GDA) has been identified to cause UV-induced keratinocyte senescence in seborrheic keratosis. Seborrheic keratosis is also frequently pigmented. Growing evidences indicate that hyperuricemia is a risk factor of acanthosis nigricans, an acquired skin hyperpigmentation. The objective of this study was to investigate role of GDA and its metabolic end product, uric acid, in hyperpigmentation of patients with seborrheic keratosis using their lesional and non-lesional skin specimen sets and cultured primary human epidermal keratinocytes with or without GDA overexpression or uric acid treatment. GDA-overexpressing keratinocytes or their conditioned media containing uric acid increased expression levels of MITF and tyrosinase in melanocytes. Uric acid released from keratinocytes was facilitated by ABCG2 transporter with the help of PDZK1 interaction. Released uric acid was taken by URAT1 transporter in melanocytes, stimulating melanogenesis through p38 MAPK activation. Overall, GDA upregulation in seborrheic keratosis plays a role in melanogenesis via its metabolic end product uric acid, suggesting that seborrheic keratosis as an example of hyperpigmentation associated with photoaging.Seborrheic keratosis, which is a benign tumor composed of epidermal keratinocytes, develops common in the elderly. Uric acid generated by upregulated guanine deaminase ( GDA) expression in keratinocytes induced by repeated ultraviolet (UV) exposure is involved in UV-induced keratinocyte senescence in seborrheic keratosis via uric acid, a guanine metabolite [GDA could contribute to hyperpigmentation in seborrheic keratosis. Thus, the objective of this study was to examine whether and how GDA and its metabolic end product, uric acid, were involved in melanogenesis and skin hyperpigmentation in patients of with seborrheic keratosis.Seborrheic keratosis is a common benign skin tumor characterized by keratinocyte proliferation. Its predilection sites are sun-exposed areas and its affected age group is the elderly ,2, suggetabolite , supporttabolite . Seborrhtabolite . Althougtabolite ,10,11,12tabolite ,14 is a tabolite ,16. We aGDA mRNA and protein have been detected in keratinocytes, but not in melanocytes [GDA in melanogenesis was examined in primary cultured adult normal human keratinocytes with or without GDA overexpression cocultured with primary cultured adult normal human melanocytes. Levels of tyrosinase mRNA and protein were increased in normal human melanocytes cocultured with GDA-overexpressing keratinocytes . Hyperpigmentation is a common clinical finding of seborrheic keratosis and melasma. In addition, UV radiation is one of main causative factors of melasma [GDA upregulation in UV-induced keratinocyte senescence [GDA contributed to the hyperpigmention, particularly related to photoaging.This study first examined whether inocytes a suggest melasma ,27,28, a melasma . We have melasma ,31,32,33 melasma ,35, indiGDA upregulation stimulated melanogenesis, its mechanism was then investigated. Melanogenesis in melanocytes was also enhanced by conditioned media obtained from GDA-overexpressing keratinocytes . It was conducted according to the Declaration of Helsinki Principles. After obtaining informed written consent from each patient, pairs of hyperpigmented and adjacent normally pigmented skin specimens taken for biopsy were used for direct comparisons through real-time PCR and immunohistochemistry.Adult skin specimens obtained from Caesarean sections and circumcisions were used to establish cell culture for keratinocytes and melanocytes ,50,51. KGDA gene [Keratinocytes or melanocytes were transfected with 25 nM CRISPR-CAS9 sgRNA for human ABCG2, URAT1, or a negative control sgRNA  using a CRISPRMAX transfection reagent . pCMV vector containing To measure cell viability, cells were incubated with MTT for 4 h. Precipitated formazan was dissolved in dimethyl sulfoxide (DMSO). The optical density was measured at 570 nm with background subtraction at 630 nm using a spectrophotometer. LDH assay performed following the manufacturer’s instruction for measuring cell cytotoxicity.2) were treated with various concentrations of recombinant GDA   up to 3 days.Melanocytes  in cell lysates or culture supernatants using a uric acid assay kit , following the manufacturer’s instructions.2) were transfected with GDA. Four hours later, cells were treated with different concentrations of allopurinol (1 and 10 μM) or Ko143 (1 and 2 μM)  for 2 days or 24 h, respectively. Cells or culture supernatants were harvested for melanocyte culture or uric acid assay.Keratinocytes (2 × 105 cells/9.6 cm2) were treated with appropriate concentrations of uric acid (Sigma-Aldrich) with or without probenecid (20 and 40 μM) (Sigma-Aldrich) for 24 h. Cells or culture supernatants were harvested for uric acid assay.Melanocytes  and 5′-cgattttcacttatatggctctga-3′ (Reverse); Tyrosinase, 5′-agcatcattcttctcctcttgg-3′ (Forward) and 5′-gcataaagactgatggctgttg-3′ (Reverse); GAPDH, 5′-tccactggcgtcttcacc-3′ (Forward) and 5′-ggcagagatgatgacccttt-3′ (Reverse).cDNA was synthesized from the total RNA using the cDNA Synthesis Kit for RT-PCR . The amount of target mRNA was quantified by real-time PCR using a Light Cycler real-time PCR machine . The relative amount of mRNAs was calculated as the ratio of each target relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences used were as follows: GDA, PDZK1, tyrosinase, and URAT1 , MITF, phospho-ERK, ERK, phospho-p38, p38, and JNK , phospho-JNK  and β-Actin . After incubating with appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Thermo Fisher Scientific), an enhanced chemiluminescence solution (Thermo Fisher Scientific) was applied and signals were captured with an image reader . The protein bands were then analyzed by densitometry.Equal amounts of extracted proteins were resolved and transferred to nitrocellulose membranes. Each membrane was incubated with one of the following antibodies against ABCG2, Supernatants of cell lysates were incubated with an anti-PDZK1 antibody and resin of Pierce™ Direct IP kit (Thermo Fisher Scientific) at 4 °C. Eluted resin-bound proteins were analyzed by immunoblotting with anti-ABCG2 or anti-ABCG2 antibody.GDA, anti-PDZK1, or anti-URAT1 antibody and 1:200 Alexa Fluor-labeled goat anti-mouse IgG , with anti-ABCG2 and Alexa Fluor-labeled goat anti-mouse IgG , or with anti-cKit , anti-uric acid (Santa Cruz Biotechnology) antibody, and Alexa Fluor-labeled goat anti-rabbit IgG . Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). Fluorescence images were evaluated using an image analysis system  and a Wright Cell Imaging Facility (WCIF) ImageJ software 1.53e (http://www.uhnresearch.ca/facilities/wcif/imagej accessed on 9 August 2021).After deparaffinization and rehydration, sections were preincubated with 3% bovine serum albumin. These sections were reacted sequentially with the an anti-p-value of <0.05 was considered significant. Statistical comparisons between two groups were performed using two-tailed Student’s unpaired t-test . For comparisons among multiple groups, one-way Analysis of Variance (ANOVA) was used. For human sample data, differences between non-lesions and lesions were assessed with the Mann–Whitney U test. Values of mean ± SD were calculated for in vitro experimental data.All statistical analyses were performed using GraphPad Prism 5.03 . A GDA upregulation stimulated melanogenesis. Regarding the mechanism of GDA-induced hyperpigmentation, GDA upregulation enhanced melanogenesis via uric acid generation. More uric acid was excreted by GDA-overexpressing keratinocytes through ABCG2 with the help of PDZK1. Excreted uric acid, which was reabsorbed by melanocytes via URAT1 upregulation, enhanced melanogenesis through p38 MAPK activation. Although other factors could be involved in GDA-induced hyperpigmentation, generation of uric acid, a metabolic end product, could contribute to GDA-induced-melanogenesis.It was described in the Materials and Methods of the original.


The relation between socioeconomic position (SEP) and obesity measured by body mass index (BMI), a measure of weight for height, has been extensively reviewed in children, showing consistent associations between disadvantaged SEP and higher BMI in high-income countries (HICs) and lower BMI in middle-income countries (MICs). Fat mass (FM), a more accurate measure of adiposity, and fat-free mass (FFM) are not captured by BMI, but have been shown to track from childhood to adulthood, and be important for cardiovascular health and functional outcomes in later life. It is not clear whether body composition is associated with SEP. We systematically reviewed the association between SEP and body composition in childhood.A systematic review was carried out following PRISMA guidelines. The protocol was pre-registered with PROSPERO (CRD42019119937). Original studies in the English language, which examined the association between SEP and body composition in childhood, were included. An electronic search of three databases was conducted. Two independent reviewers carried out screening, data extraction and quality assessment. Due to heterogeneity in results, a narrative synthesis was conducted. Heterogeneity in findings according to SEP, sex, body composition measure and country income level was investigated.50 papers were included, the majority from HICs. No papers were from low-income countries. Disadvantage in childhood was associated with greater FM and lower FFM in HICs, but with lower FM and lower FFM in MICs. When measures of FFM indexed to height were used there was no evidence of associations with SEP. In HICs, more studies reported associations between disadvantaged SEP and higher FM among girls comparative to boys.Inequalities in FM are evident in HICs and, in the opposite direction, in MICs and follow similar trends to inequalities for BMI. Inequalities in height are likely important in understanding inequalities in FFM. Childhood obesity is a globally recognised public health challenge and is a major determinant of obesity in adulthood . PreviouThe majority of evidence on inequalities in overweight and obesity in children comes from studies using BMI, a measure of weight for height which does not distinguish fat mass (FM) from fat-free mass (FFM) and therefore may under or overestimate adiposity. Measures of body composition can provide information about the location of FM and estimates of the proportion of FM to FFM. FFM includes bone mass and lean mass (LM) and is most frequently measured by bioelectrical impedance analysis (BIA). LM is a measure that excludes bone mass and is most frequently measured through dual x-ray absorptiometry (DXA) . In adulStudies using serial data in children have shown secular changes in body composition, with an increasing trend for FM index (FMI) from 1960 to 1999 in the US . In the We therefore carried out a systematic review to assess the association between SEP and measures of body composition  in children (up to and including 18 years) from general population samples. Additional aims were to assess secular changes in socioeconomic inequalities in body composition and explore heterogeneity by sex, SEP measure, body composition measure and income level of country of study.The protocol for this review, which is the second part of a larger systematic review investigating socioeconomic inequalities in adults , was regPeer reviewed papers written in the English language were included in this review if they reported an association between SEP and body composition in children (under 18 years of age) using data from an observational study including a sample from the general population. Associations between any recognised indicator of SEP  and a measure of body composition, measured at the same, or later, time point to SEP were included. As studies in this review are in children, measures of SEP, such as occupation and education, relate to parents, other markers such as overcrowding reflect the home in which the child lives, and some markers of education are based on the type of school attended. Body composition  was defined as any measurement related to total FM and FFM, location of FM and FFM or any proportion or ratio of measures of FM and FFM.CBS conducted an electronic search of three databases  from the earliest entry up until the 30th of January 2019. The search terms used are shown in Table Relevant information was double extracted using a data extraction form by CBS, AG, JB, MA, and EW. Data extracted included citation details , study details , participant details , exposure and outcome details  and statistical methods and information on adjustment for potential confounders and mediators. All available statistics relating to the association under study were extracted, along with statements of direction in text where statistics were not presented.Assessment of study quality was carried out by CBS, AG, JB, MA, and EW, using an amended version of the Newcastle-Ottawa Quality Assessment scale . QualityTwo reviewers  worked independently to complete screening, quality assessment and data extraction. Any disagreements were resolved through discussion.A meta-analysis was not possible due to the considerable variability in analytic methods used and presentation of results. As such, it was not possible to assess the degree of publication bias across studies through use of a funnel plot. Instead, a narrative synthesis was conducted, guided by the Economic and Social Research Council Methods Programme guidelines , with a Multiple relevant associations were frequently presented in a single paper. The individual association, as opposed to the paper, were thus considered the unit of analysis, similar to methods adopted by McLaren , and BalP values. Use of P values on their own only occurred if they indicated a non-significant relationship in absence of information on the direction of association.Each association reported was categorised as either a positive association (those reporting greater socioeconomic advantage associated with higher body composition measure), negative association (those reporting greater socioeconomic advantage associated with lower body composition measures), non-linear association or no association. We removed the non-linear group from the summary tables, similar to the approach of McLaren , as onlyAs outlined in the protocol, heterogeneity in associations was explored according to body composition measure , SEP measure and sex. Results from analyses using boys and girls combined were the primary results selected for summary. Where results were only presented for girls and boys separately, both associations were included in the summary results. It was not possible to investigate differences in body composition by birth year as outlined in the protocol, due to lack of information provided on birth year across studies. On extraction, it became clear that country income level should also be considered a source of heterogeneity. Studies were thus categorised into those in high-income countries (HIC), upper middle and lower middle-income countries, according to the World Bank classification in 2019 . Those pP values alone to report a non-significant result without provision of effect estimates, descriptive data or statement of direction in text.In total, 7145 papers were identified from the database searches for studies in both children and adults, with 5725 once duplicates were removed. Title and abstract screening resulted in 513 papers, with 92 papers remaining following full text screening. Searching the reference lists for additional papers returned three, bringing the total included papers to 95. Of those, 48 investigated associations between SEP and childhood body composition. A search of papers from key studies resulted in two further papers in children being identified, bringing the total number of included papers to 50 , 29–77. n = 36, 72%) with the remaining papers from MICs (n = 14). The UK and the US contributed the most papers , with 7 unique studies in both. Sample size across the papers ranged from 74 to 14,314, with a median sample size between 485 and 502.There were 38 distinct samples studied across the 50 papers. The Avon Longitudinal Study of Parents and Children (ALSPAC) and a sample from Merida, Mexico were the most commonly included studies, appearing in four and three separate papers, respectively. The majority of papers were conducted in population samples from high-income countries , or bioelectrical impedance (BIA) analysis (n = 22) to measure body composition, with two papers using both methods. Five studies used air-displacement plethysmograph with one of these also using BIA. One other paper used deuterium dilution in combination with DXA and plethysmography. The SEP variable most frequently reported was parental education (n = 25).Fat measures were considerably more frequently reported than FFM measures (in 46 papers compared to 22), with percentage body fat being the most commonly analysed (29 papers). Among papers that investigate fat-free measures, LM was the most frequently used (8 papers). The majority of papers used either dual x-ray absorptiometry (DXA) (n = 21) as were conducted in those children and adolescents over the age of ten (n = 24). One study was conducted in new-borns and was the only study to be conducted in children under the age of four.A similar number of studies were conducted in children aged, or with a mean age, between four and ten  with greater socioeconomic advantage being associated with less fat. The remaining associations in HICs all showed no overall pattern of association (24 association from 13 papers). In MICs, the majority of associations were positive (61%) with greater socioeconomic advantage associated with higher levels of fat. Only 11% (6 associations from 3 papers) reported negative associations, with the remaining associations  reporting no overall pattern.The total body fat measure most frequently reported was FM%, being used 60 times (across 29 papers), followed by FM used 38 times (18 papers), and FMI used 26 times (13 papers). In HICs, using FM or FM% yielded a slightly greater number of negative associations, where greater advantage is related to lower levels of fat,  compared to FMI (60%). In MICs, FM and FMI presented almost exclusively positive associations, where greater advantage is related to higher levels of fat . FM% exhibited more mixed results with 45% finding positive associations, 17% finding negative and the remaining 38% finding no association.Parental education was the most commonly used SEP measure across the papers, used in 53 associations across 24 papers. In MICs, composite measures of SEP were the most frequently recorded SEP measure, used in 22 associations across nine papers. Among HICs, negative associations were reported in the majority of associations (≥ 60%) for all SEP measures, with the exception of parental or household income, used in six papers, where no association was most frequently reported . In MICs, parental education, used in seven papers, yielded a higher number of positive associations  compared with composite SEP measures .Sex-specific analysis was presented in 15 papers in HICs and eight papers in MICs, with 44 and 37 associations reported, respectively Fig. . NegativWhen considering results in high quality papers only , findings were similar to the full analysis. In HICs nine associations out of 13 (69%), reported across six papers, showed greater socioeconomic advantage associated with higher levels of fat. In MICs, three out of five associations (60%), reported in two papers, found greater advantage associated with higher FM. In low-quality papers (≤3*) eight out of 13 associations (62%) from five papers reported negative associations similar to that in high quality papers. However, in MICs, the low-quality papers, contrasting with results in the full analysis, showed predominantly negative associations. However, the findings were from only two papers both conducted in the same population sample.Table Raw fat-free measures, used in 14 papers, show positive associations in 59% of analyses , more frequently than both percentage measures  and considerably more often than indexed measures . Measures that include bone in their assessment show positive associations slightly more often (54%) than those which exclude bone (42%).n = 18), with all except one such association tested in MICs. Parental occupational social class and measures of area-level SEP were used in eight and nine associations respectively, although for area-level SEP, eight were all tested in the same paper. Parental income was used three times in two papers. In MICs there was a slightly higher number of positive associations reported when using composite measures of SEP (53%) compared to education (40%). In HICs, approximately one-third of associations with both education and occupational social class were observed to be positive. In the small number of analyses including area-level SEP and income in HICs, only positive associations were seen.Parental education was the most frequently investigated SEP measure, used in 24 associations. Composite measures of SEP were also frequently used  the percentage of positive associations reported were lower than in the full analysis, with only three out of 12 associations (25%), from seven papers, finding greater socioeconomic advantage related to higher FFM, and the rest reporting no association. Similar to the full analysis, HICs reported positive associations more frequently with three out of seven associations (43%), from five papers, whilst in MICs all five associations coming from only two papers reported no overall pattern. In low quality papers (≤3*), the number of positive associations in HICs is lower compared to the full analysis with three of nine associations (33%) from four papers reporting such a finding. In MICs, the seven positive associations out of 12 (58%) were from just two papers, both using the same sample population.n = 2) and Brazil (n = 1).Five papers reported on the association between SEP and a ratio or distribution measure Table . Two papFour papers looked at the association between SEP and a measure of central fat. Two of these using the same sample found greater social advantage to be associated with decreases in trunk FM, total abdominal adipose tissue  and trunThis systematic review finds evidence of socioeconomic inequalities in body composition in childhood and adolescence, although the direction and strength of these inequalities varies by measure of SEP, measure of body composition, sex and economic development of the country of study. Evidence of negative associations were generally observed for all measures of fat. Negative associations were more frequently observed in samples from HICs compared with MICs, with positive associations only observed in studies from MICs. In HICs, negative associations were found more frequently in girls compared with boys, whilst in MICs girls more often showed positive associations. Greater socioeconomic advantage was associated with greater FFM in approximately half of the associations studied, but such associations were less common with outcome measures indexed to body size in both HICs and MICs, and in studies rated as higher quality. The review highlighted a lack of research using area-level measures of SEP, parental income and using more detailed measures of body composition, such as ratio and distribution measures.Our findings for fat mass are broadly consistent with those from reviews using BMI as the measure of adiposity. Shrewsbury and Wardle  and BarrPrevious research using anthropometric measures of obesity have reported that, in HICs, studies using parental education report inverse associations most frequently , 3 consiThere are a greater percentage of negative associations between SEP and fat measures among children compared with those found in our review of adults in HICs . This diMost studies in this review were conducted in children born post 1984, which means that in HICs they were all born into an obesogenic environment, the onset of which is generally estimated to be in the 1980s , 87. DisIn contrast to HICs, disadvantaged SEP was associated with lower levels of body fat for children in MICs. Children of advantaged SEP in these countries have greater exposure to a western lifestyle compared to those of disadvantaged SEP, and in particular greater access to more expensive and energy dense foods . TransnaWe previously reported on the association between SEP and fat-free measures in adulthood, finding predominantly no association, although with slight evidence of positive associations among women in HICs . In contGreater inequalities in FFM in childhood compared to adulthood may reflect a secular decline in levels of FFM, which are likely to be accompanied by growing inequalities, in the opposite direction to inequalities seen for adiposity. The Fels Longitudinal Study has shown mean FFMI to be lower in boys born in the 1990s compared with boys of the same age born decades before . Serial In our review, few studies used indexed measures of FFM that aim to, at least partially, remove the correlation with height, nor did they adjust for height. Among those that did, there were fewer observed positive associations. Associations of greater disadvantage and lower FFM may therefore be explained, at least partially by height, as there is evidence that disadvantaged SEP is associated with shorter height across childhood and adulthood in most populations , althougThis review was registered with PROSPERO and has been carried out according to the published protocol . The revThe generous inclusion criteria resulted in considerable heterogeneity in samples, study design and measures used. This variation, together with heterogeneity in the analytical approaches and reporting of results, prevented us from being able to conduct a meta-analysis. Additionally, the association, not paper, was used as the unit of analysis since most papers reported more than one association, meaning that in some cases one paper may contribute more weight to the overall summary of findings. The same data sets were also used by multiple authors in multiple papers.As it was not possible to conduct a meta-analysis, it was not possible to assess publication bias. It is, however, possible that publication bias exists with papers based on small sample sizes showing positive results are more likely to be published than those showing null findings. We did, however, include studies that tested the association of interest as part of a wider set of analyses instead of just focussing on those studies with a specific hypothesis on SEP differences, which may have reduced the impact of publication bias. This included papers that did not report full results where associations were found to be non-significant in preliminary analysis. However, this does mean that associations may not have appropriate adjustment for confounders.P values [P values alongside only descriptive data or description of the association in the text. As P values are influenced by the sample size of the study, lack of associations observed in such studies is likely due to a lack of statistical power. The studies included in this review were generally small, with a median sample size between 485 and 502. Additionally, because of the heterogeneity in SEP measures, outcomes, statistical approaches and the reporting of results it was not possible to make comparisons of effect size across papers, even among those studies that did use appropriate statistical methods. There may also have been overadjustment as studies have adjusted for factors which may actually be mediators rather than confounders.This review prioritised assigning patterns to associations using the effect estimates and confidence intervals, which convey more about the direction and strength of effect, and the accuracy of these estimates , 114 andP values . HoweverThere is inconsistency in the literature relating to the terminology used to describe FFM . It is nWe amended protocol slightly, due to the need to analyse heterogeneity by country income level, as more papers were identified from MICs than expected and it was clear that this was an unignorable source of heterogeneity. We had no papers included from LICs, limiting the ability to explore SEP and body composition associations in countries at an earlier stage of the nutrition transition, which would have been valuable for understanding the changing relationship of SEP and obesity with economic development.In monitoring inequalities, BMI may accurately capture fat mass at a population level in childhood, given the similarities of our findings with reviews on social inequalities in BMI. However, our review suggests contrasting findings on inequalities in FFM in HICs. This may, assuming such associations are not fully explained by inequalities in height, mean that BMI underestimates the inequalities in the health risks related to adiposity. If children from more disadvantaged SEP groups have lower levels of muscle mass and strength, as well as higher levels of fat mass, this may have important implications for inequalities in outcomes which also require good muscle function. As we have found greater evidence of inequalities in body composition in children compared with adults, tracking of body composition through the life course in more recent generations could have important implications for inequalities in physical capability in later life. Follow up of these childhood cohorts is needed to confirm whether these are secular rather than age-related changes in inequalities.It is also crucial that future research appropriately indexes body composition measures and distinguishes measures that include bone from those that do not. Inequalities in height are likely to be an important factor that can explain the observed inequalities in such measures. The results of this review also highlight the need for more research investigating the associations of SEP with ratio and distribution measures of body composition which are related to metabolic and cardiovascular disease outcomes, especially in MICs, and greater research on sex differences in both HICs and MICs. There are also gaps in research looking at the effect of area-level measures on body composition, a measure of SEP that is closely linked to the obesogenic environment, particularly in relation to FFM.Efforts should be made to address inequalities in both FM and FFM among children in HICs and MICs by reducing access to, and advertising of, fast food to children, and promoting and ensuring equal access to healthy and nutritious food. Promotion of physical activity and access to sport facilities should also be prioritised in poorer communities to address inequalities in FFM.Supplementary File 1 - PRISMA ChecklistSupplementary File 2 - Altered Newcastle-Ottawa Quality Assessment FormSupplementary Table 1


Measuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. In recent years, the detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software.https://github.com/MBoemo/DNAscent.This paper introduces DNAscent v2, software that uses a residual neural network to achieve fast, accurate detection of the thymidine analogue BrdU with single-nucleotide resolution. DNAscent v2 also comes equipped with an autoencoder that interprets the pattern of BrdU incorporation on each ONT-sequenced molecule into replication fork direction to call the location of replication origins termination sites. DNAscent v2 surpasses previous versions of DNAscent in BrdU calling accuracy, origin calling accuracy, speed, and versatility across different experimental protocols. Unlike NanoMod, DNAscent v2 positively identifies BrdU without the need for sequencing unmodified DNA. Unlike RepNano, DNAscent v2 calls BrdU with single-nucleotide resolution and detects more origins than RepNano from the same sequencing data. DNAscent v2 is open-source and available at This paper shows that DNAscent v2 is the new state-of-the-art in the high-throughput, single-molecule detection of replication fork dynamics. These improvements in DNAscent v2 mark an important step towards measuring DNA replication dynamics in large genomes with single-molecule resolution. Looking forward, the increase in accuracy in single-nucleotide resolution BrdU calls will also allow DNAscent v2 to branch out into other areas of genome stability research, particularly the detection of DNA repair.The online version contains supplementary material available at (10.1186/s12864-021-07736-6). Regions of a eukaryote’s genome may tend to replicate early or late in S-phase on average, but there is significant cell-to-cell heterogeneity that stems from both the set of origins used and time at which they fire . The higThis paper introduces DNAscent v2 which uses a new residual neural network architecture to assign a probability of BrdU to each thymidine. Overhauling the BrdU detection algorithm from a hidden Markov model to a residual neural network results in high-accuracy BrdU calls , a reference genome, and the alignment (in BAM format) of ONT reads to the genome Fig. a. The suS. cerevisiae thymidine auxotroph [The subprogram detect in DNAscent v2 detects BrdU with single-nucleotide resolution using a residual neural network consisting of depthwise and pointwise convolutions  [In addition to improving performance and adding functionality, DNAscent v2 development placed a particular focus on ease-of-use and accessibility for laboratories that may not have access to computational scientists or bioinformaticians. Origin calling with RepNano has fourteen adjustable parameters and earlier versions of DNAscent have three, but forkSense in DNAscent v2 does not require any tuning. DNAscent v2 also comes packaged with a utility that converts the outputs of detect and forkSense into bedgraphs such that BrdU and fork probabilities can easily be viewed side-by-side for each read  , and oriS. cerevisiae rDNA with 2-kilobase (kb) resolution using DNAscent v0.1 [To evaluate the performance of DNAscent detect, receiver operator characteristic (ROC) curves were plotted using nanopore sequenced unsubstituted DNA to measure false positives and DNA with four different BrdU-for-thymidine substitution rates Fig. b. DNAsceent v0.1 , but DNAent v0.1  without S. cerevisiae cells that were synchronised in G1 and released into S-phase in the presence of BrdU with no thymidine chase [S. cerevisiae cells where BrdU was pulsed for 4 minutes followed by a thymidine chase [S. cerevisiae chromosome I are shown for both experiments : LinuxProgramming language: C, C++, PythonOther requirements: GCC 6.1 or higher, CUDA 10.0 and cuDNN 7.5 (for GPU use only)License: GNU GPL-3.0Any restrictions to use by non-academics: NoneAdditional file 1 Supplementary information. The supplementary information provides technical details about how the neural networks in DNAscent v2 were designed and trained. Details are also provided for the runtime comparisons mentioned in the text.


Ebola virus disease (EVD) is a lethal disease caused by the highly pathogenic Ebola virus (EBOV), and its major symptoms in severe cases include vascular leakage and hemorrhage. These symptoms are caused by abnormal activation and disruption of endothelial cells (ECs) whose mediators include EBOV glycoprotein (GP) without the need for viral replication. However, the detailed molecular mechanisms underlying virus–host interactions remain largely unknown. Here, we show that EBOV-like particles (VLPs) formed by GP, VP40, and NP activate ECs in a GP-dependent manner, as demonstrated by the upregulation of intercellular adhesion molecules-1 (ICAM-1) expression. VLPs-mediated ECs activation showed a different kinetic pattern from that of TNF-α-mediated activation and was associated with apoptotic ECs disruption. In contrast to TNF-α, VLPs induced ICAM-1 overexpression at late time points. Furthermore, screening of host cytoskeletal signaling inhibitors revealed that focal adhesion kinase inhibitors were found to be potent inhibitors of ICAM-1 expression mediated by both TNF-α and VLPs. Our results suggest that EBOV GP stimulates ECs to induce endothelial activation and dysfunction with the involvement of host cytoskeletal signaling factors, which represent potential therapeutic targets for EVD. Ebola virus (EBOV), a member of the Filoviridae family, is an enveloped and negative-stranded RNA virus that causes severe hemorrhagic fever with high mortality rates in humans and other animals ,2. The gGP can bind to multiple cell surface molecules, conferring on EBOV the ability to infect a wide range of cell types, including immune cells such as macrophages and dendritic cells, and other cells such as hepatocytes, epithelial cells, and endothelial cells (ECs) ,11. VascExisting evidence suggests that overexpression of ICAM-1 on ECs can lead to increased vascular permeability and loss of the endothelial barrier, which is mediated by the activation of the Rho/ROCK pathway ,20 and rIn the present study, we hypothesized that suppressing the Rho/ROCK pathway and some cytoskeletal signaling molecules would prevent the overexpression of ICAM-1 and associated ECs disruption observed during EBOV infection. First, we confirmed the upregulation of ICAM-1 expression in HUVECs after exposure to Ebola VLPs bearing GP as well as TNF-α, but not Ebola VLPs lacking GP. In contrast to TNF-α treatment, Ebola VLPs bearing full-length GP induced ICAM-1 overexpression at late time points. We also found that only Ebola VLPs treatment induced significant cytotoxicity, mainly apoptosis. Moreover, screening of the cytoskeletal signaling inhibitors library identified focal adhesion kinase (FAK) inhibitors as potent inhibitors of ICAM-1 mediated by both TNF-α and Ebola VLPs bearing GP. Our results suggest that EBOV GP stimulates ECs to induce endothelial activation and dysfunction with the involvement of host cytoskeletal signaling molecules, which represent potential therapeutic targets for EBOV infection.Expression plasmids for glycoprotein (GP), nucleoprotein (NP), and viral matrix protein 40 (VP40) of Ebola virus , used for the generation of Ebola virus-like particles (VLPs), were provided by Dr. Yoshihiro Kawaoka  and Dr. Thomas Hoenen . A library of pharmacologically active cytoskeletal signaling-related compounds was purchased from Selleck Chemicals . This library contains United States Food and Drug Administration (FDA)-approved and registered drugs, as well as preclinical compounds.2 environment and were kept at a low passage number (below 20) for the experiments to maintain cell morphology and biological properties.Human umbilical vein endothelial cells (HUVECs)  were cultured in collagen-coated dishes  containing endothelial growth medium (EGM)  supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin solution, and 0.1% amphotericin B . The human embryonic kidney cell line (HEK293T) , used for the preparation of Ebola VLPs was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin solution. All cells were incubated at 37 °C in a humidified 5% COVP40/NP) were also generated by cotransfecting HEK293T cells with pCAGGS-EBOV-VP40 and pCAGGS-EBOV-NP. Transfection with pCAGGS alone served as a mock control. The collected supernatants containing Ebola VLPs and VLPVP40/NP were layered onto 20% sucrose cushions and spun at 30,000 rpm at 4 °C for 2 h. The resultant pellets were resuspended in phosphate-buffered saline (PBS) and layered again onto 20–60% sucrose gradients for ultracentrifugation at 75,000 rpm for 2 h. The obtained fractions were harvested and analyzed by Western blotting, as described below. Further, EBOV protein-rich fractions were pooled and sedimented through a 20% sucrose cushion at 75,000 rpm for 30 min. The resulting pellet was resuspended in Opti-MEM serum-free medium , giving rise to purified Ebola VLPs and VLPVP40/NP preparations. Purified Ebola VLPs and VLPVP40/NP were quantified for GP content using Western blotting with recombinant GP lacking the transmembrane domain (EBOV rGPΔTM)  as the reference standard and anti-GP as the detection antibody. Unless otherwise stated, 10× diluted VLPs, corresponding to 9.2 µg/mL of GP, were used in most of the experiments, including drug screening.Ebola VLPs bearing GPs (designated VLPs) were generated as previously described , with soVP40/NP or the mock control particles were mixed with sodium dodecyl sulfate-containing buffer , boiled for 5 min at 95 °C, and subjected to SDS-PAGE before being transferred onto a nitrocellulose blotting membrane  overnight via Western blotting. The membranes were blocked in the blocking solution, made with the wash buffer (0.2 M Tris-HCl (pH 7.5), 8.76 g/L NaCl, and 0.25% Tween 20) containing 5% skim milk, for 1 h at 25 °C. After blocking, the membranes were first incubated overnight with mouse monoclonal anti-EBOV GP antibody provided by Dr. Ayato Takada  ( before the addition of 10× diluted VLPs or the medium (control) for 48 h.For screening in 96-well plates, ECs were cultured for 24 h, as described above. Cultured media were removed, and cells were pretreated with the vehicle (DMSO or water) or the library compound for 1 h at 37 °C before the addition of TNF-α (10 ng/mL) or 10× diluted VLPs for 12 or 48 h post-treatment incubation, respectively. Cells were then fixed with 10% formalin and subjected to an immunofluorescence assay as described above. All compounds were used at a final concentration of 5 µM during the screening, and their inhibition of VLPs- or TNF-α-mediated ICAM-1 expression was determined by fluorescence measurement. Percent inhibition of ICAM-1 expression for each compound was determined by normalization to either VLPs- or TNF-α-treated wells without compounds. Following the same protocol, the experiment for the inhibition of micropinocytosis was performed with 30 min of incubation at 37 °C with 10 µM EIPA , an inhibitor of the Na+/H+ exchanger that specifically inhibits micropinocytosis [To further understand the difference in the kinetics of ICAM-1 expression between VLPs and TNF-α we questioned whether VLPs-mediated ICAM-1 expression upregulation is the result of a stimulation from the cell surface attachment or a cytokine response following the internalization. ECs were pretreated with the medium or EIPA [5- with an IC50 of 2.1 µM, PF-00562271 Besylate with an IC50 of 3.6 µM, and PF-431396 with an IC50 of 4 µM. Y15, PF-573228, and TAE226 (NVP-TAE226) were also effective with IC50 values > 5 µM and displayed low toxicity in ECs. Additionally, BI-4464 demonstrated a selective index (SI) of 22.2, indicating its highly specific inhibitory effect  with the highest IC50 value (0.9 µM), PF-00562271 Besylate with an IC50 of 2.3 µM, PF-431396 with an IC50 of 2.3 µM, and Y15 with an IC50 of 3.8 µM (50 ≥ 20 µM).To assess the potency and specificity of the selected FAK inhibitors on VLPs- or TNF-α-treated cells, we tested their dose-dependency at concentrations up to 20 µM. The cytotoxicity of the compounds was also evaluated in the same concentration range, including all nine FAK inhibitors contained in the screened library. Among them, seven and eight compounds dose-dependently inhibited VLPs- and TNF-α-induced ICAM-1 expression, respectively. As shown in y effect C. Four pf 3.8 µM A,C. GSK2In both cases, the inhibition of ICAM-1 expression had no effect on ECs viability. Contrary to our hypothesis, the images of ECs nuclei revealed that the selected compounds could suppress the expression of ICAM-1 mediated by VLPs and TNF-α in a dose-dependent manner without rescuing the cells from VLPs-induced injury .Since FAK inhibitors reduced both VLPs- and TNF-α-induced ICAM-1 expression in ECs, we checked, by Western blot analysis, whether FAK protein expression was increased upon treatment with VLPs or TNF-α. The results in VP40/NP) did not activate ECs, as shown by the absence of ICAM-1 expression , TNF-α, and interferon-γ (IFN-γ) ,38. KineThe level of ICAM-1 expression in VLP-treated ECs was not upregulated before 48 h of incubation. Connolly-Andersen et al. observed the same kinetic pattern for ICAM-1 upregulation at the transcriptional level following the activation of HUVECs with various doses of Crimean-Congo hemorrhagic fever virus (CCHFV) . Wahl-JeThe involvement of the Rho/ROCK pathway in the induction of hyperpermeability in HUVECs treated with Ebola VLPs  led us tSince the Rho/ROCK pathway controls EC dysfunction through the regulation of cytoskeleton organization , we screUnexpectedly, FAK inhibitors did not prevent Ebola VLPs-induced apoptosis in ECs, suggesting the possible existence of an additional host signaling pathway, acting either independently of FAK, or at triggering GP-mediated apoptosis at the upstream of FAK pathway associated with ICAM-1 induction. Further investigation of other identified compounds targets, including HSP and Bcr-Abl, may be key to unraveling the molecular mechanisms of GP-induced apoptosis. To the best of our knowledge, this is the first study identifying FAK as a therapeutic target for EVD pathogenesis. FAK is overexpressed in various cancers and has been found to play key roles in normal and cancer cellular functions, such as survival and proliferation . NotablyIn summary, this study showed that EBOV-like particles (VLPs)-bearing GP could induce ECs apoptotic disruption and activation, as shown by intercellular adhesion molecules-1 (ICAM-1) upregulation, without requiring internalization into the cell. Additionally, we found that the inhibitors of certain cytoskeletal signaling molecules such as focal adhesion kinase could inhibit both VLPs and TNF-α-induced upregulation of ICAM-1 expression without rescuing ECs from the GP-induced apoptotic disruption. The results suggest that Ebola GP triggers apoptosis through an additional pathway other than the ICAM-1 induction pathways. Finally, the cytoskeletal signaling pathways may serve as important targets for the development of therapeutic drugs against EBOV disease.


Heart failure (HF) with preserved ejection fraction (HFpEF) is a leading cause of hospitalizations and mortality when diagnosed at the age of ≥ 65 years. HFpEF represents multifactorial and multisystemic syndrome and has different pathophysiology and phenotypes. Its diagnosis is difficult to be established based on left ventricular ejection fraction and may benefit from individually tailored approaches, underlying age-related changes and frequent comorbidities. Compared with the rapid development in the treatment of heart failure with reduced ejection fraction, HFpEF presents a great challenge and needs to be addressed considering the failure of HF drugs to improve its outcomes. Further extensive studies on the relationships between HFpEF, aging, and comorbidities in carefully phenotyped HFpEF subgroups may help understand the biology, diagnosis, and treatment of HFpEF. The current review summarized the diagnostic and therapeutic development of HFpEF based on the complex relationships between aging, comorbidities, and HFpEF. Heart failure (HF) is divided into three forms based on left ventricular (LV) ejection fraction (LVEF): heart failure with preserved ejection fraction , heart failure with reduced ejection fraction , and heart failure with mid-range ejection fraction  . HFpEF hHFpEF is a systemic syndrome involving multiple organs . DiastolHFpEF is triggered by the cumulative expression of various risk factors and comorbidities, including age, sex , physical inactivity, obesity, AF, CAD, diabetes, dyslipidemia, hypertension, metabolic syndrome, chronic kidney disease, anemia, chronic obstructive pulmonary disease and sleep-disordered breathing . HoweverAlthough LVEF is not reduced, increased LV-filling pressure results in exertional dyspnea and exercise intolerance. If dysfunctional epicardial adipose tissue is adjacent to LV, it impairs LV distensibility and promotes HFpEF development. However, if it is adjacent to left atrium (LA), atrial myopathy is caused by electro-anatomical fragmentation and structural remodeling of LA . AF may Aging affects pathophysiological process of HFpEF. Structural and functional changes related to aging are generally believed to be significant risk factors of HFpEF . Aging rA key obstacle for exploring new pathophysiological mechanisms and testing new pharmaceutical substances is the availability of suitable animal models for HFpEF, which realistically reflect the research and clinical picture. A variety of animal models are developed with the signs of HFpEF ranging from murine models to a pig model. Most of these animal models develop HFpEF triggered by a single factor like hypertension , obesity/diabetes (db/db mouse), and aging (senescence‐accelerated mouse). Schiattarella and colleagues recently formulated a ‘two‐hit’ hypothesis, inducing HFpEF in mice by metabolic stress (feeding of a high fat diet) and mechanical stress (hypertension induced by blocking eNOS activity) as second stressor. However, another animal model, developing HFpEF owing to diabetes and hypertension, is the ZSF1 (Zucker fatty and spontaneously hypertensive) rat. This model was developed by crossing rat strains with two separate leptin receptor mutations (fa and facp), the lean female ZDF rat (+ /fa) and the lean male SHHF rat (+ /facp). Offspring being homozygous for both mutations (fa:facp) are obese and develop insulin resistance, hyperglycaemia, and mild hypertension (ZSF1‐obese). The ZSF1‐obese animals developed HFpEF signs, exercise intolerance, reduced skeletal muscle contractility and endothelial dyfunction. ZSF1 rat may serve as a suitable animal model to study pathophysiological mechanisms and pharmaceutical strategies for HFpEF.HFpEF presents a significant challenge in the diagnostic process, lacking a useful and objective one-method-fit-all approach . DiagnosCardiac magnetic resonance imaging provides structural evidence of HFpEF, such as increased epicardial adipose tissue volume and myocardial fibrosis. Cardiac catheterization is the best method to confirm increased LV-filling pressure, but being an invasive method, its application in the elderly is limited. Electrocardiography showing AF may be an available and sensitive marker of HFpEF in the elderly. Diastolic stress testing is of essential significance for diagnosing HFpEF –53. HFpE2FPEF score) ranging from 0 to 9. The odds of HFpEF doubled for each 1-unit score increase. H2FPEF score relies on simple clinical characteristics, whereas cardiac catheterization is necessary in HFA-PEFF. H2FPEF score enables the discrimination of HFpEF from noncardiac causes of dyspnea and determine further diagnostic testing in the evaluation of patients with unexplained dyspnea.HFpEF may be represented by different pathophysiological phenotypes, which require differential identification and management . DiagnosThe prevention of HF is hard and controlling risk factors may be feasible. Lifestyle modifications, such as dietary control, nutrient management, physical activity, weight loss, and cardiorespiratory fitness, have beneficial effects on the prevention of HFpEF . TreatinAddressing the risk factors and comorbidities is a significant way of preventing the development of HFpEF , 67. FirHFpEF, being one of the most challenging diseases to treat, does not respond to a one-method-fit-all approach; hence, several therapeutic methods are shown in Table Tachycardia predicts poor outcomes in patients with HFpEF . Beta-blStatins can decrease epicardial adipose tissue volume and thereby prevent systemic inflammation and myocardium fibrosis , 108. StNatriuretic peptides activate guanylyl cyclase, resulting in cyclic guanosine monophosphate (cGMP) formation and preventing myocardial fibrosis due to vasodilation and diuresis . EndogenAbnormal nitrogen monoxide-cGMP-protein kinase G (NO-cGMP-PKG) pathway may constitute a pathophysiological mechanism promoting myocardial fibrosis and diastolic dysfunction in HFpEF , 117. DiPhosphodiesterase-5 inhibitors are applied to treat precapillary pulmonary arterial hypertension (PAH) and may be considered in combined precapillary and postcapillary PAH. Sildenafil fails to significantly lower pulmonary artery pressure (PAP) in HFpEF patients without precapillary PAH and those with postcapillary PAH , 123. HoInsulin has hypoglycemic, antinatriuretic, and adipogenic effects and causes adverse outcomes in patients with HFpEF , 127. MeRegulation of incretin system includes mimicking glucagon-like peptide 1 (GLP-1) roles and inhibiting GLP-1-degrading enzyme dipeptidyl peptidase-4 (DPP-IV) . GLP-1 aAs an anti-fibrotic drug, pirfenidone suppresses the development of ventricular fibrosis and diastolic dysfunction through targeting transforming growth factor β (TGF-β) signaling pathway in pressure-overload induced HF , 146. FuCardiolipin is a significant phospholipid in the inner mitochondrial membrane, and Szeto-Schiller (SS) peptide is an antioxidant peptide binding to cardiolipin . ElamiprAs small non-coding ribonucleic acid (RNA) molecules, micro-RNAs (miRNAs), such as miR-23, miR-24, miR-125, miR-195, miR-199, and miR-214, are observed to be increased in the heart tissue of patients with HF . HypertrCardioMEMS device, a radio frequency-based wireless pressure sensor, improves cardiac outcome through continuously monitoring PAP in patients with HF , 163. LVWith its increasing prevalence and worsening prognosis, HFpEF is nearly unique to the elderly and considered a true geriatric syndrome. Compared with the tremendous progress in the diagnosis and treatment of HFrEF, HFpEF continues to be a great enigma and needs to be further studied considering the failure of HF drugs to improve its outcome . There i


The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. In addition, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients.PAFR) experiments, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. We next evaluated the downstream signaling pathway of PAFR in siPAFR-treated cells and GB-treated cells after CDDP treatment. In both cases, we observed decreased phosphorylation of ERK and Akt and increased expression of cleaved caspase-3. These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients.The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways, including regulating the activation of kinases. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). We first analyzed the correlation between PAFR expression and CDDP sensitivity in seven OSCC-derived cell lines based upon cell viability assays. Among them, we isolated 2 CDDP-resistant cell lines (Ca9-22 and Ho-1-N-1). In addition to conducting PAFR-knockdown (si Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the head and neck regions, constituting approximately 3% of all cancers . AlthougIn some cases, chemotherapy is an efficient adjuvant treatment for OSCC patients. However, the emergence of resistance to anti-cancer drugs hampers the curative effect to a large extent . CisplatPlatelet activating factor (PAF), synthesized by various types of cells, is implicated in inflammation, carcinogenesis, and tumor metastasis ,6. PAF bPAFR knockdown experiments. In addition, we found that ginkgolide B (GB), an inhibitor of PAFR, increased the CDDP chemosusceptibility, suggesting that GB may be a novel drug that could enhance CDDP chemotherapy.In the current study, we found that PAFR expression status was involved in CDDP sensitivity in OSCC cells based upon This study protocol has been approved by the Ethics Committee of the Graduate School of Medicine, Chiba University .A total of 7 human OSCC-derived cell lines  were purchased from RIKEN BioResource Center  and the Japanese Collection of Research Bioresources Cell Bank . OSCC-derived cells were cultured as previously described . We obtahttp://primer3plus.com/, accessed on 16 April 2019) was used to design the primers. The sequences of the designed primers were as follows.: PAFR  and glyceraldehyde-3-phospate dehydrogenase . The normalization of the transcript levels of target genes was previously described [PCR reaction conditions were previously described . Primer Proteins were extracted from cells using RIPA buffer  containing cOmplete ™ Protease Inhibitor Cocktail  and the protein concentration was adjusted to 1 mg/mL. Immunoblotting was performed as previously described . The pri50) values were calculated from semi-logarithmic dose-response curves by linear interpolation.The cells were treated with the indicated concentrations of CDDP  and/or GB, a PAFR inhibitor  for the indicated time periods. Cell viability was determined as previously described . Half-maPAFR siRNAs (siPAFR)   and control siRNAs (control) (Thermo Fisher Scientific) were used to transfect OSCC cells. The introduction of siRNA was conducted as previously described [Stealth 5/100 µL. Annexin V-FITC and PI were added to 100 µL of cell suspension and assayed according to the manufacturer’s instructions.The FITC Annexin V Apoptosis Detection Kit I  was used to measure apoptosis. Briefly, cells were treated with trypsin and collected. The collected cells were washed, and the cell suspension was adjusted to 1 × 10t-test or one-way analysis of variance (ANOVA), followed by a post hoc Tukey’s test (ANOVA with Tukey’s multiple comparison test). All statistical analyses were performed with Microsoft Excel . The data are expressed as the mean ± standard error of the mean.The data were analyzed using an unpaired PAFR, the seven OSCC cell lines and HNOKs were subjected to qRT-PCR and immunoblot analyses. PAFR mRNA expression was up-regulated significantly  in the four OSCC cells  compared with the HNOKs  to CDDP, we assessed cell viability after treatment with the drug A. FigurePAFR mRNA and PAFR protein in siPAFR-transfected cells decreased significantly  compared with the control cells  for 48 h. siPAFR-transfected cells had higher susceptibility to CDDP than control cells . These data suggested that PAFR expression was involved in the regulation of CDDP susceptibility in OSCC cells. To further investigate apoptosis of OSCC cells after CDDP treatment, we performed flow cytometric analysis. The apoptosis frequency of siPAFR-transfected OSCC cells was higher than that of the control group . Since the activation of ERK and Akt signaling pathways are common attributes of apoptosis or survival [PAFR-transfected OSCC cells after treatment with CDDP. We also investigated cleaved caspase-3 to confirm apoptosis. Decreased ERK and Akt phosphorylation was observed in siPAFR-transfected Ca9-22 and Ho-1-N-1 cells after treatment with CDDP. In addition, highly cleaved caspase-3, an apoptosis marker, was observed in both Ca9-22 and Ho-1-N-1 cells after treatment with CDDP . The apoptosis frequency of the cells treated with CDDP plus GB treatment was higher than that of treatment with CDDP alone . We next investigated downstream signaling of PAFR after CDDP and GB treatments. In both Ca9-22 and Ho-1-N1 cells, reduced phosphorylation of ERK and Akt and highly cleaved caspase-3 were observed in cells treated with CDDP plus GB combination therapy (First, we assessed the capability of GB as a specific inhibitor of PAFR and found that PAF-induced IL-1β expression significantly decreased after treatment with GB (Unpaired  therapy C.PAFR by siRNA (CDDP is a widely used chemotherapeutic drug used for cancer, including OSCC  however,by siRNA  and GB (by siRNA  enhancedby siRNA .In various cancers, PAFR overexpression accelerates cell proliferation, migration, and invasion relative to control cells ,26,27,28Ginkgo biloba, a Chinese herb, and used in traditional Chinese medicine for thousands of years [Ginkgolides have been isolated from of years . Currentof years . Ginkgolof years ,36. GB hof years . To dateof years ,39.We focused on GB as a selective inhibitor of PAFR, and we investigated the effect of GB on CDDP sensitivity in OSCC. To clarify whether GB enhances CDDP sensitivity to Ca9-22 and Ho-1-N-1 cell lines, cell viability assays, flow cytometric analyses, and immunoblot analyses were performed. The results showed that CDDP combined with GB reduced cell viability and increased cell apoptosis, while GB alone had no such impact . Side efThese results suggest that PAFR is involved in the regulation of CDDP sensitivity in OSCC. In addition, GB increased CDDP sensitivity through its effects on the PAFR signaling pathways. Our results suggest that GB may have therapeutic efficacy when used in combination with CDDP in OSCC.


Ubiquitously eXpressed Transcript isoform 2 (UXT—V2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination.in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA.We combine co-immunoprecipitation, ubiquitination in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXT—V1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXT—V2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus.The Ubl domain of Fbxo7 contributes to interaction with UXT—V2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway.Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson. •UXT-V2 is a canonical substrate of SCF(Fbxo7) E3 ubiquitin ligase.•Fbxo7 interacts with both UXT-V1 and UXT—V2.•UXT-V2 recruits Fbxo7 to the cell nuclei.•Fbxo7 inhibit NF-kB pathway through degradation of UXT-V2. This reFBXO7, also called PARK15, are associated with PD  and UXT-V2-HA purified from HEK293T. The reactions were incubated at 30 °C for 90 min and resolved by SDS-PAGE for immunoblotting assays.For the 2.5HEK293T cells were transfected with empty vector, EGFP or FLAG-Fbxo7 constructs and UXT-V2-HA or UXT-V1-M13G-HA, with or without ubiquitin-6xHis-myc for 36 h. The transfected cells were treated with 10 μM of MG132 6 h prior to lysis. Cells were lysed as described in last section and supernatants were subjected to immunoprecipitation (IP) with agarose-anti-HA beads. The polyubiquitinated proteins were eluted with HA peptide (300 μg/mL), and eluates were resolved with SDS-PAGE and immunoblotted. To reprobe immunoblots, membranes were incubated in a Stripping Buffer (SB)  at 37 °C for 15 min. This incubation was repeated 3 times, and the membrane was washed with TBST and blocked for 1 h with dried milk 5% in TBST. Thereafter, the membrane was tested with anti-rabbit-HRP antibody to ensure the removal of previous signals. The second probe with rabbit anti-K48 antibody was then performed. The images were captured in ChemiDoc XRS+ (BioRad), one image each 4 s during 200 s, before pixel saturation and directly used for analysis.2.6in vivo polyubiquitinated substrates were eluted by HA peptide at 300 μg/mL and stored at −80 °C. Purified deubiquitinating enzymes (DUBs) as described in , the 10.13039/501100000781European Research Council [724804], and the Lister Institute for Preventive Medicine; S.J.R. and H.L. were funded by the Biotechnology and Biological Science Research Council [BB/J007846/1]; F.R.T. was funded by FAPESP 2016/00792-2,FAPESP 2016/25798-3 and CAPES.V.S. was funded by a The authors declare no competing interests associated with this manuscript.