Single nucleotide polymorphisms in the KRT82 promoter region modulate irregular thickening and patchiness in the dorsal skin of New Zealand rabbits.

BACKGROUND: While rabbits are used as models in skin irritation tests, the presence of irregular patches and thickening on the dorsal skin can affect precise evaluation. In this study, genes associated with patchiness or non-patchiness on the dorsal skin of New Zealand rabbits were investigated to identify potential regulators of the patchiness phenotype. RESULTS: The results showed that parameters associated with hair follicles (HFs), such as HF density, skin thickness, and HF depth, were augmented in rabbits with the patchiness phenotype relative to the non-patchiness phenotype. A total of 592 differentially expressed genes (DEGs) were identified between the two groups using RNA-sequencing. These included KRT72, KRT82, KRT85, FUT8, SOX9, and WNT5B. The functions of the DEGs were investigated by GO and KEGG enrichment analyses. A candidate gene, KRT82, was selected for further molecular function verification. There was a significant positive correlation between KRT82 expression and HF-related parameters, and KRT82 overexpression and knockdown experiments with rabbit dermal papilla cells (DPCs) showed that it regulated genes related to skin and HF growth and development. Investigation of single nucleotide polymorphisms (SNPs) in the exons and promoter region of KRT82 identified four SNPs in the promoter region but none in the exons. The G.-631G > T, T.-696T > C, G.-770G > T and A.-873 A > C alleles conformed to the Hardy - Weinberg equilibrium, and three identified haplotypes showed linkage disequilibrium. Luciferase reporter assays showed that the core promoter region of KRT82 was located in the - 600 to - 1200 segment, in which the four SNPs were located. CONCLUSIONS: The morphological characteristics of the patchiness phenotype were analyzed in New Zealand rabbits and DEGs associated with this phenotype were identified by RNA-sequencing. The biological functions of the gene KRT82 associated with this phenotype were analyzed, and four SNPs were identified in the promoter region of the gene. These findings suggest that KRT82 may be a potential biomarker for the breeding of experimental New Zealand rabbits.

N-Acetylcysteine Alleviates D-Galactose-Induced Injury of Ovarian Granulosa Cells in Female Rabbits by Regulating the PI3K/Akt/mTOR Signaling Pathway.

The ovary plays a crucial role in the reproductive system of female animals. Ovarian problems such as ovarian insufficiency, premature aging, polycystic ovary syndrome, and ovarian cysts may lead to ovulation disorders, abnormal hormone secretion, or luteal dysfunction, thereby increasing the risk of infertility and abortion. Only when the ovarian function and other organs in the reproductive system remain healthy and work normally can female animals be ensured to carry out reproductive activities regularly, improve the pregnancy rate and litter size, promote the healthy development of the fetus, and then improve their economic value. The follicle, as the functional unit of the ovary, is composed of theca cells, granulosa cells (GCs), and oocytes. GCs are the largest cell population and main functional unit in follicles and provide the necessary nutrients for the growth and development of follicles. N-acetylcysteine (NAC) is a prevalent and cell-permeable antioxidant molecule that effectively prevents apoptosis and promotes cellular survival. Over the past few years, its function in boosting reproductive performance in animals at the cellular level has been widely acknowledged. However, its specific role and mechanism in influencing GCs is yet to be fully understood. The objective of this study was to examine the effects of NAC on ovarian damage in female rabbits. For this purpose, D-galactose (D-gal) was first used to establish a model of damaged GCs, with exposure to 1.5 mg/mL of D-gal leading to substantial damage. Subsequently, varying concentrations of NAC were introduced to determine the precise mechanism through which it influences cell damage. Based on the results of the Cell Counting Kit-8 assay, flow cytometry, and Western blotting, it was found that 0.5 mg/mL of NAC could significantly suppress cell apoptosis and promote proliferation. In particular, it decreased the expression levels of Bax, p53, and Caspase-9 genes, while concurrently upregulating the expression of the BCL-2 gene. Moreover, NAC was found to alleviate intracellular oxidative stress, suppress the discharge of mitochondrial Cytochrome c, and boost the enzymatic activities of CAT (Catalase), GSH (Glutathione), and SOD (Superoxide dismutase). RNA sequencing analysis subsequently underscored the critical role of the PI3K/Akt/mTOR pathway in governing proliferation and apoptosis within GCs. These findings demonstrated that NAC could significantly influence gene expression within this pathway, thereby clarifying the exact relationship between the PI3K/Akt/mTOR signaling cascade and the underlying cellular processes controlling proliferation and apoptosis. In conclusion, NAC can reduce the expression of Bax, p53, and Caspase-9 genes, inhibit the apoptosis of GCs, improve cell viability, and resist D-gal-induced oxidative stress by increasing the activity of CAT, GSH, and SOD. The molecular mechanism of NAC in alleviating D-gal-induced ovarian GC injury in female rabbits by regulating the PI3K/Akt/mTOR signaling pathway provides experimental evidence for the effect of NAC on animal reproductive function at the cellular level.

Regulation of Hair Follicle Growth and Development by Different Alternative Spliceosomes of FGF5 in Rabbits.

This study investigated the regulatory effect of alternative spliceosomes of the fibroblast growth factor 5 (FGF5) gene on hair follicle (HF) growth and development in rabbits. The FGF5 alternative spliceosomes (called FGF5-X1, FGF5-X2, FGF5-X3) were cloned. The overexpression vector and siRNA of spliceosomes were transfected into dermal papilla cells (DPCs) to analyze the regulatory effect on DPCs. The results revealed that FGF5-X2 and FGF5-X3 overexpression significantly decreased LEF1 mRNA expression (p < 0.01). FGF5-X1 overexpression significantly reduced CCND1 expression (p < 0.01). FGF5-X1 and FGF5-X2 possibly downregulated the expression level of FGF2 mRNA (p < 0.05), and FGF5-X3 significantly downregulated the expression level of FGF2 mRNA (p < 0.01). The FGF5 alternative spliceosomes significantly downregulated the BCL2 mRNA expression level in both cases (p < 0.01). FGF5-X1 and FGF5-X2 significantly increased TGFß mRNA expression (p < 0.01). All three FGF5 alternative spliceosomes inhibited DPC proliferation. In conclusion, the expression profile of HF growth and development-related genes can be regulated by FGF5 alternative spliceosomes, inhibiting the proliferation of DPCs and has an influence on the regulation of HF growth in rabbits. This study provides insights to further investigate the mechanism of HF development in rabbits via FGF5 regulation.

4D label-free quantitative proteomic analysis identifies CRABP1 as a novel candidate gene for litter size in rabbits.

In commercial rabbit breeding, litter size is a crucial reproductive trait. This trait directly determines the reproductive ability of female rabbits and is crucial for evaluating the production efficiency. We here compared differentially expressed proteins of in the ovary tissue from New Zealand female rabbits with high (H) and low (L) litter sizes by using 4D label-free quantitative proteomic technology and identified 92 differential proteins. The biological functions of these proteins were revealed through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Most distributions of GO and KEGG were related to reproduction, growth development, and metabolism. Furthermore, a novel candidate gene Cellular Retinoic Acid Binding Protein-1 (CRABP1), which was highly expressed in the L group, was selected for further biological function verification. The Cell Counting Kit-8 assay and flow cytometry analysis revealed that CRABP1 can promote granulosa cell (GC) apoptosis and inhibit GC proliferation. Furthermore, qRT-PCR and western blotting analysis revealed that CRABP1 regulates the genes (HSD17B1, Wnt-10b, FSHR, TAF4B, BMP15, and BMP6) and protein (Wnt-10b) associated with steroid hormone synthesis and follicle development. The PCR product direct sequencing method revealed single nucleotide polymorphisms in the core promoter region of CRABP1. Luciferase activity assays revealed that the transcriptional activity of the GG genotype was significantly higher than that of the TT or TG genotype. Different genotypes are accompanied by changes in transcription factors, which indicates that T-359G polymorphism can regulate CRABP1 expression. In general, we identified litter size-related genes and revealed the mechanism underlying the effect of CRABP1 on litter size. CRABP1 serves as a key factor in the reproductive capacity of rabbits and can act as a molecular biomarker for the breeding of New Zealand rabbits.

Characterization and establishment of an immortalized rabbit ovary granulosa cell line for reproductive biology experiments.

Granulosa cells (GCs) are the key components of ovarian follicles and regulate the maturation, communication, growth, and development of oocytes. GCs have great potential as human therapeutic models and in livestock breeding. In this study, we established an immortalized cell line (Im-RGCs) by transforming primary rabbit granulosa cells (Pri-RGCs) with lentivirus-mediated simian virus 40 Large T (SV40LT). Morphologically, Im-RGCs were indistinguishable from Pri-RGCs and maintained intact cell structure as observed by H&E staining. Also, Im-RGCs exhibited no significant change in cell proliferation, viability, and growth. Furthermore, GC-specific markers, such as FSHR, StAR, CYP11A1, and CYP19A1, were examined by PCR, immunofluorescence, and Western blotting. ELISA and karyotype analysis showed that Im-RGCs can synthesize steroid hormones and maintain the normal number of chromosomes during the infinite passage. Furthermore, soft-agar cloning and nude mice tumorigenic experiments indicated the absence of any malignancy transformation in Im-RGCs. In conclusion, we successfully established the immortalized granulosa cell line of rabbit follicles that can be used for biological, animal husbandry, and female reproductive research.

ChIP-Seq analysis reveals PRKACB as a target gene of HOXC13 involved in rabbit hair follicle development.

Dermal papilla cells (DPCs) are key regulators of hair follicle (HF) development and growth, which not only regulate HF growth and cycling but play a role in the pathogenesis of hair loss. The transcription factor Homeobox C13 (HOXC13) can modulate the growth and development of HFs. Nevertheless, the specific genes and pathways regulated by HOXC13 in DPCs have yet to be determined. Thus, to gain a better understanding of genomic binding sites involved in HOXC13-regulated HF development, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) was performed on rabbit DPCs with pcDNA3.1-3 × Flag-HOXC13 overexpression. A complete set of 9670 enrichment peaks was acquired by applying HOXC13-Flag ChIP. Subsequently, the peak sequence was annotated to the rabbit genome, revealing that 6.1 % of the peaks were identified within in the promoter region. Thereafter, five annotated genes were verified using RT-qPCR. The peak-associated genes were mainly enriched in signaling pathways related to HF development, such as MAPK and PI3K-Akt. Furthermore, by using a dual-luciferase reporter assay, we found that HOXC13 can target the protein kinase cAMP­dependent catalytic ß (PRKACB) promoter region (-1596 âˆ¼ -1107 bp) and inhibit its transcription, which was consistent with data obtained from ChIP-seq analysis. Overexpression of PRKACB gene significantly modulated the expression of BCL2, WNT2, LEF1, and SFRP2 genes related to HF development as determined by RT-qPCR (P < 0.01, P < 0.05). The CCK-8 and flow cytometry assays showed that PRKACB significantly inhibited the proliferation of DPCs and promoted apoptosis (P < 0.01). In conclusion, our research revealed that PRKACB has the potential to serve as a novel target gene of HOXC13, contributing to the regulation of the proliferation and apoptosis of DPCs. The process of identifying global target genes can contribute to the understanding of the intricate pathways that HOXC13 regulates in the growth of HFs.

Establishment and functional characterization of immortalized rabbit dermal papilla cell lines.

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFß2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Reproductive performance and transcriptome analysis of ovaries at different parities in female rabbits.

This study investigated the reproductive performance and ovarian molecular regulation associated with parity in commercial rabbit systems. The pregnancy data of 658 female rabbits from the first to sixth parities (P1 to P6) under the same mating pattern were analyzed, showing a significant decrease in the conception rate in P6. Compared to P1 (N = 120) and P2 (N = 105), P6 (N = 99) had significantly lower performance indices in terms of total litter size, live litter size, survival rate at birth, and weight of 3 and 5 wk old kits (P < 0.05). Using H&E staining, we found that the ovarian primordial follicle reservoir of P6 was significantly lower than that of P1 and P2, and the number of atretic follicles at P6 was significantly higher (P < 0.05). Blood (N = 30 per group) and ovaries (N = 6 per group) in P1, P2, and P6 were collected for measurement of the serum anti-oxidant capacity and indices of ovarian function by ELISA. It was found that serum glutathione, ovarian Klotho protein, and telomeres of P1 and P2 were significantly higher than those of P6 (P < 0.05). The serum levels of ROS and MDA at P1 and P2 were significantly lower than those at P6 (P < 0.05). Additionally, transcriptome analysis showed 213 up-regulated and 747 down-regulated differentially expressed genes (DEGs) between P2 and P6 ovaries. Several DEGs were related to reproduction, including CYP21A2, PTGFR, SGK1, PIK3R6, and SRD5A2. These results demonstrate the influence of parity on reproduction in female rabbits, reflected in a loss of follicle reservoir, disordered levels of anti-oxidants, and indices associated with ovarian function and molecular regulation. This study provides a basis for the strategies to increase reproductive rate in female rabbits.

The pregnancy data of 658 female rabbits from the first to sixth parities (P1 to P6) under the same mating pattern were used to assess the rate of conception at different parities. The reproductive performance and follicular development of P1, P2, and P6 female rabbits were analyzed. The results showed that conception rate was dramatically reduced in P6. Compared with P1 and P2, P6 rabbits showed evidence of lower fertility in terms of total litter size, live litter size, survival rate at birth, and weights of kits at 3 and 5-wk-old. The primordial follicle storage at P6 was significantly reduced, with greater numbers of atretic follicles compared with P1 and P2. In terms of serum glutathione, reactive oxygen species, malondialdehyde, and ovarian Klotho protein, telomeres, the anti-oxidant capacity and ovarian function at P6 was significantly affected by parity. Further, based on the ovarian transcriptomes at P2 and P6, several genes related to reproductive regulation were identified. These findings provide a basis for improving the reproductive rate of female rabbits.

Dermal PapillaCell-Derived Exosomes Regulate Hair Follicle Stem Cell Proliferation via LEF1.

Hair follicle (HF) growth and development are controlled by various cell types, including hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs). Exosomes are nanostructures that participate in many biological processes. Accumulating evidence indicates that DPC-derived exosomes (DPC-Exos) mediate HFSC proliferation and differentiation during the cyclical growth of hair follicles. In this study, we found that DPC-Exos increase ki67 expression and CCK8 cell viability readouts in HFSCs but reduce annexin staining of apoptotic cells. RNA sequencing of DPC-Exos-treated HFSCs identified 3702 significantly differentially expressed genes (DEGs), including BMP4, LEF1, IGF1R, TGFß3, TGFα, and KRT17. These DEGs were enriched in HF growth- and development-related pathways. We further verified the function of LEF1 and showed that overexpression of LEF1 increased the expression of HF development-related genes and proteins, enhanced HFSC proliferation, and reduced HFSC apoptosis, while knockdown of LEF1 reversed these effects. DPC-Exos could also rescue the siRNA-LEF1 effect in HFSCs. In conclusion, this study demonstrates that DPC-Exos mediated cell-to-cell communication can regulate HFSCs proliferation by stimulating LEF1 and provide novel insights into HF growth and development regulatory mechanisms.

Seedling maize counting method in complex backgrounds based on YOLOV5 and Kalman filter tracking algorithm.

Maize population density is one of the most essential factors in agricultural production systems and has a significant impact on maize yield and quality. Therefore, it is essential to estimate maize population density timely and accurately. In order to address the problems of the low efficiency of the manual counting method and the stability problem of traditional image processing methods in the field complex background environment, a deep-learning-based method for counting maize plants was proposed. Image datasets of the maize field were collected by a low-altitude UAV with a camera onboard firstly. Then a real-time detection model of maize plants was trained based on the object detection model YOLOV5. Finally, the tracking and counting method of maize plants was realized through Hungarian matching and Kalman filtering algorithms. The detection model developed in this study had an average precision mAP@0.5 of 90.66% on the test dataset, demonstrating the effectiveness of the SE-YOLOV5m model for maize plant detection. Application of the model to maize plant count trials showed that maize plant count results from test videos collected at multiple locations were highly correlated with manual count results (R2 = 0.92), illustrating the accuracy and validity of the counting method. Therefore, the maize plant identification and counting method proposed in this study can better achieve the detection and counting of maize plants in complex backgrounds and provides a research basis and theoretical basis for the rapid acquisition of maize plant population density.

Characterization and functional analysis of lncRNA2690 in regulating the growth cycle of the hair follicle in rabbits.

Hair follicles (HFs) achieve hair growth and renewal by periodic regeneration. Therefore, exploring the key factors affecting hair growth in rabbits is of great significance for precisely breeding Angora rabbits and improving the competitiveness of the rabbit industry. Based on the results of our previous studies, lncRNA2690 was differentially expressed in the HF cycle using lncRNA-Seq, and the full-length sequence was annotated by bioinformatics analysis. The lncRNA2690 is 363 nt long and is found on chromosome 14 from 163 321 514 to 163 321 872. The lncRNA2690 was predicted to not have the coding ability through open reading frame and CPC2, and the nuclear-cytoplasmic separation experiment showed the lncRNA2690 to be highly expressed in the nucleus (p < 0.01). The expression pattern of lncRNA2690 was further analyzed in the different HF development stages of Angora rabbits using quantitative real-time PCR. The results showed that lncRNA2690 was periodically expressed in HF development, and the expression level was found to be high in the HF resting phases. The overexpression and knockdown of lncRNA2690 were found to significantly upregulate and downregulate the expression of the genes WNT2, CCND1, BMP2, LEF1, and SIAH1 in the rabbit dermal papilla cells (p < 0.01), promoting cell apoptosis and inhibiting cell proliferation (p < 0.01). This indicated that lncRNA2690 negatively regulates the periodic regeneration of the HFs in rabbits. These results provide a basis for the further study of lncRNA2690 in the HF growth cycle of Angora rabbits.

lncRNA2919 Suppresses Rabbit Dermal Papilla Cell Proliferation via trans-Regulatory Actions.

Hair follicles (HFs) are complex organs that grow cyclically during mammals' growth and development. Long non-coding RNAs (lncRNAs) cannot be translated into proteins and play crucial roles in many biological processes. In our previous study, candidate lncRNAs associated with HF cyclic regeneration were screened, and we identified that the novel lncRNA, lncRNA2919, was significantly expressed during catagen. Here, we identified that lncRNA2919 has no coding potentiality and is highly expressed in the cell nucleus, and downregulates HF growth and development-related genes, inhibits cell proliferation, and promotes cell apoptosis in rabbit dermal papilla cells. lncRNA2919 recruits STAT1 to form a compound. As a key transcription factor, STAT1 regulates the transcriptional expression of KRTAP11-1. Our study revealed that lncRNA2919 is involved in HF cyclic regeneration through the trans-regulatory lncRNA2919-STAT1-KRTAP11-1 axis. This study elucidates the mechanism through which lncRNA2919 regulates HF growth and development and the role of lncRNA2919 as a new therapeutic target in animal wool production and human hair-related disease treatment.

DNA Methylation Mediates lncRNA2919 Regulation of Hair Follicle Regeneration.

Hair follicles (HFs) are organs that periodically regenerate during the growth and development of mammals. Long non-coding RNAs (lncRNAs) are non-coding RNAs with crucial roles in many biological processes. Our previous study identified that lncRNA2919 is highly expressed in catagen during the HF cycle. In this study, the in vivo rabbit model was established using intradermal injection of adenovirus-mediated lncRNA2919. The results showed that lncRNA2919 decreased HF depth and density and contributed to HF regrowth, thereby indicating that lncRNA2919 plays a negative role in HF regeneration. Moreover, methylation levels of the lncRNA2919 promoter at different HF cycle stages were detected through bisulfite sequencing. The key CpG site that negatively correlates with lncRNA2919 expression during the HF cycle was identified. 5-Aza-dc-induced demethylation upregulated lncRNA2919 expression, and the core promoter region of lncRNA2919 was verified on the basis of luciferase activity. Furthermore, we found that DNA methylation could prevent the binding of EGR1 to the lncRNA2919 promoter region, thereby affecting the transcriptional expression of lncRNA2919. Collectively, DNA methylation inhibits the transcriptional expression of lncRNA2919, which plays a vital role in the HF cycle and HF regrowth. These findings contribute to the basic theory of epigenetics in HF biology and provide references for further research in HF disease treatment and animal wool production.

Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit.

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits' breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2−5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.

Exosomal miRNA-181a-5p from the cells of the hair follicle dermal papilla promotes the hair follicle growth and development via the Wnt/ß-catenin signaling pathway.

Exosomal miRNAs are verified critical biomarkers, which participate in several biological processes. The growth and development of the hair follicle (HF) are typically controlled by the exosomal miRNAs via cell-to-cell communication. This study identified a high expression of miR-181a-5p in the low-passage DPC-Exos (exosomes derived from dermal papilla cell), revealing the transportation patterns of the DPC-Exos-derived miR-181a-5p entering the HFSC (hair follicle stem cell). The exosomal miR-181a-5p activates the Wnt/ß-catenin signaling pathway by targeting the Wnt inhibitor WIF1 and thereby regulates the proteins and genes related to HF growth and development. Moreover, the exosomal miR-181a-5p was found to suppress the HFSC apoptosis but promoted the HFSC proliferation. The in vitro culture of the HF organ revealed that the exosomal miR-181a-5p possesses a positive role in hair growth. Collectively, the exosomal miR-181a-5p affects the HF growth and development through the Wnt/ß-catenin signaling pathway. The exosomal miR-181a-5p might, therefore, act as the novel biomarker and therapeutic target for treating hair-related diseases and wool production in mammals.

Preparation and Synthetic Application of Naproxen-Containing Diaryliodonium Salts.

The synthesis of naproxen-containing diaryliodonium salts has been realized from naproxen methyl ester and ArI(OH)OTs activated by trimethylsilyl trifluoromethanesulfonate (TMSOTf) in a solvent mixture comprising dichloromethane and 2,2,2-trifluoroethanol (TFE). Those iodonium salts have been successfully used in the functionalization of an aromatic ring of naproxen methyl ester, including fluorination, iodination, alkynylation, arylation, thiophenolation, and amination and esterification reactions. Moreover, further hydrolysis of the obtained 5-iodo-naproxen methyl ester afforded 5-iodo-naproxen.

Characterization and functional analysis of SMAD2 regulation in hair follicle cycle in Angora rabbits.

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominatelylocalizesin the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-ß signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-ß signaling pathway.

A Treatment Combination of IGF and EGF Promotes Hair Growth in the Angora Rabbit.

The hair follicle (HF) growth cycle is a complex, multistep biological process, for which dysfunction affects hair-related diseases in humans and wool production in animals. In this study, a treatment combination of 10 ng/mL insulin-like growth factor-1 (IGF-1) and 20 ng/mL epidermal growth factor (EGF) significantly increased the elongation length of hair shafts for cultured HFs. The combined treatment of IGF-1 and EGF enhanced the proliferation of HFs and promoted HF growth and development in vitro. In vivo, the combined treatment of IGF-1 and EGF was subcutaneously injected into the dorsal skin in HF synchronized rabbits. The IGF-1 and EGF combination promoted the transition of the hair cycle from telogen to anagen and stimulated the growth of hair shafts. This IGF-1 and EGF combination maintained the structure of the HF and enhanced the cell proliferation of outer root sheaths and the dermal papilla within rabbit skin. The combined treatment of IGF-1 and EGF regulated HF-related genes, including LEF1, CCND1 and WNT2, suggesting that IGF-1 and EGF play a positive role in HF growth and development. Utilization of the combined IGF-1 and EGF treatment may assist with hair and wool production and HF related diseases in mammals.

Characterization and functional analysis of Krtap11-1 during hair follicle development in Angora rabbits (Oryctolagus cuniculus).

BACKGROUND: Keratin-associated protein (KAP), the structural protein molecule of hair fibers, plays a key role in determining the physical properties of hair. Studies of Krtap11-1 have focused only on its localization. Functional studies of Krtap11-1 in hair follicle development have so far not been reported. OBJECTIVE: This study aimed to provide evidence for the role of Krtap11-1 in skin and hair development. METHODS: Full-length cloning and analysis of Krtap11-1 were conducted to ascertain its function. Overexpression vectors and interference sequences were constructed and transfected into RAB-9 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate the hair follicle developmental stage of Krtap11-1, the expression of different tissues, and the effects on other hair follicle development-related genes. RESULTS: The full length of cloned Krtap11-1 was 947 bp. Krtap11-1 was confirmed to be a hydrophilic protein localized mostly in mitochondria. The greatest mRNA expression was observed in skin. Using a follicle synchronization model, it was found that Krtap11-1 mRNA expression levels first increased then decreased over the passage of time, principally during hair follicle catagen and telogen. Following the overexpression of Krtap11-1, mRNA expression levels of the WNT-2, KRT17, BMP-2, and TGF-ß-1 genes increased, and LEF-1 decreased (P < 0.05), the converse after the corresponding use of si-RNA interference. CONCLUSIONS: Krtap11-1 exerts a promoting effect. The results provide novel insight into the relationship between hair follicle development and Krtap11-1 gene expression.

Characterization of POU2F1 Gene and Its Potential Impact on the Expression of Genes Involved in Fur Color Formation in Rex Rabbit.

The naturally colorful fur of the Rex rabbit is becoming increasingly popular in the modern textile market. Our previous study found that POU class 2 homeobox 1 gene (POU2F1) potentially affects the expression of genes involved in fur color formation in the Rex rabbit, but the function and regulation of POU2F1 has not been reported. In this study, the expression patterns of POU2F1 in Rex rabbits of various colors, as well as in different organs, were analyzed by RT-qPCR. Interference and overexpression of POU2F1 were used to identify the potential effects of POU2F1 on other genes related to fur color formation. The results show that the levels of POU2F1 expression were significantly higher in the dorsal skin of the brown and protein yellow Rex rabbits, compared with that of the black one. POU2F1 mRNAs were widespread in the tissues examined in this study and showed the highest level in the lungs. By transfecting rabbit melanocytes with an POU2F1-overexpression plasmid, we found that the POU2F1 protein was located at the nucleus, and the protein showed the classic characteristics of a transcription factor. In addition, abnormal expression of POU2F1 significantly affected the expression of pigmentation-related genes, including SLC7A11, MITF, SLC24A5, MC1R, and ASIP, revealing the regulatory roles of POU2F1 on pigmentation. The results provide the basis for further exploration of the role of POU2F1 in fur color formation of the Rex rabbit.