The present study indicated that excessive energy enhanced fat deposition by upregulating expression of lipogenic genes in the longissimus thoracis of a Chinese fat-type pig. In addition, impairment in meat quality resulted in reduced meat tenderness and increased cross-sectional area of muscle fiber aroused by promoting glycolytic muscle fibers differentiation in muscles. These results provided a new perspective on the energy needs of Ningxiang pigs.p < 0.01), while nothing significantly changed by RE except FCR increasing (p < 0.01). EE increased the content of IMF and triglycerides (TG) (p < 0.05), L*24h and b*45min (p < 0.01), while decreasing cooking loss and meat tenderness in longissimus thoracis (LT) (p < 0.05). b*24h was significantly increased with the increase of energy level (p < 0.01). Meanwhile, EE increased the cross-sectional area (CSA) of muscle fiber and the mRNA expression of myosin heavy chain (MyHC) IIb, acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), and adipocyte fatty-acid binding protein (FABP4) (p < 0.05). In addition, throughout: A diet supplemented with excessive energy promoted IMF deposition by positively changing lipogenic potential while decreasing tenderness by increasing glycolytic muscle fibers, which together affected meat quality. In terms of growth performance and meat quality, the present study suggests that the low-energy diet is suitable for finishing Ningxiang pigs.This study investigated the effects of excessive or restrictive energy on growth performance, meat quality, intramuscular fat (IMF) deposition, and related gene expression in finishing Ningxiang pigs. A total of 36 Ningxiang pigs (43.26 ± 3.21 kg) were randomly assigned to three treatments (6 pens of 2 piglets per treatment) and fed by one of three dietary treatments until the pigs of each treatment weighed approximately 75 kg equally. The three treatments were control diet , excessive energy diet , and restrictive energy diet . Results showed that EE improved average daily gain (ADG) and feed conversion ratio (FCR) ( In animals, adipogenesis firstly occurs in visceral fat, followed by subcutaneous and intermuscular, and then is deposited in intramuscular fat last . It is wThe Ningxiang pig, as a fat-type local pig species in China, is famous for its delicious taste, which has more generous intramuscular fat (IMF), lower lightness, and higher redness than meat from the improved commercial pig . DistincFurthermore, dietary energy plays a bidirectional role in regulating the yield and quality of pork. A clear knowledge of the relationships between dietary digestible energy (DE) density and meat quality is necessary in commercial practice. However, previous studies about dietary energy concentration are mainly focused on the growth performance of swine, especially for fat-type pig species, including Ningxiang pig. In addition, the effect mechanism of energy on meat quality and fat metabolism in fat-type pig species receives much less attention at present. Given the foregoing, the Ningxiang pig was used as an animal model to investigate its growth and efficiency, as well as meat quality response to the extreme energy level at the optimal slaughter weight ,13. In aThis experiment was conducted on a commercial research pig farm  and was approved by the Animal Care and Use Committee of the Institute of Hunan Agricultural University. A total of 36 (43.26 ± 3.21 kg) Ningxiang pigs  were randomly assigned to 1 of 3 treatments (6 pens of 2 piglets per treatment). The 3 diets were control diet , excessive energy diet , and restrictive energy diet . All diets were formulated to meet the recommendations of the Chinese National Feeding Standard Type 2 for fatty growing pigs (2004) and contained similar contents of crude protein (CP) and amino acids, as shown in 3) were excised and put in 4% paraformaldehyde in PBS (pH 7.3) for further morphological detection. One pig with close to average weight was selected from each pen and fasted for 12 h. Pigs were slaughtered by standard commercial procedures. Samples of longissimus thoracis (LT) were immediately collected from the right side of the carcass, some stored at 4 °C for meat quality evaluation and others frozen at −20 °C for muscle chemical analysis. LT samples for RNA extraction were taken and treated by liquid nitrogen, and then stored at −80 °C for further analysis. Fresh samples of LT  were determined in triplicate at 45 min and 24 h by reflectance spectrophotometer . Water-holding capacity was tested using drip loss. Briefly, a 2 cm-thick LT sample was weighed. After being hung in a sealed plastic container for 24 h at 4 °C, the samples were taken out and weighed again. Drip loss was expressed as the difference (%) from the initial sample weight, based on Honikel (1998) . The weiTotal moisture, CP and IMF of LT Samples were analyzed referring to the Association of Analytical Chemists methods (AOAC 2000) . TriglycLT muscle samples were cut perpendicularly to the direction of the myofibers in cryostat (Leica CM1850) and then stained with classic hematoxylin and eosin staining. The sections were magnified 400x under light microscopy , and 8 views were captured in each section. The total fiber number (TFN) was measured by the Image-Pro Plus 4.5 software . The cross-sectional area (CSA) of muscle fiber in LT was estimated by extrapolation from the number of fibers counted over the selected fields, and the total fiber number (TFN).β-actin and GAPDH were chosen as the endogenous control gene. The PCR cycling condition was as followed: 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 60 s, and 1 cycle at 72 °C for 30 s. The relative expression was expressed as a ratio of the target gene to the control gene according to the 2 −(∆∆Ct) method, as described previously [Total RNA was extracted from samples with TRIZOL reagent ; the purity and integrity of RNA were detected by Nanodrop  and electrophoresis in 1% agarose gel, respectively. Approximately 1.0μg of total RNA was reverse-transcribed with RT Reagents . The primers were designed via Primer 5.0 software, as shown in eviously . 0.1 g freeze-dried and crushed muscle samples, mixed with 10 mL 6 M hydrochloric acid, were hydrolyzed at 110 °C for 22 h. The hydrolyzed mix was filtered to a constant volume, evaporated, diluted, and filtered with a 0.22 μm filter membrane, and then analyzed by the high-speed amino acid analyzer . p < 0.05.All data were performed using the one-way analysis of variance (ANOVA) to test homogeneity of variances via Levene’s test and followed with Duncan’s method . Results were expressed as the mean ± SEM. The level of statistical significance was set at p > 0.05; p > 0.05). The average daily gain (ADG) of the EE group was higher than that of the other two groups (p < 0.01). In addition, the feed conversion ratio (FCR) was significantly decreased with the increase of energy level (p < 0.01).The pigs in different groups were slaughtered at a similar finishing weight according to the experimental plan (p > 0.05). However, excessive energy significantly increased the contents of IMF and TG in LT , while the shear force of the EE group was significantly increased compared with the other two groups (p < 0.01). L*24h of the EE group was higher than that of the other two groups (p < 0.01). b*45min of the RE group was markedly lower than that of the other two groups (p < 0.01). b*24h was significantly increased with the increase of energy level (p < 0.01).p < 0.05). However, there were no significant differences in TFN among the three groups . acetyl CoA carboxylase (ACC) mRNA expression was upregulated in both the EE and RE group (p < 0.05) while there was no difference in the mRNA expression of PPARγ among the three groups (p > 0.05).The mRNA expression of p < 0.01), but increased Cysteine (Cys) content  content , tenderness, flavor, and IMF. In general, color indicates its primary acceptability to consumers, while tenderness is rated as the most critical palatability trait for cooked meat, followed by flavor and juiciness . High IMIn the present study, both reduced cooking loss and enhanced IMF content of the EE group were observed, which was consistent with the result of Lebret et al. 2008)  and Hocq008  and As previously stated, increased IMF content by excessive energy failed to contribute to meat tenderness, and there may be other factors also influencing shear force, such as muscle fiber condition. Morphology traits such as TFN, CSA, and FTC are major determinants of meat quality, especially tenderness ,45. It iMyHC IIb mRNA expression in LT was upregulated in the EE group, indicating that excessive energy could induce a muscle fiber transition toward more glycolytic muscle fibers. Consistently, Solomon and Lynch (1988) [CSA is mainly related to the type IIb fibers as a consequence of the abundance of type IIb fibers in LT . There ah (1988)  found thh (1988) , when thACC and FAS were assessed and found to be upregulated. Under the action of ACC, Acetyl-CoA is carboxylated to malonyl-CoA, a two-carbon unit for the synthesis of long-chain fatty acids [IMF is the amount of fat deposited inside the muscle, influencing the energy metabolism of skeletal muscle, as well as meat quality . TG, thety acids . FAS catty acids , which wty acids . In the ty acids . Considety acids . Howeverty acids . The extty acids . Accordity acids , the incFABP4 in the EE group may explain higher IMF content due to the efficient transport of fatty acids towards lipid synthesis. Alternatively, the higher expression of FABP4 mRNA will deliver more fatty acid to mitochondrial oxidation [FABP4 abundance, and IMF content in the EE group, were consistent with the fact that glycolytic muscle fibers were less oxidative and used fewer lipids as fuel substrate than other fiber types. Collectively, these observations of the present study suggested that lipid anabolic pathways, intracellular trafficking of fatty acids supported by FABP, and myofiber energetic metabolism may contribute an explanation of IMF content increasing caused by excessive energy.FABP4, considered candidate genes for IMF deposition ,68, is lxidation ,71. CoinFinally, hydrolyzed amino acids in LT were determined to evaluate the nutritional value of meat from Ningxiang pigs. A food protein with high nutritional value should contain comprehensive and well-proportioned essential amino acids (EAA), and it is best to be close to or consistent with the nutritional needs of the human body thus that EAAs will be absorbed thoroughly. These findings in all groups in this study fit the FAO/WHO/UNU   essentiaRE is comparable to CON in terms of growth performance and meat quality of Ningxiang pigs, suggesting the acceptability to lower dietary energy in Ningxiang pigs. Excessive energy led to the growth promotion of finishing Ningxiang pigs but tended to affect its meat quality negatively. Excessive energy in this study promoted IMF deposition by changing lipogenic potential to affect meat quality while causing a decrease in tenderness by increasing CSA and glycolytic muscle fibers, in particular, MyHC IIb. In addition, excessive energy had a greater impact on meat quality and IMF than restrictive energy, or rather, a more negative impact. The trial of extreme energy levels on the IMF deposition and myofiber characteristics may clarify the functional role of energy in the regulation of meat quality and provide a new perspective for the energy needs of Ningxiang pigs.


This study aimed at evaluating the association between the speed of nuclear factor-kappa B  of which presented with ≥grade 2 late fibrosis. There was a significant association between the speed of NF-κB activation in the PBMCs and an increased risk of developing ≥grade 2 late fibrosis in these patients (P = 0.004). Subgroup analysis suggested that this finding was independent of the known clinical characteristics.  The maximum nuclear p65 ratio was observed at 1 hour after ex vivo irradiation in the PBMCs from the healthy outpatients. The speed of NF-κB activation might be a potential predictor of late toxicity in cancer patients after radiotherapy. Prospective studies are needed for validation. The speed of NF- As a major treatment, radiotherapy is delivered for more than 50% of cancer patients at some time during the course of their disease . UnavoidκB) is a convergence of these pathways [κB is homodimers or heterodimers of the Rel family members, including NF-κB1 (p50), NF-κB2 (p52), c-Rel, RelA (p65), and RelB [κB is the heterodimer of p65-50 [κB is mainly cytoplasmically localized. Upon activation, NF-κB migrates into the nucleus, resulting in transcription of target genes [κB, and nuclear p65 expression is feasible to represent the degree of activation of NF-κB signalling pathway.In response to ionizing radiation, a number of signal transduction pathways are activated, including apoptosis, immune response, inflammation, differentiation, proliferation, and cell survival. Nuclear factor-kappa B  can be quantified, and the association with inflammation and cytokines has been investigated –20. SkinBetween January 2017 and July 2019, patients with HNSCC under regular follow-up care were recruited. The eligibility criteria were as follows: patients must have received curative radiotherapy, curative radiotherapy with concurrent chemotherapy, or curative surgery plus adjuvant radiotherapy with or without concurrent chemotherapy, for at least 2 years; patients were recurrence free at the time of recruitment. Data on the tumor and its treatments were collected from patient records, and personal information was gathered by self-administered questionnaires. All participants were required free from severe diabetes mellitus, uncontrollable hypertension, and other severe diseases. Controls were randomly selected from the healthy outpatients aged between 18 and 75 in the same consulting room during the same period, and those with history of cancers were not eligible.Late radiation-induced skin and subcutaneous fibrosis was measured between January 2017 and July 2019 on outpatient follow-up. Fibrosis was graded according to the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC) system  by 2 onc6 to 30 × 106 cells.The PBMCs were separated following the procedure as previously reported . Ten mL μg/ml; cat. no. ab24170; Abcam, USA) and to histone H3  [Nuclear proteins were extracted from PBMC cells using nucleoprotein kit  following the manufacturer's instructions. The protein concentration was determined by BCA protein assay kit . Any contamination of nuclear proteins in cytoplasmic fractions and vice versa was monitored by Western blot with antibodies to lysosomal-associated membrane protein 1 (LAMP1)  .Cell lysates were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membrane. The membranes were blocked with TBST buffer containing 5% powered milk at room temperature, and then were probed overnight at 4°C with primary anti-p65 . The membranes were then incubated with horseradish peroxidase-linked secondary antibodies  and were developed using the ECL chemiluminescent reagents . Next, each blot was stripped, and the membrane was reprobed with a primary antihistone H3 antibody.Blot images were captured using a chemiScope imaging system . Band densitometry was obtained using Image J . For eacWe used Western blot analysis to assess nuclear p65 expression in the PBMCs from the controls from 0 to 12 hours after 2-Gy ex vivo irradiation. We aimed at identifying the time point when maximum nuclear p65 expression occurred in the PBMCs after ex vivo irradiation.v/v) paraformaldehyde in PBS for 20 min at room temperature. Next, they were cytospin on microscopy slides for 10 minutes at 3000 rpm, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Next, the cells were blocked in PBS/5% w/v BSA, pH 7.4, for 20 minutes, and then incubated for 1 hr at room temperature with primary anti-p65  antibody. After washed with PBS, the cells were incubated with FITC-labeled secondary antibody  for 45 min at room temperature. Last, cells were stained with DAPI  for 10 min. Coverslips were mounted in Mowiol mounting medium. Image capturing was performed with an inverted fluorescence microscope  with 40x objective and a digital camera .The immunofluorescence staining was prepared following the procedure as previously reported , 25. BriThe automated image analysis was coded by Matlab  as indicated in vs. ≥65 years), sex (men vs. women), smoking history (never vs. ever), alcohol consumption (never vs. ever), TNM stage (I-II vs. III-IVa-b), chemotherapy treatment (yes vs. no), and surgery (yes vs. no). All statistical tests were two-sided. A P value of <0.05 was used for a statistical significance.All statistical analyses were performed using R, version 3.5.2. Chi-squared or Fisher's exact test was used to test differences in covariates in patients with ≥grade 2 and those with <grade 2 late skin and subcutaneous fibrosis. The Wilcoxon rank-sum test was used to compare the p65 expression difference between patients with ≥grade 2 and those with <grade 2 late skin and subcutaneous fibrosis. The difference was also assessed by using a multiple linear regression model adjusted for age, sex, smoking history, alcohol consumption, TNM stage, chemotherapy treatment, and surgery. Subgroup analyses were conducted according to clinical characteristics, including age (<65 years n = 200), 65 (32.50%) patients presented with ≥grade 2 late fibrosis  and 5 females (42%), and median age was 60 years . A total of 211 patients were recruited, of which 11 patients were excluded from subsequent analyses: PBMCs isolation failed in 2 patients; protein extraction was inadequate in 4 patients; and Western blot failed in 5 patients. Among the remaining patients (fibrosis . At the P = 0.020), alcohol consumption (P = 0.041), and tumor stage at diagnosis (P = 0.026).n = 3) showed a maximum nuclear p65 expression at 1 hour. This experiment was then repeated 3 times, and consistent results were obtained. Representative Western blot bands and distribution of the nuclear p65 expression ratio before and after irradiation are shown in Figures The nuclear p65 expression in the PBMCs from 3 controls was initially measured from 0 to 12 hours in a time interval of 2 hours after 2-Gy ex vivo irradiation. Time-related patterns of the nuclear p65 expression were similar among these samples. Almost all samples showed a maximum nuclear p65 expression at 2 hours after irradiation. We then repeated the measurement from 0 to 2 hours in a time interval of 0.5 hours, and all samples , a significant increase of p65 in the nuclei was evident. Shown are representative images .P = 0.010) by the Wilcoxon rank-sum test between patients with ≥2 grade and those with <2 grade late skin and subcutaneous fibrosis (P = 0.011) remained by multiple linear regression model adjusted for age, sex, smoking history, alcohol consumption, TNM stage, chemotherapy treatment, and surgery.Automated image analysis showed that the median p65 nuclear: cytoplasmic ratio was 0.80 (range: 0.35–1.38) for <2 grade late fibrosis, and was 0.92 (range: 0.36–1.91) for ≥2 grade late fibrosis. The nuclear p65 expression ratio was statistically different  for <2 grade late fibrosis and was 2.51 (range: 1.25–3.76) for ≥2 grade late fibrosis. Shown are representative images . The nucκB activation with the development of radiation-induced late toxicity in patients with HNSCC treated with radiotherapy. Our findings suggested that the risk of radiation-induced late skin and subcutaneous fibrosis was significantly increased in patients who had a higher speed of NF-κB nuclear accumulation in their PBMCs after ex vivo irradiation. This finding appeared to be independent of age, sex, primary site, tumor stage, surgery, or chemotherapy.In this study, we analyzed the association of NF-κB activation and late tissue toxicity in cancer patients after radiotherapy. It is of great convenience for oncologists to predict the occurrence and severity of late toxicity beforehand by an easy essay. PBMCs can be easily obtained from the peripheral blood of patients. The NF-κB activation in the PBMCs after irradiation is apparently an early response, and PBMCs were also considered as an early responding tissue [κB activation and late fibrosis. Actually, this was analogous to the radiosensitivity of patients with genetic syndromes, such as ataxia-telangiectasia and Nijmegen breakage syndromes [κB activation is associated with radiation-induced toxicity. Indeed, continuous NF-κB activation was associated with radiation-induced toxicity in rat models [κB activation correlates with the lasting time of NF-κB activation leading to late toxicity. To minimize the potential source of confusion due to long time culture of PBMCs, we did not assess the association of late toxicity with the lasting time of the NF-κB activation in the present work. The relationship between the speed of NF-κB activation, the lasting time of NF-κB activation, and late toxicity needs further investigation.We observed an association between an early NF-g tissue . Surprisyndromes , 28, sugyndromes –32. As at models –17. It iκB activation and late radiation-induced fibrosis added to current knowledge of associations between molecular radiosensitivity and late tissue toxicity. Late fibrosis is featured by a large deposition of extracellular matrix molecules  and an excessive of fibroblast proliferation [κB is only one of the many elements. Until date, a large number of studies had been conducted to identify cellular and molecular factors that might influence the occurrence and severity of late toxicity. Most of these studies investigated only a single element, whereas the relationship among these elements has not been investigated. For example, it is unknown whether fibroblast radiation sensitivity correlates with polymorphisms in these genes that lead to late toxicity. These studies had evoked interest, but no general agreement has been reached regarding any of these factors that could be sufficiently accurate for the prediction of radiation-induced toxicity for clinical use [Our finding of an association between NF-feration , 34. Theferation , 36. In feration –40. The ical use , 8, 41. Limitations in this work included the retrospective nature and heterogeneity in the treatment protocols. In addition, radiotherapy modalities were not assessed in detail due to the lack of some information and small sample size in some treatment protocol. As an explorative study, we tried all our ways to minimize any confusion. For example, we adopted a single primary endpoint, late fibrosis, because the underlying causes of late toxicity in different tissue and in different forms  were considered to be different. To minimize technical confusions, we used a method as simple as possible—the nuclear p65 expression ratio in the PBMCs before and 1 hour after ex vivo irradiation. Due to these limitations, our findings should be interpreted with caution.κB activation related to fibrosis remains unknown. Further studies will be required to understand this association.In conclusion, our analysis found that a higher speed of nuclear p65 accumulation in the PMBCs after irradiation was associated with an increased risk of developing ≥2 grade late skin and subcutaneous fibrosis in patients with HNSCC after radiotherapy. This finding adds to the growing knowledge about translation of molecular radiosensitivity into practical radiological protection, but how NF-


Burkholderia (B.) mallei is a notifiable zoonotic disease in equidae. For international trade and movement of equids, certificates of negative serological test results for antibodies against B. mallei are required. To date, the complement fixation test (CFT) is the mandatory test to issue these health certificates. The CFT is difficult to standardize and, due to its poor specificity, often leads to false-positive reactions resulting in trade restrictions with considerable financial consequences. In the present study, the new ID Screen Glanders Double Antigen Multispecies ELISA (GLANDA- ELISA)  was evaluated using 400 negative and 370 glanders positive field samples of equidae. The GLANDA-ELISA was significantly more specific (99.8%) than the CFT (97.0%). Considering the comparable sensitivities of CFT (96.5%) and ELISA (98.1%), this new GLANDA-ELISA test appears a suitable confirmatory test and a realistic alternative for serological testing of horses for trade or movement.Glanders, caused by  Burkholderia (B.) mallei is the causative agent of the zoonosis glanders of equidae. The horse, in which the infection is often chronic and asymptomatic, poses a risk for reintroducing the infection into areas where it had been eradicated. Mandatory testing and certification of freedom from glanders in the single animal before movement are intended to prevent spread of disease. Although the disease has been eradicated in most European and North American countries, regular outbreaks are reported in countries of South America and Asia  and complement fixation test (CFT) are available. The mallein test is not accepted for trade testing and is generally not recommended because of animal welfare concerns. Furthermore, malleinised animals may undergo seroconversion and then show positive reactions in additional diagnostic methods, like CFT (The “gold (reference) standard” of glanders diagnostics is the isolation of like CFT . Howeverlike CFT , 9–11. Ilike CFT . Studieslike CFT –14, whiclike CFT –18. Thuslike CFT . For exalike CFT , 14, 21.n = 370) were collected from equids during glanders outbreaks in Pakistan 2007, 2016–2020 (n = 236), and India (n = 132) in 2016 and 2017. Two samples originated from horses found positive by CFT, WB, and PCR in Germany, in 2006 and 2014. The samples from India and Pakistan originated from animals in which the infection was either confirmed by clinical signs and CFT (n = 117), or by B. mallei isolation or molecular detection of B. mallei using real-time PCR (n = 251) as recommended by OIE (True-positive field serum samples (d by OIE . The anin = 400), were collected in Germany, which is officially free from glanders. All samples were collected non-randomly during routine testing by CFT for trade or movement.True-negative samples  was used at a dilution of 1:5,000 as the secondary antibody. Detection was performed by a ready to use NBT-BCIP® staining system (Sigma). The WB was scored positive, if the banding pattern of the LPS ladder was clearly visible within the region of 20–60 Kilo Dalton (kDa).The Western blot has been described in detail in previous publications , 14. BriB. mallei antigen-coated plates with individual 8-well strips, positive and negative controls, 10x concentrated recombinant purified Burkholderia mallei protein antigen-HRP conjugate, dilution buffers, 20x wash concentrate, TMB substrate solution, and stop solution (0.5 M). Briefly, 90 μl dilution buffer and 10 μl of the serum samples were added to an antigen-coated well. After 45 ± 5 min incubation at 21 ± 5°C, wells were emptied, washed 3 times and incubated with the conjugate for 30 ± 3 min at 21 ± 5°C. Washing was repeated 3 times. Then 100 μl of conjugate (1x) were added and after incubation for 30 ± 3 min the 3-fold washing procedure was repeated. Finally, 100 μl of the substrate solution were added and after 15 ± 2 min incubation at 21 ± 5°C the reaction was stopped by 100 μl of the stop solution and OD 450 nm value was recorded. S/P% values were calculated and samples with S/P% <70% were considered as negative, and ≥70% as positive.The GLANDA-ELISA  was provided as a ready-to-use kit and was performed according to the manufacturer's instructions. The kit contained recombinant https://www.medcalc.org/). Sensitivity and specificity of test pairs were compared with McNemar's test for correlated proportions with P < 0.05 considered statistically significant.Calculations including 95% confidence interval (CI) were based on standard formulas  and doneP = 0.003) and the WB (P = 0.035) also showed a significant higher DSp than the CFT.The data and calculations for DSp and DSe are given in The frequencies of combinations of test results in known infected and non-infected populations are shown in B. mallei antigen preparations and various recombinant proteins have already been developed and evaluated  and CFT and WB (p = 0.035). In Germany, false-positive CFT results are ruled out by using the WB as a confirmatory test (However, the CFT is still the prescribed method for trade purposes to certify individual animal free from glanders . The tesory test . The WB The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because for this study, no ethical approvals were required. All blood samples were routinely collected for prescribed diagnostic purposes or official monitoring studies and subsequently made available to this study. Written informed consent was obtained from the owners for the participation of their animals in this study.ME, HS, and SM: collection, characterization of samples, and writing—original draft. ME: investigation. ME and IG: statistical data analysis. HN: supervision. ME, HS, SM, FM, IG, and HN: writing—review and editing. 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.


China has been undergoing dramatic economic development, accompanied by increased education load on the young children. This study is to investigate the prevalence, type, severity, and associated risk factors of astigmatism in school students in eastern China.In this cross-sectional school-based study, students underwent refraction using NIDEK non-cycloplegic autorefractor. Astigmatism was defined as cylinder 1.5 diopter (D) or greater, and high astigmatism was defined as cylinder 3.0 D or greaterMultivariate regression models were used to determine factors associated with astigmatism.p < 0.0001) and high astigmatism . When multivariate models were further adjusted by spherical equivalent, only gender remained statistically associated with astigmatism (odds ratio (OR) = 1.65, p < 0.0001) and high astigmatism , myopic and hyperopic refractive error were significantly associated with higher risk of astigmatism and high astigmatism .Among 4801 children  with mean age (±standard deviation) 12.3 (±3.8) years, 680 : 13.2–15.2%) had astigmatism (85% were with-the-rule) and 103  had high astigmatism. The prevalence rate of astigmatism was 7–8% in grades 4 or below, 15–16% in grades 6–8, 20% in grade 9, and 20–25% in grade 10 or above. In multivariate analyses, higher grade and male gender were associated with higher prevalence of astigmatism (all Astigmatism is common in Chinese school-age children and increases with grade. Majority of astigmatism is with-the-rule. Male gender and myopic or hyperopic refractive error are significantly associated with higher prevalence and severity of astigmatism. Astigmatism is a common vision disorder in children. Previous studies suggested that uncorrected astigmatism was associated with increased risk of myopia and amblyopia –4. EarlyThis is a cross-sectional school-based study of refractive error conducted in May 2019 in YiWu, a county-level city of about 1.2 million people in eastern China of central Zhejiang province . For thiThe information on student name, date of birth, gender and grade were obtained from the school roster. All participants underwent eye examination following the standard study protocol by the trained eye-care professionals  for common ocular diseases, tests for the distance visual acuity using retro-illuminated logMAR chart with tumbling-E optotypes followed by measuring for refractive error using table-mounted NIDEK noncycloplegic autorefractor . Three readings of refractive error were taken from each eye and the average of three readings for each eye was entered for analysis. If the difference between any of two readings from an eye was greater than 0.5 diopters, refractive error for that eye was re-taken. Students who were found having ocular diseases  or ocular injuries other than the significant refractive error were not eligible for the study.For quality control, autorefractors were calibrated every day before measuring refractive error. Approximately 5% of students were randomly chosen to repeat the test of refraction. The data were double entered into excel sheets and the differences were resolved by checking with the original paper record.The study was designed to enroll 4800 students. This sample size provides very precise estimate of prevalence rate of astigmatism with half width of its 95% confidence interval 1% assuming prevalence rate of astigmatism is 10%.The study was approved by the Institutional Review Board, and written informed consent was obtained from at least one parent or legal guardian.Spherical equivalent (SE) for each eye was calculated as sphere plus half of the cylinder. Cylindrical refractive error was expressed as positive cylinder form. To facilitate the direct comparison with other studies , 5, we dRisk factors for magnitude of cylinder power  were assessed using analysis of variance, and comparisons of prevalence rates of astigmatism and high astigmatism across levels of risk factors were performed using the chi-square test. To evaluate the independent association of the each risk factors  with cylinder power and prevalence of astigmatism, multivariate linear regression models were performed for cylinder power, and multivariate logistic regression models were performed for prevalence of astigmatism. The odds ratio (OR) and its 95% confidence interval (95% CI) for each risk factors were calculated from the multivariate logistic regression models. In the multivariate regression models, the grade instead of age was used, because age and grade were highly correlated (Pearson correlation coefficient of 0.99) and grade was slightly more associated with astigmatism than age. We performed two multivariate models, the multivariate model 1 included grade, gender and school location, while the multivariate model 2 additionally included spherical equivalent , to assess whether the potential associations of grade, gender and school location with astigmatism were due to the spherical equivalent.p < 0.05 was considered to be statistically significant.All statistical analyses were performed in SAS v9.4 and two-sided A total of 4801 school-aged students from 16 schools of YiWu city participated in the study. The student grade ranged from kindergarten to grade 12 with the number of participating students ranging from 331 to 403 in each grade , while older age (p = 0.02), male gender (p = 0.001), higher grade (p = 0.02) were significantly associated with higher prevalence rate of high astigmatism, but school location was not associated with high astigmatism (p = 0.29).In univariate analysis Table , older aP < 0.0001), with higher rates of astigmatism and high astigmatism in myopic and hyperopic children, and lower rates in emmetropic children  was significantly associated with cylinder power, prevalence rates of astigmatism and high astigmatism in a non-linear manner , male gender (p < 0.0001) were independently associated with higher cylinder power (Table p < 0.0001) and spherical equivalent (p < 0.0001) were significantly associated with higher cylinder power; the grade (p = 0.54) and school location (p = 0.18) were not significantly associated with cylinder power.In multivariate analysis for cylinder power, higher grade (er Table . Howeverp < 0.0001). When the multivariate analysis were further adjusted by spherical equivalent, male gender remained significantly associated with higher prevalence of astigmatism , but grade became not significantly associated with astigmatism (p = 0.65). Myopic spherical equivalent (OR = 20.3 for myopia − 6 D or greater) or hyperopic spherical equivalent (OR = 10.3 for hyperopic 1.0 D or greater) were significantly associated higher prevalence rate of astigmatism when compared with emmetropic spherical equivalent (− 0.5 to 0.5 D).In multivariate analysis Table  for prevp = 0.04), male gender (p = 0.001) were independently associated with higher prevalence rate of high astigmatism, while school location (p = 0.63) was not associated with high astigmatism. When spherical equivalent was included in the multivariate model, only male gender  and myopic  or hyperopic spherical equivalent (OR = 5.4 for hyperopia 0.5 or greater) were significantly associated with high astigmatism.In multivariate analysis for high astigmatism Table , higher p ≥ 0.09, online supplement Table In univariate analysis, age, gender, grade, school location and spherical equivalent were all not significantly associated with type of astigmatism . Myopic or hyperopic refractive error was also significantly associated with more severe astigmatism, the mean cylinder power being 2.50 D for students with myopic − 6.0 D or worse, and 2.26 D for students with hyperopia 1.0 D or greater as compared to 1.83 for those with spherical equivalent of 0.5 to 1.0 D  was with-the-rule. The prevalence of astigmatism increased with grade, and was higher in the male students. The myopic or hyperopic refractive error was independently associated with higher prevalence rate and severity of astigmatism.It is well-known that prevalence of astigmatism varies with race and ethnicity , 5, 7, 8p = 0.02 adjusting for age) [p = 0.08) [Our study found that male students had significantly higher prevalence rate of astigmatism (16.1% vs. 11.8%) and high astigmatism (2.8% vs. 1.4%) than female students, but there was no significant difference in types of astigmatism. In the multivariate analyses that were adjusted by the spherical equivalent levels, the prevalence rate in male students were 2.2 times that of female students for both astigmatism and high astigmatism. The Multi-Ethnic Pediatric Eye Disease Study (MEPEDS) found prevalence of astigmatism ≥ 1.5 D) was higher in Hispanic male (18.5%) than female 14.9%) (for age) . A study = 0.08) . However% (for ag = 0.08) , in non- D was hiOur study found that the majority 85%) of astigmatism was with-the-rule (defined as cylinder axis 90 ± 15 degrees). The dominance of with-the-rule as a type of astigmatism is consistent with previous studies in Chinese children in Xiamen of southeastern China (72% with-the-rule) , in Guan5% of astOur study clearly demonstrated the non-linear association between spherical equivalent and astigmatism Fig. . ChildreIn this study, we found the older age and higher grade were associated with higher prevalence rate of astigmatism. However, the association was not significant after adjusting the spherical equivalent, suggesting that significant refractive error contributed to the increasing prevalence rate of astigmatism with higher grade. The higher rate of astigmatism in our study might be due to the high prevalence rate of myopia in this study.Strengths of the study include the large sample size and the wide age range of students from kindergarten to high school students. A possible limitation includes the use of cylinder measures from NIDEK non-cycloplegic autorefractor  to determine astigmatism and to assess the spherical equivalent as a risk factor for astigmatism. The non-cycloplegic autorefraction could potentially bias our estimate of cylinder power and prevalence rate of astigmatism. However, a study did not find any statistically significant difference in axis and cylinder power of astigmatism measured before and after cycloplegia in the 4 to 17 year old children . AnotherOur study found that astigmatism is common in Chinese school-age children and prevalence rate of astigmatism increases with grade, likely due to significant refractive error. The majority of astigmatism was with-the-rule. Male gender and myopic or hyperopic refractive error were significantly associated with higher prevalence and severity of astigmatism. Future longitudinal studies are needed to investigate the possible causal relationship between myopic or hyperopic refractive error and astigmatism, and to evaluate whether intervention on refractive error can reduce the development of astigmatism.


To evaluate demographic, clinical and pathological characteristics of small renal masses (SRM) (≤ 4 cm) in a Latin-American population provided by LARCG  and analyze predictors of survival, recurrence and metastasis.A multi-institutional retrospective cohort study of 1523 patients submitted to surgical treatment for non-metastatic SRM from 1979 to 2016. Comparisons between radical (RN) or partial nephrectomy (PN) and young or elderly patients were performed. Kaplan-Meier curves and log-rank tests estimated 10-year overall survival. Predictors of local recurrence or metastasis were analyzed by a multivariable logistic regression model.p = 0.044. This disparity was not evidenced in younger patients. On multivariable model, bilaterality, extracapsular extension and ASA (American Society of Anesthesiologists) classification ≥3 were predictors of local recurrence. We did not identify significant predictors for distant metastasis in our series.PN and RN were performed in 897 (66%) and 461 (34%) patients. A proportional increase of PN cases from 48.5% (1979–2009) to 75% (after 2009) was evidenced. Stratifying by age, elderly patients (≥ 65 years) had better 10-year OS rates when submitted to PN (83.5%), than RN (54.5%), PN is performed in Latin-America in a similar proportion to developed areas and it has been increasing in the last years. Even in elderly individuals, if good functional status, sufficiently fit to surgery, and favorable tumor characteristics, they should be encouraged to perform PN. Intending to an earlier diagnosis of recurrence or distant metastasis, SRM cases with unfavorable characteristics should have a more rigorous follow-up routine. Comprehensive approach of renal cell carcinoma (RCC) is a priority in the main cancer centers. Increasing trend in incidence, particularly of incidentally detected small renal masses (SRM), has not been clearly associated to a significant decrease in global mortality. According to the SEER  database, the estimated number of new cases of RCC in the United States in 2019 is 73,820. RCC represents about 4% of all new cancer cases and about 2.5% of all cancer deaths [Latin America (LA) represents a large area with about 8.5% of the world population, most of them living in developing countries. GLOBOCAN estimated the RCC incidence and mortality in LA in 4.4/100,000 and 1.5/100,000, respectively .Nephron-sparing surgery (NSS) is the standard of care for SRM, providing equivalent oncological control and a decrease in the non-cancer related mortality , 8. BesiConsidering the paucity of information about RCC in LA, the aims of this study were to review the LARCG  database to evaluate demographic, clinical and pathologic features of SRM in this population, correlating the impact in prognosis and survival in comparison to literature.. 2018) and the study did not interfere with the treatment or follow-up of the patients.This is a multi-institutional retrospective cohort study involving the registries from 28 institutions in LA and Spain, members of LARCG . Data weWe selected patients with renal tumors up to 4 cm on pathological reports and no evidence of distant metastasis at presentation. We found 1523 patients with these characteristics which were stratified by histologic subtype criteria in clear cell, papillary, chromophobe or others  and by surgical treatment: partial nephrectomy (PN) or RN. Firstly, we excluded patients with benign histology and those with missing values for histologic subtype. Afterwards, non-surgical management cases were excluded.Demographic, clinical and pathological variables selected were age, gender, race, body mass index (BMI), smoking status, ECOG (Eastern Cooperative Oncology Group) Performance Status, Karnofsky and ASA (American Society of Anesthesiologists) classifications, symptoms at presentation, tumor size (≤ 3 cm or >  3 – 4 cm), positive surgical margins (PSM), nuclear Fuhrman grade (low or high grade), intratumoral necrosis, sarcomatoid component, extracapsular extension (ECE), multifocality or bilaterality, and admission period (before or after 2009). Patients were considered symptomatic at presentation if they had hematuria, local pain, palpable mass, paraneoplastic syndrome, neurological symptoms, acute onset varicocele and constitutional symptomatology. Nuclear grade was just established for clear cell and papillary RCC . ECE wasFollow-up was established from the date of surgery to the date of death or to the latest follow-up. Treatment indications and follow-up routine were determined at the discretion of each center. Overall deaths considered deaths due to the renal cancer and deaths from other causes. Local recurrence was defined as recurrence in the renal fossa after RN, and demonstration of recurrent enhancement within the tumor bed or enhancing suspicious lesion in the ipsilateral kidney after PN.Authors used a multiple imputation method only in variables with a limited proportion of missing data . The datt-test was used to compare continuous variables. Kaplan-Meier curves and log-rank tests were used in 10-year overall survival (OS) estimation, according age, tumor size and surgery performed. Considering that some patients underwent the surgical treatment at a referral center and then return to their communities, survival analysis were performed only for patients with follow-up ≥ 12 months. Univariable and multivariable logistic regression analysis were used to predict independent factors for local recurrence or distant metastasis. Related to these outcomes, it was considered the entire period, including all the patients regardless of the available follow-up time. The analysis was conducted using IBM SPSS®software v.24  and the packages R-Software v. 3.5 for graphical analysis. The significance level of the tests were fixed at 0.05.Continuous variables were expressed as mean, standard deviation, median and interquartile range. Categorical variables were expressed as absolute relative frequencies. They were compared with a Pearson’s chi-squared test or a Fisher’s exact test, when appropriate. Student’s A total of 1008 cases had a defined malignant histologic subtype, being 651 cases (64.6%) of clear cell RCC, 60 (6%) of papillary RCC (types I and II), 238 (23.6%) of chromophobe RCC, and 59 (6%) of other histologies. PN and RN were performed in 897 (66%) patients and 461 (34%), respectively. The proportion of PN cases increased from 48.5% (up to 2009) to almost 75% after that year. Table Regarding surgical aspects of PN cases, open and minimally-invasive approaches  were performed in 47.9 and 50.4%, respectively. Median blood loss was 200 mL (5 – 2500 mL), median warm ischemia time was 20 min, and median hospital stay was 4 days (1–37).The median follow-up time was 24 months (range: 0–289). We reported 72 (4.7%) overall deaths and 30 (2.0%) cancer-specific deaths. Local recurrence occurred in 49 (6.1%) from 804 cases and metastasis during follow-up, in 10 (1.7%) from 588 evaluable cases.There were no significant differences in 10-year OS rates comparing patients with SRM ≤ 3 cm or >  3 cm. Similarly, OS rates were not different between patients underwent PN or RN. The 5-year and 10-year OS rates for SRM ≤ 3 cm were 95.1 and 80.3%, respectively. For SRM > 3 cm, 5-year and 10-year OS rates were 94.1 and 74.8%, respectively  had better 10-year OS rates when submitted to PN (83.5%), than RN (54.5%), 044 Fig. -b. This 044 Fig. -a. ElderOn univariable analysis, extracapsular extension (ECE), ASA classification and bilaterality were associated with recurrence (data not shown). On multivariable model, bilaterality, ECE and ASA classification ≥3 were significant predictors of local recurrence. We did not identify significant predictors for distant metastasis in our series . This can be attributable to the development of minimally invasive techniques, the better expertise of the LA referral centers, and the stage migration with the raise of SRM in the last years . Sun et From 1983 to 2002, overall mortality rose in patients with RCC in the USA. In tumors < 2 cm, this rise was 0.07 to 0.2 deaths per 100,000. In tumors between 2 and 4 cm, this rise was from 0.2 to 1.5 deaths per 100,000 US population . ConsideConsidering that elderly people have more comorbidities and that RN can proportionate a higher risk of renal dysfunction , 24, we The benefits of regional or retroperitoneal lymphadenectomy are controversial, particularly in small renal masses, that rarely present positive lymph nodes. The extent and number of retroperitoneal lymph nodes removed have no defined templates. Marchioni et al  studied Ball et al.  describeFurthermore, bilaterality and ASA classification ≥3 associated with local recurrence following surgery. Patients classified with ASA ≥ 3 can present more aggressive tumors , 33. UsuRegarding nephrometry, patients with totally endophytic tumors performed RN more than three times than patients with exophytic tumors (76% vs. 24%). Due to the historic long-time design of this series, we have no confident data of nephrometry scores (e.g.: R.E.N.A.L.). We did not find differences between totally endophytic or exophytic tumors in OS neither in our recurrence or distant metastasis analysis (data present in only 449 cases).N = 244). Furthermore, the heterogeneity of the different centers and the long period of this series could lead to a selection bias of the elderly patients underwent PN or RN. Proposed protocols regarding surgical approaches have been changed during the last decades. Additionally, we did not include patients treated with non-surgical approaches, such as active surveillance, watchful waiting, or ablative treatments, which are possible options in SRM, particularly in elderly or in patients with multiple comorbidities. Due to the lack of data, we were not able to report the nephrometry score of the cases. Related to the secondary outcomes for local recurrence and distant metastasis, we were unable to define the exact moment of the event. Thus, a logistic regression model was the most appropriate for the analysis of secondary outcomes, instead of a survival analysis. Finally, the short median follow-up time of 24 months could be related to possible influences on survival outcomes. Despite these limitations, this study is one of the largest known series of patients with small renal masses in Latin America. Many of the involved institutions are referral centers or teaching institutions. They tend to have skilled uropathologists, more detailed pathology records, and more available data about oncological outcomes. The results are important to recognize particular features of SRM in LA and to counsel patients about appropriate decisions.There are limitations in our study, including the retrospective design and heterogeneity of each center. Pathologic reports were provided by different pathologists and central pathology review was performed only in Brazilian cases (RN is still overly done in the SRM setting in LA. However, NSS procedures are performed in a proportion similar to developed areas and has been increasing in the last years. RN might be considered an overtreatment for many of these patients. Even in elderly individuals, if good functional status, sufficiently fit to surgery, and presenting favorable tumor characteristics, they should be encouraged to underwent PN. Posteriorly, a more intense follow-up routine could be discussed in SRM cases with unfavorable characteristics, intend to an earlier diagnosis of recurrence or distant metastasis.


Magnesium lithospermate B (MLB) was shown to suppress oxidative stress and reduce hypertension, but the role of MLB in pregnancy-induced hypertension (PIH) remains unknown. The objective of this study was to demonstrate the effects of MLB on rats with PIH.  A total of 40 pregnant SD rats were selected, and 30 rats were orally given NG-nitro-L-arginine methyl ester  to establish PIH rat models. Rats were equally divided into four groups: control, PIH, 5 mg/kg MLB, and 10 mg/kg MLB. MLB was consecutively administered into PIH rats for one week. The effects of MLB on mean arterial blood pressure (MAP), urine protein level, inflammation, and oxidative stress together with angiogenesis were analyzed.  MLB prevented the elevation in MAP and urine protein levels induced by L-NAME. The activities of inflammatory cytokines were highly increased in serum and placental tissues of PIH rats, while cotreatment with MLB partially reversed the activities of these cytokines. MLB also recovered the expression of reactive oxygen species (ROS) in plasma of PIH rats together with levels of oxidative stress and antioxidant capacity in the placenta of PIH rats. The decreased expressions of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and NO observed in PIH rats were increased by MLB. In addition, 10 mg/kg MLB exhibited higher protective effects as compared to lower doses of 5 mg/kg.  This study demonstrated that pretreatment with MLB decreased MAP, inflammation, and oxidative stress in rats with gestational hypertension. Pregnancy-induced hypertension (PIH) is an idiopathic and common disease in obstetrics, and it usually occurs from 20 weeks of gestation to 2 weeks after delivery . The maiα, and monocyte chemotactic protein (MCP-1) were found to be significantly overexpressed in the plasma of PIH women compared with those in the plasma of normal pregnant women [However, the cause of PIH has not yet been clarified. Until now, PIH is believed to be related to genetic factors, including immune maladjustment, oxidative stress, capillary endothelial injury, and placental malnutrition . Studiesnt women .Recent research has reported that oxidative stress plays a pivotal role in the progression of PE, and elevated levels of antiangiogenic factors are associated with PIH , 8. AlteSalvia miltiorrhiza, is noted for scavenging oxygen free radicals, protecting cell mitochondria, improving energy metabolism, and regulating gene expression. The protective effects of MLB on cerebral/hepatic/myocardial ischemia/reperfusion injury and renal injury together with vascular endothelial dysfunction via regulating inflammation and oxidative stress have been extensively demonstrated [Magnesium lithospermate B (MLB), an active component of the water-soluble fraction of nstrated –14. In anstrated , 16. BasIn the present study, by using an NG-nitro-L-arginine methyl ester- (L-NAME-) induced PIH rat model, we investigated whether MLB was able to prevent the increase of MAP and proteinuria and whether the preventive effect of MLB is related to suppression of inflammation and oxidative stress.The experiments of animals have declared to follow the principles of the World Medical Association (WMA) Declaration of Helsinki.Magnesium lithospermate B (MLB) was purchased from Shanghai Green Valley Pharmaceutical Company and dissolved in normal saline. NG-nitro-L-arginine methyl ester (L-NAME) was purchased from Abcam Biotechnology and dissolved in normal saline.30 male  and 60 female  specific pathogen-free (SPF) Sprague Dawley (SD) rats were purchased from Nanjing Junke Biological Engineering Co., Ltd., and kept under a temperature- and humidity-controlled condition with a 12 h light/12 h dark cycle in the present study.th day of pregnancy.After keeping rats for one week, male and female (1 : 2) rats were kept in one cage to allow them to mate. On the second day, the vaginal secretions of female rats were collected and examined under a microscope, and the presence of sperm was considered as 0Pregnant rats were normally bred until day 15 of pregnancy, and then a total of 40 pregnant female rats were equally assigned to four groups (10 rats each group) and additionally received indicated reagents besides normal diet: (і) control, rats were fed with normal saline; (ii) PIH, rats were orally given 60 mg/kg/day L-NAME for one week ; (iii) MOn day 21 of pregnancy, the aortal blood of rats was collected, the rats were anaesthetised, and hysterotomies were performed to collect placental tissues. After the surgery, all rats were sacrificed by intraperitoneal administration with 100 mg/kg phenobarbital.The mean arterial blood pressure (MAP) was recorded on days 15–21 of pregnancy using the CODA 6 BP system .The content of 24 h urine proteins was quantified by a urine protein test kit  after collecting 24 h urine. Briefly, from day 15 of gestation, pregnant rats were kept in an automated home cage system . For measuring MAP, rats were catheterized under isoflurane anesthesia . The carotid artery was cannulated, and MAP was recorded continuously for a 2 h period after 1 h of stabilization. The urine can be automatically accumulated in the system and was collected every 24 h.α (cat. no. ab100785), IL-1β (cat. no. ab100768), IL-6 (cat. no. ab100772), and monocyte chemotactic protein (MCP-1) (cat. no. ab219045) in plasma according to the manufacturer's protocol.The whole blood collected from the aorta of rats was placed at room temperature for 2 h and then centrifuged at 2500 rpm for 10 min. The placental tissues were homogenized in PBS on ice. The placental homogenates were frozen in liquid nitrogen and stored at −80°C until further processing. The upper serum was collected and stored at −80°C for use. ELISA kits (Abcam Biotechnology) were utilized to detect the activity of TNF-The level of reactive oxygen species (ROS) in rats' plasma was analyzed by a cellular ROS assay kit  according to the manufacturer's protocol.The placental tissues were washed with PBS, dissolved with a cell lysing solution containing protease inhibitor, and then subjected to ultrasonic treatment to fully lyse the samples. After centrifugation, supernatants were collected, and the protein concentrations were determined using the Bio-Rad bicinchoninic acid (BCA) assay kit .The antioxidant capacity of the placental tissue was measured using the Trolox equivalent antioxidant capacity (TEAC) assay kit , and the levels of thiobarbituric acid reactive substances (TBARS) and NO were determined using the TBARS and nitrate/nitrite colorimetric assay kit  according to the manufacturer's instructions, respectively.μg total RNA was converted to cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser  according to the manufacturer's protocols. The following RT temperature protocol was used: 37°C for 15 min and 85°C for 5 sec. qPCR was performed using TB Green® Fast qPCR Mix  according to the manufacturer's protocols. Differential expression of mRNA was calculated using the 2−ΔΔCq method (24). The primers used were as follows: VEGF, forward 5′-ACCATGAACTTTCTGCTC-3′and reverse 5′-GGACGGCTTGAAGATATA-3′; GAPDH, forward 5′-AGTGCCAGCCT-CGTCTCATA-3′ and reverse 5′-TGAACTTGCCGTGGGTAGAG-3′. The following thermocycling conditions were used for qPCR: initial denaturation at 95°C for 30 sec and 40 cycles of 95°C for 5 sec and 60°C for 15 sec, followed by default of melt curve .The total RNA of placental tissues was extracted using TRIzol® reagent , and 1 μg) of proteins were separated by 8–12% SDS-PAGE. Separated proteins were transferred onto PVDF membranes  and then blocked with 5% nonfat milk at 37°C for 1 h, after which the membranes were incubated with the following primary antibodies (Abcam) overnight at 4°C: VEGF (cat. no. ab32152), eNOS (cat. no. ab76198), and anti-GAPDH (cat. no. ab32152). Finally, the membranes were incubated with a horseradish-conjugated secondary antibody  at room temperature for 2 h and visualized by an electrochemiluminescence system . Image J software  was utilized for densitometric analysis of western blot.After quantifying, equal amounts . Differences were calculated by Student's First of all, to observe the effects of MLB on MAP and urine proteins in PIH rats, we measured MAP and collected 24 h urine to detect the content of urine proteins from 15–21 days of pregnancy. Results showed that normal control pregnant rats maintain a stable blood pressure and urine proteins, while PIH-treated rats had elevated MAP and urine proteins in a time-dependent manner from day 16 (the second day of L-NAME treatment) to day 21 of pregnancy . The meaα, IL-1β, IL-6, and MCP-1 in both the serum and placenta of pregnant rats compared with that of normal pregnant rats, indicating the enhancement of inflammatory response. However, the cotreatment of MLB partially reduced the increased activity of these inflammatory cytokines in PIH rats. Similarly, MLB with the dose of 10 mg/kg possessed better inhibitory ability against inflammation.Then, we extracted the serum and placenta tissue of rats to detect the activity of inflammation. As shown in Next, the effects of MLB on oxidative stress in PIH rats were explored. The levels of ROS and TBARS, which are both oxidative stress markers, were observed to be highly increased in PIH rats, but their levels visibly dropped upon the presence of MLB Figures . At the Finally, we investigated the effects of MLB on angiogenic ability. The imbalance of angiogenesis is strongly associated with the etiology of hypertension and PIH . VEGF anPIH comprises of preeclampsia, eclampsia, and high MAP, all of which occurs especially during pregnancy . High MAProduced by vascular endothelial cells, NO has been proven to regulate vascular tension, dilate blood vessels, and function as a regulator of the cardiovascular system during pregnancy . L-NAME α, thereby alleviating LPS-induced endothelial dysfunction in a murine model of acute inflammation. Another report showed the reduced release of TNF-α, IL-1β, and IL-6 in myocardial ischemia/reperfusion (MI/R) rats treated with MLB [Considering the mechanism that excessive oxidative stress can cause endovascular damage, thereby triggering a series of oxidative stress and inflammatory reactions, eventually leading to PIH-related symptoms, we next investigated the alteration of cytokines involved in inflammation, oxidative stress, and angiogenesis. In fact, the inhibitory effects of MLB on inflammation and oxidative stress have been extensively demonstrated. The finding by Gao et al.  was thatwith MLB . Consistwith MLB . Inhibitwith MLB . Our reswith MLB . In the with MLB . In the Taken together, this study demonstrated for the first time that MLB exerted the preventive effect on PIH via reducing inflammation and oxidative stress, regulating the expressions of VEGF and NO, thereby restoring MAP and urine proteins in PIH rats. Therefore, MLB could potentially be used in the clinic in the third trimester for pregnant women, especially pregnant women with a history of hypertension, to prevent PIH. However, further studies will be required to uncover the underlying molecular mechanisms involved.


Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth).We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR.Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research. Wool is a source of high-quality textile raw materials derived from animals, that has a significant impact on the national economy. Improving the production of high-quality fine wool has become a hot topic in recent years. Wool growth is a very complex physiological and biochemical process, influenced by genetics, the environment, and nutrition. Wool grows from hair follicles (HF), and its yield and quality are closely related to the development of wool follicles. These are complex organs of the skin that are capable of self-regeneration, and their structure plays a very important role in their periodic growth process. Mammalian hair follicles are divided into primary hair follicles (PF) and secondary hair follicles (SF). It is the SF that is producing fine wool. Wool follicle morphogenesis involves the coordination of a series of signaling pathways that connect epidermis and dermis. The development of hair follicles is regulated by various signaling pathways, such as Wnt, sonic hedgehog (SHH), notch, bone morphogenic protein (BMP), and fibroblast growth factor (FGF). Various downstream signaling molecules, such as β-catenin, Msx1, and Msx2, are involved in hair follicle morphogenesis .CircRNAs are a novel type of noncoding RNA that regulate transcriptional and post transcriptional genes expression , 5. TheyRecently, studies have found that exposure to melatonin disturbs a key secretion signal in goat hair follicle stem cells, and consequentially disturbs normal goat hair follicle development . CircRNAAohan fine wool sheep (AFWS) is a sheep breed in China that produces excellent wool quality, with good textile process performance. Increased understanding of the function of genes involved in wool follicle development could assist in selective breeding for specific traits and thus improve wool yield and quality . In a prTo date we have only a very limited understanding of circRNA expression in AFWS follicles. To study the relationship between circRNA and changes in wool follicle at different developmental stages in sheep, RNA-Seq was used to detect the expression profiles of circRNA in skin tissue from AFWS at E90d, E120d, and Birth. Our results indicate that circRNA plays an important role in the formation of sheep wool follicles.Hematoxylin and eosin (H&E) staining at E90d showed primary- and early secondary-stage wool follicles Fig. a. From oTo examine the circRNAs expression profiles in sheep skin at different developmental stages, we performed RNA Integrity Number (RIN) tests on nine sheep skin tissue samples, three from each of the three developmental periods . The RIN values of the samples are listed in Additional file Based on the criterion of differentially-expressed circRNAs, clustering maps Fig. a were usTRPS1). It has been reported that Trps1 is involved in the growth and development of hair follicle cells [VAV3), and circ_0001520 and circ_0001524 that were mapped to source gene 3008 (TMEFF1) [SMAD1) is the source gene of circ_0006736. This gene can control the transformation of early hair follicle morphology by controlling the activity of stem cells [RFX7, the source gene of circ_0004116, in AFWS wool follicles development.Among the DEGs , circ_0004932 and circ_0004936 were mapped to gene 13,410 (le cells . Similar(TMEFF1) , 26. We em cells . The expThe function of circRNA is reflected through their source gene. It thus can be further studied by analyzing the Gene Ontology (GO) terms of their source genes. Based on statistical analysis of differentially-expressed circRNAs and their source genes , circ_0001754 from source gene 3277 (TGFBR1), circ_0008036 from source gene 25,354 (SMAD2), circ_0004032 from source gene 11,746 (SOS2), circ_0005174 from source gene 13,720 (RB1), circ_0005519 from source gene 15,130 (EZH1), and circ_0007826 from source gene 24,949 (FGFR2). A network describing the connections between the source genes and circRNAs was constructed  pathway analysis  were found. It was suggested that several circRNAs, including novel ones such as circ_0004876, circ_0005177, and circ_0026326, might play a role during hair follicle cycle , and humTo further investigate potential circRNAs mechanisms of action, we applied GO and KEGG analyses. In GO annotation, the number of DEGs between any two stages exhibiting significant differences, reflecting a cumulative effect on phase traits. It was found that source genes of the differentially-expressed circRNAs function primarily in biological processes. These included terms under Cellular Component: cellular component organization (GO: 0016043), regulation of cellular processes (GO: 0050794), cellular macromolecule metabolic processes (GO: 0044260), intracellular organelle GO: 0043229) and organelle part (GO: 0044422); and Molecular Function: binding (GO: 0005488), ion binding GO: 0043167), and heterocyclic compound binding (GO: 1901363). These findings indicate that the different source genes of circRNAs at the three developmental stages play a significant role in of wool follicle cells’ formation, playing functions related to GO terms such as regulatory of metabolic processes. Some hair follicle-related GO terms were also enriched in our study, including regulation of hair cycle (GO: 0042634), skin development (GO: 0043588), hair follicle development (GO: 0001942), regulation of epidermis development (GO: 0045682) and hair cycle process (GO: 0022405). Some of them were reported to participate in the growth of hair follicle, and might be important research targets . It was 67, and h29 and orAKT3, TGFBR1, SMAD2, SOS2, RB1, EZH1, and FGFR2) were reported to participate in the growth process of hair follicles [The KEGG is a pathway database for systematic analysis of gene function. The results we obtained suggest that multiple signaling pathways form a complex regulatory network during wool follicle development. It was reported that human papillomavirus infection , adherenollicles –43. CompAlthough some signaling pathways, such as the Hedgehog , MAPK 4, FoxO 4, TGF-β , NF-κB , TNF 4949, and WRecent studies have found that some circRNAs have multiple binding sites for miRNAs  , 58. As Our study is the first to elucidate changes in wool follicles in sheep’s fetal development. RNA-Seq analysis identified 918 differentially-expressed circRNAs. Using miRanda to predict relationships between circRNAs and miRNAs, we identified 17 pairs of circRNA-miRNA. Of the identified miRNAs, miR-370-3p, miR-432, and miR-27a were reported to be associated with hair growth. The KEGG analysis of the differentially expressed genes identified six significantly enriched pathways. These include of 55 source genes. Seven of these genes, corresponding to 35 circRNAs, are involved in regulation of wool follicle growth. Seven of these 35 circRNAs were found to be differentially-expressed. These findings might provide clues that would assist future research on the molecular mechanisms of wool growth.The AFWS used in this study were raised in the AFWS Stud Farm of Inner Mongolia Autonomous Region and fed according to the farm’s feeding plan. Twelve healthy AFWS ewes of similar age (3–5 years old), body weight (55–60 kg), and body size were selected. Estrus of the 12 ewes was synchronized, and artificial insemination was performed during September. The ewes and lambs were anesthetized with sodium pentobarbital at a dose of 25 mg/kg by intravenous injection. After samples collection, the ewes and born lambs were released, whereas the fetuses from E90d and E120d were placed, still under anesthesia, inside a closed chamber, which was filled with 20% carbon dioxide per minute. When gas concentration had reached 80%, the fetuses died. The anesthesia procedure was performed following published protocols , 67.The 2-cm-diameter skin tissue samples (about 0.5–1.0 g per fetus/lamb) were collected from the shoulder area at the three developmental stages , three individuals for each stage, nine in total. The collected samples were placed into clean RNAase-free Eppendorf tubes and stored under liquid nitrogen, pending total RNA extraction. Skin samples were also fixed in 4% formaldehyde, and paraffin sections were prepared and stained with H&E for histological observations.To extract total RNA from the nine samples, TRIzol reagent  was used. RNase-free DNase  was used to remove DNA contamination from the extracted RNA. RNA degradation and contamination were monitored by 1% agarose gel electrophoresis and RNA purity was measured at an OD260/280, using a NanoDrop ND-2000 instrument . We also assessed RNA integrity by testing the RIN of the samples.High-throughput whole transcriptome sequencing and subsequent bioinformatics analyses were performed by Annoroad Technologies  as follows: A total of 3 μg RNA per sample were used for circRNA sample preparation. The Ribo-Zero™ Gold Kit was used to remove rRNA from the samples, and different index tags were selected to build the library according to the specifications of NEB Next Ultra Directional RNA Library Prep Kit for Illumina . The specific steps of library construction were as follows: Ribosomal RNA was removed using a kit, RNase R was added to remove linear RNA. Fragmentation Buffer was added to the reaction system to fragment the RNA, and then this fragmented RNA was used as a template for first strand cDNA synthesis, using random primers (Random Hexamers). Second strand cDNA was synthesized by adding buffer, dNTPs, RNase H, and DNA Polymerase I. After purification by QiaQuick PCR kit and elution with EB buffer, the following steps were performed: repair ending, adenine addition, sequencing linker addition, and target size fragments’ recovery (approximately 350 bp) by agarose gel electrophoresis. Uracil N-glycosylase (UNG) was then added to digest the DNA strand prior to PCR amplification. Finally, agarose gel electrophoresis was used to recover the DNA fragments of the target size. The constructed library was sequenced using Illumina X Ten and PE150 sequencing strategy.Sheep genome oar_v4.0 was selected as reference genome for comparison with the RNA-Seq data. Reads were mapped to the reference genome using the BWA-MEM method, which is fast and efficient in aligning reads, and allows mapping fragment reads to genomes as well. The raw reads generated by Illumina sequencing were processed to create clean reads by several processes, including de-junction contamination and removal of rRNA. For mapping, the BWA-MEM algorithm was first used for sequence splitting and alignment. The resulting Sam files were scanned in search of PCC (paid Chinese clipping) and PEM (paid end mapping) sites, as well as GT-AG splicing signals. Finally, sequences with junction sites were re-aligned with dynamic programming algorithm to ensure the reliability of circRNA identification. CIRI , an effiP < 0.05 and absolute fold-change values of > 1.5 in any of the pairwise comparisons were considered to be significantly differentially-expressed. Upregulated and downregulated circRNA numbers were eventually obtained. The calculation formula of SRPBM is: We used SRPBM as a normalization method to quantify the expression of circRNA. The DEseq2  softwareP < 0.05.Gene Ontology and KEGG pathway analyses were used to annotate the source genes of differentially-expressed circRNAs. The Blast2GO method  was usedhttp://www.microrna.org/microrna/home.do) [To explore the functions of circRNAs, predict the targeting relationship, and thus predict which of the circRNAs function as miRNAs sponges, we used miRanda V.3.3a (home.do) . In viewGAPDH. Primers were designed and synthesized by Sangon Biotech Co., Ltd. . Total RNA was converted into cDNA using random hexamers with Transcriptor First Strand cDNA Synthesis Kit . The qRT-PCR analysis was carried out in triplicate with iTaq™Universal SYBR@ Green Supermix  on a Bio-Rad CFX96 instrument . The total 20 μL reaction mixture contained 10 μL 2 × iTaq™Universal SYBR@ Green Supermix, 1 μL cDNA, 8 μL ddH2O, and 0.5 μL each of forward and reverse primers. The following program was used: 95 °C for 10 min; 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s; 72 °C for 6 min. The 2-ΔΔCt method was used to analyze the relative expression levels of the selected circRNAs.Quantitative real-time PCR (qRT-PCR) was used to validate circRNAs expression. We randomly selected seven circRNAs for validation. The expression levels of the selected circRNAs were normalized against the expression of a housekeeping gene, To determine the resistance of the selected seven circRNAs to RNase R digestion, total RNA and RNase R  were mixed together. The mix was incubated at 37 °C for 15 min, cDNA was then synthesized, and expression level of circRNAs was finally detected by qRT-PCR.Additional file 1 : Table S1. (a). Quality report on the nine RNA samples for RNA sequencing. (b). The overall assessment of sequencing data.Additional file 2 : Table S2. Total circRNAs detected at the E90d, E120d, and Birth stages.Additional file 3 : Table S3. Differentially expressed circRNAs in the three comparison groups. .Additional file 4 : Table S4. Detailed results of GO analysis for circRNAs source genes. .Additional file 5 : Table S5. Detailed results of KEGG pathway analysis for source genes of identified circRNAs. .Additional file 6 : Table S6A. A total of 55 source genes and their corresponding 255 circRNAs were enriched in the significantly differentially-expressed KEGG pathways. Table S6B. Seven source genes and their corresponding 35 circRNAs.Additional file 7 : Table S7. Primer sequences for qRT-PCR of the seven randomly selected circRNAs.


Patients >65 years had more >IIa FIGO stage: 76% for <65 years, 84% for 65–74 years and 80% for ≥75 years (p = 0.033). Women ≥75 years had less standard procedures (40% (34/84) vs. 59% (104/177) for 65–74 years and 72% (384/530) for <65 years (p < 0.001). Only 9% (13/139) of women ≥75 years had an Aletti score >8 compared with 16% and 22% for the other groups (p < 0.001). More residual disease was found in the two older groups  than the younger group (20%) (p < 0.05). Women ≥75 years had fewer neoadjuvant/adjuvant cycles than the young and elderly women: 23% ≥75 years received <6 cycles vs. 10% (p = 0.003). Univariate analysis for 3-year Overall Survival showed that age >65 years, FIGO III  and IV  (p < 0.001), residual disease  and lymph node metastasis  were associated with lower OS. Women >65 years are more likely to have incomplete surgery and more residual disease despite more advanced ovarian cancer. These elements are prognostic factors for women’s survival regardless of age. Specific trials in the elderly would produce evidence-based medicine and guidelines for ovarian cancer management in this population.Elderly women with ovarian cancer are often undertreated due to a perception of frailty. We aimed to evaluate the management of young, elderly and very elderly patients and its impact on survival in a retrospective multicenter study of women with ovarian cancer between 2007 to 2015. We included 979 women: 615 women (62.8%) <65 years, 225 (22.6%) 65–74 years, and 139 (14.2%) ≥75 years. Women in the 65–74 years age group were more likely to have serous ovarian cancer ( Ovarian cancer is the seventh most common cancer in women  and the fourth cause of mortality by cancer in women. It accounts for an estimated 239,000 new cases and 152,000 deaths worldwide annually . Most paToday, the standard treatment for ovarian cancer involves surgery and chemotherapy. To achieve complete cytoreduction, complex surgical procedures are necessary such as salpingo-oophorectomy with hysterectomy, omentectomy, pelvic lymphadenectomy and para-aortic lymphadenectomy. Bowel resection, pancreas resection, splenectomy, diaphragmatic stripping and partial liver resection may also be required and can lead to serious complications . The benThis study aimed to evaluate the management of young, elderly and very elderly patients with ovarian cancer in a large French multicenter cohort. We also studied overall survival (OS), disease-free survival (DFS), and cancer specific survival (CSS) rates.We collected data on women who had received surgical treatment for a histologically proven ovarian cancer between January 2000 and December 2016 from eight teaching hospitals in France . All the data had been entered in a single ovarian cancer database. We excluded patients with no surgical treatment , with non-epithelial ovarian carcinoma, benign tumors, and patients with unknown FIGO stage ). The research protocol was approved by the Institutional Review Board of the Collège National des Gynécologues et Obstétriciens Français (CEROG 2016-GYN-1003).The following demographic and clinical data were collected: age, body mass index , parity and Ca125 at diagnosis. Preoperative assessment of surgical risk was performed according to the American Society of Anesthesiology (ASA) Physical Status Classification by the attending anesthesiologist at the time of surgery . The typTumors were classified according to their pathologic status. Histologic subtypes were classified as serous adenocarcinoma, endometroid, mucinous, clear-cell and mixed epithelial tumors. Patients were then graded according to the FIGO 2014 classification .Cytoreductive surgery performed before chemotherapy was called “primary debulking surgery,” and when performed after chemotherapy it was called “interval debulking surgery” or “surgery after 6 cycles.” The treatment sequence was determined on an individual basis according to the patient’s general condition, exam results , and after multidisciplinary concertation. Standard surgery for ovarian cancer consists of hysterectomy, salpingo-oophorectomy, omentectomy, pelvic and para-aortic lymphadenectomy. Bowel resection, pancreas resection, splenectomy, diaphragmatic stripping and partial liver resection may also be required to achieve complete cytoreduction.The surgical procedures were classified according to Luyckx et al.’s publication : Group 1We evaluated the complexity of the surgical procedure using Aletti’s surgical complexity score (SCS) . This scSurgery complications were assessed according to the Clavien-Dindo classification  as minor2) every 3 weeks with at least 6 cycles. The chemotherapy protocol or bevacizumab administration was modified on an individual basis after multidisciplinary concertation.Adjuvant therapies included chemotherapy and bevacizumab. Chemotherapy was based on platinum salts (CARBOPLATINE AU5 or AU6) and taxanes , DFS , and CSS  rates calculated from date of recurrence, date of death, and date of cancer-related death. We also analyzed the rates and types of Clavien-Dindo complications.t tests were used to compare two variables, and one-way analysis of variance (ANOVA) was used for more than two variables.Descriptive parameters were expressed as the mean ± standard deviation (SD). Frequencies were presented as percentages. Chi square and Fisher’s exact tests were used as appropriate for categorical or ordinal variables. For continuous variables, p-value lower than 0.05 was considered statistically significant.OS was calculated in months from the date of surgery to death (related or unrelated to cancer) or to the date of the last follow-up visit for surviving patients. CSS was calculated as the time from the date of surgery to cancer-related death, and DFS was calculated as the time from the surgery to cancer recurrence. Women who were alive and without recurrence were censored at the date of the last follow-up visit. The Kaplan–Meier method was used to estimate the survival distribution. Tick marks indicate censored data. The comparison test chosen for analysis of survival was the log-rank test. Effects were expressed as hazard ratios (HRs) with 95% confidence intervals (CIs). Cox proportional hazard models included established prognostic factors: age, FIGO status, lymph node metastasis, residual disease, Aletti’s SCS, and complications. In the multivariate analysis, missing data were considered as absent. A Of the 1171 women with ovarian cancer who received surgical treatment during the study period, 144 were excluded from analysis because of no surgical treatment (41 with only chemotherapy and seven patients without any curative treatment), a non-epithelial ovarian carcinoma or no FIGO stage reported. The distribution of the remaining 979 women in the three age groups was as follows: 615 women (62.8%) aged 65 years or younger, 225 women (22.6%) aged 65–74 years, and 139 women (14.2%) aged 75 years or older .p < 0.001). The ASA score was higher in the oldest group: 32/139 (23%) vs. 51/615 (8%) in the young group (p < 0.001). The tumor characteristics are reported in p = 0.048) (p = 0.033). There was no significant difference concerning the presence of lymph node metastasis (The mean age at diagnosis was 60.1 years (±12.7). The demographic and clinicopathologic characteristics of the whole cohort by age group are reported in = 0.048) . Patienttastasis .p < 0.05). According to the surgical procedure groups described by Luyckx [p < 0.001)). The very elderly patients had significantly fewer pelvic and para-aortic lymphadenectomies  with fewer lymph nodes removed among those who underwent a para-aortic lymphadenectomy (p < 0.05). The very elderly also had less upper-abdominal surgery  than the elderly  and young patients  (p < 0.05). Only 9% (13/139) of the very elderly had an Aletti’s SCS higher than eight compared with 16% and 22% for the elderly and young groups, respectively (p < 0.001). There was more residual disease in the two older groups (30%) than for younger women (20%) (p < 0.05).The surgical procedures are described in y Luyckx , the verp = 0.084). When chemotherapy was conducted, the very elderly were administered fewer cycles (neoadjuvant plus adjuvant) compared with the elderly and the young: 23% of the very elderly received fewer than six cycles vs. 10% in the two other groups (p = 0.003). Only 12% (11/139) of the very elderly women received bevacizumab compared with 26% (112/615) of the young patients and 20% (30/225) of the elderly (p < 0.05).Adjuvant treatments are displayed in p = 0.164 for minor complications and p = 0.567 for major complications). No significant difference in the rate of postoperative complications was observed  whether in terms of transfusion, digestive, urinary and pulmonary complications, complications of the abdominal wall, infections or repeat surgery. No patients died in the young group whereas one patient died postoperatively in each of the older groups (There was no difference in minor and major Clavien-Dindo complications in the three groups (r groups .The mean follow-up was 36 months (±29.3). Recurrences were observed in 511 of the 979 women (52.2%) with a mean time of recurrence of 26.68 months (±24.7). In the whole population, the 3-year DFS was 44% (95% CI: 39.8–48.8), the 3-year CSS was 76.4% (95% CI: 72.4–80.5) and the 3-year OS was 71% (95% CI: 67.4–75.2) . Three-yp < 0.001), residual disease  and lymph node metastasis  were significantly associated with a lower OS. The multivariate analysis is shown in Univariate analysis for the 3-year OS showed that an age over 65 years, a FIGO stage III  or IV   independently of age [The fact that elderly patients with more comorbidities have similar perioperative complications suggests that surgeons consider these patients to be frailer and perform less radical surgery to limit morbidity . Howevery of age , and they of age ,27. Withy of age , and 21.y of age ,13). Basy of age . Cloven y of age .This may explain the similar OS and CSS rates for both age groups older than 65 years and would seem to indicate that 65 years marks a threshold of two groups of ovarian cancer patients that require different guidelines of therapeutic management. However, adapting standardized treatment guidelines for women over 65 years with ovarian cancer is hampered by a lack of evidence-based medicine as elderly patients are historically under-represented in clinical trials  30,31,3,331,32. Adjuvant treatment in this population is also a subject of debate. In our study, very elderly patients received less adjuvant treatment than their younger counterparts and fewer cycles. Although several studies ,35,36,37p = 0.07), implying that patient care is influenced, at least in part, by subjective evaluation of health status. The high burden of medical comorbidities, financial and geographic barriers to care, as well as patient preferences, may influence treatment and survival [One of the strengths of our study is the high number of patients included from eight centers. However, our study has limitations that need to be considered when interpreting the data. We did not have validated cut-offs to categorize patients as “elderly” or “very elderly,” so comparison with other studies is difficult. However, there is no current consensus to define elderly women. We chose to classify women into three groups—“young” (<65 years), “elderly” (65–74 years), and “very elderly” (≥75 years)—whereas Yancik et al.  made thesurvival . Neverthsurvival ,50: a resurvival , the plasurvival , and thesurvival .The incidence of ovarian cancer is increasing with the aging of the population. Currently, elderly and very elderly women are at risk of incomplete surgery with less upper abdominal surgery and more residual disease, despite having more aggressive ovarian cancer. These elements are prognostic factors for women’s survival regardless of age. Moreover, they also have less adjuvant therapy (chemotherapy and bevacizumab) because they are perceived as being too frail to tolerate these treatments. Elderly oncology patients should undergo individual oncogeriatric assessment to determine the risk/benefit balance of therapy. To do so, specific trials dedicated to the elderly should be performed to generate evidence-based medicine and guidelines for the management of elderly patients with cancer, specifically in ovarian cancer, which requires aggressive surgery and systemic treatment.