Arctium lappa L. The anticancer effects of arctiin have been demonstrated in several studies. However, no research has been conducted on the anti-migration effect of arctiin in cervical cancer cells. The present study examined the effects of arctiin on cervical cancer cells and investigated the possible molecular mechanism. We demonstrated that arctiin exhibited low cytotoxicity and significantly inhibited cell migration and invasion in human cervical cancer cells. The S100A4 protein expression and mRNA levels were significantly reduced in HeLa and SiHa cells with arctiin treatment. Furthermore, silencing S100A4 by using small interfering RNA reduced cell migration, while overexpression of S100A4 mitigated the migration inhibition imposed by arctiin in cervical cancer cells. Western blotting revealed that arctiin significantly reduced phosphoinositide 3-kinase (PI3K) and phosphorylation of Akt in cervical cancer cells. Moreover, selective Akt induction by an Akt activator, SC-79, reverted cervical cancer cell migration and S100A4 protein expression, which were reduced in response to arctiin. Taken together, these results suggest that arctiin inhibits cervical cancer cell migration and invasion through suppression of S100A4 and the PI3K/Akt pathway.Arctiin, a lignan glycoside, is isolated from  As the fourth most common malignancy diagnosed in women worldwide, cervical cancer is a global health concern . NotablyS100A4, a member of the S100 family of calcium-binding proteins, plays a key role in the process of human tumorigenesis and metastasis . S100A4 Arctium lappa L. The anti-inflammatory effects of arctiin have been widely investigated [Arctiin, a lignan glycoside, is isolated from stigated ; its antstigated . Moreovestigated ; its celstigated . Several2. Authentication of cancer cell lines by short tandem repeat (STR) DNA profiling analysis was performed . The workflow included DNA extraction, multiplex PCR, fluorescent capillary electrophoresis, data analysis with GeneMapper v4.0 and STR profiling analysis with the DSMZ database. The results were highly similar to SiHa (DSMZ Number: HTB-35)  value: 0.94) and HeLa.P3 (DSMZ Number: JCRB0649.1)  based on comparison with the DSMZ database.Human cervical cancer cell lines: HeLa and SiHa were cultured in Dulbecco’s modified Eagle’s medium (DMEM). Human proximal tubular (PTC) cell line HK-2 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12 mix medium. We supplemented DMEM with 10% fetal bovine serum (FBS) and 100 ng/mL each of penicillin and streptomycin (P/S). Both of HeLa and SiHa cervical cancer cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 for 4 h. The viable cells were detected spectrophotometrically at 563 nm .Arctiin was bought form Sigma-Aldrich (CAS Number: 20362-31-6). HeLa, SiHa and HK-2 cells were seeded in 24-well plates and stimulated with different concentrations of Arctiin  for 24 h. After that, we removed the medium and carefully washed it with 1X PBS. MTT (Sigma) were added to each well and incubated at 37 °C in 5% CO2 for 24 h. For invasion: 10 μL Matrigel  was applied to 8 μm pore size polycarbonate membrane filters that included 10% FBS DMEM medium and then air-dried for 3~4 h in an incubator. The migrated and invaded cells were fixed by methanol for 10 min, stained with Giemsa and counted by light microscopy [HeLa and SiHa cells were treated with different concentrations of Arctiin 0, 10, 20, 40 μM) or SC-79 (10 μM) for 24 h. Then, we collected the cells by Trypsin (Gibco) and the cell migration assay in vitro was seeded in a Boyden chamber (Neuro Probe). For the migration assay: Treated cells in 0.5% FBS medium were loaded into the well of the chamber at the upper part and incubated at 37 °C in 5% CO, 40 μM oHeLa and SiHa cells were seeded into 6-cm plates and cultured for 16~18 h. After that we transfected 5 µg of the empty CS2-vector  or CS2-S100A4  into the cells and left for 6 h before carefully removing the reagent and culturing the cells with fresh 10% FBS DMEM medium overnight. The next day, the transfected cells were treated with Arctiin (0 and 40 μM) for another 24 h and analysis was performed by the migration assay and the Western blot assay.The cell lysates which were treated with Arctiin  or SC-79 (10 μM) that separated in a polyacrylamide gel and transferred onto PVDF membranes. Membranes were incubated with 5% non-fat milk in TBST for 1 h in room temperature and, subsequently, covered with a corresponding antibody against a specific protein overnight at 4 °C. Next, we removed the primary antibody and washed it with TBST buffer for 7 min, three times. Signal was detected by the ECL solution system. Antibodies used were as follows: anti-S100A4 , anti-p-JNK , anti- JNK , anti-p-ERK , anti-ERK , anti-p-P38 , anti-P38 , anti-p-Akt , anti-Akt , anti-PI3K  and anti-β-actin .HeLa and SiHa cells were treated with Arctiin  or SC-79 for 24 h, and total RNA was extracted using the TRIzol reagent. For reverse transcription, the first-strand cDNA was generated using the high-capacity cDNA reverse transcription kit. PCR amplification was performed as described previously . S100A4 t-test . Significance was set at p < 0.05. The values are the means ± standard deviation (SD) of at least three independent experiments.Statistically significant differences were calculated using the Student’s The effects of arctiin A at variCell migration and invasion assays with the Boyden chamber were used to determine the effects of arctiin on HeLa and SiHa cells. For cells treated with various arctiin concentrations (0–40 μM), the cell migration abilities of HeLa were significantly reduced by arctiin compared with the control group A,B. In aS100A4 protein and messenger ribonucleic acid (mRNA) expression were significantly reduced in HeLa cells treated with arctiin  compared with the control A,C. SimiTo determine the effect of S100A4 expression on cervical cancer cell migration, the overexpression vector of S100A4 (CS2-S100A4) was used. Boyden chamber assay and Western blotting revealed that S100A4 overexpression markedly increased cell migration activity and S100A4 protein expression in HeLa cells, respectively A–C. We cTo further analyze the mechanisms involved in arctiin-mediated S100A4 suppression, Western blotting was used to evaluate the effects of arctiin on the expression of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. Western blotting demonstrated that arctiin significantly reduced PI3K expression and phosphorylation of Akt in HeLa and SiHa cells in a concentration-dependent manner A. HoweveVarious Chinese herbal medicines or herbal products have been investigated for the treatment of cervical cancer ; the antThe S100A4 protein, a member of the S100 family, has been discovered to have a potent role in inflammation-associated diseases and the biological functions of cell differentiation, angiogenesis, apoptosis, migration, and invasion . IncreasThe PI3K/Akt pathway regulates a broad spectrum of cellular processes, including cell growth, proliferation, metabolism, motility, survival, and apoptosis . AlteratChelidonium majus resulted in the induction of apoptosis in HeLa cells through possible alterations of the PI3K/Akt and p38 signaling pathways [Reineckia carnea, exerted an anti-cervical cancer effect on HeLa cells by reducing PI3K, Akt, mTOR, and NF-κB p65 phosphorylation levels [Several compounds exert anticancer effects on cervical cancer through Akt signaling pathways . Chelidopathways . Butein,pathways . RCE-4, n levels . In a stn levels . A breasn levels . Becausen levels . In our In conclusion, our study revealed that arctiin inhibits cervical cancer cell migration and invasion through the suppression of S100A4 expression and the PI3K/Akt pathway. However, our study was limited by the lack of an in vivo animal study. Therefore, further pharmacological and clinical studies are required to verify the therapeutic potential of arctiin in cervical cancer.


Sambucus ebulus (SE), a famous traditional Iranian medicine, is grown in the north of Iran. As a traditionalmedicine with anti-inflammatory effects, SE has been utilized against inflammatory joint diseases, insect bites,infectious wounds, edema, and eczema. Type1 diabetes, is an autoimmune disease, characterized by the destructionof pancreatic beta cells by the immune system. For the first time, we investigated the effect of methanolic extract of SEon CD4+, CD8+ and regulatory T cells in experimental type 1 diabetes (T1D). In this experimental study, fifty-six C57BL\6 mice in 8 groups (G1-G8), were enrolled. Diabeteswas induced by a multiple low-dose streptozotocin (MLDS) protocol and mice were daily treated with SE extract at 200and 400 mg/kg doses, for 35 days. Fasting blood glucose was weekly measured by a glucometer. Islets insulin contentwas analyzed by immunohistochemistry. Percentage of CD4+, CD8+ and regulatory T cells and cytokines productionlevels were evaluated by flow cytometer and ELISA, respectively. The clinical symptoms of diabetes were significantly alleviated in G2 group mice which received 400 mg/kg SE extract. Immunohistochemistry analysis showed that the insulin content of islets increased in G2 group mice.Immunophenotyping analysis indicated that the percentage of CD4+ and CD8+ T cells in G2 group mice was significantlydecreased. SE extract significantly increased the percentage of regulatory T cells. The extract in G2 and G4 groupsmice significantly decreased IFN-γ and IL-17levels. The extract significantly increased IL-10 in G2 group mice.The protective effect of SE extract in MLDS-induced diabetes could be partly due to a decrease of CD4+and CD8+ T cells and an increase of Treg cells resulting in an inflammation reduction in the pancreatic islets. Type 1 diabetes (T1D) is the most prevalent chronicmetabolic disorder of younger adults in which progressivedestruction of insulin-producing β-cells leads to the lossof glucose homeostasis . AlthougBoth CD4+ and CD8+ T cells have been considered keyeffectors involved in T1D development and progression. CytotoxIslet CD4+ T cells destruct β-cells through secretionof pro-inflammatory cytokines . T helpeSambucus ebulus (SE), from the Caprifoliaceae family, extensively growsin the northern regions of Iran, has been shown to have anti-inflammatory andanti-nociceptive effects  induced-T1D.rd fraction) was concentrated using a rotary vacuum evaporator(for about 45 minutes at 35˚C) until a solid extract sample was obtained. The crudeextract was freeze-dried for complete water removal. The extract was prepared in normalsaline and tween 80 (5%) for pharmacological studies and kept at -20˚C until use.SE leaves were collected from Sari, Iran in September 2015. The voucher species of theplant has been deposited in the herbarium of the Faculty of Pharmacy, Sari University ofMedical Sciences (herbarium No. 87). The leaves were air-dried at room temperature andthen coarsely grounded before extraction. Here, 100 g of sample was fractionated bysuccessive solvent extraction at room temperature using maceration with hexane (250 ml×3)then ethyl acetate (250 ml×3) and finally methanol (250 ml×3), respectively. The methanolextract  were purchased from PasteurInstitute . The mice were housed in cages(7 mice in each cage) with a 12/12 hours light/dark cycleand access to food and tap water in a ventilated room.Temperature and humidity of the room were held at25 ± 2ºC and 55-60%, respectively. All mice were fedwith a pelletized normal commercial chow diet  for 7 days after arrival.After one-week adaptation, the animals under wentthe experiments. The study protocol was approved bythe Animal Ethics Committee of Tehran University ofMedical Sciences, Tehran, Iran (IR.TUMS.MEDICINE.REC.1398.618).Experimental autoimmune diabetes was induced usingmultiple low-dose streptozotocin (MLDS) protocol thatclosely resembles inflammatory changes within thepancreas in humans. Streptozotocin was dissolved in 0.1 M citrate buffer (pH=4.5). Thefreshly prepared STZ was injected within 10 minutes.Mice were injected intraperitoneally (i.p.) with STZ 40mg/kg daily for 5 consecutive days. Blood glucose levelsin mice were measured after 6 hours of fasting. Fastingblood sugar (FBS) concentration was measured in theblood drawn from the tail vein on days 0 (one day beforethe first dose of STZ), 7, 14, 21, 28, 35 using a glucometerTest-strips . Mice with FBSover 250 mg/dl were considered diabetic.To investigate the efficacy of SE extract in the preventionand treatment of STZ diabetes, animals were randomlydivided into 8 experimental groups (7 mice in eachgroup) as follows. G1: mice received both STZ  and SE extract (200 mg/kg) simultaneously; G2:mice received both STZ  and SE extract(400 mg/kg) simultaneously; G3: STZ diabetic micereceived 200 mg/kg SE extract 15 days after the first STZadministration; G4: STZ diabetic mice received 400 mg/kg SE extract 15 days after the first STZ administration;G5 (diabetic control group): mice received both STZ andnormal saline containing 5% tween 80 simultaneously;G6(non-diabetic control group): mice received normalsaline containing 5% tween 80; G7: healthy mice received200 mg/kg SE extract and G8: healthy mice received 400mg/kg SE extract. SE extract was i.p. administered once aday for 35 days , 19.Mice were killed by anesthesia using ketamine andxylazine combination. It is 100 mg/kg body weightfor Ketamine and 10 mg/kg body weight for Xylazineinjected i.p. Single-cells were isolated from the spleensand the erythrocytes were lysed using an isotonic solutionof ammonium chloride (pH=7.2). After washing withphosphate-buffered saline ,the cell suspensions were used for both phenotypicanalysis and cytokine evaluation.6 cells/100 µl staining buffer. Thecells were stained with anti-mouse CD4-PerCP-Cy5.5, CD8-Alexa Fluor 488, CD25- APC,andFoxp3-PE . For the analysis of regulatory CD4+ T cells, the cells weretreated by fixation/ permeabilization solution  according to themanufacturer’s instructions. The incubation time was 30 minutes at 4° C. All lymphocytessubsets were analyzed by FACS Calibur instrument . The flowcytometer data analyses were done using the FlowJo software version 7.6.1 .Splenocytes suspension was adjusted to 1×106 cells/ml of RPMI-1640 culture medium , supplementedwith 10% inactivated fetal bovine serum . The samples were stimulated with Concanavalin A  10 µg/ml and incubated for 48 hours . Thesupernatants were then collected and stored at -80◦ C until cytokines measurement. Thecytokines were quantified using ELISA kits according to the manufacturer’s instructions. The lower limit of detection for IFN-γ and IL- 10 were 31.3 pg/mland for IL-17 was 15.3 pg/ml.For determination of IFN-γ, IL-10 and IL-17 cytokines level, splenocytes were dispensedin a 24-well flat-bottom tissue culture plate  at1×102 O2) for 15-20 minutes to blocknonspecific background staining. The slides were heated in Tris/EDTA buffer pH=9.0. for 10 minutes and washed three times in PBS. The sections were blockedin PBS buffer containing 0.1% Triton  and 1% bovine serum albumin (BSA)for 60 minutes. Then, the slides were incubated with rabbit anti-insulin monoclonalantibody  overnight and then with sheep anti-rabbit IgGcoupled with peroxidase . Chromogen color development wasaccomplished with 3, 3-diaminobenzidine tetrahydrochloride . The slides werecounterstained with Harris’s hematoxylin .To investigate the effect of SE extract on insulin secretion by islets cells, weevaluated the insulin content in deparaffinated pancreatic tissue sections usingimmunohistochemistry. Briefly, the pancreaswas removed and fixed in phosphate buffercontaining 10% formalin (pH=7.2). The paraffin blocks were cut into 4-5 µm thick sections.The sections on silane-coated slides  were rehydrated and treatedwith 0.1% hydrogen peroxide . All data are presentedas the mean ± SD. Bodyweight and FBS of experimentalgroup sat different time points were analyzed by repeated-measures ANOVA. Comparing CD4+ and CD8+ T cellsubsets percentage and cytokines concentration wasperformed using one-way ANOVA followed by Tukey’stest. P<0.05 were considered to be statistically significant.Furthermore, GraphPad Prism software 5.0  was used for drawing graphs.Diabetes was successfully induced in mice (6-8 weeksold) by STZ injection. FBS concentration in diabeticmice was above 250 mg/dl on days 14 and 15. In diabeticcontrol mice (G5), FBS was significantly higher thanhealthy controls (G6) (P<0.0001). Mice in the G5 grouphad polydipsia and polyuria in comparison to the mice ofthe G6 group. Also, the body weight of the G6 mice wassignificantly higher than that of the G5 mice (P<0.0001).SE extract, at 200 and 400 mg/kg, was administreredsimultaneously with diabetes induction . The blood glucose levels in diabetic groupsstarted to increase from day day 7. Interestingly, 400 mg/kg SE extract could significantly prevent the rise of glucoselevels in the G2 mice in comparison to the G1 and G5 mice(P<0.0001). There was no significant difference in bloodglucose levels in the G3 and G4 mice which received SEboth concentrations on day 15, in comparison to the G5mice. The administration of this extract had no effect onblood glucose levels in non-diabetic mice (the G7 andG8 groups). The blood glucose levels of these mice weresimilar to those observed in the non-diabetic G6 groupwhich did not receive the extract .Furthermore, the body weight of the G1, G4, and G5mice was significantly reduced compared to the G2 andG6 mice (P<0.0001). However, the bodyweight of thehealthy groups that only received the extract (the G7 andG8) was not significantly different from that of the G2 andG6 . Body weMice treated with the 400 mg/kg SE (the G2) did notshow polydipsia and polyuria compared to diabetic mice, and were similar to non-diabeticmice .low and CD8hi cells populations were analyzed on gated cells.At least 50,000 events were counted for each sample. The gating strategy is illustrated inTo analyze the influence of SE extract on splenic lymphocytes proportions, we assessedthe percentage of both CD4+ and CD8+ T cell subsets in the studied experimental groups.First, lymphocytes were gated on a forward vs. side scatter dot plot and then Treg,CD8We observed that 400 mg/kg SE extract promoted asignificant increase in CD4+ cells in the G8 as comparedto the G6 P<0.05, . The SE high lymphocytes percentage in comparison to diabetic mice  (P<0.0001), whereas CD8low cells reduced in the G2 compared to theG1 .However, SE extract at 400 mg/kg (the G2) significantly decreased CD4+, CD8+ andCD8Our data revealed that the diabetic control group (theG5) had a significantly higher IFN-γ level than thenon-diabetic control group (the G6) P<0.05, .BesidesImmunohistochemistry (IHC) analysis of pancreatictissues in different groups of mice showed increasedproduction of insulin in the G2 mice but a significantreduction in the G1, G4 and G5 groups. The amount ofinsulin production in healthy mice receiving the extract(the G7and G8) was similar to that of healthy controlmice (the G6) .T1D is a chronic autoimmune disease that occursin genetically susceptible individuals . The disFor the first time, we showed that the SE extract wasable to prevent the development of experimentaldiabetesso that after 35 days of extract injection, FBS in diabeticmice reached the normal range. Releasing of autoantigensresults in immune cell responses, inflammation in thepancreas and increased blood glucose within 2 weeks, 21. Uncaria tomentosa, Cassiaauriculata and Bauhinia forficate  due to an increase in the number of healthy islets in thepancreas in comparison with the diabetic control group (the G5), whereas insulin productionsignificantly reduced in the G1, G4 and G5 mice in which a large number of pancreatic isletswas damaged by STZ, compared to the G6 mice. Similar findings were obtained in previousstudies using extracts , 23.Sinhi andCD8low T cells in the G2 mice. CD8+ T cells are divided into two groups basedon the level of the expression of CD8: CD8high and CD8low T cells(low T cells indicate theirrole as cytotoxic/effector T cells. These cells also produce significant amounts of IFN-γ(Both CD8+ and CD4+ T cells are involved in T1D pathogenesis and can cause the death of betacells . Direct  T cells. High leof IFN-γ. Therefoof IFN-γ.Uncaria tomentosa extract reach the percentage of Treg cells tothe percentage level of healthy mice (In the development of T1D, a defect in the function of Treg cells may be more importantthan a defect in their number . Patientthy mice .Uncaria tomentosa extract increased IL-4 and IL-5levels, suggesting that the cytokine profile could be adjusted to Th2 (Cordyceps sinensissignificantly delayed the onset of T1D and reduced its severity. This effect is attributedto an increase in the ratio of Treg to Th17 cells (Origanum vulgare extract altered the signaling pathway of Pro-Th17 toPro-Treg, and the number of Th2 and Treg cells in mice treated with this extract was higherthan that of diabetic mice (Th17 cells play an important role in T1D by increasing the destruction of islets via Th1cells. Th17 cells in the T1D pathogenesis can also stimulate TCD8 + cells . Th2 celd to Th2 . Oralad17 cells . It was tic mice .Origanum vulgare extract was ableto neutralize ROS and RNS production and prevent beta-cell apoptosis (The extract of SE also has antioxidant properties , 17. Sinpoptosis .Overall, it is probable that the protective effect ofSE extract in MLDS-induced diabetes is at least partlydue to a reduction in the process of inflammation in thepancreatic islets. These results indicate that SE can protectmice against autoimmune diabetes and in the future, it canbe valuable in treating diabetes and other autoimmuneand inflammatory diseases in humans. Of course, moreresearch is needed on this subject.


We mapped epigenome changes and identified 23,394 and 13,133 peaks in ND and T2D individuals, respectively. Out of all the peaks, 753 domains near the transcription start site (TSS) were unique to T2D. We found that T2D in atherosclerosis leads to an H3K9ac increase in 118, and loss in 63 genomic regions. Furthermore, we discovered an association between the genomic locations of significant H3K9ac changes with genetic variants identified in previous T2D GWAS. The transcription factor 7-like 2 (TCF7L2) rs7903146, together with several human leukocyte antigen (HLA) variants, were among the domains with the most dramatic changes of H3K9ac enrichments. Pathway analysis revealed multiple activated pathways involved in immunity, including type 1 diabetes. Our results present novel evidence on the interaction between genetics and epigenetics, as well as epigenetic changes related to immunity in patients with T2D and advanced atherosclerotic disease.There is emerging evidence of an association between epigenetic modifications, glycemic control and atherosclerosis risk. In this study, we mapped genome-wide epigenetic changes in patients with type 2 diabetes (T2D) and advanced atherosclerotic disease. We performed chromatin immunoprecipitation sequencing (ChIP-seq) using a histone 3 lysine 9 acetylation (H3K9ac) mark in peripheral blood mononuclear cells from patients with atherosclerosis with T2D ( Atherosclerosis accounts for more than 80% of deaths among patients with diabetes. Strong evidence from large treatment studies, such as the United Kingdom Prospective Diabetes Study (UKPDS) and Diabetes Control and Complications Trial (DCCT), supports an association between glycemic control and cardiovascular disease (CVD) risk ,2. AnimaEpigenetic mechanisms involve interactions between environmental factors  and gene expression via altering DNA methylation, non-coding RNAs, and histone modifications. These processes may persist for a lifetime and even be heritable. Histone acetylation contributes to the regulation of gene expression through its effect on conformational changes in chromatin. Histone 3 lysine 9 (H3K9ac) is a frequently acetylated site in active gene transcription under hyperglycemia. Previous investigations, including our own studies, have shown that H3K9ac is an important modification for transcription activity in glucotoxicity ,7,8.NF-kB and TXNIP in blood monocytes, pancreatic islet cells and kidney cells [Histone acetylation is a dynamic process regulated by histone acetyl-transferases (HATs) and histone deacetylases (HDACs), which add and remove acetyl groups, respectively. HATs transfer acetyl groups generated from acetyl-coenzyme A (acetyl-CoA) to lysine residues on histone tails. Glucose is one of the major sources for the production of acetyl-CoA via the tricarboxylic acid cycle ,10,11,12ey cells ,15,16. TIn this study, we compared the genome-wide profiles of H3K9ac in peripheral blood mononuclear cells (PBMCs) of atherosclerotic patients with or without T2D, to elucidate key epigenetic mechanisms underlying T2D in atherosclerosis.n = 8) or without (n = 10) T2D. The patients’ characteristics are summarized in The CPIP cohort is designed to study atherosclerosis and inflammatory or immune markers to identify mechanisms in atherosclerotic plaques that lead to the development of myocardial infarctions or strokes. The CPIP is an ongoing study since November 2005 that recruits patients who undergo carotid endarterectomy at Lund University Hospital, Malmö, Sweden. Blood samples were drawn from patients a day before the surgery. The criteria for surgery are (1) stroke, transient ischemic attack or amaurosis fugax and a stenosis degree (assessed by ultrasound) of >70%, or (2) no symptoms and a stenosis degree of >80%. For the current study, we studied blood PBMCs from 18 patients with . All experiments were performed in accordance with relevant guidelines and regulations.g for 10 min without braking to form gradients. The upper most layer was plasma, followed by the PBMC cell layer, the ficoll layer and granulocytes. Carefully, the PBMC layer was collected and washed by adding 0.9% NaCl, centrifuged at 600× g for 10 min. The washing step was repeated by adding NaCl, and followed by centrifugation at 300× g for 10 min. Finally, cells were suspended in 500 µL autologous plasma and cells were counted using a Burkes chamber. Cells were stored in freezing media containing 20% DMSO in RPMI1640 in liquid N2.Five milliliters of an EDTA-blood sample were used to isolate PBMC using Ficoll Paque Plus (GE17-1440-02) density gradient centrifugation. The total volume of blood was layered on 2.5 mL Ficoll Paque Plus and centrifuged at 1350× ChIP was performed as previously described ,17. PBMCThe purified DNA was then processed including end repair: A-tailing and barcode adapter ligation . DNA libraries were then sequenced on Illumina HiSeq 2000.−3) from pooled H3K9ac tags of patients with each individual’s input tags as control. Within each pooled sample, peaks whose termini were within 150 bp were merged into one peak. The MAnorm method was then used to compare H3K9ac enrichment across the two study groups. MAnorm took the coordinate of all peaks and aligned reads in both group samples as input. The  value of each common peak was then calculated and plotted, where M = log2 (Read density in sample 1/Read density in sample 2) and A = 0.5 × log2 (Read density in sample 1 × Read density in sample 2). A robust regression was subsequently applied to the  values of all common peaks and a linear model was derived. Finally, the linear model was extrapolated to all peaks for normalization. For each peak, a p-Value was calculated to examine the statistical significance (<0.001) of read intensity difference between the samples from the two groups. The p-value calculation was based on a Bayesian model developed by Audic and Claverie [2 fold change of the read density at a peak region between two samples, and was used for downstream analysis . Scatter plots, histograms, and box plots of ChIP-seq data were visualized using R software. Representative peaks at each gene that had significantly increased or decreased enrichment were generated by IGV software , Broad Institute of MIT and Harvard, Cambridge, MA, USA).ChIP seq was performed with 40 M effective reads. The ingroup standard deviation among replicates was set at 10% of the average read density. ChIP-seq tags generated with the Illumina HiSeq platform were de-multiplexed with the bcl2fastq utility and aligned to the human reference genome (assembly NCBI37/hg19) using BWA v0.7.10, allowing up to three mismatches per sequencing tag (default parameters). Peaks were detected using MACS2 v2.1.1  <1 × 10Claverie . The valp < 0.05 were considered to be statistically significant.Statistical analysis of ChIP-seq data was performed by a Welch’s test. A Mann–Whitney test was used for clinical comparison of the patients from the ND and T2D groups. Data presented in this manuscript are available upon reasonable request to the corresponding authors, with the exception of sensitive data according to current GDPR regulations.n = 8) or without T2D , all with advanced atherosclerotic disease. The H3K9ac peaks in each group were detected by the MACS2 peak calling method (FDR < 1 × 10−3). Differential peak enrichment was evaluated by assessing the enrichment of the corresponding region in individual samples. ChIP-seq peak calling detected 23,394 peaks in subjects without T2D and 13,133 peaks in subjects with T2D  polymorphism at rs7903146 is known to be highly associated with an increased risk for T2D from multiple large population studies [TCF7L2 rs7903146 as one of the loci with the most highly increased H3K9ac enrichment in T2D ; and HLA SNPs including HLA-B (p = 0.0004), HLA-DQB1 (p = 0.0006), HLA-DRB1 (p = 0.001), HLA-DRB5 (p = 0.01) and HLA-DQA1 (p = 0.02) (Genome-wide association studies (GWAS) in large population studies have led to the discovery of T2D-associated genetic variants loci, e.g., d TCF7L2 ,21. The d TCF7L2 , we aimed TCF7L2 . We appl = 0.02) . These dp < 0.05, Welch’s t test) in T2D included pathways related to a response to allograft rejection, cell adhesion molecules, ErbB signaling, T1D, autoimmune thyroid disease, graft-versus-host disease, endocytosis, antigen processing and presentation, Wnt signaling pathway, etc., the majority of which are linked to immune responses  in the presence of T2D [TCF7L2 was identified as a key gene to be differentially expressed in CAD patients with T2D [In this study, we investigated the modification of H3K9ac due to its association with T2D from previous studies. In T2D, despite the discovery of a large number of genetic loci associated with T2D by GWAS, the identified variants only explain a small proportion (~10%) of the heritability of T2D . A growiT2D risk . Our nove of T2D , and in with T2D . At thisHLAs with most modifications in H3K9ac enrichment in T2D. Furthermore, we also found a significant association between T2D H3K9ac enrichment and pathways related to immunity, notably, T1D as one of the major identified pathways. The HLAs are reported to account for up to 50% of the familial aggregation of T1D, with the major genetic determinants in polymorphisms of class II HLA DQ and DR [HLA that have been associated with T1D to be the most differentially enriched in H3K9ac, e.g., HLA-B, HLA-DQB1, HLA-DRB, HLA-DRB5, and HLA-DQA1. It has previously been shown that there is no fundamental difference between the immune activation and inflammation present in atherosclerosis among non-diabetics as compared to diabetics [Another surprising finding from our study is that we also identified several loci in the Q and DR . In our iabetics . Albeit Comparison of the ND vs. T2D ChIP-seq profile revealed a redistribution of H3K9ac with T2D. Previous in vitro models mimicking hyperinsulinemia showed that high insulin leads to a global increase in chromatin-associated histone acetylation, in particular at H3K9 . An earlIn conclusion, our study provides fine mapping of genome-wide histone acetylation changes in patients with T2D and advanced atherosclerotic disease. By identifying loci linked to T2D and T1D genetics, we revealed the potential mechanisms of epigenetic and genetic interactions. Furthermore, we also suggest epigenetic modifications in pathways related to immunity in T2D. These findings open the possibility that prevention of T2D-dysregulation at the chromatin level may present novel therapeutic avenues for T2D.


The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts with RB1. We show that MmuPV1 E7 interacts through its C terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting noncanonical activities of RB1, which are independent of RB1’s ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis.The species specificity of papillomaviruses has been a significant roadblock for performing  Papillomaviruses (PVs) have been isolated from a wide range of vertebrate species. They have a tropism for squamous epithelia, and individual genotypes often have a marked preference for infecting mucosal or cutaneous squamous epithelia. Approximately 440 human PVs (HPVs) have been identified, and they are phylogenetically classified into several genera . Among t7–Cell- and animal model-based studies have revealed that the E6 and E7 proteins of high-risk alpha HPVs, as well as the cSCC-associated beta HPVs, have oncogenic activities. HPV E6 and E7 encode small, cysteine-rich, metal-binding proteins. They lack intrinsic enzymatic activities, do not function as DNA binding transcription factors, and do not share extensive sequence similarities with cellular proteins. By binding to and interfering with the functionality of important, host regulatory proteins, they elicit profound alterations in cellular physiology to permit long-term viral persistence as well as viral progeny synthesis , 11. In Mus musculus papillomavirus type 1 (MmuPV1), which can be used to experimentally infect standard laboratory mice, has finally provided a viable model system to explore the importance of specific virus-host interactions in viral pathogenesis. MmuPV1 was discovered based on its ability to cause cutaneous papillomas and provides us an opportunity to better understand molecular mechanisms by which HPVs promote cutaneous disease in an in vivo animal model  signaling by interacting with the NOTCH coactivator MAML and the DNA binding SMAD2 and SMAD3 proteins that are downstream of TGF-beta signaling . Moreovein vitro in 293FT cells as described previously  genomes were used to infect Nude-FoxN1nu/nu mice at cutaneous sites, both on the ears and tail, at the same dose of 108 viral genome equivalents (VGE) that was shown previously to induce papillomas at 100% of sites infected with wild-type MmuPV1  or E7-null  analyses of carboxyl or amino terminally epitope-tagged MmuPV1 E7 . The numbers of unique and total peptides identified for each protein are shown. See reference Copyright © 2021 Wei et al.2021Wei et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The canine papillomavirus 2 (CPV2) E7 protein, which, similar to MmuPV1 E7, lacks an LXCXE domain, has been reported not only to bind RB1 but also to destabilize it . Given t1 to the S phase of the cell cycle. When RB1 is hyperphosphorylated by cyclin-dependent kinases, it permits E2Fs to function as transcriptional activators and drive S-phase progression  was constructed  as described previously . There was no apparent size difference between warts from the MmuPV1-infected wild-type mice and those from LRb1 mice based on hematoxylin and eosin (H&E) analysis (data not shown).We previously used  disease , 37. Giveviously  using thFVB mice   or truncation mutants of RB1 lacking the amino terminus (amino acid residues 379 to 928) or the C terminus (amino acid residues 1 to 792) in SAOS2 human osteosarcoma cells. HPV16 E7 was used as a control. As expected, HPV16 E7 efficiently bound wild-type RB1 as well as the two truncation mutants, both of which encode the A and B domains of RB1 that contain the LXCXE binding cleft. In contrast, MmuPV1 E7 did not efficiently interact with the 1–792 mutant RB1 that lacks the C-terminal domain of RB1  and B. HΔ84-87) and an alanine substitution at aspartate residue 90 (MmuPV1 E7D90A) were found to be defective for RB1 binding  or 108 (stock 1) VGE. Papilloma incidence was monitored biweekly for 4 months. At the endpoint, wild-type MmuPV1 at the 108 VGE dose caused papillomas at 100% of the sites infected. In contrast, the same dose of MmuPV1 E7D90A quasivirus caused papillomas at a significantly lower frequency, 12% . At the lower 107-VGE dose, wild-type MmuPV1 caused papillomas at a 65% frequency, whereas MmuPV1 E7D90A caused papillomas in only 8% of infected sites . At both doses, the papillomas arising on mice infecting with MmuPV1 E7D90A appeared at later time points  (8 VGE and 3 from 107 VGE) and 3 papillomas induced by MmuPV1 E7D90A quasiviruses  , indicating that the loss of E7’s ability to interact with Rb1 correlates with reduced size of MmuPV1-induced papillomas. We harvested and sequenced the MmuPV1 genomes present in warts arising from mice infected with the wild type and the E7D90A quasiviruses and confirmed that warts indeed contained the expected virus and that no cross-contamination had occurred. Together, the assessments of the incidence of papillomas, the time of onset of papillomas, and the size of papillomas at the endpoint all indicate that the interaction between MmuPV1 E7 and Rb1 quantitatively correlates with MmuPV1’s ability to cause papillomatosis.To assess whether E7’s ability to bind Rb1 contributes to MmuPV1 pathogenesis, we introduced the D90A mutation into the complete MmuPV1 DNA genome, which we used to make quasivirus particles in 293FT cells. We then characterized the ability of this mutant MmuPV1 versus wild-type MmuPV1 to cause papillomatosis in mice. Quasivirus stocks were confirmed to be infectious  by exposncy, 12%  (P < 0.0ed sites  (P < 0.0nk test) . At the us dose) . The sizus dose) . PapilloD90A displayed similar or different microscopic features compared to those caused by wild-type MmuPV1, we performed immunohistochemistry to assess the expression of biomarkers for papillomavirus-associated lesions. Evidence for productive viral infections within the papillomas was scored by performing immunofluorescence staining to detect the viral capsid protein L1  found E7 to be essential for promoting disease . SKP2 is rapidly degraded during G1, when RB1 is hyperphosphorylated, by CDH1 containing anaphase-promoting complex or cyclosome (APC/CCDH1)   that bintivities , includiin vivo in the context of papillomas it induces. We also observed MCM7 induction in MmuPV-1-induced papillomas arising in mice expressing the LRb1 allele and in papillomas arising in animals infected with MmuPV1 genomes expressing the RB1-binding defective E7D90A mutant. This raises an interesting question: which MmuPV1 protein(s) is responsible for the increased MCM7 expression? Experiments performed with HPVs in cell and transgenic animal-based models have all suggested that this activity is provided by E7 and is based on its ability to inactivate RB family members, and it is possible that the E7D90A mutant retains low-level RB1 binding that may be sufficient to cause some expression of E2F-responsive genes in vivo. However, our study is not the first to document E7 independent induction of hyperproliferation. Experimental infection of rabbits with an SfPV1 mutant virus that expressed an RB binding-deficient E7 mutant still caused the emergence of papillomas (67ACKE71) between its two (CXXC)2 zinc-binding domains; however, our prior AP-MS experiments failed to provide any evidence that MmuPV1 E6 binds to any of the RB family members  supplemented with 10% fetal bovine serum (FBS). SAOS-2 human osteosarcoma cells were obtained from ATCC and grown in McCoy’s 5A medium (Invitrogen) supplemented with 15% FBS. HCT116 human colon carcinoma cells were obtained from ATCC and grown in McCoy’s 5A medium (Invitrogen) supplemented with 10% FBS. NIH 3T3 murine fibroblasts were obtained from the ATCC and grown in DMEM (Invitrogen) containing 5% FBS. JB6 mouse keratinocytes  were maintained in DMEM containing 5% FBS. 293FT cells (ATCC) were maintained in DMEM with 10% FBS and 300 μg/ml neomycin (G418). Telomerase-immortalized human foreskin keratinocytes Cl398 (iHFK)  were a kThe MmuPV1 E7 open reading frame was PCR amplified from the MmuPV1 genome and cloned into N- or C-terminal FLAG/HA-CMV  and untaAffinity purification-mass spectrometry analyses of MmuPV E7 were performed as previously described . HCT116 RB1 degradation assays were performed as previously described . SAOS-2 RNA was isolated from pLenti-N-FLAG/HA mE7-, iHFK pLenti-C-FLAG/HA-16E7-, and pLenti-N-GFP-infected iHFKs and pLenti-N-FLAG/HA mE7, pLXSN-16E7, or control vector-infected primary mouse keratinocytes with the Quick-RNA Miniprep kit (Zymo Research). cDNA was synthesized with the Quantinova reverse transcription kit (Qiagen). Quantitative PCR was performed in triplicate on a Step One Plus (Applied Biosystems) thermocycler using Fast SYBR green master mix (Applied Biosystems). PCR primers are listed in 10.1128/mBio.02277-21.2TABLE S2Table S2, DOCX file, 0.01 MB.Sequences of PCR primers used in this study. Download Copyright © 2021 Wei et al.2021Wei et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the nu/nuFoxN1 mice were purchased from Envigo. LRB1 mutant mice were maintained on the FVB background and genotyped as published previously  were generated as described before . Papillomatosis was monitored over 4 months.The infection method has been described previously with some modifications . Briefly8 VGE and changed to fresh media 3 h later. After incubation at 37°C for 48 h, total RNA was extracted from infected JB6 cells using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using the QuantiTect reverse transcription kit (Qiagen). E1^E4 transcripts were detected by PCR, using p53 as a positive control. Primer sequences were described previously  incorporation by injecting BrdU . Mice were intraperitoneally injected with 250 μl stock BrdU 1 h before harvest. Tissues were harvested and processed for immunohistochemistry using a BrdU-specific antibody  as previously described .Tissues were harvested and fixed in 4% paraformaldehyde (in PBS) for 24 h and then switched to 70% ethanol for 24 h, processed, embedded in paraffin, and sectioned at 5-μm intervals. Every 10th section was stained with hematoxylin and eosin (H&E).https://www.protocols.io/view/untitled-protocol-i8cchsw.L1 signals were detected using a tyramide-based signal amplification (TSA) method . A detaiFor immunohistochemistry, tissue sections were deparaffinized in xylenes and rehydrated in 100%, 95%, 70%, and 50% ethanol and then in water. Antigen unmasking was performed by heating with 10 mM citrate buffer (pH 6) for 20 min. Blocking was performed with 2.5% horse serum in PBST for 1 h at room temperature (RT). Slides were incubated in primary antibody  at 4°C overnight in a humidified chamber. M.O.M. ImmPRESS HRP (peroxidase) polymer kit  was applied the next day for 1 h at room temperature for secondary antibody incubation. Slides were then incubated with 3,3′-diaminobenzidine (Vector Laboratories), and counterstained with hematoxylin. All images were taken with a Zeiss AxioImager M2 microscope using AxioVision software, version 4.8.2.http://www.mcardle.wisc.edu/mstat).Full scans of representative warts were performed by the UW Translational Research Initiatives in Pathology (TRIP) facility. Measurements were performed on the full-scanned images using ImageScope software (v12.4.0). All statistical analyses were performed using MSTAT statistical software version 6.4.2 (


Cottonseed is one of the major sources of vegetable oil. Analysis of the dynamic changes of fatty acid components and the genes regulating the composition of fatty acids of cottonseed oil is of great significance for understanding the biological processes underlying biosynthesis of fatty acids and for genetic improving the oil nutritional qualities.Gossypium hirsutum L.) and generated cottonseed transcriptome of the 12 time points. At 5–15 day post anthesis (DPA), the contents of polyunsaturated linolenic acid (C18:3n-3) and saturated stearic acid (C18:0) were higher, while linoleic acid (C18:2n-6) was mainly synthesized after 15 DPA. Using 5 DPA as a reference, 15,647 non-redundant differentially expressed genes were identified in 10–60 DPA cottonseed. Co-expression gene network analysis identified six modules containing 3275 genes significantly associated with middle-late seed developmental stages and enriched with genes related to the linoleic acid metabolic pathway and α-linolenic acid metabolism. Genes (Gh_D03G0588 and Gh_A02G1788) encoding stearoyl-ACP desaturase were identified as hub genes and significantly up-regulated at 25 DPA. They seemed to play a decisive role in determining the ratio of saturated fatty acids to unsaturated fatty acids. FAD2 genes (Gh_A13G1850 and Gh_D13G2238) were highly expressed at 25–50 DPA, eventually leading to the high content of C18:2n-6 in cottonseed. The content of C18:3n-3 was significantly decreased from 5 DPA (7.44%) to 25 DPA (0.11%) and correlated with the expression characteristics of Gh_A09G0848 and Gh_D09G0870.In this study, we investigated the dynamic relationship of 13 fatty acid components at 12 developmental time points of cottonseed (These results contribute to our understanding on the relationship between the accumulation pattern of fatty acid components and the expression characteristics of key genes involved in fatty acid biosynthesis during the entire period of cottonseed development.The online version contains supplementary material available at 10.1186/s12870-021-02952-4. The quality and nutritional value of plant oils are largely defined by their fatty acid compositions. Because of the low freezing point of saturated fatty acids (SFAs), vegetable oils with a high content of SFAs will coagulate, turbid, semi solidified or even solidified at a lower temperature. From health perspective, SFAs may increase the risk of cardiovascular diseases –4. PolyuFAD2 expression. Changes in fatty acid composition improved the nutritional value but may have unexpected side effects, such as reduced total oil content or seed vigor . FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. FPKM of each gene was calculated based on the length of the gene and the number of reads mapped to the gene.p-value < 0.05 and (|log2 (fold change) | ≥2 were used as the criteria for identification of DEGs among samples. Gene Ontology (GO) enrichment analysis of DEGs was implemented by the GO seq R package, in which gene length bias was corrected. GO terms with a corrected p-value less than 0.05 were considered to be significantly enriched. KOBAS software was used to test the statistical enrichment of DEGs in KEGG pathways.Differential expression analysis was performed using the DESeq R package (1.18.0) through negative binomial distribution and calculation of false discovery rate . The adjGhUBI, XM_012634824) as a reference according to Cheng et al. (2020) [Gene specific primers were designed using cDNA sequences of the target genes with the NCBI Primer-BLAST program Table . qRT-PCR. (2020) .KME values and high-weight values. The correlation networks were drawn using Cytoscape 3.6.1.The Weighted Correlation Network Analysis (WGCNA) package was used to construct the co-expression networks among DEGs associated with fatty acid synthesis in seeds from different developmental stages. The hub genes were screened on the basis of the module Additional file 1: Fig. S1. The quantitative standard curve and correlation coefficient of major fatty acid methyl esters in cottonseed determined by GC-MS. Fig. S2. The number and distribution of differentially expressed genes at 5–60 DPA. A, The number of DEGs that were up-regulated or down-regulated at 5–60 DPA. B, Venn diagram showing DEGs overlapping in different cottonseed development stages or unique to each developmental stage. Fig. S3. Heatmap comparison DEGs associated with cottonseed developmental stages. A,Venn diagram showed the different KEGGs in five stages. B and C showed the DEGs associated to the functional categories at 5, 10, 15, 20, 25 and 30 DPA respectively. B shows DEGs related to the biosynthesis of unsaturated fatty acids. Red represents high expression, and green represents low expression. Each row represents a DEG.S5, S10, S15, S20, S25and S30 represent 5, 10, 15, 20, 25and 30 DPA respectively. Fig. S4. The ratio of C18:2n-6 to C18:3n-3 during cottonseed developmentAdditional file 2: Table S1. Composition, retention times and fragment ions of the standard mixture of 37-component fatty acid methyl esters by GC-MS. Table S2. Primers used in qRT-PCR analysis.Table S3. Statistics of RNA-seq reads and mapping results.Additional file 3: Table S4. Analysis of GO enrichment for 15,647 non-redundant DEGsAdditional file 4: Table S5. Analysis of GO enrichment from 20DPA to 30DPAAdditional file 5: Table S6. Analysis of GO enrichment from 35DPA to 60DPAAdditional file 6: Table S7. Analysis of KEGG enrichment for 15,647 common DEGsAdditional file 7: Table S8. Analysis of KEGG enrichment of the DEGsAdditional file 8: Table S9. Analysis of KEGG enrichment at the 35–60 DPAAdditional file 9: Table S10. Key KEGG pathways genes enriched in six moduleAdditional file 10: Table S11. Candidate hub genes in the brown moduleAdditional file 11: Table S12. Analysis of GO enrichment in down-regulated DEGs of the 30 DPA vs 20 DPAAdditional file 12: Table S13. Number of DEGs exhibiting homeolog expression bias during cotton seed development


The article by D’Avolio and colleagues  on patieA later article on 25(OH)D concentration and risk of SARS-CoV-2 positivity was based on more than 190,000 PCR tests on patients in the U.S. with serum 25(OH)D concentration measurements from the previous 12 months on file by quest diagnostics , which raises serum 25(OH)D concentrations in hours. A successful pilot randomized controlled trial of calcifediol treatment was reported from Barcelona in 2020 . That tr3 . Thus, 0An observational study in Spain involved 537 patients hospitalized with COVID-19 between 5 February and 5 May 2020, of whom 79 were treated with calcifediol . The odd


This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.The vacuolar H  de novo and biallelic variants in ATP6V0A1 gene affect the ability of the V-ATPase complex to translocate protons and acidify the endolysosomal compartment in neurons, causing a severe neurological phenotype ranging from developmental and epileptic encephalopathy to progressive myoclonus epilepsy.Bott et al. here reports that  ATP6V0A1 presenting with a phenotype of early-onset progressive myoclonus epilepsy (PME) and ataxia and 12 cases with de novo missense variants in the same gene, with severe developmental and epileptic encephalopathy (DEE), resulting from direct impairment of endolysosome acidification and failure of lysosomal functions.Here, we report 5 individuals from 2 unrelated families with compound heterozygous variants in Ethical approval was obtained from the ethics committee of the following institutes: University of Federico II , University of L'Aquila , G. Gaslini Institute , University of Exeter (UK), Nottingham University Hospital (UK), Salpêtrière Hospital (France), University of Toronto (Canada), University of Ottawa (Canada), St. Luke's Children's Hospital (USA), Floating Hospital for Children at Tufts Medical Center (USA), Gillette Children's Specialty Healthcare Children's Hospital (USA), Children’s Hospital of Philadelphia (USA), Peking University First Hospital, Beijing (China). Written consent for the study was obtained from patients or legal representatives according to the Declaration of Helsinki. When individuals were not contacted directly, de-identified phenotypic and genomic data were used. Patient or guardian consent was given for the publication of the patient's data and clinical information. Patient II.1 was enrolled in a research study aimed at finding the genetic cause of progressive myoclonus epilepsy; Patient III.1, VII.1-X.1 and XIV.1 were referred to GeneDx for clinical whole-exome sequencing for diagnosis of suspected Mendelian disorders as previously describedBlood samples were collected and DNA was extracted using standard methods from peripheral blood lymphocytes from the indicated individuals in http://broadinstitute.github.io/picard/ Accessed 21 October 2021). Data analysis was based on the autosomal recessive mode of inheritance. Variant calling and quality score recalibration were performed using GATK (http://www.broadinstitute.org/gatk/ Accessed 21 October 2021). Variants remaining after exome data analysis containing missing data were Sanger sequenced using the BigDye Terminator version 3.1 chemistry (Applied Biosystems), run on an ABI 3730xl analyzer and analysed using Sequencher software version 4.2 (Gene Codes Corporation). Fam. II-XIV: Details on sequencing, alignment, variant calling (inherited and de novo) and variant annotation have been described previously.Fam. I: Exome sequencing was performed as previously described.Bos TaurusHomology models of the subunit a  and c  belonging to the V0 domain of the human V-ATPase has been built by Robetta server,ATP6V0A1 cDNA was kindly provided by Michael Forgac. Point mutations corresponding to the gene variants identified in the present study were introduced using a QuikChange II site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer’s instructions, and subsequently the constructs were cloned into the PiggyBac (PB) Transposon vector clone . For stable transfection, PB Transposon vector and PB Transposase vector  were transfected at a ratio of 9:1 into Neuro2a cells plated on 24-well plates (∼50 000 cells per well). One day after transfection, the cells were trypsinized and transferred as serial dilution to fresh tissue culture plates. Drug selection using 10 µg/ml puromycin started on day 3 and was continued for 2–3 weeks until the visible colonies appeared. In order to induce the transgene expression, 1 µg/ml doxycycline was added to the media starting 1 day before transfection.Human After fixation with 4% paraformaldehyde, slides were placed in blocking solution [10% normal goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS)] for 45 min at room temperature. Primary antibody staining was done at 4°C overnight in PBS with 5% normal goat serum and 0.1% Triton X-100 , using LysoTracker fluorescent dye  and Cathepsin D antibody . The slides were then washed 3 times with PBS (0.1% Triton X-100 in PBS), incubated with secondary antibody  for 1 h at room temperature in the dark, and then washed 3 times before drying and adding Vectashield/4′,6-diamidino-2-phenylinodole stain (Vector Laboratories). Coverslips were mounted with Permount (Fisher Scientific). The cells were imaged and acquired using the PANNORAMIC 250 fluorescent microscope scanner (3DHISTECH). An average of 100 cells from 3 independent experiments per each condition were analysed by a blinded investigator.2 (50 000 cells). The cells were then washed 3× in DMEM and the samples were read in a CLARIOstar Plus spectrophotometer (BMG Labtech) with dual-excitation and dual-emission spectral peaks at 324/441 and 381/541 nm. The ratio of emission was then calculated for each sample as indicated.Quantification of lysosomal pH was determined using Dextran conjugated Lysosensor Yellow/Blue DND-160 . Wild-Type and R740 mutant Neruo2a cells were grown in serum starved conditions (DMEM with antibiotics) with 1 µg/ml doxycycline to ∼80% confluence. Cells were then trypsinized, harvested and aliquoted at 100 µl into a black 96-well microplate with 5 µM Lysosensor probe for 15 min at 37°C with 5% COWhole cell lysates were collected as previously described.Caenorhabditiselegans were maintained on solid nematode growth medium seeded with E. coli OP50 at 20°C using standard methods.unc-32(ox683[unc-32::gfp +loxP]) III].mcherry at the 5′ end of endogenous lgg-1, and the introduction of a missense mutation corresponding to the substitution of the essential arginine by a glutamine in UNC-32::GFP at position 804 (numbered according to the isoform UNC-32A) into the EG9591 strain, were performed using CRISPR/Cas9 genome editing and resulted in strains AM1219 lgg-1(rm17[lgg-1::mcherry]) II and AM1233 unc-32(rm20[unc-32(R804Q)::gfp +loxP]) III/hT2[bli-4(e937) let-?(q782) qIs48] .The insertion of dpy-10 to generate cn64 rollers as phenotypic marker. Successful edits were identified by PCR screening and verified by Sanger sequencing.Genome editing was performed as previously described.mcherry at the endogenous lgg-1 locus, we targeted the 5′ end of the first exon with the guide sequence 5′-CCTTCGAATCAAAATGAAGT-3′. The repair template was synthesized by PCR to generate a double-stranded DNA fragment consisting of mCherry amplified from pAP582 (Addgene) and homology overhangs for insertion at the ATG start site using primers 5′-TAACCTTCTCTTCACACTAACCTTCGAATCAAAATGGTCTCAAAGGGTGAAGAAGATAAC-3′ and 5′-CTTCTCAAAGTTGTTCTCCTCCTTGTAAGCCCACTTCTTATACAATTCATCCATGCC-3′. The lgg-1(rm17) allele was isolated in a dpy-10(+) background and the resulting strain was back-crossed three times to N2.For inserting unc-32(ox683) allele using the guide sequence 5′-CTGCTTCATACCTTCGTCTT-3′ and a single-stranded oligonucleotide containing the CGT > CAG change, a silent T > G mutation resulting in the PvuII restriction site for genotyping, and homology arms flanking the edit as repair template 5′-TCTTGGATGTGTGTCACATACTGCTTCATACCTTCagCTgTGGGCTCTTTCATTGGCTCATGCTCGTAAGTAAAG-3′. The resulting unc-32(rm20) allele was confirmed by sequencing and kept on the hT2 balancer for strain maintenance.For generating the R804Q substitution in UNC-32::GFP, we edited the lgg-1 was performed by feeding animals with E. coli strain HT115(DE3) containing the appropriate RNAi vectors obtained from the Ahringer library and using L4440 as the empty vector control.vha-4, vha-16 and vha-17), the animals were initially grown on L4440 and transferred to RNAi plates at L4 stage.RNAi-mediated knock-down of V-ATPase genes or Animals were immobilized in 50 mM sodium azide on 3% agarose pads and imaged using a Zeiss LSM800 confocal microscope with 10× or 20× objective and Zen imaging software. For quantification of LGG-1::mCherry fluorescence levels, maximum intensity projections were generated from Z stack images and fluorescence intensity was quantified by tracing either the nerve ring for UNC-32::GFP or whole animals for LGG-1::mCherry using ImageJ software (NIH).cdc-42. The primers used are listed in Age-synchronized populations of at least 100 gravid adults were collected and snap frozen in liquid nitrogen. RNA was extracted using TRIzol (Invitrogen) and purified with QIAGEN RNeasy MinElute columns as per manufacturers’ instructions.www.graphpad.com), using the tests indicated in the figure legends. A standard confidence interval of 95% was applied in all analyses. Displayed in the figure are the mean values of all technical replicates for each of the independent experiments (displayed as single data points). Error bars represent the standard error of the mean. P-values <0.05 were considered statistically significant.Statistical analysis was performed with Graph Pad Prism version 8 for Windows , that co-segregate with the clinical phenotype  with ataxia and mental retardation, of unknown origin.henotype . From anhenotype , Table 1n = 110)n = 13 462)de novo variants c.1652G>A (p.G551E) (Fam. IV.1) and c.2219G>A (p.R740Q) (Fam. V/VI.1) heterozygous variants in ATP6V0A1  enrolled in the Italian Undiagnosed Rare Diseases Network (IURDN)  (de novo mutations (DNMs) , with frequent seizures, which were refractory to treatment, and severe mental retardation after a period of apparently normal development, with delay or loss of psychomotor milestones  , Table 1lestones , Table 1lestones . Brain blestones  and 2B, ATP6V0A1 variants, we analysed the recently published predicted damaging DNMs (n = 45 221) from the largest cohort to date of exome sequence data from individuals with severe DD (n = 31 058).ATP6V0A1, c.2219G>A recurring in 7 patients (6 of which are included in the present study), and concluded that ATP6V0A1 variants are the most common DNMs among all known lysosomal disease (LD) and LSD-associated genes in this database (To provide an estimate of the frequency of database .Saccharomyces cerevisiae V-ATPase (ScV-ATPase)ATP6V0A1, is essential for proton transport into organelles.de novo mutations overall affect organelle acidification by hindering glutamate deprotonation (p.R740Q and p. R804H), deforming the architecture of the protein region devoted to protons exchange (p.G551E), or altering the conformation of the loop that contours the exit of the luminal channel (p.S477P)  .ATP6V0A1, we generated Neuro2a cell lines stably expressing wild type or c.2219G>A (p.R740Q) mutant human ATP6V0A1 and assessed acidification of the endolysosomal compartment using the LysoSensor radiometric probe, which undergoes a pH-dependent emission shift to longer wavelengths in acidic environments. Compared to wild-type, we observed a decrease in red-shifted fluorescence signal in mutant ATP6V0A1-expressing cells, indicative of impaired protonation  fused to mCherry red fluorescent protein using CRISPR/Cas9. Consistent with a role of the V-ATPase complex in the autophagic pathway, RNA interference (RNAi) targeting unc-32 or other V0 subunits  increased LGG-1::mCherry fluorescence levels relative to control    allele show developmental arrest at early larval stages   and lyso, imp-2) , and inc  was first identified by trio-based WES as a variant of uncertain significance in one proband with gait abnormalities, stereotypic hand movements and partial loss of hand skills.de novo variant (corresponding to NM_001130021.3: p. R740Q) and two with the following biallelic mutations: a p. A512P missense variant (corresponding to NM_001130021.3: p. A505P) in compound heterozygosity with a 50-kb deletion, and a p. N534D missense variant (corresponding to NM_001130021.3: p. N527D) in compound heterozygosity with a disruption of a splice donor site. All the de novo cases reported so far displayed a phenotype of developmental and epileptic encephalopathy (DEE), with developmental delay in the first months of life and drug-resistant focal and generalized tonic-clonic seizures. Head circumference 2 standard deviation below the average was present in 50% of the children with the p. R740Q variant and one individual with the p. R804H variant, suggesting an association between microcephaly and ATP6V0A1-related diseases. With 58 DNMs  in genes of the V-ATPase complex identified in exome sequence data from over 30 000 individuals with severe DD,ATP6V0A1 and ATP6V1A) out of 281, we propose that a V-ATPase-related disease should be considered in individuals presenting with mild-to-profound developmental delays and epilepsy, with or without microcephaly. Five individuals from 2 unrelated pedigrees in our cohort carry the biallelic E149Kfs*18 frameshift and R495W substitution and manifest an early onset, progressive myoclonus epilepsy (PME) and ataxia, therefore, expanding the neurological phenotypes associated with ATP6V0A1 variants beyond the more severe developmental and epileptic encephalopathy observed in the de novo cases. Myoclonic epilepsy was also a feature in one of the biallelic cases recently reported.de novo.Here, we report 17 subjects from 14 unrelated families carrying ATP6V0A1 gene encodes the a1 isoform of the membrane-bound V0 components of the V-ATPAse complex, which of the four a-subunits is the most neuronal enriched,The de novo variants identified here cluster on the transmembrane domains of the a1 subunit and are predicted by homology modelling studies to hinder glutamate deprotonation (p.R740Q and p. R804H), deform the architecture of the protein region devoted to protons exchange (p.G551E), or alter the conformation of the luminal channel (p.S477P), suggesting that perturbation of proton translocation is fundamental to the disease  and reduced expression of Notch target genes. These findings corroborate previous reports in Drosophila indicating that the V-ATPase-mediated acidification of the endolysosomal compartment is required for the activation of Notch in endosomesWe next proceeded to study the mechanism of pathogenicity of the R740Q substitution, which accounts for over 70% of all ATP6V0A1 ortholog in C. elegans and observed reduced protein clearance in lysosomes and altered regulation of several components of the autophagic machinery in neurons, expression of global autophagy failure. Homozygote nematodes carrying the corresponding R740Q mutation showed severe developmental arrest at early larval stage, with widespread accumulation of undigested proteins in the nerve ring. In accordance with recent findings of lysosomal dysfunction and increased neuronal cell death observed in brains of ATP6V0A1 mutant mice carrying the homozygote A512P change,de novo variants.To further establish a role of this isoform in lysosomal function, we knocked down the de novo and biallelic variants in ATP6V0A1 in patients affected by DEE and PME and link the mechanism of pathogenesis to disruption of the canonical protonation function of the V-ATPase complex. Considering the increasing identification of disease-causing variants in V-ATPase genes and the critical relevance of vacuolar pump-mediated regulation of intracellular pH in cellular homeostasis in health and disease,In conclusion, here we identified Brain Communications online.fcab245_Supplementary_DataClick here for additional data file.


Neisseria meningitidis isolates submitted to the New Zealand Meningococcal Reference Laboratory during 2013–2018. Of the 347 isolates submitted for whole-genome sequencing, we identified 68 sequence types belonging to 18 clonal complexes (CC). The predominant CC was CC41/44; next in predominance was CC11. Comparison of the 45 New Zealand group W CC11 isolates with worldwide representatives of group W CC11 isolates revealed that the original UK strain, the 2013 UK strain, and a distinctive variant (the 2015 strain) were causing invasive group W meningococcal disease in New Zealand. The 2015 strain also demonstrated increased resistance to penicillin and has been circulating in Canada and several countries in Europe, highlighting that close monitoring is needed to prevent future outbreaks around the world.Genomic surveillance is an essential part of effective disease control, enabling identification of emerging and expanding strains and monitoring of subsequent interventions. Whole-genome sequencing was used to analyze the genomic diversity of all  Neisseria meningitidis, a gram-negative bacterium, is the causative agent for meningococcal meningitis and septicemia and has been associated with isolated cases, outbreaks, and epidemics worldwide  cause most life-threatening IMD (Group W CC11 (W:CC11) meningococci emerged as a global cause of IMD after an outbreak in Mecca, Saudi Arabia, in 2000 (N. meningitidis group B strain (NZMenB), defined by PorA type P1.7–2,4 and belonging to CC41/44  to perform all statistical tests. Shannon–Wiener diversity index was calculated by using vegan version 2.5.6  for routine grouping using slide agglutination or PCR (https://www.qiagen.com) or High Pure PCR Template Preparation Kit  according to the manufacturer’s protocols. A 10-µL loop of bacteria, grown overnight on Columbia blood agar plates , was suspended in 300 µL of lysis buffer and heat-killed at 56°C for 1 h. We then quantified DNA by using the Quant-iT PicoGreen dsDNA assay kit  and constructed libraries by using Nextera-XT DNA Library Preparation Kit . Paired-end sequencing of 2 × 150 bp was performed on the Illumina platform at ESR. Read data are available for download from the National Center for Biotechnology Information Sequence Read Archive (Bioproject accession no. PRJNA592848) and from PubMLST (https://pubmlst.org).We analyzed all available meningococcal isolates from 2013–2018 in New Zealand by WGS  Table 1.N. meningitidis reference sequence FAM18 (GenBank accession no. AM421808.1) and using Bowtie2 version 2.2.5 (https://arxiv.org/abs/1207.3907) to detect sequence variations among isolates with these settings: ploidy = 1, at 20× minimum depth and 70% minimum variant allele frequency. We filtered variants by using vcflib  but increased during 2014–2018 . Inflection point of the time series was identified by breakpoint analysis and using the R package strucchange . In contrast, the proportion of disease caused by group C in 2018 (10/133) is significantly less than that of 2013 .Group B meningococci continue to be responsible for most IMD cases in New Zealand. Among the 438 confirmed cases analyzed by ESR during 2013–2018 were 265 (54.7%) group B (100 NZMenB and 165 other group B) cases, 58 (13.2%) group C cases, 61 (13.9%) group W cases, and 47 (10.7%) group Y cases  Table 6.The rate of IMD continues to be highest among patients <1 year of age ; rates are second highest among children 1–4 years of age . Adults >60 years of age are more affected by group W and Y disease  Table 7.We performed WGS and in silico typing on the 347 New Zealand meningococcal isolates (288 invasive and 59 noninvasive) collected during 2013–2018. The WGS dataset includes 175 group B isolates (65 NZMenB and 110 other group B), 48 group C isolates, 2 group E isolates, 64 group W isolates, 47 group Y isolates, 1 group X isolate, and 8 nongroupable isolates. MLST analysis showed that the 347 isolates contained 49 known STs. The 10 most common STs were ST11 (85), ST154 (43), ST23 (21), ST42 (17), ST32 (16), ST213 (15), ST1655 (15), ST1572 (11), ST6058 (8), and ST22 (7). There were 39 other STs with a single isolate and 19 unassigned STs (submitted through this project). The STs identified belonged to 18 clonal complexes. A total of 93 unique strains were defined by combination of group:PorA-variable region (VR)1, PorA-VR2:FetA:CC, with a Shannon–Wiener diversity index of 3.516. Bexsero antigen sequence types analysis showed 69 unique types with 23 isolates in which type could not be determined because of the absence of required loci  Table 1.N. meningitidis FAM18 as the reference genome) showed 17 highly supported clades , 15 of which were consistent with the CC definition , a representative of CC11, was used as a reference. The mean genome depth was 152X, with 84.8%–97.7% of loci covered at >20-fold (Rates of group W disease in 2018 were higher than in 2013. Most sequenced group W isolates in 2018 (30/35) belonged to CC11. To understand how New Zealand W:CC11 isolates relate to the global group W lineages, we analyzed New Zealand W:CC11 in the context of major W:CC11 lineages. We included 196 CC11 isolates in this analysis . The mapping rate was 92.4%–99.3% when >20-fold  Table 2.We used core SNP  alignment to construct the phylogenetic relationship of New Zealand W:CC11 isolates within the global W:CC11 . PhylogeTo determine whether other isolates are closely related to the new New Zealand cluster, we searched PubMLST and found 27 additional isolates within 50 allele (the cgMLST 0.1 scheme) differences to NZ17MI0022. Because only assembled contigs were available for these isolates, we used a core-genome alignment approach to analyze their relationship within clade II of the W:CC11 isolates. All additional 27 W:CC11 isolates clustered with the New Zealand cluster with high bootstrap support  Figure 3Both phylogenetic analyses suggest that the new New Zealand cluster is part of the W:CC11 South America strain sublineage derived from the original UK strain. Because the earliest isolate of the new variant was identified in 2015, we named it the 2015 strain of the W:CC11 South America strain sublineage (the 2015 strain).Similar to the 2013 UK strain, the 2015 strain is associated with higher death rates. During 2017–2018, the 2015 strain was associated with a death rate of 17.8% (6/34 cases) in New Zealand, higher than the rate of 5.9% (10/170 cases) for other groups in the same period (p = 0.03 by Fisher exact test). The 2015 strain also disproportionally affected older adults; 26% of total 2015-strain cases affected adults >60 years of age (9/34), whereas 5.8% (7/121) of total group B cases affected adults in that age group (p = 0.01466 by Fisher exact test).<0.03 mg/L), ceftriaxone (<0.12 mg/L), and rifampin (<0.5 mg/L) (<0.06 mg/L). Of the 27 isolates belonging to the new 2015 strain, 12 displayed intermediate resistance (0.12–0.25 mg/L) and 15 were resistant (>0.5 mg/L). The 2015-strain isolates were significantly more resistant to penicillin (p<0.00001 by Fisher exact test). All 27 New Zealand 2015-strain isolates had penA allele 253, the same allele described in the Australia study   Table 8..5 mg/L)  Table 8 ia study  Table 3.N. meningitidis in New Zealand during 2013–2018 to describe its population structure after the NZMenB epidemic. We examined the rate of IMD and clonal distribution of circulating isolates. We also offer evidence that a distinct variant of W:CC11 is circulating globally and has been causing IMD since 2015.We comprehensively analyzed Meningococcal disease continues to substantially affect the health of persons in New Zealand. In comparison with other developed countries   Figure 1The distribution of group Y disease and group W disease has changed over the past decade. Incidence of group Y disease has been increasing since the late 1990s in the Americas and since 2010 in Europe  than that for 2008–2012, the 5-year period following the end of the epidemic . Since 2012, nonepidemic group B cases have regularly surpassed epidemic cases; 47% of group B cases were caused by nonepidemic strains during 2008–2011, and 63% were caused by nonepidemic strains during 2012–2018. The top 3 strains circulating within the nonepidemic group B cases were B:P1.22,14:F5–5:CC213 (13 isolates), B:P1.7,16–26:F3–3:CC32 (13 isolates), and B:P1.7–12,14:F1–7:CC1572 (12 isolates). All 3 strains are present in PubMLST. B:P1.22,14:CC213 is a common strain, however, only 2 isolates contain the FetA-VR:5–5 allele. PubMLST has 41 B: P1.7,16–26:F3–3:CC32 isolates and 6 B:P1.7–12,14:F1–7:CC1572 isolates . Taken together, these data suggest that the meningococcal population in New Zealand has become more diverse after the group B epidemic. Comparison of New Zealand with other countries is challenging because no comprehensive public database exists that contains all IMD cases with fine-typing information. The United Kingdom, however, has been depositing most of their IMD typing information into PubMLST since July 2010, and for 2013–2017, the dataset contains 2,790 records and 649 unique strains . This information suggests that the isolates that cause IMD are less diverse in New Zealand, which may reflect its smaller and more distributed population structure.The clonal expansion of a new penicillin-resistant clade of W:CC11 was first identified in Australia in 2016  Figure 3W:CC11 meningococci are emerging as a diversifying lineage with several new strains occurring in different geographic locations  Figure 3N. meningitidis has revealed in great detail the genetic diversity and population structure of circulating meningococci in New Zealand; this more refined surveillance enables the tracking of specific strains that are identifiable only by high-resolution phylogenetic analysis. By using this approach, we identified a distinct globally circulating W:CC11 strain, which would not have been possible without genomic information. Our results emphasize the value of obtaining complete genetic information for invasive pathogens and resolving their global evolutionary relationship for identifying the origin and expansion of new pathogenic variants or strains. Such information will benefit surveillance and can be used to help prevent and control future epidemics.Genomic surveillance of Additional data for genomic surveillance of a globally circulating distinct group W clonal complex 11 meningococcal variant, New Zealand, 2013–2018.More information about genomic surveillance of a globally circulating distinct group W clonal complex 11 meningococcal variant, New Zealand, 2013–2018.


Phytophthora infestans suppresses host defense by targeting Sec5. Sec5 is a subunit of the exocyst, a protein complex that is important for mediating polarized exocytosis during plant development and defense against pathogens. The mechanism by which AVR1 manipulates Sec5 functioning is unknown. In this study, we analyzed the effect of AVR1 on Sec5 localization and functioning in the moss Physcomitrium patens. P. patens has four Sec5 homologs. Two (PpSec5b and PpSec5d) were found to interact with AVR1 in yeast-two-hybrid assays while none of the four showed a positive interaction with AVR1ΔT, a truncated version of AVR1. In P. patens lines carrying β-estradiol inducible AVR1 or AVR1ΔT transgenes, expression of AVR1 or AVR1ΔT caused defects in the development of caulonemal protonema cells and abnormal morphology of chloronema cells. Similar phenotypes were observed in Sec5- or Sec6-silenced P. patens lines, suggesting that both AVR1 and AVR1ΔT affect exocyst functioning in P. patens. With respect to Sec5 localization we found no differences between β-estradiol-treated and untreated transgenic AVR1 lines. Sec5 localizes at the plasma membrane in growing caulonema cells, also during pathogen attack, and its subcellular localization is the same, with or without AVR1 in the vicinity.Plant pathogens often exploit a whole range of effectors to facilitate infection. The RXLR effector AVR1 produced by the oomycete plant pathogen  Phytophthora species are oomycete plant pathogens that are widespread and cause substantial damage in agriculture, forestry and nature . Ot. OtSec5--mutants  or Sec10ns lines , observeP. patens caulonema cells. Unfortunately, the expression of AVR1 upon induction with β-estradiol is very high Click here for additional data file.S2 FigAVR1 and AVR1ΔT pPGX8 constructs that were transformed into P. patens for homologous recombination at the PIG1 neutral locus. PIG1bR/L: DNA fragments flanking the PIG1 neutral locus. LexA:Pm35S: bacterial LexA operator fused to a CaMV minimal 35S promoter. Term: terminator. GX8: promoter of a constitutively expressed P. patens gene. XVE: a sequence encoding a chimeric transcription activator composed of the DNA-binding domain of the bacterial repressor LexA, the transcriptional activation domain VP16 and the C-terminal region of the human estrogen receptor. hygR: hygromycin resistance cassette driven by a modified CaMV35S promoter. Upon β-estradiol treatment, the XVE protein binds to the LexA operator and induces the expression of the downstream gene resulting in the production of myc and/or cerulean tagged AVR1 or AVR1ΔT [P. patens transformants to confirm homologous recombination at the PIG1 neutral locus. Each construct shown in (A) was transformed into two recipient P. patens lines (background), one containing Sec5b-GFP and the other containing Sec5d-GFP. PCR fragments are visualized by agarose gel electrophoresis. Line numbers of independent transformants (#) and primers sets with predicted sizes of the PCR fragments in brackets (bp: base pairs) are indicated.(A) Schematic representation of the r AVR1ΔT . The loc(TIF)Click here for additional data file.S3 FigSec5 silencing construct. PIG1bR/L: DNA fragments for homologous recombination into the PIG1 neutral locus. LexA:Pm35S: bacterial LexA operator fused to a CaMV minimal 35S promoter. Term: terminator. pKINID1a: promoter of a constitutively expressed P. patens gene. XVE: a sequence encoding a chimeric transcription activator composed of the DNA-binding domain of the bacterial repressor LexA, the transcriptional activation domain VP16 and the C-terminal region of the human estrogen receptor. hygR: hygromycin resistance cassette driven by a modified CaMV35S promoter and terminator. Upon β-estradiol treatment, the XVE protein binds to the LexA operator and induces the expression of the Sec5-silencing fragment. Backbone of the construct is vector pGG626 [P. patens Sec5a, Sec5b, Sec5c and Sec5d in independent Sec5RNAi moss lines two days after growth on medium with (+) and without (-) β-estradiol. Transcript levels were normalized to PpEF1α transcript levels and expressed as mean fold changes (± standard deviation) relative to the transcript level in non-induced moss tissue (set at 1). Statistical differences are indicated by asterisks (P>0.05). Background indicates the recipient strain used for transformation of the Sec5 silencing construct.(A) Schematic representation of the r pGG626 . (B) Exp(TIF)Click here for additional data file.S4 FigForty-one amino acids that are shared between StSec5, PpSec5b and PpSec5d but different in PpSec5a and PpSec5c are indicated with arrowheads. Colors shadings distinguish amino acids that are hydrophobic (skyblue), positively charged (red), negatively charged (magenta), polar (applegreen) or aromatic (cyan). Cysteines are colored pink, glycines bronze and prolines lime. Amino acids that are not conserved are not color shaded.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


K7 immunohistochemical hepatocellular expression and ductular reaction (DR) were semi-quantitatively assessed. Results were correlated with liver enzyme serum levels, cholestasis type, histological features, hepatocellular Ki67 labelling index (LI) and HepPar1 expression. Hepatocellular K7 expression was detected in 87% (81/92) cases and in all cholestatic disease types with lowest incidence in pure/mixed cholestasis and highest in incomplete bile duct obstruction (iBDO), reaching 100% in PSC. K7-positive hepatocytes had low Ki67 LI (0-5%) retaining HepPar1 expression, irrespective of disease type. PSC cases had high K7 hepatocellular expression even with intact bile ducts, a feature that may aid differential diagnosis of cholestatic syndromes. K7 hepatocellular expression significantly correlated with cholestasis type, bile duct loss and fibrosis stage. It was higher in milder acute cholestatic hepatitis showing inverse correlation with hepatocyte proliferation and serum transaminase levels. In iBDO, younger age independently correlated with high K7 expression, while serum GGT levels showed a nearly significant correlation. Correlation with DR findings implied that K7-positive hepatocytes may result through metaplasia. In conclusion, K7 hepatocellular expression is a sensitive though non-specific marker of cholestasis. It may represent a cytoprotective reaction of resting hepatocytes in cholestasis of longer duration especially in younger patients.We evaluated keratin 7 (K7) hepatocellular expression in 92 patients with common types of acute and chronic cholestatic diseases caused by bile duct obstruction/destruction or parenchymal lesions [acute hepatitis (The online version contains supplementary material available at 10.1007/s00428-021-03152-z. In normal liver, keratin 7 (K7) belongs to the spectrum of keratins that define the immunophenotype of cholangiocytes. Keratin 7 is also expressed in hepatoblasts and hepatic progenitor cells (HPC) but not expressed in normal hepatocytes constituting a marker of liver cell immaturity , 2. K7-pK7-positive hepatocytes singly or in groups are encountered in a variety of liver diseases, including viral and autoimmune hepatitis (AIH), alcoholic and non-alcoholic steatohepatitis, cirrhosis of any aetiology –10 as weCholestasis is characterised by partial or complete interruption of normal bile flow. The underlying causes are bile duct obstruction/destruction or malfunction in bile production and secretion caused by hepatocyte injury , 19. TheIn the present study, we evaluated aberrant K7 expression of hepatocytes in relation to histological and biochemical parameters in the most frequent cholestatic diseases. Our aim was to clarify the significance of K7 immunostain in defining the type of cholestasis within the context of disease type and severity, to assess its diagnostic significance and to provide further evidence regarding the underlying pathogenetic process.Eighty-seven (87) needle core liver biopsies  and 5 surgically excised liver specimens from 92 adult patients , 35 females (39%); median age 54 years, interquartile range 27) were studied. Inclusion criteria were presence of cholestasis defined by histological and/or biochemical findings based on available data on serum levels of liver enzymes  at the time of liver biopsy/surgery. Exclusion criteria were established cirrhosis and liver failure, since possible synergistic factors, such as ischemia, may contribute to aberrant K7 hepatocyte expression. Patients mean international normalised ratio (INR) was 1.15 ± 0.34 (min=0.87-max=2.81).st Department of Pathology and the Laboratory of Histology & Embryology, Medical School, National and Kapodistrian University of Athens (NKUoA) between 2012 and 2018. Demographic and biochemical data were retrieved from patient files. All cases were anonymized and permission for scientific use of patient data were obtained by the Research Ethics and Deontology Committee, NKUoA.Tissue samples were retrieved from the archives of the 1Specimens were fixed in 10% neutral formalin solution and processed according to routine protocol. Three-4 μm-thick sections were stained with Hematoxylin & Eosin, Masson trichrome and van Gieson stains. K7 expression was identified immunohistochemically . Immunostaining for hepatocyte marker HepPar1  and cell proliferation marker Ki67  were also performed on serial sections, where tissue was available. Double immunostaining for K7 and Ki67 using 3,3-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) chromogens, respectively, was performed in 5 cases of acute hepatitis and 5 cases of pure/mixed cholestasis with available tissue. K19 expression was not examined as it is reportedly absent from hepatocytes in cholestatic liver disease , while a similar trend was observed for AST levels .ALT levels were significantly higher in patients without K7 expression [mean values: 2155±387U K7(−) vs 460±428U K7(+), p=0.027). Moreover, transaminase levels were lower in cases with zone 1 K7 expression  . In Ki67/K7 double immunostained cases, Ki67 was nearly absent in K7-positive hepatocytes and was mainly detectable in K7-negative hepatocytes or non-parenchymal cells  Table . An invep=0.063, 62.5% vs 12.5%, p=0.119 respectively). There was no significant correlation between K7 expression and any of the examined parameters.Cases with moderate or severe bilirubinostasis showed more often K7 positivity as well as high K7 expression in zone 1 compared to cases with mild , as well as a significant inverse correlation between high K7 positivity with ALT levels (p=0.004) . Patients showing any percentage of BD loss compared to patients without BD loss tended to exhibit more frequently high K7 positivity in zone 1 . High K7 expression in zones 2–3 was also found more often in patients with BD loss  and in advanced fibrosis (F3) .In iBDO, K7-positive hepatocytes were almost constantly present in zone 1 (95.8%), while in zones 2–3, their presence decreased to 78% of the cases (p=0.066). The correlation was stronger in high K7 expression in zone1 or zones 2–3 . Moreover, high K7 expression, irrespective of topography, showed nearly significant correlation with GGT levels .ii.PBC vs PSCIn multivariate analysis, only age and GGT levels correlated with K7 expression. In detail, patients with high K7 expression in zone 1 and/or zones 2–3 where younger than patients with low K7 positivity . No difference was noted between PSC and PBC cases regarding high K7 positivity in advanced fibrosis. There were no differences in K7 expression in the different zones.iii.Complete bile duct obstructionAll PSC cases and 97.1% PBC cases showed K7-positive hepatocytes. Moreover, all PSC patients showed high K7 expression in zone 1 and/or zones 2–3 compared to PBC (74.3%), Almost all complete LBDO cases (7/8) showed K7 hepatocellular positivity that was high in the majority (6/8). No further statistical analysis was performed due to the small sample size.p=0.225).Supplementary Tables 4 and 5 summarise the type and grade of DR observed in parenchymal and obstructive cholestatic diseases, respectively. Briefly, in acute hepatitis, 26% (5/19) of the cases had no DR while DR type 2A was only seen in 1/19 case. No correlation was found with K7 positivity in zone 1 even in cases with high K7 expression (p=0.002).In pure/mixed cholestasis, DR type 2A was noted in 5/15 cases, all of which had high K7 zone 1 expression, while low K7 positivity (9/10) was seen in cases without DR type 2A (p=0.028).In PBC, the vast majority of cases had DR  showed DR type 2A, while in 3 cases with ductopenia and high K7 zone 1 expression, there was no DR. The latter was observed in 1 out of the two VBDS cases while the second showed grade 3 DR type 2A with high K7 zone 1 hepatocyte expression.In cLBDO, DR type 2A was seen in all cases and was grade 3 in the majority (4/6) with high K7 zone 1 hepatocyte expression , while zones 2–3 followed (73%) and had less frequent high K7 expression. Cholestasis type and BD loss were independent factors associated with K7 expression. Cases with obstructive cholestasis showed higher K7 positivity and predominance of zone 1 topography compared to those with parenchymal cholestasis. Almost all cases with BD loss expressed K7 showing significant higher frequency and higher positivity compared to those with intact BD. Zone1 K7 topography was almost constantly present in patients with BD loss while zones 2 and 3 were involved in all cases with significant BD loss. Advanced fibrosis also emerged as an independent parameter correlating with K7 hepatocellular expression, mainly of high positivity in zone 1. A similar correlation was found regarding K7 expression in zones 2 and 3, though in lower frequency. HepPar1 expression was not affected, irrespective of cholestatic disease and extent of K7 hepatocellular positivity.Acinar zone 1 appears to be most vulnerable under normal and cholestatic conditions due to the highest bile salt level exposure. Bile salt cumulative concentration most likely accounts for the high frequency and high zonal expression of K7, while bilirubinostasis and serum bilirubin levels did not exhibit any relationship with K7 hepatocellular expression in our analysis. The differences in the distribution pattern and extent of K7 expression between our study group and non-cholestatic diseases, based on the results of respective studies, suggest different pathogenetic mechanisms.Analysing each of the examined disease entities, there are certain findings to be highlighted. In acute hepatitis, an inverse correlation of K7-positive hepatocytes with histological disease severity was corroborated by an inverse correlation with serum transaminase levels. In a clinico-pathological study of autoimmune hepatitis (AIH) with acute clinical onset by Fujiwara et al., a higher number of intermediate hepatocytes and HPC was found in non-severe recovered cases compared to severe or fulminant AIH with fatal outcome . It was In drug-induced pure/mixed cholestasis, multivariate analysis showed a correlation trend between fibrosis and detectable hepatocellular K7 expression in all zones, possibly indicating a long-term impact of cholestasis on the manifestation of K7. This is supported by a previous study of 10 cases with pure cholestasis, half of them with predominant zone 1 K7-positivity and histology favouring long duration of the cholestatic lesions . The invK7 hepatocellular expression has been best studied in incomplete BDO and mainly in PBC while information referring to PSC is limited , 22, 24 Our findings highlight differences between PBC and PSC regarding K7 expressing hepatocytes. All patients with PSC and almost all with PBC showed K7-positive hepatocytes. It is noteworthy that all PSC cases including those without bile duct loss exhibited high K7 hepatocyte expression compared to only 20% of PBC cases with intact bile ducts, a feature that may prove helpful in the differential diagnosis of cholestatic syndromes without apparent BD loss in liver biopsy.Regarding the nature of K7-positive hepatocytes, based on Ki67 proliferation activity, they seem to be resting cells, retaining hepatocellular features, such as HepPar1 expression, as well as functional properties. The latter is deducted by the absence of clinical and biochemical findings of liver insufficiency in cases with almost panacinar K7 expression. According to our observations in parenchymal cholestasis, K7-positive hepatocytes do not inevitably undergo biliary transformation. The latter process, characterised primarily by ductule formation in zone 1, involves the deeper zones of the acinus especially in advanced stages of BDO (data not shown). In all cholestatic diseases, including parenchymal lesions, the vast majority of K7-positive hepatocytes most likely result from a metaplastic process and not from hepatocellular differentiation of HPC. This is supported by their strong association with DR type 2A, their presence in the cases without DR and their frequent periportal topography independent of DR location. Most likely, K7 expression in hepatocytes reflects a protective mechanism attributed to the established cytoprotective role of intermediate filaments . It may In summary, K7 hepatocellular expression in cholestatic diseases is a sensitive though not specific marker of cholestasis related to type of cholestasis, fibrosis and BD loss. Our study shows that it may represent a cytoprotective reaction of resting hepatocytes in cholestasis of longer duration without elimination of functional and other phenotypic features, especially in younger patients.ESM 1(DOCX 20.4 kb)ESM 2(DOCX 36.5 kb)ESM 3(DOCX 46.8 kb)ESM 4(DOCX 22.8 kb)ESM 5(DOC 67.0 kb)Supplementary Figure 1a & c. The majority of hepatocytes around representative portal tracts are Keratin 7 (K7)-positive in a case of PBC where ≥50% of portal tracts had a similar appearance (Score 3); b. Serial section of 1a immunostained for HepPar1. All hepatocytes, including those expressing K7, show diffuse cytoplasmic granular HepPar1 immunostaining; d. Serial section of 1c immunostained for Ki-67. K7-positive hepatocytes are negative for Ki-67 indicating that they are resting cells . (PNG 8664 kb)High resolution image (TIF 1917 kb)Supplementary Figure 2Primary biliary cholangitis: Ductular reaction grade 3 and absence of K7-positive zone 1 hepatocyte expression (x100 magnification). (PNG 6737 kb)High resolution image (TIF 1546 kb)Supplementary Figure 3Complete large bile duct obstruction: ductular reaction type 2A with score 3 K7-positive hepatocyte expression (x400 magnification). (PNG 6701 kb)High resolution image (TIF 1625 kb)