Association analysis of indels in the VISFATIN gene with five cattle breeds and their growth traits.

VISFATIN is an adipose cytokine that has been proved to correlate with growth and development traits. In a previous study from our lab, two insertion/deletions (indels; including a 35-bp insertion at its intron 4 and a 6-bp deletion in intron 5) were identified within the VISFATIN gene. To validate these indels and evaluate their association with growth traits in Chinese cattle, a total of 413 samples from four Chinese indigenous breeds and 217 samples from Chinese breeds were detected. Three genotypes (WW, WI and II) at intron 4 were detected based on the 35-bp insertion (allele I) or deletion (allele W) and showed moderate polymorphism in all samples. Two genotypes (WW and WD) at intron 5 were detected based on the 6-bp deletion (allele D) or insertion (allele W) in Xianan (XN) cattle and Jinnan (JN) cattle population but showed poor polymorphisms. Association analysis illustrated that the indel at intron 4 is significantly associated with chest girth, rump length and body weight in Ji'an (JA) cattle and the indel at intron 5 can cause a significant difference in rump length in JN cattle. To our knowledge, it is the first time it has been shown that indels within the VISFATIN gene are associated with growth traits in the two Chinese indigenous cattle breeds. These findings suggest that the VISFATIN gene can be used as a molecular marker for JN and JA cattle breeding.

Improving the Identification of Lysine-Acetylated Peptides Using a Molecularly Imprinted Monolith Prepared by a Deep Eutectic Solvent Monomer.

To overcome the identification challenge of low-abundance lysine acetylation (Kac), a novel approach based on a molecularly imprinted polymer (MIP) was developed to improve the extraction capacity of Kac peptides in real samples. Green deep eutectic solvents (DESs) were introduced and used as one of the synergistic functional monomers with zinc acrylate (ZnA). Glycine-glycine-alanine-lysine(ac)-arginine (GGAKacR) was chosen as a template and N,N'-methylenbisacrylamide (MBAA) was used as a cross-linker. The obtained GGAKacR-MIP had excellent selectivity for the template with an imprinting factor (IF) of up to 21.4. The histone digest addition experiment demonstrated that GGAKacR-MIP could successfully extract GGAKacR from a complex sample. Finally, the application to the extraction of Kac peptides from mouse liver protein digestion was studied in detail. The number of Kac peptides and Kac proteins identified was 130 and 110, which were 3.71-fold and 3.93-fold higher than those of the untreated sample. In addition, the number of peptides and proteins identified after treatment increased from 5535 and 1092 to 17 149 and 4037 (3.10-fold and 3.70-fold, respectively). The results showed that the obtained MIP may provide an effective technical tool for the identification of Kac-modification and peptide fractionation, as well as a potential approach for simultaneously identifying post-translational-modified proteomic and proteomic information.

Copper-Catalyzed Difluoroalkylation Reaction.

This review describes recent advances in copper-catalyzed difluoroalkylation reactions. The RCF2 radical is generally proposed in the mechanism of these reactions. At present, various types of copper-catalyzed difluoroalkylation reactions have been realized. According to their characteristics, we classify these difluoroalkylation reactions into three types.

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair.

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5'-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Preparation, characterization, and application of soluble liquid crystalline molecularly imprinted polymer in electrochemical sensor.

A novel soluble molecularly imprinted polymer (SMIP) without chemical cross-linker was successfully synthesized. The quinine (QN), which the structure was similar to the template, was chosen as the immobile template to improve the affinity of MIP. 4-Methyl phenyl dicyclohexyl ethylene (MPDE) was used as the liquid crystal (LC) monomer to increase the rigid of the composite. The cooperative effect of QN and MPDE was demonstrated by comparing with the conventional MIP, which synthesized without QN and MPDE. The polymerization conditions of SMIP including the ratio of MAA to MPDE, template to functional monomer, and HQN to QN were also optimized. Moreover, the characterizations of the SMIP were investigated by the transmission electron microscopy (TEM), field emission scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and nitrogen adsorption. In binding behavior, the SMIP presented the maximum adsorption capacity (0.37 ± 0.06 mmol/g) and imprinting factor (3.44 ± 0.25). And above all, the obtained polymer exhibited the solubility in the organic solution. In addition, the proposed SMIP as the electrochemical sensor exhibited a significant conductivity and sensitivity with the detection limit of 0.33 µM for HQN, the recoveries for the sample analysis varied from 97.4 to 100.8%, and the intra-day precision and inter-day precision were within 5.5% and 12.5%, respectively. It turned out that the SMIP had demonstrated more excellent potential than the traditional insoluble MIP in the development of the membrane-based electrochemical sensors.Graphical abstract.

Identification of key genes and molecular mechanisms associated with low egg production of broiler breeder hens in ad libitum.

BACKGROUND: Overfeeding reduces laying performance in broiler breeder hens, which is associated with obesity, hepatic steatosis and systemic inflammation. To unravel the underlying mechanisms governing the effect of feeding regimes on energy metabolism and egg production, a transcriptomics approach was carried out for screening differentially expressed genes (DEGs) in ovary, liver and adipose tissues of broiler chickens under ad libitum and restricted feeding. RESULTS: It showed that 289, 388 and 204 DEGs were identified in the adipose, liver and ovary, respectively. These DEGs were significantly enriched in phagosome pathway, lipid transport, activity and nutrient reservoir activity in ovary; steroid hormone biosynthesis and metabolism of xenobiotics by cytochrome P450 pathways in adipose tissue; and the metabolic pathways, peroxisome proliferator-activated receptor (PPAR) and Jak-STAT signaling pathway in liver. Estrogen receptor 1, identified as one of important hubs by constructing PPI network, was up-regulated in ad libitum group, which would make more apolipoproteins be transferred to ovary. CONCLUSIONS: High expression of VTGs, APOB, CYBB and CTSS in ovary would induce excess lipid deposit, oxidative stress and potential damage to ovulation. Our results contribute to understanding effects of feeding regimes on metabolic regulation during egg production of broiler breeder hens and also provide new evidence of metabolic regulation from integrated multi-tissue processes.

Baicalein attenuates monocrotaline-induced pulmonary arterial hypertension by inhibiting vascular remodeling in rats.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by elevated pulmonary arterial pressure (PAP) and right ventricular hypertrophy (RVH) driven by progressive vascular remodeling. Reversing adverse vascular remodeling is an important concept in the treatment of PAH. Endothelial injury, inflammation, and oxidative stress are three main contributors to pulmonary vascular remodeling. Baicalein is a natural flavonoid that has been shown to possess anti-proliferative, anti-inflammatory, anti-oxidative, and cardioprotective properties. We hypothesized that baicalein may prevent the progression of PAH and preserve the right heart function by inhibiting pulmonary arterial remodeling. METHODS: Male Sprague-Dawley rats were distributed randomly into 4 groups: control, monocrotaline (MCT)-exposed, and MCT-exposed plus baicalein treated rats (50 and 100 mg/kg/day for 2 weeks). Hemodynamic changes, RVH, and lung morphological features were examined on day 28. Apoptosis was determined by TUNEL staining, and the mRNA levels of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were detected by qRT-PCR. The changes in oxidative indicators, including malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured using corresponding commercial kits. The levels of Bax, Bcl-2, and cleaved caspase-3, and the activation of mitogen-activated protein kinase (MAPK) and NF-κB were assessed by western blotting. RESULTS: MCT induced an increase in hemodynamic parameters and RVH, which were attenuated by baicalein treatment. Baicalein also blocked MCT-induced pulmonary arterial remodeling. The levels of apoptotic (Bax/Bcl-2 ratio and cleaved caspase-3) and inflammatory (IL-6, TNF-α, and IL-1ß) biomarkers in lung tissue were lower in baicalein-treated groups. Baicalein also decreased MDA level, and increased SOD and GSH-Px activity in rat pulmonary tissue. Furthermore, baicalein inhibited MCT-induced activation of the MAPK and NF-κB pathways. CONCLUSION: Baicalein ameliorates MCT-induced PAH by inhibiting pulmonary arterial remodeling at least partially via the MAPK and NF-κB pathways in rats.

Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia.

The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes.

Nebivolol alleviates aortic remodeling through eNOS upregulation and inhibition of oxidative stress in l-NAME-induced hypertensive rats.

PURPOSE: To investigate the effect and mechanism of nebivolol on aortic remodeling in N-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. METHODS: Male Sprague-Dawley rats were treated with equal volumes of drinking water or l-NAME (60 mg/kg/day), alone or in combination with nebivolol (8 mg/kg/day) or atenolol (80 mg/kg/day) by gavage for 8 weeks. Systolic blood pressure (SBP), aortic morphometry, plasma nitric oxide (NO) levels, nitric oxide synthase (NOS) activity, and relaxation of aorta to acetylcholine were determined. Protein expression of endothelial NOS (eNOS), Akt, and NADPH oxidase (Nox) was evaluated. RESULTS: l-NAME-treated rats showed an elevated SBP associated with aortic remodeling. l-NAME-treated rats showed reduced plasma NO levels and NOS activity and increased reactive oxygen species (ROS). Protein expression of eNOS, eNOS phosphorylated at Ser1177 (p-eNOS), Akt, and Akt phosphorylated at Ser473 (p-Akt) decreased, whereas that of Nox2, Nox4, and p22phox increased in the aortas from l-NAME-treated rats. Nebivolol treatment reduced SBP and ameliorated aortic remodeling. The effects of nebivolol were accompanied by increasing NO levels, NOS activity, and expression of eNOS, p-eNOS, Akt, and p-Akt, as well as reduction of ROS generation and Nox2, Nox4, and p22phox expression. These effects of nebivolol were not reproduced by atenolol. CONCLUSION: Our data indicate a protective role of nebivolol on the high blood pressure and vascular remodeling induced by l-NAME. The beneficial vascular effect of nebivolol is mediated by the upregulation of eNOS and inhibition of oxidative stress.

[Research progress in developing reporter systems for the enrichment of positive cells with targeted genome modification].

Targeted genome editing technology plays an important role in studies of gene function, gene therapy and transgenic breeding. Moreover, the efficiency of targeted genome editing is increased dramatically with the application of recently developed artificial nucleases such as ZFNs, TALENs and CRISPR/Cas9. However, obtaining positive cells with targeted genome modification is restricted to some extent by nucleases expression plasmid transfection efficiency, nucleases expression and activity, and repair efficiency after genome editing. Thus, the enrichment and screening of positive cells with targeted genome modification remains a problem that need to be solved. Surrogate reporter systems could be used to reflect the efficiency of nucleases indirectly and enrich genetically modified positive cells effectively, which may increase the efficiency of the enrichment and screening of positive cells with targeted genome modification. In this review, we mainly summarized principles and applications of reporter systems based on NHEJ and SSA repair mechanisms, which may provide references for related studies in future.

Current trends in the development of molecularly imprinted polymers in CEC.

This review focused on the developments in the field of molecularly imprinted polymers (MIPs) for CEC since 2009. New preparation techniques of MIP-based CEC, such as, portable microchip with macroporous monolithic imprinted microchannel, and low cross-linking MIPs based on liquid crystalline monomers, were discussed. Using selected cases rather than a comprehensive review of the entire field, our goal is to highlight the studies of the interest with an emphasis on recent work, and offers suggestions for future development in the field of imprinted materials for CEC separation.

Enrichment of prime-edited mammalian cells with surrogate PuroR reporters.

Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.

ß-catenin/TCF4-induced SCUBE3 upregulation promotes ovarian cancer development via HIF-1 signaling pathway.

The precise involvement and mechanistic role of the signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) in ovarian cancer (OV) remain poorly understood. Here, leveraging comprehensive data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we unveil the selective overexpression of SCUBE3 in ovarian cancer tissues and cells. Intriguingly, elevated SCUBE3 expression levels correlate with an unfavorable prognosis in patients. Through meticulous manipulation of SCUBE3 expression, we elucidate its consequential impact on in vitro proliferation and invasion of ovarian cancer cells, as well as in vivo tumor growth in mice. Our multifaceted investigations, encompassing luciferase reporter assays, chromatin immunoprecipitation (ChIP) experiments, and mining of public databases, successfully identify SCUBE3 as a direct downstream target gene of TCF4-a pivotal positive regulator within the ß-catenin/TCF4 complex. Furthermore, utilizing a recessive mutant mouse line (kta41) harboring a functionally impaired point mutation at position 882 in the SCUBE3 gene, we uncover SCUBE3's involvement in the intricate regulation of angiogenesis and epithelial-mesenchymal transition (EMT). Strikingly, Spearman correlation coefficient analysis unveils a close association between SCUBE3 and HIF1A in OV, with SCUBE3 exerting tight control over HIF1A mRNA expression. Moreover, functional inhibition of HIF1A significantly impedes the pro-proliferative and invasive capabilities of SCUBE3-overexpressing ovarian cancer cells. Collectively, our findings underscore the pivotal role of SCUBE3 in driving ovarian cancer progression, shedding light on its intricate molecular mechanisms and establishing it as a potential therapeutic target for this devastating disease.

Anchoring tungsten oxide nanorods on TiO2 nanowires coupled with carbon for efficient lithium-ion storage.

Reasonable construction of hierarchical electrode materials is verified as a promising way to improve the electrochemical performance due to the synergistic effect between unique components and constructions. Hence, a hierarchical nanostructure composed of tungsten oxide nanorods anchored on TiO2 nanowires coupled with a carbon layer (TiO2@WOx-C NWs) was synthesized as an electrode material by exploiting the self-assembly function of dopamine and carbonization. The inner one-dimensional TiO2 nanowires served as the stable substrate with WOx anchored on the surface of TiO2 NWs and the tightly coupled carbon nanosheets, which can not only facilitate electron transport but also provide more active sites for electrochemical reactions. As a result, benefitting from the synergistic effects between three functional components and the multi-dimensional hierarchical structures, the as-prepared TiO2@WOx-C NWs displayed excellent lithium storage performance with a specific capacity of 651.4 mA h g-1 after 500 cycles at 1.0 A g-1, which is superior to most Ti-based structures. The enhanced electrochemical performance is mainly attributed to the synergistic effect of the different dimensional structures, the high capacity of tungsten oxide and the surface coating of the conductive carbon material. This work provides a simple and effective approach to designing functional hierarchical structures for energy storage and conversion.

Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.

CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.

Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells.

The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.

Yolk precursor synthesis and deposition in hierarchical follicles and effect on egg production performance of hens.

Egg production of hens is related to ovarian follicles development. The hierarchical follicle development accompanies the deposition of a large amount of yolk precursor. The aim of this study was to illustrate the effects of strain and age on yolk deposition and egg production. The experiment compared yolk synthesis, transport, and deposition in 3 groups of hens: one of a high-yield commercial hybrid laying breed (Jinghong No.1) in 2 stages (35 wk and 75 wk; JH35, JH75) and one of Chinese native breed (Lueyang Black-Boned chicken) at 35 wk (LY35). The results showed that the number of hierarchical follicles in JH35 and JH75 was significantly more than in LY35. At the same time, the yolk weight of the LY35 and JH75 was significantly higher than that of JH35. The expression of apolipoprotein A1 and apolipoprotein B genes in the liver of JH35 was higher than that of JH75. The expression of the very low-density lipoprotein receptor gene in the JH75 ovary was higher than that of the other 2 groups. The plasma concentrations of very low-density lipoprotein and vitellogenin were no significant difference among groups. The yolk deposition in hierarchical follicles based on the fat-soluble dyes measurement meant that the rate of yolk deposition of LY35 was lower than the other 2 groups. In most cases, the yolk deposition of JH75 was higher than that of the other groups, but the process showed greater fluctuation over time. These results meant that the rate and stability of yolk deposition played an essential role in affecting egg performance. In summary, both strain and age were related to egg production, but the 2 factors might impact yolk deposition and egg-laying performance differently. The egg performance may be affected by both yolk precursor synthesis and deposition for different strains, but it may be affected by yolk precursor deposition for the old laying hens.

Preparation and characterization of grafted imprinted monolith for capillary electrochromatography.

In this paper, a molecularly imprinted polymer (MIP) coating grafted to a trimethylolpropane trimethacrylate (TRIM) core material for CEC was reported. The core monolith was prepared with a solution of 20% (w/w) TRIM in a mixture of porogen and a polymerization precursor, which can generate a stable electroosmotic flow due to the formation of ionizable groups after postpolymerization hydrolization. Graft polymerization took place on the resultant TRIM monolith with a mixture of template, methacrylic acid, and ethylene glycol dimethacrylate. Strong recognition ability (selectivity factor was 5.83) for S-amlodipine and resolution of enatiomers separation (up to 7.99) were obtained on the resulting grafted imprinted monolith in CEC mode. The influence of CEC conditions on chiral separation, including the composition of mobile phase, pH value, and the operating voltages was studied. These results suggest that the method of grafted polymerization reported here allows a rapid development of MIP monolith once core materials with desired properties are available, and is a good alternative to prepare CEC-based monolithic MIPs.

Insulin-like growth factor-1 regulates follicle selection of hens by promoting proliferation and inhibiting apoptosis of granulosa cells in prehierarchical follicles in vitro.

During the reproduction stage of poultry, a single follicle is selected from a cohort of 6-8 mm small yellow follicles to initiate rapid growth and final ovulation almost daily. In the process, follicle-stimulating hormone (FSH) plays a pivotal role by interacting with intraovarian factors, including insulin-like growth Factor 1 (IGF1). The objective of this study was to analyze whether IGF1 coordinates with FSH to affect the characteristics of granulosa cells from prehierarchical follicles. After treating granulosa cells with 50 ng/mL FSH and 200 ng/mL IGF1, we detected the proliferation and apoptosis of granulosa cells using flow cytometry. The percentage of G1 phase granulosa cells was increased, and the percentage of mitotic cells and apoptotic cells was reduced under IGF1 treatment. The expression levels of the steroidogenic acute regulatory protein gene, cytochrome P450 side-chain cleavage gene and 3ß-hydroxysteroid dehydrogenase gene, which are related to steroidogenic synthesis, were reduced by cotreatment with FSH and IGF1. The expression of the cell proliferation- or apoptosis-related genes cyclin dependent kinase 2, cyclin D2, B-cell leukemia/lymphoma 2, and BCL2 like 1 and the ratio of B-cell leukemia/lymphoma 2/BCL2-associated X were increased by treatment with IGF1. There was a decrease in the expression of caspase3 after treatment with FSH and IGF1. All these results showed that IGF1 reduced the expression of genes involved in progesterone synthesis, stimulated proliferation and inhibited apoptosis in granulosa cells. Thus, IGF1 may be one of the factors involved in affecting FSH responsiveness and maintaining the undifferentiated state of prehierarchical follicles before follicle selection.

Dual-function monolithic enzyme reactor based on dopamine/graphene oxide coating for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment.

A new dual-function enzyme reactor was prepared based on a dopamine/graphene oxide coated boron affinity monolithic column, which can be used for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment. Firstly, a boron affinity monolithic column was prepared as the carrier for enzyme reactor. Secondly, the monolithic column was coated with dopamine/graphene oxide to provide higher specific surface area for the increase in the amount of trypsin bound. Then, dopamine can self-polymerize under alkaline conditions to produce multiple reaction sites. By the Schiff base reaction and Michael addition reaction with amino, sulfhydryl groups to trypsin, enzyme were immobilized on the boron affinity monolithic carrier. The enzyme activity was characterized by kinetic parameters maximum rate (Vmax) of the enzyme reaction and Michaelis constant (Km). Km of the dual-function enzyme reactors doped with PDA/GO and without PDA/GO were 34.37 and 120.93 mM, Vmax were 1.35 and 3.35 mM/min, respectively. The performance of the dual-function enzyme reactor was evaluated by protein extraction of mouse liver. After digested by the dual-function enzyme reactor, the number of peptides was 4,801, which was 960 more than the number of peptides in the solution digestion. At the same time, the dual-function enzyme reactor displayed the ability to capture cis-dihydroxy compounds specificly. A total of 55 glycopeptides were enriched in the dual-functional enzyme reactor, corresponding to 33 glycoproteins. The dual-function enzyme reactor provided repeatable performance and robust with long-term storage.