Search

Background: The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa., Results: In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses., Conclusion: This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance., (© 2024. The Author(s).)

The development of wool has a complex regulatory mechanism both influenced by genetic and environmental factors. MicroRNAs (miRNA) were involved in various biological processes of animals, and may play an important role in the regulation of wool development. In this study, we comprehensively analyzed and identified the histological parameters of hair follicles, as well as the miRNAs, target genes, pathways, and Gene Ontology terms related to wool fineness regulation and wool growth and development using HE staining and RNA-Seqs methods. Both coarse (group C, mean fiber diameter (MFD) = 22.26 ± 0.69 μm, n = 6) and fine (group F, MFD = 16.91 ± 0.29 μm, n = 6) of Gansu alpine fine-wool sheep with different wool fineness were used in this study. The results showed that the primary follicle diameter and secondary wool fiber diameter in group C were significantly higher than those in group F (P < 0.05). And the number of primary and secondary hair follicles in group C was significantly lower than that in group F (P < 0.05). Furthermore, a total of 67 DE miRNAs and 290 potential DE miRNAs target genes were screened in the skin tissues of sheep from groups F and C, and some potential target genes related to wool fineness regulation were screened, such as CDH2, KRT82, FOXN1, LOC101106296, KRT20, MCOLN3, KRT71, and TERT. These genes were closely related to Glutathione metabolism, epidermal cell differentiation, keratinization, and regulation of hair cycle. Moreover, the regulatory network of miRNAs-mRNAs suggested that miRNAs (miR-129-x, novel m0079-3p, miR-2484-z, novel m0025-5P, etc.) may play a key role in the wool development and wool fineness regulation of Gansu alpine fine-wool sheep. In summary, this study expands the existing miRNAs database and provides new information for studying the regulation of wool development in Gansu alpine fine wool sheep., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue ( p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation ( p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% ( p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% ( p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 ( DNMT1 ) and peroxisome proliferator-activated receptor gamma coactivator 1-A ( PPARGC1A ) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = -0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = -0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep.

Soil salinity constrains growth, development and yield of alfalfa (Medicago sativa L.). To illustrate the molecular mechanisms responsible for salt tolerance, a comparative proteome analysis was explored to characterize protein profiles of alfalfa seedling roots exposed to 100 and 200 mM NaCl for three weeks. There were 52 differentially expressed proteins identified, among which the mRNA expressions of 12 were verified by Real-Time-PCR analysis. The results showed increase in abundance of ascorbate peroxidase, POD, CBS protein and PR-10 in salt-stressed alfalfa, suggesting an effectively antioxidant and defense systems. Alfalfa enhanced protein quality control system to refold or degrade abnormal proteins induced by salt stress through upregulation of unfolded protein response (UPR) marker PDIs and molecular chaperones (eg. HSP70, TCP-1, and GroES) as well as the ubiquitin-proteasome system (UPS) including ubiquitin ligase enzyme (E3) and proteasome subunits. Upregulation of proteins responsible for calcium signal transduction including calmodulin and annexin helped alfalfa adapt to salt stress. Specifically, annexin (MsANN2), a key Ca 2+ -binding protein, was selected for further characterization. The heterologous of the MsANN2 in Arabidopsis conferred salt tolerance. These results provide detailed information for salt-responsive root proteins and highlight the importance of MsANN2 in adapting to salt stress in alfalfa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Saline-alkali stress is one of the main abiotic stresses that limits plant growth. Salt stress has been widely studied, but alkaline salt degradation caused by NaHCO 3 has rarely been investigated. In the present study, the alfalfa cultivar 'Zhongmu No. 1' was treated with 50 mM NaHCO 3 (0, 4, 8, 12 and 24 h) to study the resulting enzyme activity and changes in mRNA, miRNA and metabolites in the roots. The results showed that the enzyme activity changed significantly after alkali stress treatment. The genomic analysis revealed 14,970 differentially expressed mRNAs (DEMs), 53 differentially expressed miRNAs (DEMis), and 463 differentially accumulated metabolites (DAMs). Combined analysis of DEMs and DEMis revealed that 21 DEMis negatively regulated 42 DEMs. In addition, when combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEMs and DAMs, we found that phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism and plant hormone signal transduction played important roles in the alkali stress response. The results of this study further elucidated the regulatory mechanism underlying the plant response to alkali stress and provided valuable information for the breeding of new saline-alkaline tolerance plant varieties., Competing Interests: Declaration of competing interest All the authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Masson SAS.)

Background: Plant height (PH) is an important agronomic trait influenced by a complex genetic network. However, the genetic basis for the variation in PH in Medicago sativa remains largely unknown. In this study, a comprehensive genome-wide association analysis was performed to identify genomic regions associated with PH using a diverse panel of 220 accessions of M. sativa worldwide., Results: Our study identified eight novel single nucleotide polymorphisms (SNPs) significantly associated with PH evaluated in five environments, explaining 8.59-12.27% of the phenotypic variance. Among these SNPs, the favorable genotype of chr6&#95;&#95;31716285 had a low frequency of 16.4%. Msa0882400, located proximal to this SNP, was annotated as phosphate transporter 3;1, and its role in regulating alfalfa PH was supported by transcriptome and candidate gene association analysis. In addition, 21 candidate genes were annotated within the associated regions that are involved in various biological processes related to plant growth and development., Conclusions: Our findings provide new molecular markers for marker-assisted selection in M. sativa breeding programs. Furthermore, this study enhances our understanding of the underlying genetic and molecular mechanisms governing PH variations in M. sativa., (© 2024. The Author(s).)

In our previous study, circ&#95;015343 was found to inhibit the viability and proliferation of ovine mammary epithelial cells (OMECs) and the expression levels of milk fat synthesis marker genes, but the regulatory mechanism underlying the processes is still unclear. Accordingly in this study, the target relationships between circ&#95;015343 with miR-25 and between miR-25 with insulin induced gene 1 (INSIG1) were verified, and the functions of miR-25 and INSIG1 were investigated in OMECs. The dual-luciferase reporter assay revealed that miR-25 mimic remarkably decreased the luciferase activity of circ&#95;015343 in HEK293T cells cotransfected with a wild-type vector, while it did not change the activity of circ&#95;015343 in HEK293T cells cotransfected with a mutant vector. These suggest that cic&#95;015343 can adsorb and bind miR-25. The miR-25 increased the viability and proliferation of OMECs, and the content of triglycerides in OMECs. In addition, INSIG1 was found to be a target gene of miR-25 using a dual-luciferase reporter assay. Overexpression of INSIG1 decreased the viability, proliferation, and level of triglycerides of OMECs. In contrast, the inhibition of INSIG1 in expression had the opposite effect on activities and triglycerides of OMECs with overexpressed INSIG1. A rescue experiment revealed that circ&#95;015343 alleviated the inhibitory effect of miR-25 on the mRNA and protein abundance of INSIG1. These results indicate that circ&#95;015343 sponges miR-25 to inhibit the activities and content of triglycerides of OMECs by upregulating the expression of INSIG1 in OMECs. This study provided new insights for understanding the genetic molecular mechanism of lactation traits in sheep., (© 2024 Wiley Periodicals LLC.)

Given the escalating impact of climate change on agriculture and food security, gaining insights into the evolutionary dynamics of climatic adaptation and uncovering climate-adapted variation can empower the breeding of climate-resilient crops to face future climate change. Alfalfa (Medicago sativa subsp. sativa), the queen of forages, shows remarkable adaptability across diverse global environments, making it an excellent model for investigating species responses to climate change. In this study, we performed population genomic analyses using genome resequencing data from 702 accessions of 24 Medicago species to unravel alfalfa's climatic adaptation and genetic susceptibility to future climate change. We found that interspecific genetic exchange has contributed to the gene pool of alfalfa, particularly enriching defense and stress-response genes. Intersubspecific introgression between M. sativa subsp. falcata (subsp. falcata) and alfalfa not only aids alfalfa's climatic adaptation but also introduces genetic burden. A total of 1671 genes were associated with climatic adaptation, and 5.7% of them were introgressions from subsp. falcata. By integrating climate-associated variants and climate data, we identified populations that are vulnerable to future climate change, particularly in higher latitudes of the Northern Hemisphere. These findings serve as a clarion call for targeted conservation initiatives and breeding efforts. We also identified pre-adaptive populations that demonstrate heightened resilience to climate fluctuations, illuminating a pathway for future breeding strategies. Collectively, this study enhances our understanding about the local adaptation mechanisms of alfalfa and facilitates the breeding of climate-resilient alfalfa cultivars, contributing to effective agricultural strategies for facing future climate change., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

In the context of the continued advancement of the green economy transition, the proactive pursuit of carbon emissions reduction and the early attainment of carbon neutrality goals have emerged as essential components in promoting high-quality economic development. Not only does it contribute to the creation of a community of human destiny, but it is also vital to the realization of sustainable development for human civilization. A dynamic evolutionary game model, which encompasses the interactions among government, enterprises, and the public, was constructed to examine the inherent impact mechanisms of the behavior of three players on the development of a green economy under the context of energy saving and emission reduction subsidies. The results showed that the incentive and punishment mechanisms served as effective tools for harmonizing the interests of system members. Within the mechanisms, the public demonstrated a higher sensitivity to rewards, while enterprises exhibited greater responsiveness to fines. Consequently, the government could influence the behavior of enterprises by incentivizing the public to serve as a third-party inquiry and oversight body. Simultaneously, the government could encourage enterprises to expedite green technology innovation by employing a combination of incentive and punishment mechanisms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa ( Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf-stem ratio (LSR) and repression of several lignin-synthesis genes ( MsNST , MsPAL1 , MsC4H , and Ms4CL ). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa ( Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members ( MsEXPs ). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis -acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Ethnopharmacological Relevance: Fructus Choerospondiatis is the dried and mature fruit of Choerospondias axillaris (Roxb.) Burtt et Hill. It has been used for a long time in Tibetan and Mongolian medicine, first recorded in the ancient Tibetan medicine book "Medicine Diagnosis of the King of the Moon" in the early 8th century. Fructus Choerospondiatis shows multiple pharmacological activities, especially in treating cardiovascular diseases., Aim of This Review: This paper reviews the progress in research on the botanical characteristics, traditional uses, chemical constituents, pharmacological activity, clinical studies, and quality control of Fructus Choerospondiatis. This review aims to summarize current research and provide a reference for further development and utilization of Fructus Choerospondiatis resources., Method: The sources for this review include the Pharmacopeia of the People's Republic of China (2020), theses, and peer-reviewed papers (in both English and Chinese). Theses and papers were downloaded from electronic databases including Web of Science, PubMed, SciFinder, Scholar, Springer, and China National Knowledge Infrastructure.The search terms used were "Choerospondias axillaris", "C. axillaris", "Choerospondias axillaris (Roxb.) Burtt et Hill", "Fructus choerospondiatis", "Guangzao", "Lapsi", and "Lupsi"., Results: Fructus Choerospondiatis contains polyphenols, organic acids, amino acids, fatty acids, polysaccharides, and other chemical components. These ingredients contribute to its diverse pharmacological activities such as antioxidant activity, protection against myocardial ischemia-reperfusion injury, anti-myocardial fibrosis, heart rhythm regulation, anti-tumor, liver protection, and immunity enhancement. It also affects the central nervous system, with the ability to repair damaged nerve cells., Conclusion: Fructus Choerospondiatis, with its various chemical compositions and pharmacological activities, is a promising medicinal resource. However, it remains under-researched, particularly in pharmacodynamic material basis and quality control. These areas require further exploration by researchers in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2 , 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1- JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 ( CSN1S2 ), casein beta ( CSN2 ), and casein kappa ( CSN3 ) but down-regulated casein alpha s1 ( CSN1S1 ) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

High salinity is one of the detrimental environmental factors restricting plant growth and crop production throughout the world. This study demonstrated that the GARP family transcription factor MtHHO3 is involved in response to salt stress and abscisic acid (ABA) signaling in Medicago truncatula. The transcription of MtHHO3 was repressed by salt, osmotic stress, and ABA treatment. The seed germination assay showed that, overexpression of MtHHO3 in Arabidopsis thaliana caused hypersensitivity to salt and osmotic stress, but increased resistance to ABA inhibition. Overexpression of MtHHO3 in M. truncatula resulted in decreased tolerance of salinity, while loss-of-function mutants mthho3-1 and mthho3-2 were more resistant to salt stress compared with wild-type plants. qRT-PCR analyses showed that MtHHO3 downregulated the expression of genes in stress and ABA responsive pathways. We further demonstrated that MtHHO3 repressed the transcription of the pathogenesis-related gene MtPR2 by binding to its promoter. Overall, these results indicate that MtHHO3 negatively regulates salt stress response in plants and deepen our understanding of the role of the GARP subfamily transcription factors in modulating salt stress and ABA signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Changes in keratin gene expression and spatiotemporal regulation determine the compositional content and cellular localization of wool keratin, thereby affecting wool traits. Therefore, keratin gene family member 32 ( KRT32 ) was selected for a study using RT-qPCR, immunofluorescence, and penta-primer amplification refractory mutation system (PARMS) techniques. The results showed that KRT32 mRNA was highly expressed in the skin and localized to the inner root sheath (IRS), outer root sheath (ORS) and dermal papilla (DP). Sequencing results identified eight SNPs in KRT32 , and association analyses revealed that the variations were significantly associated with multiple traits in wool ( p < 0.05), including MFD, CF and MFC. The constructed haplotype combination H 2 H 3 has higher CF and smaller MFD than other haplotype combination ( p < 0.05). In conclusion, KRT32 can be used as a candidate gene for molecular genetic improvement of wool in Gansu Alpine Fine-wool sheep.

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-β) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation., (© 2024. The Author(s).)

The prevalence of substantial inflammation or fibrosis in treatment-naïve patients with chronic hepatitis B (CHB) and normal alanine transaminase (ALT) levels is high. A retrospective analysis was conducted on 559 consecutive patients with hepatitis B virus infection, who underwent liver biopsy, to investigate the value of noninvasive models based on routine serum markers for evaluating liver histology in CHB patients with normal or mildly elevated ALT levels and to provide treatment guidance. After comparing 55 models, we identified the top three models that exhibited excellent performance. The APGA model, based on the area under the receiver operating characteristic curve (AUROC), demonstrated a superior ability to evaluate significant (AUROC = 0.750) and advanced fibrosis (AUROC = 0.832) and demonstrated a good performance in assessing liver inflammation (AUROCs = 0.779 and 0.874 for stages G ≥ 2 and G ≥ 3, respectively). APGA also exhibited significant correlations with liver inflammation and fibrosis stage (correlation coefficients, 0.452 and 0.405, respectively ( p < 0.001)). When the patients were stratified into groups based on HBeAg status and ALT level, APGA consistently outperformed the other 54 models. The other top two models, GAPI and XIE, also outperformed models based on other chronic hepatitis diseases. APGA may be the most suitable option for detecting liver fibrosis and inflammation in Chinese patients with CHB.

Background: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid., Results: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress., Conclusions: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago., (© 2024. The Author(s).)

Keratin (K) is a major protein component of hair and is involved in hair growth and development. In this study, we analysed the expression, localization, and polymorphism of the K84 gene ( KRT84 ) in Gansu Alpine Fine-wool sheep using immunofluorescence, RT-qPCR, and PARMS (penta-primer amplification refractory mutation system). Haplotypes of KRT84 were also constructed and their relationship with wool traits analysed. It was revealed that KRT84 was highly expressed in hair follicles, including the inner root sheath, outer root sheath, and hair medulla and at all six lamb ages investigated from 1 to 270 days of age. Three SNPs were detected in KRT84 exon 1, and they formed three haplotypes (named H1 , H2, and H3 ) and six genotypes. Analyses revealed an association between haplotype combinations (diplotypes) and the mean fibre curvature, mean staple length, mean staple strength, mean fibre diameter, the coefficient of variation of fibre diameter, and comfort factor for these sheep. These results suggest that KRT84 is of importance in determining several key traits in Gansu Alpine Fine-wool sheep and that the gene could possibly be used as a genetic marker for wool trait selection in these sheep.

Homobox C13 (Hoxc13) is an important transcription factor in hair follicle cycle development, and its deletion had been found in a variety of animals leading to abnormal hair growth and disruption of the hair follicle system. In this study, we used immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR (RT-qPCR), and Kompetitive Allele-Specific PCR (KASP) genotyping to investigate molecular genetic characteristics of the Hoxc13 gene in Gansu alpine fine-wool sheep. The results revealed that Hoxc13 was significantly expressed during both the anagen and catagen phases ( p < 0.05). It was found to be highly expressed predominantly in the dermal papillae and the inner and outer root sheaths, showing a distinct spatiotemporal expression pattern. Two single nucleotide polymorphisms (SNPs) in the exon 1 of Hoxc13, both the individual locus genotypes and the combined haplotypes were found to be correlated with wool length ( p < 0.05). It was determined the mutations led to changes in mRNA expression, in which higher expression of this gene was related with longer wool length. In summary, this unique spatiotemporal expression pattern of the Hoxc13 gene may regulate the wool length of Gansu alpine fine-wool sheep, which can be used as a molecular genetic marker for wool traits and thus improve the breed.

Transforming growth factor (TGF)-β1 is an important multifunctional cytokine in the TGF-β signaling pathway, which is involved in the molecular regulation of multiple activities, including follicle development and ovulation in female reproductive physiology. However, the biological function of TGF-β1 in follicular development and in regulating the proliferation or apoptosis of granulosa cells in small tail Han sheep remain unclear. In this study, we analyzed the expression levels of TGF-β1 in the ovary at the follicular stage in small tail Han sheep. We further examined the effects of TGF-β1 on the viability, proliferation, and apoptosis of granulosa cells. Differential expression of TGF-β1 at the mRNA and protein levels was detected in the ovaries between the beginning of estrus and at preovulation. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU) labeling, and flow cytometry assays showed that adding 5 and 10 ng/mL TGF-β1 could improve the viability and growth rate, reduce the apoptosis rate, and reduce the expression level of the pro-apoptotic factor Bcl-2-modifying factor (BMF) in granulosa cells. Treatment of 10 ng/mL TGF-β1 at all time points (except 72 h) significantly increased the positive rate of EdU labeling compared to that of the control group. RNA interference of SMAD4 reversed the decreased apoptosis rate caused by stimulation with 10 ng/mL TGF-β1, accompanied by a corresponding increase in the BMF expression level. Collectively, these results indicate that TGF-β1 plays a role in the ovarian follicular-phase activity of small tail Han sheep by inhibiting the apoptosis of sheep granulosa cells through the SMAD4/BMF pathway to promote proliferation and vitality. This study provides new insight into the molecular mechanism underlying TGF-β1 function regulation in granulosa cells, suggests a new target for the regulation of follicle development, and expands the new field of animal reproduction regulation technology., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Keratin-related proteins (KAPs) are structural components of wool fibers and are thought to play a key role in regulating the physical and mechanical properties of fibers. Among all KAP genes ( KRTAPs ), KRTAP6 gene family ( KRTAP6-1 , KRTAP6-2 , KRTAP6-3 , KRTAP6-4 , and KRTAP6-5 ) is a very important member with high polymorphism and notable association with some wool traits. In this study, we used real-time fluorescence quantitative PCR (RT-qPCR) and in situ hybridization to investigate spatiotemporal expression of KRTAP6s . The results revealed that KRTAP6 family genes were significantly expressed during anagen compared to other stages ( p < 0.05). And it was found the five genes were expressed predominantly in the dermal papillae, inner and outer root sheaths, and showed a distinct spatiotemporal expression pattern. Also, it was found that KRTAP6-1 and KRTAP6-5 mRNA expression was negatively correlated with wool mean fiber diameter (MFD) and mean staple strength (MSS) ( p < 0.05). In summary, the KRTAP6 family genes share a similar spatiotemporal expression pattern. And KRTAP6-1 and KRTAP6-5 may regulate the MFD and MSS of Gansu Alpine fine-wool sheep wool by changing the expression.

Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in preadipocyte differentiation and adipogenesis. However, little is known about genome-wide identification, expression profile, and function of circRNAs in sheep. To investigate the role of circRNAs during ovine adipogenic differentiation, the subcutaneous adipose tissue of Tibetan rams was collected in June 2022. Subsequently, the preadipocytes were immediately isolated from collected adipose tissue and then induced to begin differentiation. The adipocytes samples cultured on days 0, 2, and 8 of preadipocytes differentiation were used to perform RNA sequencing (RNA-seq) analysis to construct the expression profiles of circRNAs. Subsequently, the function of differentially expressed circRNAs was investigated by performing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of their parent genes. Finally, a circRNAs-miRNAs-mRNAs network involved in adipogenic differentiation was been analyzed. As a result, a total of 6,449 candidate circRNAs were identified in ovine preadipocytes. Of these circRNAs identified, 63 candidate circRNAs were differentially expressed among the three differentiation stages and their parent genes were mainly enriched in acetyl-CoA metabolic process, positive regulation of lipid biosynthetic process, positive regulation of steroid biosynthetic process, and focal adhesion pathway (P < 0.05). Based on a circRNAs-miRNAs-mRNAs regulatory network constructed, circ&#95;004977, circ&#95;006132 and circ&#95;003788 were found to function as competing endogenous RNAs (ceRNAs) to regulate ovine preadipocyte differentiation and lipid metabolism. The results provide an improved understanding of functions and molecular mechanisms of circRNAs underlying ovine adipogenesis in sheep., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plant filamentation temperature-sensitive H (FtsH) proteins are ATP-dependent zinc proteases that play an important role in regulating abiotic stress adaptions. Here we explore their potential role in abiotic stress tolerance in alfalfa, an important legume crop. Genomic analysis revealed seventeen MsFtsH genes in five clusters, which generally featured conserved domains and gene structures. Furthermore, the expression of MsFtsHs was found to be tightly associated with abiotic stresses, including osmotic, salt and oxidative stress. In addition, numerous stress responsive cis-elements, including those related to ABA, auxin, and salicylic acid, were identified in their promoter regions. Moreover, MsFtsH8 overexpression was shown to confer tolerance to salt and oxidative stress which was associated with reduced levels of reactive oxygen species, and enhanced expression and activity of antioxidant enzymes. Our results highlight MsFtsHs as key factors in abiotic stress tolerance, and show their potential usefulness for breeding alfalfa and other crops with improved yield and stress tolerance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Tibetan sheep has an intricate mechanism of adaptation to low oxygen levels, which is influenced by both genetic and environmental factors. The heart plays a crucial role in the adaptation of Tibetan sheep to hypoxia. In the present study, we utilized transcriptomic and proteomic technologies to comprehensively analyze and identify the long non-coding RNAs (lncRNAs), genes, proteins, pathways, and gene ontology (GO) terms associated with hypoxic adaptation in Tibetan sheep at three different altitudes (2500 m, 3500 m, and 4500 m). By integrating the differentially expressed (DE) lncRNA target genes, differentially expressed proteins (DEPs), and differentially expressed genes (DEGs), we were able to identify and characterize the mechanisms underlying hypoxic adaptation in Tibetan sheep. Through this integration, we identified 41 shared genes/proteins, and functional enrichment analyses revealed their close association with lipid metabolism, glycolysis/gluconeogenesis, and angiogenesis. Additionally, significant enrichment was observed in important pathways such as the PPAR signaling pathway, glycolysis/gluconeogenesis, the oxoacid metabolic process, and angiogenesis. Furthermore, the co-expression network of lncRNAs and mRNAs demonstrated that lncRNAs (MSTRG.4748.1, ENSOART00020025894, and ENSOART00020036371) may play a pivotal role in the adaptation of Tibetan sheep to the hypoxic conditions of the plateau. In conclusion, this study expands the existing database of lncRNAs and proteins in Tibetan sheep, and these findings may serve as a reference for the prevention of altitude sickness in humans.

Background: Filamentation temperature-sensitive H (FtsH) is an AAA + ATP-dependent protease that plays a vital role in plant environmental adaption and tolerance. However, little is known about the function of the FtsH gene family in the most important legume model plant, Medicago truncatula., Methods and Results: To identify and investigate the potential stress adaptation roles of FtsH gene family in M. truncatula, we conducted a series of genome-wide characterization and expression analyses. Totally, twenty MtFtsH genes were identified, which were unevenly distributed across eight chromosomes and classified into six evolution groups based on their phylogenetic relationships, with each group containing similar structures and motifs. Furthermore, MtFtsH genes exhibited a high degree of collinearity and homology with leguminous plants such as alfalfa and soybean. Multiple cis-elements in the upstream region of MtFtsH genes were also identified that responded to light, abiotic stress, and phytohormones. Public RNA-seq data indicated that MtFtsH genes were induced under both salt and drought stresses, and our transcript expression analysis showed that MtFtsH genes of MtFtsH1, MtFtsH2, MtFtsH4, MtFtsH9, and MtFtsH10 were up-regulated after ABA, H 2 O 2 , PEG, and NaCl treatments. These results suggest that MtFtsH genes may play a critical role in drought and high salt stress responses and the adaption processes of plants., Conclusions: This study provides a systematic analysis of FtsH gene family in M. truncatula, serving as a valuable molecular theoretical basis for future functional investigations. Our findings also extend the pool of potential candidate genes for the genetic improvement of abiotic stress tolerance in legume crops., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

RNA N6-melthyladenosine (m6A) can play an important role in regulation of various biological processes. Chicken ovary development is closely related to egg laying performance, which is a process primarily controlled by complex gene regulations. In this study, transcriptome-wide m6A methylation of the Wuhua yellow-feathered chicken ovaries before and after sexual maturation was profiled to identify the potential molecular mechanisms underlying chicken ovary development. The results indicated that m6A levels of mRNAs were altered dramatically during sexual maturity. A total of 1,476 differential m6A peaks were found between these two stages with 662 significantly upregulated methylation peaks and 814 downregulated methylation peaks after sexual maturation. A positive correlation was observed between the m6A peaks and gene expression levels, indicating that m6A may play an important role in regulation of chicken ovary development. Functional enrichment analysis indicated that apoptosis related pathways could be the key molecular regulatory pathway underlying the poor reproductive performance of Wuhua yellow-feathered chicken. Overall, the various pathways and corresponding candidate genes identified here could be useful to facilitate molecular design breeding for improving egg production performance in Chinese local chicken breed, and it might also contribute to the genetic resource protection of valuable avian species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jia, Zhang, Liu, Xu, Xiong, Huang, Li and Li.)

Tibetan sheep are already well adapted to cold season nutrient stress on the Tibetan Plateau. Rumen, an important nutrient for metabolism and as an absorption organ in ruminants, plays a vital role in the cold stress adaptations of Tibetan sheep. Ruminal microbiota also plays an indispensable role in rumen function. In this study, combined multiomics data were utilized to comprehensively analyze the interaction mechanism between rumen epithelial miRNAs and microbiota and their metabolites in Tibetan sheep under nutrient stress in the cold season. A total of 949 miRNAs were identified in the rumen epithelium of both cold and warm seasons. A total of 62 differentially expressed (DE) miRNAs were screened using FC > 1.5 and p value < 0.01, and a total of 20,206 targeted genes were predicted by DE miRNAs. KEGG enrichment analysis revealed that DE miRNA-targeted genes were mainly enriched in axon guidance(ko04360), tight junction(ko04530), inflammatory mediator regulation of TRP channels(ko04750) and metabolism-related pathways. Correlation analysis revealed that rumen microbiota, rumen VFAs and DE miRNAs were all correlated. Further study revealed that the targeted genes of cold and warm season rumen epithelial DE miRNAs were coenriched with differential metabolites of microbiota in glycerophospholipid metabolism (ko00564), apoptosis (ko04210), inflammatory mediator regulation of TRP channels (ko04750), small cell lung cancer (ko05222), and choline metabolism in cancer (ko05231) pathways. There are several interactions between Tibetan sheep rumen epithelial miRNAs, rumen microbiota, and microbial metabolites, mainly through maintaining rumen epithelial barrier function and host homeostasis of choline and cholesterol, improving host immunity, and promoting energy metabolism pathways, thus enabling Tibetan sheep to effectively respond to cold season nutrient stress. The results also suggest that rumen microbiota have coevolved with their hosts to improve the adaptive capacity of Tibetan sheep to cold season nutrient stress, providing a new perspective for the study of cold season nutritional stress adaptation in Tibetan sheep.

The prediction of prognostic outcome is critical for the development of efficient cancer therapeutics and potential personalized medicine. However, due to the heterogeneity and diversity of multimodal data of cancer, data integration and feature selection remain a challenge for prognostic outcome prediction. We proposed a deep learning method with generative adversarial network based on sequential channel-spatial attention modules (CSAM-GAN), a multimodal data integration and feature selection approach, for accomplishing prognostic stratification tasks in cancer. Sequential channel-spatial attention modules equipped with an encoder-decoder are applied for the input features of multimodal data to accurately refine selected features. A discriminator network was proposed to make the generator and discriminator learning in an adversarial way to accurately describe the complex heterogeneous information of multiple modal data. We conducted extensive experiments with various feature selection and classification methods and confirmed that the CSAM-GAN via the multilayer deep neural network (DNN) classifier outperformed these baseline methods on two different multimodal data sets with miRNA expression, mRNA expression and histopathological image data: lower-grade glioma and kidney renal clear cell carcinoma. The CSAM-GAN via the multilayer DNN classifier bridges the gap between heterogenous multimodal data and prognostic outcome prediction., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes ( MsHSF s) in the tetraploid alfalfa genome ("Xinjiangdaye" reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSF s and tissue-wide expression for 28 MsHSF s. Based on transcriptomic data analysis, 21, 11 and 27 MsHSF s responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT-PCR, MsHSF27 / 33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSF s and for genetic improvement of stress resistance in alfalfa.

Obesity has been linked to a higher risk of pancreatic cancer. However, the mechanism by which obesity promote pancreatic carcinogenesis is still unclear. We investigated the effect of obesity on pancreatic carcinogenesis in Pdx1-Cre; LSL-Kras G12D+/- (KC) mice. Metformin was administrated to rescue the effects of obesity and NETs. The pro-tumorigenic effects of neutrophil extracellular traps (NETs) were further evaluated in vivo and vitro. We found that obesity significantly promoted the progression of murine pancreatic ductal intraepithelial neoplasia (mPanIN). The proliferation rate and epithelial-mesenchymal transition (EMT) of mPanIN ductal cells were increased in obese mice. More visceral adipocytes, PD-L1 + neutrophil infiltration and NETs formation were found in the pancreas of obese mice and visceral adipocytes could recruit neutrophils and promote NETs formation. The latter could induce an inflammatory response in ductal cells via TLR4-dependent pathways both in vivo and vitro, as demonstrated by upregulation of IL-1β. Metformin and DNase I significantly reversed the pro-tumorigenic effects of obesity and NETs in vivo and in vitro. Our study provides causal evidence for the contribution of obesity in promoting pancreatic carcinogenesis in genetic model and reveals the mechanism by NETs to regulate mPanIN progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The preadipocytes differentiation is a vital process of lipogenesis; exploring the molecular mechanisms of lipogenesis contributes to improve the meat quality and final commercial income. Lipogenesis has been widely reported in other livestock, but little is known about the gene expression profiles at different stages during preadipocytes differentiation in sheep. In this study, ovine preadipocytes were cultured in vitro and then induced to begin differentiation. Then, the gene expression profiles of preadipocytes collected on day 0 (D0), day 2 (D2), and day 8 (D8) of differentiation were analyzed by RNA-seq technology. According to the findings, 2254 differentially expressed genes (DEGs) were found in D2 vs D0; 1817 DEGs and 1902 DEGs were found in D8 vs D0 and D8 vs D2, respectively. The DEGs were found to be enriched in several biological processes, including focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, steroid biosynthesis, and MAPK signaling pathway, according to Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The regulatory network of the DEGs related to ovine preadipocytes differentiation was systematically constructed, which showed that hub genes might modulate ovine preadipocytes differentiation. In summary, preadipocyte differentiation is regulated by several key genes, including ACACB, CXCL6, SREBF1, INSIG1, APOE, GJA1, CDH11, SYNE1, PCSK1, S100A4, FN1, PLIN2, CXCL6, FN1, PTX3, and FABP3. This study provides a deeper knowledge of the roles of genes in sheep lipogenesis by revealing global gene expression profiles during preadipocyte differentiation., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Elongation factor 1 A (EF1A), an essential regulator for protein synthesis, has been reported to participate in abiotic stress responses and environmental adaption in plants. However, the role of EF1A in abiotic stress response was barely studied in Medicago truncatula. Here, we identified elongation factor (EF) genes of M. truncatula and studied the salt stress response function of MtEF1A1 (MTR&#95;6g021805)., Results: A total of 34 EF genes were identified in the M. truncatula genome. Protein domains and motifs of EFs were highly conserved in plants. MtEF1A1 has the highest expression levels in root nodules and roots, followed by the leaves and stems. Transgenic Arabidopsis thaliana overexpressing MtEF1A1 was more resistant to salt stress treatment, with higher germination rate, longer roots, and more lateral roots than wild type plant. In addition, lower levels of H 2 O 2 and malondialdehyde (MDA) were also detected in transgenic Arabidopsis. Similarly, MtEF1A1 overexpressing M. truncatula was more resistant to salt stress and had lower levels of reactive oxygen species (ROS) in leaves. Furthermore, the expression levels of abiotic stress-responsive genes (MtRD22A and MtCOR15A) and calcium-binding genes (MtCaM and MtCBL4) were upregulated in MtEF1A1 overexpressing lines of M. truncatula., Conclusion: These results suggested that MtEF1A1 play a positive role in salt stress regulation. MtEF1A1 may realize its function by binding to calmodulin (CaM) or by participating in Ca 2+ -dependent signaling pathway. This study revealed that MtEF1A1 is an important regulator for salt stress response in M. truncatula, and provided potential strategy for salt-tolerant plant breeding., (© 2023. The Author(s).)

Long non-coding RNAs (lncRNAs) play important roles in the growth and development of skeletal muscle. However, there is limited information on goats. In this study, expression profiles of lncRNAs in Longissimus dorsi muscle from Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with divergent meat yield and meat quality were compared using RNA-sequencing. Based on our previous microRNA (miRNA) and mRNA profiles obtained from the same tissues, the target genes and binding miRNAs of differentially expressed lncRNAs were obtained. Subsequently, lncRNA-mRNA interaction networks and a ceRNA network of lncRNA-miRNA-mRNA were constructed. A total of 136 differentially expressed lncRNAs were identified between the two breeds. Fifteen cis target genes and 143 trans target genes were found for differentially expressed lncRNAs, and they were enriched in muscle contraction, muscle system process, muscle cell differentiation, and p53 signaling pathway. A total of 69 lncRNA-trans target gene pairs were constructed, with close relationship with muscle development, intramuscular fat deposition, and meat tenderness. A total of 16 lncRNA-miRNA-mRNA ceRNA pairs were identified, of which some reportedly associated with skeletal muscle development and fat deposition were found. The study will provide an improved understanding of the roles of lncRNAs in caprine meat yield and meat quality., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

microRNAs (miRNAs) are involved in the regulation of biological phenomena by down-regulating the expression of mRNAs. In this study, Liaoning cashmere (LC) goats ( n = 6) and Ziwuling black (ZB) goats ( n = 6) with different cashmere fiber production performances were selected. We supposed that miRNAs are responsible for the cashmere fiber trait differences. To test the hypothesis, the expression profiles of miRNAs from the skin tissue of the two caprine breeds were compared using small RNA sequencing (RNA-seq). A total of 1293 miRNAs were expressed in the caprine skin samples, including 399 known caprine miRNAs, 691 known species-conserved miRNAs, and 203 novel miRNAs. Compared with ZB goats, 112 up-regulated miRNAs, and 32 down-regulated miRNAs were found in LC goats. The target genes of the differentially expressed miRNAs were remarkably concentrated on some terms and pathways associated with cashmere fiber performance, including binding, cell, cellular protein modification process, and Wnt, Notch, and MAPK signaling pathways. The miRNA-mRNA interaction network found that 14 miRNAs selected may contribute to cashmere fiber traits regulation by targeting functional genes associated with hair follicle activities. The results have reinforced others leading to a solid foundation for further investigation of the influences of individual miRNAs on cashmere fiber traits in cashmere goats.

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA being >200 nucleotides in length, and they are found to participate in hair follicle growth and development and wool fiber traits regulation. However, there are limited studies reporting the role of lncRNAs in cashmere fiber production in cashmere goats. In this study, Liaoning cashmere (LC) goats ( n = 6) and Ziwuling black (ZB) goats ( n = 6) with remarkable divergences in cashmere yield, cashmere fiber diameter, and cashmere color were selected for the construction of expression profiles of lncRNAs in skin tissue using RNA sequencing (RNA-seq). According to our previous report about the expression profiles of mRNAs originated from the same skin tissue as those used in the study, the cis and trans target genes of differentially expressed lncRNAs between the two caprine breeds were screened, resulting in a lncRNA-mRNA network. A total of 129 lncRNAs were differentially expressed in caprine skin tissue samples between LC goats and ZB goats. The presence of 2 cis target genes and 48 trans target genes for the differentially expressed lncRNAs resulted in 2 lncRNA- cis target gene pairs and 93 lncRNA- trans target gene pairs. The target genes concentrated on signaling pathways that were related to fiber follicle development, cashmere fiber diameter, and cashmere fiber color, including PPAR signaling pathway, metabolic pathways, fatty acid metabolism, fatty acid biosynthesis, tyrosine metabolism, and melanogenesis. A lncRNA-mRNA network revealed 22 lncRNA- trans target gene pairs for seven differentially expressed lncRNAs selected, of which 13 trans target genes contributed to regulation of cashmere fiber diameter, while nine trans target genes were responsible for cashmere fiber color. This study brings a clear explanation about the influences of lncRNAs over cashmere fiber traits in cashmere goats.

Circular RNA (circRNA) is a type of non-coding RNA generated from back-splicing the reactions of linear RNA. It plays an important role in various cellular and biological processes. However, there are few studies about the regulatory effect of circRNAs on cashmere fiber traits in cashmere goats. In this study, the expression profiles of circRNAs in skin tissue were compared between Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats, with a significant difference in cashmere fiber yield, cashmere fiber diameter, and cashmere fiber color, using RNA-seq. A total of 11,613 circRNAs were expressed in the caprine skin tissue, and their type, chromosomal distribution, and length distribution were characterized. A total of 115 up-regulated circRNAs and 146 down-regulated circRNAs in LC goats were screened compared to ZB goats. The authenticity of 10 differentially expressed circRNAs was validated by detecting their expression levels and the head-to-tail splice junction using RT-PCR and DNA sequencing, respectively. The parent genes of differentially expressed circRNA were mainly enriched in some Gene Ontology (GO) terms and pathways related to cashmere fiber traits, such as the canonical Wnt signaling pathway, which is involved in the regulation of cell promotion, stem cell proliferation, Wnt signaling pathway regulation, epithelial morphogenesis, MAPK signaling pathway, and cell adhesion molecules pathway. Eight differentially expressed circRNAs were further selected to construct a circRNA-miRNA network, and some miRNAs that were previously reported as related to fiber traits were found in the network. This study provides a deep understanding of the roles of circRNAs in the regulation of cashmere fiber traits in cashmere goats and the involvement of differential splicing in phenotypic expression according to breed and region.

The PLATZ family is a novel class of plant-specific zinc finger transcription factors with important roles in plant growth and development and abiotic stress responses. PLATZ members have been identified in many plants, including Oryza sativa , Zea mays , Triticum aestivum , Fagopyrum tataricum , and Arabidopsis thaliana ; however, due to the complexity of the alfalfa reference genome, the members of the PLATZ gene family in alfalfa ( Medicago sativa L.) have not been systematically identified and analyzed. In this study, 55 Medicago sativa PLATZ genes ( MsPLATZs ) were identified in the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatic analysis was performed, including the characterization of sequence lengths, protein molecular weights, genomic positions, and conserved motifs. Expression analysis reveals that 7 MsPLATZs are tissue-specifically expressed, and 10 MsPLATZs are expressed in all examined tissues. The transcriptomic expression of these genes is obvious, indicating that these MsPLATZ s have different functions in the growth and development of alfalfa. Based on transcriptome data analysis and real-time quantitative PCR (RT-qPCR), we identified 22, 22, and 21 MsPLATZ genes that responded to salt, cold, and drought stress, respectively, with 20 MsPLATZs responding to all three stresses. This study lays a foundation for further exploring the functions of MsPLATZs , and provides ideas for the improvement of alfalfa varieties and germplasm innovation.

Salt stress is a worldwide agronomic issue that limits crop yield and quality. Improving salt stress tolerance via genetic modification is the most efficient method to conquer soil salinization problems in crops. Crop miRNAs have been declared to be tightly associated with responding and adapting to salt stress and are advantageous for salt tolerance modification. However, very few studies have validated vital salt tolerance miRNAs and coupled potent target genes in Medicago species, the most economically important forage legume species. In this study, Mtr-miR319a, a miRNA that was identified from the previous next-generation sequencing assay of salt-treated Medicago truncatula, was overexpressed in M. truncatula and Arabidopsis thaliana, inducing the curly leaves and salt stress tolerance phenotypes. Combining the elevated expression level of Mtr-miR319a in the M. truncatula overexpression lines under normal and salt-treatment conditions, the regulatory roles of Mtr-miR319a in leaf development and salt stress adaptation were demonstrated. Several predicted target genes of Mtr-miR319a were also regulated by Mtr-miR319a and were associated with the aforementioned phenotypes in M. truncatula plants, most notably MtTCP4. Our study clarified the functional role of Mtr-miR319a and its target genes in regulating leaf development and defending salt stress, which can help to inform crop breeding efforts for improving salt tolerance via genetic engineering.

The number of words: 4645, the number of figures: 4, the number of tables: 1The outbreak of COVID-19 in December 2019 caused a global pandemic of acute respiratory disease, and with the increasing virulence of mutant strains and the number of confirmed cases, this has resulted in a tremendous threat to global public health. Therefore, an accurate diagnosis of COVID-19 is urgently needed for rapid control of SARS-CoV-2 transmission. As a new molecular biology technology, loop-mediated isothermal amplification (LAMP) has the advantages of convenient operation, speed, low cost and high sensitivity and specificity. In the past two years, rampant COVID-19 and the continuous variation in the virus strains have demanded higher requirements for the rapid detection of pathogens. Compared with conventional RT-PCR and real-time RT-PCR methods, genotyping RT-LAMP method and LAMP plus peptide nucleic acid (PNA) probe detection methods have been developed to correctly identified SARS-CoV-2 variants, which is also why LAMP technology has attracted much attention. LAMP detection technology combined with lateral flow assay, microfluidic technology and other sensing technologies can effectively enhance signals by nucleic acid amplification and help to give the resulting output in a faster, more convenient and user-friendly way. At present, LAMP plays an important role in the detection of SARS-CoV-2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest., (Copyright © 2022 Li, Ge, Sun, Fu, Cao, Feng, Meng, Peng, Liu and Zhao.)

Milk fat is the foremost nutrient of milk and a vital indicator in evaluating milk quality. Accumulating evidence suggests that microRNAs (miRNAs) are involved in the synthesis of milk fat. The miR-200c is closely related to lipid metabolism, but little is known about its effect on the synthesis of milk fat in MECs of ewes. Herein, the effect of miR-200c on the proliferation of ovine mammary epithelial cells (MECs) and its target relationship with a predicted target gene were investigated. The regulatory effects of miR-200c on the expression of the target genes and the content of triglycerides in ovine MECs were further analyzed. The results revealed that the expression level of miR-200c was differentially expressed in both eight tissues selected during lactation and in mammary gland tissues at different physiological periods. Overexpression of miR-200c inhibited the viability and proliferation of ovine MECs, while inhibition of miR-200c increased cell viability and promoted the proliferation of ovine MECs. Target gene prediction results indicated that miR-200c would bind the 3'UTR region of pantothenate kinase 3 ( PANK3 ). Overexpression of miR-200c reduced the luciferase activity of PANK3 , while inhibition of miR-200c increased its luciferase activity. These findings illustrated that miR-200c could directly interact with the target site of the PANK3 . It was further found that overexpression of miR-200c reduced the expression levels of PANK3 and, thus, accelerated the synthesis of triglycerides. In contrary, the inhibitor of miR-200c promoted the expression of PANK3 that, thus, inhibited the synthesis of triglycerides in ovine MECs. Together, these findings revealed that miR-200c promotes the triglycerides synthesis in ovine MECs via increasing the lipid synthesis related genes expression by targeting PANK3 .

Purpose: This study aims to study the depth of artery wall tumour invasion in patients undergoing surgery for pancreatic ductal adenocarcinoma., Methods: Specimens from 47 pancreatic cancer patients with major arterial (splenic, SA; celiac, CA; common hepatic, CHA) invasion were examined: 45 left (distal) pancreatectomies, including 11 celiac artery resections, and two total pancreatectomies. Dissection of tumour-invaded arteries in 25 fresh specimens was attempted ex vivo using the sub-adventitial dissection technique (SDT). Tumour invasion of 66 arteries was graded using the tumour-free distance (TFD) from the external elastic lamina (EEL): 0 = no arterial invasion; I = TFD ≥ 1 mm; II = TFD < 1 mm; and grade III = EEL invasion., Results: AJCC TNM staging was IA = 1 (2%), IB = 4 (9%), IIA = 5 (11%), IIB = 17(36%) and III = 20 (43%). Grade III tumour invasion was found in 17/47(36%) SAs, in 5/11 (45%) CAs and in 1/8 (13%) CHAs (p = 0.318). Attempted ex vivo SDT undertaken in 33 arteries from 25 specimens was complete in 16 and incomplete in 17 arteries. The median (IQR) TFD was 0.97 (0.11-2.54) mm in dissected and 0.14 (0.10, 0.14) mm in non-dissected SAs (p = 0.034). EEL tumour invasion occurred in 0/12 (0%) dissected compared to 7/13 (54%) non-dissected SAs (p = 0.005). Grades 0, I, II and III invasion were found in four (33%), two (17%) and six (50%), respectively, of 12 dissected SAs and grades II and III in six 6 (46%) and seven (54%), respectively, of 13 non-dissected SAs (p = 0.002)., Conclusions: The grading system described may form the basis for classification to further develop arterial dissection techniques for pancreatic cancer., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In our previous study, microRNA (miR)-381 was found to be the most down-regulated miRNA in skeletal muscle of Liaoning cashmere goats with higher skeletal muscle mass, but the molecular mechanism involved remains unclear. In this study, primary caprine skeletal muscle satellite cells (SMSCs) were isolated and identified. We investigated the effect of miR-381 on the viability, proliferation and differentiation of caprine SMSCs, and the target relationships of miR-381 with jagged canonical Notch ligand 2 ( JAG2 ) and phosphatase and tensin homolog ( PTEN ). Cells isolated were positive for SMSC-specific marker protein Pax7. This suggests that purified SMSCs were obtained. The expression level of miR-381 achieved a peak value on day 4 after SMSC differentiation, and miR-381 also significantly increased the expression levels of myogenic differentiation marker genes: myosin heavy chain ( MyHC ), myogenin ( MyoG ) and myocyte enhancer factor 2C ( MEF2C ) in differentiated SMSCs, the area of MyHC-positive myotubes and the myogenic index. These findings suggest that miR-381 promoted myogenic differentiation of caprine SMSCs. The CCK8 assay and EDU staining analysis showed that miR-381 mimic both inhibited the viability of SMSCs and decreased the percentage of EDU-labeled positive SMSCs. In contrast, miR-381 inhibitor had the opposite effect with miR-381 mimic. A dual luciferase reporter assay verified that miR-381 can target JAG2 and PTEN by binding to the 3'-untranslated regions (3'-UTR) of the genes. The transfection of miR-381 mimic into caprine SMSCs resulted in decreases in expression levels of JAG2 and PTEN , while miR-381 inhibitor increased the two target genes in expression. This is the first study to reveal the biological mechanisms by which miR-381 regulates caprine SMSC activities.

Fall dormancy (FD) is an essential trait to overcome winter damage and for alfalfa ( Medicago sativa ) cultivar selection. The plant regrowth height after autumn clipping is an indirect way to evaluate FD. Transcriptomics, proteomics, and quantitative trait locus mapping have revealed crucial genes correlated with FD; however, these genes cannot predict alfalfa FD very well. Here, we conducted genomic prediction of FD using whole-genome SNP markers based on machine learning-related methods, including support vector machine (SVM) regression, and regularization-related methods, such as Lasso and ridge regression. The results showed that using SVM regression with linear kernel and the top 3000 genome-wide association study (GWAS)-associated markers achieved the highest prediction accuracy for FD of 64.1%. For plant regrowth height, the prediction accuracy was 59.0% using the 3000 GWAS-associated markers and the SVM linear model. This was better than the results using whole-genome markers (25.0%). Therefore, the method we explored for alfalfa FD prediction outperformed the other models, such as Lasso and ElasticNet. The study suggests the feasibility of using machine learning to predict FD with GWAS-associated markers, and the GWAS-associated markers combined with machine learning would benefit FD-related traits as well. Application of the methodology may provide potential targets for FD selection, which would accelerate genetic research and molecular breeding of alfalfa with optimized FD., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Uridine diphosphate glycosyltransferases (UGTs) are enzymes that catalyze glycosylation modifications and play an essential role in regulating plant metabolism. Alfalfa ( Medicago sativa L.) is the most important legume in the world due to its high yields and protein content; however, the UGT genes in alfalfa have not yet been studied. Identifying UGT genes with metabolic roles in alfalfa is essential for identifying and modifying genetic traits that are relevant to yield and quality. In this study, 90 of the 239 UGT genes identified from the alfalfa "Zhongmu No. 1" genome database were found to be related to secondary metabolism, and a series of gene family characterization analyses were conducted on each. The results demonstrated that all 90 UGT genes were unevenly distributed on eight chromosomes with few introns and that tandem duplications were the crucial driving force expanding the UGT family in alfalfa. Notably, the 90 UGT genes can be clustered into ten evolutionary groups which contain specific PSPG motifs, and genes in these ten groups have specific tissue expressions. This suggests that the UGT genes in each group could have similar glycosylation roles corresponding to analogous secondary metabolites in alfalfa. Additionally, multiple cis -acting elements found in MsUGT promoter regions, such as phytohormone and flavonoids, indicate that 90 UGT members could be induced by these features, which are also related to secondary metabolism. Therefore, our study identified 90 UGT members inten evolutionary groups that are likely related to glycosylation modifications with secondary metabolites in alfalfa. These findings help uncover pivotal regulatory mechanisms associated with secondary metabolism in plant yield and quality and contribute to genetic modification and breeding in alfalfa and other plant species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yu, Jiang, Sun, Hu, Zhu, Kang, Chen, Liu, Hao, Yang, Long and Li.)

In our previous a study, circ&#95;003628 was one of the most highly expressed circular RNAs (circRNAs) in the Longissimus dorsi muscle of goats found by RNA-seq, suggesting that the circRNA may be important for caprine muscle growth and development. However, there have been no reports describing the molecular mechanisms by which circ&#95;003628 regulates the activities of goat skeletal muscle satellite cells (SMSCs). In this study, reverse transcriptase-PCR (RT-PCR) and DNA sequencing were used to validate the authenticity of circ&#95;003628, and its characteristics, expression profile and effect on goat SMSCs were also studied using real-time quantitative-PCR (RT-qPCR), EdU, CCK-8 and immunofluorescence assays. Circ&#95;003628 is partially originated from 13 exons, 12 introns and 3'-untranslated regions (UTR) of caprine Myosin Heavy Chain 1 ( MYH1 ), and 25 exons and 5' UTR of Myosin Heavy Chain 4 ( MYH4 ), as well as intergenic sequences between the two genes. A total of 77.07% of circ&#95;003628 were located in the nuclei of goat SMSCs, while 22.93% were expressed in the cytoplasm. The circRNAs were only expressed in triceps brachii , quadriceps femoris and longissimus dorsi muscle tissues in nine caprine tissues investigated, with the highest expression level in longissimus dorsi muscle. The expression level of circ&#95;003628 gradually increased during differentiation periods of goat SMSCs and reached the maximum on day 6 after differentiation. The small interfering RNA of circ&#95;003628 (named si-circ&#95;003628) inhibited the viability and proliferation of goat SMSCs, and also decreased the expression of four cell proliferation marker genes: paired box 7 ( Pax7 ), cyclin-dependent kinase 2 ( CDK2 ), CDK4 and CyclinD1 in goat SMSCs. Transfection of si-circ&#95;003628 significantly decreased the area of MyHC-labeled myotubes of goat SMSCs, as well as the expression levels of three differentiation marker genes: myosin heavy chain ( MyHC ), myogenin ( MyoG ), and myocyte enhancer factor 2C ( MEF2C ). These results suggest that circ&#95;003628 promotes the viability, proliferation, and differentiation of goat SMSCs, and they also provide an improved understanding of the roles of circ&#95;003628 in skeletal muscle growth and development in goats.

NAC (NAM, ATAF1/2, and CUC2) transcription factors compose one of the largest families of plant-specific transcription factors; they are widely involved in plant growth and development and have especially important roles in improving stress resistance in plants. However, NAC gene family members in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed genome-wide due to the complexity of the alfalfa reference genome. In this study, a total of 421 M. sativa NAC genes (MsNACs) were identified from the alfalfa “Xinjiangdaye” reference genome. Basic bioinformatics analysis, including characterization of sequence length, protein molecular weight and genome position and conserved motif analysis, was conducted. Expression analysis showed that 47 MsNACs had tissue-specific expression, and 64 MsNACs were expressed in all tissues. The transcriptomic profiles of the genes were very different, indicating that these MsNACs have various functions in alfalfa growth and development. We identified 25, 42 and 47 MsNACs that respond to cold, drought and salt stress based on transcriptome data analysis and real-time quantitative PCR (RT−qPCR). Furthermore, 22 MsNACs were found to respond to both salt and drought stress, and 15 MsNACs were found to respond to cold, salt and drought stress. The results of this study could provide valuable information for further functional analysis of MsNACs and for the improvement of stress resistance in alfalfa.

The Tibetan sheep is an indigenous animal of the Tibetan plateau, and after a long period of adaptation have adapted to high-altitude hypoxia. Many physiological changes occur in Tibetan sheep as they adapt to high-altitude hypoxia, especially in the lungs. To reveal the physiological changes and their molecular mechanisms in the lungs of Tibetan sheep during adaptation to high altitudes, we selected Tibetan sheep from three altitudes (2500 m, 3500 m, and 4500 m) and measured blood-gas indicators, observed lung structures, and compared lung proteome changes. The results showed that the Tibetan sheep increased their O 2 -carrying capacity by increasing the hemoglobin (Hb) concentration and Hematocrit (Hct) at an altitude of 3500 m. While at altitude of 4500 m, Tibetan sheep decreased their Hb concentration and Hct to avoid pulmonary hypertension and increased the efficiency of air-blood exchange and O 2 transfer by increasing the surface area of gas exchange and half-saturation oxygen partial pressure. Besides these, some important proteins and pathways related to gas transport, oxidative stress, and angiogenesis identified by proteome sequencing further support these physiology findings, including HBB, PRDX2, GPX1, GSTA1, COL14A1, and LTBP4, etc. In conclusion, the lungs of Tibetan sheep are adapted to different altitudes by different strategies; these findings are valuable for understanding the basis of hypoxic adaptation in Tibetan sheep.

Background: Extensive muscle atrophy is a common occurrence in orthopaedics patients who are bedridden or immobilized. The incidence is higher in intensive care unit (ICU) inpatients. There is still controversy about how to use neuromuscular electrical stimulation (NMES) in ICU patients. We aim to compare the effectiveness and safety of NMES to prevent muscle atrophy in intensive care unit (ICU) patients without nerve injury., Methods: ICU patients without central and peripheral nerve injury were randomized into experimental group I (Exp I: active and passive activity training (APAT) + NMES treatment on the gastrocnemius and tibialis anterior muscle), experimental group II (Exp II: APAT + NMES treatment on gastrocnemius alone), and control group (Ctl: APAT alone). Changes in the strength of gastrocnemius, the ankle range of motion, and the muscle cross-section area of the lower leg were evaluated before and after the intervention. Also, changes in prothrombin time, lactic acid, and C-reactive protein were monitored during the treatment., Results: The gastrocnemius muscle strength, ankle joint range of motion, and cross-sectional muscle area of the lower leg in the three groups showed a downward trend, indicating that the overall trend of muscle atrophy in ICU patients was irreversible. The decrease in gastrocnemius muscle strength in Exp I and Exp II was smaller than that in the control group (P < 0.05), but there was no difference between Exp I and Exp II. The decrease in active ankle range of motion and cross-sectional area of the lower leg Exp I and Exp II was smaller than that in the control group (P < 0.05), and the decrease in Exp I was smaller than that of Exp II (all P < 0.05). The curative effect in Exp I was better than in Exp II. There were no significant differences in the dynamic changes of prothrombin time, lactic acid, and C-reactive protein during the three groups (P > 0.05)., Conclusion: In addition to early exercise training, NMES should be applied to prevent muscle atrophy for patients without nerve injury in ICU. Also, simultaneous NMES treatment on agonist/antagonist muscle can enhance the effect of preventing muscle atrophy., Trial Registration: This study was prospectively registered in China Clinical Trial Registry ( www.chictr.org.cn ) on 16/05/2020 as ChiCTR2000032950., (© 2022. The Author(s).)