Rhododendron groenlandicum (Oeder) Kron & Judd; Ericaceae), present in the pharmacopoeia of the Cree of Eeyou Istchee, has shown glitazone-like activity in the 3T3-L1 adipogenesis bioassay. This activity has been attributed to phenolic compounds, which have been shown to vary in this plant as a function of insolation parameters. The goal of this study was to determine if these changes in phenolic content were pharmacologically significant. Leaves were harvested in 2006 throughout the James Bay region of Northern Quebec and ethanol extracts were tested in vitro using the 3T3-L1 murine cell line adipogenesis bioassay. This traditional medicinal plant was found active in the assay. However, there was no detectable spatial pattern in the accumulation of intracellular triglycerides, suggesting that such patterns previously observed in the phenolic profile of Labrador tea were not pharmacologically significant. Nonetheless, a reduction in the adipogenic activity was observed and associated with higher concentrations of quercetin for which selected environmental variables did not appropriately explain its variation.The traditional medicinal plant, Labrador tea ( Rhododendron groenlandicum (Oeder) Kron & Judd (Ericaceae), Labrador tea, we found the concentration of biologically active compounds to vary in Northern Quebec's Hudson and James Bay region [In a previous study on the phytochemistry of the North American medicinal plant,y region . Labradoy region –9. R. groenlandicum to be the top-ranked plant species used for the treatment of symptoms associated with type 2 diabetes (T2D) [In ethnobotanical studies conducted in six communities of the Cree Nation of Eeyou Istchee (CEI), we foundes (T2D) –13. The  R. groenlandicum was shown to possess in vitro glitazone-like activity comparable to rosiglitazone in an adipogenic assay measuring the lipid accumulation in differentiating 3T3-L1 preadipocytes [The CIHR Team on Antidiabetic Aboriginal Medicines (CIHR-TAAM), formed through collaborative work between CEI communities, the Cree Board of Health and Social Services of James Bay (CBHSSJB), and Canadian academic researchers, has screened many of the multiple plants present in the CEI pharmacopoeia –17. Of tipocytes .γ receptor agonist [γ results in an improvement in the absorption of fatty acids in differentiated adipocytes which store them as triglycerides (TG) [The antidiabetic drug rosiglitazone induces an increase in the sensitivity to insulin, acting as a PPAR agonist ; the exp agonist –22 and i agonist . Hence, des (TG) . Adipocy R. groenlandicum has been attributed to phenolic compounds [ R. groenlandicum has important implications in ensuring the quality control of traditional medicinal plants or to develop standardized natural health products (NHPs).The pharmacological activity ofompounds , 16, 23.ompounds . In deve R. groenlandicum. Our objective was to determine if the phytochemical variations observed in the species' phenolic profile are biologically significant. We evaluated the in vitro pharmacological activity of crude extracts from various localities using the adipogenesis bioassay and hypothesized that high concentrations of phenolic compounds would result in a stronger adipogenic activity.In this study, we assessed possible variations in the antidiabetic potential of R. groenlandicum. A subsample, selected randomly, of previously reported samples was used in this in vitro study.The sampling, extraction, and analytical methods for phytochemical identification and quantification are thoroughly described in Rapinski et al. , which rBriefly, mature leaves were sampled during the summer of 2006 around the communities of Mistissini, Nemaska, Eastmain, Wemindji, and Whapmagoostui, thus covering much of the northsouth gradient in Eeyou Istchee. Five accessions, each containing leaves from multiple individual plants, were collected within a 50 km radius around each community and selected for this study. Samples were air-dried and preserved in paper bags at room temperature.Samples were milled through a Wiley Mill at 40 mesh and extracted overnight in 25 mL/g of 80% EtOH by orbital shaking at room temperature at 250 RPM. The pellet was extracted overnight in 15 mL 80% EtOH. An aliquot (1 mL) of the pooled supernatants (adjusted to 50.0 mL in a volumetric flask) was prepared for High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD). The leftover crude extracts were dried using a speedVac; the trace water was removed by lyophilization using SuperModulo freeze dryer and the dehydrated extracts were stored at −80°C.μL of each extract aliquot was injected through an autosampler and detected by DAD at 290 nm, bandwidth 4, reference off. The separations were performed on a Luna C18 column . Peak identification was undertaken by cochromatographic comparison of the spectral data adopted in our in-house metabolomics spectral library [Finally, 10  library . A standμM DMX, 250 μM IBMX, and 500 nM insulin. After 48 h, the media were replaced with DMEM containing 10% FBS and 500 nM insulin for long-term differentiation. Cells were differentiated for a total of 5 days with media change every 2 days. Rhododendron groenlandicum crude extracts (75 μg/mL) and rosiglitazone  were dissolved in DMSO and added to the cells as of day 0 of differentiation. The final concentration of DMSO was kept at 0.1% throughout the differentiation period.3T3-L1 murine preadipocyte cells were grown to confluence in 24-well plates in DMEM proliferation medium containing 10% FBS. Media were changed every 2 days. At 24 h after confluence (day 0), cells were induced to differentiate with a short-term differentiation medium of DMEM supplemented with 10% FBS, 1 μL of AdipoRed reagent was added to each well and incubated for 15 minutes at room temperature. AdipoRed becomes fluorescent when partitioned in a hydrophobic compartment, namely, intracellular triglycerides (TG). The fluorescence of each well was measured with a Wallac Victor2 fluorimeter  at an excitation wavelength of 485 nm and an emission wavelength of 572 nm. The results were reported as percentage of the vehicle control, 0.1% DMSO.We measured intracellular TG content at day 5 of differentiation using the AdipoRed reagent according to the manufacturer's instructions. Methods have been previously described in Spoor et al.  and Harb1), chlorogenic acid (2), (−)-epicatechin (3), p-coumaric acid (4), rutin (5), quercetin-3-galactoside (6), quercetin-3-glucoside (7), quercetin-3-rhamnoside (12), myricetin (13), and quercetin (14) were purchased from Sigma-Aldrich  and Extrasynthese . HPLC grade water, acetonitrile, and formic acid (99% purity) were purchased from Sigma-Aldrich.For the identification and quantification of phenolic markers, (+)-catechin . Dexamethasone (DMX), bovine pancreatic insulin, 3-isobutyl-1-methylxanthine (IBMX), and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Rosiglitazone was obtained from Alexis Biochemicals .Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and bovine calf serum (NCS) were from Wisent Inc. . AdipoRed reagent was purchased from Cambrex Bio Science Walkersville Inc. .Annual estimates of bioclimatic variables, namely, annual temperature range, corresponding to our sampling year and long-term estimates for insolation variables were provided by the Canadian Forest Services of Natural Resources Canada . Long-te Rhododendron groenlandicum and rosiglitazone always induced significant increases in activity as verified by the fact that the 95% confidence interval of the mean activity (quadruplicate determinations) did not include the 100% adipogenic activity reference (p < 0.05). Differences between communities were analyzed by one-way analysis of variance. The relationships between TG content and compounds were analyzed by multiple and simple linear regressions. To represent the adipogenic activity of R. groenlandicum and the quantified compounds, principal components analysis (PCA) was performed on the matrix of these compounds using the correlation matrix. Individual samples were scored onto the PCA axes and represented with the vectors for each compounds. TG content was subsequently projected as a supplementary variable onto the principal components in order to interpret the dimensions of variability. In doing so, the calculation of distances between each of the samples and the construction principal components depends only on their phytochemical profile. Using the transition formulae described by Lê et al. [ R. groenlandicum between the two sets of variables: compounds and environmental factors. This was done using a partial-redundancy analysis  approach [α = 0.05.To reduce interassay variation, TG content was normalized relative to each assay's vehicle control, 0.1% DMSO, set at 100%.ê et al. , the cooapproach , 30. Allapproach . Results R. groenlandicum accessions has already been described and discussed in greater length in Rapinski et al. [The phytochemistry of the samei et al. . Here, w R. groenlandicum to increase the accumulation of intracellular TG in 3T3-L1 adipocytes was measured at day 5 of differentiation. Extracts increased adipogenesis, with an average content of TG of 159.0% that of DMSO , possibly due to high variability. This suggests that variations in the phytochemical profiles, observed in Rapinski et al. [There were no statistically distinct spatial patterns detected in the pharmacological activity ofp = 0.0279, Radj2 = 0.491). The distribution of R. groenlandicum samples based on their phytochemical profile was reconstructed into a reduced three-dimensional space, which represented 69.92% of the samples' variation over three statistically constructed principal components, or axes (λ1 = 3.96), 22.97% (λ2 = 2.53), and 10.96% (λ3 = 1.20) of the variation. The direction and proximity of arrows for some major markers suggest that these are highly correlated  whereby the adipogenic activity of R. groenlandicum decreases as the concentration of quercetin in the sample increases. This is consistent with observations from our own group [Indeed, we found that quantified compounds explained considerable variability obtained in this species' pharmacological activity , we found, nonetheless, that they explained an important proportion of the variation in the phytochemical profiles of R. groenlandicum, which could significantly explain TG content , their unique contribution to explaining TG content no longer was when the contribution of environmental variables, albeit small, was taken into account and removed .Our results suggest that while the geographical location does not appear to have a statistically significant impact on the adipogenic activity of crude extracts of localizedompounds . Althoug R. groenlandicum, was not found to be strongly associated with environmental variables [This confirms the caveat that environmental variables play an underlying role in affecting the content of biologically active compounds. Conversely, quercetin, the only significant compound related to changes in the adipogenic activity ofariables . This ma Hibiscus sabdariffa  was greater than the sum of its parts when polyphenols had been fractionated, isolated, and tested individually [On the other hand, our results provide support for the hypothesis that synergistic interactions may occur between compounds. For instance, the inhibitory action ofvidually . More imvidually . Finallyvidually .μM) but was not found to affect adipogenic activity [Many of the compounds quantified in this paper have shown adipogenic activity in some form or another. Quercetin-3-glucoside has been found toxic to adipocytes at relatively low concentration (50 activity . Quercetactivity . Contentactivity . Althoug R. groenlandicum. Increase in the adipogenic potential of this traditional medicine was associated primarily with lower concentrations of quercetin, but the cause for its variation will require further investigation. Nonetheless, our results do not provide enough evidence to justify the idea that specific accessions of Labrador tea may have reproducibly better adipogenic potential than others along a latitudinal gradient. Conversely, our study implies that random harvesting of R. groenlandicum in the Eeyouch territory of Northern Quebec should not have a major impact on the quality of traditional preparations or NHPs made from this plant.We have previously shown that latitude acted as a marker for the impact of environmental variables on phytochemical concentrations . Therefo


Influenza virus infection (IVI) can cause primary viral pneumonia, which may progress to acute lung injury (ALI) and respiratory failure with a potentially fatal outcome. At present, the interactions between host and influenza virus at molecular levels and the underlying mechanisms that give rise to IVI-induced ALI are poorly understood. We conducted a comprehensive mass spectrometry-based metabolic profiling of serum, lung tissue and bronchoalveolar lavage fluid (BALF) from a non-lethal mouse model with influenza A virus at 0, 6, 10, 14, 21 and 28 days post infection (dpi), representing the major stages of IVI. Distinct metabolite signatures were observed in mice sera, lung tissues and BALF, indicating the molecular differences between systematic and localized host responses to IVI. More than 100 differential metabolites were captured in mice sera, lung tissues and BALF, including purines, pyrimidines, acylcarnitines, fatty acids, amino acids, glucocorticoids, sphingolipids, phospholipids, etc. Many of these metabolites belonged to pulmonary surfactants, indicating IVI-induced aberrations of the pulmonary surfactant system might play an important role in the etiology of respiratory failure and repair. Our findings revealed dynamic host responses to IVI and various metabolic pathways linked to disease progression, and provided mechanistic insights into IVI-induced ALI and repair process. Influenza virus infection (IVI) causes annual epidemics, which result in an estimated 1 billion infections including 3–5 million severe cases and 250,000–500,000 mortality cases worldwide. Two therapeutic strategies, vaccination and antiviral drugs, are currently used to control IVI. However, vaccines need to be updated annually and are effective only if they match with the current circulating virus. Although antiviral drugs may reduce complications of IVI, they need to be given early during infection or used as prophylaxis, and certain strains of influenza viruses can develop resistance to these drugs. As a result, IVI continues to pose a major challenge to the global healthcare systems and there is an urgent need for novel treatment strategies. It has been proposed that host immune responses contributing to severe pathology need to be identified for developing new therapeutics, irrespective of the infecting strains1While the most common symptoms of IVI include fever, runny nose, sore throat, cough and fatigue, susceptible populations can have a variety of more severe complications including primary viral pneumonia, which can progress to acute lung injury (ALI) and respiratory failure with a potentially fatal outcome46911Metabolomics, the analysis of the changing metabolite levels in biological systems in response to biological stimuli or perturbations, is a rapidly evolving field in systems biology1618192022We first established the temporal pathophysiologic profiles of the mouse model in response to IVI and the results were consistent with previous characterization of the same model252627In order to obtain reliable metabolic profiles of the samples, it is important to ensure the robustness of the analytical method. We first evaluated the stability and reproducibility of the LC-MS method by performing PCA on all the samples including the 6 QC samplesp < 0.05 in one-way ANOVA with Benjamini-Hochberg Multiple Testing Correction; (ii) FC ≥ 1.5; (iii) signal intensity ≥20,000 counts (approximately three times the limit of detection of our LC/MS instrument)10H12N5O6P of the m/z 329.05 ion was calculated based on the exact mass, the nitrogen rule and the isotope pattern by Masshunter software from Agilent (http://www.hmdb.ca/), METLIN (http://metlin.scripps.edu/) and MassBank (http://www.massbank.jp/). Next, MS/MS experiments were performed to obtain structural information via the interpretation of the fragmentation pattern of the metabolite, and the characteristic fragment ion of adenosine base was clearly observed in both positive (m/z 136.06) and negative (m/z 134.04) MS/MS spectra   Agilent . Then, t spectra . The MS/ spectra .With above-mentioned method, 41, 55 and 69 differential metabolites were identified in sera, lung tissues and BALF, respectively, of which they belonged to a variety of metabolite classes . In seruwww.genome.jp/kegg/). In the overview graph of pathway analysis with MetabolAnalyst  Guidelines  in facilities licensed by the Agri-Food and Veterinary Authority of Singapore (AVA), the regulatory body of the Singapore Animals and Birds Act. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC), National University of Singapore. Mice were monitored every day after infection, and any mouse with 30% body weight loss will be euthanatized immediately.g for 10 minutes at 4 °C and the supernatant was collected and evaporated to dryness in a vacuum concentrator. The dry extracts were then resuspended in 100 μL of 98:2 water/methanol for liquid chromatography/mass spectrometry (LC-MS) analysis. Quality control (QC) samples were prepared by mixing equal amounts of serum or BALF from all the samples and processed as real samples. All samples were kept at 4 °C and analyzed in a random manner. The QC sample was run after each 8 samples to monitor the stability of the system.The procedure for serum and BALF sample preparation followed our previously published reportg for 10 min at 4 °C and the supernatant was transferred to an Eppendorf tube. Then 700 μL of prechilled methanol/water (1:1) was added to the pellet and vortexed. The mixture was again centrifuged at 17,000 g for 10 min at 4 °C and the supernatant was combined with the supernatant from previous step. The mixed supernatant was lyophilized to dryness in a vacuum concentrator. The dry extracts were resuspended in 200 μL of 98:2 water/methanol for LC-MS analysis, and 20 μL from each sample was pooled as QC samples. All samples were kept at 4 °C and analyzed in a random manner. The QC sample was run after each 8 samples to monitor the stability of the system.The procedure for lung tissue preparation followed published report with some modificationsThe metabolomics analysis followed our previously published report with some modificationsm/z 100 and 1000 at a rate of two scans per second. The ion spray voltage was set at 4,000 V for positive mode and 3,500 V for negative mode. The heated capillary temperature was maintained at 350 °C. The drying gas and nebulizer nitrogen gas flow rates were 12.0 L/min and 50 psi, respectively. Two reference masses were continuously infused to the system to allow constant mass correction during the run: m/z 121.0509 (C5H4N4) and m/z 922.0098 (C18H18O6N3P3F24).The electrospray ionization mass spectra were acquired in both positive and negative ion mode. Mass data were collected between m/z values, and the data were also normalized by internal standard. For statistical analysis, one-way ANOVA (p < 0.05) with Benjamini-Hochberg Multiple Testing Correction was employed. The fold change (FC) analysis was also performed between the group at each of the other time points relative to the control group to further filter the features and only those features with FC > 1.5 were selected as potential significantly altered metabolites. Unsupervised multivariate analysis principal components analysis (PCA) was performed with the Unscrambler-X statistical software package . Supervised multivariate analysis orthogonal partial least-squares discriminant analysis (OPLS-DA) was performed with the SIMCA-P software . The features with Variable Importance in the Projection (VIP) values greater than 1 were considered to be influential for the separation of samples in OPLS-DA analysis.Raw spectrometric data were analyzed by MassHunter Qualitative Analysis software  and the molecular features, characterized by retention time (RT), chromatographic peak intensity and accurate mass, were obtained by using the Molecular Feature Extractor algorithm. The features were then analyzed by MassHunter Mass Profiler Professional software . Only features with an intensity ≥20,000 counts (approximately three times the limit of detection of our LC/MS instrument) and found in at least 80% of the samples at the same sampling time point signal were kept for further processed. Next, a tolerance window of 0.15 min and 2 mDa was used for alignment of RT and http://www.lipidmaps.org/), HMDB (http://www.hmdb.ca/), METLIN (http://metlin.scripps.edu/) and MassBank (http://www.massbank.jp/). Next, MS/MS experiments were performed to obtain structural information via the interpretation of the fragmentation pattern of the metabolite. The MS/MS spectra of possible metabolite candidates in the databases were also searched and compared. Finally, the metabolites were confirmed by comparison with the standards when they are commercially available. For metabolic pathway analysis, Ingenuity Pathway Analysis software  and metaboAnalystThe structure identification of the differential metabolites was based on our published work2 , secretory phospholipase A2 , Perilipin A (ab3526) and hydroxysteroid (11-beta) dehydrogenase 1  . All sections were image at 5×, 10× and 40× with Mirax Midi slide scanner (Carl Ziess).Mouse lung tissues were collected at 0, 5, 9 and 13 dpi, and processed as previously describedHow to cite this article: Cui, L. et al. Metabolomics Investigation Reveals Metabolite Mediators Associated with Acute Lung Injury and Repair in a Murine Model of Influenza Pneumonia. Sci. Rep.6, 26076; doi: 10.1038/srep26076 (2016).


Formation of microaneurysms plays an important role in the development of diabetic retinopathy and nephropathy. It remains unknown if DHI can reduce these diabetic complications. In this study, diabetic db/db mice in two groups were injected with saline and DHI, respectively, for 14 weeks. Blood and tissue samples were collected to determine serum glucose, lipids and tissue structure. DHI reduced diabetes-induced body weight gain, serum cholesterol and glucose levels. In retinas, DHI blocked the shrink of whole retina and retinal sub-layers by inhibiting expression of caspase 3, matrix metalloproteinase 2 (MMP-2) and MMP-9, accumulation of carbohydrate macromolecules and formation of acellular capillaries. DHI improved renal functions by inhibiting mesangial matrix expansion, expression of vascular endothelial growth factor A, fibronectin and advanced glycation end products in kidneys. Mechanistically, DHI induced expression of glucokinase, AMPKα/phosphorylated AMPKα, insulin receptor substrate 1, fibroblast growth factor 21 and peroxisome proliferator-activated γ. Expression of genes responsible for energy expenditure was also activated by DHI. Therefore, DHI inhibits diabetic retinopathy and nephropathy by ameliorating glucose metabolism and demonstrates a potential application in clinics.Danhong Injection (DHI), a Chinese medicine for treatment of patients with coronary heart disease, inhibits primary abdominal aortic aneurysms in apoE deficient (apoE The number of diabetic patients is expected to be more than 500 million in 2030 globallyLiver plays an essential role in whole body glucose homeostasis which can be regulated by several molecules in the liver8Formation of advanced glycation end products (AGEs) is another risk factor for the development of diabetic complications12Salvia miltiorrhiza Bge, Labiatae, Danshen in Chinese) and Flos Carthami  are the well-known traditional Chinese medicines. They have been used widely and successfully to treat patients with cardiovascular diseases for a long history in China17−/−) mice which further suggests the protective effects of DHI in the vascular system21db/db mice. We then determined the involved mechanisms, in particular the effects of DHI on signaling pathways for insulin sensitivity, metabolism of glucose, lipid and energy.Both the herbal Radix Salviae Miltiorrhizae (db/db mice (~6-week-old) were i.p. injected with equal volume of saline or DHI based on body weight. C57BLKS/J wild type mice at the same age received saline injection as a normal (nondiabetic) control. The treatment was continued for 14 weeks. After treatment, retina samples from all the mice were collected and prepared retinal sections followed by HE staining. The retinal thickness and structural alterations were determined with the light microscopy. The whole retinal thickness is defined as the distance from the edge of ganglion cell layer (GCL) to the edge of retinal pigment epithelium (RPE). Compared to wild type mice, db/db mice was significantly reduced. However, treatment of db/db mice with DHI restored it to normal level indicting that DHI can maintain the integrity of retinal structure. In fact, the reduced thickness of sub-layers, such as outer plexiform layer (OPL), outer nuclear layer (ONL) and photoreceptor layer (IS + OS), in the retinas of db/db mice were restored to the levels of wild type mice, respectively  and MMP-9 can enhance the development of diabetic retinopathy due to their pro-apoptotic actions24db/db mice were substantially inhibited by DHI treatment.Cellular apoptosis is a hallmark of early diabetic retinopathy in humans and animal models. High glucose levels can induce caspase 3 (CAS-3) expression which can enhance cellular apoptosisdb/db mice can correct the diabetes-induced retinal structural abnormalities and inhibit the development of diabetic retinopathy. The action might be attributed to the anti-apoptotic effects of DHI.Taken together, the results in db/db mice against nephropathy, urine samples for a 24 h duration at the different time points of treatment were collected and determined excreted nitrogen, creatinine and proteins. To determine if DHI can also protect db/db mice appeared similar to wild type mice, the kidneys from 3 control db/db mice  demonstrated abnormalities with un-matched size and severe lipid accumulation  proteins changes .db/db mice with DHI decreased fibronectin expression in both glomeruli and tubules with a greater effect on glomeruli.Fibronectin is a high-molecular weight (~260 kDa) glycoprotein of ECM and binds ECM components, such as collagen, fibrin and heparan sulfate proteoglycans. Expression of fibronectin is often used as an index to evaluate the levels of matrix accumulation in the presence of elevated glucose levelsdb/db mice . However, DHI treatment reduced the increase to ~25% (41.6 ± 0.5 vs. 33.2 ± 0.4 g). In addition, we checked the food intake during the treatment and observed no significant difference between control db/db mice and mice receiving DHI treatment (based on the body weight).During the treatment, we routinely checked mouse body weight. db/db mice were increased with time (vs. 12.75 ± 1.1 mM). However, the increased glucose levels was substantially inhibited soon after DHI treatment (~two weeks). At the end, a moderate increased glucose levels  were observed in db/db mice receiving DHI treatment  levels were much higher in control lesterol . Treatmedb/db mouse liver and peripheral tissues was assessed. DHI had little effect on both PCK1 and G6Pase expression  and hormone-sensitive lipase (HSL) catalyze hydrolysis of TG into diacylglycerol and fatty acid, the first and rate-limiting step in TG hydrolysis although HSL can act on hydrolysis of diacylglycerol3133Reduced body weight gain may contribute to improvement of diabetic phenotypes in Taken together, the results in db/db mice inhibited both diabetic retinopathy and nephropathy. The inhibitory effect is mainly attributed to the reduction of hyperglycemia. Mechanistically, DHI increased expression of GCK, AMPKα and pi-AMPKα in the liver indicating the glycolysis is enhanced. Meanwhile, DHI induced expression of IRS1, FGF21 and PPARγ in the liver or/and peripheral tissues which can increase insulin sensitivity. Induction of genes involved lipolysis, fatty acid oxidation and mitochondrial biogenesis suggests that DHI may enhance energy metabolism. Moreover, DHI inhibited CAS-3, MMP-2 and MMP-9 expression and formation of acellular capillaries in retinas that can prevent diabetes-induced apoptosis and protect retinas against diabetes-induced damage. Inhibition of VEGFA and fibronectin expression in the kidney by DHI can reduce mesangial expansion and accumulation of AGEs or other macromolecules, and ameliorate diabetes-induced proteinuria.Both diabetic retinopathy and nephropathy are frequently observed in the patients with a long history of diabetes. The diabetic retinopathy can eventually lead to blindness while the diabetic nephropathy is the leading cause of chronic renal disease and a major cause of cardiovascular mortality in the patients. In this study, we demonstrate that administration of DHI to in vivo and in vitro, respectively384244In vivo, salvianolic acid B significantly suppresses the glomerular hypertrophy, mesangial expansion and renal fibronectin levels in STZ-induced diabetic ratsdb/db mice.Hyperglycemia stimulates biosynthesis of ECM in mesangial cells which can cause glomerular dysfunction38db/db mice. Activity of AMPKα in the liver is inversely related to the development of diabetes528Glucose homeostasis in mammals is tightly controlled through regulation of glucose production/catabolism in the liver and glucose uptake/utilization in peripheral tissues. In type 2 diabetes, decreased hepatic insulin sensitivity leads to altered glucose metabolism and thereby hyperglycemia4849db/db mice in the control group, DHI substantially reduced body weight gain after about one month treatment  and prepared as describedin vivo study with mice were approved by the Ethics Committee of Nankai University , and the methods for in vivo study were carried out in accordance with the approved guidelines. Both male type 2 diabetic  and C57BLKS/J wild type mice (6-week-old) were purchased from the Animal Center of Nanjing University . The animals were maintained at the Animal Center of Nankai University with free to access food and drinking water.The protocols for 50 of DHI was 39.5 ml/kg for mice by i.v. injectiondb/db mice were randomly divided into two groups (10 mice/group) and daily i.p. injected with saline  and DHI , respectively. Male C57BLKS/J wild type mice were i.p. injected with saline as a nondiabetic or normal control.According to the previous reports, the safety of DHI had been evaluated and the calculated LD2 chamber. Blood samples were collected and kept for 2 h at room temperature (RT). After centrifugation for 20 min at 2,000 g, the serum was transferred into a new test tube and kept at −20 °C. To determine cholesterol levels, serum samples (~150 μl/sample) were diluted with 1xPBS (1:1), and then loaded onto an automatic biochemical analyzer  to determine cholesterol levels. Serum samples were also used to determine FGF21 levels using a mouse FGF21 ELISA kit  according to the manufacture’s instruction.Blood was withdrawn from mouse tail vein after overnight fasting at the different time points after treatment and determined glucose levels with an OneTouch glucometer and test strips  according to the manufacture’s instruction. At the end of treatment, all the animals were anesthetized and euthanized in a CO2 of retina.The retinal vasculature was prepared based on the described methodTo evaluate the retinal thickness and structural alterations, eyeballs were fixed in fresh 4% paraformaldehyde/PBS for 12 h at 4 °C, and then cryo-protected in 30% sucrose/PBS overnight before quick frozen in OCT compound solution . The 5 μm frozen cross sections were prepared by a standard procedure. The sections were stained with hematoxylin and eosin (HE) for evaluation of the retinal structure. Expression of CAS-3 was determined by immunofluorescent staining as follows: the sections on cover slides were incubated with rabbit anti-CAS-3 polyclonal antibody overnight at 4 °C. After removal of the primary antibody by washing with PBS, the slides were stained with rhodamine-conjugated goat anti-rabbit IgG for 2 h at RT. After washed with PBS, the slides were re-stained with DAPI solution for nuclei. All the slides were viewed and photographed with a fluorescence microscope.At the indicated durations, the animals were housed in metabolic chambers  for 24 h to collect urine samples. The urinary protein concentrations were determined by BCA method. The content of nitrogen and creatinine in urine samples were determined with the assay kits purchased from BioSino Bio-technology and Science Inc.  according to the manufacturer’s instruction.After treatment, kidney 5 μm cross sections were prepared and conducted PAS or HE staining, and the glomerular area was quantified. The sections were also used to determine expression of fibronectin and AGEs by immunofluorescent staining. Expression of VEGFA in the kidney was determined with total cellular protein by Western blot as describedTo determine lipid content, kidney frozen sections were rinsed with 60% isopropanol and then stained with freshly prepared Oil Red O working solution (3 mg/ml in 60% isopropanol) for 45 min. The sections were then rinsed with 60% isopropanol and lightly stained for nuclei with alum hematoxylin solution. The sections were finally rinsed with distilled water, mounted in an aqueous mountant, viewed and photographed with a microscope.A piece of liver, muscle and white adipose tissue (WAT) were collected and used to extract total cellular protein followed by determination of protein expression of glucose metabolism related molecules by Western blotAfter treatment, mice retinas, a piece of liver and WAT were collected and homogenized in Trizol reagent  to extract total cellular RNA. The cDNA was synthesized with the first-stand cDNA synthesis Kit from Fermentas . Expression of CAS-3, MMP-2, MMP-9, FGF21, PPARγ, ATGL, HSL, CPT1α, LCAD, MCAD, ACOX1, Cyto-C, ATPase and PPARα mRNA was determined by real time RT-PCR using a SYBR green PCR master mix from Bio-Rad  and the primers listed in To quantify TG content, a piece of liver (~50 mg) was homogenized in ~1.1 ml 1xPBS. A portion of the homogenate (100 μl) was saved for determination of protein content which was used to normalize TG levels; 1 ml homogenate was then used to extract total lipids followed by TG quantitative analysis with an assay kit.p < 0.05 (n ≥ 5).All experiments were repeated at least three times, and the representative results are presented. Data were presented as mean ± standard errors and analyzed by a Student’s t-test using Prism (GraphPad Software). The differences were considered significant at How to cite this article: Liu, M. et al. Administration of Danhong Injection to diabetic db/db mice inhibits the development of diabetic retinopathy and nephropathy. Sci. Rep.5, 11219; doi: 10.1038/srep11219 (2015).


In conclusion, we performed an integrative analysis to offer the convincing evidence that WTD may attenuate RA partially by restoring the balance of NEI system and subsequently reversing the pathological events during RA progression.Wu-tou decoction (WTD) has been extensively used for the treatment of rheumatoid arthritis (RA). Due to lack of appropriate methods, pharmacological mechanisms of WTD acting on RA have not been fully elucidated. In this study, a list of putative targets for compositive compounds containing in WTD were predicted by drugCIPHER-CS. Then, the interaction network of the putative targets of WTD and known RA-related targets was constructed and hub nodes were identified. After constructing the interaction network of hubs, four topological features of each hub, including degree, node betweenness, closeness and k-coreness, were calculated and 79 major hubs were identified as candidate targets of WTD, which were implicated into the imbalance of the nervous, endocrine and immune (NEI) systems, leading to the main pathological changes during the RA progression. Further experimental validation also demonstrated the preventive effects of WTD on inflammation and joint destruction in collagen-induced arthritis (CIA) rats and its regulatory effects on candidate targets both  Rheumatoid arthritis (RA), as a systemic autoimmune disease, is principally characterized by the presence of inflammatory synovitis, the predominance of pro-erosive mediators, and the progressive destruction of cartilage and boneRadix Aconiti (Wu Tou), Herba Ephedrae (Ma Huang), Radix Astragali (Huang Qi), Raidix Paeoniae Alba (Bai Shao) and Radix Glycytthizae (Gan Cao), and widely produced in China in accordance with the China Pharmacopoeia standard of quality control. In clinical practice, WTD has been extensively used for the treatment of rheumatic arthritis, RA, constitutional hypotension and hemicraniaRadix Aconiti is the primary component and is believed to be effective in treating rheumatic arthritis and RA; Herba Ephedrae serves as the ministerial component to intensify the analgesic function of Radix Aconiti; Radix Astragali acts as the adjunctive component to invigorate qi , strengthen the body and reinforce the effect of Radix Aconiti and Herba Ephedrae; Raidix Paeoniae Alba and Radix Glycytthizae are both messenger drugs which can either focus the actions of the formula on a certain area of the body or harmonize and integrate the actions of the other herbs of the formula6Radix Aconiti, total glycosides and polysaccharides of Raidix Paeoniae Alba, and polysaccharides of Radix Astragali, may have excellent anti-inflammatory and anti-oxidant activities8Wu-tou decoction (WTD), as a classic TCM formula, was originally recorded in “Jin Kui Yao Lve” written by Chinese medical sage Zhang Zhongjing. It is prepared from a basic formula of five Chinese herbs, including Because a herbal formula with numerous compositive compounds is too complex to be detected solely by conventional experimental methods, there is an urgent need to develop new and appropriate approaches to address this problem. Network pharmacology, combined with pharmacology and pharmacodynamics, is a novel research field which is implicated in the application of omics and systems biology-based technologies1214Following the drug target prediction by drugCIPHER-CSRaidix Paeoniae Alba and Radix Glycytthizae shared more common putative targets with Radix Aconiti  in generalhttp://bidd.nus.edu.sg/group/cjttd/, Aug 25th, 2011). As shown in Radix Aconiti has acetylcholine activityRadix Aconiti shown in Radix Astragali, Herba Ephedrae and Radix Aconiti, is a glucocorticoid receptor, indicating the glucocorticoid activity of WTD, which was in line with the findings of previous studiesRadix Aconiti27Generally, drug indication for use is determined by functions of its affected targets. In the current study, we collected the known anti-RA drugs with the same targets of herbs in WTD from Therapeutic Target DatabaseIn order to comprehensively understand the pharmacological mechanisms of WTD, we constructed the interaction network of putative targets and known RA-related targets. According to the previous study of Li et al.To screen the major hubs, 4 topological features, 'Degree,' 'Node betweenness', 'Closeness' and 'K value' , were calculated for each hub in the network. The median values of 'Degree', ' Node betweenness ', 'Closeness' and 'K value' were 21.00, 0.13, 39.34 and 14.00, respectively. Therefore, we determined that hubs with 'Degree'>21.00, ' Node betweenness'>0.13, 'Closeness'>39.34, and 'K value'>14.00 were major hubs. As a result, 121 major hubs were identified , in which joint damage/bone destruction, inflammation/synovial pannus formation and angiogenesis are three main pathological phenotypes of RA patients. Thus, we speculated that the anti-RA effects of WTD might be associated with the roles of its targets in reversing the imbalance of NEI system and subsequently in the regulation of downstream RA-related pathways, including osteoclast differentiation, T cell receptor signaling pathway, toll-like receptor signaling pathway and VEGF signaling pathway -1/MMP-13, IL-1β/TNF-α and hypoxia-inducible factor (HIF)-1α/VEGF axes have been indicated as key players of NEI system, osteoclast differentiation, inflammation and VEGF signaling pathway involved during RA progression, respectively. Since the regulatory effects of WTD on these candidate targets have not been fully elucidated, we further performed experimental validation to address this problem based on ·day) and 3.8 g/(kg·day) WTD significantly attenuated arthritis severity in CIA rats. Additionally, the mean arthritis score . MTX also reduced significantly the inflammation score and bone destruction score of inflamed joints compared with vehicle-treated CIA rats  WTD significantly reduced the expression of MMP-1 , which is currently the largest non-commercial TCM database worldwide. TCM Database@Taiwan is based on information collected from Chinese medical texts and scientific publications, and contains more than 20,000 pure compounds isolated from 453 TCM herbs. In total, we collected the structural information of 22 compounds for Radix Aconiti, 122 compounds for Herba Ephedrae, 39 compounds for Radix Astragali, 65 compounds for Raidix Paeoniae Alba and 203 compounds for Radix Glycytthizae. The detailed information on these compositive compounds of each herb in WTD is described in Compositive compounds of each herb in WTD were obtained from TCM Database@Taiwanhttp://www.drugbank.ca/, version: 3.0). We only used those drug-target interactions whose drugs are FDA approved for the treatment of RA and whose targets are human genes/proteins. In total, we obtained 58 known RA-related targets. (2) The Online Mendelian Inheritance in Man (OMIM) databasehttp://www.omim.org/, Last updated: October 31, 2013). We searched the OMIM database with a keyword “rheumatoid arthritis” and found 7 known RA-related targets: CD244, HLA-DR1B, MHC2TA, NFKBIL1, PAD, SLC22A4, and PTPN8. (3) Genetic Association Database (GAD)http://geneticassociationdb.nih.gov/, Last updated: August 18, 2013). We used a keyword “rheumatoid arthritis” to search the GAD database. In total, we obtained 82 known RA-related targets whose association with RA was shown “Y”. (4) Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Databasehttp://www.genome.jp/kegg/, Last updated: October 16, 2012). In total, we obtained 92 known RA-related targets which appear on the RA pathway (KEGG ID: map05323) in the KEGG database. The detailed information on these known therapeutic targets is described in Known RA-related targets were obtained from four existing resources: (1) DrugBank databasePPI data were imported from eight existing PPI databases including Human Annotated and Predicted Protein Interaction Database (HAPPI)The putative targets of WTD's compositive compounds were predicted by drug-CIPHER-CS presented by Zhao and LiWe first constructed a interaction network for known RA-related targets and putative drug targets of WTD based on their interaction data obtained from eight existing PPI databases as mentioned above. Then, we applied Navigator software (Version 2.2.1) to visualize the interaction network.For each node i in interaction network, we defined four measures for assessing its topological property: (1) 'Degree' is defined as the number of links to node i; (2) 'Node betweenness' is defined as the number of shortest paths between pairs of nodes that run through node i. (3) ‘Closeness' is defined as the inverse of the farness which is the sum of node i distances to all other nodes. The Closeness centrality can be regarded as a measure of how long it will take to spread information from node i to all other nodes sequentially. Degree, node betweenness and closeness centralities can measure a node's topological importance in the network. The larger a node's degree/node betweenness/closeness centrality is, the more important the node is in the interaction networkhttp://david.abcc.ncifcrf.gov/home.jsp,version 6.7) for GO enrichment analysis. We also performed pathway enrichment analysis using pathway data obtained from the FTP service of KEGGhttp://www.genome.jp/kegg/, Last updated: Oct 16, 2012).We used Database for Annotation, Visualization and Integrated DiscoveryThe study was approved by the Research Ethics Committee of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China. All animals were treated in accordance with the guidelines and regulations for the use and care of animals of the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences.A total of 72 male SD rats (160 ~ 180 g) were purchased from Experimental Animal Center, Academy of Military Medical Sciences (production license No: SCXK 2002-001). All rats were maintained in a room equipped with an air-filtering system, and the cages and water were sterilized.2 incubator. HFLS–RA were used at passage numbers 4 to 8 in this study.HFLS-RA  was used for the in vitro experimental validation in the current study. The cells were cultured in sterile synoviocyte growth medium  supplemented with 100 U/mL 1 penicillin, 80 U/mL 1 streptomycin, 2 mM Gln-glutamine, and were maintained at 37°C in a humidified 5% COCIA was induced as our previously reported5455Radix Aconiti 6 g, Herba Ephedrae 9 g, Radix Astragali 9 g, Raidix Paeoniae Alba 9 g and Radix Glycytthizae 9 g were immersed in 2 litres of water for 2 h and then decocted to boil for 1 h. The decoction was filtered through four layers of gauze. Next, the drugs were boiled once again for 0.5 h with 2litres of water and the decoction was filtrated out with the above method. Finally, the extraction solution was made to a concentration of 1 g crude drug/mL. To clarify the chemical composition of WTD, UPLC–Q-TOF-MS analysis was conducted to identify its major compounds. The detailed strategy and results of the identification were provided in our previously published paperAccording to the original composition of WTD recorded in Chinese Pharmacopoeia 2010 edition, WTD was prepared using the following procedure. The crude drugs of For in vivo experimental validation, the route of WTD delivery was oral administration. Treatment was given daily for a period of 21 days. The dosage selection for WTD [3.8 μg/(kg·day)] was nearly equivalent to RA patient dosage daily (42 g/person/day). SD rats were divided into 6 groups with the equal number (n = 12): normal control group , CIA model control group (Vehicle), CIA rats treated with 0.95 g/(kg·day) WTD (WTD-low), 1.9 g/(kg·day) WTD (WTD-middle), 3.8 g/(kg·day) WTD (WTD-high), and 0.2 mg/kg methotrexate (MTX).For in vitro experimental validation, HFLS-RA were then incubated with different concentrations of WTD  for 24 h.Rats were observed once every day after primary immunization. Arthritis severity was evaluated by arthritis score, arthritis incidence, percentage of arthritic limbs and the time of arthritis first appeared which were performed by two independent, blinded observers. The arthritis score was the total of the scores for all 4 limbs (maximum possible arthritis score 80). Arthritis incidence values are the number positive/total number in group. In addition, the number of arthritic limbs of individual rats were counted and added to represent the number of arthritic limbs in a group. The percentage of arthritic limbs in a group was calculated as following formula:Moreover, the time of arthritis first appeared refered to the first day of the onset of the clinical symptoms of arthritis observed.Rats were sacrificed by cervical dislocation on day 21 after first immunication. Both hind limbs including the paws, ankles, and knees, were dissected, fixed immediately for 24 h in 4% paraformaldehyde, decalcified in 10% EDTA for up to 2 month at 4°C, and embedded in paraffin. Tissue sections (4 μm) were mounted on common slides for staining with hematoxylin and eosin (H&E) or safranin-O. All sections were randomized and evaluated by two trained observers who were blinded to the treatment groups and the arthritis severity of each rat. Minor differences between observers were resolved by mutual agreement. The data was expressed as mean inflammation score. All scores were based on a scale of 0–3, as previously describedAt the end of the experiment, rats were sacrificed and the left hind paws were radiographed with a digital mammography system . Radiographs of ankle and tarsus joints of each rat were evaluated for bone destruction on a scale of 0 = normal, 1 = mild changes, 2 = moderate changes, and 3 = severe changes, respectively2O2. Each section was incubated with normal goat serum for 20 min at room temperature, and then with primary antibodies against rat MMP-1  and MMP-13  respectively overnight at 4°C. After incubation with Polymer Helper for 20 min at 37°C, sections were reacted with poly-HRP anti-goat IgG for 20 min at 37°C. The sections were then stained with 3, 3-diaminobenzidine  and counterstained with hematoxylin. For the control staining, PBS was used instead of the primary antibodies.Paraffin sections (5 μm) of tissue from the knee and ankle joints were mounted on poly-L-lysine-coated slides. Immunolocalizations of MMP-1 and MMP-13 in the joints were carried out with commercial Polink-2 plus Polymer HRP Detection System For Goat Primary Antibody kits  according to the manufacturer's instructions. The paraffin sections were dewaxed by routine method and incubated for 10 min with 3% HSpecimens were examined using a Leica image analyzer and analyzed by computer image analysis  in a blinded manner. To localize and identify areas with positively stained cells, ten digital images per specimen of synovium from a knee or ankle joint were recorded, and quantitative analysis was performed according to the color cell separation. The results are expressed as the mean region of interest, representing the percentage of area covered with positively stained cells per image at a magnification of ×400.Sera from the rats on day 22 of arthritis were obtained and stored at −80°C until use. The amounts of IL-1β, TNF-α, HIF-1α and VEGF in sera were detected by ELISA assay  according to the manufacturer's protocol and absorbance was measured at 450 nm. All experiments were done in triplicate.The Western blot protocol and semiquantitative analysis were carried out following the protocol of our previous studies54P was less than 0.05.The software of SPSS version13.0 for Windows  and SAS 9.1  was used for statistical analysis. Continuous variables were expressed as S.L. and N.L. engaged in study design and coordination, material support for obtained funding, and supervised study. Y.Z. performed network analysis, designed the experimental validation and drafted the manuscript. M.B. and B.Z. performed drug target prediction. C.L., Y.S., D.W., Y.J. and Q.G. carried out the experiment validation. All authors reviewed the manuscript.Table S1Table S2Table S3Table S4Table S5Table S6Table S7


Adrenocortical carcinoma (ACC) is an aggressive tumor characterized by a high recurrence rate and poor response to treatment. This study analyzes a consecutive series of ACC patients to evaluate the prognostic value of various clinical and pathological characteristics.We retrospectively evaluated 32 ACC patients followed at our Medical Center from 1997 to 2015 and evaluated the prognostic value of age at diagnosis, gender, tumor functional status, stage, and type of treatment with respect to overall survival (OS) and disease-free survival (DFS), as determined by Kaplan–Meier curves.p = 0.007], ki-67  and advanced stage at diagnosis (III–IV)  were associated with recurrence in the 26 R0 patients in the univariate analysis. Advanced stage was an independent risk factor for recurrence in the multivariate analysis . Five-year survival was 40.0%. Positive resection margins , ki-67  and advanced stage  were associated with poor survival in all 32 patients, but only positive resection margins were an independent predictor of mortality in the multivariate analysis .ACC was associated with hormonal overproduction in 50% of cases, and patients with isolated hyperandrogenism had a better prognosis. Recurrence was observed in 12/26 (46.2%) patients with no evidence of disease after surgery. Tumor size [hazard ratio (HR) 1.32, 95% confidential intervals (CI) 1.12–1.64; ACC has a poor prognosis with a high recurrence rate. Tumor stage at diagnosis and the completeness of surgical excision are the most relevant prognostic factors. Adrenocortical carcinoma (ACC) is a rare tumor with an incidence of approximately 0.7–2 cases/million inhabitants per year –3. The cACC is one of the most aggressive solid tumors, and complete surgical resection  is the curative treatment that offers the best hope for long-term survival. However, ACC remains a challenging malignancy because of the high frequency of recurrent disease and the low rate of response to postsurgical therapies –6. The eIn spite of promising advances in molecular characterization and therapeutic approaches, the ACC outcome remains poor. Therefore, reporting the clinical experience of even a limited number of cases might be useful in attempts to provide an accurate prognosis and better care for this rare but aggressive disease.In this retrospective study, we analyzed the clinical and pathological features of a consecutive series of 32 patients affected by ACC followed at our Medical Center. These features were evaluated with respect to their relevance to outcome and prognosis.We retrospectively analyzed the clinical characteristics at presentation, treatment, and outcome of a continuous series of 32 ACC patients followed at our Medical Center from 1997 to 2015. Clinical records were reviewed and data on age at diagnosis, gender, functional tumor status , tumor size, stage at diagnosis as well as treatment details and outcome were analyzed. Some preoperative findings, as radiological reports, were not included in the analysis because they were performed by different centers and with non-standardized procedures.via the open or laparoscopic approach (on the basis of tumor size or preoperative imaging findings) at the Surgical Oncology Unit of our Medical Center.All patients underwent surgical resection of the ACC Tumor specimens were reviewed by our pathologists and staged according to the ENSAT staging system , which do,p-DDD, Lysodren 500 mg tablets, Laboratoire HRA Pharma, France) was started in 17 patients according to tumor stage and histopathological features of aggressiveness . This treatment was not given to five patients free of disease but diagnosed before 2001, when mitotane was not available at our Center  and morphological staging ; in the absence of biochemical and/or radiological signs of persistent/recurrent disease patients were considered free of disease. Adjuvant therapy with mitotane , and imaging evaluations. All patients with persistent/recurrent disease had undergone surgery or systemic therapy (mitotane and/or chemotherapy). Patients treated with mitotane underwent adrenal replacement therapy with cortisone acetate  and fludrocortisone acetate . Mitotane treatment was started at a low dosage  and thent-test was used for continuous variables. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. The results are reported with hazard ratio (HR) and 95% confidential intervals (CI). The OS and DFS curves were determined by the corresponding Kaplan–Meier curves. All tests were two-sided, and p values <0.05 were considered statistically significant. Statistical analysis was performed using SPSS software, version 13.0 for Windows .Quantitative data are shown as mean ± SD, and numbers and percentages are provided for qualitative data. Percentages were compared using chi-squared tests, and Student’s p = 0.01) and in patients with secreting tumors (n = 16) relative to patients with non-functioning tumors (n = 16) .Table p = 0.006). In particular, androgen secretion was found in seven patients (43.7%). The secretion of cortisol alone was observed in only two patients (12.5%); the secretion of both androgens and cortisol was observed in six patients (37.5%); one case (6.3%) had hyperaldosteronism at diagnosis.Among the patients with hormone-secreting tumors, most were females (15/22 or 68.2%), with only one case (1/10 or 10.0%) of functioning AAC among the male patients  and hirsutism and oligoamenorrhea in eight cases (25%). Resistant hypertension with pronounced hypokalemia was found in the patient with the aldosterone-secreting carcinoma. Two patients with secreting tumors were clinically asymptomatic. The non-functioning ACCs were revealed by abdominal discomfort and back pain in 14 (43.7%) patients. Two non-functioning ACCs were diagnosed incidentally during abdominal imaging exams performed for other indications.In the seven female patients with isolated hyperandrogenism, age at diagnosis was lower in patients with hypercortisolism  and those with non-functioning ACCs Table . In thisOpen surgery was performed on the primary tumor in 25 patients (78.1%). In the remaining seven patients (21.9%) with a preoperative diagnosis suspicious of adenoma and an adrenal mass that was small in size (<4 cm), a laparoscopic approach was used for surgery. Negative resection margins (R0) were achieved in 26 patients (81.3%). Positive margins (R1) were found in two patients (6.3%). Macroscopically residual disease (R2) was present in the four remaining patients (12.5%).p = 0.9) or ACC endocrine function .Mean tumor size was 9.5 ± 4.7 cm  with no significant difference according to gender , the ki-67 value was >10%.According to the ENSAT staging classification, only 3 patients were classified as stage I, 15 as stage II, 7 as stage III, and 7 as stage IV , tumor debulking but not complete removal of the primary tumor was achieved through surgery  R0 patients received adjuvant mitotane treatment  . Cancer recurrence was observed in 12 of the 26 R0 patients (46.2%) after a median period of 19.4 months (range 3.0–108.6): 3 of these patients underwent a second surgical intervention because of localized disease, while 9 were treated with EDP in association with mitotane as first-line systemic chemotherapy.ACC recurrence occurred in 8/17 R0 patients (47.1%) under adjuvant mitotane therapy and in 4/9 (44.4%) R0 patients not treated with mitotane Table . The recp = 0.007), ki-67 , and advanced stage at diagnosis  (Table p = 0.01)  and non-functioning ACC patients (p < 0.05) , 17 patients (53.1%) were disease-free , while 3 patients (2/15 stage II and 1/7 stage III) showed progressive disease. Twelve patients died from the disease . ACC patients with isolated hyperandrogenism had the lowest rate of progression/death for the disease compared with cortisol-secreting  was 55.8 months (95% CI 9.0–97.3). For the six patients with persistent disease after surgery, median OS was only 7.7 months (95% CI 1.2–19.9) , ki-67 , and advanced stage (III–IV) at diagnosis . At multivariate analysis, only resection margin positivity was an independent predictor of mortality  , as already reported . In pati%, as alrIn contrast to previously published studies, in which most patients had ACC in advanced stages, in recent years, a shift toward more precocious stages, with the highest percentage of patients in stage II, has been observed. This is a likely consequence of the increased diffusion of radiological imaging procedures . HoweverThe ACC recurrence rates reported in the literature are widely variable, ranging from 21 to 91% . This vaACC remains an aggressive cancer that can recur at any time, although most recurrences occur in the first 2 years after surgery . In our In the high-risk patients, some evidences indicate that adjuvant radiotherapy in the tumor bed after surgery significantly improved local control in resected ACC patients .In low-risk patients, the indications for mitotane therapy are uncertain: current recommendations suggest assigning these patients to the randomized ADIUVO trialThe optimal management of patients with recurrent ACC is not well established. A recent study showed that additional surgical procedures should be performed at first recurrence in patients with a DFS >12 months, when the tumor is completely resectable. In contrast, when recurrence occurs fewer than 6 months after disease diagnosis, the suggested treatment is aggressive medical therapy . In our Despite an increased number of surgical procedures and earlier diagnosis, the survival rate of ACC patients has not significantly improved in recent years: the Netherlands Cancer Registry shows an average overall 5-year survival rate of 32% from 1993 to 2010; this figures jumps to 62% in patients with localized disease , 15. TheOur report and clinical experience remark that ACC patient prognosis is poor and largely depends by tumor stage at diagnosis, with strong relation with postoperative stadiation (e.g. margins status). The rate of recurrence is high. No effective medical therapies are available at present, and the current treatment with mitotane and conventional chemotherapy provides small benefits in most cases.Recently, genomic studies led to the identification of genetic and epigenetic alterations characterizing subgroups of tumors with activation of specific molecular pathway patterns and with different clinical outcome. Based on these analyses, the prognostic stratification is more accurate than that obtained with the use of traditional prognostic factors. The use of genome-wide expression profile studies, microRNA, and methylation profile may allow a better prognostic prediction and promote the development of molecular-targeted therapies , 29.Our study presents some limitations, the most important being the small series of patients, the retrospective design, and the analysis of prognostic factors restricted to traditional histological markers with no insight on the molecular profile of ACC.ACC is a rare disease, more frequent in women who have a younger mean age at diagnosis and a higher rate of functioning tumors.Functioning ACC are diagnosed earlier, but only ACC patients with isolated hyperandrogenism have a better prognosis, not patients with ACC secreting cortisol .The completeness of surgery, without margin positivity, is a critical requirement to improve outcome. However, the recurrence rate is high, even when surgical resection is apparently complete. Tumor size, ki-67, and a high tumor stage are the risk factors significantly associated with ACC recurrence.MR, CS, GS, PM, RV, and GP treated the patients, gathered data, and drafted the manuscript. MAT treated the patients, carried out the surgeries, and critically reviewed the manuscript. DS and DG treated the patients and critically reviewed the manuscript. MM analyzed histological specimens and critically reviewed the manuscript. All authors contributed to the conception of the work and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine the clinical profile, burden of risk factors, and quality of care among patients hospitalized for an acute myocardial infarction (AMI) with special focus on gender differences.The study included 256 AMI patients admitted to the Coronary Care Unit of “Mother Teresa” hospital in Tirana during 2013-2014. We obtained information on patients’ demographic data, AMI characteristics, complications (heart failure [HF] and ventricular fibrillation [VF]), risk factors and medication use prior and during the AMI hospitalization. Age-adjusted Poisson regression analyses were applied to explore gender differences (women vs men) with regard to clinical profile and quality of care and results are expressed as incidence rate ratios (IRR).P = 0.042) had diabetes, (P = 0.001) developed HF (P < 0.001) or experienced a VF episode (P < 0.001). After adjusting for age, differences with regard to obesity , diabetes , HF  and VF  remained significant. There were no differences with regard to individual drug classes taken. However, women had fewer revascularization procedures than men .55.4% of patients had ≥3 risk factors, 44.5% developed HF, and 5.7% developed VF. Only 40.4% of patients received all 4 medication classes  and 46.4% had revascularization. Significantly more women than men were obese, (Women were found to have more unfavorable clinical profile, higher complication rates, and underutilization of therapy, which may be influenced by socioeconomic differences between genders and lead to a differential prognosis. Coronary heart disease (CHD) is recognized as a major cause of mortality, accounting for 1 in 5 deaths in Europe  have ledWhile during the last two decades many Western countries have experienced declines in CHD mortality and incidence rates, Albania has experienced an increase . This waDespite unfavorable trends in CHD occurrence in Albania, the proportion of gross domestic product allocated to the health care system is lower ( ~ 50%) than in many European countries . These lTherefore, we conducted this study to assess the overall burden and potential gender differences in the clinical profile, disease severity, and quality of care among patients hospitalized for an AMI in the Coronary Care Unit (CCU) of ‘Mother Teresa’ hospital in Tirana, Albania.The study included 265 consecutive patients, residents of Tirana admitted during 2013-2014 due to an AMI at the CCU of the University Hospital Center “Mother Teresa.” This is the only public hospital providing residents of Tirana with specialized cardiac care.Detailed information on age, gender, education, height and weight, systolic and diastolic blood pressure, AMI type (ST-elevation MI [STEMI] vs non ST-elevation MI), location, major complications such as heart failure (HF) and ventricular fibrillation (VF) and treatment was obtained from patients’ medical charts. Glucose and cholesterol level were measured in fasting blood samples. Information on coronary risk factors, , previous history of AMI, and drug use prior to hospitalization was obtained from patients’ reports.2 and patients were categorized as normal (<25), overweight (between 25 and 30), and obese (≥30). According to smoking status patients were categorized into never/former smokers and current smokers. Hypertension was defined as systolic blood pressure (SBP)≥140 mm Hg, diastolic blood pressure (DBP)≥90 mm Hg, or prior use of antihypertensive medications. Diabetic status was categorized as “no diabetes” (glucose levels <5.6 mmol/L), “impaired fasting glucose” (glucose levels between 5.6-6.9 mmol/L), and “diabetes” (glucose levels >6.9 mmol/L). Prior use of hypoglycemic agents  classified the patients into “diabetes” category, regardless of blood glucose levels. Hypercholesterolemia was defined as total cholesterol ≥6.2 mmol/L or prior use of statins. The study protocol was approved by the National Committee for Bio-Medical Ethics in Albania.Education was categorized into primary (up to 8 years), secondary (9-12 years), and tertiary (>12 years). Body mass index (BMI) was calculated as weight/heightt test, while distribution of categorical variables was compared using the χ2 test or Fisher exact test in cases of small sample sizes (at least one expected cell <5). Poisson regression models with robust variances were constructed to compare gender differences with regard to clinical profile and quality of care during the AMI hospitalization. These models were adjusted for age and results were expressed as incidence rate ratios (IRR) with 95% confidence intervals (CI) for women vs men (the reference category). Two-sided tests with a 0.05 level of significance were used. Analyses were performed using STATA software .Continuous variables are presented as means and standard deviations (SD) whenever their distribution was proved to be normal (Shapiro-Wilk test in STATA). Categorical variables are presented as frequency distributions (absolute numbers and corresponding proportions). Mean values of the continuous variables were compared using independent sample Mean age of participants was 64.9 (12.1) years, and 77.7% were men. There were 38.5% of patients with primary education, 43.8% with secondary, and 17.7% with tertiary education. STEMI was the dominating AMI type (87.1%). In 52.4% of the cases, AMI involved the anterior wall. Only 12.5% of patients had been hospitalized for a previous AMI.We observed a clustering of risk factors among study participants at the time of hospitalization: 20% of them had 4-5 risk factors, while 55.4% had at least 3 risk factors. Hypertension was observed in 73.6% of patients, followed by smoking (61.5%), hypercholesterolemia (57.5%), diabetes mellitus , and obesity (14.0%).The proportion of patients with AMI complicated with HF was 44.5%. Of those, 88.1% had impaired left ventricle (LV) function (defined as LV ejection fraction ≤0.45 on echocardiographic examination). An episode of VF during hospitalization was registered in 5.7% of patients .The utilization rates were higher for statins (96.6%) and aspirin (94.7%) compared to beta-blockers (55.5%) or angiotensin-converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARB) (61.5%). Only 40.4% of patients received 4 drug classes, followed by 30.6% receiving 3, 26.0% receiving 2, and 3.0% receiving only 1 class. A total of 46.4% of patients had revascularization; 44.5% underwent percutaneous coronary intervention or coronary artery bypass grafting, and only 3.8% had thrombolysis .P < 0.001). No significant gender differences were found with regard to education (P = 0.081), AMI type (P = 0.337), localization (P = 0.448), or history of AMI (P = 0.723). A higher proportion of women were obese (P = 0.041) and had diabetes (P = 0.010), whereas men more frequently smoked (P < 0.001). The clustering of risk factors was similar between men and women (P < 0.001), VF (P < 0.001), or both (P < 0.001) during hospital stay , diabetes , and smoking  remained significant. Conversely, no significant differences were observed with regard to hypertension and hypercholesterolemia . Women hP = 0.010). Fewer women received all 4 drug classes (P < 0.01) or had revascularization procedures (P < 0.001)  . Analyse< 0.001) .After adjusting for age, the uptake of each individual drug class as well as of all 4 drug classes was lower among women, although differences were not significant. Women less often had revascularization: either of any type  or invasive  . The resWe observed a clustering (≥3) of coronary risk factors in more than half of our study population. AMI was complicated with HF or VF in 48.7% of the cases, demonstrating a severe clinical expression of the disease. Only 40.4% of patients received 4 classes of medications  and 46.4% had revascularization. Our study population was also characterized by gender differences with regard to clinical profile, disease severity, and quality of care. Women were more often presenting a more severe form of the disease and received less often optimal therapy compared to men. Such differences in the treatment were independent from age and clinical severity.Information on the risk factors’ burden among coronary patients in Albania and their treatment is missing. So far only one study has shown that among 467 consecutive patients hospitalized for incident AMI or unstable angina pectoris, the proportion of obesity, hypertension, and diabetes was higher among women . These fSimilar to other international studies ,15, womeWe observed that fewer women than men received optimal treatment as recommended by AMI treatment guidelines. Similar to a previous study conducted in Canada , we obseUnderutilization of revascularization procedures among women compared to men could, to a certain extent, be explained by differences in disease expression. Women less often present with obstructive coronary artery disease -25 and iThis is the first analysis providing information on patients’ profile, quality of care, and gender differences among AMI patients in Albania, a particularly under-researched population that is still undergoing a rapid transition characterized by lifestyle changes and chronic disease patterns. The information obtained in our study highlights the need for efficient, gender-specific primary and secondary prevention measures. Our results demonstrate that the absence of prevention interventions in the general population not only increases the number of AMI cases but also deteriorates their clinical profile. This renders the treatment more challenging and patients’ prognosis poorer.Nonetheless, our study may have several limitations. Due to a relatively small sample size, we were unable to detect potential differences related to individual medication classes. However, our findings are in line with studies including a much larger number of participants. We did not have information on the absolute or relative contraindications to individual medication classes. Gender differences in the quality of care may have been related to differences in the health profile  between genders. However, such information was not available in our study and, hence, this hypothesis could not be tested. On the face of it though, there is no plausible reason to assume that there are gender differences in terms of quality of care related to different health profiles between men and women in Albania. In our study the category of “never/former” smokers included participants who did not smoke at least one year prior to the hospitalization. No detailed information on time since the last cigarette was available for those who smoked previously but stopped at some point. We regard this as another limitation of this study as time from quitting smoking is inversely associated with the risk for CHD . We alsoThe presence of many coronary risk factors and AMI clinical severity, combined with a suboptimal coronary care  can adversely influence the prognosis of coronary patients in Albania, especially when considering that the country lacks structured rehabilitation programs and standardized secondary prevention interventions. Further studies are needed to elucidate the factors influencing the clinical profile of Albanian patients and suboptimal quality of coronary care.


NAD(P)H: quinone oxidoreductase 1 (NQO1) plays a central role in catalyzing the two-electron reduction of quinoid compounds into hydroquinones. The NQO1 Pro187Ser polymorphism was found to correlate with a lower enzymatic activity, which may result in increased incidence of carcinomas including breast cancer. Previous studies investigating the association between NQO1 Pro187Ser polymorphism and breast cancer risk showed inconsistent results. We performed a meta-analysis to summarize the possible association.All studies published from January 1966 to February 2014 on the association between NQO1 Pro187Ser polymorphism and breast cancer risk were identified by searching electronic databases PubMed, EMBASE, Cochrane library, and Chinese Biomedical Literature database (CBM). The association between NQO1 Pro187Ser polymorphism and breast cancer risk was assessed by odds ratios (ORs) together with their 95% confidence intervals (CIs).Ten studies with 2,773 cases and 4,076 controls were finally included in the meta-analysis. We did not observe a significant association between NQO1 Pro187Ser polymorphism and breast cancer risk when all studies were pooled into the meta-analysis. In subgroup analysis by ethnicity, significant increased breast cancer risk was found in Caucasians . When stratified by source of control, significant increased breast cancer risk was found in population-based studies . However, in subgroup analyses according to menopausal status, quality score, and HWE in controls, no any significant association was detected.Our meta-analysis provides the evidence that the NQO1 Pro187Ser polymorphism contributed to the breast cancer susceptibility among Caucasians. Further large and well-designed studies are needed to confirm this association.http://www.diagnosticpathology.diagnomx.eu/vs/1248639991252504The virtual slide(s) for this article can be found here:  Breast cancer is the most common cancer and the second most common cause of cancer-related death in women. In 2008 there were 182,460 women diagnosed with breast cancer, and 40,480 women died of this disease ,4.http://www.ncbi.nlm.nih.gov/SNP), including the most commonly occurring C-to-T transition at nucleotide position 609 in exon 6 , which results in a proline-to-serine amino-acid substitution at codon 187 (Pro187Ser) in the protein. It was reported that the variant T allele was associated with reduced NQO1 enzymatic activity in both human cell lines and primary human tissues [NAD(P)H:quinone oxidoreductase 1 (NQO1), also known as diphtheria toxin diaphorase (DT-diaphorase), is a cytosolic flavoenzyme which is present in human epithelial and endothelial tissues. NQO1 is considered as an anticancer enzyme because it protect cells from oxidative damage by preventing quinones from entering the one-electron reduction which is catalyzed by cytochrom b5 reductase or P450 reductase to generate semiquinone free radicals and reactive oxygen species ,7. The N tissues . Further tissues -13. GiveIn the past two decades, a number of molecular epidemiological studies have evaluated the association between the NQO1 Pro187Ser polymorphism and breast cancer risk, but the results remain inconsistent. Several studies have previously suggested that the NQO1 Pro187Ser polymorphism was associated with an increased risk of breast cancer ,15. HoweWe conducted a comprehensive literature search in PubMed, EMBASE, Cochrane library, and Chinese Biomedical Literature (CBM) databases form January 1966 to February 2014 using the following search strategy: (“breast cancer”) and (“NAD(P)H:quinone oxidoreductase 1”, or “NQO1”). There was no restriction on sample size, population, language, or type of report. All eligible studies were retrieved and their references were checked for other relevant studies. The literature retrieval was performed in duplication by two independent reviewers (Qiliu Peng and Yu Lu). When multiple publications reported on the same or overlapping data, we chose the most recent or largest population. When a study reported the results on different subpopulations, we treated it as separate studies in the meta-analysis.Studies included in the meta-analysis were required to meet the following criteria: (1) case–control or cohort studies which evaluated the association between NQO1 Pro187Ser polymorphism and breast cancer risk; (2) used an unrelated case–control design; (3) had an odds ratio (OR) with 95% confidence interval (CI) or other available data for estimating OR (95% CI); and (4) the control population did not contain malignant tumor patients. Studies were excluded if one of the following existed: (1) no control population; (2) duplicate of previous publication; and (3) insufficient information for data extraction.Two authors (Qiliu Peng and Xue Qin) independently reviewed and extracted data from all eligible studies. Data extracted from eligible studies included the first author, year of publication, ethnicity, country of origin, genotyping method, source of control, matching criteria, breast cancer ascertainment, total numbers of cases and controls and genotype frequencies of cases and controls. Ethnic backgrounds were categorized as Caucasian, Asian, and Arab. When a study did not state the ethnic descendent or if it was impossible to separate participants according to such phenotype, the group reported was termed as “mixed ethnicity”. To ensure the accuracy of the information extracted, the two authors checked the data extraction results and reached consensus on all of the items. If different results generated, they would check the data again and have a discussion to come to an agreement. If these two authors could not reach a consensus, another author (Shan Li) was consulted to resolve the dispute and a final decision was made by the majority of the votes. Menopausal status was divided into premenopausal and postmenopausal and was additionally recorded for stratified analysis.The quality of eligible studies was evaluated independently by two authors (Qiliu Peng and Yu Lu) according to a set of predefined criteria Table  based onThe strength of the association between NQO1 Pro187Ser polymorphism and breast cancer risk was assessed by odds ratios (ORs) with 95% confidence intervals (CIs). The significance of the pooled OR was determined by a Z test and the p value less than 0.05 was considered significant. The association of NQO1 Pro187Ser polymorphism with breast cancer risk was assessed using co-dominant model (Ser/Ser vs. Pro/Pro and Ser/Pro vs. Pro/Pro), recessive model (Ser/Ser vs. Ser/Pro + Pro/Pro), and dominant model (Ser/Ser + Ser/Pro vs. Pro/Pro).2 based Q test was used to assess the heterogeneity among studies [Q test was PQ < 0.1, indicating the presence of heterogeneity, a random-effects model (the DerSimonian and Laird method) was used to estimate the summary ORs [Q test was PQ ≥ 0.1, indicating the absence of heterogeneity, the fixed-effects model (the Mantel–Haenszel method) was used [The χ studies ,22. If tmary ORs ; otherwi2 test. All analyses were performed using Stata software, version 12.0 .Sensitivity analysis was performed by sequential removal of individual studies. Publication bias was evaluated using a funnel plot and Egger’s regression asymmetry test. The distribution of the genotypes in the control population was tested for HWE using a goodness-of-fit χBase on our search criterion, 112 individual records were found, but only 11 full-text publications were preliminarily identified for further detailed evaluation. According to the exclusion criteria, 2 publications were excluded including 1 provide insufficient information for data extraction , and 1 wP = 0.266; Ser/Pro vs. Pro/Pro: OR = 1.015, 95% CI 0.860–1.198, P = 0.860; Ser/Ser + Ser/Pro vs. Pro/Pro: OR = 1.058, 95% CI 0.899–1.245, P = 0.498; Ser/Ser vs. Ser/Pro + Pro/Pro: OR = 1.317, 95% CI 0.919–1.887, P = 0.133). In subgroup analysis by ethnicity, statistical significant increased breast cancer risk was found in Caucasians .Begg’s funnel plot and Egger’s test were performed to assess the publication bias of literatures in all comparison models. The shape of the funnel plot did not reveal any evidence of obvious asymmetry. Then, the Egger’s test was used to provide statistical evidence of funnel plot symmetry. The results still did not suggest any evidence of publication bias . Subsequently, we performed Galbraith plots analysis to further explore the source of heterogeneity. Galbraith plots analysis showed that the study Menzel et al. [PQ values were greater than 0.10 in all genetic comparison models in overall populations, Caucasians, population-based studies, and studies consistent with HWE. However, the summary ORs in the overall populations, Caucasians, population-based studies, and studies consistent with HWE were not material changed by omitting this study, indicating that our results were robust and reliable. The results indicated that the study Menzel et al. [Heterogeneity is a potential problem when interpreting the results of a meta-analysis, and finding the sources of heterogeneity is one of the most important goals of meta-analysis ,41. In tl et al.  was the l et al. , the hetl et al.  was the However, there are still some limitations in this meta-analysis. First, in subgroup analysis by ethnicity, the included studies regarded only Caucasians, Asians, and Arabs for NQO1 Pro187Ser polymorphism. Data concerning other ethnicities such as Africans were not found. Thus, additional studies are warranted to evaluate the effect of this functional polymorphism on breast cancer risk in different ethnicities, especially in Africans. Second, our results were based on unadjusted estimates. We did not perform analysis adjusted for other covariates such as age, obesity, drinking and smoking status, use of contraceptives, environment factors, and so on, because of the unavailable original data of the eligible studies. Third, subgroup analyses were based on studies with relevant information available. Owing to the lack of detailed information in most studies, the subgroup analysis for menopausal status consisted of only two studies for each subgroup, which might not be sufficient to reach a reliable conclusion.This meta-analysis provided a more precise estimation based on larger sample size compared with the individual studies and previous meta-analysis. Our study suggested that NQO1 Pro187Ser polymorphism might contribute to breast cancer risk, especially in Caucasian populations. However, it is necessary to conduct large sample studies using standardized unbiased genotyping methods, homogeneous breast cancer patients, and well-matched controls to further validate the results of our meta-analysis. Moreover, gene-gene and gene-environment interactions should also be considered in the analysis. Such studies taking these factors into account may eventually lead to a better, more comprehensive understanding of the association between NQO1 Pro187Ser polymorphism and breast cancer risk.HWE: Hardy–Weinberg equilibrium; NQO1: NAD(P)H: quinone oxidoreductase 1; SNP: Single nucleotide polymorphism; OR: Odds ratio; CI: Confidence interval.The authors declare that they have no competing interests.QP performed the literature search, data extraction, statistical analysis and drafted the manuscript. YL, XL, ZP, RL, JS, and XQ participated in data extraction. SL supervised the literature search, data extraction, statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.


A new, fully dated total-evidence phylogeny of baleen whales (Mysticeti) shows that evolutionary phases correlate strongly with Caenozoic modernization of the oceans and climates, implying a major role for bottom-up physical drivers. The phylogeny of 90 modern and dated fossil species suggests three major phases in baleen whale history: an early adaptive radiation (36–30 Ma), a shift towards bulk filter-feeding (30–23 Ma) and a climate-driven diversity loss around 3 Ma. Evolutionary rates and disparity were high following the origin of mysticetes around 38 Ma, coincident with global cooling, abrupt Southern Ocean eutrophication and the development of the Antarctic Circumpolar Current (ACC). Subsequently, evolutionary rates and disparity fell, becoming nearly constant after approximately 23 Ma as the ACC reached its full strength. By contrast, species diversity rose until 15 Ma and then remained stable, before dropping sharply with the onset of Northern Hemisphere glaciation. This decline coincided with the final establishment of modern mysticete gigantism and may be linked to glacially driven variability in the distribution of shallow habitats or an increased need for long-distance migration related to iron-mediated changes in glacial marine productivity. Mystiwww.morphobank.org; project 687) [a priori [We address these issues through a comprehensive investigation of mysticete macroevolutionary dynamics based on the most detailed, illustrated, data matrix of extinct and extant baleen whales. We score 90 taxa—nearly all for which satisfactory material has been described—for 37 646 molecular and 272 morphological characters, covering all parts of the skeleton . To promect 687) . We analect 687)  to deriva priori ,17. Totaa priori .Figure To complement our estimate of evolutionary rates, we use the fully dated tree topology and the underlying morphological data to derive phylogenetically informed estimates of mysticete taxonomic and morphological diversity (disparity) over the past 37 Ma. These three measures chart diverse aspects of mysticete evolution during mid-Late Caenozoic from a variety of angles. Finally, we synthesize all of these metrics to derive the first comprehensive overview of mysticete macroevolution and discuss the latter in light of large-scale palaeoenvironmental and biotic change.3.3.1Mesocetus). A review of all described species is beyond the scope of this study, but see Steeman [nomina dubia.We compiled a data matrix including 90 taxa, scored for 272 morphological and 37 646 molecular characters. Of the 90 taxa, 86 represent mysticetes, 76 of which have been formally described. This constitutes roughly 41% of the described 185 mysticete species listed in the Paleobiology Database . These f Steeman  for a fiwww.morphobank.org; project 687). Twenty-five characters show a morphological transition series in three or more states and were hence ordered . Overlaps with other recent studies are indicated in the online character descriptions (in the ‘Comments’ field). For 18 taxa , the photographs used to confirm character states are not published online because the particular specimen(s) are under study by other researchers. These images will be made available once the material is formally documented. Molecular data were taken from McGowen et al. [All of our morphological scorings are based on primary observation, and illustrated with one or more of 4228 specimen photographs on MorphoBank ; and (ii) the combined morphological/molecular supermatrix, partitioned as described above. For the nucleotide data, we used the substitution models suggested by PartitionFinder, whereas the gap and transposon characters were assigned a binary model, and the morphological data the Mk+Γ model, as above. For both analyses, the coding bias for the morphological data was set to ‘informative’ to correct for the absence of constant and autapomorphic characters. Markov Chain Monte Carlo (MCMC) analyses were run in MrBayes v. 3.2.2 for 20 and 50 million generations, respectively, on the Cyberinfrastructure for Phylogenetic Research Science Gateway [Based on these preliminary results, we ran two separate analyses of: (i) the morphological partition only, using the Markov maximum-likelihood model and a gamma parameter to allow for variable rates across traits  and a mean of 54 Ma . The age of the oldest pelagicete remains controversial. A Late Lutetian or Early Bartonian age has been suggested for Basilotritus wardii from the Comfort Member of the Castle Hayne Formation of North Carolina and the Piney Point Formation of Virginia [B. wardii instead of Zygorhiza sp. for both calibrations, with the former assigned an age of 42.9 Ma ; and onanocetus –36; elecVirginia –41. HoweVirginia ,42. To tone NP16 ,38,42,43rBayes following the same settings  as described for the non-clock analyses above. Following Ronquist et al. [After the implementation of all node and tip calibrations, we ran the analysis in Mt et al.  and Pyrot et al. , we used3.4Evolutionary rates were calculated for mysticetes only. Because of our use of a single-clock model ,44, phen3.5et al. [a priori choices regarding the relevance of particular characters.As with evolutionary rates, diversity and disparity were calculated for mysticetes only. Palaeodiversity was quantified in the form of a lineage-through-time plot (in 1 Ma steps) of the fully dated tree, using the R  package et al.  . In thiet al. . Estimatatrix v. 1.0 [past v. 3.02 [are v. 1.1 [n=8 species) across all 1 Ma time bins (based on 1000 bootstrap replicates). The disparity curve resulting from this reduced sample closely follows that derived from the full dataset , thus excluding sample size bias as a source of significant error in this case.To reconstruct mysticete disparity, we used our morphological dataset to derive a Euclidean distance matrix via the software Mx v. 1.0 ,50, whic v. 3.02 . Next, w v. 3.02 . Finallye v. 1.1 ,50. A vae v. 1.1 . To testet al. [esquite v. 2.75 [Because the baleen whale fossil record contains substantial gaps  and includes many taxa known only from fragmentary material, we applied three phylogenetic corrections based on our dated topology : (i) folet al. , missinget al. ). Ancest v. 2.75 . Only unet al. [The inclusion of reconstructed character states reduced the amount of missing data from 42.9%  to just 6.8% in the phylogenetically adjusted matrix—a result remarkably similar to that of Butler et al. . Note, h4.4.1et al. [To assess the effect of forcing our data to evolve under a clock model, we conducted both dated , is the sister taxon of toothless mysticetes, Chaeomysticeti. The latter comprise a monophyletic Eomysticetidae, an undescribed new species from New Zealand  and croysticeti  or fallisensu [Mauicetus parki, Aglaocetus moreni and an undescribed new species from New Zealand (ZMT 67); and (ii) Diorocetus chichibuensis, Tiphyocetus temblorensis, Isanacetus laticephalus, as well as four species referred to Parietobalaena and an undescribed taxon from New Zealand (OU 22705). A third clade including Aglaocetus patulus, Diorocetus hiatus, Thinocetus arthritus, Pelocetus calvertensis and Uranocetus gramensis only occurs in the dated analysis  are monophyletic, and basal to the rest of the crown Mysticeti (Plicogulae sensu ), includanalysis , althoug earlier , remains4.2Llanocetus around 36.6 Ma (Late Eocene), balaenids from the other members of the crown group around 30.4 Ma and cetotheriids from Balaenopteroidea around 28.96 Ma (late Early Oligocene). Pliocogulae and crown balaenopterids (including eschrichtiines) may have diverged as early as 19.5 Ma, but low branch support makes their origins uncertain , followed by the divergence of chaeomysticetes from ncertain . On the ncertain  and pre-  [Combined phenotypic/genomic rates of evolution are fastest near the base of the tree and remain relatively high—around five to six times the median overall clock rate (0.18%)—along the entire mysticete stem lineage (grey area in ecrease) ,62, as wecrease) . These d5.Balaenoptera’portisi[B.’ ryani). Further evidence to support this view comes from the recent description of the bony labyrinth of a range of living and fossil mysticetes, which demonstrated the occurrence of a tympanal recess in eschrichtiids and crown balaenopterids, but not ‘Megaptera’miocaena [M.’ miocaena as basal relative to both living rorquals and grey whales  a shift towards bulk filter-feeding as the sole feeding strategy around 23 Ma; and (iii) a marked, climate-driven drop in diversity around 3 Ma. The origin of mysticetes coincides with a long-term cooling phase later in the Eocene, culminating in a rapid temperature drop and initiation of Antarctic glaciation near the Eocene/Oligocene boundary. These events were coeval with the gradual development of a  precursor of the modern Antarctic Circumpolar Current (ACC) [Our phylogeny suggests three major events in mysticete evolution: (i) an early adaptive radiation lasting from nt (ACC) . Our linnt (ACC) ,6,74. Hont (ACC) ,76, and nt (ACC) .As cooling continued, toothed mysticetes diversified and chaeomysticetes originated, at a time of abrupt eutrophication of the Southern Ocean around 37–36 Ma —about 2.M. parki and OU 22224; Llanocetus). Further work is needed to test whether this apparent partitioning of size niches early in mysticete evolution indeed reflects ecology, as in the case of extant cetaceans.A slowdown in the rate of morphological evolution over time was also recovered in a previous study that included both mysticetes and odontocetes, but focused on extant taxa and body size only . In addiAround the time of the Oligocene/Miocene boundary (23 Ma), both evolutionary rates and disparity became essentially static, and largely remained so until the present. Concurrently, the varied Oligocene mysticete assemblage was replaced by a more ‘modern’-looking  range of chaeomysticetes, in tandem with increased levels of localized productivity probably associated with a strengthening ACC ,76. We sWhile an essentially modern ACC probably created favourable conditions for bulk feeders , it is nParietobalaena also contributes to elevated levels of diversity around this time. This is potentially problematic, since most of the included species suffer taxonomic shortcomings. For example, the holotype of Parietobalaena palmeri is a juvenile, and many of the referred specimens are insufficiently studied; all described material attributable to Parietobalaena campiniana and Tiphyocetus is fragmentary and, in the case of P. campiniana, juvenile; and finally, the taxonomically informative periotic is insufficiently known in Parietobalaena sp. from Japan, Parietobalaena yamaokai, Diorocetus chichibuensis and Pinocetus polonicus. More detailed taxonomic studies may reveal some of these taxa to belong to the same species. This, and/or a younger date of divergence for crown Balaenopteridae  woulca 16–15 Ma) marking the Mid-Miocene Climatic Optimum [Parietobalaena-like taxa, as well as more complete sampling of the mysticete fossil record. Of particular interest are the poorly sampled Early Oligocene, which saw the initial diversification of baleen whales, and the earliest Miocene . No mysticete species has yet been described from the latter 3 Ma interval  for nowinterval . We predBalaenula, Balaenella, herpetocetines and several small balaenopterids; We interpret the sharp decline in diversity around 3 Ma as real, because of (i) its magnitude; (ii) a similar, albeit much smaller, decrease in disparity; and (iii) the concurrent decline or disappearance of small-sized forms (e.g.