Rats were examined at five or eight weeks with right-heart catheter, cardiac magnetic resonance, and autopsy. Compared to normoxic controls (23.9 ± 4.1 mmHg), right ventricular systolic pressure was elevated in Sugen–hypoxia rats at five and eight weeks . Right ventricular end-systolic volume index was increased in eight weeks Sugen–hypoxia  compared to normoxic controls (0.18 ±0.03 mlcm–2). There was progressive dilatation of the right ventricular at eight weeks Sugen–hypoxia compared to normoxic controls (0.75 ± 0.13 µlcm–2 vs 0.56 ± 0.1 µlcm–2p = 0.02). Ventricle mass index by cardiac magnetic resonance at five weeks  and eight weeks Sugen–hypoxia  were higher than normoxic controls (0.21 ± 0.04). Stroke volume, right ventricular ejection fraction, and left ventricular variables were preserved in Sugen–hypoxia. Ventricular changes during the course of illness in a pulmonary arterial hypertension rodent model can be examined by cardiac magnetic resonance. These changes including right ventricular hypertrophy and subsequent dilatation are similar to those seen in pulmonary arterial hypertension patients. Despite the persisting pulmonary hypertension, there are features of adaptive cardiac remodeling through the study duration.Cardiac magnetic resonance-derived ventricular variables are predictive of mortality in pulmonary arterial hypertension. Rodent models which emphasize ventricular function, allowing serial monitoring, are needed to identify pathophysiological features and novel therapies for pulmonary arterial hypertension. We investigated longitudinal changes in the Sugen–hypoxia model during disease progression. Sprague Dawley rats ( Stroke volume,2 RV ejection fraction,3 and RV and LV end-diastolic volume (EDV)4 have all shown to be prognostic markers in PAH patients.Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature; however, it is subsequent right ventricular (RV) failure that is the main cause of morbidity and mortality in patients. Cardiac magnetic resonance (CMR) is a non-invasive imaging tool providing high-resolution three-dimensional images of the heart. During CMR, ventricular short axis stacks are used to reconstruct a 3D image of the right and the left ventricle (LV), and ventricular volumes, mass, and function can be measured.Small animal (rodent) models are increasingly used to identify pathophysiology as well as therapies for PAH with the intention of translating findings to humans. Accurate monitoring of disease in rodents with emphasis on ventricular function  without killing the animal is needed.5 A combination of Sugen and chronic hypoxia cause pulmonary endothelial cell death and severe PH. Subsequently in 2010, Abe et al. showed that SuHx rats demonstrated evidence of severe pulmonary arteriopathy including concentric neo-intimal and complex plexiform-like lesions which closely resemble plexiform lesions seen in humans.6 Subsequently, other groups have attempted to characterize hemodynamics in a SuHx model beyond right heart catheterization alone. Vitali et al. evaluated longitudinal changes in a SuHx mouse model of PH. Echocardiographic and invasive measurements were performed after three weeks of hypoxia and after 10 weeks of recovery in normoxia. Ten weeks after hypoxic exposure, RV systolic pressure (RVSP) had decreased, but remained elevated compared to normoxic controls. However, RV hypertrophy had resolved. They observed very few angio-obliterative lesions at 10 weeks.7 De Raaf et al. used telemetry to characterize hemodynamics in SuHx rats and associated these with serial histology.8 Jones et al. demonstrated a good correlation between M-mode and Doppler Echo vs right heart catheterization in the monocrotaline rat model.9 In a study by Urboniene et al. assessing validation of high resolution echocardiography and CMR vs high fidelity catheterization in experimental PH monocrotaline rat model, non-invasive measures of RV free wall thickness/mass correlated well with post mortem measurements.10Various rodent models to recapitulate human PAH have been produced. Sugen-5416 (Sugen), an inhibitor of vascular endothelial growth factor was shown to cause a mild rise in pulmonary artery pressure in wild-type rats. Combining Sugen with another stimulus of pulmonary hypertension (PH), i.e. chronic hypoxia, Taraseviciene-Stewart et al. described the Sugen–hypoxia (SuHx) rat model in 2001.12 The same non-invasive and repeatable measurements would be of great advantage for the study of rodent models to allow a detailed understanding of ventricular structural and functional changes that occur, to enhance efficacy in translational medicine. We investigated whether CMR is feasible in a SuHx rat model of PH. Subsequently, we investigated the structural and functional changes associated with the model during disease progression. Finally, we discussed the suitability of the SuHx model for translational studies of the mechanisms of RV dysfunction in PAH.Our group has a proven track record using CMR imaging to evaluate RV function in humans with PAH.All experimental procedures were carried out in accordance with the United Kingdom Animal Procedures Act (1986) and with the US NIH publication No. 85-23, revised 1996, and ethical approval was also granted by the University of Glasgow Ethics Committee. Rodents were housed in a 12-h light–dark cycle with access to food and water ad libitum.n = 32) were divided into two groups (n = 16 in each group). Group 1: SuHx—a single dose of subcutaneous Sugen-5416  suspended in vehicle (20 mg/kg), before being placed in a hypobaric chamber (atmospheric pressure 550 mbar) for two weeks and then placed in normal room pressure (1013 mbar) for three weeks whilst PH developed. Group 2: Normoxia—maintained at normal room pressure for five weeks. In each group (n = 16), half the animals entered the CMR arm of the study (n = 8) while the other half underwent right heart catheterization for hemodynamic assessment (n = 8). Animals were assessed at five weeks and eight weeks from the beginning of the study. The study design is summarized in Male Sprague Dawley rats (three weeks)  isoflurane and then maintained at 2% (v/v) isoflurane supplemented with a constant flow of 5% (v/v) oxygen. Hemodynamic measurements were taken using an ultra-miniature Polyimide Nylon catheter capable of measuring ventricular pressure continuously . The catheter was used as per the manufacturer’s instructions with the PowerLab 35 Series data acquisition system with LabChart Pro and the pressure volume (PV) loop analysis module. For right heart pressure analysis, the catheter was inserted into the jugular vein and guided into the RV to measure RVSP.Following hemodynamic assessment, animals were culled and hearts flushed with phosphate buffered saline using a blunt needle to clear peripheral blood cells. Euthanasia consisted of an overdose of anesthetic followed by a schedule 1 kill . The heart was isolated, atria removed, and tissue fixed with 10% (v/v) neutral buffered formalin for 48 h before paraffin processing for histological analysis by immunohistochemistry. Tissue was sectioned by microtome at width of 5 µm and stained by Gomori’s trichrome staining kit  as per manufacturer’s instructions.After the heart was isolated and atria removed, the RV and LV weights were obtained to determine RV hypertrophy. Interventricular septum was considered part of the LV.Vascular thickening was determined by smooth muscle actin antibody  staining, thickening was characterized by an increase in the vessel wall diameter of more than 50% of the arterial wall or complete occlusion. The number of remodeled vessels over the total number of vessels present in a lung section was determined. Sections were analyzed in a blinded manner.CMR imaging was performed in a Bruker Biospec 7-T/30-cm  system with a gradient coil insert (400 mT/m). Using a 72-mm transmit birdcage resonator and four channel phased array rat cardiac receiver coil. Anesthesia was induced with gas flow at 2–3 l/min, and the isoflurane delivered via a vaporizer  at 3–4%. The exhaust was connected to the Omnicon F/Air device (AM Bickford). After induction, animals were maintained at 2% (v/v) isoflurane supplemented with a constant flow of 5% (v/v) oxygen. An external water jacket was used to maintain a core temperature of 37℃. During all procedures, body temperature, ECG, and respiration were monitored . Long and short axis scout images were acquired so that short axis images could be planned using a segmented, cardiac-triggered FLASH sequence. The images were acquired with a slice thickness of 1.5 mm, ensuring the entire biventricular length is covered. The CMR parameters were as follows. Slice thickness—1.50 mm, field of view—30.00 mm × 30.00 mm, image matrix—192 × 192 pixels, image resolution—156 µm × 156 µm, Flip angle—15 degrees, Echo time—2.50 ms, Rep. time—7.02 ms, number of frames—25, number of averages—6, and software version—Paravision 5.1.13Scans were coded by number and analyzed in batches by G.J who was blinded to the identity and hemodynamic results at the time of analysis. A second observer (A.U) analyzed five scans for inter-rater agreement analysis. Trabeculations and papillary muscles were considered as part of the blood pool. The epicardial and endocardial borders were manually outlined in end-diastolic and end-systolic frames using QMass . Stroke volume was determined from EDV – end-systolic volume (ESV) of the LV. Ejection fraction ((SV/EDV) × 100%) was also determined. RV and LV masses were determined by manual planimetry at diastole. Ventricular mass index (VMI) was defined as the ratio between RV to LV mass, with the interventricular septum considered part of the LV. LV eccentricity index (LVEI) was defined as the ratio between maximum anterior–posterior to septal lateral diameters of the LV and was measured at both systole and diastole. All ventricular volumes and mass measurements were indexed to body surface area.–2 and mgcm–2, respectively, indexed for body surface area. Statistical analysis was performed using SPSS  and GraphPad Prism . A significance level of 0.05 was employed for statistical tests. An analysis of variance test was used to compare RV and LV mass, volumes, and function between normoxic animals and different stages of SuHx. If there was statistical significance, a Tukey test was used for post hoc analysis. To compare different methods of VMI measurement (CMR vs autopsy), a spearmen correlation was used. Inter-rater variability for determination of LV and RV function were calculated from paired measurements of the LV ejection fraction (LVEF) and RV ejection fraction (RVEF) of two readers as interclass correlation coefficient with a two-way mixed model for absolute agreement. Results are shown as mean ± SD unless otherwise stated.Ventricular volumes and mass are given as µlcmp = 0.03; 48.89 ± 9.6 mmHg, p = 0.002, respectively). There were no significant differences in RVSP between SuHx rats at five and eight weeks. Similarly, relative RV mass measured at autopsy by RV/(LV + septum) was significantly elevated at five weeks  and eight weeks  SuHx compared to controls (0.25 ± 0.04) , there was no significant difference observed between SuHx rats at five and eight weeks (Compared to normoxic rats (23.87 ± 4.1 mmHg), RVSP was significantly elevated in SuHx rats at both five and eight weeks  . Immunohht weeks .Fig. 2.A representative long axis image (a) and a short axis cine stack (b) to (f) are shown in There was excellent agreement between the two observers  for LVEF as well as for RVEF .p = 0.003) compared to normoxic rats (0.18 ± 0.03). There were no significant differences between normoxia and SuHx at five weeks. Compared to normoxic rats (0.17 ± 0.03), RV mass index was increased in the SuHx rats at five weeks  and eight weeks . RV demonstrated progressive dilatation (increasing RVEDV index) at eight weeks of SuHx compared to normoxic rats . In RVEF, there were no significant differences between normoxic rats and five and eight week SuHx rats; however, demonstrating trends toward impairment . Stroke volume index was preserved in the SuHx model at five and eight weeks compared to normoxia (0.44 ± 0.1 vs 0.5 ± 0.04 vs 0.49 ± 0.1). VMI after five weeks  and eight weeks of SuHx  were significantly higher than normoxic rats (0.21 ± 0.04)  .Fig. 4.No differences were observed between the normoxic and SuHx groups  in terms of LVEDV index (0.73 ± 0.08 vs 0.74 ± 0.06 vs 0.75 ± 0.16), LVESV index (0.29 ± 0.05 vs 0.24 ± 0.04 vs 0.25 ± 0.03), LV mass index (0.79 ± 0.07 vs 0.82 ± 0.09 vs 0.8 ± 0.15), or LVEF (60.3 ± 7.03 vs 67.8 ±3.16 vs 66.5 ± 3.12) .Fig. 5.p = 0.006) and in SuHx rats at eight weeks  compared to normoxic rats (0.98 ± 0.08). There were no differences between LVEI at diastole between normoxia and SuHx at five or eight weeks (1.06 ± 0.05 vs 1.14 ± 0.04 vs 1.1 ± 0.06)  .Supplementary file demonstrates CMR variables between normoxia and Sugen hypoxia at five and eight weeks.r = 0.8328, 95% CI: 0.5633–0.9422, p < 0.0001). However, MR images demonstrate functional aspects of RV contraction including septal flattening and paradoxical septal motion during systole due to RV pressure overload.CMR images taken from a normoxic and SuHx animals and light microscopy images of the same animals at autopsy were compared. Although both techniques could visually demonstrate ventricular size and wall thickness (hypertrophy), CMR demonstrated functional aspects of RV contraction including septal flattening and paradoxical septal motion during systole in SuHx animals. 7 Previous CMR studies used phase contrast imaging but did not explore LV and RV volume and functional variables known to be prognostic in PH, nor look at longitudinal changes.10To the best of our knowledge, this is the first study demonstrating longitudinal biventricular mass, volume, and function in a PH animal model using CMR. Previous studies had used echocardiography to examine biventricular function and structure in small animal models of PAH.Our study demonstrated the feasibility of CMR in a small animal model of PAH with good spatial and temporal resolution and excellent inter-observer reproducibility. The future advantage of CMR over cardiac catheterization and autopsy is the ability to perform imaging serially on the same animal to look at disease progression and response to treatment without killing the animal.3 Although traditionally right heart catheterization and post mortem studies have been used, there is a need for non-invasive tests of RV function in animal models of PAH. In addition, we need to have a better understanding of the longitudinal changes in ventricular function in animal models of PAH. Although the RV is the obvious focus of attention, LV dysfunction can occur through cardiac interaction, thus simultaneous evaluation of the LV is important. Echocardiography is widely available and can be used to estimate RVSP; however, imaging of the RV with its complex geometry is difficult.The main cause of morbidity and mortality in patients with PAH is RV failure. It had been assumed that the cause of RV dysfunction was alterations to the pulmonary vasculature and therefore treatment focus had been centered on improving pulmonary hemodynamics, with the assumption that improvement in RV would follow. However, it is now evident that cardiac response to a given level of pulmonary hemodynamic overload is variable but important in the subsequent prognosis of these patients.14 The study demonstrated similar hemodynamics and RV hypertrophy compared to our study population after eight weeks. Abe et al. investigated the longitudinal hemodynamic and histological changes in the model. At 13–14 weeks after Sugen, the rats had very high RVSP (96 ± 11 vs 21) and severe RV hypertrophy .6 The varying degrees of RVSP and RV hypertrophy in these studies is likely related to the duration of exposure to hypoxia, however, could also be related to the animal strain, gender, and age at exposure.Plexiform lesions in the pulmonary vasculature are known to be the hallmark of PAH and attempts have been made to establish an animal model that closely mimics human disease. The SuHx model has been shown to cause severe PH with the development of plexiform lesions. We used a SuHx model consisting of an injection of Sugen, two weeks of hypobaric hypoxia and three weeks or normoxia. Dean et al. explored the effects of Metformin on the development of PH via aromatase inhibition, a similar SuHx model was used (Sugen + two weeks of hypobaric hypoxia and three weeks of normoxia).15 In our study, RV hypertrophy was followed by subsequent dilatation. However, stroke volume and RVEF were relatively preserved. This was despite the presence of persistently elevated RVSP. We believe this study represents the natural history of RV hypertrophy and failure, demonstrating compensated RV hypertrophy (adaptive remodeling) before progression into maladaptive remodeling with further RV dilatation and RV failure, with reduction of RV output.In humans with PAH, progressive narrowing of the pulmonary vasculature causes increased load to the RV. The RV adaptation results in increasing wall thickness (hypertrophy) and contractility (coupling). Ventriculo–arterial coupling preserves stroke volume and ventricular efficiency. The RV then dilates, increasing wall stress and oxygen consumption per gram resulting in uncoupling and reduced stroke volume.16 hypothesized that a SuHx mouse model may capture the transition from adaptive to maladaptive RV remodeling including impairment in RV function by studying PV measurements in vivo. The results suggested that RV remodeling may begin to shift from adaptive to maladaptive with increasing duration of SuHx exposure. However, for the duration of SuHx exposure used in their study, no drop in cardiac output was observed.16 We believe that our study demonstrates a period of adaptive remodeling of the RV with compensated hypertrophy with minimal RV dilatation at later stages of the study. By lengthening the exposure to SuHx, we may be able to identify the transition from adaptive to maladaptive remodeling and identify decompensated right heart failure in this model. Most patients present to clinical assessment when there are signs of more severe RV dysfunction and in contrast pre-clinical SuHx model demonstrate adaptive remodeling at least early in its disease progression. This is of importance in the design of pre-clinical studies as intervention with experimental therapeutics is likely to occur at this early adaptive stage of disease progression where RV function/stroke volume seem to be preserved despite the presence of PAH.Wang et al.18 Previous human studies have demonstrated that patients with progressive illness demonstrated lower LV systolic function compared to stable patients. Previous echocardiography studies have also demonstrated impaired LV strain and torsion in PAH patients.17 We observed preserved LV systolic function with preserved LV mass and volume variables in this small animal model of PAH. Although PAH animals demonstrated paradoxical septal motion of the septum in our study, preserved RV output probably explains the preserved LV function.In PAH, impaired LV performance is explained by low RV cardiac output and direct ventricular interaction due to inter-ventricular dyssynchrony and paradoxical septal motion.CMR is feasible in a small animal model of PAH and could be used in pre-clinical animal studies to explore bi-ventricular structural and functional changes during the course of illness. It is likely that the SuHx model demonstrates adaptive remodeling to persistently elevated pulmonary pressures which is demonstrable by preserved RV function and stroke volume with hypertrophy of the RV. Further longitudinal studies are required to assess this model in detail, especially focusing on longitudinal RV response. The search for better animal models of PH continues because our understanding of the pathobiology of disease and the development of new therapeutic strategies depends on robust animal models, but at present, no single model has all the features of human disease.19 Future studies with late gadolinium enhancement data will provide further insights into myocardial remodeling in PAH.The study assessed rats at five and eight weeks from Sugen exposure, while assessment later may have demonstrated worsened hemodynamics and RV variables mimicking disease presentation in humans. The rats were anesthetized with isoflurane, previous studies using halogenated anesthetics have been shown to impair RV–PA coupling; however, these effects were seen both in hypoxia and hyperoxia.Click here for additional data file.Supplemental material, PUL897513 Supplemental material for Understanding longitudinal biventricular structural and functional changes in a pulmonary hypertension Sugen–hypoxia rat model by cardiac magnetic resonance imaging by Geeshath Jayasekera, Kathryn S. Wilson, Hanna Buist, Rosemary Woodward, Aysel Uckan, Colin Hughes, Margaret Nilsen, A Colin Church, Martin K. Johnson, Lindsay Gallagher, James Mullin, Mandy R. MacLean, William M. Holmes, Andrew J. Peacock and David J. Welsh in Pulmonary Circulation


To the Editor:Iron deficiency anemia (IDA) is a common health problem affecting more than 2 billion people worldwide. The ability to administer high dose intravenous (IV) iron facilitates the management in clinical conditions where demands for iron are high, as well as in which oral iron is ineffective, not tolerated, or harmful.Intravenous iron has been associated with concerns of potential hypersensitivity reactions,Most trials with FDI and other IV iron formulations have been 4‐12 weeks in duration, and safety trials with a longer duration are warranted to investigate long‐term safety after re‐dosing. Herein we present a 6 month extension trial (FERWON‐EXT) enrolling patients from three randomized, comparative, open‐label trials with FDI performed in the USA ‐ the PROVIDE,The primary endpoint was the number of adverse drug reactions (ADRs). The secondary safety endpoints included incidence of adjudicated serious or severe hypersensitivity reactions, composite cardiovascular adverse events (AEs), hypophosphatemia (s‐phosphate 2.0 mg/dL), and change in Hb, s‐ferritin, and transferrin saturation from baseline to week two, and months three and six. Adjudication of serious or severe hypersensitivity reactions and composite cardiovascular AEs was performed by an independent Clinical Endpoint Adjudication Committee. The hypersensitivity terms were defined by a standardized set of Medical Dictionary for Regulatory Activities (MedDRA) terms.A group of 193 patients from the three previous RCTs were screened, of whom 103 were enrolled and 94 (91%) completed the trial. A total of 101 patients received one dose of 1000 mg FDI. One patient experienced a transient episode of back pain during the infusion and received only 350 mg.A total of seven ADRs in 5/102 (4.9%) patients were reported. No ADR term was reported more than once. The proportion of patients with ADRs (Clopper‐Pearson 95% CI) was 0.05 . No serious ADRs, or serious or severe hypersensitivity reactions were reported.Six events in 6/102 (5.9%) were confirmed as cardiovascular events, primarily in patients with CKD (four events in four patients). None of the adjudicated and confirmed cardiovascular events were assessed as related to FDI.s‐phosphate 1.0 mg/dL).Hypophosphatemia was reported in 8/102 (7.8%) and none of these patients had CKD. Of these events, one was reported as a mild, non‐serious ADR, whereas the others were not reported as clinically significant. None developed severe hypophosphatemia (s‐ferritin level increased significantly from baseline (68.2 ± 87.2 ng/mL) to week two (323 ± 227 ng/mL), month three (153 ± 188 ng/mL), and month six (157 ± 218 ng/mL) with a peak at week two  Hb level increased significantly from baseline 9.60 ± 1.52 g/dL) to week two (10.88 ± 1.29 g/dL), month three (11.38 ± 1.29 g/dL), and month six (11.06 ± 1.60 g/dL) with a peak at month three .P < .001), and hypophosphatemia often persisted at the end of follow up at day 35 (43.0% in the FCM group vs 0.9% in the FDI group).The incidence of hypophosphatemia in this trial was 7.8% which was similar to the frequency in the PHOSPHARE trials, in which patients treated with FDI had a significantly lower incidence of hypophosphatemia than those treated with FCM  or 3 months (Hb) after dosing. Note, FDI is the only IV iron with an approval in the US for dosing of 1000 mg IV iron in one infusion, and administration of single doses above 1000 mg for patients with a bodyweight >50 kg (20 mg/kg) in the EU. The efficacy of FDI is well documented in RCTs, and FDI is at least as effective as FCM in correcting IDAWhile the primary objective and endpoints of FERWON‐EXT were safety, hematopoietic responses were also assessed. Mean Hb, s‐ferritin, transferrin saturation or both, and those previously experiencing ADRs with FDI treatment were not excluded. Finally, the trial duration was 6 months, allowing for assessment of longer‐term safety.The strengths of the trial were inclusion of a broad population across a wide range of IDA etiologies, including pre‐menopausal women with menorrhagia who were otherwise healthy, and CKD patients of whom 50% were 65 years or older. Iron deficiency anemia was confirmed in all patients based upon Hb and In conclusion, these data demonstrated that re‐dosing with a single IV dose of 1000 mg FDI was well tolerated and provided a fast improvement in Hb which was sustained 6 months after dosing. All dosed patients, except one, received the full FDI infusion without interruption. Adverse drug reactionss were reported in 4.9% of the patients, and all were mild or moderate in severity and none were serious. No serious or severe hypersensitivity reaction occurred.Maureen M. Achebe has been on scientific advisory boards for Pharmacosmos A/S, AMAG, Global blood therapeutics and Fulcrum pharmaceuticals.John Glaspy has been an advisor to AMAG Pharmaceuticals.Philip A. Kalra has received personal fees and non‐financial support from Pharmacosmos A/S, grants and personal fees from Vifor Pharma, and grants from Astellas.Michael Auerbach receives research funding for data management from AMAG Pharmaceuticals.Lars L. Thomsen is employed by Pharmacosmos A/S.Sunil Bhandari has received honorarium, consultancy fees, membership advisory board, and travel funding from Pharmacosmos A/S, Vifor Pharma, and Astellas.This work was funded by Pharmacosmos A/S and the investigators/institutions received a fee per patient.


Neurological disorders are a major public health issue worldwide and are often associated with structural changes in the brain. We have previously demonstrated that iso‐α‐acids (IAAs), the hop‐derived bitter components in beer, improve memory impairment in aged and Alzheimer's disease mouse models. In this study, we evaluated the effects of IAA intake on the brain structure in healthy middle‐aged to older adults. This study was conducted under the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT) study launched by the Cabinet office of Japan.This study employed an open‐labeled, single‐arm, before and after design. Healthy middle‐aged to older adults consumed a beverage containing IAAs (3 mg/190 mL) for 4 weeks.Recently developed magnetic resonance imaging‐based brain health indicators were used to evaluate the following brain conditions: the Brain Healthcare Quotient (BHQ) based on gray matter volume (GM‐BHQ) and white matter fractional anisotropy (FA‐BHQ).In total, 25 subjects were recruited, and GM‐BHQ and FA‐BHQ were measured before and after intervention. In all subjects, no significant differences in GM‐BHQ and FA‐BHQ were observed. In subjects aged ≥ 60 years  (n = 8), GM‐BHQ was significantly increased 4 weeks after intervention compared with that before intervention.Intake of beverages containing IAAs might affect brain aging, particularly in healthy older adults, which may prevent the development of neurological disorders. Future studies employing more robust designs can elucidate the effects of IAAs on GM‐BHQ and cognitive functions. This is the first clinical trial evaluating the effects of intake of bitter component of beer, iso‐alpha‐acid, on brain structure. The brain structure before and after intervention was measured by recently developed magnetic resonance imaging‐based method. The gray matter volume of older adults was improved by the intake of iso‐alpha‐acid. Hops have long been used in brewing to add bitterness and flavor. Iso‐α‐acids (IAAs) are the main bitter components in beer produced from hops. Commercially available beer typically contains IAAs at 10‐30 ppm; furthermore, nonalcoholic beverages simulating beer contain IAAs. Our group has previously demonstrated that IAAs suppress brain inflammation and prevent cognitive impairment in Alzheimer's transgenic and aged mice.The herb hop  initiated by the Japanese cabinet office has developed the Brain Healthcare Quotient (BHQ) based on gray matter (GM) volume (GM‐BHQ) and the white matter fractional anisotropy  as the standardized health index of brain structure in healthy individuals using magnetic resonance imaging (MRI).This study is the first clinical trial evaluating the effects of IAAs on human brain health as indicated by GM‐BHQ and FA‐BHQ. We recruited 25 healthy middle‐aged to older adults, and they consumed beverages containing 3.4 mg of IAAs each day for 4 weeks. This study employed an open‐labeled, single‐arm, before and after design and was conducted under the ImPACT study.22.1We recruited 45 right‐handed healthy Japanese adults, aged 50‐70 years, who were not habitual alcohol drinkers . Exclusion criteria were listed on Table 2.2In hops, α‐acids predominantly consist of three congeners: cohumulone, humulone, and adhumulone. During the brewing process, these are isomerized into two epimeric isomers, namely cis‐ and trans‐IAAs. In the present study, we used isomerized hop extract (Hopsteiner) with 30.5% (w/v) IAAs, comprised of trans‐isocohumulone (1.74% w/v), cis‐isocohumulone (7.61% w/v), trans‐isohumulone (3.05% w/v), cis‐isohumulone (14.0% w/v), trans‐isoadhumulone (0.737% w/v), and cis‐isoadhumulone (3.37% w/v) as previously described.The test formula consisted of a 190‐mL beverage containing isomerized hop extract and containing 3.4‐mg IAAs; this beverage was ingested every day for 4 weeks, and it contained acidulant, sweetener, and flavor to mask the bitterness of IAAs. The IAA dose in this study is approximately equivalent to the IAA content in 500 mL of commercially available beer.2.3This study was performed using an open‐labeled, single‐arm, before and after design. Questionnaires for the inclusion/exclusion criteria were answered at enrollment. All eligible subjects consumed the test beverage for 4 weeks, and subjects were not blinded to the information regarding the test beverage. Subjects were instructed to maintain their regular lifestyles and avoid consumption of any foods or beverages containing hops. Compliance was monitored using subject diaries. MRI measurements were obtained before and after 4 weeks of beverage intake. On the MRI measurement day, subjects were instructed to completely avoid food and beverages containing caffeine. The data were collected at the University of Tokyo  between November 2015 and December 2015.2.4High‐resolution MRI image acquisition and GM‐BHQ and FA‐BHQ calculations were performed according to previously described methods.2.5t test. All statistics were performed using Ekuseru‐Toukei 2015 .The Kolmogorov‐Smirnov test was performed in order to check the normality of the data set. Then, changes in GM‐BHQ and FA‐BHQ were analyzed by a paired 33.1The flow of subjects through this study is shown in Figure 3.2Changes in GM‐BHQ and FA‐BHQ before and after intervention are shown in Table 4In this study, we first evaluated the effects of IAAs on brain health in humans. GM‐BHQ, but not FA‐BHQ, significantly increased following IAA intake, particularly in older subjects. GM‐BHQ reflects the volume of GM associated with an expanse of dendrites, whereas FA‐BHQ is associated with white matter plasticity.GM‐BHQ is reported to decrease with age.Furthermore, GM‐BHQ exhibits a negative correlation with body mass index.In addition to cognitive decline or dementia, GM‐BHQ is affected by psychological conditions. It was reported that GM‐BHQ was declined in the group with the subjective fatigue, which is associated with fatigue and stress.This study has several limitations. First, an open‐labeled, single‐arm, before and after design was employed, which could not preclude potential placebo effects. GM‐BHQ and FA‐BHQ are objective indexes; therefore, the placebo effects may not have significantly influenced the results. However, a study employing a more robust design is required to increase the reliability of our conclusions. Second, we did not evaluate the effects of IAAs on cognitive function or mood status with neuropsychological methods; therefore, the effect of IAA intake on cognitive functions or mood by enhancing GM‐BHQ remains unclear. Future studies are required to clarify the association between these indexes and neurological disorders and elucidate the clinical significance associated with the improvement of these indexes.In conclusion, IAA intake improves the decrease in GM‐BHQ associated with aging and contributes to brain health maintenance, which may prevent the development of neurological disorders. The IAA dose utilized in this study is easily consumed daily in alcoholic or nonalcoholic beverages. Thus, IAA intake could be a practical method to maintain brain health.M.K., S.Y., K.K., and Y.A. are employed by the funder, and Y.Y. is the member of ImPACT. The test beverage was prepared by the funder. The MRI data collection and analysis were performed by ImPACT.M.K. and Y.A. designed and conducted the study. S.Y. and K.K. supervised the study. Y.Y. conducted this study as ImPACT manager. M.K. wrote the manuscript.The study was conducted in accordance with the Declaration of Helsinki and the recommendations of Ethical Guidelines for Medical and Health Research Involving Human Subjects, Ministry of Health, Labor and Welfare. This study was approved by ethics committee of Huma R&D CO. Ltd  and the University of Tokyo .I agree to publish part of it as supplementary information.This study was approved by the ethics committee of Huma R&D Co Ltd.  and the University of Tokyo .Written informed consent was obtained from all participants.The study was registered on November 30, 2015, in the database of the University Hospital Medical Information Network (UMIN) .n/a. Click here for additional data file.


CLECL5A, SERPING1, CHI3L1 and C1R) were found to be associated with immune cell infiltration, and further study demonstrated that these four genes might drive the hypoxic phenotype of perinecrotic GBM, which affects hypoxia‐induced glioma stemness. Therefore, these might be important candidates for immunotherapy of GBM and deserve further exploration.Glioblastoma (GBM) is a malignant intracranial tumour with the highest proportion and lethality. It is characterized by invasiveness and heterogeneity. However, the currently available therapies are not curative. As an essential environmental cue that maintains glioma stem cells, hypoxia is considered the cause of tumour resistance to chemotherapy and radiation. Growing evidence shows that immunotherapy focusing on the tumour microenvironment is an effective treatment for GBM; however, the current clinicopathological features cannot predict the response to immunotherapy and provide accurate guidance for immunotherapy. Based on the ESTIMATE algorithm, GBM cases of The Cancer Genome Atlas (TCGA) data set were classified into high‐ and low‐immune/stromal score groups, and a four‐gene tumour environment‐related model was constructed. This model exhibited good efficiency at forecasting short‐ and long‐term prognosis and could also act as an independent prognostic biomarker. Additionally, this model and four of its genes ( For example, CIBERSORTx, Tumor IMmune Estimation Resource (TIMER) and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) are innovative algorithms used to calculate the infiltration of tumour‐related normal cells in tumour tissues according to genomics.In this study, we analysed the potential TME‐related prognostic signature for GBM patients by integrating of TCGA data and ESTIMATE algorithm, identifying differentially expressed genes based on their immune and stromal scores. The common genes were screened out and followed with a robust likelihood‐based survival model to identify prognostic genes associated with the GBM TME. Furthermore, using the LASSO method, we constructed an independent prognostic model containing four genes aberrantly expressed in GBM; these genes and the model were significantly associated with the immune infiltration levels and the hypoxic phenotype in GBM patients. The findings of this study will help elucidate the TME effect on GBM and provide alternative targets for immunotherapy.22.1http://gliovis.bioinfo.cnio.es/), which is a user‐friendly website for data visualization and analysis of glioma data sets. The CGGA RNA sequencing data were downloaded from CGGA data portal (http://www.cgga.org.cn/). The stromal and immune scores of TCGA GBM data set originated from the ESTIMATE algorithm, which was downloaded from the ESTIMATE website (https://bioinformatics.mdanderson.org/estimate/), which provides information regarding tumour samples based on expression data, such as scores of tumour purity, the level of stromal cells present and the infiltration level of immune cells in tumour tissues. The file reflecting the estimation of immune cells of the TCGA GBM data set was obtained from TIMER (https://cistrome.shinyapps.io/timer/), a website offering an in‐depth analysis of infiltration of immune cells in tumours. The microarray data of 416 GBM patients with clinicopathological characteristics, stromal/immune scores and estimation of immune cells from the TCGA, normalized by the ‘affy’ package, were chosen as the training set to construct the prognostic model. The validation set included data of 159 GBM cases downloaded from Gravendeel. The clinical information for the training and validation sets is shown in Table http://www.gbmseq.org/).The genomic expression and clinical data of GBM patients in TCGA and Gravendeel databases were retrieved from GlioVis , molecular functions (MF) and cellular components (CC), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted via the clusterProfiler package in R language for differentially expressed TME‐related genes.2.4Using the rbsurv, glmnet and survival packages, the robust likelihood‐based survival model and the least absolute shrinkage and selection operator regression (LASSO) analyses were conducted in the TCGA training set to construct the TME‐related model. Consequently, four prognostic TME‐related genes and their regression coefficients were obtained. Univariate and multivariate Cox hazard regression analysis showed that this gene signature model was an independent prognostic biomarker, verified using the Gravendeel validation set.Weighted gene correlation network analysis (WGCNA), an innovative method used to describe the correlation patterns among genes across microarray samples, was conducted for the training set to build the co‐expression network of the gene signature model.2.5These experiments were approved by the Human Ethics Committee of Xiangya Hospital, and informed consent was obtained from all patients. Based on polyformalin‐fixed and paraffin‐embedded tissues obtained from GBM patients, immunohistochemistry analysis was conducted as previously described.2.6P < .05 regarded as statistically significant. To assess the predictive efficiency of the signature, the time‐dependent receiver operating characteristic curves were depicted.Statistical analyses were performed with R software. Using the log‐rank test, the relationship between the critical factors  and patients’ overall survival was analysed based on the survival and survminer R package, with 2.7Dimensionality reduction and cell clustering for the single‐cell RNA‐seq data were performed based on ‘tsne’ package in R. As described by a previous study,33.1P= .082; Figure P = .062). This verified that immune/stromal scores were significantly associated with the GBM subtypes and higher scores were related to shorter survival times in GBM patients. In accordance with the results from a previous study,+ T cell was reduced in the pro‐neural GBM  and stromal scores (−3055.72 to 2016.62) of the 416 GBM patients. To elucidate the relationship between the molecular expression patterns and immune/stromal scores, both scores of the four transcriptome subtypes  and CIMP status (G‐CIMP and non–G‐CIMP) of GBMs were compared. There was a remarkable association between the immune/stromal scores and the four transcriptome subtypes. The immune/stromal scores of the mesenchymal subtype were the highest, whereas the scores of pro‐neural subtype were the lowest Figure . Immune/M Figure A.3.2P‐value < 0.05 and |log fold change|＞1. Moreover, Venn diagrams show 152 genes, differentially expressed both in the stromal and immune score groups  were screened out to construct the TME‐related model. These four genes were also validated to be differentially expressed genes in the Gravendeel data set and another independent data set in the Chinese Glioma Genome Atlas (Table P < 0.0001) , a web server for analysing the RNA sequencing expression data of tumour and normal samples, revealed that the expression of these filtered four genes was expressed at a higher level in GBM than in non–GBM tissues  Figure . The are) Figure . The per) Figure . The AUC) Figure . The co‐) Figure A‐D. The ) Figure . The num) Figure . GEPIA , CIMP status , age group  and risk level  were found to be independent predictors in the TCGA GBM data set. CIMP status , age group  and risk level  were independent predictors in the Gravendeel data set , CIMP status (non–G‐CIMP vs. G‐CIMP), MGMT status (unmethylated vs. methylated), subtype  and risk level (low vs. high) were analysed by univariate and multivariate Cox proportional hazard regression. MGMT status  , macrophages  and CD4 + T cells  , CD4 + T cells , CD8 + T cells , neutrophils  and macrophages  , neutrophils  and macrophages  , CD4 + T cells , CD8 + T cells , neutrophils  and macrophages  , CD8 + T cells , neutrophils  and macrophages   Figure . CLEC5A ) Figure A. SERPIN) Figure B. CHI3L1) Figure C. C1R di) Figure D. Risk s) Figure E. This o3.6http://glioblastoma.alleninstitute.org/static/home). This web server collected GBM samples laser‐dissected from various sites including cellular tumour, perinecrotic zone, pseudopalisading cells around necrosis, hyperplastic blood vessels in cellular tumours and proliferating microvasculature samples.PTPCR (CD45) or neoplastic cells that were collected from the tumour core, which is the location at which necrosis occurred  immunotherapy. In fact, some of them have been demonstrated to be oncogenes, and their increased expression has been found to be associated with poor survival in glioma patients. For example, CLEC5A is a member of the C‐type lectin/C‐type lectin‐like domain (CTL/CTLD) superfamily and is reported to be an M2 biomarker, associated with immunosuppression. CLEC5A overexpression decreases survival time in glioma patients.GBM occurs in the central nervous system, and the current treatment is ineffective. The TME plays a significant role in tumour malignancy and dictates therapeutic responses, including immunotherapy. Thus, a deeper knowledge of the TME in GBM can contribute to uncovering novel prognostic markers to administer precision immunotherapy. Current studies have been unable to successfully classify and analyse the components of tumour cells and the TME.Recently, several genetic biomarkers, such as O(6)‐methylguanine‐DNA methyltransferase (MGMT) methylation, epidermal growth factor receptor variant III (EGFRvIII), vascular endothelial growth factor (VEGF) and isocitrate dehydrogenase (IDH), have been well‐established in GBM.An increasing number of investigations have suggested that the therapeutic effect of immunotherapies is correlated with immunogenic TME; DCs, which function as potential antigen‐presenting cells, have a central role in regulating immunity.5In conclusion, we constructed a four‐gene TME‐related predictive signature as an independent prognostic biomarker and compared it with other probable clinical features. The risk model showed good efficiency for GBM prognosis, verified by time‐dependent ROC curve analysis and survival analysis, and it was also a useful hierarchical marker for the G‐CIMP subtype of GBM. The four genes in our model might be important for the hypoxic phenotype of perinecrotic GBM and are remarkably correlated with infiltration of immune cells in the TME of GBM, indicating their potential as candidate targets for immunotherapy. However, the detailed molecular mechanisms underlying the functions of the four genes, which significantly influence the survival of GBM patients, have to be studied further.The authors declare that they have no competing interests.Kai kai Xiao: Data curation ; Methodology (lead); Software (lead); Visualization (lead); Writing‐original draft (lead). Jun Tan: Software (supporting); Visualization ; Writing‐review & editing (supporting). Jian Yuan: Visualization ; Writing‐review & editing (supporting). Gang Peng: Funding acquisition ; Visualization (supporting); Writing‐review & editing (supporting). Wenyong Long: Funding acquisition ; Writing‐review & editing (supporting). Jun Su: Visualization ; Writing‐review & editing (supporting). Yao Xiao: Visualization ; Writing‐original draft (supporting). Qun Xiao: Methodology (supporting); Visualization (supporting). Changwu Wu: Methodology (supporting); Visualization (supporting). Chaoying Qin: Visualization (supporting); Writing‐review & editing (supporting). Lili Hu: Software (supporting); Visualization (supporting). Kaili Liu: Software (supporting); Visualization (supporting). Shunlian Liu: Software (supporting); Visualization (supporting). Hao Zhou: Software (supporting); Visualization (supporting). Yichong Ning: Software (supporting); Visualization (supporting). Xiaofeng Ding: Project administration (supporting); Visualization ; Writing‐review & editing . Qing Liu: Data curation (lead); Funding acquisition (lead); Project administration (lead); Visualization .Fig S1‐S6Click here for additional data file.Table S1Click here for additional data file.


Given the increasing availability of large biological datasets and the growing interest in transporters, we expect that the effort presented here will be critical to provide novel insights into the functions of SLCs.Solute carriers (SLCs) are the largest family of transmembrane transporters in the human genome with more than 400 members. Despite the fact that SLCs mediate critical biological functions and several are important pharmacological targets, a large proportion of them is poorly characterized and present no assigned substrate. A major limitation to systems‐level de‐orphanization campaigns is the absence of a structured, language‐controlled chemical annotation. Here we describe a thorough manual annotation of SLCs based on literature. The annotation of substrates, transport mechanism, coupled ions, and subcellular localization for 446 human SLCs confirmed that ~30% of these were still functionally orphan and lacked known substrates. Application of a substrate‐based ontology to transcriptomic datasets identified SLC‐specific responses to external perturbations, while a machine‐learning approach based on the annotation allowed us to identify potential substrates for several orphan SLCs. The annotation is available at  A thorough manual literature‐based annotation of human solute carriers (SLCs) is presented. Complemented by a substrate‐based ontology, it provides a comprehensive resource for the scientific community to get novel insights to the functions of SLCs. SLCs are responsible for the transport of a wide range of substrates, including nutrients, ions, and waste products, across plasma membrane and organellar membranes  to 320 SLCs. However, out of 382 substrate terms, only 23 terms are shared by at least 10 SLCs. This poor overlap in substrate annotation of different SLCs made the annotation unsuitable for enrichment analysis in high‐throughput datasets and underlined the urgency for knowledge standardization and organization. The Chemical Entities of Biological Interest (ChEBI) ontology contains more than 46,000 manually curated entries, each of them with assigned annotations, synonyms, chemical structure, and database and literature links. By mapping the substrates from manual annotation to ChEBI ontology terms , a contret al, et al, Next, we wanted to assess the performance of the SLC substrate ontology in identifying patterns within SLC expression values obtained from real‐world datasets. We therefore measured transcriptomics profiles of HEK293T cells upon single amino acid deprivation as well as obtained published microarray‐based transcriptional profiles of MCF7 breast cancer cells  or ReSOLUTE (www.re-solute.eu).Computational methods for the analysis and interpretation of high‐throughput omics data in biology rely heavily on the availability of well‐structured metadata complementing the increasingly large experimental datasets available. Existing classifications of SLCs, such as the HUGO Gene Nomenclature Committee (HGNC), the classification proposed in Schlessinger et al , and theet al, et al, In conclusion, we are convinced that, given the growing efforts and interest in SLCs and the ever‐increasing availability of datasets, SLC‐specific annotations and ontologies such as the ones described here will be an essential part of the toolbox required for the systematic understanding of SLC function and organization on a cellular‐, tissue‐ and organismal‐level as well as for the de‐orphanization of SLCs by orthogonal methods , nucleoside/nucleotide, orphan, peptide, vitamin and other . For symporters or antiporters, coupled ions were defined as small ions required for the transport of substrates. Subcellular localization was taken from primary literature, when immunofluorescence data obtained in human cells were available. Whenever conflicting results were reported, precedence was given to localization data obtained by co‐staining with known organelle markers.Substrates, coupled ions, transport mechanism, and subcellular localization for each of the 446 human SLCs were manually annotated from the primary literature. Substrates were defined as molecules showed to be transported by the SLC in a transport assay using reconstituted protein or gene overexpression experiments in human cells. 11 substrate classes were defined: accessory protein, amino acid, carbohydrate, ion , lipid, heavy metal  from the manual annotation were mapped to ChEBI identifiers with Ontology Lookup Service (OLS) from the European Bioinformatics Institute (EMBL‐EBI)(Jupp ftp://ftp.ebi.ac.uk/pub/databases/chebi/ontology/). The oboe R package was used for parsing of the ontology  from the ChEBI portal  . Raw sequencing reads were demultiplexed, and after barcode, adaptor, and quality trimming with cutadapt (https://cutadapt.readthedocs.io/en/stable/), quality control was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The remaining reads were mapped to the GRCh38/h38 human genome assembly using genomic short‐read RNA‐seq aligner STAR version 2.567. We obtained more than 98% mapped reads in each sample with 70–80% of reads mapping to unique genomic location. Transcripts were quantified using End Sequence Analysis Toolkit (ESAT) with Rstudio IDE version 1.0.143, ggplot2 (3.0.0), dplyr (0.7.6), readr (1.1.1), gplots (3.0.1).HEK293T were obtained from ATCC and their identity and lack of contamination with mycoplasma confirmed by STR profiling and PCR, respectively. Cells were seeded in triplicate in full media . After 24 h, media were removed and, after a wash with PBS, substituted with DMEM starvation media lacking the indicated amino acid supplemented with 10% (v/v) dialyzed FBS (Gibco cat. 26400‐044). Starvation media each lacking a single amino acid were prepared by complementing amino acid‐free DMEM media  with the other 14 amino acids . DMEM media reconstituted with all 15 amino acids and 10% dialyzed FBS as well as full media served as controls. After 16 h, media were removed, and cells were harvested in cold PBS. Total RNA was isolated using the Qiagen RNeasy Mini kit including a DNase I digest step. RNA‐sequencing (RNA‐seq) libraries were prepared using QuantSeq 3′ mRNA‐Seq Library Prep Kit FWD for Illumina (Lexogen) according to the manufacturer's protocol. Libraries were subjected to 50‐bp single‐end high‐throughput sequencing on an Illumina HiSeq 4000 platform at the Biomedical Sequencing Facility  SLCs.Ontology term enrichment was conducted using one‐sided Fisher's exact test. Benjamini–Hochberg procedure was used for multiple testing correction (5% FDR cutoff). Testing was done for upregulated . Datasets are provided in Conceptualization: UG, VS, GS‐F and EG; Ontology generation and dataset analyses experiments: EM; Data and annotation analysis: EM, UG, VS, and EG; Design and experiments: MR; experiments and transcriptomics experiments: SS; Supervision: EG and GS‐F. All authors contributed to the manuscript preparation.The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Table EV3Click here for additional data file.Table EV4Click here for additional data file.Review Process FileClick here for additional data file.


NF1 and SERPINB3/4. Overall, these findings deserve prospective investigation in the future and may help guide clinical decisions on ICIs therapy for patients with melanoma.Several biomarkers such as tumor mutation burden (TMB), neoantigen load (NAL), programmed cell‐death receptor 1 ligand (PD‐L1) expression, and lactate dehydrogenase (LDH) have been developed for predicting response to immune checkpoint inhibitors (ICIs) in melanoma. However, some limitations including the undefined cut‐off value, poor uniformity of test platform, and weak reliability of prediction have restricted the broad application in clinical practice. In order to identify a clinically actionable biomarker and explore an effective strategy for prediction, we developed a genetic mutation model named as immunotherapy score (ITS) for predicting response to ICIs therapy in melanoma, based on whole‐exome sequencing data from previous studies. We observed that patients with high ITS had better durable clinical benefit and survival outcomes than patients with low ITS in three independent cohorts, as well as in the meta‐cohort. Notably, the prediction capability of ITS was more robust than that of TMB. Remarkably, ITS was not only an independent predictor of ICIs therapy, but also combined with TMB or LDH to better predict response to ICIs than any single biomarker. Moreover, patients with high ITS harbored the immunotherapy‐sensitive characteristics including high TMB and NAL, ultraviolet light damage, impaired DNA damage repair pathway, arrested cell cycle signaling, and frequent mutations in  This study provided evidences that the genetic mutation model (named as ITS) identified a melanoma population with multiple genetic patterns of sensitivity to ICIs, who might potentially benefit from ICIs therapy. Preliminary data from three independent cohorts strongly suggested better treatment outcomes from ICIs therapy in melanoma patients with high ITS. Remarkably, the combination strategy of ITS and TMB or LDH showed better prediction efficacy compared with any single biomarker. Immunotherapeutic antibodies directed at programmed cell‐death protein 1 (PD‑1) and cytotoxic T‑lymphocyte‐associated antigen‑4 (CTLA‑4) are standard therapies for metastatic melanoma.In order to identify a clinically actionable biomarker and explore an effective strategy for prediction, we developed a genetic mutation model named as immunotherapy score (ITS) for predicting response to ICIs in melanoma based on three independent cohorts.22.1We conducted a systematic literature search on PubMed, EMBASE, and Web of Science. As a result, three eligible studies were included in this study Figure . Subsequ2.2Systematic literature search was conducted on PubMed, EMBASE, and Web of Science up to June 1, 2020. The search term was as follows:  AND  AND (WES OR “Whole Exome*” OR “Whole‐exome” OR “Whole Exome Sequencing” OR “Whole Exome Sequencings” OR “Complete Exome Sequencing”). The inclusion criteria for eligible studies were as follows: (a) clinical trials or cohort studies associated with inhibitor of PD‐1/PD‐L1, CTLA‐4, or their combination, in patients with melanoma; (b) Clinical outcomes of patients were available, including objective response, OS, and/or PFS; (c) whole‐exome sequencing(WES) was performed in the original study and the corresponding WES data were available from the databases or the articles; (d) the number of patients accessible for evaluation was more than 50; (e) studies were published in English. Reviews, case reports, editorials, meeting comments, abstracts, and letters were excluded. As a result, the Allen cohort, Snyder cohort, and Liu cohort were included in this study. The workflow of the literature search is shown in Figure 2.3https://www.cbioportal.org/). Data of the Liu cohort and Miao cohort were acquired from supplemental materials of the reported articles.https://icgc.org/). A total of 955 melanoma and 132 NSCLC patients were included in this study. The Allen cohort consisted of 110 melanoma patients treated with Ipilimumab for model construction.We downloaded the clinical information, WES, and RNA‐seq data of the Allen cohort, Snyder cohort, TCGA‐SKCM cohort, and Hellmann cohort from the cBioPortal database  or partial response (PR) to ICIs by RECIST criteria v.1.12.5χ2 test. The genes reaching the statistical significance of p < .05 were regarded as candidate variables to perform multivariate logistic regression analysis by the Backward Elimination  strategy. As a result, four mutated genes  were included in the multivariate logistic regression model , where coef was derived from the multivariate logistic model and the mutation status was equal to 1, whereas wild type was 0. Patients were divided into low (ITS = 0) and high (ITS > 0) group, where the low group showed no nonsynonymous mutation in the genes and the high group showed at least one nonsynonymous mutation in the genes. Moreover, ITS was calculated using the same formula in the Snyder cohort, Liu cohort, Hellmann cohort, and Miao cohort for validation. Receiver operating characteristic (ROC) analysis was performed to assess the predictive accuracy using “pROC” packageThe frequently mutated genes (mutation rate ≥10%) were screened out from the Allen cohort based on the WES data. Subsequently, the association between the frequently mutated genes and DCB of patients with melanoma was determined by 2.6p < .05 was considered as statistically significant.We explored the impact of TMB and ITS on survival outcomes (OS and PFS) of melanoma patients treated with ICIs in the Allen cohort, Snyder cohort and Liu cohort. In addition, we performed Kaplan–Meier survival analysis for NSCLC patients treated with ICIs. The association between ITS and overall survival of melanoma patients without ICIs therapy was also evaluated in TCGA‐SCKM cohort and ICGC‐MELA cohort. We calculated the median overall survival time and the 95% confidence interval and performed log‐rank test. Log‐rank 2.7BRAF, NRAS, and NF1 in multivariate logistic and Cox regression models. The logistic and Cox regression model were visualized using “forestplot” R package. The summary predictive effect and between‐cohort heterogeneity were estimated using “meta” R package.I2 was used to evaluate the degree of heterogeneity between the cohorts and the criteria were as follows: low (25%‐50%), moderate (50%‐75%), and high (75%‐100%).Patients were divided into high ITS and low ITS group according to whether the value of ITS is more than 0. Zero represented no mutation in the four genes, whereas 1 represented at least one mutation. We classified the patients into high and low TMB using the median value and upper quartile as thresholds, respectively. We performed univariate logistic regression analysis to evaluate the association between DCB and ITS or TMB by calculating Odds ratios (OR) and 95% confidence interval (CI). Multivariate logistic regression analysis was applied to investigate the independently predictive effect of ITS and TMB on DCB. In addition, we performed univariate Cox regression analysis to assess the effect of ITS and TMB on OS and PFS by calculating hazards ratio (HR) and 95% CI. Multivariate Cox regression analysis was applied to evaluate the independent effect of ITS and TMB on OS and PFS. ITS and TMB were adjusted by gender, M stage, LDH, TMB, mutational signatures, and mutations in 2.8We explored the feasibility and significance of biomarker combination based on ITS, TMB, and LDH for predicting response to ICIs. Considering the correlation between ITS and TMB, we first divided the meta‐cohort into subgroups with high and low TMB according to the median value of TMB. Then we evaluated the association of ITS with DCB, OS, and PFS in high and low TMB subgroups, respectively. In addition, considering the negligible association between ITS and LDH, we combined ITS and LDH as a composite biomarker for predicting DCB, OS, and PFS in melanoma patients treated with ICIs. The overall distribution of ITS, TMB, LDH, and clinical benefit in the meta‐cohort was visualized by “ggalluvial” R package.2.9In this study, we defined TMB as (nonsynonymous mutation counts)/(the whole length of exons). The correlation analysis of ITS with TMB and NAL was performed and visualized using “ggplot2” R package. The correlation degrees were defined as follows: negligible (0.00‐0.10), weak (0.10‐0.39), 0.40‐0.69 (moderate), 0.70‐0.89 (strong), and very strong (0.90‐1.00).2.10http://software.broadinstitute.org/gsea) and hallmark genesets downloaded from the Molecular Signatures Database.To further investigate the distinctive signaling pathways associated with ITS, we integrated RNA‐seq data of the Allen cohort, Snyder cohort, and Liu cohort, then performed gene set enrichment analysis (GSEA) based on the java GSEA 3.0 Desktop Application . The categorical variables were compared by chi‐squared (χ2) test or Fisher's exact test in the appropriate situation. All reported P values were two‐tailed and p < .05 was considered as statistically significant.Kaplan–Meier analysis, correlation analysis, logistic, and Cox regression analyses were conducted using SPSS software . The log‐rank test was used to compare Kaplan–Meier curves. The correlation between ranked variables was determined by Spearman rank correlation coefficient. The OR and its 95% CI were calculated by logistic regression analysis. The HR and its 95% CI were calculated by Cox regression analysis. The comparison between continuous variables was dealt with Student's 33.1We collected clinical characteristics of total 318 patients diagnosed with melanoma and treated with anti‐CTLA‐4 or anti‐PD1 therapy from previous studies. The Allen cohort,3.2χ2 test  were included in the model after running 24 steps  had GRM3 mutations, 11% (12/110) had SYNE2 mutations, and 10% (11/110) had FLNC mutations  had FLNC mutations, 14% (9/64) had SYNE2 mutations, and 14% (9/64) had GRM3 mutations  had SYNE2 mutations, 15% (21/144) had FLNC mutations, and 12% (17/144) had GRM3 mutations . Subsequently, we evaluated the association between the frequently mutated genes and DCB of melanoma patients receiving ICIs therapy by conducting t Figure . As showps Table . Then, wC Figure . In the s Figure . Overalls Figure . Overalls Figure . Overall3.3χ2 test, respectively. As a result, the proportions of DCB were all significantly higher in high ITS than low ITS group . We found that the impact of TMB on OS was inconsistent when stratified by high and low TMB in three independent cohorts. High TMB group was significantly associated with better OS in the Liu cohort  and the potential of joint prediction for response to ICIs. We observed that a part of patients with low TMB or elevated LDH level harbored DCB, some of whom showed high ITS Figure . Besides3.8p < .001; ITS with NAL: Spearman's R = 0.51, p < .001; Figure p < .001; Figure p < .001; Figure p < .001; Figure p < .001), cell cycle , PI(3)K/Akt and RTK/RAS pathways , and SERPIN family . In the DNA damage repair pathway, multiple genes involved in homologous recombination were more frequent mutated in high ITS than low ITS group, including FANCA , FANCM , FANCD2 , BRCA1 , and BRCA2 . The core genes in the mismatch repair pathway were also more frequently altered in high ITS group, including MLH3 , MSH6 , and MSH2 . ATM  and ATR  were significant altered in high ITS group, which were master controllers of cell cycle checkpoint pathways responding to DNA damage. Besides, we observed frequent aberrations in RTK/RAS signaling and PI(3)K/Akt signaling, including mutations in NF1 , ERBB2 , PI3KCA , and amplification in BRAF . Notably, ITS was not associated with deletion of PTEN , a key factor leading to immunotherapy resistance as previously reported.SERPINB3  and SERPINB4  were more frequent in high ITS than low ITS group, which might promote serpin protein misfolding to increase tumor immunogenicity.We characterized the distinctive genomic patterns associated with ITS, based on integrating somatic mutation and CNV data. First, we performed correlation analysis to examine the correlation between TMB and ITS. NAL was also included in the correlation analysis. It was shown that the correlations of ITS with TMB and NAL were at moderate degrees  on hallmark gene sets based on the RNA‐seq data of the meta‐cohort. Genes involved in inflammatory response pathways were significantly enriched in high ITS group, including TNF‐α Signaling Via NF‐ΚB, Interferon‐γ Response, Allograft Rejection, Interferon‐αResponse, IL‐2/STAT5 Signaling, and IL‐6/JAK/STAT3 Signaling Figure . Besides4Despite a number of studies have shown the powerfully predictive capability of TMB on response to ICIs,THSD7B, SYNE2, GRM3, and FLNC, making it more convenient to identify the cut‐off value than TMB. Moreover, a customized targeted sequencing panel containing THSD7B, SYNE2, GRM3, and FLNC can be designed, and the targeted next‐generation sequencing (NGS) can be conducted to determine ITS accurately, which is less‐cost and more convenient than WES.THSD7B, SYNE2, GRM3, and FLNC in lung squamous cell carcinoma and lung adenocarcinoma , mismatch repair , and cell cycle checkpoints (ATM and ATR). More frequent mutations in NF1, SERPINB3, and SERPINB4 were also observed in the high ITS group compared to low ITS group. However, we did not find the association between ITS and loss of PTEN or mutations in NRAS. Moreover, GSEA analysis revealed that tumors with high ITS showed active inflammatory response and impaired cell cycle, which were favorable for ICIs therapy. These findings could partly explain why ITS could serve as an independently genetic biomarker of ICIs therapy in melanoma. However, the biological mechanisms how the ITS‐related genes  impact ICIs response are still unclear at present. In order to preliminarily establish a biologic hypothesis, we performed a literature review and investigated the biological functions of these genes from the previous studies  as a reliable ICIs biomarker and developed a web application for calculating the TIDE of transcriptional samples.THSD7B, SYNE2, GRM3, and FLNC) mutations were functional. In other words, we did not explore the mechanism of how mutations in these genes made impact on TMB, NAL, key pathways as mentioned, and other factors associated with ICIs therapy. In addition, this retrospective study may be affected by potential confounding factors, such as the selection bias of the cohorts. It would be ideal if other independent cohorts support our investigation. Whatever the genetic mutation model requires validation in prospective clinical cohorts.It was worth noting that our study had several limitations. First, WES data from the different cohorts were based on the different platforms lacking of uniformed criteria, which may limit the definition of the cut‐off value of TMB, therefore we adopted the median and upper quartile value rather than a specific value as the threshold to decrease the bias on TMB from the different cohorts. Second, some data such as PFS were not available in the specific cohort obtained from recent publications, making the analysis incomplete in part of this study. Third, we did not differentiate whether the genes (In conclusion, this study provided evidences that the genetic mutation model identified a melanoma population with multiple genetic patterns of sensitivity to ICIs, who might potentially benefit from ICIs therapy. Preliminary data from three independent cohorts strongly suggested better treatment outcomes from ICIs therapy in melanoma patients with high ITS. Remarkably, the combination strategy of ITS and TMB or LDH showed better prediction efficacy compared with any single biomarker. These findings deserve prospective investigation in the future and may help guide clinical decisions on ICIs therapy for patients with melanoma.The authors declare that they have no conflict of interests.Lisong Teng conceived and designed the study. Junjie Jiang and Yongfeng Ding collected data, performed data analysis, and wrote manuscript. Junjie Jiang, Mengjie Wu, and Yanyan Chen drew figures. Xiadong Lyu performed literature search. Jun Lu and Haiyong Wang were involved in data interpretation and critically reviewing the manuscript. All authors read and approved the final manuscript.The authors declare human ethics approval was not needed for this study.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Fig S5Click here for additional data file.Fig S6Click here for additional data file.Fig S7Click here for additional data file.Fig S8Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Supplementary MaterialClick here for additional data file.


Oncologic patients are regarded as the population most at risk of developing a severe course of COVID‐19 due to the fact that malignant diseases and chemotherapy often weaken the immune system. In the face of the ongoing SARS‐CoV‐2 pandemic, how particular patients deal with this infection remains an important question. In the period between the 15 and 26 April 2020, a total of 1227 patients were tested in one of seven oncologic outpatient clinics for SARS‐CoV‐2, regardless of symptoms, employing RT‐qPCR. Of 1227 patients, 78 (6.4%) were tested positive of SARS‐CoV‐2. Only one of the patients who tested positive developed a severe form of COVID‐19 with pneumonia (CURB‐65 score of 2), and two patients showed mild symptoms. Fourteen of 75 asymptomatic but positively tested patients received chemotherapy or chemo‐immunotherapy according to their regular therapy algorithm (±4 weeks of SARS‐CoV‐2 test), and 48 of 78 (61.5%) positive‐tested patients received glucocorticoids as co‐medication. None of the asymptomatic infected patients showed unexpected complications due to the SARS‐CoV‐2 infection during the cancer treatment. These data clearly contrast the view that patients with an oncologic disease are particularly vulnerable to SARS‐CoV‐2 and suggest that compromising therapies could be continued or started despite the ongoing pandemic. Moreover the relatively low appearance of symptoms due to COVID‐19 among patients on chemotherapy and other immunosuppressive co‐medication like glucocorticoids indicate that suppressing the response capacity of the immune system reduces disease severity. Largely unexpected, only 78 of 1227 tested cancer patients showed a COVID‐19 infection of which only three patients were symptomatic, ranging from mild symptoms to pneumonia. These data clearly contrast the view that oncology patients are particularly vulnerable to SARS‐CoV‐2 and suggest that necessary chemotherapies could be continued or started despite the ongoing pandemic. One explanation could be the immunosuppressive effect of simultaneous systemic therapy which will be further investigated. All patients visiting one of the seven participating oncological outpatient clinics were tested for SARS‐CoV‐2, regardless of symptoms, resulting in a total of 1227 patients screened for SARS‐CoV‐2 infection.In all clinics, infection protection measures according to the local health authorities were strictly followed. The patients treated in the outpatient clinics were constantly advised to act accordingly to basic hygiene rules and social distancing as recommended by the governmental authorities. SARS‐CoV‐2 carriers were consequently shielded from other patients, but nevertheless received individual treatment. Radiological (e.g. X‐ray or CT) or nuclear medicine (e.g. PET/CT or SPECT/CT) scans of the chest were not taken of each positively tested patient, but only in patients with symptoms suggesting a clinically significant affection of the lungs.All SARS‐CoV‐2 negative patients received their therapy as planned before the virus outbreak. Thus, an ongoing therapy for all tumor patients could be assured. COVID‐19‐related symptoms such as cough, shortness of breath, loss of taste and temperature above 37.5°C were registered for each patient visiting the outpatient clinic.Throat swabs were taken using CWY076 containing virus‐inactivating reagents from CWBIO . To increase staff safety, samples were heat‐inactivated for 30 minutes at 60°C. Afterwards, RNA was isolated using the MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit in combination with an extraction protocol on MGI SP‐960 machines in a 96 well format. Extracted RNA was analyzed by RT‐qPCR using the BGI Real‐time fluorescent RT‐PCR kit for detecting 2019‐nCoV2 on Applied Bioscience ABI7500 instruments according to the vendor's manual. The target region in the procedure is located within in SARS‐CoV‐2 ORF1ab, and human GAPDH served as an internal reference for effective RNA isolation and RT‐PCR. Positive and negative controls were included in each run.P‐values < .05 were regarded as statistically significant.Differences of clinical characteristics of the patients between the two subgroups (positive and negative SARS‐CoV‐2‐PCR results) were tested for statistical significance using the Chi‐square test. Due to multiple testing, test results were adjusted using the Bonferroni method. Adjusted 3In the period between 15 and 26 April 2020, a total of 1227 patients were tested for SARS‐CoV‐2 by RT‐PCR. The demographic and clinical description of the entire population and of the positively and negatively tested sub‐populations, respectively, is shown in Table Seventy‐eight (6.4%) patients were tested positive. Of all negative tested patients, 56.8% 653/1149) were female and 43.2% (496/1149) male, compared to 66.7% (52/ 78) female and 33.3% (26/78) male in patients with positive CoV‐2 test, respectively  of the 78 positively tested patients had mild symptoms such as shortness of breath and common cold symptoms at the time of testing, but these symptomatic patients did not suffer from any active tumor disease, neither received systemic therapy. Both patients were therapy‐naive and afebrile. One additional, positive tested patient (1.3%) was diagnosed with pneumonia requiring hospitalization, but did not require assisted ventilation according to a low CURB‐65 index. This patient suffered from an active tumor disease and received chemotherapy at the time of testing. Due to the patient's medical condition, the chemotherapy had to be aborted. Recently, the patient's COVID‐19 related symptoms have resolved, and chemotherapy has been continued. All symptomatic patients showed an ECOG performance status of 0 except for the hospitalized patient who showed an ECOG performance status of 1.The majority of patients with a positive test result (75/78) were solely asymptomatic virus‐carriers 96,2%). Forty‐four (56.4%) of the positively tested patients were undergoing systemic therapy: 23 (29.5%) chemotherapy, 6 (7.7%) antihormonal therapy, 8 10.3%) chemotherapy in combination with immunomodulating antibodies (Rituximab/Obinutuzumab/Atezulizumab), 3 immunotherapy (3.8%), and two (2.6%) tyrosin kinase inhibitor (TKI)  of the patients received glucocorticoids (Dexamethason or Prednisolon) as a co‐medication. 16.7% 13/78) of positively tested were under hypertension therapy with Ramipril, and thiazide diuretics were applied in 11.5% (9/78) , followed by non‐malignant hematological diseases . In the group of solid cancer, malignancies of digestive organs were reported most , followed by breast cancer  and malignancies of male genital organs . Twenty‐five of the positively tested patients who suffered from solid cancer disease, were reported with a stage M1 (32.1%) cancer disease and 23 with M0 (29.5%) , diabetes  and nicotine abuse   M1 in patients with positive SARS‐CoV‐2 test result compared to 13% (152/1149) in the group with negative test result) and undergoing no systemic oncological therapy (36% (28/78) in the positively tested group compared to 4% (47/1149) in the negatively tested group).4Contrasting our expectation of a disadvantageous disease outcome in oncology patients, the evaluation of our data showed that the majority of the oncological patients who received a positive SARS‐CoV‐2 test result were asymptomatic carriers of SARS‐CoV‐2 despite ongoing anti‐cancer therapy. This is particularly noteworthy since it was previously assumed that these patients would have a particularly severe course or at least become symptomatic in the event of infection.It is also noteworthy that most patients with an asymptomatic course of CoV2‐infection in our study received glucocorticoids (dexamethasone or prednisolone). This is in accordance with a recent press release from the RECOVERY‐study showing that patients receiving glucocorticoid therapy had an advantageous outcome reducing deaths by one‐third in ventilated patients..In our study, radiological (e.g. X‐ray or CT) or nuclear medicine (e.g. PET/CT or SPECT/CT) scans of the chest were not regularly taken of positively tested patient. However, as several publications showed that asymptomatic COVID‐19 patients may exhibit alterations in radiological (e.g. X‐ray or CT) or nuclear medicine (e.g. PET/CT or SPECT/CT) scans of the chest  of positively tested tumor patients showed symptoms. Based on this observation, it should be investigated in the further course of this pandemic whether tumor patients are even less susceptible to the symptomatic course of a SARS‐CoV‐2 infection. To substantiate this thesis, however, further data and validation are needed, especially in larger cohorts of tumor patients.Louisa Hempel, Jakob Molnar, Sebastian Robert, Julia Veloso, Zeljka Trepotec, Sofie English, Philip Weinzierl, Valeria Milani, Kathrin Schweneker, Bastian Fleischmann, Josef Scheiber, Axel Kleespies, Wolfgang Kaminski, Dirk Hempel and Armin Piehler have no conflict of interest. Human Rights statements and informed consent: All procedures were in accordance with the ethical standard of the responsible committee of the Bavarian Chamber Of Physician (BLÄK) with the ethic committee´s approval No. 20037 and with the Helsinki Declatation of 1964 and it´s later amendments.Louisa Hempel, Dietmar Zehn, Jakob Molnar and Dirk Hempel conceived and designed the study. Sebastian Robert, Julia Veloso, Josef Scheiber and Louisa Hempel acquired the data. All authors discussed the results and contributed the final manuscript. Louisa Hempel carried out the experiment with the help from Armin Piehler, Zeljka Trepotec, and Beate Gandorfer. Valeria Milani, Katrin Schweneker, Bastian Fleischmann, Axel Kleespies, Sabine Keim, Stefanie Mederle, and Florian Ebner collected the samples. Michael Pfaffl and Benedikt Kirchner helped supervise the project.


Cordyceps militaris (CM), a valuable edible and medicinal fungus, has been used as traditional medicine to treat health conditions, as well as hyposexuality in Asian societies for over a century. Due to the high demand, several artificial cultivation methods have been developed for their biological activities. In this study, CM was cultured on medium that contained white rice and silkworm pupae, and the levels of cordycepin and adenosine, as well as its aphrodisiac effects in diabetes-induced erectile dysfunction (DIED), were evaluated. Diabetic rats were induced by streptozotocin (STZ) injection and administered orally with CM  for 3 weeks. Diabetic rats in negative and positive control groups received vehicle and sildenafil citrate (5 mg/kg), respectively. Results showed the changes in mating behaviour in which mount latency and intromission latency were significantly increased in diabetic rats, compared with the normal control group. Diabetic rats also showed a significant reduction in intracavernosal pressure (ICP) response to cavernous nerve stimulation, sperm count, testosterone level, penile nitric oxide synthase (NOS), and testicular superoxide dismutase (SOD) activities, when compared to the normal control group. Administration of CM  reversed the effects of diabetes on the mating behaviour, and the ICP responses to electrical stimulation. Moreover, the levels of penile NOS, testicular SOD activities, testosterone, and sperm count were significantly increased, and testicular malondialdehyde (MDA) levels were significantly decreased in these treated diabetic rats. Diabetic rats treated with sildenafil showed a significant induction in intromission frequency and NOS and SOD activities, as well as a marked increase in ICP responses. These results suggest that CCM exerts its aphrodisiac effect, possibly through activating testosterone production and suppressing oxidative stress to enhance erectile function in diabetic rats. Erectile dysfunction or ED has been signified as an inability of the male to achieve a penile erection, as part of the overall multifaceted process of male sexual function . ED statCordyceps militaris (CM), a valuable medicinal mushroom in the family Clavicipitaceae [Alternative approaches, such as herbal medicine, have been adopted for sexual improvement for centuries. To date, many plants have been reported to possess aphrodisiac potential, and their effects on sexual behaviour have been validated . In Asiapitaceae . Nowadaypitaceae . Due to pitaceae –11. Sevepitaceae , 12–14. pitaceae , middle-pitaceae , and agepitaceae . HoweverCordyceps militaris was obtained from the Department of Agriculture (DOA) in Thailand. CCM was prepared at the Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University. In brief, the mycelia were cultivated with modified of potato dextrose agar (MPDA) medium under stable conditions at 22°C for 2 weeks. The resultant culture was transferred to potato dextrose broth (PDB) medium, which was then incubated on rotary shaker at 22°C for 2 weeks before transferring the mycelium to sterilized rice cultured medium that contained white rice and silkworm pupae. The fruiting bodies with the inoculums were kept in 12 : 12 h light-dark at 18°C, 60-70% humidity until the mycelium had transformed into the fruiting bodies primordia. Then, the flasks were maintained at 22°C, 80-90% humidity for 64 days before the fruiting bodies had been reaped and immediately frozen at -20°C until used.v/v) and sonicated for 30 min, followed by centrifugation at 9,900 g for 15 min 2 times. The obtained supernatant was then filtered through a 0.45 μm filter membrane before injecting into the HPLC system  with a column  set at 35°C. The mobile phase was a mixture of water and methanol  with the flow rate at 1 ml/min and a UV-vis detector at 254 nm. Five standard solutions of cordycepin and adenosine  (20 μl) were prepared and injected into the HPLC to create standard calibration curves.Cordycepin and adenosine in CCM were determined according to Huang et al. (2009) with some modifications. In brief, the fruiting bodies were dried in hot-air oven . The dried samples were ground using a homogenizer, and 1.0 g of powder was added into 10 ml of methanol : water  grade and were purchased from M-CLEA Bioresource Co., Ltd. . Procedures involving animal subjects were approved by the Naresuan University Animal Care and Use Committee (NUACUC). All animals were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals  with an effort to minimize animal suffering. Rats were maintained under controlled temperature (22 ± 1°C) and relative humidity (55 ± 10%) with 12 : 12 hours of reverse light and dark cycle at Naresuan University Centre of Animal Research (NUCAR), which has been accredited by AAALACi. All rats were fed ad libitum a standard diet  and allowed free access to reverse osmosis (RO) water.In order to induce diabetes, fifty male rats received a single intraperitoneal injection of STZ  , which was dissolved in citrate acid buffer (pH 4.5). Ten male rats in the normal control group received only citrate buffer. After 72 h, fasting blood glucose (FBG) levels were checked using glucometer . Rats with FBG levels higher than 200 mg/dl were used and divided into five groups: (I) DM control, (II) DM+CCM 0.1 g/kg, (III) DM+CCM 0.5 g/kg, (IV) DM+CCM 1.0 g/kg, and (V) DM+sildenafil 5 mg/kg. CCM were weighted and blended thoroughly with a blender and were given by daily oral gavage for 3 weeks. Sildenafil citrate was given only one time 30 min before mating behaviour test.For prevention of pregnancy, female rats were subjected to bilateral oophorectomy surgery before beginning the sexual function assessment. Each adult female rat was anaesthetized by 1.5-2.0% isoflurane  combined with oxygen. The lower abdominal skin and muscle were opened vertically 1 cm, and the uterine horn was pulled out and ligated before removal of the ovary, one at a time. The uterine horn was returned to the peritoneal cavity, and the wound was closed in two layers  using sterile sutures. The skin was then disinfected with povidone iodine and covered with Fixomull Stretch®. Each rat received an intramuscular tramadol (5 mg/kg) to ameliorate postoperative pain and allowed at least two weeks for full recovery. The ovariectomized female rats were artificially brought into oestrus phase by the administration of estradiol (0.025 mg/kg) and progesterone (1 mg/kg) at 48 and 4 hours before mating, respectively.Mount latency (ML). The time interval between the introduction of the female and the first mount by the maleIntromission latency (IL). The time interval between the introduction of the female and the intromission by the maleEjaculatory latency (EL). The time interval between the first intromission and ejaculationMount frequency (MF). The number of mounts from the time of introduction of the female until ejaculationIntromission frequency (IF). The number of intromissions from the time of introduction of the female until ejaculationEjaculation frequency (EF). The number of ejaculations in a sexual cycle.Mating tests were conducted in a custom made clear glass chamber 50 × 35 × 35cm. Each male rat was allowed to habituate in the chamber for 5 min before introducing a sexually receptive female rat into the chamber. The following male sexual behaviour parameters were calculated after monitoring for 30 min:Their behaviour was recorded with the digital VDO camera  for offline analysis by two observers to ensure accuracy.-gauge needle for measuring intracavernosal pressure (ICP). The lower abdomen was opened exposing the cavernous nerve, which was then stimulated via a copper bipolar electrode connected to the PowerLab®. The cavernous nerve was stimulated by electrostimulation, starting from 0.25, 0.50, 0.75, 1, 2, 3, 4, and 5 to 10 volts at a frequency of 20 Hz for 60 sec for each voltage. The results were recorded by LabChart  connected to a computer. Since electrostimulation slightly lowered MAP, ICP was normalized by MAP as ICP/MAP. Upon completion of experiment, rats were euthanized, and their penis and left testis were immediately collected and stored at -80°C for further analysis. The relative weights of penis and testis were calculated by the following formula: (weightofpenisorlefttestis/bodyweight) × 100.After completion of mating test, each male rat was anaesthetized with 2-2.5% isoflurane combination with oxygen. The ventilation rate, pulse rate, temperature, and heart rate were monitored via PhysioSuite® . The carotid artery of rat was cannulated to measure mean arterial blood pressure (MAP) by using PowerLab® . The penile skin was removed, and a polyethylene tube was inserted with heparinized saline via a 22μl of sample was transferred into the Makler chamber and analysed under a light microscope. Sperm motility was recorded as video files for offline analysis. The number of sperm was counted and expressed as × 106 per milliliter according to the WHO manual.Blood was collected from abdominal aorta and put into the nonanticoagulated tube and stored at 4°C before sending to the Biolab Medical Clinic, Phitsanulok, Thailand, for testosterone analysis. Semen was collected from the caudal epididymis and vas deferens and diluted in 1 M phosphate buffer saline (PBS) at 37°C. Then, 10 μm membrane filter, and then the supernatant was obtained by centrifuging the sample at 100,000 g, 4°C for 15 min. The penile NOS activity was measured, following the manufacturer's instructions. The absorbance at 540 nm was determined using a microplate reader . Blank wells were used to normalize the yield.NOS activity in the penis was determined using the nitric oxide synthase assay kit . The penile tissue was weighted and homogenized with ice-cold 1 M PBS (pH 7.4) by homogenizer . The sample was centrifuged at 10,000 g for 20 minutes and filtered through 0.45 w/v), pH 7.2 . The homogenate was centrifuged at 1,500 g for 5 min. The supernatant was collected and centrifuged at 10,000 g, 4°C for 15 min. The SOD activity in the sample was then processed according to the manufacturer's instructions and determined by a microplate reader with the absorbance at 450 nm.Testicular SOD was measured using the superoxide dismutase assay kit . Testis tissue was homogenized with buffer (10% w/v), in ice-cold 1 M phosphate buffer (pH 7.4). The homogenate was centrifuged at 4,000 g, 4°C for 15 min. Sample supernatant (100 μl) was added into the vial containing 1,500 μl of 20% acetic acid (pH 3.5), 200 μl of 8.1% sodium dodecyl sulphate (SDS), and 1,500 μl of 0.8% of thiobarbituric acid (TBA). The mixture was incubated for 60 min at 95°C and immediately cooled on ice, followed by centrifugation at 10,000 g for 3 min. The resulting supernatant was then used as the enzyme source for the determination of the malondialdehyde (MDA) level by the optical density (OD) measurement of the pink complex at 532 nm. MDA values were calculated using tetramethylpiperidine (TMP) as a standard curve and expressed as nmol/mg of protein that determined absorbance at a wavelength of 562 nm. Protein level was measured using the Pierce™ BCA Protein Assay Kit .Concentrations of TBARS in testes tissues were measured according to the method of Ohkawa et al.  with somμm by using semiautomatic microtome . All slides were stained with hematoxylin and eosin (H&E), and histological changes were observed by light microscopy.The right testis was fixed in 10% neutral buffered formalin over 24 hours. The tissues were then processed with 70-100% ethanol and xylene, respectively. The infiltrated tissues were embedded in paraffin blocks and cut at 2 P values of less than 0.05 were regarded as statistically significant.The value of mating parameters, sperm concentration, NOS and SOD activities, and MDA level were presented as mean ± SEM. ICP, MAP, and testosterone levels were presented as mean ± SD. Data were analysed using one-way analysis of variance (ANOVA) followed by Dunn's post-hoc Multiple Comparison Test . Cordyceps sp. [Many species of Cordyceps are being cultivated in artificial medium for their medicinal and pharmaceutical properties. Cordycepin, a derivative of the nucleoside adenosine, has been shown to be the first and is the main active constituent isolated from ceps sp. . In thisceps sp. . Howeverceps sp. .β-cells, inducing an impairment of insulin secretion. Further development of polydipsia, polyuria, and weight loss confirmed diabetes. The body weight change of diabetic rats was lower than those in normal rats , produced either by nerves or by endothelium, plays an important role in the relaxation of corpus cavernosum smooth muscle and vasculature . In thistarpekar .SOD is a necessary antioxidant enzyme which maintains body reactive oxygen species (ROS). In this study, SOD levels were measured from the testicular tissues, and the result of disclosed SOD activity in the DM control group was significantly reduced when compared with the normal control group. Interestingly, the SOD levels in the DM+CCM  and DM+Sildenafil groups were significantly augmented when compared with the DM group . On the C. militaris could be used as herbal drug for ameliorating reproductive dysfunctions secondary to DM.One of the reproductive complications of DM in men is erectile dysfunction. In this study, diabetes had a negative effect on sexual function, penile erection, sexual organ weight, and sperm parameters with a parallel decrease in the level of testosterone of male rats. The diabetic rats that received CCM showed a significant improvement in mating behaviour, erection function, and testicular function as observed by increasing ejaculation frequency, ICP/MAP ratio, and testosterone level, respectively. These improvements may be due at least in part to bioactive compounds that could improve testosterone production and its antioxidant properties. Additionally, cordycepin itself acts as adenosine analogue mediating the vasodilation to improve erectile function. Based on our findings, cultured