RM, +CD27-NR4A1+RGS1+AHR+CD69). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (+TCF7+S1PR1+SELL+CCR7+KLF2), while retaining tissue-homing receptors . The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators . Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by +CTLA4+CSF2+TNFSF14+TIGIT tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (T RM)  comprise a heterogeneous group of peripheral non-Hodgkin’s lymphomas , 2, withRM) . Yet, in T-cells , and a s T-cells . However-γ genes , 8. Rece-γ genes , 10. Sin-γ genes . Here weThe study was conducted under a protocol approved by the Ethics Committee of the Medical University of Vienna, Austria (EK 1360/2018). After written informed consent, a 65 years old Caucasian woman with stage IVB (T3 N3 M1 B1) Mycosis fungoides (MF) was included. At time of sampling, the patient did not receive disease-specific treatment. Routine laboratory investigations revealed an LDH of 322U/L  and a CRP value of 11.47mg/dL .® Paque density gradient centrifugation . All cell suspensions were frozen at -80°C until further processing. Cell suspensions were thawed and stained with CD7 FITC, CD4 PE, CD3 APC, CD45 ECD and 7-AAD. In the case of skin and lymph node, viable CD45+CD3+CD4+ T-helper cells, other CD45+ cells, and CD45-negative cells were sorted on a FACS Aria III (BD Biosciences) at a ratio of 1:2:1. In the case of PBMC, viable CD3+CD4+ helper T-cells and remaining CD45+ cells were sorted in a ratio of 1:2. Immediately after sorting, cells were subjected to scRNA-seq processing  according to the manufacturer’s instructions.A 6mm skin punch biopsy from an MF lesion was obtained from the right flank. The biopsy was minced with scalpels and digested in collagenase IV  for 30min at 37°C to obtain a single cell suspension. Lymph node cells were obtained from a small fraction of a diagnostic biopsy from a pathologically enlarged axillary lymph node. PBMC were prepared by FicollT-cell receptor (TCR) sequencing and 5’ gene expression sequencing was performed using the Chromium Controller and Single Cell 5’ Library & Gel Bead Kit (10x Genomics) according to the manufacturer’s protocol. Sequencing was performed using the Illumina NovaSeq 4000 platform and the 150bp paired-end configuration.RNA sequencing files were preprocessed with the Cell Ranger software from 10x Genomics . After demultiplexing with the Cell Ranger command ‘mkfastq’, reads from each transcriptome library were aligned to the human reference genome assembly ‘refdata-cellranger-GRCh38-3.0.0’ using the ‘cellranger count’ pipeline. The pipeline generated both a raw UMI count matrix, including counts for all the droplets, and a filtered UMI count matrix, including only those droplets which are likely to contain at least one cell.Reads from TCR receptor libraries were assembled using the ‘cellranger vdj’ pipeline in the reference-assisted mode using the vdj_GRCh38_alts_ensembl-3.1.0 reference version. The pipeline also annotated the assembled contigs according to the reference and grouped cells into clonotypes.Filtered gene expression matrix, TCR amino acid sequences and clonotypes were then used for secondary analysis with R version 3.6.3 (2020–02–29).Seurat package  was applied to perform quality control and integrate all samples. The filtering criteria applied to each cell were the number of genes expressed (between 200 and 4000) and mitochondrial gene percentage (less than 12%) in order to discriminate for multiplets and dead cells leaking mRNA but include cell types with naturally higher mitochondrial content (such as macrophages). All cells not satisfying these criteria were discarded. Following QC, data from TCR receptor sequencing and transcriptome sequencing were merged by adding clonotype frequency and CDR3 amino acid sequences to the metadata column of the Seurat objects. All samples were aligned with the standard integration pipeline, as recommended by the Seurat developers . BrieflyCalculation of cell cycle scores was performed as implemented in the Seurat package, where gene expression of cell cycle marker genes was combined to a score. The score consisted of 43 genes primarily expressed in G1/S and 55 primarily expressed in G2/M, described in more detail by Tirosh I et al. .Trajectories and Pseudotime were calculated using Monocle 2 version 2.13 (RRID: SCR_016339) , 18. Briq‐value <0.00001 were displayed in a heat map, after eliminating ribosomal and mitochondrial genes.As model formula input for the first step of trajectory construction, differentially expressed genes between the tissues skin, blood and lymph node were used. In the next step dimensions were reduced with DDRTree and cells were ordered along the constructed trajectory and colored according to pseudotime, tissue of origin or cell cycle phase. For each branching point of the trajectory, we did branched expression analysis modelling (BEAM)  and genehttps://github.com/broadinstitute/inferCNV). The software compares gene expression values across genomic positions between case and control cells to identify genomic regions with consistent higher or lower signal intensities. We used a window of 201 for moving average smoothing and minimum of 5 cells per gene to include the corresponding gene into the analysis. CNVs were predicted with a Hidden Markov Model. We used a 6-state CNV model, attempting to predict either complete loss, loss of one copy, no change, addition of one copy, addition of two copies or addition of more than two copies.Copy number variation analysis was performed using inferCNV of the Trinity CTAT Project .The 10X Genomics datasets generated during this study are available A 65-year-old female patient suffering from MF had been in care at our department for a total of 14 years. She was originally referred in 2006 with stage IIB (T3 N0 M0 B0) disease with patches, plaques and tumors on her head and lower extremities. The Modified Severity Weighted Assessment Tool (mSWAT) showed a severity score of 8.2, indicating generally limited skin involvement. Until 2012, treatments consisted of topical corticosteroids, involved site radiation therapy (ISRT), systemic retinoid treatment (bexarotene), total skin electron beam (TSEB) radiation therapy, and gemcitabine chemotherapy, which led to temporary remission of disease. After seven years of loss to follow up, she returned to our clinic in 2019, presenting with generalized patches, plaques and tumors, and an mSWAT of 108. Skin histopathology revealed a dense subepidermal infiltrate of small to medium sized lymphocytes positive for CD2, CD3, CD4, PD-L1 and TCRβF1, partial positivity for CD5, and negative stainings for CD7, CD30 and PD-1. Ki67 staining showed a proliferation index of approximately 20% , lymphatic endothelial cells (LEC: PROX1), fibroblasts , keratinocytes (KC: KRT5) and myofibroblasts  , macrophages (MФ: CD163) and monocytes . DC-1 also contained a minor population of + CD1A+CD207 Langerhans cells that did not cluster separately (MS4A1/CD20) and NK/NKT-cells . One cluster (“other”) contained cells positive for MALAT1, suggesting damaged or dead cells  as well as two smaller clusters of +CD4 helper and +CD8A cytotoxic T-cells , with TC-2 also containing some +FOXP3 regulatory T-cells, and TC-5 consisting of proliferating (+MKI67) cells  of cells sorted from involved skin, blood and lymph node, as outlined in , EDNRA) Table S2ad cells . Among CTRA and TRB), we overlaid TCR-positive cells onto UMAP plots  and CASSQDRTLENTIYF (“TRB2”), due to a single nucleotide change (guanine to adenine). These TRB chains were mainly paired with the TRA chain CAVDHARLMF, and in <5% of clonal cells also with CALSKKPGRKAYLRT  and lymph node  within this cluster . Furthermore, we also used inferCNV to identify copy number variations between these two populations  and clusters TC-1, TC-3 and TC-4 , given many phenotypic similarities , type 2 , and type 17 (IL26) cytokines, as well as IL21 and IL32  and associated mediators  , the co-stimulatory molecule CD40LG and tumor necrosis factor-alpha (TNF) were upregulated both in lymph nodes and skin, but less so in blood (AIRE (previously associated with negative selection of self-recognizing T-cells)   , 32, butT-cells)  and TCF7T-cells)  discrimi T-cells , was uniCTLA4, PDCD1 (PD-1), HAVCR2 (TIM-3), LAG3, and CD101 (IGSF2)  highly expressed LAG3  . In line+ cytotoxic T-cells have been postulated as major anti-CTCL cells in early lesions when the disease is still confined to the skin, but lose this capacity when the disease progresses   in skin compared to lymph node tissue  were decreased compared to blood cells (ICOS (CD278)  , corrobo (CD278) , 42. Thein vitro survival of malignant T-cells (ITGAX/CD11c dendritic cells (DC-1) in skin, strongly positive for the Th2-associated markers amphiregulin (AREG), and the chemokines CXCL2, CXCL3 and CXCL8  and CTLA4, namely PVR, TNFRSF14 and CD80/CD86, respectively  were also positive for Th2-promoting chemokines such as nd CCL17 Table S2nd CCL17 .To gain further insight into the relationship of cellular fates in skin, blood and lymph node , we applq-value<10-5 were considered for further analyses. For the main branching (“cell fate 1” versus “cell fate 2”), 500 genes fulfilled this criterion  and was therefore not further analyzed (data not shown). In blood, the transcription factor KLF2 as well as the target downstream gene S1PR1 were upregulated , NR4A2, and AHR, markers associated with tissue retention of TRM cells, as well as the CTCL-typical micro-RNAs MIR155 and MIR3142 were all associated with the skin phenotype . Cells in the skin after the bifurcation (“cell fate 1”) further upregulated transcripts characteristic of TRM including CD69, CXCR4 , and these cells showed additional upregulation of Th22, TH17 and Th1-associated cytokines , while “pre-branch” cells expressed several immunoregulatory markers such as VSIR, SDC4, and ZC3H12A. In addition, “cell fate 1” cells showed upregulation of transcripts involved in cell motility including LAIR2  were upregulated. Until recently, TRM were believed to be a sessile, non-recirculating, terminally differentiated population restricted to their non-lymphoid tissue of residence, such as the skin  by a subset of skin lymphoma cells might contribute to the altered migratory behavior of these cells, but this hypothesis needs corroboration in functional studies.Reliable tracing of individual T-cell populations in humans can be a major challenge. scRNA-seq with simultaneous V-D-J sequencing of the TCR now allows for the investigation of specific T-cell clones and their transcriptomic behavior throughout various body compartments. In our patient, malignant cells of the skin showed all the characteristics of benign Ttor KLF2 , and amptor KLF2 . In bloopression . In accothe skin , 60. Thithe skin . Importativation . They coCM cells . In a xeuivalent . In linel system . In our RM cells are poised to rapidly react after antigenic re-challenge, a process that is assumed to be under stringent control by inhibitory receptors  and TIGIT, respectively, which are both synthesized by the tumor cells, and might additionally enhance local Th2 immune skewing  stage of disease. Also, due to scarcity of sample, we only had few lymph node cells available. Given the substantial heterogeneity not only between CTCL patients, but also within the malignant clone ,,68, ouhttps://www.ncbi.nlm.nih.gov/geo/, GSE165623.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Ethics Committee of the Medical University of Vienna, Austria. The patients/participants provided their written informed consent to participate in this study.Conceptualization, PB, MF, WB, CJ, KR. Software and Formal Analysis, KR, MW. Investigation, MF, WB, LS, CJ, ISK, MM, FT, SP. Writing – Original Draft, WB, CJ, KR, WW. Writing – Review and Editing, PB, MF, KR. Visualization, TR. Funding Acquisition, PB. All authors contributed to the article and approved the submitted version.This work was funded by a research grant to PMB from the Austrian Science Fund (grant number KLI 849-B). MF was supported by a research grant from the Austrian Science Fund (grant number SFB-F61.03).CJ is an employee of the Medical University of Vienna, and has received personal fees from AbbVie, Almirall, Amgen, Eli Lilly, Janssen, LEO Pharma, Mallinckrodt/Therakos, Pfizer, Novartis, Sandoz, Takeda, and UCB. CJ is an investigator for Eli Lilly and Company, Novartis and 4SC (grant paid to her institution). PB is an employee of the Medical University of Vienna, and has received personal fees from LEO Pharma, Pfizer, Sanofi Genzyme, Eli Lilly, Novartis, Celgene, UCB Pharma, Biotest, Boehringer Ingelheim, AbbVie, Amgen and Arena Pharmaceuticals. PB is an investigator for Novartis (grant paid to his institution). WB is an employee of the Medical University of Vienna and has received personal fees from Takeda, Abbvie, GSK/ViiV and Gilead. SP has received personal fees from Takeda. MM has received personal fees (speaker honoraria) from Siemens and Bristol Myers Squibb. WW is an employee of the Medical University of Vienna and has received personal fees from LEO Pharma, Pfizer, Sanofi Genzyme, Eli Lilly, Novartis, Boehringer Ingelheim, AbbVie, and Janssen.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


B-lymphoblastic lymphoma (B-LBL) with BCR/ABL mutation (Ph+ B-LBL) is a rare type of cancer in both childhood and adults. Its clinical manifestations are similar to those of other types lymphoma. However, the targeted therapy can substantially improve the outcome of Ph+ B-LBL.A 19-year-old male with blood type O, Rh+ was admitted into our hospital on August 14, 2018, due to a recurrent fever and hypocytosis for 6 months.Routine blood exam showed pancytopenia. Bone marrow sample flow cytometry (FCM) exam showed abnormal cells were 2.27% of the nucleated cells, and was classified as the abnormal early B-lineage lymphoblastic cells. FISH testing showed the BCR/ABL positive cells were 13.6%. Karyotype analysis showed the 46, XY, t. Molecular analysis of BCR/ABL mutation on ABL kinase showed that BCR/ABL T315I mutation. Patient was diagnosed with B-LBL with BCR/ABL mutation (Ph+ B-LBL).The patient was given chemotherapy with VDPI regimen .The patient achieved complete remission after 2 courses’ treatment, followed by one course of clarithromycin regimen and another two courses of VDPI regimen. Patient remains in complete remission as of March 10, 2021.In B-LBL, a BCR/ABL mutation can happen in some of these patients. It is important to guide the pathologist to perform appropriate gene mutation detection, in addition to routine Immunohistochemistry test, to ensure an accurate diagnosis and use the targeted agent for treatment. According to the literature and our results, it seems that intensive chemotherapy plus TKI regimen is effective in inducing complete remission, and allo-SCT should be used as a long-term strategy. However, the BCR/ABL mutation can happen in the B-LBL as the BCR/ABL positive B-LBL (Ph+ B-LBL) less frequently, which has been considered as a rare disease. So far, only 6 cases of Ph+ B-LBL have been reported in the literature–7 as of May 15, 2021, searched by Pub Med with the terms “B-cell lymphoblastic lymphoma” and “philadelphia” or “BCR-ABL”. A summary of the cases reported in literature is listed in Table  Extramedullary presentations made it unique and other differential diagnoses to be considered are DLBCL and Burkitt lymphoma. In the diagnosis of B-LBL, it is important to exclude Burkitt lymphoma. A careful study of the morphology and immunophenotype of the malignant cells are helpful for an accurate diagnosis. The immature lymphoblasts are usually morphologically distinct from mature lymphoid cells. In tissue biopsy, lymphoblasts of B-LBL are typically small to intermediate in size, with a high nuclear-cytoplasmic ratio, fine chromatin and inconspicuous to prominent nucleoli. We describe a case of Ph+ B-LBL, who was successfully treated with intensive chemotherapy containing tyrosine kinase inhibitor (TKI) imatinib as the targeted therapy. The patient is disease free for two and half years after diagnosis without allogeneic stem cell transplantation .Lymphoblastic lymphoma (LBL) is usually of T-cell origin and B-lymphoblastic lymphoma (B-LBL) accounts for about 10% to 15% of cases.29/L, Hb 81 g/L, Platelets 8 × 109/L, and no blast cells were found. Bone marrow sample flow cytometry (FCM) exam showed abnormal cells were 2.27% of the nucleated cells, which expressed CD10, CD19, CD34, HLA-DR, CD58, CD123, partially expressed CD13, CD33, no expression of CD38, CD117, CD15, CD20, CD3, CD4, CD8, CD7, CD56, CD11b, CD16, CD71. It was classified as the abnormal early B-lineage lymphoblastic cells. Mature lymphocytes were 17.21%, and the expressions of CD2, CD3, CD5, CD7 were normal. Large granular lymphocyte CD3+/CD57+ were 9.07%, CD3+/CD56+ NK-like T cells were 4.51%: CD3-/CD56+ NK cells were 10.17%. CD19+ B cells were 8.86%. No abnormal cells were found in the myeloid and erythriod lineages. Light chain showed multiple clonal expressions. Next-generation sequence analyses were conducted of the leukemia relevant mutations of 22 genes, including FLT3-ITD, NPM1, KIT, CEBPA, DNMT3A, IDH1, IDH2, TET2, EZH2, RUNX1, ASXL1, PHF6, TP53, SF3B1, SRSF2, U2AF1, ZRSR2, NRAS, CBL, SETBP1, ETV6, and JAK2, with the method as described in our previous publication. For the gene length longer than 150 bp, such as FLT3-ITD, alternative RT-PCR method was used for analysis. No related genes mutation was found. PET-CT showed multiple bone lesions. Femoral bone lesions puncture biopsy showed a focal area of infiltration by abnormal lymphoid cells. These cells were small, exhibiting vesicular nuclei and inconspicuous nucleoli. Immunohistochemistry (IHC) staining showed CD20(−), PAX-5(+), CD3(−), CD43(−), CD7(−), CD99(+), TdT (+), CD10 (+), CD33 (−), MPO (−), CD117 (−), CD235a (−), Ki-67 (70%+). The biopsy showed multiple fragments of bony trabeculae diffusely infiltrated by malignant lymphoid cells. Fluorescence in situ hybridization (FISH) analysis was performed on the bone lesions biopsy to exclude Burkitt lymphoma, by studying the status of the MYC gene. Using a break-apart probe, the FISH analysis showed no evidence of MYC gene rearrangement. FISH testing showed the BCR/ABL positive cells were 13.6%. Karyotype analysis showed the 46, XY, t. Molecular analysis of BCR/ABL mutation on ABL kinase showed that BCR/ABL T315I mutation.A 19-year-old male with blood type O, Rh+ was admitted into our hospital on August 14, 2018, due to a recurrent fever and hypocytosis for 6 months. Medical, family, and psycho-social history including relevant genetic information has no special issues. Physical examination of his bones, lymph nodes and other systems were unremarkable. Routine blood exam showed that WBC 1.1 × 10In view of the morphological features of the malignant cells and strong positivity for TdT, combining with the clinical manifestation and laboratory testing results especially the biopsy results, the patient was diagnosed with B-LBL with BCR/ABL mutation. The patient was given chemotherapy with VDPI regimen  and achieved complete remission (CR) after 2 courses’ treatment, followed by one course of clarithromycin regimen and another two courses of VDPI regimen. Patient also received prophylactic intrathecal chemotherapy once for each treatment course. During the chemotherapy, the patient had a mild nausea and moderate pancytopenia. No other severe adverse reactions were found. Patient remains in CR as of January 10, 2021.3 In our case, the patients had the PAX5, TdT and CD10 expression, in addition to the BCR/ABL gene mutation. For most B-LBL patients, BCR/ABL-like had been reported in different studies. Only 6 cases B-LBL patients with the BCR/ABL mutation had been reported so far, due to the rarity of disease. However, due to the uncommon of BCR/ABL gene mutation in B-LBL, some patients may not be diagnosed due to the lack of gene mutation laboratory test. So the real incidence of Ph+ B-LBL may be underestimated.The diagnosis of B-LBL has mostly depended on the characteristics of B-lymphoblast leukemia/lymphoma by IHC and a complete range of immunopheotyping, in addition to the molecular finding of the BCR/ABL gene mutation. In most cases of B-LBL, immunophenotyping shows positivity for CD19, CD79a, CD10, PAX5 and TdT, and CD20 is usually negative.+ LBL has not been standardized with consensus in the hematology/oncology field. Since multiple targeted tyrosine kinase inhibitors have been developed to specifically target the BCR/ABL mutation in leukemia/lymphoma patients, diagnosis of the subtype of the leukemia/lymphoma with the BCR/ABL mutation will be very important in regarding the patient's treatment. Although the BCR/ABL mutation B-LBL is rare, it's important to diagnose these patients in the early stage for specific therapeutic regimen and the long-term survival benefit. TKI combined with allo-SCT has demonstrated with improved prognosis for this rare disease. As for the chemotherapy regimen, it seems like that the regimen according to acute lymphocytic leukemia (ALL) protocol is preferred. In addition, it has been revealed that achieving early molecular response is associated with low risk of relapse. Among the 6 cases reported, 5 of the patients were treated with hyper-CVAD/MA regimen  combining with the TKI dasatinib. Three of 6 patients underwent the allo-SCT.At present, the treatment of PhAs for the prognosis, among these all 6 cases had been reported, 4 of the patients including 3 who underwent allo-SCT were alive at the end of follow-ups. Our case was treated with systemic intensive chemotherapy containing the TKI imatinib as targeted therapy and achieved very good results. Patient remains in CR condition for two and half years without SCT. According to the literature and our results, it seems that intensive chemotherapy plus TKI regimen is effective in inducing complete remission, and allo-SCT should be used as a long-term strategy. However, the adequate treatment strategy needs to be further defined in a large number of cases in the future.4Our case illustrates that in B-LBL, a BCR/ABL mutation can happen in some of these patients. Definitely, Ph+ B-LBL is a rare entity. However, it may get underdiagnosed due to the failure to identify the gene translocation. It is important to guide the pathologist to perform appropriate gene mutation detection by checking for Philadelphia chromosomes in all cases of B-LBL routinely, in addition to IHC test, to ensure an accurate diagnosis and use the targeted agent for treatment.Conceptualization: Xiaoning Li, Weijie Cao, Jifeng Yu.Data curation: Xiaoning Li, Weijie Cao, Suping Zhang, Li Li, Yingmei Li, Jifeng Yu.Funding acquisition: Yingmei Li, Jifeng Yu.Investigation: Dingming Wan, Jifeng Yu.Resources: Yingmei Li, Zhongxing Jiang, Dingming Wan.Supervision: Jifeng Yu.Writing – original draft: Jifeng Yu.Writing – review & editing: Jifeng Yu.


A range of natural and chemical effectors, including temperature, pressure, pH, phospholipids, and intracellular signaling molecules, substantially modulate the activity of K2P channels. In this review, we summarize the contribution of K2P channels to neuronal excitability and to potassium homeostasis in glia. We describe recently discovered functions of K2P channels in glia, such as astrocytic passive conductance and glutamate release, microglial surveillance, and myelin generation by oligodendrocytes. We also discuss the potential role of glial K2P channels in neurological disorders. In the end, we discuss current limitations in K2P channel researches and suggest directions for future studies.Two-pore-domain potassium (K2P) channels are widespread in the nervous system and play a critical role in maintaining membrane potential in neurons and glia. They have been implicated in many stress-relevant neurological disorders, including pain, sleep disorder, epilepsy, ischemia, and depression. K2P channels give rise to leaky K These channels remain open (“leaky”) across the range of physiological voltages, as well as during action potentials [Ion channels in the cell membrane, particularly potassium channels, play a vital role in the resting membrane potential of neurons and in the transmission of action potentials –5. An aptentials –8.+ channels, which feature one pore domain per subunit, K2P channels contain two pore domains per subunit. TWIKs give rise to a weakly inwardly rectifying K+ current, while TREKs are sensitive to mechanical and thermal stimuli as well as lipids. TASKs and TALKs respond strongly to changes in extra- or intracellular pH, while THIKs are sensitive to halothane.The first mammalian K2P channel, TWIK-1, was identified in humans in 1996 , 8. In cK2P channels are widely expressed in the nervous system, including the dorsal root ganglion (DRG), trigeminal ganglion (TG), spinal cord, cerebrum, and cerebellum , 11. In + conductance and maintenance of resting membrane potential. Activation of K2P channels leads to the extrusion of K+ into the extracellular space, hyperpolarizing the neuronal membrane and dampening neuronal excitability. Various endogenous and exogenous stimuli can regulate the activity of these ion channels, including membrane stretching, voltage, temperature, extra- and intracellular pH, phospholipids, and other intracellular signaling molecules generated through the activation of G protein-coupled receptors (GPCRs) [Perhaps the best-understood functions of K2P channels are passive K (GPCRs) .+, in the extracellular environment. In the brain, TASK-1 has been detected in astrocytes and oligodendrocytes, while TWIK-1 and TREK-1 are expressed in astrocytes, and THIK-1 was identified in microglia [Some K2P members are expressed not only in neurons but also in glial cells. Glia are nonneuronal cells in the nervous system that do not produce electrical impulses. Instead, they maintain homeostasis of certain ions, particularly Kicroglia .This review summarizes recent studies of K2P channel functions in neurons and glia of the central and peripheral nervous systems. These studies have relied primarily on genetic and pharmacological tools to elucidate channel function. We focus on how K2P channels in neurons help maintain resting membrane potential and regulate excitability, and on how K2P channels in glia help maintain potassium homeostasis.Some K2P channels mediate nociception and pathological pain, and knocking them out in mice increases sensitivity to noxious stimuli , 15. The+ currents: mutating Gly339 to Arg in TRESK in mice significantly reduced standing outward current in DRG neurons, without affecting resting membrane potential [TRESK was first found in the human spinal cord, then discovered in mouse cerebrum . In DRG otential , 19. At otential , 20, 21.otential . Similarotential .TRESK has been linked to migraine pathophysiology, and a frameshift mutation F139WfsX24) has been identified in patients suffering from migraine with aura  has been. The fraTRESK is also expressed in the nodose ganglia (NG), and NG neurons in diabetic rats show hyperpolarization as well as upregulated TRESK relative to NG neurons in healthy controls. , 29. SilThe fact that TRESK is expressed primarily in sensory neurons makes it a potential target for treating allodynia and migraine. Developing specific activators of this channel may expand on the range of analgesic agents currently available.TREKs belong to another subfamily of K2P channels comprising TREK-1, TREK-2, and TRAAK. TREKs are sensitive to membrane stretching, pH, arachidonic acid, and temperature, making them polymodal pain perception channels. In the DRG, TREKs are expressed in small-diameter neurons and to a small extent in medium- and large-diameter neurons . Knockin50) of 12.81 μM, and it activated TREK-2 with similar efficacy, while it did not appreciably activate TRAAK or other human K2P channels . Applying c3001a to dissociated DRG neurons produced weak hyperpolarization, increased the rheobase, and reduced the number of action potentials in small-sized neurons [Activating TREKs using the neuroprotective agent riluzole hyperpolarized the resting membrane in the superior cervical ganglion , 36. Sim neurons . Applyin+ conductance that is thermo- and mechanosensitive. The nodes of Ranvier on nearly all trigeminal Aβ-afferent nerves express both TREK-1 and TRAAK [TREK-1 and TRAAK are enriched at nodes of Ranvier on myelinated afferent nerves. These nodes display unusual action potentials and quite leaky Knd TRAAK . Knockinnd TRAAK . These cTASK-1 and TASK-3 belong to a subfamily of K2P channels sensitive to changes in extracellular pH , and thein vivo function of TASK-3 under physiological and pathological conditions began to emerge with the structure-based design of a selective TASK-3 activator, CHET3 [More details about the r, CHET3 . Adminisr, CHET3 . These gSince TASK-3 is expressed at much higher levels in the TG than DRG , future In the central nervous system, mRNAs encoding K2P channels have been detected in the cerebrum, cerebellum, brainstem, and spinal cord , 11. NeuHigh expression of TRESK has been found in several areas of the central nervous system, including the cortex, periaqueductal gray (PAG), and dorsal horn of the spinal cord . Spinal 2+ response to glutamate [Recent evidence suggests that TRESK helps regulate nocturnal dynamics of the suprachiasmatic nucleus (SCN) and light-adaptive responses . The SCNlutamate .TREK-1 and TRAAK are widely expressed across the central nervous system, with TREK-1 most abundant in the striatum, cortex and hippocampus. TRAAK is most abundant in the cortex. TREK-2 is restricted to the cerebellar granule cell layer , 52, 53.(1) TREK-1. TREK-1 can regulate the excitability of neurons, but its precise roles may depend on the type of neuron. In pyramidal neurons of the CA1 region of the hippocampus, TREK-1 deficiency depolarized resting neurons, reduced the rheobase, and increased action potential frequency [Depression: knocking out TREK-1 from several mouse models of depression alleviated depression symptoms, as measured in the forced swimming test, tail suspension test, conditioned suppression of motility test, and learned helplessness test [ess test . In factess test . Indeed,ess test , 60. Botess test , 58. TREess test . Consistess test requency . Similarrequency . The charequency . Treatinrequency , 58. Sparequency . The varIschemia and seizure: knocking out TREK-1 from mice increased their risk of ischemia due to transient bilateral occlusion of the common carotid arteries or due to occlusion of the aortic arch and left subclavian artery [(2)n artery . This isn artery . Adminisn artery These observations suggest that TREK-1 may be a useful therapeutic target in depression. Future study is needed to determine whether TREK-1 actively regulates serotonin release and is involved in the mechanism of action of selective serotonin reuptake inhibitors. TREK-1 in serotonergic neurons of the dorsal raphe has recently been implicated in the circadian photoperiod . Longer (2) TREK-2/TRAAK. TREK-2 is a downstream mediator of GABAB receptors in neurons of the entorhinal cortex, giving the channel a strong influence over spatial learning. Treating the stellate neuron of the entorhinal cortex with the GABAB receptor activator baclofen activated TREK-2, which blocked action potentials and hyperpolarized the membrane through mechanisms involving the Gai, Gao, and PKA pathways [pathways . This deα-2A adrenergic receptors as well as the Gαi and PKA pathways [In another study, norepinephrine was shown to activate TREK-2 and thereby hyperpolarize neurons at rest in the superficial layers of the entorhinal cortex. This mechanism appeared to involve pathways .In humans, FHEIG  was associated with a missense mutation in TRAAK. The recombinant mutant TRAAK channel showed a significant gain of function basally and damaged sensitivity to mechanical stimulus and arachidonic acid .TASKs are widely expressed across the central nervous system , 68. TAS− and K+ under physiological conditions. Consistent with this idea, chronic knockdown of KCC2 in rat hippocampus increased neuronal excitability by downregulating levels of TASK-3 at the membrane [In mature neurons, transmembrane chloride gradients are mainly regulated by KCC2 and NKCC1, two cation-chloride cotransporters, which, respectively, mediate outward and inward cotransport of Clmembrane . Altoget(1) Epilepsy. In the rat entorhinal cortex, application of serotonin depolarized GABAergic interneurons at rest, increased action potential firing frequency, and increased GABA release [ release . As a reKcnk9 gene coding for TASK-3 is located at chromosomal position 8q24, a locus associated with absence epilepsy [+ currents [The epilepsy , 76, altepilepsy . A mutatcurrents . Further(2) Sleep Disorder. Sleep is essential to emotional health, but the molecular processes that determine daily sleep duration and the sleep-wake cycle remain elusive. Thalamocortical neuronal networks alternate between burst activity during sleep and tonic single-spike activity during wakefulness [efulness . Inhibitefulness , 81. Simefulness . Thus, Tθ oscillation that was seen in the cortical electroencephalogram of control animals and that resembled type II θ. The deficient animals also progressed much more slowly from wakefulness to sleep, and their sleep episodes and REM θ oscillations were more fragmented [Consistent with this idea, animals expressing TASK-3 mutants showed increased nocturnal activity and shorter REM sleep , 84. Aniagmented . Knockinagmented . Exposinagmented . These rThese findings identify TASK-3 as a mediator of sleep disturbance, but how TASK-3 regulates firing patterns is still unclear. Further study is needed to investigate how TASK-3 contributes to the normal sleep cycle and how its impairment contributes to emotional disorders related to sleep disturbance, such as depression and anxiety.(3) Depression. Knocking out TASK-3 from mice impaired working memory, based on the T-maze spontaneous alternation test, and it damaged spatial memory, based on the Morris water maze test [aze test . At the aze test . These oaze test –89: knocaze test . Future Dysfunction of TASK-3 was also identified to cause neurological disorders in humans. A missense mutation in TASK-3 was identified to abolish the channel's currents and resulted in a maternally transmitted genomic-imprinting syndrome characterized by mental retardation, hypotonia, and dimorphisms in patients , 91.TWIK-1 appears to be expressed widely across the central nervous system , but fewIn dentate gyrus granule cells, downregulating TWIK-1 depolarized the cells at rest and increased their firing rate . TASK-3 via various mechanisms in the central nervous system, and K2P channels are expressed in glia. A transcriptome analysis detected the mRNAs encoding TWIK-1 and TREK-1 in astrocytes [Glia support neuronal functions trocytes , and immtrocytes . SeveralAstrocytes are star-shaped, specialized glia that differentiate from a neural stem cell pool. They primarily mediate ionic homeostasis and provide structural support to neurons. They also contribute to neurotransmitter release and synaptic development. Astrocyte dysfunction, termed reactive astrogliosis, is common in injury or disease affecting the central nervous system, including some neurological diseases related to aging: Huntington's disease, Alzheimer's disease (AD), and Parkinson's disease .+ conductance of cultured cortical astrocytes [TREK-1 and TWIK-1 in astrocytes contribute to the large Ktrocytes . While ktrocytes , depletitrocytes . In conttrocytes . On the trocytes . These dtrocytes .2+ concentration induces glutamate release via a pathway involving the exocytotic machinery [2+ concentration and glutamate transport in astrocytes. For example, the rise of intracellular Ca2+ also activates the Ca2+-activated K+ channels in astrocytes and extrudes K+ onto surrounding neurons, depolarizing them and probably resulting in glutamate release [2+ concentration in astrocytes may also trigger other changes due to activation of the same receptor on neurons. Indeed, pharmacological inhibition of certain anion channels can reduce glutamate release from astrocytes independently of the exocytotic machinery [Glutamate is one of the most important neurotransmitters that are released from excitatory presynaptic neurons and that mediate neuronal transmission. Emerging evidence suggests that astrocytes can also release glutamate to modulate synaptic plasticity, neuronal excitability, and transmitter release in physiological and pathophysiological states –105. Howachinery . However release . Using aachinery .+ uptake via K2P channels containing TREK-1, leading to a temporary increase in astrocyte volume. The volume returns to normal when bestrophin-1 allows Cl− out of the cell [2+-activated Cl− channel bestrophin-1 is responsible for slow glutamate release [αi and dissociation of Gβγ, followed by interaction between Gβγ and TREK-1, leading to the opening of glutamate-permeable TREK-1 [i-type G protein-coupled receptors including cannabinoid receptor 1, adenosine receptor A1, and μ-opioid receptor. Like TREK-1, TWIK-1 can directly interact with the Gγ subunit; in the presence of Gγ, the heterodimer TWIK-1/TREK-1, which is expressed in astrocytes [+ [βγ alters the pore region of the K2P channel. Further studies are needed to determine how TWIK-1/TREK-1 heterodimers are permeable to glutamate.Brief synaptic activity can trigger Kthe cell . TREK-1  release . Fast ree TREK-1 . This ovtrocytes , 110, becytes [+ . These dAstrocytic TREK-1 is also involved in the pathology of ischemia. In an animal model of focal ischemia induced by arterial occlusion, reperfusion was associated with upregulation of TREK-1 . Inhibit+ conductance, glutamate release, and ischemia pathology. Future work, such as using conditional knockout mice, should focus on establishing which K2P channels are expressed in astrocytes and what are their functions.These observations suggest that K2P channels, particularly TREK-1, may be involved in passive KMicroglia fulfill various functions within the central nervous system, which are related mainly to immune responses and homeostasis. Microglia monitor for damage, injury, or disease, and they contribute to synaptic pruning. Disruption of microglial function leads to several diseases, and microglia-mediated neuroinflammation, associated with synaptic loss and cognitive decline , is a ch12 receptor and hyperpolarized their membrane by ~30 mV [β from microglia [Little is known regarding the expression and function of K2P channels in microglia. THIK-1 appears to play a critical role in microglial ramification and immune surveillance in the brain . Microgly ~30 mV . Pharmacicroglia . This imThe primary function of oligodendrocytes is to insulate the neuronal axon by creating a myelin sheath, which contributes to rapid signal transduction. TASK-1 can induce currents in oligodendrocytes in response to extracellular acidification, and lack of TASK-1 depolarizes oligodendroglial precursor cells . Inhibitv and Kir channels, produce leaky K+ currents to regulate neuronal excitability. K2P channels are distributed widely in the peripheral and central nervous systems, where their expression patterns vary. In neurons, K2P channels modulate resting membrane potential and action potentials, allowing them to influence multiple biological functions. In the peripheral nervous system, TRESK, TASK, and TREK are involved in nociception and pathological pain. Activation of these channels reduces neuronal excitability and thereby alleviates allodynia in several animal models of pain, suggesting that these channels might be potent targets in pain management. In the central nervous system, activation of G protein-coupled receptors induces K2P channels to regulate neuronal excitability in ways that can influence chronic pain, ischemia, epilepsy, sleep disorder, and major depressive disorder.K2P channels, which differ from K+ and neurotransmitter homeostasis. TREK-1, TWIK-1, THIK-1, and TASK-1 are thought to be expressed in glia, but further investigation is needed to confirm the regional and cell-type distribution of K2P channels. Indeed, much more work is needed to elucidate the functions of K2P channels in glia as well as in neurons, such as through animal studies in which channels are knocked out of specific cell types under physiological and pathological conditions.While neuronal K2P channels regulate excitability, glial K2P channels are likely more involved in K


DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene ( Gene editing techniques, including zinc finger nucleases (ZFNs) ,2, transPrevotella and Francisella 1 [The CRISPR/Cas9 system is the most commonly used Cas nuclease for recognizing GC-rich sites for restriction by protospacer adjacent motif (PAM), which greatly limits its application in non-coding regions enriched with AT [isella 1 . It can isella 1 ,11,12. Iisella 1 ,14,15,16A common problem associated with CRISPR/Cas9 or CRISPR/Cas12a is the low editing efficiency, especially in stem cells such as induced pluripotent stem cells (iPSCs) ,17. AddiOrip/EBNA1, derived from the Epstein-Barr virus, has a post-synthesis function, which enables a plasmid containing Orip/EBNA1 to replicate autonomously in eukaryotic cells without integrating into the host cell genome ,26. FurtIn this study, the effect of integrating CRISPR/Cas12a with Orip/EBNA1 (COE) on gene editing was investigated. It was found that the editing efficiency of the COE system increased with the culture time and enabled the efficient editing of endogenous genes in human embryonic kidney 293T (HEK-293T) cells and iPSCs, thus providing a good alternative for efficient gene editing.KpnI  and XhoI  from the pcDNA3.1-hAsCpf1 plasmid , and the U6 promoter was amplified from px330 vector  with 2× ACE Taq Master Mix  on a Polymerase Chain Reaction (PCR) apparatus . The PCR reaction steps were as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 10 min. Additionally, AsCas12a and the U6 promoter were successively cloned into the pCEP4 vector  with T4 ligase  which contains the CMV promoter and the Orip/EBNA1 elements and can help obtain the COE plasmid  for amplification [BbsI  to construct a non-additive system as a control named Cas12a. In addition, the px330 vector containing the Cas9 element was used as another control group named Cas9. The insertion of crRNA or single guide RNA (sgRNA) binding sequences into COE/Cas12a or Cas9-based vectors was performed as previously described [http://www.rgenome.net/cas-designer/, accessed date 9 November 2021) was used. All primers were procured from Sangon Biotech .First, the AsCas12a element was cut with  plasmid . Plasmidfication . Then, p2 in a cell culture incubator (Thermo Fisher) [HEK-293T cells were purchased from ATCC  and cultured on 6 cm plates  using Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum . iPSCs  and Duch Fisher) . The res5 cells per well with high glucose DMEM cell culture medium for 24 h. When they reached 70–80% confluence, the cells were transfected with 1.5 μg of COE-GFP, Cas12a-GFP, or Cas9-GFP, respectively, according to the manufacturer’s instructions, using a Lipofectamine 2000 (Thermo Fisher). After transfection for 24 h, the medium was supplemented with 400 μg/mL of hygromycin B (Thermo Fisher) to enable drug screening, which lasted for 48 h. After transfection for 15 or 21 days, cells were collected and subjected to flow cytometry analysis as previously described [Stable Green Fluorescent Protein (GFP)-expressing HEK-293T (293T-GFP) cells were obtained by a lentivirus infection . Before escribed  to deterNRL and HBB), which included the PstI and BsrI restriction sites, were designed. Plasmid transfections were performed as previously described. After screening, genomic DNA (gDNA) was isolated on day 5, day 10, day 15, or day 20 using phenol-chloroform extraction [PstI and BsrI) for 4 h and then agarose gel electrophoresis  was performed using 1% agarose gel . The gray value of each sample was generated via the ImageJ software . All restriction enzymes were purchased from New England Biolabs.To further analyze the ability of the COE to perform gene knockout in HEK-293T cells, crRNAs for targeting two loci . On day 3, cells were selected by hygromycin B for 72 h. For the analysis of single cell-derived clones, the cells were disassociated into single cells with TrypLE Express Enzyme without Phenol Red (Thermo Fisher) at day 10 post- nucleofection. They were then seeded onto the Matrigel-coated plates with mTeSR-plus medium for 15 days. Individual colonies were selected, and genotyped PCR was performed to screen for positive clones. The PCR primer’s sequences are shown in First, 1.6× 10® II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme) according to the manufacturer’s instructions. qPCR was performed using the AceQ Universal SYBR qPCR Master Mix (Vazyme) with a CFX Connect RT-PCR detection system . The PCR primers sequence is shown in Total RNA was extracted from samples using Trizol (Thermo Fisher) extraction as previously described . For thiThe differentiation of iPSCs into cardiomyocytes was performed according to the method previously described . BrieflyCells seeded on 24-well coverslips were fixed with cold acetone  for 10 min followed by blocking with 5% bovine serum albumin  for 30 min. After washing them three times with Dulbecco’s Phosphate Buffered Saline , the cells were incubated overnight with primary antibodies at 4℃. After incubation, the antibody solution was discarded, and the sections were each washed three times at room temperature for 5 min with DPBS. Next, the cells were blocked again followed by 1 h of incubation at room temperature with appropriate secondary antibodies. Nuclei were counter-stained with SSEA-1. 4′,6′-diamidino-2-phenylindole , and 24-well chamber slides were inverted onto glass slides overlaid with 90% glycerol (Sigma-Aldrich) in DPBS as the mounting media. Additionally, the last images were examined and captured using a confocal microscope .The primary antibodies used were mouse anti-dystrophin , anti-cardiac , Nanog (Abcam), tumor-related antigen (TRA)-1-60, TRA-1-81, stage-specific embryonic antigen (SSEA)-1, and SSEA-4 all from Merck Millipore .http://www.rgenome.net/cas-offinder/, last assessed on 25 September 2021) were amplified by PCR and Sanger sequencing. All primers are shown in After genome editing, the iPSCs genomic DNA was extracted on days 40, 42, 48, 54, 60, 66, 72, and 75 using phenol-chloroform extraction, respectively. The residue of the plasmid components was tested by PCR. Additionally, the potential off-target sites predicted by an online tool  located on different chromosomes of HEK-293T were designed , and the result revealed that no genome edition had taken place in any of the seven most probable off-target sites , D-iPSCs (D-iCMs), and patient control DMD-iPSCs (DMD-iCMs) were differentiated by small molecules. The differentiation process is shown in DMD-45 in iPSCs using CRISPR/Cas9, achieving a knockout efficiency of only 8% [DMD with an efficiency lower than 5% [DMD Exon 51 with high efficiency. Of the 14 clones analyzed, 3 were positive, demonstrating that the COE system is efficient and convenient. It is worth mentioning that the episomal vector can be removed after genome editing, eliminating the influence of exogenous genes on the results.In this study, an episomal CRISPR/Cas12a gene-editing system was established based on Orip/EBNA1 (COE) and its editing efficiency in HEK-293T and iPSCs was evaluated. Under the selection pressure, the editing efficiency of this COE system in HEK-293T cells was found to increase with culture time and could reach 90%. In theory, if the time is long enough, the COE system can achieve a higher indel rate. Additionally, the DMD model of the deletion of Exon 51 was successfully constructed using this system, which proved that this system can edit efficiently in iPSCs. At present, the most common method of gene knockout in iPSCs is to produce double-strand breaks (DSB) using programmable artificial endonucleases such as ZFN, TALEN, and Cas9, but the editing efficiency was very low. In 2019, Yi-Li Min et al. successfully knocked out Exon  only 8% . Additio than 5% . These eCompared with Cas9, Cas12a recognizes A-T rich sequences, where the PAM, typically 5′-TTTV-3′, fills the gap of gene editing in genomes rich in A-T sequences. Additionally, Cas12a presents a more minimalistic system than Cas9, as Cas12a requires only a single RNA molecule, the crRNA. Meanwhile, Cas9 requires two RNA molecules (tracrRNA and a crRNA). Previous studies also showed that the pre-crRNA processing activity of Cas12a makes it an attractive candidate for multi-gene editing, which is cumbersome with Cas9. Due to these characteristics, Cas12a is becoming another powerful gene-editing tool, playing an increasingly important role in nucleic acid detection, disease modeling, gene therapy, etc. In this study, its editing efficiency was significantly improved by the use of the Orip/EBNA1 episome, which undoubtedly provides another boost for its better use in scientific research. However, the episome derived from the Epstein-Barr virus (EBVs) also has some limitations. It has been reported that the EBV vector failed to replicate in mouse and hamster cells due to Orip, which may limit its applications for gene therapy trials in animals . TherefoIn general, this gene-editing system provides a powerful platform for efficient genome editing. The COE system promotes genome editing in two ways: by allowing the continuous selection of transfected cells and long-term genome editing. The results of this study show that the editing efficiency increases over time; in theory, most crRNAs can generate indel rates of up to ~100% if the editing time is long enough. However, the sustained expression of Cas12a has created some risks as well as improved the editing efficiency. Some studies have shown that the CRISPR system can produce off-target cleavage ,43, whicAt present, nucleases can be delivered in the forms of DNA, mRNA, and protein, each with its advantages and disadvantages . The proIn this work, an episomal CRISPR/Cas12a gene-editing system was established based on Orip-EBNA1. This system can efficiently implement gene editing in the HEK-293T and iPSCs, providing a powerful tool for efficient gene editing.


Salmonella strains. Our results showed that de novo assembly did not detect the number of bacterial plasmids more accurately than reference-based assembly did. As this new hybrid mapping strategy surpassed de novo assembly in bacterial reconstruction, it was further used to identify the presence and genomic location of AMR genes among three Salmonella enterica serovar Schwarzengrund isolates. The AMR genes identified in the bacterial chromosome among the three Salmonella enterica serovar Schwarzengrund isolates included: AAC(3)-IV, AAC(6′)-Iy, aadA2, APH(4)-Ia, cmlA1, golS, mdsA, mdsB, mdsC, mdtK, qacH, sdiA, sul2, sul3, and TEM-1 genes. Moreover, the presence of TEM-1, AAC(3)-IV, aadA2, APH(4)-Ia, cmlA1, dfrA12, floR, sul1, sul3, and tet(A) genes found within three IncFIB plasmids and one IncX1 plasmid highlight their possible transmission into the environment, which is a public health risk. In conclusion, the generated data using this new hybrid mapping strategy will contribute to the improvement of AMR monitoring and support the risk assessment of AMR dissemination.It is well established that plasmids carrying multiple antimicrobial resistance (AMR) genes can be easily transferred among bacterial isolates by horizontal gene transfer. Previous studies have shown that a combination of short- and long-read approaches is effective in reconstructing accurate plasmids. However, high-quality Illumina short reads mapped onto the long reads in the context of an AMR hybrid monitoring strategy have not yet been explored. Hence, this study aimed to improve the reconstruction of plasmids, including the localization of AMR genes, using the above-described parameters on whole-genome sequencing (WGS) results. To the best of our knowledge, this study is the first to use S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) to confirm the number and sizes of plasmids detected by in silico-based predictions in  The global coronavirus disease COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 245 million people and has caused over 4 million deaths worldwide since its first identification in 2019 9 pandemi. RecentlCurrently, approximately 2.8 million people worldwide are infected with antibiotic-resistant bacteria and approximately 700,000 die per year around the globe as a result of antibiotic-resistant infections . BacteriThe introduction of third-generation sequencers  may afford one solution to this challenge. Compared to second-generation sequencing platforms that generate high throughput and shorter read lengths 150–300 bp), MinION offers longer reads (>10 kb) that can capture entire genomes including extraneous elements . Althoug0–300 bp,Salmonella bacteria from humans, animals, and food are multidrug-resistant, and infections with foodborne bacteria are thus becoming harder to treat [Salmonella enterica serovar Schwarzengrund strains with multiple plasmids isolated from animal sources [According to the latest data collected by the European Center for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA), a large proportion of to treat . As a fo sources ,18, whicSalmonella enterica serovar Schwarzengrund strains  with multiple plasmids isolated from animal sources were prepared according to our previous studies [Three multidrug-resistant  studies ,18  and sequenced on a NextSeq500 instrument  with the 150PE protocol according to the manufacturer’s instructions. Trimming of the short reads was performed with the Trimmomatic software (version 0.36) developed by Bolger et al. . The filFor MinION long-read sequencing, 1.5 µg DNA was used for library preparation employing the 1D ligation-based sequencing kit  and Native Barcoding Expansion 1-12 , which was then sequenced with a FLO-MINSP6 R9.4.1 flow cell coupled to a MinION Mk1B sequencer  connected to a MinKNOW GUI version 4.3.20 device  as recommended by the manufacturer. Base-calling of these sequences was performed with the Guppy software (version 4.2.2) developed by Oxford Nanopore Technologies . Data usC is the coverage estimated by read mapping, L is the read length, N is the total number of reads, and G is the haploid genome length.To calculate the coverage of the genome, the Lander–Waterman equation  was usedBefore de novo assembly, the quality of the short reads and long reads was checked with FastQC (version 0.11.9)  and LongThe assembly of long reads was first carried out using Unicycler with the default parameters . Mapping the short reads to the long-read assembly was performed using BWA-MEM (version 2.2.1)  with theGenomes from both the reference-based assembly and de novo assembly were screened for known plasmid genes using ABRicate (version 0.8.13)  against S. enterica serovar Braenderup H9812 was used for size determination. In brief, the genomic DNA was prepared in agarose blocks, digested with 10 units of P1 nuclease , and then separated on a 1% certified megabase agarose gel in 0.5× Tris-borate-EDTA buffer at 14 °C for 20.5 h with pulse times between 2.16 and 63.8 s using a CHEF-Mapper XA PFGE system . Plasmids less than 10 kb in size were further analyzed by running them on a 1.2% agarose gel in 0.5× Tris-borate-EDTA buffer at 120 V for 30 min with a Bio-1Kb Mass DNA Ladder  used as a marker. The gels were then stained with ethidium bromide, visualized under UV light, and photographed. The fingerprints were analyzed using BioNumerics (version 7.1).The numbers and sizes of plasmids were estimated by S1-PFGE analysis as described previously . S. enteAMR genes were identified by screening the assembled sequences (from both reference-based assembly and de novo assembly) with ABRicate against CARD databases . The locS. enterica serovar Schwarzengrund isolates were assessed by FastQC and LongQC, respectively. All samples passed the mean quality score test in FastQC (average Phred score of 20) and LongQC (average QV score of 7)  . The totS. enterica serovar Schwarzengrund isolates, SS09, SS12, and SS15 , 58 kb IncL/M, and 45 kb IncX1) and one small plasmid (4 kb Col156) within the SS12 isolate, and one large plasmid (93 kb IncFIB(K)) and one small plasmid (6 kb ColRNAI) within the SS15 isolate (The circularized plasmid contigs from each sample identified in silico  and the  isolate .AAC(3)-IV, AAC(6′)-Iy, aadA2, APH(4)-Ia, cmlA1, golS, mdsA, mdsB, mdsC, mdtK, qacH, sdiA, sul3, and TEM-1, while its IncFIB(K) plasmid (contig 2) contained AMR aadA2, dfrA12, sul1, and tet(A) genes. For the SS12 isolate, the chromosome (contig 1) carried the AMR genes mdtK, sdiA, AAC(6′)-Iy, TEM-1, aadA2, cmlA1, qacH, sul2, golS, mdsA, mdsB, and mdsC while its IncFIB(K) plasmid (contig 2) and its IncX1 plasmid (contig 4) contained AMR tet(A), dfrA12, and aadA2, and TEM-1, AAC(3)-IV, APH(4)-Ia, and floR genes, respectively. For the SS15 isolate, the chromosome (contig 1) carried the AMR genes AAC(6′)-Iy, golS, mdsA, mdsB, mdsC, mdtK, and sdiA, while its IncFIB(K) plasmid (contig 2) contained AMR AAC(3)-IV, aadA2, APH(4)-Ia, cmlA1, dfrA12, floR, qacH, sul1, sul3, and tet(A) genes.To obtain the full picture of the AMR gene content, a search on the de novo and reference-based assemblies using the CARD tool was performed . Both thSalmonella enterica bacteria constitutes a significant public health concern due to the potential transmission to humans at the end of the food chain. As AMR genes localized on plasmids can be easily transferred and spread by horizontal gene transfer, their occurrence should be closely monitored. Hence, in the current study, a strategy to reconstruct bacterial plasmids and fully characterize the presence of AMR genes and their genomic location in S. enterica serovar Schwarzengrund isolates was developed. Moreover, as a follow-up study, three multidrug-resistant S. enterica serovar Schwarzengrund isolates with multiple plasmids of different size ranges were chosen for in-depth characterization [Multidrug-resistant rization ,18.Salmonella enterica strains [In NGS data, sequencing is a critical step as it can affect the downstream analysis and interpretation processes. As this study used two assembly approaches, including a reference-based assembly of Illumina short reads mapped onto the long reads and a de novo assembly of both short and long reads, to reconstruct chromosome and plasmids, the quality of the short and long reads was first examined. Overall, after trimming and adapter removal, all sequencing reads met the recommended requirements from Illumina and Oxford Nanopore for accuracy, coverage, read length, and read counts . Moreove strains , this stNext, the reference-based assembly and de novo assembly approaches were assessed for their suitability for bacterial chromosome and plasmid reconstruction. In all isolates, both methods allowed the closing of the chromosomal contig. However, for plasmid reconstruction, the de novo assembly approach appeared to detect one additional plasmid in all isolates when compared to those identified by the reference-based assembly approach. Under the assumption that all the plasmids were detected by S1-PFGE, de novo assembly could identify a 2089 bp Col(BS512) plasmid within the SS12 isolate, while both methods had plasmids that were not recognized by the S1-PFGE and agarose gel analyses, such as the 3 kb plasmid in SS09 isolates; two plasmids of 5 and 3 kb in the SS12 isolates; and three plasmids of 8, 5, and 3 kb in the SS15 isolates identified by the de novo assembly approach, and the two plasmids of 5 and 3 kb in the SS12 isolates and two plasmids of 8 and 5 kb in the SS15 isolates detected by the reference-based assembly. According to the previous study , the assSalmonella indiana isolates [Campylobacter, while the PFGE method only detected 20 plasmids of sizes 0–45 kb [Staphylococcus aureus isolates, while PFGE identified only 151 plasmids within that size range [According to a previous study, plasmids of sizes greater than 80 kb were detected when S1-PFGE was used as a method to screen for the presence of plasmids in isolates . In thisisolates . Howeverze range . As thisSalmonella enterica serovar Schwarzengrund isolates included AAC(3)-IV, AAC(6′)-Iy, aadA2, APH(4)-Ia, cmlA1, golS, mdsA, mdsB, mdsC, mdtK, qacH, sdiA, sul2, sul3, and TEM-1, which encode resistance to aminoglycosides (AAC(3)-IV, AAC(6′)-Iy, aadA2, APH(4)-Ia), chloramphenicol (cmlA1), multiple drugs , sulfonamides , and ampicillin (TEM-1).As shown in previous studies, complete plasmid assembly is frequently impossible using short-read sequencing ; therefoTEM-1, AAC(3)-IV, APH(4)-Ia, and floR) carried by the IncX1 plasmid in this study were completely different from those reported in previous work , cmlA1, sul3, and TEM-1Bbla genes) [AAC(3)-IV, aadA2, APH(4)-Ia, cmlA1, dfrA12, floR, sul1, sul3, and tet(A), which encode resistance to gentamicin, streptomycin, hygromycin B, chloramphenicol, trimethoprim, florphenicol, sulfonamides, and tetracycline, respectively. It should be noted that gentamicin is a critically important antimicrobial, while chloramphenicol, trimethoprim, sulfonamides, and tetracycline are highly important antibiotics in human medicine, according to the latest WHO publication [Nevertheless, the presence of three IncFIB plasmids and one IncX1 plasmid found among the isolates that carried several AMR genes was particularly worrisome, as they increase the risk of spreading multiple resistance genes. Although the AMR genes (B genes) ,49,50, tB genes) . On the B genes) . Within B genes) , includilication . By usinSalmonella enterica serovar Schwarzengrund isolates that have been ignored in the past.This study compared two different approaches involved in the accurate reconstruction of the bacterial chromosome and plasmids as well as the determination of the exact location of AMR genes. As the reference-based assembly surpassed de novo assembly for bacterial plasmid reconstruction, this new hybrid mapping strategy further identified the presence and genomic location of AMR genes among


Sex hormone-dependent cancers, including breast, ovary, and prostate cancer, contribute to the high number of cancer-related deaths worldwide. Steroid hormones promote tumor occurrence, development, and metastasis by acting on receptors, such as estrogen receptors (ERs), androgen receptors (ARs), and estrogen-related receptors (ERRs). Therefore, endocrine therapy targeting ERs, ARs, and ERRs represents the potential and pivotal therapeutic strategy in sex hormone-dependent cancers. Proteolysis-targeting chimeras (PROTACs) are a novel strategy that can harness the potential of the endogenous ubiquitin-proteasome system (UPS) to target and degrade specific proteins, rather than simply inhibiting the activity of target proteins. Small molecule PROTACs degrade a variety of proteins in cells, mice, and humans and are an emerging approach for novel drug development. PROTACs targeting ARs, ERs, ERRs, and other proteins in sex hormone-dependent cancers have been reported and may overcome the problem of resistance to existing endocrine therapy and receptor antagonist treatments. This review briefly introduces the PROTAC strategy and summarizes the progress on the development of small molecule PROTACs targeting oncoproteins in sex hormone-dependent cancers, focusing on breast and prostate cancers. Great success has been achieved in drug discovery programs and human being health, particularly targeted therapy. There are two main types of targeted therapeutic drugs: monoclonal antibodies and small molecule drugs . Althougvia traditional inhibitors or non-enzymatic proteins (PROTACs can harness the endogenous ubiquitin-proteasome system (UPS) to target and degrade specific proteins , 9. PROTproteins , 11. Difproteins –15. Due Over the last 20 years, PROTACs have evolved from peptides into small molecules that can degrade a variety of proteins in cells, mice, and humans , 11, 16.via a flexible chemical linker, with one of the ligands binding to the target protein and the other binding to the E3 ubiquitin ligase, facilitating poly-ubiquitination and subsequent proteasome-mediated degradation of the target protein  box complex . Protac-To increase their cell permeability, Schneekloth et al.  designedFollowing the early pioneering work, numerous small molecule-based PROTACs have been developed to overcome poor cell permeability and stability. In 2008, Schneekloth et al.  developeSince then, small molecules have been widely used as E3 ligase ligands in PROTACs, including nutlin-3A  and IdasHomo-PROTACs are a special type of small molecule-based PTOTACs, which dimerize one particular E3 ubiquitin ligase and then induce auto-degradation. The first homo-PROTAC, the representative compound CM11, was reported in 2017 . CM11 coTo overcome the off-tissue issues, which represent one of the major limitations of PROTACs, a new generation of controllable PROTACs was devised . PhosphoSex hormone-dependent cancers, including breast, ovarian, and prostate cancers, currently contribute to a high number of cancer-related deaths worldwide . Sex horProstate cancer (PC) is the second leading cause of cancer-associated mortality in males in developed countries after lung cancer, and the incidence rate is increasing in developing countries , 74. DihUnfortunately, approximately 25% of CRPC patients do not respond to these treatments, and the vast majority of responsive patients will ultimately develop resistance. AR signal pathway can be activated in CRPC because of AR mutation, AR amplification and AR alternate splicing variants formation, which cause the failure of traditional endocrine therapy. Nonetheless, small-molecule PROTACs targeting AR, which may overcome the problem of resistance to existing drug treatments, have been recently reported and provide an attractive direction . A numbe50) of < 1 nM (PSA) and FK506 binding protein 5 (FKBP5), inhibited AR-dependent cell proliferation, and induced apoptosis in VCap cells. It also showed plasma PSA reduction potency similar to that of the traditional AR antagonist, enzalutamide, but at lower doses, and demonstrated efficacy in enzalutamide-resistant PC xenograft models  of more than 98%, even in cells expressing high levels of AR protein. After head-to-head comparisons between the currently approved AR antagonist enzalutamide and ARCC-4 in different cell models of PC resistance, the antitumor efficacy of ARCC-4, evaluated by PSA expression reduction, induction of cell apoptosis, and AR-dependent cell proliferation inhibition, outperformed enzalutamide. Interestingly, ARCC-4 effectively degraded clinically relevant ARs with different point mutants, including F876L, T877A, L702H, H874Y, and M896V, and remained active despite elevated androgen levels. ARCC-4 had better anti-proliferative effects in AR mutant cancers, whereas enzalutamide failed.In 2018, Salami et al.  synthesi50 values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1, respectively. After 24 h of treatment, ARD-69 almost completely degraded AR in LNCaP and VCaP cells, with the concentration reaching below 1 nM. ARD-69 also effectively and dose-dependently inhibited PSA, transmembrane protease serine 2 (TMPRSS2), and FKBP5 expression. Furthermore, ARD-69 inhibited LNCaP, VCaP, and 22Rv1 cell proliferation, with efficiencies 100 times higher relative to enzalutamide. Additionally, ARD-69 effectively reduced AR and PSA protein expression in VCaP xenograft tumor tissues. Altogether, these data demonstrate that ARD-69 is an extremely potent AR degrader in treating metastatic CRPC.Han et al.  designed50 values of 0.2–1 nM. In addition, AR protein levels in LNCaP and VCap cells decreased by more than 95% following treatment with 10 nM ARD-266 for 6 h. Notably, ARD-266 effectively inhibited AR-regulated gene expression in a dose-dependent manner. This was the first study to demonstrate that, even using a low-affinity ligand with nanomolar levels of E3 ligase complex, the PROTAC degrader can efficiently degrade the target protein. Development of additional AR degraders is currently underway  . The ERs50 of 1.8 nM (ESR1 mutants (Y537S and D538G) and inhibited the growth of cell lines with these mutations. It also showed significant antitumor activity in an estrogen-dependent MCF7 xenograft tumor model, with the ERα protein decreasing by more than 90% by the end of the experiment. Moreover, more significant tumor growth inhibition (131% TGI) was observed when ARV-471 was combined with a cyclin dependent kinase 4/6 (CDK4/6) inhibitor, significantly decreasing ERα protein levels. In a patient-derived xenograft model with an ESR1 Y537S mutation, ARV-471 completely inhibited growth at a dose of 10 mg/kg and significantly reduced the level of mutant ER protein, exhibiting superior inhibition compared with fulvestrant. In the future, ARV-471 will be studied as a monotherapy or in combination with other therapies, such as CDK 4/6 inhibitors.ARV-471 Table 1 f 1.8 nM . ARV-47150 values of ERD-308 in MCF-7 and T47D ER+ breast cancer cells were 0.17 and 0.43 nM, respectively, and more than 95% ERα degradation was induced at concentrations as low as 5 nM in both cell lines. Compared with fulvestrant or RAD1901, two conventional SERD molecules, ERD-308 achieved more thorough ERα degradation and more effectively inhibited cell proliferation in MCF-7 cells. Moreover, ERD-308 inhibited the expression of ER-regulated genes and the proliferation inhibition ability was much higher than that of fulvestrant and the SERM molecule raloxifene. The discovery of ERD-308 may promote the development of a completely new class of therapeutics to treat ER+ breast cancer.In 2019, Hu et al.  designed50 values of 0.8 nM, 10.5 nM, and 6.1 nM, in MCF-7 wild type, cY537S, and cD538G mutant cells, respectively. ERD-148 significantly downregulated ERα expression at concentrations as low as 10 nM in MCF-7 wild type and Y537S mutant cells. Moreover, ERD-148 significantly downregulated the mRNA level of Growth regulation by estrogen in breast cancer 1 (GREB1), an ER-regulated gene. However, ERD-148 did not inhibit the growth of ER-negative and estrogen-independent MDA-MB-231 breast cancer cells.During the structure activity relationship (SAR) studies of the PROTAC degrader ERD-308, compound ERD-148 Table 1 via the UPS. SNIPER comprises bestatin (BS), a ligand that interacts with the IAPs and an appropriate ligand for the target protein (presenili 2 (pS2) gene in ER+ estrogen-dependent MCF-7 cells. ER is degraded by proteasomes following cIAP1-mediated ubiquitylation. SNIPER(ER)-3 reduced the viability of MCF-7 cells expressing ERα, but not of U2OS and HeLa cells, which do not express ERα protein. SNIPER(ER)-3 also induced necrotic cell death, accompanied by the high mobility group box 1 (HMGB1) release, a necrosis marker, from the cells into the media. Unfortunately, BS is a nonspecific ligand that limits the bio-orthogonality and maximal potency of SNIPER(ER)s.In 2011, Itoh et al.  designed protein . Using 4 protein Table 1  protein , 86. SNISeveral IAP antagonists were subsequently incorporated into SNIPER(ER)s, and a novel SNIPER against ER, called SNIPER(ER)-87 Table 1,17e (cpd. 17e)  Table 1 in vitro and in vivo, pharmacological inhibition and gene knockout of ERRα have been shown to slow down the progression of breast cancer (The estrogen-related receptor (ERR) is an orphan nuclear hormone receptor that can produce biological functions without binding to ligands. There are three ERR subtypes: ERRα (NR3B1), ERRβ (NR3B2), and ERRγ (NR3B3). Recent studies , 96 havet cancer , 98.29  polymerase (PARP) cleavage, caspase activation, and apoptosis in 22Rv1 cells. Additionally, ARV-771 was efficacious in two different xenograft models of CRPC and resulted in tumor regression in enzalutamide-resistant 22Rv1 tumor xenografts.Inhibitors of BET proteins have recently shown growth-inhibitory activity in preclinical models of CRPC, with BET being an attractive target in CRPC . In 20163  and may not conform to Lipinski’s ‘rule of five’, which is a rule of thumb for evaluating whether a small molecule possesses pharmacological or biological properties of an orally active drug in humans .In addition, it is cumbersome to predict the degradation efficiency of small-molecule PROTACs. For target protein ligands, high binary binding affinities do not always yield efficient degradation of target proteins , suggestUnintended on-target and off-target toxicities can also affect the clinical translation of PROTACs . E3 ligaThe “hook effect” is another unavoidable problem. At high intracellular concentrations of small-molecule PROTACs, binary complexes are favored over ternary complexes, ultimately decreasing target degradation , 103. SiTwenty years after the first PROTACs were synthesized in 2001, small-molecule PROTACs have not only drawn the attention of academic researchers, but also of pharmaceutical companies. Although there are concerns over their relatively high molecular weights and low oral availability, two orally active small-molecule PROTACs, ARV-110 (an AR degrader) and ARV-471 (an ER degrader), have been studied in phase I/II clinical trials. Small-molecule PROTACs are a potential strategy for drug discovery, providing a new way to treat sex hormone-dependent cancers. Since immune checkpoint inhibitors have achieved great success against some tumors by enhancing the antitumor immunity of immune cells, researchers inspired by this have attempted to design PROTACs that can potentially strengthen antitumor immunity , 107. FuYZ, HX, and YW designed the concept of the review and participated in manuscript writing, editing, revising, prepared the figures and supervised the entire process. LS, ZW, and LL collect literature data, participated in manuscript writing and formatting. LL and YZ prepared the structure of compounds. LL and JZ participated in manuscript writing, proof editing, and graphical design. All authors contributed to the article and approved the submitted version.This work was supported by Excellent Youth Fund of Sichuan Cancer Hospital [grant number YB2021024], and Science and Technology Program of Sichuan Province [grant number 2021YFH0145], Scientific Research Project (Foundation) of the Health Planning Committee of Sichuan [grant number 20PJ108], and Scientific research project of Sichuan Medical Association [grant number S20023].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates  contained mcr-1 associated with plasmids , except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar . Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential.Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs ( The vast majority of antimicrobials used in veterinary medicine are also used in human medicine. The consumption of antimicrobial agents has increased the selection of resistant bacteria in both human and veterinary medicine. Additionally, the presence of resistance genes in mobile genetic elements has probably played a major role in the inter- and intra-species transmission of antimicrobial resistance. Enterobacterales [Use of colistin in human medicine has been abandoned, due to its toxicity when applied systemically. Nevertheless, nowadays, the emergence of multidrug-resistant (MDR) Gram-negative bacteria in hospital settings has left no other choice but to use colistin as the last-line treatment option despite its toxicity. Contrarily, in veterinary medicine, colistin sulfate has been used orally for many decades to treat infections caused by cterales . In partcterales . Additiocterales ,4,5,6. Icterales . Fortunacterales . mcr-1, being distributed worldwide [Until 2015, resistance to colistin was only associated with chromosomal mutations. More recently, different plasmid-mediated mechanisms conferring resistance to colistin have been identified ,11,12,13orldwide . The ememcr-1 and mcr-3 was described for the first time in Spain in an Escherichia coli of calf origin [E. coli genotype was endemic in the farm. In this context, whole genome sequencing combining Nanopore and Illumina technologies was applied to study the dynamics of the transmission of colistin resistance within the livestock and the farmer, the characterization of plasmids, the location and genomic context of resistance genes, and the detection of virulence factors.Back in 2017, the co-occurrence of f origin . This ismcr-1 in the 18 E. coli isolates . No additional mcr-gene variants were detected. Visible growth on MacConkey agar supplemented with colistin was observed for 18/210 fecal samples. The multiplex PCR confirmed the presence of MIC values for colistin varied, with one isolate exhibiting a MIC of 2 mg/L, 15 isolates equal to 4 mg/L, and two showing a MIC ≥ 8 mg/L . Furthermcr-1 positive isolates were sequenced. Chromosome size ranged from 4,613,927 bp (Farmer) to 5,586,543 , with an average size of 5,009,072 bp , and eight to phylotype B1 (six from cattle and two from swine). Phylogenetic analysisbased on Single Nucleotide Polymorphisms (SNPs) clustered isolates from phylotype A and B1 separately . The mosmcr–1) were retrieved after sequencing and assembling: 36 plasmids from the bovine isolates (ranging from one to five per isolate), seven from porcine (from one to two per isolate), and five from the farmer’s isolate (n = 14), which harbored the mcr-1 gene and was present in isolates from the three hosts considered. Other common replicons were IncFIB (n = 9), IncHI2 / IncHI2A combination (n = 7), IncFIC (n = 6), and replicon combination IncFIB / IncFIC (n = 6).A total of 48 plasmids bearing antimicrobial resistance genes (ARGs) , even though it had tested positive by PCR. This isolate contained the blaCTX-M-15 gene located in the chromosome. Upstream CTX-M-15 was a complete IS3 element and ΔtnpA from the ISEcp1 element, while downstream, there was ΔtnpA from the Tn2 element.After the in-silico analysis, mcr-1 gene was identified by WGS, 15 carried the mcr-genes in plasmids . Isolate 15B_22 contained two copies of the gene in two different plasmids, IncX4 and IncHI2 / IncHI2A. The remaining two isolates harboured the gene inserted in the chromosome (15B_13 and P2_16).Of the 17 isolates in which the mcr-1 gene were found within these isolates , allowing the plasmid to be self-transmissible, and the HicAB toxin-antitoxin system for plasmid maintenance and stability. Four different environments for the isolates . All of isolates . The Incisolates . While 1mcr-1 together with six other AMR genes; aadA2, aph(3′’)-Ib, aph(6)-Id, dfrA12, floR, and tetM. IncI2 and IncHI2 plasmids presented a size of 61,766 bp and 234,156 bp, respectively . Both plasmids contained the conjugative mechanism T4SS and the replication machinery. While IncHI2 yielded HipA toxin-antitoxin system, IncI2 plasmid harbored RelE/ParE, Hok, and HicAB toxin-antitoxin systems. Finally, the IncHI2 plasmid contained E. coli from cattle origin contained the highest amount of virulence genes (27)  , followen = 11) and Col (n = 2) harbored these virulence genes , tir (translocated intimin receptor), f17A (major F17 fimbriae subunit), f17G (adherence F17 fimbriae subunit); enzymes katP , ehxA (enterohemolysin), espP (plasmid-encoded extracellular serine protease); secretion-related genes espA , espB , nleA (non-LEE encoded effector A), nleB (non-LEE encoded effector B); toxins astA , sta1 (heat-stable enterotoxin ST-Ia), cdtB , and stx2 (Shiga toxin). Some of these virulence factors conferred different pathotypes, such as adherence factors mcr-1 gene in the chromosome and contained stx2 (stx2A and stx2B subunits) encoding for Shiga toxin. A complete phage D108 was found spanning a region of 86.6 Kbp that harbored the stx2 gene. Additionally, this isolate also contained several virulence factors; iha, lpfA, gad, iss, astA, cba, celb, mchB, mchC, and mchF.Isolate 15B_13 of calf origin  harbored the mcr-1-mcr-3 E. coli was previously detected, and evaluate the transmission of colistin resistance plasmids within the farm applying WGS. Our approach using long-reads to assemble, and short reads to polish (hybrid assembly), allowed to close chromosomes and circular plasmids harboring mcr-1 genes, and to study their location and genomic context among different E. coli lineages.The aim of this study was to conduct a cross-sectional thorough sampling of a farm where co-occurrence of mcr-1 positive E. coli were detected in all host species present in the facilities, including one human (the farmer). According to the farm book, pigs and calves sampled in 2017 were orally treated with colistin. Going back through the farm book, batches of both pigs and calves reared between 2015 and 2017 were consistently prescribed colistin in the same phases of their production cycle. This management practice in terms of medication regime suggests a routine use of colistin in consecutive batches, facilitating the persistence of colistin resistance mechanisms. Additionally, phenotypic and genotypic resistance to other families of antimicrobials widely used in the farm were also detected, such as β-lactams, tetracycline, aminoglycosides, and sulfonamides. mcr-1 in mobile genetic elements were associated with IncX4, IncI2, and IncHI2 plasmids, with IncX4 being the most prevalent. All IncX4 plasmids shared the same backbone, and differences were due to inserted sequences. In the case of isolate P1_10, this insertion expanded 12 kbp and comprised the tetM gene. Additionally, isolate 15B_22, presented two plasmids encoding for mcr-1 [etM), trimethoprim (dfrA12), aminoglycosides (aph(3″)-Ib, aph(6)-Id) and florfenicol (floR), as well as colistin (mcr-1). Different families of ARGs located in the same plasmid facilitated the persistence and selection of resistance to antibiotics not used in the farm. Even if colistin was withdrawn (as it happened after our visit to the farm), the use of doxycycline could co-select indirectly for the mcr-1 gene. In agreement with previous studies ,20,21,22or mcr-1 ,19,20 wimcr-1 was also integrated into the genome in two isolates of different animal origins. In one of them, the ISApl1 element was flanking mcr-1 upstream and downstream, a structure that probably facilitated the movement of the whole element by transposition. The other isolate had lost the ISApl1 element, establishing the mcr-1 as a heritable trait overcoming any possible fitness cost of plasmid maintenance [As previously described ,21,22,23ntenance . Furtherntenance ,25.E. coli isolates in phylotype A and B1, there were different MLST linages harboring the colistin resistance genes in highly similar plasmids (identity ranging from 99.96% to 100%). The farmer’s isolate (ST398) did not match the MLST type of any of the livestock isolates. Interestingly, ST398 was the only ST shared between livestock and bloodstream infections in a study carried out across the United Kingdom, underlying the zoonotic potential of some E. coli linages [mcr-1-pap2). These results suggest the transmission of resistance through mobile genetic elements between different E. coli lineages from livestock to the farmer. Several studies have demonstrated the spread of ARGs from food-producing animals to veterinarians and personnel in direct contact with animals [mcr-1-mcr-3 genes by picking a unique colony per sample, further studies including within-host diversity of mcr-1 E. coli isolates should be performed to introduce the variability of the E. coli population within a sample.Although phylogenetic analysis clustered the  linages . Convers animals ,28,29, hE. coli isolates of cattle origin showed the highest number and diversity of plasmids encoding for both ARGs and virulence genes, including stx2. Shiga toxin E. coli (STEC) serotype O81:H31 bearing stx2 have also been described previously [mcr-1 positive STEC of cattle origin and highlights the importance of food producing animals as reservoirs of ARG and virulence determinants. All the isolates from this study exhibited a MDR profile. In addition, ARGs, as well as virulence factors, were described both in the chromosome and plasmids. eviously  and are eviously ,31,32,33mcr-1 gene after in silico analysis, even though previous PCR tested positive for this gene. Presumably, the isolate lost the mcr-1 plasmid during sub-culturing steps, resulting in a false-negative result. Besides, 15A_11 isolate  was resistant to cephalosporins (cefotaxime and ceftazidime) with CTX-M-15bla inserted in the chromosome. Interestingly, upstream of the gene, there was an IS3 element, and downstream there was a truncated Tn2, indicating a possible recombination event, as has been previously described [CTX-M-15bla in livestock is concerning, since it is widely disseminated [On the contrary, isolate 15A_11 resulted negative for the escribed ,35. The eminated ,37,38, eeminated ,40,41, acdtA, cdtB, and cdtC), which are all required for bonding to the cell’s surface and for entering the cell. However, VirulenceFinder only found the cdtB gene, while cdtA and cdtC were also present when aligning the sequence with the reference genome from NCBI. In depth analysis of the sequencing data should consider if a virulence element is composed of different genes to be fully operative.Regarding the analyses of the sequencing data, it is important to be aware of the limitation of databases to obtain the most accurate results. On the basis of an example from this study, cytolethal distending toxin (CDT) is composed of three subunits . Additionally, the farmer was interviewed by the Official Veterinary Services, and the farm´s treatment book was inspected to determine the antimicrobial treatments prescribed to the two animal species from 2015 to the time of sampling.The number of samples to be collected in each of the housing facilities (three housing facilities for bovine and one for swine) was calculated to allow for the detection of a prevalence of n = 501), 57 from fattening pigs (n = 900), and one from the farmer (n = 3). Fecal samples were taken from individual animals and transported to the laboratory at 4° C on the day of sampling. The farmer sent a refrigerated fecal sample by courier within 24 h of the visit to the farm. Faces were homogenized and plated onto both MacConkey agar and MacConkey agar supplemented with colistin (2mg/L). For quality control of the colistin plates, a positive and negative control were also included. Following incubation, one presumptive colistin resistant E. coli isolate per positive sample was identified by PCR [mcr genes (mcr-1 to mcr-5) described at the time of sampling was performed by multiplex PCR, as described by Rebelo et al., [A total number of 210 fecal samples were collected: 152 from calves , cefotaxime (0.016 to 2 mg/L), ceftazidime (0.25 to 16 mg/L), nalidixic acid (1 to 128 mg/L), ciprofloxacin (0.008 to 1 mg/L), gentamicin (0.12 to 16 mg/L), streptomycin (2 to 256 mg/L), kanamycin (8 to 16 mg/L), chloramphenicol (2 to 64 mg/L), florfenicol (4 to 32 mg/L), trimethoprim (1 to 128 mg/L), sulfamethoxazole , tetracycline (1 to 128 mg/L), and colistin (0.5 to 4 mg/L). Epidemiological cut-off values were those recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MDR isolates were defined as resistance to at least three different antimicrobial families [Minimal inhibitory concentration (MIC) was carried out for 14 antimicrobial agents  in those isolates harboring families .mcr-positive isolates was extracted using QIAGEN DNeasy® Ultraclean Microbial Kit under manufacturer’s conditions. Illumina  libraries were prepared by enzymatic fragmentation and double indexing using an NGSgo kit , according to the manufacturer’s instructions. The indexed libraries were pooled, denatured, and diluted to a final concentration of 4 nM. The pooled library was sequenced on the MiSeq system (Illumina) with a 300-cycle MiSeq reagent kit v2. Illumina paired-end reads were merged into one unique file per isolate using a custom python script .DNA from In parallel, DNA was quantified using Qubit dsDNA BR assay (Invitrogen by ThermoFisher Scientific), and sequenced using MinION sequencer  in two runs of 9 samples each. Two sequencing libraries were prepared using 400 ng DNA per sample with the Rapid Barcoding Kit  according to the manufacturer’s instructions. The samples were run using MinKNOWN software (version 18.07.18). Fast5 files generated were basecalled and demultiplexed using Albacore v2.3. Reads classified as pass (minimum Phred score of 7) were used for further steps. A second round of demultiplexing was performed with Porechop , which aNanopore fastQ files were used to perform the assembly of genomes and plasmids from the 18 isolates with Flye  (v2.6), Enterobacteriaceae database (OrthoDb v10.1). SerotypeFinder [In order to characterize the presence of plasmid replicons, antibiotic resistance genes (ARGs) and virulence factors, Abricate  .Contigs were visualized with Bandage . Plasmidmcr-1-mcr-3 in E. coli from the farm could not be confirmed. Nevertheless, we isolated mcr-1 across the farm in different E. coli linages, mainly associated with plasmids of the IncX4, IncHI and IncHI2 families. The most likely mechanism for the farmer to have acquired the mcr-1 gene was the horizontal gene transfer from the calves, since plasmids from both origins were highly similar. Additionally, mcr-1 positive E. coli isolates were MDR and contained a high number of virulence genes, including stx2, demonstrating that food-producing animals can be a reservoir of such determinants, posing a risk for human health, especially for personnel at the farm.In conclusion, co-existence of