Long-term improvement was observed in > 50% of patients in both groups. These results add weight to a clinical pragmatic proposition to consider hEDS/HSD as a single entity that requires the same treatments.Diagnosing hypermobile Ehlers–Danlos syndrome (hEDS) remains challenging, despite new 2017 criteria. Patients not fulfilling these criteria are considered to have hypermobile spectrum disorder (HSD). Our first aim was to evaluate whether patients hEDS were more severely affected and had higher prevalence of extra-articular manifestations than HSD. Second aim was to compare their outcome after coordinated physical therapy. Patients fulfilling hEDS/HSD criteria were included in this real-life prospective cohort (November 2017/April 2019). They completed a 16-item Clinical Severity Score (CSS-16). We recorded bone involvement, neuropathic pain (DN4) and symptoms of mast cell disorders (MCAS) as extra-articular manifestations. After a standardized initial evaluation (T0), all patients were offered the same coordinated physical therapy, were followed-up at 6 months (T1) and at least 1 year later (T2), and were asked whether or not their condition had subjectively improved at T2. We included 97 patients . Median age was 40 (range 18–73); 92.7% were females. Three items from CSS-16  were significantly more severe with hEDS than HSD. Bone fragility, neuropathic pain and MCAS were equally prevalent. At T2 (20 months [range 18–26]) 54% of patients reported improvement (no difference between groups). On multivariable analysis, only family history of hypermobility predicted (favorable) outcome (The online version contains supplementary material available at 10.1007/s00296-021-04968-3. Joint hypermobility, the ability to move joints beyond the normal range of motion, is defined as abnormal mobility affecting multiple joints. This health condition can be asymptomatic and has a varied prevalence, 2–57% , 2. In rThe diagnosis of EDS, especially hypermobile EDS (hEDS), in which extra-articular manifestations can be subtle, is challenging . Misdiaghttps://www.ehlers-danlos.com/2017-eds-international-classification/) to provide good management and treatment for all of them. In the same vein, very recent studies consider that hEDS and HSD exist on the same physiological continuum (hEDS being a more marked form) ) > 4/10 in at least 2 extremities .- Mast cell activation syndrome (MCAS) was clinically suspected if the patient reported the coexistence of flush and/or dermographia.All patients underwent an Fig.  initial Shortly after the medical assessment, patients underwent a specialized physical therapy evaluation from our team. This assessment allowed to establish a treatment plan for physical therapy management. Regardless of the diagnosis of hEDS or HSD, the treatment included physical therapeutic approaches aimed at body awareness/proprioception as well as low-resistance/low-impact exercises in a closed or semi-closed kinetic chain to strengthen deep and stabilizer muscles. A plan for home-based exercises was established and included a cognitive behavioral approach. Patients were encouraged to re-start progressive physical activity or sports and were given some recommendations for specific activities. At T1 and T2, patients were asked to consider pain, disability, injury and empowerment together in answering the following question: “Compared to the initial assessment, how do you now consider yourself regarding pain, disability, injury and empowerment?”, with possible answers of improved/not improved.Patient characteristics are reported as frequency (percentage) or median (percentiles 25th–75th) for categorical and continuous variables, respectively. To test the ability of the 2017 diagnostic criteria to identify patients with clinically severe disease and with extra-articular involvement, we compared CSS-16 scores and prevalence of bone involvement, neuropathic pain and MCAS symptoms between the hEDS and HSD groups. We also investigated the effect on T1 and T2 outcomes of the 2017 diagnostic criteria, CSS-16, family history, bone involvement, neuropathic pain, and MCAS symptoms.p < 0.10) affected outcome at T2 (improved/not improved), the best predictors were tested on multivariable logistic regression analysis (p ≤ 0.05). Statistical analyses involved using Stata ICv14 for Windows . P ≤ 0.05 was considered statistically significant.Associations between studied parameters were tested with Mann–Whitney U-Test and chi-square test or Fisher exact test as appropriate. After univariate tests to determine which factors significantly  but not in its categorical form (no/yes: < 5/ > 5) according to 2017 criterion 1. Criteria 2017 “2” and “3” were by definition more often present in hEDS than HSD patients: the proportion of > 5 systemic manifestations (criterion 2A) was 18% and 0% (p < 10–2), positive family history (2B) 93% and 33% (p < 10–4), and musculoskeletal pain or chronic or recurrent dislocations (2C) 98% and 75% (p < 10–3). In the HSD group, 22% had another diagnosis that could be confounding with symptoms . The groups did not differ in presence of bilateral piezogenic papules of the heel .The Beighton score was significantly Table  higher fp = 0.04), but among the 16 items, only pain, motor and bleeding problems were significantly more severe in hEDS than HSD patients .Spontaneous bleeding problems were significantly more severe in patients with than without suspected MCAS , sex (p = 0.64) or Beighton score (p = 0.26). However, patients lost to follow-up at T1 had a lower CSS-16 score than those not lost to follow-up  and the proportion of lost patients was lower in the hEDS than HSD group . The situation was comparable at T2: same age (p = 0.21), sex (p = 1.00) and Beighton score (p = 0.23). Patients lost to follow-up at T2 had a slightly lower but not significantly CSS-16 score than those not lost to follow-up  and the proportion of lost patients was lower in the hEDS than HSD group .Follow-up data were available for 76 patients at T1 (78%) and 59 at T2 (61%), with a mean follow-up time of 6(6–10) and 20(18–26) months, respectively. Patients lost to follow-up at T1 did not differ from others in age .In total, 28 36.8%) patients considered their condition improved at T1 and 32 (54%) at T2 , sleep disturbance (p = 0.06), family history of hypermobility (p = 0.07) and DN4 positivity (p = 0.04) (Table p = 0.01).On univariate analyses, factors associated with better prognosis at T2 were the initial intensity of pain  and pain to criterion 2C: musculoskeletal involvement. However, we found a significantly higher prevalence of severity of spontaneous bleeding in hEDS than HSD patients. Traditionally, the bleeding symptom was associated with the vascular form of EDS. However, patients with abnormal hypermobility experience more abnormal bleeding . The bleThe 2017 criteria were proposed to avoid neglecting a potentially severe disorder and to limit overemphasizing a non-pathological variation from the norm . Thus, tWe recorded piezogenic papules separately, added as a diagnostic feature in 2017 , based oThe 2017 diagnostic criteria could not distinguish patients with more extra-articular manifestations, bone fragility, neuropathic pain, or suspected MCAS. The prevalence of anamnestic bone fragility was high (23% for a median age of 40) in our cohort, when considering that in the normal population of women aged ≥ 50 years in Europe, the prevalence of osteoporosis is 22.5% . We recoThe neuropathic pain detection score was frequently positive in our cohort, almost 50% in both groups. Chronic pain is an important problem for HSD/hEDS patients . Neuropahttps://www.ehlers-danlos.com). Therefore, it could be a bias of over-positivity of MCAS symptoms in our cohort. As suggested by Jesudas et al. [In approximately 45% of patients, symptoms were compatible with MCAS. The GoodHope study  found ths et al. , we founIn our study, we proposed the same management for hEDS and HSD patients based on an initial assessment by a physiotherapist and a semi-standardized reeducation program, coached by a physiotherapist and then progressively trusted to the patients themselves (self-care). Strong evidence for physical therapy is lacking , yet it With our program, more than 50% of patients showed improved articular symptoms at the end of follow-up. In 2013, Bathen et al.  showed iThe diagnostic category (hEDS vs. HSD) does not appear to be a prognostic factor for outcomes after physical therapy. Good clinical practice for hEDS and HSD must integrate a coordinated physical therapy program, if possible within a network of experienced caregivers, which could become the standard of care. The only factor that seemed to influence a favorable evolution was family history of hypermobility. We have no straightforward explanation for this finding. Perhaps, empowerment is facilitated by the presence of the disability in a parent or a child in the same family. Also, having another family member with the same diagnosis could induce motivation for rehabilitation.Among patients lost to follow-up, we found lower CSS-16 scores and a higher proportion of HSD diagnoses than hEDS. We can reasonably assume that the severity of symptoms and a clear diagnosis are motivational factors for attending a tertiary center.Being the only coordinated center in the French speaking part of Switzerland, our sample is representative of the hypermobile and hEDS/HSD patients in this region with a global population of 2 million. Reassuringly, the proportion of women , 17, meaOur study has limitations. This was a monocentric study in a small country, yet as described above, it is the referral center, which holds its importance. The scores used were mostly subjective or detection tools, which are convenient to use in a clinical setting. The CSS-16 is not validated, and its sensitivity and specificity for hEDS or compared to other disorders such as fibromyalgia is not known. A validation and comparison with other cohorts is needed, given the utility and easy application of this score. The global evolution is a composite self-report regarding pain, disability, injury and empowerment. We did not record evolution reports for these elements separately, and therefore could not study whether pain was decreased, for example. This plan was chosen on the basis of clinical relevance . Unfortunately, we were not able to assess the reason for lost to follow-up because it was the patients’ decision not to return to clinical care, and it would have been invasive to ask them the reason for this. The reason could be a bias in the results we present (underestimation of effects with more loss of patients with great improvement or over-estimation with more loss of patients with deterioration).Based on a clinical severity scale of 16 items, in our cohort, patients with hEDS fulfilling the 2017 diagnostic criteria and HSD patients showed globally similar severity scores except for pain, motricity problems and spontaneous bleeding, and similar spectrum of extra-articular manifestations. In addition, improvement was ≥ 50% with a coordinated physical therapy program in both groups. Altogether, these results add weight to the proposition to consider hEDS/HSD as a single entity that requires the same treatments.Supplementary file1 (PDF 22 KB)Below is the link to the electronic supplementary material.


Pueraria lobata (Willd.) Ohwi, known as kudzu, is one of the most popular traditional medicines in Asian countries. It has been widely used as a natural alternative to hormone replacement therapy for treating postmenopausal symptoms. This study aimed to investigate the estrogenic effect of P. lobata extract (PE) against postmenopausal osteoporosis in ovariectomized (OVX) rats. OVX rats were treated with PE (25–1600 mg/kg) for 8 weeks. Biochemical parameters, estradiol, and bone turnover markers  were measured in plasma samples. In addition, estrogen receptor-alpha (ER-α) protein expression and morphology of uterine were evaluated. Long-term treatment with PE did not cause liver damage in OVX rats. PE supplementation reduced body weight gain in obese rats with high lipid accumulation induced by ovariectomy. Furthermore, PE exhibited a protective effect against insulin resistance, hyperlipidemia, and hepatic lipid peroxidation. PE treatment increased uterine weight and thickness of the uterine layers in cases of uterus atrophy due to removal of ovaries. The levels of bone turnover markers, which were significantly increased in OVX rats, were decreased by PE treatment. Western blotting analysis showed that ER-α protein expression was upregulated in PE-treated rats compared with OVX rats. These results suggest that PE could be a promising alternative functional food for improving menopausal symptoms. Menopaustradiol . The antstradiol –9.Pueraria lobata (Willd.) Ohwi, known as kudzu, has been traditionally used in combination with other herbs to produce synergistic and additive effects through a multitarget approach for disease [Pueraria lobata is known to have multiple pharmacological activities that help alleviate various diseases such as liver diseases, cardiovascular diseases, diabetes, and osteoporosis [P. lobata, has been demonstrated to have preventive activity against osteoporosis in several studies using ovariectomized (OVX) animal models [P. lobata on dyslipidemia and bone metabolism in OVX rats. Based on the estrogenic activity of puerarin, we predicted that P. lobata extract (PE) could have beneficial effects on menopausal metabolic dysfunction, the endocrine system, and osteoporosis in OVX rats. Therefore, we investigated the protective effects of long-term supplementation with PE against metabolic parameters in OVX rats. In addition, white adipose tissue weight, atrophic uterus morphology, bone turnover markers, and estrogen levels were evaluated to clarify the underlying mechanisms of action of PE extract. disease . It cont disease . Puerarioporosis , 13. In l models , 15. An l models . HoweverP. lobate was soaked in 50% ethanol for 1 h and then centrifuged. The residue of P. lobate was extracted in five volumes of 50% ethanol for 1 h at 60°C. The extract was filtered and concentrated under reduced pressure. The PE was powdered in the spray drying system by mixing 30% dextrin. The content of puerarin, which is a major isoflavone in PE, was 42.38 mg/g.PE was provided by Sejun F & B Co., Ltd. . It was in the form of a spray-dried powder, which was prepared as follows. To separate the starch, dried n = 8 per group): (1) sham group, (2) OVX group, (3) OVX + PE 25 mg/kg group, (4) OVX + PE 100 mg/kg group, (5) OVX + PE 400 mg/kg group, and (6) OVX + PE 1600 mg/kg group. All animal experiment protocols in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Hongcheon Institute of Medicinal Herb (No. 19–07). PE was suspended in phosphate buffered saline (PBS) for oral administration at the desired dose in a volume of 5 mL/kg once daily for 8 weeks. The food intake and body weight of rats were measured twice a week. After 16 h of fasting on the last day of the experiment, blood was collected from the portal vein. The uterus and white adipose tissue were collected and weighed.Nine-week-old female Sprague Dawley rats, which were ovariectomized or sham-operated at 8 weeks of age, were purchased from Chemon Inc.  and maintained under controlled laboratory conditions  for 2 weeks before the experiments. The rats were randomly divided into the following treatment groups  and then stored at −80°C for assay. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), Ca, P, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels were measured using an automated clinical chemistry analyser . Free fatty acid , insulin, estradiol (E2), and serotonin levels were determined using enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer's instructions.The liver sample (120 mg) was homogenized in 1 mL of PBS and then centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant obtained was used a source for the assay. Malondialdehyde (MDA) level was determined using the thiobarbituric acid reactive substance (TBARS) assay according to the manufacturer's  instructions.The bone formation marker Osteocalcin (OC) was determined using a rat osteocalcin ELISA kit (Cusabio). The bone resorption markers, such as C-terminal telopeptide fragment of type I collagen , deoxypyridinoline , and pyridinoline , were determined using commercial ELISA kits.μg) were separated on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred on to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk for 1 h and then probed with specific primary antibodies at a final dilution of 1 : 1000. The immunoreactive protein bands were detected using an enhanced chemiluminescence western blot detection kit  and visualized using the Chemidoc Touch image system .The total protein in the uterus was extracted using RIPA lysis buffer containing phosphatase and protease inhibitor cocktails. Equal amounts of protein .p < 0.05. All statistical analyses were performed using GraphPad Prism 7.05.All data are expressed as mean ± standard error of the mean (SEM). They were analysed using the analysis of variance followed by Dunnett's multiple comparison test. Differences were considered statistically significant at As shown in The OVX rats presented with prominent atrophy of the uterus as shown in The results of plasma biochemical parameters are summarised in p < 0.001). Liver MDA levels significantly increased by 25% in the OVX group compared with the sham group. Treatment with PE significantly reduced these increases in MDA levels to values comparable to those in the sham group.To further investigate whether PE treatment improves the high lipid accumulation induced by ovariectomy, we measured the changes in plasma lipid profiles and MDA levels in the liver homogenates . The plaα was decreased in OVX rats, and PE treatment upregulated ER-α expression in a dose-dependent manner  of the femur is shown in t manner .Estrogen deficiency in postmenopausal women and OVX rats promotes metabolic syndromes . The OVXThe OVX rats present with weight gain, uterine atrophy, bone loss, and estrogen reduction, all of which commonly occur in postmenopausal women. As described previously , OVX ratα protein in the uterus and plasma E2 levels in OVX rats. ER-α is essential for the induction of uterine muscular disorganization and vaginal epithelial cell proliferation by estrogen during critical developmental stages [We also confirmed that PE supplementation elevated the expression of ER-l stages . These rkudzu roots are not highly toxic based on toxicity assessment [The supplementation with PE up to 1600 mg/kg did not change ALT and AST levels, which are biomarkers of hepatic injury. This result is similar to that of a previous study, which reported that sessment . These dα knockout female mice are reported to exhibit an obese phenotype with increased visceral adiposity, decreased energy expenditure, and insulin resistance [α is also necessary for the attenuation of body weight gain after OVX [Estrogen deprivation by ovariectomy leads to increased fat accumulation and hepatic steatosis . The ER-sistance , 24. Simfter OVX . In thisBlood Ca and P levels reflect the degree of bone loss . This stIt has been reported that blood serotonin levels increase with an increase in bone turnover in humans and in an OVX rat model of postmenopausal osteoporosis . In the α protein expression, without affecting serum levels of hepatic injury biomarkers. Moreover, PE treatment inhibited the increase in bone turnover markers such as CTX-1, OC, DYP, and PYD in OVX rats. Overall, PE might be a potential functional food for controlling menopausal symptoms.In the present study, it was demonstrated that PE exhibits phytoestrogen-like activities in OVX rats. PE supplementation increased the plasma estradiol level, uterine layer thickness, and ER-


The aim of the study is to  assess the diagnostic value of preoperative conventional radiographs for diagnosing infection associated with internal fixation devices.We prospectively collected data of patients undergoing removal of internal fixation devices for any reason. Infection was diagnosed in case of purulence, sinus tract, positive histopathology and/or positive peri-implant tissue or sonication fluid culture. In radiographs radiolucent lines, implant breakage or displacement, or periosteal reaction were assessed. White blood cell count (WBC) and serum C-reactive protein (CRP) were determined at admission.We included 421 surgeries in 380 patients , mainly indicated for infection (24.9%), nonunion (20.0%) and symptomatic implants (13.5%). Radiologic signs of infection included radiolucent lines (11.4%); implant breakage (12.4%) or displacement (10.7%); and periosteal reaction (7.1%). Infection was confirmed in 116 cases (27.6%). Only radiolucent lines (OR = 1.86 [95%CI: 1.00–3.38]) and periosteal reaction (OR = 2.48 [95%CI: 1.17–5.26]) were associated with infection, with a low sensitivity , and high specificity . Preoperative WBC and CRP had a sensitivity of 23.0 and 35.3%, and specificity of 91.7 and 89.5%, respectively.Radiological signs suggestive of infection were uncommon. Radiolucency and periosteal reaction were associated with infection, though with low sensitivity.The online version contains supplementary material available at 10.1186/s12891-021-04170-3. Diagnosing internal fixation-associated infections preoperatively may be challenging, as investigation of body fluids such as synovial fluid in periprosthetic joint infection is not possible. Conventional radiographs are usually the first diagnostic investigation before surgery, including in suspected infection , 2. HoweIn this study we evaluated the diagnostic accuracy of individual findings of conventional radiographs for the diagnosis of infections after internal fixation, and  compared its performance with that of laboratory biomarkers such as preoperative serum C-reactive protein (CRP) concentration and white blood cell (WBC) count.We reviewed consecutive patients in whom an orthopaedic internal fixation device (or part of it) initially inserted for any indication (fractures / elective orthopaedic surgery) was removed for any (presumed septic or aseptic) reason, between December 2014 and December 2017. Data was reviewed from a prospective database collected in a tertiary hospital. Internal fixation devices of any type were included, excluding arthroplasties and spinal and craniofacial fixations. Patients without recent conventional radiographs were excluded. Informed consent was obtained from all study participants included in this database study. In the case of minors (age below 18 years), the informed consent was obtained from a parent and/or legal guardian of the participant.case indicates one surgery in one patient; thus, one patient could have several cases included for analysis.Internal fixation devices were removed in 473 surgeries from 430 patients. Recent radiographs were unavailable for 52 surgeries in 50 patients, mostly due to lack of digitization in the institution’s electronic health record among patients referred from other clinics. Thirty-three patients had two surgeries and four patients had three surgeries, resulting in 380 patients and 421 surgeries for analysis  , implantAn interdisciplinary team including an infectious diseases specialist and an orthopaedic surgeon classified each case. Implant-associated infection was defined with the presence of at least one of the following criteria: (i) purulence around the implant and/or a sinus tract communicating with the implant, and/or implant on view; (ii) histology showing inflammation in periimplant tissue , 10; (IIPeriimplant tissue (bone or soft tissue) was obtained at the discretion of the operating surgeon. Tissue cultures were considered positive if (i) a highly virulent organism was identified by culture from ≥1 deep tissue sample or (ii) a phenotypically indistinguishable low-virulence pathogen , 12 was p <  0.05 (two-sided). Data was analysed using IBM SPSS Statistics v. 20. The 2015 STARD guidelines ) and periosteal reaction  were associated with infection. Sensitivity of radiolucency and periosteal reaction were low , and specificity acceptable .Table p < 0.001), as well as CRP values . The presence of infection was associated with increased WBC , but elevated CRP concentrations were more useful . The AUC for the WBC count and the CRP value were 0.639 and 0.697, respectively. Youden Indexes were highest at ≥7.120 leukocytes/mm3 for WBC count , and ≥ 4.25 ml/l for CRP concentrations .Preoperative blood analysis was performed in the week preceding surgery in 286 (67.9%) cases. WBC counts were higher among infected patients , while a positive sonication culture increased the odds of infection . The combination of tissue cultures and sonication yielded the highest sensitivity (96.6%) and diagnostic accuracy (99.1%) of any of the tests evaluated. The number of tissue samples taken was higher among cases in which infection was confirmed than in aseptic cases (3.3 [SD 2.1] samples vs. 2.2 [SD 1.8] samples). Less than three samples were taken in 233 cases (55.3%); infection was confirmed in 43 of these, in 23 based on sonication fluid cultures. Of the 68 cases without tissue samples, five were considered infected . Histological data was not available for analysis; however, no cases were diagnosed based on histological reports alone.p < 0.001), as were WBC counts . Though CRP values were higher in chronic infections than aseptic cases (p = 0.008), WBC counts were not significantly different (p = 0.167). The AUC was higher in acute infections (0.808 for WBC and 0.873 for CRP); cutoffs with maximum Youden Indexes for acute infections were ≥ 9.390 leukocytes/mm3 for the WBC count , and ≥ 8.95 mg/l for CRP concentration .Of the diagnosed infections, 30 25.9%) were considered acute, and 86 74.1%) chronic. Radiologic findings were more common in chronic infections  than in noninfected cases (26.6%). All radiologic findings were more common in nonunion cases than in consolidated cases . Only radiolucency and periosteal reaction remained significant  when comparing septic vs. aseptic nonunions . A subanalysis of the 333 cases in which the initial indication was fracture lost significance for radiologic findings on any type, though analysis was underpowered after removing over one fifth of cases. Similar results and limitations were observed when analysing only certain implant types, such as plates and/or screws.No differences were found regarding radiologic findings when comparing cases in which the initial indication for internal fixation was a fracture, compared to non-fracture cases , particularly in chronic infections  and infected nonunions .While the consensus group proposed the term “fracture-related infection” , the terPatients with acute infections or with revision surgery shortly after initial surgery  may not have developed radiological changes yet. Significance was lost when examining radiographs in acute cases. On the other hand, laboratory and microbiological tests had a higher diagnostic accuracy in acute infections; these are usually not as much a diagnostic challenge as chronic infections since they are most commonly caused by virulent pathogens. Most diagnosed infections were chronic infections suspected preoperatively, or found after revision surgery for indications such as nonunion, osteoarthritis, or removal of symptomatic devices. In these, the time between implant insertion and removal was long enough for these infections to be considered chronic.Radiological findings significantly associated with infection  reflect changes in the bone itself, while implant-related findings  were non-significant. Twenty-six cases in which infection was clinically suspected  were considered aseptic according to the predefined criteria used for this study. Clinical signs suggestive of infection are nonspecific, and the use of validated criteria including microbiology has improved diagnostic accuracy of implant-associated infections . FinallyWe cannot exclude having missed some cases of implant-associated infection. We were unable to follow the 279 cases in which infection was excluded, and some cases could have developed infection following the revision surgery: this could be because the infection was always present , or because it appeared newly as a complication following the revision surgery. However, the criteria used are based on diagnostic criteria sufficiently validated elsewhere , 11, andPrevious studies analysed radiological signs in orthopaedic infections including prosthetic joint infections, spinal infections, and paediatric acute haematogenous osteomyelitis; cases were included based on the suspicion of infection. We are not aware of any study analysing all internal fixation device retrievals, regardless of indication, as would be necessary to study diagnostic accuracy. In 1985, Al-Sheikh  comparedPeriprosthetic bone loss has been hypothesized to be mediated through osteoclast activation over several pathways originating in cytokine expression by neutrophil granulocytes . Local bWe do not analyse erythrocyte sedimentation rates (ESR) routinely anymore in our insitution, as it was replaced by newer markers such as CRP levels, reported to be more useful for the diagnosis of osteoarticular infection . Other sThe diagnostic accuracy of sonication and tissue cultures for fracture-related infection in contrast to prosthetic joint infection has been an area of recent interest , 16, 26.Our study has several limitations. First, the histopathological definition criteria (i.e. presence of ≥5 granulocytes per high-powered field ) was impIn conclusion, radiologic signs of infection are uncommon, even in cases of confirmed infection. Recent conventional X-rays should be reviewed prior to implant removal in search of subtle radiographic changes. In particular, (a) radiolucency surrounding the implant and (b) the presence of a periosteal reaction were associated with infection, with a low sensitivity and high specificity, making them useful for ruling in patients in whom infection is likely. However, though they are less common in non-infected cases, the absence of these radiological changes does not rule out infection. Therefore, we recommend obtaining (c) tissue samples for culture and histology and (d) sonication of the retrieved implants if infection is suspected, especially if new implants are to be used in the revision surgery.Additional file 1: Table, Supplementary Digital Content 1: Diagnostic accuracy of radiological and non-radiological findings for the diagnosis of chronic infection.Additional file 2: Table, Supplementary Digital Content 2: Diagnostic accuracy of radiological and non-radiological findings for the diagnosis of acute infection.Additional file 3: Table, Supplementary Digital Content 3: Diagnostic accuracy of radiological and non-radiological findings for the diagnosis of infected nonunion.


Confocal laser endomicroscopy (CLE) has advantages in detecting gastric neoplastic lesions, meanwhile it requires strict patient cooperation. Sedation could improve patient cooperation and quality of endoscopy. However, sedation is still not very popular in some resource-limited countries and regions. The purpose of this study was to compare propofol-based sedated versus un-sedated CLE in the value of diagnosing early gastric cancer (EGC) and precancerous lesions.A retrospective, cohort, single center study of 226 patients who underwent CLE between January 1, 2015 and December 31, 2017 was performed. Patients enrolled were allocated into the propofol-based sedated group (n = 126) and the un-sedated group (n = 100). The comparison of validity and reliability of CLE for identifying EGC and precancerous lesions between the two groups was performed through analyzing CLE diagnosis and pathological diagnosis. Reporting followed the STROBE guidelines.P = 0.0407). CLE with sedation performed better than without sedation in diagnosing intraepithelial neoplasia and intestinal metaplasia . For patients considered as high-grade intraepithelial neoplasia or EGC by endoscopists, they would not get biopsy during CLE but receive endoscopic submucosal dissection (ESD) subsequently, and the misdiagnosis rate of CLE was 0 % in the sedated group and 27.59 % (95 % CI: 10.30–44.91 %) in the un-sedated group (P = 0.006).The area under receiver operating characteristic curve (AUROC) of diagnosing EGC in the sedated group was 0.97 (95 % CI: 0.95 to 0.99), which was higher than that in the un-sedated group (0.88 (95 % CI: 0.80 to 0.97), Propofol based sedation was associated with improved diagnostic value of CLE for detecting EGC as well as precancerous lesions . Gastric cancer is one of the most common malignancy and leading causes of cancer mortality in the world –3, especAs the most useful tools for screening gastric cancer, a number of modern endoscopy devices and techniques, like confocal laser endomicroscopy (CLE), magnifying endoscopy and narrow-band image, have been developed to fulfill different diagnostic demands. Among these, CLE has advantages in detecting EGC and pre-malignant lesions, since it can provide a direct histological observation of the cells and subcellular regions in vivo, as well as demonstrate mucosal changes that cannot be detected by white light endoscopy (WLE) .Sedation in gastroscopy is widely accepted in high-income countries, which are found to be associated with increased patient comfort and reduced complications related with poor patient cooperation, though it causes additional medical resource consumption . As CLE This was a single-center, retrospective, cohort study, analyzing outpatient probe-based CLE database and histopathology reports from the Department of Endoscopy Unit, the First Affiliated hospital, College of Medicine, Zhejiang University between January 1, 2015 and December 31, 2017. The research protocol was reviewed and accepted by the research ethics committee of the First Affiliated Hospital, College of Medicine, Zhejiang University on June 13, 2018 (Reference Number: 750/2017). The study followed the reporting guideline-Strengthening the Reporting of Observational Studies in Epidemiology (STROBE).Patients enrolled were allocated into two groups according to their anesthesia type. Patients in the sedated group received a combination of propofol-based sedation and lidocaine-based pharyngeal anesthesia, while patients in the un-sedated group received lidocaine-based pharyngeal anesthesia only. Reports on juvenile patients (<18 years old), or patients with advanced gastric cancer were excluded.For all patients, after arrival, the electrocardiogram, non-invasive blood pressure and oxygen saturation were monitored. Lidocaine Hydrochloride mucilage  was administered orally 5 min before the beginning of CLE for pharyngeal anesthesia. For patients with sedated CLE, a 20-gauge cannula was placed in a vein in the forearm, and sedation was induced with propofol  1.5-2 mg/kg intravenously. Sedation maintained was using 5–8 mg/kg/h propofol. Patients in the un-sedated group received pharyngeal anesthesia only.CLE was conducted by two endoscopists who were with at least 5 years’ experience in performing diagnostic CLE. The procedure involved the use of a cellvizio confocal miniprobe , and a contrast agent . CLE for all participants were performed according to the standard protocol. Each CLE procedure needed to be performed with no less than 20 min. After a mucosal lesion was visualized by WLE, a total of 5ml 10 % fluorescein sodium was administered intravenously. To obtain controlled CLE images, the probe was first gently contacted to normal mucosa around the lesion, ideally showing regular round or oval glands with homogeneous epithelial cells. The probe was subsequently moved to suspicious lesion to obtain CLE image, and following it, biopsies were obtained from the area. If the lesion was considered as high-grade intraepithelial neoplasia (HGIN) or EGC by the two endoscopists under CLE, the endoscopists would not get biopsy during the CLE procedure, but suggest patients for an endoscopic submucosal dissection (ESD) directly .The CLE criteria were based on the 2011 Miami classification  intraepiThe histopathological diagnostic criteria were based on the Updated Sydney System for classification and grading of gastritis  and the Two trained research assistants collected the following data: patients’ characteristics, CLE reports by endoscopists, pathological reports by pathologists. They also collected the information of patients who were strongly suspected for HGIN or EGC and further received ESD. These patients did not get biopsy during the CLE procedure, but had pathological results of ESD specimens. So the data of CLE reports and pathological diagnoses of ESD specimens were collected.2) <90 % >10 s), hypotension (drop in systolic blood pressure of >25 %), hypertension (raise in systolic blood pressure of >25 %), bradycardia (drop in heart rate of >20 %), or tachycardia (>100 bpm).The complications appeared during CLE procedure were also recorded. Major complications were defined as need for intubation, intensive-care unit admission, resuscitation and death. Minor complications were defined as respiratory depression . Another two trained research assistants randomly extracted 10 % of them to check the completeness, accuracy, and relevance of the information.Enrolled patients were allocated into two groups: the sedated group and the un-sedated group. The diagnostic accuracy of intestinal metaplasia, intraepithelial neoplasia, gastric neoplasia lesions (IN OR EGC) and EGC that examined by CLE was compared between the two groups. Among them, IM and IN were defined as precancerous lesions.2 test was used to examine the significance of the association between two variables in a contingency table. Variables with a normal distribution were presented as mean ± standard deviation (SD), and compared using analysis of t test. A P value of 0.05 (two-sided) was considered statistically significant.No specific power calculation was performed, and the sample size of this study was determined by the number of patients recruited across site. Statistical analysis was performed by using the software STATA/MP 15.0 . Numerical data were presented as numbers (percentage). The PearsonχThe primary outcome was the comparison of the area under receiver operating characteristic curve (AUROC) of EGC and precancerous lesions that was diagnosed by CLE between the two groups. The gold standard for diagnosing EGC and precancerous lesions is the histopathological diagnosis. The nonparametric analysis was used for two-group comparison in AUROC.The second outcome was the misdiagnosis rate of CLE. For patients without biopsy during CLE and received an ESD subsequently, the misdiagnosis rate of CLE was analyzed according to their pathological results of ESD specimens.Another second outcome was the comparison of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and kappa (κ) value along with bionormal 95 % confidence intervals(95 % CI) between the two groups. Agreement was regarded as poor withκvalue below 0.4, good withκ value between 0.4 and 0.75, and excellent withκ value over 0.75.Between January 1, 2015 and December 31, 2017, a total of 253 patients were eligible for study analysis, of which 27  was excluded because of the missing pathological data; the remaining 226  were analyzed. In this study, the most severe abnormality was used as each patient’s diagnosis. For example, intestinal metaplasia associated with intraepithelial neoplasia was considered as intraepithelial neoplasia. When referring to histopathology, in the sedated group, 16 (12.70 %) patients were diagnosed as EGC, 27 (21.43 %) patients were diagnosed as intraepithelial neoplasia, 55 (43.65 %) patients were diagnosed as intestinal metaplasia and 28 (22.22 %) patients were diagnosed as normal mucosa or benign inflammatory lesions. In the un-sedated group, 18 (18.00 %) patients were diagnosed as EGC, 17 (17.00 %) patients were diagnosed as intraepithelial neoplasia, 36 (36.00 %) patients were diagnosed as intestinal metaplasia and 29 (29.00 %) patients were diagnosed as normal mucosa or benign inflammatory lesions. Detailed characteristics of the two groups are presented in Table The AUROC of CLE in diagnosing normal mucosa or benign inflammatory lesions in the sedated group was 0.80 (95 % CI: 0.71 to 0.89), which was higher than that in the un-sedated group (0.62 (95 % CI: 0.53 to 0.72), P = 0.0084)    VS 0.59 (95 % CI: 0.49 to 0.69), P = 0.0073)  , the AUROC in the sedated group was 0.89(0.83 to 0.95), which was higher than that in the un-sedated group (0.75(0.66 to 0.84)) (73) Fig. d. The AU07) Fig. e.The senP = 0.006).To those who received ESD, the agreement between main CLE finding and the ESD pathological diagnose was assessed. A total of 24 patients in the sedated group and 29 in the un-sedated group were suggested for ESD directly. The misdiagnosis rate of CLE in the sedated group was 0 %, and in the un-sedated group was 27.59 % (95 % CI: 10.30–44.91 %), which had significant difference  than that of un-sedated CLE. And for patients without biopsy during CLE procedure and received an ESD directly, the misdiagnosis rate of CLE was significantly lower in the sedated group than in the un-sedated group according to the final pathological results. To the best of our knowledge, this is the first study to demonstrate such a positive association between sedation and CLE outcomes.Sedation is a drug-induced depression in the level of consciousness, which is recommended for GI endoscopy . It coulGastric cancer is believed to arise from a series of pre-malignant lesions, through a number of stages from chronic atrophic gastritis, by way of intestinal metaplasia, through LGIN and HGIN, up to cancer . A largeIn the current study, propofol based sedation was adopted, which makes endoscopy almost painless, with a very predictable, rapid recovery process and improved patient satisfaction , 21. ComAlthough sedated endoscopy is widely accepted in high-income countries and regions , it is nOur study was limited by a few factors. Firstly, its retrospective study design was the main weakness. A prospective validation study is needed to confirm the association between sedation and improved quality of CLE. Furthermore, in the validation phase, we can also assess the safety and patients satisfactory of sedation. Secondly, the procedure time was not recorded in our databases. The procedure of CLE with propofol-based sedation might be prolonged for the patient’s great cooperation. The longer time might be correlated with the quality of mucosal inspection during CLE.In summary, this is the first study to validate the propofol-based sedation in improving the value of CLE in diagnosing EGC and precancerous lesions. Our results showed the improvement of validity and reliability of CLE in diagnosing gastric superficial cancerous and precancerous lesions though sedation. It indicates that, especially in resource-limited countries and regions, more patient undergoing CLE would benefit from sedation if more medical resources were put into this area.


The 8th edition of the American Joint Committee on Cancer (AJCC) staging led to a significant downstaging of well differentiated thyroid cancer patients. However, some patients who had been downstaged still experienced death. By using data from the thyroid cancer dataset of The Cancer Genome Atlas (TCGA), we aimed to find molecular features that could improve survival prediction.TCGA data were downloaded from cBioPortal. Restaging of cases was performed according to the pathological reports.TERT promoter mutations were no longer enriched in stage IV only, but significantly redistributed also in stages II and III. TERT mutation was the only alteration predictive of poor survival; however, in this series it was not independent from the AJCC staging. Five proteins  were significantly associated with survival, and their use as a panel refined the risk stratification independently from the AJCC staging, with a hazard ratio for a positive result of 21.2 .Out of 496 cases, 204 (41.1%) were downstaged, and the proportion of deaths increased in stages III and IV. TERT mutations will be allocated in lower stages; hence, dedicated studies should define the prognostic usefulness of these mutations in low-stage diseases.In the TCGA series, the proportion of deaths is in line with the expected survival of the latest AJCC staging, with a neat separation of risk among stages. Nevertheless, the use of protein expression can be useful in refining the stratification. Finally, after the restaging, a considerable number of tumors with  BRAF and TERT have also been associated with disease specific mortality in papillary thyroid cancer (PTC) [The American Joint Committee on Cancer (AJCC) tumor-node-metastasis staging is the most effective and used stratification system designed to predict survival of patients. The 8th edition of the AJCC staging for thyroid cancer was released at the end of 2016 but is effective from the beginning of 2018. With respect to the 7th edition, the main changes for well differentiated thyroid cancer (WDTC) were: (i) the increase of the age cutoff from 45 to 55 years, (ii) the redefinition of T3 disease, which now does not encompass minimal extrathyroidal extension (ETE) detected only on histological examination, and (iii) the downgrading of positive lymph nodes to stage II for older patients [er (PTC) ; howeverer (PTC) , we soughttps://www.cbioportal.org/) [Mutation Annotation Format (MAF) file, clinical data, and level 3 reverse phase protein lysate microarray (rppa) data of the PTC dataset of the TCGA  were dowal.org/) , 12. CliTERT promoter mutations that were available in separate files were manually added.Within the MAF file, synonymous variants were ignored. Moreover, gene fusions and The most frequent alterations were plotted using maftools v.2.2.10 Bioconductor package . The enrFor features presented as numeric variables  a ROC analysis was performed. Specifically, the Youden J statistic was used to select the best cutoff in discriminating patient’s vital status by the pROC v.1.15.3 R package . PatientAs regards rppa expression data, an exploratory ROC analysis was performed following the procedures of the caret v.6.0–84 R package . ProteinP value below 0.05 was considered significant. All analyses were performed in R environment .Survival curves were estimated by the Kaplan–Meier method, and differences among curves were tested by log-rank test using the survival v.3.1–8 R package . SurvivaOut of the 496 cases considered, 292 (58.9%) were confirmed in the same stage: 284 in stage I, three in stage II and five in stage IV, whereas 204 41.1%) cases were downstaged. Details are reported in Table .1% casesTERT promoter mutations were reported in Fig. The 20 most frequent nonsilent gene alterations including fusions and BRAF mutations were enriched in classical variant (FDR < 0.0001) and tall cell variant (TC) (FDR = 0.0007) PTC, whereas NRAS and HRAS were associated with follicular variant (FV) (FDR < 0.0001 and FDR = 0.0292 respectively). Moreover, NRAS mutations were associated with pN0 (FDR = 0.002) and ATA low-risk (FDR = 0.0077); BRAF was enriched in pN1a (FDR = 0.002), minimal ETE (FDR < 0.0001) and ATA intermediate-risk (FDR = 0.0001), whereas TERT promoter mutations were overrepresented in cases with gross ETE (FDR = 0.0018), pM1 (FDR = 0.0086) and ATA high-risk (FDR < 0.0001). In addition, TERT mutations were enriched in patients with more than 45 years (FDR = 0.006), and even strongly in those with more than 55 years (FDR < 0.0001). As regards the AJCC staging, in the 7th edition TERT was overrepresented only in stage IV (FDR < 0.0001), whereas in the 8th edition it was enriched in stages IV (FDR = 0.0061), III (FDR = 0.0049) and II (FDR = 0.0119). Finally, TERT was the only gene associated with persistence of disease (FDR = 0.0025) and death (FDR = 0.0027). Results are summarized in Table P value , ETE (P < 0.0001), age (P < 0.0001), MACIS score (P < 0.0001), TDS (P = 0.02), ERK score (P = 0.04), number of nonsilent mutations (P = 0.002) and TERT promoter mutations, both alone (P < 0.0001) or coexisting with BRAF or RAS (P < 0.0001). However, when fitting a multivariate Cox regression with the 8th edition of the AJCC, all the above-mentioned predictors were not independent.The 8th edition of the AJCC staging produced a neater separation of curves than the 7th edition and a lower lue Fig. . Among tn = 222) the majority of adverse events were in CVPTC, and no event occurred in FVPTC and TCPTC, only CVPTC (n = 143) were considered for the following analyses. The exploratory ROC analysis revealed five proteins  with an AUC greater than 0.75 in discriminating patient’s vital status. For each of these proteins, the expression value with the best Youden index was selected as cutoff. Next, patients were dichotomized according to the cutoffs, and all five proteins were significant predictors of survival. In details, poor survival was associated with a lower level of 4E-BP1_pT70, Chk1_pS345, and STAT5 alpha, and a higher level of Snail and PAI-1. Moreover, when tested in multivariate analysis with the 8th edition of the AJCC, three proteins  were independent predictors of survival, whereas for 4E-BP1_pT70 and Snail Cox regression cannot be fitted because no event occurred in one of the two groups. Details were reported in Table P < 0.0001, Fig. P = 0.0006).Since in the cases with rppa data . This fourth group closely resemble to anaplastic thyroid cancer (ATC) and was consequently named ATC-like. Herein, by using TCGA clinical-pathological and molecular data, we evaluated whether one or more molecular features could help in the patients’ risk stratification, also in the light of the AJCC staging system updating.The TCGA study on PTC provided the most comprehensive characterization of the molecular landscape of PTC ; howeverThe 8th edition of the AJCC staging system produced the downstaging of a remarkable number of cases Fig.  with theRAS-driven lesions are generally low risk as confirmed by their association with the follicular variant and the absence of lymph node involvement in this series. Lesions positive only for BRAF mutations were generally associated with an intermediate risk, presence of central lymph node metastasis and minimal ETE. TERT promoter mutations deserve a separate discussion. TERT mutations were in fact the only type of mutations enriched in patient with persistence of disease and an unfavorable outcome. On one hand they can be highly informative when detected preoperatively because they should be a bell tolling since the presence of a high-risk lesion is very likely [TERT promoter mutations with poor survival was already proved [TERT mutations are predictive of a poor outcome both alone [RAS or BRAF alterations [TERT mutated cases were distributed into lower stages. For this reason, a longer follow-up and dedicated studies with a higher number of mutated cases are needed to understand whether TERT promoter mutations can help stratify patients at higher risk within low-stage diseases.Besides the proved usefulness of gene mutations in diagnostics , they cay likely , 19. On y proved , 21 and th alone , 21 and erations . NeverthThe majority of the other features analysed in the present study were predictive of poor survival including a higher MACIS score, a lower TDS, ATA high risk and the total number of nonsilent mutations. However, none of them was independent when tested in multivariate analysis with the latest edition of the AJCC staging. By analysing protein expression, we found that five proteins, namely 4E-BP1_pT70, Chk1_pS345, Snail, STAT5 alpha, and PAI-1, had a good performance in discriminating patient’s vital status (Table Finally, the low rate of adverse events and the short follow-up period of this series could represent a limitation of the present study, thus, limiting the statistical power and requiring confirmation. Although patients with WDTC are not those with a higher risk of dying of thyroid cancer, adverse events occur also in this group of patients, albeit with a lower rate as herein observed. Moreover, the distribution of cases per stage and the proportion of deaths per each stage are in line with those reported and expected; thus, our findings are based on data that could reflect a real-life series of WDTC.In conclusion, we provided some pieces of information to be used in the stratification of patients according to the risk of death. In addition, we proposed a five-protein-based stratification strongly predictive of patients’ survival, also independently from the AJCC staging.


Platycerium stemaria. The serial exhaustive extraction method using a solvent of increasing polarity from nonpolar (hexane) to polar (water) was designed to prepare crude extracts; liquid-liquid partition was used to fractionate of active extracts. The extracts and fractions were screened for antimicrobial activity on bacteria and yeasts using the microdilution method. The antioxidant activity was done using DPPH and FRAP assays. Out of the sixteen extracts screened, four , PsMeOH(EA), and PsMeOH) exhibited potency with minimal inhibitory concentration (MIC) values ranging from 31.25 to 500 μg/mL. Out of the four extracts, two, including PsMeOH and PsMeOH(EA), exhibited DPPH radical scavenging activity with the antiradical power of 8.94 × 10−5 and 47.96 × 10−5, respectively, and ferric reducing antioxidant power values ranging from 0.34 to 61.53 μg equivalent Vit C/g of extract. The phytochemical screening of the promising crude extracts revealed flavonoids, glycosides, phenols, tannins, terpenoids, saponins, and anthraquinones. This study reports the antimicrobial and antioxidant activities of P. stemaria for the first time. The results showed that the serial exhaustive extraction approach used in this study allowed capturing the antimicrobial and antioxidant metabolites beyond the single extraction, indicating the need for a rigorous choice of an appropriate solvent and method for extracting P. stemaria. Further investigation is needed to characterize the active ingredients present in the promising extracts.Microbial infections are increasing worldwide, and the widespread emergence of antibiotic-resistant pathogens poses a severe threat to public health. Medicinal plants are well-known sources of bioactive ingredients. This study was designed to determine the antimicrobial and antioxidant activities of extracts from  Microbial infections are increasing worldwide with antimicrobial resistance emerging as one of the principal public health problems, threatening effective prevention and treatment . CurrentTraditionally, the crude extracts of different parts of medicinal plants, including root, stem, flower, fruit, and twigs, are widely used to treat various human diseases . Herbal Platycerium stemaria is an epiphytic medicinal plant of the Polypodiaceae family used to treat many ailments including cardiac palpitations, pulmonary troubles, liver diseases, genital stimulants/depressants, asthma, and infectious diseases ; 10 g of PsHex extract was dissolved in 50 mL hexane and extracted with 50 mL methanol to yield frM(PsHex) and frH(PsHex). 10 g of PsMeOH (EA) and PsMeOH was dissolved in 50 mL methanol and extracted with 50 mL hexane to yield six fractions including frH(PsMeOH(EA)), frM(PsMeOH(EA)), and P(PsMeOH (EA)) from extract PsMeOH(EA) and frM(PsMeOH), frH(PsMeOH), and P(PsMeOH) from extract PsMeOH. The afforded fractions were then concentrated using a rotary evaporator. The dried fractions were weighed and the yields calculated relative to the starting crude extracts' weight. Fractions were dissolved in 100% DMSO and progressed for antimicrobial screening.The crude extracts of Shigella flexineri NR-518 (SF), Staphylococcus aureus ATCC 43300 (SA), and Klebsiella pneumoniae ATCC 700603 (KP), and three yeasts , Candida albicans NR 29451 (CA2), and Candida albicans NR 2944 (CA3)) were used. The microbial strains were obtained from BEI resources and the American Type Culture Collection. The microorganisms were maintained on an agar slope at 4°C and subcultured before the experiment for 24 h and 48 h, respectively, for bacteria and yeasts.Three bacteria, including μg/mL for their ability to inhibit bacteria/Candida species. Briefly, ninety-two microliters (92 μL) of Muller Hinton broth (MHB) were aseptically introduced into the wells of a 96-well microplate. Eight microliters (8 μL) of each extract, initially prepared at 12.5 mg/mL, were added in wells followed by 100 μL of standardised bacterial suspension (106 CFU/mL) to obtain final volumes of 200 μL. The tests were performed simultaneously for negative control (MHB+pathogens) and sterility control . Ciprofloxacin was used as the positive control and tested at 5 μg/mL. The test was performed in duplicate, and the plates were incubated at 37°C for 24 and 48 hours, respectively, for bacteria and yeasts. The active extracts were further submitted to the dose-response study for MIC determination.The sixteen extracts were screened at 500 μL of extracts and reference drug (Ciprofloxacin) from stock solutions were introduced in the well, followed by the addition of 96 μL of bacteria inoculum standardised at 106 CFU/mL. A blank column was included for sterility control, while bacterial/yeast strains in culture medium without any inhibiting substance were negative control. The concentrations of extracts ranged from 3.905 μg/mL to 500 μg/mL, and that of Ciprofloxacin ranged from 0.562 μg/mL to 128 μg/mL. After 24 hours of incubation at 37°C, the turbidity was observed as an indication of growth. MIC was defined as the lowest concentration inhibiting the visible growth of microorganisms. All tests were performed in duplicate [The minimum inhibitory concentration (MIC) of selected extracts and fractions was determined according to the M07-A9 Clinical Laboratory Standards Institute microdilution method using 96-wells microtitre plates. Briefly, 4 uplicate .λ = 517 nm serving the methanol without DPPH solution as the blank solution. A reference solution of vitamin C (Sigma Aldrich) in methanol was used, taking 100% radical scavenging activity. The concentration of extracts ranged from 500, 250, 125, 62.5, 31.25, 15.625, and 7.812 μg/mL. The assay was performed in duplicate and two times. The scavenging percentage was calculated with the following equation:The free radical scavenging assay was performed to study the antioxidant potency of extracts, based on the scavenging activity of 1, 1-diphenyl-2- picrylhydrazyl (DPPH) using the method described by Stratil et al. . A sampl.50 and the Antiradical power (ARP), which is the inverse of the EC50.The % radical scavenging activity was used to determine the ECμg equivalent Vit C/g of extracts.The capacity of each plant extracts to reduce the ferric-ferry cyanide complex to the ferrous-ferry cyanide complex was determined as described by Stratil et al.  with sliThe presence of major phytochemical classes of compounds such as alkaloids, anthraquinones, flavonoids, glycosides, phenols, saponins, sterols, tannins, and triterpenoids were qualitatively evaluated in the plant extracts according to previously described methods .50, EC50, ARP, and plot the standard curve for vitamin C. The results were presented as the mean ± SD.The data were subjected to one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences  program. Microsoft Excel 2016 software for Windows was used to calculate, ICPsEtOH (6.002%) and PsH2O (4.32%), respectively, contrary to decoction and ethyl acetate showing weak yields as noticed with the extraction of 1.66% and 1.62% for extracts PsDec and PsEtOAc, respectively. The gradual extraction of the residue of P. stemaria with more polar solvents produced extracts with the highest extraction yield. For instance, the yield of PsH2O(H) (3.478%) was 1.58, 1.44, and 1.55 time higher as compared to PsMeOH(H), PsEtOAc(H), and PsHex, respectively.The serial exhaustive extraction system from the nonpolar to the polar solvents yielded sixteen extracts with the yield values ranging from 1.484% to 6.002%, varying as a function of extraction solvent . In the μg/mL showed that although none of the extracts inhibited Candida albicans strains, four (25%) extracts, including PsHex, PsH2O(H), PsMeOH(EA), and PsMeOH exhibited antibacterial activity in a strain-specific manner. PsHex and PsMeOH were active on S. flexineri, while PsMeOH(EA) and PsH2O(H) inhibited, respectively, S. aureus and K. pneumonia. The four extracts were progressed to a dose-response study for MIC determination. Extract PsH2O(H) was active against K. pneumonia with MIC of 31.25 μg/mL, while PsMeOH(EA) inhibited S. aureus with MIC of 250 μg/mL, and PsHex and PsMeOH inhibited S. flexineri with MIC values of 500 μg/mL.The results of the antimicrobial screening at 500 PsHex extract was active, none of the extracts (PsMeOH(H), PsEtOAc(H), and PsH2O(H)) obtained from the extraction of the initial residue displayed activity. In the contrary, the PsMeOH (EA) extract obtained from the extraction of the residue from PsEtOAc was active, while the initial ethyl acetate extract (PsEtOAc) and extract PsH2O(EA) (extracted from the residue of PsMeOH(EA)) were inactive. Finally, methanol extract PsMeOH was active, while PsH2O(M) extracted from PsMeOH residue was inactive. This analysis reveals that metabolites with antimicrobial activity are concentrated in a specific fraction in P. stemaria and, therefore, justifying the serial exhaustive extraction strategy applied to reveal their potency.Overall, the results revealed the effect of the serial exhaustive system on the activity of extracts. Indeed, while The selected extracts were later subjected to liquid-liquid partition with the aim to further concentrate the active metabolites. The ten fractions obtained were submitted to the minimum inhibitory concentration (MIC) determination as presented in PsHex extract yielded the fractions frH(PsHex) and frM(PsHex) with extraction yield of 1.7 and 0.54%, respectively, both inactive against S. flexineri. The PsMeOH extract yielded three fractions frM (PsMeOH), frH (PsMeOH), and P(PsMeOH) with the yield of 0.9, 0.4, and 0.160%, respectively. Only P(PsMeOH) was active on S. flexineri (MIC of 500 μg/mL). Similarly, the fractionation of PsMeOH(EA) also yielded three fractions among which only frM(PsMeOH(EA)) with the yield of 2.4% was active on S. aureus (MIC of 500 μg/mL). Finally, extract PsH2O(H) was separated into two fractions of 1.01% (frH(PsH2O(H))) and 2.4% (frEA(PsH2O(H))) both active on K. pneumonia with the MIC value of 500 μg/mL.The fractionation of PsHex were active, fractions from PsMeOH(EA) displayed a two-fold decrease in potency, and fractions from PsH2O(H) displayed 16-fold decrease in potency. This loss and decrease of the activity of these fractions could be related to the loss of synergistic interaction (due to separation) existing among compounds present in the mixture.In general, the liquid-liquid fractionation of active extracts led to a substantial decrease and loss of potency. In fact, while none of the fractions from The fractions having loss their antimicrobial potency, the four initial active extracts were submitted to the evaluation of their antioxidant activity using both DPPH and FRAP assays.μg/mL.The DPPH radical scavenging activity of extracts is presented in 50 values ranged from 41.69 to >500 μg/mL. Extract PsMeOH(EA) exhibited the highest DPPH radical scavenging activity with IC50 of 41.69 μg/mL as compared to PsMeOH extract (IC50 of 51.35 μg/mL). Both extracts displayed an antiradical power of 47.96 × 10−5 and 38.94 × 10−5. Indeed, the antiradical power measures antiradical effectiveness; the higher this value, the more the extract has a strong proton donor capacity to stabilize the radical. However, both extracts were less active than the ascorbic acid used as the positive control .Overall, the IC2+-extract complex increased with the concentration of extract, and this absorbance is proportional to the amount of Fe3+ ions reduced. Thus, a high absorbance would reflect a large amount of Fe2+-extract complex and, therefore, an intense reducing activity of these extracts. Using the standard curve from vitamin C, the absorbances were used to quantify the Fe3+ reducing power of different plant extracts.FRAP assay showed that there was a correlation between the concentration of the extracts and their reducing power. From the data, the absorbance of the FePsHex  and PsH2O(H)  were poorly active. On the contrary, at 250 μg/mL the activity extract PsMeOH(EA) was significantly (P ≤ 0.05) higher than those of the PsMeOH. However, at the maximum concentration of 500 μg/mL, the reducing capacities of both extracts of 58.99 μg equivalent Vit C/g of extract (PsMeOH) and 61.53 μg equivalent Vit C/g of extract (PsMeOH(EA)) were not significantly different (P > 0.05). In general, the results from both the DPPH and FRAP assays showed that PsMeOH and PsMeOH(EA) extracts presented greater antioxidant potentials. The high antioxidant effect of these methanolic extracts could be due to the diversity of compounds extracted.The selected extracts were submitted to qualitative phytochemical analysis, and the results are summarized in PsMeOH(EA) and PsMeOH with, respectively, 7 and 6 groups of metabolites showed a great abundance in phytochemicals. Extract PsHex was the poorest, with only two classes identified. In general, this test revealed the presence of flavonoids, glycosides, phenols, tannins, terpenoids, saponins, and anthraquinones. The results also show that alkaloids and sterols were absent in all extracts.The phytochemical screening presented in Platycerium stemaria used to treat various human diseases in African traditional medicines was explored. More specifically, this study was designed to identify the appropriate solvent system to concentrate antimicrobial and antioxidant metabolites present in P. stemaria as an attempt to discover new antibiotics.Today, antibiotic-resistant pathogens are a severe threat to public health. This problem becomes even worse with a decline in the supply of new antibiotics . For oldn-hexane to water to ensure the extraction of a wide range of phytochemicals with different polarities have significantly impacted the extraction yields and activity. This method was reported as suitable to improve the extraction of metabolites from various plant materials [P. stemaria, the serial exhaustive extraction is appropriate for an initial investigation.The results showed that the serial exhaustive extraction method involving the successive extraction with solvents of increasing polarity from aterials , 18. Moraterials . This meS. flexineri (PsHex and PsMeOH) and K. pneumonia (PsH2O(H)) and Gram-positive bacteria S. aureus (PsMeOH(EA)). This differential activity profile of extracts could be related to the mechanism of action of their active ingredients. More investigation into these metabolites and their possible mechanism of action could provide insightful informations. Indeed, the phytochemical analysis of promising extracts revealed a differential composition in terms of metabolite classes, including flavonoids, glycosides, phenols, tannins, terpenoids, saponins, and anthraquinones. Of note, phenols and tannins were present in all four extracts. Metabolites belonging to these classes were reported for their antibacterial activity [PsHex were inactive up to the concentration of 500 μg/mL, while fractions from PsMeOH(EA) and PsH2O(H) with MICs of 500 μg/mL displayed a 2 and 16 fold decrease in potency, respectively. The loss of potency observed likely suggests that the activity of parent extracts could be associated with synergistic interactions between two or several metabolites present in each of these crude extracts. Indeed, medicinal plants contain myriads of compounds that act together to exhibit a specific function [PsHex and PsH2O(H) were inactive up to 500 μg/mL while PsMeOH and PsMeOH(EA) exhibited potency. The observed potency corroborates the presence of various groups of antioxidant metabolites in the active extracts (PsMeOH and PsMeOH(EA)). Future metabolomics-guided investigation of these two active extracts could warrant isolation and characterization of promising metabolites for further development.The sixteen crude extracts obtained were submitted to antimicrobial screening to identify only four extracts exhibiting selective antibacterial activity against Gram-negative bacteria activity –23. The function , 25. ThePlatycerium stemaria. The investigation revealed a significant variation in extraction yield and antimicrobial activity of extracts as a function of solvent and microbial pathogen used. Moreover, among the four antimicrobial extracts identified, two extracts exhibited both radical scavenging and ferric reducing antioxidant power. These extracts also contain several antimicrobial and antioxidants metabolites identified by phytochemical screening, suggesting the need for further investigation to characterize the observed activities. Overall, this pioneering study is of particular importance in the attempt to fill the gap in phytochemical and pharmacological knowledge of P. stemaria. It showed that serial exhaustive extraction approach led to active metabolites being located beyond the single extraction, indicating that selecting the appropriate solvent and the method for extraction is very much essential to locate antimicrobial metabolites in P. stemaria. Further investigation is needed to characterize the active ingredients present in the promising extracts identified in this study.This study used a serial exhaustive extraction approach to investigate the antimicrobial and antioxidant potentials of extracts from


The purpose of this study was to investigate the feasibility of applying handcrafted radiomics (HCR) and deep learning-based radiomics (DLR) for the accurate preoperative classification of glioblastoma (GBM) and solitary brain metastasis (BM).  A retrospective analysis of the magnetic resonance imaging (MRI) data of 140 patients (110 in the training dataset and 30 in the test dataset) with GBM and 128 patients (98 in the training dataset and 30 in the test dataset) with BM confirmed by surgical pathology was performed. The regions of interest (ROIs) on T1-weighted imaging (T1WI), T2-weighted imaging (T2WI), and contrast-enhanced T1WI (T1CE) were drawn manually, and then, HCR and DLR analyses were performed. On this basis, different machine learning algorithms were implemented and compared to find the optimal modeling method. The final classifiers were identified and validated for different MRI modalities using HCR features and HCR + DLR features. By analyzing the receiver operating characteristic (ROC) curve, the area under the curve (AUC), accuracy, sensitivity, and specificity were calculated to evaluate the predictive efficacy of different methods. p < 0.05). The T1CE-based radiomic model showed the best classification performance (AUC = 0.99 in the training dataset and AUC = 0.97 in the test dataset), surpassing the other MRI modalities (p < 0.05). The multimodality radiomic model also showed robust performance (AUC = 1 in the training dataset and AUC = 0.84 in the test dataset).  In multiclassifier modeling, random forest modeling showed the best distinguishing performance among all MRI modalities. HCR models already showed good results for distinguishing between the two types of brain tumors in the test dataset . By adding DLR features, all AUCs showed significant improvement (T1WI, AUC = 0.87; T2WI, AUC = 0.80; T1CE, AUC = 0.97;  Machine learning models using MRI radiomic features can help distinguish GBM from BM effectively, especially the combination of HCR and DLR features. Brain metastases (BMs) are the most common tumors of the central nervous system (CNS), with an incidence of approximately 7–14 per 100,000 population . AccordiIt is generally accepted that magnetic resonance imaging (MRI) is an important modality for evaluating brain tumors. In patients with a history of systemic cancer and multiple lesions, the differentiation of BM from GBM may be easily achieved using conventional MRI. However, single metastases were estimated to occur in more than 25% of cases of BM . AdditioGenerally, BM and GBM are distinguished by some observable MRI features. Lesions that show nodular or ring-shaped inhomogeneous contrast enhancement and hypointense signals on T1-weighted imaging (T1WI) and hyperintense signals on T2-weighted imaging (T2WI) are visible on conventional MRI for both diseases. Compared with GBM lesions, most BM lesions are multifocal, have relatively clear boundaries, and are surrounded by severe edema. Moreover, BM lesions have smaller volumes, smaller areas of necrosis and cystic degeneration, and less enhancement than GBM lesions. Nevertheless, these methods, with limited sensitivity and specificity, are susceptible to individual subjectivity, resulting in differences among radiologists. However, the use of multiple MRI techniques is expected to significantly improve the diagnostic results. Several studies have differentiated between GBM and BM based on multiparametric MRI data, including data from advanced imaging methods such as diffusion , 12, perWhile histopathological evaluation is currently the gold standard for brain tumor diagnosis , there iPrevious studies have shown that radiomics offers important advantages in the assessment of the underlying tumor pathophysiology and improves the ability to distinguish between tumors –29. AlthThe study was approved by the Institutional Review Board (IRB) of Xiangya Hospital. According to the relevant guidelines and regulations of the retrospective study, the requirement for informed consent was waived. The study workflow overview is shown in This was a retrospective analysis of 268 patients with GBM and BM from Xiangya Hospital, Yunnan Cancer Hospital, and Nanfang Hospital, proven pathologically between January 1, 2010, and December 31, 2018. Preoperative routine plain and enhanced MRI scans were required for patients to be eligible for inclusion in the study. Patients who had a treatment history (including surgery and radiotherapy) related to GBM or BM were excluded. The 268 patients were separated into two groups: 208 (98 BM and 110 GBM) patients from Xiangya Hospital were included in the training dataset and 60 (30 BM and 30 GBM) patients from Yunnan Cancer Hospital and Nanfang Hospital were included in the test dataset. The diagnoses of all patients were confirmed histologically on the basis of the 2016 WHO classification system.All MRI examinations were conducted in the radiology department of Xiangya Hospital, Yunnan Cancer Hospital, and Nanfang Hospital with a 3.0-T MRI system. High-quality MRI images were obtained using the following protocols: axial T1WI: layer thickness = 5 mm, layer spacing = 1.5 mm, matrix = 320×256, and field of view (FOV) = 24 × 24 cm; axial T2WI: layer thickness = 5 mm, layer spacing = 1.5 mm, matrix = 384× 384, and FOV = 24 × 24 cm; and axial contrast-enhanced T1WI (T1CE): layer thickness = 5 mm, layer spacing = 1.5 mm, matrix = 320 × 256, and FOV = 24 × 24 cm. All MRI images were retrieved from the picture archiving and communication system for further image feature extraction.Through noise reduction, offset field correction, and strict object internal registration, we preprocessed each image using the public software package FSL. Histogram matching was performed such that the intensity levels between various objects were comparable. All images were assessed by two neuroradiologists (each with 5–10 years of work experience) independently. The ROIs of the entire tumor on T1WI, T2WI, and T1CE images were created manually around the enhanced part of the tumor layer by layer using ITK-SNAP software ; areas ohttps://pyradiomics.readthedocs.io/en/latest/index.html). Finally, 1106 HCR features were extracted from each MRI ROI, and 3318 HCR features were extracted from each patient.Based on the segmentation of ROIs, we performed HCR feature extraction using PyRadiomics . The higA convolutional neural network (CNN) was also used to extract and summarize MRI features. Here, we used a pretrained ResNet transferred from ImageNet, which was built by Keras. The last fully convolutional layer was reset, and each ResNet could extract 1000 features from each MRI modality.Based on the HCR and DLR features, the random forest- (RF-) based Boruta algorithm was applied to select the optimal feature groups. Boruta is a wrapper algorithm that performs robust, statistically grounded feature selection for all relevant features . By compAfter finding the best modeling method, each classifier was established and validated in the training dataset and tested in the test dataset. The HCR models of the T1CE, T1WI, T2WI, and multimodality features were built and compared; then, DLR features were added, and the above process was repeated. Through comparison of the ROC curves and AUC values, the optimal MRI modality and machine learning model were identified.t-tests were used to assess differences in continuous variables between the patient groups, and Fisher's exact tests were used to assess noncontinuous variables. The DeLong test was performed to compare ROC curves. A P value < 0.05 was considered statistically significant.All machine learning algorithms were implemented using scikit-learn and Keras libraries, and statistical analysis was carried out using SPSS . Independent The clinical characteristics of the patients in the training and test cohorts are given in p < 0.05)  .p < 0.05). Adding DLR features, T1CE showed significant improvement  and remained the best model. T1WI  and T2WI  also showed significant improvement. All details are shown in With only HCR features, T1CE showed the best distinguishing performance , which was significantly better than that of T1WI and T2WI (p < 0.05). Generally, multimodality radiomic models show better diagnostic performance than single-modality models. In our study, the multimodality model showed an AUC of 1.00 in the training dataset and an AUC of 0.84 in the test dataset, which were lower than those of the T1CE- and T1WI-based radiomic models . The combined radiomic model, based on the optimal HCR features and DLR features, showed the best overall performance, with an AUC of 0.99 in the training set and 0.97 in the test set. However, we should note that the differences in performance among the different models were not large enough to select one optimal model, specifically considering that the investigated models seemed to perform quite comparably and that the variance in AUC might be partially attributed to the small sample size. Therefore, our results can only be regarded to support our hypothesis and need to be verified in future studies.ResNet was proposed in 2015 as a CNN to solve the problem of deep networks, such as the vanishing gradient . ResiduaGenerally, the potential of multimodality MRI for application may be better than that of single-modality MRI, but there are some cases in which its performance can be limited. In this study, the simple combination of selected features from images obtained by different MRI modalities may be one cause of limited performance; we know that more heterogeneous data do not necessarily provide more valuable information. Moreover, some studies have shown that single-modality models may have predictive potential better than or equal to that of multimodality models , 38. We There are some limitations to this study that should be addressed. First, this was a retrospective analysis of a small sample. Second, the ratio of BM to GBM lesions and the variety of BM sources were not representative of the general population, which could exhibit different characteristics. Thus, a larger prospective study is needed for verification. Third, in this study, a manual method was used to segment ROIs. Although manual segmentation is usually better than automatic segmentation and two researchers participated in the segmentation process, segmentation errors still occur. Fourth, we did not compare the performance of the machine learning model with that of a human reader.In conclusion, our study suggests a radiomic machine learning method for distinguishing BM from GBM lesions preoperatively with favorable predictive accuracy and stability. In addition, this combination could yield valuable insight into tumor progression, which could also facilitate the implementation of a personalized approach in tumor management. However, the results of our study need to be used with caution; we only validated the feasibility of applying HCR and DLR for the accurate preoperative classification of GBM and solitary BM lesions.