Au@CC for catalytic degradation of hazardous water pollutant dyes viz., Eosin Y & Chromotrope 2R using mild reaction conditions. New gold Salen complex doped carbon nanocomposite Au@CC was developed by easy methodology using nano carbon cage (CC) prepared from low-priced Pyrolysis fuel oil (PFO) residue based Pitch. The UV-Vis adsorption spectroscopy results of Eosin Y and Chromotrope 2R dyes indicated complete degradation into acidic compounds which can be further mineralized to CO2 and H2O under mild reaction conditions. The heterogeneous catalyst recycled and reused successfully for four repeated experiments without loss in its adequate performance. This new sustainable and eco-friendly catalyst delivered significant degradation activity compared to existing reports and it can be further utilized for new multifunctional applications such as, radiopharmaceutical activities, heterogeneous catalysis and chiral resolution.Up to now, a very few catalysts have been developed approaching the heterogeneous catalytic degradation of Eosin Y and Chromotrope 2R dyes (Acid Red 29). The present study provides a complete perspective of recyclable nanocomposite  Eosin Y has stability, resilience, and light absorptivity3. The extensive use of Eosin Y in sensitizing industries3 and as biological stain in biomedical research laboratories and for diagnostic intention4, cause carcinogenic exposure as dye waste due to its benzene and pyran rings as well as carboxyl group. Eosin Y affects surroundings by direct discharging into wastewater due to its toxic nature and its intense color. Chromotrope 2R (Acid Red 29) is one of the azo dyes consist of azo group (−N = N−), as chromophore group with aromatic structure having, −OH and –SO3H functional groups. Azo pigment also creates water contamination and serious environmental problem due to its carcinogenic, poisonous and harmful properties9.Dyes and pigments have ubiquitous presence in modern industries such as, textile, leather, food, paper and plastic industries. The discharged effluent of these factories is evidently contaminated and critically hazardous because of its adverse outcome on the environment and society. Countermeasure of this dye-associated waste has been greater field of exploration now a day. A red fluorescent dye from xanthene family, Eosin Y, has been normally utilized in several applications such as, semiconductor’s sensitizer2, H2O and inorganic salts, which remain in the aqueous phase. Further, the intermediates are broken down into simpler, easily treatable materials before they are released into the environment. The ultimate products usually reside of low molecular weight oxygenated molecules like acetic acid, propionic acid, ethanol etc10. Currently, a variety of techniques are being used for the removal or degradation of dyes from pollutant water, e.g. adsorption, coagulation, oxidation, reduction, microbiological, photo-catalysis and biotechnology. These techniques declined to remove or destroy pollutant completely, but it offered few drawbacks such as incomplete removal, high consumption of chemical reagent, expensive, slow and generation of toxic secondary contaminants11. Modified titanium dioxide (TiO2) is possibly most practiced photocatalyst for determining environmental issues specially wastewater purification12. However, the TiO2 based photocatalysis experienced few fundamental technical hurdles that pauses its industrialization, i.e. limited utilization of visible light, small adsorption scope for hydrophobic pollutants, high aggregation tendency and recovery of the TiO2 composite after successive catalytic cycle11. The ability of biological treatment processes for decolorization of industrial effluents has limitations in that it is affected by the concentration, non-biodegradable toxic and inhibitory nature, alkalinity and elevated temperature of the dye effluents.Organic water contaminants are either moderately decomposed by using an oxidizing agent into biodegradable intermediates/important chemicals or complete mineralization into innocuous inorganic compounds such as CO13. Hydrogen peroxide is a powerful and environment friendly oxidizing agents since the product of its degradation are H2O and O214. Sodium borohydride (NaBH4) is widely used in catalytic reduction method of dyes15. Though activated carbon composite demonstrates its potential to remove the dyes, the high initial cost of carbon, coupled with problems associated with regeneration/reuse necessitates search for other developments. Owing to these reasons chemical oxidation-reduction methods are preferred. Various advanced oxidation/reduction processes of pollutant treatment have been modernized with low-cost, ecofriendlyness and high efficiency to reconcile the developing demand of direct wastewater treatment.Salen Schiff base-transition metal complexes and their composites have been broadly researched due to its potential use as a promising catalyst in a wide range of organic transformations16. To expand better awareness of the possibility of this treatment, herein, we report the development of new heterogeneous catalyst of Au(III) Salen complex onto carbon cage prepared from pyrolysis fuel oil based Pitch residue using a new and eco-friendly approach for the catalytic degradation of Eosin Y and Chromotrope 2R dyes. Gold Salen complex doped carbon nanocomposite Au@CC has promise for the degradation of dyes under mild conditions (Eosin Y: room temperature (25 °C); Chromotrope 2R: 50 °C and atmospheric pressure) using environmentally friendly oxidant (H2O2) for Chromotrope 2R and reductant NaBH4 for Eosin Y. This sustainable catalyst has immense prospective for the application of cleaning organic dye polluted water and it can be further utilized for new multifunctional applications as well.Very few reports have been published dealing with Eosin Y and Chromotrope 2R by catalytic degradation methods. Reusable, inexpensive and eco-friendly organic metal carbon nanocomposite have numerous supplementary combined features of organic moiety, metal and carbon base along with the potential for tuning the activity and selectively of nanocomposites. Combination of hetero atoms into porous carbon framework can engender active sites and most transition metals have been doped into porous nanocomposite. Transition metal nanocomposites have revealed huge importance over last decade because of their potential in applications as a solid catalyst, adsorbent, sensor, electrode, energy storage or its hostCC, CCO, CCONa and Au@CC were synthesized by simple template method using nanosilicaball (NSB) followed by air oxidation and NaOH treatment and easy deposition of gold Salen complex on hierarchical CC, respectively  and (100) planes which were correspond to 2θ value 25.3° and 42.7°, respectively @CC showed four new peaks at 2θ = 38.1°, 44.3°, 64.5° and 77.5°, which resembled to reflections of gold planes (111), (200) (220) and (311), respectively19. Some supplementary diffractions peaks of sodium metal planes observed at 2θ value 25.6°, 26.5°, 27.2°, 29.8°, 30.1°, 32.8° and 33.9°18. Though, the low-angle XRD of CCO, CCONa, Au@CC did not show any characteristic peak  to CC, the surface areas of CCO was 224 m2/g. The surface hydroxyl groups triggering and gold Salen complex incorporation to CCO, the surface area of CCONa and Au@CC observed to 258 and 239 m2/g, respectively. An increase in the pore diameter from 71 to 162 Å was observed from NSB to CC. Upon oxidation and impregnation, the values decreased further for CCO: 16; CCONa: 16; and Au@CC: 17 Å with respect to CC. The pore volume of NSB and CC was 0.290 cm3/g and 0.857 cm3/g, respectively. The pore volume of CCO, CCONa and Au@CC also increased to 0.869, 1.003 and 1.002 cm3/g, respectively @CC 1 was further monitored by solid state 13C CP-MAS NMR. It showed broad peaks in aromatic and aliphatic regions (58–189 δ ppm) @CC carbon materials are shown in Fig. CC, CCO, CCONa and Au@CC are shown in Fig. The TEM images of the CCO and CCONa, respectively. The loading of organic moiety in Au@CC was found to be 13.95% as determined from the weight loss measured by thermo-gravimetric analysis carried out in the temperature range between 27 to 800 °C @CC. The spectrum of Eosin Y showed major absorbance band at 514 nm. It clearly indicates that in Eosin Y reduction in the presence of NaBH4, the intensity of absorbance peak became weaker as the reduction continued and totally disappeared after 50 min. We have also investigated oxidative degradation of Chromotrope 2R dye using Gold Salen complex doped carbon nanocomposite. The UV-vis measurement spectrum of chromotrope 2R is shown in Fig. 2O2 mixture, the reaction slowly loss in the absorbance of the dye was observed after 55 min.The Fig. Au@CC was developed by easy methodology using nano carbon cage (CC) prepared from low-priced pyrolysis fuel oil (PFO) residue based Pitch. All the composites and products were fully characterized by X-ray diffraction (XRD), 13C cross polarized magic angle spinning (CP-MAS) NMR, thermo-gravimetric analysis (TGA), N2 adsorption–desorption isotherm, scanning electron microscopy (SEM), transmission electron microscope (TEM), Gas chromatography mass spectrometry (GCMS), Liquid chromatography mass spectroscopy (LCMS) and Fourier transform-infrared (FT-IR). The detailed characterizations are given in supplementary information. UV-Vis spectroscopy was used for catalytic study of hazardous dyes and GCMS for identification of degraded products.The gold Salen complex doped carbon nanocomposite CC indicate that the crystallinity in the porous carbons is relatively small19. Four new peaks of Au@CC designate the association of gold Salen complex verified the presence of gold complex in CC20. Additional diffractions peaks supported the presence of sodium metal planes (JCPDS No. 011173 and 772082) are labeled in Fig. 21. Metal incorporation has a considerable influence on the structural regularity of the CC materials22. Recycled Au@CC catalyst also shown identical XRD pattern after completion of catalytic cycle. It revealed that the heterogeneous catalyst Au@CC was retained and stable under catalytic degradation condition  corresponding to carbons originating from Salen complex immobilized on CC @CC 1 was further confirmed by solid state 13C CP-MAS NMR. SEM image of CC has shown uniform hollow cores of hierarchically porous CC while images of CCO, CCONa and Au@CC have shown well ordered nanoporous structure with the pores distributed consistently in the carbons. During the template synthesis process and thermal and chemical treatments, the hierarchically type framework of CC and Au@CC is well-replicated and retained their well arranged hollow morphology @CC have shown well retained nanoporous structure with the well pores distributed uniformly in the carbons upon thermo-chemical treatment @CC @CC as catalyst suggests that the benzene and pyran rings containing carboxyl group of Eosin Y destroyed and decomposed into simpler acid components which gradually will decay into its mineral components. GC-MS analysis could identify four products such as, 2-(2-formylphenyl)-2-carboxylate, 2-phenyl)2-carboxylate, 3,5-dibromocyclohex-5-ene-1,2,4-trione and -1,5-dibromo-3((Z)-2carboxyvinyl)-6-oxohexa-1,4-dien-2-olate. On the basis of degradation products, possible mechanism was suggested for catalytic degradation of Eosin Y @CC can be used to decompose dye Eosin Y. Our research findings revealed that Au@CC catalyst is more reliable and efficient compared to available literature reports4. We found 98.68% decomposition of Eosin Y within 50 min at normal pressure and room temperature, while recently practiced TiO2 based photocatalyst gave 96% degradation of Eosin Y under visible light irradiation for 180 min4. Decomposition reaction of Chromotrope 2R results into some active acid species due to degradation of aromatic structure containing azo group (−N = N−), −OH and –SO3H23. GC-MS could identify oxalacetic acid, malonic acid, oxalic and oxamic acid as the final products that are mineralized to CO2 @CC catalyst within 55 min compared to report based on decomposition of the dye using Fenton process9. Our catalytic technique is simple, effective, and recyclable and requires very less time for complete degradation of dyes.In Eosin Y degradation using −1 in different five experiments, the total conversation experienced reasonable enhancement. Required time for complete degradation of Eosin Y was 90, 70, 50, 50, and 50 min at catalyst amount 1, 3, 5, 8, 10 g L−1, respectively. Similarly, observed reaction completion time of Chromotrope 2R was 80, 70, 55, 55 and 55 min at catalyst load 1, 3, 5, 8, 10 g L−1, respectively. Thus, the optimum catalyst amount was found to be 5 g L−1, which produced maximum conversion in most favorable time of 50 and 55 min for Eosin Y and Chromotrope 2R, respectively. The UV-Vis spectra of catalyst optimization study were summarized Figs Besides, we have also conducted the catalyst optimization study by varying the amount of catalyst for the degradation of Eosin Y and Chromotrope 2R Table . In caseAu@CC, after catalytic degradation of Eosin Y and Chromotrope 2R under optimized reaction conditions  and 50 °C (Chromotrope 2R reaction). The aim was to evaluate the catalyst stability in degradation reaction, a basic necessity for its application at industrial scale. After the first catalytic reaction, the used Au@CC catalyst was separated by filtration and the nanocomposite was thoroughly washed with distilled water. The resultant nanocomposite was dried overnight in vacuum desiccators at 100 °C and this revived Au@CC catalyst was reused directly as a catalyst in the successive catalytic reaction. Four catalytic runs were carried out using the same catalyst with no observable loss of performance.An attempt was made to recycle the catalyst, Au@CC, have potential functions in catalysis owing to their outstanding organic-inorganic hybrid physicochemical properties. The sustainable catalyst acts as a dynamic heterogeneous catalyst for catalytic decomposition of hazardous water pollutant dyes viz., Eosin Y and Chromotrope 2R. The complete degradation of Eosin Y and Chromotrope 2R dyes into acidic compounds was accomplished which can be further mineralized to CO2 and H2O under mild reaction conditions, it is more environmentally responsive and acceptable compared to earlier literature reports. The recyclability of eco-friendly catalyst is another advantage of the catalytic degradation process. The study is in progress towards the development of new applications such as energy storage and labeling of gold radioisotope for radiopharmaceutical activity.In conclusion, newly developed gold Salen doped carbon nanocomposite, 4)  were employed as obtained. Petroleum residue pyrolysis fuel oil (PFO) was recieved from Yeocheon Naphtha Cracking Center . PFO based Pitch, Nano silica ball (NSB) and carbon cage (CC) were prepared according to the method reported previously using PFO based pitch residue25. All the solvents were purified by known method26.Reagents and solvents such as ethanol, sodium hydroxide, hydrogen peroxide, Eosin Y , sodium borohydride, tetraethylorthosilicate, ethylenediamine , hydrofluoric acid , Chromotrope 2R, 3,5-di-tert-butyl-2-hydroxybenzaldehyde , and hydrogen tetrachloroaurate (III) hydrate @CC, its precursors were fully accomplished by powder X–ray diffraction  in the low angle and high angle 2θ spectrum (0.5–10 and 10–80). 1H and 13C NMR were carried out by 500 and 125 MHz NMR spectrometer . Cross polarized magic angle spinning (CP-MAS) 13C NMR was used for the identification of Au@CC catalyst . Fourier transform infrared spectroscopy  was conducted using a KBr pellet facility. Microanalysis of the compounds was accomplished by CHN analyzer  and the percentage of gold associated with CC were measured by inductively coupled plasma optical emission spectroscopy . The thermal strength of the carbon nanocomposite was discovered by thermogravimetric analysis . The BET surface area was performed by nitrogen adsorption-desorption data determined at 77 K adopting volumetric adsorption technique . The micro-structural assessment of these nanocomposite were probed by scanning electron microscopy  and transmission electron microscopy . The high performance liquid chromatography mass spectroscopy  and gas chromatography mass spectroscopy  were used for the identification synthesized compounds and degraded products. The complete degradation of Eosin-Y and Chromotrope-2R dyes was monitored by UV–vis spectroscopy .The characterization of CC) was oxidized with a mixture of nitrogen and air (with 5% O2) at 698 K for 10 h to obtain CCO. The surface hydroxyl groups of CCO were triggered by refluxing it with an aqueous solution of 0.1 N NaOH for an hour @CC catalyst was synthesized by following procedure adapted from our group’s earlier report28. First, a mixture of 3,5-di-tert-butyl-2-hydroxybenzaldehyde 2  and ethylenediamine 3 (0.40 ml 5.9745 mmol) was refluxed for 6 h in 30 mL of ethanol . 1H NMR : δ 13.63 , 8.38 , 7.36 , 7.25 [CDCl3] 7.06 , 3.91 , 1.43  and 1.28  : δ 168 [C of Imine], 158 [C of Ar], 140 [C of Ar], 137 [C of Ar], 127 [C of Ar], 126 [C of Ar], 118 [C of Ar], 60 [CH2N], 35 [CCH3], 34 [CCH3], 32 [CH3] and 30 [CH3] : 2958, 2907, 2869, 2591, 1630, 1594, 1440, 1391, 1361, 1272, 1203, 1172, 1131, 1042, 974, 879, 849, 829, 773 and 710 cm−1.The Salen ligand used for the development of nol Fig. . The for3] Fig. . 13C NMRH3] Fig. . Element4 . The above mixture was refluxed for 4 h under steady stirring condition . LCMS: m/z = 723.4 [M]+. FTIR (KBr): 2978, 2912, 2802, 2744, 2710, 2676, 2573, 2519, 2418, 2279, 2056, 1601, 1487, 1344, 1085, 1034, and 820 cm−1.The gold (III) Salen complex was synthesized by refluxing an ethanolic solution of equimolar quantities of hydrogen tetrachloroaurate (III) hydrate  and ligand 5 with treated carbon cage CCONa was achieved by following method. Activated CCONa (5.0 g) nanocomposite was refluxed with an ethanolic solution of 1.38 mmol of gold (III) Salen complex 5 (1.0 g) for 12 h : δ 58–189 . Elemental analysis (Found) C: 78.68, H: 1.19, N: 2.18%. Elemental analysis and ICP results showed that Au@CC contained > 78 wt% carbon and 2.99 wt% Au after compound 5 immobilization on CC, respectively. Solid reflectance UV-Vis: 220, 280, 340 nm. FTIR (KBr): 3450, 2917, 2803, 2677, 2347, 2058, 1600, 1502, 1086 and 1034 cm−1.Functionalization of Salen complex −4 mol L−1), NaBH4  with the catalyst Au@CC (5 g L−1). The reaction mixture was stirred with 180 rpm rate for 50 min at room temperature (25 °C) and normal pressure. The oxidation reaction of Chromotrope 2R was carried out with 50 mL Chromotrope 2R (2.7 × 10−4 mol L−1), 50 mL H2O2 (10−4 mol L−1) using 5 g L−1Au@CC catalyst for 55 min at 50 °C temperature, normal pressure, and 180 rpm rate. After the completion of reaction, the catalyst, Au@CC, was separated by centrifugation. The complete degradation of Eosin Y and Chromotrope 2R was monitored using UV–visible spectroscopy (at maximum adsorption wavelength of 514 nm and 508 nm for Eosin Y and Chromotrope 2R, respectively. GCMS was used for product identification. After each reaction run was completed, the products were extracted with chloroform by mixing equal volumes of the product mixture and chloroform. The solution was sonicated for several minutes allowed to settle down layers. 0.6 μL of the extracted layer of the mixures were investigated by GC-MS. In GC-MS, the temperature of injection was 250 °C. The starting temperature was preserved at 50 °C for 5 min and then raised to 180 °C at 10 °C min−1 rate, retained at present temperature for 5 min, then increased to 250 °C at 10 °C min−1 rate, and again retained at this temperature for 5 min.Catalytic degradation of Eosin Y and Chromotrope 2R dyes was executed in a glass reactor attached with a condenser. The reduction reaction of Eosin Y was carried out under typical conditions, by combining Eosin Y (100 mL, 2.7 × 10Supplementary Information


MALDI-TOF spectrometry has not been used for urinary exosome analysis. We used it for determining UC biomarkers. From 2012 to 2015, we enrolled 129 consecutive patients with UC and 62 participants without UC. Exosomes from their urine were isolated, and analyzed through MALDI-TOF spectrometry. Immunohistochemical (IHC) analysis of another 122 UC and 26 non-UC tissues was conducted to verify the discovered biomarkers. Two peaks at m/z 5593  and m/z 5947  were identified as UC diagnosis exosome biomarkers. UC patients with detectable histone H2B1K showed 2.29- and 3.11-fold increased risks of recurrence and progression, respectively, compared with those with nondetectable histone H2B1K. Verification results of IHC staining revealed significantly higher expression of alpha 1-antitrypsin (p = 0.038) and H2B1K (p = 0.005) in UC tissues than in normal tissues. The expression of alpha 1-antitrypsin and H2B1K in UC tissues was significantly correlated with UC grades (p < 0.05). Urinary exosome proteins alpha 1-antitrypsin and histone H2B1K, which are identified through MALDI-TOF analysis, could facilitate rapid diagnosis and prognosis of UC. Urothelial carcinoma (UC), cancer of the urinary tract, is the ninth most prevalent malignancy worldwide367et al. reported that urinary exosomes from patients with high-grade bladder cancer can promote UC cell migration and angiogenesis1213Exosomes are microvesicles 30–100-nm) released by cells into surrounding biofluids, including serum and urine. These vesicles participate in intercellular communication and the exchange of materials, such as proteins, RNA, and lipids90-nm releTo verify successful exosome isolation through our sample preparation method, two established exosome protein markers, Alix and TSG101, were subjected to Western blotting and were detected in our samples that were isolated from the patients with UC, prostate cancer, UTI, and hernia . ElectroThe storage of clinical samples is crucial for acquiring reliable results. We evaluated suitable storage conditions for the microparticles that were isolated from one healthy participant in the form of urine, purified exosomes, and acid-extracted protein solution. Supplementary Table 2 shows the clinicopathological characteristics of 129 patients with UC and 62 non-UC comparison patients . No significant difference was observed between the UC and comparison groups regarding age and sex distribution . In the UC group, 48.8% of the patients had pathological stage pTa/Tis/T1, 71.3% had high-grade UC, 79.9% had maximum tumor diameters of <3 cm, 62.8% had multiple tumors, 27.7% had lymphovascular invasion, 7.8% had lymph node metastasis, and 5.4% had positive surgical margins.Two intense peaks at m/z 5593 and m/z 5947 specifically appeared in the UC group compared with the non-UC group. The sensitivity and specificity of m/z 5593 for detecting UC were 50.4% and 96.9%, respectively, with an area under the curve (AUC) of 0.736. Moreover, the sensitivity and specificity of m/z 5947 for detecting UC were 62.0% and 92.3%, respectively, with an AUC of 0.772. The sensitivity and specificity of the combination of the two peaks for detecting UC were 62.70% and 87.59%, respectively, with an AUC of 0.87.Using a pooled sample size of 202, we compared AUC of m/z 5593 and m/z 5947 with urine analysis OB (+), the current cheapest and non-invasive clinical test for diagnosing UC in the pooled sample (n = 202). The AUC of OB, m/z 5593, and m/z 5947 were 0.61 [95%CI: 0.52–0.69], AUC = 0.74 [95%CI: 0.67–0.80], and AUC = 0.77 [95%CI:0.71–0.84], respectively . To inveThe median follow-up time was 13.3 mo (2–32 mo). At the time of analysis, UC recurred in 80 of the patients (62.0%), and four of the patients died (3.1%); three of the deaths were caused by metastatic UC. In 31APEQIAADIPEVVVSLAPKHPGSRKNMACYC68 , T3LAILAAILLVALQAQAEPQARADEVAAAPEQIA37 , and A26DEVAAAPEQIAADIPEVVVSLAPKHPGSRKNMA62 . The UC marker peaks at m/z 5593 and m/z 5947 and the exosome marker at m/z 10884 were purified through LC, digested with trypsin, and analyzed through nanoLC–MS/MS. The m/z 5593 peak was identified as a fragment peptide of alpha 1-antitrypsin with the sequence S307ASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA360 . The m/z 5947 peak was identified as a peptide fragment of histone H2B1K  with the sequence K44VLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQ94 . The m/z 10884 peak was identified as a large peptide fragment of S100 calcium-binding protein A9 (S100A9) with the sequence R9NIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVIE HIMEDLDTNADKQLSFEEFI MLMARLTWAS HEKMHEGDEGP101 .The three exosome markers at m/z 3367, m/z 3441, and m/z 3483 were purified through 1D gradient Bis-Tris PAGE. The bands were excised, extracted, trypsin digested, and analyzed through nanoLC–MS/MS. These three markers were identified as peptide fragments of neutrophil defensin with sequences of AIn split-half method, the overall predictive probability of m/z 5593 was 63.6% in the training set and 72.6% in the verification set, while the overall predictive probability of m/z 5947was 66.3% in the training set and 69.0% in the verification set. In split 1/3 method, the overall predictive probability of m/z 5593 was 74.1% in the training set and 65.3% in the verification set, while the overall predictive probability of m/z 5947 was 73.8% in the training set and 65.2% in the verification set. In both split-half and split 1/3 method, the posterior probability of m/z 5593 and m/z5947 for diagnosing UC is 100%.Alpha 1-antitrypsin and H2B1K were identified based on peptide identifications with Mascot scores of ≥25 and a false discovery rate of <1%. The urinary exosome protein concentration of alpha 1-antitrypsin was 2.93-fold higher in UC group than in comparison group. (p = 0.001) The urinary exosome protein concentration of H2B1K was 4.85-fold higher in UC group than in comparison group (p = 0.005) .The IHC scores of alpha 1-antitrypsin expression were 4.40-fold higher in UC tissues than in normal uroepithelium . Moreover, the IHC scores of H2B1K expression were up to 8.20-fold higher in UC tissues than in noncancerous normal tissues . Alpha 1Cancer tissues are the most suitable sources for investigating highly sensitive and specific protein biomarkers. However, obtaining tumor tissues is invasive and occasionally difficult because of anatomical limitations. Analyzing body fluids for microparticles or biomolecules secreted by tumors is a more preferable and feasible approach for discovering biomarkers because it is noninvasive, and samples can be easily obtainedIn this study, four major peaks at m/z 3367, m/z 3441, m/z 3483, and m/z 10884 were detected among exosome samples and acted as urinary exosome markers. The peaks at m/z 3367, m/z 3441, and m/z 3483 were identified as peptide fragments of neutrophil defensin, a highly basic cationic peptide that is also known as human neutrophil peptides. Neutrophil defensins are abundant in neutrophils, epithelial cells lining the bronchial tress and genitourinary tract, and urinary exosomes1719Alpha 1-antitrypsin and H2B1K are also present in exosomes of normal urine212123et al. reported that miRNA-940, one of the regulators of alpha 1-antitrypsin expression, is lost in cancer tissues and cellset al. reported that the cell cycle phenotype, including the arrest of differentiation and increasing genomic instability, is correlated with aggressive tumors and disease progressionet al. observed dynamic changes in histone modification for parallel periods of the cell cycleAlpha 1-antitrypsin belongs to a family of serum proteinase inhibitors. Alpha 1-antitrypsin plays a critical role in modulating immunity, inflammation, apoptosis, and possibly cellular senescence programs262729Thus, one possible explanation for our lower H2B1K expression in high-grade UC tissues is that the disease progression potential was majorly determined by histone modification levels rather than histone expression levels.et al. used a panel of molecular markers, namely p53, p21, p27, ki-67, and cyclin-E1, to predict recurrence and cancer-specific survival after radical cystectomyTo date, few potential markers, such as cadherin-1The m/z 5947 peak of urinary UC exosomes identified through MALDI-TOF spectrometry provides a rapid and noninvasive strategy for direct translation into clinical practice. Furthermore, our participants represent all UC characteristics, including the tumor location, treatment, and pathological grades, thereby reflecting an actual scenario that might avoid possible selection bias, such as unequal recurrence rates among different locations and treatments, and providing a more applicable biomarker candidate for diagnosing and predicting UC.In summary, m/z 3367, m/z 3441, m/z 3483, and m/z 10884 were identified as markers for detecting urinary exosomes. These findings would facilitate rapid examination and optimization of exosome purification efficiency. The m/z 5593 and m/z 5947 peaks in urinary exosomes were identified as biomarkers for UC. The UC patients with the m/z 5947 peak had 2.29- and 3.11-fold increased risks of UC recurrence and progression, respectively. In addition, the overexpression of alpha 1-antitrypsin and H2B1K was reported in UC tissues. Our paper provides a potential rapid and analytical platform for discovering urinary exosomes and biomarkers as well as warrants further large-scale validation studies.The objective of this study was to explore the presence of peaks (peptide fragment) in urine analyzed by MALDI-TOF spectrometry that could serve as biomarkers for UC. We first determine the appropriate methods for exosome protein extraction and appropriate conditions for MALDI-TOF analysis. We consecutively collected urine samples of 129 UC patients and 62 controls. Urinary exosomes were separated, of which exosomes proteins were extracted, and analyzed with MALDI-TOF spectrometry. In parallel, the classical clinical data were obtained including age, gender, cancer staging, cancer grading, recurrence, and disease progression. The candidate peaks of which are under curve of ROC curve >0.7 were chosen for UC biomarkers. The candidate peaks were purified. Verification of these proteins for UC biomarker was carried out in two ways: internal validation of pooled exosomes samples (n = 202), and immunohistochemical stating of 122 UC tissues and 26 normal uroepithelial tissues.g for 10 min to remove debris and stored at −80 °C until the subsequent purification of urinary microparticles, and clinicpathological variables were analyzed. Disease progression was defined as distant metastasis, superficial progression to muscle invasion, or cancer-related death. Recurrence was defined as a new tumor developed after the transurethral resection of a bladder tumor, secondary primaries, progression, or distant metastasis. To confirm our discovered biomarkers expressed differently between UC and controls, iTRAQ labelling quantitative nanoLC–MS/MS was carried out for UC (n = 5) and non-UC (n = 10) groups.The human research protocols were approved by the Medical Ethics and Human Clinical Trial Committee of China Medical University Hospital, Taiwan. Informed consent was obtained from all subjects. All methods were carried out in accordance with the relevant ethic guidelines. First morning urine samples were collected from patients with hernia, urinary tract infection (UTI), prostate cancer, or UC. A protease inhibitor cocktail tablet  was added in each urine specimen (approximately 50 mL). The specimens were then centrifuged at 1000× To confirm and validate our discovered biomarkers, we categorized another set of participants into the UC (n = 122) and non-UC (n = 26) groups. Surgical specimens of their UC and non-UC tissues were analyzed through immunohistochemical (IHC) staining of alpha 1-antitrypsin and H2B1K.g for 10 min at 4 °C ; the supernatant was collected as SN1. The pellets were resuspended in an isolation solution  before 200 mg/mL dithiothreitol was added and before incubation at 95 °C for 2 min. The resuspended solution was centrifuged at 17000× g for 30 min at 4 °C, and the supernatant was collected as SN2. SN1 and SN2 were pooled and ultracentrifuged at 200000× g for 1 h at 4 °C. The supernatant was removed, and the microparticles were collected for further analysis.Urinary microparticles were prepared through ultracentrifugation, as previously described45The microparticles were harvested using an RIPA lysis buffer, and 20 μg of proteins was solubilized in Laemmli sample buffer . Proteins were separated through one-dimensional (1D) SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. After the PVDF membrane was blocked with 5% nonfat milk at room temperature for 1 h, the membranes were probed overnight at 4 °C with primary monoclonal antibodies to TSG101 , Alix , or actin . Thereafter, the membranes were probed with peroxidase-conjugated antirabbit or antimouse IgG (1:10000) for 1 h at room temperature. Immunoreactivity was detected through enhanced chemiluminescence.The microparticles were resuspended in 2.5% glutaraldehyde and applied to Formvar-coated carbon-stabilized copper grids. The grids were dried at room temperature, washed twice with phosphate-buffered saline, and then stained with 2% (w/v) uranyl acetate for 10 min. The grid was examined using a JEOL 200CX electron microscope.2O for 10 min each, and stained with silver stain.The extracted proteins were separated on 12% SDS polyacrylamide gels. Electrophoresis was performed using a power supply set at 75 V/gel and 110 V/gel for the stacking and resolving gels, respectively. All the gels were placed in a fixing solution (methanol:acetic acid:deionized water = 40:10:50) for 1 h, washed twice in ddH2O for 20 min. The gels were rinsed twice in 30% ethanol for 10 min and twice in ddH2O for 10 min. The gels were then incubated in 0.02% sodium thiosulfate for 1 min and rinsed twice with ddH2O for 1 min each. Furthermore, the gels were placed in 0.1% silver nitrate solution for 20 min, rinsed twice with ddH2O for 1 min each, and developed in 0.04% formalin  in 2% sodium carbonate with constant shaking. The development was terminated by adding 5% acetic acid after achieving the desired intensity of silver staining.The gels were first fixed in 50% methanol and 5% acetic acid in ddH4P2O7, 1 mM Na3VO4, and 1% Triton X-100. Sonication was performed for 100 s at 180 W with alternating 10-s sonication–rest for each cycle. After extraction, the insoluble sediment was removed through centrifugation at 10000× g for 15 min at 4 °C. The protein concentration of each method was measured using the Bradford assay at 562 nm. This extraction protocol was repeated for eight biological replicates  ddH2O, (2) 25% acetonitrile (ACN), (3) 50% ACN, (4) 75% ACN, (5) 25% formic acid (FA), (6) 50% FA, (7) 75% FA, (8) 98% FA, (9) 25% FA + 50% ACN, (10) 35% FA + 50% ACN, (11) 45% FA + 50% ACN, (12) 50% FA + 50% ACN, (13) RIPA buffer sonication, and (14) sonication. The RIPA buffer contained 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 50 mM NaCl, 50 mM NaF, 20 mM Naplicates .The first morning urine sample was collected from one participant. The microparticles were isolated from one fraction of the fresh urine, extracted with 75% FA solution, and immediately analyzed through MALDI-TOF spectrometry to determine the urinary exosome protein profile without storage. Furthermore, the microparticles were isolated from the remaining fraction of the urine sample, extracted with 75% FA peptide–protein solution, and stored for a long term at −80 °C. Analysis was performed after 1 and 6 mo through MALDI-TOF spectrometry.The analyte solution was mixed with saturated sinapinic acid (SA) solution (30:70 ACN:0.1% trifluoroacetic acid) at volume ratios of 1:1 or 1:5. One microliter of the analyte/SA solution was placed on the MALDI-TOF target. After analyte/SA cocrystallization, the sample plate was analyzed through MALDI-TOF spectrometry . MALDI-TOF spectrometry was operated in a linear positive ion mode with 25-kV accelerating voltage at a laser frequency of 50 Hz with a mass range of 1000–23000 Da. Peptide–protein calibrations were conducted using a peptide–protein calibration standard kit . MALDI-TOF mass spectra were processed using flexAnalysis 3.0 software .2O. Peptide bands of approximately 3000 Da were excised and from both exosome extracts and control sample (blood added into ddH2O to avoid interference of urinary proteins), extracted using 50% ACN/0.1% FA. The extracted peptides were analyzed through MALDI-TOF spectrometry to confirm the presence of peaks at m/z 3367, m/z 3441, and m/z 3483. The extracted peptides were then reduced, alkylated, trypsin digested, and finally dried for further peptide identification through nanoflow liquid chromatography (nanoLC)–tandem mass spectrometry (MS)/MS analysis. The peak identification using Bis-Tris gels has been performed with three biological replicates. Each replicate resulted from exosome extracts can have the same marker peaks at m/z 3367, m/z 3441, and m/z 3483 on MALDI–TOF  was performed to purify the marker peaks at m/z 3367, m/z 3441, and m/z 3483. Electrophoresis was performed at 75 V/gel and 110 V/gel for the stacking and resolving gels, respectively. After the separation, the gels were stained with Coomassie brilliant blue G250 and destained using ddHALDI–TOF .An LC pumping system  equipped with an LC column  was used for protein purification. The mobile phases were solvent A (5% ACN and 0.1% FA) and solvent B (100% ACN and 0.1% FA). Gradient elution at a flow rate of 250 μL/min was set as follows: 0% B for 10 min, 15–80% B for 12 min, and 80–100% B for 3 min. The eluents were monitored using a UV detector  at wavelengths of 210 nm and 280 nm. The eluents were collected at 30-s intervals. Each fraction was analyzed through MALDI-TOF spectrometry to confirm the successful purification of the marker peaks at m/z 5593, m/z 5947, and m/z 10 884. The purified peptides and proteins were subjected to in-solution digestion and nanoLC–MS/MS analysis for identification.Proteins extract of 100 μg each sample was purified as following: added with four times the volume of cold (−20 °C) acetone containing 12% (w/v) TCA, the mixture was incubated 6 hours at −20 °C, centrifuged at 12,000× g at 4 °C for 30 minutes, and then the supernatant was removed without disturbing the visible pellet. Four times the sample volume of cold (−20 °C) acetone was added to the pellet. After incubation for 4 hours at −20 °C and centrifugation at 12,000× g at 4 °C for 30 minutes, the supernatant was removed and the pellet air dried. The protein pellets were then dissolved in the solution buffer, reduced, and blocked with cysteins, as as suggested by the manufacturers. Each sample was trypsin digested  at 37 °C overnight and then labeled with the iTRAQ tags separately as UC or controls. The labeled digests were then mixed and dried for further quantitative nanoLC–MS/MS analysis.NanoLC–MS/MS was performed using a nanoflow ultra performance liquid chromatography system  coupled to a hybrid quadrupole TOF (Q-TOF) mass spectrometer . Tryptic peptide mixtures were injected using an autosampler and loaded at a flow rate of 15 μL/min on a self-packed C18 trap column  for desalting and preconcentration for 5 min. The peptides were then eluted into an analytical column  coupled to a nanoelectrospray ionization source on the Q-TOF mass spectrometer. A gradient elution of 1% ACN (0.1% FA) to 40% ACN (0.1% FA) for 90 min was conducted at a flow rate of 300 nL/min for peptide separation. Ten precursors of charge +2, +3, and +4 from each TOF-MS scan (m/z 50–2000) were dynamically selected and isolated for MS/MS fragment ion scanning (m/z 50–2000). The selected precursors were then actively excluded for 25 s. MS and MS/MS accumulation were set at 1 and 10 Hz, respectively.Tissue specimens were initially formalin fixed and paraffin embedded. All IHC staining was conducted using a Leica Bond-Max autostainer (Leica Microsystems) according to the manufacturer’s protocol. Antigen retrieval was conducted at pH 8 by using Epitope Retrieval 2 solution (Leica Microsystems) for 20 min at 100 °C. The slides were then incubated for 15 min at room temperature with primary antibodies at the following dilutions: rabbit polyclonal antitrypsin  and rabbit polyclonal H2B1K . A polymer detection system  was used.The slides were examined by a pathologist who was blinded to the outcomes and disease status of the participants. The proportion score described the estimated fraction of positive stained tumor tissues (0–100%). The intensity score represented the estimated staining intensity . The total score, the proportion score multiplied by the intensity score, ranged from 0 to 300.Statistical analysis of the MS profiling spectra from the UC and non-UC participants  was performed using ClinPro Tools software . Univariate and multivariate Cox regression analysis was used to determine the association between different clinicopathological variables and biomarkers and disease recurrence and progression. All the spectra were normalized to their own total ion count. The MS data from all the participants are presented in a two-dimensional (2D) cluster plot. A list of peaks with statistical differences between two classes that were determined using a t test and analysis of variance (ANOVA) were selected. We combined MALDI-spectra of 126 exosomes and 54 exosomes enrolled later to be a pooled sample . Split-sample validation was done in this pooled sample. We adapted split-half and split 1/3 method, where 50% or 33.33% of the sample were kept as an independent evaluation part for the m/z 5593 and m/z 5947 that was estimated on 50% or 66.67% of the sample, respectively. The split was made once and at random.The Mann–Whitney U test was used to analyze the associations between the IHC scores of the candidate proteins and different clinical parameters. Two-tailed p values of ≤0.05 were considered significant. The statistical package SPSS 20.0  was used to analyze all the clinical data.How to cite this article: Lin, S.-Y. et al. Proteome Profiling of Urinary Exosomes Identifies Alpha 1-Antitrypsin and H2B1K as Diagnostic and Prognostic Biomarkers for Urothelial Carcinoma. Sci. Rep.6, 34446; doi: 10.1038/srep34446 (2016).


Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05) in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6, and p70S6K were lower (P < 0.05) in CR vs. AL, and total rpS6 was associated with muscle protein content . Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.Short-term (5–10 days) calorie restriction (CR) downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1), however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR). Twelve-week old male Sprague Dawley rats consumed  However, skeletal muscle is also lost during CR, which may compromise successful weight management and negatively impact physical function. The loss of skeletal muscle may be due in part to the effects of CR on muscle protein synthesis  have been identified as novel regulators of skeletal muscle mass  determine the effects of prolonged CR on mTORC1 signaling, (2) determine the extent to which high protein diets modulate the mTORC1 response, (3) assess whether CR and dietary protein-mediated changes in mTORC1 signaling influence skeletal muscle, and (4) explore the potential regulatory role of miR. We hypothesized that prolonged CR would downregulate mTORC1 signaling resulting in lower fat-free mass after CR. We expected that consuming a high protein diet would prevent declines in mTORC1 signaling and spare fat-free mass. We hypothesized that CR and dietary protein-mediated changes in mTORC1 signaling would result from translational modifications secondary to altered expression of associated miR.n = 40; Charles River Laboratories) were randomized by body mass to one of four diet groups consuming ad libitum (AL) or calorie restricted  with standard (10%) or high (32%) protein (PRO) diets for 16-weeks. This investigation was part of a larger study designed to assess the influence of protein source (soy vs. milk-based protein) on intestinal calcium absorption and bone  were modified to provide 10 and 32% protein to align with the lower and upper end of the current acceptable macronutrient distribution range  , 10 mg·kg−1 xylazine , and 1.0 mg·kg−1 acepromazine ]. Small animal software  was used to determine body composition.Body mass was determined twice a week using a calibrated electronic scale . Dual Energy X-ray Absorptiometry  was used to assess body composition after the16 week feeding intervention under anesthesia [1 mL·kg−1 trypsin inhibitor and 2 μg mL−1 aprotinin. Protein concentration of the homogenate was then determined using 660 nm Protein Assay . Homogenate total protein concentration was expressed relative to DXA derived fat-free mass to provide an estimate of muscle protein content  containing 50 mM Tris–HCl (pH 7.5), 5 mM Na-pyrophosphate, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 10% glycerol (v/v), 1% Triton-X, 1 mM DTT, 1 mM benz-amidine, 1 mM PMSF, 10 μg mLSer473, mTOR, p-mTORSer2448, p70S6K, p-p70S6KThr389, rpS6, p-rpS6Ser235/236, AMPKα , and PGC-1α  at 4°C overnight. Labeling was performed using secondary antibody , and chemiluminescent reagent was applied . Blots were quantified using a phosphoimager  and Image Lab software (Bio-Rad). To confirm equal protein loading per well glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for mTORC1 associated proteins and heat shock protein 90 (HSP90) was used for AMPKα and PGC-1α. Phosphorylation and total protein were normalized to GAPDH or HSP90 and the ratio of phosphorylation-to-total protein was determined. All data are presented as fold change relative to AL 10% PRO.Phosphorylation status and total protein expression of molecular markers associated with mTORC1 signaling were determined using Western blot. Homogenates used for muscle protein content estimations were centrifuged for 15 min at 10,000 × g at 4°C, the supernatant (lysate) was collected and protein concentration analyzed (ThermoFisher). Muscle lysates were solubilized in Laemmli buffer, with equal amounts of total protein (10 μg) and separated by SDS-PAGE using precast Tris·HCl gels . Proteins were transferred to polyvinylidene fluoride membranes and exposed to commercially available primary antibodies specific to Akt, p-AktmRNA expression of genes associated with intracellular regulation of muscle mass, including mTORC1 amino acid sensing proteins , amino acid transporters (Slc38a2 and Slc7a5 TaqMan®), and energy utilization , were determined using commercial available primers and in mixed gastrocnemius samples. miR that regulate targets in the mTORC1 pathway  were analyzed using TaqMan® microRNA Assays . The roles of the mRNA and miR assessed are provided in Supplemental Table T) method ; RNA quantity and quality were assessed using a Nanodrop ND-1000 spectrophotometer . Equal amounts of total RNA were synthesized into cDNA . miR were reverse-transcribed using the TaqMan® microRNA RT kit (Applied Biosystems) with the nine miR-specific stem-loop reverse transcript (RT) primers pooled in 1X-Tris-EDTA (TE) buffer for a final dilution of 0.05X for each miR RT primer. The RT primer pool (6 μl) was added to the RT reaction mix  and 250 ng of total RNA , by combining 10 μl of diluted  sample to 150 μl of reaction master mix . The reaction was initiated when 10 μl of 10 mM oxaloacetate was added to each well. Samples were read at 412 nm. Data were normalized to protein content.Plasma amino acid concentrations were determined from blood collected by intra-cardiac puncture. Samples were tested in duplicate using HPLC and o-phthaldialdehyde post-column derivatization . Amino acid concentrations were used to determine the influence of dietary protein and energy status on mTORC1 signaling.2) for statistical analysis, but presented as original values (median ± SEM). All other data presented as mean ± SEM. The α level for significances was set at P < 0.05. Data were analyzed using IBM SPSS Statistics for Windows Version 22.0 .A repeated measures ANOVA was used to determine influence of time (Baseline vs. Week 16), energy status (AL vs. CR) and protein intake (10 vs. 32%) on body mass and composition. A univariate ANOVA was conducted to determine the influence of energy status and protein intake on muscle protein content, Western blots, mRNA and miR expression, and serum BCAA. Bonferroni adjustments for multiple comparisons were performed if significant interactions (energy-by-protein) were observed. Spearman's rank correlation coefficients were used to determine the relationship between muscle protein content, as determined by protein muscle content, and total Akt, mTOR, p70S6K, and rpS6. All mRNA and miR data were log transformed (LogP < 0.05) for CR compared to AL, regardless of protein intake  from baseline to week 16 in all rats, with no effect of energy or protein intake  in CR than AL  1.72 ± 0.11, 1.41 ± 0.08, 1.95 ± 0.05, and 3.47 ± 0.05 fold lower, respectively, in CR compared to AL rats, regardless of protein intake  with protein content. No other associations were observed.Phosphorylation status of Akte Figure . Total pe Figure . Energy e Figure . Akt (r P < 0.05) for 32% PRO vs. 10% PRO. No effect of energy or protein intake was observed for Slc7a5. Expression of amino acid sensing Map4k3 and Lars were 1.28 ± 0.06 and 1.28 ± 0.08 fold lower (P < 0.05) for CR than AL, independent of dietary protein. No effects of energy or protein intake were observed in the expression of any miR  in CR compared to AL  in CR than AL (Table −1 higher (P < 0.05) in rats consuming 32% PRO vs. 10% PRO. No energy-by-protein interactions were observed.A main effect of energy was observed for plasma branched-chain amino acids; leucine, isoleucine and valine concentrations were 25.9 ± 5.0, 19.2 ± 3.1, and 27.3 ± 5.5 μmol·LThe primary findings from this investigation were that prolonged CR  led to downregulation of fasting mTORC1 signaling activity and inhibition of protein translation and phosphorylation  of Akt, mTOR, p70S6K, and rpS6 under fasted conditions. The systematic downregulation of mTORC1 associated protein expression and activity, particularly the decline rpS6, was associated with diminished muscle protein content. These findings link the molecular regulation of mRNA translation initiation and, possibly MPS, with a measure of long-term muscle protein status in response to underfeeding. Contrary to our hypothesis, the high protein diet, fed as a percentage of total calorie intake, did not attenuate declines in mTORC1 signaling and muscle protein content compared to standard protein intake. Furthermore, the inhibition of mTORC1 associated protein translation during CR did not appear to be regulated by miR. Overall, findings from this study indicate that prolonged CR alters protein translation and downregulates mTORC1 activity, independent of associated miR. Moreover, CR appears to override the protein synthetic stimulus of increased dietary protein intake, resulting in diminished muscle protein content.The degree to which CR alters protein metabolism appears to be dependent on the magnitude and duration of the restriction  the amount of dietary protein per day than AL 10% PRO (2.4 g·d−1). Furthermore, that no physiologically relevant (≥two-fold) increase in expression of the amino acid transporters Slc38a2 and Slc7a5, with concomitant CR-induced reductions in the expression of amino acid sensing genes Map4k3 and Lars supports the theory that extracellular leucine availability was never optimal. Protein may have been stimulatory, despite CR, if food was provided at discrete times and had sufficient protein to adequately increase plasma amino acid levels.Increased extracellular amino acid concentrations, particularly leucine, upregulates amino acid transporter expression and uptake of amino acids into the intracellular amino acid pool and produces a robust increase in mTORC1 activity (Drummond et al., To the best of our knowledge this is the first investigation to assess the role of skeletal muscle miR expression on mTORC1 signaling following adaptations to prolonged CR. Recent studies have indicated miR expression play a role in the regulation of muscle mass (McCarthy and Esser, ad libitum, is not sufficient to attenuate mTORC1 adaptations to prolonged CR.In conclusion, data from this study provide a link between mTORC1 signaling, muscle protein content, and prolonged underfeeding. The systematic downregulation of mTORC1 signaling activity with CR, regardless of protein intake, was likely the result of inhibited protein translation and subsequently lower phosphorylation of Akt, mTOR, p70S6K, and rpS6. Furthermore, while the effects of CR on mTORC1 were clearly demonstrated, they appear to be independent of miR expression. These data also demonstrate that high dietary protein intake alone, when provided as a percentage of total energy and evenly distributed in food consumed JM and SP: Study design, oversight, and sample collection. LM, DR, MB, YE: Completed experiments and analyzed and managed data. LM, DR, AY, JM, RF, SP: Data interpretation and manuscript preparation. Reviewed by all authors.This material is based on the work supported by the US Army Medical Research and Material Command, the Dairy Research Institute, and the USDA under agreement No. 58-1950-4-003. LM was supported by the T32 NIDDK training grant # 5T32DK062032-23 and DR was supported by the NIA K01 award # KAG047247A-A1.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.


Nonalcoholic fatty liver disease (NAFLD) is recognized as the most common cause of liver dysfunction worldwide and is commonly associated with obesity. Evidences suggest that NAFLD might be a mitochondrial disease, which contributes to the hepatic steatosis, oxidative stress, cytokine release, and cell death. Capybara oil (CO) is a rich source of polyunsaturated fatty acids (PUFA), which is known to improve inflammation and oxidative stress. In order to determine the effects of CO on NAFLD, C57Bl/6 mice were divided into 3 groups and fed a high-fat diet (HFD) (NAFLD group and NAFLD + CO group) or a control diet (CG group) during 16 weeks. The CO (1.5 g/kg/daily) was administered by gavage during the last 4 weeks of the diet protocol. We evaluated plasma liver enzymes, hepatic steatosis, and cytokine expression in liver as well as hepatocyte ultrastructural morphology and mitochondrial function. CO treatment suppressed hepatic steatosis, attenuated inflammatory response, and decreased plasma alanine aminotransferase (ALT) in mice with NAFLD. CO was also capable of restoring mitochondrial ultrastructure and function as well as balance superoxide dismutase and catalase levels. Our findings indicate that CO treatment has positive effects on NAFLD improving mitochondrial dysfunction, steatosis, acute inflammation, and oxidative stress. Nonalcoholic fatty liver disease (NAFLD) is the accumulation of fat in the liver in patients who do not consume excessive alcohol . The terNAFLD is asymptomatic in most affected patients and characterized as the presence of more than 5% of lipid accumulation in the hepatocytes, excluding other liver disease etiologies . NAFLD can progress to an advanced form, nonalcoholic steatohepatitis (NASH), which is defined by histological findings of hepatic steatosis, with hepatocyte damage and or inflammation . AlthougIn addition to genetic risk factors, the prevalence of NAFLD is aggravated by changes in life style including physical inactivity as well as consumption of high fat and high fructose corn syrup diet . RecentlEvidence suggests that NAFLD might be a mitochondrial disease. Mitochondrial dysfunction seems to play a role in the pathogenesis of NAFLD since it affects hepatic lipid homeostasis and promotes reactive oxygen species (ROS) production and lipid peroxidation, cytokine release, and cell death –18. MiceAlthough there is no specific effective treatment available for NAFLD, dietary therapy already showed beneficial results . Adminis (Hydrochoerus hydrochaeris) (CO), the largest rodent in the world, is a rich source of PUFA and contains 17.9% of alpha-linoleic and 19.6% linoleic fat acids (FAs), which correspond to essential omega-3 (n-3) and omega-6 (n-6) FAs, respectively. In a previous study, CO reduced cholesterol blood levels in rats fed with cholesterol-enriched diets  = PP [steatosis]/PT (2) where PP is the number of points that defined steatosis [Liver fragments fixed in paraformaldehyde (4% w/v) were embedded in paraffin, sectioned at a nominal thickness of five escribed . Brieflyteatosis , 29. Thiteatosis .2O2) and nonspecific binding of the polyclonal antibodies was blocked by incubation 5% (w/v) bovine serum albumin (BSA) diluted in phosphate buffered saline (PBS). Subsequently, sections were incubated with antibodies, and these reactions were amplified using a biotin-streptavidin system . Immunoreactive products were visualized using diaminobenzidine (DAB) reagent (Dako) and counterstained with hematoxylin. We used antitumor necrosis factor alpha (TNF-α), and antitransforming growth factor beta (TGF-β)  antibodies. The estimated density per area (QA) of TNF-α and TGF-β positive cells was defined as cell numerical profile within a known test area (expressed as cells/μm2) of 3305 μm2 [Liver sections had antigen retrieval performed by citrate buffer at pH 6.0 incubation for 30 min at 60°C. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide  in 0.1 mol/L cacodylate buffer, post-fixed in 1% osmium tetroxide (Sigma-Aldrich), and embedded in Epon . Ultrathin sections (60 to 70 nm) were obtained from selected areas using an ultramicrotome , contrasted with uranyl acetate and lead citrate, and examined with a transmission electron microscope (TEM)  at 80 kV.2O2  rate, and concentrations were monitored. As an index of oxidative damage induced by lipid peroxidation, we used thiobarbituric acid reactive substances (TBARS)  method to analyze malondialdehyde (MDA) products during an acid-heating reaction. TBARS levels were expressed as MDA equivalents.Frozen liver homogenates were used for biochemical assays, as performed by Kennedy-Feitosa et al., 2016 . Superoxμg/ml oligomycin (State 4). From respiratory fluxes obtained during substrate titration protocol, respiratory control ratio (RCR) was calculated for State 3/State 4.We used high-resolution respirometry protocol according to Cortez et al., 2012 . Brieflyp value less than 0.05 was considered statistically significant.A statistical software (GraphPad Prism 6) was used for statistical analyses and graphics generation. Data is presented as means ± standard error of the mean (SEM). Analysis of variance (one-way ANOVA) was performed followed by Bonferroni test. A p < p = 0.026) in the NAFLD group (25.0 ± 6.4 mg/dL) compared to the CG group (6.8 ± 0.9 mg/dL). Four weeks of treatment with CO reduced ALT levels in 46.4% (13.4 ± 3.8). No differences were found in the levels of AST  compared to CG (1.53 ± 0.44 positive cells/fields) (β increased significantly in NAFLD group (5.50 ± 0.62 positive cells/fields) compared to CG (0.83 ± 0.47 positive cells/fields) (α expression (NAFLD + CO = 3.87 ± 0.70 positive cells/fields) (β expression (NAFLD + CO = 14.17 ± 0.83 positive cells/fields)  . Likewis/fields) . Treatme/fields)  but grea/fields) .The electron microscopy analyses of CG showed preserved organelles and numerous well developed and organized mitochondria with an intact membrane and matrix . The NAFNAFLD group showed significantly increased SOD antioxidant enzyme activity (180.9 ± 27.46 U/mg protein) compared to the CG group (70.09 ± 5.35 U/mg protein). Treatment with CO significantly decreased SOD activity (NAFLD + CO = 91.56 ± 19.42 U/mg protein) compared with NAFLD group . NAFLD gTo evaluate mitochondrial function, respirometry experiments were performed. NAFLD animals without treatment showed liver mitochondrial impairment, with a reduction in maximum ADP-stimulate respiratory rates (State 3) in oxidation protocols, CHO, and FAs reduction, compared with CG. CO treatment (NAFLD + CO) promoted a significant increase in State 3 respiration in CHO and FAs, respectively Figures . FurtherThe present study showed that CO, an alternative PUFA source, was capable of reversing liver damage associated with NAFLD in obese mice. The oil was able to reduce plasmatic levels of ALT enzyme, decrease hepatic steatosis, and improve tissue morphology and inflammation. Moreover, CO improved abnormalities in mitochondrial ultrastructure, cellular respiratory efficiency, and imbalance of antioxidant enzymes.Prolonged HFD in C57Bl/6 mice is an established model of diet-induced obesity . PreviouAminotransferase levels have been used as a marker of NAFLD . NAFLD iFat accumulation in the liver of patients with NAFLD is mainly in the form of triglycerides , 42. Triα [α are increased in patients with NASH and correlate with histological severity of liver damage [α to be increased in our model and CO significantly reduced hepatic TNF-α levels. The absence of NASH and fibrosis in our model is a limitation of this study. Nevertheless, this cytokine is known to contribute to mitochondrial dysfunction, inducing mitochondrial swelling, rarefaction of the matrix, and membrane burst interfering in mitochondrial respiratory complexes [β, a pleiotropic cytokine that plays a dual role in immune functions [β include regulation of cell proliferation, control of extracellular matrix protein production and degradation, cellular differentiation modulation, and either stimulatory or inhibitory effects on the same cell, depending on the cellular environment [α (PPARα) and inhibit factor nuclear kappa B (NF-κB) [α and interleukin (IL-6) expression in hepatocytes [+ T cells subtypes in Th17 proinflammatory T cells is dependent of association between IL-6 and TGF-β, while the anti-inflammatory Treg cells only need high levels of TGF-β stimulation to differentiate [κB (indirectly indicated by the decrease in TNF-α production) or by increasing TGF-β levels in the liver (and probably the anti-inflammatory response stimulating Treg cells differentiation). However, more analyses are necessary to confirm this hypothesis.NAFLD is associated with the production and release of proinflammatory cytokines, both systemically and locally in the liver, such as TNF-α . The perα . Serum ar damage . We founomplexes . We alsounctions . Biologiironment . It is w (NF-κB) , resultiatocytes . In adaprentiate . This meβ-oxidation [Structural and functional alterations in mitochondria have showed to contribution in the pathogenesis of NAFLD. Structural alterations encompass depletion of mitochondrial deoxyribonucleic acid (DNA) and morphological and ultrastructural changes, while functional alterations include the respiratory chain and mitochondrial xidation . The mitxidation , 55. We 2O2. With impaired mitochondrial oxidative capacity, cytosolic FAs accumulate [Oxidative stress results from an imbalance between ROS, prooxidant, and antioxidant chemical species that leads to cellular oxidative damage. The FAs oxidation is an important source of ROS in fatty livers  and oxidcumulate . The deccumulate . High cocumulate . NAFLD icumulate . TreatmeRecently, there was an increasing interest in the use of the PUFAs as a nutritional supplementation, since many studies have been proving protective effects in patients with different diseases including the ones related to obesity , 61. AltIn most of the studies, PUFA sources are fish or plants where the extraction procedures are complex and result in low yields of the product , 64, 65.We conclude that CO treatment has positive effects on the benign form of obesity-induced NAFLD improving mitochondrial dysfunction, steatosis, acute inflammation, and oxidative stress in mice.


APOL1 risk alleles, and is the classic histologic lesion resulting from Human Immunodeficiency Virus (HIV) infection of podocytes. Other viral infections, including parvovirus B19, and drugs such as interferon that perturb the immune system, have also been associated with CG. Despite significant advances, explaining such genetic and immune/infectious associations with causative mechanisms and supporting evidence has proven challenging.Collapsing Glomerulopathy (CG), also known as the collapsing variant of Focal Segmental Glomerulosclerosis (FSGS), is distinct in both its clinical severity and its pathophysiologic characteristics from other forms of FSGS. This lesion occurs disproportionally in patients carrying two APOL1 G1 risk alleles.We report the case of a healthy (HIV-negative) pregnant 36 year-old Caribbean-American woman who presented with nephrotic syndrome and fetal demise in the setting of acute parvovirus B19 infection. A series of three renal biopsies and rapid clinical course showed progression from significant podocyte injury with mild light microscopy findings to classic viral-associated CG to ESRD in less than 3 months. Genetic analysis revealed two APOL1 risk allelles, and parvoviral proteins identified in renal epithelium on kidney biopsy. These findings support the causative role of parvovirus B19 infection in the development of CG on the background of APOL1 genetic risk.This is the first published case report of CG in the setting of acute parvovirus infection in a patient with two The online version of this article (doi:10.1186/s12882-016-0330-7) contains supplementary material, which is available to authorized users. Collapsing Glomerulopathy (CG) represents approximately 12 % of focal segmental glomerulosclerosis (FSGS) histology and is more likely to present with nephrotic syndrome and rapid progression to end-stage renal disease (ESRD) compared with other FSGS variants . These eMuch of what is known about the pathogenesis of CG comes from human case reports and animal models. However, finding a central theme is difficult. Animal models suggest that HIV viral proteins, even when expressed only in podocytes, lead to both the glomerular and tubular characteristics of this lesion on particular genetic backgrounds . Other aAPOL1 genetic variants with FSGS and particularly HIV-associated Nephropathy (HIVAN) (odds ratio 17 and 29 respectively) has led to extensive effort to characterize the potential role of the protein product of APOL1 [Trypanosoma rhodesiense in addition to Trypanosoma brucei [APOL1-related risk is affected by the genotype of the donor kidney but not the recipient of a renal transplant [APOL1 genotype  for kidney disease is much lower than that for other Mendelian diseases. Of patients with this at-risk genotype, only 4 % develop FSGS  [APOL1 genotype with renal disease, suggests the need for a “second hit” as well as a likely role for other genetic or environmental factors. In this context, there may be a role of the “nephrotropic” parvovirus B19 in disease development.The discovery of the association of of APOL1 . This geof APOL1 . The isoa brucei . Its doma brucei . APOL1 iansplant , it seemansplant . There iansplant , 15. Notp HIVAN) . This lo. This finding adds plausibility to prior associations of hepatitis, myocarditis, and glomerulopathy with parvovirus infection made solely on temporal relation of viral infection and organ dysfunction coupled with the detection of parvoviral DNA in affected organ tissue.Human parvovirus B19 is a small icosahedral ssDNA virus whose genome encodes two capsid proteins, VP1 and VP2, and a non-structural protein, NS1 . Most adParvovirus B19 has been associated with CG for over 15 years, with several case reports demonstrating temporal relationship between infection and CG in both native and transplanted kidneys, but without definitive evidence of causality or explanation of its pathogenic role , 19, 20.APOL1 interaction in CG.This case report hopes to direct attention to the role of the parvovirus B19 and A previously healthy 36 year-old Caribbean woman, G4P2012, presented at 8 weeks gestation, with a 1-week history of worsening swelling in her hands and feet. Her prior pregnancies were without complication, and she took no medications other than prenatal vitamins. She had no personal or family history of kidney disease or immunodeficiency. Her prenatal workup was unremarkable and HIV test negative. Her physical exam on presentation was significant for a blood pressure of 146/95 mmHg, and 3+ pitting edema up to her knees. Approximately 1 month prior to presentation, her 4-year-old daughter had experienced a classic presentation of “Fifth disease” from parvovirus B19. On admission, an obstetrical ultrasound revealed fetal demise and she underwent a dilation and curettage procedure. Products of conception were not sent for parvovirus staining as the obstetrical team felt her presentation was entirely consistent with acute parvovirus infection.10 . Renal ultrasound revealed normal sized kidneys bilaterally and slight increase in echogenicity, but without hydronephrosis, cysts, or calcifications.Notable laboratory and imaging results included serum creatinine concentration (SCr) of 557 umol/L (6.3 mg/dL), blood urea nitrogen of 15.3 mmol/l (43 mg/dL), serum albumin 15 g/L (1.5 g/dL), total cholesterol 10.55 mmol/L (408 mg/dL), triglycerides 7.01 mmol/L (621 mg/dL), LDL 7.86 mmol/L (304 mg/dL), INR 0.96, PTT 33 s, and PT 10.7 s. A spot urine protein:creatinine ratio was 20.8 g/g. Urine microscopy revealed 2–5 white blood cells per high power field and maltese crosses were diffusely present upon polarization. Further serologic workup was negative, including ANA, anti-dsDNA, repeat HIV test, and for hepatitis B and C. Serum parvovirus B19 qPCR detected the parvoviral genome with a modest viral load of approximately 2000 copies, and parvovirus serologies both IgM and IgG were elevated demonstrating acute infection. While, “undetected” would be the reported result for unexposed individuals, the clinical virology lab reports that their sensitive qPCR assay allows for viral load quantification from approximately 200 copies to over 1X10The patient underwent renal biopsy Fig.  on day 3She was discharged home after 3 weeks of hospitalization, with tapering prednisone and an aggressive diuretic regimen. She returned 6 days later with orthostatic hypotension despite persistent anasarca (80+ pounds above her baseline). Her serum albumin was 10 g/L (1.0 g/dL). Water immersion therapy with diuresis was attempted in an effort to alleviate the patient’s edema, but had little effect.Her SCr had improved to 309 umol/L (3.5 mg/dl) by 5 weeks after presentation, but she then developed pancytopenia. Serum hemoglobin fell to 50 g/L (5 g/dL), platelet count to 15,000/uL, and ANC as low as 1700/uL with an inadequate rise in reticulocyte count. Serum lactate dehydrogenase and haptoglobin remained normal. Bone marrow biopsy showed a markedly hypocellular marrow with mild dyserythropoesis. While no viral inclusions or hemophagocystosis were found, her serum ferritin rose to 2260 ug/L. This was proposed to be from parvovirus-related inflammation, which has been reported previously . Repeat A third renal biopsy was undertaken 10.5 weeks after initial presentation to help decide whether to consider cytotoxic therapy for FSGS in a now fairly debilitated young woman. Due to the extent of disease progression on this biopsy, the decision was made to forego any cytotoxic therapy and to initiate dialysis for uremia and volume management.APOL1 risk alleles . For completeness, the following 50 additional genes with published association with glomerular diseases were also assessed: ACTN4, ADCK4, ALG1, ALMS1, ANLN, APOA1, APOL1, ARHGAP24, ARHGDIA, ATGB4, B2M, CD151, CD2AP, CFH, COL4A3, COL4A4, COQ2, COQ6, CRB2, CUBN, DGKE1, EMP2, FGA, FN1, GLA, INF2, ITGA3, ITGB4, LAMB2, LMX1B, LYZ, MEFV, MYH9, MYO1E, NEIL1, NLRP3, NPHS1, NPHS2, PAX2, PDSS2, PLCE1, PLCG2, PMM2, PTPRO, SCARB2, SMARCAL1, TRPC6, WDR73, WT1, ZMPSTE24. There were no loss of function  variants, nor were there any rare non-synonomous variants with predicted deleterious consequence in these genes.Given the uncommon severity of this case and the possibility of African ancestry given her Caribbean origin , the posAPOL1 genotype were made available to transplant physicians during transplant evaluation and clinical genetic testing was suggested to the patient’s donor (sister) but she declined. As there are no established guidelines in place for APOL1 testing of donor organs, the donation was accepted. With over 6 months of follow-up, her renal function fortunately remains excellent, with <500 mg proteinuria, and no anemia.Over the subsequent 2 months on hemodialysis, the patient became anuric. Her serum albumin climbed to 3.0 g/L . She was evaluated for transplantation, and successfully received a living donor kidney transplant from her sister 8.5 months after initial presentation and 6 months after starting hemodialysis. Our findings of the patient’s APOL1 genotype. It remains to be seen if the APOL1 at-risk genotype is essential for development of this lesion following parvovirus infection. Importantly, CG is not the only renal lesion seen in those with the at-risk APOL1 risk genotype [This case report adhered to CARE  are more severe than expected in an immunocompetent host. Secondly, this occurred in the setting of a low viral load during acute infection. Her viral load failed to clear promptly; however, it has been suggested that parvovirus DNA can be detected for months to years after acute infection in a normal person . Worth nAPOL1 G1 risk alleles. We believe that with parvovirus B19 infection identified as one potential “second hit” in those with APOL1 risk alleles, perhaps clinical consideration of silent infection in other cases of “idiopathic” CG should be considered to aid in further data accumulation on this fascinating topic.This case provides evidence of parvoviral proteins in renal epithelial cells and a strong case for the role of parvovirus B19 infection in the pathogenesis of CG in a patient genotyped to show homozygous ANA, antinuclear antibody; dsDNA, double-stranded DNA; ESRD, End Stage Renal Disease; FSGS, Focal Segmental Glomerulosclerosis; HIV, Human Immunodeficiency Virus; HIVAN, HIV-associated Nephropathy; qPCR, quantitative polymerase chain reaction; SCr, serum creatinine


AbstractProterothrix (Proctophyllodidae: Pterodectinae) is described from the Large NiltavaNiltavagrandis (Blyth)  in northeast India . Proterothrixchachulae Constantinescu, sp. n. differs from all known species of the genus by having in males the aedeagus with bilobate tip. The morphological description is supplemented with molecular characterisation of a fragment f near the 5` terminus of the mitochondrial COI gene.A new species of the feather mite genus  Niltava Hodgson belongs to the family of Old World Flycatchers (Passeriformes: Muscicapidae), currently includes six valid species and is distributed in Indo-Malayan biogeographic region ; Analges sp., Proctophyllodeselegans Atyeo and Braasch, 1966, Proterothrix sp., Therisalges sp., Trouessartia sp., and Xolalges sp. ex Niltavasundara Hodgson .The bird genus c region . Feather Hodgson . An unidProterothrix Gaud, 1968 (Proctophyllodidae: Pterodectinae) includes 28 species known exclusively from the Old World  (The genus regions) . In the Proterothrix have been identified to date on birds from the orders Coraciiformes (Alcedinidae) and Passeriformes . According to modern concepts, this genus, along with seven more genera belongs to the “Phroterothrix generic group” incorporating archaic pterodectines with setae ps3 anterior to adanal suckers in males (megacaula (3 species), schizothyra (4 species) and wolffi (19 species)  . The newPageBreakethanol. Later, in laboratory conditions, mite specimens were cleared in 90% lactic acid for 24 hours, and mounted on microscope slides in Hoyer’s medium. Drawings were made using an Olympus CX21 microscope, with a camera lucida drawing device. The bird specimens were identified according to Proterothrix is given according to the current format used for species of pterodectine mites  in January 2014. The birds were captured using mist–nets, identified and visually checked for the presence and collection of mites and released back to the wild. Mite specimens were collected manually with a needle and placed in tubes with 96% ne mites  and the The full set of measurements is given for a holotype  and a range of measurements for corresponding paratypes. All measurements are in micrometers (μm). The holotype and paratypes of the new species are deposited in the Acarological Collection of the “Grigore Antipa” National Museum of Natural History, Bucharest, Romania .Proterothrixchachulae sp. n.  were used to isolate DNA using DNAeasy Tissue Kit (Qiagen). All three specimens used for molecular analyses were mounted and kept as reference vouchers for morphological examination. The specimens preserved in ethanol 96% were transferred in 180μl ATL Buffer with 20 μl of Proteinase K and incubated overnight at 56ºC on a shaking thermoblock. After 24h, 5μl of Proteinase K were added and incubation was continued until 72h. For the rest of the protocol we followed the manufacturer specifications and the modification suggested by Three paratype specimens of COI gene was used as DNA barcode region, amplified by PCR with the degenerate primers bcdF05 (5`- TTTTCTACHAAYCATAAAGATATTGC-3`) and bcdR04 (5`- TATAAACYTCDGGATGNCCAAAAAA-3`), according to PCR products were isolated from samples containing visible bands and sent for sequencing to Macrogen .The 663bp fragment near the 5` terminus of the K2P distance model  ; INDIA: Meghalaya, Jaintia Hills, Shnongrim village, ; 1151 m; subtropical forest; collector D. K. B. Mukhim.Male holotype (ANA672), 6 male , ANA748(P1♂)) and 6 female  paratypes, 27.01.2014, from the Large se separated by 36 (35–42). Scapular shields narrow. Humeral shields narrow, separated from outer sclerotized areas of epimerites III. Setae cp situated ventrally, setae c2 filiform, situated dorsally, both pairs on striated tegument. Subhumeral setae c3 lanceolate, 18 (18–20) × 6 (6–8). Hysteronotal shield with anterior margin concave, anterior angles rounded, distance from anterior margin to bases of setae h3 174 (180–186), greatest width in anterior part 74 (72–80), surface with small circular lacunae. Opisthosomal lobes roughly trapezoidal, short, each with angular median expansion on posterior margin, setae h3 situated slightly posterior to setae h2. Terminal cleft V–shaped, 16 (16–20) in length; margins of terminal cleft without membranes. Supranal concavity clearly outlined, triangular. Setae f2 slightly posterior to bases of setae ps2. Setae h1 near lateral margins of opisthosoma. Setae ps1 filiform, length 6 (5–8), situated near lateral margins of opisthosomal lobes, anterior to bases of setae h3. Setae c1 present, setae h3 flattened and enlarged in basal part, shorter than h2. Dorsal measurements: c2–d2 68 (64–68), d2–e2 70 (72–78), e2–h3 42 (40–46), d1–d2 33 (31–34), e1–e2 24 (20–26), h1–ps2 10 (7–11), h2–h2 36 (34–36), h3–h3 20 (20–22), ps2–ps2 44 (46–48).MALE Figs . Scapula4a  long, extending to level of setae ps3. Genital papillae situated at level of genital arch. A pair of small ovoid sclerites located near tips of genital arch. Two pairs of adanal shields  represented by small triangular plates, setae ps3 situated on median pairs. Anal suckers 12 (11–12) in diameter, corolla indented, with 8 small teeth. Ventral measurements: 3a–4b 20 (18–22), 4b–4a 30 (28–30), 4a–g 30 (27–32), g–ps3 36 (37–40), ps3–ps3 17 (16–18), ps3–h3 36 (36–40).Epimerites I fused into a V, posterior end connected with epimerites II by transverse sclerotized bands. Epimerites II long, with posterior ends free. Coxal field I closed, coxal field II open, coxal fields III almost closed, coxal fields IV with narrow sclerotized areas at bases of trochanters IV. Epimerites IVa present, well developed, their anterior tips bearing bases of setae 4a Fig. . Genitale of tarsus I filiform. Setae mGII thickened basally, with filiform apex, setae d of tarsi II, III much shorter than corresponding setae f. Tarsus IV 23 (22–24) long, with apical claw-like process, setae d, e button-like, seta d bigger in diameter than e, situated in basal and apical parts of segment, respectively , ω1II 9 (8–10), φI 56 (54–60), φII 46 (42–44), φIII 25 (22–26), φIV 22 (18–20).Legs I longer than legs II, femora I and II with ventral crest Fig. . Seta e ely Fig. . Length PageBreakPageBreakPageBreakand length of hysterosoma 270–280. Prodorsal shield entire, anterolateral extensions with rounded tips, lateral margins without deep incisions, posterior margin almost straight, length 117–121, width 107–110, surface with small circular lacunae in anPageBreakterior part and bigger ovate and circular lacunae in posterior part , for her support of our research of ectoparasites of birds from Meghalaya (India).Proterothrixchachulae sp. n. belongs to the wolffi species group by having almost closed coxal fields III in males and parallel-sided terminal cleft in females. Males of the new species differ from all known males of the genus by having the aedeagus with bilobate tip. Among all species of the genus, Proterothrixchachulae is closely related to Proterothrixcyornis Mironov and Tolstenkov, 2013 from Cyornistickelliae Blyth (Passeriformes: Muscicapidae) by having the pseudorutelar lobes with acute lateral extensions in both sexes. Males of both species have the opisthosomal lobes roughly trapezoidal and short, setae h3 flattened and enlarged in basal part, a similar general proportions of aedeagus and epimerites IVa well developed, their anterior tips bearing bases of setae 4a. Proterothrixchachulae differs from that species by the following features: in males, the prodorsal shield has posterior margin with wide blunt-angular extension, the opisthosomal lobes have angular median expansion on posterior margin, a pair of small ovoid shields is located at the tips of the genital arch, seta d is bigger in diameter then e on tarsus IV; in females, the prodorsal shield bears in anterior part a few small circular lacunae, the lobar shield is without ornamentation, and the setae h3 have about 1/3 from the length of terminal appendages. In males of Proterothrixcyornis, the prodorsal shield has posterior margin slightly convex, the opisthosomal lobes have posterior margin with a small median invagination, small sclerites near the tips of genital arch are absent, seta d and e on tarsus IV are subequal in diameter; in females, the prodorsal shield bears big ovate lacunae in anterior part, the lobar shield bears few circular lacunae in anterior part, and the setae h3 have about 1/5 from the length of terminal appendages.Representative DNA sequences. GenBank accession numbers for molecular voucher: ANA747 P2 male KY594726; ANA748 P1 male KY594724; ANA749 P1 female KY594725.COI) gene for two male and one female paratypes. In the resulted alignment we identified 8 variable sites. Two haplotypes were identified: H1 (ANA_748_P1_M and ANA_749_P1_F) and H2 (ANA_747_P2_M).We sequenced a 660-pb fragment of the mitochondrial cytochrome c oxidase subunit I (K2P model is 0.8% (SE 0.3). All observed nucleotide substitutions were synonymous and did not change the amino acid sequence.Intraspecific genetic distance between the analyzed specimens using Analgoidea species like Proctophyllodescetti (0.87%) (This reported genetic distance in the nucleotide sequence of the DNA barcode is comparable with genetic distances found for other  (0.87%) .


The effect of process parameters such as resorcinol concentration (100–500 g/L), current density (2–10 mA/cm2), Na2SO4 concentration (0–20 g/L), and reaction temperature (25–45°C) was analyzed on electrochemical oxidation using response surface methodology (RSM). The optimum operating conditions were determined as 300 mg/L resorcinol concentration, 8 mA/cm2 current density, 12 g/L Na2SO4 concentration, and 34°C reaction temperature. One hundred percent of resorcinol removal and 89% COD removal were obtained in 120 min reaction time at response surface optimized conditions. These results confirmed that the electrochemical mineralization of resorcinol was successfully accomplished using BDD anode depending on the process conditions, however the formation of intermediates and by-products were further oxidized at much lower rate. The reaction kinetics were evaluated at optimum conditions and the reaction order of electrochemical oxidation of resorcinol in aqueous medium using BDD anode was determined as 1 based on COD concentration with the activation energy of 5.32 kJ/mol that was supported a diffusion-controlled reaction.Electrochemical oxidation of resorcinol in aqueous medium using boron-doped diamond anode (BDD) was investigated in a batch electrochemical reactor in the presence of Na The other uses include the manufacture of specialty chemicals, explosive primers, antioxidants, flame retardants, UV stabilizers, dyes, agricultural chemicals, fungicidal creams and lotions, meta-amino phenols, hair dyes, and pharmaceuticals  thin film is a new electrode material superior to TiO2 and H2O which may result in complete mineralization , The aim of this study was to investigate the electrochemical oxidation of resorcinol in aqueous medium using boron-doped diamond (BDD) anode in the presence of Na2SO4 (Riedel-de Haën), HgSO4 (Merck), methanol (Merck), and Merck Spectroquant® 14541 COD cell tests were purchased in extra pure grade and used without purification. Double distilled water was produced using water still (GFL-2008) and ultrapure water system (Millipore Simplicity® UV) with the resistivity of 18.2 MΩ.cm@25°C, TOC < 5 ppb.Resorcinol (Merck), Na2. Batch electrochemical reactor was equipped with programmable Goodwill PST-3201 power supply, Heidolph RZR 2021 mechanical mixer, thermometer, Memmert WB 22 heating/cooling water bath and Heidolph PD 5206 peristaltic pump. Reaction medium was mixed at 750 rpm. Samples were withdrawn from the electrochemical reactor at regular periods for the analytical measurements.DURAN® batch electrochemical reactor  with heating/cooling jacket used in this study. The net reaction volume was 600 mL. Three plates DIACHEM® boron-doped diamond (Nb/BDD) anode  and 4 cylindrical (ϕ = 12.0 mm) iron cathode were used as electrodes with 15 mm anode/cathode spacing. Iron electrodes were purchased from local sources. Total electrode surface area was 280 cmR2 = 0.9991. pH was measured using WTW inoLab BNC720 model pH meter/conductivity meter. Merck Spectroquant® 14541 COD cell tests and Nova 60 water/wastewater photometer were used for the COD analysis.HPLC analysis of resorcinol were conducted using Inertsil ODS-3  column in a Shimadzu Prominence LC-20AD Liquid Chromatography equipped with DGU-20A5 degasser, CBM-20Alite System Controller, LC-20AD gradient pump, SIL-20A auto sampler, CTO-20A column oven, and SPD-20A UV/Vis detector. Mobile phase consisted of methanol and water (75/25) at a flow rate of 1.0 mL/min. UV detection was performed at 270 nm wavelength. The column temperature and injection volume were set as 40°C and 30 μL, respectively. The regression coefficient of the calibration curve for resorcinol was Response surface methodology (RSM) is a mathematical and statistical method used in designing experiments, building models, evaluating the effect of process variables, and searching optimum operating conditions for the responses , Na2SO4 electrolyte concentration (0–20 g/L), and reaction temperature (25–45°C); and the responses were chemical oxygen demand (COD) removal, mass transfer coefficient (km), RSM is a very powerful tool that provides detailed mathematical and statistical analysis of the experimental results. In the literature, RSM was applied for the experimental design and process optimization in electrochemical oxidation of various types of wastewater containing textile dyes, pharmaceuticals, paint, dairy effluents, pulp and paper, phenols, phenol derivatives, landfill leachate, organic acids, and pesticides in most common using Box-Behnken design (BBD) and central composite design (CCD) , yCOD, ykm, yα, and yEC are COD removal, mass transfer coefficient, x1, x2, x3, and x4 are process variables (factors) of resorcinol concentration (mg/L), current density (mA/cm2), Na2SO4 electrolyte concentration (g/L), and reaction temperature (°C), respectively.The effect of resorcinol concentration, current density, Nax1, x2, x3, and x4 were evaluated for design matrix using Design-Expert© 10 and no aliases were found for the response surface quadratic models. The lack of fit test is valid for this study according to degrees of freedom (df) values where minimum 3 df for lack of fit and 4 df for pure error is recommended for a valid lack of fit test. The variance inflation factors (VIF) measures how much the variance of the model coefficient increases due to the lack of orthogonality in the design. The ideal VIF value is 1.00 and if the design has multilinear constraints multicollinearity exists to a greater degree with VIF > 10. The VIF values were obtained as 1.00 and 1.01 for COD removal, km, Process variables F-value is the test for comparing the sources mean square to the residual mean square and p-value is the probability of seeing the observed F-value for factor effects. Model F-values of 2.44, 3.08, 2.35, and 10.30 imply the quadratic models are significant for COD removal, km, P-values less than 0.05 indicate the model terms are significant and signal/noise (s/n) ratio >4 is desirable for adequate precision. Significant model terms have a real effect on the response and lack of fit. S/n ratios of 6.775, 5.870, 7.952, and 12.951 imply adequate signals and P < 0.05 indicate the significance for the quadratic models that encompasses 95% confidence interval for the design space. In Table F-values of 0.95 and 1.39 indicate the insignificance for COD removal and mass transfer coefficient; and F-values of 92.01 and 13.40 indicate the significance for 2SO4, 6.8–9.9 mA/cm2 current density and 26–34°C reaction temperature in Figures 22SO4 electrolyte increased the COD removal efficiency as well. Most of the electrochemical processes are based on indirect oxidation because direct oxidation of organic pollutants is very slow at inert anodes due to reaction kinetics, limiting reactions, and mass transport limitations  in surface and contour plots can be seen in Figures −6 and 6.93 × 10−6 m/s with standard deviation of 1.51 × 10−6 m/s which are consistent to those reported in the literature .Electrochemical process is controlled by the mass transport, secondary reactions involve and COD removal follows an exponential trend due to mass transport limitations results in instantaneous current efficiency (ICE) decrease (α > 1).2 current density.In Figures r with standard deviation of 62.6 kWh/kg CODr using Equation (13).In electrolysis, electrical charge involves for the chemical changes in non-spontaneous oxidation and reduction reactions (ΔG > 0) and in most cases supplied electrical energy is larger than the Gibbs free energy change of the reactions. Therefore, energy consumption in electrochemical processes should be optimized for energy efficient processes. In the batch runs, energy consumption values were evaluated between 39.0 and 302.7 kWh/kg COD2SO4 concentration, and the decrease in the current density. The energy efficient reaction temperature was found between 28 and 42°C.Figures 2 current density, 12 g/L Na2SO4 concentration and 34°C reaction temperature by analyzing Figures r energy consumption in 95% confidence interval. The specific batch run was conducted at response surface optimized conditions and the results are presented in Figure −1 for resorcinol oxidation and COD removal, respectively. These results confirmed that the electrochemical mineralization of resorcinol was successfully accomplished using BDD anode depending on the process conditions, however the formation of intermediates and by-products were further oxidized at much lower rate. Brillas and Martínez-Huitle (•OH) than the aromatic pollutants.Maximization of COD removal efficiency was preferred for the process optimization, and optimum process parameters were determined as 300 mg/L resorcinol concentration, 8 mA/cmz-Huitle  also rep3PO4 electrolyte concentration, pH 2, 30 mA/cm2 current density and 25°C reaction temperature. Nasr et al.   and reaction rate constant (k) from batch reactor data. In Figure R2 = 0.96) as 1 and 0.0145 min−1, respectively. Liu et al.  can be linearized into Equation (15) for the evaluation of reaction order  can be defined with Arrhenius equation (Equation 16) and that can be linearized into Equation (17) when overall reaction rate is expressed with Equation (14).The reaction rate constant (k) was calculated between 25 and 45°C reaction temperatures and plotted vs. 1/T with linear regression (R2 = 0.97). The activation energy (Ea) and the frequency factor (A) were evaluated at 300 mg/L optimum resorcinol concentration as 5.32 kJ/mol and 0.1169 min−1, respectively. The activation energy depends on the nature of the reaction and fast reactions generally have small Ea values. It is known that the activation energy for a diffusion-controlled homogeneous reaction is < 40 kJ/mol  anode was investigated in a batch electrochemical reactor. The following conclusions were drawn from the study:m), The effect of process parameters on COD removal, mass transfer coefficient  were calculated between 0.33 × 10−6 and 6.93 × 10−6 m/s.Mass transfer coefficients  anode in electrochemical oxidation of phenolic effluents that can be found in domestic and industrial wastewater.PD conducted the literature review, performed the experiments, analyzed the results, and prepared the draft manuscript. BK guided in design of experiments, response surface methodology, process optimization and reaction kinetics, and revised the manuscript. BK is the supervisor of this research.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.