In Belgium, confinement measures were introduced on the 13th of March 2020 to curb the spread of the coronavirus disease (COVID-19). These measures may affect health behaviours of the population such as eating habits, physical activity and alcohol consumption, which in turn can lead to weight gain resulting in overweight and obesity, increasing the risk of several chronic diseases, but also of severe COVID-19. The purpose of this study is to assess the impact of confinement measures on health behaviours and their associations with weight gain.n = 28,029). The association between self-reported weight gain and health behaviour changes, adjusted for gender, age group and household composition was assessed through OR’s (95% CI) calculated with logistic regression models, using post-stratification weights.Data were derived from the second national COVID-19 health survey. Data were collected between the 16th and the 23rd of April 2020. The recruitment of participants was based on snowball sampling via Sciensano’s website, invitations via e-mail and social media. The study sample includes participants aged 18 years and over with no missing data on the variables of interest  and 1.29 (1.04–1.60), respectively), among those who increased their consumption of sweet or salty snacks (OR = 3.65 (3.27–4.07)), among those who became less physically active (OR = 1.91 (1.71–2.13)), and among those who increased their alcohol consumption (OR = 1.86 (1.66–2.08)).The most important correlates of weight gain during confinement were an increased consumption of sweet or salty snacks and being less physically active. These findings confirm the impact of diet and exercise on short term weight gain and plead to take more action, in supporting people to achieve healthier behaviours in order to tackle overweight and obesity, especially during the COVID-19 pandemic. In Belgium, several confinement measures were introduced by the National Security Council on the 13th of March 2020 with the aim of curbing the spread of the coronavirus disease (COVID-19) [Confinement measures may impact health behaviours, such as eating habits and physical activity, both in a positive and in a negative way , 3. If sOne of the key risk factors for weight gain that may be affected by confinement measures is unhealthy eating habits. Snacking is the intake of specific foods, often nutrient-poor, energy-dense, between traditional meals. Epidemiological studies have found a positive association between snacking and weight gain among adults , 11. OftBeing more often at home during confinement, may give easier access to snacks – often also a cheaper alternative to healthier options – and sugar-sweetened beverages, and extra occasions to consume them.Furthermore, a high consumption of food prepared out-of-home has also been related to weight gain , 14. TheAnother important health behaviour that may be impacted by the confinement is physical activity. Potential reasons for a decrease in physical activity include the fact that people are recommended to stay at home which reduces their movements. Besides, the closure of indoor sport facilities, as well as the combination of work and homeschooling may be additional factors for reducing physical activity . The assThe last risk factor for weight gain that may be impacted due to the COVID-19 confinement is the consumption of alcohol. Studies have generally shown that light to moderate alcohol consumption is not associated with obesity, but heavy drinking and binge drinking are . This caThe purpose of this study is to assess the short-term impact of the COVID-19 confinement measures on health behaviours, such as eating habits, physical activity and alcohol consumption, and on the change in body weight among adults in Belgium. Further, the associations of these health behaviours with weight gain during confinement were determined.To evaluate the impact of the confinement measures on the mental health, health behaviours and weight status of the population, Sciensano, the Belgian institute of public health, organised a series of online health surveys. The first COVID-19 health survey was launched 3 weeks after the start of the confinement period (the 2nd of April), the second survey took place 2 weeks later (the 16th of April), the third one started on the 28th of May, the fourth on the 24th of September and finally the fifth on the 3rd of December 2020. All five surveys were developed using LimeSurvey version 3 and were available online for 1 week. The launch of the surveys and the call for participation were announced on the website of Sciensano and of other organisations , and at the COVID-19 press conference, through the press and on social media. Recruitment was based on snowball sampling : particiThe data for the purpose of this study were derived from the second COVID-19 health survey that included specific questions on health-related behaviours. After exclusion of participants with missing data on the sociodemographic covariates and health behaviour indicators, the final study sample contained 28,029 individuals aged 18 years and older. Since the study sample was biased at the level of region , gender (overrepresentation of women), age group (underrepresentation of the youngest (18-24 years) and oldest (65+ years)) and educational attainment (underrepresentation of the low educated), post-stratification weights taking these elements into consideration were applied. The post-stratification technique uses information on the composition of the population from another data source to rebalance the sample. Population in terms of gender, age, province of residence and educational attainment benchmark were obtained with reference to the “Labour Force Survey” carried out by Statbel in 2018 [Table 2) was calculated based on self-reported height and weight. The weight status was classified as underweight, normal weight, overweight and obesity using WHO BMI cut-offs [The outcome measure was self-reported ‘weight gain’ over 6 weeks during confinement Table . The fivcut-offs .Amongst the sociodemographic variables measures in the survey, we identified the variables which could have a possible impact on weight gain. Retained sociodemographic covariates were: gender (men and women), age group , education attainment (secondary school diploma or less versus higher education), household composition ; couple with child(ren); living alone with child(ren); living with parents, family, friends; other) and employment status . This was the case for all covariates, except for educational attainment (low versus high), and therefore the latter was no longer taken into account. The weighted distribution (in percentage) of the covariates and the health indicators among the study population was determined in a weighted frequency table. Next, the percentage of population reporting weight change over 6 weeks during confinement was reported by weight status. The association between self-reported weight gain and weight status was assessed through a logistic regression analysis, adjusting for gender, age group, household composition, employment and quality of life. Odds Ratio (OR), the 95% confidence interval (CI) and the P-values are reported in the text.In first instance, it was tested if the covariates were individually associated with weight gain , additional stratified analyses by those covariates separately were also conducted. All the analyses were performed with SAS® 9.4 [Logistic regression models were used to determine the associations between self-reported weight gain as the dependent variable and health behaviour change indicators as independent variables , adjusted for gender, age group, household composition, employment status, quality of life and the health behaviour indicators. Crude and adjusted ORs with 95% confidence intervals (CIs) and SAS® 9.4  using thTable Figure P-value< 0.0001) and those with obesity (OR = 2.23 (1.98–2.52), P-value< 0.0001), and lower for underweight persons (OR = 0.30 (0.19–0.46), P-value< 0.0001).Compared to persons in the normal  weight range, the odds of gaining weight was higher for overweight persons (OR = 1.72 (1.56–1.91), adj = 1.39 (1.15–1.68), P-value< 0.001). However, persons who decreased their consumption of sugar-sweetened beverages also had higher adjusted odds of weight gain (ORadj = 1.29 (1.04–1.60), P-value = 0.018). The highest odds of weight gain was observed for persons with an increased consumption of sweet or salty snacks in these 6 weeks (ORadj = 3.65 (3.27–4.07), P-value< 0.0001). Furthermore, persons who were less physically active in the 6 weeks of confinement were almost twice as likely to gain weight (ORadj = 1.91 (1.71–2.13), P-value< 0.0001). Persons who were more active in these 6 weeks had lower odds of gaining weight in the adjusted model only (ORadj = 0.84 (0.74–0.96), P-value = 0.013). Finally, an increased consumption of alcohol in these 6 weeks was positively associated with weight gain (ORadj = 1.86 (1.66–2.08), P-value< 0.0001). Persons who decreased their consumption had a lower adjusted odds of weight gain (ORadj = 0.85 (0.72–0.99), P-value = 0.043).Table adj = 1.28 (1.02–1.61), P-value = 0.036, and ORadj = 1.14 (0.85–1.51), P-value = 0.381, respectively) and men who were more physically active in these 6 weeks had lower adjusted odds of weight gain than women (ORadj = 0.77 (0.61–0.98), P-value = 0.033, and ORadj = 0.91 (0.79–1.05), P-value = 0.195, respectively).Overall, the associations between changes in health behaviours and weight gain were similar for men and women, although two differences were observed Table : women wadj = 2.52 (2.03–3.12), P-value< 0.0001). The association between increased snacking and weight gain also appears to strengthen with age (ORadj = 3.92 (3.29–4.68), P-value< 0.0001). An increased consumption of food prepared out-of-home appears only to be influencing weight gain in the middle age group (ORadj = 1.27 (1.00–1.60), P-value = 0.048), while a decrease in sugared-sweetened beverages appears to be mostly associated with weight gain in older adults (ORadj = 1.55 (1.09–2.20), P-value = 0.016).Some of the associations between health behaviours and weight gain appear to differ in intensity across age groups Table . In partThis study assessed the association between weight gain and changes in health behaviours, such as nutritional habits, physical activity and alcohol consumption, during the 6-weeks confinement period. More than a quarter (28.6%) of the adults reported weight gain over this period in Belgium. Persons who already suffered from overweight or obesity reported weight gain more frequently. Weight gain during confinement has also been reported in other studies: 22% of adults in the US sampled by Facebook reported gaining weight during self-quarantine due to COVID-19  and 49% An increased consumption of sweet or salty snacks and being less physically active during this period both appear to be important health behaviour changes associated with weight gain during the confinement period. These behaviours were also found to be major risk factors in other studies , 29, 30.The proportion of persons who indicated having increased their consumption of food prepared out-of-home in the first 6 weeks of confinement is low. Nevertheless, an increased consumption of alcohol was found to be a risk factor for weight gain in 6 weeks during confinement, especially in the older age groups. The closure of bars and restaurants undoubtedly had an impact on the consumption of alcohol, especially for social drinkers and youngsters who could not go out anymore whereby their lower consumption. However, confinement also results that people will drink more (often) alcohol at home.Confinement and other COVID-19 related restrictions substantially altered the social, physical and economic environments in which people lived, which resulting in a modification of health behaviours for many. While some people had the social, economic and educational resources to make healthier (food) choices, other people adopted less healthy (food) behaviours and gained weight as a result , 32. In Besides a healthy diet, staying active during confinement is also an important health behaviour, not only for controlling the weight status, but also for the well-being and the quality of life . In BelgThis study has several strengths. Firstly, the online tool made it possible to react rapidly to the crisis. The first COVID-19 health survey was launched only 3 weeks after the confinement. A web survey not only has financial advantages, but also logistical ones  whereby high quality data were instantly available , 37. MorThe results from this study may help the government to determine specific strategies to prevent a further increase in the prevalence of overweight and obesity if a similar crisis occur or new confinement measures are introduced due to COVID-19 in the future. This is important since overweight and obese people not only have an increased risk of morbidity  and premature death, but recent studies have shown that obesity increases the risk of severe COVID-19 which may result in an increased pressure on the health care system.


Objective: The present study investigates a score based on baseline C-reactive protein (CRP) and fibrinogen values (FC score) in 173 consecutive glioblastoma (GBM) patients.Methods: The optimal cut-off value for fibrinogen and CRP was defined as 3.5 g/dl and 3.0 mg/L, respectively, according to previous reports. Patients with elevated CRP and fibrinogen were classified with a score of 2, those with an elevation of only one of these parameters were allocated a score of 1, and those without any abnormalities were assigned a score of 0.Results: No significant differences in age, gender, tumor area, molecular pathology, physical status, or extent of resection were identified among the three groups defined by this score. Univariate survival analysis demonstrated that a high baseline FC score (≥1) is significantly associated with a shortened overall survival (OS) . A multivariate Cox regression analysis considering age (>65/≤65), extent of resection (GTR/STR), MGMT promoter status (hypermethylated/non-hypermethylated), and FC score (0/≥1) confirmed that an elevated FC score (≥1) is an independent predictor of shortened OS .Conclusions: The baseline fibrinogen and CRP score thus serves as an independent predictor of OS in GBM. Further investigations of the role of inflammation in the prediction of a prognosis are needed. Glioblastoma (GBM) is still the most devastating malignancy of the central nervous system. GBM represents 15.8% of all brain and CNS tumors –5. FurthSystemic inflammation is suggested to be associated with shortened survival in patients with cancer , 12. FibThe purpose of the present study was to assess the relationship between the preoperative fibrinogen and CRP score and survival among a homogeneous population of GBM patients who underwent surgical resection and started full radiochemotherapy.n = 208) were excluded for the following reasons: only conventional stereotactic or VarioGuide  biopsies without additional cytoreductive surgeries were performed, multiple intracranial lesions were present, the patients had partial or no clinical follow-up (≥1 month), the patients underwent surgery for recurrent GBM, and the patients had no availability of baseline serum CRP or plasma fibrinogen -enhanced MRIs were routinely performed within 48 h before surgery.White-light resection was performed under neuronavigation guidance . When the surgeon believed that gross total resection (GTR) of the tumor was achieved, hemostasis was employed. Afterward, the resection cavity was examined using 5-aminolevulinic acid (5-ALA) , and areas that were suspected to contain remaining tumor tissue were demarcated and resected. Post-operative MRIs were obtained within 72 h after surgery by a senior neuroradiologist to determine the extent of the resection . Gross tThe histological evaluation was performed according to the World Health Organization 2016 diagnostic consensus criteria . For thiRetrospective data acquisition was performed using the laboratory information system Lauris . Venous blood samples were routinely collected within 24 h prior to the surgical resection of GBM. These laboratory examinations were performed at constant time points, which made it possible to analyze patient survival and progression rates. The standard examination before surgery included complete blood count, kidney, and liver tests. The coagulation profile  was also examined for every patient. The baseline plasma fibrinogen level was determined by the Clauss method, which involves adding a standard and high concentration of thrombin  to platelet poor plasma. This fibrinogen concentration is determined based on a reference curve. The serum C-reactive protein values were obtained by turbidimetric immunoassays with a CRPL3 reagent .The following general preoperative characteristics of the patients were recorded: age, sex, Karnofsky Performance Status (KPS), American Society of Anesthesiologists (ASA), body mass index (BMI), and medical history of diabetes mellitus type II and secondary malignant neoplasms.Tumor characteristics were analyzed based on a measurement of the midline shift, tumor area, maximum extent of the peritumoral oedema, and location classified according to the topography using the Sawaya grading system –29. DefiCRP was dichotomized into low (<3.0 mg/L) and high (≥3.0 mg/L) according to previous prospective data evaluating the baseline CRP in patients with cancer . A normaPost-surgery treatment protocols were evaluated by the local tumor board review. Follow-up MRIs were routinely performed every 3 months . The Kart-test for metric variables to compare the FC score groups. Only two-sided p-values were reported. Kaplan–Meier charts of OS and PFS were also calculated. Differences between the high (≥1) and low (0) FC score groups were analyzed using a log-rank test. A p < 0.05 was defined as statistically significant. Furthermore, a multivariate Cox regression analysis was performed to analyze the PFS and OS. Data were organized and analyzed using SPSS© for MacOS 10.15 version 25.0 .We used the Fisher's exact test (two-sided) for nominal variables and Student's A total of 173 newly diagnosed GBM patients with available pretreatment fibrinogen and CRP records who underwent surgical resection and started post-surgical adjuvant therapy  were analyzed. The proportions of patients with an FC score of 0, 1, and 2 were 60.7, 26.6, and 12.7% respectively. The median CRP and fibrinogen levels were 0.92 mg/L (range: 0.2–64.5) and 3.0 g/L (range: 0.4–5.5), respectively. The median age was 62 years (range: 18–85 years). Demographic parameters, physical status, body-mass-index (BMI), extent of resection , MGMT promoter status, IDH-1 status, completion of post-operative treatment protocols, and type of treatment did not differ between the groups in terms of FC score (two-tailed Fisher's exact test). Comorbidities which are potential confounders with regard to the levels of inflammatory markers were also analyzed. Secondary malignant neoplasms, smoking, obesity, diabetes mellitus, connective tissue disorders, and blood-borne infectious diseases  were homogeneously distributed among the FC-score groups. Further details of the baseline characteristics, established predictors, and treatment informations are summarized in p = 0.045, p = 0.022, p = 0.27).The median OS for the entire cohort was 19.0 months [95% confidence interval (CI): 17.2–20.8]. Patients with an FC score of 0 had a median OS of 21.0 months (95% CI: 17.6–24.4), FC score 1 patients had a median OS of 18.0 months (95% CI: 17.0–19.0), and FC score 2 patients had a median OS of 16.0 months  was 21.0 months (95% CI: 17.2–20.8). Eighteen patients featured only an elevation of CRP. The median OS in this subgroup was 19.0 months (95% CI: 13.9–24.0). Patients with increased fibrinogen combined with or without increased CRP (n = 50) had a median OS of 16.0 months (95% CI: 17.5–24.4). The median OS in patients without any increased acute-phase protein (p < 0.001), non-hypermethylated MGMT promoter status , and FC score ≥1  as significant and independent predictors of shortened OS (p = 0.317). The multivariate Cox regression analysis of PFS with consideration of the FC score (≥1/0), age (>65/≤65), extent of resection (STR/GTR), and MGMT promoter status (non-hypermethylated/hypermethylated) identified only an unmethylated MGMT promoter  as an independent predictor of shortened PFS.A univariate Cox regression analysis of OS was performed for known predictors of OS in GBM and the FC score in the entire cohort. An age of >65 at diagnosis, a baseline FC score ≥ 1, subtotal resection, and a non-hypermethylated MGMT promoter status were significantly associated with shortened OS. We conducted a multivariate Cox regression analysis of OS considering age (>65/≤65), FC score (≥1/0), KPS (<70/≥70), extent of resection (STR/GTR), and MGMT promoter status (non-hypermethylated/hypermethylated). The multivariate Cox regression analysis identified age >65 , 32 patients had MGMT promoters that were hypermethylated and a median OS of 27.0 months (95% CI: 18.8–35.3) compared to the 32 patients who had MGMT promoters that were unmethylated and a median OS of 15.0 months (95% CI: 12.5–17.5). After stratifying patients whose MGMT promoters were hypermethylated, those with a tumor area <967.4 mm2 had an increased median OS of 38.0 months (95% CI: 23.8–52.2). By stratifying patients whose baseline FC scores were low (0), those with a hypermethylated MGMT promoter were shown to have an increased median OS of 27.0 months (95% CI: 7.1–46.9). The addition of the criterion “GTR” to this group of patients showed an increase of 17.0 months in the median OS.Using the data analysis presented above, an overall survival stratification tree was constructed and is shown in t-test. p = 0.31].KPSs were homogeneously distributed among both the FC score groups (0 vs. ≥1). Patients with a high FC score ≥1 had a slightly poorer KPS at their 3-, 6-, and 12-months follow-ups. Statistical significance was not observed in the Student's In this study, we evaluated the FC score as a prognostic dual biomarker score for GBM patients. We observed significantly decreased overall survival times in patients with an increased FC score (≥1) at the time of GBM diagnosis. An elevated FC score can be measured via determination of the plasma fibrinogen level and the serum CRP level prior to surgery for GBM and was found to be an independent risk factor for shortened survival. Our findings suggest the potential importance of assessing the prognosis of GBM by combining clinicopathological characteristics with initial inflammatory status.In our cohort, 26.6% of patients presented with elevated fibrinogen plasma concentrations, and 12.7% of patients presented with elevated CRP serum concentrations, indicating that increased circulating concentrations of classical inflammatory response elements are common among GBM patients. There is emerging evidence that inflammatory response is associated with clinical outcomes in patients with various cancer entities, such as non-small cell lung cancer, ovarian cancer, and glioma –43. The in vivo  inflammatory biomarker screening in GBM has not yet been described. The baseline FC score ≥ 1 was significantly associated with a shortened OS, especially in patients with an unmethylated MGMT promoter. The stratification of patients for overall survival by tree-structured investigation revealed that the FC score ≥1 does not predict survival in patients with a hypermethylated MGMT promoter. Hübner et al.  found thMGMT promoter hypermethylation was found to be the only independent predictor for prolonged PFS in our multivariate cox regression analysis. One reason for this result might be the smaller number of patients with increased serum CRP or plasma fibrinogen levels compared to GBM patients with normal inflammatory parameters. Secondly, there was a slightly poorer clinical course among the patients with a high baseline FC score (≥1), which was reflected by a decrease of the KPS in our data. Therefore, the shorter OS, despite having no statistically significant association with PFS in the high FC score (≥1) group, might be explained by the poorer functional status that was reflected over the course of the KPS at the follow-up examinations in this GBM group. Furthermore, patients with a decreased and non-ambulatory functional status often do not pass the regular magnetic resonance imaging follow-up examinations as frequently as ambulatory patients with a good KPS. This bias might also influence the analysis of tumor progression and is a frequent limitation of retrospective oncological studies.The present study has several limitations. Patient data were acquired retrospectively. The number of patients included in the high FC score (≥1) group was low because of the short time period that was chosen for analysis. However, this time period was chosen to rule out a lack of data on established prognostic molecular markers, such as IDH mutations and the MGMT promoter status in the cohort. However, we used highly selective inclusion criteria, such as an analysis of patients who underwent surgical resection and started post-operative chemo- and radiotherapy for primary GBM, to achieve a reliable analysis. Furthermore, the present investigation represents a single-center experience only. Despite the limitations, the results showed that the baseline FC score could be a fast and potential low-cost biomarker to predict overall survival and facilitate sufficient risk stratification and individualized interdisciplinary treatments for GBM patients. However, future investigations are needed to validate these findings in a large cohort study.FC score is a potential biomarker that can independently predict overall survival in GBM. Our investigation emphasizes the need for further studies evaluating the role of inflammation in the prognosis of GBM.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics committee of the Rheinische Friedrich-Wilhelms-University, Bonn, Germany. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Data acquisition was performed by SA, MS, and JW. Study design, methodology, statistical analysis writing, and creation of figures were performed by JW and EG. Supervision of the study was done by EG. Proofreading was done by AG, PS, UH, EG, and HV. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nanohigh-performance liquid chromatography-tandem mass spectrometry (nano-HPLC-MS/MS) was used to identify differentially expressed proteins (DEPs), and bioinformatics was used to investigate the protein function. Proteomic data were validated by western blot analysis. Oxytocin-induced writhing responses and abnormal serum levels of PGE2 and PGF2α were reversed following the administration of GGD. A total of 379 DEPs were identified; 276 were identified between the control group and the model group, 144 were identified between the model group and the treatment group, and 41 were identified as DEPs that were common to all groups. Bioinformatics revealed that the DEPs between the control group and the model group were mainly associated with cellular component biogenesis and binding processes. The DEPs between the model group and the treatment group were mainly involved in the protein binding and metabolic process. The expression levels of HSP90AB1 and the phosphorylation levels of ERK, JNK, and P-p38 in the uteri of rats in the three groups were consistent with the proteomic findings; MAP kinases  are known to be involved in the production of inflammatory cytokines and oxytocin signaling while HSP90AB1 is known to be associated with estrogen signaling. Collectively, these data indicate that GGD may exert its protective function on PD by regulating the inflammatory response and signaling pathways associated with oxytocin and estrogen.Ge-Gen decoction (GGD) is widely used for the treatment of primary dysmenorrhea (PD) in China. However, the mechanisms that underlie this effect are unclear. We investigated the protective mechanism of GGD in a rat model of PD using label-free quantitative proteomics. The model was established by the administration of estradiol benzoate and oxytocin. Thirty rats were divided into three groups (ten rats/group): a control group , a model group (PD rats), and a treatment group (PD rats treated with GGD). The serum levels of prostaglandin E2 (PGE2) and pros Primary dysmenorrhea (PD) refers to recurrent menstrual cramps caused by uterine contractions , 2. ThisShang Han Za Bing Lun and is composed of Pueraria lobata (Ge Gen), Ephedrae (Ma Huang), cinnamon twig (Gui Zhi), Paeonia lactiflora (Shao Yao), ginger (Sheng Jiang), Glycyrrhizae radix et rhizome (Zhi Gan Cao), and red dates (Hong Zao) in a specific ratio (4 : 1 : 3 : 3 : 2 : 2 : 4) [Paeonia lactiflora, Glycyrrhizae Radix et Rhizoma, and ginger could exert antidysmenorrhea effects [cinnamon twig, have also been reported to inhibit uterine contractions induced by oxytocin [Ephedrae, has been shown to relax smooth muscle and activate β-adrenoceptors [Herbal medicine has been used in China for many years to treat PD –10. Ge-G: 2 : 4) , 14. Pre effects . Cinnamioxytocin . Furtheroceptors , 14. HowIn this study, we used label-free quantitative proteomics to investigate the effects of the GGD administration in a rat model of PD.GGD was obtained by the Jiangsu Province Hospital of Chinese Medicine  and consisted of seven components . The priThirty clean, healthy, mature, and unmated Wistar female rats  were obtained from Cavens Laboratory Animal Co., Ltd. . Next, we used established methods to create painful models of menstruation  . Rats weWrithing scores were used to evaluate the writhing reaction of rats in response to pain, as described previously –18. The α, TNF-α, and IL-8 were then measured with specific ELISA kits, as described in the manufacturer's guidelines.Blood samples were collected from the retroorbital plexus of each rat following the administration of GGD. Serum levels of PGE2, PGF2The uteri of three rats per group were collected. The uterine tissue was first cut into smaller pieces and mixed with radioimmunoprecipitation assay (RIPA) buffer prior to three rounds of mechanical homogenization on ice (3 s per round). Samples were then centrifuged, and the supernatants were separately transferred into new microcentrifuge tubes for analysis. The bicinchoninic acid (BCA) assay was then used to determine the protein concentration in each supernatant. Proteins were then diluted with urea solution (8 M) followed by further incubation for 1 h at 37°C. Thereafter, the mixture was transferred into 10 K Microcon centrifugal filter units  and centrifuged to remove urea. Finally, the proteins were alkylated by iodoacetamide (55 mM) for 20 min at room temperature (in the dark), digested with sequence-grade modified trypsin , and lyophilized.μL), loaded into a trap column , and subsequently separated and loaded onto an analytical column  using a linear gradient of 5–38% formic acid (0.1%) for 120 min. Throughout the study, we used an electrospray voltage of 2 kV between the sprayer and the ion inlet of the mass spectrometer.Peptides were treated with formic acid was used to analyze the tandem mass spectra in relation to the UniProtKB database for Rattus norvegicus. The search parameters for the mass tolerances of the fragment and parent ions were 0.05 and 7 ppm, respectively. The fixed modification was carbamidomethylation, while the variable modifications were deamidation (NQ), oxidation (M), and acetylation (Protein N-term). Peptides were filtered with a 1% false discovery rate (FDR) and one unique. The relative abundance of peptides and proteins were compared using analysis of variance (ANOVA). The mean abundance of all peptides was normalized by the application of medians. A protein with a fold change > 1.5, and the presence of least 2 unique peptides with a P value <0.05, was considered to be differentially expressed protein (DEP).MaxQuant software (http://kobas.cbi.pku.edu.cn), Gene Ontology , and the Clusters of Orthologous Groups . We also used STRING (http://string-db.org/) to create an interaction network for the DEPs.Once identified, DEPs were then analyzed by reference to three key databases: Kyoto Encyclopedia of Genes and Genomes . β-Actin was used as a loading control. After washing three times with Tris Buffered saline Tween (TBST) buffer, the membranes were incubated for 1 h at room temperature with goat anti-rabbit antibody . Enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific Inc.) was then used to detect protein bands showing positive immunoreactivity. ImageJ software  was used to compare the relative optical densities of each band of interest.Proteins from the uteri of rats in each treatment group were extracted using RIPA lysis buffer . We then used the BCA assay to estimate the concentration of each protein extract. Proteins were then separated by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with nonfat milk (5%), and the PVDF membranes were incubated overnight at 4°C with the following antibodies: rabbit anti-P-JNK , rabbit anti-JNK , rabbit anti-P-ERK , rabbit anti-ERK , rabbit anti-P-P38 , rabbit anti-P38 , rabbit anti-HSP90AA , rabbit anti-HSP90AB , and anti-t-test. One-way ANOVA was used to make comparisons between multiple groups. Results are expressed as the mean ± standard deviation. Statistical significance was assumed at P < 0.05.SPSS version 19.0 software  was used for all statistical analyses. Differences between the two groups were analyzed by Student's P < 0.05; P < 0.05, The writhing scores of rats in the model group were significantly higher than those in the control group (α in each group of rats. As shown in P < 0.05) in the serum levels of PGE2 and PGF2α. However, the administration of GGD resulted in a remarkable reduction in the serum levels of PGE2 and PGF2α (P < 0.05). These findings suggested that GGD treatment could attenuate the oxytocin-induced writhing response.We then measured the serum levels of PGE2 and PGF2Label-free quantitative proteomics was used to investigate the protective mechanisms of GGD. A total of 1968 protein groups, 4273 proteins, 10352 peptides, and 9311 unique peptides were successfully identified by LC-MS/MS among the three groups. The details of 379 DEPs are shown as Venn diagrams in http://www.omicsbean.cn/, Gene for health, Shanghai, China) was used to analyze the DEPs with regards to pathways and biological processes (BP). GO annotation of the DEPs between the model group and the control group identified a total of 276 cell component (CC) terms, 337 molecular function (MF) terms, and 1579 BP terms (P < 0.05) are shown in Figures OmicsBean . However, the levels of these proteins were significantly reduced following the GGD administration. These findings were consistent with those derived from proteomic analysis. In addition, the serum levels of TNF-α and IL-8 were increased in the model group  . To the α, two naturally occurring PGs, are regarded as the most crucial pain factors in PD [α; these effects were suppressed by GGD, thus suggesting that GGD may inhibit oxytocin-induced uterine contraction by regulating the levels of PGE2 and PGF2α. It is well known that PD is associated with increased levels of PGF2α [The main bioactive compounds of GGD were standardized previously via the application of high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) . A previrs in PD –22; thesrs in PD –22. Our of PGF2α –22. Howeof PGF2α . Increasof PGF2α , 25. TheOur proteomic experiments identified a total of 379 DEPs. GO and KEGG analyses further revealed that the DEPs identified between the model group and the treatment group participated in various BPs and pathways, including cellular component biogenesis, protein binding, and metabolic pathways. These GO terms and pathways may play critical roles in the occurrence and development of PD. Several proteins were observed to recover after the administration of GGD. Protein-protein interaction (PPI) analysis further showed that these proteins were involved in signaling pathways related to oxytocin and estrogen and performed their functional roles collectively in specific networks.α and IL-8). These results indicated that GGD may ameliorate oxytocin signaling and the inflammatory response in PD by reducing the activities of MAP kinases.Previous work has shown that inflammation is involved in PD, and that inflammatory cytokines are produced when inflammation occurs –29. MAP HSP90, a highly abundant molecular chaperone, has been shown to participate in the maintenance of proteostasis , 37. HSPPueraria lobata, Ephedra, Radix Paeoniae Alba, and Ramulus Cinnamomi. Previous studies have reported that puerarin exerts an inhibitory effect on the production of NO, PGE2, and proinflammatory cytokines [Ephedra is also known to exert analgesic effects; the ephedrine and pseudoephedrine contained in Ephedra can activate the p38 MAPK pathway and inhibit the release of proinflammatory factors [Radix Paeoniae Alba exerted an analgesic effect in an animal model via anticholinergic receptors. In another study, Zhu et al. [Radix Paeoniae Alba inhibited cyclooxygenase, and that total glucosides from the paeony can play an anti-inflammatory role via the arachidonic acid metabolism. Pharmacological experiments have also demonstrated that Ramulus Cinnamomi exhibits antibacterial, anti-inflammatory, antiviral, antitumor, antipyretic, analgesic, antidiabetic, and antiplatelet aggregation effects. A previous study revealed that essential oil prepared from Ramulus Cinnamomi can exert anti-inflammatory effects by reducing the production of NO and PGE2 [The main active ingredients of GGD that show therapeutic effects against PD are considered to be ytokines , 45. Eph factors –48. In a factors  found thu et al.  reportedand PGE2 , 52.This study provides evidence to support the protective mechanisms of GGD against PD. GGD exhibited significant analgesic effects in the treatment of PD by regulating oxytocin and estrogen signaling pathways in a rat model of PD. Our data suggest that the mechanisms underlying the protective effects of GGD on PD include the downregulation of HSP90AB1 and the reduced phosphorylation of three MAPKs . Our data provide guidance and a useful foundation for investigating new treatments for PD.


To explore the correlation of flash dual source computed tomography perfusion imaging (CTPI) and regional lymph node metastasis of non-small cell lung cancer (NSCLC), and to evaluate the value of CT perfusion parameters in predicting regional lymph node metastasis of NSCLC.120 consecutive patients with NSCLC confirmed by postoperative histopathology were underwent flash dual source CT perfusion imaging in pre-operation. The CT perfusion parameters of NSCLC, such as blood flow (BF), blood volume (BV), mean transit time (MTT) and permeability (PMB) were obtained by the image post-processing. Then microvessel density (MVD), luminal vascular number (LVN), luminal vascular area (LVA) and luminal vascular perimeter (LVP) of NSCLC were counted by immunohistochemistry. These cases were divided into group A  and group B  according to their pathological results. The CT perfusion parameters and the microvessel parameters were contrastively analysed between the two groups. Receiver operating characteristic (ROC) curve was used to assess the diagnostic efficiency of CT perfusion parameters in predicting regional lymph node metastasis of NSCLC in pre-operation.P < 0.05), while BV, LVN, LVP and MVD were no significant difference (P > 0.05). Correlation analysis showed that BF was correlated with LVA and LVP (P < 0.05), while BV, MTT and PMB were not correlated with LVN, LVA and LVP (P > 0.05). All the perfusion parameters were not correlated with MVD. According to the ROC curve analysis, when BF < 85.16 ml/100 ml/min as a cutoff point to predict regional lymph node metastasis of NSCLC, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value were 60.8, 81.7, 71.5, 75.6 and 69.5% respectively.Group A presented significantly lower LVA, BF and higher MTT, PMB than Group B (Flash dual source CT perfusion imaging can non-invasively indicate the luminal vascular structure of tumor and BF can be used as one of the important indexes in predicting regional lymph node metastasis of NSCLC in pre-operation. Lung cancer has been the leading cause of cancer death, in which non-small-cell lung accounts for more than 80% of the total . So accuThis study has been approved by the ethics committee of Zunyi Medical University. And each patient or the patient’s family was fully informed and signed the informed consent before performing the pre-operative CT examination. From January 2015 to May 2019, 120 consecutive patients with NSCLC confirmed by histopathology were enrolled in the study according to the following criteria: (i) These cases successfully underwent CT perfusion imaging before surgical resection; (ii) No treatment before CT perfusion imaging, such as chemotherapy or radiotherapy; (iii) The time interval between CT perfusion imaging and surgery was less than 2 weeks; (iv) No serious cardiac, pulmonary or renal dysfunction [CT perfusion scan was performed by using SOMATOM Definition Flash dual source CT scanner  with dose reduction techniques including. CT scan involved two steps. First, an unenhanced CT scan was performed with following scan parameters: 100 kV, 100 mAs, 0.5 pitch, 512 × 512 mm matrixs and slice thickness was 8 mm. CT scanning covered the entire lung. Second, a perfusion CT scan which based on the plain scan was performed after injecting contrast medium (iohexol) 2–4 s in B20f smooth Eva image reconstruction method with following parameters: 80 kV, 120 mAs, 3 mm reconstruction slice thickness . The scanning range included the upper and lower 3.5 cm of lung tumor and the patient was breathless during the scan. 50 ml iohexol (300 mgI/ml) was injected via an antecubital fossa vein at a flow rate of 6 ml/second, followed by 20 ml of saline flush . In our First, transmitting perfusion imaging data to workstation . Then, we use the volume perfusion computed tomography (VPCT) body software to obtain blood flow (BF), blood volume (BV), mean transit time (MTT), permeability (PMB) of NSCLC. We usually choose the thoracic aorta as the reference vessel and set the region of interest (ROI). If there was no thoracic aorta at the lesion level, the carotid artery or brachiocephalic trunk will be selected as the reference vessel. And the maximum level without artifact interference was selected to delineate the ROI around the boundary (2–3 mm from the edge) of the tumor by hand, avoiding the necrosis area, calcification areas and blood vessels. Of 120 cases, 3 cases of necrosis and 1 case of calcification. We asked two similar level radiologists with more than 6 years of experience in interpreting CT perfusion imaging to do the experiments. Neither radiologists knew anything about the patient. We take the average of them as the final data. If the difference between their data is more than 10%, the perfusion data will be recalculated.After surgery, tumor tissue specimens were fixed in the inflated state by 10% buffered formalin, and embedded in paraffin. The tissue samples (3 μm paraffin slice) that came from the maximum section of tumors which diagnosed to be NSCLC by hematoxylin and eosin staining were immunohistochemically stained by using the PV 2-Step method  with CD34  monoclonal antibody according to the following steps: (i) removing paraffin of the tissue slices; (ii) the slices were incubated in 3% nonionic hydrogen peroxide for 10 min and then washed with phosphate-buffered saline (PBS) three times (2 min each time); (iii) addition of anti-human CD34 monoclonal antibody in a 1:50 dilution for 1 h at room temperature and then washed with PBS three times (2 min each time); (iv) addition of reagent 1(Polymer Helper) for 20 min at room temperature, then washed with PBS three times (2 min each time). Then addition of reagent 2 (Polyperoxidase- anti-mouse/ribbit IgG) as the method of reagent 1; (v) the slices were then stained with DAB solution and subsequently counterstained with hematoxylin; (vi) the slices were then rinsed with distilled water, dried and mounted. The tissue slices were stained with smooth muscle actin (SMA) in the same method as CD34 staining. The control group was set up with PBS solution instead of the first antibody respectively.2  was chosen randomly for the purposes of counting and measure MVD, LVN, LVP and LVA. MVD were counted in the chosen field at high magnification (× 200). Endothelial cell, cell cluster that was apparently separate from peripheral tissues and branch construct with discrete breaks which highlighted in CD34 staining wrere counted as microvessels. The final MVD value was obtained from the average MVD value in the above six fields. To be considered vascular, it is necessary to have a distinguishable lumen and one or more layers of pericytes and smooth muscle cells stained with smooth muscle actin. Software (image pro plus 6.0) was used for vascular measurements. If the slices exist following situations: (i) the pathological tissue slip off; (ii) there are air bubbles; (iii) uneven staining; (iv) the background is not clean because the non-specific staining, will be excluded. We asked two similar level pathologists with more than 15 years of experience to count each slice respectivel. If the difference between their data is more than 10%, the data will be recalculated. Then we take the average of the two or four results is the final histologic data.Each slice was scanned at low magnification (× 40 or × 100) to determine six “hot spot” areas where the number of microvessels was at maximum including four peripheral areas and two center areas. In each area, one field of 0.20 mmP < 0.05, the difference is considered to indicate a statistical significance. All data, including the perfusion parameters  and the microvessel parameters  were underwent consistency test within inter-observer and intra-observer. All continuous variables were underwent normality test. Normal distribution data were expressed as mean ± SD, and abnormal distribution data were represented as median ± interquartile range (IQR). The two-tailed Student t test or Wilcoxon test was used to compare the perfusion parameters and the microvessel parameters between the group A and the group B. Pearson or Spearman correlation analysis was used to investigate the relationships between the perfusion parameters and the microvessel parameters. The diagnostic efficiency of CT perfusion parameters in predicting regional lymph node metastasis of NSCLC was assessed by using receiver operating characteristic (ROC) curve .We using statistical software (SPSS 18.0) to perform all statistical analyses. When P < 0.05), but no statistically significant differences in age, gender, location, pathological type and lesion size (P > 0.05).Of these cases, group A includes 58 cases  and group B includes 62 cases . For pN-stage, there were 62, 23, 35 and 0 cases in pN0, 1, 2 and 3, respectively. For pT-stage, there were 51, 46, 23 and 0 cases in pT1, 2, 3 and 4, respectively. In group A, the primary tumor is from 1 cm to 3 cm in length and the short diameter of lymph node was less than or equal to 10 mm in 11 cases (25 lymph nodes), there were 31, 20, 7 in well-differentiated, moderately differentiated and poorly differentiated, respectively. While in group B, the primary tumor is from 1 cm to 4 cm in length and the short diameter of lymph node was greater than 10 mm in 8 cases (12 lymph nodes), there were 58, 1, 3 in well-differentiated, moderately differentiated and poorly differentiated, respectively  and CT perfusion parameters  were underwent consistency test within inter-observer and intra-observer. The Kappa value of the perfusion parameters within inter-observer and intra-observer is 0.86 and 0.81 respectively, and the Kappa value of the microvessel parameters within inter-observer and intra-observer is 0.85 and 0.82 respectively.P = 0.027, 0.006, 0.011 and 0.048, respectively). There were no significant difference in BV, LVN, LVP and MVD (P > 0.05) , while BV, MTT and PMB were not correlated with LVN, LVA and LVP (P > 0.05). All the perfusion parameters were not correlated with MVD (P > 0.05) . According to the ROC curve analysis, when BF < 85.16 ml/100 ml/min as a cutoff point to predict regional lymph node metastasis of NSCLC, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value were 60.8, 81.7, 71.5, 75.6 and 69.5% respectively  for BF was 0.746  is a good index marker for the diagnosis of metastatic lymph nodes, but there is no consensus on the optimal threshold of SUVmax, and some non-metastatic lymph nodes can also have high uptake of 18 F-FDG. it may also lead to false positive and false negative , 20. In CTPI can provide qualitative and quantitative hemodynamic information, and it can reflect noninvasively the angiogenesis of tumor , 21, 22.It was reported that CTPI plays an important role in evaluating lymph node metastasis of cancer in pre-operation , 24. ThiROC curve was used to evaluate the value of the dual source CT perfusion parameter BF in predicting regional lymph node metastasis of NSCLC. The result showed that BF was valuable in predicting regional lymph node metastasis of NSCLC in pre-operation. And the possibility of NSCLC with regional lymph node metastasis may be suggested when BF < 85.16 ml/100 ml/min. For these patients, the regional lymph node dissection should be performed more carefully, systematically and extensively to avoid missing the small metastatic lymph nodes.This study showed that the luminal vascular parameters were the important indexes for the evaluation of tumor angiogenesis and were related to the regional lymph node metastasis of NSCLC. Flash dual source CT perfusion imaging can non-invasively indicate the luminal vascular structure of tumor and BF can be used as one of the important indexes in predicting regional lymph node metastasis of NSCLC in pre-operation. So the dual source CT perfusion imaging can be an effective supplement of traditional morphological image in diagnosing regional lymph node metastasis of NSCLC.Small sample size, a scanner from a single vendor and single-center study are the main limitations of this study. Therefore, further work is to expand the sample size and conduct multi-center research to improve the value of the CT perfusion parameters in predicting regional lymph node metastasis of NSCLC.


Halteria grandinella, a cosmopolitan unicellular eukaryote, is composed almost entirely of gene-sized nanochromosomes with extremely short nongenic regions.How to achieve protein diversity by genome and transcriptome processing is essential for organismal complexity and adaptation. The present work identifies that the macronuclear genome of  Halteria grandinella, a cosmopolitan unicellular eukaryote, is composed almost entirely of gene-sized nanochromosomes with extremely short nongenic regions. This challenges our usual understanding of chromosomal structure and suggests the possibility of novel mechvanisms in transcriptional regulation. Comprehensive analysis of multiple data sets reveals that Halteria transcription dynamics are influenced by: (i) nonuniform nanochromosome copy numbers correlated with gene-expression level; (ii) dynamic alterations at both the DNA and RNA levels, including alternative internal eliminated sequence (IES) deletions during macronucleus formation and large-scale alternative splicing in transcript maturation; and (iii) extremely short 5′ and 3′ untranslated regions (UTRs) and universal TATA box-like motifs in the compact 5′ subtelomeric regions of most chromosomes. This study broadens the view of ciliate biology and the evolution of unicellular eukaryotes, and identifies Halteria as one of the most compact known eukaryotic genomes, indicating that complex cell structure does not require complex gene architecture.How to achieve protein diversity by genome and transcriptome processing is essential for organismal complexity and adaptation. The present work identifies that the macronuclear genome of  Ciliates are regarded as one of the most diverse and highly differentiated groups of unicellular eukaryotes, based on their nuclear dimorphism , complex epidermal ciliation, and strong swimming ability , 2. Thes3–9–Halteria grandinella is a highly specialized ciliate distributed worldwide and very common in all freshwater ecosystems , much research on morphology, taxonomy, ecology, and cell biology has been performed  with a 2,800-bp mean chromosome size   and usually only code for a single gene. Even the smallest chromosome in Oxytricha (430 bp) possesses a single well-positioned nucleosome  genome of  After removal of low-quality and nearly identical contigs, mitochondrial contigs , and contamination from bacterial genomes, the final assembly of the Halteria macronuclear genome is ∼64 Mb and includes 40,570 contigs. Only 13 out of 40,570 contigs have non-self matches that are ≥99% identical (matches that are ≥100 bp), indicating that the assembled genome is haploid. The haploid size of the Halteria MAC genome is similar to that of other ciliates with nanochromosomes, such as Oxytricha, Stylonychia, Euplotes, Nyctotherus, Urostyla, Paraurostyla, and Tetmemena, each of which has a 50 to 70 Mb MAC genome  of two-telomere capped chromosomes is just 2,485 bp, which is close to the length of a regular transcript (below). This length is much smaller than that found in Oxytricha trifallax , which has a similar well-assembled highly fragmented macronuclear genome (If we focus on the well-assembled chromosomes (2-telomere contigs), we see an assembly of tiny chromosomes. Including the length of telomeres (20 to 28 bp on each chromosome end), the r genome .Gene prediction and genome annotation. A total of 28,203 genes were predicted, 16,267 of which have functionally annotated orthologs in other species, with the remaining 11,936 predicted as hypothetical protein-coding genes without functional information. A total of 11,800 chromosomes contain at least one gene, most of them (77%) only encoding a single gene , which is 64,614 bp in length and also encoded in the longest chromosome  . Download Copyright © 2021 Zheng et al.2021Zheng et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Oxytricha, but we could not detect the ∼10-bp periodicity reported therein  composition of each nucleotide for the first 200 positions of all single-gene coding chromosomes. (A) All chromosomes. (B) Chromosomes with blast hits in the forward direction. (C) Chromosomes with blast hits in the reverse direction. Download Copyright © 2021 Zheng et al.2021Zheng et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Copy numbers of macronuclear chromosomes are correlated with expression level. By mapping the Illumina sequencing reads back to the genome assembly, we obtained an average sequencing coverage of contigs of 108×. At such high coverage, if the copy numbers of macronuclear chromosomes of Halteria are uniform, the coverage distribution should be approximately Gaussian in form, with deviations largely resulting from random errors in sequencing and assembly. However, the distribution of sequencing coverage of contigs, which is expected to reflect chromosomal copy numbers, is strongly skewed, much like the typical distribution of transcriptome sequencing (RNA-seq) reads coverage (reflecting wide-range expression levels) in most species  and Paramecium tetraurelia    . As showP value < 2.2e−16). Thus, chromosomes with higher copy numbers tend to have higher expression levels. For O. trifallax, which also has nanochromosomes and positively correlated copy number and expression level  database in 100 quantiles ranked from low to high by their copy numbers. Genes with the top 10% highest copy numbers have obviously higher mapping rate within the COG database . This is consistent with our observation that the distribution of minor-allele frequencies peaks at 0.5 , but thiariation .Halteria cells are partitioned unequally into daughter cells, gradually increasing deviations from the initial 1:1 ratio of allele pairs expected following the last sexual reproduction event. Such asexual chromosomal copy drift is expected to increase with the number of clonal cell divisions, explaining why clonal lab strains of many ciliates cannot be maintained for long periods   .Halteria is composed of polyploid nanochromosomes with nonuniform copy numbers. In support of the chromosomal-drift hypothesis, there is a positive correlation between the estimated allele frequencies and sequencing coverage  are eliminated  software that can detect alternative DNA deletion events in ciliates  DNA, as the latter would have rendered the nonexcised version (the default status of MIC) as the minor form.By mapping all genomic sequencing reads to the macronuclear genome assembly, we inferred 2,172 alternatively excised IESs with pointer-sequence structure using ADFinder . These alternatively excised IESs reflect rare and alternative DNA splicing events during the macronuclear genome development. The average length of these IESs is 101 bp . Chromosraurelia , in whic10.1128/mBio.01964-20.4FIG S4Halteria genome. Download FIG S4, TIF file, 0.5 MB.Length distribution of 2,172 alternatively excised IESs in the Copyright © 2021 Zheng et al.2021Zheng et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Among 2,172 cryptically excised IESs, only 370 (17%) are contained in coding regions, suggesting that cryptically excised IESs are enriched in flanking regions (noncoding regions) of nanochromosomes. The major nonexcised versions in coding regions are highly expressed, with the average RNA-seq coverage at 324×. More strikingly, their minor excised versions are also successfully transcribed, with 75% of them having successfully mapped RNA-seq reads .Paramecium species, cryptically excised IESs are not enriched in coding regions    are have lengths that are multiples of 3 (3n IESs) (67.8% for IESs inserted between codons) , much moaurelia) . For thoaurelia) ; therefoaurelia) , 53, 54.GCTCAAC, 3′: TCAACAG), although they are in different locations. The pointer-sequences of these IESs contain the 3′ boundary motif TCAACAG and can be represented by the motif “N(3–5)GGNTCAACAG” (where N represents A/T/G/C) , and generally have a length difference divisible by three (85.08%). Contig 8469 (a single-gene chromosome) is shown as an example . Here, 5A/T/G/C) . All lenA/T/G/C) . AlthougWidespread, low-frequency spliced introns. By mapping all RNA-seq reads to the genome assembly, we inferred 23,188 GT…AG type introns, 0.34 introns per gene on average. Compared with other eukaryotes, Halteria has relatively short introns, 59% of which are between 30 and 40 bp  and Stentor coeruleus (15 bp)  , 56, whi (15 bp) . The fir (15 bp) .Halteria has a slight deficit of introns with lengths that are multiples of 3 . These 3n introns represent 29.3% of the total, in contrast with 37.9% and 32.8% for 3n + 1 and 3n + 2 introns, respectively. This deficiency of 3n introns in Halteria is less extreme than that in Paramecium tetraurelia; only 18.7% of Paramecium introns are reported as 3n introns, while 42.3% and 39.0% are 3n + 1 and 3n + 2 introns, respectively (Paramecium introns with 3n length tend to cause premature translation termination (PTC) in the event of intron retention (59.2%)  , whereasHalteria revealed the positions of introns to be preferentially skewed toward 5′ ends (SRR578166) data sets of Oxytricha trifallax reveals a similar distribution of intron positions in the two species   overlap at least one other intron in 1,475 genome locations. Of these, 3,130 of 3,593 alternatively spliced introns share a donor (GT) or acceptor (AG) site with at least one other intron, and there is a strong preference for sharing the same donor (GT) rather than acceptor (AG) site.In contrast with the limited intron alternative splicing found in spliced) , 59, HalBy analyzing the length difference between pairs of introns sharing the same donor or acceptor site, we found that 66.84% of the additional sequence between two overlapped introns is divisible by three, which is much higher than the random expectation of 33.33%, and the proportion of 3n introns among total introns, which is 29.3%. This implies that the removal of alternatively spliced introns in this species tends to leave the original reading frame intact.Finally, we quantified the mapped RNA-seq reads for all mRNA isoforms to determine the major isoform (the most abundant spliced variant) in each intron location. On average, the frequency of the minor form is 7.7% of the major form. Again, although this large-scale alternative mRNA splicing, along with alternative IES deletion, may increase the proteomic complexity, it remains unclear whether this is adaptive, maladaptive, or effectively neutral , 44.Transcriptional initiation. After removing the telomeres, the 5′ subtelomeric regions of the coding strand of single-gene coding chromosomes, which are potential locations for transcription-start sites, were searched for potential regulatory sequence motifs using the MEME software , 0 ≤ L ≤ 30 (TSS located <30 bp downstream of the motif region), and L > 30 (TSS located >30 bp downstream of the motif)  revealed 88% of them to be <30 bp in length. According to the distance L = Pe motif) .Halteria, which is consistent with the high PTSS value for this group being potentially biased upward due to low expression, resulting in deficient read mapping, i.e., the PTSS value in the L > 30 group may not reflect real transcription start sites. Notably, a relatively small fraction of the genes in the L > 30 group have orthologs in other ciliates  (compared with the L < 0 and 0 ≤ L ≤ 30 groups), suggesting that the nonconserved genes tend to have lower expression levels  was selected for further analysis of the locations of the TSSs, TATA box-like motifs, and translation start codons. As shown in  to TSSs . In huma to TSSs . The extHalteria-specific genes may be in the evolutionary process of forming or losing a stable TATA box. Based on previous research of anomalous base compositions in subtelomeric regions of Oxytricha  and transcription (intron deletion). These alternative deletions may produce functional gene products, but may also simply reflect erroneous splicing. Finally, the analyses of transcriptional initiation/termination of nanochromosomes reveal conserved TATA box-like motifs and short untranslated regions (∼10 bp) in the 5′ subtelomeric regions, and extremely compact untranslated 3′ regions (∼49 bp). The mean UTR lengths of Halteria resemble those reported in Oxytricha trifallax , Qingdao, China, and was identified by its morphological features and small subunit ribosomal ribonucleic acid (SSU-rRNA) gene sequence. A single cell was picked, washed, and cultivated in cell culture flasks with filtered and autoclaved pond water. All downstream experiments, including DNA and RNA extractions, were based on clones produced by asexual reproduction of this clone-founding cell. Escherichia coli strain DH5α was applied as the food source. The ciliate culture for DNA and RNA extraction was cultivated for 7 days at 23°C.A E. coli and other bacteria. Cells were harvested by centrifugation at 300 × g for 5 min. DNA was extracted using phenol chloroform extraction followed by ethanol precipitation. RNA extraction was performed with the RNeasy Plus minikit  following the manufacturer’s instructions.Before the DNA and RNA extraction, cells were starved for 2 days and ampicillin was added to reduce contamination by The DNA library was constructed with NEBNext DNA library prep master mix set for Illumina  following the manufacturer’s instructions. High-throughput sequencing was performed on an Illumina Hiseq 2500 platform. The paired-end sequencing produced 20G of clean data after quality control and removing adapters (read length: 150 bp).The RNA libraries were generated using NEBNext Ultra RNA library prep kit for Illumina  following the manufacturer’s instructions. Four RNA-seq replicates were sequenced. High-throughput sequencing was performed on Illumina Hiseq 2500 platform and produced 10G of clean data for each RNA library after quality control and removing adapters (read length: 150 bp).Halteria telomere are considered high-quality and were retained without contamination filtering. Contigs without telomeres were filtered based on the GC content. Contigs with GC content higher than 60% were considered contamination and were removed. Contigs with no telomeres, low sequencing coverage (coverage < 2×), and lengths < 500 bp were considered low quality, and were removed.Raw sequencing reads were trimmed using Trimmomatic with the following parameter: “ILLUMINACLIP:adapter_path:2:30:10 LEADING:10 TRAILING:10 SLIDINGWINDOW:4:20” . The genHalteria and bacterial genome.Fifty thousand randomly selected bacterial genomes downloaded from the NCBI database were used as the target database. The filtered genome assembly was split into 426,052 150-bp-long fragments and aligned with the target database. No fragment was mapped to the bacterial database, indicating that this genome assembly does not contain chimeric contigs resulting from false recombination between The micronuclear (MIC) sequence contamination was estimated by checking the sequencing coverage of all sites using RSEM software . GenomicN50, and other genomic statistics . In this script, all matched genes in the Swiss-Prot database for each query  were identified and extracted by searching for the start and stop codons of the longest potential open reading frame. The output file of this script was used in the model training of AUGUSTUS software. Detailed functional information of each gene was obtained using a perl script “entrez_information_extract.pl” .The RNA-seq reads and genome contigs were aligned using Tophat2 to predict introns . Before de novo gene prediction was performed using AUGUSTUS (version 2.5.5) . In the file containing parameters, the conventional TAA and TAG codons were modified to encode glutamine instead of the translation stop codon, as in other hypotrichous ciliates (Oxytricha and Stylonchyia) (The n 2.5.5) . The AUGonchyia) . Becauseonchyia) . These tonchyia) . The preonchyia) .de novo predicted genes based on AUGUSTUS and predicted genes based on the Swiss-Prot database were combined to form the final result of gene predictions. Another perl script “combine_database_and_denovo_annotation.pl”  was designed to unite the genes predicted by the Swiss-Prot database and by AUGUSTUS. For the genes shared by both predictions, those predicted by the Swiss-Prot database were retained in the final prediction file. The alignment between all predicted genes and the COG (Clusters of Orthologous Genes) database was performed using blastx method with default parameters . For these internal-broken reads, each can simply signify a splicing event. The core code of ADFinder was attached in our source code list . The detailed description, purpose, and usage of ADFinder is shown in our GitHub webpage (https://github.com/weibozheng/ADFinder). The RNA and DNA sequencing coverage of each splicing site was determined by mapping reads with recombined segments produced by the following methods. The “excised” version (with the IES deleted) was inferred by the conjoining within genomic sequence of the 125-bp 5′ upstream and 125-bp 3′ downstream region of the IES. The “nonexcised” version was inferred in two ways according to the length of IES: (i) if the presumed IES length The software ADFinder was designed to filter the paired-end reads, align reads with genome assembly, and analyze the resulting alignment (unpublished software). This software is written in python and utilizes the bowtie2 software  in read alignments. The local mode of bowtie2 allows each read to align with the reference genome with a soft clip . In the output file of bowtie2 software, the CIGAR field of soft-clipped reads is shown in the format “110M40S” (“110 matches + 40 mismatches”) or “110S40M” (“110 mismatches + 40 matches”), suggesting that this read is partially mapped. This kind of partially mapped read is essential in identifying the splicing event in the reference genome. Then, soft-clipped reads in alignments were used to generate the splice sites. If one read was mapped as a soft-clipped read at two or more different genomic locations, all matching records of this single read were analyzed. For example, if a read was matched to a contig at the x10.1128/mBio.01964-20.6FIG S6FIG S6, TIF file, 0.4 MB.Schematic plot of spliced/nonspliced versions of a genomic location containing an alternative IES splicing event. Download Copyright © 2021 Zheng et al.2021Zheng et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Halteria grandinella and two other ciliates  was conducted by blastx using protein identity > 30% and overlap region > 60% as criteria.The motif searching software MEME was used for searching the motifs in 400-bp 5′ subtelomeric regions . Bowtie2RRYP01000000, genomic reads: SRX5087334, RNA-seq reads: SRX5097405, SRX5097406, SRX5097407, and SRX5097408). All source codes in our research are available in this GitHub webpage: https://github.com/weibozheng/Halteria_genome_research.The final genome assembly, genomic reads, and RNA-seq reads have been deposited in GenBank (genome assembly:


Circulating IgA anti-citrullinated protein antibodies (ACPA) associate with more active disease, but a previous study implied that salivary IgA ACPA is related to a less severe disease. Therefore, we aimed to characterize the IgA ACPA response in the saliva and serum in relation to clinical picture and risk factors among patients with rheumatoid arthritis (RA).n = 196) and healthy blood donors (n = 101), included in the cross-sectional study “Secretory ACPA in Rheumatoid Arthritis” (SARA), were analyzed for ACPA of IgA isotype, and for subclasses IgA1 and IgA2 ACPA in paired saliva and serum samples using modified enzyme-linked immunosorbent assays (ELISA) targeting reactivity to a cyclic citrullinated peptide (anti-CCP). Cutoff levels for positive tests were set at the 99th percentile for blood donors. Antibody levels were related to clinical characteristics, radiographic damage, smoking habits, and carriage of HLA-DRB1/shared epitope (SE).RA patients (r = 0.455). The presence of salivary IgA ACPA was associated with a higher erythrocyte sedimentation rate (ESR), 28-joint disease activity score, tender joint count, and patient global assessment at the time of sampling. None of the antibodies was associated with smoking, SE, or radiographic damage.IgA ACPA in the saliva was found in 12% of RA patients, IgA1 occurred in 10%, and IgA2 in 9%. In serum, IgA ACPA was found in 45% of the patients, IgA1 in 44%, and IgA2 in 39%. Levels of IgA ACPA in the saliva correlated significantly with serum levels of IgA (Salivary IgA ACPAs were detected in a subset of RA patients in association with higher disease activity. This suggests that mucosal ACPA responses in the oral cavity may contribute to disease-promoting processes in RA. Mucosal surfaces, rather than the joints, are proposed as sites of initial triggering events in rheumatoid arthritis (RA) development, particularly in anti-citrulline protein antibody (ACPA)-positive disease , 2. ACPAIgA antibodies play important roles at mucosal surfaces, capable of both pro- and anti-inflammatory effects, e.g., opsonizing pathogens, neutralizing toxins, and inhibiting microbial adhesion to epithelia , 8. IgA In serum, monomeric IgA dominates, whereas in secretions, IgA is mainly dimeric. Dimeric IgA is produced by subepithelial plasma cells, and during the trans-epithelial transportation into the lumen, a protein chain, the secretory component (SC), is attached to form secretory IgA (SIgA) –10. SIgAPorphyromonas gingivalis  which has the ability to citrullinate peptides in the oral cavity [P. gingivalis have been demonstrated to associate with ACPA in RA patients [P. gingivalis has been shown to induce arthritis in rats [Circulating IgA ACPA is found in 30–50% of RA patients and has been shown to associate with cigarette smoking as well as a more severe disease , 20. In patients , and ora in rats . IgA sub in rats , where R in rats .In this study, we aimed to investigate salivary and circulating IgA ACPA antibodies, including subclasses, in relation to disease characteristics and risk factors in RA patients.n = 101) were recruited as controls from the local blood donor center and referred to the Rheumatology Clinic for blood and saliva sampling. The ethics review board in Uppsala, Sweden, approved of the study, and all participants signed a written informed consent . Participants were required to provide at least 0.5 mL of the saliva during the 10-min sampling time; otherwise, they were excluded from the study.One hundred ninety-six patients with established RA from the County of Dalarna, Sweden, were included in the cross-sectional, observational, “Secretory antibodies in Rheumatoid Arthritis” (SARA) study with enrolment 2012–2013. RA patients were randomly selected among planned follow-up visits at the Rheumatology Clinic, Falun Hospital, Sweden. Healthy blood donors  enzyme-linked immunoassays (ELISA) tests  were modified to analyze IgA, IgA1, and IgA2 ACPA in the saliva and serum, as well as for serum SC and IgG ACPA. All samples were analyzed in duplicate, and reanalysis was carried out if the coefficient of variation between the duplicate samples was > 20%. The cutoff levels for a positive test of saliva ACPA  were set at the 99th percentile among saliva samples from blood donors, and cutoff levels for serum ACPA  were set at the 99th percentile in serum among blood donors.g at 4 °C for 10 min) to remove non-soluble material. The supernatant was diluted to a final concentration of 1:20. The secondary antibody in the salivary IgA ACPA assay  was diluted 1:200. Secondary antibodies for the subclass analyses  were diluted 1:300. Incubation and washing were carried out according to the manufacturer’s instructions. Absorbance was read at 450 nm .The saliva was thawed at room temperature and centrifuged . Anti-CCP and anti-CAP analyses were performed in parallel. Anti-CAP background levels were subtracted from anti-CCP values -measurements for anti-CCP and anti-CAP), and the cutoff values for a positive test were set to 0.78 for IgA ACPA, 0.70 for IgA1 ACPA, and 0.39 for IgA2 ACPA, corresponding to the 99th percentile of the 101 healthy blood donors. Total IgA in the saliva was analyzed with enzyme immunoassay tests from the IBL International .Serum samples were analyzed for IgG ACPA according to the manufacturer’s instruction (Euro Diagnostica AB), with cutoff set at 25 U/mL. Serum IgA and SC ACPA analyses were performed as previously described . In brieFor IgA1 and IgA2 ACPA analyses, serum was diluted 1:50 and the subclasses were detected using anti-IgA1 and anti-IgA2 polyclonal mouse IgG anti-human Fc-region-specific antibodies, conjugated with HRP . Secondary antibodies were diluted 1:500 in the IgA1 assay and 1:400 in the IgA2 assay. Seven-step serial dilutions of high-leveled serum samples were used as a calibrator, and the results were expressed as AU/mL. Incubation and washing were carried out as instructed by the manufacturer. Absorbance was read at 450 nm . Cutoff limits for IgA1 ACPA were set to 27 AU/mL and for IgA2 ACPA 179 AU/mL.Total IgA in serum was analyzed with a PEG-enhanced immunoturbidimetric method, using Siemens Atellica CH930 .At inclusion, the participants answered a questionnaire regarding smoking habits . At the The prevalence of radiographic erosions was assessed from written reports by experienced radiologists in routine clinical care between 1982 and 2013. Results were dichotomized into having erosions or not.Genotyping of HLA-DRB1 was performed by Sanger sequencing . Shared epitope (SE) was defined as HLA-DRB1*01, *0401, *0404, *0405, *0408, *0409, *0410, *0413, *0416, *0419, *0421, or *10.p values < 0.05 were considered significant. Antibody test results below the reference curve were given a value corresponding to half of the lowest detected value among patients and controls. Regarding saliva samples, negative delta values  were set to zero.Statistical analyses were performed using SPSS v. 26.0 , and two-tailed U test was used to relate antibody status in the serum and saliva versus clinical variables, inflammatory markers, HAQ, and levels of other ACPAs. Spearman’s rho correlation coefficient (r) was used to test correlations between antibody levels in the saliva and serum. Linear regression analysis was used to evaluate associations between disease activity and levels of salivary IgA ACPA adjusting for levels of serum IgA ACPA, disease duration, and treatment. By including 196 RA patients of which 12% tested positive for IgA ACPA in the saliva, we achieved 80% power at alpha = 0.05 to detect a difference in DAS28 of 0.9 units between patients positive vs. negative for IgA ACPA in saliva.As IgG ACPA has been known to associate with poorer prognosis , compariThe 196 RA patients included in the SARA study had a mean disease duration of 12.2 years and 80% were women. Additional baseline characteristics of patients and controls are shown in Table In the saliva samples, 24 (12%) patients were positive for IgA ACPA, 19 (10%) for IgA1, and 18 (9%) for IgA2. Six saliva samples were positive for all tested IgA ACPA in the saliva (IgA+/IgA1+/IgA2+), 8 were positive for both IgA ACPA subclasses (IgA1+/IgA2+), 11 were positive for only IgA1 (IgA1+/IgA2−), and 10 were positive for only IgA2 (IgA1−/IgA2+) . IgA subclasses as well as SC ACPA occurred predominantly in the IgG ACPA-positive subset of the patients. IgG ACPA co-occurred in 87 out of 88 (99%) IgA ACPA-positive patients, 85 out of 86 (99%) IgA1 ACPA-positive patients, 73 out of 76 (96%) IgA2 ACPA-positive patients, and all SC ACPA-positive patients (100%). Twenty-eight patients were positive for all tested ACPAs in serum . Sixty were positive for both IgA ACPA subclasses (IgA1+/IgA2+), 26 positive for only IgA1 (IgA1+/IgA2−), 16 positive for only IgA2 (IgA1−/IgA2+) , IgA ACPA in 88 (45%), and SC ACPA in 42 (21%) RA patient sera. Serum IgA1 ACPA was detected in 86 (44%) of the patients and in 76 (39%) for IgA2 , IgA1 (r = 0.434), and weakly to the other serum ACPA isotypes; IgA2 (r = 0.277), SC ACPA (r = 0.29), and IgG (r = 0.342), all at the p < 0.001 level  as described in the “Comparisons of disease activity measures were restricted to the serum IgG ACPA-positive patient subset (p = 0.031), DAS28 , and tender joint count (TJC)  compared to patients negative for IgA ACPA in the saliva. Also, patients positive for IgA ACPA in the saliva had higher HAQ  and patient global assessment (PGA)   , and when adjusting for levels of serum IgA ACPA, the association was borderline significant (p = 0.071). DAS28 was associated with levels of salivary IgA ACPA also when adjusting for disease duration and treatment with biologic drugs and/or glucocorticoids at the time of saliva sampling (p = 0.021). When analyzing the impact of the secretory rate of the saliva (mL/min), there was no correlation between levels of salivary IgA ACPA and secretory rate .In linear regression analysis, salivary ACPA levels are associated with DAS28 (p = 0.005) and PGA  than patients negative for IgA ACPA. Similar findings were obtained regarding IgA1 and IgA2 ACPA , C-reactive protein (CRP) , DAS28 , and HAQ   after diagnosis. The presence of radiographic erosions did not differ significantly between groups according to saliva or serum IgA, IgA1, or IgA2 ACPA status (Supplementary Table p = 0.006) while “current smoking” was not. SE was detected in 163 of 196 (83%) of the RA patients and in 47 of 101 (47%) of controls (p < 0.001).Smoking habits and carriage of SE in patients and controls are shown in Table None of the salivary ACPAs was associated with smoking status (data not shown) or pack-years Fig. b.Fig. 4p < 0.001). When only IgG ACPA-positive patients were included in the analyses, no difference in SE carriage was detected according to serum status of IgA, IgA1, IgA2, or SC ACPA  or SC ACPA Fig. a. HoweveCPA Fig. c.To our knowledge, this is the largest study to date addressing salivary ACPAs in RA. We characterized the IgA ACPA and IgA ACPA subclass response in paired saliva and serum samples and investigated possible clinical implications. Our main findings are that IgA ACPA responses in the oral cavity as well as in the circulation associate with a more clinically active disease.In our previous pilot study, IgA ACPA in the saliva occurred in 22% of RA patients and was associated with signs of a milder disease . The preIn serum, IgA1 ACPA was detected in 44% and IgA2 ACPA in 39% of the patients. A previous study on circulating RF IgA subclasses by Otten et al. found circulating IgA1 RF (73%) to be twice as common as IgA2 RF (36%) in RA . SpeculaCirculating SC ACPA has in previous studies been found to associate with increased ESR and CRP levels in early RA , 18. AlsCigarette smoking, causing local irritation of respiratory mucosa, is a possible inducer of ACPA production in RA  and assoThe strengths of this study are the large number of included patients and the paired saliva and serum samples. A limitation is that the radiographic outcome was based on written reports from radiologists, but not subjected to formal scoring. This approach obviously reduces the level of detail, but we still regard that clinically relevant erosive disease could be identified in this way. Also, the cross-sectional design introduces the risk that treatment attenuated possible clinical differences and possibly influenced antibody levels. Finally, the lower mean age among controls may have influenced IgA cutoff levels, as it may associate with age in non-RA patients .For future directions, longitudinal studies are preferred to investigate the possible predictive value of IgA ACPAs in the saliva and serum.This large cross-sectional study confirms the presence of salivary IgA ACPA in a small subset of RA patients, in association with a higher disease activity and functional impairment. This suggests that mucosal ACPA responses in the oral cavity may contribute to disease-promoting processes in RA.Additional file 1 : Supplementary Table 1. Radiographic joint erosions versus ACPA isotypes. Abbreviations: ACPA anti-cyclic citrullinated peptides, SC secretory component containing.