Monocytes (Mos) and macrophages (Mφs) are key players in the innate immune system and are critical in coordinating the initiation, expansion, and regression of many autoimmune diseases. In addition, they display immunoregulatory effects that impact inflammation and are essential in tissue repair and regeneration. Juvenile idiopathic arthritis (JIA) is an umbrella term describing inflammatory joint diseases in children. Accumulated evidence suggests a link between Mo and Mφ activation and JIA pathogenesis. Accordingly, topics regarding the signals and mechanisms regulating Mo and Mφ activation leading to pathologies in patients with JIA are of great interest. In this review, we critically summarize recent advances in the understanding of how Mo and Mφ activation is involved in JIA pathogenesis and focus on the signaling pathways and mechanisms participating in the related cell activation processes. Juvenile idiopathic arthritis (JIA) is the leading chronic rheumatic disease of childhood and occurs in children before their sixteenth birthday with symptoms of articular inflammation for at least 6 weeks . Based oThe etiology and pathogenesis of JIA are still elusive. Joint infiltrating inflammatory cells, residential synovial fibroblasts and osteoclasts, antibodies targeting autoantigens, and various inflammatory mediators are some of the critical players that promote the chronic inflammatory process ,4,5,6. IMos/Mφs are important players in the innate immune system and are critical in coordinating the initiation, expansion, and resolution of many autoimmune diseases . They poThe origin and ontogeny of synovial Mφs are incompletely characterized. Studies suggest that tissue-resident Mφs may be derived from the yolk sac, fetal liver Mos, or bone marrow ,14. Whil++CD16− expression and represent 80–90% of the Mos in the bloodstream of healthy individuals [+CD16++ Mos and intermediate CD14++CD16+ Mos account for a much smaller proportion of circulating Mos and can expand considerably under inflammatory conditions [++CD16+ Mos among both the circulating and synovial Mos is expanded in patients diagnosed with enthesitis-related arthritis (ERA) [Schmidt, Cren and Gaur investigated the distribution of Mo subsets in paired SF and blood samples from patients with oligoarticular JIA and ERA. These authors discovered that while classical CD14++CD16− Mos dominate in the circulation, intermediate CD14++CD16+ Mos were highly enriched in oligoarticular JIA and ERA patient SF [++CD16+ synovial Mos can be induced by cytokine-rich SF and are found with similar patterns across Mo subsets, reports have suggested that the increased CD16 expression in these cells may likely result from the cytokine milieu of the synovial space and not the recruitment of intermediate CD14++CD16+ Mos from circulation due to their unique features [Monocytes are a heterogeneous population of leukocytes circulating in the blood until they are recruited to tissues upon signaling. The expression levels of the surface molecules CD14 and CD16 determine the distinct subpopulations of human Mos ,24. Clasividuals . Nonclasnditions . For exais (ERA) ,27. Recetient SF ,29,30. Afeatures ,31. Specfeatures ,29.Macaubas et al., the Mo lineage is expanded in patients with active sJIA [+CD16+ Mo subsets was not altered compared to that in healthy controls [++CD16- Mos and CD14+CD16+ Mos are activated in sJIA [Srivastava et al. to contribute to the apoptosis resistance of Mos [As demonstrated by ive sJIA ,32. Whilcontrols ,32. Both in sJIA . The ince of Mos .+CD16+ Mos form cell clusters and exhibit proangiogenic behavior [++CD16+ Mos from arthritis patients with naïve T cells skewed the T cells toward pathogenic Th17 cells via the production of IL-23. The increased frequency of IL-17-producing natural killer (NK) cells, CD4, and gamma-delta T cells in patients with ERA was also recently proposed to result from the expansion of intermediate CD14++CD16+ Mos due to their role as the major producer of IL-23, a key cytokine in the pathogenesis of ERA [Seemingly important players in JIA, intermediate Mos are noted for their high surface levels of class II molecules, CD40, CD54, and CD74, which are capable of inducing T cell stimulation and proliferation ,35. Uponbehavior . In a rhs of ERA .Plasticity and heterogenicity are the hallmarks of Mos/Mφs. Polarization is believed to influence disease progression by altering effector function  and has Schmidt et al. suggested that the inflammatory features of those who suffered from oligoarticular JIA may not fit into the traditional dichotomous polarization categories and should be considered according to their unique pattern [The polarization pattern of Mos/Mφs and its consequences in patients with oligoarticular JIA remain largely unclear. In a recently published report,  pattern . Specifi pattern . Corresp pattern ,41,44. I pattern . It is t pattern . Similar pattern ,46. Alth pattern ,45,47,48+ Mφs has been suggested as soluble CD163 can suppress the activation and proliferation of T lymphocytes upon stimulation [Immunoregulatory Mφ activity was found in ERA patients, evidenced by the expression of CD163 ,47. CD16mulation . The antmulation ,52. CollCytokines play a critical role in the activation and polarization of Mos/Mφs. As summarized in GM-CSF, a hemopoietic growth factor, is secreted by myeloid cells, T and B lymphocytes, and tissue residential cells, such as fibroblasts, chondrocytes, osteoblasts, and epithelial and endothelial cells. GM-CSF interacts with the GM-CSF receptor and activates the Janus kinase (JAK)2-STAT5- suppressor of cytokine signaling (SOCS) as well as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3 kinase, and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), resulting in the activation of tissue-resident cells and the recruitment of inflammatory cells . The proTNFα is a pleiotropic cytokine that promotes the expression of adhesion molecules, inflammatory cytokines, prostaglandin E2, collagenase, and collagen by synovial cells . As Mos/Macaubas et al. recently discovered impaired IFN/STAT1 signaling in sJIA Mos during active disease, skewing them away from a classically activated phenotype [IFNγ, the sole member of type II IFNs, is produced by multiple cell types, including T cells (Th1 cells in particular), B cells, Mos/Mφs, and NK cells . It reguhenotype . Howeverhenotype .IL-10 is a critical cytokine in the negative feedback loop that limits inflammation and increases phagocytosis . IL-10 iToll-like receptors (TLRs) belong to the group of protein recognition receptors and are type I transmembrane proteins serving as the first-line defense against microbes. They recognize both invading pathogens and endogenous danger molecules and are crucial in bridging innate and adaptive immunity . Upon liStudies in experimental models have documented the ability of microbial TLR ligands to trigger arthritis in animals . IncreasBy definition, the presence of autoantibodies, RF and anti-citrullinated protein antibodies (ACPA) is the hallmark of RF-positive JIA . RFs areThe hypoxic nature of JIA synovium and the induction of chemokine CCL20 in JIA synovial Mos within the hypoxic milieu of inflamed joints suggests that hypoxia likely increases inflammatory cell infiltration and contributes to the development of local inflammation ,86. As tSchulert et al. discovered that miR-125a-5p was highly upregulated in active and new-onset sJIA patients and correlated with their systemic features but not the degree of joint involvement [The polarization of Mos/Mφs in sJIA was also found to be driven by epigenetic factors, such as negative transcriptional regulation by microRNAs ,52,90. Tolvement . Througholvement . Furtherolvement . Specifiolvement . Moreoveolvement . miR-155olvement . Compareolvement . Togetheolvement . In addiolvement ,94.Activated Mos and Mφs in JIA synovial space secrete a variety of proinflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-12, IL-18, and IL-23 ,95. WhilThe importance of TNFα in inflammatory arthritis has been highlighted in numerous clinical observations and experimental settings. In addition to its autocrine feedback role in propagating an inflammatory response ,57,58, TCepika et al. discovered that the stimulation of Mos in patients with sJIA resulted in an increase in activin receptor signaling, which in turn inhibited IL-1β secretion without altering the accumulation of intracellular IL-1β within sJIA Mos [Innate proinflammatory cytokines, such as IL-1, IL-6, IL-18, and TNF, account for many of the features observed in sJIA . SpecifisJIA Mos . MoreovesJIA Mos ,105. ThesJIA Mos ,108,109.matrix metalloproteinase (MMP)-9 and its tissue inhibitors of metalloproteinases-1, in synoviocytes and chondrocytes [-/- transgenic mice [IL-6 is a multifunctional cytokine that drives JIA development via immune response regulation, hematopoiesis, and bone metabolism . IL-6 eldrocytes ,111. IL-drocytes . Moreovedrocytes . Interesdrocytes . Patientdrocytes . As the nic mice , a prominic mice ,117.In patients with active sJIA, the level of IL-18 is significantly elevated . As docuIL-12 and IL-23 are mainly produced by inflammatory myeloid cells and are critical mediators influencing the differentiation of Th1 and Th17 cells . While ISpecifically, increased numbers of Th1 and Th17 were reported in the synovial space of patients with oligoarticular JIA and ERA ,127. IL-IL-10 is a potent cytokine that represses proinflammatory responses and limits unnecessary tissue damage caused by inflammation . IL-10 hIncreased levels of chemokines were found in JIA SF, favoring the migration of Mos to the inflamed tissue while promoting their activation and differentiation. While synovial fibroblasts are important sources of proinflammatory cytokines and chemokines , synoviaVEGF is the most critical regulator of angiogenesis and mediates inflammatory and bone-destructive activities ,139. SynBased on the clinical relativity and the current knowledge on the pathogenesis of Mos/Mφs in JIA, different strategies have been explored or are currently under investigation for JIA patients and/or have been tested in arthritic animal models. The induction of anti-inflammatory human Mos is a unique property of glucocorticoids . In arthBiological therapies targeting the Mo/Mφ-producing cytokines TNFα, IL-1β, and IL-6 are now recommended as the standard of care for JIA patients with an advanced disease course . While tThe effectiveness of existing anti-rheumatic regimens and novel therapeutic agents targeting Mos/Mφs in patients with inflammatory arthritis has been investigated. A brief summary of Mo/Mφ-related therapies is shown in The present review summarizes recent understanding of the phenotype of Mos/Mφs and the mechanisms regulating their activation to elicit inflammation in patients with JIA. As discussed, the activity and phenotype of Mos/Mφs play an important role in pathogenesis across different JIA subtypes. Recruited to the joints by chemokine MCP-1/CCL2 and RANTES, Mos infiltrate the inflamed synovium and add to the tissue-resident Mφ pool orchestrating a local and global inflammatory reaction resulting in tissue damage. Upon the stimulations of cytokines, immunocomplexes, TLR ligands, the hypoxic microenvironment, and microRNAs, a mixed polarization of Mφs toward an inflammatory phenotype was mediated via the JAK-STAT, MAPK, NFκB, TRIF, and HIF signaling pathways. Proinflammatory cytokines, including IL-1β, IL-6, IL-12, IL-18, IL-23, and TNFα were secreted to stimulate activation of osteoclasts and synovial fibroblasts and to promote T lymphocyte polarization. VEGF, in addition, drives synovial angiogenesis, contributing to JIA pathogenesis .While investigations into strategies targeting Mo recruitment, Mo/Mφ polarization, cell depletion, and cytokine blockade have been recently performed , uncover


This study aimed to identify the factors underlying the metastasis of breast cancer and sentinel lymph nodes and to screen and analyze the risk factors of sentinel lymph node metastasis to provide a reference and basis for clinical work.A total of 99 patients with breast cancer were enrolled in this study. These patients received treatment in our hospital between May 2017 and May 2020. The general information, characteristics of the color Doppler echocardiography, molybdenum, conventional pathology, and molecular pathology of the patients were collected. Factors influencing sentinel lymph node metastasis in breast cancer patients were retrospectively analyzed.P < 0.05). The number and locations of tumors, quadrant of tumors, regularity of tumor margins, presence of blood flow signals, presence of posterior echo attenuation, presence of calcification, histological grade, molecular typing, and mutations of BRAF, ATM, and PALB2 were irrelevant factors (P > 0.05).In this study, age, tumor diameter, BI-RADS category, pathology type, expression profiles of CK5/6, EGFR, and CK19, and TP53 and BRAC1/2 mutations were independent risk factors for sentinel lymph node metastasis in breast cancer (In conclusion, age, tumor diameter, BI-RADS category, invasive type, expression of CK5/6, EGFR, and CK19, and mutations in TP53 and BRAC1/2 were positively correlated with sentinel lymph node metastasis. These independent risk factors should be given more attention in clinical studies to strengthen the management and control of sentinel lymph node metastasis in high-risk breast cancer and support early chemotherapy or targeted therapy. Breast cancer is a malignant tumor that commonly occurs in the lactiferous ducts and mammary gland epithelium. Currently, it has the highest incidence rate of lung cancer worldwide . The senA total of 99 patients with breast cancer were enrolled in this study. These patients received treatment in our hospital between May 2017 and May 2020. They were divided into two groups according to whether the sentinel lymph nodes in frozen pathology were metastatic during radical mastectomy or subtotal mastectomy, with 49 or 50 patients in each group. The general information of the two groups is presented in Table The inclusion criteria are as follows : (1) womThe exclusion criteria are as follows: (1) patients with inflammatory breast cancer or invasive cancers, such as tubule carcinoma, apocrine adenocarcinoma, and lipid-rich carcinoma; (2) patients with other types of malignant tumors; (3) male breast cancer or occult breast cancer; (4) advanced breast cancer with distant metastasis; and (5) patients with incomplete clinical data, auxiliary examination, or pathological results from other hospitals. The clinical study was approved by the hospital ethics committee and performed in accordance with the Helsinki Declaration. The family members were informed and voluntarily signed the informed consent form.The general data, color ultrasound characteristics, molybdenum targets, pathological features, and molecular pathology data of breast cancer patients were collected and collated by two investigators in the case system of our hospital. Subsequently, two investigators checked the data, including color ultrasound characteristics. The molybdenum targets and pathological features were determined by the Department of Ultrasound and Pathologists. The traditional blue dye method was used for sentinel lymph node biopsy tracing. For patients with micrometastasis, the scope of lymph node dissection was expanded, that is, dissection of axillary lymph nodes, subclavian lymph nodes, and intermuscular lymph nodes. The general data included age, tumor diameter, tumor number (single or multiple), tumor location (left or right), and the quadrant where the tumor was . The color ultrasound data included whether the tumor margin was regular, whether there was a blood flow signal, and whether the posterior echo was attenuated, as well as the BI-RADS category. Molybdenum target features included regular tumor margins, presence of calcified foci, and BI-RADS category. Routine pathological features included histological grades , pathological types , and molecular types . Routine pathology data also contained immunohistochemical staining for specific proteins across keratin 5/6 (CK5/6), cadherin E (E-cadherin), multidrug resistance gene 1 (MDR-1), epithelial growth factor receptor (EGFR), and keratin 19 (CK19). The expression score was based on the positive area of the protein (0–4) × staining intensity (0–3) , 13. TP5U rank sum test. Enumeration data were expressed as [n (%)] and analyzed using the χ2 test. Logistic regression analysis was used for statistical analysis, and the measurement data were converted into binary counting data for calculation. All data were double-tailed with 95% confidence intervals, and statistical significance was set at P < 0.05. The receiver operating characteristic (ROC) curve was constructed using R language to draw the area under the curve (AUC). The closer the AUG value is to 1, the stronger is the accuracy of this indicator. AUC between 0.5 and 0.7 indicated general accuracy, while the value > 0.7 suggested strong accuracy [IBM SPSS 20.0, statistical software was used for data analysis. The measurement data that followed a non-normal distribution were represented as medians and subject to the Mann–Whitney accuracy .P > 0.05). Age and tumor diameter were associated with sentinel lymph node metastasis (P < 0.05), as shown in Table There was no statistically significant difference in tumor number, tumor location, or tumor quadrant between the two groups (P > 0.05), while the BI-RADS category was associated with sentinel lymph node metastasis (P < 0.05), as shown in Table There was no statistically significant difference in tumor margin, blood flow signal, and posterior echo attenuation between the two groups (P > 0.05), whereas the BI-RADS category was associated with sentinel lymph node metastasis (P < 0.05), as shown in Table There was no statistically significant difference in tumor margin and calcification between the two groups , PR (++), and HER (−) were determined as the features of luminal A breast cancer, ER (+ − ++), PR (+), HER (+ − ++) as luminal B, ER (−/+), PR (−), and HER (+++) as HER2 overexpression in breast cancer, and ER (−), PR (−), and HER (−) as basal-like breast cancer, as shown in Fig. P > 0.05), while the pathologic type was associated with sentinel lymph node metastasis (P < 0.05), as shown in Table There was no statistically significant difference in histological grade and molecular typing between the two groups , median E-cadherin score 8 (6–11), median MDR-1 score 3 (1–6), median EGFR score 10 (8–12), and median CK19 score 9 (6–12) in the lymph node metastasis group. The corresponding figures in the other groups were 3 (1–6), 7 (5–11), 3 (1–5), 7 (4–10), and 3 (1-5), respectively , while CK5/6, EGFR, and CK19 were associated with sentinel lymph node metastasis, as shown in Table There was no statistically significant difference in E-cadherin and MDR-1 scores between the two groups P > 0.0, while CP > 0.05). However, TP53 and BRAC1/2 were associated with sentinel lymph node metastasis (P < 0.05), as shown in Table BRAF, ATM, and PALB2 levels were not significantly different between the two groups (P < 0.05), as shown in Table Logistic regression analysis showed that age, tumor diameter, BI-RADS category, pathological type, CK5/6, EGFR, CK19, TP53, and BRAC1/2 were independent risk factors for sentinel lymph node metastasis of breast cancer (The area under the ROC curve (AUC) was observed in the two groups with respect to age, tumor diameter, BI-RADS grade, pathological type, CK5/6, EGFR, CK19, TP53, and BRAC1/2 for sentinel lymph node metastasis (Fig. The “Multivariate logistic regression analysis of risk factors for sentinel lymph node metastasis” and the “ROC curve of independent risk factors for sentinel lymph node metastasis in breast cancer” sections indicate that age, tumor diameter, BI-RADS score, pathological type, CK5/6, EGFR, CK19, TP53, and BRAC1/2 are independent risk factors affecting sentinel lymph node metastasis of breast cancer, which may provide some guidance on sentinel lymph node metastasis.According to the latest epidemiological data, the incidence of breast cancer in China is as high as 42.57/100,000, accounting for 19.2% of all malignant tumors in women. It has surpassed lung cancer as a malignant tumor with the highest incidence . For treChen et al.  pointed Among the influencing factors analyzed using color Doppler ultrasonography and molybdenum target characteristics, BI-RADS grade was directly related to sentinel lymph node metastasis, possibly because the higher the ultrasound grade, the higher the clinical stage of breast cancer, and the higher the probability of sentinel lymph node metastasis. This is related to the biological behavior of breast cancer. However, the regularity of the tumor margin, blood flow, and calcification foci did not affect lymph node metastasis.Pathological characteristics can directly affect the proliferation and metastasis of malignant tumors, in which the degree of differentiation, growth pattern, and biological behavior of tumor cells can be used as important indicators to evaluate the clinical stage and prognosis of breast cancer . In thisThe expression of breast cancer-related proteins plays a key role in the occurrence and development of breast cancer. CK5/6, E-cadherin, MDR-1, EGFR, and CK19 are common immunohistochemical indicators and drug resistance indicators –30. CK5/Gene mutations are the focus of clinical research on the prognosis of breast cancer. In this study, the mutation rates of TP53, BRAC1/2, BRAF, ATM, and PALB2 in patients were statistically analyzed using first-generation sequencing. In this study with a limited sample size, it was found that TP53 and BRAC1/2 may affect sentinel lymph node metastasis; however, validation with a larger sample size is needed.In this study, we identified independent risk factors for sentinel lymph node metastasis in breast cancer based on general data, color ultrasound, molybdenum, and pathological features. This indicates that color ultrasonography, molybdenum target, and pathology techniques can be used as important diagnostic and prediction methods for sentinel lymph node metastasis of breast cancer and can be used to build a prediction model, serving as a guide for Chinese community hospitals. In conclusion, age, tumor diameter, BI-RADS grade, invasive type, expression of CK5/6, EGFR, and CK19, and mutations in TP53 and BRAC1/2 were positively linked to sentinel lymph node metastasis of breast cancer as independent risk factors. Clinical studies should focus on these risk factors in order to strengthen the management and control of sentinel lymph node metastasis in high-risk breast cancer and perform better in early chemotherapy, radiotherapy, or targeted therapy.


Lycoris is used as a garden flower due to the colorful and its special flowers. Floral coloration of Lycoris is a vital trait that is mainly regulated via the anthocyanin biosynthetic pathway. In this study, we performed a comparative transcriptome analysis of Lycoris radiata petals at four different flower development stages. A total of 38,798 differentially expressed genes (DEGs) were identified by RNA sequencing, and the correlation between the expression level of the DEGs and the anthocyanin content was explored. The identified DEGs are significantly categorized into ‘flavonoid biosynthesis,’ ‘phenylpropanoid biosynthesis,’ ‘Tropane, piperidine and pyridine alkaloid biosynthesis,’ ‘terpenoid backbone biosynthesis’ and ‘plant hormone signal transduction’ by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The candidate genes involved in anthocyanin accumulation in L. radiata petals during flower development stages were also identified, which included 56 structural genes  as well as 27 key transcription factor DEGs . In addition, a key structural gene namely LrDFR1 of anthocyanin biosynthesis pathway was identified as a hub gene in anthocyanin metabolism network. During flower development stages, the expression level of LrDFR1 was positively correlated with the anthocyanin content. Subcellular localization revealed that LrDFR1 is majorly localized in the nucleus, cytoplasm and cell membrane. Overexpression of LrDFR1 increased the anthocyanin accumulation in tobacco leaves and Lycoris petals, suggesting that LrDFR1 acts as a positively regulator of anthocyanin biosynthesis. Our results provide new insights for elucidating the function of anthocyanins in L. radiata petal coloring during flower development. Plant pigments, such as anthocyanins, carotenoids and chlorophylls, play important roles in affecting the appearance of flower, fruit and seed coloring . As an iArabidopsis (O-glucosyltrasnferase (UFGT) are important in anthocyanin biosynthesis -based anthocyanin engineering. For example, regulatory roles of MYB TFs in anthocyanin biosynthesis such as Production of Anthocyanin Pigmentation 1 , PeMYB2/11/12, PsMYB114L, FtMYBF18, EsMYB90, and FhMYB5 depend on DFR expression  and WD40 repeat protein TTG1 regulate anthocyanin biosynthetic gene expressions  complex, B-box, bZIP, MYC, NAC, WRKY, bHLH, MADS-box, and WD could also coordinate anthocyanin biosynthesis initiation by binding to the promoter regions of structural genes . For exaressions . Similarressions . Strawbend FaANS . Furthernd FaANS . DhMYB2 d petals . TherefoLycoris species belongs to Amaryllidaceae family, and is a perennial bulb plant native to Northeast Asia, including China, South Korea, and Japan. It consists of about 20 species, of which 15 species and one variety are distributed in China  were identified  was used as a rapid technique to uncover DEGs, biosynthesis pathways, and TFs related to specific traits in plants . In thisLycoris radiata (L’Her.) Herb. plants were grown in Experimental Plantation of Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China. According to the studies reported previously , FL1 , FL2 (fully opened flower stage) and R (senescent flower stage), as shown in L. radiata tissues, including scape, stamen, pistil, flower stalk, and petal samples were obtained during flowering time, while leaf, root, as well as bulb samples from the same plants were obtained during the vigorous vegetative growth stage. The fresh samples were harvested and instantly frozen in liquid nitrogen, then kept at –80°C until use.eviously , three bL. radiata flowers was performed following the protocol of –1 HCl) and then incubated overnight in the dark at 4°C with gentle shaking. After centrifugation for 10 min at 12,000 rpm, the supernatant was diluted four times with acidic methanol and the absorbance was measured at 530 and 657 nm using a UV-1600 spectrophotometer . The concentration of anthocyanins was calculated using the following formula: QAnthocyanins = (A530 – 0.25 × A657) × FW–1, where QAnthocyanins is the amount of anthocyanins, A530 and A657 is the absorption at the indicated wavelengths and FW represents the weight of the fresh sample [g].Extraction and determination of anthocyanins of de novo assembly using the Trinity software  following the manufacturer’s protocol. The quality and quantity of the RNA were examined by the Agilent 2100 Bioanalyzer . Samples with RNA Integrity Number (RIN) ≥ 7 were subjected to cDNA library construction using the TruSeq Stranded mRNA LTSample Prep Kit . Sequencing of the cDNA libraries was done on the Illumina sequencing platform (Illumina HiSeq™ 2500) by Shanghai OE Biotech. Co. Ltd. . Reads were cleaned by removing adapters, as well as low-quality and ambiguous regions, then subjected to software .Alignment of the assembled unigenes was done against public databases including National Center for Biotechnology Information (NCBI) non-redundant protein (Nr) and nucleotide (Nt) database, the Swiss-Prot protein database, Gene Ontology (GO) database, Protein Family (Pfam) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Eukaryotic Ortholog Groups (KOG) database, and eggNOG  database.p-value < 0.05 algorithms  normalized against FPKM via the pheatmap package in R.The expression level of unigenes was calculated using fragments per kilobase per million fragments mapped (FPKM) method . Identifgorithms . To visuL. radiata, we utilized the getorf database (mini-size 150) to find the open reading frame (ORF)   and thenme (ORF) . MoreoveLrDFR1 was based on putative ORFs of unigenes from the RNA-seq database. Primers . Afterward, those T-vectors were transferred into DH5α competent cells  for amplification. The overexpression vectors of LrDFR1 were established by linking their ORFs into a linear plant transformation vector, pBinGFP4, using the One Step Cloning Kit . Then the 35S:LrDFR1 recombinant vectors were transformed into Agrobacterium tumefaciens EHA105 competent cells.Cloning of  Primers  were synLrDFR1-GFP was transformed into the Agrobacterium tumefaciens strain EHA105, and transferred into Nicotiana benthamiana epidermal cells . For staining of the nuclei, 10 mg/mL 4′6-diamidino-2-phenylindole (DAPI) was infiltrated into N. benthamiana leaves 6 h before observation.The pBinGFP4 vector with al cells . Cultivawas used . AssessmN. benthamiana leaves were decolorized in a solution of ethanol: glacial acetic acid (3:1). A dimethylaminocinnamaldehyde (DMACA) reagent staining solution  was then added for staining.Staining of proanthocyanidin was conducted as described by A. tumefaciens harboring 35S:LrDFR1-GFP construct and the control pBinGFP4 vector were prepared for injecting into Lycoris petals, respectively. The recombinant Agrobacterium strains were cultured in YEB broth containing 50 μg mL–1 kanamycin and incubated at 28 °C. Then, the collected recombinant Agrobacterium strains were resuspended to OD600 of 0.6 in a buffer with 10 mM 2-(4-Morpholino) ethanesulfonic acid, 10 mM MgCl2, and 120 μM acetosyringone. Transformed Lycoris petals were stored for 48 h in the dark after which they were transferred to a phytotron at a constant photon flux density of 100 μmol m–2 s–1. With 5 days cultivation, Lycoris petals were obtained for anthocyanin level assessment and RNA extraction.The TIP41-like protein (TIP41) according to previous study on L. aurea  or a significant t-test . All the results are presented as the mean ± standard deviation (SD).Statistical analyses were done by SPSS version 10.0 software . The significant difference among sets of data was determined by one-way analysis of variance (ANOVA) with Duncan’s multiple range test  stages , and a total of 644.93 million raw reads as well as 96.73 Gb raw bases were obtained. After eliminating the adaptor, poor-quality sequences, and ambiguous reads, 634.09 million clean reads and 89.86 Gb clean bases were retrieved from 12 samples , 29,476 (33.65%), 37,487 (42.8%), and 22,318 (25.48%) unigenes were annotated to the Nr, Swiss-Prot, eggNOG, and Pfam databases, respectively. Taken the entire public databases together, a total of 41,534 (47.42%) unigenes could be successfully annotated . To elucL. radiata petal color transitions, six pair-wise comparison groups  were conducted  was found between FB and R libraries, of which 10,958 and 12,244 genes were down-regulated and up-regulated, respectively  and LrDFR2 (DN42380) indicated a significant positive correlation with the total anthocyanins content in petals during the flower development stages, while LrFLS (DN37334) indicated a significant negative correlation with the total anthocyanins content , AP2/ERF (1), bHLH (1), bZIP (1), NAC (1), NF-X1 (1), and Trihelixs (4) genes, likely act to improve anthocyanin synthesis during L. radiata petal development stages. However, the 17 negative regulators, including Alfin-like (1), AP2/ERF (1), GATA (1), GRFs (2), bHLHs (2), MYBs (2), C2H2s (2), C3Hs (4), MADS (1), and NAC (1), might act as repressors in L. radiata anthocyanin biosynthesis  involved in the accumulation of anthocyanins were identified, including 10 positive regulators and 17 negative regulators . These 1ynthesis . Notablyynthesis .DFRs, four MYBs, two WD40s, two 4CLs, one F3′H, one UFGT, one CHS, one ANS, one FLS, and one CHI were selected as hub genes based on their connection position in the network modules, expression pattern and functional annotation (LrDFR1 (DN43960) and LrFLS (DN37334) could be regarded as key hub genes for participating anthocyanin biosynthesis. Two MYBs (DN45447 and DN33872), two NACs (DN39353 and DN39797), one C3H (DN42291), and one GATA (DN30983) TF genes were identified as hub genes in regulating anthocyanin biosynthesis , indicating that the RNA-seq data were credible and accurate.To validate the accuracy and transcription profiles revealed by the RNA-seq data, 32 unigenes were selected for qRT-PCR assays. The relative expression levels of these 32 genes were normalized to the expression of LrDFR1 gene (DN43960) from L. radiata. The full-length cDNA of LrDFR1 is 1113 bp in length and it encodes a 370 amino acid protein with a molecular weight of 41.67 kDa (Agapanthus praecox (75.33%), Muscari armeniacum (74.74%), and Hyacinthus orientalis (72.72%)  . Multipl(72.72%)  and the (72.72%)  in the L(72.72%) . qRT-PCRn petals . Moreovetage FL1 . These fLrDFR1 in tobacco epidermal cells to assess subcellular localization of LrDFR1. As shown in LrDFR1 in regulating anthocyanin as well as proanthocyanidin biosynthesis in L. radiata, an LrDFR1-overexpressing plasmid was transfected into Lycoris petals and tobacco epidermal cells  (Lilium spp.)  , lilies um spp.) , Magnoliprengeri , Paeoniactiflora , Paeoniadelavayi , and Sillittorea . For betfunction , 5.L. radiata petals during flower development stages  were highly expressed in petals at FL1 and FL2 stages (UFGT (DN44965), which glycolyzes anthocyanidin into anthocyanin  and one FLS (DN37334)   were fouDN37334) . In anth changes . qRT-PCR samples . Our resTranscription factors play critical functions in flavonoid biosynthesis, by regulating expression of structural genes. For example, the class of TFs identified were previously implicated in regulation of petal color formation in roses . ParticuAP2/ERF, bHLH, bZIP, C2C2, HSF, MYB, NAC, TIFY, and WRKY families were predicted  was highest in FB, followed by FL1, FL2, and R, which was contrary to the total anthocyanin content trend. Conversely, the expression of LrMYB1 (DN33872) exhibited a similar trend to the total anthocyanin content in the L. radiata petals (MYBs (DN45447 and DN51496) negatively regulated anthocyanin accumulation, whereas LrMYB1 (DN33872) was identified as one of the eight hub genes may positively regulate anthocyanin accumulation in L. radiata  and one positively correlated LrbHLH1 (DN48856) were identified , wounding, drought, and different light intensities (LrMYB1 (DN33872) and LrbHLH1 (DN48856), of which the expression was positively correlated with LrDFR1, LrCHS, LrCHI, F3′H, LrUFGT and LrANS genes during petal development stages , which has a highly preserved NADPH-binding domain “VTGAAGFIGSWLIMRLLERGY” as well as a substrate-binding domain in plants  DFR into white-flowered petunia varieties, transgenic plant flowers accumulate non-native pelargonidin, which results in novel brick red-flower varieties  involved in anthocyanins biosynthesis in transgenic Lycoris petals  can be found below: NW and ZW designed the research and wrote the manuscript. NW performed most of the experiments and data analysis. XS and FZ collected the experimental materials. TW assisted with the data analysis. WZ provided helpful comments on the manuscript. ZW provided guidance on the whole study and contributed with valuable discussions. All authors read and approved the final manuscript.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli strains, including Shiga toxin-producing E. coli O157:H7, are known to be resistant to tellurite. This resistance is involved in ter operon, which is usually located on a prophage-like element of the chromosome. The characteristics of the ter operon have been investigated mainly by genome analysis of pathogenic E. coli; however, the distribution and structural characteristics of the ter operon in other E. coli are almost unknown. To clarify these points, we examined 106 E. coli strains carrying the ter operon from various animals. The draft genomes of 34 representative strains revealed that ter operons were clearly classified into four subtypes, ter-type 1–4, at the nucleotide sequence level. Complete genomic sequences revealed that operons belonging to three ter-types  were located on the prophage-like elements on the chromosome, whereas the ter-type 2 operon was located on the IncHI2 plasmid. The positions of the tRNASer, tRNAMet, and tRNAPhe indicated the insertion sites of elements carrying the ter operons. Using the PCR method developed in this study, 106 strains were classified as type 1 (n = 66), 2 (n = 13), 3 (n = 8), and 4 (n = 17), and two strains carried both types 1 and 2. Furthermore, significant differences in the minimum inhibitory concentration (MIC) of tellurite were observed between strains carrying ter-type 4 and the others (p < 0.05). The ter-type was also closely related to the isolation source, with types 2 and 4 associated with chickens and deer, respectively. This study provided new insights related not only to genetic characteristics of the ter operons, but also to phenotypic and ecological characteristics that may be related to the diversity of the operon.Tellurite is highly toxic to most bacteria owing to its strong oxidative ability. However, some bacteria demonstrate tellurite resistance. In particular, some  Tellurite toxicity is related to the generation of reactive oxygen species (ROS) (Escherichia coli (STEC) belonging to O157:H7 and other serotypes such as O26:H11, O103:H2, O111:H8, O121:H19, O145:H28, and O45:H2, which are responsible for numerous foodborne disease outbreaks, are known to possess tellurite resistance. Therefore, when STEC is efficiently isolated from patient and food samples, selective agar plates containing potassium tellurite (K2TeO3), such as cefixime-tellurite sorbitol MacConkey (CT-SMAC) agar and CHROMagar STEC are routinely used in laboratories.Tellurium (Te) is a rare element on Earth  and is fes (ROS) , which tes (ROS) , and conarsABC)  (narGHIJ)  (katG)  (zwf) (ter) operon  are directly involved in tellurite resistance  , cysteinB, IscS) , nitratenarGHIJ) , superoxB, soxS) , hydroge) (katG) , telluri telAlB) , and gluB) (zwf)  have bee) operon  is invols strain , 1988. Asistance . Genome  species , 2.ter operon. For example, the STEC O157:H7 Sakai strain carried the ter operon on a prophage-like 86-kb element, named SpLE1 on the chromosome (ure), and iron-regulated gene A (IrgA)-homolog adhesin (iha)  . Comparaerotypes . Some STsistance . A uropa plasmid . These rterA-positive E. coli strains isolated in Japan were used in this study   at 37°C for 18–20 h and then inoculated on deoxycholate hydrogen sulfide lactose (DHL)  or CHROMagar STEC  agar plates. E. coli strains from chickens were isolated from colibacillosis infected sites (such as under the skin and organs) of chickens on farms and slaughterhouses. The swabs were directly inoculated onto DHL agar plates. After incubation at 37°C for 18–20 h, E. coli-like single colonies were picked from each plate. DNA for PCR was purified using the Wizard® Genomic DNA Purification Kit . E. coli species were confirmed by PCR using E. coli-specific gyrB sequences   . The theE. coli O and H serotyping (Og- and Hg-typing) were conducted as described previously . Illumina short-read libraries were prepared using 100 ng of extracted DNA using the Nextera DNA Library Prep Kit, and paired-end reads were generated using the MiSeq Reagent Kit (v3-600) on MiSeq (Illumina). MinION long-read libraries were prepared from 300 ng of genomic DNA using the Rapid Barcoding kit (SQK-RBK004) and were sequenced using R9 flow cells (FLO-MIN106) on MinION (Oxford Nanopore Technologies) to produce > 100 Mb of data per strain. The Illumina data were preprocessed using Trimmomatic (v0.36) to remove the adapter and low-quality sequences (PRJDB10561.DNA for genome sequencing was extracted and purified using the Qiagen Genomic DNA Preparation Kit (Qiagen) or Maxwellequences . The quaequences  a hybridequences . The seqin silico Molecular Cloning software . Plasmids were identified using PlasmidFinder 2.1   of the seven genes were used to construct a phylogenetic tree.Multilocus sequence typing (MLST) was performed using the internal sequences of seven housekeeping genes (nd recA) . The prind recA) . Sequenc2TeO3) ranging from 0.125 to 512 μg/mL, according to the general steps of the agar dilution method , cattle (34 strains), pigs (6 strains), chickens (8 strains), and deer (32 strains) were collected (see terA-positive E. coli strains contained eae-positive STEC (positive for either or both stx1 and stx2) (42 strains), eae-negative STEC (28 strains), enteropathogenic E. coli (EPEC) (negative for both stx1 and stx2 and positive for eae) (4 strains), and others  (32 strains).In total, 106 cted see . There wter operons  containing six genes terZ, terA, terB, terC, terD, and terE , terA , terB (456 bp), terC , terD (579 bp), and terE (576 bp) showed that type-3 and type-4 formed independent groups for all genes, whereas type-1 and type-2 roughly formed their respective groups for terZ, terA, terD, and terE  were highly conserved with SpLE1 in the O157:H7 Sakai strain  were almost identical, except for the upstream region in Og168:Hg19 and Og9:Hg19 , a fatty acid biosynthesis gene cluster, site-specific tyrosine recombinase genes, and some transposase genes, including the IS256 family.The 6,442 bp . However103:Hg14 . Conservter-type 2 from Og136:Hg16, Og183:Hg18, Og161:Hg4, Og183:Hg42, and Og84:Hg2 was located on IncHI2 plasmids, including the conjugative system (tra genes) and some antibiotic resistant genes , with sizes ranging from 165,509 to 249,877 bp .The operon of 9,877 bp . Except ter operons used in this study, which were extracted from the genomes of strains other than E. coli, were located on plasmids, except for a Salmonella-derived ter-type 2 operon, and Proteus- and Yersinia-derived operons, which are on the chromosome  were the insertion sites for the elements of the ter-type 1 and 3 operons. Although the types of elements carrying the ter-type 1 and 3 operons were different, a homology (98% amino acid sequence) was confirmed in the integrase genes located on the elements inserted in tRNAMet (ter-type 4 (Og54:Hg45 and Og22:Hg16 strain) were highly homologous (upper 97% amino acid sequence) with the integrases in the other integrating elements or the locus of enterocyte effacement that was inserted in the tRNAPhe positions in seven major STEC genomes , type 2 (n = 13), type 3 (n = 8), and type 4 (n = 17), except for two strains belonging to Og84:Hg2, which were positive for both ter-type 1 and 2 , whereas eae-negative E. coli strains, including STEC and non-STEC, were positive for ter-type 2 (n = 13), type 3 (n = 8), and type 4 (n = 17), in addition to ter-type 1 (n = 21) (Based on the sequences of the ach type . Using P 1 and 2 . All eae(n = 21) .terA-positive E. coli strains (one representative strain of the same O:H genotype and the same ter-type was selected from 106 strains) is shown in ter-types were distributed in various lineages of E. coli including A, B1, B2, E, D, A × B1, and ABD , which was tellurite-sensitive. The genome sequence of this strain had an IS5 inserted on the terZ due to which the gene was disrupted  in ter-type 4 was resistant to 64–128 μg/mL   .ter operon carrier elements on the chromosomes were roughly conserved for each of the ter-types, 1, 3, and 4  and elements with the other type of integrase gene were inserted into tRNAMet    , 6. The elements , and allg9:Hg19) . Althougter-type 2 operons were located on the IncHI2 plasmids of E. coli strains, and were also found on the IncHI2 plasmids carried by Y. pseudotuberculosis and K. pneumoniae . In recent years, large amounts of antibacterial drugs have been used in broiler production, and the resulting emergence and spread of drug-resistant bacteria has become a global public health concern , except for 4 strains belonging to type 1   see . These rter-type 4 operon showed significantly lower MICs of potassium tellurite compared to the other three types. The function of Ter proteins has been suggested to involve their mutual association, as they interact at the interface of the inner plasma membrane and the cytosol . Of the isolation sources used in this study, deer were the only animals from the wild environment, and their feces were collected from a wide area ranging from Hokkaido in the north, to Kyushu in the south of Japan. In addition to tellurite resistance, the ter operon is also involved in resistance to infection by various bacteriophages and to membrane-pore forming colicins  was identical to those of Og121:Hg19 (TS23_1 strain) (data not show). However, the resistance level of the latter strain (16–32 μg/mL of potassium tellurite) was roughly lower than that of the former (64–128 μg/mL). A previous study has also shown that even O157:H7 serotype strains differ in their MICs of potassium tellurite   . The aboter operons responsible for tellurite resistance among various serotypes and sources of E. coli. Comparative analysis clearly distinguished the ter operon sequences into four subtypes, namely, ter-type 1–4 operons. The amino acid sequences of the ter-type 4 operon genes were distant from those of the other types, further, strains with the ter-type 4 operons showed significantly lower MICs compared to the other types. Furthermore, genomic analysis using long reads indicated the structural details of the integrating elements carrying the ter operons and insertion sites on the chromosome. Genomic comparison and phylogenetic analysis showed that at least three highly conserved elements were spread horizontally within E. coli species. Type 2 was found to be located on plasmids distributed in E. coli as well as in some Enterobacteriaceae species. The ter-type was also closely related to the source of isolation, the cause of which was unknown; however, types 2 and 4 were associated with chickens and deer, respectively. This study provided new insights related not only to genetic characteristics of the ter operons in E. coli, but also to phenotypic and ecological differences that may be related to them. In the future, by clarifying the unknown function of the Ter proteins, it is expected that the relationship between the diversity of the ter operon and the phenotypic and ecological characteristics revealed in this study will be clarified.This study compared the details of 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 in the article/TN, AI, and SI conceived and designed the experiments. TN performed the experiments. TN, TK, and TT analyzed the data. TN and AI contributed to reagents, materials, analysis tools, critical revision of the manuscript for important intellectual content, and wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Early and effective glycemic control can prevent or delay the complications associated with type 2 diabetes (T2D). The benefits of glucagon-like peptide-1 receptor agonists (GLP-1RAs) are becoming increasingly recognized and they now feature prominently in international T2D treatment recommendations and guidelines across the disease continuum. However, despite providing effective glycemic control, weight loss, and a low risk of hypoglycemia, GLP-1RAs are currently underutilized in clinical practice. The long-acting GLP-1RA, semaglutide, is available for once-weekly injection and in a new once-daily oral formulation. Semaglutide is an advantageous choice for the treatment of T2D since it has greater efficacy in reducing glycated hemoglobin and body weight compared with other GLP-1RAs, has demonstrated benefits in reducing major adverse cardiovascular events, and has a favorable profile in special populations . The oral formulation represents a useful option to help improve acceptance and adherence compared with injectable formulations for patients with a preference for oral therapy, and may lead to earlier and broader use of GLP-1RAs in the T2D treatment trajectory. Oral semaglutide should be taken on an empty stomach, which may influence the choice of formulation. As with most GLP-1RAs, initial dose escalation of semaglutide is required for both formulations to mitigate gastrointestinal adverse events. There are also specific dose instructions to follow with oral semaglutide to ensure sufficient gastric absorption. The evidence base surrounding the clinical use of semaglutide is being further expanded with trials investigating effects on diabetic retinopathy, cardiovascular outcomes, and on the common T2D comorbidities of obesity, chronic kidney disease, and non-alcoholic steatohepatitis. These will provide further information about whether the benefits of semaglutide extend to these other indications. ThisGlycemic management in patients with T2D has become more individualized, and there are now several different treatment options available, with various factors influencing the most appropriate choice for individual patients. Glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs) are a well-established class of glucose-lowering agents that act on multiple pathophysiological defects in T2D, providing effective glycemic control, weight loss, and a low risk of hypoglycemia, with a well‑characterized safety profile . In addivia its new oral formulation.This article will review the place of GLP-1RAs in therapy and, within this class, specifically discuss some clinical considerations around the use of the long-acting GLP-1RA, semaglutide, when given subcutaneously or 1c target after 3–6 months, another glucose-lowering medication should be added  (if estimated glomerular filtration rate [eGFR] is adequate) with proven efficacy to reduce the risk of CV events , 11. Thiuminuria , 11. Of  CV risk .1c, body weight, and systolic blood pressure with a lower requirement of rescue therapy when a GLP-1RA was added in combination with an SGLT2i vs. SGLT2i monotherapy alone , it appeared that over time GLP-1RAs were being prescribed to patients with progressively more advanced disease, with significant increases in baseline age, diabetes duration, presence of CVD, and insulin use in patients receiving GLP-1RA therapy during the study period  adverse effects  or onceotection  and liraotection ). Subseqotection , dulagluotection , and semotection ) to reduotection , and thi1c and weight reductions compared with exenatide ER 2.0 mg     and withctively) . Since tctively) –43.It is known that some patients prefer oral over injectable medications , 45, andAs a class, GLP-1RAs have a well-defined safety profile. The most commonly reported adverse events (AEs) are GI-related effects, including nausea, diarrhea, and vomiting, which are generally mild-to-moderate in severity and transient in nature . In geneSubcutaneous semaglutide can be dosed at any time on the day of the weekly injection, with or without meals . For oraIn pharmacokinetic studies, subcutaneous or oral semaglutide did not have clinically relevant effects on the exposure of other widely used medications, such as warfarin, metformin, digoxin, atorvastatin/rosuvastatin –55, or tIn a drug–drug interaction study, levothyroxine exposure was increased by 33% when co-administered with oral semaglutide 14 mg, which may be due to delayed gastric emptying and increased levothyroxine absorption . MonitorIn population pharmacokinetic and exposure−response analyses, the exposure range following oral semaglutide was wider than for subcutaneous dosing but with a considerable overlap between oral semaglutide 7 and 14 mg and subcutaneous semaglutide 0.5 and 1.0 mg . The eff2) (2) (2)  , 21, 23 2) Table 1.2) (2) , 63, 64. (2) (2) , 26.2)   . Superio 6 CVOTs . Indeed,via possible direct cardio-nephroprotective mechanisms, such as improved endothelial dysfunction, reduced oxidative stress, and reduced inflammation   . The priThere is a complex interplay between T2D and liver disease, particularly non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), which are common in patients with T2D . The mecGLP-1RAs appear to be well-tolerated in patients with hepatic impairment, and dose adjustments are not necessary Table 1 Novel therapies are in demand for the treatment of NAFLD, and early studies suggested that GLP-1RAs may reduce liver inflammation and fibrosis . Potentip < 0.001).A phase II trial recently evaluated the effects of once-daily subcutaneous semaglutide  vs. placebo in 320 patients with NASH . Treatmevia weight loss. However, semaglutide is also associated with improvements in insulin resistance, hepatic lipotoxicity, and hepatic inflammation. In pre-clinical models, improvements in inflammation with liraglutide were shown to be independent of weight reduction, as was prevention of initiation of fibrosis  vs. placebo for 12 weeks, and this was associated with reduced appetite and food cravings, better control of eating, and lower preference for fatty, energy-dense food  but without T2D were randomized to once-daily subcutaneous semaglutide , once-daily subcutaneous liraglutide , or placebo, in combination with dietary and physical activity counseling, with the primary endpoint of percentage weight loss at week 52. Estimated mean weight change was –2.3% for the placebo group and ranged from –6.0% with subcutaneous semaglutide 0.05 mg to –13.8% with subcutaneous semaglutide 0.4 mg after 52 weeks . Furthermore, mean body weight reductions with semaglutide at a dose of 0.2 mg or higher were significantly greater than with liraglutide (–7.8%).A phase II dose-finding trial evaluated the efficacy and safety of once-daily subcutaneous semaglutide in promoting weight loss . In tota2 or BMI ≥27 kg/m2 with at least one weight-related comorbidity , while patients in STEP 2 had to have a BMI ≥27 kg/m2 and T2D. The primary endpoint of STEP 1–5 is the change from baseline to end of treatment in body weight; the proportion of patients achieving a body weight reduction of ≥5% is a co-primary endpoint in STEP 1–3 and 5. In the completed STEP trials, semaglutide 2.4 mg as an adjunct to lifestyle intervention led to mean body weight losses of ~15–17% over 68 weeks in patients without T2D , with a smaller mean weight loss of 9.6% seen in patients with T2D over the same period (STEP 2). At week 68, 86–89% of patients without T2D achieved ≥5% body weight loss , with 69% of patients with T2D achieving this threshold (STEP 2). Across all studies, semaglutide 2.4 mg also demonstrated benefits beyond weight loss on cardiometabolic parameters and patient-reported outcomes (These findings paved the way for the phase III STEP (Semaglutide Treatment Effect in People with obesity) program, which is currently investigating body weight changes following treatment with once-weekly 2.4 mg subcutaneous semaglutide . This gloutcomes –84.In addition to the STEP program, the effect of semaglutide treatment on CV outcomes is being assessed in adults aged ≥45 years with overweight or obesity. The SELECT phase III trial (NCT03574597) is investigating whether once-weekly subcutaneous semaglutide (up to 2.4 mg) can reduce MACE vs. placebo in approximately 17,500 people with overweight or obesity and established CVD with a follow-up of approximately 5 years .1c reduction in patients with pre-existing diabetic retinopathy  is an ongoing CVOT evaluating the effects of once-daily oral semaglutide (up to 14 mg) vs. placebo in 9,642 patients with T2D and CVD, cerebrovascular disease, symptomatic peripheral artery disease, or CKD . The pri1c and body weight compared with other GLP-1RAs and favorable clinical characteristics make semaglutide, either subcutaneous or oral, an advantageous choice for T2D treatment. Oral semaglutide provides an additional treatment option for patients and physicians who may be reluctant to initiate or intensify therapy by injection, and this may also help to increase earlier GLP-1RA utilization.The benefits of GLP-1RAs are becoming increasingly recognized in international T2D recommendations and, along with other agents targeted at T2D pathophysiology, such as SGLT2is, their initiation early in the disease trajectory is advocated. The higher efficacy of semaglutide in reducing HbAWhere unanswered questions remain about the impact of semaglutide on outcomes, ongoing trials are underway to provide additional clarity. Effects on diabetic nephropathy and retinopathy are being assessed for subcutaneous semaglutide, and whether there are any positive CV benefits of oral semaglutide will also be determined. The management of comorbidities that are increasingly common in patients with T2D, such as obesity and liver disease, need to be better addressed; in this respect, ongoing trials will provide further information about whether the benefits of semaglutide extend to these other indications.All authors contributed to the article and approved the submitted version.This article was supported by Novo Nordisk, who was provided with the opportunity to perform a medical accuracy review.FG has received research support from Eli Lilly, Lifescan, and Takeda, and has provided advisory services to AstraZeneca, Boehringer Ingelheim, Eli Lilly, Lifescan, Merck Sharp & Dohme, Novo Nordisk, Roche Diabetes Care, and Sanofi. BG has provided advisory services to AstraZeneca, Bayer, Boehringer Ingelheim, Eli Lilly, Merck Sharp & Dohme, and Novo Nordisk, and has received lecture honoraria from Bristol Myers Squibb and the above-mentioned companies.The authors declare that this article received funding from Novo Nordisk. The funder had the following involvement in the article: medical writing support.


Fragmented mitochondrial (mt) genomes and extensive mt gene rearrangements have been frequently reported from parasitic lice (Insecta: Phthiraptera). However, relatively little is known about the mt genomes from the family Philopteridae, the most species-rich family within the suborder Ischnocera.Falcolipeurus suturalis and compare it with the mt genome of F. quadripustulatus. Phylogenetic relationships within the family Philopteridae were inferred from the concatenated 13 protein-coding genes of the two Falcolipeurus lice and members of the family Philopteridae using Bayesian inference (BI) and maximum likelihood (ML) methods.Herein, we use next-generation sequencing to decode the mt genome of F. suturalis is a circular, double-stranded DNA molecule 16,659bp in size that contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and three non-coding regions. The gene order of the F. suturalis mt genome is rearranged relative to that of F. quadripustulatus, and is radically different from both other louse species and the putative ancestral insect. Phylogenetic analyses revealed clear genetic distinctiveness between F. suturalis and F. quadripustulatus (Bayesian posterior probabilities=1.0 and bootstrapping frequencies=100), and that the genus Falcolipeurus is sister to the genus Ibidoecus (Bayesian posterior probabilities=1.0 and bootstrapping frequencies=100).The complete mt genome of Falcolipeurus and provide useful genetic markers for systematic studies of bird lice.These datasets help to better understand gene rearrangements in lice and the phylogenetic position of The online version contains supplementary material available at 10.1186/s13071-021-04776-5. The typical insect mitochondrial (mt) genome is a circular, double-stranded DNA molecule of about 1220kb in length that contains 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA), and two ribosomal RNAs (rRNA) , 5, Meno7Bothriometopus macrocnemis , an, anF. suly baits .Fig. 1Fecox1 and rrnS sequences of F. suturalis were used as initial references for assembly in Geneious v 11.1.5  [The quality of extracted genomic DNA was tested by agarose gel electrophoresis  and DNA ize=5bp) . Genome F. quadripustulatus [AT)/(A+T), GC-skew=(GC)/(G+C) [Protein-coding and rRNA genes were identified by alignment to homologous genes of previously sequenced mt genome of the vulture louse stulatus  using thstulatus . tRNA gestulatus  and tRNAstulatus , with mastulatus . Nucleotstulatus . AsymmetC)/(G+C) .Haematomyzus elephantis, and eight species of sucking lice, were used for phylogenetic analysis [930Heterodoxus macropus (GenBank: AF270939), used as an outgroup [nad2 because it is unidentified in the H. elephantis mt genome) were aligned individually using MAFFT [A total of 23 mt genomes from lice, including ten species from the Philopteridae, three species from the Trichodectidae, one elephant louse, nalysis 9, 3032, woutgroup . Amino ang MAFFT , and subng MAFFT . The ining MAFFT . ML analng MAFFT . PhylogrF. suturalis, Degeeriella fulva, Degeeriella aquilarum, Nosopon chanabense, Colpocephalum impressum, Laemobothrion Laemobothrion vulturis, and Laemobothrion Laemobothrion maximum) can parasitize the tawny eagle A. rapax .Seven louse species  body slender in deep dark brown colour; (2) head longer than wide with pointed anterior and slightly broad posterior; (3) dorsally, 3 dark brown spots on each side of preantennal area and 1 dark brown horizontal stripe near antennal area; (4) antennae sexually dimorphic, male antennae stout and long on segment 1, female antenna slender and short on all 5 segments; (5) thorax widened from top to bottom; (6) dorsal thorax with 1 pronotum and 2 pteronotums, pronotum larger than pteronotum, 1 stout short seta on posterior margin of each pteronotum; (7) ventral thorax, mesosternum with 1 pair dark brown spots near midlegs; (8) 3 pairs of legs slender, forelegs small, midlegs and hindlegs progressively larger; (9) abdomen longer than wide; (10) dorsal abdomen deep dark brown colour, 2nd segment slightly narrower than thoracic pteronotum and with 2 divided tergite plates; (11) tergites on dorsal abdominal segments 3 to 6 slightly narrow in middle on both male and female; (12) male with 2 divided tergite plates laterally and female with 1 tergite on each dorsal abdominal segment of 7 and 8; (13) ventral abdomen with 9 segments and in white colour, 1 pair of dark brown spots laterally on each segment of 2 to 9; (14) female with 1 small dark brown spot in inverted triangle shape in middle of ventral abdominal segments 8 and 9.F. suturalis (GenBank accession: MW696813). All 37 mt genes typical of metazoan mt genomes were present. Gene arrangement was distinct from that of F. quadripustulatus [A=27.8%, T=44.8%, C=11.1%, G=16.3%. All mt genes were encoded on the heavy strand, except tRNA-Arg. Three overlapping regions in the mt genome were observed: nad4L/nad1, tRNA-His/tRNA-Asp and tRNA-Asp/tRNA-Arg, ranging from 4 to 8bp overlaps  was obtained from the Illumina HiSeq 2500 platform, raw sequencing of 15,178,382 paired reads. Reads were cleaned  and assembled. The longest assembled contig was 16,659bp in size and was the complete mt genome of stulatus  and otheA+T and G+C content of the complete mt genome was 73.0% and 27.0%, respectively, consistent with the nucleotide content of lice reported in previous studies [Total  studies , 15  or TTG as their initiation codons. Four genes  use ATA, five  use ATG, three  use ATT, and one (cytb) uses TTG  use TAA, three genes  use TAG, nad1 uses TA, and nad5 genes use T  and F. quadripustulatus . In the F. suturalis mt genome, rrnL was located between rrnS and tRNA-Ala, while rrnS was between tRNA-Asn and rrnL   is located between cox2 and nad5 and non-coding region (NC3)  is located between tRNA-Gly and nad3 , and the least difference was found in cytb (13.2%)  are inverted in both Falcolipeurus species relative to the putative ancestral insect. Only two gene blocks are shared between Bothriometopus and the putative ancestral insect: tRNA-Glynad3 and atp8atp6 [Campanulotes species and the ancestral insect: atp8-atp6 [cox3, tRNA-Tyrcox2, and tRNA-LeuCUNnad4L, are shared between Falcolipeurus and Ibidoecus [nad4tRNA-LeuUUR and tRNA-Glynad3, are shared between Falcolipeurus and Campanulotes. In the Philopteridae, only one gene block, tRNA-Ilecox1, is shared across all Philopterids, excepting I. bisignatus, Columbina, and Columbicola. Such a lack of conserved gene arrangements in the mt genome of bird lice complicates the accurate reconstruction and identification of rearrangement events across their history [The mt genome arrangements of both sect Fig.. Compareions Fig.. Of the atp8atp6 , while otp8-atp6 , 14, 33.bidoecus . Two gen history .Fig. 4GeF. suturalis and F. quadripustulatus were identified consisting of at least one translocation  was found in the F. quadripustulatus mt genome [F. suturalis mt genome. Gene duplications have also been reported in the mt genomes of several families in the class Insecta, such as the Reduviidae and Thripidae [One tRNA gene (tRNA-Gly) was lacking, while the duplication of three genes . The branch leading to the two Falcolipeurus species is much longer than the branch of two Columbina species. The genus Falcolipeurus is more closely related to the genus Ibidoecus than to other genera  sequences of 127 species from the four louse suborders, showing the Philopteridae to be paraphyletic . YoshizaMt genome sequences are effective molecular markers for the study of phylogenetic and systematic relationships at various taxonomic ranks across the phylum Arthropoda, including for ectoparasites 48. RecenF. suturalis and compared it with the mt genome of F. quadripustulatus. Its gene order is rearranged relative to other Falcolipeurus species, and represents a new pattern within the Phthiraptera. These novel datasets will help to better understand the gene rearrangements and phylogenetic position of Falcolipeurus and provide useful genetic markers for systematics and phylogenetic studies of bird lice.The current study presents the entire mt genome of Additional file 1: Table S1. Primers used for assembly validation.Additional file 2: Figure S1. PCR amplicons from the mitochondrial genome of Falcolipeurus suturalis. Amplicons generated with the F. suturalis primers. M: DL8000 DNA marker, 1: Validation_01, 2: Validation_02, 3: Validation_03, 4: Validation_04, 5: Validation_05, 6: Negative control.


Tumor metastasis to bone is a common event in multiple forms of malignancy. Inflammation holds essential functions in homeostasis as a defense mechanism against infections and is a strategy to repair injured tissue and to adapt to stress conditions. However, exaggerated and/or persistent (chronic) inflammation may eventually become maladaptive and evoke diseases such as autoimmunity, diabetes, inflammatory tissue damage, fibrosis, and cancer. In fact, inflammation is now considered a hallmark of malignancy with prognostic relevance. Emerging studies have revealed a central involvement of inflammation in several steps of the metastatic cascade of bone-homing tumor cells through supporting their survival, migration, invasion, and growth. The mechanisms by which inflammation favors these steps involve activation of epithelial-to-mesenchymal transition (EMT), chemokine-mediated homing of tumor cells, local activation of osteoclastogenesis, and a positive feedback amplification of the protumorigenic inflammation loop between tumor and resident cells. In this review, we summarize established and evolving concepts of inflammation-driven tumorigenesis, with a special focus on bone metastasis. Inflammation is an evolutionarily conserved mechanism to protect organisms from invading pathogens, to clear damaged cells, and to repair injured tissues, ultimately aiding in restoring homeostasis . A primeTumor-promoting inflammation has been proposed as an emerging hallmark of cancer by Hanahan and Weinberg in 2011 . First oα and interleukin (IL)-6, with both of them playing significant roles in malignancies [In further consideration, inflammation is the connective link between cancer and its major risk factors including smoking, obesity, hyperglycemia, alcohol consumption, UV light, radiation, as well as chronic bacterial, viral, or parasitic infections, as shown in several studies . All of gnancies ,34,35.Once a tumor is growing, cancer cells may undergo necrosis as a result of limited nutrient and oxygen supply. Necrotic cells release several molecules that serve as alarming danger-associated molecular patterns (DAMPs), which are not always anti-tumorigenic and protective but may foster inflammation or alter local immune responses by recruitment and modulation of immunosuppressive cells ,37,38,39β, IL-6, IL-8, TNF-α, and a number of chemokines such as CC-chemokine ligand (CCL)2, CCL5, and CCL18 [Another hallmark of cancer is metastasis , which mnd CCL18 . Local tnd CCL18 . Finallynd CCL18 .In summary, inflammatory processes are a hub in cancer, being involved in virtually all steps of tumorigenesis, from initiation and promotion of primary tumor growth, to EMT, invasion, dissemination, and establishment of solid metastases . In addiA prime example of metastasis is the dissemination and local outgrowth of tumor cells to and within the bone . The seqThe clinical manifestation of bone metastasis is a result of an imbalance between bone formation and bone resorption accompanied by ECM remodeling, leading either to direct destruction of bone tissue (osteolytic metastases) or the production of new bone of poor quality (osteoblastic metastases), both of which contribute to a significantly increased fracture risk in affected individuals . Bone isA fine-tuned balance of bone formation and resorption maintains the normal physiology of healthy bone tissue, resulting in a stable bone mass . In the Bone metastases impose a significant individual and socio-economic burden and manifest in skeletal-related events (SRE) such as fractures, hypercalcemia, nerve compression syndromes, pain, and immobility ,63. Currβ and insulin-like growth factors, thus alluring cancer cells to settle and grow [β, TNF-α, etc.), as well as cellular and soluble mediators that have attained increasing attention in recent years.  and cellular mediators , etc. [Historical observations of bone involvement in patients with breast cancer by the British surgeon Stephen Paget led him to propose the “seed and soil” theory  with theand grow ,67,68. Iand grow . Notablyand grow . In addiand grow . To delit years. . Certainm), etc. ,73,74,75α), chemokines , and additional factors such as parathyroid hormone-related protein (PTHrP) produced by stromal, immune, or tumor cells directly or indirectly stimulate osteoclastogenesis, thus accelerating the vicious cycle [Osteolysis is a hallmark of bone metastases secondary to BrCa and characterized by a massive overactivation of osteoclastic bone resorption . Osteoclus cycle ,66,76,77us cycle ,78. RANKus cycle . In lineus cycle .α [Osteoblastogenesis is not only inhibited by tumor-derived factors due to inactivation of pivotal signaling pathways or by induction of osteoblastic and osteocytic apoptosis as shown for Dickkopf-1, IL-6 and TNF-α ,82,83. Iα ,85,86,87α . Increasα . Taken tα, CCL2, CCL5) upon EMT activation and during metastatic outgrowth [α and IL-1β are strong inducers of IL-6 in tumor cells, a cytokine that in turn promotes intraosseous tumor growth, osteoclastogenesis, and osteolysis [Macrophages are central components of the innate immune system with distinct roles in inflammation and tissue homeostasis. Bone marrow resident macrophages contribute to HSC niche maintenance and bone repair . The tumutgrowth ,94,95,96utgrowth ,98. Macrteolysis .A further key function of macrophages is the engulfment and digestion of apoptotic cells, a process referred to as efferocytosis, that normally leads to production of anti-inflammatory cytokines. However, efferocytosis of tumor cells initiates NF-κB and signal transducer and activator of transcription 3 (STAT3) signaling, thus activating proinflammatory cytokine production, especially CXCL5. This chemokine, in turn, accelerates efferocytosis and PrCa development ,101. CXCObesity is tightly linked to chronic inflammation  and assoβ expression, which consequently fuels PrCa cell engraftment within the bone marrow [Adipocytes and osteoblasts within the bone marrow share common stromal progenitor cells. While the bone marrow adipocyte number increases with age, the number of osteoblasts progressively declines, implying an imbalance in differentiation between the two lineages with growing age . Moreovee marrow ,125,126.e marrow . Moreovee marrow ,123. Hene marrow .β production, which fuels BrCa tumor growth and metastasis [CAFs are heterogeneous tumor-promoting cells of the stromal part and emerging players within solid tumors and implicated in EMT, modulation of ECM, angiogenesis, tumor progression and survival, and metabolic reprogramming of tumor cells, as well as chemotherapy resistance ,128,129.tastasis . Hence, tastasis . CAFs-detastasis . These oAs previously described, different cell types are involved in cancer metastasis to bone. All of them are able to produce a broad panel of soluble mediators ,140, somα is a classical pro-inflammatory cytokine produced by activated monocytes, macrophages, CAFs, adipocytes, and tumor cells, among others [α mediates both pro- and anti-tumorigenic effects in a context-dependent fashion. However, a tumor-supporting role is believed to predominate as TNF-α has been shown to support tumor progression and metastasis, angiogenesis, and resistance to immunotherapies [α is implicated in primary stages of bone-metastatic malignancies including BrCa and PrCa, where it is often overexpressed and supports maintenance of stem-like cells [α and further mediators such as CCL2, whose expression by tumor cells and macrophages is directly stimulated by TNF-α, thus sustaining a tumor-promoting effect [α-activated mesenchymal stromal cells activate a proinflammatory chemokine signature that recruits neutrophils, which in turn stimulate the initiation of a metastatic reprogramming of tumor cells [α leads to reduced OPG production, thereby stimulating osteoclastogenesis and tumor growth. Neutralization of TNF-α suppresses osteolysis by the BrCa cell line MDA-MB-231, an effect that involves a reduction of CXCL12/CXCR4 signaling [TNF-g others . TNF-α mherapies ,110,142.ke cells . Increaske cells . Strong g effect . Moreoveor cells . TNF-α lignaling ,147.β is a key mediator within the inflammatory TME, where it is produced by several cell types including fibroblasts, adipocytes, TAM, and tumor cells [β is a member of the IL-1 family, secreted as an inactive pro-protein and cleaved by inflammasome-activated caspase-1 into a mature, biologically active form [β/IL-1R signaling induces a wide range of effects which are mediated by activation of signaling proteins such as NF-κB [β has been shown to be significantly upregulated in most primary tumors such as breast, prostate, colon, lung, head and neck cancer, and melanoma and promotes tumor cell migration, angiogenesis, immune response, and metastasis formation [β and tumor-derived IL-1β significantly correlate with tumor invasiveness and bone metastasis in BrCa and PrCa, which translates into a poor prognosis [β has been identified as a potential biomarker for predicting the risk of developing bone metastases in patients with BrCa and is an emerging therapeutic target in bone-homing malignancies [β promotes cancer metastasis to bone by increasing EMT and tumor cell invasion and migration to the bone marrow and adipose tissue [β together with TNF-α stimulated the reactivation and proliferation of tumor cells within an osteoblastic matrix [β induces lipolysis of adjacent adipocytes and the expression of metastasis-related and inflammatory factors such as matrix metalloproteinases (MMP), COX-2, CCL2, IL-6, TGF-β, and IL-8 which promote tumor growth and bone metastasis [β signaling, either genetically or pharmacologically using the neutralizing IL-1β antibody canakinumab or the IL1R antagonist Anakinra, significantly reduced experimental BrCa and PrCa bone metastases [α release, further indicating the potential of targeting IL-1β in breaking the vicious dialogue between tumor and resident bone cells [The pro-inflammatory cytokine IL-1or cells ,148. IL-ive form . IL-1β/Iormation . High lerognosis ,154,155.gnancies . IL-1β pe tissue ,158. In c matrix . Moreovetastasis ,160,161.tastases ,162,163.ne cells .β and one of its main downstream transcription factors, NF-κB, has been demonstrated to favor BrCa stem cell colonization and outgrowth within bone [α4β1 integrin upregulation, recruits osteoclast precursors, accelerates local osteoclastogenesis, and drives the progression into full-blown osteolytic macrometastases [A direct link between bone marrow-derived IL-1hin bone . Indeed,hin bone . A stronhin bone . Concernhin bone ,169,170.tastases ,171. Contastases . These oSeveral tumor cells including BrCa and PrCa cells as well as local cells of the TME such as adipocytes, macrophages, or osteoblasts are able to produce IL-6, IL-8, and IL-11. In turn, tumor cells also induce inflammatory cytokine production in osteoblasts ,87,172. The role of chemokines and chemokine receptors has gained increasing attention over the past 20 years, and CXCL12 is one of the most studied . MediatiCCL2 or MCP-1 is an inflammatory cytokine produced by osteoblasts, osteoclasts, tumor, endothelial, and stromal cells, and is further stimulated within the TMA by bone marrow adiposity, inflammation, and the presence of tumor cells or tumor-derived factors such as PTHrP ,224,225.Similar to CCL2, CCL5 is weakly expressed in healthy breast tissue, but its expression increases in BrCa, especially in advanced stages, where it is also significantly enriched in the plasma of affected patients ,244. By β and TNF-α [α [β, IL-6, and COX-2 within osteolytic lesions, respectively [COX-2 catalyzes the production of prostaglandins from arachidonic acid, is implicated in several steps of tumorigenesis, and is induced in inflamed and cancerous tissues by proinflammatory cytokines including IL-1nd TNF-α ,250. TheTNF-α [α . IncreasTNF-α [α ,108,250.TNF-α [α ,253,254.TNF-α [α . Neutralectively ,259,260.Inflammation and cancer are inseparably linked. Inflammatory mediators drive tumor initiation, progression, and metastases as well as chemoresistance at several levels, making them attractive therapeutic targets. Bone metastases secondary to BrCa and PrCa are a serious, incurable, long-term complication and associated with the risk of fracture, immobility, and pain. Currently, standard therapeutic options are limited to the prevention of SRE and pain palliation by surgery, radiotherapy, and the use of bisphosphonates and the monoclonal RANKL-antibody denosumab. Research needs to further delineate the importance of inflammatory mechanisms during specific stages of bone metastasis and to expand these investigations by characterizing the specific role of additional cellular and soluble players, such as neutrophils or osteocytes, and by revealing unidentified mutual interactions. Moreover, a precise definition of when and how these mediators contribute to bone metastases at different stages and in different cancer subtypes is necessary. This is of special interest as the therapeutic success of targeting inflammatory mediators is likely to be dependent on additional parameters such as host metabolism, environmental factors, cancer phenotype, the specific cellular composition of stromal, immune and tumor cells within the metastatic niche, and the therapeutic window.


The etiology of subacute combined degeneration (SCD) of the spinal cord is closely associated with vitamin B12 (VitB12) deficiency. The clinical manifestations of SCD are complex and vary substantially. Due to some SCD patients with atypical manifestations and concomitant autoimmune disorders, the probability of misdiagnosis and missed diagnosis is still relatively high in the early stage. We report the cases of two patients who were missed or misdiagnosed at another hospital because of the normal initial VitB12 level and partial overlap of clinical manifestations, finally diagnosed as SCD with atypical manifestations and concomitant autoimmune disorders, pharyngeal-cervical-brachial Guillain-Barre syndrome in Case 1 and SCD with autoimmune thyroiditis in Case 2. After undergoing corresponding treatment, death was reported in Case 1 and improvement in Case 2. Analysis of the clinical manifestations and investigation of the underlying pathogenesis in such patients could help improve the rate of early diagnosis and allow timely treatment of SCD, thereby preventing disease progression and poor clinical outcomes. Subacute combined degeneration (SCD) of the spinal cord is a rare disease characterized by demyelination of dorsal and lateral columns due to vitamin B12 (VitB12) deficiency. SCD with comorbidities such as autoimmune diseases is less reported, of which concomitant presence increases the difficulty in diagnosis. Here, we report two cases of SCD accompanied by autoimmune diseases.A 59-year-old man was admitted to the Department of Neurology of the Shanghai Eighth People's Hospital in November 2019 due to a 1-month history of repeated falls and urination/defecation disorders. In September 2019, he was diagnosed with lumbar disc protrusion at another hospital owing to progressive bilateral lower-limb numbness and unsteady walking for 1 year and waist pain for 6 months. His serum VitB12 level was normal and he underwent minimally invasive surgery (percutaneous endoscopic interlaminar lumbar discectomy) without improvement at the time. He had a 30-year history of drinking , and denied a history of nitrous oxide abuse or anesthesia.12/L  4.3-5.8*1012/L); hemoglobin (Hb), 82 g/L (NR 130-175 g/L); hematocrit (HCT), 23.2% (NR 37-50%); mean corpuscular volume (MCV), 114.9 fL (NR 82-100 fL); mean corpuscular hemoglobin (MCH), 42.5 pg (NR 27-34 pg); red blood cell distribution width (RDW), 62.9% (NR 11.9-14.5%); VitB12, 30 pM (NR 138-652 pM); folic acid (FA), 16.11 nM (NR 7.0-46.4 nM); homocysteine (HCY), 118 μM (NR 5-15 nM); and serum copper, 13.56 μM (NR 7.12-21.29 μM). Anti-nuclear antibody (ANA), anti-cardiolipin antibody (ACA), anti-neutrophil cytoplasmic antibody (ANCA), rheumatoid factor (RF), syphilis, HIV, and tumor markers were negative. Lumbar puncture and cerebrospinal fluid (CSF) examination yielded normal results; autoantibody and oligoclonal bands were negative.A physical examination at our hospital showed that the patient had a clear mind and multiple sites of pallor in the face, sclera, and lips. Muscle tension and strength were normal in the upper bilateral limbs and decreased in the bilateral lower limbs . Tendon reflexes were present (++) in bilateral upper limbs and absent in bilateral lower limbs. Pinprick sensation was decreased below the T6 level; vibration, motor, and position sensations were decreased in both lower limbs. The Babinski and Chaddock signs were positive on the left side. Blood tests showed: red blood cells (RBCs), 1.93×10Electromyography showed reduced conduction velocity of multiple sensory and motor nerves in both lower limbs; no evoked potentials were induced, and P40 of the left tibial nerve was not induced. Thoracic vertebral magnetic resonance image (MRI) showed long T2 signals in both dorsal and lateral columns at T5-T6, i.e., the “small character” sign . CervicaThe patient was diagnosed with SCD, megaloblastic anemia, and cervical and L5-S1 lumbar intervertebral disc protrusion. The patient gradually stopped drinking after hospitalization and received daily intramuscular injections of mecobalamine  and oral folic acid , VitB12 , and VitB6 . Following treatment, the symptoms improved and all biochemical examination results returned to normal. Intramuscular mecobalamine was administered every other day for 4 weeks and was gradually replaced with oral mecobalamine. The patient could walk with assistance and urinate/defecate normally.However, the patient was re-admitted to our department 1 month later due to worsening lower-limb weakness for 1 week. Re-examination showed VitB12 >1000 pM and positive serum anti-intrinsic factor antibody. Gastroscopy and biopsy showed type A chronic atrophic gastritis  and 3. IIn 2020, the patient was repeatedly admitted to our department for worsening of lower-limb weakness, during which VitB12 treatment was continued, and VitB12 levels were maintained normal. In March 2020, the patient was treated in another hospital for bilateral upper-limb weakness, cough, and difficulty in chewing for 3 days. However, his symptoms did not improve after intramuscular mecobalamine, citicoline sodium infusion, and oral VitB and FA.Campylobacter jejuni infection, which was treated with gentamicin. Electromyography 9 days after onset showed increased motor latency and decreased amplitude of the terminal median nerve. Reduced motor-nerve conduction velocity, decreased amplitude, and delayed F wave were seen. Increased motor latency of the terminal ulnar nerve, with normal conduction velocity, was found. Fibrillation potentials were detected in the distal upper limbs. No abnormalities were found after repeated electrical stimulations.After 4 days, he was admitted to our department. Before disease onset, he had diarrhea, which improved after 2 days of oral berberine. Physical examination showed that the patient had a clear mind, but a tender neck, feebleness, hoarseness during speaking, and difficulty in chewing, coughing, shrugging, and raising his head. The lifting of the soft palate was bilaterally poor, and the uvula was in the middle. Both upper-limb muscle tension and strength were reduced , and tendon reflexes were positive. Lower-limb muscle tension and strength were identical to the previous examination. No pathological signs were found. Routine blood tests showed normal results. Stool culture 7 days after onset showed 2O; white blood cells, 1×106/L; and total protein, 488 mg/L. Blood GQ1b and GT1a antibodies were positive, while GM1, GM2, GM3, GT1b, GD1a, and GD1b antibodies were negative. Paraneoplastic antibodies (Hu/Yo/Ri), CSF oligoclones, and AQP4 were negative.Cranial MRI showed no abnormalities. Cervical and thoracic vertebral MRI showed similar changes in the cervical vertebrae as in the previous examinations, along with T10-11 intervertebral disc bulging, thoracic vertebral degeneration, and partial recovery of the T5-T6 spinal cord lesion . Lumbar The patient was diagnosed with pharyngeal-cervical-brachial (PCB) Guillain-Barre syndrome (GBS) in SCD. Intravenous γ-globulin (0.4 g/kg) was administered for 5 days beginning 9 days after onset, and on day 12, the following changes were seen: sudden dyspnea with repeated diarrhea; finger oxygen desaturation index, 93%; heart rate, 110 bpm; decreased consciousness; and moderate coma. Oral tracheal intubation and mechanical ventilation were performed immediately. In addition, 1 g methylprednisolone was injected intravenously for 3 days, and gradually tapered until discontinuation. By day 20, the patient could open his eyes when called, had bilaterally reduced light reflexes, and stable vital signs. Anti-infection and supportive treatments were continued, with little effect. Due to the heavy financial burden, his family voluntarily gave up the treatment, and the patient passed away.A 70-year-old woman was admitted to our department with bilateral lower-limb numbness and unsteady walking for 1 month. In January 2020, she was diagnosed with anxiety disorder at another hospital, due to loss of appetite and bilateral lower-limb numbness for 3 months. She received Deanxit treatment, without improvement.At our hospital in February 2020, she reported a 1-month history of weight loss, diarrhea alternating with constipation, and multiple episodes (>10/d) of irradiative, electric shock-like pain in both lower limbs, which lasted for several seconds and then subsided. She denied a history of nitrous oxide abuse or anesthesia.12/L (NR 4.3-5.8*1012/L); Hb, 81 g/L (NR 130-175 g/L); HCT, 29.90% (NR 37-50%); MCV, 114.5 fL (NR 82-100 fL); MCH, 35.2 pg (NR 27-34 pg); RDW, 69.4% (NR 11.9-14.5%); VitB12, 61 pM (NR 138-652 pM); FA, 45.3 nM (NR 7.0-46.4 nM); serum copper, 16.31 μM (NR 7.12-21.29 μM); serum anti-intrinsic factor antibody, positive; HCY, normal; ANA, ACA, ANCA, and RF, negative; syphilis and HIV, negative.Physical examination showed normal consciousness, slow reactions, low emotions, and skin desquamation at multiple sites of the hands and feet. We found normal muscle tension and slightly reduced muscle strength in the limbs , with positive tendon reflexes . Pinprick and vibration sensations were decreased in both lower limbs. The Babinski sign was detected in both lower limbs; the Chaddock sign was present in the left lower limb. The Romberg sign was positive. Blood tests showed the following: RBCs, 2.7×10Thoracic vertebral MRI showed long T1 and T2 signals in both dorsal and lateral columns of T5-T6, i.e., the “small character” sign . ElectroThe patient was diagnosed with SCD, malignant anemia, and autoimmune thyroiditis (AITD). Daily intramuscular injection of mecobalamine (1 mg/) and oral FA, VitB12, and VitB6 were administered for 2 weeks. The VitB12 level returned to normal. Lower-limb muscle strength (grade IV+ to V-) and deep sensation improved, tendon reflexes were bilaterally positive (++), and urination/defecation were normal. The patient could stand without help. She was referred to the Endocrine Department for AITD treatment.We have reported two rare cases of SCD with atypical manifestations and complicated with autoimmune disorders (Supplementary Table S1). Wong summarized the diagnostic criteria for VitB12 deficiency (In Case 1, the normal initial VitB12 level led to initial misdiagnosis, which means VitB12 may not be a reliable marker for SCD . In addiCase 2 also had an atypical onset. Moreover, AITD was present as a comorbidity and manifested as hypothyroidism, which can induce emotional disturbance and gastrointestinal symptoms, and mask sensory manifestations in early SCD. Approximately 16% of AITD patients have concomitant malignant anemia, with parietal-cell damage, inhibition of gastric acid secretion, and gastric mucosal atrophy, leading to reduced VitB12 absorption (As shown in the two cases in this report, the role of autoimmune dysfunction in SCD has been largely overlooked . NeumannCampylobacter jejuni infection (PCB GBS in SCD is especially rare. Its clinical manifestations partially overlap with those of SCD and are easily misdiagnosed as SCD progression. In Case 1, electromyography and autoantibody tests revealed the correct diagnosis as severe PCB GBS . Among pnfection . The lownfection . This paThe comorbidity of AITD as in Case 2 is also relatively rare. This patient had diarrhea alternating with constipation, malignant anemia, and weight loss. These symptoms may be attributable to SCD-induced autonomic nerve dysfunction and overgrowth of small intestinal bacteria due to hypothyroidism, autoimmune dysfunction, or immunosuppression . The aboIn conclusion, for SCD patients with concomitant autoimmune disorders, the probability of misdiagnosis and missed diagnosis is still high, and autoimmune mechanisms can play an important role in the pathogenesis of SCD. More studies are required to investigate the underlying mechanisms. In the reported cases, we considered VitB12 deficiency to be a “result” rather than a “cause”. Patients comorbid with multiple autoimmune diseases have immune disease diathesis, and they produce a variety of autoantibodies, including anti-ganglioside antibody, anti-thyroid autoantibody, anti-intrinsic factor antibody, and others. The anti-intrinsic factor in both of our two patients was positive, which resulted in the malabsorption and deficiency of VitB12. The mechanism of autoimmune diseases as comorbidities may be related to many genetic susceptibility genes, including human leukocyte antigens type 2 ,18. Futu