Osteosarcoma (OS) is a highly prevalent bone malignancy among adolescents, accounting for 40% of all primary malignant bone tumors. Neoadjuvant chemotherapy combined with limb‐preserving surgery has effectively reduced patient disability and mortality, but pulmonary metastases and OS cells' resistance to chemotherapeutic agents are pressing challenges in the clinical management of OS. There has been an urgent need to identify new biomarkers for OS to develop specific targeted therapies. Recently, the continued advancements in genomic analysis have contributed to the identification of clinically significant molecular biomarkers for diagnosing OS, acting as therapeutic targets, and predicting prognosis. Additionally, the contemporary molecular classifications have revealed that the signaling pathways, including Wnt/β‐catenin, PI3K/AKT/mTOR, JAK/STAT3, Hippo, Notch, PD‐1/PD‐L1, MAPK, and NF‐κB, have an integral role in OS onset, progression, metastasis, and treatment response. These molecular classifications and biological markers have created new avenues for more accurate OS diagnosis and relevant treatment. We herein present a review of the recent findings for the modulatory role of signaling pathways as possible biological markers and treatment targets for OS. This review also discusses current OS therapeutic approaches, including signaling pathway‐based therapies developed over the past decade. Additionally, the review covers the signaling targets involved in the curative effects of traditional Chinese medicines in the context of expression regulation of relevant genes and proteins through the signaling pathways to inhibit OS cell growth. These findings are expected to provide directions for integrating genomic, molecular, and clinical profiles to enhance OS diagnosis and treatment. This process includes the interaction among multiple molecules and proteins to transmit information through a series of molecules such as signaling molecules, signaling receptors, and signal transduction factors.To offer a thorough overview of the recent research advances on the mechanisms and clinical investigations of OS‐targeted signaling pathways, we examine each of the OS‐related signaling pathways in this review. TCM has become a hot spot for OS treatment because of its broad action, low side effects, and multiple targeting functions.2Recently, research evidence has emerged showing that the dysregulation of various intracellular signaling pathways is closely related to the onset and progression of OS, and to treat OS, it is crucial to research the signaling pathways linked to its development. The PI3K/AKT/mTOR signaling pathway, one of the crucial pathways in cancerous cells, has been demonstrated to have the ability to enhance the cell cycle, inhibit apoptosis, and promote cellular proliferation, invasion, and metastasis. The modulation of OS cellular proliferation, apoptosis, and vascular neogenesis, among other processes, is critically controlled by the JAK/STAT3 axis, a classical intracellular signaling pathway. Numerous mammalian cells and organs are subject to homeostatic modulation by the Hippo signaling pathway, which also controls OS cellular proliferation, apoptosis, invasiveness, and metastasis. The immunosuppression and weakening of immune cell killing effect on tumor cells lead to immune escape of OS cells, consequently culminating in the occurrence and development of OS. Abnormal activation of the MARK signaling pathway leads to loss of apoptosis and differentiation of OS cells, which is intimately correlated with the malignant transformation and abnormal proliferation of OS cells. The OS's ability to proliferate, differentiate, and resist medication is influenced by the Notch signaling pathway. Furthermore, an important factor regulating OS cell proliferation, metastasis, and drug resistance is the NF‐κB pathway. This section examines the possible biomarkers for OS as well as the modulatory functions of the aforementioned signaling pathways.2.12.1.1In 1984, a new signaling pathway intimately correlated with the activity of tyrosine kinase encoded by the viral oncogene was identified. In 1988, Cantley and Downes' team discovered the virus‐associated tyrosine kinase as PI3K, which generates PI3K via the mechanism of phosphorylating the 3‐OH group on the inositol ring.The PI3K/AKT/mTOR pathway regulates various cellular functions by integrating multiple extracellular signals.Phosphatase and tensin homolog (PTEN) is an antagonist of PI3K. PIP3 functions as a second messenger and binds to the intracellular signaling protein AKT through the pleckstrin homology (PH) domain. After binding, the complex undergoes conformational changes, causing AKT to translocate from the cytoplasm to the cell membrane.mTOR is an important Ser/Thr protein kinase present downstream of PI3K/AKT.2.1.2In OS, the PI3K/AKT/mTOR pathway frequently experiences changes. The high frequency of PI3K mutations and/or their increased catalytic function in tumor cells is a key mechanism of proliferation and metastasis in OS.The PI3K axis is primarily activated by AKT.PTEN chromosomal loss (antagonist of PI3K/AKT) is a common molecule in OS.mTOR is a bridge to multiple downstream signaling pathways that could be triggered by a variety of upstream regulatory variables.2.22.2.1Drosophila) and Int‐1 were homologous and encoded the same protein, both of which were abbreviated as Wnt.2+ pathway are examples of nonclassical Wnt signaling pathway.The Wnt gene was discovered during the process of exploring the transcriptional mechanism of mouse mammary tumor virus and was initially named Int‐1.The major components of the Wnt/β‐catenin pathway include ligands (Wnt family proteins), receptors for Wnt family proteins (the primary receptor Frizzled [Fzd] protein and the coreceptor low‐density lipoprotein receptor‐related protein 5/6 [LRP‐5/6]), Dishevelled (Dsh), axin (Axin), glycogen synthase kinase 3β (GSK‐3β), casein kinase 1 (CK1), colorectal adenomatous polyposis coli (APC), and β‐linked protein (β‐catenin).β‐Catenin is a key signaling protein factor in the classical Wnt signaling pathway.2.2.2OS development occurs due to the activation or silencing of several signaling pathways.The positive rate of β‐catenin in OS tissues is 70%, which differs from that in paraneoplastic tissues. β‐Catenin enables the invasion of OS cells and increased β‐catenin content in the nucleus can promote the epithelial‐to‐mesenchymal transition (EMT) of OS cells and increase stem cell formation, both of which could enhance the migration and invasiveness of tumor cells as well as the growth and spread of OS cells.2.32.3.1The JAK/STAT signaling pathway regulates a variety of cytokines and is crucial for immune and metabolic regulation as well as biological activities including cellular division, differentiation, and apoptosis.Different extracellular signals can activate the JAK/STAT3 signaling pathway in vivo, mainly through the binding of extracellular cytokines  and growth factors  to cell surface receptors as ligands, which dimerize the receptor molecules and induce the polymerization of intracellular glycoprotein 130 (gp130) with the α‐subunit on the receptor.2.3.2The most significant STAT3 upstream protein is JAK. Additionally, the most predominant class of indirect STAT3 inhibitors that work by preventing STAT3 activation is comprised of JAK inhibitors. Only a few studies have evaluated the role of JAK in OS. In recent years, the JAK/STAT3 signaling pathway has been extensively studied, and researchers have proven that inappropriate activation of JAK/STAT3 in OS is always linked to physiological events such as defective tumor cell migration, invasiveness, metastasis, and vascular neogenesis.OS cell lines highly express STAT3 and p‐STAT3, and inhibition of STAT3 activation can downregulate Cyclin D1 expression and further inhibit the proliferative ability of OS cells.Couto et al.OS‐associated macrophages (tumor‐associated macrophages) promote metastasis and EMT of OS cells by elevating the expression levels of p‐STAT3,Metastatic OS involves the formation of a very complex and robust vascular system through angiogenesis. More specifically, metastatic OS cells can form tumor blood vessels directly by mimicking angiogenesis without relying on the involvement of endothelial cells.2.42.4.1Drosophila hippo gene was discovered in 2003, Pan and Hariharan's group discovered that Hippo inactivation regulated tissue development in a negative manner. This brought attention to the Hippo signaling pathway.When the Drosophila Hippo homolog), transcriptional coactivator with PDZ‐binding motif (TAZ), Yes‐associated protein 1 (YAP), Mob kinase activator 1 (MOB1), Salvador homolog 1 (SAV1), and large tumor suppressor 1/2 (Lats1/2).Typical Hippo signaling pathways include the mammalian STE20‐like protein kinase 1/2 , dendritic cells (DCs), monocytes, B cells, and T cells. PD‐1/PD‐L1 mediates immune responses through T cells: APCs and tumor cells both express PD‐L1 and T‐cell dysfunction and even failure may occur when PD‐1 binds to PD‐L1 in T cells. PD‐L1 overexpression in tumors allows self‐escape from cytotoxic T cells (CD8+)‐mediated cell killing.The steps of the antitumor immune cycle include the release of tumor antigens (Ags), Ag presentation, trigger and activation, T‐cell trafficking to the tumor, T‐cell infiltration, T‐cell recognition of cancer cells, and the killing of malignant cells. PD‐1/PD‐L1 affects immunity mainly by impairing the activity of cytolytic T cells.2.5.2The level of PD‐1/PD‐L1 expression correlates with the prognosis of tumors and is substantially elevated in OS tissues. PD‐1 and PD‐L1 were found to be expressed in 47 and 53% of biopsy specimens, respectively, according to studies examining PD‐1, PD‐L1, and associated immunomarkers in samples of individuals with OS.When OS cells exhibit high immunogenicity, follicular helper T cells overexpress PD‐1 and underexpress the antitumor substance IL‐21, thereby attenuating the ability of CD8+ T cells to release interferon‐γ (IFN‐γ) and degranulation, which, in turn, reduces the toxicity of T cells to OS cells.PD‐1/PD‐L1 trans‐signaling pathways mediating the immune escape of tumor cells in OS involve relevant signal transduction mechanisms, which have received much attention from researchers. Blocking STAT3 elevated the expression levels of DCs that are triggered by the innate immune system, enhances the production of chemokines and cytokines in tumor tissues, triggers tumor T‐cell responses,miRNAs are closely associated with PD‐L1 expression.2.62.6.1There are two types of Notch signaling pathways: classical and nonclassical. The classical Notch signaling pathway mainly includes Jagged/Jagged ligands (Jagged 1 and Jagged 2), Delta‐like ligands , receptors , as well as TF C‐promoter‐binding factor (CBF), and its coactivators.2.6.2Cell migration‐inducible protein (CEMIP) exerts oncogenic effects in several cancers.The Notch signaling pathway affects tumor drug resistance by reducing intracellular drug accumulation and regulating EMT, leading to miRNA dysregulation, apoptosis gene disruption, and regulating tumor stem cells.P53 is the most well‐studied oncogene and is intimately linked to diverse biological behaviors like growth arrest, senescence, apoptosis, and DNA repair.2.72.7.1MAPK is a tightly controlled signaling pathway with Ser/Thr protein kinase activity that performs an instrumental function in diverse biological processes in living organisms, it can be activated by stimuli or cellular signals such as physical stress and inflammatory responses.2.7.2VDAC1 independently functions as a prognostic indicator in OS and patients with high VDAC1 expression exhibit a low survival rate. VDAC1 promotes OS cell proliferation and apoptosis through the MAPK signaling pathway.2.82.8.1NF‐κB belongs to the Rel family of eukaryotic TFs and consists of two subunits: p50 and p65.2.8.2In OS, the NF‐κB pathway performs an indispensable function in modulating tumor growth, metastasis, and chemoresistance. Aberrant expression of NF‐κB is closely associated with OS. Li et al.Contemporary research on NF‐κB‐associated mechanisms in OS is centered on the modulation of miRNAs and lncRNAs that are possible upstream modulators of NF‐κB and could exert their oncogenic or their inhibitory properties on tumors by affecting the levels of STAT3 expressed in OS cells.3The current treatment for patients with OS is mainly neoadjuvant chemotherapy, including high‐dose methotrexate, doxorubicin, and cisplatin, and surgical excision of the lesion with adjuvant chemotherapy, but the treatment for OS is still slightly inadequate compared with the advantages of chemotherapy combined with targeted therapy for lung cancer and breast cancer. This part aims to discuss the molecular biological pathogenesis of OS, from both small molecule inhibitors and TCM, which are strongly correlated with the onset and advancement of OS, and to explore the corresponding therapeutic targets and downstream pathways to provide strong evidence for further research.3.1A class of organic compounds with molecular weights less than 1000 Da known as small molecule inhibitors can target proteins, reduce protein activity or block biochemical reactions, and are widely used in the study of signaling pathways.3.1.1LY294002, the first broad‐spectrum PI3K inhibitor, has a broad inhibitory effect on PI3K‐related pathways in OS cells, and it is often used in combination treatment with other oncogenic drugs in clinical studies to improve drug efficacy and to decelerate the developed drug resistance.With the current development of drugs targeting the mTOR pathway and the limited benefit of monotherapy, multidrug combinations will become the main treatment modality for OS in the future. In human OS cells and mouse OS models in vitro, simultaneous inhibition of PI3K and mTOR targets effectively induced cellular apoptosis, whereas single‐targeted inhibition of PI3K or mTOR was ineffective.3.1.2The enzyme COX2 celecoxib can inhibit β‐catenin from translocating to the nucleus by activating the GSK3β signaling pathway, decreasing the abnormal activation of the β‐catenin pathway, and inhibiting the growth, invasive, and migratory capacities of OS cells by increasingly suppressing the Wnt/β‐catenin pathway.Accumulating evidence suggests that microRNA (miRNA) dysregulation and the Wnt/β‐catenin pathway together trigger cancer development, metastasis, and drug resistance.3.1.3Existing research on hindering OS development by inhibiting the JAK/STAT3 pathway is centered on the inhibitory effects of JAK2 inhibitors and STAT3 inhibitors. AG490, a selective inhibitor of JAK2, targets the JAK signaling pathway in Saos‐2 cells and reduces the proliferative and migratory capacities of OS cells by decreasing the phosphorylation level of STAT3 and impeding its activation, which eliminates the stimulatory effect of THAP9‐AS1 on cellular processes.Similar to the synthesis‐specific small‐molecule, LLL3,3.1.4In xenograft OS, Verteporfin is known to inhibit YAP/TAZ signaling and suppress the proliferation, migration, and invasiveness of spontaneous OS owing to the deficiency of Rb1 and Trp53 in cells that express Ctsk by blocking the Hippo pathway.3.1.5In vivo experiments have shown that DAPT (GSI‐IX), a common synthetic γ‐secretase inhibitor, specifically blocks γ‐secretase and prevents the activation of the Notch signaling pathway.3.1.6In an in situ OS transplantation model, iron chelators induce apoptosis in OS cells owing to their disruption of the homeostatic balance of intracellular iron and activation of the MAPK pathway.3.1.7The multipotent cytokine macrophage migration inhibitory factor (MIF) inhibitor 4‐iodo‐6‐phenylpyrimidine impedes the binding of MIF with CD74 and p65 and significantly reduces the proliferation and metastasis of OS cells by inhibiting the NF‐κB pathway.3.2TCM has numerous active monomeric components and their mechanisms of action are complex and diverse. Relying on the synergistic modulation of multiple components, targets, and pathways, TCM plays multiple therapeutic functions in the onset, progression, and metastasis of OS. In recent years, in the study of OS‐targeted signaling pathways, in addition to human‐assembled small molecule inhibitors, more and more studies on natural products have been conducted in recent years Figures  and 4.3.2.1Sodium cantharidinate is a semisynthetic derivative of cantharidin hydrolyzed by sodium hydroxide.Bcl‐2 is a major target molecule for studying the molecular mechanisms of apoptosis. The members of the Bcl‐2 protein family encompass proapoptotic proteins  and antiapoptotic proteins (Bcl‐xL and Bcl‐2), which control the release of mitochondria‐associated apoptotic factors by controlling the mitochondrial outer membrane permeability. Aloperine,3.2.2GSK‐3β can phosphorylate β‐catenin at four N‐terminal sites and is thus a marker of β‐catenin phosphorylation. Following phosphorylation, β‐catenin is degraded by the proteasome through ubiquitination.In vitro trials have illustrated that oleuropein inhibits the proliferation and invasion of OS cells, promotes apoptosis, and exerts good anticancer effects. Additionally, it reduces the expression of the cytoplasmic total β‐catenin in OS cells, reduces the translocation of β‐catenin into the nucleus, and blocks the Wnt/β‐catenin pathway. Notably, the reduction of Wnt‐specific TOP/FOP luciferin reporter gene expression indicated an attenuated transcriptional activity of the TF TCF/LEF in the Wnt/β‐catenin pathway. Furthermore, the expression of the Wnt target genes MMP2 and MMP9 was downregulated to inhibit the migratory invasive ability of cells.3.2.3Curculigo orchioides Gaertn, suppressed the growth and metastasis of OS cells by inhibiting the JAK/STAT3 pathway.Besides the human‐assembled small‐molecule inhibitors, natural products have recently gained increasing attention, and their extracts have monomeric components that can inhibit JAK/STAT pathway. In vivo studies suggest that curcumin remarkably and dosage‐dependently lowers the levels of p‐JAK2 and p‐STAT3 expressed in MG63 cells. Also, curcumin suppresses the proliferative, migratory, and invasive capacities of MG63 cells, induces G0/G1 phase arrest, and promotes apoptosis through the inhibition of the p‐JAK2/p‐STAT3 pathway.3.2.4A thorough analysis of the function played by curcumin in OS revealed that Notch 1/2/3 gene signaling was activated in most of the OS cell lines and that the proliferation and metastasis of OS cells were suppressed when Notch 1 signaling and downstream gene expression were downregulated.3.2.5Notably, MAPK pathway proteins play a central function in regulating the expression of MMPs.The MAPK pathway is regulated by ERK, JNK, and p38 MAPK kinase, which are activated and phosphorylated to regulate downstream MMP gene expression, subsequently promoting the occurrence and progression of OS.3.2.6Punicalagin can interfere with 19 human OS cell lines, including U2OS, MG63, and Saos2. It significantly reduces proliferation and increases apoptosis of OS cells while downregulating IL‐6 and IL‐8 expression levels.4Presently, immunotherapy and targeted therapies are the primary focus of the efforts to develop novel treatment regimens for OS. While mounting evidence on cellular and molecular levels indicated that numerous distinct signaling pathways and genes are implicated in the onset and advancement of OS, the druggable fraction of these markers is extremely limited. The principal drug targets for existing drugs include insulin‐like growth factor (IGF)‐R, EGFR, PDGF/PDGFR, mTOR, VEGFR, and Aurora kinase pathways linked to cellular growth, immune checkpoint pathways linked to immunity, relay immune cell therapy, and tumor vaccines Figure .4.14.1.1The IGF system includes three main ligands , three corresponding tyrosine kinase receptors  and six distinct IGF‐binding proteins (IGF‐BPs1‐6). They promote cell growth and antiapoptosis, mainly through the MAPK pathway and PI3K/protein kinase B pathway, thereby promoting tumor development and progression. Overexpression of IGF1R has been shown in canine OS and is strongly linked to tumor staging and dismal outcome.Cixutumumab (CIX) is an anti‐IGF‐1R monoclonal antibody that functions by binding to IGF‐1R with strong affinity, thereby blocking the interface between IGF‐1R and its ligand and inducing internalization and degradation of IGF‐1R.Human epidermal growth factor receptor 2 (HER‐2) is a tyrosine kinase receptor and a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. HER‐2 is remarkably correlated with tumor growth, and targeted therapies for HER‐2 have been used in some solid tumors.Multitarget TKI of EGFR are the fastest advancing class of drugs for targeted therapy of OS. Its mechanism of action is to inhibit the autophosphorylation of intracellular zone receptor tyrosine kinase or competitively suppress the binding of receptor tyrosine kinase ATP to the EGFR site, blocking downstream signaling and is primarily used in the clinical management of advanced non‐small cell lung cancer (NSCLC). Studies on OS have shown that gefitinib significantly reduces the survival of OS cells from starvation stress and can be used as an adjuvant agent to enhance the anti‐OS activity of adriamycin and methotrexate.4.1.2The EGFR family belongs to type I transmembrane tyrosine kinase receptors, including EGFR/ErbBl/Her‐1, ErbB2/Her‐2, ErbB3/Her‐3, and ErbB4/Her‐4. The EGFR also performs a fundamental function in numerous types of cancers. In NSCLC, the driver mutations consist of EGFR mutations, particularly exon 20 insertions and point mutations.Cetuximab, a monoclonal antibody against EGFR, blocks EGFR pathway signaling mainly by competitively suppressing the binding action of EGFR to related ligands, which can kill EGFR‐positive OS cells by enhancing the cytolytic function of NK cells and inhibit distant tumor invasion and angiogenesis.4.1.3PDGF/PDGFR is a significant signaling pathway with remarkable involvement in the proliferation and differentiation of osteoblasts and osteoclasts. Additionally, PDGF/PDGFR performs a central function in the process of tumor angiogenesis by activating downstream signaling molecules through the formation of dimeric complexes by PDGF and PDGFR binding to trigger receptors.Imatinib is a potent TKI that functions by targeting PDGFR signaling and was originally used to treat chronic myeloid leukemia.4.1.4mTOR is a downstream regulator of PI3K, which is implicated in multiple regulatory functions like cell survival, proliferation, metastasis, and vascular regeneration. Studies have confirmed that mTOR signaling is aberrantly activated in OS and its inhibitors can exert antitumor effects by inhibiting cell growth and proliferation.Ridaforolimus rapamycin and its analogs are first‐generation inhibitors of mTOR, inhibiting mTORC1 kinase activity by binding to FKBP‐1.Everolimus, a 40‐O‐(2‐hydroxyethyl)‐rapamycin derivative, impairs the proliferative potential of OS cells by stopping the G1 phase of the cell cycle. A phase II clinical trial illustrated that the proportion of patients with high‐grade OS treated with sorafenib in conjunction with everolimus increased from 27 to 45% of patients with a median PFS duration of 6 months compared with sorafenib alone, thus showing that the survival benefit of sorafenib combined with everolimus for high‐grade OS was superior to sorafenib monotherapy.In OS xenograft animals, second‐generation dual mTOR inhibitors with weak antitumor activity included AZD8055, which inhibits both mTOR1 and mTOR2. Nonetheless, additional new small molecules inhibitors, like dual PI3K/mTOR antagonists  and dual mTOR/DNA‐PK inhibitors (CC‐115), are still being studied in clinical trials to determine their efficacy.4.1.5Angiogenesis is primarily modulated by VEGF/VEGFR signaling,Sorafenib is a specific TKI that targets the VEGFR pathway. Heymann et al.Cabozantinib, a VEGFR2 and MET inhibitor, was evaluated in a phase II clinical trial involving 43 patients with OS, 39 of whom had pulmonary metastases, the 6‐month PFS rate was 33% and only five patients had partial responses, a finding similar to that of the sorafenib trial.4.1.6The hallmark of tumor cells is the loss of cell cycle regulation, resulting in uncontrolled cell proliferation. Aurora kinase has functions such as regulating cell mitosis, and abnormal expression or activation of aurora kinase results in the transformation of normal cells into tumor cells.4.2Over the past 10 years, immunotherapy has revolutionized the way that cancer is treated, and immunotherapy for OS has developed at a breakneck pace. Major cancer immunotherapies include peripatetic immune cell therapy, checkpoint inhibitors, and cancer vaccines, among others. Several solid tumor types can be treated with checkpoint inhibitors. Clinical investigation of other adaptive immune cell therapies and cancer vaccines in solid tumors is ongoing.4.2.1Researchers have recently developed monoclonal antibodies that are directed against CTLA‐4 and PD‐1/PD‐L1, and they have improved effectiveness in treating malignancies like melanomaA phase II clinical trial of camrelizumab in conjunction with apatinib for advanced OS also failed to meet the 6‐month PFS preset goal.Another important checkpoint is CTLA‐4, a transmembrane glycoprotein receptor expressed in regulatory T cells and memory T cells that inhibits antitumor immune response by interacting with CD80/86 on DCs.4.2.2Relay immune cell therapy is another area of immunotherapy that is rapidly evolving, and CAR‐T therapy is at the heart of this. CAR‐T cells are highly specific and targeted for cytotoxicity, using the immune system's capacity to track down, spot, and eliminate cancerous cells.TIL is another form of passaged cell therapy, and studies have shown satisfactory objective response rates of 40 to 70% in patients with metastatic melanoma to TIL.4.2.3Tumor vaccines are used to achieve antitumor effects by inducing a strong immune response against tumors. A widely used tumor vaccine is the DC vaccine, which is an APC that induces the proliferation of cytotoxic T lymphocytes to achieve antitumor effects.5Currently, the signaling pathways related to the development of OS are increasingly being investigated by humans, whether it is PI3K/AKT/mTOR signaling pathway, Wnt signaling pathway, JAK/ STAT3 signaling pathway, Hippo signaling pathway, Notch signaling pathway, PD‐1/PD‐L1 signaling pathway, MAPK signaling pathway, or NF‐κB signaling pathway (Table The development of pulmonary metastasis from OS is not something anyone wants to foresee, but the highly active and highly metastatic nature of OS cells leads to early metastasis to distant tissues and can lead to a range of malignant symptoms, which may further impact the prognosis and the rate of survival for OS patients. Although the patients’ prognoses in OS have not changed significantly in recent decades, and pulmonary metastasis of OS remains a major challenge to curing OS and improving patient survival, various therapeutic approaches have made some progress and have yielded important clinical benefits. Targeted therapies for OS are safer and more effective than chemotherapy. For OS‐targeted therapy, the development of more potent medications and the identification of biomarkers with higher sensitivity and specificity remain the major priorities Table . With coThere are few relatively satisfactory clinical chemotherapy regimens available, and collaborative multiparty studies are needed to identify a chemotherapy regimen that will improve patient prognosis, while basic, translational, and clinical research teams collaborate, leading to the development of new prognostic markers and new therapeutic targets. No single immunotherapy has been definitively shown to be effective in OS, and the clinical efficacy of a single immunotherapy is inadequate, but there are clear studies demonstrating an immune link between OS progression and immunity, so in‐depth studies of immune mechanisms and the development of new immunotherapies are a priority in immunology research. Pain management is also important, not only to improve symptoms but also to benefit patients physically and mentally and their recovery. As immunotherapy and gene therapy are expected to provide more opportunities and possibilities for the treatment of OS, the combination strategy of multiple therapeutic approaches is a hot topic of current research..This review also focuses on the signaling targets involved in the preventive and curative effects of TCM on OS through the modulation of related protein and gene expression in signaling pathways. Although the research results of TCM for OS are encouraging, most of the current experimental studies focus on the impact of the drugs on OS cells in the context of inhibiting proliferation or promoting apoptosis, and only a few research reports have specifically examined the impact on metabolism and immunity, which is relatively single‐directional research, and it is currently difficult to achieve clear clinical efficacy. Regarding the action mechanism of TCM in OS therapy, most of the TCM compounds are still under the general framework of multitarget and multipathway synergy, and their specific targets have not been fully elucidated. Given the complex and multitarget components of TCM, more extensive research is required. to realize the synergistic effects of multiple TCM extracts to achieve multitarget, multipathway, and multifaceted treatment of OS. Moreover, future studies should focus on comprehensively revealing the mechanism of action of herbal components and compound formulations from a holistic perspectiveWriting—original draft: Ji Ziyu. Writing—original draft, visualization, funding acquisition: Shen Jianlin. Methodology, writing—review and editing, funding acquisition: Yi Qian. Conceptualization, writing—review and editing, supervision, funding acquisition: Liu Huan. Draw figure: Yujian Lan. All authors have read and approved the final manuscript.The authors declare no conflicts of interest.Not applicable.


The human T-cell leukemia virus type 1 (HTLV-1) is the only known human oncogenic retrovirus. HTLV-1 can cause a type of cancer called adult T-cell leukemia/lymphoma (ATL). The virus is transmitted through the body fluids of infected individuals, primarily breast milk, blood, and semen. At least 5–10 million people in the world are infected with HTLV-1. In addition to ATL, HTLV-1 infection can also cause HTLV-I-associated myelopathy (HAM/TSP). ATL is characterized by a low viral expression and poor prognosis. The oncogenic mechanism triggered by HTLV-1 is extremely complex and the molecular pathways are not fully understood. However, viral regulatory proteins Tax and HTLV-1 bZIP factor (HBZ) have been shown to play key roles in the transformation of HTLV-1-infected T cells. Moreover, several studies have shown that the final fate of HTLV-1-infected transformed Tcell clones is the result of a complex interplay of HTLV-1 oncogenic protein expression with cellular transcription factors that subvert the cell cycle and disrupt regulated cell death, thereby exerting their transforming effects. This review provides updated information on the mechanisms underlying the transforming action of HTLV-1 and highlights potential therapeutic targets to combat ATL. Orthoretrovirinae and is endemic to the southwestern part of Japan, South America, the Caribbean, the Middle East, Australo-Melanesia [The human T-cell leukemia virus type 1 (HTLV-1), also known as human T-lymphotropic virus type 1, was the first human retrovirus discovered. It belongs to subtype Delta of the subfamily elanesia . HTLV-1 elanesia ,4. In adelanesia . To undeelanesia . This regag, pro, pol, and env, which encode proteins essential for viral replication [The HTLV-1 genome consists of a small positive ss(+) RNA with a size of approximately 9 kB. After infection, HTLV-1 is integrated into the host DNA as a provirus, similarly to HIV-1. Both the sense strand and the anti-sense strand of the integrated provirus can be transcribed . The virlication ,11,12,13lication  and indulication , cellulalication , biofilmlication , and extlication ,19. Viralication , as detalication . It has lication . HTLV-1 lication , and flulication . Studieslication . Neverthlication . The meclication  for furtATL develops in 5% of individuals infected with HTLV-1 and is characterized by lymphadenopathy, skin lesions, hypercalcemia, and severe involvement of organs, such as the central nervous system, lungs, liver, spleen, and bone marrow. Survival prognosis is poor and may be as short as 6 months. ATL patients respond poorly to traditional anti-leukemia chemotherapy. Rather, more favorable responses have been achieved by combined treatment with zidovudine (AZT)plus interferon (IFN)  or allogOne of the most important factors in the transformation and pathogenesis of HTLV-1 is the multifunctional viral protein Tax. This protein has the ability to interact directly with a variety of cellular proteins, including transcription factors, cell signaling proteins, cell cycle regulators, apoptotic proteins, and DNA damage response factors. Relevant to the aim of this review is the assumption that Tax positively modulates the expression of Bcl3 through activating the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway . Even thIn addition to regulating viral transcription, Tax regulates the expression of several signaling molecules involved in the processes of oncogenesis, proliferation, immune response, and apoptosis. Among these, the nuclear transcription factor NF-κB plays a central role in coordinating the various cellular signals. The prototypical NF-κB complex corresponds to a heterodimer of the p50 (NFKB1) and RelA (p65) members of the NF-κB/Rel family of transcription factors . Tax indHTLV-1 infection can remain latent for years before full disease onset, suggesting that there are sophisticated mechanisms regulating the on/off switching of viral protein expression, but also the activation of genes related to the T-cell receptor/NF-κB and signaling related to immune surveillance, such as HLA and FAS . In addiTax is not the only factor responsible for the various changes observed in viral expression and cell fate during infection. As already indicated in Other accessory proteins play a selective role in HTLV-1 leukemogenesis. The p30 counteracts viral transformation by inhibiting the export of tax/rex mRNA from the nucleus , while pTaken together, these data fit with the apparent paradox of a dual role of Tax in apoptotic cell death in the early phases preceding the completion of the transformation process triggered by HTLV-1. To shed light into this subject, some of us pioneered the experimental model of HTLV-1 infection in vitro several years ago. Our model consisted of a long-term culture of PBMCs from healthy donors after exposure to irradiated HTLV-1-infected cells as viral donors . We feltA recent study investigated the association between the early transcription of the positive viral strand, viral burst, and expression of pro- and anti-apoptotic genes in two naturally infected T-cell clones from patients transduced with a Tax-responsive timer protein . The resInterestingly, apoptosis may play a role in the awakening of the virus from latency. It has been provocatively hypothesized that, in cells that are prone to apoptotic RCD in response to infection, apoptosis itself may awaken viral latency and promote viral replication through the direct upregulation of caspase 9. This protein, in addition to having a proapoptotic effect, may form a complex with Sp1-p53 and activate viral LTR . On the Simplified schematics of the complex and elusive mechanisms leading to immortalization and transformation processes induced in HTLV-1-infected cells are summarized in Although the mechanisms of HTLV-1 immortalization/transformation have been extensively studied, it has not been possible to find a unique, suitable candidate treatment. This may be due to the large number of targets that could potentially be hit. The disease starts after the specific HTLV-1 infection of CD4+ cells. Therefore, hypothetically, a wide range of potential targets could be found within the viral genome/proteins, as well as among the host cell genes transcribed during infection that are highly subjected to virus-induced dysregulation. In particular, the specific cell signaling activated by HTLV-1 in infected cells includes multifunctional factors/complexes that could serve as targets for pharmacological intervention. Accordingly, several inhibitors of host cell signaling have recently been proposed in in vitro/ex vivo studies as potential anti-ATL therapeutics. These include apigenin, as an inhibitor of the aryl hydrocarbon receptor and transcription factor that enhances the cytotoxicity of antiretroviral drugs, and dimethyl fumarate as an inhibitor of the so-called CBM complex ,87. AnotStudies conducted in ATL patients have shown that an important goal is to induce cells to undergo apoptosis or generally move toward RCD, in response to therapy. Among the various ways to undergo RCD, interaction with the autophagy pathway should be considered. Autophagy may actually play a dual role in RCD, either initiating cell death or maintaining it . The autPotential targets could be identified not only in genes directly involved in cell signaling but also among regulators of gene expression. Recently, 12 miRNA associated with the regulation of key cell signaling genes in ATL cases have been identified, thanks to a bioinformatics approach . Based oAs mentioned in the previous section, even latency reversion may represent a potential strategy for a novel HTLV-1/ATL-targeted therapy. Latency could possibly be reversed by activating the expression of viral antigens that are likely to be recognized by the immune system. This would allow the recruitment of effector cells capable of knocking out the virus. Recently, a treatment to reverse latency has been proposed by using the histone deacetylase inhibitors (HDACi) panobinostat and romidepsin . These iA summary of the main suggestions for HTLV-1-targeted therapy is provided in CRISPR/Cas9 genome editing is a novel technology that uses a guide RNA (gRNA) to precisely cleave double-stranded DNA at a specific site. After cleavage, double-stranded DNA breaks in human cells are normally repaired by the error-prone NHEJ pathway . This caAlthough retroviral proviruses are largely restricted to HIV-1, HTLV-1 offers more sites for gRNA targeting than HIV-1 due to its highly conserved viral genome with remarkable sequence homogeneity. Moreover, as shown in Unfortunately, although some progress has been made in slowing its progression, ATL is still an incurable disease. However, in recent years, considerable progress has been made in defining the mechanisms at the molecular and genetic levels associated with the events underlying the complex viral/cellular system that leads to the selection of the transformed clone determining the onset of ATL in HTLV-1-infected patients. The definition of these mechanisms has already led to the identification of new therapeutic targets and corresponding agents that can act on them. Therefore, this seems to be an indication of how new therapeutic strategies may be found to counteract ATL in the near future. Nevertheless, a precise prediction of if and when it will actually be possible to use therapeutic treatments that can prevent or control ATL and potentially transform HTLV-1 infection into a chronic disease is currently not possible.


Fertility preservation in cancer patients is currently based on either assisted reproductive technology or fertility-sparing surgery. Loss of fertility may be caused by excisional surgery associated with an adnexal or uterine pathology or secondary to gonadal insufficiency caused by chemotherapy or radiation. The counseling of these patients is very important, being carried out jointly by the oncologist, gynecologist, and reproductive medicine specialist. Reproductive surgery usually requires avoiding laparotomy to significantly reduce the formation of adhesions and trauma or tissue damage. This is done using standard laparoscopic surgery or robotic surgery (computer-assisted laparoscopy), a method increasingly used and accessible to all specialists who want to maintain the fertility of their patients with various oncological diseases. Adolescents and young adults (AYA) with cancer are often unaware that their life-preserving treatments can also threaten their future fertility potential –3. Oncofet al. concluded that post-chemotherapy AMH levels could be a relatively accurate predictor of ovarian function recovery, as indicated by the resumption of menstruation, in breast cancer patients with chemotherapy-induced amenorrhea [In women, chemotherapy is associated with a decrease in anti-Müllerian hormone (AMH) levels and an increase in follicle-stimulating hormone (FSH) levels, indicating a decline in ovarian reserve and potential impairment of fertility , 7. Decrenorrhea .et al. [et al. [et al. [et al. [et al. [et al. [Current studies revealed the risk-stratified priority in cancer patients to optimize care among various malignancies; thus, Smith et al.  on breas [et al.  on urolo [et al.  and Pass [et al.  on onco- [et al.  and Colo [et al.  on gynecFor patients with oncological diseases, the surgical options that preserve fertility are mainly addressed to four major types of gynecological cancers . In recent years, some recommendations and innovative experimental research have been implemented on fertility conservation techniques in these patients.For early stages of cervical cancer, cold knife tanning, loop electrosurgical excision procedure (LEEP), simple or radical trachelectomy with a permanent strap and sentinel node mapping, or extra fascial hysterectomy with ovarian preservation is recommended. In advanced diseases where chemotherapy is necessary, preservation of oocytes or ovarian tissue could be performed. Gonadotropin-releasing hormone (GnRH) agonists have also been used for their potential to temporarily suppress ovarian function during cancer treatment, thereby reducing the risk of ovarian damage and preserving fertility. By suppressing ovarian function, GnRH agonists may protect the ovaries from the toxic effects of chemotherapy or radiation therapy . If radiTreatment for the early stages of endometrial cancer requires hysteroscopic tumor resections and progestin therapy with mandatory endometrial sampling every 3-6 months. If cancer regression occurs, therapy will be stopped to allow conception; otherwise, an ovarian preservation hysterectomy is recommended. In the latter case, a gestational carrier can use assisted reproduction technology with the patient's oocytes .In the case of ovarian cancer, conservative surgery includes unilateral/bilateral salpingo-oophorectomy with lymphadenectomy and uterine conservation, depending on the extemporaneous anatomopathological result. If the situation recommends the use of adjuvant chemotherapy, the potential gonadotoxicity could cause a degree of ovarian failure of 3-10%. Due to the increased risk, cryopreservation of oocytes can be considered only in patients with unilateral damage, and in the early stages, an option may be the artificial ovary . This onThe progress of fertility-preserving surgical techniques in gynecologic cancers has been associated with progression-free survival rates and overall survival compared with more radical surgeries and in full accordance with patients' reproductive desires without compromising their safety.For female patients, embryo/oocyte cryopreservation before starting anticancer therapies is the first option to be discussed, but if the chemotherapy is urgent, surgical ovarian tissue cryopreservation may be indicated. A surgical ovarian transposition procedure is recommended before pelvic radiotherapy; temporary ovarian suppression with luteinizing hormone-releasing hormone agonists during the chemotherapy is a simple at-home pharmacological intervention . The nexThe last few years have represented a new way of thinking about fertility surgery in patients with certain types of cancer, both about introducing innovative surgical techniques  and some major discoveries such as tissue transplantation, thawed cryopreserved ovary, artificial ovary, and uterine transplant . This re®/MEDLINE database scan.In the present study, we performed a narrative review with a search on the following topics: (1) “fertility preservation,” (2) “oncofertility,” (3) “fertility-sparing surgery,” (4) “cancer patient” - published in peer-reviewed journals and written in English within a ten-year timeframe. We applied the search with filters: Clinical Trial (CT), Randomized Controlled Trial (RCT), and Clinical Case Series (CCS). The review methods of the search were previously established and involved PubMed® 2013 .The following data were extracted: author (s), year, country of publication, the aim of the study, study design, and main results. Two authors (V.N.V. and R.G.B.) extracted and analyzed the data. Over 100 studies were identified and screened for eligibility. According to the topic search, data extracted included demographic variables, number of participants in the study, treatment, side effects profile, and associated comorbidities. Twenty-nine papers were included in the present study, centered on the main topics included in the search. The statistical analysis used Microsoft Excel® search retrieved only 2943 all-time results, 2117 from the ten-year span between 2012 and April 2021, with 117 meta-analyses and systematic reviews, and 53 randomized controlled trial and clinical trials. The combined search terms “fertility preservation” and “oncofertility” revealed 337 results, with 4 CT/RCT; “fertility preservation” and “fertility-sparing surgery” retrieved 2250 results with 43 CT/RCT.Despite the plethora of published papers featuring the keywords “fertility preservation’’ and ‘’cancer patient” during the preliminary search, the PubMedThe evaluation of the last ten years refined to RCT/CT observed the constant interest in young cancer patient topics and a scarcity of data regarding the fertility-sparing surgery and oncofertility topic .The importance of preserving fertility is given by the overall survival rate of children treated for cancer that exceeds 80%, which explains the growing number of survivors . The theCryopreservation of ovarian tissue is a technique for preserving fertility addressed to girls and women before initiating gonadotoxic treatments. It is the only possible method for prepubertal girls. The technique was proposed in 1996 and is aimed especially at young patients with various oncological conditions: leukemia, Hodgkin's lymphoma, bone tumors, nephroblastoma, retinoblastoma, and neuroblastoma. It has the advantage that it is immediately available and does not require the administration of hormonal drugs. The preserved ovarian tissue is slow-frozen or vitrified and thus can be used later to restore reproductive and endocrine function in the absence of cancer recurrence , 27. Howet al. emphasized the time-sensitive nature of fertility preservation, as it requires a delay in cancer treatment initiation [Moreover, the research of Letourneau itiation . This deitiation –34 becauitiation , 36, whiOvarian transposition is a surgical procedure that allows fertility preservation and is recommended for women with gynecological oncological conditions  and urological or hematological diseases that require pelvic or craniospinal radiotherapy . AlthougGiven the secondary gonadotoxicity of aggressive neoadjuvant chemotherapy regimens, the time factor defines these patients, who tend to lose their fertile potential quickly. Thus, cancer patients must start their systemic anticancer treatment immediately, in parallel with evaluating the opportunity to initiate oncofertility-related procedures. The fertile prognostic of young cancer patients was negatively influenced by less surgical training in oncologic surgery and few oncology clinical trials .Patients with cervical cancer with tumors <2 cm (IA2-IB2 FIGO) undergoing the radical vaginal trachelectomy - Dargent procedure have a survival rate identical to those undergoing radical hysterectomy. Patients with tumors >2 cm cannot be prescribed the Dargent procedure due to the increased risk of central pelvic recurrence (approximately 17%). The need for parametrectomy is debatable in patients at low risk of parametric invasion due to urinary and digestive complications and the subsequent risk of miscarriage or premature birth .According to the NCCN, for stage IA1 without lymphovascular space invasion (LVSI), simple cone resection or hysterectomy is recommended. In contrast, for stages IA1 with LVSI, IA2 - IB1, simple/radical trachelectomy in association with sentinel node biopsy or pelvic lymph dissection is indicated . The SEEet al. reported a >97% overall survival rate or absence of progression at 5 years in patients with tumors <2 cm who opted for fertility preservation through simple trachelectomy or cone resection with laparoscopic identification of the sentinel node ± pelvic lymph dissection [et al. indicated radical trachelectomy in the presence of IB1 stage LVSI [Plante ssection . Bentiveage LVSI . In contage LVSI .The correct selection of patients , the experience of the surgical team, the correct choice of surgical procedure, the technical possibilities of the center of excellence in gynecological oncological surgery, are elements of favorable prognosis on the preservation of fertility.Patients who have received FSS for cervical cancer may later experience several risk factors, such as decreased fertility rate, increased risk of miscarriage, cervical failure, risk of chorioamnionitis, premature rupture of membranes, and the onset of preterm labor . If cervIn patients who have undergone cone resection, a vaginal birth may be attempted in the absence of residual disease or obstetrical contraindications, as opposed to trachelectomy in which a cesarean section must be performed as the small scarred cervix is at risk of rupture. , 54. ThuEndometrial cancer (EC) is the fourth most common cancer in women, being the most frequent gynecological cancer . Conservet al. evaluated the safety of fertility in young women with atypical endometrial hyperplasia (AEH) or grade 1 endometrial cancer (EC). The EC's oral treatment with progestin (medroxyprogesterone acetate and megestrol acetate) is accompanied by a recurrence rate of 30.7% and a pregnancy rate of 52.1%. Other treatment options include LNG-IUD, megestrol acetate plus metformin [et al., on a group of 418 patients with AEH and grade 1-2 endometrioid EC with minimal or no myometrial invasion on MRI, shows that hormone therapy with LNG-IUD is better than the MPA-containing regimen [In a review of the literature, Garzon etformin , 59, andetformin . Thus, tetformin . Gullo'setformin . Novikov regimen .Despite this evidence, the lack of randomized clinical trials on the efficacy and safety of treatment in EC in relation to the risk of worsening oncological disease explains the cautious way to preserve fertility in these patients . For obeGnRH agonists, such as leuprolide acetate, goserelin, and triptorelin, can be used to induce a temporary menopausal state, suppress endogenous estrogen production, and potentially preserve fertility. By reducing the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland, GnRH agonists effectively suppress the ovarian production of estrogen, which is essential for the growth and progression of endometrial cancer. Lower estrogen levels create an unfavorable hormonal environment for cancer cells, potentially leading to disease regression and preservation of fertility .Metabolic syndrome and insulin resistance are associated with an increased recurrence rate in patients with AEH and EC who have resorted to fertility-sparing procedures .After primary conservative treatment for patients with recurrent EC and EAH who wish to preserve their uterus after complete remission, resumption of treatment that preserves fertility may be an option as it allows the completion of pregnancy and delivery .Ovarian cancer is the most aggressive and with the highest mortality among gynecological cancers . FertiliAfter staging, FSS in patients with EOC will address stage IA of grade 1/2 of serous, mucinous, or endometrioid tumors or stage IC grade 1. For EOC in stage IA grade 3 or IC1/IC2 grade 1, an option is given by bilateral salpingo-oophorectomy, preservation of the uterus, and the use of donated oocytes . In patiet al., using a multicenter database, identified 56 babies born to 40 malignant ovarian germ cell tumor (MOGCT) survivors from 110 MOGCT patients [Two retrospective studies of 79 patients and 27 patients with non-epithelial cancers , regardless of tumor stage and histological type, showed that fertility preservation surgery with adjuvant chemotherapy was followed by a high fertility rate , 70. Thepatients .Sexually stromal cord tumors diagnosed in young women benefit from FSS, with a risk of premature ovarian failure, a situation that would require cryopreservation of ovarian tissue . The AGOet al. presented a case of a 33-year-old woman diagnosed with synovial cell sarcoma of the vulva. Following radical hemi-vulvectomy with bilateral lymph dissection and subsequent brachytherapy (20 Gy) and external radiotherapy (50 Gy), her fertility was preserved, leading to a later successful childbirth [Vulvar cancer accounts for 4% of gynecological malignancies, with a peak incidence of in situ vulvar carcinoma in the 40-49 age group. The anatomopathological form in women under 45 is vulvar squamous cell cancer , 76. Theildbirth .In patients under 40, the incidence of breast cancer is over 7% of all oncological conditions. The main options for fertility preservation in women with breast cancer are oocyte or embryo cryopreservation. Also, patients who want to maintain their fertility before breast radiotherapy have two options: either to use shields for the adnexal area or to transpose the ovaries, although the radiation doses that reach the genital organs are small without affecting ovarian function. Some women choose mastectomy as their first intention, precisely to avoid radiotherapy and its possible effects on fertility , 80. Obset al. [A recent systematic review by Lambertini et al.  aimed toet al. [However, many authors argue that the available evidence is insufficient to determine the impact of GnRH agonists on fertility preservation, and further investigation is needed –84. The et al.  suggesteDelaying a pregnancy or lowering the age of onset of cancer has revolutionized medicine by developing various surgical interventions to preserve fertility. The correct intervention choice depends on the cancer's location, type, and stage. Thus, regarding the chances related to fertility, an important role belongs to the counselors, a process that must be initiated by the gynecologist and continued by the surgeon, oncologist, chemotherapist, radiotherapist, and psychologist so that in the end, the integrative role to belong to the specialist of reproductive medicine that will balance the benefits versus risks for each case. This is how the multidisciplinary approach to oncofertility is defined. Fertility-sparing surgery has been, is, and will be an additional challenge for cancer care and fertility. The influence can be found at the level of any link within this multidisciplinary team, mainly in initiating aggressive gonadotoxic therapy.


Recently, the identification of the phytomelatonin receptor PMTR1 in Arabidopsis has been considered a turning point in plant research, with the function and signal of phytomelatonin emerging as a receptor-based regulatory strategy. In addition, PMTR1 homologs have been identified in several plant species and have been found to regulate seed germination and seedling growth, stomatal closure, leaf senescence, and several stress responses. In this article, we review the recent evidence in our understanding of the PMTR1-mediated regulatory pathways in phytomelatonin signaling under environmental stimuli. Based on structural comparison of the melatonin receptor 1 (MT1) in human and PMTR1 homologs, we propose that the similarity in the three-dimensional structure of the melatonin receptors probably represents a convergent evolution of melatonin recognition in different species.Phytomelatonin is a pleiotropic signaling molecule that regulates plant growth, development, and stress response. In plant cells, phytomelatonin is synthesized from tryptophan  N-acetyl-5-methoxytryptamine) is an evolutionarily conserved chemical molecule that was first discovered in the bovine pineal glands in 1958  signaling or via mitogen-activated protein kinase (MAPK) cascades  to form N-acetylserotonin, which is then converted into phytomelatonin by N-acetylserotonin methyltransferase (ASMT) in aquatic algae or caffeic acid-3-O-methyltransferase (COMT) in land plants (Plants possess both a major biosynthetic pathway (MP) and an alternative biosynthetic pathway (AP) for phytomelatonin . Phytomed plants   and 6-hydroxymelatonin (6-OHM) by the cytochrome P450 enzyme, phytomelatonin can be metabolized into other metabolites in an enzymatic or other biochemical manner . The preN1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) (2O2 results in the oxidative cleavage of its indole ring and produces AFMK (N-acetyl-5-methoxykynuramine (AMK) with the help of electron/hydrogen-abstracting radicals or by reacting with indoleamine 2,3-dioxygenase (IDO) (N1-acetyl-5-methoxy-3-nitrokynuramine (AMNK) in the presence of nitric oxide (NO) or 3-acetoamidome-thyl-6-methoxycinnolinone (AMMC) in the presence of peroxynitrite (N-acetylserotonin deacetylase (ASDAC), which is also responsible for the conversion of N-acetylserotonin into serotonin  . Similarces AFMK . AFMK isse (IDO) . This reynitrite . It has erotonin .Arabidopsis  have been shown to facilitate the transportation of melatonin into the mitochondria, where it induces cell apoptosis . Melatonuberosum . WhetherArabidopsis (TAA)  . Tryptopis (TAA) . Consequis (TAA) . Moreoveis (TAA)  , tobacco (Nicotiana benthamiana), maize (Zea mays), and cassava are localized in the plasma membrane and are required to perceive phytomelatonin signaling in salt, immunity, drought, and leaf senescence, respectively  under a guard cell-specific promoter (GC) loses the circadian rhythm-mediated stomatal movement  significantly inhibited seed germination  genes in the ABA biosynthetic pathway  and inhibited the expression of IAA17, AXR3, and NAC, which decreased the expression of SEN4 and S4G12 and delayed leaf senescence  that is responsible for starch degradation in tomatoes , are known as pattern recognition receptors (PRRs) . Plants Rhizoctonia solanacearum and enhanced the resistance of apple trees to brown spot disease (Pseudomonas fluorescens (Pst DC3000) infection increased the concentration of endogenous phytomelatonin in A. thaliana, and exogenously applied phytomelatonin induced the production of defense signals, such as NO and SA, that are responsible for plant defense against Pst DC3000 infection (Botrytis cinerea in cherry tomato fruit and enhanced the resistance to tobacco mosaic virus (TMV) in Solanum lycopersicum by activating the SA pathway  plays an essential role in basal defense and systemic acquired resistance (SAR) partly by promoting the closure of the stomata, which limits the entry of pathogens . Phytome disease . It has nfection . Similar pathway .PR1, ICS1, and PDF1.2, which were found to decrease in snat1 mutants compared to wild-type Arabidopsis during pathogen infection , pathogenesis-related gene 1 (PR1), and pathogenesis-related gene 5 (PR5)  . However2 assimilation for photosynthesis and water loss during respiration, the stomata are regarded as the main sites for foliar pathogen invasion. Plants have evolved a mechanism to close the stomata after sensing PAMPs/MAMPs in order to restrict pathogen invasion, known as stomatal defense or stomatal immunity  and BAK1 (Brassinosteroid Insensitive 1-Associated Kinase 1) immune receptor signaling pathways , which can improve the leaf photosynthetic efficiency, alleviate oxidative stress, and slow down drought-induced leaf senescence  were upregulated, while chlorophyll catabolism genes  were significantly downregulated  were significantly upregulated   . It alsoFv/Fm) .+ leads to oxidative stress-induced membrane damage   .MPK3, MPK4, and MPK6) was induced by phytomelatonin during salt stress in cucumbers , phytomelatonin showed similar function in stomatal closure and signaling to ABA. These signaling processes include the GPA1-mediated activation of NADPH oxidase-dependent ROS production, Ca2+ transient influx, and cascades that are activated by MAPK (pmtr1 mutants lost circadian stomatal closure, but remained sensitive to ABA-induced stomatal closure. Given that phytomelatonin affects ABA metabolism and that ABA is known to regulate osmotic stress response (Similar to the classical model of ABA regulation for stomatal closure, it was found that  closure . In Arab by MAPK . However by MAPK . Further by MAPK . It has response , their cArabidopsis, AtPMTR1 was the first putative phytomelatonin receptor discovered in plants that regulates stomatal closure. More recent related studies have shown that phytomelatonin-mediated seed germination and seedling growth, leaf senescence, and resistance to several biotic and abiotic stresses are dependent on PMTR1. Therefore, PMTR1-mediated phytomelatonin signaling may play varied roles in plant growth, development, and stress resistance.Phytomelatonin is widely present in plants and functions throughout their entire growth and development stages. In However, there are still two overarching scientific issues that need further investigation in future work, as follows:2+, have been discovered, but their regulatory mechanisms are still not fully understood. In the future, research will need to focus on the mechanisms that link PMTR1 receptors and MAPK cascades in order to illustrate how activated and phosphorylated MPK3/6 are released when phytomelatonin signaling stops. Moreover, it remains unclear how PMTR1 recognizes and transduces phytomelatonin signals. Determination of the crystal structure of PMTR1 will shed light on the interactions between PMTR1 and its ligands. Furthermore, future work should also explore whether there are other receptors that mediate phytomelatonin signaling. Ultimately, the identification of other players that interact with PMTR1 and the components that integrate phytomelatonin signaling with other plant hormones should be carried using quantitative proteomics and liquid chromatography–tandem mass spectrometry analysis, which will greatly enhance our understanding of the cross talk between phytomelatonin and other signals.PMTR1-mediated signaling pathways and regulatory mechanisms. At present, only GPA1 and MAPK signaling members and signaling molecules, such as ROS and CaPMTR1 in crops through genetic modification and molecular breeding should be explored to improve the stress resistance of crops.The application of PMTR1 in agricultural production and crop breeding. Existing studies have shown that phytomelatonin plays an important role in plant growth, development, stress response, and many other functions that are dependent on the PMTR1 receptor. Therefore, changing the expression levels of QC and XP conceived this study. DK, NC, and XP wrote the manuscript. WZ, LL, and XP analyzed the data. XP, MG, and QC revised the manuscript. All authors contributed to the article and approved the submitted version.


Chromosomal instability (CIN), an increased rate of chromosomal segregation abnormalities, drives intratumor heterogeneity and affects most human cancers. In addition to chromosome copy number alterations, CIN results in chromosome(s) (fragments) being mislocalized into the cytoplasm in the form of micronuclei. Micronuclei can be detected by cGAS, a double-strand nucleic acid sensor, which will lead to the production of the second messenger 2′3′-cGAMP, activation of an inflammatory response, and downstream immune cell activation. However, the molecular network underlying the CIN-induced inflammatory response is still poorly understood. Furthermore, there is emerging evidence that cancers that display CIN circumvent this CIN-induced inflammatory response, and thus immune surveillance. The STAT1, STAT3, and NF-κB signaling cascades appear to play an important role in the CIN-induced inflammatory response. In this review, we discuss how these pathways are involved in signaling CIN in cells and how they are intertwined. A better understanding of how CIN is being signaled in cells and how cancer cells circumvent this is of the utmost importance for better and more selective cancer treatment. Distinguishing between CIN and aneuploidy is key to understand their independent contributions to tumor evolution and growth. CIN typically yields a heterogenous tumor cell population and provides cells with an ability to undergo selective evolution. However, when CIN rates are low, tumors can still be highly aneuploid with very little heterogeneity, potentially reducing their evolutionary capacity  or within a single tumor . One of the processes fueling intratumor heterogeneity is chromosomal instability (CIN) /stimulator of interferon genes (STING) Fig. , extensiet al. demonstrated that cGAS and a downstream type I interferon response are activated by chromatin bridges more than by micronuclei  affect its ability to mount a cGAS or other DNA sensor-dependent cell-intrinsic inflammatory response. For instance, Zierhut In addition to cytosolic DNA originating from the nucleus, other triggers can also activate tumor cell-intrinsic inflammatory signaling, such as mitochondrial DNA /signal transducer and activator of transcription (STAT) 1 and 3 network as well as canonical and non-canonical nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) signaling have been reported to be the major downstream players of CIN-induced inflammatory signaling. Although these factors are often reported to operate in separate pathways, it is becoming increasingly clear that these signaling cascades are very much intertwined Fig. .Fig. 2ThSTAT1 is a central mediator of both type I (ɑ and β) and type II (ɣ) interferons (IFNs), regulating an antiviral and immune defense transcriptional response and is considered to play a central role in antitumor immunity , i.e., segmental aneuploidies, as a predictor for the survival of patients after immunotherapy in a melanoma cohort; the somatic CNA levels were lower in patients with long-term survival compared to patients with short-term survival   and STAT1 signaling, respectively. These antagonistic effects of cell-intrinsic inflammatory signaling might explain why cancers rarely show loss of function mutations of cGAS and STING  CIN. Alternatively, reactivation of STAT1-mediated immune signaling in CIN+ cancers could provide a strategy to selectively treat aneuploid cancers. However, before such therapies will reach the clinic, further in vivo work and clinical trials confirming these hypotheses are required.Importantly, the interaction between CIN


Due to the significant increase in overall survival rates of childhood cancer, more awareness has been raised for the long-term consequences of treatment, including infertility. Currently, patients and their families are offered information regarding the risk of gonadal damage by paediatric oncologists and fertility counselling by fertility specialists regarding fertility preservation. However, the experiences of childhood cancer patients with oncofertility care are underreported. The available evidence reported in this review shows that female patients and survivors are variably satisfied with fertility information and report challenges in communication. They prefer to receive general information at diagnosis and detailed information later. Regrets are reported after refusal of fertility preservation. Patients and survivors are concerned about future children’s health, effect on relationships and lack of control over fertility. With the results from this review,  standards for information for paediatric cancer patients and families may be developed to improve fertility information and counselling for current and future childhood cancer patients and survivors. Background: Childhood cancer patients and their families are increasingly offered oncofertility care including information regarding their risk of gonadal damage by paediatric oncologists, fertility counselling by fertility specialists and fertility preservation options. However, experiences regarding oncofertility care are underreported. We aimed to summarize the available evidence of experiences of female childhood cancer patients and survivors regarding oncofertility care. Methods: Manuscripts were systematically identified using the PubMed and Embase database. From, respectively, 1256 and 3857 manuscripts, 7 articles were included and assessed, including risk of bias assessment. Outcome measures included data describing experiences of female childhood cancer patients and survivors, regarding fertility information, counselling and/or preservation. Results: Female patients and survivors are variably satisfied with fertility information, report challenges in communication with healthcare professionals and prefer to receive general information at diagnosis and detailed fertility information later. Regrets after fertility counselling are underreported, but are associated with refusing fertility preservation. Lastly, regardless of counselling, female patients and survivors report fertility concerns about their future children’s health and effect on relationships. Conclusion: Currently, the satisfaction with oncofertility care varies and female patients or survivors report regrets and concerns regardless of receiving fertility information or counselling. These results may help to improve the content of fertility information, communication skills of healthcare professionals and timing of counselling. A . A 26]. The search was updated on 4 March 2023, using the PubMed and Embase database and using the initial search in PubMed in With the updated PubMed search, 97 additional articles were identified, of which 45 abstracts were selected for full-text screening . Using tPublished manuscripts were included if they described female childhood cancer patients or survivors and their experiences  after fertility information, counselling or preservation at diagnosis, during or after end of treatment in a paediatric cancer setting . ChildhoRisk assessment of bias in non-intervention studies and qualitative research [From the included studies, the study characteristics, including study design, patient demographics and tumour types, age at study and at diagnosis, data collection and outcome measures were extracted. Outcomes are categorized in (1) experiences and satisfaction with oncofertility information and counselling, (2) regrets after fertility information and counselling and (3) concerns regarding patients’ fertility impairment, after fertility information, counselling or preservation. Fertility information is considered counselling when information is given by a fertility expert, such as a gynaecologist. Fertility information given by any other healthcare professional, such as a paediatric oncologist or nurse practitioner, is not considered counselling . The quaresearch ,35, incln = 19; 6 months–3 years after diagnosis) [n = 17; 3 months–14 years after diagnosis) [n = 110), defined as being off therapy for a variable time (0–2 months to >2 years) or >5 years after diagnosis [Four included studies reported data on both female and male cancer patients and/or survivors ,27,28,29agnosis) , both paiagnosis ,37. The iagnosis ,37,38. Tiagnosis ,37,38, siagnosis ,27,36,38iagnosis , renal tiagnosis ,28,36 aniagnosis ,37, or siagnosis ,37,38, fFor 75 of the 146 eligible female patients and survivors, it was reported that they recalled receiving fertility information, and 5 recalled that they had been counselled at diagnosis by a fertility expert  Table 3Table 3. n = NR) was at diagnosis [n = NR) welcomed the information, regardless of gonadal damage risk or fertility preservation options [n = NR) even reported they believed that the offer of fertility preservation was an expression of professional belief that they had a future [n = NR) preferred ‘broad’ information around diagnosis, provided that it was age-appropriate and participants (n = NR) appreciated the opportunity to ask questions and have a more detailed discussion later, if needed [n = NR) indicated that they would have found it helpful if information was offered repeatedly and in an unambiguous, low key manner [n = NR) appreciated the involvement of only a small number of professionals and family members in fertility discussions [The majority of female childhood cancer patients and survivors (61–96%) preferred more information on the effect of cancer treatment on their fertility at an early stage ,29,36,37iagnosis ,36. Pati options , some (na future . Particif needed . Furthery manner . Particicussions . n = NR) had difficulty understanding fertility information, even after they had received counselling and regardless of whether they accepted or refused fertility preservation options [Reported reasons for dissatisfaction included problems in communication with healthcare professionals; information provided in an insensitive manner; not revisiting the topic of fertility and insufficient, incorrect, unclear or confusing information regarding participants’ expected future fertility status or fertility preservation options ,27,29. Wn = 3) or uncertainty regarding fertility status, they contemplated the circumstances of the offer made, including timing of the offer, and questioned their decision [Regrets of female CCS after fertility counselling were underreported in the literature . Only Crdecision . It was decision  (p. 385)decision  (p. 385)n = 11, 100%) had concerns regarding the effect of potential fertility damage on future romantic relationships [n = NR) feel additional distress due to the perceived need to disclose their potential infertility early in a relationship, and this distress is associated with the fear of abandonment [Four studies reported on concerns childhood cancer patients and survivors have related to the expected fertility-related side effects of cancer treatment  13,28,3,37. Concn = 21), and seem to be associated with refusal of fertility preservation [The overall survival rate of childhood cancer has improved significantly ,2, whichervation . No concervation ,28,36,37n = 18; including two female CCS—aged 4 and 5 years at diagnosis), suggestions of CCS to improve fertility care included providing an informative leaflet or web-based information and access to fertility specialists, giving information as early as possible, but also at various stages during treatment and subsequent check-ups allowing patients/survivors the opportunity to discuss fertility at different stages of their lives [n = 3) indicated that they would have liked to know more about their fertility status and whether or not they might be able to have children [While the results remain consistent between most studies, the availability of and differences between oncofertility programmes ,37, a yoir lives . Ellis echildren . In linechildren ,40.n = NR) [n = 19) and their parents: three survivors reported challenges in communication with professionals, resulting in uncertainty regarding their future fertility [Dissatisfaction with fertility care was most often related to poor communication and/or incorrect, confusing, unclear or insufficient information. The relatively complex information regarding fertility and fertility preservation in women has been reported to be confusing and leading to difficulty understanding the information (n = NR) . Besidesn = NR) ,27,36,37n = NR)  reportedertility . n = 17) [n = 60). Decisional regret is shown to be correlated with decisional conflict (p < 0.0001) [Information on regrets in childhood cancer patients and survivors with and after fertility counselling is hardly available in the published literature. As regrets were only discussed in one article with a relatively small sample n = 17) , regrets , regretn = 11, 100%) [n = NR) experienced distress and frustration due to the (perceived) lack of control over their impaired fertility [n = NR) report concerns both when a child wish occurs [Fertility-related concerns reported by female childhood cancer patients and survivors include the risk of infertility 8/13, 62%), entering menopause prematurely, impact on the normal progression of puberty  , effect , 62%, en1, 100%)  reportedertility . These certility . Similarh occurs ,41, but h occurs . These ch occurs ,40,41. Ih occurs .This systematic review provides the first analysis of a subgroup of the population, female CCS. Male  or (younn = 43) included ≤75% female participants or participants diagnosed after 18 years, or did not provide a sub-analysis of female participants. In paediatric oncology, patients older than 18 are also treated according to paediatric treatment protocols. Therefore, patients aged 18 years were also included in this review. Due to the qualitative nature of several articles, these articles may have been subjectively biased and statements of included participants may not be generalizable and may differ from participants from other age groups or countries. We expect the results to vary between different risk groups . Only one study in this review stratified between risk groups [n = 5) believed that they had a low risk of gonadal damage, but this was not confirmed or further specified [Some gaps of knowledge still remain regarding female experiences of fertility counselling. Few studies have studied the experiences and perspectives regarding fertility care of female childhood cancer patients or survivors specifically. Many identified articles  and providing counselling by fertility experts in higher risk patients to ensure that oncofertility care is tailored to patients, regarding their age, gender, wishes and needs ,13. ThesIn conclusion, childhood cancer patients and survivors are variably satisfied with oncofertility care. Improvements can be made in providing personalized fertility risk information, repeating this information during treatment and survivorship, improving communication by the physician and stimulating fertility counselling by fertility experts. Regrets after declining fertility preservation have been reported as well as fertility-related concerns, regardless of counselling, including concerns for the health of future offspring. Larger studies are needed, reflecting on the experiences, concerns, regrets and satisfaction regarding fertility information, counselling and/or preservation, to improve the quality of oncofertility care.


Giardia duodenalis is a gastrointestinal protozoan ubiquitous in nature. It is a confirmed zoonotic pathogen, and cattle are considered a source of giardiasis outbreaks in humans. This study aimed to evaluate the prevalence and multilocus genotype (MLG) of G. duodenalis in dairy cattle in Central Inner Mongolia. This study was based on the small subunit ribosomal RNA (SSU rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed on 505 dairy cattle fecal samples collected in 2021 from six sampling sites and four age groups in Central Inner Mongolia to determine the prevalence and MLG distribution of G. duodenalis. The PCR results of SSU rRNA revealed that the overall prevalence of G. duodenalis was 29.5% (149/505) and that the overall prevalence of the diarrhea and nondiarrhea samples was 31.5% (46/146) and 28.5% (103/359), respectively; the difference was not significant (p > 0.05). SSU rRNA sequence analysis revealed that G. duodenalis assemblage E  was primarily detected and that assemblage A  was detected in 13 samples. The G. duodenalis—positive samples were PCR amplified and sequenced for gdh, tpi, and bg, from which 38, 47, and 70 amplified sequences were obtained, respectively. A combination of G. duodenalis assemblages A and E were detected in seven samples. Multilocus genotyping yielded 25 different assemblage E MLGs, which formed six subgroups. To the best of our knowledge, this is the first report regarding G. duodenalis infection in dairy cattle in Inner Mongolia, China. This study revealed that Inner Mongolian cattle pose a risk of giardiasis transmission to humans and that the distribution of local cattle G. duodenalis assemblage E MLGs is diverse. The findings of this study can bridge the knowledge gap in the molecular epidemiological investigation of giardiasis in Central Inner Mongolia. Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, was first described in 16812 and is a group of ubiquitous pathogenic gastrointestinal protozoans3. The hosts include humans, companion animals, livestock, and wildlife4. Human waterborne protozoan parasitic outbreaks worldwide has increased from 325 in an almost 100-year period5 to 199 between 2004 and 20106 and at least 381 outbreaks between 2011 and 20167. In the latter two records, Giardia was confirmed as the pathogen in 70 (35.2%)6 and 141 (37%) cases7, respectively. It is estimated that 8 × 108 global cases of giardiasis occur annually2. Giardiasis is a notifiable disease by the Centers for Disease Control and Prevention in the USA2. Cattle are considered the source of waterborne giardiasis outbreaks in humans4. Calves are more susceptible to acute infections caused by G. duodenalis and adult cattle often do not exhibit clinical signs and tend to have mild G. duodenalis infections. However, adult animals help maintain persistent infections in cattle and environmental contamination, thereby leading to the spread of giardiasis8.G. duodenalis was classified into at least eight genetically distinct but morphologically identical lineages, i.e., assemblages A–H11. Of these, assemblages A and B have a broad host spectrum, infect most vertebrates, and have a high zoonotic risk14. Assemblages C–H have a high host specificity15, with assemblages C and D mainly found in canine animals, assemblage E mainly found in hoofed livestock and wildlife, assemblage F found in cats, assemblage G found in rodents, assemblage H found in seals and other marine mammals14, and assemblages C, D, E, and F identified in human patients12. Assemblage E is the most common genotype in cattle globally20, followed by assemblages A and B16, in addition assemblages C, D15, and F have also been reported in cattle15. In addition, the frequency of infection with zoonotic assemblages A and B from calves was reportedly higher than that with assemblage E, suggesting that calves are associated with a greater risk of transmitting G. duodenalis infection to humans24.Based on genetic studies using molecular typing techniques, G. duodenalis detected using molecular methods was ~ 22% globally, and the difference between the highest pooled prevalence (55.4% in Canada) and the lowest pooled prevalence (4.2% in Iran) of giardiasis in different geographic areas is high14. Recently, in China, molecular epidemiological surveys of giardiasis in cattle have been conducted in several provinces in recently with differences in prevalence48. Inner Mongolia is located on the northern border of China, spanning 28° 52′ in longitude from east to west with a linear distance of > 2400 km and spanning 15° 59′ in latitude from north to south with a linear distance of 1700 km. Presently, only the prevalence and molecular characterizations of G. duodenalis in 108 beef cattle from one farm in the Southwest Alxa Left Banner have been investigated in Inner Mongolia24. Hence, this study aimed to investigate the prevalence of G. duodenalis in dairy cows in Central Inner Mongolia.The pooled prevalence of bovine g for 10 min and the precipitate was used for DNA extraction.From March to September 2021, 505 fresh fecal samples were randomly collected from six dairy farms in the vicinity of Tumd Left Banner, Horinger County, Togtoh County, Dalad Banner, and Hanggin Rear Banner  in Central Inner Mongolia. We have annotated six dairy farms on the map using phptpshop software  using Premix Taq™ (TaKaRa Taq™ Version 2.0 plus dye)  and 1 μL extracted DNA as the template. SSU rRNA–positive DNA was subsequently amplified via the nested PCR of bg50 , gdh51 , and tpi52 . Subsequently, 5 μL PCR products were analyzed via 1.5% agarose gel electrophoresis, and all the PCR products positive for the four genes were sent to a commercial company  for sequencing.The genomic DNA of 505 fecal samples was extracted E.Z.N. A® Stool DNA Kit  according to the manufacturer’s protocol and stored at − 20 °C for subsequent experiments. The small subunit ribosomal RNA (SSU rRNA) genehttp://www.ncbi.nlm.nih.gov) using MEGA 7.0 software (http://www.megasoftware.net/) and the obtained results were analyzed using the BLAST online platform. To comprehensively investigate the relationship among the different isolates, phylogenetic analyses were performed using a concatenated dataset of bg, gdh, and tpi sequences. The specimens successfully subtyped at all the three loci were included in the multilocus genotype (MLG) analysis of G. duodenalis, wherein MLG types were identified. Phylogenetic trees were constructed using the neighbor-joining algorithm based on a matrix of evolutionary distances calculated using the Kimura 2-parameter model via MEGA 7.0 software. To assess the robustness of the clusters, 1000 bootstrap replicates were used.The sequences were aligned with reference sequences downloaded from GenBank  was obtained using SPSS Statistics 21.0  to compare This study was carried out in strict accordance with international standards as published in the “Guide to the feeding, management and use of experimental animals” (8th Edition) and follows the “Regulations on the management of experimental animals” and other relevant laws and regulations. The biomedical research ethics committee of Inner Mongolia Agricultural University specifically approved this study (No. 2020[081]). In addition, permission was obtained from the farm owners before the specimens were collected, and all efforts were made to minimize suffering.G. duodenalis, 149 positive samples were amplified via PCR of 505 samples, with an overall prevalence of 29.5% (149/505). The overall prevalence of diarrhea and nondiarrhea samples was 31.5% (46/146) and 28.7% (103/359), respectively , 24.5% (27/110), 21.7% (26/120), 31.8% (35/110), 35.3% (6/17), and 12.5% (1/8). The difference in the Tumd Left Banner 1 field was highly significant compared with that in the other fields ; Horinger County field had also showed a significant difference . The prevalence G. duodenalis in the diarrhea samples from the six sampling sites was 32.7% (18/55), 33.3% (4/12), 27.5% (11/40), 33.3% (13/39), 0 (0/0), and 0 (0/0) , 47.6% (50/105), 34.7% (43/124), and 26.0% (45/173). The difference in preweaned calves was highly significant compared with that in the other age groups ; the difference in postweaned calves was highly significant . The prevalence of G. duodenalis in the diarrhea samples of the four age groups were 0 (0/30), 44.1% (30/68), 30% (6/20), and 35.7% (10/28)  for G. duodenalis assemblages A and 133  for assemblage E. Assemblage E was detected in all the four age groups. Only assemblage E was present in Hanggin Rear Banner field and in some age groups in other fields. Assemblage A was not observed in preweaned calves at any of the sites .The phylogenetic analysis of gdh Fig. , tpi  (Table The sequences of s) Table . PhylogeGiardia duodenalis is an important intestinal parasite. To date, numerous studies worldwide have reported G. duodenalis infection in cattle14. Bovine giardiasis is also widespread in China48. Herein, a molecular epidemiological investigation of G. duodenalis was conducted involving 505 dairy cattle fecal samples from six sites in Central Inner Mongolia. The findings filled the gaps in the data regarding G. duodenalis infection in dairy cattle in Inner Mongolia as well as reconfirmed the existence of G. duodenalis infection in animals in Inner Mongolia55. The overall prevalence of G. duodenalis was 29.5% (149/505) in this study, which exceeded the global pooled prevalence of 22%14 and was higher than the pooled prevalence of 14.1% in Chinese cattle14. In China, the prevalence of G. duodenalis in this study was only lower than the prevalence of G. duodenalis in cattle in Shanghai 32, Sichuan 34, and Guangdong 38 and higher than the prevalence data reported in Xinjiang 4; Ningxia 37 and 24; Heilongjiang 26, 27 and 28; Jilin 28; Liaoning 28; Hubei 29; Shandong  and 30; Shaanxi 31; Sichuan 33; Beijing 35; Gansu 36 and 37; Guangdong 39; Hebei and Tianjin 40; Henan 41; Jiangsu 42; Jiangxi 43; Qinghai 45; Tibet 46; Yunnan 47; Taiwan 48 and that reported in the only survey on G. duodenalis in Inner Mongolia 44. However, it is difficult to compare prevalence data because they are influenced by a range of factors, including study design, diagnostic method, geographical conditions, total number of samples, age of animals, and sampling season4. The high prevalence of this study may also be related to the high density of local cattle farming. In addition, there were significant differences in the overall prevalence of G. duodenalis among the sampling sites in this study. Furthermore, positive samples were detected in all the age groups, indicating that all the age groups of cows are susceptible to G. duodenalis38.G. duodenalis was correlated with the presence of diarrhea in the sampled animals. Additionally, G. duodenalis was not detected in the diarrhea samples of preweaned calves. This observation is inconsistent with the combined global data, as the latter reported a significant correlation between G. duodenalis infection and cattle having diarrhea and preweaned calves14. The causes for diarrhea in animals are complex, with the pathogens including various viruses, bacteria, and parasites56. In addition, in G. duodenalis infection, the appearance of symptoms is also related to the stage of the infection. However, in the diarrhea samples in the present study, the prevalence of G. duodenalis in postweaned calves was significantly higher than that in the other age groups. In addition, the overall prevalence in the preweaned calves was significantly lower than that in the other age groups and that in postweaned calves was significantly higher than that in the other age groups in all the samples, which does not reflect the decrease in G. duodenalis infection rates with increasing age as reported in the literature42. The high prevalence of G. duodenalis in postweaned calves has also been reported in China29, Norway57, Germany58, USA60, and Canada61. However, there are also several studies reporting a relatively high prevalence of G. duodenalis infection in preweaned calves43. Certainly, there are some inconsistencies in the ages of preweaned and postweaned calves, or the age of the sampled cows is not clearly stated in previous studies. If calves aged < 6 months are defined as preweaned calves, the postweaned calves in this study will be classified as preweaned calves, but this is not consistent with the current situation of cattle farming in China. If this was the case, the prevalence of G. duodenalis in preweaned calves in this study was 29.3% (61/208), which is inconsistent with the higher G. duodenalis prevalence in preweaned calves (aged < 6 months) reported in the pooled global data14. As mentioned earlier, several factors influence the prevalence of G. duodenalis infection, age distribution, and diarrhea occurrence, such as the immature and susceptible immune system of younger animals60, asymptomatic immunocompetent individuals62, and the increased resistance due to nonspecific immunity acquired via breast milk63.Herein, neither the overall prevalence nor the prevalence in the different sampling sites of SSU rRNA sequences were identified as 13 assemblage A and 133 as assemblage E, with assemblage E being the dominant genotype. This finding is consistent with the studies reporting that assemblage E is the most common genotype in cattle worldwide20. Combining the sequence analysis results of 38 isolates of gdh, 47 isolates of tpi, and 70 isolates of bg, the genetic diversity of these positive G. duodenalis isolates was determined. The results revealed their genetic diversity, and seven isolates were identified to exhibit inconsistent assemblage. This finding is similar to the results reported previously in China and abroad64. It is also consistent with the results reported for 89,139 cattle from 48 countries in seven regions, with assemblage E being the most common, followed by assemblages A and A + E14. Transmission via environment  may play a key role in parasite epidemiology65. Assemblages A and B are considered zoonotic24, and assemblage E has also been reported in humans in Egypt66, Brazil67, and Australia68. In China, cows are considered significant reservoirs of human giardiasis69. The results of the present study suggest that dairy cattle in Inner Mongolia pose a risk of causing G. duodenalis infection in humans.Overall, 146 plausible G. duodenalis assemblage genes revealed that preweaned calves only contained assemblage E, whereas postweaned calves contained assemblages E and A. Seven assemblage A + E were present in postweaned calves (n = 3), young cattle (n = 2), and adult cattle (n = 2). This finding is consistent with the report of preweaned calves containing only assemblage E in Sichuan, China33, and postweaned calves containing assemblages E, A, and A + E34. Furthermore, it is inconsistent with reports mentioning that assemblages E and A were detected in preweaned calves and postweaned calves in China41, USA60, and Europe70. A high prevalence of assemblage A in preweaned calves has also been reported4. Assemblage A infection has been reported in dairy cattle of all ages72, and assemblage E is also been reportedly common in adult cattle47. In addition, assemblage E is more common in calves than in adult cattle73. Herein, only assemblage E was found in the Hanggin Rear Banner field; all the other fields were found to have assemblages E and A, and three of them were also found to have assemblage A + E. In China, reports on cattle G. duodenalis assemblage varied with different sites. Assemblage E was only detected in Hubei Province29, Beijing35, Gansu37, Inner Mongolia44, and Qinghai45; assemblages E and A were detected in Shaanxi31, Jilin28, Jiangxi43, Tibet46, and Yunnan47; assemblages E and A + E were detected Hebei and Tianjin40; assemblages E, A, and A + E were detected in Xinjiang4, Liaoning28, Sichuan34, Guangdong39, Henan41, and Jiangsu42; assemblages E, B,37, and A were detected37 in Ningxia; assemblages E, A, B, and A + E were detected in Heilongjiang28 and Shanghai32; and assemblages E and D were detected in Taiwan48. However, the available data in China do not reflect the geographical distribution pattern of G. duodenalis assemblages.Herein, the comparison of cattle belonging to different age groups for four of G. duodenalis assemblage remain insufficient. The characteristics of individual loci of G. duodenalis often lead to inconsistent genotyping results16. The MLG model was used to better understand the diversity of human and animal G. duodenalis in different geographic regions, which can help reveal the potential and dynamic transmission of zoonosis74. Herein, 26 isolates containing the gdh, tpi, and bg were combined to obtain 25 different assemblage E MLGs with six subgroups. Consistent with the results of previous studies, numerous MLGs were identified in assemblage E. Additionally, G. duodenalis isolates that were classified in the same assemblage may be classified as distinct MLGs75.Reports on genetic variations in G. duodenalis infection in humans. In addition, Giardia are commonly found on fruits76, and vegetables78, and in various types of water89 in other regions of China. Therefore, further studies need to investigate the molecular epidemiology of cattle keepers and neighboring water sources in Inner Mongolia to evaluate the transmission dynamics of G. duodenalis, to adopt effective strategies to prevent and control G. duodenalis transmission among dairy cattle and humans in Inner Mongolia.The results of the present study suggest that dairy cattle in Inner Mongolia pose a risk of causing G. duodenalis infection in dairy cattle in Inner Mongolia, thereby filling a gap in the molecular epidemiological data regarding giardiasis in Central Inner Mongolia. The results reconfirmed previous findings in other parts of China that G. duodenalis infection is common in dairy cattle. The livestock-specific G. duodenalis assemblage E was the dominant assemblage; however, zoonotic assemblage A was also present in Inner Mongolia. The distribution of bovine G. duodenalis assemblage E MLGs was diverse.To the best of our knowledge, the present study is the first to report