Rationale
Paper spray (PS) has been developed as a method of choice for point‐of‐care analysis in many real cases, where its applications can be further expanded with delicate high‐throughput design. ...To achieve this goal, we developed a new PS regime, with the assembly of an induced high voltage into the ion source. Compared with regular DC high voltage, the newly developed setup is capable of high‐throughput, simple configuration and rapid switching between individual papers without complicated electric/mechanic design.
Methods
A device of high‐throughput induced PS (IPS) was designed by using a two‐dimensional (2D) rotating platform equipped with a circular glass plate. The paper substrate was placed on the circular glass plate and separated from the electrode. The method avoids physical contact between the electrode and the sample. Charged droplets were generated at the paper tip once an induced high voltage was applied to a wet paper.
Results
A relatively rapid analytical speed of 2.6 s per sample was achieved via IPS‐MS. Rapid quantification of amitriptyline (AMT) in complicated matrices was obtained within 1 min using an isotope internal standard method. Limits of detection for AMt in urine, FBS and blood were calculated to be 1.04, 0.84 and 1.33 ng/mL, respectively. In addition, high‐throughput IPS‐MS can be used for chemical reaction monitoring.
Conclusions
We have demonstrated the versatility of high‐throughput IPS‐MS in ambient ionization, which successfully simplified the experimental installation and facilitated the experimental operation. Therefore, we believe that high‐throughput IPS‐MS analysis will be widely used for discovering drugs and screening reactions, and the present design has the potential for applications in paper chip‐MS analysis.
A previous whole-genome association analysis identified
(
), which encodes a type of histone demethylase, as a candidate gene associated to goat fecundity.
gene knockout mouse disrupts gametophyte ...development, suggesting that it has a critical role in reproduction. In this study, goat
mRNA expression profiles were determined, insertion/deletion (indel) variants in the gene identified, indel variants effect on
gene expression assessed, and their association with first-born litter size analyzed in 2326 healthy female Shaanbei white cashmere goats.
mRNA was expressed in all tissues tested (heart, liver, spleen, lung, kidney, muscle, brain, skin and testis); the expression levels in testes at different developmental stages 1-week-old (wk), 2, 3 wk, 1-month-old (mo), 1.5 and 2 mo indicated a potential association with the mitosis-to-meiosis transition, implying that
may have an essential role in goat fertility. Meanwhile, two novel intronic indels of 16 bp and 5 bp were identified. Statistical analysis revealed that only the 16 bp indel was associated with first-born litter size (
< 0.01), and the average first-born litter size of individuals with an insertion/insertion genotype higher than that of those with the deletion/deletion genotype (
< 0.05). There was also a significant difference in genotype distributions of the 16 bp indel between mothers of single-lamb and multi-lamb litters in the studied goat population (
= 0.001). Consistently, the 16 bp indel also had a significant effect on
gene expression. Additionally, there was no significant linkage disequilibrium (LD) between these two indel loci, consistent with the association analysis results. Together, these findings suggest that the 16 bp indel in
may be useful for marker-assisted selection (MAS) of goats.
Myostatin (MSTN) is a powerful negative regulator of skeletal muscle mass in mammalian species that is primarily expressed in skeletal muscles, and mutations of its encoding gene can result in the ...double-muscling trait. In this study, the CRISPR/Cas9 technique was used to edit MSTN in Shaanbei Cashmere goats and generate knockout animals. RNA sequencing was used to determine and compare the transcriptome profiles of the muscles from three wild-type (WT) goats, three fibroblast growth factor 5 (FGF5) knockout goats (FGF5+/- group) and three goats with disrupted expression of both the FGF5 and MSTN genes (FM+/- group). The sequence reads were obtained using the Illumina HiSeq 2000 system and mapped to the Capra hircus reference genome using TopHat (v2.0.9). In total, 68.93, 62.04 and 66.26 million clean sequencing reads were obtained from the WT, FM+/- and FGF5+/- groups, respectively. There were 201 differentially expressed genes (DEGs) between the WT and FGF5+/- groups, with 86 down- and 115 up-regulated genes in the FGF5+/- group. Between the WT and FM+/- groups, 121 DEGs were identified, including 81 down- and 40 up-regulated genes in the FM+/- group. A total of 198 DEGs were detected between the FGF5+/- group and FM+/- group, with 128 down- and 70 up-regulated genes in the FM+/- group. At the transcriptome level, we found substantial changes in genes involved in fatty acid metabolism and the biosynthesis of unsaturated fatty acids, such as stearoyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydratase 2, ELOVL fatty acid elongase 6 and fatty acid synthase, suggesting that the expression levels of these genes may be directly regulated by MSTN and that these genes are likely downstream targets of MSTN with potential roles in lipid metabolism in goats. Moreover, five randomly selected DEGs were further validated with qRT-PCR, and the results were consistent with the transcriptome analysis. The present study provides insight into the unique transcriptome profile of the MSTN knockout goat, which is a valuable resource for studying goat genomics.
Rationale
Although it is claimed that desorption electrospray ionization (DESI) causes less ion suppression effects than electrospray ionization (ESI), a related investigation with quantification ...measurement of ion suppression effects is absent. Herein, a comparative analysis of ion suppression effects between DESI and ESI was conducted, with the aiming of quantitatively studying the ion suppression effect.
Methods
CTVA mixtures, a constant concentration of tioconazole with varied concentrations of atenolol, were analyzed by ESI and DESI. The ion suppression effect was characterized by the signal loss of tioconazole in the mixture compared to the signal intensity of tioconazole without interference (denoted as (Isingle − Imixture)/Isingle). According to the variations in the experimental conditions (such as flow rate, solvent composition, substrate material, capillary inner diameter, sheath gas velocity), ion suppression effects in DESI and ESI were compared.
Results
With the increasing flow rate, the ion suppression effect in DESI became weaker, while the opposite trend was obtained for ESI. As for capillary inner diameter, a smaller inner diameter resulted in weaker ion suppression effects in DESI and ESI. The solvent composition affected the ion suppression effect, and the PTFE substrate presented the weakest ion suppression effect among the five substrate materials.
Conclusions
Though the ion suppression effect in DESI and ESI was shown to relate to experimental conditions, DESI had less effect than ESI under the same experimental conditions in most cases. Moreover, DESI displayed stronger matrix‐tolerant ability than ESI which is also attributed to the weaker ion suppression effect.
Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method ...in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.
Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the ...CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes.
Insulin-like growth factor 2 mRNA-binding protein 2 (
IGF2BP2
, also called
IMP2
) plays an essential role in the development and maturation of germ cells and embryos and is a candidate gene for goat ...litter size, based on a previous genome-wide selective sweep analysis. In this study, the mRNA expression level of
IGF2BP2
was found to be significantly higher in a single-lamb group than in a multi-lamb group. Insertions/deletions (indels) within the goat
IGF2BP2
gene, including P4-Ins-13bp and P5-Del-12bp, were verified in 918 Shaanbei White Cashmere (SBWC) female goats. The minor allelic frequencies (MAFs) of P4-Ins-13bp and P5-Del-12bp loci were 0.349 and 0.295, respectively. Analysis using the Chi-square (
χ
2
) test showed that the genotype (
χ
2
=14.479,
P
=0.006) distribution of P4-Ins-13bp was significantly different between the single-lamb and multi-lamb groups. Correlation analysis demonstrated that P4-Ins-13bp was significantly associated with goat litter size (
P
=0.022), and individual goats with the homozygous deletion/deletion (DD) genotype produced more litters than other goats. Therefore, considered as a potential molecular marker significantly related to lambing traits, the P4-Ins-13bp mutation of the goat
IGF2BP2
gene can be used in goat breeding with practical molecular marker-assisted selection (MAS) to optimize female reproduction and improve economic efficiency in the goat industry.
The
(
) gene, as the major gene responsible for growth, plays a vital role in myogenesis. Meanwhile, the relationship between copy number variation (CNV) of this gene and growth traits in goats ...remains unclear. Therefore, this study investigated four aspects: bioinformatics analysis, mRNA expression (n = 6), CNV detection (n = 224), and association analysis. The findings indicated that the gene had a large number of conserved motifs, and the gene expression level was higher in fetal goats than in adult goats. Three CNV loci were selected from the database, among which CNV1 was located in the bidirectional promoter region and was associated with goat growth traits. CNV analysis showed that CNV2 and CNV3 of the
gene were associated with growth traits such as body weight, heart girth, height at hip cross, and hip width (
< 0.05), with CNV1 loss genotype being the superior genotype, and CNV2 and CNV3 median and gain genotypes of being superior genotypes. This finding further confirms that the
gene is the dominant gene for growth traits, which will serve as theoretical guidance for goat breeding.
Direct voltammetric determination of oxytetracycline (OTC) at a zinc cation-exchanged montmorillonite modified glassy carbon electrode (Zn-Mt/GCE) was described. The OTC yielded one well-defined ...oxidation peak at the Zn-Mt/GCE. The experimental parameters, which influence the voltammetric responses of OTC, e.g., the pH value, scan rate and accumulation condition, were optimized. The oxidation peak current changed with the OTC concentration over the range from 0.80 to 40 mu M. The detection limit was 0.12 mu M for an accumulation time of 4 min. The coefficient of variation, determined at 10 mu M OTC, was 3.4% (n = 10). Using this method, OTC in the real food and feedstuff samples was determined.
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