Optic neuritis is (ON) is believed to be an immune-mediated disease; however, the association between optic neuritis and autoimmune diseases remains unclear. This study aimed to identify the incidence rate and adjusted hazard ratio (aHR) of autoimmune diseases in patients with optic neuritis.This nationwide, population-based, retrospective cohort study collected patients’ data between 1999 and 2013 from the National Health Insurance Research Database in Taiwan. A total of 9,235 patients were included. Using 1:4 propensity scoring, 1,847 patients were enrolled in the optic neuritis group and 7,388 in the non-optic neuritis group according to age, sex, comorbidities, and corticosteroid use. Follow-up was started from the index date and the endpoint was a diagnosis of new-onset autoimmune diseases including, myasthenia gravis (MG), psoriatic arthritis (PsA), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ankylosing spondylitis (AS).The Kaplan-Meier curves depicted that patients with optic neuritis had a higher cumulative incidence of autoimmune diseases than patients without optic neuritis. Cox proportional hazard regression showed that patients with optic neuritis were at a high risk of autoimmune diseases , including MG , SLE , and AS . Subgroup analysis provided that patients with optic neuritis aged below 65 years  or who were females  had a significantly increased risk of autoimmune diseases compared to respective controls. The use of corticosteroids reduced the risk of autoimmune diseases in patients with optic neuritis .Patients with optic neuritis presented with a high risk of autoimmune diseases such as MG, SLE, and AS, especially patients with optic neuritis who were young or females. Corticosteroids attenuated the link between optic neuritis and subsequent autoimmune diseases. Optic neuritis is a demyelinating optic neuropathy affecting one or both optic nerves. The incidence of optic neuritis ranges from 0.83 to 5.36 per 100,000 . Optic nAlthough the pathophysiologic mechanism remains ambiguous, several anatomical features may be related to optic neuritis. The limited space of the optic canal makes the optic nerve susceptible to compression when swelling. Nerves were profoundly affected by compression. Besides, the inflammatory cells can easily infiltrate through the subarachnoid space, pia, and pial mater around the optic nerve –3. MoreoSince autoimmune diseases are mainly characterized by T cell dysregulation and B cell response against self-antigens in tissues or organs  with disThis nationwide, population-based, retrospective, cohort study used data from the Longitudinal Health Insurance Database (LHID), which includes 1 million people randomly sampled from the National Health Insurance Research Database (NHIRD) to represent over 99% of 23 million population in Taiwan. Claims data from January 1, 2000 to December 31, 2012 were used as the data source. The study was approved by the Institutional Review Board of Chung Shan Medical University, Taiwan, R.O.C. (number CS15134).n = 2031). Diagnosis of optic neuritis was based on typical symptoms of the patient, including orbital pain while moving the eyes, acute onset of visual loss, decreased visual acuity, and defect of the visual field. A magnetic resonance imaging (MRI) scan was performed for patients with monosymptom or clinically isolated syndrome to confirm the diagnosis. Optic neuritis was identified as either isolated optic neuritis, optic neuritis with association of multiple sclerosis, or antibody-related optic neuritis such as NMOSD typically with aquaporin-4 antibody (AQP4-Ab) seropositivity and simultaneous immune-mediated myelitis, and MOG optic neuritis, typically with myelin oligodendrocyte glycoprotein seropositivity, bilateral optic disk edema, and markedly steroid responsive (n = 165). The follow-up period was defined as the duration from the index date to the date of a newly diagnosed autoimmune diseases, withdrawal from insurance, or December 31, 2013, whichever came first.The study population included patients with optic neuritis in LHID from January 1, 2000 to December 31, 2012 . ASD < 0.1 indicated the similarities of the characteristics in both groups. The cumulative incidences of autoimmune diseases in both groups were plotted with Kaplan–Meier methods, and a log-rank test was performed to test the significance. The hazard ratio (HR) for autoimmune diseases was evaluated by the Cox proportional hazard model. SPSS version 18.0 was used to analyze all statistical data. A In the optic neuritis group, 51.4% were women and 48.6% were men. Before propensity score matching, prominent differences were observed between two distributions, chronic liver disease, and corticosteroid use, in the optic neuritis group than the non-optic neuritis group (ASD > 0.10). However, after the propensity score had been calculated, there were no differences in the baseline characteristics and comorbidities between the two groups (ASD < 0.10) .p = 0.02)  and 3.24 (95% confidence C.I.: 2.81–3.74) per 1,000 person-years in patients with and without optic neuritis, respectively. Poisson regression showed that patients with optic neuritis had a higher relative risk  for autoimmune diseases compared to patients without optic neuritis . Further = 0.02) .The Cox proportional hazard regression illustrated that patients with optic neuritis had a higher risk for autoimmune diseases than patients without optic neuritis . Generally, females had a higher risk of developing autoimmune diseases compared to males . Patients aged between 40 and 64 years  and patients with comorbid chronic liver disease  were at a significantly high risk of autoimmune diseases .Subgroup analysis revealed the subpopulations of patients with optic neuritis that were more susceptible to autoimmune diseases, in which patients with optic neuritis patients aged below 65 years  or who were females  were prone to autoimmune diseases. Moreover, the use of corticosteroids had a protective effect on autoimmune diseases in patients with optic neuritis  . Sub-outOptic neuritis has been recently considered as an immune-mediated diseases with speculated autoimmune pathogenesis, as evidenced by studies providing that seropositive autoantibodies subtypes including myelin oligodendrocyte glycoprotein antibody (MOG-Ab) and seropositive aquaporin-4 antibody (AQP4-Ab)  typicallAs for the associations between optic neuritis and autoimmune diseases, cohort study in the United Kingdom  of 10,93The estimated incidence rate of SLE varies from 1 to 25 per 100,000 person-years in North America, South America, Europe, and Asia with a predilection for Asians and Africans –30. In oPredictor variables of autoimmune diseases included age, sex, and chronic liver diseases. Particularly, SLE is diagnosed at a mean age of 35 years, and RA typically occurs in middle-aged individuals –34. On tIt is believed that optic neuritis is an immune-mediated disease triggered by inflammation and causes axon demyelinating injury. Optic nerve degeneration and visual loss are both pathological changes of optic neuritis . ActivatThere are several strengths in our study. First, this is a nationwide population-based study, in which a large sample size was , 43–45 qIn conclusion, this cohort study elaborated on prominent risk of autoimmune disease in patients with optic neuritis, especially patients with optic neuritis who were young or females. Notably, the use of corticosteroids attenuated the link between optic neuritis and autoimmune diseases. Clinicians should consider this association as a guide in managing patients with optic neuritis. Further investigations with a longer follow-up period are necessary to ensure the relationship between optic neuritis and autoimmune diseases.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.This study was approved by the Institutional Review Board of Chung Shan Medical University (number CS15134). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.KM, C-ML, WZ, and P-HC participated in the research design and writing of the manuscript. KM, YY, and YD collected the data and wrote the original draft. KM and Y-HW participated in data collection and statistical analysis, and JW was a project administrator and revised the manuscript. All authors contributed to the study and have 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.


The system was catalyzed by Pt@Ag NFs to change the color of 3,3′,5,5′-tetramethylbenzidine (TMB) from colorless to blue, and the morphology and chemical properties of Pt@Ag NFs were characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. Under the optimized conditions, a linear relationship between the absorbance and UA concentration was in the range of 0.5–150 μM (R2 = 0.995) with a limit of detection of 0.3 μM (S/N = 3). The method can be applied to detection of UA in actual samples with satisfactory results. The proposed assay was successfully applied to the detection of UA in human serum with recoveries over 96.8%. Thus, these results imply that the UA assay provides an effective tool in fast clinical analysis of gout.A ultrasensitive assay for the determination of uric acid (UA) based on Pt@Ag nanoflowers (Pt@Ag NFs) was constructed. H A ultrasensitive assay for the determination of uric acid (UA) based on Pt@Ag nanoflowers (Pt@Ag NFs) was constructed. Many diseases are caused by diet and lifestyle, so as a remedy, all asymptomatic gout patients should be encouraged to change their lifestyle. This requires the development of a suitable cost-effective monitoring system to provide adequate feedback and treatment guidelines for gout patients.Uric acid (UA) is the final product of human sputum metabolism,7 There is a direct positive correlation between UA levels and the future risk of gout. Specifically, as the concentration of UA increases, the risk of crystal formation increases, thereby increasing the patient's susceptibility to gout.8Gout is an inflammatory reaction that occurs primarily in the joints and forms UA crystals that cause pain.9 fluorometry,10,11 chromatography,12 electrochemical methods,13,14 chemiluminescence15 and colorimetry.16 Furthermore, the colorimetric method is widely used because of its simple operation, fast analysis speed, high sensitivity in these methods. Currently, there are two main colorimetric methods for the detection of UA. One is a colorimetric assay based on precious metal  nanomaterials, in which uric acid can induce nanoparticle aggregation,17 etching and anti-etching18,19 to cause color changes. However, the dispersion of the nanomaterial is unstable and precipitates easily, which causes the color of the solution to change or even disappear. Many external factors also may cause undesirable changes in the state of nanomaterials,20 resulting in unsatisfactory results. The other one is the catalytic oxidation strategy, which bases on the peroxidase-like properties of nanomaterials, such as porous metal–organic framework MIL-53 (Fe),21 graphite carbonitride nanosheets22 and cobalt selenide nanosheets.23 They can change color by H2O2 catalytic peroxidase substrate. The peroxidase mimic enzymes have the advantages of low cost, good stability, simple preparation, controllable structure and composition, and adjustable catalytic activity. Therefore, this kind of nanomaterials have become an ideal and important colorimetric detection tool.There are many approaches to detect uric acid, such as, capillary electrophoresis,24,25 More importantly, the bimetallic nanostructures have synergistic and controllable catalytic properties compared with their mono-metallic nanostructures.26 So far, nanomaterials of various shapes have been synthesized by chemical methods, such as nanowires,27 nanotubes,28 nanorods29 and nanowires.30 Platinum nanomaterials have attracted much attention due to their excellent catalytic ability and high utilization rate, and have been applied to many fields such as catalysis, battery, sensing and medical treatment.31–34 Silver nanoflowers have large specific surface area and good electrical conductivity, especially peroxidase-like properties, which provides an excellent substrate for the construction of the assay.35–37 A new type of Pt@Ag nanoflower (Pt@Ag NFs) was prepared by adhesion of platinum nanoparticles with high catalytic activity on the surface of silver nanoflowers to further improve the ability of reducing H2O2.Precious metal nanomaterials have a wide range of applications in catalysis and assays due to their unique physical and chemical properties.2O2 , resulting in a solution color changing from colorless to blue. The color change of the solution depends indirectly on the amount of uric acid and can be judged by an ultraviolet-visible spectrophotometer and the naked eye.This paper presents a method for preparing Pt@Ag NFs using bovine serum albumin as raw material. A colorimetric assay using the peroxidase-like activity based on Pt@Ag NFs was developed to detect UA with sensitivity and high selectivity. Pt@Ag NFs catalyze the colorimetric system of TMB and H3), chloroplatinic acid (H2PtCl6·6H2O), bovine serum albumin (BSA), ascorbic acid (AA), 3,3′,5,5′-tetramethylbenzidine (TMB), cysteine (Cys) and glucose (Glu) were purchased in Sinopharm Chemical Reagent Co., Ltd. .Silver nitrate (AgNO2O2), KNO3, Mg (NO3)2, NaCl, CaCl2 were obtained from Beijing Chemical Industry Co., Ltd. . All reagents are analytical grade and do not require further treatment. Experimental water was ultrapure water .Uric acid (UA), uricase, dopamine (DA), and glutathione (GSH) were obtained from Sigma-Aldrich . Urea, hydrogen peroxide . The morphology of the Pt@Ag NFs were characterized by transmission electron microscopy . X-ray photoelectron spectroscopy (XPS) was carried out on EscaLab 250Xi with Al K α X-ray radiation as the source for excitation. Biochemical analysis was measured using biochemical analyzer .37 Briefly, BSA  and AgNO3  aqueous solution were mixed and stirred at room temperature for 10 min, then AA  was added into the above solution drop by drop. The color of the solution turned light grey, indicating that Ag NFs had been successfully formed. H2PtCl6·6H2O  was then mixed with the Ag NFs solution and the mixture was stirred at room temperature for a further 10 min until the color turned dark grey. This indicates that Pt@Ag NFs was successfully synthesized, and the obtained product was repeatedly washed three times with water and ethanol, respectively. Finally, the precipitate was re-dissolved in 20 mL of ultrapure water for later use.The innovative synthesis of Pt@Ag NFs is the key step to this experiment.−1 uricase and 50 μL different concentrations of UA in a 35 °C water bath for 15 min. Then 200 μL, 16 mM TMB, 30 μL Pt@Ag NFs and 1520 μL, 50 mM HAc–NaAc buffer of pH 4.0 were added to the above solution. Shake well to bring the mixed solution into full contact, and incubate for another 40 min in a 55 °C water bath, and collect the data using an UV-vis spectrometer.150 μL of Britton–Robinson buffer  was mixed with 50 μL, 100 μg mLThe serum samples of gout patients were collected from the Nanchang University Fourth Affiliated Hospital . The samples treatment was relatively simple. The samples were treated with 4000 rpm centrifugation for 15 min, and the supernatant diluted with phosphate buffer saline . The analysis of each serum sample was repeated three times.2O2. Pt@Ag NFs have peroxidase-like activity, which further catalyzes the decomposition of H2O2 to form hydroxyl radicals (˙OH). ˙OH oxidizes the peroxidase substrate TMB (colorless) to oxidized TMB . These two steps can be expressed by 2O2 were compared. As shown in 2O2 was mixed with TMB and Pt@Ag NFs (dark blue curve), a similar phenomenon can be observed, and the absorbance increases. This indicates that Pt@Ag NFs catalyzes H2O2 and TMB in the same way as UA, uricase and TMB. The results of the whole experiment demonstrated that UA was first catalyzed by uricase to produce H2O2, and then further catalyzed H2O2 by Pt@Ag NFs, and the color of reaction substrate TMB changed. Therefore, it can be seen that there is a significant color change from colorless to blue in In order to verify the principle of the colorimetric assay, the UV-vis spectra of TMB and the mixture of UA, uricase, Pt@Ag NFs and H38–407/2, Pt 4f5/2, Ag 3d5/2, and Ag 3d3/2, respectively, verifying the formation of Pt and Ag.Pt@Ag NFs play a crucial role in the detection of colorimetric uric acid assays. The structure and morphology of Pt@Ag NFs were studied by transmission electron microscopy. A − A0)/A . Next, the catalytic activity of Pt@Ag NFs catalyst was optimized to obtain the best sensing response of UA assay. These parameters include pH, incubation temperature, incubation time, Pt@Ag NFs volume and TMB concentration. As can be seen from It is known from previous literature that the optimal pH, incubation temperature and incubation time for uricase-catalyzed UA reaction are 8.5, 35 °C and 15 min, respectively. Relative activity was used to judge the optimal value of reaction parameters, which can be represented by (A and UA concentration (0.5–150 μM) and the limit of detection (LOD) was 0.3 μM (S/N = 3). The corresponding linear equation is expressed as: ΔA = 0.0051C + 0.0453 (μM) and the correlation coefficient (R2) is 0.995. For comparison, In the analysis and detection, the sensitivity of the biological enzyme assay is one of the most important factors. In order to verify the sensitivity of Pt@Ag NFs assay for detecting UA, a series of concentrations of UA standard solutions were detected under optimal conditions. With the increase of UA concentration (0.5–150 μM), the absorbance of the solution at 652 nm also increases . MoreoveTo verify the selectivity of the assay in detecting uric acid, we tested several potential interfering substances in human serum, including cysteine, ascorbic acid, glucose, and several inorganic ions. Under the optimum conditions, the following experiments have been carried out. One is the signal of only UA and interfering substances mixed the detection system (n = 3) of 5 samples is less than 2.6%. These results show that colorimetric assay for the detection of UA has great practical value in clinical application.In order to study the feasibility of UA assay, we used the proposed method and biochemistry analyzer to test the serum samples of 5 gout patients. The serum sample was centrifuged at 4000 rpm for 3 min. Then, 5.6 μL serum mixed with uricase, 4-aminoantipyrine (4-AAP), and 3,5-diphenylamine disodium salt (MADB). And the samples were put in the biochemical analyzer and measure at 660/800 nm. The measurement values of the two methods are almost identical , and theIn conclusion, a new assay based on Pt@Ag NFs was constructed to detect UA by the simple colorimetric method. UV-vis spectroscopy showed that Pt@Ag NFs had high sensitivity with the limit of detection for 0.3 μM and high selectivity in the detection of UA. The proposed assay was successfully applied to the detection of UA in human serum with the recoveries in the range of 96.8–103.3%, which has the potential of the simple and fast UA detection platform for point-of-care diagnostics.The study was approved by the ethics committee of Jiangxi Medical College, and their guidelines were followed throughout this study. The serum samples involved in our research were from gout individuals. Informed consent was obtained from all human participants.The authors declare that they have no competing interests.


Reproductive failure is a major economical drain on cow-calf operations across the globe. It can occur in both males and females and stem from prenatal and postnatal influences. Therefore, the cattle industry has been making efforts to improve fertility and the pregnancy rate in cattle herds as an attempt to maintain sustainability and profitability of cattle production. Despite the advancements made in genetic selection, nutrition, and the implementation of various reproductive technologies, fertility rates have not significantly improved in the past 50 years. This signifies a missing factor or factors in current reproductive management practices that influence successful fertilization and pregnancy. Emerging lines of evidence derived from human and other animals including cattle suggest that the microbial continuum along the male and female reproductive tracts are associated with male and female fertility—that is, fertilization, implantation, and pregnancy success—highlighting the potential for harnessing the male and female reproductive microbiome to improve fertility in cattle. The objective of this narrative review is to provide an overview of the recent studies on the bovine seminal and vagino-uterine microbiome and discuss individual and interactive roles of these microbial communities in defining cattle fertility. One of the most fundamental aspects of successful and profitable beef and dairy cattle operations is maintaining a strong reproductive efficiency within the herd. Advancements in reproductive technology that have been made in the past few decades have been profound, providing producers with Expected Progeny Differences (EPDs) to assist with genetic selection, estrous synchronization to pair with artificial insemination, and even cryopreservation of gametes from genetically superior animals . In addiin utero microbial colonization  residing within the male and female reproductive tracts has been less characterized as a variable that may influence reproductive efficiency in cattle. Emerging evidence derived from human and vertebrate animals suggest that the reproductive microbiome is important for reproductive health and fertility . The assnization , we alsovia culture-independent, high-throughput sequencing  male fertility. In addition, Koedooder and colleagues conducted a narrative review of the microbiota composition of both the male and female reproductive tracts  or unsatisfactory (n = 13) after breeding soundness exams (BSE) and sperm analysis  as well as post-breeding (n = 12). Overall, our results indicate that there is a rich and complex microbiota present in yearling beef bulls. Fusobacteriota (36.3%), Bacteroidota (30.4%), Firmicutes (17.1%), and Actinobacteriota (14.9%) were identified as the major phyla. Our qPCR results showed that the overall mean total bacterial concentrations in the semen samples collected at d 112 and post-breeding was estimated to be about 1.1 billion 16S rRNA gene copies per ml semen  and Proteobacteria (23%) are the most predominant phyla constituting the bovine cervical microbiota of healthy cattle ; Table 2iversity .Fusobacterium and Porphyromonas spp. are abundant within the uterine microbial community along with Bacteroides, Streptococcus, and Bacillus genera  hematogenous spread of microbes from the gut and oral microbiota; (2) natural ascension of vaginal bacteria to the upper genital tract through the cervix or the introduction of the vaginal bacteria into the uterus via assisted reproductive technology-related procedures; (3) transmission of bacteria via the seminal microbiota  Discovering the origin of seminal microbiota and factors influencing it; (2) Determining if the seminal microbiota influences sperm metabolism, motility, or morphology; (3) Elucidating the interaction of the semen microbiota with the female reproductive tract and its effects on fertility; (4) Identifying the core seminal microbiota present in healthy individuals; and (5) Further characterization of factors affecting the uterine microbiome and other reproductive microbiomes  and their effects on fertility and fetal development. The long-term goal of discovering the role of the reproductive microbiome is to aid in the development of microbiome-targeted strategies such as next-generation probiotics and a synthetic microbial community that can be used along with assisted reproductive technologies to enhance fertility and improve pregnancy in cattle herds. When the claimed role of temporary combined male and female microbiome in fertilization and subsequent embryo development is further supported by in vitro and in vivo studies, then, the first logical step would be to revisit the current artificial insemination (AI) practice. Bovine semen straws used for AI are stored with antibiotics, which are intended to eliminate all viable bacterial cells in the sperm and seminal fluid. Thus, a new way of storing semen straws without antibiotics might be needed as this will enable the semen associated commensal microbiota to be deposited into the uterus and may ultimately improve pregnancy rates relating to AI. Next, future research should be directed to develop synthetic semino-uterine microbial consortium (SSUMC) based on the seminal and uterine bacterial species that are associated with positive bovine sperm quality and enhanced fertilization. In-utero inoculation of the SSUMC before or along with AI may enable microbiome-mediated fertilization and pregnancy outcome improvement. Current bovine AI practice with a rigid quality and microbial contamination control protocol, established use of liquid nitrogen for storage, and mechanisms of intrauterine placement is well suited to store and deliver synthetic microbial consortium into the female reproductive tract in cattle.Although the taxonomic composition of the microbial communities residing within the male and female reproductive tract has been characterized in cattle, the functional features of these microbiomes and their role in defining the reproductive health and male and female fertility are yet to be elucidated. Therefore, there is a need to explore the reproductive microbiome using more targeted, and mechanistic approaches including shotgun metagenomic sequencing, metabolomics, and In terms of the reproductive microbiome in female cattle, many fundamental questions regarding the vagino-uterine microbiome and its interaction with the seminal microbiota, its involvement in fertility, pregnancy, and developmental programming remain to be understood. To harness the female reproductive microbiome-mediated fertility, future studies should focus on the following areas: (1) Identification of the healthy vaginal and uterine microbiota associated with a positive pregnancy outcome; (2) Understanding of the role of vagino-uterine microbiota in maintaining reproductive health and fertility; (3) Characterizing the interaction of vagino-uterine microbiome with seminal microbiome and its involvement in shaping the respective male and female urogenital microbiota; and (4) Defining the impact of the maternal reproductive microbiota during pregnancy on fetal programming and early life microbiome development.Given that female fertility and pregnancy outcomes may be influenced by the seminal and uterine microbiome, and interactions between the two microbial communities, it is necessary to apply a holistic approach and consider both male and female reproductive microbiome in future research aiming to develop microbiome-targeted strategies for improved fertility in cattle.in utero fetal microbial colonization. Thus, the microbiome of the male and female reproductive tract holds tremendous potential to improve bovine reproductive health, fertility, and offspring development. Developing strategies to improve fertility and reproductive efficiency in beef cattle by harnessing the seminal and vagino-uterine microbiome and their interaction has important implications for the modern cattle industry in an era defined by increasing scrutiny of antimicrobial use, and genetic selection and management refinement may have reached a plateau.The microbial communities residing within the male and female reproductive tract are important in reproductive health in cattle. Female fertility and pregnancy outcomes may be influenced not only by its own reproductive tract microbiota but also by the semen-associated microbes that enter the uterus, and the interactions between the seminal and uterine microbiome. In addition to influencing reproductive performance, the impact of the female reproductive tract microbiome seems to extend beyond the pre-conception and early embryonic development stages and may have paramount impact on fetal and offspring development and health, considering the involvement of microbiome in DOHaD and existence of SL and SA: conceptualizing and outlining the review. SL and EW: conducted the literature search. SL and SA: drafting the initial manuscript. SL, EW, CD, LR, and SA: manuscript writing, revision, editing, and finalizing. All authors contributed to the article and approved the submitted version.The work presented in this study was financially supported in part by the North Dakota Agricultural Experiment Station as part of a start-up package for SA.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.


Despite exciting advances in organic chemistry, access to these skeletons is still highly challenging. Using synthetic biology to engineer microbes provides an innovative alternative to bypass synthetic challenges. In this study, we constructed two truncated artificial pathways to efficiently produce terpentetriene and ent-kaurene, two representative clerodane and ent-kaurane diterpenes, in Escherichia coli. Both pathways depend on the exogenous addition of isoprenoid alcohol to reinforce the supply of IPP and DMAPP via two sequential phosphorylation reactions. Optimization of these constructs provided terpentetriene and ent-kaurene titers of 66 ± 4 mg/L and 113 ± 7 mg/L, respectively, in shake-flask fermentation. The truncated pathways to overproduce clerodane and ent-kaurane skeletons outlined here may provide an attractive route to prepare other privileged diterpene scaffolds.The clerodane and  Diterpenoids, of which there are over 34,000 members (http://terokit.qmclab.com), have attracted great attention from chemists and biologists due to their intriguing chemical structures and broad pharmacological functions –4. The v5 isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced in the cell via one of two pathways: i) the mevalonate (MVA) pathway includes seven steps from acetyl-CoA (A-CoA); and ii) the 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) pathway includes eight steps starting from the condensation of pyruvate and ᴅ-glyceraldehyde 3-phosphate (G3P) [cis-abienol, and prenylated tryptophan [Engineering microbes via synthetic biology provides new opportunities to produce terpenoid carbon skeletons. All terpenoids are derived from the minimum Cte (G3P) –14  –18. Howete (G3P) –18. Recete (G3P) ,19. In tte (G3P) . Notablyyptophan ,19,22–23ent-kaurane diterpenoids are two categories of diterpenoids that are widely distributed in terrestrial plants, fungi, and a few bacteria and possess broad pharmacological bioactivities [ent-kaurane diterpenoids. In this paper, we report the reconstruction of truncated artificial pathways to overproduce two representative clerodane and ent-kaurane diterpenes, terpentetriene and ent-kaurene, in E. coli. The titers of terpentetriene and ent-kaurene were optimized to 66 ± 4 mg/L and 113 ± 7 mg/L, respectively, in shake-flask fermentation.The clerodane and tivities –3. Repretivities –27. How phoN and ipk from Shigella flexneri and Thermoplasma acidophilum, respectively, were codon-optimized and synthesized for overexpression in E. coli [E. coli, which balances IPP and DMAPP in vivo, was also included in our construct. To initially test the efficiency of this two-step artificial pathway, we constructed strains DL10001  and DL10002  [ent-kaurane diterpenes in E. coli.Following the Williams design,  E. coli . Isopentand idi) . Compareent-kaurene are labdane-related diterpenes and biosynthetically constructed by two sequential DTSs from the common C20 linear allylic diphosphate GGDP [Kitasatospora griseolosporeus MF730-N6 in 1985 [syn-labda-13-en-8-yl+ diphosphate intermediate  after a survey of the existing genome sequence databases, however, we discovered a strain, Kitasatosporia griseola DSM 43859, without a genome sequence disclosed, from the strain library of CGMCC. Using the primers designed from the sequences of cyc1 and cyc2, we fortunately obtained two genes of expected lengths, which we named tdps and ttes, by polymerase chain reaction (PCR). The sequencing results of tdps and ttes showed extremely high identities with those of cyc1 and cyc2   for a truncated artificial pathway to produce the clerodane diterpene, terpentetriene, were fully collected.Terpentetriene and ate GGDP . Terpent in 1985 ,30. In trmediate , which, ent-kaurene resembles that of terpentetriene. First, a class II DTS catalyzes the cyclization of GGDP into a diphosphate intermediate, ent-copalyl diphosphate (ent-CPP). Next, a class I DTS further cyclizes ent-CPP into the target tetracyclic skeleton, ent-kaurene (ent-CPP synthase (eCDPS) gene was cloned from Streptomyces sp. NRRL S-1813, which was an alternative ent-kaurenol-derived antibiotic platensimycin producer [ent-kaurene synthase (BjKS) gene was from Bradyrhizobium japonicum, a bacterial symbiont of soybean that is known to produce the ent-kaurene-derived phytohormone gibberellin [bjks gene was codon-optimized and synthesized for overexpression in E. coli. We used the same ggdps from K. griseola DSM 43859 in this construct. Thus, the full pathway to ent-kaurene, possessing six genes , was completed.The biosynthetic pathway towards -kaurene . In thisproducer –35, whilberellin –37. The ent-kaurene from ISO and DMAA  leading to the production of terpentetriene, each with a strong and inducible T7 promoter, were cloned into pETDuet-1 plasmid to form pLD10010, which was transformed into E. coli BL21 (DE3) to create strain DL10003. In another expression system, the upstream two genes, phoN and ipk, were cloned into pETDuet-1 plasmid, while the other four genes  were cloned into pRSFDuet-1 plasmid. Both plasmids were co-transformed into E. coli BL21 (DE3) to create strain DL10004. Using the same protocol, another two strains, DL10005 with a single plasmid expression system and DL10006 with a two-plasmid expression system, that included the whole truncated artificial pathway  for ent-kaurene production were created.After collecting all the essential genes, we initially designed two different expression systems for producing terpentetriene and and DMAA . To decrE. coli BL21 (DE3), all strains successfully produced new peaks in the HPLC profiles after a 3-day fermentation. Larger scale (3 L) fermentations of DL10004 and DL10006 led to the isolation of 45 mg and 90 mg of terpentetriene and ent-kaurene, respectively, whose 1H and 13C NMR spectra supported their chemical structures  of terpentetriene and (3-fold) of ent-kaurene than single plasmid expression system . DL10004 and DL10006 were therefore selected for subsequent fermentation optimization.Next, DL10003–10006 were fermented in Lysogeny broth (LB) medium supplied with 1% glycerol, 6 mM ISO/DMAA 3:1, and 0.1 mM isopropyl β-ᴅ-1-thiogalactopyranoside (IPTG) inducer. In comparison with the negative control of wild-type ent-kaurene, a series of feeding experiments was carried out with various concentrations of ISO/DMAA. When strains DL10004 and DL10006 that were cultured in LB medium reached an OD600 of 0.6, 0.1 mM IPTG and different amounts of ISO/DMAA were added. After 3-day fermentations in the absence of ISO/DMAA, both strains only produced small amounts of terpentetriene and ent-kaurene , suggesting a low expression level of the endogenous MEP pathway in E. coli  and ent-kaurene (53 ± 2 mg/L) were observed in the presence of 25 and 10 mM of DMAA, respectively. These results demonstrated that i) the introduced two-step artificial pathway could efficiently convert exogenous supplemented ISO and DMAA into a pool of hemiterpenes; and ii) IDI effectively balances the proportion of IPP and DMAPP. Given that the commercial DMAA is cheaper than ISO, our result in merely using DMAA should be helpful in decreasing the overall cost for the production of other terpenes using this two-step artificial pathway [To determine the ideal concentrations of exogenous supplementary ISO/DMAA for producing terpentetriene and  E. coli . When IS pathway .ent-kaurene production. Given that the carbon source is vital for overproducing natural products in E. coli, and glycerol is one of the most frequently used carbon sources, we first tested for optimal glycerol concentrations for terpentetriene and ent-kaurene production. Strains DL10004 and DL10006 were cultured in 50 mL LB medium and supplied with 0%, 1%, 2%, 5%, and 10% (v/v) glycerol, respectively. As showed in ent-kaurene in DL10006. The production of terpentetriene and ent-kaurene, however, decreased significantly when more glycerol (5% or 10% (v/v)) was added, suggesting that higher glycerol concentrations might be harmful for the host cell, which was also supported by the less cell pellets harvested.Three orthogonal experiments were run to examine the effects of varying glycerol and IPTG concentrations and fermentation time, all in an effort to optimize terpentetriene and ent-kaurene. As shown in ent-kaurene when 0.5 mM IPTG was added, no significant decrease of the ent-kaurene yields under the other two IPTG concentrations (72 ± 4 mg/L for 0.1 mM and 71 ± 2 mg/L for 0.25 mM). From an economic point of view, 0.1 mM IPTG was therefore selected as the optimal concentration for both strains.We next explored optimal IPTG concentrations  on the production of terpentetriene and E. coli, we set up a maximum 7-day experimental timeline for determining the titers of terpentetriene and ent-kaurene. As shown in ent-kaurene was gradually increased until day 5 before decreasing to day 7. At day 5, the titer reached 113 ± 7 mg/L for ent-kaurene. Taken together, the optimal fermentation conditions of both terpentetriene and ent-kaurene were fully established.Finally, we tested the fermentation time course of DL10004 and DL10006. Based on the above established fermentation conditions, including the ratios of ISO/DMAA and the concentrations of glycerol and IPTG, we performed a sequence of parallel assays to determine the best fermentation length. Considering the inherent growth feature of ent-kaurane diterpenes, terpentetriene and ent-kaurene, in E. coli by exogenously feeding ISO/DMAA to reinforce the supply of IPP and DMAPP. We optimized the ratio of ISO/DMAA, concentrations of glycerol and IPTG, and fermentation time to enhance the production of terpentetriene and ent-kaurene. As a result, strain DL10004, an engineered terpentetriene producer with a two-plasmid expression system, reached the titer of 66 ± 4 mg/L under the optimal conditions of supplementary 25 mM DMAA, 1% glycerol, and 0.1 mM IPTG for a 7-day shake-flask fermentation in LB medium. Strain DL0006, an engineered ent-kaurene producer with a two-plasmid expression system, reached the titer of 113 ± 7 mg/L under the optimal conditions of supplementary 10 mM DMAA, 2% glycerol, and 0.1 mM IPTG for a 5-day shake-flask fermentation in LB medium. Compared with the reported optimal combination of efflux pumps for ent-kaurene production [ent-kaurane carbon skeletons but also offers a blueprint for coupling with emerging chemoenzymatic strategies and biocatalysis in preparation of high value diterpenoid natural products.In this study, using the truncated artificial pathways, we overproduced two clerodane and oduction , the titFile 1Experimental part and supplementary figures and tables.


Delayed cancer diagnosis and inefficient cancer prognosis determination are problems faced in cancer diagnosis and treatment. MicroRNAs (miRs), especially miR-212, have shown a promise in cancer diagnosis and prognosis. Herein, we performed a systematic review and meta-analysis to assess the prognostic and diagnostic value of miR-212 level in cancer and evaluated its association with patient characteristics.A fully electronic literature search using related keywords was performed in PubMed, Scopus, Web of Science, Embase, and ScienceDirect databases by June 6, 2021, with no time or language restriction. Meta-analysis was performed to pool survival prognosis data using hazard ratio (HR), association using odds ratio (OR), and diagnostic data using sensitivity, specificity, and diagnostic odds ratio (DOR). Sub-group analysis and meta-regression were performed as appropriate.Results of 28 studies on 1880 patients showed a poor cancer prognosis with high levels of miR-212 in pancreatic ductal adenocarcinoma , and a poor cancer prognosis with low levels of miR-212 in other cancers (HR = 2.514 [2.162–2.923]). Higher alpha-fetoprotein (AFP) level and Edmondson-Steiner grade were factors associated with miR-212 low level incidence. Diagnostic odds ratio 10.688 (3.644–31.348) and SROC AUC of 0.84 confirmed high diagnostic performance of miR-212.Our systematic review and meta-analysis results confirm miR-212 high value in cancer prognosis and diagnosis. High level of miR-212 showed poor prognosis in PDAC and low level of miR-212 showed poor prognosis in other cancers. in conclusion, miR-212 could be a novel potential biomarker in cancer diagnosis and prognosis.The online version contains supplementary material available at 10.1186/s12935-022-02584-0. Cancer is a leading cause of death by causing about 10 million deaths in 2020 worldwide [MicroRNAs (miRs) are a main member of small non-coding mRNAs; they act as modulatory tools that work post-transcriptionally to regulate mRNA translation by attaching to the 3'-untranslated region . GeneticA systematic review and meta-analysis were performed and reported according to The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement .Without any time or language restriction, we searched Pubmed, Scopus, Web of Science, Embase, and ScienceDirect on June 6, 2021, using Medical Subject Headings (MeSH), entry terms, and related keywords to miR-212 and cancer. The search was ameliorated by hand search in google scholar and references of included studies. The detailed search strategy is provided in Additional file Original human research papers were included in this study. For prognostic evaluation papers providing hazard ratio (HR) or enough data to estimate HR of cancer death comparing patients with low and high levels of miR-212 were included, for patient characteristics association with miR-212 level evaluation papers providing odds ratio (OR) or enough data to calculate OR of patient characteristics comparing patients with low and high level of miR-212 were included, and for diagnostic evaluation papers providing numbers of true and false positive and negative samples using miR-212 level as determinant or enough data to estimate these were included in this study.First author name, publication year, country of study, type of assessed miR-212, cancer type, tumor stage, Specimen, and miR-212 assay method were extracted from all studies. For articles included in the prognostic evaluation, crude and adjusted HRs were extracted, and in case of no reported HR, it was extracted from the Kaplan–Meier curve using the Guyot method . For assQuality assessment was performed using the Newcastle–Ottawa scale (NOS) for the assessment of the quality of nonrandomized studies in meta-analyses , 10 for 2 and p-value [2 < 50 and p-value > 0.05 fixed-effects model was used to pool data; in other conditions, random-effects model was applied. In order to solve observed inconsistency in the included studies email was sent to the corresponding author.Cochran’s Q test and Higgins’s I p-value , 13 wereFor prognostic evaluation, Comprehensive Meta Analysis software  was used to calculate pooled HR. For studies not reporting HR, we digitized the Kaplan–Meier curve using Web Plot Digitizer . For oveFor association evaluation of patient characteristics and low miR-212 level incidence, OR calculation and pooling from the number of patients based on characteristics in low miR-212 and high miR-212 was performed using Comprehensive Meta-Analysis software.®) software  was used to pool the studies [For diagnostic evaluation, the numbers of true positive, false positive, false negative and true negative were extracted from articles or calculated using article-provided sensitivity and specificity. To lower the heterogenicity and threshold effect induced by various optimal cut-off determination methods used in different studies, sensitivity and specificity and number of true and false positive and negative were also extracted based on the numbers obtained by Web Plot Digitizer from ROC curves utilizing two methods: higher Youden Index , which i studies . Pooled  studies . No signPerformed search provided 874 records, including 173 articles from Pubmed, 295 from Scopus, 187 from Web of Science, 192 from Embase, and 27 from Science Direct. After the removal of duplicate records, 521 articles were left. Title and abstract of articles were screened, resulting in the exclusion of 470 studies. The full textes of 51 remaining articles were assessed resulting in exclusion of 21 articles . Ultimately 28 studies were included in the systematic review and meta-analysis.Totally 1880 patients from 28 studies were included in this systematic review and meta-analysis. Pooled analysis of prognostic dimension of miR-212 on cancer survival was performed on 1479 patients from 18 studies, pooled analysis of the association between low miR-212 level incidence and patient characteristics were performed on 1438 patients from 19 studies, and pooled analysis of the diagnostic value of miR-212 in cancer diagnosis was performed on 416 patients from 8 studies.Studies included in the prognostic evaluation Fig. 2 = 68.884 was investigated through sub-group analysis  for PDAC and 2.514  for other cancers , specificity 0.790 0.748–0.827), positive likelihood ratio 3.249 (1.849–5.709), negative likelihood ratio 0.378 (0.226–0.631), and diagnostic odds ratio 10.688 (3.644–31.348) and SROC AUC of 0.84 confirming high diagnostic performance of miR-212  in predicting OS. To eliminate heterogeneity, we used subgroup analysis which detected PDAC studies as the heterogeneity source. Subgroup analysis illustrated a poorer prognosis of low miR-212 levels in cancers other than PDAC (HR = 2.514); however, PDAC was an exception (HR = 0.408). But why a specific miR is increased in one cancer and decreased in another? It should be noted that one miR potentially can interact with 200 genes [Our prognostic assessment showed that the HR of low levels of miR-212 to its high levels was 2.081  and assessing them in specific biofluids  can remarkably increase clinical diagnostic accuracy , 27. CotThe present review has some limitations: all prognostic articles were from China; thus, more studies are needed in other races to generalize the reported findings. Also, miR-212 diagnostic accuracy and prognostic value differ by type of cancer and even cancer subtypes.In conclusion, miR-212 can help us diagnose cancers with a prolonged latency period and determine cancer prognosis.Additional file 1: Detailed search strategy.Additional file 2:Table S1, S2 and Figure S1–S43.


Intracellular signaling pathways are essential to establish embryonic patterning, including embryonic axis formation. Ectodermal patterning is also governed by a series of morphogens. Four ectodermal regions are thought to be controlled by morphogen gradients, but some perturbations are expected to occur during dynamic morphogenetic movement. Therefore, a mechanism to define areas precisely and reproducibly in embryos, including feedback regulation of signaling pathways, is necessary. In this review, we outline ectoderm pattern formation and signaling pathways involved in the establishment of the pre-placodal ectoderm (PPE). We also provide an example of feedback regulation of signaling pathways for robust formation of the PPE, showing the importance of this regulation. Embryonic patterning is one of the most crucial steps for constructing a complex body shape from a simple egg. The fundamental concept of embryonic fate determination involves localization of signaling molecules inside an egg and differential activation of pathways in each embryonic area, directing localized expression of specific genes. To establish cell fates precisely, strict regulation of signaling strength in each area is essential . There aXenopus gastrula, several proteins such as chordin, noggin, and follistatin allow formation of BMP gradients. Both FGF and Wnt signaling are important for anterior–posterior neural patterning. Wnt antagonists , secreted from anterior mesendoderm, induce anterior neural structure, including the brain .,36.5,36]A morphogen gradient of BMP signaling is essential to establish embryonic patterning, as in dorsoventral axis formation . SimilarXenopus embryos, signaling from DLMZ during gastrulation is important, whereas the signal from intermediate mesoderm, as well as adjacent ectoderm is important for maintenance of the NC region, indicating the necessity of stage-dependent inhibition of BMP signaling for NC formation  . . Sproutyignaling ,106. In ry (MHB) . In chic placode , suggestRipply3, Tbx1 and Six1. As shown above, Ripply3 suppresses FGF signaling, suggesting that RA and FGF signaling form a negative feedback loop via these genes [RA signaling functions cooperatively with FGF signaling. RA nuclear receptor, RARa2, reduced the expression of se genes . Pitx2c se genes . In oticse genes .In this review, we discussed the role of signaling pathways in PPE formation. In particular, we focused on BMP and FGF signaling and showed examples of their feedback regulation in PPE patterning. Signal adjustment is obviously important not only to form clear boundaries, but also to pattern narrow areas robustly. In particular, feedback adjustment contributes to noise suppression, which reduces signal fluctuation, and contributes to robust acquisition of patterns.Further analysis is needed to fully elucidate the mechanisms of formation of the PPE region, the NP, the NC, and the epidermis. In addition, other experimental approaches may be important: one of them is to artificially change the feedback cycle by changing the intron length of a target gene and examining the effect on PPE formation . Further


Seseli L., which consists of 125–140 species distributed in the Old World from western Europe and northwestern Africa to China and Japan, is one of the largest and most taxonomically difficult genera of Apiaceae Lindl. Although several previous studies have been conducted on Seseli based on limited morphological characteristics and molecular fragments, a robust and comprehensive phylogeny of Seseli remains elusive. Plastomes provide abundant genetic information and have been widely used in studying plant phylogeny and evolution. Consequently, we newly generated the complete plastomes of eleven Seseli taxa. We combined plastome data and morphological characteristics to investigate the phylogeny of Seseli.The genus Seseli plastomes were variable. Several appropriate mutation hotspot regions may be developed as candidate DNA barcodes for evolution, phylogeny, and species identification of Seseli. The phylogenetic results identified that Seseli was not a monophyletic group. Moreover, the eleven newly sequenced Seseli taxa did not cluster with S. tortuosum , where S. delavayi clustered with Eriocycla belonging to the tribe Echinophoreae and the other ten belonged to Selineae. The comparative plastome and morphological characteristics analyses confirmed the reliability of the phylogenetic analyses and implied the complex evolution of Seseli.In our study, we observed that the genome length, gene numbers, IR/SC borders, and repeat composition of the eleven Seseli. We suggest that “a narrow sense” of Seseli will be meaningful for further study and the current taxonomic system of Seseli needs to be revised. In summary, our study can provide new insights into the phylogenetic relationships and taxonomic framework of Seseli.Combining molecular and morphological data is efficient and useful for studying the phylogeny of The online version contains supplementary material available at 10.1186/s12870-022-03919-9. Seseli L. is one of the largest genera of Apiaceae Lindl.    should be transferred into Eriocycla  . In the  fruits) . For Erieristics . Moreoveljuykov] . Howevermentclasspt{minimaSdobnina . Most ofdentical , and dif species . TherefoSeseli is not a monophyletic group, which is the same as other large genera of Apiaceae  and is one of the most taxonomically complicated genera within Apiaceae  and Apieae (S. webbii Coss.)  with high support . S. yunnanense, S. mairei, S. mairei var. simplicifolia, S. squarrulosum, and S. incisodentatum formed a robust Subclade C , in which S. yunnanense + S. mairei, clustered with S. mairei var. simplicifolia with strong support , and S. squarrulosum and S. incisodentatum clustered together . S. valentinae and Seseli montanum L. formed a robust Subclade D . Seseli coronatum Ledeb. and S. eriocephalum were in Subclade E, in which S. coronatum was more closely related to the Ligusticum subclade (Ligusticum thomsonii + Ligusticum mucronatum) but with moderate support , and S. eriocephalum was located at the base of Subclade E with strong support .For CDS-based phylogenetic trees, the ML and BI trees were highly consistent in topology Fig. . S. delaeae Fig. . In Selides Fig. . S. glabS. delavayi was also far from the other Seseli taxa and clustered with Eriocycla nuda within the tribe Echinophoreae with strong support . The other Seseli taxa belonged to Selineae. Nine Seseli taxa, being S. arenarium M.Bieb., S. hartvigii Parolly & Nordt, S. andronakii Woronow ex Schischk., S. grandivittatum (Sommier & Levier) Schischk., S. serpentinum B.L.Burtt ex H.Duman & E.Doğan, S. alexeenkoi Lipsky, S. globiferum Vis., S. leptocladum Woronow, and S. tortuosum formed the robust “S. tortuosum” clade . Eight Seseli taxa, being S. corymbosum Boiss. & Heldr., S. paphlagonicum Pimenov & Kljuykov, S. lehmannii Degen, S. ponticum Lipsky, S. rupicola Woronow, S. resinosum Freyn & Sint., S. dichotomum Pall. ex M.Bieb., and S. gummiferum Pall. ex Sm. formed the robust “S. gummiferum” clade with high support in BI (PP = 1.00) but weak support in ML (BS = 67). Five Peucedanum taxa and Saposhnikovia divaricata formed a clade . Then, these three clades clustered together with weak support . S. yunnanense, S. mairei, S. mairei var. simplicifolia, S. squarrulosum, and S. incisodentatum formed a robust clade . S. coronatum clustered with S. glabratum . S. eriocephalum formed a single branch. S. transcaucasicum (Schischk.) Pimenov & Sdobnina clustered with Libanotis sibirica (L.) C. A. Mey. , while S. intramongolicum clustered with S. marashicum E.Doğan & H.Duman , then, the two subclades clustered together. S. valentinae and Ligusticum mucronatum formed a clade .The concatenated nrDNA dataset included 984 aligned characters. The ML and BI trees were nearly identical in topology, and several branches with quite low support (< 50% bootstrap support) were treated as parallel branches Fig. S. S. delaS. glabratum had the shortest SSC region  and the longest ycf1 gene extended into the IRa region  among the eighteen plastomes. Subclade B: S. intramongolicum had the longest part of ycf2 gene extended into the IRb region  and an extreme distance of trnL-CAA gene from the JSA  among the eighteen plastomes. Subclade C: S. yunnanense and S. mairei had highly similar structures, while S. mairei var. simplicifolia had 63 bp between the ycf2 gene and the JLB. S. squarrulosum and S. incisodentatum had similar structures: the JLB was within the rpl23 gene and the JLA was located between the trnI-CAU and trnH-GUG gene. Subclade D: S. valentinae and S. montanum had relatively similar structures, in which S. valentinae had the farthest distance between the trnH-GUG gene and the JLA  among the eighteen plastomes. Subclade E: Ligusticum thomsonii and Ligusticum mucronatum had similar structures, the ycf2 gene of S. coronatum was distant from the JLB (446 bp), while the JLB of S. eriocephalum was within the rps3 gene and the JLA located between the rpl22 and trnH-GUG gene. Echinophoreae: S. delavayi and Eriocycla nuda had the JLB within the rpl2 gene and the JLA located between the rpl23 and trnH-GUG gene , IRb/SSC (JSB), SSC/IRa (JSA), and IRa/LSC (JLA) Fig. . Taxa wi region 1,501 bp arps16-trnQ-UUG, rpoB-trnC-GCA, petA-psbJ, rpl32-trnL-UAG, ycf1, ycf2), while the sequence divergence of taxa within the same subclade was relatively low.The eighteen plastome sequence divergence analysis suggested that the coding regions were more conserved than the non-coding regions, and the IR regions were more conserved than the single copy regions Fig. . Taxa wiLibanotis buchtormensis to 68,313 bp in S. eriocephalum  had lower RSCU values of UAG (RSCU = 0.71) and higher values of UAA (RSCU = 1.65) than others. Overall, usage of codons showed bias in different subclades, and similarity in different taxa within the same subclade  and each furrow . However, these five taxa were different in their ribs and calyx teeth: S. yunnanense, S. mairei, and S. mairei var. simplicifolia had ribs narrowly keeled or rounded, while S. squarrulosum and S. incisodentatum had median and lateral ribs keeled and filiform, marginal ribs winged. The first three taxa had calyx teeth obsolete, while S. squarrulosum had small calyx teeth and S. incisodentatum had broadly triangular calyx teeth. S. valentinae in Subclade D, had mericarp ovoid or oblong-ovoid, dorsally compressed, densely puberulent, endosperm flat on commissural side, calyx teeth small and triangular, all ribs equal, obtuse-keeled, and vittae 1 in each furrow, 2 on commissure. For Subclade E, S. coronatum had mericarp oblong, compressed dorsally, sparsely puberulent, endosperm flat on commissural side, calyx teeth obsolete, median and lateral ribs keeled and filiform, marginal ribs winged, and numerous vittae in commissure (8–12) and each furrow (3–5). S. eriocephalum had mericarp oblong, slightly dorsally compressed, densely tomentose, calyx teeth obsolete, ribs thick, marginal ribs slightly winged, and vittae 1 in each furrow, 2 on commissure. S. delavayi in Echinophoreae, had mericarps ovoid, slightly dorsally compressed, densely white hispid, endosperm slightly concave on commissural side, calyx teeth present and triangular, all ribs equal, keeled and filiform, hidden by indumentum, and vittae 1 in each furrow, 2 on commissure.We mapped the mericarps of the eleven ees Fig. . S. glabSeseli. The genome length, gene numbers, IR/SC borders, and repeat composition were variable, which further implied the complexity of plastomes evolution and the non-monophyly of Seseli. Then, we reconstructed the phylogeny of Seseli based on plastomes and nrDNA sequences. Both CDS-based and nrDNA-based phylogenetic trees indicated that the Seseli taxa did not form a monophyletic group, which was consistent with previous studies using molecular fragments [Seseli taxa did not cluster with S. tortuosum, in which S. delavayi clustered with Eriocycla belonging to Echinophoreae and the others belonging to Selineae. The comparative plastome analyses and morphological characteristics confirmed the reliability of the phylogenetic analyses and implied the complex evolution of Seseli. We suggest that “a narrow sense” of Seseli is meaningful for further study and the current taxonomic system of Seseli needs to be revised.In this study, we newly generated eleven plastomes to investigate the plastome features and evolution of ragments –21. The Seseli. In addition, twelve mutation hotspot regions  longer than 200 bp with high Pi values were selected as candidate DNA barcodes for phylogenetic analysis and species identification of Seseli. Among them, matK gene, ccsA gene, and trnH-psbA region have been used as universal DNA barcodes in studying plant phylogeny [We observed that the genome structure  and GC content of these eleven plastomes were conserved, and there was no gene rearrangement or loss among these eleven plastomes. These conservations are commonly in other genera of Apiaceae , 29, whihylogeny –41. We wSeseli taxa did not form a monophyletic group and were divided into several branches. The “S. tortuosum” clade and the “S. gummiferum” clade were separated with high support, which was consistent with a previous study [Peucedanum taxa and a monotype genus, Saposhnikovia Schischk., while they did not cluster with S. transcaucasicum, S. marashicum, and the eleven newly sequenced Seseli taxa. After determining the morphological characteristics of the taxa belonging to the “S. tortuosum” and “S. gummiferum” clade  and unequal, calyx teeth very minute, mericarps ovoid or oblong, and ribs prominent. According to the literature and the results of Lyskov et al. [S. gummiferum” clade were morphologically different from the “S. tortuosum” clade   Table S. In addiSeseli species are regional endemics and the composition of the type section of Seseli  has not been clearly defined, which leads to imperfect and incomplete classification among Seseli species [Peucedanum, Ligusticum, and Libanotis) related to Seseli were also non-monophyly [Seseli harder. It is worth noting that many phylogenetic and taxonomic problems of Peucedanum and Ligusticum have been effectively resolved after considering a narrower sense [Seseli, we suggest that it is important to treat Seseli in a narrow sense. Thus, according to our results, the “S. tortuosum” clade could be referred to as the narrow sense of Seseli (within Selineae), containing common features such as leaf segments linear to lanceolate, bracts nearly absent, bracteoles linear to lanceolate, rays unequal, calyx teeth very minute, mericarps ovoid or oblong, ribs prominent, and consisting of the following species: S. tortuosum, S. arenarium, S. hartvigii, S. andronakii, S. grandivittatum, S. serpentinum, S. alexeenkoi, S. globiferum, and S. leptocladum.Most  species . Moreoveonophyly , 28, 30,er sense , 43. OveSeseli are distributed into three tribes: Selineae, Pimpinelleae, and Apieae [S. diffusum belonging to Pimpinelleae has been treated as Psammogeton difusum (Roxb ex Sm.) Rech.f. ex Pimenov [S. webbii belonging to Apieae has been treated as Canaria tortuosa (Webb & Berthelot) Jim.-Mejías & P.Vargas [Seseli taxa still belong to Selineae. However, in our study, we found that S. delavayi was furthest from the main branches of Seseli (within Selineae), but clustered with Eriocycla nuda and belonged to Echinophoreae [S. delavayi had a similar plastome structure and SC/IR borders to Eriocycla nuda. It also possessed the most different morphological characteristics from other Seseli taxa but is similar to Eriocycla species: pubescent throughout, peduncles elongate, bracts 5-7, bracteoles longer than 2 × pedicels, prominent calyx teeth, and fruit densely white hispid  should be transferred into Eriocycla, while Pimenov [Eriocycla taxa as synonyms of Seseli taxa based on type specimens. In our study, it was clear that the type species of Seseli, S. tortuosum, was located in Selineae, while the type species of Eriocycla, Eriocycla nuda, was nested in Echinophoreae. Additionally, S. tortuosum and Eriocycla nuda were morphologically dissimilar  [Eriocycla should be identified as a separate genus based on our molecular and morphological evidence. Additionally, introducing more Eriocycla taxa would be useful to establish a robust phylogenetic framework for Eriocycla and resolve the taxonomic problem of S. delavayi.Taxonomic controversies between gtjareva  proposed Pimenov  treated  yellow) . TherefoSeseli taxa within Selineae failed to form a monophyletic group but were divided into five subclades. The comparative plastome analyses and morphological characteristics confirmed the reliability of our phylogenetic analyses and implied the complex evolution of Seseli. For the comparative plastome analyses, Seseli taxa belonging to different subclades showed distinguishing SC/IR borders, sequence divergence, and codon usage, which implied the complexity of plastome evolution and the non-monophyly of Seseli. Additionally, Seseli taxa had significantly different morphological features. For example, S. yunnanense, S. mairei, S. mairei var. simplicifolia, S. squarrulosum, and S. incisodentatum had glabrous mericarps, while other Seseli taxa had more or less hairy mericarps: S. glabratum had sparsely papillose, S. intramongolicum had densely papillose-pubescent, S. valentinae had densely puberulent, S. coronatum had sparsely puberulent, and S. eriocephalum had densely tomentose. In addition, mericarp in transverse section was slightly dorsally to laterally compressed in S. intramongolicum but strongly dorsally compressed in S. coronatum. Even though these taxa had different morphological characteristics, they also possessed several common characteristics: bracts absent or 1-2, bracteoles numerous, calyx teeth almost obsolete, and mericarp ovoid or oblong, which were very important taxonomic characteristics of Seseli [Seseli was indeed a taxonomically complex genus.f Seseli . These mS. glabratum clustered with Libanotis buchtormensis, Libanotis spodotrichoma, and Saposhnikovia divaricata in the CDS-based phylogenetic tree, while it clustered with S. coronatum in the nrDNA-based phylogenetic tree, S. valentinae clustered with S. montanum in the CDS-based phylogenetic tree, while it was clustered with Ligusticum mucronatum in the nrDNA-based phylogenetic tree. These incongruences might be the result of hybridization, introgression, and incomplete lineage sorting (ILS) [Seseli.The incongruences between nrDNA-based and plastome-based phylogenetic trees often appear in plant phylogenetic analyses , 46–48, ng (ILS) . Moreoveng (ILS)  proposedSeseli taxa due to the lack of adequate morphological and molecular data of S. tortuosum. We recommend that comprehensive studies of morphological characteristics and molecular phylogeny should reduce the uncertainties in the taxonomy of Seseli. Overall, in our study, we verify the non-monophyly of Seseli based on both plastomes and nrDNA sequences, and this provides a foundation for studying the evolution, phylogeny, and taxonomy of Seseli.It is impossible for us to conduct taxonomic treatments of several Seseli taxa. We observed that the genome length, gene numbers, IR/SC borders, and repeat composition of the Seseli plastomes were variable. Several appropriate mutation hotspot regions might be developed as candidate DNA barcodes for evolution, phylogeny, and species identification of Seseli. Thirty-seven plastome CDS and 59 nrDNA sequences were used to perform the phylogenetic analysis of Seseli. The phylogenetic results identified that Seseli was not a monophyletic group. Moreover, the eleven newly sequenced Seseli taxa did not cluster with S. tortuosum , where S. delavayi clustered with Eriocycla belonging to Echinophoreae and the other ten belonged to Selineae. The comparative plastome and morphological characteristics analyses confirmed the reliability of the phylogenetic analyses and implied the complex evolution of Seseli. We suggest that “a narrow sense” of Seseli will be meaningful for further study and the current taxonomic system of Seseli needs to be revised. Overall, our study can provide new insights into the phylogenetic relationships and taxonomic framework of Seseli.In this study, we newly sequenced, assembled and annotated complete plastomes of eleven S. mairei, S. mairei var. simplicifolia, S. yunnanense, S. squarrulosum, S. incisodentatum, S. glabratum, S. intramongolicum, S. valentinae, S. coronatum, S. eriocephalum, and S. delavayi, were collected from the wild  and immediately dried with silica gel for subsequent treatment. These taxa belong to four of the five sections  according to the treatment of Flora Republicae Popularis Sinicae (FRPS) [Fresh and mature green leaves from adult plants of eleven taxa, namely e (FRPS) . The forSeseli taxa with primers ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’), ITS-5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) [2O. We used the software DNAstar-SeqMan to edit and obtain the newly assembled ITS and ETS sequences [Total genomic DNA was extracted from silica gel-dried materials using the modified CTAB method . Then, wAAGG-3’) , 18S-ETSAAGG-3’) , and UmbAAGG-3’) . Polymerequences .Seseli taxa were generated by Illumina platform, generating 150 bp paired-end reads at Novogene . The raw data was filtered through fastP v0.15.0 (-n 10 and -q 15) to ensure high quality [rbcL gene sequence of S. tortuosum (MW662022) as the seed sequence. Genome annotation was performed using Plastid Genome Annotator (PGA) [S. montanum (KM035851) as the reference. Manual adjustment compared with related species’ plastomes was conducted in Geneious v9.0.2 [Seseli plastomes were detected using Mauve Alignment [For plastomes, raw data of the eleven newly sequenced  quality . After q quality , with deor (PGA)  with S. s v9.0.2 . Then, ws v9.0.2  to draw uosum MW6022 as thSeseli taxa have been submitted to the GenBank under accession numbers ON975056-ON975066, ON980800- ON980810, and ON980787-ON980797  to detect simple sequence repeats (SSRs) of the eleven Seseli plastomes with thresholds (the minimum number of SSRs) of 10, 5, 4, 3, 3, and 3, for mono-, di-, tri-, tetra-, penta-, and hexanucleotide SSRs, respectively.The online REPuter program  was usedDnaSP version 6.12.03  was usedSeseli species are divided into three tribes: Selineae, Pimpinelleae, and Apieae [Aegopodium podagraria L. and Carum carvi L. belonging to the tribe Careae as the outgroup to root the phylogenetic tree, according to the results of Wen et al. [et al. [et al. [Previous studies have shown that d Apieae . Thus, in et al. . The nam [et al.  and Wen  [et al. . All taxSeseli.Eighty common CDS of 37 Apiaceae taxa were extracted, respectively aligned, and concatenated as the plastome CDS dataset, using PhyloSuite v1.2.2 . PreviouMaximum likelihood (ML) analysis was conducted using RAxML v8.2.8  based onSeseli plastomes from this study. The remaining seven plastomes were from published data .Comparative plastome analyses were based on phylogenetic results, and a total of eighteen plastomes belonging to Selineae and Echinophoreae were selected. Of the eighteen, eleven were the newly generated S. yunnanense as the reference.The online program IRscope  was usedEighty common single-copy coding sequences of the eighteen plastomes were extracted, and then codon usage analysis and relative synonymous codon usage (RSCU) values  were calSeseli taxa were collected from the field and were then photographed using a stereomicroscope (Nikon SMZ25). The mericarp terminology followed Kljuykov et al. [Seseli taxa and other related taxa involved in this study was obtained during our field observation, consulting type specimens, and previous literature review.Mericarps of the eleven v et al. . MorpholAdditional file 1: Figure S1. The nrDNA-based phylogenetic tree constructed by Bayesian inference (BI) and maximum likelihood (ML) with the posterior probabilities of BI and the bootstrap values of ML above the branches, respectively, (*) represents maximum support in both two analyses, (-) represents those nodes not occurring in the ML strict consensus tree. The black triangle indicates the type species of Seseli, S. tortuosum.Additional file 2: Table S1. Collection locality and voucher information of the eleven Seseli taxa.Additional file 3: Table S2. GenBank accession numbers of DNA sequences used in this study. The accession numbers of DNA sequences from our lab were in bold.Additional file 4: Table S3. Gene contents of the eleven Seseli plastomes.Additional file 5: Table S4. The repeat sequences distribution in the eleven Seseli plastomes.Additional file 6: Table S5. Simple sequence repeats (SSRs) distribution in the eleven Seseli plastomes.Additional file 7: Table S6. Nucleotide diversity (Pi) of coding and non-coding regions.Additional file 8: Table S7. Codon usage and relative synonymous codon usage (RSCU) values of protein-coding genes of the eighteen plastomes.Additional file 9: Table S8. Morphological data of the taxa belonging to the “S. tortuosum” and “S. gummiferum” clade.Additional file 10: Table S9. Morphological data of the eighteen taxa in Selineae and Echinophoreae involved in this study.


Although the majority of infected CD4+ T-cells adopt a resting phenotype, antigen stimulation may result in bursts of viral expression. The antigen-dependent “on-off” viral expression creates “conditional latency” that when combined with ineffective host responses precludes virus eradication. Epidemiological and clinical data suggest that the continuous attempt of the host immunity to eliminate infected cells results in chronic immune activation that can be further exacerbated by co-morbidities, resulting in the development of severe disease. We review cell and animal model studies that uncovered mechanisms used by HTLV-1 to usurp and/or counteract host immunity.Human T-cell Leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other inflammatory diseases. High viral DNA burden (VL) in peripheral blood mononuclear cells is a documented risk factor for ATLL and HAM/TSP, and patients with HAM/TSP have a higher VL in cerebrospinal fluid than in peripheral blood. VL alone is not sufficient to differentiate symptomatic patients from healthy carriers, suggesting the importance of other factors, including host immune response. HTLV-1 infection is life-long; CD4 Its cure nature ,11,12,13e nature ,15,16. TThe genomic organization and nucleotide sequence of HTLV-1 isolates are highly conserved. To classify HTLV-1 into different subtypes (subtypes A-G) with characteristic geographic distribution, variations in the sequence of the HTLV-1 long terminal repeat (LTR) sections have been used . The pre+ T-cell and uninfected cells. The most common routes of transmission are mother-to-infant, sexual intercourse , and, rarely, blood transfusion (whole blood products and sharing syringes) and organ transplants [HTLV-1 infection occurs primarily through cell-to-cell contact between the virus-infected CD4nsplants ,20. CellRisk factors of HTLV-1 vertical transmission are mainly associated with breastfeeding and additional factors, such as vulnerable socioeconomic position, with the rate of vertical transmission ranging from 3.9% to 22% in endemic areas . Of noteAnother major route of transmission is sexual intercourse in both genders, as HTLV-1-infected cells are present in genital secretions, such as vaginal mucus or secretions and semen . Many in+ T-cells facilitating HTLV-1 transmission [Increased HTLV-1 transmission may occur in individuals infected with other sexually transmitted diseases because these infections induce inflammatory reactions that recruit lymphocytes, which have a high proportion of CD4smission . In addismission , increassmission . It is p4 HTLV-1-infected cells is necessary to establish a transfusion-transmitted HTLV-1 infection [HTLV-1 transmission can occur through whole blood transfusions or white blood cell contaminated plasma ,28,29. Infection . Sharingnfection ,33,34,35HTLV-1 transmission also may occur during allograft transplantation . Despite+ T-cells [Although HTVL-1 can infect various cell types, such as dendritic cells, B cells, macrophages and T-cells, the virus preferentially induces clonal expansion of CD4 T-cells ,44 and h T-cells ,46,47. A T-cells ,49, HTLV T-cells ,51,52. V+ cells of HAM/TSP patients exhibit spontaneous proliferation with an increased production of proinflammatory cytokines such as interferon (IFN)-γ, TNF-α, IL-1 and IL-16 and neurotoxic cytokines IFN-γ and TNF-α, which are found in high concentrations in the spinal fluid of HAM/TSP patients [HTLV-1 infection is associated with diseases that are often accompanied by changes in immune responses (4–16). ATLL is often associated with severe immune suppression, while HAM/TSP is accompanied by chronic inflammation. CD4 cells regulate immune responses, but due to viral infection, their function is altered, causing changes in inflammatory responses and immune tolerance. Increased Treg cell function and production of IL-10 and TGF-b trigger the immunosuppressive phenotype observed in patients ,54. In Hpatients ,56,57. DThe type-I interferon response is induced by viral infection ,60,61,62gag, pro, pol, and env [env and the 3′ long terminal repeat (LTR), contains four partially overlapping open reading frames (orfs) expressing regulatory proteins [mRNAs and by internal initiation codons [Orf-I produces the p12 protein, which is proteolytically cleaved at the amino terminus to generate the p8 protein, while differential splicing of mRNA from orf-II results in production of the p13 and p30 proteins [After entry of the virus into a host cell, the viral RNA is reverse transcribed into double-stranded DNA, which integrates into the host chromosomal DNA and results in lifelong infection. The HTLV-1 integrated genome (provirus) contains the characteristic retroviral structural and enzymatic genes  and env . In addiproteins  that aren codons ,71,72,73proteins ,75. The proteins ,77,78. Nproteins ,80.orf-I gene [orf-1 from methionine to a threonine [rex-orf-I, uses an initiation codon in exon 2 and the acceptor splice site at position 6383 to encode a protein of 152 amino acids referred to as the Rex-orf-I protein of 17 kDa. In this mRNA, the first coding exon of the Rex protein is joined in frame to p12/p8. The distinct functional motifs implicated in p12 function are conserved in the amino acid sequence of the putative protein rex-orf-I, and thus could possibly compensate for the role of p12 in viral persistence and immune dysregulation. Interestingly, it was shown that the HTLV-1C subtype does not encode the f-I gene . In HTLVhreonine . AlthougOrf-III and orf-IV encode for the Rex and Tax proteins that are essential for viral expression and production, respectively, and an antisense mRNA transcribed from the 3′ LTR that generates the HTLV-1 basic leucine zipper (HBZ) protein [ protein ,84,85,86HTLV-1 bZIP factor, HBZ, have been shown to have pleiotropic functions connected to viral pathogenesis. Many early studies focused on the viral transcriptional activator, Tax. In addition to being required for induction of the 5′ viral long terminal repeat, and thus the expression of viral sense strand genes, Tax has been shown to regulate the expression of NF-kB- and CREB-responsive genes, cellular pathways central to immunity [The prognosis for ATLL is still bleak and HAM/TSP remains an intractable disease. The two regulatory proteins of HTLV-1, Tax, and the immunity ,88. Tax immunity ,92,93,94immunity , and hasTax/Rex doubly-spliced RNA in the nucleus [However, while Tax expression is high in early infection, it is often suppressed at later timepoints, likely because Tax is highly immunogenic and renders infected cells vulnerable to cytotoxic T-cells ,95,96,97 nucleus , whereas nucleus . Other m nucleus , methyla nucleus ,104,105. nucleus . HoweverRecent ex vivo studies in T-cell clones showed that Tax can be expressed in bursts  that can+ T-cell depletion, as a means to demonstrate their importance in non-human primates, has demonstrated that their decrease accelerates primary HTLV-1 infection [The CTL response is a critical component of the host immune response against viral infection. CTLs directed toward HTLV-1 predominantly recognize the Tax antigen, and anti-Tax CTLs have been suggested to contribute to the control of expansion of infected cells ,120,121.nfection .orf-I encodes a 99 amino acid p12 protein which can be proteolytically cleaved at the amino terminus to generate the p8 protein [Orf-I mRNA is expressed early after virus entry and is critical for establishing and maintaining viral infection in vivo [HTLV-1  protein . The two in vivo ,126,127.orf-I influenced T-cell proliferation by activating the nuclear factor of activated T-cells (NFAT), which is dependent on calcium-binding proteins for its dephosphorylation and nuclear import, to increase T-cell proliferation [2+/calmodulin-dependent phosphatase calcineurin, triggering NFAT’s nuclear import. Because p12 can modulate the cytosolic calcium levels, it can also activate NFAT independent of TCR signaling [HTLV-1 persists primarily through the proliferation of infected cells. The viral p12 protein localizes to the endoplasmic reticulum (ER) through a noncanonical ER retention signal . In the feration ,130,131.ignaling . NFAT isignaling . Since pignaling . In a caignaling . In addition, early studies demonstrated another function in the ER of p12. P12 binds to the IL-2R β chain in a region critical for JAK1 and JAK3 recruitment, and the interaction of p12/p8 with the immature IL-2R leads to an increase in Signal Transducer and Activator of Transcription 5 (STAT5) phosphorylation and DNA binding activity and decreases the cellular requirement for IL-2 . Further+ T-cells, also called cytotoxic T lymphocytes (CTLs). Because CTLs recognize viral peptide:MHC-I complexes on target cells, many viruses have evolved proteins to interfere with this pathway [+ T-cells infected with HTLV-1 mutant viruses  demonstrated that a decrease in surface MHC-I was seen only in cells infected with virus that predominantly expresses the p12 protein HTLV-1G29S [WT) was necessary for the protection of infected CD4+ cells from CTL lysis [The presentation of antigens via the MHC class I (MHC-I) processing pathway plays a critical role in the development of host immunity against pathogens. All nucleated cells express MHC-I on their cell surface. MHC-I molecules present antigen peptides to the TCRs on effector CD8 pathway . The MHC pathway . Early w pathway . These i pathway . Immatur pathway . AlthougLV-1G29S . This saTL lysis . By prev+ T-cells, making them resistant to autologous NK cell killing [+ T-cells did not express ligands for NK cell activating receptors NCR and NKG2D, further contributing to the reduced adherence of NK cells to HTLV-1-infected cells [I in primary CD4+ T-cells was sufficient to cause downregulation of surface ICAM-1 and ICAM-2. NK cells detect and destroy cells expressing low surface MHC-I levels. Thus, reduced MHC-I cell-surface expression enables infected cells to evade CTL killing but makes them targets for NK cells. NK cells directly kill target cells by delivering cytotoxic proteins (perforin and granzyme B) to their targets. When NK cells recognize a target, a lytic immune synapse is established through integrins like LFA-1 on the NK cell, and its ligand intercellular adhesion molecule 1 (ICAM-1) on the target cell . Early s killing . Pretreaed cells . This storf-I expression, as the surface expression of both MHC-I and ICAM-1 increased following Pom treatment in primary CD4+ cells infected with wild type HTLV-1 but not primary CD4+ cells infected with a mutant orf-I knockout HTLV-1 virus [+ cells greatly enhanced the infection of both wild type and orf-I knockout virus [The immunomodulatory drug Pomalidomide (Pom), used as part of the standard treatment for multiple myeloma and recently approved for the treatment of Kaposi Sarcoma ,145, inc-1 virus . Additio-1 virus ,147,148.ut virus . Althougut virus . While aut virus ,151. TheT-cells are critical in mediating the protective immune response to pathogens. The localization of p8 on the surface of T-cells was shown to decrease T-cell activation by inhibiting proximal T-Cell Receptor (TCR) signaling . The recIt is well-documented that HTLV-1 is transmitted via cell-to-cell contact and that cell-free virus is poorly infectious and rarely detected in the blood plasma of HTLV-1-infected individuals ,155,156.Interestingly, the vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, co-immunoprecipitated with p8, and imaging showed partial areas of co-localization of VASP and p8 on the plasma membrane and in membrane protrusions . The knoN26) infected monocytes similar to wild type virus, with a proviral load of three to four copies per cell and high supernatant p19 levels. In contrast, mutant viruses expressing only p12 (HTLV-1G29S) or no p12/p8 (HTLV-1p12KO) had lower proviral loads of > one copy per cell and no detectable supernatant p19 produced [G29S and HTLV-1p12KO did not establish persistent infection, while HTLV-1WT and HTLV-1N26 did [orf-I expression is associated with the inhibition of inflammasome activation, with increased CD47 “don’t-eat-me” signal surface expression in virus-infected cells and the decreased monocyte engulfment of infected cells [The role of p12/p8 in monocyte function is unclear. It was shown that HTLV-1 virus knocked-out for Orf-I protein expression was severely impaired in its ability to replicate in dendritic cells . Furtherproduced . This is1N26 did . Orf-I aed cells . Similar to the E5 protein of the bovine papilloma virus, both p12 and p8 can bind to the proton pump V-ATPase through the 16 kilodalton subunit ,173,174.orf-II gene encodes for two proteins: p30, a 241-residue nuclear/nucleolar protein expressed from a doubly-spliced mRNA, and p13, an 87-residue protein coded by a singly-spliced mRNA corresponding to the carboxy-terminal portion of p30 [+ and CD8+ T-cells, a small percentage is observed in all three monocyte subsets defined by CD14 and CD16 expression [The n of p30 ,73,75. Hn of p30 ,186,187,pression , suggest+ T-cells isolated from HTLV-1-infected patients [Interferons (IFN-Iα and IFN-Iβ) play a critical role in mediating innate and adaptive antiviral immunity. This is accomplished predominantly through their impact on cell activation, cell proliferation, and apoptosis. Activation of the IFN response increases the expression of over 300 genes encoding antiviral and immunoregulatory proteins ,190,191.patients ,196.tax/rex mRNA in the nucleus, as well as by repressing LTR-mediated transcription [Early studies demonstrated that the HTLV-1 p30 protein could work as a latency factor by retaining newly transcribed cription ,111. It cription ,63. TLRscription ,198,199.cription . Followicription ,201. Theorf-II by a singly-spliced mRNA corresponding to the carboxy-terminal portion of p30 [The viral protein p13 is produced from n of p30 ,73,75. Un of p30 ,203,204.n of p30 ,206. ROSn of p30 .+ T-cell activation induces a synchronized program of mitochondrial biogenesis and remodeling [The effect of p13 on mitochondrial function could also affect the host immune response to the virus. Several studies have revealed important roles for mitochondria in immune responses . By indumodeling . HTLV-1 modeling ,212,213.orf-I and orf-II regions in HTLV-1-infected individuals [Unlike Tax, Rex and HBZ, the HTLV-1 regulatory genes p12, p8, p30, and p13 are not absolutely required for virus replication or immortalization of human primary T-cells in vitro ,77,78. Tividuals ,217,218.orf-I is essential for infectivity in the macaque model and the requirement of orf-I for viral infectivity in macaques parallels HTLV-1 infectivity of dendritic cells in vitro [orf-I mutant viruses support the importance of p12/p8 expression and CD8+ cells in viral persistence [+CD25+ T-cells. However, when mice were infected with virus ablated for orf-I expression, HTLV-1p12KO infection only occurred after reversion of HTLV-1p12KO back to wild type [orf-I, species specific requirements of orf-II and hbz for viral infectivity [Several studies demonstrated that primary human T-cells immortalized with molecular clones lacking p12 or p30 grew less efficiently than the wild type molecular clone and are more dependent on IL-2 ,79,80. Ein vitro . No revesistence . In a huild type . Similarild type . In addiectivity  suggest + cytotoxic T-cell (CTL) [orf-I expression for counteracting NK and CTL responses was validated in macaques by the depletion of either CD8 and NK cells (CD8/NK) or CD8 cells alone prior to virus exposure. HTLV-1 orf-I knockout virus is un-infectious in macaques, but following the depletion of CD8/NK, viral infectivity was restored and all animals were persistently infected with detectable mutated viral DNA in tissues [orf-I knockout HTLV-1 [Increases in the HTLV-1 proviral load and persistent infection are likely linked with the virus’s ability to evade the host immune response. As stated above, p8 and p12 are dispensable for viral replication in vitro ,126,224,ll (CTL)  responsell (CTL) ,225 and ll (CTL) ,152,168. tissues . Similart HTLV-1 . These dt HTLV-1 . The fret HTLV-1 . Interest HTLV-1 . orf-I knockout HTLV-1 was not restored by clodronate treatment prior to virus exposure. Interestingly, orf-I expression was associated with defective efferocytosis in part linked to its upregulation of CD47, the “don’t-eat-me” signal on infected cells [orf-I expression by transiently protecting engulfed cells from degradation may facilitate the spread of virus by migratory efferocytosis to tissues. In addition, defective efferocytosis could create a durable and vicious inflammatory response that is unable to clear the virus by inducing further inflammation [Monocyte/macrophage depletion by clodronate prior to viral exposure to HTLV-1 wild type was associated with a faster seroconversion in all macaques, but antibody levels were not sustained, suggesting a possible role of monocytes in persistent infection . The infed cells . These fammation  and reguammation . Indeed,ammation . This stWhile the function of the viral regulatory proteins in modulating the T-cell response is actively being studied, little is known about the role these proteins play in modulating the HTLV-1 humoral response. In a study looking at a cohort of HTLV-1 exposed transfusion recipients, it was noted that antibodies to core, envelope and tax protein appeared within 30–60 days following primary HTLV-1 infection . In mostMany viral vaccines are directed toward blocking virus entry into target cells. The HTLV-1 envelope (Env) protein is necessary for infection, highly immunogenic and the primary target of neutralizing antibodies . ResultsAs discussed above, NK cells play an important role in controlling viral persistence. Thus, eliciting anti-HTLV-1 antibodies may be important for clearance by antibody-dependent cellular cytotoxicity (ADCC). An early study examining ADCC and NK cell activity from newborns, infants and adults suggests that these activities can protect against the transmission of mother-to-child . A more + cells, as observed in infected individuals with high virus burdens [HTLV-1 counteracts host NK and CTL activity and usurps monocyte and dendritic cell immunity . The con burdens ,247,248.orf-I-mediated immune dysregulation caused reactivation of the virus, allowing its recognition by the host immune system [Although an HTLV-1 preventative vaccine is feasible, no candidate vaccine has ever proceeded to clinical trial. Vaccine development efforts have used recombinant vaccinia virus vectors, protein immunization, DNA vaccine vectors, and peptide vaccines ,260,261.e system . Unfortue system , there we system . Integrae system ,265,266.In addition, the data suggest that a preventive HTLV-1 vaccine should either prevent infection upfront or eliminate the virus very early on to avoid the establishment of a reservoir that host immunity is unable to clear. Given the HTLV-1 modes of transmission, virus vulnerability to neutralizing antibodies is uncertain. The engagement of less canonical host responses such as ADCC and efferocytosis, based on the ability of NK and monocytes to recognize and effectively dispose of infected cells, may be necessary for an HTLV-1 vaccine to prevent the establishment of infection.