Since its introduction in 1991, laparoscopic splenectomy (LS) has become the gold standard in elective spleen surgery in many centres. However, there still lack the report of long-term outcomes of LS with the large-scale cases. The aim of the present study was to analyze the short- and long-term outcomes of LS in a single institution over 16 years, and to compare the perioperative outcomes of totally laparoscopic splenectomy (TLS) and hand-assisted laparoscopic splenectomy (HALS) for splenomegaly.Between November 2002 and December 2018, 486 consecutive patients undergoing elective LS were enrolled in this study, including 222 TLS and 264 HALS. The intraoperative, postoperative, and follow-up data were retrospectively analyzed.P = .013). Compared with TLS, HALS had a shorter operative time (P = .000), lower intraoperative blood loss (P = .000), comparable conversion rate (P = .271), and morbidity (P = .922) for splenomegaly > 17.0 cm. During the follow-up period, the overall respond rate for immune thrombocytopenia was 77.8%, and the esophagogastric variceal bleeding rate was 6.9% in 320 patients with hypersplenism secondary to hepatic cirrhosis.The 5 most common indications were hypersplenism (71.0%), immune thrombocytopenia (14.8%), splenic benign tumor (4.5%), splenic cyst (2.9%), and splenic malignant tumor (2.9%). The mean operative time, intraoperative blood loss, and length of stay were 149.4 ± 63.3 minutes, 230.1 ± 225.1 mL, and 6.7 ± 3.2 days, respectively. The morbidity, mortality, reoperation, and conversion rate were 23.0%, 0, 0.4%, and 1.9%, respectively. Portal vein system thrombosis (PVST) was the most frequent complication with an incidence of 19.8%. The incidence of PVST in HALS was higher than that in TLS (23.9% vs 14.9%, LS is a safe, feasible, and effective procedure with satisfactory short- and long-term outcomes. HALS is a reasonable technique in patients with massive spleens. Following that inspiring initial experience, laparoscopic approaches to splenic surgery have been demonstrated to be safe and feasible by numerous cases. Compared with open splenectomy (OS), LS had advantages of less estimated blood loss, less requirements for transfusion, lower postoperative morbidity rate, faster recovery, and improved quality of life.,3 LS is primarily used for elective resection in patients with benign spleen diseases, including primary hematological diseases, immune thrombocytopenia (ITP), spleen hamartoma, and hypersplenism.In 1991, Delaitre and Maignien Several studies have demonstrated that TLS for massive splenomegaly had longer operation time, more blood loss, and higher conversion rate than TLS for normal-sized spleens.,6 In 1995, Kusminsky et al introduced the technique of hand-assisted laparoscopic splenectomy (HALS). This technique allows hand-assisted manipulation and dissection of the spleen, manual control of large vessels, and removal of an intact spleen through the hand port. Therefore, the introduction of hand-assisted technique has broadened the scope of LS to massive splenomegaly.With rapidly advancing in laparoscopic techniques, totally laparoscopic splenectomy (TLS) is nowadays considered as the gold standard for normal to moderately enlarged spleens. However, the adoption of TLS in patients with massive splenomegaly secondary to liver cirrhosis and portal hypertension introduces more difficulties than OS because of the enormous size of the spleen and existence of varicose vessels and coagulation disorders.Although LS is widely performed in many centers, there still lack the report of long-term outcomes of LS with large-scale cases, and controversy still remains regarding the best approach for patients with massive splenomegaly. The first TLS and HALS of our institution were successfully performed in November 2002 and March 2006, respectively. Up to now, we had completed more than 500 cases of elective or emergency LS. This has inspired us to present our experience of LS over a 16 years period. The aim of the present study was to analyze the short- and long-term outcomes in a series of 486 elective LS (including TLS and HALS) from a single institution. Furthermore, we compared the perioperative outcomes of TLS and HALS for patients with splenomegaly, which maximum diameter of spleen greater than 17.0 cm.22.1A retrospective cohort study, using a prospectively collected database, included all consecutive patients undergoing elective LS (including TLS and HALS) in our institution from November 2002 to December 2018. The inclusion criteria of this study were:(1)patients with primary or secondary spleen diseases who underwent elective LS in our institution;(2)Child-Pugh class A or B;(3)no organic lesions in the heart, lung, kidney, or other important organs.Patients who could not tolerate pneumoperitoneum had severe disease in other systems that affected their daily life, preoperative imaging examination has found thrombus in the portal vein system, underwent emergency LS due to splenic rupture or without complete clinical data were excluded. Written informed consent was obtained from each participant in accordance with the Declaration of Helsinki. The Ethics Review Board of the first affiliated hospital of Nanchang University approved this study (no. 2020B0017).2.2A total of 486 patients were enrolled in this retrospective study, including 222 (45.7%) TLS and 264 (54.3%) HALS. The annually number of cases was shown in Figure 2.3,9 Perisplenic ligaments were dissected with ultrasonic dissector  or LigaSure vessel sealing system , and 3 different methods were used for managing the splenic pedicle, including ligation by snare, secondary pedicle division, and endoscopic linear vascular stapler (Endo-GIA).In TLS, patients received general anesthesia and were placed in the right lateral decubitus position. Generally, 3 or 4 ports were used. A 10 mm trocar was placed at the lower umbilicus for telescope. The main manipulation 12 mm trocar was placed in the left subcostal midclavicular line and the auxiliary 5 mm trocar was placed at the subxiphoid position. Another 5 mm trocar was placed in the left axillary line for the assistant if necessary. The procedure of anterior or lateral approach LS was detailedly described in the literatures.For the HALS procedure, patients were placed in a right semi-lateral recumbent position. A subxiphoid midline incision approximately 6 to 8 cm in length was performed, in which the hand port was inserted. The left hand of the surgeon was inserted intraperitoneally through the hand port to help complete the surgery. A 10 mm trocar was placed in the lower umbilicus for telescope. The main manipulation 12 mm trocar was placed at the left midclavicular line below the inferior margin of the spleen. Firstly, gastrocolic ligament and splenogastric ligament were divided using ultrasonic dissector or LigaSure. The lesser sac was opened, and the splenic artery was identified and ligated above the body of the pancreas. Subsequently, splenocolic ligament and splenorenal ligament were dissected. A tunnel behind the splenic hilum was established with the left hand, and the splenic pedicle was handled as related in TLS procedure. Then, the short gastric vessels and splenophrenic ligament were divided. When using snare to management the splenic pedicle, all the perisplenic ligaments should be dissected firstly. Finally, the resected spleen was placed into a retrieval bag and extracted from the midline incision. The peritoneal cavity was irrigated and examined for any active hemorrhage, and a drain was placed in the splenic bed.2.49/L. Patients with hypersplenism were given a platelet transfusion intraoperatively if the platelet counts less than 50 × 109/L. Antibiotic was routinely given at induction of anesthesia, and continued for several days after surgery depending on the patient's condition. All patients received routine care and postoperative monitoring. Antiplatelet agents  were administrated to prevent thrombus when the platelet count was more than 600 × 109/L. Ultrasonographic screening for portal vein system thrombosis (PVST) was performed on the seventh postoperative day. All patients received every 3 to 6 months follow-up at outpatient clinics or by a telephone interview.Before surgery, the spleen size was estimated in imaging studies (ultrasound/CT/MRI). Patients with ITP received oral prednisolone and immunoglobulin G for 3 to 5 days, starting at least 1 week before LS to increase their platelet counts to 50 × 102.5t test. Qualitative data were presented as number and percentage and compared using chi-square test or Fisher's exact test. Results were statistically significant with P-value < .05.The short-term outcomes included operative time, intraoperative blood loss, need for blood transfusions, intraoperative autotransfusion, conversion, reoperation, length of stay, morbidity, and mortality (within 30 days after surgery). The long-term outcomes included respond rate of patients with ITP and esophagogastric variceal bleeding rate with hypersplenism. Data were analyzed with SPSS 22.0 for windows . Quantitative data were presented as mean ± standard deviation (SD) and compared using Student's 33.1P = .087), hypertension (P = .480), diabetes mellitus (P = .152), cardiac disease (P = .163) between the TLS group and HALS group. Most patients with hematologic benign diseases and splenic benign tumor underwent TLS. However, patients with hypersplenism mostly underwent HALS. One hundred thirty five patients underwent elective LS with additional operation, including 36 TLS with additional operation (TLS plus group) and 99 HALS with additional operation (HALS plus group). The types of additional operation are summarized in Table The patients’ characteristics in TLS and HALS group were presented in Table s P = .15, cardiac3.2P = .287), intraoperative autotransfusion rate (P = .134), conversion (P = .375), reoperation (P = .288), and morbidity (P = .442) between TLS and HALS group; TLS group exhibited a longer operative time (P = .000), but had a lower intraoperative blood transfusion rate (P = .000) and a shorter postoperative hospital stay (P = .000), compared with HALS group. Among elective LS with additional operation, TLS plus and HALS plus group were comparable in intraoperative autotransfusion rate (P = .224), conversion (P = .118), reoperation (P = 1.000), morbidity (P = .695), and postoperative hospital stay (P = .110); TLS plus group exhibited a longer operative time (P = .000), more intraoperative blood loss (P = .014), and higher intraoperative blood transfusion rate (P = .013), compared with HALS plus group. Nine patients  were converted to open surgery. The cause of conversion was bleeding (n = 7), needing distal pancreatectomy (n = 1), and needing partial hepatectomy (n = 1). There were 101 (21.0%) patients who underwent intraoperative autotransfusion. Two  patients required reoperation because of postoperative hemorrhage. There was no perioperative death.Three methods were used to manage the spleen pedicle in our series (except conversion), including ligation by snare (n = 21), secondary pedicle division n = 34), and Endo-GIA (n = 422). The mean operative time, intraoperative blood loss, and length of stay were 149.4 ± 63.3 minutes, 230.1 ± 225.1 mL, and 6.7 ± 3.2 days, respectively. Perioperative outcomes of 4 subgroups  were listed in Table 4, and EnAccessory spleen was found in 46  patients. The location of accessory spleen included splenic hilum (n = 22), greater omentum (n = 8), gastrosplenic ligament (n = 6), splenocolic ligament (n = 5), pancreatic tail (n = 3), and small bowel mesentery (n = 2). Accessory spleens were resected together with the spleen in patients with hematologic benign diseases and splenic malignant tumor.P = .013). According to the Clavien-Dindo classification, 6 patients were grade I, 100 grade II, 4 grade IIIa, and 2 grade IIIb.As shown in Table 3.3P = .537), intraoperative blood loss (P = .919), conversion rate (P = 1.000), morbidity (P = .802), and postoperative hospital stay (P = .204) between the 2 approaches.In TLS, 63 (28.4%) patients underwent the lateral approach and 159 (71.6%) patients underwent the anterior approach. As shown in Table 3.4P = .000), and less intraoperative blood loss  compared to TLS. The conversion rate  and morbidity  of HALS were comparable with that of TLS. However, the postoperative hospital stay of HALS was significantly longer than that of TLS . No reoperation and mortality were observed in both groups.The perioperative outcomes of TLS and HALS  for splenomegaly (maximum diameter greater than 17.0 cm) were listed in Table 3.5,11 47 (65.3%) patients achieved complete response (CR), 9 (12.5%) response (R), 16 (22.2%) no response (NR). The total therapeutic response (CR + R) rate was 77.8% (56/72).Up to December 2019, 72 patients with ITP were followed-up for 2 to 181 months (average: 89.5 months). Response to splenectomy was assessed at the last available follow-up. According to the criteria of the International Working Group endorsed by the American Society of Hematology guidelines,Among 345 patients with hypersplenism, 25 (7.2%) patients were lost to follow-up, the other 320 (92.8%) patients were follow-up for 10 to 160 months (average: 71.9 months). Theirs’ white blood cell and platelet counts were all rose to above normal level after the operation. During the follow-up period, esophagogastric variceal bleeding (EGVB) recurred in 22 (6.9%) patients. All these patients underwent endoscopic therapy, and 3 patients died from acute upper digestive tract rebleeding. Ten (3.1%) patients occurred secondary liver cancer, and 4 died from secondary liver cancer.4–15 The reported morbidity following LS varied from 0% to 35.7%, with mortality varied from 0% to 3.9%, the conversion rate varied from 0% to 4%, and the reoperation rates varied from 0% to 6.7% in the literatures. In November 2002, our team firstly performed LS for a 35-years old woman with ITP. Since then, the volumes of LS were gradually increased in our institution, especially after the introduction of HALS in 2006. During the past 16 years, our indications of LS have broadened from benign hematological disorders to massive even supermassive splenomegaly secondary to hepatic cirrhosis. Meanwhile, the procedure has expanded from single LS to LS combination with esophagogastric devascularization. Herein, we presented the short- and long-term outcomes of 486 elective LS. In our series, the mean operative time, intraoperative blood loss, and length of stay were 149.4 minutes, 230.1 mL, and 6.7 days, respectively. The morbidity, mortality, reoperation, and conversion rate were 22.8%, 0, 0.4%, and 1.9%, respectively. Obviously, these perioperative outcomes were consistent with those reported by the literatures.Since its introduction in 1991, LS has gained worldwide acceptance with many advantages over OS. With the development of laparoscopic techniques and instruments, the indication of LS had gradually expanded from normal size spleen to massive splenomegaly. Although LS was a routine procedure in many centres, only few reports with more than 300 cases. In our opinion, there were no significant difference between the 2 approaches, and the choice of approach should be depended on the surgeon's experience and concomitant conditions. Three methods were used to manage the spleen pedicle in our series, and they have their own advantage and disadvantage. Endo-GIA transection is a simple, effective, and time-saving method, but it is more expensive than other 2 methods. Ligation by snare is an economical and effective method; however, it needs to completely dissect the perisplenic ligments and fully mobilize the spleen, which is difficult under the circumstance of perisplenic adhension and massive splenomegaly. Secondary pedicle division strategy is a highly cost-effective method. Nevertheless, its disadvantages are technique challenging and higher risk of bleeding. Whatever method is used, we recommended to ligate the splenic artery in patients with splenomegaly as early as possible, which could decrease the volume of spleen and lower the risk of massive hemorrhage during operation.In regard to surgical techniques, safely dissecting the perisplenic ligments and managing the spleen pedicle are the most important manipulations for successful LS. Usually, there have 2 surgical approaches for LS, including anterior approach and lateral approach. The lateral approach provides better exposure of the splenic hilum and the pancreatic tail because the abdominal viscera are retracted away from the upper-left quadrant by gravity, allowing easier dissection of the splenic hilar structures. Recently, a systematic review and meta-analysis suggested that lateral approach is superior to anterior approach with the advantage of better access, more secure hemostasis, less conversion to open surgery, less morbidity, earlier recovery, and shorter length of hospital stay. As a non-surgical intervention, PSE is an effective option for patients who are not surgical candidates. PSE owns several advantages over conventional LS including decreased the incidence of PVST and preservation of splenic tissue function to protect against infections. However, the effect of PSE is strongly dependent on the infarcted splenic volume, a relative insufficient embolization extent may lead to the recurrence of hypersplenism; and this procedure still has a high risk and can cause substantial complications. At present study, the most frequent indication was hypersplenism secondary to hepatic cirrhosis (71.0%). All patients’ white blood cell and platelet counts were all rose to above normal level after LS. The EGVB rate was 6.9% after a mean 71.9 months follow-up. Therefore, the clinical effect of LS for hypersplenism was verified in our series.Hypersplenism is a clinical syndrome characterized by an enlarged, overactive spleen. Currently, splenectomy and partial splenic embolization (PSE) are the most popular treatment for hypersplenism. LS can eliminate hypersplenism-induced blood cell destruction, prevent EGVB, and decrease portal pressure and reverse hypersplenism. comparing laparoscopic to open splenectomy for portal hypertension. However, portal hypertension caused by liver cirrhosis is considered as a contraindication for LS in the clinical practice guidelines of the European Association for Endoscopic Surgery (EAES). It also recommends HALS for massive splenomegaly to avoid conversion to OS and complications. Targarona et al conducted a comparison between conventional LS and HALS for splenomegaly  and concluded that HALS was associated with shorter operative times, less morbidities, and shorter hospital stays. Wang et al compared the outcomes of TLS (n = 20) and HALS (n = 19) for splenomegaly (maximum diameter greater than 17 cm) and hypersplenism due to cirrhosis, the results showed that TLS had a longer operative time, more estimated blood loss, more patients requiring transfusion, and more complications than HALS. Recently, a meta-analysis of HALS versus LS for splenomegaly showed that the operative time was significantly shorter, blood loss volume and conversion rate were significantly lower in the HALS group than those in the LS group. However, no significant difference was observed in hospital stay length, blood transfusion, time to food intake, complications, or mortality rate between the 2 groups. Based on our results, we recommended HALS for splenomegaly > 17 cm because of HALS with a shorter operative time, less intraoperative blood loss, and comparable conversion rate and morbidity. Furthermore, HALS was convenient for extracting the enlarged spleen and performing esophagogastric devascularization for patients with esophageal and gastric varices.As we known, LS for hypersplenism has always been a technique challenging due to the limited working space, increased risk of bleeding, the potential risk of increasing conversion, operative time, and morbidity. Therefore, there is some controversy regarding laparoscopic operations of markedly enlarged spleens and patients with portal hypertension. Some studies supported the laparoscopic approach, clearly demonstrating the benefits of LS even in the case of massively enlarged spleens. This is very well documented in the system review published by Cai et al,26 At present study, accessory spleen was found in 9.5% patients and resected together with the spleen in patients with hematologic benign diseases and splenic malignant tumor. No patient had recurrence related to accessory spleen.Accessory spleens represent the most common anatomic abnormality and are present in 15% to 30% of children. They most likely originate from mesenchymal remnants that do not fuse with the main splenic mass. Accessory spleens are most commonly located medial to the splenic hilum, adjacent to or within the pancreatic tail or in the splenorenal ligament. Rarely they may be located elsewhere in the abdomen. Surgeons must be cognizant of these locations and routinely check for their presence at the time of planned total splenectomy, because a missed accessory spleen can lead to recurrence of ITP or hereditary spherocytosis. There were several reports of laparoscopic accessory splenectomy after initial splenectomy in the management of recurrent hematologic diseases. It is reported that about 80% of ITP patients respond to splenectomy, and 66% of patients can sustain the response with no additional therapy for more than 5 years.,29 LS can provide better short-term results, such as less blood loss and blood transfusion, quicker resumption of oral intake, shorter hospital stays, and comparable long-term results to those of OS for ITP.,31 Tastaldi et al reported 109 patients with ITP underwent LS, the initial response rate, and long-term response rates at a median 62 months follow-up were 90.8% and 68%, respectively. They also found that there was no statistically significant difference when LS performed before or after second-line medical therapies, and only a robust increase in platelet counts on short-term being associated with long-term response. Xu et al presented a retrospective analysis of 114 patients with ITP who underwent LS from 2001 to 2013. The 140-month response rate to LS was 68%. Multivariate analysis showed that age and postoperative platelet count were independently associated with response rate. Among those 72 patients with ITP in our study, CR was found in 65.3% and R occurred in 12.5% of the cases within a mean follow-up of 89.5 months; that is, a total of 77.8% of the patients responded to splenectomy performed in accordance with the guidelines.Therapy-resistant ITP was the second frequent indication of LS in our series. Splenectomy is recommended as the mainstay second-line treatment for adult ITP. The indications include: the patient has failed glucocorticoid treatment and has been diagnosed with ITP for more than 6 months; the maintenance dose of prednisone exceeds 15 mg/day, or the patient has a contraindication to glucocorticoids. It is reported that massive splenomegaly, splenic weight, and splenic vein diameter are independent risk factors for PSVT after LS.–37 Our results showed that the incidence of PVST in HALS group was significantly higher than that in TLS group, probably because there were more patients with hypersplenism in HALS group. Although mostly patients with PVST following LS were asymptomatic, its high incidence justifies ultrasonographic screening on the seventh postoperative day. For patients at high risk, we suggested that the usage of low-molecular-weight heparin within the first 5 days after surgery, followed by oral warfarin or aspirin for 1 month for the prevention of PVST. Postsplenectomy thrombocytosis may increase the risk of thromboembolism, especially in patients with hematological malignancies or myeloproliferative disorders. It is necessary for surveilling the platelet counts after LS, and we recommend a threshold of 600 × 109/L to begin antiplatelet agents .PVST was the most frequent complications in our series with the incidence of 19.8%, which accounted for 85.7% of the overall morbidity. Recent reports have described an incidence of PSVT after LS > 50%.There were some limitations for the present study. First of all, it was a retrospective study; therefore, selection bias may unavoidable in this study. Second, the choice of either TLS or HALS was mainly based on the surgeon's judgement. Several different surgeons performed the LS in our institution, which may have had an impact on the results of the study. Third, this study is based on a single-center experience, which may lead to the lack of representativeness. Therefore, a multi-center, prospective, and randomized study is necessary for further study.5In conclusion, our results showed LS is a safe, feasible, and effective procedure with satisfactory short- and long-term outcomes. HALS is an alternative technique in patients with massive spleens.Conceptualization: Xiaowei Fu, Yong Li, Weidong Xiao.Data curation: Zhengjiang Yang, Shuju Tu, Wanpeng Xin, Haiming Chen, Xueming Li.Formal analysis: Xiaowei Fu, Zhengjiang Yang, Weidong Xiao.Funding acquisition: Weidong XiaoInvestigation: Zhengjiang Yang, Shuju Tu, Wanpeng Xin.Software: Xiaowei Fu, Zhengjiang Yang.Supervision: Yong Li, Weidong Xiao.Writing – original draft: Xiaowei Fu, Weidong Xiao.Writing – review & editing: Weidong Xiao.


O-heterocyclic, complex, and hydroxanthones) and six biological activities . Moreover, the natural product-likeness and the drug-likeness of marine xanthones were also assessed. Marine xanthone derivatives are rewarding bioactive compounds and constitute a promising starting point for the design of other novel bioactive molecules.The marine environment is an important source of specialized metabolites with valuable biological activities. Xanthones are a relevant chemical class of specialized metabolites found in this environment due to their structural variety and their biological activities. In this work, a comprehensive literature review of marine xanthones reported up to now was performed. A large number of bioactive xanthone derivatives (169) were identified, and their structures, biological activities, and natural sources were described. To characterize the chemical space occupied by marine-derived xanthones, molecular descriptors were calculated. For the analysis of the molecular descriptors, the xanthone derivatives were grouped into five structural categories (simple, prenylated,  The marine environment occupies more than half of the Earth’s surface and harbors the largest pool of biodiversity. Among others, marine microorganisms produce specialized metabolites used mostly in interspecies competition and defense from predators. The harsh conditions found in the sea spurs the development of specific biosynthetic pathways that produce metabolites bearing novel scaffolds, quite different from those found in terrestrial sources . SpecialAmong the most relevant chemical classes of specialized metabolites isolated from the marine biodiversity, xanthones are a class of oxygen-heterocycles containing a heterocycle containing a dibenzo γ-pyrone moiety ,5,6. Dep−1 (log S); and carbon saturation by the fraction of sp3 carbons (Fsp3). Besides providing a numerical expression for chemical features, molecular descriptors allow tracking the suitable pharmacodynamics and pharmacokinetics properties, i.e., allow pursuing drug-likeness. Sets of rules or filters have been proposed over time in order to predict pharmacokinetic behavior. The most common set is the Lipinski′s rule of five [The chemical space occupied by a collection of substances is usually mapped by molecular descriptors . Molecul of five , but oth of five , Ghoose  of five , Egan [1 of five , and Gle of five . More re of five . Due to  of five . The app of five .In this work, we review 169 bioactive marine xanthone derivatives and present their structures, biological activities, and marine sources. The chemical space occupied by bioactive marine xanthones is mapped and framed according to the NP-likeness and drug-likeness concepts.®, Web of Science®, and Google Scholar® without any temporal restriction. The keywords used were “marine AND xanthone*”.The bibliographic research was conducted using ScopusO-heterocyclic groups, such a pyran or furan ring, were classified as “O-heterocyclic” compounds. Xanthones bearing O-heterocyclic and isoprenyl groups were classified as “O-heterocyclic” because they have a higher similarity with this category. Dimeric, pseudo-dimeric (one xanthonic and a hydroxanthone nucleus connected by a C-C bond), and glycosylated xanthones were loosely classified as “complex”. Dihydro-, tetrahydro-, and hexahydroxanthones were included in the “hydroxanthone” category , “hydroxanthones” (9.9%), and “prenylated” (6.4%)  and “complex” (28.7%) groups were the most prevalent, followed by “” (6.4%) a.Aspergillus genus . Half of the reported bioactive marine xanthones (53%) were isolated from endophytic microorganisms associated with macroorganisms, like mangroves (39 marine xanthones), sponges (38 marine xanthones), algae (16 marine xanthones), corals (12 marine xanthones), jellyfish (1 marine xanthone), and seaweed (1 marine xanthone).The bioactive marine xanthones were mostly isolated from marine fungi, namely from fungi belonging to the nus 41%, b. Only aAs specialized metabolites, xanthones are often used as chemical defense agents. The most prevalent described activities were antitumor (43.5%) and antimicrobial , antifungal (12.4%), and antiviral (10.6%) which provides some sort of protection to other competitive or predator marine organisms c. IntereBioactive marine xanthones are produced by and act in living organisms, and to fulfill their specific biological task, they are structurally optimized by nature. Therefore, defining their chemical space is important for designing new molecules with desirable properties and pharmacological potential.To describe the chemical space occupied by bioactive marine xanthones, several molecular descriptors, embracing different molecular, physico-chemical, and topological properties, were calculated using the RDkit (release 2021_03_5 Q1 2021) and SwissADME . For eac−1. “Complex” xanthones presented the highest mean value (621.7 g·mol−1) and the highest value dispersion (standard deviation of 85) due to the inclusion of several dimeric and glycosylated structures. On the other side, “simple” xanthones and “hydroxanthones” have the lowest MW mean values  as these xanthones are composed by the xanthonic nucleus with simple substituents (such as hydroxyl or methyl or methoxy group).Molecular size can be expressed in terms of MW, which is a predictor for pharmacokinetics behavior because bioavailability usually decreases as the molecular size increases . In term3) and on the number of rotatable bonds (RB). A high number of RB and/or Fsp3 means that the molecule has conformation flexibility which results in a less planar, less rigid, and more complex three-dimensional shape. Both RB and Fsp3 are important for determining oral bioavailability [3 values  as saturated bonds are only present in substituent groups , but the latter showed higher value dispersion . “Hydroxanthones” have the highest Fsp3 values , but they have the lowest number of RB , meaning that saturated bonds belong to the cyclic system of the hydroxanthonic nucleus  to the xanthone itself , meaning that the additional O-heterocyclic moiety does not contribute significantly to lipophilicity. “Prenylated” xanthones have the highest log P value , significantly higher than the “O-heterocyclic”. “Complex” xanthones (median of 2.57) present the highest dispersion of log P values (standard deviation of 1.2), putting in evidence their structural diversity. The increase or decrease lipophilicity of marine xanthones is dependent on the substitution pattern, namely on the presence of hydrophilic or lipophilic substitutions. The size of the marine xanthone was not correlated with increasing lipophilicity as different sized molecules have quite similar log P values  and compound 157 .Lipophilicity, assessed by log P, is a key parameter that affects both pharmacodynamics and pharmacokinetics . “HydroxP values h, such a2 for “prenylated” up to 110.7 Å2 for “hydroxanthones”), with the exception of complex xanthones that have significantly higher values  , has been used to predict the permeability of drugs . Differe03.9 Å2) e. “ComplO-heterocyclic” > “prenylated” > “complex”. “Hydroxanthones” were the most soluble group , while “complex” xanthones were the most poorly soluble group   f. “Simplf −5.84) j and sizf −5.84) k. The pof −5.84) f. Solubif −5.84) l.NP-likeness allows measuring the similarity of a molecule to natural products . The NP-O-heterocyclic”, “simple”, and “prenylated” xanthones meet most of the criteria defined by classical rules, except for the Gleeson rules. On the contrary, “complex” xanthones violate at least one criterion in all the considered rules  with the most representative biological activities . As the number of xanthones reported for each biological activity is different, the results should only be compared when the number of reported molecules is similar .Considering the reported biological activities of marine xanthones, the relationship between these and the molecular descriptors was established. −1 and another mode of 636.6 g mol−1) (−1) and log S values (modes of −4.23 and −5.84) , raising the hypothesis that more rigidity might be an important aspect for the antibacterial activity  a. The prg mol−1) b, which g mol−1) k. Howeveg mol−1) c. Similad −5.84) a,b and ad −5.84) c. The feactivity d.−1)  a. Antidi−1) a, and an−1) b and of −1) c. AntiviThe most representative heteroatom in marine xanthones is the oxygen atom, distributed by phenols (present in 96.5% of the reported marine xanthones), methoxy groups (67.8%), alcohols (63.7%), esters (58.5%), ketones (26.9%), and carboxylic acids (7.0%) . Other h50) lower than 10 µM varied depending on the tested cell lines. For instance, the number of xanthones screened against HL-60, MCF-7, and Huh7 cells was almost the same, but the number of most potent xanthones was higher identified against HL-60 cells. None of the marine xanthones assayed against C4-2B, 22RV1, and RWPE-1 presented an IC50 lower than 10 µM, while for MGC-803, the most part presented an IC50 lower than 10 µM.A total of 74 marine xanthones described in literature were evaluated for antitumor activity, measuring their growth inhibitory activity in different tumor cell lines . The cer−1 varied depending on the tested bacteria. Marine xanthones revealed a great selectivity for growth inhibition of Gram-positive bacteria as the percentage of marine xanthones with MIC lower than 4 is significantly higher in these types of bacteria (28% Gram-positive vs. 8% Gram-negative). This could be ascribed to a target potentially involving the peptidoglycan layer that is present in Gram-positive bacteria and absent in Gram-negative bacteria. The majority of marine xanthones were evaluated against S. aureus and E. coli , Colletotrichum (8 xanthones), Candida (5 xanthones), and Microbotryum (4 xanthones) were the most frequent genus.A total of 21 marine xanthones were evaluated for the antifungal activity against 17 different fungi, measuring the growth inhibitory activity of different fungi . Among tA total of 18 marine xanthones were evaluated against different viral targets of H1N1 (10 xanthones), HSV-2 (10 xanthones), HSV-1 (7 xanthones), HIV-1 (3 xanthones), EV71 (2 xanthones), H3N2 (2 xanthones), TMV (1 xanthone) .A total of 19 marine xanthones were evaluated for antidiabetic activity using two different approaches: the assessment of α-glucosidase or protein tyrosine phosphatases inhibition activity and the assessment of the induction of the pancreatic β-cells proliferation in a zeafish model .A total of 9 marine xanthones were evaluated for anti-oxidant activity through the DPPH assay and ABTS or trolox equivalent antioxidant capacity (TEAC) assay .A total of 8 marine xanthones were evaluated for anti-inflammatory activity by measuring the inhibitory activity against cyclooxygenase (COX), by measuring the inhibition of inflammatory response induced by nitric oxide (NO) and NF-κΒ (factor nuclear kappa B), and by measuring the decrease in IL-6 cytokine production on LPS-stimulated macrophages .Trypanosoma brucei, Trypanosoma cruzi, Leshnmania donovani, and Plamodium falciparum. Nine marine xanthones were evaluated for their aquatic pathogens biocide activity against Vibrio sp.The remaining biological activities were classified as miscellaneous . Seven mAspergillus sp., which normally live in an endophytic relationship with microorganisms .As far as we know, 169 bioactive marine xanthone derivatives were reported in the literature up to 2021. They were isolated from microorganisms, mainly from O-heterocyclic” xanthones are those that better resemble NPs and the ones that better fulfill the drug-likeness criteria. Therefore, hydroxanthones” and “O-heterocyclic” xanthones represent the most promising starting point for a hit-to-lead expansion.The chemical space occupied by bioactive marine xanthones was described through molecular descriptors. For each structural category, the distribution of the MW, Fsp3, number of RB, Log P, TPSA, and Log S values were described and analyzed. The descriptors were framed accordingly to the NP and drug-likeness concepts. Among the different structural categories of xanthones, “hydroxanthones” and “50 < 10 µM) were identified mostly against HeLa , A549 , HL-60 (acute myeloid leukemia), HCT-116 (colon carcinoma), and MCG-803 (Gastric mucinous adenocarcinoma) cells lines. The most potent antibacterial marine xanthones (MIC < 4 µg mL−1) were identified predominantly against Gram-positive bacteria, namely against S. aureus, B. subtilis, and E. faecalis.In terms of biological activities, a total of 13 different activities were reported for marine xanthones. The antitumor and antibacterial activities were the most predominant. Potent antitumor marine xanthones (ICXanthones isolated from marine and terrestrial organisms share a similar biosynthetic pathway. However, marine-derived xanthones have not been as exploited as terrestrial-derived xanthones in traditional drug discovery campaigns. The numerous and relevant bioactive xanthones isolated from the marine environment could inspire the development of new drugs. The data concerning this review allow us to go deeper in understanding molecular properties, at different levels, of such an important family of marine NP.


Here, we report differential dominance (from recessive to overdominant) of the putative null tβhnM18 allele in 2 behavioral measures in Buridan’s paradigm  and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tβh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tβh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The  Differential dominance occurs when genes associated with several phenotypes (pleiotropic genes) show different modes of inheritance  depending on the phenotype. This study reveals that differential sensitivity to gene expression levels can mediate differential dominance, which can be a significant challenge for standard transgenic techniques commonly used to elucidate gene function. Pleiotropy is a central feature in genetics with pervasive implications for evolution –5. PleioDifferential dominance, found to be ubiquitous in quantitative and population genetics studies ,9,19, maIn the simplest case, the 2 alleles in question are a wild-type and a null mutant allele. In this arrangement, any differential dominance effects must be due to differential sensitivity of the phenotypes to gene dosage or gene expression levels or both. Therefore, such a situation is a good study case for investigating both the practical consequences for functional genetics studies and the mechanisms underlying the differential dominance phenomenon.tyramine-β-hydroxylase (tβh) [Drosophila, the X-linked tβhnM18 mutant has been an important tool to understand the role of TA and OA in many behaviors such as egg laying [Because of the promiscuous role of biogenic amines in many different behavioral and physiological processes, the genes coding for their synthesis enzymes are prime candidates for pleiotropy and, hence, differential dominance. The biogenic amine octopamine (OA) is structurally and functionally related to vertebrate noradrenaline –29. OA ise (tβh) . OA playse (tβh) –35. In sse (tβh)  and relase (tβh) , but alsse (tβh) ,39. OA ase (tβh) ,41. In Dg laying –44, aggrg laying ,46, fligg laying –50, and g laying .tβhnM18 male mutants, with a complete depletion of OA, display reduced aggression: Their fight initiation latency is increased, while lunging and holding frequencies are decreased [Shits phenocopies the tβhnM18 mutants, indicating that the reduced aggression does not result from developmental defects in the mutants [tβhnM18 mutant flies by expressing tβh in a small subset of octopaminergic neurons [Loss-of-function ecreased . Further mutants ,52–54. I neurons . These r neurons .tβh-associated differential dominance and conducted rescue experiments using behavioral phenotypes as disparate as sugar sensitivity [In the present work we studied sitivity ,56 and wsitivity ,58. In Bsitivity ,60. Intesitivity –64 or flsitivity . The gaisitivity ,67. The tβhnM18 , oamb , honoka , hsp-tβh , Octβ2R3.22Δ and Octβ2R4.3Δ , and w+;;UAS-tβh  were obtained from Henrike Scholz, Cologne, Germany; Hiromu Tanimoto, Martinsried, Germany; Andreas Thum, Konstanz, Germany; Martin Schwärzel, Berlin, Germany; and Amita Seghal, Chevy Chase, Maryland, US. TyrRf05682 , TyrRII29Δ , and TyrRII-TyrR124Δ were kindly provided prior to publication by Edward Blumenthal, Milwaukee, Wisconsin, US , oamb , as described elsewhere [tβhnM18 mutant line with its original control line, which was also obtained from Henrike Scholz.The l levels . Mutantslsewhere . In ordehsp-tβh and elaV-GAL4;tub-GAL80 crosses, which were kept at 18°C without humidity control.Flies were kept on standard cornmeal/molasses food in a 12/12 h light/dark cycle at 60% relative humidity and 25°C except for 2-anesthetized and sorted by sex , and their wings were clipped at two-thirds of their length. If not stated otherwise, animals recovered in the food vials overnight. Individuals were captured using a fly aspirator and transferred into the experimental setup on the following day.After hatching, experimental flies were collected into new food vials for 2 days. The day before testing, flies were COhsp-tβh flies were heat shocked for 30–45 min at 37°C with 3–4 h of recovery time at 25°C before testing. elaV-GAL4;tub-GAL80;UAS-Tdc2 flies were heated at 33°C overnight, with 30 min of recovery time at room temperature before testing.http://buridan.sourceforge.net). If a fly jumped off the platform, it was returned by a brush, and the tracker was restarted. All data were obtained from 5 min of uninterrupted walk or the first 5 min of a 15-min walk. See doi: 10.17504/protocols.io.c7vzn5 for fly preparation.We used the Buridan’s setup to test fly locomotion; details are described in  (RRID:SChttps://github.com/jcolomb/CeTrAn/releases/tag/v.4) as previously described in [Data were analyzed using CeTrAn v.4 (ribed in . Brieflyribed in . Stripe ribed in ). TherefBuridan’s paradigm appears to be particularly sensitive to differences in genetic background . TherefoSugar response was measured as described elsewhere . Brieflyp < 0.05), so we used the parametric 2-way ANOVA followed by Tukey HSD post hoc test and Welch 2-sample t test, respectively, or non-parametric paired Wilcoxon rank sum test with Bonferroni correction and Wilcoxon rank sum test, respectively. The p-value was additionally corrected for 2 repeated measurements of data from Buridan’s paradigm. The sample size of each group is indicated within the graphs. Default alpha value was set to 0.005 [The mean walking speed was calculated out of medians (see “Buridan’s paradigm” above) and plotted with the standard error of the mean. Sucrose response and stripe deviation are shown as boxplots representing the median (bar), the 25%–75% quantiles (box), and data within (whiskers) and outside (outliers as black dots) 1.5 times interquartile range. Statistical analyses were performed in R (RRID:SCR_001905). Walking speed data followed normal distribution whereas stripe deviation did not .As some aspects of Buridan’s paradigm have been shown to be highly sensitive to genetic background  and the tβhnM18 mutation provide evidence for 3 different modes of inheritance, depending on the phenotype analyzed, conforming to the definition of differential dominance.Taken together, we find that flies heterozygous for the tβh gene. In other words, the failure of the heterozygous flies to behave like wild-type flies may be due to the reduced gene dosage. To avoid such reduced gene expression in trans-heterozygous animals and to, instead, mimic the hemizygous wild-type males, we started our rescue experiments by driving an X-linked UAS-tβh transgene. We drove expression of the rescue construct in different tissues in tβh mutant males: in all cells (Actin-GAL4), in all neurons (nSyb-GAL4), in non-neuronal tyraminergic cells (Tdc1-GAL4), in tyraminergic neurons (Tdc2-GAL4), and in octopaminergic neurons (NP7088-GAL4). All of those lines drive expression throughout development and in adulthood . However, we are not aware of a tβhnM18,hsp-tβh X chromosome.Taken together, the results from heat-shock-induced expression of ckground  phenocopckground  throughotβh gene dosage, where only a narrow range of gene expression supports wild-type behavior. To test this hypothesis, we increased the acute expression of the tβh and Tdc  enzymes in wild-type animals , the stripe deviation was affected in only the 2 TA receptor mutants TyrRf05682 and honoka, in opposite directions  and other epigenetic effects, e.g., via differential maternal or paternal transfer of the gene in question . The homozygous females and hemizygous males show identical phenotypes in all parameters tested, both in the mutant and the wild-type genotypes  and find phenotypes indicating differential dominance, no dosage compensation takes place as this process occurs in males in Drosophila and not in females [tβh gene expression may be lower in heterozygous animals than in either wild-type males or wild-type females. Importantly, this lower level of gene expression does not have the same effect in all phenotypes, leading to varying inheritance. Finally, the mutant allele in the heterozygous females always came from the mutant father, while a wild-type mother  provided the other X chromosome, such that any heterogeneity in the inheritance cannot come from heterogeneity in parent-of-origin imprinting effects, either. Thus, the only remaining explanation from the design of these experiments is that the pleiotropic tβh locus indeed confers differential dominance to the alleles we used here  to network function.Several studies have suggested that OA and TA may operate as an opponent system ,83–86. ITdc affected walking speed.Both the spatial rescue Figs  and 3 anTo further explore this possibility, we manipulated TA and OA signaling individually, via OA receptor knock-out in the non-recessive walking parameter experiments . In thestβh gene increased fixation behavior of the flies, even beyond wild-type levels. Perhaps most strikingly, median stripe deviation was the lowest value for every single driver line we tested  increased walking speed, while removing both (homozygous mutants) decreased walking speed  could not be starker in walking speed . While it proved exceedingly difficult to decrease stripe fixation to control levels (observed in only 1 out of 13 manipulations), adding or removing ng speed . Confirmtβh rescue constructs . ,45. 24,4tβh gene in its genomic locus in heterozygous animals (and hence with certainly wild-type spatiotemporal expression levels; tβh gene is involved in all the phenotypes we studied, despite multiple failed rescue experiments in the walking phenotypes. Specifically, the autosomal or gonosomal location of the rescue construct affected rescue results via male dosage compensation (Figs In this work, we not only introduced the wild-type allele of the ion Figs  and 3, bion Figs . These dDrosophila are pleiotropic, nor how many of them display differential dominance. However, we have recently observed differential dominance in at least 1 other gene, the transcription factor FoxP [While pleiotropy was not found to be universal , it is ntor FoxP .


The present study selected four genes strongly related to autoimmunity. Their expression was found to be significantly altered in melanoma patients according to a multi-validation procedure carried out on 1948 patients. Such genes may represent suitable molecular targets to further investigate the role autoimmunity may play in melanoma setup and development. Our data suggest that autoimmunity may play a beneficial role in melanoma set up, at least to some extent.NOD2, BAX, IL-18 and ADRB2 were found to be significantly different in melanoma vs. controls and discriminate melanoma from controls in an extremely effective way, either as single molecules  or as a profile, according to the PCA analysis. Patients showing high-expression of NOD2 and of IL-18 also show a significant survival improvement as compared to low-expression patients. (4) Conclusions. Four genes strongly related to autoimmunity show a significant altered expression in melanoma samples, highlighting the role they may play in melanoma.(1) Background. Immune response dysregulation plays a key role in melanoma, as suggested by the substantial prognosis improvement observed under immune-modulation therapy. Similarly, the role of autoimmunity is under large investigation in melanoma and other cancers. (2) Methods. Expression of 98 autoimmunity-related genes was investigated in 1948 individuals . Data were derived from four independent databases, namely, GEO in the selection phase, and Ist Online, GEPIA2 and GENT2, in three sequential validation-steps. ROC analyses were performed to measure the ability to discriminate melanoma from controls. Principal Component Analysis (PCA) was used to combine expression data; survival analysis was carried out on the GEPIA2 platform. (3) Results. Expression levels of  Autoimmunity is activated when the immune system recognizes target cells as non-self. Under normal conditions and activity, the immune system reacts towards foreign molecules that are potentially harmful; for example, against molecules associated with viruses, bacteria and parasites. Under such conditions, structures recognized as self undergo immune tolerance . On the Melanoma is reported as being strongly related to autoimmune responses. In PubMed-indexed manuscripts we measured the occurrence of autoimmunity keywords in manuscripts related to 28 different cancer types, according to the methodology reported in Materials and Methods. Such occurrence is here reported as a “Knowledge-based Autoimmunity Score” (kb-AS). https://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS1375) accession date 8 April 2021. p value is also indicated. AUC indicates the ability to discriminate melanoma from nevi samples; its value ranges from 0.5 to 1, with 1 indicating 100% ability to discriminate controls from disease cases.Ninety-eight autoimmunity related-genes were selected from the literature ,18,19 anp < 0.0001. Six genes match these criteria, namely: NOD2, BAX, IL-18, ADRB2, ITGAV and MYO9B. The ROC analysis for each gene is reported in p values. The AUC is > 0.93 in all cases, indicating an extremely high ability to differentiate melanoma samples from controls.Severe requirements were selected from the values reported in NOD2, BAX, IL-18, ADRB2, ITGAV and MYO9B) was then investigated in an independent public database, namely IST Online. Such database contains several different cancers datasets and normal tissues datasets; the melanoma dataset contains 208 melanoma patients and the healthy skin dataset, used here as control, contains 147 healthy skin samples. Data collected on the GEO dataset were fully validated; in fact, all six genes confirmed significant different expression levels in melanoma vs. healthy skin, in the IST Online dataset (p < 0.0001). The used threshold values are indicated as dashed lines in The expression of the six genes , a public database reporting expression data from 461 melanoma patients and 558 healthy skin controls. Four out of six genes showed a significantly different expression in melanoma vs. healthy skin, as depicted in During the finalization of the present study, Ist Online database became not publicly available any longer. We then decided to validate the six genes in two additional public databases. As a second validation, the expression levels of NOD2, BAX, IL-18 and ADRB2 were investigated in skin cancer samples from the GENT2 database (available at http://gent2.appex.kr/gent2/ accession on 28 May 2021), which collects data from 310 skin cancers samples and 201 controls. All four genes show a strongly significant differential expression in skin cancers as compared to the appropriate controls. Namely NOD2 shows a LOG2 FC (Fold change expressed in Log2) expression of −1.34, p < 0.001; BAX shows a LOG2FC expression of 1.3, p < 0.001; IL-18 shows a LOG2FC expression of 3.09, p < 0.001; ADRB2 shows a LOG2FC expression of 1.6, p < 0.001.As a third-round validation, the expression levels of the four genes NOD2, BAX, IL-18 and ADRB2) was then analyzed according to the PCA dimensionality reduction tool in the GEPIA2 database. As reported in We have previously used Principal Component Analysis (PCA) to identify the best genes-combination able to discriminate melanoma from controls within a large selection of nicotinamide receptors  and cytoNOD2, BAX, IL-18 and ADRB2 achieves an extremely high efficacy to discriminate controls from melanoma samples, with AUC = 0.983. In addition, 88.9% of observed nevi were correctly classified and 93.3% of observed melanoma were correctly classified.As an additional evaluation, multiple logistic analysis and ROC analysis were carried out on the four genes combined expression profile, in the 45 melanoma and 18 nevi samples derived from the GEO GDS1375 dataset. NOD2, BAX, IL-18 and ADRB2 is significantly altered mostly in cancer types showing the highest autoimmunity scores, further supporting the possible role of such genes in the interplay between autoimmunity and cancer.We then further characterized the gene-expression of the four validated genes in 28 different cancer types. NOD2 or IL-18 showed a significantly better survival, with a Hazard Ratio (HR) of 0.58 and 0.52, respectively, compared to low expression patients . In addition to the possible diagnostic role reported in the previous figures, such data indicate that NOD2 and IL-18 expression levels may also have a prognostic value in melanoma.The survival associated with the expression values of each gene was then analyzed according to the GEPIA2 database. Patients expressing high levels of patients ; the anaA possible role of autoimmunity in melanoma is currently under intense investigation. Autoimmunity is generally recognized as a possible severe side effect of melanoma therapy, upon interferon-  or ipiliNOD2, BAX, IL-18 and ADRB2, NOD2 and IL-18 appear to be particularly interesting from a prognostic point of view since their expression was also found to be related to a significant survival improvement 1.2, further supporting the role we recently underlined ion channels play, as possible melanoma therapeutic targets [s system . As reces system , ADRB2 prthritis . Polymorrthritis  and Rheurthritis . ADRB2 hmelanoma ; the pre targets ,60.BAX is a proapoptotic molecule, member of the BCL-2 family, with a recognized role to control immune tolerance and prevent autoimmune disorders in mice [BAX in melanoma patients, supporting it as a possible autoimmunity-related target in melanoma. in mice . Its com in mice , supportThe link between autoimmunity and cancer is largely investigated but it is still controversial whether autoimmunity helps or blocks cancer set up or development ,63,64. FNOD2, IL-18 and ADRB2, since their expression is shown to be almost abolished in melanoma patients. Interestingly, the multiple logistic analysis shows that the AUC = 0.98 computed for the four-genes combined profile   and melanoma-associated antigens (MAGEs). The co-occurrence of vitiligo, an autoimmunity-based disease, has been associated with better prognosis in melanoma patients  and a pr profile , is muchfor BAX) . This deFinally, when the expression analysis was extended to several cancer types other than melanoma, the four genes were found significantly altered especially in cancer types mostly related to autoimmunity, according to the knowledge-based Autoimmunity Score (kb-AS) computed in the present study. Some pitfalls may be recognized in the procedure followed to measure kb-AS. The first relates to the continuous growth of the PubMed database, which implies the possibility that the kb-AS may (or will) change over time. Further, less common cancer types were not investigated. We are aware that the searches carried out were not extensive, and we will address this issue in more details and larger completeness in a study focused on this specific aspect. However, we believe the current form of the kb-AS is sufficiently indicative to recognize at least the most common cancer-types showing the known highest relation to autoimmunity.Ninety-Eight Autoimmunity-Related Genes Were Taken for the Literature ,18,19. TThe known relation of 28 cancer types to autoimmunity was measured as follows: PubMed searches were carried out looking for the occurrence of the words relating to each cancer type in All-fields. Such a procedure was carried out as a stand-alone search or in the presence of autoimmunity-related words. Therefore, the co-occurrence of cancer-related words and autoimmunity-related words was measured. Words used to identify each cancer type and to identify autoimmunity are reported below.The number of manuscripts presenting the co-occurrence, divided by the total number of manuscripts related to each cancer-type, expressed as percentage, was then reported as the “knowledge-based Autoimmunity Score” of cancers (kb-AS). The cancer types investigated were the 28 cancer types reported in the GEPIA2 database. The words related to each cancer type, searched in PubMed All-fields, are as follows:Cervical = “Cervical squamous cell carcinoma” OR “Endocervical adenocarcinoma”;Esophag = “Esophageal carcinoma” OR “Esophagus carcinoma”;Pheoc-Para = “Pheochromocytoma” OR “Paraganglioma”;Endometrial = “Uterine Corpus Endometrial carcinoma” OR “endometrial carcinoma” OR “endometrial cancer” OR “Endometrium cancer” OR “Uterine carcinosarcoma”;Rectum = “Rectum adenocarcinoma” OR “rectum carcinoma” OR “rectum cancer”;Adrenocortical = “Adrenocortical carcinoma”;Prostate = “Prostate adenocarcinoma” OR “prostate carcinoma” OR “prostate cancer”;Mesothelioma = “Mesothelioma”;Ovary = “Ovarian serous cystadenocarcinoma” OR “Ovarian carcinoma” OR “Ovarian adenocarcinoma”;Head and Neck = “Head and Neck squamous cell carcinoma” OR “Head and Neck carcinoma”;Gliobl = “Glioblastoma”;Sarcoma = “Sarcoma”;Liver = “Liver hepatocellular carcinoma” OR “liver carcinoma”;Bladder = “Bladder carcinoma”;Testis = “testicular Germ cell tumors” OR “testicular cancer” OR “testis cancer “ OR “seminoma”; Colon = “Colon adenocarcinoma”;Kidney = “Kidney carcinoma” OR “kidney cancer”;Breast = “Breast carcinoma”;Lung = “Lung adenocarcinoma” OR “Lung carcinoma”;Glioma = “Glioma”;Leukemia = “Leukemia”;Gastric = “Gastric adenocarcinoma” OR “stomach adenocarcinoma”;Pancreati c = “Pancreatic adenocarcinoma” OR “pancreatic carcinoma”;Melanoma = “Melanoma”;Chol = “Cholangio carcinoma”;Thyroid = “Thyroid carcinoma”;Lymphoma = “Lymphoma”;Thymoma = “Thymoma”.The autoimmunity related words with wild cards indicated by the asterisks were: “autoimmun*” OR “autoantigen*” OR “selfantigen*” OR “selftolerance”.https://www.ncbi.nlm.nih.gov/gds/), accessed on 8 April 2021 reporting the actual expression values in 63 samples (45 melanoma-patients vs. 18 nevi-patients). Calculations such as mean, Mann–Whitney test and ROC analysis were then carried out. ROC analysis is a binary test; it was used here to measure how effective the expression-level of any given gene to discriminate healthy from melanoma-biopsies is. The computed Area Under Curve (AUC) value ranges from 0.5 to 1, indicating 50% to 100% discrimination ability.The expression of 98 autoimmunity-related genes was evaluated in the melanoma GDS1375 dataset, from the GEO public database (http://ist.medisapiens.com/) accessed on 6 May 2021, having 208 melanoma samples and 147 healthy-skin samples. Different to the GEO database, IST Online does not show actual numbers; rather it expresses data as scatter plots, where each dot is one patient. Genes showing expression levels in melanoma significantly different to controls, according to the Fisher’s exact test, were then considered validated and were selected for the following validation step.The first validation step was carried out by analyzing expression levels of the selected genes in the independent IST Online database  were considered fully validated.During the finalization of the present study, the IST Online database, previously public for many years, became unavailable. In order to assure a full reproducibility of the results, we therefore decided to carry out additional validation steps. A second round validation step was carried out on the public database GEPIA2, available at n/#index  accessedNOD2, BAX, IL-18 and ADRB2) were investigated in control and in skin cancer samples , taken from the GENT2 database at the link http://gent2.appex.kr/gent2/ accessed on 28 May 2021. The gene profile tools were used, indicating as types: “Tissue”, terms: “Gene symbol”, and as keyword the four genes symbol; data were then extracted from the table reporting T-test and Log2 fold changes results.The four selected genes  dataset, according to a methodology previously described [Survival analysis in melanoma patients was investigated according to GEPIA2 at NOD2, BAX, IL-18 and ADRB2 were analyzed in all cancer types present in GEPIA2 database, available at http://gepia2.cancer-pku.cn/#index [p value < 0.0001 were selected as the significance threshold.The expression levels of n/#index  accessedp value < 0.0001. Fisher’s exact test was carried out to evaluate sample distribution above or below a given threshold, in data from Ist Online database. The statistical package GraphPad Prism version 9.3.0 was used .The analyses were performed on 1948 human samples from four independent databases. The samples distribution was as follows: 45 melanoma and 18 nevi samples from GEO database; 208 melanoma and 147 healthy-skin samples from IST Online database; 461 melanoma and 558 healthy-skin samples from GEPIA2 database and 310 melanoma and 201 healthy skin samples from GENT2 database. Mean, two tails t Test, Mann–Whitney test and ROC analysis were carried out, with a significance-threshold set at In conclusion, the present study identifies four genes strongly related to autoimmunity. Their expression was found to be significantly altered in melanoma patients according to a multi-validation procedure carried out on 1948 patients from four independent databases. We believe such genes may represent suitable molecular targets to further investigate the role autoimmunity may play in melanoma setup and development. Such data suggest that autoimmunity may play a beneficial role in melanoma set up, at least to some extent.


Thunnus species or Katsuwonus pelamis can be used as canned tuna. Here, we authenticated 90 canned tuna products, including 25 cat food samples, by sequencing two mitochondrial regions, 16S rRNA (16S) and the control region (CR). BLAST analysis revealed that Sarda orientalis, Euthynnus affinis, Auxis rochei, and Auxis thazard are all used as substitutes for legitimate tuna products. We found that 63.33% of investigated samples are true canned tuna, i.e., contain Thunnus species or skipjack tuna. We advocate that the Taiwanese government publishes an official standardized list of fishes, especially so that scientific, Chinese and vernacular names can be assigned unambiguously based on a “one species-one name policy”, thereby clarifying which species can be used in seafood products such as tuna. Furthermore, we feel that the large-scale and long-term monitoring of canned tuna products is warranted to fully assess the extent of tuna product adulteration in Taiwan.Fish of the tribe Thunnini represent a significant proportion of the stock caught by the fishing industry, with many of these fishes being collectively called tuna. However, only certain species can be used legally as an ingredient in canned tuna products, depending on regional food regulations. In Taiwan, only  Katsuwonus pelamis) (in Chinese: 正鰹) and yellowfin tuna  (in Chinese: 黃鰭鮪) [Approximately 17% of the global human population’s intake of animal protein in 2017 constituted fish . In 2018se: 黃鰭鮪) ,5.Thunnus (in Chinese: 鮪屬), Katsuwonus (in Chinese: 正鰹屬), Auxis (in Chinese: 花鰹屬), Euthynnus (in Chinese: 巴鰹屬), and Allothunnus (in Chinese: 細鰹屬). Fishes of this tribe can be generally termed “tuna”. For example, Auxis rochei is called bullet tuna (in Chinese: 圓花鰹), Euthynnus alleteratus is little tunny (in Chinese: 小巴鰹), and Allothunnus fallai is slender tuna (in Chinese: 細鰹). However, the Chinese translation of tuna is 鮪 in Taiwan or 金槍魚 in Mainland China, which specifically refers solely to Thunnus spp. Previously, many different scombrids were used as an ingredient in “canned tuna”, even if they did not belong to the tribe Thunnini. For instance, Sarda (in Chinese: 齒鰆屬) spp. were once widely used in canned tuna because they possess a similar taste and texture to it [Thunnus and skipjack tuna are widely recognized as legal canned tuna species. To align with international standards, the Taiwan Food and Drug Administration allows skipjack tuna to be used in 鮪 (for tuna)-labeled canned tuna products, even though it does not belong to the genus Thunnus, but other “pseudo-tunas” can no longer be used legally as a canned tuna ingredient.The tribe Thunnini comprises five genera: re to it . ImportaSeafood mislabeling is profuse worldwide ,19,20,21Traditional morphology-based identification is rarely applied to seafood because many products undergo physical  or chemical  processing before being sold. These aspects of food processing typically eliminate diagnostic morphological characters needed for species authentication. Fortunately, molecular authentication based on nucleic acid sequence similarity can overcome this limitation. DNA can be obtained from a tiny piece of tissue and it is more resistant to degradation and food processing. Therefore, DNA-based authentication is being widely employed to identify species in seafood ,28,29,30Thunnus fishes are important elements of sashimi and sushi. The development of freezing technology and booming global trade in the early 1970s has transformed the bluefin tunas (T. thynnus (in Chinese: 大西洋黑鮪), T. maccoyii (in Chinese: 南方黑鮪), and T. orientalis (in Chinese: 太平洋黑鮪)) from a cat food into a delicacy served at high-end restaurants [Thunnus fishes, attaining the largest size and greatest price. However, increased consumption has also threatened their stocks, which are decreasing and the status of all three species is deemed Critical (IUCN). Today, regional fishery management organizations are responsible for managing and monitoring tuna fishing in order to keep it sustainable [The increasing global popularity of Japanese cuisine has markedly increased market demand for tuna, since taurants . Bluefintainable .Thunnus fishes, especially bluefin tuna, makes them very vulnerable to mislabeling. Previous molecular authentication studies on sushi reported that many fishes are used as substitutes for Thunnus species, including escolar (Lepidocybium flavobrunneum) (in Chinese: 鱗網帶鰆), salmon  (in Chinese: 安大略鱒), banded rudderfish (Seriola zonata) (in Chinese: 環帶鰤), great amberjack (Seriola dumerili) (in Chinese: 杜氏鰤), skipjack tuna, little tunny, as well as various shark species [Thunnus species varies, prompting high-priced bluefin tuna or bigeye tuna (T. obesus) (in Chinese: 大目鮪) to be substituted for a cheaper species such as yellowfin tuna. Notably, enforcement of fishery management can drive reverse substitution, whereby high-priced bluefin tuna is sold as cheap yellowfin tuna or Thunnus fishes are labeled as skipjack tuna to enable market entry of illegal catch [The soaring demand for  species ,35,36,37al catch ,38,39.Thunnus fishes, but Xiong et al. [Although DNA-based methods are very powerful tools for authenticating fish products, food processing, and especially canning, can limit their applicability. To date, conventional DNA barcoding remains the most widely deployed authentication approach, whereby a ~650-bp region of the mitochondrial gene encoding for cytochrome c subunit I (COI) is sequenced as a bioidentification “barcode” ,41. Howeg et al.  and Hwang et al.  detectedThunnus fishes, if the ingredients list contained Thunnus spp. or skipjack tuna, or if an image on the label indicated the can harbored Thunnus fishes. We recorded information, typically written in Chinese, on brand, manufacturer or importer, place of manufacture, labeling, and ingredients. If the cans were imported from the USA or Japan, the respective English or Japanese labels were also recorded , if the company website claimed the product was made from recorded . The samrecorded , and theThunnus spp., we PCR-amplified a fragment of the mitochondrial control region  from the same DNA sample. CR amplification was conducted in a mixture containing 5 ng template DNA, 12.5 μL of 2× Taq PCR MasterMix , and 12.5 μmol of each forward and reverse primer—Forward, Tuna-CR_F; Reverse, Tuna-CR_R1 or Tuna-CR_R2 [DNA was extracted from each of the 90 tissue samples using DNA Extraction Kit S . PCR amplifications of the mitochondrial 16S rRNA fragment (16S) (85 bp) were performed in a mixture containing 5 ng template DNA, 12.5 μL of 2× Taq PCR MasterMix , and 12.5 μmol of each forward and reverse primer. We used primers designed by Horreo et al.  and modina-CR_R2 —made up Edited 16S sequences were first aligned using ClustalW in MEGA11 , and the3 bootstrapping replicates in MEGA11 [All CR sequences used in the study of Mitchell and Hellberg (2016) were downloaded to serve as reference sequences, and then our CR fragments and reference sequences were aligned in ClustalW. A neighbor-joining (NJ) analysis was then conducted based on Kimura two-parameter (K2P) distances and 1 × 10n MEGA11 . Accordin MEGA11 , monophyhttps://fishdb.sinica.edu.tw/ (accessed on 26 October 2021)). Although the Chinese symbols 鰹魚could broadly refer to any species from the genera Auxis (in Chinese: 花鰹屬), Euthynnus (in Chinese: 巴鰹屬), and Katsuwonus (in Chinese: 正鰹屬), skipjack tuna is the only species generally termed 鰹魚 and that can be used legally in Taiwan to make canned tuna  (in Chinese: 東方齒鰆) or displaying the ambiguous vernacular name 煙仔虎, which can refer to either skipjack tuna or oriental bonito  (in Chinese: 巴鰹), Hap_C is skipjack tuna, Hap_D and Hap_F are Thunnus species, Hap_H is either longtail tuna (T. tonggol) (in Chinese: 長腰鮪) or bigeye tuna (T. obesus), and Hap_G is either bullet tuna or frigate tuna (Auxis thazard) (in Chinese: 扁花鰹).We successfully amplified the 16S fragment from all 90 samples, resulting in eight haplotypes . All hapThunnus fishes. However, the success rate of CR amplification from those samples was quite low . The aligned CR dataset  is never associated with oriental bonito, so it is unreasonable for the symbol for oriental bonito to be placed in parentheses following “鮪魚” (representing “tuna fish”) on the label for this sample. We observed a similar issue for sample T4, since neither skipjack tuna nor oriental bonito can be regarded as a type of “鮪” (Thunnus spp.). Since the ingredient statement on the label fails to clearly indicate which species is contained in the can, it is difficult to judge whether or not these two samples are mislabeled. Notably, many of the products identified as exhibiting inconsistent labeling were also found to be mislabeled. The mislabeling rate of canned products for human consumption was 38% (25/65). Mislabeling was even more pervasive among cat food products, with a rate of 68% (17/25). The main reason for this high mislabeling rate of cat food products is that many claim to contain Thunnus fishes but are in actual fact made from skipjack tuna. The overall mislabeling rate for the 90 tested products of this study is ~47% (42/90).Excluding the canned cat food samples that were all accurately labeled, 25 of the remaining 65 canned tuna products were mislabeled and a further three were potential mislabeling cases. Our BLAST analysis confirmed that sample T36 contained Thunnus fishes or skipjack tuna, and 20 out of 25 cat food products are true canned tuna. Overall, the proportion of true canned tuna products is about 63.33% (57/90) in this study.Of the 65 human food products we tested, 37 (57%) legitimately contained either Thunnus species or skipjack tuna as an ingredient, obviously mislead customers into believing these products contain true canned tuna. For this study, we solely relied on the information on Chinese labeling, but we also noted conflicting information between Chinese and source-language labeling of imported products. For example, the ingredient statement in Japanese of sample T42 clearly declares that it is made from albacore tuna , but its Chinese label only states that it contains Thunnus fishes (in Chinese: 鮪魚). Similarly, the Japanese label of sample T59 indicates skipjack tuna as an ingredient (in Japanese: かつお), but its Chinese label specifies yellowfin tuna (in Chinese: 黃鰭鮪)  on their labels but also list scombrids other than se: 黃鰭鮪) . Such cohttps://fishdb.sinica.edu.tw/chi/chinesequer1.php (accessed on 26 October 2021)) as a standard list of fishes in Chinese corresponding to scientific nomenclature. This Dictionary indeed clarifies that the Chinese symbols 東方齒鰆  correspond to Sarda orientalis, but it does not include other Chinese vernacular names. Thus, any official “one species-one name” standard should not only contain scientific and Chinese names, but also incorporate vernacular names.The “one species-one name” policy is critical to the authentication of fishery products . Clearlyp < 0.01).Notably, our success rates for amplifying the two mitochondrial DNA fragments differed considerably—100% for 16S, but only 16% for CR. The canning process is known to damage DNA molecules, with Quinteiro et al.  documentThunnus comprises very closely related species, some of which display genetic introgression [Thunnus fishes, their exact species identity remains unclear. Though conventional DNA barcoding can distinguish Thunnus fishes [Apart from haplotypes Hap_A and Hap_C, a single species was not identified by BLAST for the other 16S haplotypes. There are a number of possible reasons for that outcome. First, DNA degradation through the canning process limits molecular authentication based on longer sequences, such as via conventional DNA barcoding. Accordingly, shorter DNA segments must be targeted, but they contain less information and so are less likely to unambiguously assign a specific species . Second,gression ,59. Conss fishes ,61,62, is fishes . GenBanks fishes ,64,65, aThunnus fishes or skipjack tuna. Although oriental bonito was never found in canned cat food products, the other three substituted fishes were identified in both human and cat food samples. These same four species have also been reported as illegitimate tuna substitutes in other studies [Thunnus products in other studies [In this study, we found that many canned tuna products in Taiwan are made from oriental bonito, kawakawa, bullet tuna, or frigate tuna instead of legitimate  studies ,50,66,67 studies ,68, we dThunnus composition of canned tuna products in Taiwan remains unclear. Identifying canned tuna products to species level is important because certain Thunnus fishes have higher mercury levels [Our NJ analysis of CR sequences, including five haplotypes generated in this study, further revealed that both yellowfin tuna and longtail tuna are used in canned tuna products. Yellowfin tuna is one of the commonest canned tunas ,69, so iy levels , posing y levels , or smalp < 0.01). Accordingly, we implore the responsible authorities to clearly define the terms for use in canned product labeling.We observed the terms 白身鮪魚 or 鮪魚白肉 commonly in the ingredient statements of our cat food samples , reflectAlthough we found that 63.33% of our samples are true canned tuna, this outcome may not reflect the actual adulteration level of canned tuna products in Taiwan. First, we selected only one small piece of tissue from each can, but the mixing of tuna species in cans has been found in the European market . An asseThunnus fish or skipjack tuna. In many cases, the labels of the sampled canned tuna products would confuse customers as to what species they contain. Either they contain species such as oriental bonito that do not conform to Taiwanese legislation, or ill-defined terms such as 白身鮪魚 or Chinese vernacular names are used. We assert that a standard list of scientific names and their corresponding Chinese and vernacular names conforming to the “one species-one name” principle, as well as clear definitions of terms for use in canned tuna labeling, is crucial to tackling fish product adulteration. We found that ~37% of investigated canned tuna products contain illegitimate species. In many cases, the manufacturers have substituted so-called “pseudo-tunas”, such as oriental bonito, kawakawa, bullet tuna, and frigate tuna, for legal species, i.e., Thunnus species and skipjack tuna. Our study demonstrates that a pair of primers targeting a short segment (85 bp) of 16S performs well in amplifying DNA extracted from canned food samples. However, the limited information content provided by this short sequence hampered molecular identification to species level, especially given the close phylogenetic relationships and potential for gene flow among Thunnus species. Moreover, the CR fragment we targeted largely proved uninformative, likely owing to the extreme DNA fragmentation caused by high heat treatment during the canning process. Our previous study of seafood adulteration in conveyor-belt sushi restaurants revealed no case of tuna fraud in such establishments [We report an overall mislabeling rate of 46.67% among the 90 samples of this study, with 63.33% of sampled canned products being true canned tuna legitimately made from ishments , so such


Healthy lifestyle and rehabilitation may mitigate late effects after cancer treatment, but knowledge about lifestyle and rehabilitation information needs among long-term young adult cancer survivors (YACSs) (≥ 5 years from diagnosis) is limited. The present study aimed to examine such information needs among long-term YACSs, and identify characteristics of those with needs.The Cancer Registry of Norway identified long-term YACSs diagnosed with breast cancer, colorectal cancer, non-Hodgkin lymphoma, leukemia, or malignant melanoma at the age of 19–39 years, between 1985 and 2009. Survivors were mailed a questionnaire, in which respondents reported their information needs on physical activity, diet, and rehabilitation services 5–30 years post-diagnosis. Descriptive statistics and logistic regression analyses were used to examine the prevalence of information needs and associated factors.Of 1488 respondents (a response rate of 42%), 947 were included. Median age at diagnosis was 35 years (range 19–39) and median observation time since diagnosis was 14 years (range 5–30). In total, 41% reported information needs for information about physical activity, 45% about diet, and 47% about rehabilitation services. Information needs were associated with higher treatment intensity, increasing number of late effects, and an unhealthy lifestyle.A large proportion of long-term YACSs report information needs regarding lifestyle and/or rehabilitation more than a decade beyond treatment. Assessments of such information needs should become a part of long-term care of these cancer survivors.The online version contains supplementary material available at 10.1007/s00520-021-06418-z. Young adults aged 19–39 years when diagnosed with cancer will often face unique challenges, such as disruptions to their education and career paths, and establishing relationships, form a family, and ensure financial stability, compared to those diagnosed in later adult age . SuccessA healthy lifestyle may reduce risks of late effects, potentially improving long-term health among cancer survivors . In ordeA large proportion of young adult cancer survivors (YACSs) report various information needs related to health outcomes and lifestyle during the first years after diagnosis   (stages I–III), colorectal cancer (CRC), non-Hodgkin lymphoma (NHL), leukemia (LEUK), or malignant melanoma (MM) , between 1985 and 2009, during young adulthood (19–39 years of age). Relatively good prognosis and risk of late effects are reasons why these cancer diagnoses were chosen for inclusion. YACSs treated for other cancer types relevant for young adults, such as Hodgkin lymphoma, cervical cancer, and testicular cancer, were not included because these survivors were included in other concurrent studies at our department at the time of survey.YACSs were excluded if more than one cancer diagnosis or distant metastases were registered in the CRN, if the participants reported to be on cancer treatment at the time of survey, if they reported recurrence of cancer, or if they did not respond to questions related to treatment and/or about information needs. Survivors after non-metastatic MM treated with minimal surgery served as a reference group for treatment intensity.Information on gender, date of birth, cancer diagnosis, and date of diagnosis were obtained from the CRN. This information was used to calculate age at diagnosis, age at survey, and time from diagnosis to survey.In total, the questionnaire consisted of 302 items of which 162 items were compulsory. The topics covered were socio-demographic background, late effects, health care use and needs, information needs, work ability and financial burden, physical health, mental health, fatigue, lifestyle, health-related quality of life, and health literacy. The majority of the measures were covered by validated instruments –18.Participants received the questionnaire by mail, together with study information, an informed consent form, and a pre-paid return envelope. Non-responders received one reminder after 5 months.Perceived information needs on lifestyle advice and rehabilitation services were assessed by three single questions: “Do you want advice on physical activity/exercise?”; “Do you want dietary advice?”; and “Do you want information about rehabilitation services?” The response categories for each question were “Yes,” “No, have no need,” and “No, have had need, but received enough information.” To identify characteristics of those with information needs, we chose to compare those who responded “Yes” with those who responded “No, have no need.”We obtained information about marital status (living as a couple versus not), education , and work situation (within work force/being a student versus not). Socio-economic status (SES) was assessed by combining marital status, education, and work situation. To be included in the high SES group, at least two of the three following conditions had to be fulfilled: living as a couple, college/university, and being within work force/being a student.Treatment was self-reported and categorized into (1) “minimal surgery restricted to localized MM” , (2) “surgery and/or radiotherapy only,” (3) “systemic treatment only,” and (4) “systemic treatment combined with surgery and/or radiotherapy.” Information on late effects was obtained by asking if the participants had experienced any of 16 listed late effects . The total number of late effects was summarized for each participant and categorized into groups with 0, 1–2, 3–4, and ≥ 5 late effects. Chronic fatigue and psychological reactions were excluded from the list, since these conditions were measured by separate, validated instruments.Do you have problems with tiredness?”; “Do you have difficulty concentrating?”) scored from 0 to 3, with increasing total score (0 to 33) implying higher levels of fatigue. Internal consistency  for the population included in this analysis was 0.92 for fatigue scale. To identify chronic fatigue, scores of each item were dichotomized  and chronic fatigue was defined by a dichotomized sum score ≥ 4 with ≥ 6 months duration [Fatigue was assessed by the Chalder Fatigue Questionnaire (FQ) . FQ consduration .Little interest or pleasure in doing things,” “Feeling down, depressed, or hopeless”) scored from 0 to 3, with increasing sum score (0 to 27) indicating higher level of depressive symptoms. Cronbach’s alpha for PHQ-9 was 0.88 in the present population. Anxiety symptoms were measured by the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS-A) [I feel tense or “wound up”,” “I get a sort of frightened feeling as if something awful is about to happen,” “Worrying thoughts go through my mind”), scored from 0 to3, with increasing sum scores (0 to 21) indicating higher level of anxiety symptoms. Cronbach’s alpha for HADS-A was 0.83 in the present population.Depressive symptoms were assessed using the Patient Health Questionnaire-9 (PHQ-9) . PHQ-9 c(HADS-A) . This su2) ≥ 30) was calculated from self-reported height and weight [Physical activity/exercise was assessed by a modified version of the Godin Leisure Time Exercise Questionnaire . Being pd weight . Being aP value ≤ 0.05 was considered statistically significant.Continuous variables are presented by means, standard deviations (SD), medians, and ranges, and categorical variables by numbers and percentages. Three logistic regression analyses identified characteristics of YACSs with information needs on (1) physical activity, 2) diet, and 3) rehabilitation, compared to those without such needs. Statistically significant variables associated with each of the three types of needs in univariate analyses were included as explanatory variables in three separate multivariable logistic regression analyses  responded to the survey, of which 541 were excluded for the present analyses Below is the link to the electronic supplementary material.


Maximizing osteointegration potential of three-dimensionally-printed porous titanium (3DPPT) is an ongoing focus in biomaterial research. Many strategies are proposed and tested but there is no weighted comparison of results.We systematically searched Pubmed and Embase to obtain two pools of 3DPPT studies that performed mechanical implant-removal testing in animal models and whose characteristics were sufficiently similar to compare the outcomes in meta-analyses (MAs). We expanded these MAs to multivariable meta-regressions  to verify whether statistical models including reported scaffold features  or post-printing treatments  could explain the observed differences in treatment effects (expressed as shear strength of bone-titanium interface).“Animal type”  and “type of post-treatment” (treatment performed after 3D printing) were moderators providing statistically significant models for differences in mechanical removal strength. An interaction model with covariables “pore-size” and “porosity” in a rabbit subgroup analysis  was also significant. Impact of other moderators (including “time” and “location of implant”) was not statistically significant.Our findings suggest a stronger effect from porosity in a rat than in a sheep model. Additionally, adding a calcium-containing layer does not improve removal strength but the other post-treatments do. Our results provide overview and new insights, but little narrowing of existing value ranges. Consequent reporting of 3DPPT characteristics, standardized comparison, and expression of porosity in terms of surface roughness could help tackle these existing dilemmas. In this model, the regression coefficient (RC) for the animal type “rabbit” was estimated to be 2.7 times higher than for the “rat” model (intercept = 5.6009) and the “sheep” model was estimated to be 4.7 times lower. These findings are shown in Fig. p-value for the test of moderators  as well as that of a model involving the interaction of “pore-size” and “animal type”. These combinations raised the accounted heterogeneities (R2) to 38.55 and 52.78%, respectively. However, the likelihood ratio test comparing all three models found no superiority of the extended models; thus, the simpler “animal type only” model was favored. To offer a better understanding of their relationship, Fig. To extend this MA to a meta-regression, we first conducted an exploratory AICc-based ranking of moderators but ultimately performed step-down selection based on the offering of a significant p-value (p = 0.0130) for the test of moderators [F  = 5.0371], with the likelihood ratio test favoring it over a reduced “pore-size only” or “pore-size and porosity but no-interaction” model. We noted that “pore-size” and “porosity” were correlated (0.79), making it impossible to fully distinguish between both. This model accounted for 66.44% of the heterogeneity (R2) and RC = − 0.0022 [p = 0.0803 ]. This finding is illustrated in Fig. Because “rabbit” was our most represented model, we performed subgroup analysis to exclude the aforementioned effect of “animal type” and further investigate the role of other possible moderators. Here, a multivariable regression model with the interaction of covariables “pore-size” and “porosity” provided the most significant  p = 0.010 for the2 = 72.2%) and a between-study variance (τ2) of 3.8944  for which a mixed-effects model was applied again. The data, in turn, provided an SMD of 1.63 . The forest plot of MA2 is shown in Fig. 2 to 36.6% and kept the SMD at 1.64  = 2.5932, p = 0.0432). The likelihood ratio test favored this model over a combined “animal type and treatment type” model, and it provided an R2 of 44.58%. The RCs for the individual “treatment types” were not significant.Regarding MA2, we included the single moderator “treatment type” to provide the model with the best Many reports have been published concerning 3DPPT , 14, 25,Here, we conducted an SR and 2 separate MAs. The SMDs calculated (2.79 for MA1 and 1.63 for MA2) were not the focus of our search. They express a difference in the mechanical removal strength between porous and non-porous 3D-printed titanium implants and between post-treated and non-post-treated 3DPPT implants, expressed as the difference in the of SDs of TE; these differences are reasonable and have never been challenged, particularly concerning the former. However, the asymmetry in the funnel plots Figs. b should In MA1, we found that the “animal model” as a moderator may explain a difference in TE. Because our TE indicates improvement in the mechanical removal strength when making the implants porous vs not porous, less improvement is expected when implanting a porous implant in a sheep than in a rat. For the rabbit model, we had many more observations than those for rats or sheep (26/32 observations), and the TE values enclosed a wider reach  on the bone-titanium interface strength of solid titanium is well established –37. A po2 values. An explanation could be the discrepancy between grouping different animal models at the MA level and extreme genetic similarity of the individual animals at the study level [p < 0.0001) but still significant. The SMDs from MA1 and MA2 both show high between-study variance values, indicating that the estimated effects differ across studies. Another explanation could be an overlooked effect due to variation in the observation methods . However, because the included studies were small (12 samples at best), there is no straightforward explanation for the heterogeneity and we should conclude that both MAs are sensitive to biasing animalities [However, because certain caveats apply to our study, our findings should be interpreted with caution. First, substantial heterogeneity was found among the included studies, as demonstrated by the high Idy level . The Cocmalities . Second,malities . Howevermalities . In MA2,malities . Third, malities . Howevermalities . A full We performed two separate MAs with moderator analyses to determine whether statistical models including reported scaffold features  or post-printing treatments  could explain the observed differences in the treatment effect. Our findings suggest a stronger effect from porosity in a rat model than that in a sheep model. Additionally, adding a calcium-containing layer does not improve the mechanical removal strength but the other post-treatment types do. Our results provide an overview and some new insights but little narrowing of the existing value ranges. We would advocate more research involving comparing implantation to similar, “standardized” control groups and expressing “pore-size” and “porosity” in terms of surface roughness to help address existing dilemmas, along with the consequent reporting of 3DPPT characteristics.