Palbociclib is a highly selective, reversible, oral inhibitor of cyclin‐dependent kinases 4 and 6 that is approved to treat hormone receptor‐positive/human epidermal growth factor receptor 2‐negative advanced breast cancer. An open‐label, single‐arm, Japanese phase 2 study was conducted to investigate the efficacy and safety of palbociclib plus letrozole as first‐line treatment in 42 postmenopausal patients with estrogen receptor‐positive/human epidermal growth factor receptor 2‐negative advanced breast cancer. The probability of progression‐free survival originally reported at 1 year was 75.0% , but median progression‐free survival was not attained at the primary analysis. In this report, updated efficacy and safety results with a longer follow‐up period are presented. The median duration of treatment in the updated analysis was 33.0 months . The probability of progression‐free survival at 1 year was 75.6% . Median progression‐free survival was 35.7 months . Objective response rate and disease control rate were 47.6%  and 85.7% , respectively. Common treatment‐related adverse events  were neutropenia , leukopenia , and stomatitis . Treatment‐related febrile neutropenia was reported in one patient. In general, no clinically meaningful deterioration in health‐related quality of life was observed. Palbociclib plus letrozole remained effective and tolerable in Japanese postmenopausal patients with estrogen receptor‐positive, human epidermal growth factor receptor 2‐negative advanced breast cancer in this updated analysis. With longer follow‐up of an open‐label, single‐arm, Japanese phase 2 study conducted to investigate the efficacy and safety of palbociclib plus letrozole as first‐line treatment in postmenopausal patients with estrogen receptor‐positive/human epidermal growth factor receptor 2‐negative advanced breast cancer, median progression‐free survival was 35.7 months . Palbociclib plus letrozole remained effective and tolerable in Japanese postmenopausal patients with estrogen receptor‐positive/human epidermal growth factor receptor 2‐negative advanced breast cancer. If necessary, the first palbociclib dose reduction was from 125 to 100mg/d, and the second dose reduction was from 100 to 75mg/d. After the study transitioned to a postmarketing clinical study, the patients switched from the study drug to commercially available palbociclib.2.3The primary outcome was 1‐year PFS. Secondary outcomes included PFS, objective response rate (ORR), disease control rate (DCR), duration of objective response (DOR), 1‐year survival, OS, safety, and patient‐reported HRQoL. Safety was recorded during treatment and for 28 days after treatment; severity of AEs was graded using the Common Terminology Criteria for Adverse Events (CTCAE) v4.0 and classified according to the Medical Dictionary for Regulatory Activities . Patient‐reported HRQoL was measured using the Functional Assessment of Cancer Therapy‐General (FACT‐G)2.4All efficacy analyses were performed using the full analysis set, which comprised all patients who received >1 dose of palbociclib. Efficacy was also assessed in the baseline disease characteristic subgroups of visceral or nonvisceral metastases; length of disease‐free interval (DFI) since completion of prior treatment ; prior or no prior endocrine therapy; prior or no prior chemotherapy; age (<65years or ≥65 years); and Ki67 staining (≤20% or >20%). The 1‐year PFS probability and 90% CI were estimated using the Kaplan‐Meier method. Median PFS, OS, and associated 95% CIs were also estimated using the Kaplan‐Meier method. ORR was defined as the percentage of patients who had either complete response (CR) or partial response (PR) based on RECIST criteria. DCR was the percentage of patients with CR, PR, or stable disease lasting ≥24 weeks. DOR was analyzed in patients in the full analysis set with OR and defined as the time from first OR to first objective disease progression or death. Treatment duration of subsequent therapy was estimated using the Kaplan‐Meier method. Change from baseline in HRQoL scale scores was summarized by descriptive statistics (mean±SD). As a post hoc analysis, an examination of the time to definitive deterioration (TTD) in FACT‐B total score was conducted using the Kaplan‐Meier method. A meaningful change in QOL is defined as a change in baseline score equal to or greater than the established minimally important differences: for FACT‐B, this is 7‐8 points.33.1A total of 42 patients were enrolled in the study. Patient demographics and baseline disease characteristics have been previously published3.2Study drug exposure is summarized in Table3.3At the data cutoff date, the 1‐year probability of PFS was 75.6% 90% CI, 62.4‐84.7). The Kaplan‐Meier estimated median PFS was 35.7months . In the full analysis set, the ORR was 47.6%  and the DCR was 85.7% . In the response evaluable set (n = 36), the ORR and DCR were 55.6%  and 83.3% , respectively. OS data remain immature; median OS was not reached at the date of this data cutoff  received subsequent systemic anticancer therapies , 3.5 , and 3.8  months, respectively. Five patients received an everolimus‐containing regimen  as any line of subsequent therapy. Median duration of subsequent everolimus‐containing treatment was 4.1 months . The median time to first use of subsequent chemotherapy was not yet reached  at the data cutoff date Figure . The tim3.5The most commonly reported AEs were similar to those previously described Table , and no 3.6Overall changes from baseline to end of study treatment in FACT‐B and TOI total scores remained consistent Figure , indicat4With extended follow‐up, palbociclib plus letrozole remains an effective and well‐tolerated therapy in postmenopausal Japanese women with ER+/HER2– ABC. In general, the data reported here are consistent with those of the PALOMA‐2 extended follow‐up results; with a median duration of follow‐up of 37.6 months , median PFS in the palbociclib arm of PALOMA‐2 was 27.6 months .Palbociclib plus letrozole remained well tolerated in Japanese patients. No new safety signals were observed in Japanese patients with longer follow‐up. Hematologic AEs were the most frequently reported AEs with extended follow‐up in both this Japanese phase 2 study and in PALOMA‐2.Japanese patients with nonvisceral metastases demonstrated greater benefit with palbociclib plus letrozole than did patients with visceral metastases; median PFS was approximately 2.5years longer in patients with nonvisceral metastases (46.7 and 16.7months). A similar PFS difference between patients with visceral and nonvisceral metastases was also observed in other clinical trials. The proportion of patients with nonvisceral metastases was comparable in the Japanese phase 2 study and PALOMA‐2 . In PALOMA‐2, median PFS was >1year longer in patients with nonvisceral metastases compared with patients with visceral metastases .Most Japanese patients (87%) received endocrine therapy as their first subsequent therapy after disease progression on palbociclib, and approximately 30% received endocrine therapy as their second subsequent therapy. Only 3 patients (13%) received chemotherapy as their first subsequent therapy. Single‐agent fulvestrant was the most common subsequent treatment. In PALOMA‐2, 60.8% and 36.6% of patients received endocrine therapy and chemotherapy, respectively, as the first subsequent treatment after discontinuation of palbociclib,Generally, no clinically meaningful deterioration in FACT‐G, FACT‐B, and TOI total scores and FACT‐B subscale scores was observed in Japanese patients in this study. This finding is similar to that observed in the overall PALOMA‐2 study population with extended follow‐up, in which the FACT‐B total score was maintained over time with palbociclib plus letrozole, and changes from baseline did not significantly differ between the palbociclib and placebo arms.This study is subject to certain limitations, including its single‐arm, open‐label design and small sample size, and therefore may be subject to biases. However, with extended follow‐up, treatment of Japanese patients with ER+/HER2– ABC with palbociclib in combination with letrozole yielded a median PFS of almost 3 years. Palbociclib plus letrozole was confirmed as safe and effective, without clinically meaningful deterioration in patient‐reported HRQoL, in postmenopausal Japanese patients with ER+/HER2– ABC. Furthermore, as the majority of patients received hormonal therapy as subsequent therapy after disease progression with palbociclib plus letrozole in this study, it will provide the opportunity for future analysis of the efficacy of subsequent therapies.M. Takahashi, H. Iwata, S. Hashigaki, Y. Muramatsu, Y. Umeyama, and M. Toi made substantial contributions to the study conception and design. M. Takahashi, N. Masuda, R. Nishimura, K. Inoue, S. Ohno, H. Iwata, and M. Toi participated in data acquisition. S. Hashigaki performed data analysis. All authors made substantial contributions to the interpretation of data. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved the final version of the manuscript to be published; have participated sufficiently in the work to take public responsibility for appropriate portions of the content; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Authors SH and YU are employees of Pfizer R&D Japan. YU also owns stock in Pfizer Inc. YM is an employee of Pfizer Japan Inc. HI received honoraria from Chugai, Daiichi‐Sankyo, AstraZeneca, and Pfizer; manuscript fees from Eisai; and research funding from MSD, Chugai, Daiichi‐Sankyo, and Boehringer Ingelheim. KI’s institution received research funding from Parexel, Puma Biotechnology, Pfizer, Novartis, MSD, GSK, Chugai, and Daiichi‐Sankyo. NM received honoraria from Chugai, Pfizer, Eli Lilly, Eisai, Takeda, and AstraZeneca; is a board member of the Japan Breast Cancer Research Group Association; and his institution received research funding from Chugai, AstraZeneca, Kyowa‐Kirin, MSD, Novartis, Pfizer, Eli Lilly, Eisai, and Daiichi‐Sankyo. SO received honoraria from Chugai, AstraZeneca, Eisai, Pfizer, Taiho, Kyowa‐Kirin, Nippon Kayaku, and Novartis, and research funding from Taiho and Eisai. RN received honoraria from Pfizer, Novartis, and Chugai. MTa received honoraria from Pfizer, AstraZeneca, Eli Lilly, and Eisai; and research funding from Taiho, Kyowa‐Kirin, and Eisai. MTo received honoraria from AstraZeneca and Eli Lilly; research funding from Taiho, Kyowa‐Kirin, Shimadzu, AFF Technology, Bizcom Japan, C&C Research Laboratories, Astellas, AstraZeneca, and Chugai; scholarship endowments from Chugai, Eisai, and Pfizer; and is a member of the board  of the Japan Breast Cancer Research Group association, Kyoto Breast Cancer Research Network, and Organisation for Oncology and Translational Research.Supplementary MaterialClick here for additional data file.


The exact pathogenesis of polycystic ovary syndrome (PCOS), the most common neuroendocrine disorder in women of reproductive age, has not been fully elucidated. Recent studies suggested that chronic inflammation and neurotransmitter disorder involved in the progress of PCOS. Troxerutin, a natural flavonoid, was reported to possess neuroprotective effect in several disease models by inhibiting inflammation or enhancing neurotrophic factor. In this study, we investigated the possible protective effect and mechanism of troxerutin in a dihydrotestosterone (DHT)-induced rat model of PCOS. The PCOS rat models were treated with troxerutin at a dose of 150 mg/kg or 300 mg/kg for up to 4 weeks. Results showed that 300 mg/kg troxerutin significantly decreased the body weight gain and improved the pathological changes of ovary induced by DHT. Meanwhile, the elevated gonadotrophin-releasing hormone (GnRH), gonadotrophin and testosterone in the serum of PCOS rats were reduced with the treatment of troxerutin. The expression of kisspeptin and NKB in arcuate nucleus and their receptors kiss1r and NK3r in GnRH positive neurons of median eminence were markedly decreased in troxerutin-treated rats. Of note, the GnRH inhibitory regulator GABA and stimulatory regulator glutamate were also restored to the normal level by troxerutin. The present study indicated that troxerutin may exhibit a protective effect in PCOS rat model via regulating neurotransmitter release. Polycystic ovary syndrome (PCOS) is a reproductive endocrinopathy with the prevalence estimated to be 6% ~ 20%(depending on the different diagnostic criteria used), making it the most common endocrine condition in women of reproductive age . The diaThe hypothalamus-pituitary-gonad (HPG) axis plays a central role in the exquisite neuroendocrine regulation of reproduction. Hypothalamic secretion of gonadotrophin-releasing hormone (GnRH), has been robustly established as the key pathway that controls reproductive function . Axons oGnRH is influenced by extrinsic factors such as neurotransmitters and neuropeptides, any alteration of the GnRH regulatory neurotransmitters, such as inhibitory gamma-aminobutyric acid(GABA) and stimulatory glutamate (Glu), may result in reproductive endocrine dysfunction . ActuallTroxerutin, a trihydroxyethylated derivative of the naturally occurring plant bioflavonoid rutin, also called vitamin P4. It has been widely identified in plants consumed as part of our daily diet, such as tea, coffee, cereals, and a variety of fruits and vegetables, and exerts multifarious biological activities, such as anti-oxidant and anti-inflammatory effects and exerts beneficial effects on animal models of different central nervous system diseases , 17. TroPostnatal 14 days female SD rats were purchased from the Breeder center of Rodents . The rats were housed under controlled lighting  and temperature (24 °C) with free access to food and water for a week of acclimatization. All experimental protocols used in this study were approved by the Animal Care and Ethical Committee of Xuzhou medical University. Postnatal 21 days rats weighing approximately 50-55 g, were randomly divided into four groups: sham group (each rat was implanted with a tube without DHT), PCOS group (each rat was implanted with a silastic tube filled with 5α- DHT at dose of 7.5 mg under anesthesia), PCOS+ 150 mg/kg troxerutin group and PCOS+ 300 mg/kg troxerutin group . Due to the high water-solubility of troxerutin, the gastrointestinal system can absorb it well without toxicity. Troxerutin interacts with tRNA by external binding manner with low binding affinity, and also binds with DNA through groove binding manner. Sham and PCOS groups were injected daily with equivalent 0.9% saline which was used to dissolve troxerutin. DHT was procured from sigma, St. Louis, Mo and troxerutin (purity > 95%) was from Baoji Fangsheng Biotechnology Co., Ltd., Baoji, China. The rats were housed individually after surgery.At the terminal of the study, rats were decapitated and trunk blood was collected and centrifuged at 3000 rpm for 15 min. Plasma was separated and stored at − 80 °C until analyzed for biochemical and hormonal analysis. Ovaries were cleaned in saline and managed fat free. Ovaries were fixed in 10% buffered formalin for 48 h and paraffin-embedded. Paraffin-embedded tissue sections were de-waxed, sectioned (6 μm) and stained with hematoxylin and eosin (H&E). Numbers of follicles were classified and counted in every 40th ovarian section for 10 ovaries per group. Follicles were counted according to the following definition. Primordial follicle was surrounded by a single layer of squamous granulosa cells. Primary follicle contained a single layer of cuboidal granulosa cells. Secondary follicle contained two or more layers of granusola cells, but no antral space in the granulosa layer. Antral follicle displayed multi-layers of granusola cells with the presence of follicular antrum.Vaginal smears were taken daily at 09:00 am from the 18th to the 28th day after the first day of DHT treatment,which were the terminal 10 days of the study. Microscopic analysis  was carried out to determine the stage of the estrous cycle by the predominant cell types in vaginal smears following methylene blue staining . Proestrus consists of enlarged,round,nucleated epithelical cells; estrus consists of a large number of cornified squamous epithelical cells; metestrus consists of two kinds of cells: epithelial cells and leukocytes with approximately same amount; and diestrus is characterized by a small amount of cells with the predominance of leukocytes.Body weight was measured every week after implantation for a total of 4 weeks.The serum concentration of LH, FSH and testosterone were measured via Enzyme Linked Immuno Sorbent Assay (ELISA) with the help of commercial kits  and the procedure was followed as given in the kit catalog. The concentration of LH, FSH and testosterone was estimated by the standard curve.To identify the hypothalamic GnRH status, hypothalamus was dissected out. The rat brain was carefully taken out and temporarily put on ice with the ventral surface upward. Thalamus is the ellipsoidal part at the center of the ventral brain. With the middle of tuber cinereum and optic chiasma as the center, prechiasmal border as the anterior edge, postmammillary border as the posterior edge, hypothalamus (4x4x2mm) was carefully dissected out. Hypothalamus tissue were homogenized to obtain protein samples. Then the protein (25 μg) was separated by SDS-PAGE and transferred to membranes. The membranes were incubated with mouse monoclonal anti-GnRH antibody  and mouse anti-GAPDH antibody . After being washed with TBST three times, the membranes were incubated with IRDye-labeled secondary antibodies in TBST for 2 h. The bands on the membrane were scanned with an Odyssey infrared scanner  and the density of the bands was analyzed with ImageJ software.Chromatographic analysis was carried out to evaluate the levels of two kinds of GnRH regulatory neurotransmitters GABA and glutamate in the brain/pituitary. Weigh the brain and add in the mix of methanol and water (1:1) at the ratio of 100 mg/ml to prepare homogenate. The brain homogenate was centrifuged at 12000 rpm for 30 min at 4 °C. The mix of supernate and 80 μL acetonitrile were centrifuged at 12000 rpm for 30 min at 4 °C. The new supernate was derivatized, mixed with 80 μL borate saline buffer, 160 μL water and 80 μL F-moc, filtered by 0.45 μm filtration membrane and immersed in 40 °C bath for 5 min. Then, the samples were used for the measurement of GABA and glutamate using chromatographic analysis. All standard biomarkers used for identification purpose in chromatographic studies were procured from Zhongke Co, Ltd., Beijing, China. High-performance liquid chromatography (HPLC) was performed on a Waters e2695 system  equipped with a hypersil ODS column . The mobile phase A was a mix of natrium aceticum (pH 4.8),water and tetrahydrofuran with the ratio of 410:85:5, and mobile phase B was pure acetonitrile. Flow rate was 1 ml/min. The column temperature was set at 30 °C. Samples were detected at the wavelength of 265 nm. Injection volume was 20 μL.After intracardiac perfusion with normal saline followed by fixation with 4% cold paraformaldehyde (PFA), the rat brains were separated and postfixed in 4% PFA for 6 h at 4 °C. Then the brains were incubated in 30% sucrose- 100 mM sodium phosphate buffer (pH 7.4) for 48 h at 4 °C. Serial coronal sections (20 μm) were made from the bregma anterior-posterior − 2.0 mm to − 3.30 mm. Totally 60 sections were collected per brain. At least three sections per brain were selected for NKB, Kisspeptin, GnRH/NK3R and GnRH/Kiss1r immunofluorescent staining, respectively.For immunofluorescence, the primary antibodies rabbit anti-NKB , rabbit anti-kisspeptin , rabbit anti-Kiss-1r , rabbit anti-NK3r and mouse anti-GnRH  were used. After incubating with the antibodies for 24 h at 4 °C, the sections were washed with PBS and then were treated with goat anti-mouse IgG (H + L) Alexa Fluor®555 or 488 -conjugated or goat anti-rabbit IgG (H + L) Alexa Fluor®488  secondary antibodies. According to the manufacturer’s instructions, DAPI  was used to label nucleus. For negative controls, sections were incubated with PBS instead of the primary antibodies. Fluorescence images were captured using a Zeiss Axioskop 40 microscope . Image Pro-Plus 6.0 software was used for semi-quantitative measurement of immunofluorescent density. Values (three slides for each brain) of integral optical density (IOD) in individual cells represented the quantity of objective protein and were calculated using the following equation: Σ IOD/Σ DAPI.P < 0.05 for all tests.All statistical analyses were performed with SPSS software (version 16.0), and the data were analyzed using the one-way Analysis of Variance (ANOVA). The data were expressed as the mean ± s.e.m. Statistical significance was set at P < 0.01), 3rd(P < 0.01) and 4th (P < 0.001) week than the age-matched sham rats which denoted normal body weight, indicating that PCOS group have gained more body weight, hence a PCOS-like over weight feature. In comparison to PCOS group, treatment with troxerutin 150 mg/kg did not induce any significant changes. Nevertheless, the body weight of PCOS+ 300 mg/kg troxerutin group were markedly reduced as compared to that of PCOS group at 2nd (P < 0.05),3rd(P < 0.01) and 4th (P < 0.05) week, suggesting that troxerutin alleviated increased body weight in PCOS. Since ovarian histological change is another feature of PCOS, we also investigated whether troxerutin improved histological structure in rat ovaries. As demonstrated in Fig. P < 0.001; P < 0.001), and troxerutin 300 mg/kg inhibited the markedly low percentage of primary follicles in PCOS rats(P < 0.05). As compared to the multi-cystic follicles in ovaries of PCOS rats, the number of cystic follicles in troxerutin groups showed tendency toward decreasing. As Fig. We first investigated whether troxerutin administration has any influence on body weight in DHT-induced PCOS rat models. The time course of troxerutin administration and evaluation of phenotypes were illustrated Fig. . Body weP < 0.001; P < 0.05), which was in accordance with the characteristic LH and testosterone elevation in patients with PCOS. Though there was no significant difference in FSH levels among all four groups, administration of troxerutin to rats for 4 weeks significantly reversed the elevated serum LH  and testosterone levels , suggesting that troxerutin has a beneficial effect on the aberrant gonadotrophin and testosterone in PCOS rats.Serum LH, FSH and testosterone levels were measured at the fourth week by ELISA Fig. . PCOS raP < 0.01), however, troxerutin 300 mg/kg markedly decreased the level of GnRH compared to the PCOS rats and high Glu (P < 0.01) compared to sham rats. Although no significant difference was observed between troxerutin 150 kg/mg group and PCOS group, administration of troxerutin 300 kg/mg to rats for 4 weeks successfully reversed the notably low GABA(P < 0.05) and high Glu (P < 0.01) levels in the hypothalamus compared to PCOS group.GnRH could be influenced by regulatory neurotransmitters such as the major inhibitory GABA and stimulatory Glu. As depicted in Fig. P < 0.001; P < 0.001), troxerutin 300 mg/kg treatment significantly reversed the increase of kisspeptin and NKB IOD induced by DHT . Meanwhile, the changes of the expression of kiss1r and NK3r in ME were also observed. The numbers of GnRH/kissr1/NK3r positive cells increased in the PCOS group compared to the sham group, however, they were significantly decreased in the 300 mg/kg troxerutin-treated rat  respectively.Kisspeptin and NKB secreted from the KNDy neurons in ARC, are considered to be novel GnRH stimulatory neurotransmitters and stimulate expression of kisspeptin receptor (kiss1r) and neurokinin B receptor (NK3r) of GnRH neurons in the median eminence (ME). Here, we carried out immunofluorescence staining to observe whether troxerutin administration alters the expression of kisspeptin/ kiss1r and NKB/ NK3r in their respective regions Fig. . The IODPCOS is the most common endocrine condition affecting approximately 20% of reproductive -aged women. Though some ovulation induction agents, such as letrozole, clomiphene citrate and metformin, have been used to improve reproductive outcomes in clinic, the side effects of these drugs should not be ignored , 25. ThuGnRH pulses stimulate the synthesis and secretion of LH and FSH from anterior pituitary. It is well known that although produced in the same cell named gonadotroph, LH and FSH synthesis is regulated by different frequency of GnRH pulses, with LH favored by fast pulse frequencies(> 1 pulse per hour) and FSH favored by slow pulse frequencies(< 1 pulse per 2–3 h). As for PCOS, a neuroendocrine hallmark is persistent and rapid GnRH pulses, which favor pituitary synthesis of LH and contribute to the increased LH levels . LH is aCompelling evidence provided the strong support for brain being the culprit in PCOS , 41. TheTaken above, these results indicated that the protection of troxerutin against PCOS partially might be due to its ability to regulate hypothalamic GABA, Glu, kisspeptin/kiss1r and NKB/NK3r. Although it remains determined whether this troxerutin-caused reversal is permanent, the present study highlighted its therapeutic potential for PCOS. More in depth research will be necessary to determine whether troxerutin targeted at hypothalamic neurotransmitters alone is a promising therapeutic approach for the treatment of PCOS.


We aimed to establish a nomogram for lung cancer using patients' characteristics and potential hematological biomarkers.Principle component analysis was used to reduce the dimensions of the data, and each component was transformed into categorical variables based on cutoff values obtained using the X‐tile software. Multivariate analysis was used to determine potential prognostic biomarkers. Five components were used in the predictive nomogram. Internal validation of the model was performed by bootstrapping of samples, while external validation was performed on a separate cohort from Shandong Cancer Hospital. The predictive accuracy of the model was measured by concordance index and risk group stratification. Decision curve analysis was performed to evaluate the net benefit of the models.One hundred patients in the Discovery group and 111 patients in the Validation group were retrospectively analyzed in this study. Forty‐seven indexes were sorted into eight subgroups. Five components based on cox regression analysis were enrolled into the predictive nomogram. The nomogram prediction of the probability of 3‐ and 5‐year overall survival was in great concordance with the actual observations. Of interest, the nomogram allowed better risk stratification of patients and better accuracy in predicting patients' survival compared with pathological tumor‐node‐metastasis staging system.A nomogram was established for prognosis of lung cancer, which can be used for treatment selection and clinical care management. We established a nomogram for survival prediction of lung cancer, which can be used for treatment therapy selection and clinical care option. The nomogram allowed better distinction between survival curves compared with pathological tumor‐node‐metastasis staging system. Despite the rapid advancement in diagnosis and treatment technologies in last decade, the prognosis of lung cancer remains poor.9/L and 450 × 109/L, and the proportion of patients with elevated PLTs ranged between 6.9% and 58.5%. In addition, several studies have analyzed PLT count and PLT‐to‐lymphocyte ratioAccumulating studies have employed clinical factors such as hypertension history, diabetes history,Nomogram is a commonly used tool to evaluate the prognostic biomarkers in oncology and medicine. With their ability to generate an individual numerical probability of a clinical event by integrating diverse prognostic and clinical variables, nomograms allow the development of potential biologically and clinically integrated models that bring us steps closer toward personalized medicine.22.1A total of 100 lung cancer patients receiving pneumonectomy at Qilu Hospital between 2012 January and December of Shandong University were assigned as the Discovery group. A separate cohort of 111 patients at Shandong Cancer Hospital between 2013 January and 2014 January was assigned as the Validation group. Patient characteristics were collected including gender, age, smoking and drinking habits, high blood pressure and diabetes history, and family history of lung cancer. Tumor characteristics (TC) included size, site, pathology type, lymph node ratio, differentiation, and pTNM stage. In addition, blood measurements of patients before surgery were also collected, including: (a) routine blood biomarkers such as Hb and blood count of red blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils; (b) coagulation indicators such as PLTs, prothrombin time, international normalized ratio, activated partial thromboplastin time, fibrinogen and thrombin time; (c) glucolipid metabolism (GM) indicators such as cholesterol, triglyceride, high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), and glucose; (d) liver function (LF) indicators including glutamic‐oxalacetic transaminase, glutamic‐pyruvic transaminase, alkaline phosphatase, total protein, albumin, globulin, and albumin‐to‐globulin ratio; (e) biochemical and electrolyte indicators such as K, Na, Ca, lactate dehydrogenase (LDH), and carbon dioxide combining power; and (f) renal function (RF) indicators including uric acid, creatinine, and blood urea nitrogen.Patients were followed up every 6 months, and the inclusion criteria of patients were as follow: (a) patients who received pneumonectomy; (b) patients who were pathologically diagnosed and confirmed as lung cancer; (c) patients with complete follow‐up data. Patients were excluded if they had received neoadjuvant radiotherapy and chemotherapy, or if they refused to attend the study. Relapse, metastasis, and death time were recorded. This study was approved by the Ethics Committees of Shandong Cancer Hospital and Qilu Hospital. All patients included in this study provided signed informed consents.2.2P value approach using the X‐tile software (http://www.tissuearray.org/rimmlab).P ≤ .1 were included in the multivariate Cox regression. The significant variables from the multivariate analysis were included into a backward step‐down process with an Akaike information criterion to build the nomogram. The nomogram was evaluated using 1000 bootstraps resampling of patients in the Discovery and the Validation groups. Calibration of the nomogram for 3‐ and 5‐year overall survival (OS) was performed by comparing the predicted survival with the observed survival after bias correction. The predictive performance of the model was evaluated using the receiver operating characteristic (ROC) curve together with concordance index (C‐index). In addition, we conducted a group‐stratified analysis of the total risk score to compare the discriminative ability of the nomogram with that of the pTNM staging system in the Discovery and the Validation cohorts. Decision curve analysis (DCA) was performed to evaluate the net benefit of the models. Statistical analysis was performed using the IBM SPSS statistics version 24.0 (SPSS) and using the rms and Hmisc statistical packages in R version 3.1.2 (http://www.r-project.org).Mean substitution method was used to impute missing data. The method replaces missing values with an average value of non‐missing elements of the corresponding variable33.1A total of 211 patients, including 145 males and 66 females, were retrospectively analyzed in this study  results. All the indexes were sorted according to their clinical significance into the following eight categories: patients' characteristics (PC), TC, routine BE measurements, coagulation function indicators (CF), GM, LF indicators, biochemicals and electrolytes (Bio), and RF indicators. Principle component analysis was applied on the collected indexes and 18 principle components with eigenvalues >1 were extracted for survival analysis, two of which were from the PC category, four from TC, two from BE, two from CF, two from GM, three from LF, two from Bio, and one from RF category. The component scatter plot in Figure 3.3P = .001), TC2 (P = .068), BE1 (P = .014), BE2 (P = .003), CF2 (P = .010), GM1 (P = .011), GM2 (P = .069), LF1 (P = .021), LF3 (P = .073), Bio1 (P = .017), and RF (P = .065) in 5‐year survival prediction. These indexes were included in a multivariate analysis, which identified the following indexes as potential biomarkers for 5‐year OS prediction: TC1  [1.280‐7.510]), TC2 , BE2 , GM2 , and Bio1   and the net benefit of using the model to risk‐stratify patients (y axis) relative to predict the 3‐ and 5‐year survival, the DCA graphically presented that the new scoring system was better than traditional pTNM system  and 5‐year OS prediction . In the Validation group, the discriminative ability of the new scoring system remained superior to that of the pTNM staging system for 3‐year OS prediction  and 5‐year OS prediction . Three risk groups were better stratified by the new scoring system in both the Discovery and the Validation cohorts, compared with the pTNM staging system. And the DCA graphically presented that the new scoring system was better than traditional pTNM system.Missing data are a common problem in clinical trials and are often inadequately handled in the statistical analysis even in the top tier medical journals.To the best of our knowledge, this is the first prognostic nomogram for lung cancer that considers patients' clinical characteristics in addition to circulating blood biomarkers. We believe that this nomogram provides comprehensive information for patients, which could provide a better guidance for clinical therapy. Based on this tool, potential higher risk patients with poor survival could be more precisely selected for a specific treatment strategy.Finally, this study has some limitations that need to be addressed. First, some important indicators which have been reported to be significant in survival prediction, such as D‐dimer, C‐reactive protein, and circulating tumor biomarkers, were not included in this study due to missing data in our database. Second, this tool is limited by small population and the retrospective nature of the data collected.In conclusion, we have developed a promising nomogram for predicting survival in lung cancer patients after esophagectomy. The nomogram is based on both clinical and circulating biomarkers, and provides a strong prognostic superiority over pTNM staging system in lung cancer patients. The nomogram can help clinicians to make better predictions of patient survival and to give improved individualized treatment recommendations for lung cancer treatment.None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


We aimed to compare the intraocular pressure (IOP) measurements by a dynamic Scheimpflug analyzer (Corvis ST), a non-contact tonometer, and an ocular response analyzer after hyperopic small-incision lenticule extraction (SMILE).NCT), Corvis ST , and ocular response analyzer (Goldmann-correlated intraocular pressure [IOPg] and cornea compensated IOP [IOPcc]). A linear mixed model was used to compare the IOPs and biomechanical values among methods at each time point.Thirteen patients who underwent hyperopic SMILE in one eye were enrolled prospectively. IOP and corneal biomechanical parameters were measured preoperatively and at 1 week, 1 month, and 3 months postoperatively with a non-contact tonometer . Postoperatively, IOPNCT and bIOP were higher than IOPg and IOPcc. bIOP was independent of cornea thickness at last follow-up visit, whereas it correlated significantly with corneal biomechanics similar to the other three IOP values.IOPbIOP is a relative accurate measure of IOP after hyperopic SMILE. Cornea refractive surgery corrects the refractive error by removing a part of the corneal tissue, and consequently changes both the corneal shape and corneal biomechanics. Previous studies have identified this procedure to change the intraocular pressure (IOP) measurements , 2. AmonThe principle of hyperopic correction, which differs from that of myopic correction, is to make the central cornea steeper. This is achieved with SMILE by creating a concave lenticule that is thinnest in the central area. Liu  reportedThe current study aimed to explore the effect of hyperopic SMILE on IOP assessment using different measurement methods.Thirteen patients with hyperopic 13 eyes) were enrolled prospectively between March 2017 and June 2018 at the Eye and ENT Hospital of Fudan University , with astigmatism up to 3.0 D; the difference between manifest and cycloplegic refraction was no more than 1.0 D.Patients with abnormal topography, dilated pupil size less than 7 mm, history of intraocular surgery, and glaucoma were excluded.Preoperative examinations included slit lamp examination, objective and subjective refraction assessments, uncorrected distance visual acuity (UDVA) measurement, corrected distance visual acuity (CDVA) measurement, corneal tomography with a rotating Scheimpflug camera , and fundus examination.IOPNCT(non-contact IOP): The non-contact tonometer  was used. One average value was automatically calculated from 3 measurements. Reproducibility of measurements was identified previously [eviously .The Corvis ST  is a Scheimpflug-based dynamic corneal tonometer, which incorporates the corneal biomechanics and IOP parameters. The system uses an algorithm to calculate biomechanically corrected IOP (bIOP) and compensates for changes in corneal thickness and stiffness .These four values were derived from ocular response analyzer . ORA uses an air puff to deform the cornea. Because of its viscoelastic nature, the cornea resists the air puff, resulting in different values for the inward and outward flexing, which is termed as corneal hysteresis (CH). The corneal resistance factor (CRF) represents the resistance of cornea . Each eyTwo corneal biomechanical parameters derived from Corvis ST were analyzed. A1 Time (first applanation time), and HC DA  were the most repeatable and reproducible parameters . SP-A1 (All measurements were performed by the same examiner (FD) to decrease inter-observer variability and were taken at approximately the same time of day.All surgeries were performed by the same surgeon (ZXT). After standard sterile draping, all patients were treated with the VisuMax laser  with repetition rate of 500 kHz and pulse energy of 30 nJ. The following settings were used for hyperopic SMILE: the cap diameter was 8.8 mm and the thickness 120 μm; the optical zones ranged between 5.3–6.3 mm, with a 2-mm transition zone; a single 2.0-mm side cut was made at the 12 o’clock position with an angle of 90°.The detailed steps of SMILE have been described previously . Total sAll surgeries were performed successfully, with no intraoperative or postoperative complications.Postoperatively, the patients were instructed to use fluorometholone eye drops 8 times a day, and to reduce the usage frequency by 1 every 3 days . Artificial tears were prescribed for 3 to 4 weeks, for use as needed.Patients were examined at 1 week, 1 month, and 3 months postoperatively. At each follow-up visit, the visual acuity, subjective refraction, corneal topography, and IOP measurements were performed using three devices.Kolmogorov–Smirnov test was used to check the normality of data. Linear mixed-model analysis of variance with post hoc least significant difference multiple comparisons were used to compare the postoperative IOP measurements between different visits and different methods at the same visit. The Spearman rank correlation was used to assess the corneal biomechanical parameters obtained from the Corvis ST and to determine potential postoperative factors affecting the postoperative IOP measurements. P <  0.05 was considered statistically significant.All data were recorded and analyzed using SPSS . First, the .All patients completed the 3-months follow-up visit. The safety index (postoperative CDVA/preoperative CDVA) was 0.96 ± 0.12, and the efficacy index (postoperative UDVA/preoperative CDVA) was 0.93 ± 0.14 at the last visit. ; bIOP was 2.32 ± 0.85 mmHg higher than IOPg (P = 0.02); IOPNCT and bIOP values showed no difference. At 1 month postoperatively, bIOP was 3.60 ± 0.89 mmHg higher than IOPg (P = 0.004) and 3.32 ± 0.86 higher than IOPcc (P = 0.005), and IOPNCT was 2.56 ± 0.50 mmHg higher than IOPg (P = 0.001). No difference was found between IOPNCT and bIOP values. At 3 months postoperatively, bIOP was the highest IOP value . IOPg and IOPcc values showed no difference at all postoperative visits.The IOP values at different points of time are shown in Table NCT remained stable from before surgery to 1 month after the surgery , and decreased 3.15 ± 0.48 mmHg at postoperative 3 months compared with the preoperative values .Except bIOP, compared with the preoperative values, the other three measurements were lower postoperatively. IOPP = 0.03), and decreased until 1 month postoperatively . IOPg decreased by 4.30 ± 1.13 mmHg (P = 0.007) at 1 week postoperatively and remained stable thereafter. Among all 4 measurements at postoperative 3 months postoperatively, IOPg showed the greatest difference between pre- and postoperative values  . CH, A1 Time and HC DA showed no significant difference among the follow-up visits.The biomechanical parameters from ORA and Corvis ST are shown in Table 4P <  0.05). IOPg and IOPcc were correlated with CRF  and postoperative CCT . bIOP was independent of preoperative CCT as well as postoperative CCT and correlated with A1 Time  and HC DA  at the last postoperative follow-up visit. IOPNCT at the last postoperative follow-up visit correlated with homologous CRF, A1 Time, and HC DA as well as preoperative IOPNCT   most closely matched the approximate preoperative IOP values, whereas the other three estimated IOP values were lower after hyperopic SMILE. Similar results were observed after myopic LASIK and myopic SMILE , 17. LeeIn this study, IOPg and IOPcc remained constant at all postoperative follow-up visits, and both values correlated with CCT as well as cornea biomechanics. This was consistent with the results of Mollan, , but conNCT was the most commonly used clinical parameter. In this study, IOPNCT decreased by 3.15 ± 0.48 mmHg after hyperopic SMILE, greater than bIOP, but less than IOPg and IOPcc. Although Wolfs et al. reported a positive correlation of IOP with CCT, [NCT values in this study. In addition, IOPNCT at last follow-up visit correlated with preoperative IOPNCT and some corneal biomechanical properties, suggesting that corneal biomechanics but not CCT may be of greater importance when predicting IOPNCT after hyperopic SMILE. Additional studies are warranted to further validate these findings.IOPith CCT,  we foundThis study has several limitations. The study lacks corresponding measurements with Goldmann applanation tonometer, which is considered the gold standard reference for IOP measurements. The enrolled cases had astigmatism, which did not lead to pure hyperopia correction, though cornea thickness and biomechanics are major factors affecting IOP and mixed astigmatism might probably have some effect on the results. In addition, the sample size in the current study is small, and a larger sample size and longer study duration are needed.NCT decreased after hyperopic SMILE, and its value correlates with preoperative IOPNCT as well as corneal biomechanical properties. bIOP seems to be an accurate parameter to assess postoperative IOP.In conclusion, IOP


Sperm characteristics and biochemical components were correlated by Spearman’s rank correlation coefficient in the whole population and in each group. The levels of TESTO and E2 were positively correlated with sperm quality in particular, and E2 was correlated with fertility index expressing the number of sperm free of ultrastructural defects evaluated by TEM. On the contrary, the indices of iron metabolism  were positively associated with low sperm quality and sperm necrosis, particularly in leukocytospermia and varicocele groups, pathologies in which an inflammatory status and oxidative stress condition are present. The study of the seminal plasma composition deserves attention because the levels of the various components seem to be associated with specific reproductive pathologies.The evaluation of the seminal plasma plays a relevant role in the definition of male infertility and in assisted reproduction outcomes; for this reason, it would be recommended to find biochemical markers able to characterize sperm pathology. In this study, 53 infertile patients  and 10 fertile men were selected. Spermiogram was performed by light microscopy, and sperm ultrastructure was evaluated by transmission electron microscopy (TEM) mathematically elaborated. Testosterone (TESTO), estradiol (E2), ferritin (FERR), iron (Fe), transferrin (TRSF), triglycerides (TRG), cholesterol (CHOL), and isoprostanes (F Seminal plasma is the protein-rich fluid part of the semen and it plays an important role in sperm metabolism as well as sperm function. The analyses of seminal enzyme activities and the concentrations of biochemical elements have been performed in men and animals –4 in ordThe evaluation of seminal components may be used to characterized sperm quality and pathological conditions related to male infertility . Many moTransferrin is reported to be an iron-binding protein that regulates free iron levels in biological fluid in the body . In the Ferritin is an intracellular protein that stores iron in the atoxic form, and it is found in the extracellular environment as a result of cellular synthesis and secretion. Seminal ferritin is found in abundance in the seminal plasma, and Sertoli cells produce approximately 70% of its contents .The investigation of other different semen biochemical components may explain their role in seminal pathologies. Recently, Murgia et al.  indicateMany hormones are also found among the constituents of seminal plasma. When compared with blood serum levels, concentrations of testosterone in seminal plasma are lower by almost one order of magnitude, while estradiol is in some instances higher . Other aOur previous paper reported that increased albumin, total proteins, ferritin levels, and γ-glutamyl transferase and creatine kinase activities concomitant with decreased amounts of alkaline phosphatase and folic acid were associated with reduced sperm parameters. These results suggested their possible involvement in male infertility . In huma2-isoprostanes (F2-IsoPs), a specific class of IsoPs, have been indicated as a marker of sperm immaturity in semen of infertile patients with varicocele − precursor ion (m/z 569), was detected and referred to the internal standard PGF2α-d4 (m/z 573 → m/z 303) [F2-isoprostanes (Fity(ies) . Here, tl ethers . For 8-im/z 303) .R) coefficient was calculated to measure the correlation between variables for all enrolled patients and separately for each group. A P value < 0.05 (two-tailed) was considered statistically significant. All analyses were performed by IBM SPSS Statistics Software v.25.Data were reported as medians and interquartile range (IQR 75°–25° centile). The comparisons between groups  were evaluated by Kruskal-Wallis test followed, only for significant cases, by Dunn’s post hoc test for multiple comparisons. Spearman rho (P < .001). Finally, in varicocele patients, immaturity percentage was significantly elevated (P < .001) with respect to that detected in fertile, idiopathic infertility, and leukocytospermia groups . The values of CHOL, E2, TESTO, TRSF, and TRG did not show differences in the considered groups. In leukocytospermia group, the concentration of FERR was significantly increased than that observed in fertile, varicocele, and idiopathic infertility groups (P < .001) and the Fe level was higher than that detected in idiopathic infertility group (P < .05).F2-IsoP levels correlated positively with sperm immaturity . Positive correlations between the TRSF (P < .01) and TRG (P < .05) levels and sperm concentration were observed. FERR showed negative correlations with sperm morphology, vitality, and fertility index (P < .05) and positive correlations with sperm necrosis (P < .01) and Fe (P < .05); Fe compound also positively correlated with sperm necrosis (P < .05). Interestingly, TESTO displayed positive correlations with the sperm vitality (P < .05) and E2 level (P < .01) and negative correlations with TRG (P < .01) and sperm apoptosis .In order to understand the possible correlations among the studied variables, we used the Spearman rank correlation coefficient considering the whole population of interest Table . Regardiity Fig. ; P < .01sis Fig. ; P < .05Different correlations between biochemical indices and sperm parameters were analyzed in each studied group (Table P < .05) and TESTO (P < .01); CHOL positively correlated with TRG (P < .001).In fertile men, E2 positively correlated with fertility index  and TESTO negatively correlated with sperm necrosis (P < .05); both TESTO and E2 negatively correlated with FERR (P < .05). TRSF had positive correlations with CHOL and TRG (both P < .01). Sperm immaturity positively correlated with F2-IsoPs (P < .05). Sperm necrosis is positively correlated with CHOL (P < .05).In leukocytospermia group, E2 positively correlated with TESTO (P < .01), and both E2 and TESTO negatively correlated with TRSF . FERR displayed a positive correlation with sperm necrosis (P < .05) and negative correlation with sperm motility (P < .05). Finally, F2-IsoPs had a positive relationship with sperm immaturity (P < .01).In infertile patients affected by varicocele, E2 showed positive correlation with TESTO  were positively associated with low sperm quality and negatively with TESTO and E2 levels, particularly in leukocytospermia and varicocele groups, pathologies in which an inflammatory status and oxidative stress condition are generally present .The relationship among TESTO, FERR, and inflammation is well known in the literature –46 even FERR has been localized in human Sertoli and Leydig cells , 52 and 2-IsoP varicocele and sperm immaturity observed also in previous studies [2-IsoPs can be considered a semen biochemical marker of sperm immaturity and varicocele.This study confirmed the close relationship between increased levels of F studies , 54; sinNoteworthy is the behavior of CHOL and TRG that in the first analysis seems to be irrelevant, either as marker of pathologies either in correlations performed in the whole studied population. When the correlations between variables were carried out in each group separately, we observed that in the case of leukocytospermia, CHOL and TRG positively correlated with sperm necrosis. Previous studies suggested that elevated lipid levels in seminal plasma might have adverse effect on sperm quality , and semPatients with idiopathic infertility showed an altered sperm quality as the other infertile groups; however, they did not show correlations between semen parameters and seminal plasma markers highlighted for leukocytospermia and varicocele groups. This observation could be explained by the intrinsic meaning of idiopathic infertility since it is difficult to group patients with homogeneous characteristics. Indeed, in semen of many men who are currently diagnosed as having unexplained male infertility, oxidative stress was detected, but other genetic, epigenetic, or environmental unknown causes could be supposed , 59.2-IsoPs to sperm immaturity. We are aware that the number of cases is low and should be extended in a future study; however, these preliminary data indicated that the seminal plasma composition deserves attention because the levels of the various components seem to be associated with specific reproductive pathologies.In conclusion, infertile men with varicocele, leukocytospermia, and idiopathic infertility differed in seminal fluid composition. TESTO and E2 levels appeared to be necessary for a regular spermatogenesis and play a role in sperm quality; FERR, Fe, and TRSF appeared to be linked to inflammatory conditions and sperm necrosis as well as FThe knowledge of the role of seminal molecules in male infertility will help in the development of targeted treatments.


To compare effects of physical exercise and dietary therapy (PED‐t) to cognitive behavioral therapy (CBT) in treatment of bulimia nervosa (BN) and binge‐eating disorder (BED).n = 23) were put on a waiting list. Outcome measures were the eating disorder examination questionnaire (EDE‐Q), Clinical Impairment Assessment (CIA), Satisfaction with Life Scale (SWLS), Beck Depression Inventory (BDI), and numbers in remission at posttreatment, and at 6‐, 12‐, and 24‐months follow‐up.The active sample (18–40 years of age) consisted of 76 women in the PED‐t condition and 73 in the CBT condition. Participants who chose not to initiate treatment immediately (Both treatment conditions produced medium to strong significant improvements on all outcomes with long‐term effect. The PED‐t produced a faster improvement in EDE‐Q and CIA, but these differences vanished at follow‐ups. Only PED‐t provided improvements in BDI, still with no between‐group difference. Totally, 30–50% of participants responded favorable to treatments, with no statistical between‐group difference.Both treatments shared a focus on normalizing eating patterns, correcting basic self‐regulatory processes and reducing idealized aesthetic evaluations of self‐worth. The results point to the PED‐t as an alternative to CBT for BN and BED, although results are limited due to compliance and dropout rates. Replications are needed by independent research groups as well as in more clinical settings. All outcomes were measured by self‐reports at baseline (T1), after treatment (T2), and at 6‐ (T3), 12‐ (T4) and 24‐months (T5) posttreatment. Additionally, an experienced clinician assessed by interview the diagnostic criteria for BN and BED according to the definition by DSM‐5 , being the manual, which PED‐t relies on, we did not find it safe to include persons with a BMI above 35. A comorbid axis I psychiatric disorder that precluded the eating disorder focused treatment and needed more extensive care was excluded. More thorough description of inclusion and exclusion criteria is provided in the open‐access published protocol , and a BMI between 17.5 and 35 kg mParticipants were recruited through general practitioners (GP's), magazines, newspapers, national TV, social media, posters, handouts, and through webpages of the national patient organizations for EDs. We interviewed participants by phone 2–6 weeks prior to the baseline measures and randomization, and all participants and their GP's had to sign an informed consent before final inclusion.2.2http://www.randomizer.org) with block size of eight, and concealed allocation to either group CBT or group PED‐t, and they performed allocation by listing the randomization results by id‐number. The project leader conveyed the allocation to the participants after they had completed the baseline measures. Participants recruited during ongoing treatment or who for some reason were unable to enter therapy immediately were put on a waitlist for 16 weeks and thereafter randomized to one of the two groups. This group served as a reference group for the two therapies at pre‐ and post‐treatment. All outcomes, anonymously coded by ID‐number, were transferred to SPSS files by blinded statistical staff before the final statistical analyses.Two researchers not affiliated with the study produced the randomization list  validly assesses the frequency and severity of ED features to produce ED diagnoses according to the DSM‐5  global score for a Norwegian cohort of healthy female controls is 1.25 (1.10), while corresponding national clinical cutoff for probable BN and BED are 2.62, and 2.63, respectively . Reported purging episodes are based on scores from EDE‐Q item 16–18 .The EDE‐Q 6.0   measures ED‐elicited personal, social, and cognitive impairment  measures current (past 2 weeks) self‐reported symptoms of depression  is a short 5‐item scale measuring overall contentment with life compared to personal standards and expectations, using a 5‐point Likert scale ranging from 1 (never true) to 7  , typically for reasons like work/studies, moving too far away, injuries or illness, pregnancy, not willing to attend physically to the testing facilities, and not responding to email, mail or electronic questionnaires.2.6t‐tests or Mann–Whitney test. Attrition rates were analyzed separately for each time  with independent t‐test or chi‐square analyzes as appropriate. A significance level of p < .05 was used for all bivariate comparisons.The power calculation for this trial is provided elsewhere  number of waitlist participants was excluded from group comparisons due to low statistical power, but are presented as a reference group in the figure and tables. Linear mixed regression models were built to estimate the coefficients for the between (PED‐t vs. CBT) and the within factors (baseline vs. any of the three posttest measures). This analysis yields relatively unbiased estimates despite drop out given that data are missing completely at random or missing at random. Moreover, it can be safely used without conducting beforehand multiple imputations  representing the overall therapy difference, Time (repeated measures) representing change across measurements, and the Group × Time interaction in order to detect treatment differences at certain time points only. The between‐group analyses used the baseline values as a covariate to increase the statistical power , and the Satterthwaite approximation to settle a more appropriate degrees of freedom. For that reason, we provide 99% confidence bands for the estimated means.g effect‐sizes for continuous data were calculated as a ratio of the estimated mean difference (based on the mixed model) to the observed pooled SDs. Values around .2, .5 and .8 were interpreted as weak, medium and strong, respectively , diagnostic reasons , having severe comorbidity , other self‐chosen reasons , or other competing treatment .Of the 418 women who responded during the recruitment period (March 2014–August 2016), 229 did not fulfill the inclusion criteria, 164 were randomized to therapy groups, and finally 76 and 73 initiated therapy in PED‐t and CBT, respectively  attendance rate to therapy was 80.6% (11.4) in PED‐t, and 82.1% (45.7) in CBT. In PED‐t the compliance rate to exercise sessions, as supervised in therapy + unsupervised at home  was 69.8% and 56.7% for resistance‐ and interval exercises, respectively. There were significant differences between diagnoses at baseline, with higher body weight and BMI among those with BED, and higher EDE‐Q global score and number of purging episodes among those with BN (Table The mean percentage (as 80.6% 1.4 in PEn = 37) a higher mean (CI95) baseline score for depression was found compared to completers (n = 112), . Significantly more CBT‐participants were lost to follow‐up (LFU) at 6‐months follow‐up (PED‐t = 19 (24.4%), CBT = 37 (47.4%), p = .005), 12‐months follow‐up (PED‐t = 20 (25.6%), CBT = 39 (50.0%), p = .003), and 24‐months follow‐up (PED‐t = 20 (25.6%), CBT = 25 (38.5%), p = .03). Overall, there was no difference in LFU across the BN and BED diagnoses (p > .02); however, more with BN in CBT were LFU at all follow‐ups compared to BN in PED‐t (p < .03). There were no differences (p > .06) between attending participants at 6‐months follow‐up  and LFUs  with respect to the scoring at posttest, as was the case at 12‐months follow‐up with respect to 6‐months follow‐up scores  (p > .05), and at 24‐months follow‐up with respect to 12‐months follow‐up scores  (p > .05).The numbers of dropout from treatment were not significantly different across groups or ED diagnoses. Among those who dropped out at posttest . The same relate to EDE‐Q subscales weight concern , shape concern , eating restraint ., and eating concern . Additionally, both therapy groups reported a significant alleviation of binge eating from baseline to posttreatment (p < .001 in both groups), and from baseline to each follow‐up . Both therapy groups also reported reduction in episodes of self‐induced vomiting from baseline to posttreatment (p = .003 in PED‐t and p = .002 in CBT) and from baseline to most follow‐ups' , except at T4 in PED‐t (p = .02) and at T3 in CBT (p = .12). There were no significant changes in the number of episodes with laxative use (p > .02), but both groups reduced the number of episodes with driven exercise from baseline to posttreatment (p < .001 in both groups), and from baseline to all follow‐ups , except at T3 in CBT (p = .09).Compared to baseline, both therapy groups improved in EDE‐Q global score at posttreatment and at all follow‐ups  indicating a general improvement across the follow‐ups. However, the more important Group and Group * Time effects were not significant . Planned comparison tests nevertheless indicated an advantage of PED‐t above CBT at the first posttest (T2) exclusively in EDE‐Q global score  and the EDE‐Q subscale “body weight concern” . Analyzing Time as a linear trend indicated a general decline across the follow‐ups , but it was not significantly different by Group .The fixed effect of Time, Group, nor Group * Time effects were significant for binge eating and purging episodes, and no planned between‐group comparison test were significant either.Neither the fixed effect of 3.23.2.1p < .001; CBT p = .001) and at follow‐ups  compared to baseline, with no differences between the groups   and all follow‐ups , compared to baseline. The changes in the CBT group approached, but were not significantly different posttreatment (p = .02), with no differences to baseline at follow‐ups . There was no difference in numbers in partial remission between the two groups at any time   and at follow‐up tests  was significant. The Time  and Group * Time  effects were not significant. The planned comparison tests  , and neither did it significantly co‐vary by group .The fixed effect 3.43.4.1p < .003 and T5 p < .001, Table Ci 99%) with BDI score above clinical cutoff was reduced in PED‐t at T2 with a marginal statistical significance , but with no further differences to baseline at follow‐ups. There were no significant changes in number of participants with scores above cutoff in CBT .Compared to baseline, participants in PED‐t displayed reduced BDI scores posttreatment (p < .001) and at follow‐ups , whereas Time  and Group * Time  was not. The planned comparison tests showed a tentative advantage from PED‐t above CBT posttreatment (p = .016), but not in the remaining posttests. The linear trend analysis of Time was not significant , and did not significantly co‐vary with group . We found no differences between groups by time in percentage of participants scoring above BDI clinical cutoff (p > .11).The fixed effect 3.53.5.1p < .003) and at follow‐ups  and of Group * Time  was not significant, whereas the effect of Time approached significance . The trend analysis of Time was not significant .The fixed effect of 4Overall, we found support to our hypothesis, as the PED‐t performed equal to CBT in alleviating symptoms of BN, BED, as well as in improving subjective well‐being and psychosocial impairment. In addition, PED‐t reduced symptoms of depression. We also found support for the hypothesis that the PED‐t produced a more rapid treatment response , however, contradicting our hypothesis, was the lack of difference between groups in drop out from treatment.The effect size of changes within groups was with medium to strong effects, hence, implying changes with important clinical impact. This also relates to the acute differences identified between groups posttreatment in EDE‐Q global score, body weight concern and clinical impairment. Nevertheless, both therapies performed well in an outpatient group setting, with a summarized response rate  to therapy of ~50% after PED‐t and ~30% after CBT. This corresponds well with previous findings on treatment outcome from group‐CBT, that is, ~0–52% remission after group‐CBT  and practical experiences of body functionality (PED‐t). The dissimilarity makes future search for possible common mechanisms of change highly interesting. Neurobiological and cognitive factors may be considered, given that these factors have been associated with effects of physical activity (Lubans et al., Strengths to this study are the RCT‐design with 24 months follow‐up, the equal length and duration of the treatments, and the unlikely diffusion of effects from previous or concurrent treatments. Limitations in need to be mentioned are the low compliance rate to PED‐t exercise module, the large number of CBT‐participants lost to follow‐ups as well as the failure to control for therapist factors, and for other kind of treatments that may have been sought in the follow‐up period. Concerning the loss of CBT‐participants, we argue the statistical model handles this most favorable, with full information maximum likelihood methods reducing any risk of biased data (Witkiewitz et al., 4.1The effects of PED‐t are overall on par with the well‐established CBT for BN and BED. Its focus on physical exercise and diet may appeal to many sufferers. In addition, PED‐t may represent an alternative for those who do not want CBT, or in cases where CBT is unavailable. This can be accommodated given the high availability of professionals with expertise in exercise medicine and nutrition compared to CBT‐certified therapists. Following the logic behind the construction of the PED‐t it may be worthwhile to test the incremental effects of combining PED‐t and CBT. Replications of the present findings are needed both by independent research groups and within new and preferably more clinical contexts. Equally needed is the exploring of mechanisms of change.


Dear Editor,MYC gene rearrangements or MYC (MYC proto‐oncogene) and BCL2 (B‐cell lymphoma 2) dual translocations.Central nervous system (CNS) involvement in diffuse large B‐cell lymphoma (DLBCL) patients correlates with dismal outcomes, and the detection sensitivity of conventional diagnosis of lymphoma is restricted.Pim‐1 proto‐oncogene , lysine methyltransferase 2D , BCL2 (27.5%), myeloid differentiation primary response 88 , B‐cell translocation gene 2 , and tumor protein p53 . Majority of altered genes were involved in four important pathways including epigenetic regulation , BCR (breakpoint cluster region gene) and NF‐kB (Nuclear factor‐kappa B) signaling pathway , apoptosis/cell cycle , and immunity . SNV was the most frequently observed type of alteration, whereas CNV had the tendency to be enriched in patients with lower CNS‐IPI including BCL2 and MALT1 (mucosa‐associated lymphoid tissue lymphoma translocation protein 1) amplification. Interestingly, cell‐free DNA (cfDNA) concentration in CSF‐in DLBCL patients showed a trend of increase from low to high level CNS‐IPI. By dividing patients into 0‐3 and 4‐6 CNS‐IPI, we observed a significant elevation in CSF‐cfDNA concentration, suggesting CSF‐cfDNA might be an indication of CNS involvement . Genomic landscape of this DLBCL cohort in systemic tumor tissue is shown in Figure t Figure . Plasma‐t Figure .In the 20 CSF‐cfDNA‐positive patients, a comparison of gene alterations (GAs) in matched systemic tumor tissue, CSF, and plasma was performed Figure . DespiteBTG2 S31N, was found in the PCNSL cohort.Compared to GAs only found in tumor tissue  were identified in more than 20% of total PCNSL cases. Meanwhile, 31 DLBCL patients with high risk for CNS had at least one alteration in the BTG2, PIM1, DUSP2, ETV6, and CXCR4 genes from CSF and/or plasma, including 14 patients with level4‐6 CNS‐IPI. Ten high‐risk DLBCL patients (nine were CSF‐cfDNA‐positive) were later confirmed CNS involvement by magnetic resonance imaging (MRI) or flow cytometry (FCM). Among them, seven patients were identified with multiple alterations of the five CSF‐CNS genes in CSF (Figure Then we look into the CSF signature of high‐risk DLBCL in the gene level rather than mutation level. Here, we referred the genes with alterations found exclusively in CSF of high‐risk DLBCL as CSF‐CNS genes. As shown in Figure F Figure . MoreoveLimitations of this study included the lack of CSF and plasma samples from low‐risk group to assess the status of CSF‐CNS genes. Close monitoring of CNS relapse in the high‐risk cohort will be also of great help to further validate our observation. Potential blood contamination or normal cfDNA in the CSF sample might also have some impact on the result interpretation.In summary, our study provided evidence for the association between CSF‐cfDNA concentration and CNS‐IPI score, highlighting the importance of CSF‐cfDNA in the detection of CNS tumors in DLBCL. Five CSF‐CNS genes in CSF was found to be associated with CNS risk in DLBCL patients, which deserve further investigation to determine their relevance among the diagnosis, treatment, and outcome.The National Science Foundation of China; Grant Number: 81970176; Natural Science Foundation of Guangdong Province; Grant Number: 2019A1515011943.Study design: WXX, GY, and HHQ. Data acquisition: WXX, SCG, LMY, ARY, WXX, and CLB. Data analysis: WXX, HHX, GY, RY, and QO. Manuscript writing: WXX, HHQ, RY, XW, and YWS. Study supervision: HHQ.Ruoying Yu, Qiuxiang Ou, and Xue Wu are the shareholders or employees of Geneseeq Technology Inc. Canada. Xiaoxia Wang and Yang W. Shao are the shareholders or employees of Nanjing Geneseeq Technology Inc. The remaining authors have no conflict of interest to declare.SUPPORTING INFORMATIONClick here for additional data file.SUPPORTING INFORMATIONClick here for additional data file.


Human papillomavirus (HPV)‐positive oropharyngeal squamous cell carcinoma (OPSCC) have better prognosis and treatment response compared to HPV‐negative OPSCC. This study aims to noninvasively predict HPV status of OPSCC using clinical and/or radiological variables.Seventy‐seven magnetic resonance radiomic features were extracted from T1‐weighted postcontrast images of the primary tumor of 153 patients. Logistic regression models were created to predict HPV status, determined with immunohistochemistry, based on clinical variables, radiomic features, and its combination. Model performance was evaluated using area under the curve (AUC).Model performance showed AUCs of 0.794, 0.764, and 0.871 for the clinical, radiomic, and combined models, respectively. Smoking, higher T‐classification (T3 and T4), larger, less round, and heterogeneous tumors were associated with HPV‐negative tumors.Models based on clinical variables and/or radiomic tumor features can predict HPV status in OPSCC patients with good performance and can be considered when HPV testing is not available. Due to the retrospective design, informed consent was waived.2.12]) at our Institute between January 2010 and December 2015, were considered for this study. Patients were excluded when pretreatment MRI of the primary tumor was not available (n = 38), image quality was poor (n = 7), tumors were undetectable on MRI (n = 17), a second head and neck tumor was present (n = 1), or when HPV status of the tumor was missing (n = 24). This resulted in a total of 153 patients eligible for this study.A total of 240 consecutive patients with histologically proven primary OPSCC, treated with CRT (70 Gy radiation with three planned cycles cisplatin‐based chemotherapy [100 g/mN > 0) or node‐negative disease (N = 0). Differences in clinical variables between HPV‐positive and HPV‐negative tumors were assessed by applying the Fisher exact test and independent t‐test for age. P values of <.05 were considered statistically significant.Age, gender, smoking status, tumor subsite, and TNM classification (TNM seventh edition), were collected for each patient. T‐classification and N‐classification was determined in multidisciplinary consensus based on clinical and radiological information, including MRI, ultrasound staging with fine needle aspiration cytology, and, when available, PET images. Smoking status was classified into the categories nonsmoker, current smoker, and former smoker (quit more than 2 years prior to diagnosis) at the initial visit to the outpatient clinic. T‐classification was dichotomized in low (T1 + T2) or high T‐classification (T3 + T4). N‐classification was dichotomized in node‐positive  using a standard head and neck coil (SENSE‐NV‐16). The imaging protocol included T1‐weighted (T1W), T2‐weighted (T2W), postcontrast 3D T1W, perfusion, and diffusion‐weighted sequences. Imaging details are summarized in Table 2.4Signal intensities for each individual MRI scan were normalized (with zero mean and unit SD) prior to further analysis to reduce intensity variations between MRI scans obtained from different patients. Image resampling to isotropic voxels of 1.0 mm was performed using B‐spline interpolation. Image discretization was applied to allow quantification of texture images in fixed bin width of five. In total, 1184 radiomic features per patient were calculated from the postcontrast 3DT1W MRI within the primary tumor volumes using the open‐source package PyRadiomics 2.2.0,U test in features with regard to the different MRI field strengths. Features with an interclass correlation coefficient greater than 0.75 and a significance level equal to or above .05 in the Mann‐Whitney U test were considered stable. From the selected stable features, collinear features (Pearson correlation coefficient > 0.9) were removed, where for each pair the feature that has the largest mean absolute correlation is deleted. The remaining 77 features  were randomly allocated to a training/validation subset and 40% (n = 62) to a test subset, stratifying for HPV status and MRI magnet strength (1.5 or 3.0 T).Then, separate logistic regression modelsFeature dimensionality is reduced by applying a sequential backward wrapper feature selection approach (recursive feature elimination). This method obtains the optimal feature set for the given classifier (in this case logistic regression) by iteratively removing the weakest feature assessed by its feature importance score. The optimal set of features is used to train the model.λ, 0.005‐200), a parameter for the complexity of the model, and the number of features  were tuned based on the four training performances obtained during cross‐validation. Area under the curve (AUC) was calculated as measure of model performance, where the loss function is minimized. The loss function was defined as In the training phase, Bayesian optimization was used to obtain optimal hyperparameters employing 1000 iterations of 4‐fold cross‐validation on a 75% (n = 68) training and 25% (n = 23) validation set. During this process, the regularization parameter  of 500 randomly selected samples (with replacement) of the test subset. Median AUC and the 95% confidence interval (95% CI) of these 500 iterations were then calculated to reflect the model performance that can be attained of HPV prediction. All analyses were implemented in python 3.5 and SPSS version 25.0 (SPSS Inc. Chicago). The complete machine learning pipeline is shown in Figure A clinically applicable nomogram was constructed from the clinical logistic regression model using R software package RMS (version 3.6.3).3Table P = .007), less likely to smoke (P < .001), and had a lower T‐classification  compared to patients with HPV‐negative tumors. For node‐positive disease (P = .051) and male gender (P = .067), these differences were borderline significant at the 5% level. Tumors of the soft palate (P = .017) were significantly more frequent in HPV‐negative tumors.OPSCC patients with HPV‐positive tumors were younger . This underscores that the clinical model, besides the good overall performance, is biologically plausible.The discriminatory MRI features in the radiomic‐based models probably reflect differences in tumor biology between HPV‐positive and HPV‐negative tumors. HPV‐positive tumors are characterized by less‐invasive exophytic growth, nonkeratinizing histopathology, genetic stability, and well‐defined surroundings.Although no direct comparison was made, our MR‐based predictive radiomic model suggests similar performance (AUC = 0.76) compared to CT.The models in this article are less sensitive (88%) and specific (71%) compared to pathological methods , where former smokers stopped for at least 2 years prior to diagnosis. Only postcontrast 3DT1W MRIs were used in this study to limit the number of features with our available cases. Other MR sequences might give additional radiomic features for prediction of HPV status and is a topic for further study. In a preliminary study, we included all available MRI sequences, revealing mainly radiomic features from the postcontrast 3DT1W sequence, suggesting that other sequences would not contribute to the eventual predictive models. Finally, time‐consuming manual tumor delineations were used for feature extraction, which introduces interobserver variability. Stable features with regard to delineations were selected to minimize the effect of interobserver variability in the eventual models. Ideally, this interobserver variability should be eliminated. Automated tumor delineation algorithms by, for instance, convolutional neural networks may overcome interobserver delineation variability.5This study shows that logistic regression models based on clinical variables, MR‐based radiomic features, or a combination of clinical and radiomic features can accurately predict HPV status in OPSCC patients. Although a model based on clinical and radiomic features performs best, the clinical model would be the method of choice due to its ease of implementation. These models have a place in determination of HPV tumor status in settings where tumor biopsy material, tumor samples, immunohistochemistry, and/or DNA polymerase chain reaction techniques are not available. HPV testing is becoming more a routine in hospitals, but not everywhere, especially not in the past when the importance of HPV status of the tumor was not known. Medical images, on the other hand, are widely available due to the advantage of storage capability of medical images for a long time, making it a good alternative to assess HPV tumor status.H.J.W.L.A. is a stockholder of Sphera and Genospace, outside submitted work. All other authors declare no conflict of interest.AppendixS1: Supporting informationClick here for additional data file.


Zanthoxylum armatum collected from wild and cultivated populations in Nepal. TPC was determined by Folin–Ciocalteu colorimetric method using gallic acid as standard, and various concentrations of the extract solutions were measured at 760 nm. TFC was calculated by aluminum chloride colorimetric assay. Quercetin was used as standard, and the absorbance was measured at 510 nm. The antioxidant potential of the different extracts was estimated by DPPH free radical scavenging assay, and the absorbance was measured at 517 nm. The highest TPC value was 226.3 ± 1.14 mg GAE/g in wild fruits, and the lowest was 137.72 ± 4.21 mg GAE/g in cultivated seeds. Similarly, the highest TFC value was 135.17 ± 2.02 mg QE/g in cultivated fruits, and the lowest was 76.58 ± 4.18 mg QE/g in cultivated seeds. The extracts showed variable antioxidant properties. The fruits exhibited excellent antioxidant properties with IC50 values of 40.62 μg/mL and 45.62 μg/mL for cultivated and wild fruits, respectively. Similarly, the IC50 values of the bark were 63.39 μg/mL and 67.82 μg/mL, respectively, for cultivated and wild samples. And the least antioxidant capacity was shown by the seeds extract with IC50 values of 86.75 μg/mL and 94.49 μg/mL for wild and cultivated seeds, respectively. The IC50 value of the standard ascorbic acid was 36.22 μg/mL. Different extracts of Z. armatum contain considerable amount of phenols and flavonoids, including antioxidant properties, suggesting the potential use of this species in pharmacy and phytotherapy as a source of natural antioxidants.Natural antioxidants present in several medicinal plants are responsible for inhibiting the harmful effects of oxidative stress. These plants contain polyphenols and flavonoids that act as free radical scavengers and reduce oxidative stress and may be an alternative remedy to cure various harmful human diseases. This study aims to quantify the total phenolic and flavonoid contents (TPC and TFC) and antioxidant properties of methanol extracts of fruits, seeds, and bark of an important medicinal and aromatic plant,  Medicinal plants have been used in several indigenous herbal practices since very old times to cure several diseases. Herbal medication still continues to serve as an important health care system even today despite the greater advancements in modern medication systems in the recent years. Their long uses in the folk medicine and their safer implications in human health have generated much interest in them, especially in developing countries. It has now been established that medicines derived from plant products are safer than their synthetic counterparts .Plant and plant-based products are the natural sources of different phytochemicals such as phenols, flavonoids, alkaloids, glycosides, lignins, and tannins. Phenols and flavonoids are the most common phytoconstituents of different fruits, vegetables, and medicinal and aromatic plants, which are responsible for antioxidant activities . Due to Zanthoxylum armatum DC (family: Rutaceae), commonly called as Timur in Nepal, is an aromatic perennial shrub or a small tree up to 6 m height with dense glabrous foliage and straight prickles. It is distributed from Kashmir to Bhutan, north-east India and Pakistan, Laos, Myanmar, Thailand, China, Japan, North and South Korea, North Vietnam, and Taiwan [d Taiwan . In Nepad Taiwan . It is ud Taiwan –14. The d Taiwan , 15, 16.d Taiwan –21.Z. armatum regarding their antioxidant properties [Z. armatum collected from wild and cultivated populations. The correlation between total phenolic and flavonoid content and antioxidant activity with the habitat conditions could help to establish foundation for further studies focusing on the development of safer and inexpensive natural antioxidants for their use in therapeutic and pharmaceutical preparations.A lot of experiments have been carried out in operties . But theZ. armatum were collected from wild and cultivated populations from Salyan district of west Nepal during May 2018. The plants were collected with the permission of Department of Plant Resources, Ministry of Forests and Environment, Government of Nepal, in accordance with article no. 10(B) of Plant Resource Research Procedure 2013 and revised 2016. This plant is not mentioned in CITES and protected plant list of Nepal. The plant was identified by Nirmala Phuyal. Herbarium of voucher specimens was prepared and deposited at National Herbarium and Plant Laboratories (KATH); NPZA 20-NPZA 50. The samples were cleaned and shade dried for a week before the extraction procedure.The fresh fruits, seeds, and bark of The dried samples were then powdered separately in a grinder. Known weight of the powdered samples was loaded in thimble and put inside the Soxhlet apparatus. They were then successively extracted with methanol by the hot Soxhlet extraction method. The apparatus was run for 72 hours till the colored solvent appeared in the siphon for obtaining the crude extracts of the samples. After complete extraction, the solvent was evaporated in a rotary vacuum evaporator at 65°C under reduced pressure. The obtained extracts were then dried in a water bath. The dried extracts were sealed inside 20 mL sterilized culture tubes and stored in refrigerator at 2–8°C for further analysis .μg/mL) were prepared from the standard solution. To each concentration, 5 mL of 10% Folin–Ciocalteu reagent (FCR) and 4 mL of 7% Na2CO3 were added making a final volume of 10 mL. Thus, the obtained blue colored mixture was shaken well and incubated for 30 min at 40°C in a water bath. Then, the absorbance was measured at 760 nm against blank. The FCR reagent oxidizes phenols in plant extracts and changes into the dark blue color, which is then measured by UV-visible spectrophotometer. All the experiments were carried out in triplicates, and the average absorbance values obtained at different concentrations of gallic acid were used to plot the calibration curve.Total phenolic contents (TPC) in the fruits, seeds, and bark extracts were determined by Folin–Ciocalteu colorimetric method as described by Singleton et al.  with somμg/mL) were prepared. The procedure as described for standard gallic acid was followed, and absorbance for each concentration of the extracts was recorded. The samples were prepared in triplicate for each analysis, and the average value of absorbance was used to plot the calibration curve to determine the level of phenolics in the extracts. Total phenolic content of the extracts was expressed as mg gallic acid equivalents (GAE) per gram of sample in dry weight (mg/g). The total phenolic contents in all the samples were calculated by the using the formula:C = total phenolic content mg GAE/g dry extract, c = concentration of gallic acid obtained from calibration curve in mg/mL, V = volume of extract in mL, and m = mass of extract in gram.Various concentrations of the extracts  of quercetin was prepared by dissolving 4 mg of quercetin in 1 mL of methanol. This standard solution was diluted serially to make various concentrations of 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, and 1 mg/mL solutions. 1 mL quercetin of each concentration was added to the test tube containing 4 mL of distilled water. At the same time, 0.3 mL of 5% NaNOStock solutions of 4 mg/mL concentration in methanol of the extracts were prepared, and they were diluted serially to make different concentrations  solutions. Similar procedure as described for quercetin was followed for the extracts also, and the absorbance was measured by spectrophotometer at 510 nm. Readings were taken in triplicate, and the average value of absorbance was used to calculate the total flavonoid content. The flavonoid content was expressed as quercetin equivalent (mg QE/g) using the linear equation based on the standard calibration curve.In vitro antioxidant activities of the extracts were determined using the DPPH free radical scavenging assay described by Nithianantham et al.  with somDPPH solution (0.1 M) was prepared by dissolving 0.39 mg of DPPH in a volumetric flask, dissolved in methanol, and the final volume was made 100 mL. Thus, prepared purple-colored DPPH free radical solution was stored at −20°C for further use.μg/mL solutions of each extract were prepared.Stock solution of different extracts of 1 mg/mL was prepared by dissolving required quantity of each extract in required volume of methanol. From the sample stock solution, 25, 50, 75, and 100 50 values) of the extracts were calculated from graph as concentration versus percentage inhibition. Radical scavenging activity was expressed as percentage of inhibition. IC50 value is the concentration of sample required to scavenge 50% of DPPH free radical. Measurements were taken in triplicate. IC50 of the extracts indicates the corresponding concentration in which the radical scavenging potential is 50%. The IC50 of the extract and standards were determined graphically.To the sample solutions of different concentration, 1 mL DPPH solution was added and incubated at room temperature for 30 min in dark. A control was prepared by mixing 1 mL methanol and 1 mL DPPH solution. Finally, the absorbance of the solutions was measured by using a spectrophotometer at 517 nm. Ascorbic acid was used as the standard. 50% inhibitory concentrations .The radical scavenging activities of the extracts are expressed in terms of their ICZ. armatum were determined by Folin–Ciocalteu (F–C) method using gallic acid as the standard. The absorbance values obtained at different concentrations of gallic acid were used for the construction of calibration curve. Total phenolic content of the extracts was calculated from the regression equation of calibration curve  and expressed as mg gallic acid equivalents (GAE) per gram of sample in dry weight (mg/g).Total phenolic contents in different extracts of fruits, seeds, and bark of TPC values were higher in the fruit and bark extracts than the seed extracts. The highest TPC value was observed for the fruits followed by the bark, and the lowest was for the seeds extracts. TPC value of the cultivated fruit extract was 226.3 ± 1.14 mg GAE/g and that for wild fruit was 185.02 ± 2.15 mg GAE/g. Similarly, the value was 185.15 ± 1.22 mg GAE/g for wild bark and 171.13 mg GAE/g for cultivated bark. And for the wild seeds, the TPC value was 167.74 ± 2.63 mg GAE/g and that for cultivated was 137.72 ± 4.21 mg GAE/g .Z. armatum fruits was found to be 21.68 ± 0.44 mg/g and that of the bark was 16.48 ± 1.33 mg/g. Similarly, in another study, the TPC value of methanol extract of fruits was 366.3 mg of GAE/g [Z. armatum may contribute significantly to the antioxidant properties. Because of these properties, this plant might have been used in several traditional herbal medications.This is in accordance with the study of Barkatullah et al. , where tof GAE/g . The pheof GAE/g . PresencThe antioxidant response of phenolic compounds varies remarkably, depending on their chemical structure . In addiY = 0.0011x; R2 = 0.992) and expressed as mg quercetin equivalents (QE) per gram of sample in dry weight (mg/g). The TFC values also showed similar trends with that of TPC values. The highest TFC value was obtained for the fruits followed by the bark, and the lowest was for the seeds extracts. The highest TFC value was 135.17 ± 2.02 mg QE/g for cultivated fruit extract, and for wild fruit, it was 103.7 ± 1.39 mg QE/g. The TFC values were 111.2 ± 3.67 mg QE/g and 91.27 ± 3.13 mg QE/g for cultivated and wild barks, respectively. Similarly, the lowest TFC value was 76.58 ± 4.18 mg QE/g for cultivated seeds, and for wild seeds, it was 92.71 ± 3.14 mg QE/g  values or in other words, the amount required to scavenge 50% DPPH free radicals. The mean percentage of DPPH free-radical scavenging activity at different concentrations of extracts is shown in Antioxidant activity of the fruits, seeds, and bark of 50 value of ascorbic acid was 36.22 μg/mL. The higher IC50 value indicates lower radical scavenging activity or lower antioxidant potential. The fruits extracts had the highest antioxidant capacity compared to the seeds and bark extracts. The IC50 value of the fruits extracts was close to that of the standard, i.e., 40.62 μg/mL and 45.62 μg/mL, respectively, for the cultivated and wild fruits. Similarly, the IC50 value was 86.75 μg/mL and 94.49 μg/mL, respectively, for wild and cultivated seeds, showing least antioxidant properties. And the bark showed moderate antioxidant capacity with the IC50 value of 63.39 μg/mL and 67.82 μg/mL for cultivated and wild extracts, respectively. The antioxidant potential of fruits and bark extracts were higher in cultivated than in wild samples, whereas in seeds extracts, it was higher in wild than cultivated samples.Ascorbic acid used as the standard and the different extracts showed variable antioxidant properties. The ICZanthoxylum armatum has been evaluated by various previous studies [50 value of the methanolic bark extract of Z. armatum was 149.26 μg/mL [μg/mL in the present study.The antioxidant potential of different parts of  studies , 31–35.  studies , while i26 μg/mL , but it Z. armatum may be due to the presence of polyphenols, flavonoids, and phenolic compounds, and most of the antioxidant activity of plants is because of the phenols [The radical scavenging activity of different extracts of  phenols . Natural phenols . Apart f phenols . These b phenols .Antioxidants are tremendously important substances, which possess the ability to protect the body from damage caused by free radical-induced oxidative stress. Plant polyphenols act as reducing agents and antioxidants by the hydrogen-donating property of their hydroxyl groups .Z. armatum were considerably good. However, these parameters were remarkably better in fruit and bark extracts as compared to the seed extracts. Some of the wild samples showed excellent results, and some of the cultivated samples showed better results. The differential TPC, TFC contents, and antioxidant properties from different habitat may plausibly be due to geographical variations in chemical constituents. The results of the present study suggested that the fruits, seeds, and bark of Z. armatum could be the potent source of natural antioxidants because of their phenolic and flavonoid contents and their remarkable scavenging effects on DPPH. So, this plant could be of greater significance in preventing several harmful human diseases. Further studies should be directed towards the extensive in vivo antioxidant activities of the plant and the relationship of individual phenolic compounds to antioxidant with different mechanisms and isolation, screening, and characterization of individual compounds responsible for antioxidant properties to authenticate their probable uses as sources of natural antioxidants as well as to validate their traditional uses in several medicinal practices.Total phenolic, flavonoid contents, and antioxidant properties of the fruits, seeds, and bark extracts of