Polymorphonuclear neutrophils (PMNs) play an essential role in the innate immune response, and their number increases after prolonged inflammatory diabetic wounds and prolonged wounds in older rats. The expression of CD80 and CD86 on PMNs confirms their participation in acquired immunity, wherein these molecules are involved in antigen presentation. MIP-2) and MIP-1α by real-time polymerase chain reaction (PCR) in diabetic wound, which was healed by a camel milk peptide (CMP). The animals were allocated to the following wounded groups: control, diabetic (DM), and diabetic treated with CMP (DM-CMP).  We investigated CD80 and CD86 expression on PMNs by flow cytometry and analyzed the mRNA expression of neutrophil chemoattractants macrophage inflammatory protein-2 (MIP-2 mRNA expression in DM rats. However, neutrophil number at wounded sites of DM rats declined at day 1 after wounding as compared to that in control rats. MIP-2 mRNA expression and neutrophil number were restored in CMP-treated diabetic rats.  Alkaline phosphatase, gamma-glutamyl transpeptidase, and lactate dehydrogenase levels were elevated in DM rats but decreased in peptide-treated rats. The expression of CD80 and CD86 was significantly higher in DM rats with prolonged wounds than in control rats. The expression of both markers was restored to normal levels in diabetic rats treated with CMP. RT-PCR analysis revealed the upregulation in  Prolonged wound stress induced toxicity in DM rats and significantly increased the expression of CD80 and CD86 on PMNs. CMP peptide ameliorated the levels of toxicity markers, CD80 and CD86, and chemoattractant molecules in diabetic rats. HoweverDuring the inflammatory stage of wound healing, neutrophils are responsible for microbial clearance at the site of wound infection. Further, they also contribute to antigen presentation, phagocytosis, and production of inflammatory cytokines and growth factors . Hence, 2+, exocytosis , and respiratory burst.Interleukin-8 (IL-8) is an important interleukin that promotes phagocytosis following recruitment of neutrophils and macrophages at wounded site. It is also a potent promoter of angiogenese and induces a series of physiological responses in target cells that are needed for migration and phatocytosis, including upregulation in the level of intracelluar of CaMacrophage inflammatory protein- (MIP-) 2, also known as chemokine ligand (CXCL2), is one of the CXC chemokines that alters the recruitment and activation of neutrophils through the p38/mitogen-activated protein kinase-dependent signaling pathway via binding to its specific receptors, CXCR1 and CXCR2. This protein is synthesized in different cells such as macrophages, monocytes, epithelial cells, and hepatocytes during pathogenic invasion or tissue injury. As MIP-2 plays an important role in the regulation of neutrophil infiltration and microabscess formation, information on its functions and signaling network may generate novel ideas to cease chronic inflammation and associated complications [In general, neutrophil number gradually decreases at wounded sites as healing progresses. These cells disappear in the remodeling phase when inflammation and/or bacterial load is reduced. However, we have previously noted that PMN population gradually increases in diabetic rats  and oldeα and Fas, resulting in improved pancreatic ß-cell structure and function. WP has been shown to be able to regulate impaired wound healing normally [Rayner et al.  suggestenormally . Moreovenormally . In addinormally .g for 20 min. The milk was acidified to pH 4.3, and the casein obtained was collected. The supernatant  was further saturated with ammonium sulfate. The WP was collected and dialyzed for 48 h at 4°C using a Spectra/Pro® Membrane, MWCO 6000–8000 Da. The WP was finally lyophilized using a Unitop 600 SL  and stored at −20°C until use [Camel milk was centrifuged at 5000 × ntil use . A 2.5% ntil use .n = 15). Two groups, wounded normal (CN+) and wounded diabetic (DM), were daily administered with the vehicle solution of phosphate-buffered saline  by gastric intubation for 2 days (n = 5), 4 days (n = 5), or 7 days (n = 5). The third group included wounded diabetic rats that were daily treated with CMP. Rats of the third group were supplemented with CMP in a dose of 100 mg/kg of body weight via gastric intubation. The fourth group comprised wounded diabetic rats daily treated with 10 kDa of hydrolyzed WP at a dose of 100 mg/kg body weight (1000 μL/rat) by gastric intubation for 2 days (n = 5), 4 days (n = 5), or 7 days (n = 5).Male adult albino rats (about 6 months old) were obtained from the Department of Zoology, College of Sciences, King Saud University, Riyadh. The rats were allocated to four groups  in Saudi Arabia. No specific permissions were required for research activities in this farm, as the study did not involve endangered or protected species. All procedures were conducted in accordance with the standards set forth in the guidelines for the care and use of experimental animals by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Institutes of Health (NIH). The study protocol  was approved by the Animal Ethics Committee of King Saud University, Riyadh.Diabetes was induced by a single peritoneal dose of freshly dissolved streptozocin  at 50 mg/kg body weight in 0.1 mol/L citrate buffer (pH 4.5). Control group rats were injected with citrate buffer. After 14 days of STZ injection, rats with glucose level ≥ 220 mg/dL after overnight fasting were carefully chosen as diabetic rat models.The wound models were performed at least one month after diabetes induction. After being anesthetized, the rat's back was shaved and sterilized. The wound model in this study was established as previously described  with somμg anti-CD80-FITC and CD86-PE. Cells were analyzed by the MACS Quant Analyzer (MACS) Miltenyi Biotec and Cell Quest software . All results were expressed as percentage of positive cells in the respective gate.Blood samples were obtained in 7.5 mL heparin-coated tubes  and analyzed within 2 h. Cells were tested in whole blood samples. For fluorescence-activated cell sorting (FACS) analysis of the whole blood, an erythrocyte FACS lysing solution obtained from Becton-Dickinson  was diluted 1 : 10 in bidistilled water. For cytofluorometry, fluorescent-tagged (fluorescein isothiocyanate (FITC) or phycoerythrin (PE)) antibodies were used. For CD80 and CD86 detection, PMNs in the whole blood were stained with 2 The levels of IL-8 in serum samples from experimental groups were determined using the ELISA kits purchased from Abcam Systems, UK. IL-8 concentration was determined using a spectrophotometer at 450 nm wavelength according to the manufacturers' instructions.The data of wound diameter were analyzed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test to compare various groups.γ-glutamyl transferase (GGT), and lactate dehydrogenase (LDH) levels were evaluated for this purpose.Oxidative stress is one of the principle phenomenon involved in the damage incurred by any toxicant or infection. Hence, the study of toxicity markers is vital for toxicological assessment. Alkaline phosphatase (ALP), ALP is an important marker to assess the liver function and the extent of severity of any infection in vivo. We analyzed ALP level in the liver tissues of treated animals and found that group II animals showed a 25% elevation in ALP level. Animals from group III demonstrated a 29.22% increase in ALP level, while those from Group IV showed a decrease in ALP activity by 14.08%. The decrease in ALP activity was more evident in animals from group V  .GGT is a reliable marker to assess the extent of infection and toxicity burden in vivo after treatment with chemicals or drugs. GGT activity significantly increased by 438.44% in group II rats, whereas group III rats showed an increase of 91.19% in GGT activity. Among the peptide-treated groups, group IV showed a decrease of 65.46%, and group V rats demonstrated a decrease of 72.97% in GGT activity as compared with group II rats .LDH activity increased in group II and III rats by 89.06% and 99.68%, respectively. However, group IV rats showed a decrease in LDH activity by 23.47%, while the activity reported for group V decreased by 39.05% as compared to that observed in group II rats .The results shown in We observed a marked decrease in the number of PMNs in the wounds of diabetic rats, consistent with healing impairment. In this group, inflammatory cell number declined in the dermal region after the first 24 h from wounding. Both neutrophil number and inflammatory cell number were restored in the dermal areas of diabetic rats treated with CMP. Wound closure rate was slower in diabetic rats than in control rats. CMP was found to restore the wound closure rate to normal level .IL-8 induces chemotaxis in target cells, neutrophils, and other granulocytes and facilitates migration toward the site of infection. Although IL-8 level was not significantly elevated, its concentration was higher in diabetic rats than in control and CMP-treated diabetic rats .MIP-2 in wounded rats from all groups when neutrophil number peaked at day 1 postwounding as compared to rats from the control group. Although the mRNA expression of MIP-2 significantly increased in diabetic and CMP-treated diabetic rats, the levels were downregulated in all rat groups after 7 days of incision  [Much information is yet to be gathered on the precise role of each immune cell in the various stages of wound healing . Here, wβ) . Furtherβ) .PMNs are important effector cells associated with the host defense and inflammation. They have a short half-life and undergo spontaneous apoptosis in vivo as well as in cultures . Recent mechanism involving β2 integrin macrophage antigen- (Mac-) 1-dependent adhesion [MIP-2 is a macrophage inflammatory protein instrumental in eliciting neutrophil response. Thus, the upregulation in MIP-2 expression may strongly induce the migration and chemotaxis of neutrophils and facilitate the secretion of inflammatory cytokines from macrophages . Our resadhesion , 37. Fro


The aim of this study was to assess the predictive value of five different intrauterine adhesion (IUA) evaluation systems for live birth rate following transcervical resection of adhesion (TCRA).This retrospective study included 128 women with IUA who desired for spontaneous conception after TCRA. All the patients were retrospectively scored by the American Fertility Society (AFS) classification, European Society of Gynecological Endoscopy (ESGE) classification, March’s classification (March), Nasr classification (Nasr) and Chinese IUA diagnosis classification criteria (Chinese). The predictive value of these evaluation systems was determined by receiver operating characteristic (ROC) curves and area under a ROC curve (AUC).P <  0.001). All five scoring systems were efficient to predict live birth rate. Among them, Nasr classification showed the highest AUC (0.748) with the best predictive value. Multivariate logistic regression analyses showed that Nasr classification had the highest OR . And, Nasr’s classification system also showed highest sensitivity (81.8%) and negative predictive value (96.7%) when divide the system into mild IUA vs. moderate and severe IUA.The correlation coefficients of AFS, ESGE, March, Nasr and Chinese classification and the live birth rate were 0.313, 0.313, 0.288, 0.380, and 0.336, respectively. Among women with hypomenorrhea and amenorrhea, as well as women with no infertility, the severities determined by all five evaluation systems were correlated with live birth rate (AFS, ESGE, March, Nasr and Chinese classification were demonstrated to be capable of predicting live birth following TCRA although the predictive capacities might be limited, and Nasr classification showed the highest predictive value of live birth.The online version contains supplementary material available at 10.1186/s12958-021-00697-1. Intrauterine adhesion (IUA) is also called Asherman’s syndrome (AS), which is defined by the presence of adhesions in the uterine endometrium, and lead to hypomenorrhea, amenorrhea, subfertility, miscarriage and abnormal placentation . The domIt is a common sense that severity of IUA is the most critical factor affecting their reproductive outcomes. Numerous evidence suggested that the conception rate, recurrence rate were boosted in women with severe IUA than in mild type , 8. TherTherefore, to figure out an optimal evaluation system for predicting the reproductive prognosis of IUA following hysteroscopic adhesiolysis, we systematically evaluated the predictive value of live birth rate based on five different evaluation systems.This was a retrospective cohort analysis of data from 150 patients with IUA who received transcervical resection of adhesion (TCRA) during January 2014 to December 2014 at Sun Yat -Sen Memorial Hospital. This study was approved by the local Ethics Committee . Informed consent and permission of acquisition of clinical data was obtained from each participant. Information including the age, history of miscarriage, the numbers of the intrauterine operations, the preoperative and postoperative endometrial thickness in the late proliferation, preoperative menstrual amount, change of menstrual amount postoperatively, the number of TCRA, hysteroscopic finding and reproductive outcomes after the surgery were collected through searching the medical record system and follow-up phone calls.The inclusion criteria of IUA patients were as following, (1) women aged ≤40 years old, (2) all patients received TCRA by the same experienced surgeon, (3) all patients was diagnosed as IUA based on three-dimensional transvaginal ultrasound and hysteroscopy, (4) patients with a desire for spontaneous conception and attempted to conceive in the following 2 years after TCRA.The exclusion criteria were as following, (1) breastfeeding women, (2) women with severe systemic diseases such as uncontrolled hypertension and heart disease, diabetes, liver or renal dysfunctions, immune disorders, (3) women with presence of risk factors affecting reproductive outcomes such as obesity, history of gestational diabetes, and gestational hypertension diseases, (4) women with tubal or male factors induced infertility who may require assisted reproductive techniques, (5) women with amenorrhea caused by neuroendocrine disorders, thyroid dysfunctions or endometrial tuberculosis, (6) patients with endometrial polyps, uterine submucosalmyoma or other type of uterine myoma (diameter ≥ 3 cm).The diagnosis of IUA was based on three-dimensional transvaginal ultrasound and hysteroscopy. All patients received TCRA by the same surgeon who had more than 10-year experience on hysteroscopy. All women had general anesthesia during the surgery. The surgical procedure of each woman was picture recorded in the local data center. The Olympus S70 hysteroscopy equipment was used, and either sodium chloride or mannitol was provided as perfusion medium. Adhesiolysis was performed under the direct observation under hysteroscopy and monitored under ultrasonography if necessary. The adhesion tissue was cut by needle electrodes or ring electrodes. The remaining normal endometrium tissue was protected and untouched. The surgical procedure would be finished if adhesion tissue was separated and normal uterine cavity was restored. A heart-shaped COOK intrauterine balloon stent  was inserted into and kept inside uterine cavity after TCRA until the third withdrawal bleeding. All patients were treated with sequential estrogen  and progesterone  therapy for consecutive 3 cycles. The reproductive outcomes of those patients were obtained from medical record and patients were followed up through phone call interview.Here in this study, five IUA diagnosis classification criteria were compared, including The American Fertility Society classification of intrauterine adhesions  , EuropeaWomen’s reproductive outcomes were checked through medical record and phone call interview. Clinical pregnancy was determined as the observation of an intrauterine pregnancy sac with live fetus through ultrasound at gestational 6–7 weeks. Live birth was determined as delivery of at least one live baby after 28-week gestation. The delivery of twin babies by one mother was defined as one live birth. Live birth rate is defined as the proportion of women with live birth out of women with a desire for spontaneous conception.P <  0.05 was considered as statistically significant. Normal distribution was tested for continuous variables. Descriptive parameters with a normal distribution were expressed as mean ± standard deviation (SD). Continuous variables were compared using the independent t-test. Dichotomous data were compared using χ2 test, or using Fisher’s exact test as appropriate. Ranked count data were dealt using Rank-Sum test. Correlation between dichotomous data were examined with logistic regression analysis and Spearman rank correlation analysis, and the odds ratop (OR) value and 95% confidential interval (95% CI) were calculated. Receiver Operating characteristic (ROC) curves were created for each classification system to determine their abilities to predict live birth.All statistical analyses were performed using SPSS 20.0 software . A two-tailed probability value of n = 35) and those with miscarrriage (n = 22) and ectopic pregnancy (n = 3). There were 68 women end up with live birth, and among them, 30 women had Cesarean section and the remaining 38 women had spontaneous vaginal delivery.As illustrated in Fig. P = 0.032).Baseline clinical characteristics of IUA women with and without live birth are documented in Table P <  0.001, see Table Table In the present study, the severities of 128 cases with IUA were evaluated using AFS, ESGE, March, Nasr, and Chinese classification system, respectively. As clearly demonstrated in Table P <  0.001). Moreover, the correlation between live birth rate the all five evaluation systems were adjusted for menstrual pattern before hysteroscopy and history of infertility. As demonstrated in Table P <  0.001). such correlations were not observed among with normal menses and those with infertility history. Although each classification system was related with the live birth rate, the correlation coefficients was small to medium, indicating that other factors may also contribute to the live birth outcome. Among them, Nasr classification showed the highest correlation coefficient .The correlations between these five evaluation systems and live birth rates were determined by Spearman rank correlation analysis. The correlation coefficients of AFS, ESGE, March, Nasr, Chinese classification and the live birth rates were 0.313, 0.313, 0.288, 0.380, and 0.336, respectively  with 14-year follow-up , whereasTo our knowledge, this is the first study to compare the predictive value of live birth rate based on the five evaluation systems including AFS, ESGE, March, Nasr and Chinese classifications. However, this study is limited by its retrospective design and relatively small number of patients included. Besides, certain clinical characteristics, including the gravidity, parity, and body mass index was not obtained due to its retrospective nature. Further, we should also acknowledge the the evaluation for women with multiple TCRA may not represent the original severity of IUA, and may bring potential bias on the interpretation of the outcomes. And finnally, the AUC of most classification systems was not satisfactory enough (AUC <  0.7), indicating other risk factors may also contributing to the live birth outcomes.AFS, ESGE, March, Nasr and Chinese classifications of IUA were demonstrated to be capable of predicting live birth following TCRA, although the predictive capacities might be limited. Among them, Nasr classification might be the optimal evaluation system for predicting live birth following TCRA through natural conception.Additional file 1: Table S1. The American Fertility Society (AFS) classification of intrauterine adhesions, 1988. Table S2. Classification by March 1978. Table S3. European Society of Gynecological Endoscopy (ESGE) classification of IUAs (1995 version). Table S4. Scoring by Nasr 2000. Table S5. Chinese intrauterine adhesions diagnosis classification criteria in 2015.


Gallus gallus, is considered ancestral for all Aves. However, other rearrangements including a duplicated control region and neighboring genes have been reported in many representatives of avian orders. The repeated regions can be easily overlooked due to inappropriate DNA amplification or genome sequencing. This raises a question about the actual prevalence of mitogenomic duplications and the validity of the current view on the avian mitogenome evolution. In this context, Palaeognathae is especially interesting because is sister to all other living birds, i.e. Neognathae. So far, a unique duplicated region has been found in one palaeognath mitogenome, that of Eudromia elegans.Bird mitogenomes differ from other vertebrates in gene rearrangement. The most common avian gene order, identified first in Crypturellus, Rhea and Struthio as well as ND6 pseudogene in three moas. The copies are very similar and were subjected to concerted evolution. Mapping the presence and absence of duplication onto the Palaeognathae phylogeny indicates that the duplication was an ancestral state for this avian group. This feature was inherited by early diverged lineages and lost two times in others. Comparison of incongruent phylogenetic trees based on mitochondrial and nuclear sequences showed that two variants of mitogenomes could exist in the evolution of palaeognaths. Data collected for other avian mitogenomes revealed that the last common ancestor of all birds and early diverging lineages of Neoaves could also possess the mitogenomic duplication.Therefore, we applied an appropriate PCR strategy to look for omitted duplications in other palaeognaths. The analyses revealed the duplicated control regions with adjacent genes in The duplicated control regions with adjacent genes are more common in avian mitochondrial genomes than it was previously thought. These two regions could increase effectiveness of replication and transcription as well as the number of replicating mitogenomes per organelle. In consequence, energy production by mitochondria may be also more efficient. However, further physiological and molecular analyses are necessary to assess the potential selective advantages of the mitogenome duplications.The online version contains supplementary material available at 10.1186/s12864-020-07284-5. Gallus gallus [ND5 and tRNA-Phe genes followed by random losses of one copy of duplicated items. Due to the prevalence of the Gallus gallus gene order in other birds, this rearrangement is generally believed to be an ancestral state for all Aves. In consequence, it is called common, standard or typical.Animal mitochondrial genomes are characterized by compact organization and almost invariable gene content, so any changes in them are especially interesting because they can be associated with major transitions in animal evolution , 2. The s gallus  turned oND5 and tRNA-Phe genes, which seems to be particularly susceptible to duplication.However, the growing number of avian mitochondrial genomes sequenced in recent years has revealed that other gene orders may also be present in a frequency higher than it was previously thought. To date, several distinct variations of mitochondrial rearrangements have been reported in a lot of representatives of many avian orders: Accipitriformes , 5, BuceThe most fully duplicated region  has been found only in one representative of palaeognaths, namely Eudromia elegans [Struthio, Rhea, Casuarius, Dromaius and Crypturellus. The new data help to elucidate the evolution of the Palaeognathae mitogenome in terms of duplication events, and also have implications for mitogenomic evolution in Aves as a whole.So far, a duplicated region   to 26 bp for Struthio camelus  amplification. Based on the obtained sequences of ilomelos . This ge species . The tanCasuarius casuarius and Dromaius novaehollandiae, an attempt to amplify the CR1/CR2 fragment was also made. Similar to other taxa, species-specific D-F and D-R primers  between these sequences along the 84-bp alignment , this insertion is preceded by a fragment (with 433–450 bp) almost identical with the first ND6 copy followed by a highly degenerated region. This similar sequence pattern prompted us to search for potential ND6 pseudogenes within the 5′ spacers of CRs in Anomalopteryx didiformis, Emeus crassus and Dinornis giganteus. The comparison of 5′ CR sequences with appropriate ND6 genes of these species revealed a significant similarity between the aligned sequences  to 97.8% (Crypturellus tataupa). The difference is mainly located at their 3′ ends, except for Rhea taxa, whose control regions differ also at their 5′ ends  and next, Rheiformes (Rheidae) diverged. Dinornithiformes (Dinornithidae + Emeidae) is grouped with Tinamiformes (Tinamidae), whereas Casuariiformes (Dromaiidae + Casuariidae) is sister to Aepyornithiformes (Aepyornithidae) + Apterygiformes (Apterygidae). Almost all nodes are very well supported. The least significant are two nodes: one clustering Casuariiformes, Aepyornithiformes and Apterygiformes, and the other encompassing the palaeognath lineages separated after the divergence of Struthio and Rhea. Nevertheless, these two nodes obtained the highest posterior probability in MrBayes analysis, i.e. 1.0 and support in the Shimodara-Hasegawa-like approximate likelihood ratio test (SH-aLRT) equal to 93 and 78, respectively.Three phylogenetic methods applied for the mitogenomic sequences resulted in a consistent topology Fig. . The earIn order to eliminate a potential artefact related with the compositional heterogeneity in the third codon positions of protein-coding genes, we created phylogenetic trees based on the RY-coding alignment Fig. . The treRhea clade was grouped with Casuariiformes + Apterygiformes. However, the support of this grouping was very weak and occurred only in MrBayes tree and IQ-TREE consensus bootstrap tree based on the alignments without seven and eight highest rate categories, respectively. A greater Bayesian support (0.95–0.97) was obtained by the node encompassing rheas with Casuariiformes in the topology t3 based on the alignments after removing three, four and five highest rate categories. This topology was also produced in MrBayes using the alignment without eight highest rate categories and in IQ-TREE for the alignments without four, five and six highest rate categories. However, the node support was generally weak. The topology t4 was produced only by PhyloBayes for the alignments without two, three, four, five, seven and eight highest rate categories. As in the topology t1, the Rhea clade was also sister to all other palaeognaths excluding Struthio, but Casuariiformes were clustered with the rest non-ostrich palaeognaths, not directly with Aepyornithiformes and Apterygiformes. The posterior probability values of the clade including palaeognaths sister to rheas did not exceed 0.8. The topology t5 differed from the others because Struthio camelus was placed within other Palaeognathae and the external position was occupied by Dinornithiformes + Tinamiformes, whereas Rhea was grouped with Casuariiformes. This topology was obtained for the alignments without three (in IQ-TREE) and six highest rate categories (in MrBayes and IQ-TREE). Nevertheless, the controversial nodes were poorly supported.Moreover, we performed phylogenetic analyses based on ten alignments, from which we sequentially excluded partitions characterized by the highest substitution rate  and maximum likelihood (ML) methods Fig. .Fig. 6RP > 0.982 of this state for the last common ancestors of all palaeognaths and non-ostrich palaeognaths. The last common ancestor of the remaining groups, i.e. Dinornithiformes, Tinamiformes, Casuariiformes, Aepyornithiformes and Apterygiformes, could also contain a duplication with P = 0.899. However, the last common ancestor of Casuariiformes, Aepyornithiformes and Apterygiformes lost this duplication (P = 0.914). In turn, the last common ancestor of Dinornithiformes and Tinamiformes still had the duplicated region (P = 0.983), which was probably lost in Tinamus, whereas Anomalopteryx, Dinornis and Emeus maintained only a pseudogenized ND6.Two methods applied to the topology t1 clearly indicated that the last common ancestor of palaeognaths contained a duplicated region in its mitogenome Fig.  a and b.P = 0.996) as well as the last common ancestor of Rheiformes, Casuariiformes, Aepyornithiformes and Apterygiformes (P = 0.906). Among four latter orders, only Rheiformes maintained the duplication, whereas the last common ancestor of the other orders had a mitogenome without the duplicated region (P = 0.914).According to the topology t2, the last common ancestors of all palaeognaths and non-ostrich palaeognaths also had the duplication in their mitogenomes with the probability of at least 0.982 Fig.  c and d.ND6 pseudogene in Anomalopteryx, Dinornis and Emeus are equivocal. Therefore, we conducted analyses in which we assumed that mitogenomes of these genera had already lost the duplication  and Turnix velox (MK453380.1). However, our annotation and reannotation of another mitogenomes from Alca torda (CM018102.1), Sterna hirundo (CM020500.1) and Uria aalge (MN356418.1) revealed the presence of the most fully duplicated avian region (GO-FD) or its variant (GO-FDr), in which the second control region is remnant , Ciconiiformes (Ciconia nigra), Gaviiformes , Podicipediformes (Podiceps cristatus) and Sphenisciformes (Spheniscus demersus) (Table Apus apus (Apodiformes), Corythaixoides personatus (Musophagiformes) and Podiceps grisegena (Podicipediformes)  and Puffinus lherminieri (Procellariiformes) mitogenomes deposited in GenBank database revealed the presence of GO-FD  as well as Ketupa blackistoni and Ketupa flavipes (Strigiformes) after reannotation of their duplicated gene rearrangements , representing the typical avian gene order , so there is enough space for duplicates. Moreover, the length of this fragment corresponds well to those proved to contain two CRs in 46 representatives of avian orders closely related to Eurypygiformes, i.e. Charadriiformes, Gaviformes, Gaviiformes, Pelecaniformes, Phoenicopteriformes, Procellariiformes, Suliformes and Suliuformes [Eurypyga helias. This fragment should contain an additional but remnant CR and likely some pseudogenes, because no amplicon was obtained in diagnostic CR1/CR2 reactions  and obtained in this study indicate that mitogenomes with sequenced duplications are distributed in 32 out of 43 avian orders . It includes the repetition of zed Fig. . This rezed Fig. . The lenzed Fig.  because Whether the duplicated region is only a neutral trait or can provide a real selective advantage is an interesting question. Previous studies have suggested that bird species having mitogenomes with the duplicated control region can be characterized by longer life-span , 121 as The obtained results indicate that duplicated control regions with adjacent genes are more common in Palaeognathae mitochondrial genomes than it was previously thought. What is more, this feature was most likely present in the last common ancestor of this avian group. Once the duplication occurred, it was preserved during the evolution of Struthioniformes, Rheiformes and some Tinamiformes. The duplicated regions were subjected to concerted evolution, which resulted in homogenization of some parts of duplicated regions and degeneration of others. Reconciliation of the mitogenome-based phylogenetic tree with a probable species tree based on nuclear markers suggests that the evolution of mitogenomes in Palaeognaths could have involved different mitogenomic variants in one cell, i.e. heteroplasmy, in a short period of time.The distribution of mitogenomes with duplications across the avian phylogenetic tree implies that the last common ancestors of not only Palaeognathae but also major Neognathae groups and even all known Aves could have had a mitogenomic duplication, which was then inherited by many modern lineages. The presence of duplicated regions in mitogenomes can be a neutral feature associated with the mechanism of replication and recombination or can give a selective profits, e.g. more effective energy production by mitochondria. However, it needs further studies in many avian representatives.Blood, muscle and feather samples from avian species were obtained thanks to courtesy of European zoological gardens, universities, foundations, aviculture parks, animal rehabilitation centers and private breeding facilities Table . They weChrysolophus pictus  and 4–25 for Neoaves representatives  or ND5/CR1 fragments   or CR2/12S fragment (for Crypturellus tataupa) . Finally, the whole duplicated mitogenomic regions of Struthio camelus, Rhea americana, Rhea pennata and Crypturellus tataupa were amplified in four overlapping fragments , following program was used: 94 °C for 5 min; 94 °C for 30 s, 56–62 °C for 30 s, 72 °C for 120 s repeated 35 times; and 72 °C for 5 min. In the case of all other fragments the reaction conditions were as follows: 94 °C for 5 min; 94 °C for 30 s, 56–62 °C for 30 s, 72 °C for 180 s repeated 35 times; and 72 °C for 5 min. For each amplified fragment, the appropriate amount of the PCR reaction mixtures was cleaned with the use of Clean-up Kit (A&A Biotechnology) to obtain the final volume of 100 μl with the concentration of at least 50 ng/μl. The two DNA strands of the cleaned PCR products were sequenced using the Primer Walking method . Overlaps between three or four fragments amplified for each species were sufficient to assemble the whole mitogenomic regions including duplicated elements with the use of appropriate avian reference mitogenomes containing GO-FD  gene orders.The PCR amplifications were performed in 25 μl reaction mixture containing 50 ng of the DNA template, 1 U DreamTaq Green DNA Polymerase (Thermo Fisher Scientific), 2.5 μl of 10 x buffer, 0.6 μl of 10 mM dNTPs, and 0.6 μl of each primer (10 μM). In the case of diagnostic fragments , Anseranas semipalmata (NC_005933.1), Crax daubentoni (NC_024617.1), Gallus gallus (NC_001323.1) and Numida meleagris (NC_034374.1). The sequence records were downloaded from the GenBank database. The analyses were based on nucleotide sequences of 13 protein coding genes, genes for 12S and 16S rRNAs, as well as 22 tRNAs. Sequences of the control region (CR) were excluded due to their high variation. The sequences were aligned in MAFFT using a slow and accurate algorithm L-INS-i with 1000 cycles of iterative refinement [Phylogenetic relationships between Palaeognathae were inferred based on all available 19 complete or almost complete mitochondrial genomes Table . Five refinement . The resfinement  and sitefinement . The conWe applied three phylogenetic approaches in the phylogenetic analyses: the maximum likelihood method in IQ-TREE , as wellThe ModelFinder program associated with IQ-TREE , 134, prIn MrBayes, we assumed 11 substitution models for the appropriate partitions as proposed by PartitionFinder . HoweverIn PhyloBayes, we applied the CAT-GTR + Γ5 model with the number of components, weights and profiles of the model inferred from the data. Two independent Markov chains were run for 100,000 generations with one tree sampled for each generation. The last 50,000 trees from each chain were collected to compute posterior consensus trees after reaching convergence, when the largest discrepancy observed across all bipartitions (maxdiff) was 0.0055.To reduce a potential compositional heterogeneity in sequences related with AT or GC bias, we recoded respective nucleotides for purines (R) and pyrimidines (Y) in the third codon positions of protein-coding genes. In MrBayes and PhyloBayes, we adopted the assumptions as described above, whereas in IQ-TREE, we applied 11 partitions with individual substitution models proposed by ModelFinder , Rhea pennata (b), Rhea americana (c) and Crypturellus tataupa (d). The numbering of amplicons separated in the agarose gel corresponds to the reaction numbered in Table Fig. S2. Sequence comparison of two copies of tRNA-Pro, ND6from, tRNA-Glu genes and control region, found in this study as duplicated, with appropriate sequences of mitogenomes previously deposited in GenBank and showing a typical avian gene order. The alignments are shown for Struthio camelus (a), Rhea americana (b), Rhea pennata (c) and Crypturellus tataupa (d). Dots indicate residues identical in the obtained copies with those in the single sequence previously published. Genes for tRNA-Pro are marked in yellow coloring, NADH dehydrogenase subunit 6 in cyan coloring, tRNA-Glu in grey shading, and control regions in red fonts. In the case of Crypturellus tataupa species only two obtained copies are compared to each other because the previously published mitogenome of these taxa is incomplete and it does not contain any of the analyzed genes or the control region. The sequences were aligned with MUSCLE [Fig. S3. Sequences of mitogenomic fragments amplified and sequenced in this study for Struthio camelus (MH264503), Rhea americana (MK696563), Rhea pennata (MK696564) and Crypturellus tataupa (MK696562). Genes for tRNA-Leu are marked in bold, NADH dehydrogenase subunit 5 in magenta coloring, cytochrome b in red coloring, tRNA-Thr in green coloring, tRNA-Pro in yellow coloring, NADH dehydrogenase subunit 6 in cyan coloring, tRNA-Glu in grey shading, 12S rRNA in blue fonts, tRNA-Val in brown fonts, 16S rRNA in green fonts, tRNA-Phe are in bold and underlined, and control regions in red fonts. Fig. S4. Cropped agarose gels of potential NUMTs amplified for Dromaius novaehollandiae (a) and Casuarius casuarius (b) with the use of primers designed for detection of CR1/CR2 fragments. The numbering of lanes corresponds to the reaction numbered in Table Fig. S5. Comparison of selected sequence associated with reannotation of Palaeognathae mitogenomes. Eudromia elegans gene for tRNA-Pro with the last 200 nucleotides of the control region (a); Emeus crassus, Dinornis giganteus and Anomalopteryx didiformis control region 5′ spacers (b); Probosciger aterrinus (MH133970.1), Eolophus roseicapilla (MH133971.1) and Cacatua moluccensis (MH133972.1) ND6 pseudogenes (c); Anomalopteryx didiformis ND6 gene with control region 5′ spacer (d); Emeus crassus ND6 gene with control region 5′ spacer (e); Dinornis giganteus ND6 gene with control region 5′ spacer (f). Sequences rich in G and A residues are marked in a red box. The sequences in b and c were aligned with MUSCLE [Fig. S6. Sequence comparison of two Eudromia elegans control regions. Dots indicate residues identical in the compared copies. Dots indicate residues identical in the compared sequences. Sequence repeats present in both control regions are marked in red and blue boxes. The sequences were aligned with MUSCLE [Fig. S7. Influence of removing partitions with the highest substitution rate on alignment and tree parameters: the number of parsimony informative sites and mean distance (a) as well as mean support values (b). The mean phylogenetic distance was obtained from MrBayes tree. The mean support values were calculated from posterior probabilities received in MrBayes and PhyloBayes as well as SH-aLRT and non-parametric bootstrap percentages obtained in IQ-TREE. The posterior probabilities were scaled to 100%. Fig. S8. Maximum parsimony  and maximum likelihood  reconstruction of ancestral states and mapping of mitogenomic duplications onto the Palaeognathae tree topology t1 based on sequences of mitochondrial genes  and the Palaeognathae tree topology t2 supported by nuclear markers in coalescent-based approaches . In contrast to Fig. Anomalopteryx didiformis, Emeus crassus and Dinornis giganteus already lost mitogenomic duplication. The area of colors at nodes in b and d corresponds to the probability of the given state, single or duplicated region. Two-colored branches correspond to the equal probability of two states, single or duplicated region. The probability value for a more likely state was also given at these nodes. Mk1 model was applied for ML approach. Fig. S9. Annotated and reannotated duplicated gene orders in mitogenomes of Neoaves and Galloanserae deposited in GenBank for: Alca torda (CM018102.1), Antrostomus carolinensis (MN356120.1), Calypte anna (CM016612.1), Cariama cristata (CM020379.1), Catharus ustulatus (CM020378.1), Colius striatus (MN356125.1), Ketupa blackistoni (LC099104), Ketupa flavipes (LC099100.1), Leptosomus discolour (MN356135.1), Merops nubicus (CM020464.1), Melanereps aurifrons (CM022134.1), Morus serrator (GU071056.1), Nyctibius grandis (CM023771.1), Pterocles burchelli (MN356340.1), Pterocles gutturalis , Puffinus lherminieri (MH206162.1), Rynchops niger (MN356248.1), Sterna hirundo (CM020500.1), Thalassarche chlororhynchos (MN356342.1), Uria aalge (MN356418.1) and Urocolius indicus (MN356373.1). Genes for NADH dehydrogenase subunit 5 are marked in magenta coloring, cytochrome b in red coloring, tRNA-Thr in green coloring, tRNA-Pro in yellow coloring, NADH dehydrogenase subunit 6 in cyan coloring, tRNA-Glu in grey shading, 12S rRNA in blue fonts, tRNA-Phe in bold fonts, and control regions in red fonts. Pseudogenes are in italics and underlined. Microsatellites present within Pterocles gutturalis mitogenomes between tRNA-Thr gene and tRNA-Pro pseudogene or tRNA-Thr gene and ND6 pseudogene are marked in brown. Fig. S10. Sequences of mitogenomic fragments in Neoaves and Galloanserae mitogenomes amplified and sequenced for: Alca torda (MK263222), Apus apus (MW151827), Cathartes aura (MN629891), Chrysolophus pictus (MW151829), Ciconia nigra (MH264509), Corythaixoides personatus (MW082596), Gavia arctica (MK263210), Gavia stellata (MK263209), Eurypyga helias (MW208859), Podiceps cristatus (MN629890), Podiceps grisegena (MK263194), Scopus umbretta (MW151828), Spheniscus demersus (MH264510) and Uria aalge (MK263188). Genes for NADH dehydrogenase subunit 5 are marked in magenta coloring, cytochrome b in red coloring, tRNA-Thr in green coloring, tRNA-Pro in yellow coloring, NADH dehydrogenase subunit 6 in cyan coloring, tRNA-Glu in grey shading, 12S rRNA in blue fonts, tRNA-Phe in bold fonts, and control regions in red fonts. Pseudogenes are in italics and underlined. Fig. S11. Representative cropped agarose gels of the following amplicons: cytb/12S rRNA obtained for Rhynochetos jubatus (a), Eurypyga helias (d) and Trogon collaris (f); CR1/CR2 obtained for Rhynochetos jubatus (c) and Trogon collaris (g); CR/12S rRNA obtained for Rhynochetos jubatus (b) and Eurypyga helias (e); cytb/CR obtained for Eurypyga helias (e). The numbering of amplicons separated in the agarose gel corresponds to the reaction numbered in Table Fig. S12. Reconstruction of ancestral states and mapping of mitogenomic duplications onto the Aves three tree topologies under a more liberal assumption for assignment of duplication states. The colored thick lines of the trees correspond to maximum parsimony reconstruction, whereas values at nodes mean the probability of a more likely ancestral state  provided by maximum likelihood reconstruction. The trees obtained by Prum, et al. [Fig. S13. Original uncropped and unprocessed agarose gels of: CR1/CR2 amplicons obtained for Struthio camelus (a), which were also shown in the Fig. Rhea pennata (b) which were also shown in the Fig. Rhea americana (7 lanes to the left of DNA Ladder) (c), which were also shown in the Fig. Crypturellus tataupa (6 lanes to the right of the DNA Ladder) (d), which were also shown in the Fig. Dromaius novaehollandiae (e), which were also shown in the Fig. Casuarius casuarius (f), which were also shown in the Fig. cytb/12S rRNA amplicons obtained for Rhynochetos jubatus (g), which were shown in the Fig. 12S rRNA amplicons obtained for Rhynochetos jubatus (h), which were shown in the Fig. Rhynochetos jubatus (i), which were shown in the Fig. cytb/12S rRNA amplicon obtained for Eurypyga helias (j), which was shown in the Fig. cytb/CR and CR/12S rRNA amplicons obtained for Eurypyga helias (k), which were shown in the Fig. cytb/12S rRNA amplicon obtained for Trogon collaris (the first lane prior the first DNA Ladder) (l), which was shown in the Fig. Trogon collaris (m), which were shown in the Fig. h MUSCLE  in MEGA h MUSCLE . Fig. S3h MUSCLE  in MEGA h MUSCLE , whereash MUSCLE . Fig. S6h MUSCLE  in MEGA h MUSCLE . Fig. S7elus MH2603, Rhea , et al.  in c. ThAdditional file 2: Table S1. Characteristics of primers and PCR reactions for amplification of CR1/CR2 fragments. Sequences of primers, their internal laboratory numbering and naming as well as positions according to reference mitogenomes are included. Reactions that failed are marked with an asterisk. Reactions whose products were finally sequenced are marked in grey shading. Table S2. Characteristics of control regions in Palaeognathae species based on mitochondrial fragments obtained in this study and previously published mitogenomes. Table S3. Characteristics of alignments between ND6 copies for various avian taxa. Table S4. Substitution models, partitions and their relative rate in MrBayes analysis. Table S5. Results of tests comparing various palaeognath tree topologies. The topology t1 corresponds to the best tree found for the full alignment. The topologies are shown in Fig. p-value from an approximately unbiased test (AU), the bootstrap probability calculated from the multiscale bootstrap (NP), the bootstrap probability calculated in the usual manner (BP), Bayesian posterior probability calculated by the BIC approximation (PP), p-value of the Shimodaira-Hasegawa (SH) and the weighted Shimodaira-Hasegawa tests (WSH), Bayes factor (BF) expressed in natural logarithm units as differences between marginal likelihoods of the given and the topology (t1). Table S6. Number of known mitogenomic sequences and species in terms of duplication for individual avian orders. Table S7. Avian species in which GO-FD gene order was identified in their mitogenomes. Previous annotations assuming single, i.e. without duplication, version were also included. Incomplete mitogenomes are marked with an asterisk. Table S8. Substitution models and partitions used in IQ-TREE analysis. Table S9. Characteristics of primers and PCR reactions for amplification of mitochondrial fragments from Neoaves and Galloanserae representatives. Sequences of primers, their internal laboratory numbering and naming as well as positions according to reference mitogenomes are included. Reactions that failed are marked with an asterisk. Reactions whose products were finally sequenced are marked in grey shading. Table S10. Substitution models and partitions used in IQ-TREE analysis for RY-recoded sequences.


A large number of patients suffer recurrence after curative resection, and mortality from colon cancer remains high. The role of systemic inflammatory response, as reflected by neutrophil-to-lymphocyte ratio (NLR), in cancer recurrence and death has been increasingly recognized. This study aimed to analyze long-term oncologic outcomes of Stage II-III colon cancer to examine the prognostic value of NLR using a propensity score analysis.A total of 375 patients with colon cancer underwent radical surgery between 2000 and 2014 at Tokyo Medical University Hospital. Long-term oncologic outcomes of these patients were evaluated according to NLR values. A cut-off NLR of 3.0 was used based on receiver operating characteristic curve analysis. Primary outcomes were overall survival (OS) and relapse-free survival (RFS). An analysis of outcomes according to tumor sidedness was also performed.n = 135) and 87.0% in the higher NLR group (n = 135), showing a significant difference (p = 0.042). Five-year RFS rates were 87.8% in the lower NLR group and 77.9% in the higher NLR group, also showing a significant difference (p = 0.032). Among patients with left-sided colon cancer in the matched cohort, 5-year OS and 5-year RFS rates were 95.2 and 87.3% in the lower NLR group (n = 88), respectively, and 86.4 and 79.2% in the higher NLR group (n = 71), respectively, showing significant differences .Patients with lower NLR values (“lower NLR group”) were more likely to have lymph node metastasis compared to those with higher NLR values (“higher NLR group”) before case matching. After case matching, clinical outcomes were similar between the two groups. There were no significant differences in 5-year OS and 5-year RFS rates between the two groups before case matching based on propensity scores. After case matching, 5-year OS rates were 94.5% in the lower NLR group (The NLR is an important prognostic factor for advanced colon cancer, especially for left-sided colon cancer. However, many patients suffer recurrence after curative surgical resection, and mortality rates from colon cancer remain high. The role of systemic inflammatory response in cancer recurrence and death has been increasingly recognized . In addiThe present study aimed to analyze long-term oncologic outcomes of patients with Stage II-III colon cancer at our institute to examine the prognostic value of NLR using a propensity score analysis.Medical records of 422 patients who underwent radical surgery for Stage II-III colon cancer between 2000 and 2014 at Tokyo Medical University Hospital were retrospectively reviewed. Of these, 47 patients without NLR values were excluded, and the remaining 375 patients were divided into two groups based on NLR values (lower NLR and higher NLR groups). In general, patients were admitted to our hospital two days before operation, and NLR values were obtained on the day of admission. According to receiver operating characteristic (ROC) curve analyses and previous reports, a cut-off value of 3.0 was set for both mortality and recurrence  = 0.521, 95%CI 0.449–0.592; recurrence: AUC = 0.502, 95%CI 0.423–0.581). This study was approved by the institutional review board of Tokyo Medical University Hospital.All patients underwent curative surgery. Gross specimens were opened up along the antimesenteric border, and lymph nodes were harvested from the mesocolon. The specimens were then laid out on a board and fixed in 10% formalin.Postoperative systemic adjuvant chemotherapy is generally performed for pStage III colon cancer at our institute. In the present study, adjuvant chemotherapy was performed in 19 of 216 patients (8.8%) with pStage II disease and 86 of 202 (42.5%) patients with pStage III disease. Oxaliplatin-based and 5-fluorouracil-based regimens were most commonly used.Median follow-up period was 73.2 months . For follow-up, patients with Stage II or III tumors were examined for up to five years postoperatively. Specifically, tumor marker measurements were performed every three months for the first two years, and both tumor marker measurements and CT scans were performed every six months for the next three years. When the first recurrence occurred, the recurrence site and date were recorded.p < 0.05.Primary outcomes were 5-year overall survival (OS) and 5-year relapse-free survival (RFS). OS was defined as the interval between the date of operation and the date of either death or the end of the observation period. Patients alive at the end of follow-up were censored. RFS was defined as the interval between the date of operation to date of recurrence, or death from underlying disease. Observations were censored when patients died for a reason other than colon cancer. Survival characteristics were assessed using the Kaplan-Meier method and were compared using the log-rank test. Propensity score analyses were performed to adjust for heterogeneity between two groups by NLR. Multivariate logistic regression was used to generate a propensity score predicting condition by NLR (NLR > 3.0 or NLR ≤ 3.0). The following six covariates were included: age, sex, body mass index (BMI), tumor size, pathological T-stage, and pathological lymph node metastasis. Each patient was assigned an estimated propensity score, which represented the patient’s predicted probability of the NLR status. We specified matching IDs based on the propensity scores. According to the matching IDs, we divided the patients into two groups by their NLR values, in which patients were paired by similarities in their characteristics. Then, propensity score matching was performed. Each patient with a NLR < 3.0 (lower NLR) was matched to a patient with a NLR ≥ 3.0 (higher NLR) and had the closed propensity score on the logit scale with a caliper of 0.05. Standardized mean difference (SMD) was calculated to evaluate variable balance after propensity matching. Propensity scores were used for regression adjustment, in which the treatment effect was estimated by adjusting for the impact of background covariates in a regression model. We also performed analyses by dividing patients into two groups according to tumor sidedness . All statistical analyses were performed using SPSS software . The level for statistical significance was set at n = 375). There were 230 patients in the lower NLR group and 145 patients in the higher NLR group. The median NLR was 3.0  after case matching .The median NLR was 2.5  for the entire cohort (Table p = 0.006). No significant differences were observed in other covariates.Baseline characteristics are summarized in A matched analysis was performed according to propensity scores to adjust for heterogeneity in the lower NLR and higher NLR groups, with six covariates . Distributions of propensity scores before and after case matching are shown in Fig. n = 375), hazard ratios (HRs) for OS and RFS in NLR > 3.0 versus NLR < 3.0 were 1.546 (95%CI 0.879–2.718) and 1.419 (95%CI 0.910–2.215), respectively , respectively, and 87.1 and 77.7% in the higher NLR group (n = 145), respectively, with no significant differences   and the higher NLR group  (p = 0.042) . A significant difference was also observed in 5-year RFS rate after case matching betw0een the lower NLR group (87.8%) and the higher NLR group (77.9%) (p = 0.032) .The analysis of survival data after case matching revealed a significant difference in 5-year OS rate between the lower NLR group , respectively, and 87.8 and 76.5% in the higher NLR group (n = 64), respectively, showing no significant difference  , respectively, and 86.4 and 79.2% in the higher NLR group (n = 71), respectively, showing significant differences   guidelines recommend adjuvant chemotherapy for high-risk Stage II and Stage III colon cancer . High-riThis study has some limitations. First, we used a single-center retrospective design. However, to minimize selection bias, we analyzed the data according to propensity scores. Second, our data did not include records of whether patients had hematologic or autoimmune disease, which may have influenced preoperative NLR values. Third, no molecular assessment was performed in this study, e.g., to determine microsatellite instability. A prospective study will be needed to examine confounders and further clarify the prognostic value of NLR.The present study demonstrated that NLR could be used as a prognostic factor for advanced colon cancer, especially for left-sided colon cancer.


Methotrexate  is a folic acid reductase inhibitor used to treat autoimmune diseases and certain types of cancer. Testicular toxicity resulting from MTX is a significant side effect that may cause subsequent infertility. The present study was conducted to examine the ameliorating effects of vitamin B17 (VitB17) against testicular toxicity induced by MTX in male rats. A total of 50 male albino rats were equally divided into five groups . In methotrexate group (MTX), a significant decrease was observed in body weight and the testicular weight, as well as the levels of plasma testosterone, luteinizing hormone and follicle-stimulating hormone compared with control. The sperm count, viability, morphology index, total motility, and progressive motility also decreased in MTX rats compared with control. Furthermore, the levels of reduced glutathione, catalase, and superoxide dismutase, as well as proliferating cell nuclear antigen protein expression, in the testicular tissue decreased in MTX compared with control. In addition, MTX caused a significant increase in DNA and tissue damage compared with control. However, VitB17 ameliorated these effects, indicating that it has a preventative and curative effect against MTX-induced reproductive toxicity in male rats. The protective effect of VitB17 may be associated to its antioxidant properties as it possibly acts as a free-radical scavenger and lipid peroxidation inhibitor, as well as its protective effect on the levels of GSH, SOD, and CAT. The testes are known to incur injury resulting from exposure to both chemotherapeutic and toxic environmental agents –4. As chMethotrexate  is a folic acid antagonist that has antineoplastic characteristics . MTX achPrunus family and some members of the Rosaceae family, including apricots, apples, almonds, peaches, cashews, and macadamias [Prunus armeniaca seeds can induce apoptosis [Medicinal plants are good sources of exogenous antioxidants which might be considered as the new alternative approach to ameliorate pathological alterations in oxidative stress-related pathology –17. Vitacadamias , 19. Vitcadamias , 21. Duecadamias , 23. A npoptosis . VitB17 poptosis . HoweverThe animals in the study appeared healthy and did not show clinical signs of disease, and no mortality was recorded in the either the control group or the group receiving only VitB17 during the experiment's duration. However, various side effects were observed in animals injected with MTX, such as loss of body weight, lack of activity, weakness, and yellowish body hair. A 15 ± 3.2% mortality rate was recorded in the MTX group, while a 12 ± 1.5% mortality rate was recorded for the animal cotreated group (VitB17+MTX). Interestingly, a 18 ± 2.5% mortality rate was recorded for animals initially treated with MTX and then posttreated with VitB17 (MTX+VitB17).P < 0.05) decrease was recorded in the relative body weight (RBW) and relative testes weight (RTW) for MTX compared with control and VitB17. However, there was a significant increase in these parameters for co- and posttreated groups compared with MTX group.The data summarized in mean ± SE of 5 observations. Significant difference from the control group at ∗P < 0.05. Significant difference from the methotrexate group at #P < 0.05. Relative organ weight = (Organ weight/Body weight) x 100.Data are expressed as As would be expected due to the known toxicity of cancer drug methotrexate, the rat sperm were adversely affected in the group given methotrexate alone (group 3) compared with the control and vitamin B17 groups. All measures  showed that the sperm had deteriorated after methotrexate treatment .However, the result in the vitamin B17 group alone (group 2) was more surprising. Rats dosed with vitamin B17 alone showed improved sperm as measured by a raised sperm count, greater movement, improved morphology, and a decrease in the number of abnormal sperm observed. This was surprising given amygdalin's known poison potential through its cyanide content . MeanwhiP < 0.05) decrease in serum total testosterone, LH, prolactin, and FSH in treated rats with methotrexate when compared with control group  and seminiferous tubules with a regular cycle of spermatogenesis in the rat testicular sections taken from control rats and rats treated with vitamin B17 only , while the other spermatogenic cell types showed negative reaction Figures , and 6. Today, there are many different kinds of chemotherapy that are used for cancer treatments. It is therefore important to search for therapies which can reduce the side effects of anticancer treatments without altering their efficacy or increasing toxicity or damage in target organs , 5, 7. VMost cases of male infertility are due to an altered sperm count or disruptions in the motility and/or morphology of sperm cells , 26. OurOur results revealed significant decreases in serum total testosterone, LH, FSH, and prolactin in MTX compared with control. The lower serum testosterone level in MTX-treated rats could be attributed to the impaired Leydig cells. This finding agrees with Sainath et al.  who repo2O2, and NO, which suggests that vitamin B17 extract has free-radical scavenging and antioxidant properties. Our results revealed a significant increase in testicular DNA fragmentation in rats treated with MTX. However, as shown by the results in co- and posttreated rats, VitB17 significantly decreased testicular DNA fragmentation compared with MTX. Therefore, it can be concluded that VitB17 has a strong potential for use as a therapeutic adjuvant to MTX to prevent gonadotoxicity. In this regard, our results agree with Padmanabhan et al., who reported MTX-induced cytotoxicity and genotoxicity in the germ cells of mice [Oxidative stress plays an important role in the pathogenesis of MTX-induced testicular damage . It lead of mice . Our res of mice . Adminis of mice  who findMTX (Methotrexate®) was obtained from Hospira UK Ltd. (United Kingdom), and VitB17  (CAS number 29883-15-6) was obtained from Cayman  and purity ≥98%.Fifty male albino rats (weighing 140 ± 10 g and aged 11–12 weeks) were bred in the animal facility at Qassim University to be used in this study. Rats were housed in Qassim University's animal house in a controlled and pathogen-free environment (25°C) with free access to water and a standard chow diet. The experiment was conducted as per the standard guidelines for animal studies after obtaining approval from the Institutional Animal Ethics Committee .n = 10 animals per group [control group in which animals did not received any treatment; vitamin B17 (VitB17) group in which rats received VitB17 (175 mg/Kg body weight/day)  orally by stomach tube for four weeks according to Mutar et al. [via an intravenous injection of 100 mg/kg sodium pentobarbital and subjected to a complete necropsy.A total of 50 rats were equally divided into five groups with r et al. ; methotrr et al. ; cotreatBlood samples were individually collected from the inferior vena cava of each rat in nonheparinized glass tubes to estimate the blood parameters. Blood serum was separated by centrifugation at 4000 rpm for 10 minutes. The collected serum was stored at -20°C until analysis. Testes and epididymides were carefully removed, cleaned of adhering connective tissue in cold saline, and weighed. One testis from each pair was quickly stored at -80°C until homogenization for biochemical analysis; the other testis was fixed with neutral buffer formalin solution for histopathological and immunohistochemical examinations. Meanwhile, the epididymides were prepared for fertility evaluation .The serum level of total testosterone was measured using a solid-phase competitive chemo-luminescence enzyme immune assay  . Serum lThe testes and epididymides were carefully removed, cleaned of adhering connective tissue in cold saline, and weighed. The epididymides were prepared for fertility evaluation that assessed the sperm count, spermatozoa motility parameters, and sperm morphology using a computer assisted semen analysis  with an Olympus microscope  . A totalw/v) separately in ice-cold 1.15% KCl in sodium/potassium phosphate buffer  in a Potter-Elvehjem-type homogenizer. The homogenate was centrifuged at 10,000 g for 20 minutes at 4°C, and the resultant supernatant was used for the enzyme assays.Testes tissues were weighed, cut, and homogenized  were mounted on gelatin/chromalum-coated glass slides and stored at room temperature until further processing. Some paraffin sections were used for haematoxylin and eosin (H&E) staining via the routine method [Testes from the different groups were fixed with 10% neutral buffered formalin solution for 24–48 hours. The fixed specimens were then dehydrated, cleaned and embedded in paraffin. Paraffin sections (5 e method .μm) using Avidin–Biotin-Peroxidase immunohistochemical method  with PCNA monoclonal antibody  [Distribution of proliferating cell nuclear antigen immunoreactivity- (PCNA-ir) stained nuclei in kidney tissue was examined in deparaffinized sections  .We determined the PCNA labeling index (PCNA-LI) in the PCNA immunoreactive slides by examination under a light microscope with a magnification 200x and with the help of the Image J analysis software.P values less than 0.05 were considered significant. All statistical analyses were performed using the SPSS Statistical Version 16 software package .Results were analyzed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) tests to compare between the different groups. Data were presented as the mean ± SEM. Administration of VitB17 had a protective and ameliorative effect against MTX-induced testicular toxicity. The protective effect of VitB17 may be associated to its antioxidant properties as it possibly acts as a free-radical scavenger and lipid peroxidation inhibitor, as well as its protective effect on the levels of GSH, SOD, and CAT.


Aedes aegypti is a major vector of arboviruses, including Yellow Fever, Dengue, Chikungunya, and Zika. Vector control is difficult due to this mosquito high reproductive capacity.Successful mating of female mosquitoes typically occurs once, with the male sperm being stored in the female spermatheca for every subsequent oviposition event. The female spermatheca is responsible for the maintenance, nourishment, and protection of the male sperm against damage during storage. Following comparative RNA-seq analyses of spermathecae obtained from virgin and inseminated females, eight transcripts were selected based on their putative roles in sperm maintenance and survival, including energy metabolism, chitin components, transcriptional regulation, hormonal signaling, enzymatic activity, antimicrobial activity, and ionic homeostasis. In situ RNA hybridization confirmed tissue-specific expression of the eight transcripts. Following RNA interference (RNAi), observed outcomes varied between targeted transcripts, affecting mosquito survival, egg morphology, fecundity, and sperm motility within the spermathecae.Ae. aegypti. Using RNAi we characterized the role of eight spermathecal transcripts on various aspects of female fecundity and offspring survival. RNAi-induced knockdown of transcript AeSigP-66,427, coding for a Na+/Ca2+ protein exchanger, specifically interfered with egg production and reduced sperm motility. Our results bring new insights into the molecular basis of sperm storage and identify potential targets for Ae. aegypti control.This study identified spermatheca-specific transcripts associated with sperm storage in  Aedes aegypti (Diptera: Culicidae) is a major disease vector responsible for the transmission of arboviruses, such as Yellow Fever, Dengue, Chikungunya, and Zika. From its pantropic distribution and its role in the transmission of such pathogens, with dengue fever alone being responsible for over 100 million cases annually with 2.5 billion people at risk, attempts to control Ae. aegypti is carried out across much of the tropics and subtropics  and theA. mellifera [+/Ca+2 exchange caused by the KD of AeSigP-66,427 thus support the idea of an ionic basis for control of sperm motility and longevity [AeSigP-66,427 in glandular and epithelial cells, as well as the gametes, and is supported by our findings of the in situ hybridization  and by the presence of the transcript in the spermathecal content.Ion imbalance in the spermathecae has been previously linked to the presence of non-motile sperm in ellifera , 48. Theongevity , 47–49. +/K+ ATPases in Locusta migratoria [2+ ionic channels play an important role in vitellogenin (Vg) uptake in insects, including Ae. aegypti [AeSigP-66,427 Ae. aegypti KD females, there was no evidence of egg development even after blood feeding. We hypothesize that lack in egg development in these females was due to a disruption, at least temporarily, in the acquisition of nutrients from the blood meal [Oocytes accumulate yolk protein precursors whose uptake is directly regulated by Nagratoria . Additio aegypti , 52. Anyood meal .Aedes aegypti is a primary vector for many human diseases. The successful ability of this mosquito as disease vector is directly associated with its high reproductive output. Mosquitoes mate only once, and a single mating event provides enough sperm to fertilize the eggs for the entire reproductive life of the female [As major vectors of important human pathogens, mosquitoes impose an enormous burden on public health. e female . The speAe. aegypti, and highlights aspects of the critical balance between spermatheca gene expression regulation, male sperm viability, and overall insect fertility. In spite of the non-linear and target-dependent effects from our KD experiments, our results provide evidence of the role played at least for some of the selected transcripts in female survival, egg production, and fecundity. Knockdown of the Na+/Ca2 exchanger AeSigP-66,427 in particularly provided strong evidence for the role of this transcript in sperm motility and fecundity. Because the mosquito spermatheca directly influences sperm viability and thus female fertility, understanding of underlying mechanisms related to sperm maintenance and survival by the spermatheca will likely identify potential targets for intervention and vector population control.This study provides a unique catalog of spermathecal transcripts from virgin and inseminated This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use was approved by the Johns Hopkins University Institutional Animal Care & Use Committee (IACUC) (Protocol M018H18), the Johns Hopkins Institutional Biosafety Committee (IBC) (Protocol #DN0305070116), and the Ethics Committee of Universidae Federal de Viçosa (UFV-Protocol 561/2016).Aedes aegypti females  were obtained from a colony maintained in the Departamento de Biologia Geral, Universidade Federal de Viçosa (DBG/UFV), Brazil. For our studies, stored eggs were allowed to hatch in dechlorinated tap water under a 12 h photoperiod at 25 °C ± 0.5 °C. Mosquito larvae were fed with turtle food (Reptolife®), and upon emergence, adults were fed on 10% sucrose solution ad libitum. Adult females were separated on the day of emergence into two cages/groups, one cage containing only females  and another cage containing both males and females , at a two-to-one ratio of males to females. Seven days after emergence, the spermathecae . The read length average was 300 bp. RNA library preparation and sequencing were performed at the Kansas State University’s Integrated Genomic Facility.RNA libraries were prepared with 400 ng of high-quality total RNA using the TruSeq RNA Sample Preparation v.2 kit (Illumina®) diluted to 10 nM/μL (according to manufacturer instructions) and divided into two libraries  per pool. Each pool was sequenced using the MiSeq reagent v3 kit (600 cycles/paired-ends) in MiSeq Illumina (Ae. aegypti coding sequences (version 3.3) and assembled using PSI- BLAST and CAP3 pipeline [P-value package version 3.3.0 from R software [Transcriptome analysis of whole spermathecae was performed according to Ribeiro et al. , 55. Bripipeline . Coding pipeline . Each sppipeline  were appsoftware . The norsoftware . The RPKsoftware . To compsoftware . Proteinsoftware .Ae. aegypti (Rockefeller strain) available in the Department of Immunology and Microbiology (DIM) at the Johns Hopkins University . Following hatching in distilled water, larvae were fed with Cat Chow (Purina®) and maintained under the conditions indicated above. Another group of females was again separated into two groups (virgin and inseminated) and dissected 7 days after emergence. One hundred virgin and inseminated females (300 spermathecae each) were dissected in RNAse-free 0.1 M PBS (pH 7.6), and the total RNA was extracted using TRIzol® . To tease out gene expression in sperm present in the spermathecae, expression levels of the same selected transcripts were also assessed in the luminal content of the spermathecae from inseminated females. For this, reservoirs of 100 inseminated females were disrupted by hand (using forceps), transferred to 1.5 mL microtubes, containing 100 μL of PBS solution, and centrifuged at low speed (below 3000 rpm/956 g for 10 s) to avoid cell damage. The supernatant with the reservoir contents was subjected to total RNA extraction, cDNA synthesis, and expression quantification together with the virgin and the inseminated spermathecae.Validation of RNA-seq was performed using RT-PCR to assess the expression profiles of the eight selected transcripts , midguts, and ovaries from virgin (non-blood fed) and inseminated (blood fed) females. Carcasses, midguts, and ovaries were collected from 5-day old virgin or inseminated females. Developed ovaries were collected 2 days after the blood meal following feeding on an anesthetized mouse, and as described in the RNAi experiments section below. Total RNA was extracted separately for each sample belonging to the two pools (virgin vs. inseminated), and each pool contained tissues from ten females. Each RNA sample was treated with DNAse I (Invitrogen), precipitated in ethanol/ammonium acetate solution, and finally suspended in RNAse-free water. After RNA quantification , 1st strand cDNA was obtained for each sample using Superscript III (Invitrogen) with random hexamers (Thermo Fisher Scientific) and 500 ng of RNA per sample. cDNAs were treated with RNA H (New England Biolabs) for 10 min at 37 °C, and stored at − 70 °C until use.Relative gene expression profiles were assessed in real time, using SYBR Green PCR Master Mix  in 20 μL reactions containing 300 nM of each primer, and 100 ng each cDNA. PCR were run using MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL)  in the StepOne™ Real-Time PCR System . The amplification conditions were 94 °C for 2 min, 94 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Each reaction was performed in triplicate using two biological replicates.-ΔΔCt Method [AAEL009496) was used as an endogenous reference, and the results were normalized using the virgin spermathecae control group.The relative expression profiles were determined using Real Time Quantitative PCR and the 2t Method . The ribAe-92,048), for the chitin associated transcripts , transcriptional regulation , hormonal regulation (AeSigP- 4002), enzymatic activity , antimicrobial activity , and ion homeostasis . Selection of transcripts for KD was based on their presumed role in maintaining sperm viability during storage within the spermathecae, as well as aspects of mosquito fitness, including survival, fecundity, oviposition or number of laid eggs, morphological characteristics of eggs, and number of eggs hatched  at VectorBase (version 5.2). For each identified coding sequence, we attributed a link to its profile on VectorBase  kit, with 5 μM of the designed primers  were obtained from the DIM, Johns Hopkins University and Departamento de Biologia Geral (DBG/UFV). Here, mosquito rearing was done as described in the section “Sample preparation and RNA sequencing.” Newly-emerged females were separated from males to prevent mating. For injection, virgin females were anesthetized on ice, placed over a glass slide covered with filter paper, and injected in the mesothorax with 69 nL of a 3 μg/μL solution of dsRNA in PBS [A in PBS , 68, usiConsidering that the rate of gene silencing is highly variable and dependent upon multiple factors, including transcript and protein turnover rates, a time course of transcript levels at 24, 48, 72, and 96 h post-injection was performed by RT-PCR. Total RNA was extracted from the spermathecae of 10 individuals per each group of dsRNA (eight transcripts) and used for 1st strand cDNA synthesis. RT-PCR was performed as describe elsewhere –69. dsRNEffects of dsRNA microinjections on the expression profiles of spermathecal genes were assessed by using duplicate pools of the spermathecae of ten females 2 days after injection, in the case of virgin females, or 2 days after mating, in the case of inseminated females. Mosquito dissection, RNA extraction, DNAse I treatment, and RT-PCR expression was performed as described in the previous section.The survival analysis of the injected females was done on data collected from three independent replicates with 100 females each. Females were kept in plastic cages and fed with cotton soaked with 10% sugar solution ad libitum. To check the mating effect on female survival, the females were allowed to mate on day one after dsRNA injection for 24 h. After this period, males and females were separated. Dead females were counted and removed daily for 10 days to assess the effect of gene KD on mosquito survival. A schematic design of the phenotypic experiments is shown in Additional file To assess the effects of dsRNA injection on blood-feeding behavior, 100 females were separated into a cage and allowed to mate with males. After 24 h, the males were removed from the cage. The next day, all the females in the cage were allowed to blood-feed for 30 min on mice anesthetized with 10% ketamine hydrochloride  and 2% Xylazine hydrochloride  (diluted 1:4) (with mouse rotation every 10 min) in accordance with the UFV Ethics Committee (Protocol 561/2016). After the blood meal, the fully engorged females were sorted, and provided with 10% sugar solution ad libitum and used for fertility and fecundity analyses.Two groups of 10 blood-fed females were individually transferred to 50 mL plastic tubes with filter paper soaked in 10 mL of distilled water and covered with fabric nets. These females were offered 10% sugar solution soaked in cotton ad libitum, for 4 days. After 4 days, the filter paper with the eggs was removed for posterior fecundity assays. Females that did not lay eggs were dissected, and their spermathecae and ovaries were photographed using a light microscope Olympus BX50 coupled to a camera, Moticam 580 at DBG/UFV. Images were compared to the control.The eggs laid by the injected females from the previous experiment were counted, aligned, and stuck to a white tape and photographed. The egg length and total area were measured in the digital images using the ImagePlus software®. The measurements were performed twice to avoid any experimental error. We took these measurements to check how each dsRNA injection affects the egg phenotype and whether the changes in the egg phenotype affect the egg viability in comparison with the control.The viability of eggs laid by the injected females was assessed using four pools of 100 eggs, each placed in plastic cups with 100 mL of distilled water and turtle food. Eggs were allowed to hatch for 2 days  and the number of live larvae was counted (number of emerged larvae/100 eggs). Fertility was assessed as the number of hatched eggs from 100 eggs. Eggs were randomly sampled.The spermathecal phenotype was also assessed following dsRNA injections. To this end, spermathecae collected from groups of 10 injected virgin and inseminated females were fixed in fixative solution  for 2 h. Fixed samples were rinsed in PBS, dehydrated in an ascendant series of ethanol (70–100%), and embedded in a Historesin embedding kit . Thin sections (4 μm thick) were stained with hematoxylin and eosin (HE) and dried. The stained sections (4 μm) were mounted with Eukitt® Quick-hardening mounting medium  and photographed under the light microscope Olympus BX50 coupled to a camera, Moticam 580 at the Departamento de Biologia Geral/UFV. Additionally, an assessment of sperm motility was performed to account for effects of the KD in the gametes within the spermathecal reservoir. On the third day after the blood feeding, five females of each dsRNA-injected group were randomly separated from the cage and dissected in PBS. The spermathecae of each female were transferred to cleaned glass slides  with PBS and covered with cover slips. Sperm motility was visually inspected in freshly dissected spermathecae also using the Olympus BX50 at room temperature, classified as motile or non-motile compared with the control group (dsEGFP). For non-motile cases, reservoirs were gently mechanically disrupted, freeing the sperm. This part of the experiment was performed in duplicates for each dsRNA and recorded accordingly  of Ae. aegypti female injected with dsRNA targeting the Na+/Ca2+ seven days after the blood meal.Additional file 7: Movie S2. Sperm motility inside of the reservoir one day after mating. Spermathecae of inseminated Ae. aegypti female controls injected with dsRNA targeting EGFP.Additional file 8: Movie S3. Reduced sperm motility inside of the reservoir one day after mating. Spermatheca of inseminated Ae. aegypti female injected with dsRNA targeting the Na+/Ca2+.Additional file 9: Movie S4. No sperm motility inside of the reservoir five days after a blood meal. Spermatheca of inseminated Ae. aegypti female injected with dsRNA targeting the Na+/Ca2+.Additional file 10: Movie S5. Sperm their motility re-established once the reservoir is disrupted or broken. Spermathecae of inseminated Ae. aegypti female injected with dsRNA targeting the Na+/Ca2+ at day five after blood feeding.The in situ hybridization was performed using the FISH Tag RNA Red Kit, with Alexa Fluor 594 dye . For probe synthesis, 1 μg of the purified PCR product generated in the purification step of the dsRNA production (as described above for the RNAi experiments) was used, according to the manufacturer’s protocol. The spermathecae (virgin and inseminated) were dissected, fixed for 1 h, washed in distilled water, and incubated in 500 μL of labeling solution (10% probe solution in 1X PBS) overnight. The whole tissue was washed three times with PBS and stained with 4′, 6-diamidino-2-phenylindole in 0.5 g/mL solution (DAPI) (1:1000)  at 23 ± 2 °C for 1 h in the dark. It was then mounted in 50% sucrose solution and photographed under the fluorescence microscope, Olympus BX53 microscope, coupled to an Olympus DP 73 digital camera, using a WU filter . As the control, we used the EGFP as template for the probe synthesis and checked it in both the virgin and the inseminated spermathecae.t-test with a 95% confidence interval and α = 0.05.Data were analyzed using GraphPad Prism v.6 software  and plotted as bar graphs. The data were tested for normal distribution using Shapiro-Wilk normality test with α = 0.05. Survival analysis was performed by the Kaplan-Meier method, and differences were detected by Log-rank (Mantel-Cox) test with α = 0.05. In order to compare either expression levels and knock down phenotypic effects between virgin and inseminated spermathecae, the comparative analysis was performed by one-way analysis of variance (ANOVA) and paired analysis with the two-tailed Additional file 1. Tables of comparative gene expression analyses between virgin and inseminated Ae. aegypti spermathecae. Results show summaries of DEG and respective functional annotations, selected transcripts used in RNAi experiments, and phenotypic assessments.Additional file 2. Expression profiles and fitness effects from knockdown of selected transcripts including survival, oviposition rates, fecundity, and morphological effect on Ae. aegypti female ovaries.Additional file 3. Histological sections of the spermathecae of Ae. aegypti spermathecae .Additional file 4. Sequence and alignment information of eight selected transcripts targeted for RNAi. Primer sequences used for dsRNA synthesis as well as amplicon sizes for RT-PCR assessment of expression profiles are shown.Additional file 5. Hyperlinked excel spreadsheet with reassembled coding sequences and reads mapped with RPKM>1 from Ae. aegypti virgin and inseminated spermathecomes.


The survival time of patients with early pancreatic cancer (PC) is still disappointing, even after surgical resection. PC has an extremely poor prognosis. Herein, we aimed to investigate the survival effect of postoperative radiotherapy (PORT) on resected stage I to II PC.A large eligible sample of patients was identified from 2010 to 2015 from the Surveillance, Epidemiology, and End Results (SEER) registry. Survival analysis was conducted to evaluate the efficiency of PORT. Propensity score matching (PSM) analysis was used to reduce selection bias and to make the groups comparable.A total of 3219 patients with resected stage I to II PC was included after rigid screening. The median overall survival (OS) was 26 months with PORT (n = 1055) versus 21 months with non-PORT (n = 2164) before matching (p<0.001). By multivariable analysis, PORT remained a favorable prognostic predictor for OS. In PSM analysis, receiving PORT was associated with improved OS . After further meticulous exploration, only the stage IIB subgroup benefited from PORT (p<0.001). This result was due to the positive lymph node state (N+), whose mortality risk was cut by 23.4% (p<0.001) by PORT.Addition of PORT to the treatment of patients with resected stage I to II PC conveys a survival benefit, particularly among those with N-positive or stage IIB disease. Pancreatic cancer (PC) has emerged as the deadliest malignant cancer in the United States, with the lowest five-year overall survival rate of only 9%. It has become the fourth leading cause of tumor-related death and accounts for 3% of estimated new cases of cancer with an unpromising median survival of 12.4 months . Even acBecause the majority of patients are diagnosed when the disease is advanced, the percentage of those who are evaluated as “resectable” is less than 20% . HoweverThe SEER database, established by the National Cancer Institute (NCI), provides incidence and survival data from various locations and sources throughout the United States, representing 28% of the national population. We present the latest, most precise, and most comprehensive analysis based on SEER data in resected stage I to II pancreatic cancer patients whose postoperative chemotherapy information was available to explore the role of PORT on survival in this population.http://seer.cancer.gov/seerstat) to access the 1975 to 2016 SEER Research Data. The user ID is “11628-Nov2018.” Since we needed to know patients’ specific treatment information, we applied for “Radiation/Chemotherapy Databases” as well.We registered our account with the official SEER site and downloaded the SEER*Stat software (We used SEER*Stat software (version 8.3.6) to extract eligible cases. We selected the SEER database “Incidence-SEER 18 Regs Custom Data , Nov 2018 Sub (1975–2016 varying)”, and selected “pancreas” for the ‘‘SEER Site Recode” variable. A limit was prescribed as “8140/3: adenocarcinoma. NOS” to the ‘‘Site and Morphology. ICD-O-3 Hist/behav” variable. Staging data were collected by limiting “Stage-7th edition. Derived AJCC Stage Group, 7th ed. (2010–2015)” to stage I to II. To avoid the invalidity of some variables of interest, only “positive histology” patients were included in the current study. In SEER, we selected “active follow-up” item, and the following three types of patients were excluded: 1) “Autopsy Only” or “Death Certificate Only”; 2) in situ cancer of the cervix uterus only; 3) not originally in active follow-up but in active follow-up now (San Francisco-Oakland only). Complete follow-up dates were also guaranteed to be available. Entries with “Autopsy only” and “Death certificate only” in the “Other. Type of reporting source” variable were excluded, as were those with “unknown” in “Cause of Death (COD) and Follow-up. Survival time.”The exclusion criteria were as follows: (1) patients who did not receive cancer-directed surgery or did not know whether the surgery had been performed; (2) patients with unknown primary tumor site; (3) patients who had a survival <3 months; (4) patients who received other radiotherapy techniques, except external irradiation; (5) patients who did not know the sequence of radiotherapy and surgery or those who received radiation therapy prior to, both before and after, or during surgery; and (6) patients with unknown race, tumor sizes and insurance type, or any of these factors. Of note, we excluded patients who died within less than three months after diagnosis to prevent a bias for OS in the PORT arm. Those with a short OS may lose the opportunity to receive PORT, which may contribute to an unsatisfactory outcome in the non-PORT analysis arm.All data management and statistical analyses were performed using SPSS software . Survival analyses were performed using the Kaplan-Meier (K-M) curve analysis method for the entire cohort. The log-rank test was used to identify differences in survival curves. Multivariate analyses calculated by Cox proportional hazard model were conducted to identify predictors of survival. Statistical significance was assumed at a threshold of P<0.05.Since none of the included patients were randomized into different groups, we conducted a PSM analysis, which was performed using a logistic regression model to lessen the potential selection bias between the PORT and non-PORT groups. PSM was performed based on nearest neighbor matching at a 1:1 ratio, with the value of matching tolerance as 0.0001. The current study used a PSM model to match predefined covariates, including age at diagnosis, sex, race, T stage, N stage, primary site, chemotherapy, type of insurance and type of surgery. Among them, age was matched according to the method of database collection (every five years as a period). Survival analysis was conducted in the matched population.We identified 7665 resected stage I to II pancreatic cancer patients from the SEER database who met the inclusion criteria , 45.9% (95% CI 44.1%-47.7%), and 19.6% (95% CI 17.8%-21.4%), respectively. Our K-M analysis results also revealed that older age, male sex, high AJCC stage, lymph node involvement, lack of chemotherapy, larger tumor size, and Medicaid benefits were associated with worse OS, as shown in The PSM process achieved a balance between the two groups (n = 782 for each group). Details of the covariates in the groups after PSM are shown in Though the OS curves became closer after PSM  and non-PORT (n = 739) in stage II subgroups were 25 months (95% CI 22.3–27.7) and 23 months (95% CI 21.2–24.8) (P = 0.002), respectively. Furthermore, poor OS was significantly associated with higher N stage (p<0.001), no chemotherapy (p<0.001), larger tumor size (P = 0.006), older age (P = 0.028) and type of insurance (P = 0.009) after K-M analysis. Older age , receiving PORT  and chemotherapy , higher N stage , larger tumor size  and type of insurance (P = 0.009) remained prognostic factors for OS in the multivariate Cox regression analysis.To elucidate the underlying factors that are sensitive to PORT among stage II patients, we meticulously analyzed subgroups of stage IIA and stage IIB first. As shown in The most obvious difference between stage IIA and stage IIB is whether there is lymph node involvement. Given the previous result of stage I, we considered that lymph node state may play a key role in patient prognosis, and further research is to be conducted. To investigate whether N-status contributes to an improved OS in stage IIB patients, we performed further analyses, and the final result is shown in SEER is a large, complex, multi-institutional database that affords many advantages to researchers. In this study, we used SEER to identify an eligible sample of patients with resected early-stage (I to II) pancreatic cancer in an attempt to examine the benefit of adding PORT to the treatment protocol for this population. We stratified our analysis to allow greater depth to identify more clearly which subgroups might benefit from PORT. Due to the shortcomings of the database, however, we had to utilize appropriate statistical means to minimize the resulting impacts.Results demonstrated that addition of PORT was associated with an OS benefit in resected stage I to II PC on the whole. To produce more sound results and make the two groups meaningfully comparable, PSM was performed. Our outcome of preferable OS among patients with PORT versus non-PORT remained significant after PSM. We found that only stage IIB benefited from PORT, which prompted us to query whether lymph node involvement (N+ subgroup) helped explain this result. Subsequent research confirmed the hypothesis that only the N+ group acquired longer OS after PORT.Considering the conflicting results of previous randomized clinical trials, the role of PORT in resected pancreatic cancer remains debatable , 11. TheAfter further subgroup analysis, PORT was fortunately found to reduce the risk of death in lymph node-positive patients by 23.4%. Lymph nodal status is the key to distinguishing prognosis of different stages. There was no significant benefit to OS in PORT group versus non-PORT group in either stage I (T1-2N0M0) or stage IIA (T3N0M0). Only patients evaluated as stage IIB (node-positive) achieved better survival from PORT. Current clinical trials had insufficient power to look into the survival benefit in patients with node-positive disease. However, prior analyses based on shared databases have reported results similar to ours. Moody et al. reviewed the SEER data (1988–2003) and demonstrated that only resected stage IIB disease (T1-T3N1M0) obtained better OS from PORT . UnfortuThere have been many prior studies of the SEER database reporting survival benefits from PORT for the resected population , 17–24. The National Cancer Data Base (NCDB) in the United States is another large population-based database. There is a certain amount of NCDB research about adjuvant radiotherapy in resected pancreatic cancer , 25–27. In many studies supported by large-repository data, PSM is used to solve endogenous population problems and to create highly comparable comparison groups. The current study adopted this statistical method to minimize treatment selection bias. Although there have been many similar encouraging studies providing valuable information, the present study has several design advantages. First, this study performed PSM at a ratio of 1:1, which significantly increases the efficacy of the research. Second, we limited the population to patients with stage I to II resected PC, which eliminates the effects of the T4 subgroup, which involves a vital artery. Third, the distribution of baseline characteristics before and after matching was clearly compared in tabular form, demonstrating that all baseline characteristics of each subgroup were well balanced after PSM. Fourth, through stratified analysis, we delineated which groups benefit from PORT. Our study shows that only N-positive patients obtain benefits from PORT, which is consistent with conclusions from other studies , 14, 26.Although the SEER database is a powerful tool to briefly characterize national trends and collect detailed data such as pathologic or treatment information that takes advantage of a large patient population, there are still some limitations to this study. It is confounding that the SEER database fails to offer complete and detailed information of therapy experience . Furthermore, the prognosis for different PORT strategies varies widely. These factors, regarded as potential pitfalls, might alter the results of the study. In addition, information on variables, such as operative margins and patient functional status, which play an important role in OS outcomes, was not collected in the SEER database and could influence postoperative treatment selection and introduce treatment bias. Fortunately, given our strict access principles, we feel that this study, based on our dataset design, represents a very valid and persuasive result.Although PORT is associated with improved OS for stage I to II patients as a whole, we observed that the trend appears to be generated by stage IIB or N-positive resected patients in a large national database using PSM. Most patients, especially those with pancreatic head cancer, significantly benefit from PORT. These data merit consideration and the final results of ongoing RTOG 0848 clinical trial are eagerly awaited. We believe that PORT will be accepted by more patients and clinicians in the future.