The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

MVIP: multi-omics portal of viral infection.

Virus infections are huge threats to living organisms and cause many diseases, such as COVID-19 caused by SARS-CoV-2, which has led to millions of deaths. To develop effective strategies to control viral infection, we need to understand its molecular events in host cells. Virus related functional genomic datasets are growing rapidly, however, an integrative platform for systematically investigating host responses to viruses is missing. Here, we developed a user-friendly multi-omics portal of viral infection named as MVIP (https://mvip.whu.edu.cn/). We manually collected available high-throughput sequencing data under viral infection, and unified their detailed metadata including virus, host species, infection time, assay, and target, etc. We processed multi-layered omics data of more than 4900 viral infected samples from 77 viruses and 33 host species with standard pipelines, including RNA-seq, ChIP-seq, and CLIP-seq, etc. In addition, we integrated these genome-wide signals into customized genome browsers, and developed multiple dynamic charts to exhibit the information, such as time-course dynamic and differential gene expression profiles, alternative splicing changes and enriched GO/KEGG terms. Furthermore, we implemented several tools for efficiently mining the virus-host interactions by virus, host and genes. MVIP would help users to retrieve large-scale functional information and promote the understanding of virus-host interactions.

MaGenDB: a functional genomics hub for Malvaceae plants.

Malvaceae is a family of flowering plants containing many economically important plant species including cotton, cacao and durian. Recently, the genomes of several Malvaceae species have been decoded, and many omics data were generated for individual species. However, no integrative database of multiple species, enabling users to jointly compare and analyse relevant data, is available for Malvaceae. Thus, we developed a user-friendly database named MaGenDB (http://magen.whu.edu.cn) as a functional genomics hub for the plant community. We collected the genomes of 13 Malvaceae species, and comprehensively annotated genes from different perspectives including functional RNA/protein element, gene ontology, KEGG orthology, and gene family. We processed 374 sets of diverse omics data with the ENCODE pipelines and integrated them into a customised genome browser, and designed multiple dynamic charts to present gene/RNA/protein-level knowledge such as dynamic expression profiles and functional elements. We also implemented a smart search system for efficiently mining genes. In addition, we constructed a functional comparison system to help comparative analysis between genes on multiple features in one species or across closely related species. This database and associated tools will allow users to quickly retrieve large-scale functional information for biological discovery.

"One-Pot" CuCl2-Mediated Condensation/C-S Bond Coupling Reactions to Synthesize Dibenzothiazepines by Bi-Functional-Reagent N, N'-Dimethylethane-1,2-Diamine.

The efficient "One-pot" CuCl2-catalyzed C-S bond coupling reactions were developed for the synthesis of dibenzo[b,f][1,4]thiazepines and 11-methy-ldibenzo[b,f][1,4]thiazepines via 2-iodobenzaldehydes/2-iodoacetophenones with 2-aminobenzenethiols/2,2'-disulfanediyldianilines by using bifunctional-reagent N, N'-dimethylethane-1,2-diamine (DMEDA), which worked as ligand and reductant. The reactions were compatible with a range of substrates to give the corresponding products in moderate to excellent yields.

Effect of in ovo injection of serotonin on the behavior and hormone level in laying hens.

Feather pecking is a typically abnormal behavior that significantly impacts breeding efficiency and animal welfare in the egg production sector. Serotonin (5-HT) is essential for neuronal development and behavioral regulation. This study evaluated the effects of birds' behavioral development (including feather pecking) and changes in serum hormones in chickens followed in ovo injection of 5-HT. On day 11, incubated eggs were injected with 5-HT at 0 (saline control), 5 ug (low) or 15 ug (high) (n = 166 per treatment). The hatched female chicks were raised under similar conditions up to 20 weeks of age (n = 60 per treatment). Birds' behaviors were recorded using a digital video recording system. The time to first vocalize and first move, along with the duration of vocalization and escape attempts during the isolation test, during isolation test were analyzed on day 1, and week 4, 8, 12, 16 and 20. Blood samples were collected followed behavioral tests (n = 5/treatment). The expression of 5-HTR1A genes in the hypothalamus was measured by real-time PCR. Compared to controls, 5-HT administrated pullets had greater body weight (P < 0.05) with an improved feed conversion rate (P < 0.05) as well as higher serum concentrations of norepinephrine (NE) regardless of their doses. In addition, serum dopamine (DA) concentrations were lower in both high- and low-dose pullets at 8 and 12 weeks of age (P < 0.05). Also, a decrease in fearfulness response was observed based on the test to vocalize and duration of vocalization (P < 0.05). Further, this exhibited a lesser frequency of total aggressive behavior compared with the chicks in the control group, especially at 8 weeks of age (P < 0.05), where it is associated with elevated serum 5-HT concentration and upregulated hypothalamic expression of 5-HTR1A (P < 0.05). The changes of these hormone concentrations and gene expressions suggested that 5-HT accumulation in early embryonic stages may alter both the adrenergic and serotonergic systems, which could further regulate the isolation behavior and improve birds' growth performance to a certain extent.

White Isthmus Transcriptome Analysis Reveals the Mechanism of Translucent Eggshell Formation.

The presence of translucent eggshells is a type of egg quality issue that impacts egg sales. While many researchers have studied them, the exact mechanisms behind their formation remain unclear. In this study, we conducted a transcriptomic differential expression analysis of the isthmus region of the oviduct in both normal egg- and translucent egg-laying hens. The analysis revealed that differentially expressed gene pathways were predominantly concentrated in the synthesis, modification, and transport of eggshell membrane proteins, particularly collagen proteins, which provide structural support. These findings suggest that variations in the physical structure of the eggshell membrane, resulting from changes in its chemical composition, are the fundamental cause of translucent eggshell formation. This research provides a theoretical reference for reducing the occurrence of translucent eggs.

Positive effects and mechanism of mulberry leaf extract on alleviating fatty liver hemorrhagic syndrome in laying hens.

This experiment was conducted to investigate the effects of mulberry leaf extract (MLE) on alleviating fatty liver hemorrhagic syndrome (FLHS) in laying hens. The 576 Jing Fen laying hens of 56 weeks of age with good health and similar weights (1.76 ± 0.17 kg) were randomly divided into 6 groups, with 8 replicates in each group and 12 chickens in each replicate. The experiment lasted 56 d. The control group was fed a corn-soybean meal diet. The FLHS group was fed a high energy-low protein (HELP) diet, and the other four experimental groups were fed HELP diets supplemented with 0.04, 0.40, 0.80, and 1.20% MLE, respectively. The results showed that HELP treatment significantly induced liver injury, which indicated that the FLHS model was successfully established. MLE supplementation could alleviate the FLHS by reducing the liver index, abdominal fat percentage, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) in the serum (P < 0.05), and subsequently increase the egg production rate (P < 0.05). The laying hens fed 0.8% MLE exhibited the greatest production performance (P < 0.05) and could improve serum lipid levels. In addition, the genes associated with fatty acid synthesis (ACC, HMGR and SREBP-1C) were downregulated (P < 0.05), and genes related to fatty acid oxidation (CPT1A, AMPK, and ATGL) were found to be upregulated (P < 0.05). Supplementation with 1.2% MLE significantly reduced the relative abundance of Firmicutes and Desulfurized Bacillus (P < 0.05) and significantly increased the relative abundance of Fecal Bacillus (P < 0.05). In conclusion, MLE may regulate the mRNA expression of lipid metabolism-related genes through the AMPK signaling pathway and improve cecal microbiota balance and serum lipid levels to alleviate FLHS in laying hens and subsequently improve egg production performance.

Identification and biochemical characterization of a carboxylesterase gene associated with ß-cypermethrin resistance in Dermanyssus gallinae.

Dermanyssus gallinae is a major hematophagous ectoparasite in layer hens. Although the acaricide ß-cypermethrin has been used to control mites worldwide, D. gallinae has developed resistance to this compound. Carboxylesterases (CarEs) are important detoxification enzymes that confer resistance to ß-cypermethrin in arthropods. However, CarEs associated with ß-cypermethrin resistance in D. gallinae have not yet been functionally characterized. Here, we isolated a CarE gene (Deg-CarE) from D. gallinae and assayed its activity. The results revealed significantly higher expression of Deg-CarE in the ß-cypermethrin-resistant strain (RS) than in the susceptible strain (SS) toward α-naphthyl acetate (α-NA) and ß-naphthyl acetate (ß-NA). These findings suggest that enhanced esterase activities might have contributed to ß-cypermethrin resistance in D. gallinae. Quantitative real-time PCR analysis revealed that Deg-CarE expression levels were significantly higher in adults than in other life stages. Although Deg-CarE was upregulated in the RS, significant differences in gene copy numbers were not observed. Additionally, Deg-CarE expression was significantly induced by ß-cypermethrin in both the SS and RS. Moreover, silencing Deg-CarE via RNA interference decreased the enzyme activity and increased the susceptibility of the RS to ß-cypermethrin, confirming that Deg-CarE is crucial for ß-cypermethrin detoxification. Finally, recombinant Deg-CarE (rDeg-CarE) expressed in Escherichia coli displayed high enzymatic activity toward α/ß-NA. However, metabolic analysis indicated that rDeg-CarE did not directly metabolize ß-cypermethrin. The collective findings indicate that D. gallinae resistance to ß-cypermethrin is associated with elevated CarEs protein activity and increased Deg-CarE expression levels. These findings provide insights into the metabolic resistance of D. gallinae and offer scientific guidance for the management and control of D. gallinae.

Integrated transcriptomic and metabolomic analyses reveal potential regulatory pathways regulating bone metabolism pre- and postsexual maturity in hens.

At the onset of sexual maturity, the increasing circulating estrogen stimulates the formation of medullary bone, which provides available calcium for eggshell formation. The bone loss of laying hens is caused by the continuous dynamic changes of structure bone leading to bone fragility and susceptibility. The degree of medullary bone mineralization in sexual maturity is positively correlated with bone quality in the late laying stage. This study aimed to explore the molecular regulation mechanism of bone metabolism pre- and postsexual maturity in hens based on the joint analysis of transcriptome and metabolome. A total of 50 Hy-line Sonia pullets with comparable body weight at 13 wk were selected. Eight pullets were killed at 15 wk (juvenile hens, JH) and 19 wk (laying hens, LH), and LHs were killed within 3 h after oviposition. Differentially expressed genes and metabolites in tibia were analyzed based on transcriptome and metabolome, and then combined to construct the relevant metabolisms and hub genes. In the LH hens, plasma levels of estrogen and tartrate-resistant acid phosphatase were significantly elevated by 1.7 and 1.3 times. In addition, the midpoint diameter, bone mineral density and bone mineral content of the tibia and femur were higher at 19 wk of age. A total of 580 differentially expressed genes were found between the JH and LH group in the tibia, including 280 up-regulated, and 300 down-regulated genes in the LH group. Gene set enrichment analysis (GSEA) showed that the intracellular biosynthesis and secretion of matrix vesicles were significantly enrichment in the LH hens. A total of 21 differential metabolites were identified between JH and LH group. Estradiol valerate positively correlated with L-theanine, tryptophan betaine, dopamine, and perindopril. Joint analysis showed that the top 20 hub genes were enriched in cholesterol biosynthesis and phospholipid metabolism, which played a key regulatory role in bone metabolism during pre- and postsexual maturity. These results provide a theoretical foundation for maintaining efficient egg production and reducing bone health problems in laying hens.

Further screening of SNP loci of eggshell translucency related genes and evaluation of genetic effects.

Eggshell translucency is a widespread issue in the field of egg quality. Previous research has established that the heritability of eggshell translucency is relatively low or moderate. Scientists have also successfully identified SNP loci related to eggshell translucency on different chromosomes by using gene chips and single-variant GWAS. However, the specific impact of single or multiple genes on the trait of eggshell translucency remains unknown. In an effort to investigate this, we examined 170 SNPs associated with eggshell translucency obtained by our research group. We selected 966 half-sibling laying hens from 2 generations in 3 pure lines: Dwarf Layer-White, Rhode Island Red-White Strain, and Rhode Island Red. Eggs were collected from each hen over a period of 5 consecutive days, and eggshell translucency was measured using a grading method in which the hens were divided into 2 groups: an opaque group and a translucent group. We collected blood samples from the laying hens and extracted DNA. Time of flight mass spectrometry (TOF-MS) was used for genotyping to identify SNP loci that influence the trait of eggshell translucency. The results of our analysis revealed that using TOF-MS in 3 chicken strains, we were able to eliminate loci with low gene polymorphism, genetic effect contribution less than 1%, and deviation from Hardy-Weinberg equilibrium. Ultimately, 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were identified on chromosomes 1, 5, and 19. Additionally, nine candidate genes (DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A) were found to be associated with these SNPs. No linkage disequilibrium relationship was observed between the 2 pairs of SNP loci on chromosomes 1 and 19. Based on previous studies on the formation mechanism of eggshell translucency, we hypothesize that NUCB2, FTL, and ACACA genes may be affecting the eggshell structure through different mechanisms, such as increase the water permeability or make thin of eggshell membrane, which promote moisture or part of other egg contents and ultimately lead to the formation of eggshell translucency. These findings validate and identify five SNP loci that regulate the translucency trait, and provide molecular markers for breeding non-translucent populations. Furthermore, this study serves as a reference for further investigation of the genetic regulatory mechanisms underlying eggshell translucency.

Melatonin alleviates endoplasmic reticulum stress to improve ovarian function by regulating the mTOR pathway in aged laying hens.

Granular cell apoptosis is a key factor leading to follicular atresia and decreased laying rate in aged laying hens. Endoplasmic reticulum stress (ERS) induced cell apoptosis is a new type of apoptosis pathway. Previous studies have shown that the ERS pathway is involved in the regulation of follicular development and atresia, and can be regulated by mTOR. Melatonin (MEL) can protect the normal development of follicles, but the precise mechanism by which MEL regulates follicular development is not yet clear. So, we investigated the potential relationship between MEL and ERS and mTOR signaling pathway in vivo through intraperitoneal injection of MEL in aged laying hens. The results show that the laying rate, ovarian follicle number, plasma MEL, E2, LH, FSH concentrations, as well as the mRNA expression of mTOR signaling-associated genes TSC1, TSC2, mTOR, 4E-BP1, and S6K in old later-period chicken control (Old-CN) group was significantly decreased (P < 0.01). In contrast, the ERS-related of plasma and granular cell layer mRNA expression of Grp78, CHOP, and Caspase-3 was significantly increased (P < 0.01). While both of the effects were reversed by MEL. Then, aging granulosa cells were treated with MEL in vitro, followed by RNA seq analysis, and it was found that 259 and 322 genes were upregulated and downregulated. After performing GO enrichment analysis, it was found that DEGs significantly contribute to the biological processes including cell growth and apoptosis. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to mTOR signaling and endoplasmic reticulum (ER) stress, involving genes such as GRB10, SGK1, PRKCA, RPS6KA2, RAF1, PIK3R3, FOXO1, DERL3, HMOX1, TLR7, VAMP7 and INSIG2. The obtained results of RT-PCR showed consistency with the RNA-Seq data. In summary, the underlined results revealed that MEL has significantly contributed to follicular development via activating the mTOR signaling pathway-related genes and alleviating ERS-related genes in laying hens. The current study provides a theoretical background for enhancing the egg-laying capability of hens and also providing a basis for elucidating the molecular mechanism of follicular selection.

Comparative Analysis of the Ultrastructure, Bubble Pores, and Composition of Eggshells of Dwarf Layer-White and Guinea Fowl.

The decrease in eggshell quality seriously affects production efficiency. Guinea fowl (GF) eggs possess strong eggshells because of their unique crystal structure, and few systematic studies have compared laying hen and GF eggs. Sixty eggs were collected from both 40-week-old Dwarf Layer-White (DWL-White) laying hens and GF, and the eggshell quality, ultrastructure, bubble pores, and composition were measured. The results showed that the DWL-White eggs had a higher egg weight and a lower eggshell strength, strength per unit weight, thickness, and ratio than the GF eggs (p < 0.01). There were differences in the mammillary layer thickness ratio, the effective layer thickness ratio, the quantity of bubble pores (QBPs), the ratio of the sum of the area of bubble pores to the area of the eggshell in each image (ARBE), and the average area of bubble pores (AABPs) between the DWL-White and GF eggs (p < 0.01). The composition analysis demonstrated that there were differences in the organic matter, inorganic matter, calcium, and phosphorus between the DWL-White and GF eggs (p < 0.01). There were positive associations between the mammillary knob number in the image and the QBPs and ARBE and a negative correlation with the AABPs in the DWL-White eggs (p < 0.01). This study observed distinctions that offer new insights into enhancing eggshell quality.

Melatonin alleviates endoplasmic reticulum stress and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens.

Follicular atresia in chickens reduces the number of follicles that can further develop, leading to decrease egg laying. Endoplasmic reticulum stress (ERS) can initiate a unique pathway inducing the apoptosis of follicular granulosa cells, thus reducing egg laying. Melatonin (MEL) is involved in the regulation of follicle development, ovulation, and oocyte maturation, and is closely related to follicle fate. Mammalian target of Rapamycin (mTOR) signaling pathway plays an important role in cell growth regulation, and that there is a possible crosstalk between melatonin and mTOR activity in granular cells maturation and ovulation. This study aimed to investigate whether MEL inhibits ERS and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens. Frist, we established an in vitro ERS cell model using tunicamycin (TM). The results showed that different concentrations of TM exhibited dose-dependent inhibition of cell activity and induction of granulosa cells (P<0.01). Therefore, we chose 5 µg/mL of TM and a treatment time for 6 h as the optimal concentration for the following experiments. Then we investigate whether melatonin can inhibit ERS. TM treatment decreased the cell viability and Bcl-2 expression, increasing ROS levels and the mRNA expression of Grp78, ATF4, CHOP, PERK, eIF-2α, and BAX (P<0.01), whereas TM+MEL treatment significantly inhibited these changes (P<0.01). Then we explored whether melatonin protects follicular granulosa cells from ERS-induced apoptosis through the mammalian target of rapamycin (mTOR) signaling pathway by regulating ATF4, we found that ATF4 knockdown inhibited ERS by decreasing the expression of ERS-related genes and proteins and activating mTOR signaling pathway by increasing the protein expression of p4E-BP1 and pT389-S6K (P<0.001), while these changes were promoted by TM+si-ATF4+MEL treatment (P<0.01). These results indicate that MEL could alleviate TM-induced ERS by regulating ATF4 to activate mTOR signaling pathway in follicular granulosa cells, thus providing a new perspective for prolonging the laying cycle in chickens.

Effects of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, and uterine characteristics of hens during the late laying period.

This study aimed to systematically determined the effect of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, uterine morphological characteristics, and gene expression of hens during the late laying period. At 74 wk, 260 Hy-Line Brown layers were randomly divided into 2 groups of 130 birds each and in duplicates. Both a regular (16L:8D) and an ahemeral light cycle (16L:12D) were provided to the hens. The oviposition pattern in an ahemeral cycle shifted into darkness, with oviposition mostly occurring 3 to 5 h after light out. Production performance was unaffected by light cycle (P > 0.05). Nonetheless, compared to the normal group, the ahemeral group exhibited increased egg weight, eggshell weight, eggshell percentage, yolk percentage, eggshell thickness, and eggshell strength (P < 0.05). There were rhythmic changes in the uterine morphological structure in both cycles, however, the ahemeral group maintained a longer duration and had more uterine folds than the normal group. In the ahemeral cycle, the phases of the CLOCK and PER2 genes were phase-advanced for 3.96 h and 4.54 h compared to the normal cycle. The PHLPP1 gene, which controls clock resetting, exhibited a substantial oscillated rhythm in the ahemeral group (P < 0.05), while the expression of genes presenting biological rhythm, such as CRY2 and FBXL3, was rhythmically oscillated in normal cycle (P < 0.05). The ITPR2 gene, which regulates intracellular Ca2+ transport, displayed a significant oscillated rhythm in ahemeral alone (P < 0.05), while the CA2 gene, which presents biomineralization, rhythmically oscillated in both cycles (P < 0.05). The ahemeral cycle caused 2.5 h phase delays in the CA2 gene compared to the normal cycle. In conclusion, the 28 h ahemeral light cycle preserved the high condition of the uterine folds and changed the uterine rhythms of CLOCK, PER2, ITPR2, and CA2 gene expression to improve ion transport and uterine biomineralization.

[Erythropoietin levels in patients with multiple sclerosis complicated with anemia].

OBJECTIVE: To explore the potential decrease of serum erythropoietin (EPO) level in patients with multiple sclerosis (MS) complicated with anemia. METHODS: The serum EPO levels were detected in the patients with MS complicated with anemia (MS group, n=31), patients with iron deficiency anemia (IDA group, n=33), and healthy subjects (normal control group, n=80) by enzyme-linked immunosorbent assay (ELISA). Blood routine test, reticulocyte count, hemoglobin, and indexes of liver and kidney function were also detected. RESULTS: The serum EPO level in MS group was significantly lower than those in IDA group [(101.3±17.6)U/L vs.(166.1±8.7)U/L, P<0.01]. Moreover, the serum EPO level decreased as the severity of anemia in the MS group increased: it was (95.7±9.6), (101.7±8.1), and (123.7±9.3) U/L in patients with mild, moderate, and severe anemia, respectively (P<0.05). Other indicators including blood routine findings, reticulocyte count, hemoglobin, and liver and kidney function parameters showed no significant difference between the MS group and the IDA group (P>0.05). CONCLUSIONS: The serum EPO level decreases in patients with multiple sclerosis complicated with anemia, and the decreasing levels are related with the severity of anemia. Thus EPO therapy may be beneficial for these patients.

Direct Nucleophilic Attack/Addition Cyclization and C-H Bond Activation Reactions to Synthesize 3-Benzyl-/3-Benzyl-2-phenyl-benzo[4,5]imidazo[2,1-b]thiazoles.

The economic and practical strategies of direct nucleophilic attack/addition cyclization and C-H bond activation reactions to synthesize 3-benzyl-/3-benzyl-2-phenyl-benzo[4,5]imidazo[2,1-b]thiazoles via (Z)-(2,3-dibromoprop-1-en-1-yl)benzene/(3-bromoprop-1-yn-1-yl)benzene, 1H-benzo[d]imidazole-2-thiols and halobenzenes have been developed. With the optimized reaction conditions, the nucleophilic attack/addition cyclization reaction (Cs2CO3, MeCN, 90 °C, 24 h) and C-H bond activation reaction [Pd(OAc)2/PPh3, p-xylene, 135 °C, 24 h] could tolerate various electron-donating and electron-withdrawing groups and afford new benzo[4,5]imidazo[2,1-b]thiazoles in good to excellent yields (up to 93% yield).

Transcriptomic analysis reveals the molecular mechanisms underlying osteoclast differentiation in the estrogen-deficient pullets.

Several previous reports have suggested that estrogen (E2) is a vital signal responsible for the regulation of skeletal homeostasis and bone remodeling in mammals. E2 could efficiently accelerate the growth of medullary bone in pullets during sexual maturity. Furthermore, the low E2 level can strengthen the mechanical bone functions in female hens. However, mechanistic studies to describe the effects of E2 on bone in pullets during the initiation of the puberty period are remaining elusive. Therefore, the aim of this study was to explore the effect of inhibiting E2 biosynthesis on the biomechanical properties and its molecular mechanism during sexual maturity of pullets. In this study, a total of 90 Hy-line Sonia pullets with comparable body weight at 13 wk of age were selected and categorized into 2 separate groups. Daily, 0.5 mg/4 mL of letrozole (LZ) was orally administered to the treatment (TRT) group and 4 mL of saline to the control (CON) group of pullets for 6 wk. Compared with the CON group, a lower plasma E2 level was observed in the TRT group. Furthermore, plasma P, Gla protein (BGP), and 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) levels were markedly suppressed, whereas the plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) levels were significantly elevated. Moreover, the cortical bone thickness and breaking strength of the tibia and femur, the bone mineral density of the humerus, and the bone mineral content of the humerus as well as the femur were increased significantly. The expression levels of 340 differentially expressed genes (DEGs) differed significantly between the CON and TRT group in the tibia at 19 wk of age. Among them, 32 genes were up-regulated, whereas 308 were down-regulated in the TRT group. The variations in candidate genes associated with osteoclast differentiation and cell adhesion may indicate that LZ inhibits E2 biosynthesis, consequently, reduces osteoclast differentiation by suppressing inter-cellular communication and cells attaching to extracellular matrix components. Taken together, the present study demonstrated that inhibiting E2 synthesis during sexual maturity of pullets decreased osteoclast differentiation and considerably enhanced bone quality.

Corrigendum: Advances in eggshell membrane separation and solubilization technologies.

[This corrects the article DOI: 10.3389/fvets.2023.1116126.].

Advances in eggshell membrane separation and solubilization technologies.

Eggshell membranes (ESM) contain 90% protein, 3% lipids, 2% sugars, and small amounts of minerals such as calcium and magnesium. Of the 90% of proteins present, 472 proteins species have been identified. ESM provide the initial mineralization platform for eggshell formation, and can be used for to produce adsorbents, cosmetics, and medical products because of their special physical structure and chemical composition. The special physical structure of the eggshell membrane, with disulfide bonds between and within the protein molecules and the cross-linking of lysine-derived and heterochain chains between the eggshell membrane, makes the membrane very difficult to dissolve, with a maximum solubility rate of only 62%. Also, the insolubility of ESM limits its development and use also any related research. Based on the physical structure and chemical composition of the eggshell membrane, this paper reviews the latest research on eggshell membrane separation and membrane protein solubilization to provide a reference for promoting the separation, dissolution, and rational development and use of the avian eggshell membrane.

Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway.

Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3 (PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine (8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1 (FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.