The exact mechanism of tumour necrosis factor α (TNF‐α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co‐cultured with TNF‐α to explore the effect of TNF‐α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti‐TNF‐α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF‐α at a concentration of 20 ng/mL up‐regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti‐TNF‐α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency‐induced osteoporosis. Postmenopausal women experience bone loss and further form osteoporosis, referred to as postmenopausal osteoporosis (PMOP).Numerous studies have shown that the decrease of postmenopausal oestrogen levels can increase self‐inflammatory cytokines and inflammatory bodies, including IL‐1, IL‐6 and tumour necrosis factor α (TNF‐α), thus promoting an increased number of osteoclasts. Among these inflammatory factors, the level of TNF‐α increased most significantly, and in vitro experiments showed that IL‐1 and IL‐6 alone could not increase the number of differentiated osteoclasts.Purine energy signals are associated with P2 receptors and have been confirmed to regulate the balance between bone resorption and formation. Genetic studies have found a correlation between P2 polymorphism and increased risk of osteoporosis. For example, animals with P2 gene knockout develop bone deformities.TNF‐α promotes the progress of osteoporosis, and the P2 receptor also increases as osteoclasts increase. Therefore, it is hypothesized that TNF‐α can affect the P2 receptor, thereby increasing osteoclasts and aggravating the progression of osteoporosis. Previous studies have found that TNF‐α increases the number of P2Y2 receptors and suppresses osteoblast differentiation. However, the effect of the P2 receptor on osteoclasts affected by TNF‐α remains unclear.Another potential mechanism of osteoclast differentiation could be confirmed by studying the effects of TNF‐α on P2 receptor, osteoclast differentiation and bone resorption of mouse bone marrow haematopoietic stem cells (BMHSCs). This study mainly focuses on the effect of TNF‐α on BMHSCs. We found that TNF‐α significantly promoted the differentiation of BMHSCs into osteoclasts and up‐regulated the expression of specific purine receptors. Inhibition of this receptor significantly inhibited the effect of TNF‐α on BMHSCs. In addition, the results suggest that TNF‐α up‐regulates P2 receptor by activating the Akt signalling pathway, which affects the regulation of calcium ion in BMHSCs, and further affects the differentiation of osteoclasts, enhances bone resorption and aggravates the progress of osteoporosis. The results of this study provide a potential mechanism for the occurrence and development of osteoporosis and provide basic research results for future therapeutic targets.22.1Animal experiments were carried out in accordance with the guidelines of the Medical Ethics Committee of Shanghai Changzheng Hospital. Eight‐week‐old female C57BL/6J mice were randomly divided into three groups (n = 9/group). The sample number was based on a preliminary experiment : control group ; model group ; and experimental group . Eight weeks later, three mice were randomly selected from each group for the following experiments: (a) serum, separated by centrifugation from blood taken from the eyeball, was detected and analysed using an ELISA (Bio Legend); (b) microscopic CT scanning was performed on bilateral femur and tartrate‐resistant acid phosphatase (TRAP) staining , HE staining and immunohistochemical staining were performed on bilateral tibia. The remaining mice in each group (n = 5) were used for cell experiments.2.22 for 24 hours, the supernatant was centrifuged  to obtain osteoclast precursor cell precipitate. Then the cells were treated with M‐CSF (30 ng/mL) and RANKL (50 ng/mL). The optimal stimulating concentration of TNF‐α was 20 ng/mL, and the maturation time of osteoclasts was 5.5 ± 1.1 days.BMHSCs were isolated from the bone marrow of femur and tibia of 8‐week‐old female C57BL/6 mice and cultured in 10 mL medium . After culturing in a cell incubator at 37°C and 5% CO4/mL. When the cells grew to about 50% confluence, they were infected for 24 hours by adding P2X7 receptor shRNA lentivirus particles (Santa Cruz Biotechnology) to the culture medium. The control group cells were sham infected by adding shRNA‐P2X7‐negative‐control (shRNA NC) for 24 hours. Different samples were collected after culturing for different times to run qRT‐PCR detection, Western blot, TRAP staining , cyclopeptide staining, immunofluorescence staining and absorption indentation test of bone slices (cultured continuously for 14 days). TRAP+ cells with more than three nuclei were counted as osteoclasts. For the in vitro work, the experiment was repeated with three independent biological samples, each done in triplicate.Osteoclast progenitor cells were inoculated into 6‐well tissue culture plates at a density of 3 × 102.2.13‐104 cells/well in a 96‐well plate in an incubator. After 24 hours, 10 μL Cell Counting Kit‐8  solution was added to each well for another reaction of 4 hours. Then to ensure the liquid colour in the well was evenly mixed, the plate was gently shaken for 1 minute. The absorbance at 450 nm was measured using the Infinite F200/M200  enzyme labelling instrument.The cells were cultured in different media with a density of 102.2.2−ΔΔCt method.The cells were lysed with Trizol (Invitrogen), and RNA from the cells was purified by dissolution of chloroform, low temperature centrifugation, precipitation of isopropanol and alcohol washes. Using a SYBR Premix kit (Takara) and the manufacturer's instructions, RNA reverse transcription was performed to synthesize DNA, and amplification was performed using an ABI 7500 Real‐Time System (ABI) by means of incorporation of SYBR Green fluorescent dye as described in the protocols. Beta actin was used as a reference gene to detect the changes in cellular P2 receptors and osteoclast markers using the 22.2.34/mL and continuously cultured . Fourteen days later, the surface indentation area of bone slices was observed and measured by TRAP staining and fluorescence microscopy, and the area of bone indentation was quantified.In order to investigate the effect of TNF‐α stimulation on the function of osteoclasts by increasing P2X7 receptor, osteoclasts were divided into three groups according to the density of 3 × 102.2.43 cells in each group were cultured for 5 days, then fixed with 4% neutral formaldehyde for 20 minutes and rinsed three times with PBS. Immunofluorescence staining was performed using DAPI, phalloidin staining  and receptor antibody immunofluorescence staining.In order to clarify the effect of TNF‐α on osteoclast differentiation and P2X7 receptor, immunofluorescence was used to detect the number of osteoclasts and P2X7 receptor of osteoclast progenitor cells in the presence or absence of TNF‐α. A total of 102.2.5Protein samples of cells or tissues were separated with SDS‐PAGE (7.5%‐10% polyacrylamide gel), and protein imprinting was polymerized on a vinylidene fluoride membrane  via an electroporation membrane. Then 5% skimmed milk powder was used to seal the membrane in TBS brine for 1 hour. The membrane was then incubated with a specific antibody (1:400) at 4°C for 16 hours (Santa Cruz Biotechnology), followed by a second antibody (1:5000) at room temperature for 4 hours (Santa Cruz Biotechnology), The antigen‐antibody complex was observed using an enhanced chemiluminescence analysis (Thermo Scientific).5 cells/well) were inoculated into 6‐well plates and divided into two groups: RANKL‐treated group and Rankl + TNF‐α‐treated group (20 ng/mL). The cells were detected by Western blotting at 5, 15, 15, 30, 45, 55 and 60 minutes, and the phosphorylation of Akt, PI3k, MAPK, c‐fos, IKB and NF‐kB was observed. Through different stimulation culture methods, the expression level of TRAP, Cathepsin K (CTSK), MMP‐9, C‐Scr, and markers related to osteoclast production were detected by Western blotting. The primary antibody included mouse‐derived anti‐actin, anti‐Akt, PI3k, MAPK, c‐fos, IKB, NF‐kB phosphorylated antibodies, cathepsin K, C‐Scr, Trap, NFATc1 and other marker protein antibodies. The secondary antibody was anti‐rabbit IgG.The effects of TNF‐α on NF‐kB, MAPK and AKT pathways in osteoclast progenitor cells were detected by Western blot. Osteoclast progenitor cells . Two‐tailed unpaired one‐way ANOVA was used to compare between more than two groups. Bars are the mean ± SD of countable data of biological samples. Results were considered statistically significant if 33.1P < .05). Based on qRT‐PCR detection, the P2 receptors were shown to increase to different degrees in the process of osteoclast differentiation of BMHSCs stimulated by different concentrations of TNF‐α; the largest increases occurred with P2X7 receptor . Western blot analysis showed that deletion of shRNA‐P2X7 receptor was effective at different time points (2 and 5 days) Figure . BMHSCs 3.3In addition to the PI3k/Akt signalling pathway, activation of MAPKs and the NF‐kB pathway plays an important role in differentiation of osteoclasts. To evaluate the effects of TNF‐α on the PI3k/Akt, NF‐kB, and MAPK pathways after incubation with RANKL, we firstly examined the phosphorylation of major subfamilies of the signalling pathways, such as phosphorylated PI3k (p‐PI3k), Akt (p‐Akt), ERK (p‐ERK) and C‐fos (p‐c‐fos) by Western blot analysis. Among the major subfamilies, p‐ERK and p‐c‐fos levels did not change significantly, but p‐PI3k and p‐Akt showed a significant increase upon TNF‐α stimulation Figure . NFATc1,3.4In order to clarify the effect of TNF‐α on PMOP osteoclasts, we performed ovariectomized (OVX) or sham operation in 8‐week‐old female mice. Three days later, OVX mice were treated with anti‐TNF‐α or saline, and the sham operation group was treated with the same dosage of physiological saline twice a week for 8 weeks.The results of Micro‐CT analysis showed that compared with the sham‐operated control group, the trabecular structure of OVX mice treated with anti‐TNF‐α was significantly improved compared with the untreated group Figure . The res4Destruction of the dynamic balance between osteoblasts and osteoclasts is an important pathological mechanism of PMOP. Through detection of the purine receptor gene in postmenopausal women, some scholars have found a correlation between purine receptor polymorphism and bone mineral density.Some studies have shown that low concentrations of TNF‐α (1‐5 ng/mL) promote osteoblast differentiation through the NF‐κB signal pathway,Previous studies showed that the P2 receptor is related to inflammation and cellular immune response, as well as energy metabolism.However, the relationship between purine receptors and osteoclasts has not been clearly confirmed.Previous studies have suggested that purine receptors are affected by ATP,For postmenopausal women, oestrogen deficiency can induce T cells to secrete more TNF‐α. Both in vivo and in vitro experiments showed that TNF‐α promoted the differentiation of osteoclasts by up‐regulating the P2X7 receptor of BMHSCs. Therefore, the P2X7 receptor is a potential effective therapeutic target against PMOP and is of research significance. Therefore, to some extent for PMOP, inhibiting the P2X7 receptor of BMHSCs can inhibit the differentiation of osteoclasts and delay bone loss. However, the effect of the P2X7 receptor on the immune response and inflammation of BMHSCs needs further research.This study focused on osteoclasts and their progenitor‐hematopoietic stem cells, and simply but systematically studied the effects of postmenopausal tumour necrosis factor‐α on their differentiation and bone resorption and identified an important role and mechanism of the P2X7 purine receptor.In this study, we found that TNF‐α significantly up‐regulates the P2X7 receptor of BMHSCs by activating the AKT signalling pathway, regulating calcium metabolism, promoting BMHSCs to differentiate into more osteoclasts, affecting the dynamic balance between osteoblasts and osteoclasts, and aggravating the progress of osteoporosis. Therefore, inhibiting P2X7 receptor significantly reduces the effect of TNF‐α on BMHSCs and reduces the formation of osteoclasts. This study found that P2X7 was a potential target for anti‐osteoporosis therapy and provided a basis for future research.The authors declare no financial conflict of interest with each other involving this study.Jiajia Lu: Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Resources (lead); Software (lead); Writing‐original draft (lead). Zhibin Zhou: Conceptualization (supporting); Data curation ; Formal analysis (supporting); Investigation ; Project administration (supporting); Supervision (supporting); Visualization (supporting); Writing‐review & editing (supporting). Jun Ma: Conceptualization (supporting); Data curation (supporting); Investigation (supporting); Project administration ; Validation (supporting); Writing‐review & editing (supporting). Nan Lu: Data curation (supporting); Project administration (supporting); Supervision (supporting). Lei Zhu: Conceptualization (supporting); FormalAnalysis (supporting); Methodology (supporting); WritingReviewEditing (supporting). Di Du: Conceptualization (supporting); Formal analysis (supporting); Methodology (supporting); Project administration (supporting); Resources (supporting); Supervision (supporting); Writing‐review & editing (supporting). Aimin Chen: Conceptualization (lead); Supervision (lead).My manuscript does not contain data from any individual person.


Microvesicle particles (MVP) secreted by a variety of cell types in response to reactive oxygen species (ROS)-generating pro-oxidative stressors have been implicated in modifying the cellular responses including the sensitivity to therapeutic agents. Our previous studies have shown that expression of a G-protein coupled, platelet-activating factor-receptor (PAFR) pathway plays critical roles in pro-oxidative stressors-mediated cancer growth and MVP release. As most therapeutic agents act as pro-oxidative stressors, the current studies were designed to determine the role of the PAFR signaling in targeted therapies -mediated MVP release and underlying mechanisms using PAFR-expressing human A549 and H1299 non-small cell lung cancer (NSCLC) cell lines. Our studies demonstrate that both gefitinib and erlotinib generate ROS in a dose-dependent manner in a process blocked by antioxidant and PAFR antagonist, verifying their pro-oxidative stressor’s ability, and the role of the PAFR in this effect. We observed that these targeted therapies induce MVP release in a dose- and time-dependent manner, similar to a PAFR-agonist, carbamoyl-PAF (CPAF), and PAFR-independent agonist, phorbol myristate acetate (PMA), used as positive controls. To confirm the PAFR dependency, we demonstrate that siRNA-mediated PAFR knockdown or PAFR antagonist significantly blocked only targeted therapies- and CPAF-mediated but not PMA-induced MVP release. The use of pharmacologic inhibitor strategy suggested the involvement of the lipid ceramide-generating enzyme, acid sphingomyelinase (aSMase) in MVP biogenesis, and observed that regardless of the stimuli used, aSMase inhibition significantly blocked MVP release. As mitogen-activated protein kinase  pathways crosstalk with PAFR, their inhibition also significantly attenuated targeted therapies-mediated MVP release. These findings indicate that PAFR signaling could be targeted to modify cellular responses of targeted therapies in lung cancer cells. Lung cancer is currently the second most prevalent type of cancer in both men and women in the United States, which continues to be a major health and socioeconomic issues ,2,3. SevAmong targeted therapies, receptor tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib have been used to treat NSCLC patients ,21,22. TPtafr; PAFR), expressed on a variety of cell types including tumor cells  for 10 min on a shaker, and absorbance was read at 570 nm with the Synergy H1Mf plate reader. Treatment groups were normalized with the control (0.1% DMSO) group.Cell survival was measured by the sulforhodamine-B (SRB) assay as per our previous report . The A54The apoptosis induction was assessed by quantitative caspase 3/7 activity assay using kit’s manual protocol, as described by us previously . For thi−∆∆Ct method.The A549 cells were transfected using lipofectamine 3000 with three separate clones  of PAFR siRNA as per the manufacturer’s protocol. To optimize the condition, following transfection PAFR knockdown was evaluated at 24 and 48 hours’ time points by analyzing the relative PAFR mRNA expression. For this, the total RNA was extracted from A549 cells by TRIzol extraction method, and extracted RNA was quantified with Nanodrop One spectrophotometer . High-Capacity cDNA Reverse Transcription kit was used to transcribed RNA samples to cDNA for the analysis of the PAFR mRNA expression using an SYBR green-based, quantitative fluorescent PCR method as per our previous reports ,66. The g for 20 min at 4 °C to remove cells, and cell debris. Then, the supernatants were collected and centrifuged the cell-free solution again at 20,000× g for 70 min at 4 °C. The pelleted MVPs were resuspended with 100 μL of filtered PBS and used for the nanoparticle tracking analysis (NTA). The size and concentration of these MVPs were detected by using a NanoSight 300 (NS300) instrument . MVPs counts were normalized with the cell count of each treatment [The extraction and analysis of MVP were done from the culture supernatants of NSCLC cell lines as per our previous reports ,35,36. Ireatment ,35,36. Sp < 0.05 was considered to indicate a statistically significant difference between the tested groups.Statistical analysis was assessed by GraphPad Prism software version 7.0 . All in vitro experiments were repeated, independently, at least three times. Data were analyzed by Student’s t-test (to compare between two groups) or one-way ANOVA (for more than two groups) with post hoc Tukey or Bonferroni multiple comparison tests. The value of


In particular, we show that any theoretically admissible coefficients for a pair of noninbred individuals can be produced by a finite pedigree with inbred founders. Coupled with our computational methods, implemented in the R package ribd, this allows for the first time computer analysis of general constructibility solutions, thus making them accessible for practical use.We study an extension of the standard framework for pedigree analysis, in which we allow pedigree founders to be inbred. This solves a number of practical challenges in calculating coefficients of relatedness, including condensed identity coefficients. As a consequence we expand considerably the class of pedigrees for which such coefficients may be efficiently computed. An application of this is the modelling of background inbreeding as a continuous effect. We also use inbred founders to shed new light on  A standard convention in pedigree analysis is that the pedigree founders are assumed to be noninbred. While this is natural in many settings, it is sometimes a severe limitation leading to unjustified approximations and biased results  allele sharing between pedigree members. Alleles are said to be IBD if they have the same origin in some fixed reference population, typically the pedigree founders Thompson . It is iinbreeding coefficientf measuring the expected amount of IBD within an individual, will never reach 1 exactly; this is achievable only in the limit of an infinite pedigree. As a result it is impossible to represent completely inbred individuals in software following the standard conventions.A striking consequence of the conventional assumptions is that large classes of pedigrees are beyond reach of current software. For example, no software that we know of is capable of computing a complete set of IBD coefficients for the siblings shown in Fig. Another feature inadequately handled by existing methods is background inbreeding. This refers to the fact that all living creatures are related if their ancestry is traced far enough. Hence all individuals are in one sense inbred, including the designated founders of any real-life pedigree. It is well known that background inbreeding may seriously distort pedigree coefficients in wild pedigrees  members of a and b are identical by descent (IBD) if they descend from the same allele carried by a common ancestor of a and b within We define a a and b is the kinship coefficienta is IBD with a random allele from b at the same locus. Moreover, if a and b have a child c, the inbreeding coefficientc’s autosomes that are autozygous, i.e., where the paternal and maternal alleles are IBD. The kinship/inbreeding coefficient was first studied by Wright  of non-overlapping pedigree paths from s to a and b respectively, with path lengths |u| and |v|.The simplest measure of relatedness between two pedigree members y Wright , who proa and b their IBD coefficientsIBD triangle shown in Fig. admissible if it is not in the inadmissible region. (Note that the boundary points are admissible.) Fig. For noninbred individuals entclass1pt{minimaThompson  discoverentclass1pt{minimacondensed identity coefficientscondensed identity states shown in Fig. A complete characterisation of the expected IBD sharing at a single locus, of any two individuals, is given by the Jacquard . These aIn this section we briefly review Karigl’s recursive algorithm for computing condensed identity coefficients Karigl , and we generalised kinship coefficienta, b, c, as the probability that if a random allele is sampled from each of them, at the same autosomal locus, all alleles are IBD. Similarly, we define a, b, c, d, the two from a and b are IBD and the two from c and d are IBD.Define the a and b we find by conditioning on the 9 identity states thatKarigl showed that the nine identity coefficients can be expressed as linear combinations of the generalised kinship coefficients defined above. The easiest way to see this is to start with the inverse relations. For example, for any individuals o Karigl  for detaa is a nonfounder with parents p and m, and b, c, d are (not necessarily distinct) nondescendants of a.a and b are different founders (and c and d any members) the assumption of unrelatedness implies thatFor the computation of generalised kinship coefficients, Karigl  gave thea and b respectively. To verify the first of these formulas, suppose i, j, k are alleles sampled with replacement from a. We proceed by conditioning on the event that a is autozygous, i.e., that her alleles are IBD, which has probability Now we consider the situation when founders are allowed to be inbred. This has no impact on the general recursions ; only thhttps://CRAN.R-project.org/package=ribd). In addition to the nine identity coefficients, the package offers separate functions for generalised kinship coefficients, IBD coefficients , and standard kinship coefficients. Founder inbreeding is allowed in all cases. Furthermore, ribd contains algorithms for computing X-chromosomal kinship and identity coefficients, as well as various two-locus coefficients.We have implemented the modified algorithm presented in the previous section in the R package ribd, which is freely available  parent-offspring if their shared parent is inbred.The presence of inbreeding in pedigree founders can have a large effect on the genetic relationships within the pedigree. Fig. An interesting feature of founder inbreeding is exemplified by pedigrees 1 and 2 in Fig. In this section we improve on two results of Karigl regarding the constructibility of kinship and IBD coefficients. The main idea is to use inbred founders to mask most of the complexity. It turns out that this can always be carried out in such a way that a finite pedigree suffices. As a consequence, all of our constructions may be analysed and verified with the ribd package.The following theorem is due to Karigl :(Karigl) Any number finite pedigree with individuals a and b such that arbitrarily close  to ad infinitum, can What Karigl actually proved was that for any We now introduce a class of simple relationships, which we will use repeatedly in what follows. Essentially, these are half-cousin relationships, but where we allow the shared ancestor to be inbred. As we will see, this enables the construction of any kinship coefficient, but with the crucial advantage of hiding all the infinite parts in a single founder inbreeding coefficient.a and b of a pedigree half cousins with founder inbreedingf if they are connected through a single non-collapsing path in f. The path length is called the separation of a of b.Members f was produced, is irrelevant for our purposes of computing relatedness coefficients. Hence we regard this individual as a founder with an assigned inbreeding coefficient, as illustrated in Fig. The actual genealogy of the inbred path member, i.e., how the inbreeding coefficient m and founder inbreeding f, and no further relationships or inbreeding involving the path members. It is convenient to include in this notation the cases f.We denote by At first glance half cousins may seem like a small class of relationships. But as the next theorem shows, they in fact cover the entire spectrum of kinship coefficients:Any number m is finite, the kinship coefficient of any half cousin relationship m the map m runs through m and f are uniquely determined by Observe that when mentclass2pt{minimf is irrelevant. Finally, the endpoint We emphasise that Theorem Any number The endpoint For each  formula  we find Fig. We end this section with an observation about breeding schemes with stationary inbreeding levels.a and b are half cousins with separation m and founder inbreeding For each m, with valueThe point is that An example is shown in Fig. Karigl’s constructibility theorem for IBD coefficients can be stated, in our terminology, as follows Karigl :(Karigl) Any admissible point in the IBD triangle is constructible as IBD coefficients in the limit of an infinite pedigree.Karigl’s proof of this theorem relies on a combination of several limit processes. The resulting pedigree in general contains 4 infinite parts, making it unsuitable for computer implementation. In contrast, by using half cousins with inbred founders, we are able to give a different construction which is always finite, and can be easily analysed in appropriate software.m, n be nonnegative integers, and Let The definition is illustrated in Fig. Any admissible point in the IBD triangle is constructible as a double half cousin relationship with inbred founders.a and b whose fathers have kinship coefficient a and b are noninbred, and that their IBD coefficients are given byConsider two individuals Thompson  these eqa and b are double half cousins in The point is now that by Theorem For explicit values of e insert  into thee insert  in the pentclass1pt{minimaentclass1pt{minimaNote that if the fathers in a double half cousin relationship have infinite separation   \documenConversely, suppose Finally, suppose }f2=λ in  it is clm and n. Note that when m and n are finite, Figure Within the interior of each S in fact belongs to three regions: The point Q in Fig. different relationship with exactly these IBD coefficients.For our final example we turn to a popular case in the literature of pedigree analysis, namely the relationship of quadruple half first cousins. It is well known that the IBD coefficients of this relationship are U and D are defined in the proof of Theorem m and n imply that Q lies in the region By Theorem mentclasspt{minimaWe can verify Example The most immediate consequence of this work and its implementation in ribd, is a substantial expansion of the class of pedigrees for which identity coefficients can be exactly computed. This pertains to any pedigree containing a completely inbred individual, or in fact, any member with a non-dyadic inbreeding coefficient. Several examples of such pedigrees are provided by previous figures in this paper, for instance Figs. Our work has important applications in the analysis of human pedigrees. Suppose we wish to check if a model depending on pedigree coefficients is sensitive to background inbreeding. With ribd this is straightforward, simply by assigning a suitable sequence of inbreeding values to the founders, and re-calculating the coefficients in each case. With earlier methods, the only alternative would be to model background inbreeding explicitly, by adding ever-larger genealogies to each founder. While theoretically possible to a certain extent, such an approach would be highly inefficient and ill-suited for studying the background inbreeding as a continuous variable.The results of Sect. While we have focused on autosomal relatedness coefficients in this paper, the ideas presented transfer easily to X-chromosomal coefficients. To our knowledge ribd is the only package with a complete set of functions for computing kinship and identity coefficients both for the autosomes and the X chromosome, as well as a variety of other single-locus and two-locus coefficients. Table We end the discussion by examining two possible extensions of the ideas presented in this work.pair of related founders. Unfortunately this does not suffice in general, as shown by the following counter-example.It is natural and interesting to seek a further extension of our approach, allowing pedigree founders to be not only inbred, but also related to each other. This would be particularly relevant for pedigrees in isolated populations, where the assumption of unrelatedness between all founders is unrealistic. However, the complexity of multi-person relatedness poses serious challenges for such an extension in full generality. For example, consider the algorithm in Sect. a, b and c. We claim that these three-way relationships are identical in terms of the pairwise relationships, but have different generalised kinship coefficients. Indeed, in both pedigrees a is a half sibling of b and a half-uncle of c. The relationships between b and c are also inseparable, being uncle-nephew in the pedigree on the left hand side, and half siblings in the pedigree to the right .Fig. a, b and c are founders, and we want to compute identity coefficients for some pair among their common descendants. Seeking a simple modification of the boundary conditions  inbreeding coefficient of an individual. To illustrate, consider the two cases of half sisters in Fig. mentclass2pt{minimThere is one important special case, however, where the (single-locus) inbreeding coefficient in fact captures the complete genetic constituency of the individual, namely when In this paper we have studied an extension of the conventional approach to pedigree analysis, in which we allow the assignment of inbreeding coefficients to the founders. The motivation is to enable a more compact representation of many pedigrees, while retaining sufficient information for exact computation of relatedness coefficients. This is particularly useful in cases where the true ancestries of certain pedigree members are unknown or unsuitable for computer modelling, such as completely inbred individuals. We believe that our implementation in ribd is the first software capable of computing identity coefficients in such pedigrees, even as simple as that in Fig. We also showed that pedigrees with inbred founders are especially potent in constructibility problems for relatedness coefficients. Previous solutions by Karigl  requiredhttps://CRAN.R-project.org/package=ribd) and runs on all platforms. Importantly, ribd is part of the ped suite of packages for pedigree analysis, giving the user access to a large range of tools for creating, manipulating and visualising pedigrees, as well as likelihood computations and simulations. Pedigrees can be be loaded from text files in standard pedigree format, or made from scratch using built-in utility functions. Founder inbreeding is a core feature of the ped suite, allowing the ideas introduced in this paper to be explored in a variety of contexts.The R package ribd is available from the CRAN repository (


In this context, a GO-TNF based ink was prepared and directly incorporated within the binary photoactive layer, in different volume ratios (1%–3% ratio to the blend) for the effective realization of inverted ternary organic solar cells (OSCs) of the structure ITO/PFN/PTB7:GO-TNF:PC71BM/MoO3/Al. The addition of 2% v/v GO-TNF ink led to a champion power conversion efficiency (PCE) of 8.71% that was enhanced by ~13% as compared to the reference cell.A novel solution-processed, graphene-based material was synthesized by treating graphene oxide (GO) with 2,5,7-trinitro-9-oxo-fluorenone-4-carboxylic acid (TNF-COOH) moieties, via simple synthetic routes. The yielded molecule N-[(carbamoyl-GO)ethyl]-N′-[(carbamoyl)-] (GO-TNF) was thoroughly characterized and it was shown that it presents favorable highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels to function as a bridge component between the polymeric donor polyoxy]benzodithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl)carbonyl] thienothiophenediyl}) (PTB7) and the fullerene derivative acceptor -phenyl-C Due to the highly increased global demand for low-cost energy generation over the last three decades, significant research efforts have been made towards the development and progress of organic solar cells (OSCs), in order to boost their competitiveness over silicon technology ,2. OwingIn this context, several polymeric donor:fullerene-based acceptor combinations have been flourished providing a rapid increase in the OSC devices efficiency over 9% ,5,6,7. OUnlike to the typical binary OSC configuration, that is based on a donor–acceptor bulk heterojunction (BHJ) blend, the ternary one contains a third component which can function as: (i) second donor, (ii) second acceptor, and (iii) non-volatile additive . The ope71BM) for the realization of inverted ternary OSC devices. GO-TNF consists of graphene oxide (GO) as core and TNF side groups linked with ethylenediamine (EDA) aliphatic spacers. Since the energy levels of the synthesized graphene-based molecule and these of PTB7 and PC71BM perfectly match, GO-TNF ink was incorporated in different ratios ranging from 1% to 3%. Upon the incorporation of GO-TNF, charge transfer operational mechanism dominated (cascade effect), while the photovoltaic performance was boosted in all ternary devices compared to the reference cell. The champion device, containing 2% v/v GO-TNF ink, exhibited a significant enhancement by ~13%, leading to a power conversion efficiency (PCE) of 8.71%. This increase is mainly due to the improvement of the nanomorphology between the donor:acceptor blend’s interfaces, as well as to electron mobility enhancement.This study discusses for the first time the design and synthesis of a novel graphene-based material (GO-TNF) through simple chemical processes as well as its direct incorporation in ink form within the binary active layer . PTB7 was purchased from Solaris Chem , while PC71BM and PFN were both purchased from Solenne BV . Finally, MoO3 and Al were bought from Kurt J. Lesker , while the glass-ITO substrates were purchased from Naranjo Substrates .Initially, 9-oxo-fluorene-4-carboxylic acid 97%, 1,2-ethylenediamine (EDA) puriss. p.a., absolute, ≥99.5% (GC), graphite synthetic, H2SO4, 95–97%) and fuming nitric acid , yielding 2,5,7-trinitro-9-oxo-fluorene-4-carboxylic acid (TNF-COOH). Afterwards, the carboxyl group of TNF was chlorinated using thionyl-chloride (SOCl2), to get 2,5,7-trinitro-9-oxo-fluorene-4-acyl-chloride (TNF-COCl). The linkage of TNF-COCl with 1,4-ethylenediamine (EDA) was held via a typical nucleophilic substitution reaction to obtain TNF-EDA. In the second parallel step, GO was prepared via a modified Hummers’ method  (PFN) as the ETL, a PTB7:PC−1. UV-vis absorption spectra were taken using a Shimadzu UV-2401 PC spectrophotometer, over the wavelength range of 270–800 nm. The photoluminescence (PL) measurements of the devices’ active layers were carried out at room temperature and resolved by using a UV grating and a sensitive, calibrated and liquid N2-cooled CCD camera in the wavelength range from 600 to 950 nm. The excitation source employed was a He-Cd CW laser at 325 nm with a full power of P0 = 35 mW. Raman measurements were performed at room temperature using a Horiba LabRAM HR Evolution confocal micro-spectrometer, in backscattering geometry (180°), equipped with an air-cooled solid-state laser operating at 532 nm with 100 mW output power. The laser beam was focused on the samples using a 10× Olympus microscope objective , providing a ~55 mW power on each sample. XRD patterns were collected on a Panalytical Expert Pro X-ray diffractometer, using Cu Kα radiation (λ = 1.5406 Å). Thermogravimetric analysis (TGA) was performed on 5–10 mg samples over the temperature range from 40 to 800 °C at a heating rate of 10 °C/min utilizing a Perkin-Elmer Diamond Pyris model under N2 atmosphere. The morphology of the surfaces was examined with an Atomic Force Microscope . SEM images were taken through a JEOL JSM-7000F field emission scanning electron microscope. Cyclic voltammetry measurements were conducted on an Autolab PGSTAT302N. The photovoltaic performance of the devices was evaluated at room temperature within glove box (MBRAUN) conditions ((O2 < 0.1 ppm), moisture-free (H2O < 0.1 ppm)), and under standard illumination conditions with an Air Mass 1.5 Global (A.M. 1.5 G) solar simulator at an intensity of 1000 Wm−2 using an Agilent B1500A Semiconductor Device Analyzer, calibrated through a reference monocrystalline silicon solar cell supplied by Newport Corporation. The external quantum efficiency (EQE) measurements were conducted immediately after device fabrication, using an integrated system  and a lock-in amplifier with a current preamplifier, under short-circuit conditions. To enhance the credibility of our measurements, the solar simulator (a Xenon lamp) spectrum was calibrated using a monocrystalline photodetector of known spectral response. OSC devices were measured using a Xe lamp and an optical chopper at low frequencies (~200 Hz) in order to maximize the sound/noise (S/N) ratio. At least ten identical devices with six photovoltaics cells each were fabricated so that the reproducibility of the J-V characteristics is ensured.ATR FT-IR (transmittance) experiments were carried out with a Bruker Vertex 70v FT-IR vacuum spectrometer equipped with a A225/Q Platinum ATR unit with single reflection diamond crystal which allows the infrared analysis of unevenly shaped solid samples and liquids through total reflection measurements, in a spectral range of 4000–700 cm−1, which is attributed to O-H stretching vibration of the OH- moieties. Furthermore, stretching vibration of C=O moieties is appeared at 1706 cm−1, while the remaining graphitic domains (C=C) stretching vibration are shown at 1568 cm−1. In addition, C-O-H bending vibration due to COOH groups are presented at 1394 cm−1. The peaks at 1143 cm−1 and 1027 cm−1 represent C-OH stretching vibration of the hydroxide domains and the stretching vibration of C-O-C groups, respectively. On the other hand, GO-TNF (red line), exhibits a broad peak of low intensity at 3331 cm−1, indicating a N-H stretching vibration. Next, a peak at 1697 cm−1 is attributed to C=O stretching vibration deriving from the carbonyl moiety of trinitrofluorenone. Moreover, two peaks occurred at 1652 cm-1 and 1575 cm−1 are due to amidic C=O stretching vibration. NO2 asymmetric and symmetric stretch vibrations are shown at 1525 cm−1 and 1334 cm−1, respectively, as well as the peak at 1446 cm−1 corresponds to the aliphatic a-CH2 bending vibration of ethylenediamine moiety. Finally, C-N stretching vibration of the ethylene diamine moieties appears at 1105 cm−1. ATR FT-IR spectra of the intermediate TNF and EDA-TNF are reported in the SI.ATR FT-IR spectra of GO and GO-TNF, in powder form, are presented in In −1, while G bands at ~1580 cm−1. However, a difference in the relative intensity ratio (ID/IG) was observed from 0.92 for GO to 1.04 for GO-TNF, indicating that the linking between GO and TNF increased disorder and defects in the graphitic lattice [Raman spectra of GO and GO-TNF are shown in  lattice .v/v GO-TNF ink was added, PL intensity quenching is significant owing to the better energy offset between the LUMO levels of PTB7 and GO-TNF that enhances the charge transfer mechanism. In our case, the incorporation of GO-TNF ink with an optimum concentration of 2% v/v, facilitates exciton dissociation at the PTB7:PC71BM interface thus leading to a higher number of electrons that can be collected by the cathode, which is in agreement with the champion current density value achieved so far (17.65 mA cm−2) [Photoluminescence (PL) measurements were conducted to evaluate charge transfer mechanism upon the incorporation of GO-TNF within the active layer, and the respective PL spectra are depicted in mA cm−2) .The crystallinity of pristine GO and GO-TNF was investigated by X-ray diffraction (XRD) in a 2θ range from 5° to 60° . GO disp3CN 0.1M, with a scan rate of 10 mVs−1, between the potential sweep window of −2 V to +2 V, as demonstrated in To determine the energy levels of GO-TNF, cyclic voltammetry measurements were carried out using an electrolytic solution of TBAPF6 in CHThe HOMO level was approximately −5.66 eV as calculated by the oxidation peak onset 0.53 V, while its LUMO level was extracted from the onset of the reduction peak (0.85 V) and was −4.13 eV.The morphology of GO-TNF was examined using field emission scanning electron microscopy (FE-SEM). Representative SEM images of its flakes coated on silicon substrates are demonstrated in 71BM photoactive layer  performance  of 17.65 mA/cm2 and a power conversion efficiency (PCE) of 8.71% that show an improvement of ~10% and ~13% respectively when compared to the reference device.All ternary devices showed an improved performance, especially the device containing 2% scJ improvement due to the incorporation of GO-TNF, external quantum efficiency measurements were conducted to determine the calculated scJ. v/v GO-TNF ink.To further confirm the experimental scJ increase, as calculated from the respective J-V curves depicted in scJ values of the OSCs from the integration of the EQE spectra. The calculated scJ was found to be −15.72 mA cm−2 and −16.78 mA cm−2 for the binary and the 2% GO-TNF ink content ternary devices, respectively, which are within the standard deviation from the scJ obtained from the J-V curves. It should be also noted that any concentration of GO-TNF ink higher than 3% wt. resulted in a short circuit, probably due to the occurrence of local shunts. This undesired effect could be linked with the concentration of GO-TNF ink in the blend that becomes enough to allow a direct bridging with the ITO electrode.It can be seen that the EQE enhancement is in full accordance to 71BM/MoO3/Al for holes and ITO/PFN/PCDTBT:GO-TNF:PC71BM/Ca/Al for electrons, respectively. The evaluation of the charge carrier mobilities was based on the Mott–Gurney equation [rε is the relative dielectric constant, ε0 is the permittivity of free space, µ is the charge carrier mobility, V is the applied voltage, biV is the built-in potential, and d is the active layer thickness.To get a more accurate insight into the influence of GO-TNF blend into the charge transfer process in the ternary approach, hole-only and electron-only cells were fabricated to calculate the hole and electron mobility, respectively. Measurements were based on space charge limited current method. Hole-only cells and electron-only cells were fabricates using the architecture indium tin oxide ITO/PEDOT:PSS/PTB7:GO-TNF:PCequation :JSCLC=98J-V2 characteristics under dark conditions for (a) electron-only and (b) hole-only devices, respectively, where the black line refers to the control device (PTB7:PC71BM), while the red line corresponds to the champion ternary one PTB7:GO-TNF (2%): PC71BM. According to J-V2 characteristics, although hole mobility did not present any significant change upon the addition of GO-TNF, the respective electron mobility has shown a significant improvement, passing from 7.80 × 10−5 cm2 V−1 s−1 to 9.93 × 10−5 cm2 V−1 s−1  surfaces were both examined by atomic force microscopy (AFM), with a scan size of 1 mm by 1 mm, as displayed in scJ increase, indicating that the possibility of energetic disorders formation is smaller in case of the ternary device. On the other hand, ocV slightly changed upon the addition of GO-TNF, which is normal, since GO-TNF was incorporated in very low concentrations that cannot significantly affect ocV values.The morphology of the reference PTB7:PC71BM, in efficient ternary OSCs. Due to its ideal energy levels, it was directly incorporated, as the third component, within the binary photoactive layer providing a significant improvement in current density of the champion device (2% v/v GO-TNF ink) by ~10%. Respectively, the PCE value of the same device was higher by ~13%, leading to a champion efficiency of 8.71%. Our efforts proved that the rational design of graphene-based molecules could provide the opportunity for novel materials synthesis with tunable properties to be incorporated as additives, even as interlayers into organic, as well as hybrid solar cells, thus contributing to the realization of new generation high performance PV systems.Herein, we reported the synthesis and characterization of a new graphene-trinitrofluorenone derivative, named GO-TNF, as well as its operational role as the cascade material between the LUMO energy levels of PTB7 and PC


Various methods were used to verify the effect of bazedoxifene on VECs, including a cell viability assay, a wound healing assay, immunofluorescence staining, and western blotting. Our results showed that TNF-α could induce inflammatory damage to VECs, which manifested as upregulated expression of CD40, increased production of ROS, enhanced adhesion of THP-1 cells to VECs, and impaired viability and migration of VECs, while bazedoxifene could significantly reduce the endothelial damage caused by TNF-α. In addition, we found that an siRNA targeting CD40 dramatically alleviated the VEC damage induced by TNF-α. Therefore, we explored the potential relationship between bazedoxifene and CD40. Our data suggest that bazedoxifene has a protective effect against VEC damage induced by TNF-α and that its underlying mechanism may be related to the regulation of CD40.The inflammatory response and oxidative stress play key roles in the formation and development of atherosclerosis. Bazedoxifene is a new IL6/GP130 inhibitor recommended by the FDA for clinical use as a selective estrogen receptor modulator. However, its role in cardiovascular diseases has been poorly studied. In our study, we explored the mechanism of bazedoxifene's protective effect against inflammatory injury of vascular endothelial cells (VECs) stimulated by TNF- Atherosclerosis (AS) is a chronic inflammatory disease occurring in large and medium arteries and can cause progressive stenosis of diseased vessels. AS in the coronary and carotid arteries can lead to myocardial infarction, cerebral ischemia, and, in severe cases, death . The patα, IFN-γ, IL-6, etc.) and promote the expression of a variety of inflammatory genes, including E-selectin, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) [κB, MAPK, JAK/STAT, and PI3K/AKT pathways [α induces excessive ROS production and subsequent necrotic core formation in vascular lesions, inhibits VEC migration, increases mononuclear tissue factor expression, and activates platelet aggregation [CD40, a member of the tumor necrosis factor superfamily, is a type I transmembrane protein receptor that plays an important role in AS . Accordi(ICAM-1) . Increaspathways . In addipathways , 10. ROSpathways , 12. Forregation –16. TherκB signaling [α-induced endothelial inflammation by targeting CD40, indicating that bazedoxifene may be a new therapeutic drug for AS.Bazedoxifene is a novel IL6/GP130 inhibitor that has been approved by the FDA as a selective estrogen receptor modulator for the treatment of osteoporosis in postmenopausal women . Recent ignaling . HoweverκB p65  antibodies were obtained from Cell Signaling Technology . Anti-phosphor-ERK  was bought from Sigma . Anti-phosphor-STAT3-Tyr705  was bought from absin (China). And anti-STAT3  and anti-ICAM-1  were obtained from Servicebio .Anti-CD40 , anti-AKT , anti-GAPDH , and anti-ERK  antibodies were purchased from Proteintech . Anti-AKT-phosphor-Ser473  and NF-2 at 37°C. VECs were used to perform experiments from the third passage. VECs were washed with phosphate-buffered saline (PBS) twice when the cell density in a culture flask reached more than 80%. Then, cell contacts were disrupted with 0.25% trypsin, and VECs were subsequently centrifuged after the trypsinization process. The VECs were diluted with complete medium for further experiments.Human umbilical VECs were purchased from Central South University. VECs were cultured in DMEM  supplemented with 10% fetal bovine serum  and maintained in a relatively humidified atmosphere containing 5% CO5 cells/mL, and 100 μL of cell suspension was added to each well. VECs were pretreated with bazedoxifene  for 30 min, and TNF-α  was added to the medium for another 24 h of culture. MTT solution was added to each sample, and the cells were incubated for another 4 h. Subsequently, the blue-violet crystals were dissolved in 150 μL formalin solution. Finally, the samples were measured with a microplate reader at 490 nm.The MTT method was used to test cell viability. VECs were cultured in 96-well plates at a density of 1 × 10α was added to the medium for another 24 h of culture. THP-1 cells were stained with 5 μmol/L BCECF-AM  away from light for 30 min, and then, THP-1 cells were added to the VECs for coculture for another 30 min. Subsequently, the wells were washed with PBS twice to the remove cells that did not adhere to the VECs. Monocyte-endothelial cell adhesion was assessed and quantitatively analyzed by the Operetta High Content Imaging System .VECs were seeded into 96-well plates for 24 h. After pretreatment of VECs with bazedoxifene for 30 min, TNF-α was added to the medium, and the cells were cultured for 24 h. Dihydroethidium  is an indicator of ROS permeability, and ROS were labeled with 5 μg/mL dihydroethidium in the dark for 30 min. The generation of ROS was analyzed by the Operetta High Content Imaging System in the Cy3 channel.VECs were first plated in a 96-well culture plate for 24 h. Subsequently, they were pretreated with bazedoxifene for 30 min. Then, TNF-α was added to the medium, and the cells were cultured for 24 h. After 24 h, the wound was measured by inverted fluorescence microscopy.VECs were inoculated into a 6-well cell culture plate. Scratches of the same width were made in the VECs with a pipette tip when the cells were 100% confluent. The VECs were washed with PBS twice. After VECs were pretreated with bazedoxifene for 30 min, TNF-To silence CD40, VECs seeded in 6-well plates were transiently transfected with a small interfering RNA (siRNA) targeting CD40 or a negative control siRNA (Nc-siRNA) for 48 h. Nc-siRNA or siRNA was diluted with the reagents of the riboFECT™ Transfection Kit according to the manufacturer's instructions. The silencing effect of the siRNA was assessed by western blotting after transfection.According to the manufacturer's instructions, total cell lysates were extracted with RIPA lysis buffer containing 0.1% PMSF  and denatured at 100°C for 10 min. Total protein was separated by SDS-polyacrylamide gel electrophoresis  and then transferred onto PVDF membranes at 100 V for 80 min. Subsequently, the membranes were blocked with TBST containing 5% nonfat dry milk at room temperature for 60 min and incubated with the corresponding antibody overnight at 4°C followed by a horseradish peroxidase-conjugated secondary antibody (1 : 10000) for 60 min. Immunoreactive proteins were identified by Clarity™ Western ECL Substrate . Densitometric analysis of the band intensity was performed using Image Lab software (Bio-Rad).ΔΔCt- method. The sequences of primers were listed as follows: IL-6 forward 5′-GCAATAACCACCCCTGACCCAA-3′ and reverse 5′-GCTACATTTGCCGAAGAGCC-3′; GAPDH forward 5′-TGACTTCAACAGCGACACCCA-3′ and reverse 5′-CACCCTGTTGCTGTAGC CAAA-3′.Total RNA was extracted from VECs by using TRIzol reagent . Real-Time PCR was performed using the ABI 7300 Real-Time PCR system with the SYBR Green Real-Time PCR Kit . The relative expression of IL-6 was analyzed by the 2t-test was used for direct comparisons between two groups. The differences among the groups were compared using one-way analysis of variance (ANOVA). The least significant difference t-test (LSD-t) was used for pairwise comparisons after ANOVA. P < 0.05 was considered statistically significant.Statistical analysis was performed with SPSS software (version 16.0). The data are shown as the mean ± SD. Unpaired Student's α can be used to establish a stable inflammation model in VECs at a concentration of 10 ng/mL to 50 ng/mL [α  to confirm its effect on VEC activity. As shown in α had no significant effect on VECs. When the concentration of TNF-α was higher than 25 ng/mL, TNF-α decreased the activity of VECs in a dose-dependent manner. Therefore, we selected 25 ng/mL TNF-α to establish a model of VEC damage and explored the pharmacological effect of bazedoxifene on VECs stimulated by TNF-α. In our research, we used the MTT assay to evaluate the cytotoxicity of bazedoxifene. As shown in μmol/L, bazedoxifene reduced VEC activity in a dose-dependent manner, while low concentrations of bazedoxifene had no significant effect on cell activity. Subsequently, we attempted to confirm whether bazedoxifene can reverse the damage to VECs induced by TNF-α. In line with our expectations, 4 μmol/L bazedoxifene reversed the TNF-α-induced decrease in VEC viability  at the protein level, and bazedoxifene markedly reduced the upregulation of p-p65 expression triggered by TNF-α. Our results also showed that bazedoxifene alone could inhibit the expression of p-p65. We also evaluated the IL-6 mRNA level using Real-Time PCR. Interestingly, bazedoxifene did not inhibit the increase in IL-6 mRNA expression induced by TNF-α (To investigate the effect of bazedoxifene on TNF-by TNF-α . These dα-treated groups. In addition, the combination of siRNA-CD40 with bazedoxifene further reduced the expression of CD40 compared with single treatment (CD40 is a transmembrane protein, and its overexpression can induce the activation of multiple downstream AS-related pathways. Therefore, we further confirmed the role of CD40 in endothelial inflammation by a loss-of-function experiment. As shown in reatment .α, suggesting that bazedoxifene may play a protective role in VEC function by targeting CD40 (α treatment, inhibited monocyte adherence to VECs, and reduced ROS levels after TNF-α administration (Figures α administration, indicating that these effects of bazedoxifene on VECs are CD40-dependent.In this study, we found that the siRNA targeting CD40 and bazedoxifene were able to reverse the impairment in the migration of VECs induced by TNF-ing CD40 . We also Figures . Interesα could establish a stable VEC inflammation model [α used to establish the model may be different by specific cell conditions and techniques. In our research, TNF-α at a concentration of 25 ng/mL decreased cell viability to 80% approximately. Therefore, we selected 25 ng/mL TNF-α to establish an inflammation model of VECs. Bazedoxifene, a third-generation selective estrogen receptor modulator, is clinically used for the treatment of osteoporosis. Recent studies have confirmed that bazedoxifene is also an effective IL6/GP130 inhibitor [α-induced endothelial dysfunction. According to our data, low-dose bazedoxifene had no significant effect on the viability of VECs, while high-dose bazedoxifene showed some cytotoxicity. Therefore, bazedoxifene at different concentrations was applied to VECs to explore its potential pharmacological effects. Our data indicated that bazedoxifene significantly improved the survival rate of VECs after stimulation with TNF-α. Therefore, we believe that bazedoxifene may be an effective drug for the treatment of inflammatory damage to VECs and may protect the endothelium from the damage caused by various inflammatory diseases, such as AS.A large number of studies have shown that TNF-on model –25, 28. nhibitor , 29, 30.nhibitor . Howeverα in VECs. Given the role of the CD40/CD40L system in the inflammatory response and oxidative stress, bazedoxifene may play an anti-AS role by regulating CD40.CD40/CD40L is a pair of complementary transmembrane proteins that are mainly expressed in a variety of immune and nonimmune cells, including vascular smooth muscle cells, macrophages, lymphocytes, and VECs . ActivatκB p65 subunit from the cytoplasm to the nucleus; upregulate the expression of VCAM-1, ICAM-1, and other proinflammatory genes; promote the infiltration of inflammatory cells; and induce oxidative stress [α-induced activation of JAK/STAT3, MAPK/ERK, PI3K/AKT, and NF-κB in VECs. More importantly, bazedoxifene protected VECs from TNF-α-induced damage, suggesting that bazedoxifene may play a protective role in cardiovascular diseases through regulating the complex network connecting CD40, STAT3, AKT, ERK, and NF-κB signaling.CD40/CD40L may trigger multiple signaling cascades, including the JAK-STAT, PI3K/AKT, and MAPK/ERK pathways, which cause the translocation of the NF-e stress , 33–36. e stress . Therefoe stress . The anae stress , 37, bazα-induced VEC damage model. However, these effects were reversed by bazedoxifene. More importantly, bazedoxifene treatment did not further alter VEC migration, VEC viability, monocyte adherence, and ROS production in CD40 knockdown VECs after TNF-α administration, suggesting that the effects of bazedoxifene on VEC function are mediated via CD40.Increased CD40 and CD40L expression is found in human atherosclerotic plaques, and inhibition of CD40 via gene knockout in mice dramatically reduces the area of atherosclerotic plaques, promotes the stability of plaques, and decreases the risk of plaque rupture . Recentlα-induced endothelial damage model through anti-inflammatory and antioxidant pathways. Our work provided evidence that bazedoxifene protects the vascular endothelium by inhibiting the activation of the NF-κB p65, MAPK/ERK, PI3K/AKT, and JAK/STAT3 pathways induced by TNF-α. Bazedoxifene attenuates ROS production and the adhesion of monocytes to the damaged endothelium and restores the migration ability of VECs upon TNF-α treatment. The inflammation-mediated damaging effect of TNF-α on VECs can be abolished by knocking down CD40, indicating that bazedoxifene exerts its protective effect partially through targeting CD40. Our study was the first to investigate whether bazedoxifene's cardiovascular effects could be harnessed to treat endothelial dysfunction and to demonstrate for the first time that bazedoxifene targets CD40 to protect VECs from inflammatory damage. Our study also has extensive implications for the diagnosis and treatment of AS. However, this study also has some limitations. Our data indicate that bazedoxifene plays a role in endothelial protection through CD40, but they cannot explain the pharmacological mechanisms of bazedoxifene.In summary, we showed that bazedoxifene may play an endothelial protective role in a TNF-


This study aimed to investigate the possible incidence of visual light perceptions (VLPs) during radiation therapy (RT). We analyzed whether VLPs could be affected by differences in the radiation energy, prescription doses, age, sex, or RT locations, and whether all VLPs were caused by radiation. From November 2016 to August 2018, a total of 101 patients who underwent head-and-neck or brain RT were screened. After receiving RT, questionnaires were completed, and the subjects were interviewed. Random forests (RF), a tree-based machine learning algorithm, and logistic regression (LR) analyses were compared by the area under the curve (AUC), and the algorithm that achieved the highest AUC was selected. The dataset sample was based on treatment with non-human units, and a total of 293 treatment fields from 78 patients were analyzed. VLPs were detected only in 122 of the 293 exposure portals (40.16%). The dataset was randomly divided into 80% and 20% as the training set and test set, respectively. In the test set, RF achieved an AUC of 0.888, whereas LR achieved an AUC of 0.773. In this study, the retina fraction dose was the most important continuous variable and had a positive effect on VLP. Age was the most important categorical variable. In conclusion, the visual light perception phenomenon by the human body during RT is induced by radiation rather than being a self-suggested hallucination or induced by phosphenes. The authors found that sleeping animals experienced changes due to radiation within less than 1 second of exposure [There is a long history of research on radiation-induced visual light perceptions (VLPs). In 1895, after discovering the X-ray, Wilhelm Röntgen conducted an experiment to determine whether X-rays could generate phosphenes, and verified that X-rays could generate VLPs; however, he could not definitively confirm that phosphenes were induced by X-ray irradiation [exposure ,3.et al. discovered that the phosphene mechanisms of eye proton therapy (PT) might be related to those of space radiation. The authors concluded that phosphenes could be produced by two mechanisms: indirect interaction of Cherenkov light in the eye due to scattering and direct simulation, or the excitation of excessive free radicals in nerve fibers located in the posterior part of the eye, close to the retina [For the first time during a space flight, the astronauts of the 1969 Apollo Mission reported experiencing VLPs, such as light flashes. The main contributory factors possibly included cosmic rays in outer space that directly stimulated the retinal photoreceptors or VLPs induction in the occipital cortex of the brain . In 2016e retina .Thus, VLPs were mostly experienced by either astronauts  or patieMachine learning algorithms incorporate statistics to identify patterns that could be used to make predictions in the dataset , and havThis study was conducted with an aim to identify the main factor that induces VLPs in patients who receive radiation therapy. We compared logistic regression and random forest analyses to select the best-performing method for predicting the outcome in terms of the receiver operating characteristics (ROC) curve and to discuss the relationship between the outcome and causative factors as these algorithms fit the dataset to not only predict the outcome but also to ascertain how the main factors modulate the outcome.This prospective cohort case-control study was approved by the Institutional Review Board of Taipei Veterans General Hospital (2016-09-025C). Between November 2016 and March 2017, we screened 101 patients for study participation, and 23 were subsequently excluded after failing the visual perception light switch test and the visual perception radiation field time point verification test. During the fractional treatment, after the completion of the questionnaire, the timepoint verification tests of light and VLPs were conducted with the consent of the subjects. The test was performed by delivering the treatment with all lights turned off in the treatment room and asking the patient to identify whether they had experienced VLPs. The purpose of this test was to exclude VLPs caused by the treatment room light or the field light during irradiation. The research workflow and patient disposition schema are shown in The 78  participants included 48 patients who were receiving brain irradiation and 30 head-and-neck RT patients. The brain and head-and-neck RT patients were further subdivided into two groups according to their dose schemes and radiation fields as: an experimental group with VLPs (n = 39) and control group without VLPs (n = 39). Subsequently, 6/10 MV photon beams were delivered to patients in the experimental group using A-brand or B-brand dual-energy medical linear accelerators, and the dose rate (DR) was limited to 400–1600 MU/min. Subjects were asked to complete the study questionnaires and interviews after they had received RT. The treatment plan of the corresponding patient in the treatment planning system (TPS) was then reviewed according to the results of the questionnaire.Patients who were eligible for study participation included men or women aged 20–85 years, with various levels of education, who were fully conscious, had no mental disorders, could communicate with medical staff and fill out questionnaires, demonstrated normal visual function, and were irradiated in either the brain or the head-and-neck area.Based on the questionnaire results, the E-TPS and the V contouring software was used to identify the area that induced VLPs during irradiation by reviewing and analyzing the patients’ output dose, DR, beam angle, intra-fractional dynamic radiation field, and isodose lines, which delineate the visual path and irradiation field of the brain. After the completion of the questionnaire, information was collected in the TPS to analyze the corresponding dose distributions.The timepoint for the switch light test, with the participant’s consent, was specified as the completion of the questionnaire in the study process flowchart. The duration for which the lights are turned on will be determined in conjunction with each participant’s treatment plan, mainly before and after the initiation of RT, whereas the lights will only be turned off during the actual RT of the subjects. Therefore, comparing the response to the questionnaire that was previously filled out by each subject with the VLPs response that was recorded during the treatment/the sub-treatment was used to check whether each participant’s VLPs would change as a result of the treatment. White and yellow bulbs were used in the room, the lamp seat was distributed on the ceiling of the treatment room where the machine is installed, and the indoor bulbs (brightness 27 Watts) are situated 2–4 m away from the participants.From this test, we discovered that the subjects could be further assigned to three categories. The first category included patients who experienced VLPs regardless of whether the lights were on or off. The second category included patients who did not experience any VLPs regardless of whether the lights were on or off. The third category included patients whose VLPs disappeared once the lights were turned off. Consequently, participants in the third category were excluded from the analysis to ensure that they would not be a source of bias in the data collection. In addition, the field timepoint verification test with VLPs was performed by turning the treatment room light on and off during photon irradiation and asking the patients to notify the staff by raising their hands when they experienced any VLPs in the treatment field.In the TPS, we adopted the point-dose method to analyze the fraction dose (FD) that was delivered to certain parts of the optic path to facilitate a comparison on specific points at different areas of the optic path . To detet-test for continuous variables and the chi-square test for categorical variables, and differences were considered statistically significant if the p-value was less than 0.05.Statistical analyses were conducted by using SAS 9.4  and RStudio version 1.2.5001 . The frequencies and percentages were calculated for categorical variables, and the means and standard deviations were computed for continuous variables. Between-group comparisons of the baseline characteristics of participants with and without VLPs were analyzed by the Student’s p-values can be obtained directly from the regression output. The difference between them is that linear regression analyzes continuous dependent variables, and LR is used for binary or multinomial dependent variables and it estimates the probability of occurrence of the interested outcome [Logistic regression (LR) has a similar model as linear regression, and the effect estimates and  outcome ,21.In the random forest model, we incorporated the random forest package developed by Breiman and Culter in the R environment . The ranWe used four measures, including accuracy, sensitivity, specificity, and area under the curve (AUC), to evaluate the prediction performance. Accuracy, sensitivity, and specificity are defined as follows:ectively .x-axis represents 1 − specificity and y-axis denotes sensitivity. The curves with higher sensitivity and specificity are close to the upper left corner of the diagram, and those with less sensitivity and specificity are close to the diagonal line. Previous studies have suggested that the AUC is a better indicator for comparing and measuring the performance of classification algorithms [Sensitivities and specificities are shown in the ROC curve diagram wherein the gorithms . This isgorithms .t-test to examine whether the means showed significant differences between continuous variables and VLPs. The means of all variables, except FD, showed significant differences.A total of 293 samples filled by 78 participants were collected, and 41.64% of the participants had experienced VLPs. For categorical variables, we used chi-square tests to examine whether two variables were independent. As shown in Furthermore, we compared random forests to LR and selected the best model according to the comparison of AUCs. To avoid overfitting of predictive performance, it is common in machine learning to randomly divide the original dataset into a training set and a test set, wherein only the training set is incorporated to develop a prediction model, and the test set is used to evaluate the real predictive performance. We randomly divided the original dataset into 80% as the training dataset, with 235 observations, and 20% as the test set, with 58 observations. The random forests built 5,000 trees and randomly sampled three variables at each split in each tree. In the results, random forests outperformed LR in both training and test sets in terms of accuracy, sensitivity, and AUC, although the specificity was lower than in the LR model . In the To further depict the importance of variables, x-axis of individual plot, respectively. The y-axis shows the changes in the fraction of vote for probability of VLPs in each variable. The continuous variables are retina FD and FD, and they exhibit significant nonlinear relationships. As shown in This study focused on whether the VLPs experienced by patients receiving RT was caused by phosphenes or was an actual result of radiation. The basis of this research theory is to construct the visual light perception phenomenon during the RT of the brain and head and neck. This study analyzed whether VLPs could be affected by different radiation energy, prescription doses, age, sex, or RT locations, and whether all VLPs were caused by radiation. Phosphenes can be considered to be a kind of light hallucination, which is characterized by the ability to sense light when no visible light enters the eyes. The normal VLPs of the human body is produced by light conversion. In addition, phosphenes can be produced in other ways without light stimulation; for example, they also appear in the research records of transcranial magnetic stimulation .The direct priming mechanism of the retina is the same as that of visible light . In our In our subject group, categorical variables of patients younger than 65 years had a higher incidence of visual perception than those from patients older than 65 years . The queThe primary beams used in this study are high-energy X-ray photon beams, the characteristics of which are different from those of the proton, heavy-ion, and electron beams. From our research questionnaire and statistics , it seemRadiation was induced in the eyes mostly by the indirect interaction between radioactive decay and nucleus . In the A previous study suggested that patients would experience VLPs regardless of whether the eyes were open or closed, which is consistent with our questionnaire results. Furthermore, our results indicate that the retina FD is significantly correlated with the incidence of VLPs, which is contradictory to the conclusion in the literature that most of the VLPs happen in the optic path areas beyond the retina ,31.Finally, the limitations of this study mainly include the shape or the type of light, the time coordinate axis of induced VLPs during treatment, and other variables that were not evaluated in this study. The CR observed in radiotherapy is affected by predictable factors in patient imaging, such as radiation beam characteristics, tissue thickness, entrance/exit geometry, curved surface effect, curvature, and imaging angle. Experiments have found that changes in CR signal have varying degrees of influence on tissues . HoweverRandom forest is a good and stable methodology in binary classification. In the comparison of two models measured by accuracy and AUC, random forests outperform LR in terms of the predictive ability. In this study, the most important continuous variable was the retina FD, and the most important categorical variable was age. The VLPs phenomenon in the human body during RT was indeed induced by radiation rather than being a self-suggested hallucination or induced by phosphenes. Radiation-induced VLPs often demonstrates complicated conditions and warrants special attention. Whether radiation-induced VLPs can lead to side effects, such as visual impairment, requires further investigation.S1 Dataset(CSV)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Ebola virus infections lead to severe hemorrhagic fevers in humans and nonhuman primates; and human fatality rates are as high as 67%–90%. Since the Ebola virus was discovered in 1976, the only available treatments have been medical support or the emergency administration of experimental drugs. The absence of licensed vaccines and drugs against the Ebola virus impedes the prevention of viral infection. In this study, we generated recombinant baculoviruses (rBV) expressing the Sudan virus (SUDV) matrix structural protein (VP40) (rBV-VP40-VP40) or the SUDV glycoprotein (GP) (rBV-GP-GP), and SUDV virus-like particles (VLPs) were produced by co-infection of Sf9 cells with rBV-SUDV-VP40 and rBV-SUDV-GP. The expression of SUDV VP40 and GP in SUDV VLPs was demonstrated by IFA and Western blot analysis. Electron microscopy results demonstrated that SUDV VLPs had a filamentous morphology. The immunogenicity of SUDV VLPs produced in insect cells was evaluated by the immunization of mice. The analysis of antibody responses showed that mice vaccinated with SUDV VLPs and the adjuvant Montanide ISA 201 produced SUDV GP-specific IgG antibodies. Sera from SUDV VLP-immunized mice were able to block infection by SUDV GP pseudotyped HIV, indicating that a neutralizing antibody against the SUDV GP protein was produced. Furthermore, the activation of B cells in the group immunized with VLPs mixed with Montanide ISA 201 was significant one week after the primary immunization. Vaccination with the SUDV VLPs markedly increased the frequency of antigen-specific cells secreting type 1 and type 2 cytokines. To study the therapeutic effects of SUDV antibodies, horses were immunized with SUDV VLPs emulsified in Freund’s complete adjuvant or Freund’s incomplete adjuvant. The results showed that horses could produce SUDV GP-specific antibodies and neutralizing antibodies. These results showed that SUDV VLPs demonstrate excellent immunogenicity and represent a promising approach for vaccine development against SUDV infection. Further, these horse anti-SUDV purified immunoglobulins lay a foundation for SUDV therapeutic drug research. Currently, the latest devastating outbreak of the Ebola virus disease in the DRC is ongoing. As of today, a total of 3250 cases were reported, including 3133 confirmed and 117 probable cases, of which there have been 2174 fatalities with a mortality rate of 67% (WHO situation report https://www.who.int/csr/don/24-october-2019-ebola-drc/en/). Ebola virus infections lead to severe hemorrhagic fevers in humans and nonhuman primates, with human fatality rates of up to 90% . The firEbolavirus [The Ebola virus is a non-segmented negative-strand RNA virus belonging to the family Filoviridae and the genus olavirus . The genolavirus . GP, theolavirus . Severalolavirus ,12, DNA olavirus ,14, humaolavirus , rabies olavirus ,17, cytoolavirus , Venezueolavirus  and viruolavirus . There aolavirus  and chAdolavirus . At the olavirus  and immuolavirus . VLPs represent a promising vaccine platform for a diverse array of viruses that include influenza virus, rotoviruses, noroviruses, HIV, hepatitis B virus, parvoviruses, rift valley fever virus, human papillomavirus and filoviruses ,26,27. VThere are currently no approved specialized drugs or vaccines to protect against SUDV disease outbreaks, and thus there is an urgent need for the development of an efficacious, safe and economically viable vaccine or therapeutic antibody to control SUDV infections. Here, we report that production of SUDV VLPs has been accomplished in insect cells by the co-infection with recombinant baculoviruses rBV-GP-GP and rBV-VP40-VP40, and evaluate the ability of SUDV-VLPs to induce SUDV-specific humoral and cellular immune responses in vaccinated mice. Further, horses were immunized with SUDV VLPs, and horse serum was purified to prepare purified immunoglobulins and the purified immunoglobulins had neutralizing activity.The 293T and Huh7 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum and penicillin-streptomycin . These cells were maintained at 37 °C. The Spodoptera frugiperda (Sf9) cells  were cultured in suspension in SF-900 II serum-free medium  supplemented with 10% FBS and penicillin-streptomycin. These cells were maintained at 27 °C.Female BALB/c mice, 6–8 weeks old, were purchased from Changchun Institute of Biological Products . All experiments involving mice adhered to the principles of the Welfare and Ethics Committee of the Military Veterinary Research Institute at the Academy of Military Sciences. The mice were provided ad libitum access to sterilized water and food throughout the study and were vaccinated under BSL-2 conditions. Healthy male horses 2–6 years old and 400–500 kg were provided by Red Hill Military horse farm . Horse studies were conducted with prior approval from the Animal Welfare and Ethics committee of the Institute of Military Veterinary, Academy of Military Medical Sciences (permit number SCXK-2012-017), according to Horse Quarantine and Immunization Protocols for Equine Serum Production.E. coli DH10Bac competent cells to generate recombinant bacmids. Sf9 cells were transfected with recombinant bacmids using Cellfectin® II Reagent  according to the manufacturer’s instructions. Transfected cells were incubated at 27 °C for 4 d and harvested, the recombinant baculoviruses rBV-GP-GP and rBV-VP40-VP40 by collecting the tinfected-Sf9 cells and supernatant.The cloning and construction of the recombinant bacmids, pFastBacDual-GP-GP, and pFastBacDual-VP40-VP40, were carried out. Briefly, we synthesized the full-length GP and VP40 genes  accordinE. coli BL21(DE3)  and the transformants selected on Luria-Bertani (LB) agar plates with 100 μg/mL kanamycin. A single clone was picked and inoculated into 4 mL of LB medium with 100 μg/mL kanamycin at 37 °C for overnight growth. Expression was induced with the addition of 0.4 mM IPTG when the optical density at 600 nm (OD600) reached 0.6. The proteins were purified using a HisPurTM Ni-NTA Spin Purification kit  according to the manufacturer’s instructions. To visualize the expression of the purified proteins, samples were resolved on a 12% SDS-polyacrylamide gel (SDS-PAGE) and stained with Coomassie blue, and protein concentrations were determined using a bicinchoninic acid (BCA) assay  followed by analysis at an absorbance of 570 nm; bovine serum albumin  was used as the protein standard.The SUDV GP (158~368 aa) and VP40 proteins were generated by a prokaryotic expression system. Briefly, the synthesized genes encoding the SUDV GP (158–368 aa) and VP40 proteins were separately inserted into the prokaryotic expression vector PET 30a (+) by using BamHI and NotI, in-frame with the 6×His-tag on the C- and N-terminus to construct PET 30a (+) -GP-his-C/N and PET 30a (+) -VP40-his-C/N. After the recombinant plasmids were confirmed by restriction digest, the two recombinant plasmids were transformed into Polyclonal antisera against SUDV GP (158~368 aa) or VP40 were prepared by immunizing BALB/c mice with 10 μg purified GP or VP40 recombinant proteins twice at 2-week intervals, and harvesting the mouse serum, which were mouse polyclonal antisera against SUDV GP (158~368 aa) or VP40.The Sf9 cells were infected with the fourth generation recombinant baculovirus rBac-GP-GP or rBac-VP40-VP40 for approximately 48 h, infected Sf9 cells were fixed with 4% paraformaldehyde for 15 min at the room temperature. Infected-Sf9 cells were washed with PBS and blocked with PBS containing 5% BSA . Then, the cells were incubated with a 1:100 dilution of a mouse polyclonal antisera against SUDV GP or a mouse polyclonal antisera against SUDV VP40 (generated in our lab) for 1 h at 37 °C, and then washed with PBS and incubated with FITC-conjugated goat anti-mouse IgG  containing 1% diluted Evans blue for 1 h at 37 °C; then the infected Sf9 cells were washed with PBST (containing 0.05% Tween-20) three times and were observed with a fluorescence microscope. The control cells were incubated with the two primary antisera at the same time. Baculovirus titers were determined using a BacPAKTM Baculovirus Rapid Titer kit .g. The crude VLPs were then concentrated by ultracentrifugation at 30,000× g for 1 h, and the pellets were resuspended in PBS before purification with a 10–30–50% discontinuous sucrose gradient. Bands between 30–50% sucrose were collected and resuspended in endotoxin-free PBS, and the VLPs concentrations were determined using a BCA assay  followed by analysis at an absorbance of 570 nm. The VLP preparations were not tested for endotoxin after production.To generate SUDV VLPs, Sf9 cells were co-infected with recombinant baculoviruses rBV-GP-GP and rBV-VP40-VP40 at different ratios of 1:1, 1:2, 1:2.5, 1:3, 2:1, 2.5:1, or 3:1. The SUDV VLPs were harvested at 4-d post-infection. To purify the VLPs, culture supernatants were harvested and spun at 2000× Purified SUDV VLPs were processed and examined by Western blotting and transmission electron microscopy. For Western blotting, aliquots containing 10 μg of total protein were diluted with reducing buffer and denatured by heating at 95 °C for 10 min. Proteins were separated in 12% acrylamide gels, before they were transferred onto polyvinylidene fluoride (PVDF) membranes  under denaturing conditions. For protein detection, two polyclonal antisera were used: mouse anti-SUDV GP polyclonal antisera and mouse anti-SUDV VP40 polyclonal antisera were mixed at a dilution of 1:1500 as a primary antibody for blotting; a goat anti-mouse IgG HRP-conjugated antibody  was used at a dilution of 1:5000 as a secondary antibody. Negative staining transmission electron microscopy (TEM) was used to analyze the shape and size of purified SUDV VLPs. In short, 30 μL of sucrose gradient-purified SUDV VLPs were fixed for 15 min on carbon-coated formvar grids, grids were washed with 30 μL PBS and then treated with 1% phosphotungstate acid for 5 min. Grids were left to air dry and observed by using a HITACHI H-7650 transmission electron microscope.n = 6 per group) and were vaccinated intramuscularly. Mice in group one were vaccinated with PBS, mice in group two were vaccinated with Montanide ISA 201 VG (ISA 201) adjuvant , mice in group three were vaccinated with 20 μg of SUDV VLPs-only, and mice in group four were vaccinated with 20 μg of SUDV VLPs mixed with an equal volume of Montanide ISA 201 VG (ISA 201) adjuvant, and all groups were vaccinated twice at 3-week intervals . The mouse sera were collected at two-weeks after every immunization. One week after the booster immunization, splenocytes from 3 mice of each group were isolated. In batch two, nine mice were randomly distributed into three groups (n = 3 per group)  and were vaccinated intramuscularly. One week after the primary immunization, the inguinal lymph nodes were collected from 3 mice.A total of two batches of BALB/c mice  were purchased from the Changchun Yisi Laboratory Animal Technology Co., Ltd.  and immunized. In batch one, 24 mice were randomized into four groups , 2–6 years old, 400–500 kg in weight and without detectable antibodies against SUDV detected by indirect ELISA, were supplied by Red Hill Military horse farm. The horses were multipoint injected subcutaneously in the rear area with 1.0, 2.0, 3.0, 3.0, or 4.0 mg of purified SUDV-VLPs for a total of 5 times, primary immunization mixed with an equal volume of Freund’s incomplete adjuvant  and booster immunization mixed with an equal volume of Freund’s complete adjuvant, maximum immune volume does not exceed 4 mL, with boosting at two week intervals . The horse sera were collected before each immunization and stored at −20 °C for further studies.2SO4. Optical density (OD) values were measured at a wavelength of 450 nm (OD450). After each incubation step, ELISA plates were washed five times with PBST. Mean OD values were considered positive if the OD values were more than two times the value of the negative control.The mice and horse serum samples were collected two weeks after each immunization. Levels of SUDV GP-specific antibodies were detected by indirect ELISA. Briefly, 100 μL of purified -prokaryotic expressed SUDV GP was coated on ELISA plates at a concentration of 10 µg/mL overnight at 4 °C, and then the plate was blocked with 2% BSA for 2 h at 37 °C. The serum was added (100 μL/well), and it was 2-fold serially diluted in 2% BSA; plates were subsequently incubated for 1.5 h at 37 °C, and 100 μL of HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-horse IgG , diluted 1:10,000 in 2% BSA was added to each well. After one hour of incubation at 37 °C, 100 μL of 3,3′,3,5′-tetramethylbenzidine (TMB)  substrate solution was added to each well and then was stopped with the addition of 50 μL of 0.5 M HTM 3000 Transfection Reagent . After 2 days, the supernatant containing the pseudotyped viruses was harvested, and the titer was determined in Huh7 cells as previously described [50 of pseudotyped viruses for 0.5 h at 37 °C and were then added to Huh7 cells. After 4 h, the inoculum was removed and replaced with fresh media. Then, cells were lysed at 48 h with cell lysis buffer, that was followed by the addition of 100 μL of luciferase substrate  to determine luciferase activity. The luciferase activity of the samples was measured with the Infinite M200 Microplate Spectrophotometer . The percent inhibition rate was calculated as previous showed [A pseudotyped virus neutralization assay was performed to test the neutralzing antibodies. Recombinant lentiviral vectors that express SUDV glycoprotein and carry a luciferase reporter gene were produced as described previously , with soescribed . For tes2 for 48 h, the frequencies of splenocytes producing IFN-γ or IL-4 were measured using mouse ELISpot kits  according to the manufacturer’s instructions. Spot-forming cells (SFCs) were enumerated by an automated ELISpot reader .One week after the booster immunization in batch one, three mice from each group were randomly selected and euthanized. Their spleens were harvested into a tissue culture dish, and each spleen was roughly minced and pressed through a 5-mL syringe. The cells were filtered through a 40-μm filter  and centrifuged at 2000 rpm for 10 min at the room temperature. The cells were processed by resuspension in a red blood cell lysis buffer and centrifuged at 2000 rpm for 10 min at twice the room temperature. The splenocytes were harvested and washed with RPMI 1640 medium  containing 10% FBS . Then, the splenocytes were cultured in RPMI 1640 medium containing 10% FBS and stimulated with or without the purified SUDV-GP antigen (10 μg/mL). Following incubation at 37 °C in 5% COThe levels of cytokines in the supernatant of stimulated splenocytes were detected by commercial ELISA. Splenocytes were stimulated as described above and were then incubated for 48 h. The cell culture supernatant was collected by centrifugation at 3000 rpm for 15 min, and the manufacturer directions were followed to detect IL-2, IL-4, IL-10, IFN-γ, or TNF-α by ELISA kits .Inguinal lymph node samples were harvested from batch two vaccinated mice 7 days after the primary immunization. Lymphoid cells were harvested into a tissue culture dish, and were roughly minced and pressed through a 5 mL syringe. The cells were filtered through a 40-μm filter . Then, prepared in PBS with 2% FBS and were stained with equal volumes of 1:250 dilutions of anti-CD19-APC and anti-CD40-FITC antibodies  for 30 min at 4 °C; the labeled B cells were then washed twice with PBS with 2% FBS and analyzed using a FACSAria TM Cell Sorter .For equine antisera, the blood was taken from the jugular vein of #392 immunized horses, and the sera were collected. The horse serum was diluted 8-fold with PBS and then added to a saturated ammonium sulfate solution until the ammonium sulfate concentration was 50%. The solution was allowed to stand at 4 °C for 3 h, and it was centrifuged at 5000 rpm for 20 min. After removing the supernatant, the precipitate was dissolved in PBS, and saturated ammonium sulfate was added until the concentration of ammonium sulfate was 33%. The solution was allowed to stand at 4 °C for 3 h, and centrifuged at 5000 rpm for 20 min. The precipitate was dissolved in PBS and dialyzed against PBS at 4 °C for 18 h to remove the ammonium salt.The treatment of all mice was in accordance with the Welfare and Ethical guidance of Laboratory Animals of China (GB 14925-2001). The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Veterinary Medicine of the Military Academy of Sciences .E. coli BL21(DE3) cells. Recombinant proteins SUDV GP and VP40 were experessed in cells after added 0.4 mM IPTG. The proteins were purified by 6×His-tag affinity chromatography and detected by SDS-PAGE analysis (The sequences encoding the GP (158–368 aa) and VP40 proteins of SUDV were cloned into the prokaryotic expression vector PET 30a (+), resulting in the plasmid PET 30a (+) -GP-his-C/N and PET 30a (+) -VP40-his-C/N . Correct insertion of the sequences in the recombinant plasmid was confirmed by restriction digest mapping analysis and DNA sequencing. The recombinant plasmids PET 30a (+) -GP-his-C/N and PET 30a (+) -VP40-his-C/N were transformed into analysis . The purTo identify the successful rescue of the recombinant baculovirus and effectively expressed the SUDV GP and VP40 proteins, the fourth generation of recombinant baculovirus were detected by IFA. Sf9 cells infected with recombinant baculovirus rBac-GP-GP or rBac-VP40-VP40 were incubated with mouse anti-GP polyclonal or anti-VP40 polyclonal antisera respectively, and then incubated with FITC-conjugated goat anti-mouse IgG. The immunofluorescence results showed that Sf9 cells infected with recombinant baculovirus rBac-GP-GP or rBac-VP40-VP40 were shown in specific green D,E and nSUDV VLPs were generated by co-infecting with recombinant baculoviruses rBV-GP-GP and rBV-VP40-VP40 in Sf9 cells. The SUDV VLPs were harvested from the culture supernatant and purified as described in the Material and Methods. To further characterize the structure of the SUDV VLPs, we examined purified material by transmission electron microscopy. A negative staining study revealed that the SUDV VLPs were found to mimic the naive virus in structure and size; they were approximately 80 nm in diameter and 800–1500 nm in length A.The identity of the protein component of the purified SUDV VLPs was assessed by Western blot analysis with mouse anti-SUDV GP polyclonal antisera and mouse anti-SUDV VP40 polyclonal antisera. Both VP40 and GP proteins are present in the VLPs B. These To detect a SUDV-VLP-induced humoral immune response in vaccinated BALB/c mice, indirect ELISA and virus neutralization assay were performed to evaluate the production of SUDV GP-specific antibodies and neutralizing antibodies. At the fifth week of the immunization, the sera from mice treated with 20 μg SUDV VLPs mixed with an ISA 201 adjuvant group had high titers of SUDV specific IgG antibodies with endpoint titers up to 1:81,920 C, virus-ELISpot assays were used to evaluate the antigen-specific cellular immune response in vaccinated mice. Since 20 μg VLPs-only did not stimulate the production of specific antibodies and neutralizing antibodies, the VLPs-only group was removed in the cellular immune response experiment. The results from the IL-4 ELISpot assay are shown in To further investigate the antigen-specific cellular immune responses induced by SUDV VLPs, cytokines secreted by splenocytes were measured by commercial ELISA kits. Splenocytes were harvested from mice one week after the booster immunization and stimulated with Prokaryotic-expressed SUDV GP (158~368 aa). Cytokines secreted into the supernatant by splenocytes were assessed. Type 1 cytokines such as IL-2 A, IFN-γ +CD40+) was significantly higher in the 20 μg SUDV VLPs mixed with ISA 201 adjuvant group than in the PBS group and ISA 201 adjuvant group.To study whether the vaccine can induce B cell activation, inguinal lymph nodes were collected from the mice vaccinated with PBS, ISA 201 adjuvant and 20 μg SUDV VLPs mixed with ISA 201 adjuvant. The result showed  that theThe humoral immune response in horses was assessed at 2, 4, 6, and 8 weeks after immunization B. Serum The mechanism of natural immunity against EBOV remains unclear. The efficacy of different types of vaccines may vary depending on the vaccine platform: antibody is the major immune correlate factor of the rVSV-ZEBOV vaccine . MeanwhiThe impact of SUDV outbreaks in recent years, and the potential for viral spread to non-endemic regions or countries, makes the development of safe and efficacious vaccines urgent.Previous findings revealed that SUDV VLPs could be readily assembled by the co-expression of baculoviruses expressing GP, NP and VP40 in insect cells . Here, wIn our study, we generated recombinant baculaviruses rBV-GP-GP and rBV-VP40-VP40 using the pFastBacDual vector to increase the protein production utilizing the dual promoter. To determine the optimal proportion of two recombinant baculaviruses rBV-GP-GP and rBV-VP40-VP40, we also tried to co-infect Sf9 cells with recombinant baculaviruses rBV-GP-GP and rBV-VP40-VP40 at ratios of 1:1, 1:2, 1:2.5, 1:3, 2:1, 2.5:1, and 3:1. At a ratio of 2.5:1, the morphology of VLPs was similar to the native virus, as assessed by TEM A. The WeThe immunization results depicted the failure of the group vaccinated with 20 μg of SUDV VLPs-only to stimulate the production of humoral immune responses in mice, while the group vaccinated with 20 μg of VLPs mixed with an ISA 201 adjuvant could stimulate the production of humoral immunity in mice. Presumably, immunization with 20 μg of SUDV VLPs-only is an insufficiently low dose of an immunogen that cannot stimulate the mice to produce an immune response, and the use of an ISA 201 adjuvant with SUDV VLPs might cause low-dose immunogens to initiate a response, and using with adjuvant can save antigen amount C. The saBoth humoral and cellular responses are indispensable for providing protection. IFN-γ is a Th1-type cytokine involved in the antiviral action of cellular immune responses and IL-4 is mainly produced by Th2 cells and is associated with humoral immune responses. The SUDV VLPs could effectively stimulate Th1 and Th2 cytokine production in vaccinated mice A,B. The In addition, we have conducted research on therapeutic antibodies with the goal of preparing antibodies for the post-exposure treatment of SUDV. Horses were selected as immunized animals in our study because horse anti-immunoglobulins have been used in the treatment of various viral infections ,38,39. SWe tested the SUDV VLPs immunogenicity and neutralizing activity of hours purified IgG by using pseudo typed viruses, and efficacy studies still need to be performed. These SUDV VLPs vaccine and horse purified IgG provide ideas for the development of vaccines and therapeutic antibodies that could prevent and treat SUDV infections. The Ebola epidemic is still spreading, vaccination is an effective means of preventing and controlling the outbreaks, and effective antibodies represent key drugs for the treatment of Ebola patients. Therefore, further Ebola vaccine and therapeutic antibody research are needed.