and RUNX1 and LOC101111988 were significantly differentiated between VTS and Oura-type Tibetan sheep (OTS). The duplication of RUNX1 may facilitate the hypoxia adaptation of OTS and HTS in Qinghai-Tibetan Plateau, which deserves further research in detail. In conclusion, for the first time, we represented the genome-wide distribution characteristics of CNVs in Tibetan sheep by resequencing, and provided a valuable genetic variation resource, which will facilitate the elucidation of the genetic basis underlying the distinct phenotypic traits and local adaptation of Tibetan sheep.

Chromosome-level assembly of Gymnocypris eckloni genome.

Gymnocypris eckloni is widely distributed in isolated lakes and the upper reaches of the Yellow River and play significant roles in the trophic web of freshwater communities. In this study, we generated a chromosome-level genome of G. eckloni using PacBio, Illumina and Hi-C sequencing data. The genome consists of 23 pseudo-chromosomes that contain 918.68 Mb of sequence, with a scaffold N50 length of 43.54 Mb. In total, 23,157 genes were annotated, representing 94.80% of the total predicted protein-coding genes. The phylogenetic analysis showed that G. eckloni was most closely related to C. carpio with an estimated divergence time of ~34.8 million years ago. For G. eckloni, we identified a high-quality genome at the chromosome level. This genome will serve as a valuable genomic resource for future research on the evolution and ecology of the schizothoracine fish in the Qinghai-Tibetan Plateau.

Synthesis of hierarchical and flower-like TiO2 nanowire microspheres as biocompatible cell carriers.

Fibrous materials are of great interest in the development of tissue regenerative matrix. However, synthesis of inorganic fibrous microspheres as cell carriers is a great challenge. In this study, we for the first time report on the synthesis of novel hierarchical and flower-like TiO2 nanowire (NW) microspheres as biocompatible cell carriers. TiO2 NW microspheres were synthesized through in situ alkali hydrothermal treatment of the TiO2 nanoparticle (NP) microspheres and their microstructure, formation mechanism and in vitro biocompatibility were evaluated. SEM observations show that the resulting TiO2 NW microspheres were constructed by a large number of NWs with the diameter of 10-20 nm and exhibited a flower-like and hierarchical morphology with the diameter of 400-600 µm. XRD patterns indicate that TiO2 NW microspheres were constructed by both rutile and anatase phase of TiO2. FT-IR spectra reveal that Ti-O-Ti bonds were involved in TiO2 NW microspheres. In vitro biocompatibility was evaluated by seeding the fibroblast L929 cells on the microspheres. A conventional MTT assay quantitatively indicates that the TiO2 NW microspheres favored adhesion and proliferation of cells and were biocompatible, while SEM observations qualitatively confirmed that the cells were well grown on the surface of TiO2 NW microspheres. Thus, the as-synthesized TiO2 NW microspheres would be applicable to novel and biocompatible cell carriers.

An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene.

In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.