NPM1, DNMT3A, and IDH2 mutations were more common while biallelic CEBPA and IDH1 mutations were less seen in e‐AML patients. s‐AML patients carried a higher frequency of KMT2A‐AF9. In treatment response and survival, e/s‐AML conferred a lower complete remission (CR) rate and shorter duration of event‐free survival (EFS) and overall survival (OS) compared with young patients. In multivariate analysis, s‐AML was an independent risk factor for OS but not EFS in the whole cohort. Importantly, intensive therapy tended to improve the survival of e/s‐AML patients without increasing the risk of early death, and hematopoietic stem cell transplantation (HSCT) could rescue the prognosis of s‐AML, which should be recommended for the treatment of fit patients.To illustrate the clinical and genetic features of elderly and secondary acute myeloid leukemia (AML) patients, we compared 145 elderly AML (e‐AML) and 55 secondary AML (s‐AML) patients with 451 young de novo AML patients. Both e‐AML and s‐AML patients showed lower white blood cell (WBC) and bone marrow (BM) blasts at diagnosis.  In addition, age is another important clinical feature, which exerts negative effect on the disease. More importantly, AML is increasingly considered as a senile disease, which was reported of a median age of 66 in the United StatesMore importantly, the treatment of e/s‐AML remains controversial. Various modalities, such as hypomethylation agents as exemplified as decitabine and azacitidine, and low doses chemotherapy were tried in this group of patients; however, no therapeutic regimen was proved to be significantly superior to traditional chemotherapy. To some extent, the treatment decision was strongly dependent on the fitness of AML patients.In this study, we examined genetic alterations and post‐treatment minimal residual diseases (MRD) in order to illustrate their distribution and prognostic impact in e/s‐AML and to provide treatment recommendations for those patients.22.1From January 2013 to December 2017, a total of 651 adult patients (18 years old or above) with newly diagnosed non‐M3 AML at Shanghai Institute of Hematology (SIH) were executively enrolled in this study, among which, 55 patients were diagnosed as s‐AML . Cytogenetic risk stratification was based on 2017 European LeukmiaNet (ELN) recommendations.This study was approved by the ethic committee of Ruijin Hospital. All patients had given informed consent for both treatment and cryopreservation of bone marrow (BM) and peripheral blood according to the Declaration of Helsinki.2.22 or daunorubicin 45‐60 mg/m2, D1‐3; cytarabine 100 mg/m2 D1‐7) were given as the initial induction therapy. If CR was achieved, four cycles of high‐dose cytarabine (2 g/m2) was given as consolidation. For e‐AML (60 years and older) and s‐AML patients, the treatment was mainly decided by the physician in consideration of the fitness of patients and risk of disease. Fit patients received treatment similar to young patients but reduced cycles of consolidation to 2 cycles of high‐dose cytarabine; unfit patients received “3 + 7” regimens with reduced dose, hypomethylation treatment or palliative treatment according to the physician's decision.For young de novo patients (younger than 60 years old), standard intensive “3 + 7” induction regimens  was defined by the criteria of the International Working Group.33.1P = .041), most of whom had a previous history of breast carcinoma (43%). Older age (P < .001), lower white blood cell (WBC) count (P = .031), hemoglobin , and BM blasts (P < .001) were observed in s‐AML as compared with young patients. Similarly, elderly patients showed lower WBC (P = .036) and BM blasts (P = .009) at diagnosis. In WHO subtype distribution, higher frequency of pure erythroid leukemia was seen in s‐AML (P = .013). Both e‐AML and s‐AML patients received less intensive induction, but more hypomethylation treatment and palliative treatment than younger patients .The baseline characteristics of patients were shown in Table 3.2P = .011). Favorable cytogenetic alterations were less frequent in both elderly and secondary patients  as compared with young patients.In cytogenetic classification, elderly patients had a significantly higher proportion of intermediate risk cytogenetics . A higher frequency of NPM1 (P = .003), DNMT3A (P = .015), and IDH2 (P = .004) mutations, but lower frequency of biallelic CEBPA  and IDH1 (P = .038) mutations were observed in elderly patients. In addition, s‐AML patients carried KMT2A‐AF9 (P = .007) more frequently when compared with young de novo patients . Moreover, s‐AML patients with KMT2A‐AF9 were prone to having higher BM blasts (P = .068)  Table .3.2.1P < .001; e‐AML vs young, 60.7% vs 83%, P < .001). Additionally, a higher frequency of ED  was observed in e‐AML  and tended to have a lower incidence of positive MRD  than those treated with other therapy categories. In addition, e/s‐AML patients receiving intensive therapy tended to have a lower ED rate than those who were treated with palliative treatment .Among patients achieving CR, 258 young, 56 elderly, and 23 secondary AML patients had a definite LAIP feature before treatment, and the MRD of whom could be monitored. The frequency of positive MRD was higher in s‐AML than in young patients   . A similar tendency was observed in s‐AML . Combining e‐AML and s‐AML patients together, patients receiving intensive therapy were prone to having a longer EFS and OS than those treated with decitabine‐based hypomethylation therapy  and palliative treatment  , while it was not associated with EFS. Notably, the independent prognostic impact of s‐AML on OS was lost when HSCT was not regarded as a censored event, suggesting that HSCT may abrogate the adverse impact of s‐AML on survival to a certain extent . Generally, e/s‐AML patients had inferior EFS and OS compared with young patients  countries.Our study demonstrated that e/s‐AML patients have distinct clinical features compared with young de novo AML patents, such as lower WBC and BM blasts at diagnosis, which indicate that both elderly and secondary AML are less proliferative diseases, partly because they may have either transformed from MDS or experienced an undetected MDS period. Consistently, R. Coleman Lindsley et alFLT3‐ITD, TP53, RUNX1, ASXL1 aberrations, and KMT2A rearrangements are associated with adverse prognosis, while patients with biCEBPA mutations, RUNX1‐RUNXT1T1, and CBFβ‐MYH11 seem to have a relatively good outcome.KMT2A‐AF9 and DNMT3A mutations and less favorable ones including RUNX1‐RUNXT1T1, CBFβ‐MYH11, and biallelic CEBPA. Furthermore, genetic aberrations including NRAS, DNMT3A, IDH1 mutations, and CBFβ‐MYH11 conveyed prognostic information independently in e‐AML or s‐AML patients. However, some significant genetic alterations such as TP53, TET2, ASXL1, and RUNX1 mutations were not routinely tested in our center and accordingly not available in this retrospective study. Tsai et alPTPN11, NPM1, RUNX1, ASXL1, TET2, DNMT3A, and TP53 genes, but had less WT1 mutations. In addition, DNMT3A and TP53 mutations were independent adverse prognostic factors for elderly patients. Other studiesDNMT3A, ASXL1, and TET2 were more frequent in e‐AML patients, which was thought to be associated with age‐related clonal hematopoiesis and inferior survival. S‐AML patients were reported to carry less NPM1 mutations and FLT3‐ITD.ASXL1 and NRAS mutations.The genetic and molecular heterogeneity of AML have been widely acknowledged and integrated to optimize the prediction of clinical outcomes for AML patients. Previous studies demonstrated that Consistent with previous studies,Recently, a study reported that CPX‐351 could improve the response rates and survival of patients aged 60 to 75 with s‐AML compared with standard 3 + 7 treatment.In summary, the incidence of e/s‐AML is increasing and will be more common in the future, which merits our attention. Both elderly and secondary AML presented with distinct clinical, cytogenetic, and molecular features, whose prognosis remains dismal compared with young de novo patients, with a significant shorter EFS and OS. Intensive therapy could improve the prognosis of e/s‐AML patients to a certain degree and should be recommended for patients as long as the conditions are appropriate. HSCT could abrogate the adverse prognostic impact of s‐AML and should be considered for the treatment of fit s‐AML patients. More importantly, prospective clinical trials with new drugs are warranted in this special group of patients.The authors declare no conflict of interest.Table S1. Gene abnormalities and clinical aspects.Table S2. Univariate analyses for CR and ED.Table S3. Univariate analyses for event‐free survival and overall survival.Table S4. Multivariate analysis of OS in total cohort with HSCT not censored.Click here for additional data file.


Bronchopulmonary dysplasia (BPD) is a devastating lung condition that develops in premature newborns exposed to prolonged mechanical ventilation and supplemental oxygen. Significant morbidity and mortality are associated with this costly disease and effective therapies are limited. Mesenchymal stem/stromal cells (MSCs) are multipotent cells that can repair injured tissue by secreting paracrine factors known to restore the function and integrity of injured lung epithelium and endothelium. Most preclinical studies showing therapeutic efficacy of MSCs for BPD are administered either intratracheally or intravenously. The purpose of this study was to examine the feasibility and effectiveness of human cord tissue‐derived MSC administration given via the intranasal route. Human umbilical cord tissue MSCs were isolated, characterized, and given intranasally (500 000 cells per 20 μL) to a hyperoxia‐induced rat model of BPD. Lung alveolarization, vascularization, and pulmonary vascular remodeling were restored in animals receiving MSC treatment. Gene and protein analysis suggest the beneficial effects of MSCs were attributed, in part, to a concerted effort targeting angiogenesis, immunomodulation, wound healing, and cell survival. These findings are clinically significant, as neonates who develop BPD have altered alveolar development, decreased pulmonary vascularization and chronic inflammation, all resulting in impaired tissue healing. Our study is the first to report the intranasal delivery of umbilical cord Wharton's jelly MSCs in experimental BPD is feasible, noninvasive, and an effective route that may bear clinical applicability. Intranasal delivery of human umbilical cord mesenchymal stromal cells improves lung growth and development in a rat model mimicking neonatal lung disease. Bronchopulmonary dysplasia (BPD) is the most common cause of morbidity and mortality in extremely premature neonates. Unfortunately, current therapies for BPD are limited. Preclinical studies have shown that mesenchymal stem cell (MSC) treatment can restore alveolar growth, enhance vascular development, and stimulate tissue repair. Most of these studies have administered the cells via the intravenous/tracheal route. Results of this study show, for the first time to the authors' knowledge, that the intranasal delivery of MSCs for BPD is effective in restoring lung alveolar growth and vascular development. Importantly, this study provides evidence that this noninvasive approach may be given separately or as an adjunct/alternate to other routes.1Preterm birth is a major health concern affecting approximately 12% of all deliveries in the United States and costing over $26 billion every year.Regenerative medicine is an emerging field that seeks to remedy complex disease states by “stimulating previously irreparable organs to heal themselves.”22.1Umbilical cord Wharton's jelly tissue was collected from a healthy term human newborn. After tissue enzymatic digestion, cells were expanded on culture dishes under standard conditions. Wharton's jelly cells met the minimum criteria for MSCs, as described in our previous publication.5 cells were seeded onto 100 mm tissue culture dishes and grown under standard conditions with media changes every 2‐3 days. At 24 hours prior to administration, cells were checked for confluency (80%‐90%) and the media was changed for the last time. On the day of administration, the media was removed from the dishes and the cells were washed twice with phosphate buffered saline. TrypLE Express enzyme  was added to the dishes for 5 minutes to allow the cells to detach and an equal volume of αMEM  was added to the dishes to deactivate the TrypLE. The cell suspension was transferred to a conical tube, counted, and distributed into 20 μL aliquots containing 5 × 105 cells and phosphate buffered saline . We derived our dose based on the systematic review and meta‐analysis conducted by Augustine et al .Preparation of MSCs was achieved as follows, 5 × 102.2Guide for the Care and Use of Laboratory Animals and experiments were carried out in compliance with the Helsinki Declaration.All animal protocols were approved by The University of Texas Health Science Center (UTHSCSA) Institutional Animal Care and Use Committee. The animals received care in compliance with the 2O, Portland, ME) with cage changes (every 48 hours).Timed pregnant female Sprague‐Dawley rats were obtained from Charles River Laboratories at E14‐E15 days of gestation. Animals were singly housed with 12‐hour light/dark cycles, standard rodent laboratory diet and water was provided ad libitum. Dams were provided with nesting material at E18‐E19 onwards and received DietGels (Clear H2) for 21 days. The remaining BPD groups were exposed to 4 days of continuous hyperoxia (60%) using a BioSpherix animal housing chamber .On postnatal day 4, newborn rat pups were randomly assigned into four groups: (a) room air (RA), (b) BPD, (c) BPD treated with αMEM as a vehicle (BPD + Veh), and (d) BPD treated with mesenchymal stromal cells (BPD + MSCs). RA animals were survived at normoxia 21% O% O2 for 2.3Intranasal delivery of cells or vehicle was achieved using a modified version of the methods as described by Hanson et al.2.42 exposure followed by thoracotomy. Carcasses were kept on ice until tissue processing was completed. Lungs were excised, and the right lung was ligated, removed, and flash frozen in liquid nitrogen for protein and RNA analysis. The left lung was inflation fixed with 4% paraformaldehyde instilled through the trachea at 20 cm pressure for 2 minutes. After 24 hours, tissues were transferred to 70% ethanol prior to histologic analysis. Lungs were cut into four levels and oriented with alternating surfaces exposed prior to creation of 5 μm paraffin embedded sections. The heart was excised and the apex flash frozen, the remaining heart tissue was fixed in 10% neutral buffered formalin. The atria of the hearts were removed, and the remaining tissue oriented such that transverse sections of the ventricles could be obtained after paraffin embedding. All histological sectioning and staining were performed by UTHSCSA's Pathology Laboratory.Animals were euthanized by COLung slides were stained with hematoxylin and eosin stain, von Willebrand Factor, Masson's Trichrome, and α‐smooth muscle actin. Digital slides were created using the Aperio CS2 Digital Pathology Slide Scanner . Measurements were made using the following software: Aperio ImageScope software version 12.3.3, Pathomation (Belgium), MIPCloud (Iran), Image J, and Digimizer (Belgium).2.5Five micron‐thick sections were cut from paraffin‐embedded lung tissue, deparaffinized, and stained with a monoclonal antibody specific to intact human mitochondria  as described in Allard et al.2.62.6.1To obtain a comprehensive assessment of the lungs, we measured mean linear intercepts in a total of 270 random fields  on each animal.2.6.2To discern airspace enlargement, we adapted the techniques described by Jacob et al.2.6.3Pulmonary vessels were point counted on H&E stained lung sections measuring 727 μm × 482 μm and imaged at ×20 magnification. Nine viewing lung fields were randomly created for each animal. The total number of vessels was then averaged over the number of viewing fields analyzed to generate the presented values (vessels/viewing field). Findings were confirmed by randomly choosing four animal lungs per group (save BPD + Veh) for manual counting of von Willebrand factor stained vessels. Counts were done in 20 randomly selected lung fields measuring an area of 423 μm × 530 μm at a ×20 magnification.2.6.4External diameters of pulmonary vessels ranging from 50‐200 μm were analyzed for medial wall thickness (MWT). The following equation was used to calculate MWT:  / ) × 100.Immunostaining for α‐smooth muscle actin (α‐SMA) was used to assess pulmonary vasculature muscularization. A horizontal or vertical line was made across the shortest diameter of pulmonary vessels measuring 50‐200 μm. Thickness of the α‐SMA stained portion  was calculated over the external diameter of the vessel. All animal lungs in each group were evaluated by calculating α‐SMA thickness in five random vessels. Furthermore, positive staining for alpha‐SMA was quantified on all lungs using the Positive Pixel Count Algorithm . Data are reported as positivity .2.6.5Masson's trichrome staining was done to evaluate collagen deposition in the perivascular, peribronchial, and alveoli. Ten fields were observed per lung at a magnification of ×20. ImageJ was used to determine percent staining in a section measuring 2181 μm × 1446 μm.2.7A ratio of the thickness of the right ventricle to left ventricle+ septum on H&E stains of the heart was used as an index of right ventricular hypertrophy (RVH). To measure RVH, a series of seven horizontal lines spaced at 12 μm was placed over the heart images, and the length of the septum and ventricles were calculated.2.8Total RNA was extracted from flash frozen tissue using TRIzol reagent and Phasemaker tube system (Thermo Fisher). Briefly, ~20 mg of tissue was homogenized in 1 mL of TRIzol using a beadbeater (BioSpec) for 60 seconds followed by 60 seconds rest on ice, then another 60 seconds of homogenization. The tissue homogenate was briefly spun to eliminate bubbles and transferred to the Phasemaker tubes. RNA extraction was then completed using the TRIzol manufacturer's instructions. Synthesis of cDNA was achieved using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Thermo Fisher) with 2 μg of RNA. qRT‐PCR reactions were created using cDNA, EasyOligo custom primers , and SYBR Green PCR Master Mix (Thermo Fisher). The BioRad CFX384 Touch Real‐Time PCR Detection System  was used for PCR data acquisition. The program settings were as follows: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute, followed by a melt curve . Starting quantity of RNA was calculated in CFX Manager Software (BioRad) using the relative standard curve method and data were normalized using β‐Actin as a housekeeping gene. Primers were designed using the protocol described in Thornton and Basu 2010 and are summarized in Supplementary Table 2.9Pathway‐specific gene expression was also analyzed using the RT2 Profiler PCR Array for Rat Endothelial Cell Biology . In total, 96 genes from pathways associated with angiogenesis, vasoconstriction/vasodilation, inflammation, apoptosis, cell adhesion, coagulation, and platelet activation were profiled . Per manufacturer's directions, a total of 250 μg of tissue lysate was incubated in a nitrocellulose membrane array overnight at 4°C. Signals were visualized with chemiluminescence Western blot and developed on X‐ray film. The arrays were scanned, and optical densities were quantified using Image J software (NIH). The protein macroarray analysis included 79 cytokines/factors and included lung tissue protein samples from a subset of the three groups .3P value <5% signifying statistical significance. Power analysis calculations were based on MLI differences between BPD and BPD + MSC groups in a published study demonstrating MSC efficacy in a study by Thebaud et al.The data generated by the described experiments was evaluated using standard statistical approaches. Continuous data were expressed as mean ± SEM. Intergroup differences for parametric data were tested with one‐way ANOVA and Tukey's comparison test and nonparametric data with Kruskall Wallis followed by Dunn's test. Data were analyzed using STATA,  and a 44.1Umbilical cord Wharton's jelly‐derived cells adhered to tissue culture dishes and displayed the characteristic cell surface antigens for MSC classification . Kaplan‐Meier curve is provided in Supplementary Figure A total of 35 animals were used in the experiments . Survival rates among the groups were comparable  and alveolar tissue distribution. No difference was found comparing mean linear intercept in RA and BPD + MSC groups or BPD with BPD + Veh .Exposure of hyperoxia created the characteristic histologic findings in human BPD. Figure 4.5P = .07), the pattern suggests that compared to RA rats, hyperoxic exposed animals trended towards a reduction in the number of blood vessels , whereas treatment with MSCs restored vessel number (1.9 ± 0.2 blood vessels/field).To assess the effects of hyperoxic injury on vasculature development, the number of pulmonary vessels were counted on H&E stained lung sections Figure A. AlthouP < .01; Figure P < .01compared to BPD; P = .5 compared to RA).To confirm the above findings, von Willebrand factor staining was conducted in the following cohorts: RA, BPD, and BPD + MSC animals. Similar to H&E results, hyperoxia‐induced BPD animals demonstrated a lower number of blood vessels when compared with RA animals  was determined in H&E lung sections. MWT was elevated in BPD and BPD + Veh animals .4.9MSC treatment increased vascular endothelial growth factor (VEGF) mRNA expression in lung homogenates compared to RA rats but did not differ from the BPD animals  and NPPB   in the expression profile of two genes: 4.11P = .011). MSC treatment in this experimental model of BPD appears to impact inflammation when the genes were grouped into six processes . Figure To investigate the effects of MSCs on lung protein expression we performed a protein macroarray. Of the 79 factors/cytokines tested, RA only expressed 30 proteins while BPD and BPD + MSC expressed 61 and 58 proteins, respectively. When compared to RA and BPD + MSC, BPD had four elevated proteins: CX3CL1, TNFα, TIM‐1, and RGM‐A. On the other hand, when compared to RA and BPD, MSCs showed an enriched expression of G‐CSF, WISP‐1, and prolactin. The lungs of MSC treated mice had significantly lower levels of BPD injury related to proteins associated with immunomodulation , cell survival (osteoprotegerin), and wound healing . Although proteins pertaining to fibrogenesis and anti‐vascular processes were different between RA and BPD + MSC groups, there was no significant difference between the BPD and BPD + MSC groups. The single protein that demonstrated a difference between the BPD and BPD + MSC group  was G‐CSF  intranasally is feasible and a noninvasive approach for delivery to the lungs, (b) can potentially restore lung alveolarization and vascularization in a moderate model of BPD, and (c) the restorative effects may, in part, be due to a concerted effort on angiogenesis, immunomodulation, wound healing, and cell survival.The rationale for using MSCs as novel agents for BPD is based on their ability to secrete factors that contribute to tissue repair. MSCs are known to stimulate blood vessel formation, blunt inflammation, and promote alveolar epithelial cell survival and proliferation. While most preclinical BPD studies report the therapeutic action of these cells in BPD using an intratracheal and intravenous route, our project is the first to report intranasal delivery is a viable and efficacious approach . For appropriate membrane repair, a series of steps must occur including an influx of calcium, activation of vesicular exocytosis, and upregulation of annexin production.The immunomodulatory properties of MSCs make them novel and invaluable agents for the repair of lungs injured by chronic inflammation. Studies propose MSCs subdue immunologic responses by secreting soluble factors known to be immunosuppressive.Wound healing is a multifaceted process that requires a balanced response between re‐epithelialization, inflammation, and tissue granulation. After tissue injury, epithelial cells rapidly begin repairing the wound via secretion of MMPs and LIF.Intranasal delivery of therapeutic agents may represent a less invasive strategy for treating critically ill neonates. Relatively few studies have investigated the efficacy of intranasal administration of therapeutic agents for lung disease, and to our knowledge none of the current literature has investigated the use of intranasally delivered human umbilical cord Wharton's jelly tissue MSCs for BPD. The current study suggests that intranasally delivered MSCs may represent a promising regenerative therapy for treating chronic lung disease in preterm neonates. However, as with all studies the current work should be interpreted with considerations for the limitations of the study. First, although the rodent model is well established and provides valuable insight into the pathophysiology of BPD, it is not a premature model and therefore does not represent the full milieu of illness experienced by preterm neonates. In retrospect, we also would have added a cohort of RA animals that received MSCs. In this way, we could have further demonstrated safety. Given the heightened production of numerous proteins following MSC therapy brings into question whether we needed to provide three intranasal doses. We cannot exclude that these results are a consequence of examining the proteomic expression 1 day after the last administration. Given the pleotropic effects of many of these cytokines another drawback is that the biologic processes we assigned may oversimplify their role/action in the body. Similar to intravenous and intratracheal routes, administering one dose of intranasal MSCs may suffice and may not imbalance or produce factors that when secreted chronically may potentially be harmful. In the future, our experiments will incorporate the efficacy of intranasal MSCs in a severe model of BPD and assess pulmonary function testing, fibrosis, and animal survival. Furthermore, we also plan to examine for MSC infiltration in other tissues including the brain, spleen, kidneys, and heart. These studies will be furthered by considering potential strategies to optimize or improve the proposed route of administration, such as: the timing and frequency of cell‐based or cell‐free therapies and the standardization of the regenerative preparation.6In summary, we provide evidence that the intranasal route of human umbilical cord tissue MSC delivery for BPD is effective in restoring lung alveolar growth and vascular development. Importantly, this study demonstrates that this noninvasive route of cell‐based administration may be given separately or as an adjunct/alternate to other routes. Our findings are translationally notable, given that premature neonates who develop BPD may no longer be intubated (but on noninvasive respiratory modes) nor require intravenous access . Ultimately, further studies will be necessary, however, this approach can theoretically be tested in other models of rodent lung injury.The authors indicated no potential conflicts of interest.A.M.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of the manuscript; C.W. and L.W.: conception and design, data analysis and interpretation, manuscript writing; J.J.: collection and/or assembly of data, final approval of the manuscript; M.J., M.N., K.Q., A.C., and T.S.: collection and/or assembly of data; M.P.: collection and/or assembly of data, illustrations; Y.A.: manuscript writing; S.M.: collection and/or assembly of data, data analysis and interpretation; P.H.: conception and design, manuscript writing, final approval of the manuscript; S.A.: data analysis and interpretation, final approval of the manuscript.Supplementary Figure 1: Sample method used to calculate Alveolar Tissue Distribution. Using MIPCloud hematoxylin and eosin lung sections were uploaded and standardized thresholds (200) and region of interest were obtained. The program computes the phasefraction . Scale bar = 100 μm.Click here for additional data file.Supplementary Figure 2: Human umbilical cord cells met minimum mesenchymal stromal cell (MSC) criteria. Umbilical cord cells had the characteristic fibroblast‐like morphology and adhered to plastic under standard culture conditions and expressed (magenta) specific cell surface antigen markers CD73, CD 90, and CD 105, while negative controls are seen in grey. Scale bar = 40 μm.Click here for additional data file.Supplementary Figure 3: Kaplan‐Meier survival curve. Survival outcomes between the 4 groups did not demonstrate statistical differences. RA = room air; BPD = bronchopulmonary dysplasia; Veh = vehicle, MSC = mesenchymal stromal cell.Click here for additional data file.Supplementary Figure 4: Body weight curve showed no difference among groups. RA = room air; BPD = bronchopulmonary dysplasia; Veh = vehicle, MSC = mesenchymal stromal cell.Click here for additional data file.Supplementary Figure 5: Xenotransplantation of human umbilical cord MSCs via the nasal route migrated to the lungs in rats with hyperoxic injury. Immunohistochemistry of rat lung sections stained for human mitochondrial antibody . Depicted are lung sections for 5 randomly chosen animals in the BPD + MSC cohort. Bars denote 50 μm.Click here for additional data file.Supplementary Figure 6: Alpha smooth muscle actin (SMA) staining of pulmonary blood vessels and hematoxylin stained hearts. No difference noted between the groups in pulmonary vessel muscularization nor right ventricle remodeling; n = all animals/group. RA = room air control; BPD = bronchopulmonary dysplasia; BPD + MSC = bronchopulmonary dysplasia treated with mesenchymal stomal cells. Scale bar for SMA = 10 μm and heart sections = 200 μm.Click here for additional data file.Supplementary Figure 7: RT‐PCR data of rat lung homogenates. IL‐interleukin, TIMP‐tissue inhibitors of metalloproteinases, TGF‐transforming growth factor, VEGF‐vascular endothelial growth factor. Data are shown as median with IQR. RA = room air control; BPD = bronchopulmonary dysplasia; BPD + MSC = bronchopulmonary dysplasia treated with mesenchymal stomal cells. N = all animals/group. * P < 0.05 compared to RA.Click here for additional data file.Supplementary Figure 8: Sample protein array membranes for room air (RA) control, hyperoxia‐induced lung injury (BPD), and BPD animals treated with mesenchymal stromal cells (BPD + MSCs). (A) blots (B) all plots (C) statistical methodology for processes. Labels below were taken from vendor (R&D) pamphlet.Click here for additional data file.Supplementary Table 1 List of forward and reverse RNA primers. BAX = BCL2 associated x; Casp = caspase; IL = interleukin; TIMP = tissue inhibitor of metalloproteinases; VEGF = vascular endothelial growth factor; TGF = transforming growth factor.Click here for additional data file.Supplementary Table 2 List of genes tested in the RT2 Profiler PCR Array for Rat Endothelial Cell Biology.Click here for additional data file.Supplementary Table 3 Advantages/disadvantages of the intranasal route for delivery of human umbilical cord mesenchymal stromal cells.Click here for additional data file.Supplementary Table 4 References for groups of biologic processes.Click here for additional data file.


To evaluate the antitumor capability and to investigate the underlying molecular mechanism of paclitaxel.First, cck‐8 and apoptosis assays were used to determine survival and apoptotic effects of HS 737.T cells under treatment of paclitaxel. Next, RNA‐seq and bioinformatics were used to determine the differentially expressed genes and to analyze the pathway involved. Quantitative real‐time polymerase chain reaction was used to verify the accuracy of some differentially expressed genes (DEG). ClueGO was used to decode and visualize functionally grouped GO terms of differentially expressed genes, and to map the DEG protein–protein interactions (PPI) network. Western blotting was used to check the expression of target genes, the cleavage of Caspase‐3 and PARP1, and the phosphorylation level of p53. Finally, transcriptomics, bioinformatics, and RNAi were used to estimate the antitumor capability and to identify the underlying mechanisms of paclitaxel in GCTB.Our data revealed that paclitaxel had significant time‐dependent effects on the viability and induced apoptosis of HS 737.T cells. RNA‐seq and bioinformatics analysis showed that apoptosis, death receptor signaling pathway, TNF signaling pathway, and TP53 regulated transcription of cell death genes pathway were closely associated with paclitaxel in the treatment of GCTB. Western bolt results revealed that paclitaxel induced cleavage of Caspase‐3 and PARP1, and increased the phosphorylation level of p53 in HS 737.T cells. RNAi results showed that the expression level of TP53INP1 was significantly decreased in HS737.T cells (the decrease was more than 70%). In addition, we found that the inhibitory ratios of paclitaxel on HS737.T cells deficient in TP53INP1 were less than in HS737.T cells with empty vector . Hence, our data revealed that TP53INP1 regulated paclitaxel‐driven apoptosis in HS737.T cells.Paclitaxel can significantly repress cell proliferation and induce apoptosis of HS 737.T cells through activating Caspase‐3, PARP1, p53, and TP53INP1. Paclitaxel may be an effective drug in the management of GCTB. A giant cell tumor of bone (GCTB) is an osteolytic, locally aggressive, rarely metastasizing bone tumor. Many GCTB are benign connective tissue neoplasms, such as stromal cells, osteoclast‐like giant cells, and tumor‐associated monocytes/macrophagesIt is well known that the transformation of cancer cells leads to an increasing trend of apoptosis. Therefore, apoptosis of multinucleated giant cells and stromal cells may increase in GCTB to regulate tumor regressionTaxus brevifolia, exhibits potent efficacy against both breast and ovarian cancers. Mechanistically, paclitaxel selectively binds to tubulin and subsequently stabilizes microtubules, thus inhibiting cell division. Interestingly, metastatic bone lesions have been reported to better respond to a combination of paclitaxel and docetaxel than to a variety of single chemotherapeutic agents. While these clinical observations point to a beneficial effect of paclitaxel on bone lesions, the underlying mechanisms of its action remain to be established. Numerous studies have demonstrated that paclitaxel has a significant role in the modulation of the cell cycle and apoptosis in various tumor cells. Nevertheless, the mechanisms by which paclitaxel modulates the anti‐proliferation and apoptosis of GCTB remain obscure.Paclitaxel, which is derived from the bark of the Pacific yew tree, Apoptosis plays a crucial role in anticancer and disease defense. In general, the caspase‐dependent apoptosis is driven by interior or exterior stimuliIn the present study, to further elucidate the anti‐tumor activity and mechanisms of paclitaxel on GCTB, we used RNA‐Seq, RT‐qPCR, and western blot, to reveal differentially expressed genes in the giant cell tumor cell HS 737.T under paclitaxel treatment. We then used bioinformatics to analyze the pathway that the differentially expressed genes involved. Finally, we combined transcriptomics, bioinformatics, and RNAi to investigate the underlying mechanisms of paclitaxel on GCTB.Lipofectamine 3000 Transfection Reagent, RT reagent Kit, and quantitative polymerase chain reaction (qPCR) kits were from Thermo Fisher Scientific . The CCK‐8 kit was provided by Dojindo Molecular Technologies . Restriction enzymes EcoRI‐HF and AgeI‐HF were provided by New England Biolabs . pLKO.1‐TRC cloning vector was a gift from David Root .Paclitaxel was obtained from Selleck . Cell culture products were from Biological Industries . Puromycin, streptomycin and penicillin, Human GCTB Hs 737.T  were grown in Dulbecco's modified eagle medium plus 10% FBS, l00 μg/mL streptomycin and penicillin.Cell viability was checked with a CCK‐8 kit. Hs 737.T cells were exposed to paclitaxel at different concentrations (0–20 ng/mL). After 6–48 h incubation, 20 μL CCK‐8 agent was added to each well; after incubation for 1.5 h, signals were measured using a Microplate Absorbance Reader .To investigate the impact of paclitaxel on apoptosis of GCTB, Hs 737.T cells were treated with or without paclitaxel for 12 h. Next, adherent cells were trypsinized and stained with an Annexin V‐FITC Apoptosis Staining/Detection Kit  following the manufacturer's instructions. The apoptotic cells were quantified by flow cytometer .RNA‐Library preparation and sequencing were conducted by Novogene . RNA libraries were constructed with the Illumina mRNA sample preparation kit  following the manufacturer's directions. Sequencing was performed using the Illumina NextSeq500 platform .P‐value < 0.05, fold change > 2). Systematic and integrative analysis of DEG was conducted using DAVID bioinformatics resources as previously describedet al.We used the DESeq to perform differentially expressed gene (DEG) analysis .The mRNA levels of interesting genes were checked by quantitative real‐time polymerase chain reaction (RT‐qPCR) using the SYBR Green Real‐Time PCR Kit . The qPCR primers specific for the target genes are presented in Table Total or intracellular protein were extracted from Hs 737.T cells treated and untreated with paclitaxel, and separated by SDS‐PAGE, then transferred to PVDF membrane. The specific antibodies of the immunoblotting were as follows: anti‐β‐actin, anti‐PARP1 , anti‐cleaved Caspase‐3, anti‐DDIT4, anti‐TRAIL, anti‐phospho‐p53 (S15), and anti‐TP53INP1 . The immunoblot signals were measured and quantified using the Chemiluminescence image analysis system .Lipofectamine 3000 Transfection Reagent . Lentiviral particles were generated and infected into HS 737.T cells. Stable integration of lentivirus was achieved through selecting culture‐media plus 2 mg/mL puromycin for 48 h. The knockdown efficiency of TP53INP1 was verified by RT‐qPCR or western blot analysis.To generate Hs 737.T cells deficient in TP53INP1, we used the protocol published previouslyt‐test, ANOVA, and Dunnett's and Tukey's multiple comparison test were used to evaluate the statistical significance of the differences. Results were presented as means ± s.e.m. of triplicate experiments. Results were considered statistically significant with P‐values < 0.05, P‐values < 0.01, and P‐values < 0.001.Statistics were obtained using Graphpad prism 7. Cell viability experiments demonstrated that paclitaxel was effective in inhibiting cell proliferation and survival of HS 737.T. The IC50 value of the paclitaxel‐treated HS 737.T cell was 9.78 ng/mL Fig. a. At theCompared with the untreated cells, apoptotic cells were obviously increased after HS 737.T cells were exposed to paclitaxel at 5, 10, and 20 ng/mL for 24 h, with apoptotic ratios of 27.03%, 37.90%, and 52.16%, respectively  of HS 737.T treated with paclitaxel. Among the 286 differentially expressed genes, upregulated and downregulated genes were 168 and 118, respectively  were found by RNA‐seq and differentially expressed genes analysis .As shown in Fig. A functional map of the upregulated gene protein–protein interactions (PPI) based on significant or group analysis revealed that apoptosis, TNF signaling pathway, TP53 regulated transcription of cell death genes pathway, and death receptor signaling pathway was the important regulatory node in paclitaxel‐driven apoptosis of HS 737.T cells, (the node size indicates significant enrichment), and the core components were related to apoptosis and viability of tumor cells, including TP53INPI, Caspase 10, TNFSF10, and PIK3R3 Fig. d,e.P‐value < 0.001, fold change >2) in HS 737.T cells treated with paclitaxel, compared with the negative control, as analysis by RNA‐Seq demonstrated  is an anti‐proliferative and pro‐apoptosis protein involved in cell stress responseAs vital anti‐tumor drugs, most chemotherapeutic agents rely on the capability to induce apoptosis of tumor cells. To further develop paclitaxel as an effective anti‐GCTB agent in the clinic, we need to better comprehend the mechanisms through which paclitaxel drives apoptosis of GCTB. Based on the results of the present study, a model of the mechanism through which paclitaxel triggers apoptosis of HS 737.T cells was proposed Fig. . Our datThere are some limitations of this study. First, we studied the apoptosis mechanisms of paclitaxel based on only one kind of GCTB cell line. Further studies could use more cell lines to investigate the commonness and mechanisms of paclitaxel‐driven apoptosis of GCTB. Second, we lacked data from animal experiments. Future research should consider experiments on transplanted tumors in animals.In summary, by combining transcriptomics, bioinformatics, and RNAi (Lentivirus shRNA), we revealed systemic transcriptomics changes in HS 737.T cells under paclitaxel treatment and the therapeutic mechanism of the treatment. Results of RNA‐seq and bioinformatics analysis showed that apoptosis, death receptor signaling pathway, TNF signaling pathway, and TP53 regulated transcription of cell death genes pathway, which was the top 20 most enriched pathway and involved in cell viability, were closely associated with paclitaxel in the treatment of GCTB. Combined with transcriptomics and RNAi, TP53INP1 signaling pathways were ultimately found to be closely related to paclitaxel in the treatment of GCTB. Our study implied that paclitaxel might be valuable for the repression of GCTB.Table S1 Primers specific for quantitative real‐time polymerase chain reactionTable S2 shRNA primers for TP53INP1Click here for additional data file.


Autism spectrum disorder is commonly co‐diagnosed intellectual disability, language disorder, anxiety, and epilepsy, however, symptom management is difficult due to the complex genetic nature of ASD.We present a next‐generation sequencing‐based case study with both de novo and inherited genetic variants and highlight the impact of structural variants on post‐translational regulation of protein expression. Since management of symptoms has classically been through pharmaceutical therapies, a pharmacogenomics screen was also utilized to determine possible drug/gene interactions.FOXP1 3′ untranslated regulatory region using exome sequencing. Additionally, inherited variants that likely contribute to the current and potential future traits were identified within the COMT, SLC6A4, CYP2C19, and CYP2D6 genes.A de novo variant was identified within the This study aims to elucidate how a collection of variant genotypes could potentially impact neural development resulting in a unique phenotype including ASD and epilepsy. Each gene's contribution to neural development is assessed, and the interplay of these genotypes is discussed. The results highlight the utility of exome sequencing in conjunction with pharmacogenomics screening when evaluating possible causes of and therapeutic treatments for ASD‐related symptoms. Deletions result in mild craniofacial abnormalities; a long narrow cranium, high and broad forehead, and short nose with broad bridge  of 22.1This study was approved by the University of Minnesota Ethics Committee. As an educational investigation, this study was considered exempt from requiring additional Internal Review Board approval. Samples from three family members were provided from a previous collection, with written consent to be used in this study.2.2The patient (Patient 1), a 20‐year‐old male, was diagnosed with ASD by DSM‐IV criteria at age 2 years. He was born to nonconsanguineous parents, his father age 39, and his mother age 33 at the time of his birth. The patient's parents noted environmental risk factors including exposure to flood conditions in the home in early pregnancy and high levels of maternal stress throughout gestation. During pregnancy, his mother experienced significant bleeding at 36 weeks. He was born by cesarean section due to transverse lie, no other complications were noted during delivery. His sibling, a younger sister, and both parents are neurotypical. The proband, Patient 1, showed first signs of regression when he stopped tracking objects at 8 months. When he did not begin speaking at 12 months, he began speech and occupational therapy under the diagnosis of “autistic tendencies.” Patient 1 went on to speak short one‐syllable words and took his first steps at 13 months. Patient 1 was evaluated for hypertelorism, macrocephaly, prominent forehead, frontal hair upsweep, long narrow face, pronounced jaw, down‐slanting palpebral fissures, short nose, and frontal bridge. No dysmorphic craniofacial characteristics were reported. Patient 1 suffers from severe anxiety, hyperstimulation, and depression. He experiences difficulty transitioning and exhibits self‐injurious behavior when angry. These ASD‐related symptoms remain unresolved despite pharmaceutical treatment. Following formal diagnosis of “ASD and language disorder” at 2 years of age, Patient 1 was placed on multi‐vitamin supplementation along with the following: risperidone and aripiprazole without effect, methylphenidate, amphetamine/dextroamphetamine, dexamphetamine, catapres to treat hyperactivity, and gabapentin for anxiety. For anxiety a low dose clonazepam, and sertraline HCl have been tried, and he is currently taking citalopram. At the age of 17 years, following anesthesia for wisdom tooth removal, he began to experience seizure‐like activity. Frequency was initially weekly but improved with treatment of lamotrigine and lacosamide to every other month. Patient 1 recovered quickly from seizure‐like activity and did not experience loss of consciousness during episodes. Onset of seizure‐like activity was marked by slurring of speech and eyes rolling to the right. Several focal spikes in the front temporal lobe have been recorded on EEGs, and MRI results were normal. Parents report no family history of neurological disorders.2.3https://www.ncbi.nlm.nih.gov/clinvar/).Genomic DNA was isolated from peripheral blood, obtained following informed consent, using QIAamp DNA Blood Maxi Kit , quantified using Nanodrop‐8000 and stored at −20°C. Agilent XT libraries were captured using Agilent All Exon V5 + UTR kit. Libraries were pooled, and Exome sequencing was performed on a 125 bp paired‐end run in Illumina's HiSeq2500 next‐generation high‐throughput sequencing system using v4 chemistry. Variants identified through exome sequencing were submitted to the NCBI ClinVar database  was performed using pair‐end synchronization (pe‐sync) and FastQC tools in the Galaxy tools pane. Poor base call quality reads and adapter contamination were filtered using Adapter Removal  genomes in the 1,000 Genomes Project were used as controls to filter out common overlapping mutations  (NM_032682.5:c.3413_3414del) (NM_032682.5:c.3413_3414dup) was evaluated using minimum free energy, and centroid algorithms accessed through the Vienna RNA package 2.0. Minimum Free energy thermodynamic analysis provided predicted secondary structures (RNAfold) to evaluate the effects of variation on secondary structure  and PTEN , with β‐actin  used as a loading control, and a FOXP1 overexpression lysate  used as a positive control. Protein concentration was determined using the Pierce BSA colorimetric assay (Thermo#23227). Samples (20 μg) were loaded on a 12% Bis Tris gel. Western blots were viewed using a Li‐Cor Odyssey imaging system (FC‐0470).Samples were obtained from Patient 1 and both parents following informed consent and peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll‐Paque gradient method. Nuclear extraction was completed following the manufacturer's recommended protocol (Abcam#113474), and the cytoplasmic fraction was used for western blot. To eliminate genomic contamination, each sample was treated with DNase 1 prior to electrophoresis. Western blots were completed using antibodies for 2.8https://www.ncbi.nlm.nih.gov/clinvar/).To assess possible side effects of pharmaceutical therapy enlisted in this case, a pharmacogenomics screen was completed. The RightMed comprehensive test  was administered to determine possible unforeseen drug‐gene interactions that could result in an increase in the severity of symptoms or toxicity in the individual. Relevant variants identified through pharmacogenomics testing were submitted to the NCBI ClinVar database . Interestingly, clinical annotation identified FOXP1 as a candidate gene associated with the symptoms “Intellectual Disability with Language Impairment and Autistic Features”  genomes in the 1,000 Genomes Project data  at the same location, within the 3′ UTR of FOXP1 (NM_032682.5:c.3413_3414del) (NM_032682.5:c.3413_3414dup). Figure To validate our results, we performed PCR‐amplified Sanger sequencing using DNA from the proband, parents, and sister. Both mother and sister exhibited the common population sequence obtained from the NCBI ClinVar database, including 12 tandem (TG) repeats in the 3′ UTR of the 3.2FOXP1’s 3′ UTR likely forms a complex secondary structure that regulates accessibility of RNA binding proteins and microRNA binding sites. We hypothesized that the 3′ UTR variant observed in Patient 1 may substantially alter FOXP1 mRNA secondary structure and therefore impact FOXP1 post‐transcriptional regulation. We queried the Vienna RNA secondary structure prediction software  variants identified above. Single base pairs were analyzed to identify regions that would self‐hybridize to form classical stem loop structures that could be targeted by RNA interacting proteins by predicting the minimum free energy (MFE) state of each sequence. Stability of the mRNA transcript is determined by hydrogen bonding between paired nucleotides  was analyzed, the ΔG value was indicated to be −34.0 kcal/mol. and the predicted structure was preserved. However, when the TG deletion (Patient 1) was analyzed, it was predicted that the secondary structure would be significantly altered, and the stem‐loop would be destabilized with a ΔG of −33.0 kcal/mol.FOXP1 3′ UTR variations. Centroid analysis was completed based on the Boltzmann‐weighted structure ensemble using the RNAfold webserver  would be associated with a decreased expression of FOXP1, while an overexpression could indicate decreased binding of microRNA‐486‐5p. Since microRNA‐486‐5p interacted with both FOXP1 and phosphatase and tensin homolog (PTEN) transcripts, we hypothesized that alteration in FOXP1 interaction could lead to a microRNA‐486‐5p directed alteration of PTEN expression. Patient 1 exhibited a substantial (2.2‐fold) decrease in FOXP1 protein, and elevated expression of PTEN (1.4‐fold) , levels for both proteins were significantly different than the control with reported p < 0.0001. In his father's case (NM_032682.5:c.3413_3414dup), there was a much smaller difference in the level of FOXP1 (1.4‐fold) decrease, and PTEN protein levels were not significantly different than the control levels observed in the mother.To explore stantial .2‐fold d3.4Pharmaceutical therapies are commonly used to manage behavioral symptoms associated with ASD. However, due to variants that influence metabolism, unforeseen drug‐gene interactions may occur including toxicity , the catechol‐O‐methyltransferase (COMT) gene (OMIM#116790) (NM_000754.3:c.472G>A), the coagulation factor II (F2) gene (NM_000506.4:c.97G>A), and the Solute Carrier Family 6 Member 4 (SLC6A4) gene (OMIM#182138) (NM_001045.5:c.‐1917_‐1875del43 and NM_001045.5:c.‐1936G>A). A summary of the variants reported in this case can be seen in Table Among initial incidental findings, the gene and phenotype summary identified variants in the CYP2C cluster and NUDT15 (OMIM#615792) (NM_018283.3:c.415C>T) variants are strongly associated with therapies involving warfarin and thiopurines    superfamily of TFs, coding for proteins associated with neural development, metabolism, and immune function   genes and FOXP1 . From that report we discovered Patient 1 carries an inherited dominant variant in the F2 gene that is associated with a significantly increased risk of thrombosis   homeostasis is involved in sleep cycle regulation, appetite, thermoregulation, pain perception, respiration, and bowel function  is encoded by the CYP2C19) inhibitor fluconazole can result in serotonin toxicity when taken with citalopram. Citalopram is a substrate for CYP2C19 and inhibition of metabolism can result in toxicity  with SSRIs is another mechanism of SS, where an accumulation of serotonergic drugs  leads to toxicity through decreased drug metabolism  is caused by a progressive increase in serotonin levels in the central and peripheral nervous systems (Wang, Vashistha, Kaur, & Houchens, These findings emphasize the importance of assessing both environmental and genetic risk factors and to evaluate possible drug‐gene interactions when enlisting pharmaceutical therapies. This study identified complex genetic variants that may represent possible causative drivers for ASD. Additionally, incidental findings through pharmacogenomic screening identified several variants that potentially have substantial implications for symptom management, emphasizing the importance of such screenings. Proper expression of TFs is of particular importance during pre‐natal and early post‐natal development, and deficits in neuronal migration have been implicated in autism, schizophrenia, and epilepsy development; however, the direct mechanism is yet unknown (Li et al., None to declare. Click here for additional data file.


To evaluate the association between pinguecula excision and subsequent improvement in dry eye syndrome.  We included 30 consecutive patients with primary nasal pinguecula and dry eye symptoms undergoing ocular surgery for the first time. Criteria for pinguecula excision surgery were nasal location, yellowish color, and protrusion of conjunctiva at least 2 times thicker than adjacent normal conjunctiva as measured by anterior segment optical coherence tomography. Our primary outcomes were 3-month postoperative changes in tear film breakup time (TBUT), Schirmer test, and a dry eye symptom score.  30 eyes from 30 different patients (12 men and 18 women) underwent pinguecula excision and conjunctival autografting using fibrin glue. The mean age was 42.5 ± 8.35 (range 28–63) years. The preoperative protrusion ratio of pinguecula was 2.33 ± 0.28 (range 2.00–2.90). Mean preoperative TBUT, Schirmer test, and dry eye symptom scores were 5.10 ± 1.27 seconds, 6.07 ± 2.27 mm, and 2.80 ± 0.76 points. Mean postoperative 3-month TBUT, Schirmer test, and dry eye symptom scores were 7.80 ± 1.13 seconds, 7.27 ± 2.02 mm, and 0.30 ± 0.47 points, respectively. The median pre- and postoperative changes were found to be statistically significant by Wilcoxon signed-rank tests for TBUT, Schirmer test score, and dry eye symptom score.  Surgical excision of pinguecula and conjunctival autograft using fibrin glue is an effective and safe method to improve symptoms of dry eye syndrome. A pinguecula is a round, yellowish, elevated fleshy tissue on the bulbar conjunctiva. It is more common in nasal limbal conjunctiva and often contains deposits of protein, fat, or calcium . AlthougThe primary reason pinguecula is excised is for cosmesis. Argon photocoagulation has shown good results for small, nonvascularized pinguecula , 4, wherIn our clinical practice, similar to published reports after pterygium excision, we have consistently found improvement in patients' dry eye symptoms after pinguecula excision. Interestingly, there is scant literature on the association between pinguecula and dry eye. The contribution of this study is that we examine the relationship of pinguecula and dry eye pre- and postoperatively in a quantitative manner using accurate high-resolution OCT data and changes in tear film breakup time (TBUT), Schirmer tests, and dry eye symptom scores.This study is a retrospective chart review of patients having excision of high-grade primary pinguecula for cosmetic reasons and/or irritation. We included 30 consecutive patients meeting inclusion criteria from May 2016 to April 2017. Inclusion criteria were patients with a noninflamed, vascularized nasal pinguecula, dimensions greater than 2.0 mm × 2.0 mm, and thickness greater than 2 times that of the adjacent conjunctiva. Tissue thicknesses were measured using AS-OCT  . The pinAll surgeries and pre-/postoperative evaluations were performed by a single surgeon (J. W. K.) using a standard technique . Under tp-value of less than 0.05 was considered statistically significant.We measured preoperative and 1- and 3-month postoperative changes of TBUT, Schirmer test, and dry eye symptom scores. 3 months was considered to be a sufficient amount of time for post-op healing and prednisolone acetate drop washout. The TBUT was measured by asking the patient to look nasally and then staining the superotemporal bulbar conjunctiva with fluorescein . The breakup time of the corneal tear film after one blink was recorded , 8. TBUTFrom May 2016 to April 2017, 30 eyes of 30 patients (12 men and 18 women) underwent pinguecula excision and conjunctival autografting using fibrin glue. Their mean age was 42.5 ± 8.35(range 28–63) years. Preoperative ophthalmic examination showed a yellowish, raised pinguecula with significant vascularization in each case and a mean protrusion ratio of 2.33 ± 0.28 (range 2.00–2.90). Histologic examination of excised tissues confirmed subepithelial solar elastosis with thinning of the overlying epithelium. The mean postoperative follow-up period was 12.27 ± 1.34 (range 10–15) months. After pinguecula excision with conjunctival autograft, it showed vascular shrinkage and flattening of the conjunctival surface . There wr = −0.552, p=0.002) and Schirmer test score  and positively correlated with preoperative dry eye symptom score  and postoperative change in dry eye symptom score  (The protrusion of the pinguecula was negatively correlated with lower TBUT (p=0.005) .There is a significant amount of research on the association of pterygium and dry eye syndrome , but verPrevious reports showed improvement in tear film stability after pinguecula and pterygium excision , 13–16. Importantly, we found no clinically significant complications after our pinguecula excision surgeries. At a mean follow-up period of 12.3 months, there was not a single recurrence, and all patients were satisfied with their results. With respect to our surgical approach, we preferred using a conjunctival autograft with sutureless fibrin adhesive application rather than a conjunctival rotational flap or reapproximation because the latter technique often requires conjunctival sutures and may result in stretching tension to the wound. The sizes of our conjunctival autograft were relatively small; however, in instances when conjunctiva may be needed for future surgery , an amniotic membrane graft could also be considered as an alternative to our approach.Our study has a number of limitations. The study is a retrospective analysis and was not designed to prove the causality of pinguecula excision in improving dry eye syndrome. The study does not include grading of fluorescein staining, an important measure in the study and treatment of dry eye syndrome. We attempted to control for confounders by excluding patients with active inflammation and including a prednisolone acetate washout period of more than 10 weeks. However, we did not include a nonsurgical control group and so symptom improvement may be due to a placebo effect. Lastly, the sample size was not sufficiently large to perform subgroup analyses to examine if the effect varied across different patient demographics.In conclusion, we accurately measured the protrusion of pinguecula by comparing the relative thickness with adjacent normal conjunctiva using AS-OCT, and we found that protrusion was statistically correlated with preoperative dry eye symptom scores and that the surgical excision of symptomatic pinguecula improved not only cosmesis but also improved dry eye syndrome without any instances of recurrence or other serious complications with properly selected patients. Given the high prevalence of pinguecula , 16, its


ECS), a function of area, CV and refractoriness, and AF vulnerability in patients undergoing AF ablation.The multiple wavelets and functional re‐entry hypotheses are mechanistic theories to explain atrial fibrillation (AF). If valid, a chamber's ability to support AF should depend upon the left atrial size, conduction velocity (CV), and refractoriness. Measurement of these parameters could provide a new therapeutic target for AF. We investigated the relationship between left atrial effective conducting size (LAECS were: (a) left atrial body area (A); (b) effective refractory period (ERP); and (c) total activation time (T). Global CV was estimated as √A/T. Effective atrial conducting size was calculated as LAECS=A/(CV×ERP). Post ablation, AF inducibility testing was performed. The critical LAECS required for multiple wavelet termination was determined from computational modeling. LAECS was greater in patients with persistent vs paroxysmal AF . AF was inducible in 14/39 patients. LAECS was greater in AF‐inducible patients . The difference in LAECS between inducible and noninducible patients was significant in patients with persistent (P = .0046) but not paroxysmal AF (P = .6359). Computational modeling confirmed that LAECS > 4 cm was required for continuation of AF.Activation mapping was performed in patients with paroxysmal (n = 21) and persistent AF (n = 18) undergoing pulmonary vein isolation. Parameters used for calculating LAECS measured post ablation was associated with AF inducibility in patients with persistent, but not paroxysmal AF. These data support a role for this method in electrical substrate assessment in AF patients.LA Nevertheless, a key concept of these hypotheses is that a critical mass of tissue is required to facilitate the perpetuation of AF.22.1Adult patients with AF undergoing first‐time ablation were eligible for inclusion. The research protocol conformed to principles outlined in the Declaration of Helsinki and ethical approval was granted by the National Research Ethics Service. Written informed consent was obtained from all participants before the study. Patients with prior atrial ablation were excluded. Following femoral venous access, a decapolar  catheter was positioned in the coronary sinus. Left atrial access was obtained via a trans‐femoral approach following a trans‐septal puncture. A circular mapping catheter  and an 8Fr ablation catheter  were advanced into the left atrium via two 8.5Fr SR0 sheaths. All patients were anticoagulated intraprocedurally targeting an activated clotting time of >300 milliseconds.2.2Before ablation, all patients underwent left atrial activation and voltage mapping using the Carto electro‐anatomic mapping system  during coronary sinus pacing at a cycle length of 600 milliseconds. For patients who presented in AF, direct current cardioversion was performed. Wide‐area encirclement of the pulmonary veins was performed in power‐controlled mode using a contact force‐sensing ablation catheter. Entrance and exit block were demonstrated to confirm pulmonary vein isolation and additional ablation performed in the case of reconnection. Following successful pulmonary vein isolation, the ablation catheter was positioned in the center of the posterior left atrial wall. Once adequate contact force (>3 g) and catheter stability were achieved, a pacing protocol was applied consisting of an 8‐beat drive train at a basic cycle length (BCL) of 600 milliseconds and an extra stimulus with coupling intervals reducing from 450 milliseconds in 10 millisecond steps. The atrial effective refractory period (ERP) was defined as the longest extra stimulus coupling interval failing to capture the atrium.2.3Following ERP measurements, an AF induction protocol was performed by pacing from the ablation catheter at the left atrial posterior wall. The protocol consisted of sensed doubles , sensed triples S1 = 600 milliseconds; S2 =ERP + 50 milliseconds, S3 =ERP + 50 milliseconds, decreasing in 10 millisecond steps to atrial ERP) and incremental pacing, decreasing in 10 millisecond intervals from 450 milliseconds to loss of 1:1 atrial capture.2.4A) was calculated by subtracting the area of the isolated pulmonary veins, the left atrial appendage, and the mitral annulus from the total area of the left atrial shell  was calculated by subtracting the earliest from the latest activation time point as annotated on the isochronal activation maps. Using A and T as the characteristic length and time, respectively, we defined the characteristic CV asCarto3 maps were analyzed offline and used to calculate left atrial body atrial area, total activation time, and global CV. Left atrial body area  was then calculated asLeft atrial effective conducting size .K1 was modified to reproduce the average ERP of the inducible persistent AF patients in this study (GK1 = 1.8 mS/cm2), to attain ERP = 231 milliseconds. AF was initiated by cross‐field stimulation. The numerical simulations were performed with CARPentry (https://carp.medunigraz.at/carputils/). To model the effects of pulmonary vein isolation, tissue at the left and right‐hand edges of the domain were set to be nonconductive (0.001 S/m). The effect of reducing left atrial body area by 10 to 60 cm2 was modeled by setting between 0.5 to 3.0 cm at each side of the domain to be nonconductive. To model the effects of changes in ERP , IK1 conductance was modified. Following Mitchell et al, we modeled ERP prolongation by sotalol in the range 290 to 330 milliseconds.Two dimensional (2D) and three dimensional (3D) computational modeling. For the 2D model, an 11 × 10 cm atrial tissue model was meshed using quadrilateral elements . Electrical propagation was modeled using the mono‐domain equation, with isotropic conductivities of 0.3 S/m, giving CV= 77 cm/s. The cellular ionic current was modeled using the Luo‐Rudy cellular model, with ionic conductances modified to reproduce atrial cellular properties following Virag et al (2. The pulmonary veins were clipped (https://www.paraview.org) and meshes created using MMG tools software (http://www.mmgtools.org/). Endocardial atrial fiber direction information was mapped from an atlas geometry,K1 to produce ERP values in the range of 230 to 270 milliseconds. Ablation was modeled during arrhythmia simulations by setting ablation region conductivities as nonconductive (0.001 S/m). The following ablation patterns were applied: (a) pulmonary vein isolation alone via wide‐area encirclement of the pulmonary veins and (b) pulmonary vein isolation together with roof and inferior lines to model posterior wall box isolation. To vary the area of the isolated tissue the inferior line was applied at increasingly inferior locations on the posterior wall.3D simulations were performed on three left atrial anatomies generated from cardiac magnetic resonance sequences representing a range of left atrial body surface areas: 75, 103, and 140 cm2.6U test for independent samples. Categorical variables were compared using χ2 test. P < .05 was considered statistically significant.Data analysis were performed using SPSS statistics  and Prism . Continuous variables were expressed as a mean ± standard deviation. Comparison of means between groups was performed using the Mann‐Whitney 33.1Thirty‐nine patients  undergoing first‐time AF ablation were studied (Table 3.22. Mean ERP (BCL = 600 milliseconds) was 262.6 ± 57.2 milliseconds. Mean left atrial total activation time was 97.1 ± 27.7 milliseconds. Calculated mean CV was 1.0 ± 0.4 m/s. Calculated mean LAECS was 3.7 ± 1.8 cm . Left atrial total activation time was significantly longer in patients with persistent AF than paroxysmal AF  but there were no significant relationships between ERP , CV  or wavelength  and AF category.LA3.2.2ECS was significantly greater in patients in whom AF was inducible than patients in whom AF was not inducible  but there were no significant relationships between ERP , CV  or wavelength  and AF inducibility. There was no significant relationship between AF type and ability to induce AF .Overall, LA3.2.32 vs 91.9 ± 16.4 cm2; P = .0346) and wavelength was significantly shorter  for persistent AF cases who were inducible compared to those who were noninducible  but not paroxysmal AF  cases. Wavelength is plotted against left atrial body area in Figure ECS isolines. In contrast, patients with persistent AF who were inducibly localized below the 4 cm isoline (ie LAECS > 4 cm).Left atrial body area was significantly greater  or 240 milliseconds . The median ERP and area values are also significantly different between inducible and noninducible cases.For the smallest geometry, ERP values of 250 to 270 milliseconds resulted in LAECS and terminated AF in 67% of simulation set‐ups using the inferior line that resulted in the lowest LAECS. Figure 2 and corresponding LAECS of 5.82 cm. Figure ECS of 3.94 cm, and termination of AF. In contrast, Figure ECS remaining above 4 cm (LAECS = 4.18 cm). By additionally increasing ERP to 250 milliseconds, through modeling the effects of sotalol, LAECS is decreased less than 4 cm (LAECS = 3.84 cm) and AF terminates  in individual patients to predict response to treatments for arrhythmias.Prior studies assessing the relationship between electrical remodeling and atrial dilatation have, however, yielded inconsistent results. Ravelli et al4.2The concept of “atrial substrate size” was originally communicated by Moe in terms of a dimensionless quantity termed the fibrillation number.ECS. This metric differs from the fibrillation number previously describedECS significantly greater in persistent AF patients with inducible vs noninducible AF (Figure ECS also notably identified a population of patients with persistent AF who were not AF‐inducible after PVI and who, from an electrophysiological standpoint, appear to behave more like paroxysmal AF patients. Although this parameter ignores heterogeneity in refractoriness,ECS in this study is easily obtainable within the timeframe of an ablation procedure and according to the present data is likely to represent a useful and clinically applicable metric for assessing AF vulnerability irrespective of the clinical AF categorization.Given the interaction between atrial size, refractoriness, CV, and AF vulnerability, we incorporated these parameters into a new metric termed LAF Figure F. LAECS 4.3ECS < 4 cm, a trigger‐based ablation strategy may be most appropriate. In this study, LAECS < 4 cm incorporated the vast majority of patients with paroxysmal AF which is in line with success rates of pulmonary vein isolation in this population.ECS < 4 cm were noninducible after pulmonary vein isolation, suggesting that isolation of PV triggers could be successful in this subpopulation. Conversely, in patients with LAECS > 4 cm, additional substrate modification is likely to be required. In these patients, initial trigger‐dependent AF may have evolved to multiple wavelet/re‐entry based AF due to progressive structural and electrical remodeling.ECS (Figures 6D and ECS could be achieved by positioning the PV wide‐area encirclement lines more toward the center of the chamber, by isolating the LA posterior wallECS sufficiently to terminate AF and this is only achieved through an additional increase in ERP. As such, knowledge of LAECS could be used to guide ablation during the initial ablation procedure or to guide the selection of additional ablation vs. antiarrhythmic drugs for the management of recurrent arrhythmia after initial ablation. Such management strategies require investigation in prospective randomized trials.This study highlights the physiological difference between categories of AF and suggests a possible methodology by which arrhythmia management strategies could be individualized for patients post‐pulmonary vein isolation. In patients with LA5ECS in a statistical model including other clinical parameters associated with AF vulnerability and recurrence. ERP was measured at a single site in the left atrium which may not be fully reflective of the heterogeneity in refractoriness seen in patients with persistent AF. Some patients were maintained on antiarrhythmic drugs periprocedurally. While the effects of these in terms of CV and refractoriness will be accounted for in the LAECS concept, it is not possible to predict how discontinuation of medications post procedure may affect the vulnerability to AF in a particular patient from these data. Therefore, LAECS measured in the study may not reflect the true LAECS once antiarrhythmic drugs are either discontinued or resumed post procedure. This study focused on multiple wavelet mechanisms of AF only. It is possible that this hypothesis may hold for other forms of re‐entry in including micro‐entrant circuits and rotors however this was not explored in the modeling work carried out here. Similarly, we did not examine the potential roles of the right atrium in arrhythmogenesis in these patients.This was a prospective simulation/clinical study to evaluate a new clinically applicable concept. Since there were a low number of recurrence events in the cohort, it was not possible to formally test the predictive power of LA6ECS was significantly associated with the ability to induce AF in patients post first‐time ablation for persistent, but not paroxysmal AF. These data support the mechanistic hypotheses of re‐entry in the population of persistent AF patients studied. The LAECS metric may be useful for predicting AF vulnerability and guiding arrhythmia management in such patients.LAProfessor O’Neill has received research support and honoraria from Biosense Webster and has received consultation fees from Medtronic, Biosense Webster, St. Jude/Abbott and Siemens. Dr. Niederer has received research support from St. Jude/Abbott, Boston Scientific, Roche, Pfizer and Siemens.Concept/design, data collection, data interpretation/analysis and critical revision of article: SW. Data collection, data interpretation/analysis, statistics, drafting of article: LO’N. Data collection, critical revision of article: CR. Data collection: JJ. Data collection: AM; Data collection: BR; Critical revision of article: RM; Critical revision of article: IS; Critical revision of article, statistics: JW. Data collection: MW. Critical revision of article: SN; Data collection: CS; Concept/design, data collection, critical revision of article, approval of article: MO’N.


To report the first case in Togo of Biermer's disease associated with intrauterine growth retardation (IUGR) in a 39-year-old pregnant woman. 2P0 (a spontaneous abortion at 3 months), was referred to hematology on 17th March 2017 for anemia at 26 weeks of amenorrhea (WA). She had received martial treatment with ferrous fumarate 66 milligrams daily. At 26 weeks, the uterine height was 16 centimeters, and there was good fetal vitality. During ultrasound, there was a harmonious development of the fetus, but it was lower than that for the gestational age at 10th percentile based on fetal biometry, and anemia was at 65 g/l. She was then referred to hematology where she was found to have pancytopenia with macrocytic aregenerative anemia at 47 g/l (MCV at 109 fl), neutropenia at 1.02 g/l, and thrombocytopenia at 58 g/l. The myelogram found megaloblastosis at 53%, collapsed serum B12 vitamin at 61.7 pg/ml, normal serum folate at 9.9 ng/ml, increased serum homocysteine to 51.44 μmol/l, and elevated LDH. The search for intrinsic anti-factor antibodies was positive. Digestive endoscopy noted fundal atrophy. The patient was given vitamin B12 injection; at the 7th day, hemoglobin was observed at 94 g/l, then a normalization of the blood count after 3 weeks, and a good evolution of the pregnancy with delivery at 38 WA of a newborn, female, weighing 1960 g with 500 grams of placenta, with a size of 40 cm, and a cranial perimeter of 29 cm. The child had stunted weight growth .  The patient with phenotype AA, born on 20/02/1978, G Biermer's disease as a maternal cause of IUGR and infantile hypotonia is a reality in Togo. It requires management in patients and especially during pregnancy to avoid neurological complications in children born from mothers with this disease. Biermer's disease (BD) also called pernicious anemia is an autoimmune atrophic gastritis with dryness of Castle's intrinsic factor (IF) responsible for vitamin B12 deficiency. It is usually diagnosed in the presence of megaloblastic anemia, neurologic symptoms, or atrophic gastritis . ThroughIn Togo, a recent study reported cases of Biermer's disease in its classic presentation . In the th March 2017 for an exploration of severe chronic anemia. She was pregnant and at 26 WA. She was a second gesture, nulliparous, and antecedent of spontaneous abortion at 3 months. She was seen in gynecology at 13 weeks of amenorrhea (WA). She had a regular cycle, the date of the last menstruation well known confirmed by the ultrasound of the first trimester. Physical examination revealed conjunctival paleness and moderate asthenia. The prenatal checkup blood count showed anemia at 7 g/l, the deep drop the parasitological examination of the stools were without abnormalities; and the electrophoresis of hemoglobin (Hb) showed a normal Hb AA. She was not an alcoholic; she had received martial treatment with ferrous fumarate 66 milligrams per day. At 26 WA, clinical examination showed the persistence of mucous pallor and asthenia, a weight of 57 kg, a height of 163 cm, a blood pressure (BP) of 120/60 mm·Hg in both arms, and negative proteinuria. Obstetrically, the uterine height was 16 cm, and there was good fetal vitality. During ultrasound, there was a harmonious development of the fetus, but it was lower than that for the gestational age at 10th percentile based on fetal biometry. The amniotic fluid amount was normal. The uterine artery Doppler and the fetal brain Doppler were normal. The control hemogram had objectified anemia at 65 g/l. In view of etiological research of anemia, this patient was referred to hematology before the signs of IUGR complication.The patient, born on 20/02/1978, was referred to hematology of the Campus Teaching Hospital by the gynecologist on 17μmol/l (3.36–20.44 μmol/l), and elevated LDH (6044 IU/l) > 13 normal. Immunological marking of anti-intrinsic factors (IF) performed at the CERBA Laboratory in France was positive. Upper gastrointestinal endoscopy with biopsies noted fundal atrophy with erosive bulbitis. Anatomopathological examination showed chronic atrophic gastritis without Helicobacter pylori. The anti-IF antibody had not been researched on biopsy specimens. The diagnosis of BD had been posited with certainty. IUGR was secondary to this chronic anemia related to BD.The clinical examination in hematology had found the relatively compensated anemic syndrome. The general condition was good. There were no digestive or neurological signs. After hematological examination, pancytopenia with macrocytic aregenerative anemia 47 g/l (MCV at 109 fl) (reticulocytes at 23 g/l), leukopenia at 3.4 g/l, neutropenia at 1.02 g/l, and thrombocytopenia at 58 g/l were observed. The myelogram by sternal puncture found megaloblastosis at 53%. On a biochemical basis, the serum vitamin B12 assay showed a collapsed vitamin B12 at 61.7 pg/ml (208–963.5), normal serum folates at 9.9 ng/ml (7.2–15.4), increased serum homocysteine to 51.44 μg daily during 10 days and then a monthly injection for lifetime). Under this treatment, reticulocyte crisis was noted at the 7th day (reticulocytes at 94.3 g/l) with a hemoglobin rate of 94 g/l and a gradual normalization of the blood count after 3 weeks. On the gyneco-obstetrical level, good evolution of the pregnancy was noted, which resulted in a delivery on 3rd June 2017 by caesarean section at 38 WA of a newborn, female, of 1960 grams with 500 grams of placenta. The size of the newborn was 40 cm with a cranial perimeter of 29 cm. In October 2018, the child had a stunted weight loss because at 16 months, the child weighed only 7 kg for a height of 69 cm. However, she had good neurological development.The patient was given vitamin B12 deep intramuscular injection  is an autoimmune atrophic gastritis, predominantly fundic, responsible for vitamin B12 deficiency  by malabsorption of the latter . These aμmol/l. HHCh is among the congenital hypercoagulable states and is a long-known vascular disease risk factor. The discovery that HHCh may also be responsible for several pregnancy complications has only recently been made [On the obstetrical side, recent studies have shown the effects of homocysteine on the fetus. Indeed, homocysteine results from the transmethylation of methionine. Its metabolism depends primarily on three enzymes and several vitamin cofactors. Genetic abnormality in these enzymes or deficiency of these vitamins leads to hyperhomocysteinemia (HHCh). HHCh is usually biologically defined by a fasting value >15 een made . Studieseen made .N-homocysteinylation of neuronal proteins, in particular, associated with the cytoskeleton. This modification induces the aggregation of proteins, a phenomenon involved in many neurodegenerative diseases. The combination of these different mechanisms sheds new light on the developmental defects and cognitive disorders associated with early methyl donor deficiency, highlighting the importance of “fetal programming” in the occurrence of certain neurological pathologies [Also, methyl donors (B12 and folates) regulate the monocarbon cycle, which plays a key role in epigenetic/epigenomic regulation by methylation . Methyl Very recently, Savall et al. in 2015  presenteBiermer's disease as a very rare maternal cause of intrauterine growth retardation and infantile hypotonia is becoming a reality in Togo. Vitamin B12 therapy should be provided lifelong to prevent relapses of neurological conditions and fetal consequences that may be irreversible. We advocate special care from birth for children born to mothers with this disease.


Viruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching.Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced.Slides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Staining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units.This kit was used in practical virology classes for the Biological Sciences course , and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment. Viruses are the most numerous entities on Earth, and they are found in the majority of ecosystems . Over a Acanthamoeba polyphaga mimivirus (APMV), the first described amoeba giant virus  30010) cultured at 32 °C in 175 cm2 cell culture flasks with 50 mL peptone-yeast extract with glucose (PYG) medium supplemented with 25 mg/ml amphotericin B  and 500 U/ml penicillin . The amoebae were infected with at a multiplicity of infection (MOI) of 0.01. Cells were incubated until the expected CPEs appeared in culture, and the media was collected by two centrifugation steps. A. castellanii cells and cellular debris were first removed by centrifugation (400 x g for 10 min), and the particles were purified by centrifugation  through a sucrose cushion (40–50%), suspended in PAS, and stored at − 80 °C. KAUV, OBRV, and FTSV were propagated individually in Vermamoeba vermiformis (ATCC50237) cultivated at 32 °C in PYG medium with a MOI of 0.01. After the appearance of CPEs, the cells and supernatants were collected, with sterile serological pipettes, stored in sterile conical tubes, and the viruses were purified through ultracentrifugation with a sucrose cushion.TPV, CVG, PDV, and Niemeyer virus (NYMV) were propagated individually using 2 at 37 °C in Eagle’s minimum essential medium , supplemented with 5% fetal bovine serum , 2.5 μg/mL amphotericin B, 500 U/mL penicillin , and 50 μg/mL streptomycin .African green monkey kidney BSC-40 cells (ATCC CRL-2761) and Vero cells (ATCC CCL-81) were maintained in an atmosphere with 5% CO2 in a large 175 cm2 bottle. Subsequently, for YFV, MAYV, and CHIKV, the cell infection supernatant was transferred to tubes and centrifuged at 3,000 x g for 5 min. The clarified supernatants from these centrifugations were stored at − 70 °C. VACV and CPXV were purified on a sucrose gradient as previously described [YFV (vaccine strain 17DD), MAYV (Strain BeAr20290), CHIKV , VACV group I (Isolate Caragola eye I), VACV group II (Isolate Caragola eye II), and CPXV (strain Brighton Red) were individually multiplied in Vero cells in MEM that contained 1–2.5% FBS, 0.25 μg/mL amphotericin B, 100 U/mL penicillin 100 U/mL , and 100 μg/mL streptomycin at 37 °C in an atmosphere with 5% COAliquots of purified virus were diluted 1:10  or 1:20 (NYMV), and 10 μL of the appropriate dilution was placed on the center of glass slide. The liquid was spread using circular movements to obtain an approximately 1-cm diameter drop. The slide was kept at room temperature until the liquid dried on the surface. Subsequently, the virus was fixed by adding 200 μL methanol over the center of the slide and stained with crystal violet for 15 min. After staining, the slides were washed in running water and dried at room temperature. Once dried, the stained region was covered by a 13 mm glass coverslip and affixed to the slide with Canada balsam .A. castellanii cells in PYG medium were cultured in cell culture flasks (25 cm2). After the amoebae adhered to the flask, NYMV was inoculated with a MOI of 1. After 12-h infection, cells were removed from the flask and centrifuged at 885 x g for 10 min and stained with hemacolor reagents . Once dried, the stained region was covered with a 13 mm glass coverslip using Canada balsam.Approximately 1 million 5 cells/well) were plated on sterile 13 mm coverslips, grown in MEM with 5% FBS for 24 h, and maintained at 37 °C in an atmosphere with 5% CO2. The next day, the cells were infected with viruses and observed until the characteristic CPE appeared (lysis plaques). Once CPEs were detected, the coverslips were fixed in formaldehyde (3.7% v/v) for 2 h and stained with crystal violet. After staining, the coverslips were washed, dried, and affixed to the glass slides using Canada balsam. For viruses from both VACV groups and CPXV, lysis plaque visualization was performed by infecting BSC-40 cells. The same procedure described above was followed to prepare this slide.Two cell lines were used for visualization of virus plaque-forming units. For MAYV, CHIKV, and YFV, Vero cells (2 × 105 cells/well) were plated on sterile 13 mm coverslips, grown in MEM with 5% FBS for 24 h, and maintained at 37 °C in an atmosphere that contained 5% CO2. The cells were infected with CPXV (MOI: 10), and once CPEs were detected, the coverslips were fixed in formaldehyde (3.7% v/v) for 2 h and stained with the solution rich in eosin from the hemacolor kit for 10 min. After staining, the coverslips were washed, dried, and affixed to glass slides using Canada balsam.BSC-40 cells , and it will soon be made available at a low cost to elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment about virology.We designed a slide microscope kit named “Virus Goes Viral” that explores giant virus particles and some aspects of animal virus interactions with cells, with the aim to provide an innovative approach in virology teaching. Slides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Additional file 1: Figure S1. The cytopathic effects caused by different viruses on Acanthamoeba castellanii.Additional file 2: Figure S2. The cytopathic effects caused by different viruses on Vermamoeba vermiformis.Additional file 3. Infographics related to Virus Goes Viral educational kit.