There was no growth retrieval, after exposure to chalcone N9 alone, in a long-term assay to determine the cumulative population doubling (CPD) of human oral cancer cells. A PCR array evaluating 168 genes related to cancer and inflammation, demonstrated striking actions for N9, which altered the expression of 74 genes. Altogether, our results point out quinoxalinic chalcones, mainly N9, as potential strategies for oral cancer treatment.The present study aimed to characterize the effects of quinoxaline-derived chalcones, designed on the basis of the selective PI3Kγ inhibitor AS605240, in oral cancer cells. Three lead compounds, namely N9, N17 and N23, were selected from a series of 20 quinoxaline-derived chalcones, based on an initial screening using human and rat squamous cell carcinoma lineages, representing compounds with at least one methoxy radical at the A-ring. The selected chalcones, mainly N9 and N17, displayed marked antiproliferative effects, via apoptosis and autophagy induction, with an increase of sub-G1 population and Akt inhibition. The three chalcones displayed marked  The oral squamous cell carcinoma (OSCC) is the most common type1. Regardless of the vast number of studies and the development of new and less toxic treatment regimens, in addition to the advances in diagnosis tools, the survival rates have not significantly changed over the past decades2. The five-year survival rate of patients with OSCC remains below 50%; besides, around 70% of advanced-stage cases are incurable3. For these reasons, OSCC remains a significant worldwide disease burden4. The poor outcome can partly be related to the development of resistance to radiation and chemotherapy, with loco-regional and distant failures2, or the occurrence of a second primary tumor5. Therefore, novel and effective therapeutic options for treating these tumors are needed.Head and neck cancers (HNSCC) encompass tumor types arising from many sites in the upper aerodigestive tract. More than 90% of cases are squamous cell carcinomas, which occur most frequently in the oral cavity, oropharynx and larynx6, presenting a broad spectrum of biological activities, such as anti-cancer, antioxidant, anti-inflammatory, antibacterial and antimalarial7. Chemically, these compounds are open-chained molecules composed of two aromatic rings joined by three unsaturated α, β carbons and one carbonyl group8. The simple structure and the easy process for obtaining these compounds make them interesting for structure-activity relationship (SAR) studies7. Numerous substituents were linked to the chalcone scaffold, and different series of effective synthetic analogs with therapeutic potential for many cancer types were obtained. These structural modifications produced a great variety of compounds with different mechanisms of action9. Previous data showed that compounds with a quinoxaline ring in their structure have the ability to inhibit the angiogenic process10, and to induce caspase-dependent apoptotic cell death11. Some additional antiproliferative mechanisms also support the notion that such compounds might be potential candidates for cancer treatment10. Previous studies from our group and co-workers demonstrated that different chalcones derived from quinoxaline and based on the selective PI3Kγ inhibitor AS605240, showed a great capacity to inhibit cell proliferation and to reduce the viability of glioma cell lines13. With this in mind, the present study aimed to evaluate the action of twenty quinoxaline-derived chalcones in different OSCC cell lineages. Attempts have been made to characterize the anti-cancer activity of the most effective compounds, presenting at least one methoxy radical on the A-ring, focusing on the mechanisms of action underlying its effects in OSCC cells, alone or in combination with classical anti-cancer drugs in clinically relevant treatment protocols.Molecules based on natural products have a relevant role in oncology drug discovery, and several natural product-derived compounds present beneficial effects when combined with classical chemotherapeutic drugs. Chalcones  are a group of natural precursors of flavonoid biosynthesis in high plantsThe first set of experiments was conducted to select the quinoxaline-derived chalcones with the greatest cytotoxic effect on OSCC lines, based on the reduction of cell viability assessed through the MTT assay. Human HN30  around 50% after 48 h, according to assessment of both cell lines. The IC50 values were greater than 30 μM for most tested compounds. Interestingly, the chalcones with higher cytotoxic potential display at least one methoxy group at the phenyl A-ring of its structure , chalcone N17  and chalcone N23 (3′-OCH3). Hereafter, this study was designed to define the potential cellular targets of lead compounds, and for comparison and combination with standard chemotherapies clinically used in the treatment of OSCC.As a first approach, HN30 cells were treated with chalcones N9 , N17  and N23 , for 48 h, and a clonogenic assay was performed. When compared to the control, a significant decrease in colony formation was observed after treatment with chalcones N9 (15.61 µM) and N17 (14.27 µM)  on the flow cytometry (cell size) was observed after the treatment with N9 and N17  and mitogen-activated protein kinases (MAPK). The signaling pathway phosphatidylinositol 3-kinase/protein kinase B/ mammalian target of rapamycin (PI3K/Akt/mTOR) is involved in the control of cell cycle, survival, proliferation, metabolism and motility, which are important hallmarks of cancerion Fig. . Akt phoion Fig. . These r24. To evaluate the effects of the quinoxaline-derived chalcones on Erk 1/2 and p38 activation, we used the same protocol described previously for Akt phosphorylation analysis. The chalcones N9, N17and N23 did not alter the status of Erk 1/2 or p38 activation in HN30 cells  are also involved in cell growth, proliferation, survival, autophagy and apoptosis. JNK, Erk 1/2 and p38 MAPK are key regulatory proteins in these processeslls Fig. , implyinlls Fig. .9. To verify if the selected quinoxalinic chalcones interfere with the cell cycle progression of OSCC, HN30 cells were treated with chalcones N9, N17 and N23, then marked with BrdU and analyzed with flow cytometry, after 48 h. An increase in sub-G1 population was observed, especially when cells were treated with N9 and N17  in different schemes of combination with cisplatin, docetaxel and 5-fluoruracil, using subthreshold concentrations, at different time-points. The selective PI3Kγ inhibitor AS605240 was included in this analysis, as it was used as the prototype for the synthesis of the quinoxaline-related chalcones used herein. The tested chalcones or AS605240, at subthreshold concentrations, incubated alone or in combination, failed to alter the viability of HN30 cells, as evaluated in the MTT assay , by testing the long-term antiproliferative effects of lead chalcones, AS605240 and the standard chemotherapy drugs, alone or in combination, for 18 days. A set of groups displayed a clear cell growth shortly after treatment, which remained increased throughout all the monitoring period, showing refractoriness to the treatments  in the MTT viability assay. A selectivity index (SI) was estimated on the basis of inhibition percentages obtained in non-tumor cells (HaCaT and Vero cells) versus tumor HN30 cells, as adapted from Tamokou et al.27. Data presented in the Supplementary Table Cancer chemotherapy generates several and important adverse events, mainly related to the effects on normal cells presenting high proliferation rates28. The identification of these genes and pathways can point out new targets and treatment options for OSCC. To evaluate the effects of the selected quinoxaline-derived chalcones on the modulation of classical cancer pathways, we performed a PCR array comprising 84 genes. HN30 cells were treated with the lead compounds N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM), for 48 h, followed by the assessment of genes related to cell proliferation, apoptosis, cell cycle, angiogenesis, invasion and metastasis. It was possible to observe that the three chalcones modulated the expression of 51 different genes, from a total of 84 genes  might present dual effects on cancer, depending on the activated pathways and the tumor type58. Curiously, the chalcone N9 induced the upregulation of other genes of the TNF family, namely cd40, fasg, ltbr, tnf and tnfaip2, which might also display dual effects in the tumor cell death58. However, in the light of the available literature data, at this moment, we are not able to justify the anti-tumor effects displayed by N9 based on the upregulation of some inflammation-related genes, including casp1, csf1, csf2, il1b, il1r, il1r1, myd88, nod1, tlr1, tlr2 and tlr6. Indeed, there is much to be investigated regarding the connection between these inflammatory molecules and cancer. The chalcones N17 and N23 led to the positive modulation of casp2 and casp7, whereas the compound N9 increased the expression of casp1 and casp8, what might help to explain, at least partly, the distinct ability of the lead chalcones to modulate apoptosis, as demonstrated by different approaches in our study.On the basis of the narrow relationship between cancer and inflammationin vitro anti-proliferative activities against OSCC. The cytotoxicity of these compounds on OSCC cells was mainly related to the induction of apoptosis and inhibition of Akt, besides the regulation of various genes related to tumorigenesis and inflammation. In parallel, cells that survived the acute stress caused by the chalcones triggered autophagy, probably as an escape mechanism to survive. The combination of the lead compounds with classical chemotherapy agents exhibited marked acute and long-term anti-tumor effects, with a good selectivity index. Intriguingly, the anti-tumor actions were prominently observed for the chalcones N9 and N17 that present two and three methoxy groups on the ring A, when compared to the subtle effects displayed by the monomethoxylated chalcone N23. It is possible to suggest that the quinoxalinic chalcones N17, and mainly N9, might represent promising compounds for the treatment of OSCC, although the in vivo efficacy of these compounds remains to be confirmed in future in vivo studies.Despite the advances in multimodality therapy, the overall survival of patients with OSCC remains poor and new treatment options are necessary. This study revealed that quinoxalinic-derived chalcones present marked 1H NMR, 13C NMR and elemental analysis. 1H NMR spectra revealed that all structures were geometrically pure and configured E (JHα-Hβ = 15.2–16.4 Hz).Twenty chalcones were prepared by aldolic condensation using quinoxalinic benzaldehyde 3 synthetized from 3,4-diaminobenzoic acid as presented in Supplementary Figure 1), as previously described by Melero-Pérez et al.59. Compound 1 was then reduced to its primary alcohol (2) with LiAlH4 after oxidized to aldehyde (3) with pyridine-chlorochromate in dichloromethane  were prepared by magnetic agitation of acetophenone (2 mmol), methanol (30 mL), KOH 50% w/v (5 mL) and benzaldehyde 3 (2 mmol), at room temperature for 24 h. Distilled water and chloric acid 10% were added in the reaction for total precipitation of the compounds. The compounds were then obtained by vacuum filtration and later recrystallized in dichloromethane and hexane. The compounds N2 (R = 4′-OCH3), N3 , N4 (R = 4′-Br), N5 (R = H), N7 (R = 4′-NO2), N9 , N10  and N12  were previously described by Mielcke et al. (2012)34. The compounds N15 , N16 , N17 , N18 , N19 (R = 2-OCH3), N20 , N23 (R = 3′-OCH3) and N36  were described by Winter et al. (2014)36. The compounds N24 (R = 4-imidazol), N33 (R = 4-CN), N34 (R = 3-CN) and N37 (R = 4-F) are new chalcones.The chalcones were prepared by aldolic condensation between quinoxalinic benzaldehyde and different acetophenones. The reagents used for the synthesis of the benzaldehyde, as a well as the acetophenone were obtained commercially . The quinoxalinic benzaldehyde was obtained by condensation between 3,4-diaminob enzoic acid and glyoxal at reflux with ethanol and acetic acid, generating the acid derivated , infrared spectroscopy (IR), 1 H and 13 C nuclear magnetic resonance spectroscopy (NMR), and for the unpublished ones, also elementary analyses. Melting points were determined with a Microquimica MGAPF-301 apparatus. NMR (1 H and 13 C NMR) were recorded on Varian Oxford AS-400 (400 MHz), using tetramethylsilane as an internal standard. Elementary analyses were obtained with a CHNS EA 1110. Percentages of C and H were in agreement with the product formula . The purity of the synthesized chalcones was analyzed by thin-layer chromatography (TLC) using Merck silica pre-coated aluminum plates 200 mm in thickness with several solvent systems of different polarities. Compounds were visualized with ultraviolet light (254 and 360 nm) and using sulfuric anisaldehyde solution followed by heat as developing agent and purified by recrystallization from hexane and dichloromethane. 1 H NMR spectra revealed that all the structures were geometrically pure and configured E .E)-1-(4-(1H-imidazol-1-yl)phenyl)-3-prop-2-en-1-one. Yellow solid, m.p. 233-234 °C1H NMR (CDCl3) δ 7.27 , 7,40 , 7,46  7.77 , 7.87 , 7.88 , 8.03 , 8.04 , 8.12 , 8.28 , 8.86  ppm. 13C NMR (CDCl3) δ 116.75, 123.80, 124.24, 128.54, 129.75, 130.06, 130.71, 131.37, 133.56, 135.43, 136.20, 138.45, 140.32, 143.32, 145.08, 146.23, 147.08, 188.72 ppm. Anal. Calcd for C20H14N4O: C 73.61, H 4.32, N 17.17. Found: C 73.33, H 4.62, N 17.20. Yield: 77%.(E)-4-acryloyl)benzonitrile. Yellow solid, m.p. 201-202 °C. 1H NMR (CDCl3) δ 8.80 , 8.30 , 8.15 , 8.08 , 8.00 , 7.95 , 7.70–7.68 , 7.69  ppm. 13C NMR (CDCl3) δ 116.16, 123.45, 125.20, 126.15, 128.88, 130.25, 131.25, 132.72, 137.32, 142.59, 143.03, 143.11, 146.93, 147.14, 187.43 ppm. Anal. Calcd for C18H11N3O: C 75.78, H 3.89, N 14.73. Found: C 75.72, H 3.69, N 14.56. Yield: 78%.(E)-3-acryloyl)benzonitrile. Yellow solid, m.p. 188-189 °C; 1H NMR (CDCl3) δ 7.16 , 7.44 , 7.48 , 7.65 , 7.71 , 8.00 , 8.05 , 8.15 , 8.30 , 8.85  ppm; 13C NMR (CDCl3) δ 115.76, 119.61, 122.22, 124.49, 128.44, 129.70, 130.21, 130.52, 136.61, 139.19, 142.91, 143.13, 144.85, 145.08, 145.8, 188.74 ppm; Anal. Calcd for C18H11N3O: C 75.78, H 3.89, N 14.73. Found: C 75.72, H 3.69, N 14.56. Yield: 80%.(E)-1-(4-fluorophenyl)-3-prop-2-en-1-one. White solid, m.p. 189-190 °C. 1H NMR (CDCl3) δ 8.88 , 8.37 , 8.20-8.16 , 8.03 , 8.01 , 7.67  ppm. 13C NMR (CDCl3) δ 117.27, 124.24, 128.54, 129.75, 130.71, 131.37, 133.56, 136.20, 138.75, 142.60, 145.09, 146.23, 147.08, 169.05, 184.25 ppm. Anal. Calcd for C17H11FN2O: C 73.37, H 3.98, N 10.07. Found: C 73.52, H 3.99, N 10.23. Yield: 75%..HN30  and HaCaT  cell lines were gently donated by Dr. Décio dos Santos Pinto Jr. . The Japanese Cancer Research Resources Bank  supplied the SCC158 cell line . Vero cells  were acquired from the Cell Bank of Rio de Janeiro . The cells were grown in culture flasks with Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 0.1% amphotericin B and 1% penicillin/streptomycin. The cells were maintained at 37 °C, in a relative humidity of 95%, at a 5% COFor screening purposes, the cell viability was evaluated by 3--2,5-diphenyl tetrazolium bromide (MTT) assay. This assay is based on the ability of living cells to reduce the tetrazolium bromide salt -2,5-diphenyltetrazolium bromide]) in formazan crystals by the mitochondrial enzyme succinate dehydrogenase. SCC-158 and HN-30 cells were seeded in 96-well plates at the density of 1 × 10³ and 5 × 10³, respectively. After 24 h, the cells were exposed to increasing concentrations (0.29 µM to 38.42 µM) of twenty different quinoxaline-derived chalcones (Table The effects of the selected compounds N9 (7.81 µM), N17 (7.14 µM), N23 (8.61 µM) were also tested in combination with standard chemotherapeutic drugs, currently used in clinics for treating OSCC, namely cisplatin (10 μM), 5-fluorouracil (7.69 μM) and docetaxel (0.015 μM). In this experimental set, the selective PI3Kγ inhibitor AS605240 (at 10 μM), used as the basis for synthesis of quinoxaline-derived chalcones, was also evaluated. The chalcones or the reference drugs were tested alone or in combination and the viability of the cell lines HN30, HaCat and Vero was analyzed by the MTT assay at 24 h, 48 h and 72 h.For all experiments, at least three independent experiments (in triplicate) were performed. The results were expressed as the percentage of cell viability in relation to the negative control with 0.01% DMSO.th day after incubation, the cells were fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. The colonies with more than 50 cells were considered as survivors. They were identified under a microscope; photographs were taken for subsequent quantification of the number of colonies in the treatment and control groups. A minimum of three independent experiments (in duplicates) were carried out.In this study, we assessed the number of cell colonies that were able to recover after the exposure to the selected chalcones. For this purpose, the growth and the proliferative characteristics of HN30 cells were determined after incubation with the selected quinoxaline-derived chalcones N9 , N17  and N23  or with the positive control drug rapamycin (100 nM) and the starving in Hanks’ Balanced Salt solution (HBSS) for 48 h. After this period, the surviving cells were re-plated in six-well plates (100 cells/well), without treatments. After 7 days, the culture medium was changed. On the 124 cells/well. The following day, they were treated with the chalcones N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM). After 48 h, the cells were fixed with 4% paraformaldehyde, and stained with 300 nM of 4′,6-diamidino-2-phenylindole solution . The cells were photographed in a fluorescence microscope with appropriate filters , at 32X magnification. The images were analyzed using Image Pro-Plus 6.0 software . To evaluate the nuclear morphometry and the nuclear irregularity index (NII), five parameters were used  and the nuclei were classified as irregular (I), large and regular (LR), large and irregular (LI), small and regular (SR), small (S), or small and irregular (SI) according the methodology by Fillipi-Chiela et al.60.DAPI is a fluorescent dye that preferentially stains dsDNA, allowing distinction of live cells and apoptotic cells. HN30 cells were seeded in 24-well plates at the density of 1 × 104 cells/well) and treated with the compounds N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM), for 18 and 24 h. The chemotherapy agent cisplatin  was used as a positive control. At the appropriate time-points, the cells were detached with 0.5% trypsin/EDTA solution . The cells were resuspended with annexin binding buffer, and incubated with 1 μL of annexin V-FITC plus 3 mM of propidium iodide (PI) . Following incubation at 37 °C, for 15 minutes, the cells were analyzed by flow cytometry in Attune® Acoustic Focusing Cytometer . Data analysis was performed with the FlowJo Single Cell Analysis Software V10 .To characterize the cell death process caused by the quinoxaline-related chalcones, HN30 cells were seeded on 24-well plates (3 × 104 cells/well) were plated in 6-well plates, and treated with the chalcones N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM) for 5, 15 or 30 min. Alternatively, the cells were treated at the same time intervals with the selective PI3Kγ inhibitor AS605240 (30 μM). The HN30 cells were detached with 0.5% trypsin/EDTA solution  and incubated with Phosflow Cytofix Buffer® at 37 °C, for 10 min. After washing, the cells were permeabilized with Phosflow Perm Buffer III® for 20 min, on ice. Subsequently, the antibodies anti–pp38, anti–pAKT and anti-pERK1/2 were incubated for 30 min in the dark, on ice. The analysis was made in an Attune® Acoustic Focusing Cytometer . Data was analyzed with FlowJo Single Cell Analysis Software V10 .This experimental set was conducted to verify the effects of the selected compounds on the activation of MAP kinases ERK 1/2 and p38, or the PI3K-related serine/threonine kinase AKT. For this purpose, HN30 cells (6 × 104 cells/well), and treated with the chalcones N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM). After 48 h of treatment, the cells were incubated with AO  for 15 min, and followed by visualization under a fluorescence microscope .Acridine orange (AO) is a fluorescent dye that stains (in red) the acidic vacuolar organelles (AVOs), mainly late autophagosomes/autolysosomes, in contrast with the green fluorescence from the whole cell. For the qualitative assay, HN30 cells were plated in 24-well plates (1 × 104 cells/well) and treated with the chalcones N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM), for 48 h. The cells were stained with AO (1.5 µg/mL), and analyzed in an Attune® Acoustic Focusing Cytometer . After 48 h, the cells were re-plated in a drug-free medium. The quantification of positive AVOs and red fluorescence was done at 72 and 120 h.To quantify the percentage of cells with AVOs and the intensity of the red fluorescence, HN30 cells were plated in 24-well plates , N17 (14.27 µM) and N23 (17.22 µM), for 48 h. The cells were incubated with BrdU (10 µM) for 2 h (from 46 h to 48 h after treatment). Then, the cells were detached with 0.5% trypsin/EDTA solution , fixed with BD Cytofix® and permeabilized with BD Cytoperm Buffer®, followed by treatment with DNase and staining with fluorescent anti-BrdU and 7-AAD, according to the manufacturer’s protocol . Data was acquired using an Attune® Acoustic Focusing Cytometer , and analyzed with FlowJo Single Cell Analysis Software V10 . The cell cycle phase and DNA synthetic activity of cells was determined by the evaluation of total DNA expression (7-AAD) and incorporated BrdU levels (anti-BrdU FITC).HN30 cells were seeded at 6 × 104 cells/well. After 24 h, the cells were treated with the chalcones N9 (7.81 µM or 15.61 µM), N17 (7.14 µM or 14.27 µM) and N23 (8.61 µM or 17.22 µM), AS605240 (10 or 30 μM), cisplatin (10 μM), 5-fluorouracil (7.69 μM) and/or docetaxel (0.015 μM), alone or in combination. After 48 h, the cells were counted in an automated cell counter , and re-plated at the same density, in drug-free medium. The counting was repeated every 2–4 days (according to the cell confluence), and the cells were re-plated at the same density for 18 days. The CPD was calculated using the equation PD = [log N(t) * logN(to)]/log 2, where N(t) is the number of cells/well at the time of the count, and N(to) is the initial number of cells. The sum of the PDs was plotted versus the time of the culture.The long-term effects of a single treatment with the lead chalcones, alone or in combination schemes, were assessed by using the cumulative population doubling (CPD) assay. Briefly, HN30 cells were seeded in 24-well plates, at the density of 1 × 106 cells) were treated with the chalcones N9 (15.61 µM), N17 (14.27 µM) and N23 (17.22 µM), for 48 h. The total RNA was isolated with the RNeasy® mini kit (Qiagen®) and DNase digestion step was conducted with on-column DNase digestion step kit (Qiagen®), according to manufacturer’s recommendations. The yield and the quality of RNA samples was determined spectrophotometrically. One μg of total RNA sample was reverse transcribed using the First Strand cDNA kit RT2 (Qiagen®) in a final volume of 20 μL.The effects of the lead chalcones on several genes related to cancer and NF-κB-mediated cell signaling were assessed via PCR array. PCR array represents a method with high reproducibility, sensitivity, wide linear dynamic ranges, and specificity, similar to real-time PCR. For this experimental set, HN30 cells  was used to analyze the mRNA levels of 84 genes related to cell proliferation, apoptosis, cell cycle, angiogenesis, invasion and metastasis. The kit Human NF-κB Plus Signaling Pathway PCR array  was used to analyze mRNA levels of 84 genes related to signal transduction mediated NF-κB activation, according to the manufacturer’s instructions. Both kits are pre-designed cataloged pathway-focused PCR Arrays (Qiagen®). For data analysis, the 7500 Real-Time Systems Software v.2.0.6 was used .The amplification protocol consisted of an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, renaturation and extension at 60 °C for 1 min. The expression of each gene was quantified based on Ct (threshold cycle), i.e. the number of cycles at which the fluorescence signal crosses the threshold. All of the genes represented by the array exhibited a single peak in the fusion curve, characteristic of this product. The analysis of gene expression was performed using the Excel-based PCR array Data Analysis Software (Qiagen®) .et al.61. The gene stmn1 was selected considering its similar regulation by the three chalcones. The primer was selected based on the publication by Zhang et al.62.Quantitative real time PCR according to the method described by Sgnaolin Supplementary Information


SAA and apolipoprotein AI (apoAI) concentrations, paraoxonase-1 (PON-1), cholesteryl ester transfer protein (CETP), and lecithin-cholesterol acyltransferase (LCAT) activities were measured in the serum and/or HDL2 and HDL3 subfractions. SAA concentrations were higher in T2DM compared to controls: serum  versus 15 mg/L ; p = 0.002), HDL2  versus 0.4 mg/L ; p < 0.001), and HDL3,  versus 6 mg/L ; p < 0.001). Serum-PON-1 activity was lower in T2DM compared to that in controls  versus 41,109 U/L (5690); p = 0.043). CETP activity was higher in T2DM versus controls in HDL2 (232.6 μmol/L (14.1) versus 217.1 μmol/L (25.1); p = 0.001) and HDL3 (279.5 μmol/L (17.7) versus 245.2 μmol/L (41.2); p < 0.001). These results suggest that individuals with T2DM have increased SAA-related inflammation and dysfunctional HDL features. SAA may prove to be a useful biomarker in T2DM given its association with elevated CVD risk.Persons with type 2 diabetes mellitus (T2DM) have an elevated risk of atherosclerosis. High-density lipoproteins (HDL) normally protect against cardiovascular disease (CVD), but this may be attenuated by serum amyloid A (SAA). In a case-control study of young females, blood samples were compared between subjects with T2DM ( The prevalence of type 2 diabetes mellitus (T2DM) in younger adults is increasing, such that in the UK between 1991 and 2010, the prevalence of T2DM rose from 5.9% to 12.4% in persons < 40 years of age . There aUnder normal circumstances, HDL is proposed to be antiatherogenic, displaying anti-inflammatory and antioxidant properties through its utilisation of several associated proteins and enzymes. These include apolipoprotein AI (apoAI) that is essential for reverse cholesterol transport (RCT) and paraoxonase-1 (PON-1), which has the ability to prevent oxidative damage of both HDL itself and low-density lipoprotein (LDL) . Also, c3 subfraction [HDL has been reported to become dysfunctional under inflammatory conditions, and the release of serum amyloid A (SAA), an inflammatory marker whose concentration can increase 1000-fold in response to injury or inflammation, associates with HDL, in particular, the smaller HDLfraction . The assfraction , 14–16.T2DM is associated with increased inflammation, and studies have reported increased serum-SAA in subjects with T2DM, compared to that in healthy controls , 18. As Favourable ethical opinion was obtained from the Research Ethics Committee of Adelaide and Meath Hospital and St James' Hospital, Dublin, Ireland. Written informed consent was obtained from each participant included in the study, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.n = 42) or controls without T2DM (n = 42), matched for age and body mass index (BMI). Participants with T2DM were prescribed metformin (26), sulfonylurea (7), dipeptidyl peptidase-4 (DDP4) inhibitors (4), glucagon-like peptide-1 (GLP-1) analogue (8), and insulin (5), with 13 of the T2DM subjects being managed with diet alone.A total of 84 subjects, without clinical evidence of CVD, were recruited. All participants were female, either with T2DM  at 3000 rpm for 15 minutes at 4°C. The resulting supernatant was removed and immediately frozen in 1.3 mL aliquots at −80°C, until required.2).Baseline measurements included fasting serum levels of glucose, insulin, total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol, which were measured using standard enzymatic assays on an automated ILab-600 biochemical analyser . HbA1c was measured in serum by ion exchange HPLC. Height (cm) and weight (kg) were collected using a stadiometer and calibrated scales and used to determine BMI  as described by McPherson et al. [2 and HDL3, respectively.ApoAI concentration in HDLn et al. . The coe2, and HDL3, specifically detecting SAA1. As per the manufacturer's instructions, analysis was performed on a TRITURUS ELISA system . Prior to analyses, samples were diluted as follows: serum, 1 : 150; HDL2, 1 : 10; and HDL3, 1 : 100. The CVs for SAA were 2.8%, 3.6%, and 5.6% (interspecific) and 8.0%, 9.5%, and 11% (intraspecific) for SAA, HDL2, and HDL3, respectively.The commercially available ELISA procedure  was used to measure SAA concentration in serum, HDLμL was pipetted into a U/V 96-well plate . The Beer-Lambert Law was used to calculate concentration:A is the absorbance (nm), ε is the extinction coefficient (2.95 × 104 M−1 cm−1), C is the concentration (M), and L is the path length (1 cm). The CVs for CDs were 11.8%, 5.8%, and 8.3% (interassay) and 2.4%, 1.0%, and 0.6% (intra-assay) for LDL/VLDL, HDL2, and HDL3, respectively.The concentration of conjugated dienes (CDs) was evaluated by measuring the absorbance at 234 nm using a spectrophotometric plate reader . Briefly, samples were diluted 1/10 using a sodium chloride solution (density 1.006 g/mL) and 300 μL), HDL2 (200 μL), and HDL3 (20 μL) were adjusted to 2700 μL with assay buffer , followed by the addition of 300 μL of 10 mM phenyl acetate. 300 μL of this solution was then dispensed in duplicate into a 96-well UV plate. The production of phenol was monitored spectrophotometrically at 25°C at λ = 270 nm in a plate reader for 5 minutes at 10-second intervals. Change in absorbance was noted after 3 minutes. The following equation was used to determine arylesterase activity (U/L):A is the change in absorbance, Vt is the total volume of the sample in mL (3.0 mL), T is the time in minutes, ε is the extinction coefficient (1310 M−1 cm−1), and Vs is the sample volume in mL . One unit of arylesterase activity (U) was defined as 1 μmol of phenol generated per minute. The CVs for PON-1 were 3.8%, 8.0%, and 4.2% (interassay) and 6.4%, 3.1%, and 2.3% (intra-assay) for serum, HDL2, and HDL3, respectively.This method was an adaptation from the methodology according to Hasselwander and colleagues . Paraoxo2 and HDL3 using commercially available fluorometric assays, as per the manufacturer's instructions . A FLUOstar OPTIMA plate reader   was used to measure the rate of CE transfer from the synthetic donor particles to the acceptor molecule. The fluorescence intensity was then determined by subtracting the blank fluorescence intensity from each sample and read against the standard curve. Units are expressed in μmoles/L for absolute values. LCAT results were reported as a ratio of the two emission intensities (470/390 nm), where the nonhydrolysed substrate excitation was read at Em = 470 nm and the hydrolysed substrate emission fluorescence was read at Em = 390 nm using a FLUOstar OPTIMA plate reader. The CVs for CETP were 3.7% and 6.2% (interassay) and 1.6% and 1.0% (intra-assay), for HDL2 and HDL3, respectively. The CVs for LCAT were 4.4% and 0.7% (interassay) and 0.9% and 0.9% (intra-assay) for HDL2 and HDL3, respectively.CETP and LCAT activities were analysed in HDLt-test was used to determine between group differences and correlations were performed using Pearson's coefficient. Nonstandardised results are presented, where concentrations of HDL2- and HDL3-associated proteins were calculated per volume of serum, with apoAI-standardised results appearing in Appendix 1 in Supplementary Material available online at https://doi.org/10.1155/2017/1314864.Statistical analyses were performed using SPSS version 21 . Individual variables were assessed for normality of distribution and, if required, were logarithmically transformed. Results were expressed as mean (standard deviation (SD)) if normally distributed or geometric mean (interquartile range (IQ)) if logarithmic transformation. An independent p > 0.05 for all comparisons. However, T2DM subjects had higher levels of fasting glucose, 7.78 mmol/L  versus 4.94 mmol/L ; p < 0.001, and HbA1c, 7.26%  versus 5.42% ; p < 0.001, and lower HDL-C, 1.03 mmol/L  versus 1.20 mmol/L ; p = 0.004, compared to the control subjects.Subject characteristics are shown in 2 or HDL3, p > 0.05 for both comparisons (No significant difference was found between T2DM subjects and control subjects with regard to apoAI concentration in either HDLparisons .p = 0.002), HDL2  versus 0.4 mg/L ; p < 0.001), and HDL3  versus 6 mg/L ; p < 0.001)  versus 15 mg/L ; < 0.001) .p = 0.043). However, when subjects taking insulin or GLP-1 analogues were excluded, serum-PON-1 activity was not significantly different between the groups (p > 0.05). In HDL2 and HDL3, there was no difference in the activity of PON-1 (p > 0.05 for both comparisons)  ), arisons) .A subanalysis excluding those taking insulin and/or GLP-1 analogues found no significant change in any reported associations except between group analyses of serum-SAA and serum-PON-1 activity, which was no longer significant.2 was higher in subjects with T2DM compared to that in control subjects (232.6 μmol/L (14.1) 217.1 μmol/L (25.1), p = 0.001). This was also the case in HDL3, with CETP activity being higher in the T2DM subjects compared to that in the controls (279.5 μmol/L (17.7), 245.2 μmol/L (41.2), p < 0.001) (CETP activity in HDL< 0.001) .2 or HDL3, p > 0.05, for both comparisons , HDL2 , and HDL3 . Fasting glucose was positively correlated with SAA concentration in serum  and HDL3 . HbA1c was positively correlated with SAA concentration in serum  and HDL3 . Age and HDL-C did not show any correlation with SAA concentration (p < 0.05). There was a strong positive correlation between serum-SAA and HDL2-SAA  and a much stronger relationship between serum-SAA and HDL3-SAA subfraction .Correlations compared data from all 84 subjects . BMI shop > 0.05). The concentration of HDL2-SAA was not related to the activity of HDL2-PON-1, HDL2-CETP, or HDL2-LCAT (p > 0.05). The concentration of HDL3-SAA was also not related to the activity of HDL3-PON-1 or HDL3-LCAT (p > 0.05). However, results illustrated a positive correlation between HDL3-SAA association and HDL3-CETP activity . Given HbA1c was significantly associated with serum-SAA, correlations between HbA1c- and HDL-associated proteins and enzymes were considered  and HDL3 . Positive associations between both LDL/VLDL-CD and serum-SAA and LDL/VLDL-CD and HDL3-SAA were detected , although there was no significant difference in CD concentration for LDL/VLDL, HDL2, or HDL3 (p > 0.05) between T2DM and controls (data not shown).Correlations between SAA- and HDL-associated enzymes are presented in nsidered . Only CESubjects with T2DM have increased inflammation and an enhanced risk of developing atherosclerosis and subsequent CVD, with those who develop T2DM before 45 years of age being at greater risk of macrovascular complications than those developing T2DM after 45 years of age . OverallOur study focused solely on young premenopausal females with T2DM. Normally, premenopausal women benefit from the protective properties of oestrogen against developing CVD . HoweverThe subjects in our study were not taking statin drugs, which reduced the potential confounding impact of this class of medication on lipid profiles and their influence on decreasing SAA-related inflammation . Neverth2-SAA and HDL3-SAA of 20% and 23%, respectively, in subjects with type 1 diabetes mellitus (T1DM). Unfortunately, due to limited sample size and power, it was not possible for the current study to investigate this relationship further. However, one could assume that this relationship would extend to subjects with T2DM, although this would require further exploration in a larger T2DM population.BMI was measured and found to be similar between case and control groups, thus minimising its potential confounding influence, given that increased adiposity and associated obesity are known to be associated with increased inflammation, specifically SAA-related inflammation . As expe2-SAA and HDL3-SAA in subjects with T1DM without a change in apoAI concentration. Although their study was conducted in those with T1DM, the mechanistic effect may result from something beyond simple displacement of apoAI by SAA in diabetes. Furthermore, others have reported that apoAI levels alone were insufficient for the prediction of diabetes incidence with the actual apoAI/HDL-C ratio being more informative [Lower HDL-C levels were reported in subjects with T2DM, while apoAI concentrations were comparable between T2DM and control subjects. This would suggest that the HDL particle size was reduced in subjects with T2DM compared to that in controls, which is suggestive of a proatherogenic phenotype since the HDL particle size has been reported to be inversely associated with CVD risk . Evidencormative . Since t2 and HDL3 subfractions were higher in subjects with T2DM, supporting previously reported findings [3-SAA provide some support for increased oxidative stress within LDL/VLDL subfractions where SAA levels are high and which normally HDL would reduce atherogenesis risk [r = 0.906; p < 0.001) observed between serum-SAA and HDL3-SAA adds further support that HDL3 is the preferred acceptor of SAA [2-SAA and HDL3-SAA, together with a nonsignificant increase in serum-SAA in those with T1DM compared to controls, suggesting a common increase in inflammation in both types of diabetes.SAA concentrations within serum and HDLfindings , 35. Whefindings , 36, 37.sis risk . The higr of SAA , suggestr of SAA . They re2 and HDL3, which may result from a lack of change in apoAI concentration that is required for the stabilisation of PON-1 and its association with HDL. Kopprasch and colleagues suggested the duration of T2DM could affect PON-1 activity [PON-1 is an antioxidant enzyme whose binding to HDL particles is stabilised by apoAI . PON-1 aactivity . Given tactivity . Recentlactivity  and in sactivity . Jayaramactivity . The con2 and HDL3, similar to that reported by Ståhlman and colleagues [3-SAA and HDL3-CETP suggests that higher SAA levels in T2DM lead to increased CETP activity similar to previous reports [3 more readily with SAA than HDL2. CETP activity was also found to correlate with HbA1c suggesting poor glycaemic control would result in a smaller HDL particle size.Our study has shown that CETP activity was increased in both HDLlleagues . Our datlleagues , 48, suplleagues . A stron reports , furtherThe effects of T2DM on LCAT activity remain controversial –52, and Several subjects with T2DM were taking medication known to affect lipid levels, which may influence confounding factors, but without further investigation, it cannot be certain if these drugs directly influenced HDL subfraction functionality.Good glycaemic control is a recommended clinical measure for those with T2DM, as elevated HbA1c leads to increased complications with levels lower than 7% recommended. However, in our study, 33% of those with T2DM had HbA1c levels greater than 7%, which could contribute to potential confounding; however, the small number did not allow us to perform a subanalysis based on HbA1c levels.The lack of ability to detect any significant difference in PON-1 activity between T2DM and controls in HDL subfractions could be due to several factors, such as limited sample size, the age of onset, and duration of diabetes.This study has focused only on female participants and as such may not be a reflection of similar observations of a similar male population.2 and HDL3. Previous studies have tended to focus on vascular and diabetic complications in persons with diabetes >45 years. We studied younger individuals with diabetes (without apparent CVD) to explore if biochemical phenotypes associated with increased risk of CVD and diabetic complications were present at this earlier age.In conclusion, our data suggests that SAA may be a useful biomarker of inflammation in subjects with T2DM and provides some further mechanistic support to explain why individuals with T2DM have increased risk of atherosclerosis and CVD, partly through the reduced antiatherogenic properties of both HDLSupplementary material Appendix 1: Results standardised to apoAI.


The objective of this study is to elucidate the effect of anagliptin on glucose/lipid metabolism and renoprotection in patients with type 2 diabetic nephropathy.Twenty-five patients with type 2 diabetic nephropathy received anagliptin 200 mg/day for 24 weeks, and 20 patients who were switched to anagliptin from other dipeptidyl peptidase-4 (DPP-4) inhibitors were analyzed regarding primary and secondary endpoints. The primary endpoint was change in hemoglobin A1c (HbA1c) during treatment with anagliptin. Additionally, we evaluated changes in lipid data , blood pressure (BP), urinary albumin to creatinine ratio (UACR), liver-type fatty acid-binding protein to creatinine ratio (ULFABP) and renal function (estimated glomerular filtration rate and serum cystatin C) as secondary endpoints.After switching to anagliptin from other DPP-4 inhibitors, the levels of HbA1c in the 20 participants showed no significant change, 7.5%±1.2% at 24 weeks compared with 7.3%±0.9% at baseline. The levels of the log10-transformed UACR were significantly reduced from 1.95±0.51 mg/g creatinine (Cr) at baseline to 1.76±0.53 mg/g Cr at 24 weeks after anagliptin treatment (p<0.01). The percentage change in the UACR (Δ%UACR) from baseline to 24 weeks was also significantly lower by −10.6% (p<0.001). Lipid data, systolic BP and renal function were not changed during anagliptin treatment. Additionally, ULFABP in eight participants, who had ≥5 µg/g Cr at baseline, was significantly decreased from baseline (8.5±2.8 µg/g Cr) to 24 weeks  after anagliptin treatment, and the percentage change in the ULFABP during anagliptin treatment was −58.1% (p<0.001).Anagliptin induced no significant change in HbA1c, lipid data, systolic BP and renal function. However, anagliptin reduced the UACR and ULFABP, although without a corresponding change in HbA1c, indicating direct action of anagliptin on renoprotection in patients with type 2 diabetic nephropathy. Previous clinical reports already exhibit that dipeptidyl peptidase-4 (DPP-4) inhibitors ameliorate diabetic nephropathy, such as albuminuria, in a glucose-lowering effect.Our current study demonstrates that administration of anagliptin, which is switched from other DPP-4 inhibitors, in patients with type 2 diabetes with nephropathy showed that the levels of urinary albumin to creatinine ratio were significantly reduced after 24 weeks. However, the levels of hemoglobin A1c in patients showed no significant change during the treatment.We also found that treatment with anagliptin significantly decreased urinary liver-type  fatty acid-binding protein excretion after 24 weeks.Anagliptin may exert beneficial effects for renoprotection in patients with type 2 diabetes with nephropathy in a glucose-lowering-independent manner.The prevalence of diabetes mellitus has been increasing worldwide in recent years. Long-term diabetes results in vascular changes and dysfunction, and its complications are the major causes of morbidity and mortality in patients. Among diabetic vascular complications, nephropathy is recognized as a leading cause of end-stage renal disease (ESRD) and an independent risk factor for cardiovascular diseases (CVD).Multifactorial management, including diet therapy and glycemic, blood pressure (BP) and lipid control, is recommended for diabetic nephropathy.A total of 48 participants with type 2 diabetes (30 men and 18 women) were selected for the present study from outpatients who visited the Department of Endocrinology and Metabolism at Kanazawa Medical University Hospital. The entry criteria included (1) age ≥20 years old, (2) type 2 diabetes with hemoglobin A1c (HbA1c) ≥6.0%, (3) urinary albumin to creatinine (Cr) ratio (UACR) ≥30 mg/g Cr in spot urine for screening of diabetic nephropathy, and (4) treatment with diet, exercise therapy and oral antidiabetic agents (glimepiride ≤2 mg/day or gliclazide ≤40 mg/day or glibenclamide ≤1.25 mg/day). The exclusion criteria were (1) type 1 diabetes, (2) treatment with insulin therapy, (3) severe diabetic metabolic complications such as ketoacidosis, (4) severe liver dysfunction, (5) hemodialysis, (6) severe chronic heart failure, (7) pregnant or nursing women and those who might be pregnant, and (8) any patient whom the investigator judged to be inappropriate for this study. Patients were given detailed explanations of the study protocol. Informed consent was obtained from each patient. The study protocol was approved by the Ethical Committee of Kanazawa Medical University. The trial was registered with the University Hospital Medical Information Network (UMIN No 000012802).The present study was an open-label, prospective study. At the start of the study, anagliptin 200 mg/day was added to other oral antidiabetic agents such as sulfonylurea (SU), metformin, an α-glucosidase inhibitor (α-GI), pioglitazone and a sodium-glucose cotransporter 2 (SGLT2) inhibitor or, when participants received other DPP-4 inhibitors, the DPP-4 inhibitor was switched to anagliptin 200 mg/day. In addition, in some cases, the anagliptin dose was increased to up to 400 mg/day after 12 weeks, if the physician judged glucose control in the patients to be insufficient. Participants were assessed for the following parameters before the start of the study, 12 and 24 weeks after the addition of anagliptin or when switching to anagliptin: No changes were made to the type and dose of glucose-lowering agents, renin–angiotensin system (RAS) inhibitors such as ACE inhibitors (ACEIs), angiotensin receptor II blockers (ARBs) or spironolactone during the study period. These agents had been prescribed for at least 3 months before the study.After performing a screening of UACR ≥30 mg/g Cr in spot urine, diabetic nephropathy was finally diagnosed by an UACR ≥30 mg/g Cr in the first urine in the early morning. The primary endpoint of the study was change in HbA1c during the treatment with anagliptin. Additionally, we evaluated the changes in lipid data (low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) levels), UACR and urinary L-FABP excretion as secondary endpoints.−1.094×age−0.287 in men and as 194×serum creatinine−1.094×age−0.287×0.739 in women.Blood samples were collected in the morning after an overnight fast. First urine in the early morning sample was collected at the home of participants, and urine was carried in a cooling box to the hospital. HbA1c was measured using an automated analyzer, HLC-723 G11 . Serum LDL-C and HDL-C levels were measured using enzymatic methods . Serum TG levels were measured using enzymatic assays . Urinary albumin was measured by immunonephelometry using a kit from NITTOBO MEDICAL . Because of their skewed distribution, the UACR data were log10-transformed before analysis. The results of the analysis were back-transformed to obtain geometric means of the UACR of the 24-week value to the baseline value; the values then were expressed as percentage change in the adjusted geometric mean of the UACR ratios of the 24-week value to the baseline value. Serum and urinary Cr were measured using enzymatic assays , and the estimated glomerular filtration rate (eGFR) was calculated as 194×serum creatinineStatistical analyses were performed with a StatView V.5 system  for Windows. All values are summarized as the mean and SD unless otherwise indicated. Differences in the percentage change in the UACR and urinary L-FABP before and after administration of anagliptin were assessed by a paired t-test. A Wilcoxon signed-rank test was performed as appropriate for comparison of the two groups. The unpaired t-test was performed as comparison of the two groups on HbA1c, lipid data, BP, body mass index (BMI) and renal function. The correlation of two variables was analyzed by single linear regression analysis. Statistical significance was defined as p<0.05.Patient disposition is shown in 2 and 26.6±3.3 kg/m2, and the duration of diabetes was 15.1±7.6 and 12.4±4.8 years, respectively, in the two groups. Baseline HbA1c levels were 7.3%±0.9% and 7.8%±0.9%, and fasting glucose levels were 153.7±38.5 mg/dL and 182.8±80.5 mg/dL. Lipid data were 91.5±25.4 mg/dL and 104.2±17.6 mg/dL for LDL-C, 50.2±13.9 mg/dL and 46.8±11.0 mg/dL for HDL-C, and 165.6±98.7 mg/dL and 123.2±46.6 mg/dL for TG, respectively. Alanine aminotransferase levels were 20.3±9.8 IU/L and 21.0±6.4 IU/L, and uric acid levels were 5.5±1.3 mg/dL and 4.6±1.3 mg/dL, respectively. The median eGFR and serum cystatin C at baseline were 74.0±18.4 mL/min/1.73 m2 and 66.3±23.9 mL/min/1.73 m2, and 0.95±0.23 mg/dL and 0.98±0.35 mg/dL, and the UACR values at baseline were 206.4±343.9 mg/g Cr and 172.3±139.4 mg/g Cr, respectively. We assessed urinary L-FABP excretion in the participants showing more than 5 µg/g Cr at baseline, and the median urinary L-FABP excretion was 8.5±2.8 and 6.4±0.95 µg/g Cr, respectively. At entry of study, in each of the two groups, 80% of the participants had microalbuminuria, and macroalbuminuria was noted in 20% of the individuals. All participants received oral antidiabetic agents including SU (55% and 40%), metformin (75% and 100%), an α-GI (20% and 0%), pioglitazone (10% and 0%) and an SGLT2 inhibitor (5% and 0%) at baseline. Twenty participants received a DPP-4 inhibitor, which includes sitagliptin , teneligliptin , alogliptin , vildagliptin  and linagliptin . Fifteen participants (75%) in the group of switch (n=20) received antihypertensive therapy at baseline. Of the 15 participants, 13 participants (87%) received RAS inhibitors such as ACEIs (n=4), ARBs (n=9) or spironolactone (n=2) at baseline, with two participants receiving dual RAS blockade, using ACEI and ARBs, or ARBs and spironolactone. Fourteen participants (70%) in the group of switch were treated with lipid-lowering therapy. Of the 14 participants, 12 participants received statins (85%).Baseline clinical and biochemical characteristics in two groups (shown as a group of switch (n=20) and a group of addition (n=5)), as well as concomitant background therapies, are shown in In a group with additional treatment with anagliptin , HbA1c was significantly decreased at 12 and 24 weeks  from baseline (7.8%±0.9%), respectively (see online s10.1136/bmjdrc-2017-000391.supp1After treatment with anagliptin switching from other DPP-4 inhibitors (n=20), HbA1c was not significantly changed, 7.4%±1.1% at 12 weeks and 7.5%±1.2% at 24 weeks, compared with baseline HbA1c (7.3%±0.9%) . The UACThe single linear regression analysis between Δ%UACR and age, duration of diabetes, ΔHbA1c, ΔLDL-C, Δsystolic BP or ΔBMI for 24 weeks, and HbA1c, UACR, BMI and eGFR at baseline, did not show significant correlation . In addiThe present study showed that the administration of anagliptin for 24 weeks significantly decreased the UACR from the baseline in a glucose-independent, lipid-independent and BP-independent manner. In addition, treatment with anagliptin significantly reduced the levels of urinary L-FABP excretion in the participants, who had more than 5 µg/g Cr at baseline, independent of the change in HbA1c. This is the first report showing a renoprotective effect of anagliptin on patients with type 2 diabetes with nephropathy.DPP-4 is an enzyme that cleaves and inactivates incretin hormones capable of stimulating insulin secretion from pancreatic β cells. DPP-4 inhibitors are now widely used for the treatment of type 2 diabetes. Previous reports have shown that currently available DPP-4 inhibitors, including anagliptin, exert a glucose-lowering effect in patients with diabetes,et al,et alPrevious clinical studies have shown a beneficial effect of DPP-4 inhibitors in diabetic nephropathy. Sitagliptin reduced albuminuria in several uncontrolled trials and a small randomized controlled trial, and the reduction of albuminuria was independent of the decrease in HbA1c,et alet al.There are few reports regarding comparative data among DPP-4 inhibitors on the renoprotective effect, including reduction of albuminuria. Fujita et al reported that urinary L-FABP of more than 5 µg/g Cr may be a predictive marker for renal and cardiovascular prognosis in patients with type 2 diabetes without advanced nephropathy.Urinary L-FABP is one of the markers for tubulointerstitial damage and an oxidative stress marker. Araki et alet alet alet alet alExperimental studies have suggested a renoprotective role of DPP-4 inhibitors in various models of chronic kidney disease (CKD), including diabetic nephropathy, which may be independent of lowering glucose levels. The renoprotective effect of DPP-4 inhibitors in diabetic nephropathy may be exerted through an increase in active GLP-1 or through the inhibition of DPP-4 itself. Previous reports show that GLP-1 receptor agonists may prevent disease progression in diabetic nephropathy through direct effects on the GLP-1 receptor in renal cells including glomerular endothelial cells and monocytes/macrophages.There were several limitations in our study design. It was a non-controlled observational study that occurred over a short time period, and the number of participants was small. DPP-4 cleaves a lot of substrates (peptides) including GLP-1, GIP, SDF-1α, brain natriuretic peptide (BNP) and so on. Although we could not show the changes in the levels of these peptides following anagliptin treatment, these changes may be involved in renal protection observed in this study. In addition, we could not evaluate oxidative stress or inflammation to assess the mechanism of the beneficial effect on the diabetic kidney. Therefore, the mechanism by which anagliptin reduced the UACR and urinary L-FABP excretion in a glucose-lowering independent manner is unclear. Further study is necessary to elucidate these points.In conclusion, in the present study of just 24 weeks’ duration, anagliptin caused a decrease in the UACR and urinary L-FABP, which are prognostic markers for CKD and CVD, and the decrease was independent of any change in HbA1c. Therefore, anagliptin may have potential for halting the progression of diabetic nephropathy and the development of CVD through a renoprotective effect.10.1136/bmjdrc-2017-000391.supp2


Standardized treatment in pediatric patients with Hodgkin’s lymphoma (HL) follows risk stratification by tumor stage, erythrocyte sedimentation rate and tumor bulk. We aimed to identify quantitative parameters from pretherapeutic FDG-PET to assist prediction of response to induction chemotherapy.n = 42) or -C2 treatment protocol (n = 8). Total metabolic tumor volume (MTV) in pretherapeutic FDG-PET was defined using a semi-automated, background-adapted threshold. Metabolic  and heterogeneity parameters  were derived. Early response assessment (ERA) was performed after 2 cycles of induction chemotherapy according to treatment protocol and verified by reference rating. Prediction of inadequate response (IR) in ERA was based on ROC analysis separated by stage I/II (1 and 26 patients) and stage III/IV disease (7 and 16 patients) or treatment group/level (TG/TL) 1 to 3.Retrospective analysis in 50 children with HL  consecutively treated according to EuroNet-PHL-C1 . Among all PET parameters, MTV best predicted IR; ASP was the best heterogeneity parameter. AUC of MTV was 0.84  in stage I/II and 0.86 (0.7–1.0) in stage III/IV. In patients of TG/TL 1, AUC of MTV was 0.92 (0.74–1.0); in TG/TL 2 0.71 (0.44–0.99), and in TG/TL 3 0.85 (0.69–1.0). Patients with high vs. low MTV had IR in 86 vs. 0% in TG/TL 1, 80 vs. 29% in TG/TL 2, and 90 vs. 27% in TG/TL 3 .In this explorative study, high total MTV best predicted inadequate response to induction therapy in pediatric HL of all pretherapeutic FDG-PET parameters – in both low and high stages as well as the 3 different TG/TL.EA2/151/16 (retrospectively registered).Ethics committee number: The online version of this article (10.1186/s12885-018-4432-4) contains supplementary material, which is available to authorized users. Hodgkin’s lymphoma (HL) accounts for about 7% of pediatric malignancies and about 1% of childhood cancer-related deaths in the United States , 3. F18-Despite this important role of FDG-PET/CT, no initial PET based parameter has been identified so far to predict response at ERA in pediatric HL patients – including the standardized uptake value (SUV) as the most common quantitative FDG-PET parameter . NeverthAccordingly, the aim of this study was to identify metabolic or heterogeneity parameters from pretherapeutic FDG-PET/CT to predict inadequate response (IR) in pediatric patients with HL. PFS or OS were not selected as endpoints in this study because both are not only determined by initial characteristics/risk profile (which was the main focus of this study) but also by the extent of treatment (and namely the performance of RT).n = 18; male, n = 32; median age, 14.8 years; range, 4.0 to 18.0 years) treated according to EuroNet-PHL-C1 (n = 42) or -C2 treatment protocol (n = 8) between 2007 and 2017 [n = 2). These protocols are consecutive multinational standardized treatment protocols for pediatric patients with HL (lymphocyte-predominant subtype excluded). All patients undergo two cycles of induction chemotherapy (OEPA) before ERA with FDG-PET/CT is performed. This is followed by further chemotherapy  and, most importantly, determines the necessity of additional radiation therapy of the initially involved regions . In TG/TL 2 or 3, additional LRA is conducted after the second treatment segment in patients with IR at ERA to decide on further intensification of the subsequent radiation therapy [This retrospective study included 50 consecutively examined children with classical HL  or non-enhanced low-dose CT . PET raw data was reconstructed iteratively with TOF analysis . Projection data was reconstructed with 4 mm slice thickness .PET/CT imaging was performed using the tracer FDG and a dedicated PET/CT device  with Philips Astonish TF technology. FDG was administered intravenously using a weight-adapted activity  based on recommendations provided by the European Association of Nuclear Medicine (EANM) . A test Quantification of FDG-PET data was performed with dedicated software . All analysis was performed blinded to the results of ERA; however, the identification of HL lesions comprised all clinical and imaging data available in the pre-treatment setting including the final tumor stage as defined by interdisciplinary consensus . The combined MTV of the entire FDG-avid HL lesion load of the patient  was delineated using a semi-automatic, background-adapted algorithm , 16  and total lesion glycolysis  were calculated for the whole MTV. Heterogeneity parameters were derived including the asphericity (ASP) , 18, entt41) and a fixed absolute threshold of SUV = 2.5 (MTV2.5).For comparative analysis  calculated as follows:Statistical analysis was performed using SPSS 22 . Descriptive parameters were expressed as median, IQR and range or 95%-confidence interval (95%-CI), unless otherwise specified. Optimal cut-off values for quantitative FDG-PET parameters to distinguish IR from AR were defined by receiver operating characteristic (ROC) curves with respective areas under the curve (AUC). The optimal cut-off value was defined as the point on the ROC curve with the minimal distance U test. The relationship between a high MTV or high ASP, respectively, the patient’s tumor stage (I/II vs. III/IV) or TG/TL, and the result of ERA (IR vs. AR) was further assessed with log-linear analysis. Statistical significance was assumed at a p ≤ 0.05.Patients were divided into groups of stages I/II versus III/IV and, alternatively, based on the assigned treatment group or treatment level (TG/TL) 1 versus 2 versus 3 according to the respective treatment protocol. Differences in MTV and ASP between these groups were investigated with Mann-Whitney One of 50 patients had stage I, 26 patients had stage II, 7 patients had stage III, and 16 patients had stage IV disease (Table p = 0.01). Comparison with stage I was not performed as it only included one patient. ASP in stage I was 22.2%, in stage II it was 137.9% (87.4 to 179.1%), in stage III 195.5% (121.7 to 236.4%), and in stage IV it was 224.9% (190.1 to 306.3%); no significant differences were detected. Among the remaining metabolic and heterogeneity parameters, only TLG was significantly different between stage II and III (p = 0.009).Median MTV was 7.0 ml in stage I (one patient only), 154.0 ml  in stage II, 386.2 ml (137.9 to 537.8 ml) in stage III, and 350.6 ml (207.4 to 555.9 ml) in stage IV patients . ASP in TG/TL 1 was 136.8% (77.4 to 206.3%) compared to 136.2% (91.2 to 165.3%) in TG/TL 2 (p = 0.95) and TG/TL 3 with 231.0% (189.7 to 290.3%). ASP in TG/TL 3 patients was significantly higher compared to TG/TL 1 or TG/TL 2 patients (each p < 0.01). Among the remaining parameters, a significant difference between TG/TL 1 vs. 2 was measured for SUVmax, SUVmean, SUVpeak, TLG, contrast, and local homogeneity (each p < 0.05). Significant differences between TG/TL 2 vs. 3 were observed for TLG, entropy, contrast, local homogeneity, energy, and CSH (each p < 0.05).The median MTV in TG/TL 1 was 87.4 ml (29.3 to 138.9 ml) compared to TG/TL 2 with 177.4 ml (103.4 to 221.0 ml) and TG/TL 3 with 375.8 ml (213.9 to 555.3 ml). MTV was significantly higher in TG/TL 3 vs. TG/TL 2 vs. TG/TL 1 patients (each p < 0.001) but not between tumor stage (I/II vs. III/IV) and MTV or response to therapy (both p > 0.05).MTV showed the highest AUC of all PET parameters; AUC for MTV in patients with stage I/II was 0.84 , AUC in patients with stage III/IV was 0.86 (0.7 to 1.0). Using the respective optimal cut-off value , patients with high vs. low MTV showed IR in 78.9 vs. 12.5% in stage I/II as well as 90.0 vs. 23.1% in stage III/IV. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) to predict IR were 94, 64, 88, and 79% in stage I/II compared to 75, 91, 77, and 90% in stage III/IV (Tables p < 0.01) but not between tumor stage and ASP or response to therapy (both p > 0.05).Among heterogeneity parameters only, ASP provided the highest AUC in patients with stage I/II of 0.65 0.43 to 0.88) and in stage III/IV of 0.74  but not the relation between TG/TL and MTV or the response to therapy (both p > 0.05).The average AUC across all TG/TL was highest for MTV; AUC was 0.92 0.74 to 1.0) for TG/TL 1, 0.71 (0.44 to 0.99) for TG/TL 2, and 0.85 (0.69 to 1.0) for TG/TL 3. Patients with high vs. low MTV had IR in 85.7 vs. 0% in TG/TL 1 , 80.0 vs. 28.6% in TG/TL 2 , and 90.0 vs. 27.3% in TG/TL 3 . Sensitivity, specificity, NPV, and PPV of MTV to predict IR in TG/TL 1 were 100, 83, 100, and 86% compared to 80, 71, 71, and 80% in TG/TL 2 and 75, 89, 73, and 90% in TG/TL 3 (Tables  to 1.0 fp < 0.01) but not between TG/TL and ASP or response to therapy (both p > 0.05).Among the heterogeneity parameters, ASP provided the highest AUC in patients within TG/TL 1 of 0.78 0.49 to 1.0) compared to 0.5 (0.2 to 0.8) in TG/TL 2 and to 0.7 (0.48 to 0.93) in TG/TL 3 (Tables 9 to 1.0 Patients with either high ASP or MTV vs. patients with both low MTV and ASP had IR in 85.7 vs. 0% (TG/TL 1), 71.2 vs. 0% (TG/TL 2), and 76.9 vs. 25% (TG/TL 3).The aim of the present study was to investigate the predictive value of several quantitative parameters derived from pretherapeutic FDG-PET in pediatric HL regarding the occurrence of IR at ERA after induction therapy.The total MTV of all nodal and extranodal HL lesions of the patients predicted IR with high but varying accuracy within all three TG/TL . Furthermore, the optimal cut-off to distinguish patients with IR or AR was distinctly different between either stages I/II versus III/IV  or between the three TG/TL (> 80 ml vs. > 160 ml vs. > 410 ml). This reflects an increasing average MTV especially between stage I/II and stage III/IV disease as one expects given the supposed increase in involved regions – while, interestingly, the average MTV in stage III and stage IV was similar  helps to predict PFS and OS . Both CoFurther metabolic parameters performed slightly (TLG) or considerably worse (different SUVs) compared to the MTV. This is in accordance with a study by Bouallègue et al. who did not find an association between SUVmax, SUVmean or SUVpeak with the early metabolic response in 57 patients (mostly adults) with HL or non-Hodgkin lymphoma . The obsThis retrospective explorative study is limited by the investigation of only 50 patients  which necessitates a larger study with an independent patient cohort to validate the presented results and to further elucidate the predictive value of single parameters within the relevant subgroups. Furthermore, as only one patient had stage I, no specific conclusions can be drawn for this stage in particular.The response to induction therapy at ERA was used as endpoint in the current study but is only a surrogate for patient outcome – as opposed to the PFS or OS. Indeed, the predictive value of a high pretherapeutic MTV independent from the German Hodgkin Study Group (GHSG) risk group has been recently demonstrated retrospectively in 267 adult patients with early stage HL . NeverthIn this explorative study, a high total MTV best predicted IR to induction therapy in pediatric HL of all pretherapeutic FDG-PET parameters. This was true in both low and high stages as well as the three different TG/TL. Among the investigated heterogeneity parameters, only ASP may be sufficiently predictive of IR to serve as a supplemental parameter to the MTV and further improve the predictive accuracy. The influence of inter-method MTV variability of different delineation approaches on their predictive accuracy requires further investigation.Additional file 1:t41) and a fixed absolute threshold (MTV2.5) in comparison with the background-adapted semi-automated delineation presented in the main manuscript (MTVBG) [Supplemental materials. Results of additional analysis on delineation with a fixed relative threshold (MTV (MTVBG) . (DOCX 3


Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4.T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain.We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups  and three control groups . Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of Mice vaccinated with different immunization regimens  elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls.A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization. Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with an extremely broad host range inclusive of almost all warm-blooded animals, including human [T. gondii infection is usually asymptomatic, but not in immunosuppressed patients . Infection can spread widely, involving the brain, eyes, lymph nodes, can damage multiple organs, and even cause death. T. gondii infection in pregnant women can be transmitted to the fetus via the placenta, leading to miscarriage, premature birth, stillbirth, fetal malformation, and eye toxoplasmosis [ng human . Toxoplang human . T. gondlasmosis .T. gondii life cycle: tachyzoites, cysts, and oocysts [T. gondii surface antigen 1 (SAG1) induces effective and durable humoral and cellular immune responses in immunized mice and is regarded as a standard compared to other antigens [There are three infectious stages in the  oocysts . Tachyzo oocysts . Current oocysts . Therefo oocysts , 7. For antigens .T. gondii expresses several proteases, including serine, cysteine, and aspartic proteases, which play an important role in infection [T. gondii expresses 5 nonmitochondrial rhomboid intramembrane proteases, TgROM1 to TgROM5. ROM proteins affect the cleavage of microneme protein (MIC), which plays an essential role in T. gondii invasion [T. gondii and uniformly distributed on the parasite surface [T. gondii effectively invades host cells, TgROM4 plays an important role in regulating microneme protein 2 (MIC2), MIC3, MIC6, and apical membrane antigen 1 (AMA1) [T. gondii in mice [nfection –11. Rhominvasion , 13. TgRinvasion , 13. TgR surface , 13. Whe1 (AMA1) –16. Li e in mice . We ther in mice , 19.T. gondii [Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing importance in vaccine research , 21. In . gondii , 26.T. gondii infection. Furthermore, we assessed the protective efficacy of different vaccination strategies .In the present study, we analyzed the ROM4 protein to identify potential antigenic epitopes using a bioinformatics approach. A eukaryotic expression plasmid pEGFP-C1-ROM4 (pROM4) was constructed and used in a DNA prime-peptide boost vaccination regimen to immunize mice and assess protection against http://tools.immuneepitope.org/mhcii) online service was used to analyze the half-maximal inhibitory concentration (IC50) values of peptides that bind to the major histocompatibility complex (MHC) class II molecules of ROM4.The TgROM4  nucleotide (GenBank ID: AY704175.1) and amino acid sequences (GenBank ID: AAU11320.1) were obtained from GenBank. Protein epitopes determine antigen specificity , 28. TheFemale BALB/c mice (7–8 weeks old) were purchased from the Shandong University Laboratory Animal Center . All mice were maintained under specific-pathogen-free conditions. All animal experiments were approved by the Ethics Committee on Animal Experiments of the Medical School of Shandong University.T. gondii RH strain was used to challenge BALB/c mice. Tachyzoites were extracted from human foreskin fibroblast cells 1 h before injection to ensure freshness prior to challenge. About 8 × 109 tachyzoites were used to create soluble tachyzoite antigens (STAg) after isolation by centrifugation, and resuspended in sterile PBS as previously described [6tachyzoites were used to extract total RNA with TRIzol Reagent . After tachyzoites were lysed with 1 ml TRIzol, 0.2 ml chloroform was added, and the homogenate was separated into three layers after centrifugation. RNA was precipitated from the upper aqueous layer with isopropanol and then was washed to remove impurities. RNA was resuspended in RNase-free water and incubated in a water bath at 55–60 °C for 10–15 min. The RNA was reverse-transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s protocol The escribed , while aT. gondii tachyzoite cDNA . XhoI and EcoRI restriction sites are underlined, respectively. The ROM4 ORF was amplified by PCR from T. gondii tachyzoite cDNA . XhoI and KpnI restriction sites are underlined, respectively.The whole SAG1 open reading frame (ORF) was amplified by polymerase chain reaction (PCR) from XhoI, EcoRI and XhoI, KpnI restriction sites. The new recombinant plasmids were then used to transfect HEK 293-T cells.The SAG1 and ROM4 DNA fragments were respectively inserted into a pEASY-T1 vector . After sequencing, SAG1 and ROM4 were respectively subcloned into the eukaryotic expression plasmid pEGFP-C1  to form pEGFP-C1-SAG1(pSAG1) and pEGFP-C1-ROM4 (pROM4), respectively using the Escherichia coli DH5α. After being verified by PCR, double restriction enzyme digestion and double stranded sequencing, recombinant plasmids were extracted using an endotoxin-free mega kit following the manufacturer’s instructions , and stored at −20 °C until use. pSAG1 and pROM4 concentrations were determined by A260/A280 measurement.Recombinant pSAG1 and pROM4 were transformed into ROM4 peptide 405–424 (YALLGALIPYCVEYWKSIPR) was synthesized by SynPeptide Co Ltd , and purity was confirmed by analytic HPLC.5/well) were transferred into Costar 6-well culture plates . When HEK 293-T density reached 80–90%, pEGFP-C1, pSAG1 and pROM4 were transfected with Lipofectamine 2000 reagent  according to the manufacturer’s protocol. Plasmids (2.5 μg/well) were mixed with Lipofectamine 2000 reagent (7 μL/well) in DMEM, incubated at room temperature for 20 min, and then added drop by drop on to HEK 293-T cells. After incubation for 6 h, the medium was exchanged with medium containing 10% FBS. After 48 h, the cells from different groups  were observed using fluorescence microscopy under a blue laser.HEK 293-T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with streptomycin (100 mg/ml), penicillin (100 IU/ml) and 10% fetal bovine serum (FBS), at 37 °C in a humidified atmosphere with 5% CO2. Before transfection, HEK293 cells (about 1–2 × 10All BALB/c mice were divided randomly into six groups (27 per group). Mice were immunized twice at 2-week intervals with PBS (100 μl), pEGFP-C1 (100 μg), pSAG1 (100 μg), pROM4 (100 μg), peptide (100 μg), or pROM4 (100 μg)/peptide (100 μg) by intramuscular injection. All groups were immunized four times. The last group was injected with pROM4 the first two times and with peptide the last two times. T. gondii RH strain (1 × 104 tachyzoites). Changes in health status were observed and survival times recorded. The remaining mice were infected intragastrically with 20 cysts of T. gondii PRU strain. At 1 month after challenge, brains were removed and homogenized in 1 ml PBS. The number of cysts in each brain was determined by counting three samples of 10 μl from the homogenate via an optical microscope, and the average value was used to evaluate the effect of the vaccine.Two weeks after the final immunization, seven mice per group were euthanized and splenocytes harvested under aseptic conditions for cytokine detection. Ten mice from each group were challenged intraperitoneally with T. gondii IgG, IgG1, and IgG2a antibodies were detected using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates (Costar) were coated with STAg (10 μg/well) and incubated at 4 °C overnight. Plates were washed three times with special ELISA solution and blocked with PBS containing 1% BSA for 2 h at room temperature, followed by three washes. Plates were then incubated with PBS-diluted mouse sera for 1 h at 37 °C. After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (diluted 1:4000 in PBS-1% BSA), IgG1 (1:2000), and IgG2a (1:2000) for 1 h at 37 °C, washed with ELISA solution, with orthophenylene diamine (Sigma) and 0.15% H2O2 added. Plates were then incubated in the dark for 30 min at 37 °C, and the reaction stopped by adding 2 M H2SO4. The optical density was measured at 490 nm using an ELX800 ELISA reader . All samples were run four times.Serum samples were collected from all mice prior to each immunization and 2 weeks after the final injection. Anti-6 cells/ml. Then, 1 × 105 spleen cells was added to each well in a 96-well plate with 10 μl non-specific irritant Con A (5 μg/ml) and cultured at 37 °C in 5% CO2. Cell-free supernatants were harvested and assayed for interleukin-2 (IL-2) and IL-4 at 24 h, IL-10 at 72 h, and IL-12 (p70) and interferon (IFN)-γ at 96 h. The concentrations of cytokines were measured by ELISA according to the manufacturer’s instructions . The detection limits of the assays for IFN-γ, IL-2, IL-4, IL-10 and IL-12 (p70) were 2.1 pg/mL, 3.5 pg/mL, 2.3 pg/mL, 1.97 pg/mL and 2.24 pg/mL, respectively. All samples were run four times.Spleens were isolated from seven mice per group 2 weeks after the last immunization and used to form a cell suspension adjusted to 1 × 10P < 0.05) was observed among treatments, a Tukey’s studentized range test was used for post-test comparisons.SPSS 17.0  was used for statistical analysis. The mean of total IgG, IgG1, IgG2a, cytokine levels and cyst numbers among the different groups were analyzed and determined by one-way analysis of variance (ANOVA). Mouse survival time was compared using the Kaplan-Meier method. When a significant difference  (P < 0.05) . No statistical differences were found between PBS and pEGFP-C1, pROM4 and peptide (P > 0.05).The levels of P < 0.05). IgG2a levels in mice immunized with pSAG1 were increased by 10.3% compared with mice injected with pROM4, but with no statistical difference (P > 0.05). There was no statistical difference in IgG2a levels between the pROM4 and peptide groups (P > 0.05).The levels of IgG subclass (IgG1 and IgG2a) at 2 weeks after the last injection were assayed to determine whether a Th1 and/or Th2 response was elicited Fig. . An appaP < 0.05), respectively, compared to PBS injected groups (Table P < 0.05). IFN-γ levels in mice immunized with pSAG1 were increased by about 15% compared with pROM4, but there was no statistical difference. No difference in IFN-γ levels was found between the PBS and pEGFP-C1 groups. Significantly higher levels of IL-2 and IL-12(p70) were generated from mice vaccinated with pSAG1, peptide, pROM4 or pROM4/peptide, compared to mice immunized with PBS or an empty plasmid (P < 0.05). IL-2 levels in pROM4/peptide immunized mice increased by 38%, 62%, and 63%, compared to pSAG1, pROM4 or peptide groups (P < 0.05), respectively, and the highest IL-12 level also was detected in the pROM4/peptide group (P < 0.05) (Table P > 0.05).Culture supernatants of immunized splenocytes were obtained 2 weeks after the last injection and IFN-γ, IL-2, IL-12(p70), IL-4 and IL-10 activity determined. IFN-γ levels in mice immunized with pSAG1, pROM4, peptide or pROM4/peptide were increased about 12-fold, 10-fold, 9-fold, and 15-fold . Moreover, mice immunized with pROM4/peptide exhibited the latest onset of symptoms  and the longest survival time (18 days) among all groups (P < 0.05).Survival days after challenge by 1 × 10T. gondii PUR strain to evaluate the protective effect of the DNA vaccine. Brain cysts were reduced in mice vaccinated with pSAG1, pROM4, peptide, or pROM4/peptide compared to mice injected with PBS or pEGFP-C1 (P < 0.05) (Table P > 0.05), or between the PBS and pEGFP-C1 groups (P > 0.05).Two weeks after the last injection, all the mice were challenged intragastrically with 20 cysts from the 5) Table . The cysT. gondii. Following bioinformatics analysis, the ROM4 gene was cloned into the eukaryotic expression plasmid pEGFP-C1, and recombinant plasmids transfected into HEK 293-T cells. The results showed that the recombinant plasmid pROM4 was efficiently transcribed and expressed in eukaryotic cells.In the present study, ROM4 B-cell and T-cell epitopes were analyzed using DNAStar software and online services. This approach yielded several potential ROM4 T-cell epitopes, suggesting that it may serve as a vaccine against T. gondii infection [T. gondii response [T. gondii immunity [T. gondii antibodies, and inhibiting parasite and host cell adhesion, especially during vaccine-induced protective immunity [Cellular immunity mediated by T cells plays an important role in nfection . To deveresponse , 31. Cytimmunity , 32. CD4immunity , 33. Theimmunity , 35. In immunity , 37.T. gondii-specific IgG antibodies. IgG, IgG2a, IL-2, IL-12 and IFN-γ levels in immunized mice were higher than in negative controls, and IgG1, IL-4, and IL-10 levels were similar between all groups. These results suggest that pSAG1, pROM4 and peptide mainly induced a Th1 immune response. In addition, IgG and IgG2a levels in pSAG1 immunized mice were higher than in pROM4 immunized mice. This is not in accord with the results of the bioinformatics analysis, suggesting that bioinformatics analysis is predictive and that the predicted protein antigenic epitopes need to be verified by an experimental approach.In the current study, IgG antibody levels in immunized mice were significantly higher than in negative controls, illustrating that the vaccine constructed here induces a sufficient amount of T. gondii tachyzoites and cysts. The results showed that the onset of symptoms were later in immunized mice, which had prolonged survival times and reduced brain cysts compared with negative controls. Zhang et al. [To assess the protective efficacy of the vaccine, BALB/c mice were infected with g et al.  construcg et al. .T. gondii challenge [T. gondii DNA vaccines, which produced a stronger immune response and better protection compared to priming with polypeptide and boosting with DNA.DNA vaccines often induce insufficient protective immunity against hallenge . Prime-bhallenge , 22. Thehallenge . Synthethallenge , 41. Menhallenge  used theThe prime-boost vaccination usually include two strategies: homologous boosting and heterologous boosting. Researches display that homologous boosting is relatively inefficient at boosting cellular immunity than that of heterologous boosting,. So-called heterologous boosting means that different antigen-delivery systems are used . In the T. gondii ROM4 is a potential DNA vaccine candidate against toxoplasmosis. A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among the immunization regimens, indicating it may be a promising approach to generate an efficient protective immune response and prevent T. gondii infection.In conclusion,


AbstractRhodactina is a small sequestrate genus in Boletaceae with two described species, R.himalayensis and R.incarnata. Phylogenetic analyses of a three-gene dataset including atp6, tef1 and rpb2 of Rhodactina species along with selected Boletaceae species showed that all Rhodactina species formed a monophyletic clade, sister to the genera Spongiforma and Borofutus in subfamily Leccinoideae with high support. All of the taxa in the clade have a similar chemical reaction in which basidiospores turn purplish, purplish red to violet or violet grey when in contact with potassium hydroxide. The molecular analyses also showed that all Rhodactina specimens collected from Ubon Ratchathani province, northeastern Thailand, belong to a new species. Morphologically, the new species is different from others by having a markedly prominent hilar appendage and a terminal hilum on its basidiospores. Thus, the new species, Rhodactinarostratispora, is introduced with detailed macroscopic and microscopic descriptions and illustrations. Rhodactina Pegler & T.W.K. Young was first described in 1989 with R.himalayensis Pegler & T.W.K. Young, from northwestern India, as the type species. Typical PageBreakcharacters of the genus are a whitish to pinkish puffball like basidiomata lacking both stipe and columella, violet brown to purple brown or pale pink to red hymenophore when mature, combined with purplish to purplish red, dextrinoid basidiospores with longitudinal ridges, lack of both clamp connections and cystidia. The genus was originally classified based on morphological characters in the family Gautieriaceae Zeller as the spore ornamentation was originally viewed as similar to the genera Gautieria Vittad and Austrogautieria E.L. Stewart & Trappe , both of which have been reported to occur in northern Thailand .The new PageBreak(colour reactions) of peridium, hymenophore and microscopic structures were determined using 5 % (w/v) aqueous potassium hydroxide, 28–30 % ammonium hydroxide or Melzer’s reagent. Microscopic structures were observed from dried specimens, rehydrated in 5% KOH or 1 % ammoniacal Congo red. For each collection, a minimum of 50 basidiospores and 20 basidia were randomly selected and measured at 1000× with a calibrated ocular micrometer using an Olympus CX31 microscope. Spore dimensions include ornamentation. The notation ‘(n/m/p)’ represents the number of basidiospores n measured from m basidiomata of p collections. Dimensions of microscopic structures are presented in the following format: (a–)b–c–d (–e), in which c represents the average, b the th5 percentile, d the th95 percentile and extreme values a and e are shown in parentheses. Q, the length/width ratio, is presented in the same format. Sections of the peridium surface were made radially and perpendicularly to the surface, halfway between the centre and the side of basidiomata. All microscopic features were drawn free hand using an Olympus Camera Lucida model U−DA fitted to the microscope cited above. For scanning electron microscopy (SEM), small fragments of dried hymenophore were mounted directly on to an SEM stub with double-sided tape. The samples were coated with gold for 60 seconds using SPI-Module Sputter Coater, examined and photographed at 15–20 kV with a FIB Quanta 200 3D scanning electron microscope .The macroscopic description was based on detailed field notes and photos of basidiomata. Colour codes followed atp6, tef1 and rpb2 were amplified by polymerase chain reaction (PCR) technique. For the amplification of atp6, ATP6-1M40F and ATP6-2Mprimers were used  and incubated at 37 °C for 1 h, followed by inactivation at 80 °C for 15 min. Sequencing was performed by Macrogen Inc. (Korea and The Netherlands) with PCR primers, except for atp6, for which universal primers M13F-pUC(-40) and M13F(-20) were used; for tef1, additional sequencing was performed with the two internal primers, EF1-1577F and EF1-1567R  phylogenetic tree inference was performed using RAxML  and introns were removed prior to alignment based on the amino acid sequence of previously pubng MAFFT  on the sng RAxML  on the Cng RAxML . The phyBoletaceae and was 2429 characters long (TreeBase number 21933). The authors could not obtain tef1 and rpb2 sequences from R.incarnata (CMU25116) nor rpb2 sequence from R.himalayensis (CMU25117). The specimens were in relatively poor condition and genomic DNA was highly degraded. The 3-gene phylogram indicated that all selected collections of the new taxon R.rostratispora formed a monophyletic group with high bootstrap support sister to R.incarnata within the Rhodactina clade .THAILAND, Ubon Ratchathani Province, Trakan Phuet Phon District, Don Khok Tam Lae community forest, From Latin “rostrati–” meaning having beaked prow or a solid projection and “spora” meaning spores, referring to the basidiospores having a markedly prominent and large hilar appendage.Basidiomata small to medium-sized 0.8–2.5(4.5) cm diam., subglobose to ovoid with a rudimentary elongated basal attachment, with greyish white to pale brown rhizoids at the base and going up along the surface of basidiomata to about half of the height. Peridium surface (outer peridium) fibrillose to arachnoid, off-white to pinkish white (7A2–3 to 9A2), dull, moist, cracked in places. Peridium very thin, 0.1–0.2(0.4) mm thick. Hymenophore cartilaginous, completely enclosed, whitish orange to reddish orange (7A3–4 to 8A5–6) at first becoming orangey red to red (9D–E8 to 10D–E8) with age, then dark red when very old, irregular; Stipe-columella absent. Taste fungoid. Odour absent when young, very strongly fruity alcoholic when old.Macrochemical reactions: hymenophore turned dark purplish (15F8) to greyish violet (19D3) with 5% KOH, slightly greyish violet (19D3) with NH4OH.Basidiospores [404/8/8] (11.5–)12–13.6–15(–16) × (10–)10.5–11.7–13(–14), Q = (1–)1.04–1.16–1.3(–1.4), from the holotype, (12–)12–13.5–15.2(–16) × (10–)10–11.6–13.2(–14) µm, Q = (1–)1–1.02–1.33(–1.4), N = 50, ellipsoid to broadly ellipsoid with longitudinal ridges, stellate in polar-view, thick-walled (1–1.5 µm thick), yellowish to orangey hyaline to reddish yellow at first, reddish to brownish yellow with age in water, slightly purplish and slightly more reddish to brownish in 5% KOH, slightly purplish hyaline in NH4OH, slightly dextrinoid to dextrinoid in Melzer’s reagent; ornamentation (7)8–9 solid ridges regularly and longitudinally arranged under light microscope, sometimes anastomosing under SEM, 2–3 µm tall and 2–2.5 µm wide at the base; hilar appendage prominent, 2.5–5 µm long with a terminal hilum. Basidia 4–spored, (26–)26.1–32.3–36(–36) × (8–)8–9.5–11(–11) µm , clavate to cylindrical, hyaline in water, 5% KOH and NH4OH, yellowish hyaline in Melzer’s reagent; sterigmata broken by spore release, stout, 3–4 µm long. Cystidia none observed. Hymenophoral trama 60–130 µm thick, irregular, with a narrow, central layer of subparallel to loosely interwoven, 3–7(8) µm wide, thin-walled hyphae, slightly gelatinised, hyaline in water, 5% KOH and NH4OH. Peridiopellis a tomentum 45–120 µm thick, poorly differentiated, composed of thin-walled, 3–10 µm wide hyphae, anastomosing at places and covered with yellowish brown incrustations on the surface at places, otherwise hyaline in water, 5% KOH and NH4OH, inamyloid. Clamp connections not seen in any of the tissues.PageBreakDipterocarpustuberculatus, D.intricatus, D.obtusifolius, Shoreaobtusa, S.siamensis and Eucalyptus sp.Subepigeal, solitary to gregarious (4–7 basidiomata), or fasciculate by 2–5 basidiomata, on sandy soil in dipterocarp forest dominated by Currently found only from Ubon Ratchathani province, northeastern Thailand.Rhodactinarostratispora.—THAILAND, Ubon Ratchathani Province, Trakan Phuet Phon District, Don Khok Tam Lae community forest, 15°35'40.2"N–105°06'37.8"E, elev. 150 m., 28 July 2015, S. Vadthanarat 169, ; ibid. 15°35'41.5"N–105°06'35.4"E, elev. 150 m., 28 July 2015, O. Raspé 1055, ; ibid. 15°35'48.3"N –105°06'35.9"E, elev. 150 m., 6 August 2015, S. Vadthanarat 206, ; ibid. 15°35'52.4"N–105°06'41.2"E, elev. 150 m., 6 August 2015, S. Vadthanarat 208, ; ibid. 15°35'56.1"N–105°06'38.9"E, elev. 150 m., 6 August 2015, S. Vadthanarat 212, ; ibid. 15°36'2.6"N–105°06'36.7"E, elev. 150 m., 14 May 2017, S. Vadthanarat 376, ; Ban Huay Fai community forest, 15°32'42.7"N–105°10'16.3"E, elev. 160 m., 15 July 2017, S. Vadthanarat 406, .R.himalayensis. – THAILAND, Chiang Mai Province, Doi Suthep-Pui National Park, forest behind Channel 9 TV station, 4 August 2000, Saisamorn Lumyong, Pipob Lumyong, Rarunee Sanmee and B. Dell 2254 (CMU25117).PageBreakR.incarnata. – THAILAND, Chiang Mai Province, Sanpatong District, Mae Wang, Conservation forest, Sanpatong-Ban Guard Rd., 24 July 2002, Saisamorn Lumyong, Pipob Lumyong, Rarunee Sanmee and Zhu L. Yang 45209 .Rhodactinarostratispora is characterised by its basidiospores having a markedly prominent hilar appendage , with a terminal hilum; ornamentation consisting of (7)8–9 longitudinal ridges, and (11.5–)12–13.6–15(–16) × (10–)10.5–11.7–13(–14) µm. R.himalayensis has larger basidiospores (15–20 × 12.5–18 µm) without prominent hilar appendage, with fewer [(5)6–7(8)], broader ridges, while R.incarnata has a similar spore size (10–13 × 10–12 µm) and the same number of spore ridges [(7)8–9(10)] as the new species, but it does not have the prominent hilar appendage.R.rostratispora specimen (S. Vadthanarat 208), abnormal spores were observed. Those spores were elongated, 21–24 × 4–8 µm, thick-walled, narrowly fusiform to bacilliform, with or without longitudinal ridges, more or less constricted in the middle. They were usually found attached to apparently normal basidia with four sterigmata.In one PageBreakPageBreakR.rostratispora is characterised by its ridged basidiospores having a markedly prominent hilar appendage with a terminal hilum, which is not found in other Rhodactina species  and also their hilar appendage lacks a terminal hilum. Macroscopically, those species differ from R.rostratispora in that both Rossbeevera and Turmalinea have basidiomata often turning blue to greenish blue when bruised, which has never been reported in any Rhodactina species  Wolfe, Austroboletusmalaccensis (Pat. & C.F. Baker) Wolfe and Austroboletustristis (Pat. & C.F. Baker) Wolfe (In the examination of solution . Spongifn 3% KOH . A chemin 3% KOH . The rea species . Therefor) Wolfe .R.rostratispora were old when collected, with dark red hymenophore and had a very strong fruity, alcoholic odour. The odour seems to be present in old basidiomata only (S. Vadthanarat 212 and one basidiomata of S. Vadthanarat 406). One possible explanation to the alcoholic smell is that sterigmata are broken from spore release and any remaining cytoplasm in the basidia could leak into the cavities of the hymenophore and be fermented. Fermentation by yeasts might be possible due to the cracking of the peridium, allowing contact of the hymenophore cavities with ambient air. As mammals and marsupials are known to be the main spore dispersal vectors of truffle-like fungi . However, in the forest where the new species was found, some scattered Eucalyptus trees were also observed. As Eucalyptus species have been reported to be ectomycorrhizal trees (e.g. Eucalyptus trees found in the forest could also possibly be host of R.rostratispora. However, Eucalyptus is not indigenous to Thailand; several species have been planted since the early 1900s (Rhodactina species seem to be indigenous to Thailand and Eucalyptus not, they are less likely to be ectomycorrhizal partners. Further study is needed, however, to confirm the range of ectomycorrhizal host tree species of R.rostratispora. Borofutus and Spongiforma, the most closely related genera of Rhodactina, are also ectomycorrhizal associates with trees in Dipterocarpaceae. The only known Borofutus species, B.dhakanus is ectomycorrhizal with Shorearobusta (Spongiforma species, S.thailandica was reported as associated with Dipterocarpus sp. and Shorea sp. in primary forest while S.squarepantsii was reported as associated with unidentified dipterocarp trees (The three Thailand . They prly 1900s . As Rhodrobusta . As for rp trees .


Staphylococcus aureus is part of the human epithelial microbiota; however, it is also a pathogen. The acquisition of mobile genetic elements plays a role in the virulence of S. aureus isolates and contributes to treatment failures. This report details the draft genome sequences of 14 clinical S. aureus isolates. Staphylococcus aureus is part of the microbiota found on the skin of humans and other animals. It is also a known pathogen that causes mild to severe infections, as well as toxin-mediated diseases (S. aureus (MRSA) is a growing concern in the health industry, and MRSA isolates are categorized based on each isolate’s source as either hospital-acquired MRSA (HA-MRSA), community-acquired MRSA (CA-MRSA), or livestock-associated MRSA (LA-MRSA). Multilocus sequence typing is employed to determine an isolate’s sequence type (ST), which indicates the genetic background and denotes the characteristics of the isolate common to the group. The ST5 lineage is globally distributed and well known for acquisition of virulence factors and antibiotic resistance genes contained on mobile genetic elements   were used to generate indexed libraries. Libraries were pooled and sequenced using the MiSeq version 2 500-cycle reagent kit, yielding 2 × 250-bp paired-end reads (Illumina).http://mira-assembler.sourceforge.net/docs/DefinitiveGuideToMIRA.html), resulting in the average coverages indicated here for each isolate: UCI3, 56×; UCI9, 82×; UCI11, 72×; UCI19, 47×; UCI21, 66×; UCI24, 62×; UCI27, 40×; UCI43, 46×; UCI45, 53×; UCI46, 73×; UCI48, 46×; UCI52, 33×; UCI56, 36×; and UCI64, 52×. Following assembly, only contigs >1,500 bp in length having a coverage of >66% of the average genome coverage were retained. Additionally, when the assembly tool indicated that a contig was part of a potentially repetitive element, the contig was required to be >2,000 bp for inclusion in the assembly.Draft genome assemblies were generated using MIRA version 4.0.2 (LKYU00000000; UCI9, LKZA00000000; UCI11, LKZC00000000; UCI19, LKZK00000000; UCI21, LKZM00000000; UCI24, LKZP00000000; UCI27, LKZS00000000; UCI43, LLAI00000000; UCI45, LLAK00000000; UCI46, LLAL00000000; UCI48, LLAN00000000; UCI52, LLAR00000000; UCI56, LLAV00000000; and UCI64, LLBD00000000.The draft genome sequences obtained for these isolates were entered into DDBJ/ENA/GenBank with accession numbers as follows: UCI3,