Control microcosms denied AMF hyphal access to the patch compartment. When permitted access to the patch compartment, the fungus proliferated extensively in the patch and transferred substantial quantities of N to the plant. Moreover, our data demonstrate that allowing hyphal access to an organic matter patch enhanced total plant N and P contents, with a simultaneous and substantial increase in plant biomass. Furthermore, we demonstrate that organic matter fertilization of arbuscular mycorrhizal plants can foster a mutually beneficial symbiosis based on nitrogen transfer, a phenomenon previously thought irrelevant.Arbuscular mycorrhizal fungi (AMF) can transfer nitrogen (N) to host plants, but the ecological relevance is debated, as total plant N and biomass do not generally increase. The extent to which the symbiosis is mutually beneficial is thought to rely on the stoichiometry of N, phosphorus (P) and carbon (C) availability. While inorganic N fertilization has been shown to elicit strong mutualism, characterized by improved plant and fungal growth and mineral nutrition, similar responses following organic N addition are lacking. Using a compartmented microcosm experiment, we determined the significance to a mycorrhizal plant of placing a  When granted access to an organic matter patch, arbuscular mycorrhizal fungi were found to obtain significant amounts of nitrogen (N) from the patch and transfer a substantial amount to their partner plant. The patch‐derived N contributed significantly to, and increased, total plant N. Total plant phosphorus (P) increased also, as did total plant mass. This study is the first to demonstrate simultaneous increases in N, P and biomass of plants with arbuscular mycorrhizal hyphal access to a patch of organic matter. The arbuscular mycorrhizal (AM) association is the most common type of mycorrhizal symbiosis and forms between c. two‐thirds of all land plant species and soil fungi in the phylum Glomeromycota. The fungus receives photosynthetically fixed carbon (C) while, in return, the fungus confers a number of benefits to its associated host plant, the most well‐established being that of increased acquisition of phosphorus (P)  barrier, which prevented root access but allowed AMF hyphal access. The ‘No Arbuscular Mycorrhizal Access’ (NAMA) units used a 0.45 μm mesh  that prevented the access of both roots and AMF hyphae to the patch compartment. This 0.45 μm mesh barrier does not retard the diffusion of solutes from the patch compartment to the plant compartment, but the AMF mycelium cannot encounter the organic matter patch directly in NAMA microcosms. Into the bottom of each compartment, four holes were drilled and covered with 20 μm mesh to permit drainage. Both compartments of the microcosms were filled with a 1:1 (v/v) mix of silica sand and AgSorb® [a calcinated, attapulgite clay soil conditioner, Oil‐Dri, Cambridgeshire, UK  were surface sterilized in a 5% (w/v) calcium hypochlorite solution, after which they were germinated on filter paper in a sterile Petri dish. At week 0, 2‐week‐old seedlings were transferred into each plant compartment, 4 cm from the mesh aperture . The plant compartment was watered daily, as required, with de‐ionized water.Except for an aperture through which the plants grew, microcosms were enveloped in aluminium foil to reduce the influx of contaminating organisms. The microcosms were planted on 30 August 2013, and maintained in a heated, lit glasshouse and re‐randomized weekly to avoid environmental artefacts. From planting to harvesting, the experiment ran for 23 weeks.Glomus intraradices inoculum comprising macerated P. lanceolata and Trifolium repens L roots inoculated with G. intraradices  and growth medium (sand and AgSorb® mix described above) was added. The mycorrhizal inoculum was 9 months old when added to microcosms. While we acknowledge that changes in the nomenclature of AMF species have been recommended , a complex N and P source which encourages AM establishment  in a matrix of 0.8 g homogenized algal matter . The patch contained 59 mg N (8.85 mg of which was 15N), 26 mg P, 413 mg C and the C:N ratio of the organic matter patch was 7:1.After 16 weeks of plant growth in the microcosms, patches of organic litter were added to the patch compartment, 6 cm away from the mesh aperture between compartments. Each patch contained 0.075 g of 98 Atom% The organic patch was mixed into 20 g of the silica sand:AgSorb® mix, which was then placed into the PVC pipe, filling the bottom 5 cm. The remaining 2 cm of PVC core was filled with the sand: Agsorb® growth medium only. The PVC pipe was placed into the patch compartment to a depth of 7 cm, such that the top of the core was flush with the level of the growth medium in which it sat.−1 made from NH4Cl and KNO3. Briefly, 0.2 g algal material was mixed with 10 mL de‐ionized water and incubated for 60 min at 50 °C. This preparation was then centrifuged at 5000 g for 15 min, after which the supernatant was decanted. Labile nitrate was measured as detailed in Cataldo et al. (w/v) salicylic acid in >96% (v/v) H2SO4 was added. After cooling to 20 °C the flask received 19 mL of 2 M NaOH to raise the pH above 12. Absorbance was measured at 410 nm, after samples had cooled to 20 °C. Labile ammonium quantification required the use of the solutions ‘A’ and ‘B’, with details of preparation given below. A 0.05 mL aliquot of supernatant was mixed with 1 mL ‘solution A’, 0.25 mL ‘solution B’ and 2.5 mL de‐ionized H2O. Both solutions ‘A’ and ‘B’ were prepared using de‐ionized water. Solution A contained 20 g trisodium citrate dihydrate, 17 g salicylic acid, 5 g NaOH and 0.2 g sodium nitroprusside, made up to 500 mL. Solution B, also made up to 500 mL, contained 5 g NaOH and 0.4 g dichlorosyonurate. Absorbance was measured by spectrophotometer at 650 nm.Labile nitrogen as ammonium or nitrate in the algal patch was quantified by spectrophotometer  and calculation from standard curve, created using standards containing 10 mg N Lo et al. . BrieflyP. lanceolata was separated into shoots and roots, and dried at 80 °C for 48 h. A subsample of the extracted roots was retained to assess root length colonization by the AMF. After drying, root and shoot material was ground and homogenized in a ball mill , for analysis by Isotope Ratio Mass Spectrometry .At 23 weeks after planting, the systems were destructively harvested. The et al. Phosphorus content was measured using X‐ray fluorescence spectrometry (XRF). Briefly, dried plant material was milled and homogenized as described above, before being pressed into a pellet and analysed with a portable X‐ray fluorescence spectrometer (as Reidinger w/v) KOH, acidified in 1% (v/v) HCl, stained with acid fuchsin and then stored in destain solution . All procedures were incubated at 20 °C, as per the ‘no heating’ variation of the method, detailed by Kormanik & McGraw . Hyphal extraction was carried out by the modified membrane filter technique of Staddon et al , and hypet al .All data were analysed using SPSS 21 , utilizing Levene's test for equality of variance. Data for HLD in bulk versus plant compartments were analysed using Paired‐Sample T Tests, while all other data were analysed using Independent‐Samples T Test. Data were transformed to satisfy Kolmogorov–Smirnov and Shapiro–Wilk tests of normality. Percentage data were square root‐arcsine transformed before analysis.T1,37 = 4.33, P < 0.001). This increase in plant dry weight was driven by an increase in both the shoot and root mass, which increased by 62% and 73%, respectively, compared with those plants whose AMF partner was not permitted access to the organic patch  in the plants whose AMF partner was denied access to the patch. The presence of 15N in the No AMF Access microcosm plants . P. lanceolata benefitted greatly from this AMF contribution acquired from the patch as demonstrated by the 68% increase in shoot N content when AMF had access to the patch compartment, corroborated by an 80% increase in root N content , suggesting that the AMF were adept at exploiting a newly available patch of organic matter and transferring the N acquired to their plant partner. Allowing AMF hyphal access to the patch resulted in 4.69 mg extra N in the roots and 4.61 mg in the shoots which greatly outweighs the 0.001124 mg of ammonium‐N and 0.0003976 mg nitrate‐N extractable from the patch.Allowing AMF hyphal access to the patch greatly increased the plant uptake of T1,34 = 3.31, P = 0.002), but the shoot P concentration was not affected by allowing AMF access to the patch. The increase in root P concentration was not substantial enough to change the total plant P concentration , but combined with an increased root mass, root P content increased by 135%. Similarly, despite no increase in P concentration, shoot P content was 94% greater in AMF access plants than in no AMF access plants . Mean AMA plant N:P was 2.18 ± 0.15, compared with mean NAMA plant N:P of 1.84 ± 0.10.Root P concentration increased by 28% when AMF had access to the patch (−1 DW) were found in the organic matter patches where AMF were denied access, hyphal length densities (HLD) were significantly greater  in the treatments that permitted AMF hyphal access to the organic matter patch (1.54 ± 0.19 m g−1 DW). Hyphal growth in the plant compartment was 21% greater when the AMF partner was denied access to the patch compartment than when access was permitted  suggesting that the C supply from the plant was limited and that the fungus was optimizing distribution of its hyphal network – into the patch compartment instead of the plant compartment. Calculating total hyphal length  shows that the AMA microcosms supported in excess of three times the hyphae seen in the NAMA microcosms (Fig. Although low levels of fungal hyphae (0.01 ± 0.01 m g15N content in the growth medium outside the patch did not change between treatments, even in the plant compartment, suggesting that 15N lost from the patch was either lost as volatile constituents to the atmosphere, or that the AMF was very successful at acquiring N from the patch. Unfortunately it was not possible to quantify the root length colonization because of disintegration of the root material during the clearing process.et al.G. intraradices was adept at acquiring N from patches of algal material and transferring a significant fraction of patch N to the partner plant. The quantity of nitrogen transferred, and the increased contribution made by the ERM in AMA microcosms support the argument that AMF can be a significant conduit for plant N uptake, a position not universally supported in the literature (e.g. Reynolds et al.et al.et al. 15N in the plant shoots (Table 15N in roots remains in the intraradical mycelium of the AMF. We assumed no fractionation of 15N and 14N during uptake by the fungus or transfer to the plant. Ectomycorrhizal fungi may transfer 14N preferentially to a partner plant, so the mycelium becomes relatively 15N enriched (Hobbie & Colpaert We show for the first time that both total N content and total dry weight of plants increased as a result of allowing AMF access to an organic matter patch. Our data show that an organic N source can elicit the ‘strong mutualism’ scenario predicted by the ‘trade balance model’ of Johnson , wherebyet al. (Lolium perenne patches, as used by Leigh et al. (et al. (et al.15N detected in the plant tissue from NAMA microcosms (Table μm membrane from patch to plant compartment is implicated, but higher N and P levels in AMA treatments confirm the importance of AMF mediated nutrient transfer. Although it remained inside the PVC tube, the algal powder settled and mixed with the sand and AgSorb® during the course of the experiment, and became inseparable from the latter by the time the microcosms were harvested. As such, the retrieval of the patch at the end of the experiment was not possible. This prevented patch analysis to determine the extent of decomposition.The increased contribution of patch N to the plants' total N when AMF were allowed access the patch was similar to that shown by Leigh et al. , suggesth et al. . Where t (et al.  makes ou. et al.. Rapid eet al. et al. et al. et al. et al. et al. et al. et al. (The contribution of patch N to total plant N varies among different studies using similar experimental systems: from <7% (Hodge & Fitter . et al.  showed net al. et al. (G. intraradices proliferated in high P patches, while reducing P uptake from low P areas. Hyphal proliferation in the high‐N, high‐P patch compartment and reduced AMF growth in the plant compartment in AMA microcosms (Fig. Increased P content in AMA plants suggests that the patch represented a significant source of P for the fungus (see also Barrett . et al.  demonstrsms Fig.  suggestset al. Reduced hyphal length in the plant compartment of AMA microcosms may suggest C limitation, as previous studies with similar experimental design (Hodge Previously, experimental evidence for strong mutual benefit of AMs was obtained only by inorganic N addition to mycorrhizal plants. Our findings demonstrate that AMF can provide considerable benefit to plant N and P nutrition following the addition of organic matter, followed by substantial increases in biomass, both for plant and fungus.The authors have no conflict of interest to declare.


Our study indicates that breast tumors present enhanced oxidative/nitrosative stress, with concomitant augmented antioxidant capacity when compared to the adjacent normal breast. These data indicate that breast cancers may be responsible for the induction of a prooxidant environment in the mammary gland, in association with enhanced TNF- Redox imbalance is a process reported in most of the chronic diseases . The conCancer cells constantly experience moderate to high levels of oxidative stress, but curiously moderate to high oxidative stress does not cause immediate cell death . This fa in situ oxidative stress than the proximal or distant nontumoral tissues. On the other hand, this fact suggests that cancer can potentially induce oxidative damage in surrounding normal cells.Tumors with enhanced proliferative capacity, as breast cancer, produce high levels of RS during their chronic cycles of ischemia, reperfusion, and angiogenesis, resulting in exceeding growth signaling . It is rTherefore, nontransformed epithelial cells located adjacently to the tumoral tissue may experience variable concentrations of RS generated by the constitutive activation of mitogenic pathways arising from surrounding tumor cells ; howeverAlthough growing evidence highlights the occurrence of persistent oxidative stress in breast tumors, most of studies have not focused on reporting the redox modifications of breast cancer regarding its healthy counterpart tissue and whether there is a relationship between the tumor oxidative status and the systemic redox profiling. In this context, we proposed to map the oxidative and inflammatory profiles of fresh nonfixed breast tumors and their paired adjacent mammary nontumoral tissue, as well as their respective plasma. To reach these goals, we designed our analysis employing high-sensitivity oxidative stress approaches to investigate the functional redox changes that occurred in tumor microenvironment and its correlation with the circulating levels of proinflammatory/oxidative mediators.A series of 321 women with breast cancer were screened from March 2011 to December 2012 at Londrina Cancer Institute, Londrina, Paraná, Brazil. A total of 50 women were included based on the inclusion and exclusion criteria. Inclusion criteria embraced women bearing unilateral tumor with histopathological diagnosis of primary ductal infiltrative carcinoma of the breast, before starting the chemotherapeutic regimen. Exclusion criteria included current smoking, hepatic, cardiac, or renal dysfunction, obesity, use of drugs, hypertension, autoimmune disorders, and diabetes, among other chronic conditions.Adjacent mammary tissue and tumoral tissue were surgically resected at the moment of tumor withdrawal according to standard procedures, before chemotherapy starting. The adjacent tissue was collected from the most distant point in relation to the tumoral tissue (3 to 4 cm of distance from the macroscopic tumor). Adjacent breast was confirmed as nontumoral by conventional histopathological analysis. Heparinized blood was further collected for analysis. Samples were kept frozen at −86°C until analysis, by at most 2 weeks.All recommendations of the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria  were folClinicopathological data of cancer patients was collected from medical records and included age at diagnosis, TNM staging, tumor histological type, histological tumor grade, lymph nodal status, tumor size, and presence of distant metastasis.Nitrotyrosine (NT) is a residue formed by the action of peroxynitrite (derived from the reaction of nitric oxide and superoxide anion) on proteins. Paraffin-embedded sections were heated , deparaffinized, and rehydrated. Sections were treated at room temperature with 2% bovine serum albumin and incubated overnight at 4°C with primary mouse anti-human antibodies against NT-labeled residues , previously validated for human samples . The secFrozen tissue samples were thawed, precisely weighted, and homogenized in sterile saline phosphate buffer 10 mM pH 7.4, at a final concentration of 100 mg tissue/mL. The mixture was centrifuged at 5000 ×g, 4°C during 10 minutes. Supernatants were collected and kept in ice bath until analysis. All described methods used this concentration of tissue homogenate, except when some specific dilution is highlighted. Heparinized blood samples were centrifuged at 1500 ×g, 4°C during 5 minutes. Plasma samples were separated for further analysis. All measurements were conducted at the same day of sample processing.μL of tissue homogenate (100 mg/mL) were added to 750 μL of saline phosphate buffer 10 mM pH 7.4, with addition of 10 μL of t-butyl 3 mM solution. For plasma analysis, 200 μL of sample was mixed with 780 μL of saline phosphate buffer 10 mM pH 7.4, with addition of 20 μL of t-butyl 3 mM solution. Readings were carried out in a Glomax luminometer . The results were expressed as relative light units (RLU) and the entire curve profile was used as indicator of lipoperoxidation. The area under the curve (AUC) was obtained by area integration.Lipoperoxidation of adjacent and tumoral tissue was evaluated as previously published, with some adaptations . AliquotμL were mixed with 50 μL of FOX reagent. Samples were incubated for 30 minutes in the dark at room temperature and the absorbance of the supernatant was measured at 550 nm and the results were expressed as μM hydroperoxide/mg tissue.Lipid hydroperoxide concentrations in samples were estimated by the FOX method as published by Victorino et al. . Plasma μL were added in 2 tubes. Test tubes received 1 mL of dinitrophenylhydrazine (DNPH) 10 mM and blank tubes received 1 mL of HCL 2.5 M. Tubes were incubated during 1 hour in ice bath. After that, samples were successively incubated with 1.25 mL of trichloric acetic acid 20% and 10% in ice bath during 20 minutes each, with centrifugation between incubations (1400 ×g/15 minutes). Supernatants were discarded and pellets were twice treated with 1 mL of an ethanol/water solution (1 : 1). The final precipitates were dissolved in 1 mL of guanidine 6 M and were left for 24 hours at 37°C. Carbonyl content was calculated by obtaining the spectra at 355–390 nm of DNPH-treated samples, employing one blank tube for each test. The obtained peaks were employed to calculate carbonyl concentration using a molar extinction coefficient of 22 M−1cm−1. Results were expressed as nmol/mL/mg tissue. Myeloperoxidase was detected by a commercial ELISA kit following manufacturer's instructions , and the data were expressed as U anti-MPO/mL.Carbonyl content was measured as estimate of oxidative injury in proteins . Tissue μM trolox/g tissue [TRAP was measured in pure breast tissue homogenates (100 mg/mL) or plasma samples (diluted 1 : 50). For TRAP calculation, the induction time of the sample (time for which the sample antioxidants can inhibit the ABAP action) was compared to that of the standard antioxidant (trolox) and expressed as g tissue .μL) were deproteinized, and the supernatants were recovered and incubated with cadmium granules. After 10 minutes, the Griess reagent (Sigma) was added to 200 μL of the supernatants, and the reactions were incubated for 10 minutes at room temperature. The absorbance was read at 550 nm using a standard microplate reader . The final results were expressed as μM nitrite/mg tissue.NO was estimated by measuring nitrite as previously described . HomogenμL of plasma samples, tissue homogenate, or standard solution reacted with 100 μL of 0.5 M perchloric acid. Samples were centrifuged for 5 minutes, 5000 ×g at 4°C. About 180 μL of supernatant was recovered to react with 100 μL of thiobarbituric acid for 30 minutes, at 95°C, and transferred to ice bath to stop reaction. 100 μL of 1 M NaH2PO4, pH 7.0, was added to stabilize sample pH. Further, samples were centrifuged for 10 minutes, 5000 ×g at 4°C. Mobile phase was composed of 65% 50 mM KH2PO4 buffer, pH 7.0, and 35% methanol HPLC grade. Readings were executed at 535 nm during 12 minutes with isocratic flow of 0.8 mL/minute and results were expressed as MDA peak height.MDA determinations were made using equipment HPLC-20AT Shimadzu equipped with a LC20AT pump and SPDM20A UV, diode array absorbance detector employing a C18 reverse phase column, as described by . Aliquotα levels  were determined in aliquots of 200 μL of plasma or tissue homogenates by using commercial kits. Homocysteine was expressed as μmol/L and TNF-α as pg/mL.Homocysteine  and TNF- post hoc Bonferroni's test for the lipid peroxidation curves and by Student's paired t-test for the other parametric parameters. Nonparametric data was analyzed by Mann-Whitney or Wilcoxon matched-pairs tests. Correlations among parameters in plasma and tumoral tissue were also performed using Pearson or Spearman tests. All data were checked using the Grubbs test  to eliminate significant outliers (p < 0.05). p < 0.05 was considered statistically significant.All analyses were conducted in triplicate sets. Statistical analysis was performed using GraphPad Prism 5.0, Microsoft Office Excel 2007, and OriginLab 7.5 software. Results were expressed as arithmetic means and errors of the means. Differences among groups were assessed by two-way analysis of variance (ANOVA) withp < 0.0001). Other lipid peroxidation-derived metabolites did not vary . Semiquantitative analysis of nitrotyrosine (p = 0.0025).The protein-induced oxidative modifications are represented in tyrosine  showed aα (p < 0.001), tumoral nitrotyrosine versus plasmatic NO (p = 0.0456), and plasmatic versus tumoral carbonyl contents (p = 0.0302).We further performed Spearman analysis to investigate whether there was some correlation between the oxidative status of tumors and its respective plasma obtained from the same patient . All parIt is known that oxidative stress is active during the carcinogenic process and correlates with disease prognosis in breast cancer patients , 20. In α, NO, nitrotyrosine, and the carbonyl content between tumor and its matched plasma sample. The mammary tissue presents a large content of adipocytes, which provides abundant feedstock for the occurrence of lipid peroxidation reactions. Polyunsaturated fatty acids that contain two or more double bonds are more susceptible to peroxidation [Protein oxidation also was elevated in tumoral tissue possibly as a consequence of nitrosative stress. Our results showing high levels of TNF besides increased levels of NO and nitrotyrosine suggest a possible cross talk between nitrosative stress and inflammatory mediators in tumor tissue. This cross talk is enforced by the significant correlation between TNF-xidation . Severalxidation . In thisxidation . The anaxidation . Low lipxidation , 26.In association with the reduced lipid peroxidation profile of tumors, we further found unaltered levels of lipid peroxidation products in tumor samples, as hydroperoxides (FOX) and malondialdehyde (MDA), suggesting that other low molecular weight substances may affect the lipoperoxidative status of cells. These findings indicate that, in some instance, tumors are protected against lipid peroxidation, potentially by accumulating membrane and intracellular antioxidants that neutralize this process. This fact was corroborated by the enhanced antioxidant capacity detected in tumor samples when compared to normal adjacent tissue, which explains the reduced lipid peroxidation chain observed in breast tumors. Therefore, high antioxidant content inside breast tumors may be able to retard the initiation of lipid peroxidation process, which may have a regulatory role in cell adaptation to oxidative changes.In spite of the enhanced antioxidant capacity of tumors, we found significant augmented protein carbonylation in the tumoral tissue (but not in the adjacent mammary breast). The carbonylation reaction is an irreversible posttranslational modification that occurred in protein structure that results from the reaction between amino acid residues with the low molecular weight aldehydes generated during the lipid peroxidation process –30 and iWe further detected increased homocysteine levels in breast tumors, suggesting the attack of thiol residues by RS originated in tumor microenvironment. Homocysteine regulates cell cycle, apoptosis, and oxidative stress responses . A direcα, are concomitant with augmented protein carbonylation [α level seem to comprise a network communicating with each other in the breast cancer microenvironment.In cancer cells, enhanced activity of proinflammatory cytokines leads to RS production . A recennylation  and corrnylation , corroboα and NO levels in tumor samples. NO is a multifunctional molecule in cancer. Our data indicate a nitrosative function for this molecule inside breast tumor, as nitrotyrosine was found only in the tumor analysis and not detected in the nontumorous counterpart. In addition, the TNF-driven pathway is constitutively activated in breast cancer [α, NO/nitrotyrosine, and carbonyl content. Altogether, these data support that the systemic prooxidative status reported for several studies in breast cancer patients may be correlated with tumor-driven inflammation.The prooxidative status found here in the tumoral tissue was probably sustained by its proinflammatory nature, as shown by TNF-t cancer , inducint cancer  and modut cancer . Our preα. Therefore, in spite of the reduced size of the tumor mass, it seems that the breast tumor microenvironment  is endowed with an enormous capability to promote profound modifications in the host organism, resulting in the persistent inflammatory status provoked here by TNF-α, NO, and RS production. This fact cannot be satisfactorily explained by the immune response against the tumor, since women presenting local breast disease have systemically established a Th2 immune status [A recent study from our group  has demoe status .Another contributing factor to this redox scenario may be the components of tumor stroma. Tumor-associated fibroblasts can affect metabolically their adjacent cells, and cancer cells use the oxidative environment as advantage to obtain nutrients from the surrounding environment . FurtherIn conclusion, this set of redox alterations found in breast tumors seems to be necessary to ensure the hallmarks of cancer biology, since RS have been implicated in oncogene activation, genomic instability, chemotherapy resistance, and the metastatic process . Further


In this work, proteins in extracellular polymeric substances extracted from anaerobic, anoxic and aerobic sludges of wastewater treatment plant (WWTP) were analyzed to probe their origins and functions. Extracellular proteins in WWTP sludges were identified using shotgun proteomics, and 130, 108 and 114 proteins in anaerobic, anoxic and aerobic samples were classified, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activity and binding activity. The results exhibited that the main roles of extracellular proteins in activated sludges were multivalence cations and organic molecules binding, as well as in catalysis and degradation. The catalytic activity proteins were more widespread in anaerobic sludge compared with those in anoxic and aerobic sludges. The structure difference between anaerobic and aerobic sludges could be associated with their catalytic activities proteins. The results also put forward a relation between the macro characteristics of activated sludges and micro functions of extracellular proteins in biological wastewater treatment process. In the surfaces of microbial cell and aggregate, a major player exhibit important physiological functions during the formation and growth of microbial aggregates, which are extracellular polymeric substances (EPS). It is a complex high-molecular-weight mixture of polymers which most main compositions are extracellular proteins and polysaccharides. Exopolysaccharide components have been well elucidated, which mainly includes glucans, fructans alginate, xanthan, Pel and Psl1356Proteins separation and purification from EPS are essential to further investigation because of their complex compositions. Recently, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the extracellular proteins, and then the gel pieces were analyzed by mass spectrometry (MS) through further processing13In this work, the proteins in EPS extracted from the anaerobic, anoxic and aerobic tanks of wastewater treatment plant (WWTP) were separated and purified. The objective of this study is to identify the extracellular proteins by shotgun proteomics, as well as attempt to investigate their origins and functions.Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Acidobacteria, Actinobacteria, Planctomycetes and Nitrospira were the majority in the three samples. Meanwhile, the classified OTUs and relative abundance were analyzed at genus level to understand the microbial community structure in anaerobic, anoxic and aerobic sludges (Terrimonas (8.38%), Caldilinea (7.71%), Nitrospira (6.89%), Sulfobacillus (5.62%), and Clostridium (5.53%) in the aerobic sludges sample. Terrimonas (23.02%), Caldilinea (12.42%), Pirellula (7.19%) and Planctomyces (6.69%) were the dominant functional genuses in the anoxic sludges sample, whereas Terrimonas (11.26%), Nitrospira (10.78%), Clostridium (10.15%) and Caldilinea (5.98%) were the majority in the anaerobic sludges sample. As shown in Terrimonas and Caldilinea were the dominant genuses in the three samples. Genus Caldilinea was the predominantly members of the phylum Chloroflexi. Chloroflexi play an important structural role in sludge floc formation and can also cause serious sludge bulking or foaming when they are over proliferationNitrosomonas and Nitrospira involved in nitrosation and nitrification processes in biogeochemical nitrogen cycling. However, Genus Nitrosomonas in aerobic sludge (0.09%) was much lower than anaerobic (1.12%) and anoxic (1.17%) sludges, which implied that the concentration of ammonia nitrogen in aerobic tank was decreased significantly. Abundant Genus Nitrospira revealed that the dominant ammoxidation process was nitrification rather than nitrosation in aerobic tank. Genus Pirellula and Genus Planctomyces belong to phylum Planctomycetes, and they are obligate aerobic bacteria. Many aerobic bacteria were present in anaerobic and anoxic tank, which could be associated with the influent substrate and a few aerobic areas existed in these tanks.In order to search and indentify extracelluar proteins of anaerobic, anoxic and aerobic sludges in database, the operational taxonomic units (OTUs) were obtained at phylum level . A total sludges . The domldilinea .42%, Pir−1 were assigned to the O–H or N–H stretching vibrations. The peak at 1654 cm−1 was assigned to the C = O stretching vibration of amide I. The N–H deformation vibration of amide II was observed at 1532 cm−1, whereas the peak at 1230 cm−1 was due to the C–N stretching vibration of amide III. The bands at 1131 cm−1, 1082 cm−1, 1041 cm−1, and 983 cm−1 were attributed to the C–O and C–C stretching as well as the C–O–C and C–O–H deformation vibrations of the polysaccharide structures. The bands at 1082 cm−1 and 1041 cm−1 may be also associated with the P = O stretching, C–P–O and C–O–H stretching vibrations, respectively. The peak at 983 cm−1 was also likely to be related to O–P–O stretches. The results indicated that the extracellular protein samples could mix/bind carbohydrate and phosphorylated macromoleculesThe FT-IR spectra of extracelluar proteins from anaerobic, anoxic and aerobic sludges are shown in 1314The MS/MS spectra of protein samples were automatically searched against the protein database according to the dominant phyla of microbial communities and 149, 129 and 131 proteins were identified in anaerobic, anoxic and aerobic sludge samples, respectively. 130, 108 and 114 proteins in the identified proteins of anaerobic, anoxic and aerobic sludge samples were classified, respectively, according to GOA in terms of biological process, cellular component and molecular function4 (3.1%), 1 (0.9%) and 3 (2.6%) proteins in anaerobic, anoxic and aerobic sludge samples were derived from extracellular region, respectively, and the identified names are exhibited in et al.1920232425Most extracellular proteins primarily located in the cell and cell part, which implied that the main extracelluar proteins were derived from cell lysis. It was consistent with the result described by Silva Clostridium mainly contains a large group of anaerobic bacillus, which was also abundant in aerobic sludge. The presence of the anaerobic environment could be ascribed to the tightly aggregation of microorganisms. It indicated that the two sludges’ structures were various, and a probable reason was the diversities of catalytic activity proteins in the sludges. Excessive extracellular enzymes could deteriorate the stability of aggregate structures from the following probably aspects: (1) EPS was hydrolyzed or degraded, causing to EPS fracture and aggregate structures damage, which resulted to sludge floc dispersion; (2) Microbial attachment was suppressed, reducing the sludge floc compactness43031Anaerobic and aerobic sludges exhibit distinct aggregate architectures, biochemical processes, and surface characteristics. Anaerobic condition creates porous activated sludge, whereas aerobic condition produced more compact flocs28Extracellular proteins extracted from WWTP sludges were identified by shotgun proteomics, and 130, 108 and 114 proteins were classified in anaerobic, anoxic and aerobic sludges, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activity and binding activity, indicating that their main roles in activated sludges were multivalence cations and organic matters binding, as well as catalysis and degradation. Both the amount and proportion of catalytic activity proteins in anaerobic sludge were higher than those in aerobic sludge. The results implied that the catalytic activity proteins in anaerobic sludge and aerobic sludge contributed to their different floc structures.Activated sludges were collected from the anaerobic, anoxic and aerobic tanks of Jiguanshi WWTP, Chongqing, China. The samples were stored in darkroom and at 4 °C before DNA and EPS extraction. DNA was extracted using 3S column centrifugal environmental sample DNA extraction kit . 16S rRNA gene polymerase chain reaction amplification, Illumina MiSeq sequencing, and data analysis were performed by Personalbio Biotech  Co., Ltd. Primers 520F (5-AYTGGGYDTAAAGNG-3) and 802R (5-TACNVGGGTATCTAATCC-3) which target V4 regions of bacterial 16S rRNA genes were selected.g for 20 min at 4 °C. At last, the supernatant was filtered through 0.45 μm filter and stored at 4 °C for further experiments.The EPS in activated sludge was extracted using cationic exchange resin (CER) method. In general, 200 mL of sludge suspention and CER (70 g/g VSS) were transferred to a conical flask. The extraction was carried out in a shaking incubator at 250 rpm for 2 h at 4 °C. Then the suspension was centrifuged at 10000 g for 20 min at 4 °C, and the supernatant was removed. The residual TCA in the precipitates was washed using acetone and centrifuged at 12000 g for 3 min, and the supernatant was rejected. The washing step was performed 3 times at 4 °C. At last, the precipitates were collected for enzymatic hydrolysis.The protein in EPS was separated by the modified method of trichloroacetic acid (TCA) precipitationet al.4HCO3 twice. Finally, the protein suspension with 2 μg of trypsin in 40 μl of 25 mM NH4HCO3 incubated overnight at 37 °C to protein digestion. The resulting peptides were filtrated and collected for further analysis.Extracellular protein digestion was conducted according to the method of Wisniewski m/z 300–1800) was followed by twenty MS/MS events of the most intense ions using dynamic exclusion .The LC system  was used for desalting and separation of tryptic peptides mixtures. In this system, samples were desalted on reverse phase (RP) trap columns , and then separated on a RP column . The mobile phase A was 0.1% formic acid in HPLC-grade water and the mobile phase B was 0.1% formic acid in acetonitrile. 20 μg of tryptic peptide mixtures was separated at a flow rate of 2 μL/min with a linear gradient  in the columns. A mass spectrometer  was equipped with a micro-spray interface and connected to the LC setup to detecte the eluted peptides. The MS/MS spectra were set so that one full scan mass spectrum  using the BioworksBrowser rev. 3.1 . Protein identification results were extracted from SEQUEST out files with BuildSummary. The mass tolerance allowed for the precursor ions was 2.0 Da and fragment ions was 0.8 Da, respectively. The protein identification criteria were based on Delta CN (≥0.1) and cross-correlation scores with the minimum values of Xcorr: one charge ≥1.9, two charges ≥2.2, and three charges ≥3.753536http://www.uniprot.org/uniprot/) according to the protein accession numbers.MS/MS spectra were automatically searched against the protein database based on the dominant phyla of microbial communities . The extracellular protein precipitates were lyophilized and mixed with IR grade KBr powders to prepare the pellets to measure.How to cite this article: Zhang, P. et al. Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics. Sci. Rep. 5, 12041; doi: 10.1038/srep12041 (2015).


A relatively new method of electrocautery, the radiofrequency bipolar hemostatic sealer (RBHS), uses saline-cooled delivery of energy, which seals blood vessels rather than burning them. We assessed the benefits of RBHS as a blood conservation strategy in adult patients undergoing multilevel spinal fusion surgery.n = 38) matched for variables related to blood loss. Transfusion-related costs were calculated by two methods.In a retrospective cohort study, we compared blood utilization in 36 patients undergoing multilevel spinal fusion surgery with RBHS  to that of a historical control group (p = 0.002), and over the entire hospital stay, red cell utilization was 51% less  and plasma use was 56% less  in the RBHS group. Platelet use was 0.1 ± 0.5 and 0.3 ± 0.6 units/patient in the RBHS and control groups, respectively (p = 0.07). The perioperative decrease in hemoglobin was less in the RBHS group than in the control group , and hemoglobin at discharge was higher in the RBHS group . The estimated transfusion-related cost savings were $745/case by acquisition cost and approximately 3- to 5-fold this amount by activity-based cost.Patient characteristics in the two groups were similar. Intraoperatively, blood loss was 55% less in the RBHS group than in the control group (810 ± 530 vs. 1,800 ± 1,600 mL; The use of RBHS in patients undergoing multilevel spine fusion surgery can conserve blood, promote higher hemoglobin levels, and reduce transfusion-related costs. Given the recognized risks [–3]] and] and3]] Studies have shown that bleeding is reduced when RBHS is used during liver resection , total n = 38) that was propensity-matched for important variables related to blood loss. These variables included the number of spinal levels fused, type of surgical procedure , the nine specific surgeons performing the procedures, and the percentage of patients undergoing revision spinal surgery. Patients in the matched control group were selected from the operating room medical information system (ORMIS) database and underwent surgery between August 2012 and January 2013. Patients in the RBHS group were selected as consecutive surgical cases during July and August of 2013, when the RBHS was introduced. Exclusion criteria were preoperative coagulopathy, thrombocytopenia, and refusal of blood product transfusion.After receiving approval from the Johns Hopkins Hospital institutional review board, we performed a retrospective cohort study, whereby 36 consecutive patients undergoing multilevel spinal fusion surgery with the RBHS device  were compared to a historical control group  and intraoperative autologous blood salvage  were used in both study groups. An adult-size 225-mL bowl was used for blood salvage, and salvaged blood was processed and transfused if the collected volume exceeded 500 mL. Intraoperative hemoglobin (Hb) triggers were used according to the usual practice at our institution for this type of surgery (9–10 g/dL intraoperatively and 8–9 g/dL postoperatively). FFP and PLTs were transfused according to the clinical judgment of the anesthesiologists and surgeons. Typically, FFP and PLTs were given if the transfusion requirements exceeded ½ of the patient's total blood volume (≈5 units RBCs).Blood utilization data were obtained from two separate databases. Intraoperative data were obtained from the electronic anesthesia records . A detailed description of this database and its validation have been published previously . Whole We calculated transfusion-related cost by two different methods. The first method is based on the average cost to acquire blood components from blood suppliers in the mid-Atlantic region (acquisition cost). These costs were calculated as $220/RBC unit, $50/FFP unit, and $600/PLT unit. The second method is the activity-based cost of blood, as described by Shander et al. , which α of 0.05 and power of 0.8, we required an estimated sample size of 32 patients per study group.We performed a sample size calculation based on the known blood utilization for these types of cases at our institution . To dett tests. Dichotomous variables were analyzed by the chi-squared test. All tests were two-tailed. All data are presented as mean ± SD, and p < 0.05 was used to define significance.Differences in the mean measured values between groups were analyzed by Student's The RBHS and control groups were similar for the variables of age, sex, body mass, number of spinal levels fused, and preoperative hemoglobin Table . The perp = 0.002). Likewise, the volume of colloid used was 63% less (p = 0.04), and the volume of returned salvaged blood was 54% less (p = 0.003) in the RBHS group than that in the control group (Table p = 0.04), and the hemoglobin concentration at discharge was 0.8 g/dL higher in the RBHS group (p = 0.01) than that in the control group. These changes in hemoglobin levels are summarized in Figure Intraoperatively, estimated blood loss was 55% less in the RBHS group than in the control group p = 0.00. Likewisp p = 0.0 than thap = 0.009) and 83% fewer units of FFP (p = 0.002) than did the control group. The two groups did not differ significantly in PLT requirements. Over the entire hospital stay, the RBHS group received 51% fewer units of RBCs (p = 0.01) and 56% fewer units of FFP (p = 0.03) than did the control group .We also compared the transfusion requirements in the two groups Table . Intraop p = 0.00 than didp = 0.35).The incidence of morbid events and the composite outcome of any morbid event were similar in the two groups Table . The difWhen we multiplied the acquisition costs for each unit of blood component in US dollars by the number of units saved (difference between groups) during the entire hospital stay, we found that use of the RBHS device saved an average blood cost of $745/patient  from K2M (paid directly to institution/employer) and DePuy Spine (paid directly to institution/employer).SF was involved with design of the study, statistical analysis, and with manuscript preparation. JW was involved with the data interpretation, statistical analysis, and manuscript preparation. ID assisted with data collection and analysis. ZG, PN, and KK assisted with data interpretation and manuscript preparation. All authors read and approved the final manuscript.SF is the Medical Director of the Patient Blood Management Program at Johns Hopkins Hospital. ZG is the Chief of Spine Surgery at Johns Hopkins Hospital. PM is the Director of Transfusion Medicine at the Johns Hopkins Hospital.


Sleep disorder induced by acute stress has always been an important topic for study among the general population. However, the mediating effect of social support between acute stress and sleep disorder has rarely been reported before.A total of 2,411 grass-root military personnel were randomly selected by cluster sampling, and administered the Chinese Military Personnel Sleep Disorder Scale, Military Acute Stress Scale and Social Support Rating Scale.r = 0.209 ~ 0.465, P < 0.01); The total score of social support scale was positively correlated with the total score of acute stress scale and the total score and factor scores of sleep disorder scale . The analysis of mediating effects showed that lack of social support partially mediated between acute stress and the factors of sleep disorder. The analysis of structural equation model showed that acute stress not only had a direct effect on sleep disorder , but also on lack of social support ; lack of social support had a direct effect on sleep disorder .The total score of acute stress scale was positively correlated with the total score and factor scores of sleep disorder scale (Acute stress and lack of social support are two significant factors of sleep disorder in grass-root military personnel. Well-established social support could alleviate sleep disorder induced by acute stress. Lack of social support was a partial mediator between acute stress and sleep disorder. Stress has been a significant predisposing cause for various major fatal diseases. Military stress is defined as a special emotional state under extraordinary military circumstances, which might physiologically and psychologically exert negative influences upon individuals . Acute sP < 0.01), and the correlation coefficient between factors and the general scale ranged from 0.72 to 0.82 (P < 0.01); the test-retest coefficient of the general scale and factors ranged from 0.62 to 0.80 (P < 0.01); Cronbach’α coefficients ranged from 0.62 to 0.87; split half coefficients ranged from 0.55 to 0.83. These verification results have demonstrated that this scale had excellent reliability and validity, meeting psychological assessment criteria.Social Support Rating Scale : there aP < 0.01), and the correlation coefficient between factors and the general scale ranged from 0.40 to 0.83 (P < 0.01); the test-retest coefficient of the general scale and factors ranged from 0.62 to 0.88 (P < 0.01); Cronbach’α coefficients ranged from 0.35 to 0.67; split half coefficients ranged from 0.59 to 0.85. These verification results have demonstrated that this scale had excellent reliability and validity, meeting the psychological assessment criteria.Chinese Military Personnel Sleep Disorder Scale : there aP < 0.01), and the correlation coefficient between factors and the general scale ranged from 0.70 to 0.85 (P < 0.01); the test-retest coefficient of the general scale and factors ranged from 0.38 to 0.91 (P < 0.01); Cronbach’α coefficients ranged from 0.61 to 0.93; split half coefficients ranged from 0.47 to 0.86. These verification results have demonstrated that this scale had excellent reliability and validity, meeting the psychological assessment criteria.Military Acute Stress Scale : there aAll participants were divided into groups of about 30 to 50 individuals and were group-tested by automatic testing device. One research fellow made the leading remarks before the procedure, and three research fellows monitored the procedure, which lasted about half an hour. All participants were given the informed content before testing.This study was approved by the Human Research Medical Ethics Committee at No. 102 Hospital of PLA. Informed consents were obtained from all participants. Details regarding the study methods have been reported previously.All participants were screened for histories of psychological diseases, organic diseases and drug dependence. They were not requested to fill out the name of the responders to dispel misgivings. All questionnaires which were continuously, randomly or arbitrarily filled, or whose lying score was higher than the average plus 1.96 standard error, were excluded. All 2,490 pieces were recollected, among which 79 were excluded based upon standards mentioned above, making questionnaire validity rate 96.8%.P < 0.01 was considered as statistically significant.Pearson correlation analysis and stratified regression analysis were performed in the platform of SPSS version 17.0. The stratified regression analysis was carried out as follows. Based upon the study by Zhonglin et al. , regressTable P < 0.01). It also shows the total score of acute stress scale was positively correlated with social support (P < 0.01); the total score of acute stress scale was positively correlated with the total score and factor scores of sleep disorder (P < 0.01).Table Based upon the study by Zhonglin et al. , Table 3x2/df = 30.963 (P = 0.000), GFI = 0.931, AGFI = 0.877, CFI = 0.938, RMSEA = 0.112. This model was modified by assuming covariation between e1 and e6, e3 and e8, e6 and e8, e8 and e9, and better fitting indexes were generated as follows:x2/df = 7.596 (P = 0.000), GFI = 0.985, AGFI = 0.968, CFI = 0.989, RMSEA = 0.042 (see Figure Maximum likelihood method was employed to calculate the major fitting indexes as follows: The correlation analysis in this study demonstrated that acute stress and social support were positively correlated with sleep disorder and its factors, which might suggest that acute stress and lack of social support exert impact upon sleep quality, according well with other studies. Spoor-maker et al. concluded that sleep disorder constituted a prominent issue in acute stress, and more serious consequences could be brought about by acute stress events plus sleep disorder , 12. BotThe results of stratified regression analysis showed that social support partially mediated between acute stress and sleep disorder, which was further confirmed by structural equation model construction. Both results verified that acute stress could directly lead to sleep disorder, and also could indirectly lead to sleep disorder through the mediating effect of social support. This conclusion is consistent with other previous studies. Yu et al. found that social support mediated between stressful life events and psychological symptoms . AnotherThis study has demonstrated through mediating effect analysis and structural equation model construction that social support partially mediated between acute stress and sleep disorder, and well-established social support system and less acute stress could greatly improve sleep quality. This study is of great significance in providing references for improving sleep quality in grass-root military personnel and enhancing military combating capacity.


Scientific Reports5: Article number: 1354510.1038/srep13545; published online: 08272015; updated: 02092016In the Supplementary Information file originally published with this Article, parameter k6 was omitted from Table 2. This error has been corrected in the Supplementary Information that now accompanies the Article.In addition, the model source codes were omitted.There was also an error in Fig. 1, where reaction ‘k5’ now reads ‘2*k5’. The correct These errors have now been corrected in both the PDF and HTML versions of the Article.


Hyperprolactinemia has been related to autoimmune diseases. Herein, we describe a case of a female with a prolactin producer pituitary macroadenoma who developed severe polymyositis one month after its removal. The patient had very high levels of CPK and muscle biopsy showed remarkable inflammatory infiltration. Steroid therapy was followed with total recovery. To the best of our knowledge, this is the first case reported of acute polymyositis after pituitary macroadenoma exeresis. Polymyositis is a chronic autoimmune disease that causes proximal muscle weakness as well as remarkable functional limitation. Its aetiology is unknown; it has been suggested, however, that prolactin could play a role in its development. This is based on the fact that high levels of this hormone have been found in up to 25% of patients with polymyositis ; it has μL (4–12), platelets of 287 K/μL (150–450), ASAT of 37 U/mL (13–39), ALAT of 35 U/ML (7–52), glucose of 98 mg/dL (65–100), creatinine of 0.7 mg/dL (0.6–1.2), and prolactin of 75 ng/mL (2–29). Head MRI now showed a pituitary macroadenoma which was removed by endoscopic transnasal exeresis. After this procedure, prolactin levels returned to normal values.A 39-year-old woman without any relevant background started having an intermittent headache in 2008, which improved with common analgesics. Six months later, she noticed amenorrhea, with repeated negative pregnancy tests. Her gynaecologist ordered both blood tests and head magnetic resonance imaging (MRI) that showed hyperprolactinemia and a pituitary microadenoma. He prescribed cabergoline 1.5 mg twice a week with a good response for two years, when the patient noticed visual deficit and increased headache intensity. On admission to our hospital, the only alteration found on physical examination was bitemporal hemianopsia. Blood tests reported haemoglobin of 14.8 g/dL (13–15.7), WBC of 6.7 K/μL (4–12), and platelets of 287 K/μL (150–450). Antinuclear antibodies were positive with speckled pattern (1 : 320) and anti-Jo1 antibodies were negative at 2.1 U/mL (<6.5). No other antibodies were tested. CPK was 7377 U/mL (37–177), ASAT was 891 U/mL (13–39), and ALAT was 609 U/mL (7–52). Respiratory acidosis was also detected with pCO2 of 46 mm Hg in arterial blood which necessitated the patient to be put under noninvasive positive pressure ventilation. Muscle MRI showed remarkable inflammatory alteration with diffuse bilateral myofibrillar intensity of all muscle groups evaluated, from the pelvis to the distal quadriceps. Muscle biopsy of vastus lateralis demonstrated notable inflammatory infiltration with atrophy, degenerative changes, and myocitolysis.Just one month after surgery, the patient started with progressive muscle weakness that became worse in the following two weeks until it left her prostrate in bed. A heart rate of 120 beats/min, a respiratory rate of 20/min, blood pressure of 90/50 mm Hg, and temperature of 37°C were found upon arrival to the emergency room. She was extremely weak, to the extent that she was able to neither keep her head up nor swallow. Blood tests now showed haemoglobin of 9.7 g/dL (13.0–15.7), WBC of 9.8 K/Taking the above data into account, treatment was started with 8 mg of IV dexamethasone every 24 hrs, which resulted in a progressive decrease of serum's CPK levels until their normalisation; the patient, however, had to be kept on totally enteric nutrition through a gastrostomy for one month, after which she recovered her deglutition ability. At the time of writing, the patient is completely asymptomatic and is under treatment with 100 mg azathioprine.This case shows a clear temporal relationship between the extraction of the prolactinoma and the onset of disease. Is it possible, however, that, in addition to a simple association, there could be a causal relationship between these phenomena?There is a vast amount of information regarding the presence of hyperprolactinemia in rheumatic autoimmune diseases . This isInterestingly, prolactin concentrations at slightly higher levels than the physiological range but not those vastly superior induce an increased production of IgG by mononuclear cells from SLE patients , thus crThus, even though it is possible that the chronic exposure to slightly higher levels of prolactin in a genetically susceptible individual could have contributed to the onset of polymyositis, the actual manifestation of the disease did not take place until the removal of the tumor, which begs an obvious question: why was the disease dormant until precisely that moment? In this regard, dopamine agonists have shown a therapeutic effect in SLE and, less consistently, in RA and psoriasis , 10. It Hence, the possibility exists that, in this case, the presence of cabergoline could have acted as therapy for an incipient autoimmune disease. Even though dopamine-agonist could have helped to maintain a quiescent state of the disease, both tumor removal and treatment withdrawal were probably the trigger for the full-blown manifestation of the disease. Certainly, this speculation, although undoubtedly interesting, is based on the role of prolactin in other autoimmune diseases, whilst not in polymyositis.Be that as it may, this case is interesting inasmuch as it represents the first description of acute onset polymyositis after removal of a pituitary macroadenoma in the English literature.


Eulemur species, we identified two key study groups ‐‐ one comprised of species expressing FSD and increased female scent marking, the other comprised of species (from a recently evolved clade) showing equal status between the sexes and the more traditional pattern of sexually dimorphic behaviour. Comparing females from these two groups, we show that FSD is associated with more masculine androgen profiles. Based on the widespread prevalence of male‐like features in female lemurs and a current phylogeny, we suggest that relaxation of hormonally mediated FSD emerged only recently and that female masculinization may be the ancestral lemur condition, an idea that could revolutionize our understanding of the ancient socioecology and evolution of primate social systems.Female social dominance (FSD) over males is unusual in mammals, yet characterizes most Malagasy lemurs, which represent almost 30% of all primates. Despite its prevalence in this suborder, both the evolutionary trajectory and proximate mechanism of FSD remain unclear. Potentially associated with FSD is a suite of behavioural, physiological and morphological traits in females that implicates (as a putative mechanism) ‘masculinization’ via androgen exposure; however, relative to conspecific males, female lemurs curiously show little evidence of raised androgen concentrations. By observing mixed‐sex pairs of related  Eulemur clade, point to FSD as a likely ancestral, hormonally mediated condition Lemur catta)4: androst‐4‐ene‐3,17‐dione), are either dramatically or moderately elevated during pregnancy, providing a basis for organizational effects on developing offspring; otherwise, T in adulthood, rather puzzlingly, follows the typical mammalian pattern of much reduced concentrations in females relative to males2329303132Crocuta and Lemur, respectively – are the sole extant members of their genera.In the best known cases of FSD – displayed by the spotted hyena  and by prominent anogenital glands that often are more elaborate than are those of conspecific males  produce olfactory signals in adulthood that are more chemically complex than are those of conspecific males, whereas females from species that are putatively egalitarian and show sexual ‘co‐dominance’  do notE females relative to co‐E females might be indicative of an unusually important role in the former for scent marking, which would be consistent with female behavioural masculinization. Ideally, a potential mechanism of FSD would explain the entire suite of masculinized characteristics in females, as well as the observed patterns of individual, sexual, and species variationEulemur species might implicate uniform organizational effects in females via exposure to prenatal steroids, whereas the disassociation between masculinized morphological and behavioural traits across Eulemur species could be explained by variability in adult, activational effects of steroids.Interestingly, all ic males 1234. DesEulemur derives from studies in which researchers had measured various outcomes (e.g. feeding priority versus agonistic ‘wins’), under varying socioecological conditions, both in captivity and in the wild342628E females and the absence of ‘masculinized’ behaviour in co‐E females.Because the attribution of either FSD or sexual co‐dominance in E females directed more dominance  and aggressive  behaviour toward their respective male partner than vice versa  than did dom‐E females  and no difference between the females of either group . By confirming the lack of intersexual dominance in E. fulvus species, as well as describing reduced aggression and scent marking in co‐E females, we established two study groups of females from closely related species portraying different degrees of female masculinization, one group with morphological masculinization only, the other group with both morphological and behavioural masculinization.Supporting the inferences derived from prior chemical data, we also found that dom‐4 and T, and the estrogen, estradiol ‐triene‐3,17‐diol). All four analytes typically reveal measurable sex differences in mammals and have been shown to correlate, particularly in males, either negatively (e.g. 5‐HT) or positively (e.g. androgens and estrogens) with the expression of aggression and dominanceNext, we compared the hormonal profiles between and, more importantly, within the sexes of our two study groups. We measured serum concentrations of serotonin (5‐HT: 5‐hydroxytryptamine), the androgens ACrocuta63031Lemur723Eulemur study groups showed the typical mammalian sex difference in T , aged 9–29 (mean + S.E.M.: 20.32 + 0.98) years, representing six species of Eulemur . The anihromatic , furtherLemur catta, the members of the various Eulemur species exhibit strictly seasonal estrous cycles, with seasons in the Northern Hemisphere being shifted by six months from those in Madagascar23Eulemur at the DLC generally begins in October, with the peak in births occurring in March and weaning being completed by the end of May. So as to avoid individual variability owing to differences in the timing of female reproductive cycles, we studied the animals across a 3‐month period from June to September 2010, examining behaviour and its endocrine correlates during the nonbreeding season.As in 2), and were exposed to natural daylight and the local photoperiod. During the warmer months, some of the animals had access to larger, forested enclosures (1.5–27.2 acres), often with several other non‐Eulemur species occupying the same habitat. The housing has been described previouslyEulemur mongoz are fed folivore chow , whereas the other Eulemur species are fed Old World monkey chow . The diets of all the animals are supplemented with a mixture of fruits and vegetables. Those animals that semi‐free‐range can additionally supplement their normal diet with local vegetation and with insects foraged from the forest.Our focal pairs of animals were housed in large, indoor/outdoor enclosures .Animals at the DLC are well habituated to the presence of humans, so behavioural observation can occur from short distances (e.g. 1 m), even within the forest. Because each of our focal pairs was housed within a well‐defined area and separately from other A comprehensive ethogram included dominance behaviour (e.g. supplant), aggression , affiliation , and scent marking . We entered the behavioural data, with a time stamp for each event, directly into hand‐held, portable computers . We used either an ‘actor‐behaviour’ format (for scent marking) or an ‘actor‐behaviour‐recipient’ format , which allowed us to record the frequency, directionality, and duration of all interactions between the male and female pair members. All four observers were trained prior to data collection and were tested routinely for inter‐observer reliability (mean ± S.D. = 88.7 ± 0.1%). To further minimize the effect of variation in the data potentially owing to multiple observers, each dyad was observed an equal number of times by each observer over the course of the study.n = 42 male and n = 42 female samples). On blood‐draw days, the animals that previously had been corralled into their indoor enclosures were netted, transported to a veterinary procedure room located in the same building, and processed individually, to minimize the time delay between capture and blood draw (mean ± S.E.M. = 5.00 ± 0.68 min). Handling occurred primarily in the morning . Using a 23‐gauge needle and syringe, we drew blood samples (3 cc) from the femoral vessels of awake, manually restrained animals, all of which were habituated to these procedures. We immediately transferred the blood samples to serum separator tubes , allowed them to clot at room temperature, centrifuged them at 1500 × g for 20 min, and stored the decanted serum at −80°C.With the assistance of DLC veterinary personnel, we obtained from each subject one blood sample per month, for a total of three samples per subject in the nonbreeding season  kits . We validated the EIA assays for analyte recovery by spiking a known amount of analyte into a pooled serum sample and comparing the observed and expected results. We validated the EIA assays for linearity by running a serial dilution of the pooled serum and comparing the slopes against the standard curves. For all assays, recovery ranged from 85% to 105% and each dilution curve was parallel to the appropriate assay standard curve. The 5‐HT assay has a sensitivity of 5 ng/ml using a 25‐μl dose, with an intra‐ and inter‐assay coefficient of variation (CV) of 5.4% and 6%, respectively. The A4 assay has a sensitivity of 0.04 ng/ml using a 25‐μl dose, with an intra‐ and inter‐assay CV of 5.23% and 8.7%, respectively. The T assay has a sensitivity of 0.02 ng/ml using a 50‐μl dose, with an intra‐ and inter‐assay CV of 7.9% and 7.3%, respectively. The E2 assay has a sensitivity of 10 pg/ml using a 50‐μl dose, with an intra‐ and inter‐assay CV of 7.7% and 8.7%, respectively. We performed all assays in duplicate. If any individual sample's duplicate CV exceeded the intra‐assay CV it was re‐run in a subsequent assay.We determined serum concentrations of 5‐HT, AE vs. co‐E). We conducted these analyses separately for both the behavioural and endocrine data. To account for small sample sizes and lack of normality in the data, we used the two‐tailed Mann‐Whitney U test for all comparisons.We averaged each individual's data points across the study. In the event that an individual's assay result was below the level of detectability, we used the minimum sensitivity value for that assay in our calculations. We tested for sex effects within each study group or type of dominance structure  and for effects of dominance structure within the female sex (i.e., dom‐Eulemur research under the supervision and guidance of C.M.D. J.M.A.P. analyzed the data and prepared the figures. C.M.D. and J.M.A.P. wrote the manuscript.C.M.D. conceived of and conducted the comparative strepsirrhine work on female dominance, genital masculinization, and olfactory communication. J.M.A.P. designed and conducted the comparative


Taeniopygia guttata) that had been dosed with 0.0, 0.1, and 0.6 parts per million (ppm) dietary methylmercury and measured changes in blood total mercury concentrations (THg) in relation to reductions in lean mass. Birds lost 6–16% of their lean mass during the fast, and THg increased an average of 12% and 11% in the 0.1 and 0.6 ppm treatments, respectively. Trace amounts of THg in the 0.0 ppm control group also increased as a result of fasting, but remained extremely low. THg increased 0.4 ppm for each gram of lean mass catabolized in the higher dose birds. Our findings indicate that methylmercury is mobilized from lean tissues during protein catabolism and results in acute increases in circulating concentrations. This is a previously undocumented potential threat to wild migratory birds, which may experience greater surges in circulating methylmercury than demonstrated here as a result of their greater reductions in lean mass.The pollutant methylmercury accumulates within lean tissues of birds and other animals. Migrating birds catabolize substantial amounts of lean tissue during flight which may mobilize methylmercury and increase circulating levels of this neurotoxin. As a model for a migrating songbird, we fasted zebra finches ( Methylmercury is a toxic and bioavailable form of mercury that can be prevalent in aquatic and terrestrial food webs22In migrating birds, organs and muscles are rapidly catabolized during flight, losing as much as 50% of their pre-flight mass75911Taeniopygia guttata) to a prolonged fast and measured changes in their blood mercury concentrations in relation to changes in their lean mass. We expected fasting to increase blood mercury level and that the increase would be proportional to the loss of lean tissue.As a model for the lean mass dynamics that occur in migrating songbirds613F3,40 = 0.36, p = 0.78).Dosing resulted in average blood total mercury concentrations  of 0.987 (±0.213 SD) and 5.749 (±1.092 SD) μg/g (hereafter ppm) in the 0.1 and 0.6 ppm groups, respectively. Control birds had only traces of blood THg . Males and females did not differ in blood THg concentration, lean body mass, or the amount of lean body mass lost during the fast . Birds lost 0.626–2.385 g (mean: 1.545 g ± 0.409 SD) of lean mass during the fast, which represented 6–16% of their starting lean mass (mean: 12% ± 3 SD).Starting lean mass and change in lean mass were not different among treatment groups  and 0.6 ppm treatments  by an average of 12% and 11% above starting concentrations, respectively , but concentrations remained extremely low (mean = 0.009 ± 0.005 ppm).Fasting significantly increased blood THg in the 0.1 ppm .When we tested treatments separately, there was a marginally significant relationship between change in blood THg and change in lean mass for the 0.6 ppm group , but not for the 0.1 ppm  or 0.0 ppm groups  analysis  as in824318Postabsorptive birds were weighed, blood sampled , scanned in the QMR, and then held in indoor cages without food or water for 18–21 h. Following the fast, birds were weighed, scanned in the QMR, and blood sampled from the opposite wing. All procedures were approved and overseen by, and carried out in accordance with the regulations of, the College of William & Mary Institutional Animal Care and Use Committee.We measured THg (ppm wet weight) in the blood, which is representative of methylmercury in birds23p > 0.17) and the final model included treatment group and change in lean mass. We also tested the relationship between change in blood THg and change in lean mass within each treatment group independently using GLMs. Change in lean mass, change in fat mass, and change in blood THg data were log + 1 transformed prior to analyses to better meet assumptions of homoscadicity and normality. Tests were performed using R and statistical significance was accepted when p < 0.05.We used multivariate analysis of variance (MANOVA) to assess whether sex had any effect on pre-fasting blood THg concentrations, pre-fasting lean mass, or the amount of lean mass lost during the fast because sex ratios were different among treatment groups. Pre-fasting lean mass and change in lean mass after fasting were compared among treatment groups also using MANOVA. Blood THg before and after the fast was compared within each treatment group using paired t-tests. We used a general linear model (GLM) to test the relationship between change in blood THg and change in lean mass. The initial model included treatment group, change in lean mass, change in fat mass, and all two-way interactions as factors. Non-significant terms were then sequentially dropped .


Supplemental Digital Content is available in the text Estrogen receptor alpha 36 (ERα36), a truncated variant of ERα, is located in cytoplasm and membrane that is different from other nuclear receptors of ERα family. ERα36 is involved in progression and treatment resistance of a variety of carcinomas. However, the clinical and prognostic significance of ERα36 in renal tumors have not been fully elucidated.Here, renal tumor tissues from 125 patients were collected and immunohistochemical stained with ERα36 antibody. ERα36 expression level and location in these cases were analyzed for their correlations with clinical characteristics. The differential diagnosis value was also assessed for benign and malignant renal tumors, as well as its prognostic value.The results showed that membrane ERα36 expression was rarely detected in benign tumors but predominantly observed in malignant renal tumors. Kaplan–Meier analysis indicated that significant correlations of high ERα36 level and ERα36 membrane expression were correlated with both poor disease-free survival and overall survival. Univariate and multivariate analysis confirmed that both ERα36 high expression and membrane location can serve as unfavorable prognostic indicators for renal cell carcinoma.It is thus concluded that membrane ERα36 expression is valuable for differential diagnosis of malignant renal tumors from benign ones. Both ERα36 high expression and membrane location indicate poor prognosis in renal cell carcinoma. The lack of biomarkers for prognosis estimation may lead to poor clinical response.6 Hence, it is required to investigate the predictive biomarkers for differential diagnosis and targeting therapies for renal tumors.Most primary renal tumors are malignant, but it is difficult for a differential diagnosis of benign renal tumors from malignant ones, because of the complicated histological characters in renal tumors.7 The animal models of renal cancer that were established with estrogens exposure also confirmed that hormone/estrogen receptor (ER) complex participated in renal cell carcinoma initiation and progression.10 Two types of ERs, ERα and ERβ were investigated in clinical cases in previous studies.14 However, immunohistochemistry (IHC) study of tissue microarray (TMA) showed that ERα immunoreactivity was less than 10% of tumor cell nuclei.16 Another study found that estrogen-activated ERβ acted as a tumor suppressor in renal cell carcinoma.12 However, gene expression analysis of ER targeted genes in renal cell carcinoma demonstrated that ER signaling was closely associated with tumor progression.18 Therefore, hormone/ER signaling-related cancer progression is probably mediated by another ER variant.Emerging proofs indicate that estrogens and their receptors play critical roles in various cancers and it is speculated that human kidney maybe also affected.19 It is participated in non-genomic estrogen signaling to promote cell proliferation.21 The expression of ERα36 is correlated poor prognosis in many kinds of carcinoma.24 In this study, we assessed the expression of ERα36 by IHC in renal tumors, and its association with clinicopathologic characteristics as well as clinical outcome. We further evaluated its differentiation and prognostic significance in renal tumors.ERα36 is a truncated variant of ERα, which was reported located in membrane and cytoplasm, rather than nuclei.The retrospective study cohort consisted of 125 patients with primary renal tumors, who underwent surgical resection in the Affiliated Hospital of Qingdao University Medical College, and 401st Hospital, Shandong, China, between 2001 and 2013. Informed consent was obtained from each patient according to the research proposals approved by the local ethics committee of Qingdao University and 401st Hospital. Eligibility criteria included written informed consent and availability of tumor tissue, and follow-up data. For each patient, the following clinicopathologic information was collected, including age, sex, tumor size, TNM stage, presence of histological tumor necrosis, and Fuhrman grade. Clinical information was obtained by reviewing the medical records, by telephone or written correspondence, and by reviewing the death certificate. Follow-up information was updated every 6 months by telephone interview or questionnaire letters and was last done in January 2015.24 ERα36 expression levels in 5 renal tumor tissues were studied by immunoblotting and qRT-PCR assays,19 which confirmed the IHC staining specificity . TMA was created from the formalin-fixed, paraffin-embedded tissue blocks of the patients. All samples were reviewed histologically by hematoxylin and eosin (HE) staining, and representative areas were marked on the paraffin blocks away from necrotic and hemorrhagic materials. Sections from the TMA blocks were cut at 4 μm. Primary antibody against human ERα36  was applied for immunohistochemistry analysis. Antigen retrieval was performed in citrate buffer pH 6.0, then the sections were incubated overnight at 4°C with the primary antibody at 1:200. Next, they were rinsed with phosphate buffer solution (PBS) and incubated with the horseradish peroxidase-conjugated secondary antibody, followed by a rinse in PBS, incubation with diaminobenzidine staining, and counterstaining with hematoxylin blue. The negative control sections were incubated with control IgG in equal concentrations to the primary antibody, and known positive human breast cancer tissue was performed as positive control.The IHC study was performed as previously described.25 The score for each sample was multiplied the staining intensity  and the percentage of tumor cells  at each intensity level, ranging from 0 (the minimum score) to 12 (the maximum score). The membrane/cytoplasm positive staining was determined by the subcellular location of the ERα36 positive granules. Generally, ERα36 positive granules, which arranged as cellular outlines, were diagnosed as membrane positive, whereas those with brown intracytoplasmic granules were diagnosed as cytoplasm positive. The IHC results were evaluated by 2 pathologists without the knowledge of patient outcome.Representative IHC images in renal cell carcinoma tissues were collected at 40× objective with BX51 microscope  and DP72 Camera . The IHC staining level was assessed with German semiquantitative scoring system.26 The correlation of ERα36 and other potential clinical variables were assessed using Fisher exact test.28 Kaplan–Meier analysis with log-rank test was applied to compare survival curves.29 A univariate/ multivariate analysis was done using Cox proportional hazards model. Hazard ratios and their corresponding 95% confidence intervals were computed to provide quantitative information about the relevance of results of statistical analysis.30 All statistical tests were 2 sided and differences with a P value of 0.05 or less were considered to be statistically significant.All data were analyzed using SPSS 19.0 software. The categorization was analyzed with the receiver-operating characteristic curve (ROC).A total of 99 patients with renal cell carcinoma were analyzed for ERα36 expression, as well as another 26 cases of diagnosed benign renal tumor. Immunohistochemical staining showed that the pericarcinous renal tissues were observed with low ERα36 immunoreactivity. ERα36 expression was rarely observed in nephron Figure A, but fohigh cases was observed between malignant and benign tumors ; low (score ≤4) , clinical stage (P = 0.029), and necrosis (P = 0.018). ERα36 high expression was correlated with larger tumor size, late clinical stage and more necrosis in tumor tissue. However, we failed to detect significant correlations between ERα36 expression level and other clinical characteristics, including age, sex, resection procedure, histological subtype, and Fuhrman grade.The relationships between ERα36 expression levels and clinical features in renal cell carcinoma were listed in Table P = 0.002). More necrosis was observed in membrane ERα36 expression cases. No significant correlation was found between ERα36 location and other clinical characteristics. Moreover, no significant correlation was observed between ERα36 expression level or subcellular location and ERα66 expression .Furthermore, the relationships between ERα36 location and clinical features were shown in Table P = 0.042) and disease-free survival  in renal cell carcinoma  and DFS (P = 0.025) . During the follow-up period, carcinoma progression was found in 14 patients (14.1%). Kaplan–Meier curves were analyzed to show that ERα36 high expression was statistically correlated with both poor overall survival , high ERα36 expression was observed on the plasma membrane and cytoplasm of renal cancer specimens.38 The plasma membrane-localized ERα36 was proposed to transduce membrane-initiated estrogen signaling.39 When estradiol binds to the cell surface receptor, a rapid generation of cAMP is stimulated. The non-genomic estrogen signaling is transduced to activate RNA and protein synthesis,34 which regulates various physiopathological processes for carcinogenesis and progression,40 such as promoting cell proliferation and invasion.41 Thus, membrane located ERα36 and related signaling maybe responsible for tumor progression of renal cell carcinoma. However, further studies for the mechanism are required in the future.More importantly, membrane ERα36 expression is correlated worse prognosis relative to cytoplasm positive, which indicated that non-genomic estrogen signaling mediated by ERα36 may be involved in renal cell carcinoma progression. Different from those traditional nuclear receptor variants, ERα36 is located on membrane and cytoplasm as reported in previous studies.42 such as the differential diagnosis of renal tumors with tubulopapillary features includes metanephric adenoma and papillary renal cell carcinoma.2 Accurate classification relies on careful examination of clinical and pathological features and immunohistochemical characteristics. Here, we evaluated ERα36 subcellular location for renal tumor classification and found that ERα36 membrane location was rarely observed in benign tumors, which provide useful criteria for accurate diagnosis differentiation in renal tumors.Accurate classification is crucial for both diagnosis and therapeutic intervention in renal tumors. However, majority of renal tumors have unusual morphology that renders classification challenging,Different ERα variants play important roles for estrogen signaling dysregulation. No significant correlation was observed between ERα36 and ERα66 in our study. However, other ERα variants (such as ERα46) were not included in our IHC study because of the limitation of specific antibody for them. Further study is still needed for the interaction between different variants. Taken together, membrane located ERα36 may act a critical role for renal cell carcinoma initiation and progression. IHC staining for ERα36 can provide valuable information for diagnosis, prognostication, and personalized treatment of renal tumors.