Background: With the increasing number of new cancer cases and mortality rates, cancer has become a serious global health problem, but there are no ideal cancer biomarkers for effective diagnosis. Currently, mounting evidence demonstrates that lncRNAs play a fundamental role in cancer progression. BBOX1 anti-sense RNA 1 (BBOX1-AS1) is a recently clarified lncRNA and has been identified as dysregulated in various carcinomas, and it contributes to poor survival in cancer patients.Methods: We thoroughly searched six databases for eligible articles published as of 27, April 2023. The association of BBOX1-AS1 expression levels with prognostic and clinicopathological parameters was assessed by odds ratios (OR) and hazard ratios with 95% CIs. Additionally, we further validated our results utilizing the GEPIA online database.Results: Eight studies comprising 602 patients were included in this analysis. High BBOX1-AS1 expression indicated poor overall survival (OS)  when compared with low BBOX1-AS1 expression. Furthermore, BBOX1-AS1 expression was positively correlated with lymph node metastasis  and advanced tumor stage  for cancer patients. Moreover, BBOX1-AS1 was remarkably upregulated in 12 malignancies, and the elevated BBOX1-AS1 expression predicted poorer OS and worse disease-free survival (DFS) confirmed through the GEPIA online gene analysis tool.Conclusion: The findings highlight that BBOX1-AS1 was significantly associated with detrimental overall survival, disease-free survival, lymph node metastasis and tumor stage; thus, it could act as a novel promising biomarker to predict the clinicopathological characteristics and prognosis for various cancers. With rising morbidity and mortality, cancer has become a serious global health concern, and an additional economic burden is attributable to improving cancer survival worldwide . The GLO+ T-cell responses in colorectal tumors (Long non-coding RNAs (lncRNAs) are defined as RNAs with longer than 200 nucleotides that lack protein coding potential . Additiol tumors . TherefoBBOX1 anti-sense RNA 1 (BBOX1-AS1) is a recently clarified lncRNA and has been identified to be dysregulated in several carcinomas, which include lung cancer , cervicaWe thoroughly searched six databases, which included SinoMed, Web of Science, EBSCO, Cochrane Library, PubMed, and Ovid for eligible articles published up as of 27 April, 2023. The following search keywords were adopted to identify relevant publications: “long non-coding RNA BBOX1-AS1” OR “BBOX1 anti-sense RNA 1” OR “BBOX1-AS1” OR “lncRNA BBOX1-AS1.” Furthermore, the references in these retrieved articles were also manually searched to examine potentially eligible studies. Only manuscripts written in English or Chinese were included.Inclusion criteria: 1) BBOX1-AS1 expression was identified in cancerous tissues and corresponding non-cancerous tissues; 2) all patients did not receive preoperative anticancer treatment; 3) the approach of detecting BBOX1-AS1 expression was qRT-PCR; 4) studies reported clinicopathological characteristics or prognostic outcomes such as OS, disease-free survival (DFS), and Kaplan–Meier (K-M) curves to calculate the hazard ratios (HR) (95% CI) indirectly; and 5) patients were divided into high- and low-expression groups, according to BBOX1-AS1 expression levels.Exclusion criteria: 1) studies absent of clinicopathological characteristics or prognostic outcomes; 2) duplicated articles; 3) case reports, review articles, meta-analysis, editorials, non-human studies and conference reports; and 4) studies based on the public database.After reviewing all the eligible articles, two investigators (GYL and YZW) independently extracted the requisite data, and all disagreements were resolved by discussing with two additional reviewers (LD and TY). The necessary information was carefully extracted from each study: author’s last name, publication year, cancer types, total number of patients, the number of patients in the high- and low-BBOX1-AS1 expression groups, detection methods, outcomes, the HR and corresponding 95% CIs for OS, follow-up time, and extraction methods for OS data. The HR (95% CI) for OS was calculated using the Engauge Digitizer 4.1 software indirectly through K-M curves if the eligible articles failed to offer the HR (95% CI) directly . If bothhttp://gepia.cancer-pku.cn/)  . Surviva2 statistics was applied to calculate heterogeneity among all included studies. The fixed-effects model was used for data analysis if heterogeneity was absent (I2< 50%). Otherwise, the more appropriate random-effects model was adopted. A p-value <0.05 was considered to be statistically significant.Data management was conducted with the EndNote 20.2 software. All analysis were carried out with the Review Manager 5.3 software. The effect of BBOX1-AS1 levels on OS, tumor differentiation, lymph node metastasis, tumor stage, tumor size, patient age, and gender were assessed by odds ratio (OR) and HRs with 95% CIs. Cochrane Higgins IBy searching six databases (up to 27 April 2023), a total of 81 publications concerning the prognosis of BBOX1-AS1 and cancer patients were retrieved preliminarily. Among the 81 pieces of literature, 60 duplicate studies were excluded and 21 studies were selected for abstract screening. Nine publications were excluded since they were retraction articles, cell-based experiments, reviews, or research studies based on public databases. Then, we carefully removed another four studies because they lacked sufficient data regarding clinicopathologic characteristics or prognosis for analysis. Finally, eight studies from 2020 to 2023 were considered eligible for the meta-analysis. The selection flowchart for the included publications is depicted in These studies were published between 2020 and 2023, and the sample sizes ranged from 40 to 135. A total of 602 patients were divided into the low- and high-BBOX1-AS1 expression groups, with 299 and 303 cases in each group, respectively. The studies were performed in different regions of China and six types of malignancies were examined: lung cancer, cervical cancer, ESCC, gastric cancer, esophageal carcinoma, and hepatocellular carcinoma. Among these included studies, seven estimated the correlation between BBOX1-AS1 and OS of patients. 2 = 53%, p = 0.0001)    (versus <60 years (young) and tumor size ≥ 5 cm (large size) versus <5 cm . The pooled results demonstrated high BBOX1-AS1 expression was not significantly associated with patient age     and more0.00001) . However = 0.26)  and pati = 0.77) . Subsequ = 0.99)  and tumo = 0.07) .p < 0.00001). A fixed-effects model was applied to evaluate the HR of these studies because of low heterogeneity (I2 = 15%). The result suggested that the upregulation of BBOX1-AS1 predicted poor OS among various malignancies (p < 0.00001). A fixed-effects model was utilized since all these stratified analysis manifested low or no heterogeneity.Seven studies with data from 562 patients reported the association between BBOX1-AS1 as a prognostic biomarker of malignancies and OS. Pooled HR was 2.30  (p < 0.05)  . Besides < 0.05) .Recently, a burgeoning number of studies discovered that lncRNAs were dysregulated in diverse human malignancies. Many may act as novel potential markers for early diagnosis, therapy, and prediction of prognosis in cancers. Hence, in recent years, numerous reviews, clinical trials, basic experiments, and systematic review publications, such as our meta-analysis, have been performed to gradually demonstrate the correlation between lncRNAs and diverse tumor prognoses . Furtherp < 0.00001). Furthermore, subgroup analysis demonstrated the same result despite cancer types, sample sizes, and extracted methods. Additionally, high BBOX1-AS1 expression significantly was connected with lymph node metastasis  and more advanced tumor stage  in cancer patients. Clinically, a study involving 10,126 cases demonstrated that lymph node metastasis was tightly correlated with a high incidence of regional recurrence and distant metastasis, which directly lowered the survival rate of cancer patients (p = 0.26), patient age , patient gender , and tumor size . Meanwhile, the GEPIA database was applied to confirm our results as extensively as possible. Elevated BBOX1-AS1 expression levels were also established in 12 different types of malignancies. Moreover, increased BBOX1-AS1 expression was associated with poor OS in LGG, MESO, and BRCA and with worse DFS in LGG, MESO, and sarcoma. Taken together, this consequence demonstrated that BBOX1-AS1 could be applied as a novel biomarker for prognosis or detection of various malignancies.In this meta-analysis, we identified the relevance between BBOX1-AS1 expression and human malignancies’ prognostic parameters. The results illustrated that an increase in BBOX1-AS1 was dramatically related to unfavorable OS  and tumor differentiation (I2 = 58%). Since all included studies were from different hospitals and examination approaches used by individual studies might not be uniform, these may likely contributed to heterogeneity. Consequently, an appropriate random-effects model had to be utilized, which might somewhat degrade the accuracy of results. Fourth, studies published only in English or Chinese were included, whereas studies exploring the relationship between BBOX 1-AS 1 and survival in cancer patients in other languages were omitted. Fifth, some HRs were extracted indirectly through rebuilding the K-M curves, which might inevitably cause possible deviations. Notwithstanding the intrinsic deficiencies, our results render robust evidence that overexpressed BBOX1-AS1 indicates poor prognosis in cancer patients.Nevertheless, several limitations in this study should be noted as well. First, as a novel biomarker, the earliest study demonstrating the association between BBOX1-AS1 and cancers was published in 2020. Although we thoroughly searched six databases, only eight studies were finally enrolled for this analysis after careful screening. This may hence be one of the explanations for the relatively small amount of literature included in our study. However, the GEPIA database and subgroup analysis were adopted to strengthen our results, and they demonstrated that our results were reliable. Second, therapeutic regimens may also be considered to play a vital role in cancer patient survival. Thus, different treatments, to some extent, may impact the calculation of HR or OR values. Third, there was moderate heterogeneity in some analyses such as lymph node metastasis (Iin vitro/in vivo validation of the promotion of BBOX1-AS1 in different malignancies and its effects on tumor pathology could be investigated in future studies.In conclusion, our meta-analysis demonstrated that overexpressed BBOX1-AS1 is exceedingly correlated with worse OS, DFS, lymph node metastasis, and advanced tumor stage in carcinomas. Specifically, BBOX1-AS1 can be served as a novel promising biomarker for predicting the clinicopathological characteristics and prognosis for various cancers. Furthermore,


While a certain level of inflammation is critical for humans to survive infection and injury, a prolonged inflammatory response can have fatal consequences. Pattern recognition Toll-like receptors (TLRs) are key players in the initiation of an inflammatory process. TLR2 is one of the most studied pattern recognition receptors (PRRs) and is known to form heterodimers with either TLR1, TLR4, TLR6, and TLR10, allowing it to recognize a wide range of pathogens. Although a large number of studies have been conducted over the past decades, there are still many unanswered questions regarding TLR2 mechanisms in health and disease. In this review, we provide an up-to-date overview of TLR2, including its homo- and heterodimers. Furthermore, we will discuss the pro- and anti-inflammatory properties of TLR2 and recent findings in prominent TLR2-associated infectious and neurodegenerative diseases. Escherichia coli, whereas TLR2 is known to form heterodimers with either TLR1 or TLR6, depending on the ligand [Over the past few decades, a great deal of knowledge has been gained about human innate and adaptive immunity and its key players, including the pattern recognition receptors (PRRs). These germline-encoded receptors can rapidly identify the specific molecular structures on the surface of pathogens, so-called pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs), linking non-specific immunity to specific immunity ,2. To dae ligand ,12. In ae ligand ,14,15.In this review, we aim to provide an up-to-date overview of TLR2 dimerization, including homo- and heterodimerization, and propose a model to further investigate these dimerization mechanisms. Additionally, the expanded role of TLR2 with its pro- and anti-inflammatory capabilities, and the recent findings in prominent TLR2-associated (neuro) inflammatory diseases will be discussed.Drosophila melanogaster Toll receptor originally discovered in 1985, are well conserved and primarily associated with pattern recognition [TLRs, which are the mammalian orthologues of the ognition ,16,17. Tognition ,19,20. Iognition ,21.Helicobacter pylori LPS recognition [Escherichia coli, and FSL-1, which represents a pathogen-associated molecular pattern of the LP secluded from Mycoplasma fermentans or Mycoplasma salivarium [TLR2/1 heterodimers can sense triacylated lipopeptides (LPs) from Gram-negative bacteria or mycoplasma, such as lipoarabinomannans and lipomannans, whereas TLR2/TLR6 heterodimers recognize diacylated LPs, including lipoteichoic acid (LTA) from Gram-positive bacteria and mycoplasma ,22,23,24ognition ,26. Addiognition . Interesognition . Besidesognition ,29. Commlivarium ,30,31. Mlivarium ,33,34,35livarium ,37. TherLigand-induced dimerization is the starting point for TLR2 signal transduction. Dimerization brings the TIR domains of the cytoplasmic tails into close proximity, providing a platform for signaling by TIR domain-containing adaptor molecules . TLR2 prMycoplasma pneumoniae and Streptococcus pneumoniae [Mycobacterium tuberculosis induces TLR2 homodimerization, which triggers anti-inflammatory type responses that cause increased activation of the mammalian p38 MAPK [The existence of TLR2 homodimers has been mentioned in a number of publications in recent years, however, the function of this homodimer is still controversial ,43,44,45eumoniae . In a dieumoniae . Su et aeumoniae . Contrarp38 MAPK . Furtherp38 MAPK . Taken tHelicobacter pylori or LPS and found a significant upregulation of interleukin-1β (IL-1β) in TLR2/10-transfected HEK293 cells [Listeria monocytogenes, and they unveiled that the heterodimer of TLR2/TLR10 was the one behind the activation of NF-κB [Helicobacter pylori and concluded that the TLR2/TLR10 heterodimer upregulates NF-κB activation to a greater extent than other TLR2 heterodimers [Ochrobactrum intermedium is a potent TLR2/TLR4-inducing agonist, which they additionally confirmed by molecular docking analysis and fluorescence resonance energy transfer [The ability of TLR2 to dimerize not only with itself but also with a variety of TLRs, greatly expands the spectrum of detectable pathogens. Ozinsky et al. were among the first to discover that TLR2 relies on heterodimerization with either TLR1 or TLR6 to initiate adequate cell activation and a pro-inflammatory response ,21. Stru93 cells . In anotof NF-κB . Nagashirodimers . A recenrodimers . Howeverrodimers . Furtherrodimers . The exitransfer . NeverthParacoccidioides brasiliensis infection led to an increase in TLR2 and in parallel, α3β1 integrin levels were downregulated in human lung epithelial cells [Additional co-receptors, called accessory receptors, are often required to augment TLR2 ligand delivery, pattern recognition, and other functions see  12,21,6,621,60. al cells . These rArabidopsis thaliana or Chlamydomonas reinhardtii [Vaucheria frigida aureochrome 1, was C-terminally fused to the full-length TLR4. This innovative system allows precise on/off switching under temporal and spatial control, which may be an interesting approach to elucidate TLR2 homodimerization and signaling.Conventional techniques to further identify and characterize dimerization and signaling mechanisms, such as pharmacological control including ligand exposure, knock-out models, or biochemical assays, may not always be the most appropriate method. For example, models based on TLR2 knock-outs emphasize the importance of TLR2 but do not provide insight into the detailed signaling mechanism or interactions with other co-receptors. However, new experimental models, such as optogenetics, are emerging that do not rely on ligand binding to activate cellular receptors. Optogenetics was first introduced by Deisseroth in 2006 in the field of neuroscience, where light-sensitive ion channels were being utilized to remotely control action potentials and thereby neuronal networks . In shornhardtii ,84. Our nhardtii ,86. For Scutellaria baicalensis Georgi inhibited inflammation in rats with chronic obstructive pulmonary disease via the TLR2/MyD88/NF-κB pathway [Bacteroides fragilis in B cells and T cells, resulting in the production of IL-10 and interferon-γ (IFN-γ) [TLR2 activation can evoke pro-inflammatory and anti-inflammatory responses that are highly dependent on the cell type and the cell compartment in which it is expressed, the ligand, and the co-receptors. A complete overview of all cells expressing TLR2 and the functional impact of TLR2 activation is beyond the scope of this review. However, to illustrate the diverse and complex effects of TLR2 activation, a selection of recent findings will be mentioned in this section. While the well-known heterodimerization of TLR2 with TLR1 or TLR6 leads to the classical pro-inflammatory response in different cell types, the anti-inflammatory reaction serving as an immune regulator seems to be more heterogeneous. A study from 2021 showed that a high number and blockwise distribution of certain methyl esters of pectins were responsible for a TLR2/1-dependent anti-inflammatory effect . Interes (IFN-γ) ,90,91. M (IFN-γ) . Soluble (IFN-γ) . In this (IFN-γ) .Listeria monocytogenes infection in vitro and in vivo (mouse model) is generally TLR2 dependent and that immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs [Although pattern recognition appears to be the most common activity of TLR2, it is not its only biological function. Our recent publication indicated that TLR2 also plays an important role in cell adhesion and migration of monocytes . More spmal TLRs . Table 1TLR2 and its signaling have been implicated in a variety of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, asthma, and multiple sclerosis ,108,109.Streptococcus pneumoniae induces platelet activation via TLR2 and that its inhibition completely abolished platelet aggregation, implicating TLR2 in the thrombotic complications of sepsis [Staphylococcus aureus bacteremia and found that TLR2 downregulation and high IL-6 and IL-10 levels, indicative of immune dysregulation during early bacteremia, may be linked to mortality [Sepsis is described as a systemic dysregulated immune response of the body to an infectious reaction caused by pathogens, including bacteria, viruses, fungi, and parasites ,111. Thif sepsis . Anotherortality . Furtherortality . Consistortality . In the ortality . Taken tCoronaviridae family of β-coronaviruses, which are positive-stranded, enveloped RNA viruses [Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has challenged human health and public safety in recent years, is a member of the  viruses . Clinica viruses ,126. Whi viruses ,128. Zhe viruses . Consist viruses . However viruses . Notably viruses . These dTLR2 is expressed not only on a variety of immune cells, but also on neuronal and glial cells in the central, peripheral, and enteric nervous systems, enabling them to act as immune cells ,134. As The most prevalent neurodegenerative disease in the aging population, AD, is pathologically characterized by the accumulation of Aβ plaques, hyperphosphorylated tau in neurofibrillary tangles, and consequently, enhanced neuroinflammation ,143. SinPD is the most prominent form of α-synucleinopathies and the second most common neurodegenerative disease . The patOne of the major discoveries in medicine over the last twenty years has been that the immune system and subsequent inflammatory processes are associated not only with a few specific diseases but also with a broad range of mental and physical health conditions ,162. In


Hepatocellular Carcinoma (HCC) continues to pose a substantial global health challenge due to its high incidence and limited therapeutic options. In recent years, the Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) pathway has emerged as a critical signaling cascade in HCC pathogenesis. The review commences with an overview of the JAK/STAT pathway, delving into the dynamic interplay between the JAK/STAT pathway and its numerous upstream activators, such as cytokines and growth factors enriched in pathogenic livers afflicted with chronic inflammation and cirrhosis. This paper also elucidates how the persistent activation of JAK/STAT signaling leads to diverse oncogenic processes during hepatocarcinogenesis, including uncontrolled cell proliferation, evasion of apoptosis, and immune escape. In the context of therapeutic implications, this review summarizes recent advancements in targeting the JAK/STAT pathway for HCC treatment. Preclinical and clinical studies investigating inhibitors and modulators of JAK/STAT signaling are discussed, highlighting their potential in suppressing the deadly disease. The insights presented herein underscore the necessity for continued research into targeting the JAK/STAT signaling pathway as a promising avenue for HCC therapy. It continues to be a substantial global health concern due to its increasing incidence and the limited therapeutic options available ,2,3. Accorldwide . The higIn the context of therapeutic interventions for HCC, surgical resection and local ablation therapy are preferred for early-stage HCC. Nonetheless, the incidence of recurrence within a five-year period following such therapeutic interventions can be as high as 70% ,3. In thGiven the profound diversities intrinsic to the molecular landscape of HCC and the formidable challenges entailed in devising universally effective therapeutic strategies for this disease, the identification of molecular signaling pathways perturbed in HCC assumes fundamental importance in developing therapeutic targets as a personalized medical approach ,9. In thA multitude of pro-inflammatory cytokines, including various interleukins, growth factors, and interferons, are elevated in livers with chronic inflammation ,12, favoThis review paper aims to provide a comprehensive overview of the current state of knowledge regarding the role of the JAK/STAT pathway in HCC. We will explore the implications of JAK/STAT pathway dysregulation in HCC, which is triggered by a variety of cytokines. Additionally, we will discuss the results from recent preclinical and clinical studies that target the JAK/STAT signaling cascade as a potential therapeutic approach for treating HCC.The JAK/STAT pathway is a vital cellular cascade that controls a wide range of processes, including cell proliferation, differentiation, and apoptosis ,17. Its The activation of this pathway begins when specific cytokines, such as interleukins, interferons, or growth factors, bind to their corresponding cell surface receptors . These rThere are four types of JAK proteins, JAK 1, 2, 3, and tyrosine kinase 2 (TYK2), as well as seven types of STAT proteins, STAT 1, 2, 3, 4, 5A, 5B, and 6. Although their roles differ slightly, they share common structures. Each JAK protein consists of seven homology domains known as Janus homology (JH) domains . These sSTAT proteins consist of several domains: N-terminal domain, coiled-coil domain, DNA-binding domain (DBD), linker domain, SH2 domain, and transcription activation domain (TAD) ,29. The Various ligands trigger the initiation of the JAK/STAT signaling pathway, with cytokines being one of the most representative examples. Cytokines are small molecules secreted by a variety of cells that facilitate interactions and communication between cells . They plThe IL-2 family cytokines, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, engage their respective receptors, which consist of heterodimeric or heterotrimeric complexes composed of various cytokine-specific receptor chains. Upon ligand binding, these receptors undergo conformational changes that facilitate the recruitment and activation of JAK family members, primarily JAK1 and JAK3 ,44. ThesThe IL-6 family of cytokines, including IL-6, IL-11, IL-27, IL-31, and leukemia inhibitory factor (LIF), engages their cognate receptors, which generally consist of gp130 as a common receptor subunit, along with specific ligand-binding receptor chains ,48. IL-6The IL-10 family includes IL-10 itself, as well as IL-19, IL-20, IL-22, IL-24, and IL-26. These cytokines engage specific receptor complexes, typically composed of receptor subunits such as IL-10R1, IL-10R2, and IL-22R1 . Upon liThe β common cytokine family includes cytokines such as IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which share a common β subunit (CD131) in their receptor complexes . The ligHematopoietic GFs, including erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), and thrombopoietin (TPO), engage their respective receptors to initiate signal transduction cascades. These receptors typically consist of single or multiple subunits, with some sharing the common β subunit (CD131), and predominantly activate JAK2, leading to the activation of specific members of STATs ,77,78,79Type 1 IFNs, including IFN-α and IFN-β, are produced in response to viral infections and engage a heterodimeric receptor complex composed of IFNAR1 and IFNAR2 subunits. Upon ligand binding, this receptor complex triggers the activation of JAK1 and TYK2. These JAKs primarily phosphorylate STAT1 and, to a lesser extent, STAT2 . On the Initiation of JAK/STAT signaling occurs when the aforementioned ligands, primarily cytokines, bind to transmembrane receptors. The types of transmembrane receptors involved in the JAK/STAT pathway include cytokine receptors, G-protein coupled receptors, and growth factor receptors . Given tClass I receptors feature conserved pairs of cysteines connected by disulfide bonds. This class of receptors is further divided into the glycoprotein 130 (gp130) family, the γ chain family, the β chain family, the single-chain family, receptor tyrosine kinases, and other interleukin receptors. Within the glycoprotein 130 (gp130) family, members include the IL-6R family , the G-CSF receptor, and the leukemia inhibitory factor receptor chain (LIFR). The interaction of gp130 with JAK1, JAK2, and TYK2 leads to the activation of STAT1, STAT3, and STAT5. Additionally, the γ chain family encompasses receptors for the IL-2 family . Another subset of Class I receptors is the β chain family, which involves IL-3R, IL-5R, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor . The sinThe JAK/STAT signaling pathway is crucial for transmitting signals from cell surface receptors, primarily those of cytokines, to the nucleus, where it regulates the expression of numerous genes involved in various cellular processes. To maintain appropriate cellular responses, the pathway requires tight regulation. In addition to the numerous activators of this signaling pathway, including various growth factors and cytokines, there are negative regulators that play a vital role in inhibiting the JAK/STAT pathway. There are three categories of negative regulators: suppressors of cytokine signaling (SOCS), protein inhibitors of activated STAT (PIAS), and protein tyrosine phosphatases (PTPs) .The SOCS family encompasses eight distinct protein members: cytokine-inducible SH2 domain protein (CISH), SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7. The SOCS proteins bind to the activation loop within the kinase domain of JAK or the cytoplasmic tail of the receptor. For instance, SOCS1 binds to activated JAKs, while SOCS3 binds to tyrosine residues on receptors. This binding efficiently blocks the phosphorylation of the receptor by JAKs . AdditioThe JAK/STAT signaling pathway, activated by numerous cytokines and growth factors, serves as a central communication hub in cellular activities ,88. CompCCND1 (encoding Cyclin D1), BCL2, BIRC5 (encoding Survivin), HIF-1α, VEGF, CD133 (encoding Prominin-1), NANOG, IL-10, TWIST, MMP9, etc. [The signals from the JAK/STAT pathway are carried out by the STAT transcription factors. In HCC, STAT3 is the major oncogenic player in various aspects of carcinogenesis. Genes that are transcriptionally activated by STAT3 in HCC include P9, etc. ,97,98,99P9, etc. ,100.IL6ST encoding the gp130 receptor can lead to phosphorylation of STAT3 in the absence of ligand binding [IL6ST, however, are found in less than 3% of HCC patients  proteins function as inhibitors of the JAK/STAT signaling . SOCS hiThe livers of HCC patients are often infiltrated with different types of inflammatory cells that secrete various types of cytokines, growth factors, chemokines, and other molecules ,112. DysIL-6 family cytokines, which activate the JAK/STAT signaling pathway, have been linked to the initiation and progression of HCC . Both T Interleukin-10 (IL-10) family cytokines play a pivotal role in the progression of liver diseases. Beyond their diverse immunoregulatory functions, IL-10 influences susceptibility to HCV infection and hepatic fibrogenesis ,124. A sDespite sharing structural similarities and utilizing similar or partially identical receptors, different members of the IL-10 family exhibit distinct biological functions ,130. IntIL-20 signals through its binding to either IL-22R/IL-20R2 or IL-20R1/IL-20R2. Both of these heterodimer receptor complexes activate the JAK/STAT pathway. Interestingly, the presence of constitutive IL-20R2 expression on hepatocytes alone is enough to activate STAT3 ,136. IL-IL-23 belongs to the IL-12 family of cytokines and acts as a crucial molecular link between tumor-promoting pro-inflammatory processes and the impairment of adaptive immune surveillance . Upon biInterleukin-17 (IL-17), which exhibits increased expression in HCC, can facilitate the invasion and migration of HCC cells ,143. TheAs discussed above, the upregulation of JAK/STAT signaling significantly contributes to the increased proliferation, migration, and invasion of HCC cells. Furthermore, this up-regulation leads to enhanced cell survival and metastasis. Given that the JAK/STAT signaling pathway exerts strong tumor-promoting effects on HCC, it is reasonable to consider inhibiting the pathway for the treatment of HCC. In vitro investigations have consistently indicated that inhibiting JAK/STAT activity results in increased apoptosis in HCC cells, thereby reducing cell survival. Similarly, in vivo preclinical studies have also convincingly demonstrated that blocking JAK/STAT activity can effectively impede tumor growth or even delay the progression of tumors.The SOCS proteins act as endogenous inhibitors of the JAK/STAT signaling pathway. Frequent observations of hypermethylation or histone modification in the promoter regions of SOCS have been made in HCC ,147. TheJAK1 was identified as a target of MicroRNA 30e (miR-30e). Transfection of HCC cells (HepG2 and Huh-7) with miR-30e led to decreases in phosphorylated levels of JAK1 and STAT3. Additionally, overexpression of miR-30e resulted in reduced cell proliferation, migration, and invasion, along with increased apoptosis in the HCC cells . PharmacEtomidate inhibits phosphorylation and activation of JAK2 by competing with ATP for binding to the ATP-binding pocket of JAK2. Application of etomidate to HCC cells (HepG2 and Huh-7) suppressed phosphorylation of JAK2 and STAT3, resulting in the inhibition of cell proliferation, migration, and invasion, coupled with enhanced apoptosis . SimilarTargeting STAT proteins also exerts similar anticancer effects in HCC cells. Pravastatin, a nonpeptide small molecule, effectively impedes STAT1 phosphorylation upon the activation by cytokine-induced pathways. Notably, pravastatin effectively dampened the phosphorylation of STAT1 induced by IFN-γ . The proNapabucasin (BBI608) is a novel small-molecule inhibitor of STAT3. It has been shown to impair cancer stemness in various types of cancers, such as colorectal, pancreatic, and prostate cancers ,157. In Studies have demonstrated that miR-515-5p directly binds to the 3′-untranslated region (3′-UTR) of interleukin 6 (IL-6). Treatment of HCC cells (HepG2) with miR-515-5p suppressed the JAK/STAT signaling pathway along with reduced IL-6 expression. Overexpression of miR-515-5p inhibited migration and invasion of HepG2 cells that were transplanted into nude mice . SimilarSOCS3 is a suppressor of the JAK/STAT signaling. A long noncoding RNA known as C1QTNF1 antisense RNA 1 (C1QTNF1-AS1) maintains SOCS3 activity by binding with miR-221-3p, which downregulates SOCS3. In nude mice transplanted with HCC cells (HepG2 or Huh-7), ectopic expression of C1QTNF1-AS1 led to suppression of HCC tumor growths in vivo which was accompanied by a decrease in the expression of phosphorylated STAT3, as well as an increase in SOCS3 expression . JAK2 anDehydrocrenatidine is a potent JAK family kinase inhibitor. HCC cells treated with dehydrocrenatidine exhibited a significant suppression in the phosphorylated level of JAK2. HepG2-xenograft mice treated with dehydrocrenatidine showed a significant reduction in tumor volume, leading to increases in animal survival. Of note, the treatment led to significantly reduced EMT and CSC phenotypes in HCC . An azasAdditionally, attempts to inhibit STAT3 have been made in HCC murine models. Mice transplanted with Hepa1-6 cells were intraperitoneally injected with napabucasin, a STAT3 inhibitor, at a dose of 20 mg per kilogram of body weight, or vehicle. Napabucasin treatment significantly reduced tumor volumes compared to the control group. Notably, approximately 25% of mice showed tumor regression when examined 21 days after administration . OPB-311In line with the results from xenograft models of HCC, chemically induced autochthonous HCC models also showed inhibition of the JAK/STAT signaling suppressed HCC development. For example, rats harboring autochthonous HCC were treated with pravastatin, an inhibitor of STAT1 phosphorylation. The treatment led to significantly smaller tumor sizes in the livers. Immunohistochemistry studies revealed that the level of cellular proliferation in HCC from rats treated with pravastatin was significantly diminished compared to that from untreated mice .The significant role of the JAK/STAT signaling pathway in HCC suggests that targeted inhibition of this pathway could hold therapeutic potential for treating patients with HCC . VariousConsidering the tumor-suppressive effects of pravastatin in preclinical animal models of HCC , the STADanvatirsen  is an antisense oligonucleotide that binds to STAT3 mRNA, thereby inhibiting the translation of STAT3 mRNA . In patiIn vitro and in vivo animal studies have demonstrated that napabucasin has a strong antitumor effect on HCC, as the STAT3 inhibitor suppresses cell viability and proliferation, as well as downregulating expression of stemness genes ,159. BasSimilarly, due to its effective suppression of tumor growth in murine HCC models , OPB-311It is important to note that while inhibition of the JAK/STAT signaling pathway has shown promise in preclinical studies, its efficacy in patients with advanced HCC remains uncertain. Current clinical trials focused on this strategy are still in their early stages, and more studies are needed to better assess the effectiveness of targeting this signaling pathway. In summary, the ongoing clinical studies targeting the JAK/STAT pathway emphasize the complexity of this approach in HCC treatment and underscore the necessity for further investigation to identify more effective treatment strategies.In the realm of clinical translation, it is noteworthy that while targeting the JAK/STAT pathway in hepatocellular carcinoma (HCC) holds great promise, ongoing clinical trials focused on this strategy are still in their infancy. Despite the well-established significance of aberrant JAK/STAT signaling in HCC pathogenesis, along with preclinical observations of the anticancer effects of targeting the pathway in HCC, the journey toward clinical implementation requires a more systematic approach and refinement. Notably, adverse effects routinely observed in administering anticancer agents are particularly problematic in patients with HCC, given their pre-existing liver dysfunction due to sustained ailments from underlying chronic hepatitis and cirrhosis ,2.To explain the failure of therapeutic drugs that demonstrated antitumor effects in preclinical models of HCC to achieve effectiveness in clinical studies, several complex factors must be considered. In vitro and animal models of HCC do not perfectly mimic the human tumor microenvironment . FactorsWhile current clinical studies targeting the JAK/STAT signaling pathway might not be yielding optimistic results, it is crucial to point out that these studies are still in the early stages. As researchers navigate the complexities of this pathway and its clinical translation, it is imperative that these efforts be met with the clinical successes they deserve. By shedding light on the underlying mechanisms driving JAK/STAT pathway activation and its contributions to HCC pathophysiology, we aim to pave the way for the development of novel JAK/STAT targeted therapies that hold promise in effectively combating this aggressive malignancy. Further, it is noteworthy that combining JAK/STAT targeted therapy with immunotherapy might hold promise, as both approaches involve targeting dysregulation of inflammatory and immune responses in the tumor microenvironment.As an emerging therapeutic paradigm, the immune checkpoint has garnered attention as an attractive target for immunotherapy ,181. TheDespite initial enthusiasm, targeting immune checkpoints in isolation has failed to demonstrate sustained therapeutic efficacy in HCC ,182. Con


Historically, long term intravenous (IV) antimicrobial therapy has been considered superior to oral (PO) for the management of BJIs. In recent years, this concept has been challenged with studies showing similar efficacy and safety between PO and IV therapy. The purpose of this study is to evaluate areas for improvement within Johns Hopkins Medicine (JHM) in utilizing oral antibiotics for the treatment of BJIs as outlined in the OVIVA trial.A multicenter, retrospective study was performed of adult patients with BJIs discharged from The Johns Hopkins Hospital and Johns Hopkins Bayview Medical Center followed by the JHM OPAT Service between February 1, 2021 and February 1, 2022. The following was collected on all patients: demographics, comorbidities, site of infection, OPAT-related adverse events (AEs), 30-day readmissions, emergency department (ED) visits, source control, microbiological data, antibiotic regimen, and treatment outcomes. The primary outcome is the proportion of patients treated for BJIs with OPAT who would have qualified for oral antibiotics according to OVIVA trial criteria. Secondary outcomes include the rate of OPAT-related AEs and treatment outcomes . Descriptive statistics were used to summarize demographics and the primary and secondary outcomes.Among the 350 patients with BJIs, 221 patients met inclusion criteria. For the primary outcome, 169 (76.4%) met oral eligibility criteria with sulfamethoxazole-trimethoprim, tetracyclines, and cephalosporins being the most common PO options. There were 35 total OPAT-related AEs (24 [14.2%] in the PO eligible group and 11 [21.2%] in the PO ineligible group). There were 15 readmissions (11 [6.5%] in the PO eligible group and 4 [7.7%] in the PO ineligible group) and 17 ED visits (13 [7.7%] in the PO eligible group and 4 [7.7%] in the PO ineligible group). For 6-month treatment failure, there were 29 total occurrences (23 [13.6%] in the PO eligible group and 6 [11.5%] in the PO ineligible group).Based on OVIVA trial criteria, the majority of evaluated patients could have utilized oral antibiotics, which could have resulted in fewer OPAT-related AEs, readmissions, and ED visits.Sara C. Keller, MD, MPH, MSPH, Pfizer: Advisor/Consultant


GRAS, named after GAI, RGA, and SCR, is a class of plant-specific transcription factors family that plays a crucial role in growth and development, signal transduction, and various stress responses.GRAS gene family, a genome-wide identification and bioinformatics analysis of the banana GRAS gene family was performed based on information from the M. acuminata, M. balbisiana, and M. itinerans genomic databases.To understand the biological functions of the banana MaGRAS, 59 MbGRAS, and 58 MiGRAS genes in bananas at the whole-genome scale, and 56 homologous genes were identified in the three banana genomes. Banana GRASs can be classified into 10 subfamilies, and their gene structures revealed that most banana GRAS gDNAs lack introns. The promoter sequences of GRASs had a large number of cis-acting elements related to plant growth and development, phytohormone, and adversity stress responsiveness. The expression pattern of seven key members of MaGRAS response to low-temperature stress and different tissues was also examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microRNAs-MaGRASs target prediction showed perfect complementarity of seven GRAS genes with the five mac-miRNAs. The expression of all seven genes was lowest in roots, and the expression of five genes was highest in leaves during low-temperature stress. The expression of MaSCL27-2, MaSCL27-3, and MaSCL6-1 was significantly lower under low-temperature stress compared to the control, except for MaSCL27-2, which was slightly higher than the 28°C control at 4 h. The expression of MaSCL27-2, MaSCL27-3, and MaSCL6-1 dropped to the lowest levels at 24 h, 12 h, and 4 h, respectively. The MaSCL27-4 and MaSCL6-2 expression was intermittently upregulated, rising to the highest expression at 24h, while the expression of MaSCL22 was less variable, remaining at the control level with small changes.In the present study, we identified 73 GRAS family has an important function in low-temperature stress in bananas. This study provides a theoretical basis for further analyzing the function of the banana GRAS gene and the resistance of bananas to cold temperatures.In summary, it is tentatively hypothesized that the  GRAS is involved in almost the whole process of plant growth and development and plays a key role in root and axillary bud development, meristem formation, male gamete development, and gibberellin and light signal transduction . There aGRAS gene family has been reported in several plants species including Arabidopsis thaliana (33), rice (60)  (GRAS genes were grouped into 14 subgroups in rice and 8-16 subgroups in poplar (The identification of the ice (60) , poplar ice (60) , water lice (60) , tomato ice (60) , pepper ice (60) , mung beice (60) , barley ice (60) , soybeanice (60) , and teaice (60) . The GRAice (60) , and thed LISCL) . Furthern poplar , mung ben poplar , barley n poplar , soybeann poplar , and othGRAS family vary, which may lead to different functions of different subgroups. It is known that PAT is involved in the signal transductions of phytochromes; for example, AtPAT1 and AtSCL21 play a positive regulatory role in the signal transductions of phytochromes A in Arabidopsis  is one of the most popular and economically important fruits in the world. Cultivated banana is generally susceptible to external and unstable environmental changes such as exposure to cold stress. The minimum temperature for banana growth and development is 13 °C. When the temperature drops to 5 °C, the leaves begin to suffer from cold damage, at 2.5 °C the leaves are severely damaged, and at 0 °C the plants freeze to death. The lower the temperature or the longer it lasts, the more severely the plants suffer. To further investigate the banana GRAS family and improve the resistance of bananas to cold, this study was conducted to identify the GRAS transcription factor family in the Musa acuminata, M. balbisiana, and M. itinerans genomic databases based on previous studies on the GRAS family. A total of 73 MaGRAS, 59 MbGRAS, and 58 MiGRAS genes were identified. The functional diversity of the banana GRAS family was inferred based on cis-acting elements, and the presence of low-temperature response elements in most of the banana GRAS genes was found, which was hypothesized to have important functions in banana low-temperature stress. Collinearity analysis indicated that tandem and segmental duplications (SD) might be the main reason for the expansion of the banana GRAS family.The banana  at 28 °C, and to a photoperiod of 16/8 h . After cold treatment (4 °C), the first fully expanded top leaves were harvested at time points 0, 4, 8, 12, 18, and 24 h. The first leaf (expanded leaf) and different tissue parts  of the control plants at 28 °C were snap frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80 °C and used for total RNA extraction, qRT-PCR expression analysis, and RLM-RACE test materials.2.2M. acuminata var. DH-Pahang . Hidden Markov model files of the GRAS structural domains were downloaded from the Pfam (http://pfam.xfam.org/), and these three genomic data of bananas were searched  using the HmmSearch program in the Hmmer 3.0 software to obtain preliminary candidate proteins after redundancy removal. The structural domains of the banana MaGRAS, MbGRAS, and MiGRAS candidate proteins were detected by NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) tool and SMART (http://smart.embl-heidelberg.de/), and the sequences without GRAS structural domains were removed, resulting in 73, 59, and 58 members, respectively. After NCBI-Blastn/Blastp comparison, and with reference to the Arabidopsis GRAS nomenclature and the position of the chromosome (chr) where each member is located, each member was named sequentially. Segmental duplications and tandem duplications genes of the GRAS family in bananas were analyzed using TBtools  database and transcription factor database (http://planttfdb.cbi.pku.edu.cn/) as comparison sequences. Using the Neighbor-joining method of MEGA 6.06, phylogenetic trees with a total of 265 GRAS amino acid sequences were constructed for three species of banana, M. acuminata, M. balbisiana, and M. itinerans, Arabidopsis, and rice, with the running parameters of Poisson model, complete deletion, and bootstrap value set to 1,000 replicate tests. Other parameter values defaulted.A total of 33 2.3https://web.expasy.org/protparam/) and SignaIP 4.1 Server . The conserved motifs of banana GRAS protein sequences were analyzed through the MEME  online website. Their promoter transcription factor binding sites were predicted using the PlantTFDB (http://planttfdb.cbi.pku.edu.cn/) with parameters set to p-value ≤ 1e-6. The data obtained was compared, analyzed, and finally plotted by TBtools software.Sequences 2000 bp upstream of the transcription start site of the lantCare  (http://lantTFDB  online w2.5http://plantgrn.noble.org/v1_psRNATarget/) was used for the prediction analysis of miRNAs regulating the MaGRAS gene family of banana. The expected value was set to 0–3, and the others were the default parameter values.The plant miRNA from the miRbase database and the miRNA mature body sequence identified from the banana transcriptome were used as the database, and the psRNATtarget  online aTotal RNA from the roots, pseudostem, and leaves of ‘Tianbaojiao’ transplants with satisfactory integrity and purity were mixed, and reverse transcribed by RLM-RACE using the GeneRacerTM Kit  to obtain cleavage site verification cDNA. Cleavage primers of target genes were designed using DNAMAN 6.0 (Lynnon Biosoft), with primers shown in The PCR reaction system was 25 µL, Dream TaqTM Green PCR Master Mix (2X) 12.5 μL, ddH2O 9.5 μL, template cDNA 1 µL, and 1 µL each of the upstream and downstream primers. The amplification procedure was 94 °C predenaturation for 3 min, 94 °C denaturation for 30s, 55 to 60 °C annealing for 30s, 72 °C extension for 30s, 34 cycles, and the last 72 °C extension for 10 min. The amplified products were detected by 1% agarose electrophoresis, and the target bands were recovered by Gel/PCR Extraction Kit. After being ligated into the pMD 18-T vector and transformed into DH5α sensors for bacteriophage PCR, several single colonies were picked for sequencing after TA cloning. The sequencing of the samples was entrusted to Shanghai Boshang Biotechnology Co.2.6MaGRAS gene family were further selected, and qPCR-specific primers were designed on both sides of the cleavage site. The key members of the MaGRAS gene family were analyzed for their specific expression in different tissue sites of bananas and under low-temperature stress. Primer design was performed using DNAMAN 6.0 with a target fragment size of 80–160 bp, and the primer sequence and annealing temperature are shown in According to the results predicted by psRNATarget prediction, the key members of the CAC was used as the internal reference gene  were used for calculation analysis and mapping. IBM SPSS Statistics for Windows, version 26.0  was used for significance analysis of the expression level of each gene member.We took samples of roots, false stems, and leaves of transplanting seedlings under 28 °C control (CK) and 4 °C cold treatment for 4 h, 8 h, 12 h, 18 h, and 24 h, and then extracted the total RNA of each sample according to the operating instructions of RNAprep Pure Plant Kit . The concentration, purity, and integrity of the extracted RNA were determined by 1.0% non-denaturing agarose gel electrophoresis and a UV spectrophotometer. RNA processed at different tissue and various time points was reverse-transcribed to cDNA as a template for qPCR using PrimerScriptTM RT Reagent Kit  reverse transcription kit , according to the manufacturer’s instructions. nce gene , and U6 33.1MaGRAS genes contained multiple transcripts; Ma03_g02280 and Ma08_g09130 had three transcripts each, and Ma07_g05950 and Ma11_g18690 had two transcripts each. The resulting members were named sequentially according to the genome database annotation, coding sequences chromosomal mapping information, and the NCBI-Blastp/Blastn alignment results, and with reference to the Arabidopsis GRAS nomenclature method.NCBI-CDD and SMART yielded 73 MaGRAS, 59 MbGRAS, and 58 MiGRAS family members. Coding sequences (CDS) alignment analysis using NCBI-BLAST revealed that some ExPASy’s online tool ProtParam was used to analyze the basic physicochemical properties of the proteins encoded by the banana GRAS family . All had only one member except for S333, S658, S1345, S1584, and S3004, which had two members each, and S784, which had three members  in MaGRAS, distributed on all chrs except chrUn_random. There were 28 segmental duplication gene pairs in MbGRAS with no tandem duplication, and at least 2 segmental duplication genes were present on each chr. There were 16 segmental duplication gene pairs and 1 tandem duplication gene pair (Mi_g027597 and Mi_g027598) in MiGRAS, unevenly distributed over 20 scaffolds. GRAS covariance analysis of the three genomes of bananas revealed 121 homologous gene pairs between MaGRAS and MbGRAS, 95 homologous gene pairs between MaGRAS and MiGRAS, and 70 homologous gene pairs between MbGRAS and MiGRAS. In addition, many GRAS genes were found to be relatively conserved among bananas, and 56 of the 73 MaGRAS genes were homologous in both MbGRAS and MiGRAS. Moreover, banana MaGRAS was also homologous in rice and mimosa, with co-linearity between 12 AtGRAS and 9 MaGRAS and between 19 OsGRAS and 24 MaGRAS.To investigate the gene duplication events in the GRAS family were calculated and analyzed whether the family was under natural selection pressure and traced its duplication time during evolution . Whereas the remaining members had a complete GRAS superfamily or GRAS conserved domains. In addition to the common GRAS domain, some members also contained other domains. MaSCR-2a had a PHA03307 superfamily functional domain, MiSCL3-3 had a PRK14698 superfamily and a PHA03247 superfamily functional domain, whereas MaSLR1-2, MaSLR1-3, MaSLN1, MaSLR1-1, MbSLN1, MbSLR1-2, MbSLR1-1, MiGAI-1, and MiSLN1 all had a DELLA functional domain. That might be related to their diversity of functions.The 20 conserved motifs of the banana GRAS proteins were analyzed using the MEME online website and it was found that each MaGRAS contained motif 1, motif 2, motif 4, and motif 6. Except for a few MaGRAS proteins with partial deletions, the rest contained motif 5, motif 7, motif 8, motif 9, and motif 11. Motif 14 and motif 20 were only present in the LISCL subfamily, motif 15 and motif 16 were only present in the AtPAT1 subfamily, and motif 18 was only present in the AtSCR subfamily. Most of the MbGRAS proteins contained motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, motif 7, motif 8, motif 10, motif 11, motif 15, and motif 16. Motif 14 and motif 17 were present only in the AtSCL4/7 subfamily, whereas motif 20 was only present in the AtPAT1 subfamily. Most proteins of the MiGRAS contained the conserved motif, motif 1, and motif 13, besides motif 15 in the LISCL subfamily, motif 16 and motif 20 in the HAM subfamily, and motif 18 in the AtPAT subfamily. The c-terminus sequence of the GRAS proteins was highly conserved and consisted of five motifs in sequence, namely, HLR I, VHIID, LHR II, PFYRE, and SAW 5, present in motifs 1–9, motif 11, and motif 17 of MaGRAS, motifs 1–8, motif 10, and motif 15 of MbGRAS, and motifs 1–7, motif 9, motif 10, and motif 13 of MiGRAS. The majority of motifs of GRAS protein appeared at the C-terminus, suggesting that the family proteins are highly conserved at the C-terminus. The N-terminus motifs, on the other hand, were more discrete, indicating that there may be dissimilarities in the function of the banana GRAS family.3.4Arabidopsis and GRAS proteins from MaGRAS, MbGRAS, and MiGRAS, 265 GRAS protein sequences from bananas, Arabidopsis, and rice were used to construct phylogenetic trees using the Neighbor-joining method of the software MEGA 6.06. The typical GRAS domain minimum length is about 350 amino acids, and some members of the GRAS family in bananas, rice, and Arabidopsis have a GRAS domain of less than 350 amino acids and are not reliable, so these members were excluded when constructing the phylogenetic tree . It is speculated that the expression of banana GRAS may be regulated by a variety of factors. Different cis-elements and their number of loci with the same or similar functions were classified and counted , and MaSCL4-3 and MaSCL8-3 contain the largest number (25). The banana GRAS family contains five hormone-response elements, namely, abscisic acid, auxin, gibberellin, methyl jasmonate, and salicylic acid response elements. All members except MaSCL1-1a, MaSCL1-1b, and MaSCL1-1c contain at least one hormone response element. Most members contain three or more, and 22 members contain five hormone response elements. Further analysis of phytohormone response elements revealed that the largest number of cis-elements related to the response of MeJA, with 82% of the members containing at least 6, and some even 18 (MaSCL4-2 and MaSLR1-3). This was followed by the ABA response elements, with 73% of the members containing it. Finally, SA (59%), GA (57%), and AUX (43%) were found. According to the abovementioned analysis, it shows that each member of the banana GRAS family gene is involved in different hormone signaling pathways and has a different division of labor in the hormone response.Banana GRAS gene family, namely, anaerobic induction (ARE), hypoxia-specific induction (GC-motif), defense and stress (TC-rich repeats), low temperature (LTR), drought-induced MYB binding point (MBS), and injury response elements (WUN-motif). Besides, multiple cis-acting elements are also present in the banana GRAS promoter regulatory sequence, both specific and related to tissue growth and development. These include a root-specific element (motif 1), seed-specific regulatory element (RY-element), cell cycle regulatory element (MSA-like), circadian regulatory element (circadian), palisade mesophyll cell differentiation regulatory element (HD-Zip1), endosperm expression element (GCN4_motif), endosperm-specific negative expression regulatory element (AACA_motif), meristem expression (CAT-box) regulatory element, MYB binding point (MBSI) of flavonoids biosynthesis genes, and cornicin metabolism regulatory element. The aforementioned results suggest that the banana GRAS family may not only regulate plant growth and development but also respond to stress.During their growth and development, plants have complex environmental conditions, and they will face various biological and abiotic stresses, such as low temperature, drought, injury, and pathogens. Multiple stress response elements exist in the promoter region of the banana 3.5.2GRAS, the transcription factor binding site (TFBS) in its promoter region was predicted and analyzed  were present in 43 members, followed by MIKC_MADS (419) and AP2 (341), both present in 42 members; the least were C3H, CPP, Trihelix, and WRKY, all with only one binding site. A total of 23 transcription factor families were also present in the promoters of both MbGRAS and MiGRAS genes, and they both had 20 transcription factor families identical to MaGRAS. Trihelix was present only in MaGRAS and MbGRAS, WRKY was present only in MaGRAS and MiGRAS, MYB_related was present only in MbGRAS and MiGRAS, and HSA, E2F/DP, and Nin-like were present only in MaGRAS, MbGRAS, and MiGRAS, respectively. In addition, there were some differences in the type, number, and distribution of transcription factors in the promoters of each member of the banana GRAS family, for instance, 11 transcription factor family binding sites in the MbSLR1-1 promoter. However, no transcription factor was absent in the MaSCL9-1, MaNSP2, MaCIGR1-4, MbSCR-1, MiSHR2-2, and MiNSP2 promoters; and there were 321 transcription factor binding sites in MbSCL18-2. However, only one transcription factor-binding site was included in MaSHR2-2, MaSCL4-2, MaNSP1, MaGAI-1, MbSCL21, MbSCL4-5, MbNSP1, MiSCL8-4, MiNSP1, and MiCIGR1-3. Therefore, it is speculated that the GRAS family plays multiple roles in banana growth and development and that different members may play different roles.To explore the interaction of transcription factors and banana analyzed Figure 63.6MaGRAS family members were predicted using psRNATarget online prediction software. The analysis showed that when the E value was 0–3, 14 members were shown to be targets of 38 miRNAs. Among them, MaSCL27-1, MaSCL27-2, MaSCL27-3, MaSCL27-4, MaSCL6-1, MaSCL6-2, MaSCL22, MaSCL15-1, MaSCL15-2, and MaSCL15-3 were shown to be targets of 22 microRNAs of the miR171 family. MaSCL15-3 was also targeted by five miR408. MaCIGR2-1 can be targeted by 3 miR172, and MaNSP2 was shown to be a target of 10 microRNAs of the miR171 family  targeted by miR171 was analyzed in different tissue parts of ‘Tianbaojiao’ transplants using CAC as an internal reference gene , and their expression patterns were more similar in each tissue site. Overall, MaGRAS genes showed low levels of expression in both roots and high levels in leaves and pseudostems, with most members reaching the highest expression in leaves.The expression of seven key nce gene Figure 83.8MaGRAS members under low-temperature stress are shown in MaGRAS members were downregulated at 8 h of low-temperature treatment in ‘Tianbaojiao’ transplants compared with the 28 °C control. In addition, the expression patterns of the seven MaGRAS during low-temperature stress compared with the 28 °C control level can be divided into two categories. In the first category, GRAS expression levels were positively regulated by intermittent upregulation with increasing low-temperature stress. MaSCL27-4, MaSCL6-2, and MaSCL22 were upregulated or significantly upregulated at 4 h, 12 h, and 24 h, and MaSCL27-1 was significantly upregulated from 12 h to 18 h. The second category was the continuous negative regulation of GRAS expression level with the increasing degree of low-temperature stress. The expression of MaSCL27-3 and MaSCL6-1 was always lower than that of the control without low-temperature treatment, and the expression was lowest at 24 h and 4 h, respectively, while the expression of MaSCL27-2 was continuously lower than that of the control after 4 h and dropped to the lowest at 12 h.The results of the relative expression analysis of different MaSCL27-1, MaSCL27-2, MaSCL27-3, MaSCL27-4, MaSCL6-1, MaSCL6-2, and MaSCL22, and mac-miR171a-3p.1, mac-miR171a-3p.2, mac-miR171a.2, mac-miR171c.1, and mac-miR171b.2 were analyzed under low-temperature stress at 4 °C, using U6 as an internal reference gene  during the pre-low-temperature stress period. To some extent, miRNAs are confirmed to negatively regulate the expression of target genes at the post-transcriptional level.The expression patterns of nce gene Figure 944.1M. acuminata, M. balbisiana, and M. itinerans, 73 MaGRAS, 59 MbGRAS, and 58 MiGRAS genes, respectively, were identified by bioinformatics methods. Four of the MaGRAS genes had more than one transcript , with significant amplification of membership compared to various species such as Arabidopsis (33), water lily (38), and tomato (53). In addition, the GRAS gene number in M. acuminata banana was the highest, which may be the largest by this genome. Chromosome localization information showed that MaGRAS and MbGRAS genes were unevenly distributed on all the banana chrs, and only one GRAS gene was found on chr2, while most members were distributed on chr4 and formed short clusters of tandem repeats. Most MiGRAS genes were distributed on all scaffolds, but a few scaffolds had multiple GRAS genes.The GRAS gene family regulates plant growth and development, including GA signaling, root and axillary shoot development, and response to abiotic stress. They have been identified in a variety of plants, but have been reported relatively rarely, in bananas, especially as genome-wide identifications. Based on genomic data of MaGRAS, MbGRAS, and MiGRAS genes, respectively, had no intron. GRAS genes without intron also occupied a large proportion of other species. For example, 82.2%, 67.6%, 55%, 81.3%, and 77.4% of Prunus mume , AtHAM2 (AtSCL22), and AtHAM3 (AtSCL6) are involved in the development of apical meristem and axillary meristem of Arabidopsis , 12 times (40 genes), 7 times (15 genes), and 4 times (8 genes) in Arabidopsis, poplar, rice, and pepper, respectively , and only one tandem repeats in MiGRAS (2 genes), representing short clusters. It is speculated that these genes may be the product of the banana’s long evolutionary process and that they play an important role in its reproduction. It has been described that bananas may have undergone three genome-wide duplication events during their evolution, namely, α, β (approximately 75 million years ago), and γ (approximately 100 million years ago)  . The dup4.3GRAS genes in bananas have a low-temperature response element, which is not active under normal conditions but can be activated when plants are subjected to low-temperature stress, and plays an important role in plant cold resistance response. In addition, six members of the banana GRAS family were found to be regulated by miR171. Plant miRNAs are widely involved in the regulation network of response to low-temperature stress , SCL6-III(SCL22), and SCL6-IV(SCL6) of the GRAS family (MaGRAS were targeted by multiple members of the miR171 family, suggesting that they may play a role in the banana cold-resistance response. In addition, GRAS is involved in the response of various plants to stress. Ye Jiejun (GRAS gene expression in citrus plants was significantly upregulated under low-temperature stress, and the increase of GRAS gene expression was positively correlated with plant resistance to low temperature. Guo Peng et al (ZmSCL7 in maize under 4 °C stress, and the results showed that ZmSCL7 was significantly induced by low temperature. LaSCL18 can actively respond to low-temperature stress, and its downregulated expression trend appeared after 12 h of low-temperature treatment but was always higher than the control level, indicating that this gene may be related to the low-temperature tolerance of LaSCL18 (GRAS gene expression in ‘Tianbaojiao’ transplants during low-temperature stress, it was found that the MaGRAS gene expression pattern during low-temperature stress was not a simple linear change relationship, but a response of multi-directional regulation. MaSCL27-2, MaSCL27-3, and MaSCL6-1 showed negative regulation in response to low temperature, while the other four members showed intermittent positive regulation. In particular, the expression level of MaSCL27-4 reached nearly eight times that of the control after 24 h of low-temperature treatment, and promoter prediction analysis found that MaSCL27-4 contained two low-temperature response cis-acting elements, suggesting that MaSCL27-4 may play a major role in the process of low-temperature response in bananas. Therefore, the GRAS family of bananas may participate in the cold-resistance response of bananas and play an important role in the cold-resistance process.According to promoter identification, nearly half of the e stress , and miRe stress . MiR171 S family . The miRe Jiejun  found thng et al . used No LaSCL18 . By analGRAS binding region were completely complementary and paired, which was very similar to the way and function of siRNA degrading target mRNA by means of RNAi. This was consistent with the results of Llave et al., which showed that miR171 and GRAS internal sequences of Arabidopsis were completely complementary. The RLM-RACE verification results showed a clear targeting relationship between miR171 and MaGRAS, which was basically consistent with the software prediction results and research reports. Further analysis of the regulation mode of miR171 and its target genes under low-temperature stress showed that MaSCL22, MaSCL6-1, and MaSCL27-4 were negatively correlated with miR171. These miRNAs regulate the post-transcriptional level expression of the three MaGRAS members in response to low-temperature stress by cleaving target mRNA. The response patterns of the other four members were not completely negatively correlated with miR171, but significantly inhibited each other in the early stage, while there was a certain degree of synergistic and complementary inhibition in the later stage, which fully indicated the complexity of post-transcriptional regulation of miRNA and mRNA in response to low-temperature stress in plants. The regulatory functions of miRNA and the regulatory modes of target genes are diverse.In general, miRNAs mainly exist in the form of single chains, which are not completely complementary to the binding region of target mRNA in action, resulting in mismatch, inhibition of translation or degradation of mRNA, and regulation of gene expression at a post-transcriptional level without affecting the stability of mRNA. The online prediction results of miRNA showed that miR171 and its target 5MaGRAS, 59 MbGRAS, and 58 MiGRAS genes were identified based on the genomic data of Musa acuminata, M. balbisiana, and M. itinerans. The banana GRAS gene promoter has more light, hormone, and stress response elements, indicating that this family may have an important role in plant growth and development and in stress endurance. MiR171 targets seven MaGRAS genes and there are differences in the mode of regulation of these seven genes by miR171. In addition, tissue-specific expression analysis showed that the expression of all seven MaGRAS regulated by miR171 was downregulated in ‘Tianbaojiao’ transplants at 8 h of low-temperature treatment compared with the 28 °C control, indicating that low-temperature stress affects the expression of GRAS genes, which in turn may have important effects on the growth and development of banana and fruit set. In this study, we investigated the regulatory mode of miR171 and its target gene MaGRAS, and, in future research, we will further analyze how miR171 and MaGRAS genes interact to regulate the resistance of bananas to cold temperatures.In this study, a total of 73 The original contributions presented in the study are included in the article/ZL, NT, and DL designed and coordinated the study. NT and DL conducted the experiment and wrote the research paper. DL and SZ prepared the materials and helped complete experiments. NT analyzed the data. MT drew out the pictures. ZL revised the research paper. All authors agree to be accountable for all aspects of the study in ensuring that questions related to the accuracy or integrity of any part of the research work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.


Escherichia fergusonii strains from porcine nasal swab samples collected from 50 swine farms in China. All the E. fergusonii isolates were highly resistant to tigecycline with minimal inhibitory concentration (MIC) values of 16–32 mg/L, and all contained the tet(X4) gene. In addition, 13–19 multiple resistance genes were identified in these isolates, revealed by whole-genome sequencing analysis. The tet(X4) gene was identified as being located in two different genetic structures, hp-abh-tet(X4)-ISCR2 in five isolates and hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26 in one isolate. The role of efflux pumps in tigecycline resistance was evaluated by using inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The MIC values of tigecycline showed a 2- to 4-fold reduction in the presence of CCCP, indicating the involvement of active efflux pumps in tigecycline resistance in E. fergusonii. The tet(X4) gene was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquisition of tigcycline resistances in the transconjugants. Whole-genome multilocus sequence typing (wgMLST) and phylogenetic analysis showed a close relationship of five isolates originating from different pig farms, suggesting the transmission of tet(X4)-positive E. fergusonii between farms. In conclusion, our findings suggest that E. fergusonii strains in pigs are reservoirs of a transferable tet(X4) gene and provide insights into the tigecycline resistance mechanism as well as the diversity and complexity of the genetic context of tet(X4) in E. fergusonii.Tigecycline is a last-resort antibiotic for the treatment of infections caused by multidrug-resistant bacteria. The emergence of plasmid-mediated tigecycline resistance genes is posing a serious threat to food safety and human health and has attracted worldwide attention. In this study, we characterized six tigecycline-resistant  The emergence and spread of bacterial antimicrobial resistance pose a serious threat to food safety as well as human and animal health . Of specEnterobacteriaceae [Escherichia coli and Klebsiella spp. [plsC, rpsJ, trm, tet(A), and tet(M) have been found to decrease tigecycline susceptibility [Bacteroides spp. [Tigecycline resistance has emerged over recent years, mostly identified among extensively drug- and carbapenem-resistant isolates ,5,6. Oveeriaceae , which alla spp. ,14,15,16tibility ,18,19,20tibility . The Tetdes spp. ,23,24, htet(X4) gene is a novel plasmid-mediated high-level tigecycline resistance gene discovered in Enterobacteriaceae and Acinetobacter isolates from animals and humans in China in 2019 [tet(X4) has mainly been discovered in E. coli from animal origin and sporadically in several other bacteria species, such as Klebsiella pneumoniae, Shigella flexneri, Shigella boydii, Shigella sonnei, Aeromonas caviae, Acinetobacter sp., and Escherichia fergusonii [tet(X4) in different bacterial species is important to uncovering the resistance mechanism and its potential effects on human health, but this has not yet been thoroughly investigated. The  in 2019 , includi in 2019 , which prgusonii ,25,27,28E. fergusonii isolates from porcine nasal samples obtained across 50 farms in Fujian, China, including analyzing the efflux pump activity in relation to tigclycline resistance, genetic context of tet(X4) genes, as well as transferability and phylogenetic relationship of the strains in order to gain insight into the possible public-health impact of tigecycline-resistant E. fergusonii from pigs.The aim of this study was to characterize six tigecycline-resistant E. fergusonii by 16S rRNA gene sequencing and exhibited MIC values for tigecycline between 16–32 mg/L  from six different pig farms were identified as –32 mg/L .In general, 13–19 acquired antimicrobial resistance genes were identified in all isolates by ResFinder, which encodes resistance to nine different antimicrobial classes, including beta-lactam, sulphonamide, aminoglycoside, disinfectant, macrolides-lincosamides-streptogramines (MLS), fluoroquinolone, trimethoprim, tetracycline, and phenicol .PlasmidFinder predicted plasmid types IncFIA(HI1), IncFIB, IncHI1A, IncHI1B(R27), IncX1, and IncY in six isolates . MLST tytet(X4) gene. In addition to tet(X4), tet(B) without mutation was identified in isolate 2022GZP273, and tet(A), as well as tet(M) with the same mutations, was found in all the other isolates (tet(X4) gene was located in a classic genetic context, hp-abh-tet(X4)-ISCR2, while a new genetic context of tet(X4), hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26, was observed in isolate 2022GZP273 (All strains contained the isolates . For fiv22GZP273 .hp-abh-tet(X4)-ISCR2, has been mainly identified in plasmids in E. coli and less frequently in other bacteria species -ISCR2 share the same downstream structure as those in pHNCF11W-tetX4 (GenBank accession number CP053047), and the same upstream structure as pQZZ116-tetX-190K (GenBank accession number CP095844)  .hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26, ISCR2 was truncated by ISEc57 and linked by a copy of IS26 -ΔISCR2-ISEc57 without IS26 (GenBank accession numbers CP047578 and CP047572)  .The MIC value of CCCP is 8 mg/L for all isolates. After addition of 2 mg/L CCCP, there was a fourfold decline in the MIC value for the tigecycline of one isolate (2022GZP491) and half a decline in the MIC values for the tigecycline of the remaining five isolates , indicating the presence of active efflux pumps mediating tigecycline resistance in all isolates .The known efflux pumps contributing to tigecycline resistance, AcrAB-TolC, AcrZ and NorM, were identified in the genome sequences of all isolates. In addition to the known efflux pumps, several efflux pumps and porins associated with multi-drug resistance, such as OmpF outer membrane porin, MarA and its local repressor MarR, were also identified in all isolates .tet(X4) genes from all E. fergusonii strains to a plasmid-free recipient E. coli J53. Antimicrobial susceptibility testing revealed that the acquisition of the tet(X4) genes by E. coli J53 caused at least a 32-fold increase for tigecycline (−7 to 2.6 × 10−6 transconjugant per recipient cell in E. fergusonii strains (Polymerase Chain Reaction (PCR) results confirmed the successful transfer of the ecycline . The con strains .The wgMLST and phylogenetic analysis showed that isolates 2022GZP462, 2022GZP331, 2022GZP221, 2022GZP175, and 2022GZP491 were all highly related, while they were distantly related to 2022GZP273 .E. fergusonii is an opportunistic pathogen infecting humans and animals [E. fergusonii from livestock have been reported to be an underrated repository for antimicrobial resistance, especially with regards to mcr-1 gene [E. fergusonii significantly increases its importance to public health [ animals . It caus animals , and it  animals . E. fergr-1 gene . Thus, tc health ,34.E. fergusonii isolates have only been reported in two samples in China, a pig feces sample and a chicken feces sample [tet(X4) gene, which was located in the classic genetic structure hp-abh-tet(X4)-ISCR2. Similar to these findings, for five out of six E. fergusonii isolates in the present study, the tet(X4) gene was located in hp-abh-tet(X4)-ISCR2. Interestingly, the genetic structure of tet(X4) in these E. fergusonii isolates shared either the same upstream or downstream sequences, indicating that a recombination process at an earlier stage is likely to have happened at fixed sites in E. fergusonii. Importantly, the wide distribution of the hp-abh-tet(X4)-ISCR2 in broad bacterial species indicates its high transferability, which has also been observed in this study and which might cause an expansion of tigecycline resistance.Tigecycline resistance has been found in many species. However, tigecycline-resistant s sample ,35. In thp-abh-tet(X4)-ΔISCR2-ISEc57-IS26, was observed in an E. fergusonii isolate. In this structure, the downstream ISCR2 was truncated and inserted by ISEc57 and was associated with IS26. ISCR2 is likely to have been truncated by different IS elements, such as IS26, IS1D, IS1R, ISEc57, and ISKpn19 [hp-abh-tet(X4)-ΔISCR2-ISEc57 has only been observed in two E. coli isolates. Notably, the unit was found to be associated with one more IS26 in the E. fergusonii isolate in this study, suggesting that the emergence of this structure in E. fergusonii was a newer event at a molecular level. IS26 can mediate the formation of a hybrid plasmid between tet(X4)-positive and -negative plasmids [CR2, ISEc57 and IS26 might be a major driving force in the rapid expansion of tet(X4) in E. fergusonii. Notably, a novel genetic structure,  ISKpn19 . Howeverplasmids . Thus, ttet(X4) gene, efflux pumps were observed to be a contributor to tigecycline resistance in E. fergusonii isolates in the current study. The contribution of efflux pumps to tigecycline resistance in different bacterial species has been investigated in many studies [E. fergusonii has not been elucidated. In this study, by searching through genome sequences, we identified various RND-type efflux pumps  that existed in all E. fergusonii isolates, which have been confirmed to be associated with tigecycline resistance [E. fergusonii was further revealed by the efflux pump inhibitor, CCCP. CCCP has been shown to have a good activity against RND-type efflux pumps associated with tigecycline resistance [E. fergusonii isolates also partly contribute to the high level of tigecycline resistance. However, in addition to tet(X4) and RND-type efflux pumps, which were confirmed to contribute to tigecycline resistance in E. fergusonii, we cannot exclude the fact that other factors may also contribute to phenotypical tigclycline resistance, such as mutations in tet(A) and tet(M) genes, OmpF outer membrane porin, MarA and its local repressor MarR, which have been described as contributing to overexpression of the AcrAB efflux pump and may indirectly lead to phenotypical tigclycline resistance in E. fergusonii [E. fergusonii.Besides the  studies ,37,38. Hsistance ,38,39. Trgusonii ,41,42,43E. fergusonii isolates harboring the same genetic structure of tet(X4) were closely related, despite being obtained from different farms. This finding could indicate that E. fergusonii in pigs are reservoirs of the tet(X4) gene and that tet(X4)-positive E. fergusonii may have been transmitted between different pig farms in China. Since E. fergusonii isolates in this study were all multi-drug-resistant, the emergence of transferable tet(X4) in E. fergusonii from pigs needs to be monitored and further investigated, as it may spread along the food chain to humans.Unexpectedly, the five In August 2019, a total of 250 porcine nasal swab samples were collected from a total of 50 farms in Fujian, China. Five samples were collected from each farm and pooled for strain isolation. A low-temperature box with ice was used to transport samples from the field to the laboratory for further processing. The samples were incubated in buffered peptone water (BPW) broth for 18 to 24 h and then inoculated onto Luria–Bertani  agar plates with 2 mg/L tigecycline. Colonies were identified using 16S rRNA gene sequencing using universal primers  .http://www.eucast.org/clinical_breakpoints/, (accessed on 5 March 2021). E. coli ATCC25922 was used as a control, and all experiments were performed with three biological replicates.MIC of tigecycline  was determined by broth microdilution . The resE. fergusonii isolates was sequenced on Illumina Novaseq-PE150 150-bp paired-end reads . The initial data quality inspection was performed with FastQC , after which reads were filtered and trimmed using Cutadapt (v1.17) to discard the low-quality reads that contained ambiguous nucleotides or a quality score lower than 20 [https://enterobase.warwick.ac.uk/, (accessed on 20 May 2022) [http://blast.ncbi.nlm.nih.gov/Blast.cgi, (accessed on 21 May 2022). Clonal analysis was assessed by MLST 2.0 . PlasmidFinder V2.1 was used to identify plasmid replicon types [The whole genome of a total of six  than 20 . The genay 2022) . The preay 2022)  and furton types .The activity of the efflux pump system on tigecycline MIC was tested using efflux pump inhibitor CCCP . The MICE. fergusonii isolates, a minimum spanning tree was constructed in Enterobase using the RapidNJ algorithm and the whole genome multilocus sequence typing (wgMLST) (wgMLST scheme available on EnteroBase) scheme [In order to assess the relatedness of the six ) scheme .tet(X4) was assessed by performing the conjugation experiment, using solid mating on a filter . The sodium azide-resistant E. coli strain J53 was used as a recipient strain [The transferability of t strain .Briefly, recipient and donor strains were cultured overnight in LB broth, and then the cells were harvested, washed with saline, mixed together in a ratio of 1:1, and spotted onto a 0.45 µm pore size filter (Millipore) on LB plates to be cultured for 20 h. They were also spotted individually on LB plates as controls. The transconjugants were selected on LB plates containing 150 mg/L sodium azide and 2, 4, 8, or 16 mg/L of tigecycline after being cultured for 24–48 h. The test was conducted at least three times. Control spots were transferred to the same selective media to make sure that no growth was observed.The conjugation frequency was calculated as the ratio of transconjugants over the number of recipients. The transfer of the plasmid was confirmed by PCR, with the primers listed in The Illumina sequence data were deposited in the Enterobase database under the barcode numbers ESC_ZA4185AA, ESC_ZA4186AA, ESC_ZA4187AA, ESC_ZA4188AA, ESC_ZA4189AA, and ESC_ZA4190AA.tet(X4) in the tigecycline resistance of E. fergusonii. Our study revealed that E. fergusonii in pigs are reservoirs of tet(X4) gene and that they may have been transmitted between different pig farms in China, which poses potential hazards to associated pork products’ safety and a public health risk and which thus requires continuous investigations.To summarize, this study for the first time reports the involvement of efflux pumps and transferable diverse genetic structures of


ACBP genes have been well-studied in a variety of species including Arabidopsis, soybean, rice and maize. However, the identification and functions of ACBP genes in cotton remain to be elucidated. In this study, a total of 11 GaACBP, 12 GrACBP, 20 GbACBP, and 19 GhACBP genes were identified in the genomes of Gossypium arboreum, Gossypium raimondii, Gossypium babardense, and Gossypium hirsutum, respectively, and grouped into four clades. Forty-nine duplicated gene pairs were identified in Gossypium ACBP genes, and almost all of which have undergone purifying selection during the long evolutionary process. In addition, expression analyses showed that most of the GhACBP genes were highly expressed in the developing embryos. Furthermore, GhACBP1 and GhACBP2 were induced by salt and drought stress based on a real-time quantitative PCR (RT-qPCR) assay, indicating that these genes may play an important role in salt- and drought-stress tolerance. This study will provide a basic resource for further functional analysis of the ACBP gene family in cotton.Members of the acyl-CoA-binding protein (ACBP) gene family play vital roles in diverse processes related to lipid metabolism, growth and development, and environmental response. Plant  BnACBP, the first plant ACBP gene, was discovered in Brassica napus [ACBP genes have now been found in a variety of plant species, but the number of ACBP genes in different species varies considerably. Six ACBP genes have been found in Arabidopsis thaliana [Oryza sativa [Zea mays [Glycine max [Arachis hypogaea [B. napus [ACBP gene family has been reported in cotton to date.Plant acyl-CoA-binding proteins (ACBPs) comprise a highly conserved family that bind to a variety of acyl-coenzyme A esters with high specificity and affinity through an acyl-CoA-binding (ACB) domain . Based oca napus . Subsequca napus . Many ACthaliana , 6 in Ora sativa , 9 in ZeZea mays , 11 in Gcine max , 15 in Ahypogaea , and 19 ACBP genes are involved in a wide range of cellular processes including modulation of lipid metabolism, regulation of gene expression, and regulation of plant growth and development [BnACBP altered the fatty acid composition in Arabidopsis developing seeds [AtACBP1 and AtACBP2 have been shown to be involved in lipid transfer. Simultaneous mutation of AtACBP1 and AtACBP2 results in embryo lethality [ACBP genes can be essential for specific environmental conditions such as drought, high salinity or low temperature. AtACBP2 is induced by drought and ABA. Overexpression of AtACBP2 confers transgenic Arabidopsis enhanced drought tolerance [Extensive studies show that plant elopment ,2,3. Forng seeds . AtACBP1ethality . Furtherolerance .G. hirsutum  and G. barbadense , which are cultivated worldwide for their economic value, evolved from an occasional hybridization between the A- and D-genome ancestors, followed by chromosome doubling. The diploid G. arboreum  and G. raimondii  are closely related to the A- and D-genome ancestors, respectively [ACBP genes have been well-characterized in A. thaliana, particularly with regard to the regulation of lipid metabolism and stress tolerance [ACBP genes in cotton has not been identified to date. In this study, we performed a genome-wide characterization of the ACBP gene family in four cotton species. The phylogenetic relationships, gene structure, conserved motifs, gene duplication, cis-acting regulatory elements, and expression patterns in different tissues and in response to drought and high salinity were comprehensively analyzed. Our results will provide the basic resource for further functional analysis of the ACBP gene family.As a major cash crop, cotton not only provides natural fiber for the textile industry, but is also an important source of vegetable oil and protein. The allotetraploid ectively . Althougolerance ,12, the G. arboreum  [G. raimondii  [G. barbadense  [G. hirsutum  [https://cottonfgd.net/, (accessed on 17 September 2022)) [https://www.ebi.ac.uk/interpro/entry/pfam/PF00887/, (accessed on 17 September 2022)) [Gossypium species with an e-value < 1 × 10−10. The resulting hits were further validated in the presence of ACBP domain by the SMART tool ) [https://web.expasy.org/compute_pi/, (accessed on 19 September 2022)).The genome data of I, v1.0) , G. raimI, v2.0) , G. barbU, v2.0) , and G. U, v1.1)  were dowr 2022)) . The Hidr 2022)) . The molACBP genes identified in four Gossypium genomes and previously reported in Arabidopsis and rice [Gossypium genomes. The ten conserved motifs were identified using MEME online software ) [The ACBP domain sequences of and rice  were useand rice  using 10r 2022)) .ACBP genes was extracted from GFF gene annotation files and then visualized with the MapChart software [ACBP gene was analyzed by the method reported in the pear sugar transporter family genes [G. hirsutum genome were aligned using an all-vs-all local BLAST search with an e-value < 1 × 10−10. MCScanX software [The chromosomal location information of software . The duply genes . For exasoftware  was usedGhACBP genes (1500 bp upstream) were retrieved from G. hirsutum genome sequences [cis-acting regulatory elements in the promoter region of each GhACBP gene were predicted using the PlantCARE online tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 23 September 2022)) [The promoter sequences of r 2022)) .GhACBP genes in different tissues were determined based on public transcriptome data of genotypes TM-1, 11-0509 and Emian22 [The expression profiles of  Emian22 ,27,28. T Emian22 . FurtherG. hirsutum cultivar Lumian451 were grown in commercial soil at 28 °C with a photoperiod of 16 h light/8 h dark. Two-week-old seedlings were gently uprooted, rinsed, and cultivated in Hoagland’s solution for two days. These seedlings were then randomly divided into three groups for 200 mM NaCl, 15% PEG6000 and 100 μM ABA, respectively. Total RNA was isolated from the leaves. RT-qPCR was carried out as described by Chen et al. (2022) [GhACBP genes were calculated based on the 2-ΔΔCt method [Healthy seeds of the . (2022) . The expt method . The RT-ACBP genes in cotton, a genome-wide identification was carried out using HMMER searches with the ACBP domain (PF00887) as a query in the protein database of four Gossypium species, G. arboreum, G. raimondii, G. barbadense, and G. hirsutum. A total of 62 non-redundant ACBP genes were identified, including 11, 12, 20, and 19 in G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, respectively. Based on previous reports in Arabidopsis and rice, the ACBP genes of G. arboreum, G. raimondii, G. barbadense, and G. hirsutum were named GaACBP1 to GaACBP11, GrACBP1 to GrACBP12, GbACBP1 to GbACBP20, and GhACBP1 to GhACBP19, respectively. The detailed information of the Gossypium ACBP genes, including gene locus, chromosome location, exon number, sequence length, MW, and pI are listed in Gossypium ACBP proteins ranged from 85 (GrACBP11) to 679 (GbACBP9 and GbACBP10), with corresponding MWs varying from 9.59 (GrACBP11) to 74.61 (GbACBP9) kDa. Their pI ranged from 3.98 (GrACBP2) to 9.48 (GbACBP16) (To mine bACBP16) .ACBP genes in cotton, a total of 74 ACBP genes, including 62 Gossypium ACBP genes, 6 AtACBP genes from A. thaliana and 6 OsACBP genes from O. sativa, were used to construct an ML phylogenetic tree using the MEGA11 software [ACBP genes were classified into four distinct clades, namely small ACBPs, large ACBPs, ankyrin-ACBPs, and kelch-ACBPs  in kelch-ACBPs clade had motifs 1, 2, 3, 4, 5, and 8. In the small ACBPs clade, 18 Gossypium ACBP proteins had only motif 5, and the remaining 12 members except GhACBP19 (missing motif 9) contained motifs 2, 9, and 10  were distributed at both ends of chromosomes, such as GaACBP2 at the top of chromosome Chr06 and GbACBP1 at the bottom of chromosome A09. In conclusion, the Gossypium ACBP genes were unevenly distributed on their chromosomes, with most ACBP genes located at both ends of the chromosomes.According to the sequenced genome data, the 62  species . SpecifiGossypium ACBP genes, gene duplication analysis was performed in the four Gossypium species using the MCScanX program [ACBP genes were detected in G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, respectively. Specifically, 17 pairs of GhACBP genes, involving 16 GhACBP genes, were segmental duplications, and 2 pairs (GhACBP12/GhACBP13 and GhACBP17/GhACBP18) were tandem duplications within the G. hirsutum genome , ethylene response (ERE), anoxic response (ARE), and drought response  are present in most GhACBP genes, whereas the element for GA response (TATC-box) is present in GhACBP1, GhACBP6, and GhACBP12. Interestingly, the divergence of regulatory elements occurred in all duplicated GhACBP genes. For example, only one out of five cis-acting elements for hormone response was shared by the GhACBP1/GhACBP2 duplicate pair , while GhACBP11, GhACBP14, and GhACBP19 were highly expressed in the Leaf . Expression analysis shows that GhACBP12, GhACBP13, GhACBP17, and GhACBP18 are highly expressed in 10 and 20 dpa embryos  was shown to be highly expressed in developing cotton embryos. Overexpression of G. barbadense ACBP6 significantly increased oil content in transgenic yeast. In addition, the expression level of ACBP6 in G. barbadense acc. 3–79 (33.79% seed oil content), was remarkably higher than that in G. hirsutum cv. Emian22 (24.97%) during almost the entire seed development process, suggesting that ACBP6 may play a decisive role in the accumulation of high oil content [GhACBP13, like AtACBP6 and BnACBP6, may influence the fatty-acid composition in cotton seeds.Accumulating evidence showed that many tabolism ,3. AtACBpe seeds . BnACBP6 flowers ,33. Overseed oil . In this embryos , suggest content . Based oACBP genes, particularly the ankyrin-ACBPs clade, have been implicated in several stress responses, including salt stress and drought stress [AtACBP1 and AtACBP2. AtACBP1 was induced by NaCl and mannitol treatments. Transgenic Arabidopsis plants overexpressing AtACBP1 showed reduced tolerance to salt and mannitol treatments, whereas the acbp1 mutant exhibited the opposite phenotype [AtACBP2 was induced by drought and ABA. Overexpression of AtACBP2 increased drought tolerance and enhanced sensitivity to ABA treatment in Arabidopsis [GmACBP3 and GmACBP4 belong to the ankyrin-ACBPs clade and share 98.02% amino acid sequence identity [GmACBP3, GmACBP4, and their alternatively spliced isoforms were all increased in soybean roots [GmACBP3 or GmACBP4 were more sensitive to salt stress compared to wild plants, while transgenic plants overexpressing the alternatively spliced isoforms were more salt-tolerant [GhACBP genes, GhACBP1 and GhACBP2, which are induced by salt and drought stresses (GhACBP1 and GhACBP2 in abiotic stress tolerance.Plant t stress ,8,12. Inhenotype . AtACBP2an roots . Transgetolerant . In thisstresses . GhACBP1