Henoch–Schönlein purpura (HSP) is a small-vessel vasculitis that has been extensively studied in children, but little is known about its natural history in adults. There is no consensus regarding the treatment of glucocorticosteroids use for HSP. The efficacy of glucocorticoid for preventing from severe complications or relapse is also controversial in HSP.A 21-year-old male was admitted to the hospital due to abdominal pain for more than 20 days, hematochezia for more than 10 days, and rash for 2 days.The diagnosis of HSP is based on the European League against Rheumatism and the Paediatric Rheumatology European Society in 2006.The patient received glucocorticosteroids treatment for 17 days at the time of first hospitalization.The abdominal pain and hematochezia completely disappeared on the 6th day after the use of glucocorticosteroids, and purpura completely disappeared on the 8th day.Our patient has a good response to glucocorticoid. Glucocorticosteroids may be effective for the treatment of HSP. The proportion of males and females in children is close to 2:1. By comparison, it is more rare in adults with an estimated annual incidence of 1.3/100,000 cases. Compared with HSP in children, HSP in adults have more serious clinical manifestations and worse outcomes. Clinical characteristics of HSP include abdominal pain, gastrointestinal bleeding, nonthrombocytopenic palpable purpura, arthritis, and renal involvement. Most of them occur in autumn and winter, but its pathogenesis and causes are still unclear. In 2006, the diagnostic criteria published by the European League against Rheumatism and the Paediatric Rheumatology European Society which include palpable purpura in combination with at least one of other manifestations . There is no consensus on treatment of glucocorticoid for HSP in adults. The efficacy of glucocorticoid in preventing from severe complications or relapse is also controversial in HSP.,9Henoch–Schönlein purpura (HSP) is the most common systemic vasculitis in children.In this case report, we showed that an adult patient with HSP presenting with abdominal pain and gastrointestinal bleeding had a good response by glucocorticoid.2On February 12, 2019, a 20-year-old male was admitted to our department due to abdominal pain for more than 20 days, hematochezia for more than 10 days, and rash for 2 days.9/L (3.5–9.5 × 109/L); percentage of granulocyte (GR%), 89.9% (40–75%); platelet count (PLT), 465 × 109/L (100–300 × 109/L); gamma-glutamyl transpeptidase (GGT), 82.79 U/L (8–78 U/L); carbohydrate antigen 125, 74.76 U/mL (0–35 U/mL); and serum sodium, 132.0 mmol/L (137–147 mmol/L). Abdominal color Doppler ultrasound showed cholestasis and cholecystitis. Chest and abdominal computed tomography scans showed localized thickening of small intestinal wall in the right lower abdomen, a small amount of pericardial effusion, pelvic effusion, and abdominal lymph node enlargement , 38.4 × 109/L; GR%, 89.5%; red blood cell (RBC), 4.16 × 1012/L (4.3–5.8 × 1012/L); hemoglobin (Hb), 120 g/L (130–175 g/L); C-reactive protein (CRP), 25.68 mg/L (≤10 mg/L); hematocrit, 35.8% (40%–50%); and PLT, 327 × 109/L. Fecal occult blood test was positive. Electrocardiogram showed sinus tachycardia. Therefore, according to the European League against Rheumatism and the Paediatric Rheumatology European Society criteria in 2006, HSP was diagnosed. He was initially treated with somatostatin 3000 μg and pantoprazole sodium 40 mg.On February 12, 2019, he was admitted to our department. His previous medical history included haemorrhoidectomy. Physical examinations showed soft abdomen, deep tenderness in the middle abdomen without rebound pain, and diffuse purpuric rashes on his limbs. Laboratory tests showed WBC, 30.4 × 109/L; GR%, 88.5%; RBC, 4.66 × 1012/L; Hb, 135 g/L; erythrocyte sedimentation rate (ESR), 22 mm/h (0–15 mm/h); CRP, 39 mg/L; PLT, 403 × 109/L; albumin, 23.1 g/L (40–55 g/L); serum calcium (Ca), 1.97 mmol/L (2.08–2.6 mmol/L); procalcitonin, 0.281 ng/mL (0–0.05 ng/mL); D-dimer, 24.11 mg/L; immunoglobulin A, 3.20 g/L (0.7–4 g/L); immunoglobulin G, 8.47 g/L (7–16 g/L); immunoglobulin M, 0.37 g/L (0.4–2.3 g/L); complement C3, 1.081 g/L (0.75–1.55 g/L); and complement C4, 0.185 g/L (0.1–0.4 g/L). Hepatitis B and C virus were negative. A dermatologist's consultation suggested discontinuation of suspected drugs, oral use of desloratadine 5 mg/d, ebastine 20 mg/d, and melilotus extract tablets 3600 mg/d, external use of calamine lotion 6 times a day, intravenous infusion of compound glycyrrhizin 60 mL/d, and glucocorticoid therapy if appropriate. Octreotide was given intravenously with a dosage of 0.3 mg every 12 hours. Levofloxacin was given intravenously with a dosage of 0.5 g/d. Calcium gluconate was given intravenously with a dosage of 10 mL/d. Human albumin was given intravenously with a dosage of 10 g.On February 13, 2019, the patient had dark red bloody stool twice again with the total amount of about 400 mL. His heart rate was about 125 beats per minute. Laboratory tests showed WBC, 34.3 × 109/L; GR%, 91.0%; PLT, 408 × 109/L; and Hb, 138 g/L. Considering that gastrointestinal bleeding episodes persisted, glucocorticoid therapy was given after the informed consent written by his father. Methylprednisolone was given intravenously with a dosage of 80 mg twice a day.On February 14, 2019, the patient had hematochezia twice with the total amount of about 600 mL. Laboratory tests showed WBC, 39.2 × 109/L; GR%, 90.1%; RBC, 3.75 × 1012/L; Hb, 112 g/L; ESR, 27 mm/h; Ca, 1.94 mmol/L; and Streptolysin O, 238.1 IU/mL (0–20 IU/mL). Antinuclear antibody and fecal parasites examinations were negative. On February 16, 2019, the dosage of methylprednisolone was reduced to 40 mg twice a day.On February 15, 2019, his abdominal pain was improved obviously and heart rate was dropped to normal with an average of 80 beats per minute. He intermittently had hematochezia with the total amount of about 400 mL. Laboratory tests showed WBC, 32.2 × 109/L; GR%, 77.1%; RBC, 3.93 × 1012/L; Hb, 118 g/L; and ESR, 13 mm/h. On February 19, 2019, his abdominal pain and hematochezia disappeared. Oral methylprednisolone was employed with a dosage of 20 mg twice a day. Subcutaneous injection of octreotide was given with a dosage of 0.1 mg every 8 hours.On February 18, 2019, laboratory tests showed WBC, 21.0 × 10On February 21, 2019, his condition was relatively stable. Gastroscopy showed chronic superficial gastritis with bile reflux . Researchers also suggested that the relapse was strongly associated with a high ESR value.The precipitating factor for relapse in our case might be a recent history of infection, which presented with cough, sputum, and runny nose with an increased ESR level on laboratory tests Fig. . The uppIn conclusion, our patient with HSP had a good response to glucocorticoid therapy. However, current evidence in regard to the efficacy of glucocorticoid or relapse is lacking. Further researches are necessary to explore the relationship between the use of glucocorticoid and prognosis of HSP.Conceptualization: Xingshun Qi.Formal analysis: Xiaozhong Guo, Xingshun Qi.Methodology: Fangfang Yi, Zhaohui Bai, Yingying Li.Supervision: Zhaohui Bai, Xiangbo Xu, Xingshun Qi.Writing – original draft: Fangfang Yi.Writing – review and editing: Xingshun Qi, Xiangbo Xu Zhaohui Bai.Xingshun Qi orcid: 0000-0002-9448-6739.


Their structures were elucidated by spectral methods. This is the first time that arginine has been found to be involved in the biosynthesis of bufotenines in parotid of toad. The cytotoxic activities of these compounds have been assayed against A375 and A549 cell lines by the MTT method; however, they showed no cytotoxic activities.A new indole alkaloid named bufobutarginine ( Bufo bufo gargarizans Cantor and B. melanostrictus Schneider. In traditional Chinese medicine, toad venom possesses a wide range of biological effects and has long been used for the treatment of heart failure, tumors, sores, and pains in clinical settings + . Hydrolysis with 6 M hydrochloric acid provided arginine, which was identified by TLC with l-arginine standard + ; c 0.9, H2O); UV (H2O) λmax (log ε): 217 (4.38), 281 (3.56), 380 (3.52) nm; IR (KBr) νmax: 3385, 2930, 1633, 1578, 1468, 1456, 1384, 1188 cm−1; 1H NMR and 13C NMR spectral data, see 2O): Δε194nm − 4.14, Δε202nm + 1.82, Δε212nm − 3.43.1 (1 mg) was heated with 6 M HCl (0.5 mL) in a sealed tube at 100 °C for 8 h. A portion of the resulting solution was subjected to TLC on silica gel GF254 with n-butanol–HOAc–EtOH–H2O (4:1:1:2) as a developing solvent. Ninhydrin reagent was used for the detection of amino acids on the TLC plate [Compound LC plate .1 was analyzed using Waters 2695 HPLC with a 2478 UV detector. HPLC experiments were performed on Chirex 3126 column  at a flow rate of 1.0 mL/min eluted with 1 mM CuSO4. Column temperature was maintained at room temperature. Detection was carried out at 254 nm. l- and d-arginine were products of Aladdin (China).The hydrolysate of in vitro cytotoxic activities test on two human tumor cell lines were performed using MTT methods in a 96-multiwell microtiter integrated system. Compounds 1–4 were dissolved in DMSO to make stock solutions, then diluted in cell culture medium at different concentrations, and used immediately. The positive control was 5-fluorouracil (5-FU) dissolved in the same solution. In all assays, the final concentrations of DMSO in the culture medium were less than 0.01%. The cells were cultured in a RPMI-1640 medium  and supplemented with 10% fetal bovine serum (FBS)  and 0.03% l-glutamine (Gibco) at 37 °C in 5% CO2. Tumor cells were seeded at 5 × 104 cells/well in 96-well plates . After overnight incubation, different concentrations of 1–4 were added, and the final concentrations were 0.1, 1, 10, 100, and 200 μM, respectively. Incubated for 0, 12, 24, and 48 h, cell growth was measured at different time points in the 3--2,5-diphenyltetrazolium bromide (MTT) assay with a plate reader  [The


Combination testing in checkerboard assays revealed that aminomethyl spectinomycin-antibiotic combinations had mainly an additive effect against the susceptible biodefense pathogens. The in vivo efficacy of compound 1950 was also demonstrated in mice infected with B. mallei (FMH) or F. tularensis (SchuS4). These results suggest that aminomethyl spectinomycins are promising new candidates for development of therapeutics against biodefense bacterial agents.New antibiotics that are active against multi-drug-resistant strains and difficult-to-treat bacterial infections are needed. Synthetic modification of spectinomycin, a bacterial protein synthesis inhibitor, has been shown to increase antibacterial activity compared with spectinomycin. Aminomethyl spectinomycins are active against Gram-negative and Gram-positive bacterial pathogens. In this study, the ability of aminomethyl spectinomycins to treat biothreat pathogens is examined by MIC profiling, synergy testing, and in vivo efficacy experiments. Compound  Neisseria gonorrhoeae until resistance arose, and it was withdrawn from frontline use in the United States + 472.2459, found 472.2459. Elemental analysis theoretical composition for empirical formula C22H34FN3O7∙3 HCl∙2.5 H2O: C, 42.46; H, 6.74; N, 6.75; Cl, 17.09; F, 3.05. Found formula for C22H34FN3O7∙3 HCl∙2.5 H2O: C, 42.21; H, 6.86; N, 6.52; Cl, 16.73; F, 2.85.R)-3′-(N-(4-ethylbenzyl)aminomethyl)dihydrospectinomycin trihydrochloride (1951)3′-: δ 7.44 , 7.40 , 4.74 , 4.33–4.20 , 4.01 , 3.94 , 3.69 , 3.50 , 3.38 , 3.17 , 2.81 , 2.76 , 2.68 , 1.82 , 1.22 . 13C NMR : δ 146.68, 130.10, 128.77, 127.39, 93.20, 92.35, 72.12, 69.80, 67.27, 66.01, 65.48, 61.58, 59.60, 58.54, 51.11, 48.76, 40.06, 30.54, 30.35, 27.97, 19.84, 14.84. HRMS-ESI calculated for C24H39N3O7 [M+H]+ 482.2788, found 482.2873.R)-3′-(N-(3-phenylpropyl)aminomethyl)dihydrospectinomycin trihydrochloride (2241)3′-: δ 7.40 , 7.32 , 4.89 , 4.67 , 4.15 , 4.01 , 3.90 , 3.84 , 3.43 , 3.22 , 3.10 , 2.77 , 2.74 , 2.63 , 2.05 , 1.88 , 1.78 , 12.5 . 13C NMR : δ 128.80, 128.53, 128.47, 126.50, 96.35, 93.23, 70.02, 67.32, 66.19, 65.72, 61.69, 59.77, 58.64, 50.22, 48.83, 48.15, 42.79, 40.04, 31.84, 30.61, 30.46, 26.59, 23.19, 19.88. HRMS-ESI calculated for C24H39N3O7 [M+H]+ 482.2866, found 482.2860. Elemental analysis theoretical composition for the empirical formula C24H39N3O7∙3 HCl∙2.3 H2O: C, 45.58; H, 7.43; N, 6.64; Cl, 16.82. Found formula for C22H34FN3O7∙3 HCl∙2.3 H2O: C, 45.36; H, 6.96; N, 6.54; Cl, 16.66.R)-3′-(N-(4-chlorobenzyl)aminomethyl)dihydrospectinomycin trihydrobromide (2324)3′-: δ 7.48–7.37 , 4.30–4.16 , 3.97 , 3.88 , 3.73–3.62 , 3.50–3.45 , 3.33 , 3.26 , 3.21–3.12 , 2.75 , 1.83–1.67 , 1.15 . 13C NMR : δ 135.28, 131.57, 129.34, 128.53, 93.16, 92.32, 72.18, 69.56, 67.33, 65.86, 65.28, 61.43, 59.45, 58.43, 50.76, 49.03, 40.02, 30.60, 30.37, 19.87. HRMS-ESI calculated for C22H34ClN3O7 [M+H]+ 488.2164, found 488.2169.−1 and serially diluted twofold. Approximately 5 × 104 CFU of bacteria in CAMHB  were added to each well and plates were incubated at 37°C for 24–48 h. The MIC was determined as the lowest concentration of the compound that prevented growth of the bacterial strain. The MIC50 and MIC90 values were determined as the concentration of the amSPCs at which growth was inhibited in 50 and 90%, respectively, of the strains that were tested.The MICs were determined as recommended by CLSI in 96-well plates with compound at an initial concentration of 200 µg ml1950 were also tested in combination with other antibiotics or polymyxin B nonapeptide (PMBN). For the dual antibiotic studies, a checkerboard serial dilution of antibiotics was completed in 96-well plates so that, when possible, the MIC of an individual antibiotic was in the middle of the dilutions . The bacteria were applied to the plates and incubated as described above. The fractional inhibitory concentration (FIC) index was calculated with the following formula: FIC = (A/MICA) + (B/MICB), where MICA and MICB are the MIC of each drug individually and A and B are the MICs of each antibiotic in combination. Effects were determined to be synergistic (FIC ≤ 0.5), additive (0.5 < FIC ≤ 1.0) indifferent (1.0 < FIC ≤ 4.0), or antagonistic (FIC > 4.0).The MICs of spectinomycin and compound 1950  and PMBN  were completed in a 96-well plate. F. tularensis LVS or B. pseudomallei strain Bp82 were applied to the plate as described above and incubated at 37°C with 5% CO2 for 40–48 h. The MIC of spectinomycin or 1950 was recorded for each concentration of PMBN.For the PMBN combination studies, a checkerboard serial dilution of spectinomycin or compound B. mallei strain FMH in CAMHB was diluted to an OD600 of 0.01 and incubated, with shaking, at 37 °C for 6 h to allow the culture to reach the mid-exponential phase of growth. The culture was then diluted to a concentration of 5 × 105 CFU ml−1. Compound 1950, spectinomycin, or an equal volume of DMSO/diluent was added such that the final concentration of compound/antibiotic was 50 µg ml−1 and DMSO was 0.5%. The samples were incubated at 37°C with aeration. A sample was removed from the culture at 0, 1.25, 2.5, 4, 6, and 24 h for serial dilution and enumeration of the bacteria on sheep blood agar (SBA) plates. Significant differences between the groups at each time point were determined by multiple t tests with the Holm-Šídák method to correct for multiple comparisons in GraphPad Prism 7.An overnight culture of Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. Six- to eight-week-old BALB/c mice  were used for all experiments.4 CFU B. mallei strain FMH in 20 µl. Treatment with amSPC 1950 (25 or 50 mg kg−1) or saline was initiated at 1 h post-infection. Treatments were administered twice daily (BID) via s.c. injection in 200 µl for 14 days. Mice were closely monitored for morbidity and mortality. On day 40, five mice were removed from the 1950-treated groups (25 and 50 mg kg−1) for necropsy and were marked as censored subjects for the survival curve. Mice that survived until day 60 were euthanized and necropsy was completed. The lungs, livers, and spleens of mice were removed, homogenized, and CFU g−1 of tissue was determined.Mice received an intranasal (i.n.) instillation of 3 × 104 CFU B. mallei strain FMH in 20 µl. Treatment with 1950 , administered s.c. in 200 µl, was initiated at 1, 6, 24, or 48 h post infection and continued for 14 days. Control mice were administered saline or spectinomycin  starting at 1 h post infection. Mice were closely monitored for morbidity and mortality for 42 days.To determine if a delay in initiation of treatment had an effect on survival of mice post-infection, mice received an i.n. inoculum of 2 × 103 CFU F. tularensis strain SchuS4 in 20 µl. Treatment with compound 1950 (50 or 75 mg kg−1) or spectinomycin (50 mg kg−1) in 200 µl was initiated at 1 h post-infection and continued BID for 14 days. Mice were closely monitored for morbidity and mortality for 55 days. In a second experiment, mice were infected with 900 CFU F. tularensis SchuS4 and s.c. administration of saline, spectinomycin (50 mg kg−1), or 1950 (50 mg kg−1) in 200 µl was initiated at 1 h post-infection and continued three times daily (TID) for 14 days. Mice were closely monitored for morbidity and mortality for 37 days.Mice were infected i.n. with 1 × 10Significant differences in survival between groups were determined by log-rank (Mantel–Cox) test in GraphPad Prism 7. Significant differences in bacterial load were determined by Mann–Whitney test in GraphPad Prism 7.B. mallei, Brucella spp., F. tularensis, B. anthracis, and B. pseudomallei. The four amSPCs investigated had lower MIC50 and MIC90 values than those observed for spectinomycin for all species tested, except B. pseudomallei  were most susceptible to the amSPCs; the 29 strains tested had an MIC range of 0.4–12.5 µg ml−1  and B. pseudomallei Bp82  to 8 µg ml−1. The addition of 16 µg ml−1 PMBN reduced the MIC of 1950 from 4 to 1 µg ml−1. These data indicate that there are possible combination therapies that can improve the efficacy of amSPCs against biodefense pathogens.We also tested polymyxin B nonapeptide (PMBN) in combination with spectinomycin and 82 Table . PMBN bi1950 and spectinomycin to kill B. mallei in vitro was assayed over a 24 h time frame . These data indicate that amSPC 1950 is bactericidal against B. mallei.The ability of amSPC 1950 was selected for murine studies due to its improved half-life compared with spectinomycin and previously demonstrated efficacy against other pathogens [1950 could protect BALB/c mice from an i.n. infection with 3 × 104 CFU B. mallei strain FMH (10 x LD50). Treatment with 1950  was initiated at 1 h post-inoculation and continued for 14 days. The mice that received no treatment succumbed to infection by day 5 post-infection. Treatment with 1950 resulted in an increased survival of infected mice; 10/10 mice that received 1950  and 8/10 of mice that received 1950  survived until day 40 post-infection  and one mouse that received 1950  survived until day 60 post-challenge. The survival of mice that received either dose of 1950 was significantly different from the survival of the untreated control mice (P < 0.0001), but there was no difference in survival between the two treatment groups.In addition to its in vitro potency, amSPC athogens , 9. We dion Fig. . On day 1950 treatment groups  on day 40. Only two mice that received 1950  had detectable levels of bacteria in their lungs, livers, and spleens  had bacteria in their livers and spleens while two of these mice also had bacteria in their lungs. The decreased bacterial load in the mice that received 1950  is indicative of a dose-dependent response. Furthermore, the bacterial load in the spleens of mice that received 1950  was significantly different from those that received 1950  (P = 0.0317). The tissue burden in the mice that survived until day 60 post-infection in the 1950  group (n = 3) and the 1950  group (n = 1) was also determined , two mice had bacteria in their lungs, livers, and spleens and one mouse only had bacteria in its spleen on day 60. In the one mouse that survived in the 1950  group, bacteria were present in its lungs, liver, and spleen. There was a trend that the bacterial load in the mice increased from day 40 post infection to day 60 post infection for the 1950  and 1950  treatment groups, although the only significant difference was in the spleen between the 1950  day 40 and 1950  day 60 groups (P = 0.0357). This suggests that there is an expansion of the bacteria that survived the 14-day treatment regimen.The bacterial load in the lungs, livers, and spleens were determined in mice from the ens Fig. . In contned Fig. . Of the B. mallei mouse infection  was initiated at 1, 6, 24, or 48 h post infection and continued for 14 days (BID). Untreated control mice had 2/10 mice survive until day 42 post-infection. Initiation of treatment at 24 or 48 h post-infection resulted in survival that was similar to the untreated control mice, 1/10 and 2/10, respectively. In comparison, treatment initiation at 1 or 6 h post-infection resulted in survival of 6/8 or 6/10 mice, respectively. The mice that received 1950 at 1 or 6 h post-infection had a significant increase in survival compared with the untreated control mice (P < 0.05). Initiation of spectinomycin  at 1 h post-infection resulted in the survival of 6/10 mice, which was similar to the survival observed after 1950 treatment. These data indicate that 50 mg kg−1 is an effective dose of 1950 when administered up to 6 h post-infection.We also tested whether the time of treatment initiation post-infection altered the outcome in a ion Fig. . Mice we1950 could protect BALB/c mice from an i.n. infection with 1 x 103 CFU F. tularensis strain SchuS4 (10 x LD50). Treatment with 1950 (50 or 75 mg kg−1) or spectinomycin (50 mg kg−1), administered s.c., was initiated at 1 h post-infection and continued for 14 days (BID). Untreated mice (n = 10) succumbed to the infection by day 5 and the spectinomycin-treated  mice succumbed to the infection by day 8  resulted in survival of 3/10 mice at day 55 post-infection which was significantly different from the untreated control (P < 0.0001) and the spectinomycin control  protected less than half of the mice from F. tularensis infection, we also tested whether administration of 1950  could improve survival  succumbed to infection by day 5 and mice that received spectinomycin  succumbed to infection by day 8. Administration of 1950  resulted in the survival of 4/10 mice, which was significantly increased compared with the untreated control mice (P < 0.0001) and the spectinomycin control mice (P = 0.0315). Together, these data suggest that amSPC 1950 is a more effective treatment than spectinomycin in the in vivo F. tularensis model.Since treatment with val Fig. . After iBrucella spp., B. mallei, and F. tularensis. Although there was a slight improvement of activity against the Gram-positive pathogen B. anthracis compared with spectinomycin, the MIC values were higher than those which would be preferred for a B. anthracis therapeutic. Of the antibiotic combination therapies tested, we determined that most possessed additive or indifferent rather than synergistic effects with compound 1950, and that the previously noted synergy with macrolides and spectinamides was not analogous to the amSPCs [1950, but not with spectinomycin, against B. anthracis. This effect was not observed when tested in Gram-negative bacteria, so the issue may be related to the differences between Gram-positive and Gram-negative susceptibilities and uptake of these antibiotics or specific to B. anthracis itself. In addition, while streptomycin and spectinomycin bind to the 16S rRNA, they have distinct binding sites, so competition is not expected to be the cause [This study demonstrates that amSPCs possess useful antibacterial activity against multiple biothreat pathogens. The modifications to the spectinomycin core rendered these compounds more active than spectinomycin against the Gram-negative pathogens e amSPCs . It is whe cause .1950 has an improved half-life compared with spectinomycin, and was able to protect mice from S. pneumoniae or N. gonorrhoeae infections [1950 against two intracellular pathogens, B. mallei and F. tularensis, which have innate antibacterial resistance [Chlamydia trachomatis in cell culture, we anticipated that these compounds could gain access to the intracellular B. mallei or F. tularensis in vivo [90 was lower for B. mallei than for F. tularensis, the challenge strains for the two species had the same MIC (12.5 µg ml−1). Despite these similar MIC values, compound 1950 had a greater efficacy against B. mallei than it did against F. tularensis, even when treatment initiation was delayed from 1 to 6 h post-infection. The majority of mice that succumbed to B. mallei did so after the treatment with 1950 ended; this corroborates with the increase in bacterial load from day 40 to day 60 post infection in 1950-treated mice. However, almost half of the mice that succumbed to F. tularensis infection did so in the first 14 days post-infection, the time frame for 1950 treatment. These data indicate that compound 1950 reached therapeutic levels in B. mallei-infected cells and was able to reduce, but not eliminate, the B. mallei infection while the compound was unable to effectively lower F. tularensis below lethal quantities. A longer treatment regimen may have improved the survival of B. mallei-infected mice, but would likely have no effect on the survival of F. tularensis-infected mice.AmSPC compound fections , 9. Heresistance , 14, 16. in vivo , 9. Whil1950 had a greater improvement in efficacy in vivo compared with spectinomycin in F. tularensis-infected mice than in B. mallei-infected mice. The survival of mice administered compound 1950 after F. tularensis infection was significantly better than the survival of mice that received spectinomycin, the dose was administered BID or TID. The difference in the MICs of spectinomycin (64 µg ml−1) and 1950 (12.5 µg ml−1) against the challenge F. tularensis strain, SchuS4, explains this difference in survival of mice. The in vitro time-kill assay demonstrated that spectinomycin killed B. mallei faster than compound 1950, despite similar MIC values . However, the survival of mice administered 1950 at 1 h post-infection with B. mallei FMH was not significantly different from that of the mice that received spectinomycin, although the 1950-treated mice had a slightly higher survival percentage (75% compared with 60%). This demonstrates amSPCs may have improved activity compared with spectinomycin against some pathogens, like F. tularensis.Compound Overall, we demonstrated that amSPCs have some useful activity against Gram-negative biothreat pathogens in vitro and in vivo. We further demonstrated the potential for spectinomycin analogs as broad-spectrum antibiotics that function against Gram-negative pathogens of clinical and biodefense importance. Efforts are ongoing to improve the potency of the amSPCs, such as boosting their ability to penetrate Gram-negative pathogens which would lead to a greater level of protection against biothreat pathogens.Supplemental Material


In the present study, we compared the plasma and brain tissue concentrations of MK-801 after intraperitoneal (i.p.) or subcutaneous (s.c.) administration at a dose of 0.1 mg/kg in male ICR mice. Moreover, these data present the optimization of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the analysis of MK-801 in biological samples. Procedures for the preparation of brain tissue and plasma samples and instrumental analysis are described. This article is related to a research article entitled “Effects of the N-methyl-d-aspartate receptor antagonist, MK-801, on spatial memory and influence of the route of administration” [MK-801, a N-methyl- The concentration of MK-801 in the brain was significantly higher after s.c. administration than after i.p. administration at all time points until the 120-min time point ; Fig. 4AIdentical effects were observed in blood plasma after MK-801 administration ; Fig. 4Bmax) of MK-801 in brain tissue and blood plasma were almost 2-fold higher when administered via the s.c. route than the i.p. route -MK-801 hydrogen maleate) was dissolved in sterile saline (0.9% NaCl). MK-801 concentrations in the brain tissue and plasma were determined in mice receiving single i.p. or s.c. doses of MK-801 at 0.1 mg/kg 15 and 30 min, and 1, 2, and 4 hours before decapitation. Each group consisted of 3 animals for the retrieval of blood and brain samples. The brain was divided into two hemispheres, and 6 measurements were obtained per point.2.3The concentrations of MK-801 in brain tissue and plasma were measured using UPLC-MS⁄MS. Blood was collected in heparin-coated tubes and centrifuged at 3000 rpm at 4 °C for 10 min to separate the plasma. The brain was gently removed and homogenized in ice-cold Milli-Q water at a w/v ratio of 1:5 in 20-second bursts using a Cole Parmer 130-Watt ultrasonic processor set to 40 kHz. The obtained homogenate was centrifuged at 16500 rpm for 10 min at 4 °C. The supernatant was then decanted, and the pellet was homogenized in the same volume of Milli-Q water as before. The obtained homogenate was centrifuged at 16500 rpm for 10 min at 4 °C. The supernatants were combined and stored at −80 °C until use.Brain tissue extract (300 μL) were mixed with 700 μL of 2% formic acid aqueous solution (v/v), vortexed und centrifuged at 13000 rpm for 10 min. 100 μL of mouse plasma were mixed with 900 μL of 2% formic acid aqueous solution (v/v), vortexed and centrifuged at 13000 rpm for 10 min. 950 μL of the supernatant was load onto conditioned Strata X  SPE cartridge (Phenomenex), washed 2 times with 1mL of 5% methanol aqueous solution and eluted with 900 μL of 1% formic acid solution in acetonitrile (v/v). The eluent was evaporated using a Genevac EZ-2 at 40 °C, reconstituted in 200 μL of mobile phase solution , and subjected to UPLC-MS/MS analysis. Calibration was performed against external calibration standard solutions.UPLC was carried out using the Waters Acquity UPLC system equipped with the Acquity BEH C8 column . The mobile phase consisted of A (aqueous 0.1% formic acid) and B (acetonitrile) at a flow rate of 0.25 mL/min, using a gradient elution: Initial – 50% B, 2.5 min–98% B, 4.5 min–98% B, 4.7 min −50% B, 6 min–50% B. The injection volume was 5 μL.m/z 222.2 to 205.2 . Quantitative analysis was achieved using QuanLynx 4.1 software .MS/MS analysis was performed on a Micromass Quattro MicroTM tandem mass spectrometer in positive ion mode using multiple reaction monitoring of the transition from 2.4(+)-MK-801 hydrogen maleate -(+)-5-Methyl-10,11-dihydro-5H-dibenzo cyclohepten-5,10-imine hydrogen maleate) was purchased from Sigma Aldrich . Ethanol was purchased from LAKO Ltd. . 0.9% physiological saline was purchased from Fresenius Kabi . Acetonitrile and methanol were obtained from Merck .2.5All data were expressed as the mean ± standard error of the mean (SEM). MK-801 concentrations in brain tissue and blood plasma were analyzed using unpaired Student's t-tests. P values less than 0.05 were significant. The statistical calculations were performed using the GraphPad Prism 3.0 software package .


Bronchial asthma is an inflammatory disease of the airways. Beclomethasone dipropionate/Formoterol (BDP/F) and Fluticasone furoate/Vilanterol (FF/V) are two of the most effective LABA/ICS combinations for managing persistent bronchial asthma. Aim of the study was to compare the outcomes achieved in mild-to-moderate asthma patients assuming BDP/F 100/6 μg b.i.d. (Group A) or FF/V 92/22 μg once-daily (Group B) for 12-months. No head-to-head long-term comparison is available at present.1% predicted values; the exacerbation and hospitalization rates; days of hospitalization; GP and/or specialist visits; days of inactivity; courses of systemic steroids and/or antibiotics were recorded at baseline and after 3, 6 and 12 months of both treatments. The overall adherence to treatments was also calculated. The propensity score method was used for matching and comparing the two cohorts of patients; Anova and Wilcoxon tests were used for checking the trends and time-to-time comparisons over the period; statistical significance was accepted for p < 0.05.Data were automatically and anonymously obtained from the institutional database: FEVp < 0.001), even if it was significantly higher and time-dependent in group B. The mean (±SE) exacerbation rate/patient changed from 0.63 (±0.13) at baseline to 0.53 (±0.12) after three; to 0.58 (±0.13) after six, and to 0.60 (±0.18) after twelve months in group A (p = ns), while from of 1.05 (±0.16) at baseline, to 0.28 (±0.07) after three; to 0.33 (±0.08) after six, and to 0.18 (±0.08) after twelve months in group B (p < 0.001), respectively. The mean hospitalization rate/patient changed from 0.25 ± 0.07 at baseline to 0.15 (±0.06) after three; to 0.08 (±0.04) after six, and to 0.13 (±0.05) after twelve months in group A (p = ns), while from 0.30 (±0.07) at baseline to 0.08 (±0.04) after three; to 0.10 (±0.05) after six, and to 0.03 (±0.03) after twelve months in group B (p < 0.001), respectively. Also mean duration of hospitalization and days of inactivity were in favour of FF/V treatment over time (in both cases p < 0.001). GP’s visits were reduced by both treatments . Steroid and antibiotic courses were significantly reduced by both treatments, even if more systematically in group B . Moreover, changes in all outcomes considered proved time-dependent during the FF/V treatment only, particularly over the second semester. Finally, the overtime adherence to treatment was higher by 22 days during FF/V .The PS-matching process returned a cohort of 40 group A patients matched with 40 patients of group B, fully comparable for demographics, clinical characteristics, and comorbidities. The improvement in lung function was significant in both groups (Both the ICS/LABA combinations proved effective, even if characterized by different patterns of effectiveness either in terms of lung function and of long-term clinical outcomes. Only the once-daily inhalation of combined FF/V 92/22 μg once-daily optimized systematically the exacerbation and hospitalization rates in mild-to-moderate asthma, together with all other outcomes over time. The effectiveness of FF/V 92/22 once-daily μg proved progressive and time-dependent over the twelve-month period of the study, and associated to a higher adherence to treatment. Bronchial asthma is a chronic inflammatory disease of the airways which is characterized by airflow limitation, usually reversible spontaneously or following therapy, bronchial hyper-responsiveness and accelerated decline in lung function, and the occurrence of exacerbations .The excessive presence and activation of inflammatory cells within the mucosal, muscular and vascular structures of the airways are the underlying mechanisms responsible for asthma, which cause the release of inflammation mediators and the remodeling of the airways. Clinical manifestations of asthma consist of recurrence of cough, dyspnea, wheezing , and chest tightness . These mAccording to WHO estimates, 235 million people suffer from asthma. The Italian National Institute of Statistics (ISTAT) survey on health and use of health services estimated a prevalence of asthma of 4.2%  in Italy in 2012 [1), variability of peak expiratory flow (PEF), reversibility of airway obstruction, exacerbation rate, quality of life. Four levels of asthma severity are recognised: mild intermittent, mild persistent, moderate persistent and severe persistent.Severity of the disease is evaluated on frequency of symptoms, value of forced expiratory volume in 1 s  can be added. The combination of these two categories of drugs is the recommended therapeutic strategy for persistent asthma [The therapeutic strategy includes two main categories of drugs: the controller medications which must be assumed regularly to keep the disease under control, and the rescue medications which relieve the acute bronchoconstriction and related symptoms. Since asthma is an inflammatory disease, inhaled corticosteroids (ICS) are the most effective controller medications currently available and represent the first choice of treatment, to which long-acting betat asthma .Two of the most recent LABA/ICS combinations for persistent bronchial asthma are the Fluticasone furoate/Vilanterol (FF/V) 92/22 μg delivered via the Ellipta device –7 and thAlthough several studies investigated both the effectiveness and the safety of these two ICS/LABA combinations singularly, no long-term comparison is still available to our knowledge.The aim of the present study was to estimate and compare the outcomes achievable by mild-to-moderate asthma patients assuming BDP/F 100/6 μg b.i.d. to those of patients assuming FF/V 92/22 μg once-a-day over a twelve-month treatment.The study was an observational, retrospective analysis on asthmatic patients referring over the period February–September 2015 to the Lung Unit of the Specialist Medical Centre (CEMS), Verona, Italy.1 in Litres and FEV1 as % predicted), and comorbidities of the patients were recorded. All patients were followed for 12 (±2) months. FEV1 values; number of relapses and of related hospitalizations; duration of hospitalization (in days); number of general practitioner (GP) and/or specialist visits; days of inactivity; and number of courses of systemic steroids and antibiotics were recorded over the study period at baseline and after 3, 6, and 12 months of both treatments. Baseline values for outcomes were corresponding to values assessed over the three months preceding the index date for selections. Furthermore, as both the inhaler devices used are provided with a precise dose counter, the patients’ adherence to both treatments was also recorded monthly since the index data , and expressed in % inhalations vs the expected number of inhalations at each time of the study.Data were obtained automatically and anonymously from the institutional, UNI EN ISO 9001–2008 validated database, and the classic Boolean algebraic formula were used for selections . Selecti1 (%) and presence of comorbidities, was used. Moreover, the propensity score matching was performed without replacement, i.e. each of 40 patients of the Group B was matched with only one patient of the Group A.In order to compare the outcomes achieved in the two groups of patients, the propensity score matching method (PS)  was usedData reported at baseline and after three months of both treatments correspond to those already published in a previous study which was limited to a twelve-week observational period on the same cohort of patients . Data cop < 0.05.The analysis of variance was used to check the four-point trend  recorded in each treatment group for all outcomes. Finally, the extent of changes achieved in both treatment groups by each outcome considered was also compared at the same times by Wilcoxon test. Statistical significance was accepted for th, 2016. The patients’ consent to participate was not inserted because data were obtained automatically and anonymously.The study was approved by the R&CG Ethical Committee during the session officially held on January 111 in litres (L) was 2.4 (±0.09) in group A, and 2.5 ((±0.12) in group B. Mean (±SE) FEV1% pred. was 82.2% (±1.14) and 81.9% (±2.00) in group A and B, respectively. Patients with perennial allergy were 61.0% (47/77) in group A, and 62.5% (25/40) in group B, while those with seasonal allergy were 39.0% (30/77) in group A, and 37.5% (15/40) in group B, respectively. The percentage of patients with established comorbidities was 37.7% in group A, and 42.5% in group B. The following comorbid diseases were equally reported in both groups: arterial hypertension, kyphoscoliosis, obesity, severe depression, AIDS, diabetes mellitus, severe osteoporosis, and ischemic heart disease. In particular, arterial hypertension was the most prevalent comorbidity in both groups: 12.5% in group A, and 10.4% in group B, respectively.Clinical data of 77 patients treated with BDP/F 100/6 μg b.i.d (Group A) and of 40 patients treated with FF/V 92/22 μg once-a-day (Group B) were obtained. Characteristics of the entire cohort and of the PS-matched cohort at baseline are summarized in Table 1 and comorbidities, returned a cohort of 40 group A patients of the entire cohort matched with 40 patients of group B. The demographics and clinical characteristics of the PS-matched cohort at the baseline are described in Table 1% pred. was 82.4% (±1.63) in group A and 81.9% (±2.00) in group B. The presence of comorbidities was balanced (42.5%) in both groups  at baseline to 87.08% (±1.58) after 3 months; to 89.98% after 6 months, and to 91.88% after 12 months (Anova = p < 0.001) in group A, while in group B, mean (±SE) FEV1% pred. changed from 81.93% (±2.00) at baseline, to 89.50% after 3, to 90.9% after 6, and to 99.1% after 12 months of treatment (Anova = p > 0.001). Even if the overall trends of lung function proved significantly improved with both treatments, treatment B induced a FEV1% pred. improvement in the second semester of the study which was significantly higher than that obtained with treatment A (t test: p < 0.01)  in group B, respectively. In this case, the reduction of the rate achieved with treatment B was substantial and significant over the entire study period  exacerbation rate per patient was 0.63 (0.13) at baseline; 0.53 ±0.12) after 3; 0.58 (0.13) after 6, and 0.60 (0.18) after 12 months in group A (Anova: p = ns), while the corresponding rate in group B was 1.05 (0.16) at baseline; 0.28 (0.07) after 3; 0.33 (0.08) after 6, and 0.18 (0.08) after 12 months of treatment , respectively. The difference was statistically significant in favour of group B (t paired test: p < 0.04) over the second semester  rate of asthma-induced hospitalizations per patient was 0.25 (0.07) at baseline; 0.15 (±0.06) after 3; 0.08 (0.04) after 6, and 0.13 (0.05) after 12 months in group A (Anova: p = ns), while the corresponding mean number in group B was 0.30 (0.07) at baseline; 0.08 (0.04) after 3; 0.10 (0.05) after 6, and 0.03 (0.03) after 12 months   Fig. .p < 0.001), respectively. The difference in favour of treatment B was extremely clear over the second semester of treatment  at baseline; 0.28 (0.12) after 3; 0.48 (0.38) after 6, and 0.60 days (0.31) after 12 months (Anova: p = ns) in group A, while the corresponding mean duration in group B was 0.88 (0.31) at baseline; 0.08 (0.04) after 3; 0.10 (0.05) after 6, and 0.03 days (0.03) after 12 months (Anova: p = 0.11) in group A, while the corresponding duration in group B was 3.35 (0.63) at baseline; 0.60 (0.19) after 3; 1.10 (0.21) after 6, and 0.83 days (0.39) after 21 months , respectively. The difference was statistically significant in favour of group B over the entire period of treatment  at baseline; 1.53 (0.27) after 3; 1.40 (0.27) after 6, and 1.45 days (0.58) after twelve months (Anova: p < 0.007) in group A, while the corresponding number in group B was 1.53 (0.24) at baseline; 0.38 (0.12) after 3; 0.55 (0.09) after 6, and 0.25 (0.06) after 12 months , respectively. The frequency of GPs’ visits was significantly lower during the second semester of treatment B (t test: p < 0.01)  at baseline; 0.85 (0.15) after 3; 0.55 (0.13) after 6, and 0.63 (0.15) after 12 months , respectively. The difference was in favour of group B for all the three times of follow up . The difference between the treatments proved highly significant in favour of group B over the entire study period  at baseline; 0.68 (0.11) after 3; 0.65 (0.08) after 6, and 1.05 (0.13) after 12 months (Anova: p = ns), while the corresponding number in group B was 0.70 (0.16) at baseline; 0.28 (0.07) after 3; 0.35 (0.08) after 6, and 0.23 (0.08) after 12 months (Anova: p < 0.004) in group A, while the corresponding number in group B was 1.08 (0.16) at baseline; 0.33 (0.08) after 3; 0.30 (0.08) after 6, and 0.13 (0.08) after 12 months (Anova: p < 0.001), respectively  at baseline; 0.73 (0.13) after 3; 0.48 (0.11) after 6, and 0.30 (0.09) after 12 months  at baseline; 0.63 (1.10) after 3; 0.43 (0.12) after 6, and 0.40 (0.11) after 12 months (Anova: 0.047), while the corresponding number in group B was 1.03 (0.13) at baseline; 0.35 (0.08) after 3; 0.33 (0.08) after 6, and 0.15 (0.08) after 12 months (Anova: p < 0.001), respectively Fig. .Fig. 9ChThe adherence to prescribed treatments calculated in terms of expected doses over the period (stemming from the index date) was of 82.2% at three; 81.7% at six, and 80.8 at twelve months in group A, while of 93.3% at three; 91.7 at six, and 90.6 at twelve months in group B, respectively. In other words, an average of 132 doses (approximately corresponding to 66 days of treatment) in group A, and to 44 doses were skipped (corresponding to 44 days of treatment) in group B.No relevant side effect was reported in both groups of patients. Transient hoarseness was recorded in 5 patients in group B and in three patients of group A, while transient tachycardia was recorded in two patients of group A and in one patient in group B.A variable degree of airway obstruction related to a variable extent of underlying airway inflammation usually characterizes bronchial asthma. In persistent mild-to-moderate asthma a therapeutic strategy based on the regular assumption of ICS, or ICS/LABA is recommended in order to prevent and/or avoid the occurrence of asthma exacerbations.Results of different treatments can be affected by several factors, such as: the pharmacological peculiarities of the molecules prescribed; the daily regimen ; the usability of inhaler devices adopted for the drug(s) delivery; the patient’s adherence to treatment; the existence of comorbidities; the cost of treatment, and the indices considered in the study .The present observational, retrospective, matched study, aimed to compare outcomes achievable in mild-to-moderate asthma patients assuming FF/V once-daily or BDP/F for 12-months, represents the very first head-to-head comparison between these two LABA/ICS combinations in asthma to our knowledge, and here clinical outcomes are assessed over a long-term period.Actually, in a previous pharmaco-economic study, a short-term cost-analysis carried out over twelve weeks suggested the superiority of FF/V 99/22 once-daily via Ellipta when compared to DP/F b.i.d. via Nexthaler . This su1% predicted significantly also in the present study. The net improvement achieved in group B proved once again significantly higher, but also progressive, according to a time-dependent trend, particularly over the second six months of treatment.In terms of lung function, both treatments confirmed effective in improving FEVA novel evidence came out from the present study: beyond lung function, all main clinical outcomes proved clearly in favour of FF/V once-daily when compared to BDP/F b.i.d. Actually, the long-term treatment likely contributed to enhance and magnify the extent of FF/V clinical convenience, previously only suggested during a short-term therapeutic strategy . In partEven if the two compared ICS/LABA combinations are active and regarded as equally effective in persistent asthma –10, none2- receptors in favour of Vilanterol represent crucial aspects from this point of view [On the other hand, Formoterol and Vilanterol as well as Beclomethasone dipropionate and Fluticasone furoate are characterized by different pharmacokinetics and pharmacodinamics –17. The  of view –17. Actu of view , these aper sé to explain the better and the time-dependent asthma control achievable with this treatment. To note that this result should be regarded as independent of the inhaler devices used by patients  as both characterized by a quite similar handling and an equal number of steps needed for inhalation actuation [The once-daily assumption has been supposed to foster the patients’ adherence during long-term therapeutic strategies , 19, 20.ctuation , 22.Finally, hospitalization and exacerbation rates, as well as patients’ absenteeism and medical referrals represent the main components of asthma annual costs –22. The The present study has some limits. One is represented by the relative small number of subjects included, even if well matched by means of the propensity score. Moreover, the study consists in a mono-centric investigation, even though patients were from all Italian regions, anonymously selected. On the other hand, some points of strength just consist in the automatic selection of subjects from a unique database, associated to the use of the propensity score matching method which assures a strictly objective system for comparison between the two subjects’ samples. Finally, the patients’ adherence to treatments was not calculated according to the usual criteria adopted during usual clinical trials. Anyway, the registration of the number of doses monthly remaining in both devices (each provided with a precise dose-counter) rendered information collected in real life pretty reliable and acceptable.The present study showed that the once-daily inhalation of combined Fluticasone furoate/Vilanterol 92/22 μg once-daily for twelve months consents an enhanced and time-dependent efficacy in terms of lung function and of all main clinical outcomes when compared to BDP/F 100/6 μg b.i.d. in mild-to-moderate asthma. Stemming from the extent of the systematic improvement of clinical outcomes achieved over the FF/V 92/22 μg treatment, the corresponding long-term economic consequences are easily and quantitatively presumable.


PtCu– and PdCu–mordenite allow for isothermal reaction at 200 °C for the stepwise methane to methanol conversion with higher yields under isothermal conditions than after high temperature activation. PtCu- and PdCu–mordenite allow for isothermal reaction at 200 °C for the stepwise methane to methanol conversion with comparably high yields. In contrast to traditional Cu-zeolites, these materials are more reactive under isothermal conditions than after high temperature activation. Methane is an abundant feedstock that can be extracted inexpensively from shale gas and other sources.I/CuII redox couple.–1 for Cu-MOR and 125 μmol g–1 for Cu-CHA.–1, assumed to be achieved over the mono-μ-oxo site.–1.Methane monooxygenase enzymes transform methane to methanol at low temperature with active dicopperTo date, most studies use monometallic copper-zeolites based on different topologies and material compositions for the transformation and these have so far not showed increased activity under isothermal conditions. Based on the well-known interplay between copper and platinum group metals,8.5 with 4.3 wt% Cu was prepared by three-fold ion exchange of Na-MOR (Si/Al = 8.5) using Cu(OAc)2. A series of PtCu-MOR8.5(Pt/Cutheory) materials was prepared by suspending Cu-MOR8.5 in a solution containing the Pt(NH3)4(NO3)2 precursor, subsequently water was evaporated to yield the final material. Measured Pt and Cu contents can be found in Table S1.8.5(0.35) was prepared from a parent Cu-MOR8.5 with 2.4 wt% Cu. The Pt/Cu- and Pd/Cu-ratios were more thoroughly evaluated using samples with Si/Al = 6. Materials were prepared by impregnation of an analogously prepared Cu-MOR6. H2PtCl6 dissolved in water or PdCl2 dissolved in an aqueous HCl solution (30 wt%) were used, allowing for rather predictable loading of the platinum over a broad concentration range.Cu-MOR8.5 yielded 0.8 μmol g–1. Note that after an isothermal reaction, Cu-MOR only releases MeOH during extraction with steam at 200 °C  and at 450 °C (30.1 μmol g–1). The low activity of copper-zeolites at 200 °C activation and reaction is in agreement with the inactivity of Cu-ZSM-5 under similar conditions.6 (1 wt% Pt) and Pd-MOR6 (2 wt% Pd) yielded 4.5 μmol g–1 and 2.0 μmol g–1  using PtCu-MOR8.5(0.35) under isothermal conditions at 200 °C. On the other hand, the yield decreased with increasing platinum content after 450 °C activation from 30.1 μmol g–1 for Cu-MOR8.5 to 2.7 μmol g–1 for PtCu-MOR8.5(0.35). At an intermediary activation temperature of 350 °C the platinum-rich zeolites were less active, but the effect is not as pronounced as observed at 450 °C activation. Thus, PtCu-MOR with increasing Pt/Cu-ratio provides higher activity under isothermal conditions, while the opposite is true after 450 °C activation. These results show the positive effect of platinum for the isothermal conversion. The beneficial effect of added platinum becomes even more obvious, when the yield is normalized to the amount of copper present in the material , whereafter the yield decreased with increasing Pt/Cu-ratio to yield 3.3 μmol g–1 at Pt/Cu = 1.1. Thus, there is an optimal Pt/Cu-ratio providing maximum reactivity. At low Pt/Cu-ratios the effect of platinum introduction is low, while at higher Pt/Cu undesired agglomerates can be formed during activation or reaction. In analogy to Cu-MOR,6 reached a yield of 22.0 μmol g–1, which further increased to 31.7 μmol g–1 for PtCu-MOR6(0.12), after which it decreased to minimum of 6.1 μmol g–1 for PtCu-MOR6(0.4). At 36 bar, the yield decreased monotonously from 43.1 μmol g–1 for the parent Cu-MOR6 until finally no methanol was extracted for PtCu-MOR6(1.1). These results indicate that an overall beneficial result can be obtained using higher methane pressures (e.g. for PtCu-MOR6(0.12)), yet increasing pressures and Pt-loading result in lower yields compared to the parent Cu-MOR6.Next, the effect of Pt/Cu-ratio was studied at different methane pressures after activation in oxygen and reaction with methane at 200 °C followed by off-line extraction with liquid water to test the performance for the isothermal conversion . Parent –1 for PdCu-MOR6 (0.3). A slightly lower yield (6.5 μmol g–1) was obtained for PdCu-MOR(1.0), while Pd/Cu = 1.2 and 3.0 yielded ∼2.4 μmol g–1. The presence of Pd in PdCu-MOR6(0.3) was confirmed by EDX  and PdCu-MOR6(0.30) revealed the formation of a small amount of formaldehyde as only side-product, which is formed during the extraction of PdCu-MOR6(0.30)  and PdCu-MOR6(0.30) was compared after activation in oxygen using XPS. The noble metals are enriched on the surface with a Pt/Cu ratio of 0.5 and a Pd/Cu ratio of 0.7 (Table S36(0.30) is somewhat higher than that of PtCu-MOR6(0.15), which can be caused by the higher metal loading. Note that activation at 450 °C resulted in a lower yield of 20 μmol g–1 for PdCu-MOR6(1.0) compared to 27 μmol g–1 for the parent Cu-MOR6 under identical conditions  Fig. S5. The sur Table S3. The rat Table S4. Thus, t8.5 materials after activation at 200 °C (8.5(0.02) showed larger particles (ø = 2.2 nm) compared to PtCu-MOR8.5(0.08) (ø = 1.1 nm), PtCu-MOR8.5(0.20) (ø = 0.65 nm) and PtCu-MOR8.5(0.35) (ø = 0.80 nm). However, if a sample undergoes 450 °C activation, agglomerated particles were observed, which can be responsible for the low activity after high temperature activation 4(NO3)2 and dried at 110 °C. The materials mostly showed an additional reflection at about 2θ = 13° and further additional peak in case of H2PtCl6, which consequently comes from a platinum-containing phase  and also PdCu-MOR6(0.3) over four cycles (–1 for PtCu-MOR6(0.15) and 6.5–7.8 μmol g–1 for PdCu-MOR (0.3) was achieved. These yields are similar to those obtained by off-line extraction in liquid water, which are 8.6 μmol g–1 and 7.2 μmol g–1, respectively. The materials are recyclable for at least four cycles. Active PtCu-MOR6(0.15) after four successive cycles at 200 °C, however, showed rather small particles with a mean particle diameter of 3.2 nm without large agglomerated nanoparticles (Fig. S14Next, the recyclability of the materials was tested. Extraction in liquid water of PtCu-MOR Fig. S13. To prevr cycles . A stabl Fig. S14.Conclusively, we have pioneered the use of PtCu-MOR and PdCu-MOR materials for the stepwise isothermal conversion of methane to methanol. Upon platinum- or palladium-introduction into the system, the activity after activation and reaction at 200 °C significantly increased. In contrast to all prior approaches for the stepwise methane to methanol conversion, these materials are more active after activation at low temperature than after high temperature activation. Although promising results were obtained, the best yields of Cu-zeolites after high temperature activation are still to be reached under isothermal conditions. The character of the bimetallic Cu-oxo clusters facilitate reduction of the Cu-oxides, which enables reactivity of less activated sites. These bimetallic oxide materials allow for higher activity than other reported materials under isothermal conditions at ambient pressures. We strongly believe that this study will stimulate new research and eventually such bimetallic oxide materials can be a further step to finding the holy grail of catalysis.There are no conflicts to declare.Supplementary informationClick here for additional data file.


O-β-d-glucopyranosyl α,4,2′,4′,6′-pentahydroxy–dihydrochalcone (1) is a powerful antiglycation compound previously isolated from Eysenhardtia polystachya. The aim was to investigate whether (1) was able to protect against diabetic nephropathy in streptozotocin (STZ)-induced diabetic mice, which displayed renal dysfunction markers such as body weight, creatinine, uric acid, serum urea, total urinary protein, and urea nitrogen in the blood (BUN). In addition, pathological changes were evaluated including glycated hemoglobin (HbA1c), advanced glycation end products (AGEs) in the kidney, as well as in circulation level and pro-inflammatory markers ICAM-1 levels in diabetic mice. After 5 weeks, these elevated markers of dihydrochalcone treatment  were significantly (p < 0.05) attenuated. In addition, they ameliorate the indices of renal inflammation as indicated by ICAM-1 markers. The kidney and circulatory AGEs levels in diabetic mice were significantly (p < 0.05) attenuated by (1) treatment. Histological analysis of kidney tissues showed an important recovery in its structure compared with the diabetic group. It was found that the compound (1) attenuated the renal damage in diabetic mice by inhibiting AGEs formation.Previous studies have shown that accumulation of advanced glycation end products (AGEs) can be the cause of diabetic nephropathy (DN) in diabetic patients. Dihydrochalcone 3′- Type 2 diabetes mellitus is a cause of mortality due to complications such as diabetic nephropathy (DN), which is the main cause of end-stage renal failure . DN is cEysenhardtia polystachya  at m/z 468.4130, C21H24O12 requires 468.4110; UV λ max (MeOH) nm 260, 303, 375; IR νmax (KBr) cm−1: 3448, 2932, 1645, 1586, 1519, 1441, 1182, 1071; The 1H-NMR espectra  showed the following data: δH 2.75 , 2.36 , 5.25 , 7.49 , 6.65 , 9.10 , 6.09 , 10.60 , 12.24 , 13.50 ; Glu: δH 4.80 , 3.87 , 3.54 , 3.28 , 3.20 , 3.76 , 3.45 ; 13C NMR  δC: 128.7 (C-1), 131.6 , 118.2 , 160.7 (C-4), 44.30 (C-α), 78.97 (C-β), 192.94 ; 109.4 (C-1′), 164.2 (C-2′), 102.6 (C-3′), 166.2 (C-4′), 111.5 (C-5′), 165.7 (C-6′); Glu: δC 108.6 (C-1″), 72.3 (C-2″), 78.0 (C-3″), 69.7 (C-4″), 76.8 (C-5″), 60.4 (C-6″).The study was conducted on healthy adult male C57BL/6J mice, weighing about 25–30 g. Before and during the experiment, animals were fed a standard laboratory diet  with free access to water. Mice were procured from the bioterium of ENCB and were housed in microloan boxes cages in a controlled environment (temperature 25 ± 2 °C). Animals were allowed to acclimate for a period of three days in their new environment prior to the study. Before commencing the experiment, litter in cages was renewed three times a week to ensure hygiene and maximum comfort for the animals. The experiments reported in this study followed the guidelines stated in “Principles of Laboratory Animal Care” . All animal procedures were performed in accordance with the recommendations for the care and use of laboratory animals (756/lab/ENCB).After 15 min of administrating an intraperitoneal injection with 120 mg/kg nicotinamide , mice were made diabetic by administering a single intraperitoneal injection of freshly prepared streptozotocin (STZ) (60 mg/kg b.w. i.p.) in 0.1 M citrate buffer at pH 4.5. The animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycaemia. After 10 days of diabetes development, moderately diabetic mice having persistent glycosuria and hyperglycaemia (blood glucose > 200 mg/dL) were used for further experimentation . After tForty-eight mice were divided into eight groups of six animals and had free access to water and food, and then animals were treated for five weeks as follows: Group 1: normal mice, Group 2: diabetic control mice, Group 3: Dihydrochalcone (25 mg/kg/day p.o) treated diabetic mice, Group 4: Dihydrochalcone (50 mg/kg/day p.o) treated diabetic mice, Group 5: Dihydrochalcone (100 mg/kg/day p.o) treated diabetic mice, Group 6: Mentformin (AG) (200 mg/kg/day p.o) treated diabetic mice as a standard drug.g and stored at −80 °C for further assays. After the kidneys were removed, weighed and washed with phosphate buffer saline (PBS pH 7.4). The right kidneys were homogenized in ice-cold PBS, and stored in liquid nitrogen for further biochemical and molecular assays. The left kidneys were removed and used for histopathological examination.After 5 weeks of treatment, diabetes mice were placed inside individual metabolic cages for 24 h and urine was collected. Body weight, kidney weight, food, and water intake were recorded; after 12 h fasting, the blood samples were collected from the tail vein. The animals were then sacrificed by asphyxiation using carbon dioxide. The serum was centrifuged for 20 min at 877× The plasma glucose concentration was determined using an enzymatic colorimetric method using a commercial kit . Total urinary protein levels were measured using a Rat Urinary Protein Assay Kit . In serum, creatinine was evaluated using a QuantiChrom™ Protein Creatinine Ratio Assay Kit , urea, uric acid and blood urea nitrogen test (BUN) were measured using Assay Kits  according to the manufacturer’s instructions. Kidneys were dissected and weighed (wet weight).g at 4 °C for 10 min to remove packed cells and plasma. Using two volumes of water packed cells were lysed. Then was added one volume of carbon tetrachloride to the hemolysate, and refrigerated at 4 °C overnight. Lysate was centrifuged at 27,000× g at 4 °C, for 30 min supernatant removed and used to analysis. Colorimetric estimation was measured according to the procedure of Parker et al. [Blood samples were obtained by ocular venipuncture into tubes with EDTA and centrifuged at 800× r et al. . One mL g and the pellet obtained was resuspended in 2 mL sucrose and centrifuged again then both supernatant obtained were mixed. The proteins were precipitated by adding trichloroacetic acid (TCA) in equal volume and centrifugated at 5 °C at 900× g, then protein pellet was added 1 mL methanol to eliminate the lipid fraction. Then, washing the insoluble protein using 10% cooled TCA and centrifuged: after the residue was solubilized in 1 mL of 1 N NaOH and the AGEs concentration was evaluated fluorometrically with an emission at 440 nm and excitation at 370 nm, and the results were indicated as relative fluorescence units (RFU)/mg protein [The kidneys were homogenized in 2 mL of 0.25 M sucrose, followed by centrifugation at 5 °C with 900×  protein .Mouse plasma was centrifuged to collect supernatants. AGEs levels in plasma were used to determine Absolute Fluorescence  and Advanced Glycation Index   using a After 5 weeks of treatment, frozen cortex was chopped on ice, then flushed through a disposable filter  with cold saline solution and collected. The solution was then flushed through filter (70-mm). The glomerular solution was obtained by inversion of the filter (70-mm), and afterward was flushed with a cold saline solution, followed by spinning at 6000 rpm at 4 °C for 15 min. The supernatant was eliminated and the pellets were suspended in 1 mL of Trizol. The presence of glomeruli was supported in a microscopy. Protein obtained from kidney cortex were used to evaluate the level of the expression of (ICAM)-1  according to ELISA kit instructions.Kidney fragments were fixed in 10% neutral buffered formalin solution, dehydrated in ethanol, embedded in paraffin, and sectioned at 5 μm thickness using a rotary microtome. After dehydration, sections were stained with hematoxylin and eosin (HE). To evaluate the histopathological damage, each image of sections was examined for microscopic observations .p < 0.05.The data are presented as mean ± standard deviation. The significance of the differences was analyzed by one-way ANOVA with the Dunnett’s test using GraphPad Prism 7.0 for Windows . The value of statistical significance was established at


Myzus persicae (Sulz.) (Hemiptera: Aphididae) during probing and settling. The choice and no-choice tests , respectively) revealed that stereochemistry of thujone was important for biological activity (β-thujone caused changes in aphid behavior while α-thujone did not) and that cyclopentane ring modifications and functional groups addition gave derivatives that possessed stronger and more durable deterrent effects. The most effective modification was the incorporation of a lactam moiety into the β-thujone molecule. Application of β-thujone lactam limited aphid settling for at least 24 h, caused restlessness in aphids and a delay or failure in reaching phloem phase by M. persicae. β-Thujone lactam can be considered a deterrent of medium potency with activity expressed at preingestive phase of aphid probing. Other compounds did not restrain aphid stylet penetration in non-phloem tissues but slightly limited sap ingestion , and restrained aphid settling for a period of less than 24 h . Thujone is a natural biologically active monoterpene ketone component of essential oils of numerous plants. The aim of the study was to evaluate the effect of β-thujone and β-thujone derivatives bisulfite adduct, lactone, oxime, and lactam application on behavior of  Silica gel  column chromatography with a mixture of hexane, acetone and chloroform (3:1:1 v/v) as eluent was used to purification of lactam (6). Gas chromatography analyses were carried on an Agilent 7890A  equipped with FID detector and H2 as carrier gas. Substrates and products of reaction mixtures were separated on capillary columns: HP-5 (30 m × 0.32 mm ID × 0.25 μm film) with followed oven temperature program 110 °C, 200 °C (10 °C min−1), 300 °C (30 °C min−1) (3 min) for lactam (6) and CP-ChiralSil-L-Val (25m × 0.25 mm ID × 0.12 μm film) with followed oven temperature program 80 °C, 120 °C (1.5 °C min−1) (2 min), 200 °C (15 °C min−1) (3 min) for oxime (5). 1H, 13C and two-dimensional  NMR spectra were recorded for CDCl3 on a Bruker Avance DRX 300 MHz. Chemicals shifts were referenced to the residual solvent signal . Melting points were determined on a Boetius apparatus.Reaction reagents: hydroxylamine hydrochloride and eviously ,31. Anal2)  dissolved in methanol (5 mL) was added to 7 mL of aqueous solution of hydroxylamine hydrochloride  and sodium acetate . The reaction mixture was stirred for 48 h at room temperature, followed by the oxime (5) which was extracted with diethyl ether (3 × 25 mL). The combined organic phases were washed by aqueous saturated solution of sodium bicarbonate, dried on anhydrous MgSO4, and concentrated under vacuum. The pure crystal of β-thujone oxime (5)  was obtained. The physical and spectral data of this product are given below:The β-thujone (β-Thujone oxime (5). Colorless crystals; m.p. 48–50 °C, lit. 53–54 °C heptan-4-one). Colorless crystals, m.p. 85–88 °C, lit. 89–90 °C [1H-NMR  δ: 0.31 , 0.52 , 0.91 and 0.94 2CH-), 1.12 2CH-), 1.19 , 2.36 , 2.46 , 3.85 , 6.08 ; 13C-NMR  δ: 9.11 (C-7), 18.60 and 19.09 ((CH3)2CH-), 21.59 (CH3-2), 23.36 (C-1), 24.04 (C-6), 31.02 (C-5), 35.55 ((CH3)2CH-), 46.16 (C-2), 172.32 (C-4).89–90 °C ; 1H-NMR M. persicae was maintained on Chinese cabbage Brassica rapa L. ssp. pekinensis L. M. persicae probing behavior was monitored on B. rapa ssp. pekinensis (the plant it was reared on), which was considered as a control plant. Aphids and plants were kept in the laboratory at 20 °C, 65% relative humidity, and L16:8D photoperiod. The stock colonies were kept as follows: an adult aphid was placed on a plant and allowed to reproduce for 24 h. Then the female was removed and the remaining larvae were allowed to develop. Young, 2–3 days old  viviparous apterous females were selected for experiments. The plants used in the bioassays were 5–6 weeks old.The laboratory culture of M. persicae were placed between the leaves at the center of the Petri dish (the distances between two leaves approximately 2.0 cm). Aphids were offered a choice between treated and control leaves. Aphids that settled on each leaf were counted at 1, 2 and 24 h intervals after the beginning of the experiment. This experiment was replicated 8 times for each treatment . The number of aphids that settled on treated leaves was compared to the number of aphids that settled on control leaves 1, 2, and 24 h after treatment, separately for each time point; data were analyzed using Student t-test (p ≤ 0.05).Aphids settle on a plant only when they accept it as a food source. Therefore, the number of aphids that settle and feed on a given substrate is a good indicator of host quality. A conventional settling choice test was conducted to evaluate the deterrent activities of studied extracts after Powell et al. . This biAphid probing behavior was monitored using the technique of electronic registration of aphid probing in plant tissues, known as EPG . The anawww.statsoft.com.All EPG parameters describing aphid probing behavior were calculated manually and individually for every aphid using the EPG analysis Excel worksheet created for this study by one of the authors (Anna Wróblewska-Kurdyk). Subsequently, the mean and standard errors were determined. Aphid behavior on leaves treated with β-thujone was compared to aphid behavior on control plants. Aphid behavior on leaves treated with β-thujone derivatives was compared to aphid behavior on control plants and on β-thujone treated plants, separately. Mann–Whitney U-test was used for these comparisons. All statistical calculations were performed using StatSoft, Inc. (2014) STATISTICA , version 12,


PPARγ, Pgc-1α and Fndc5 genes in gastrocnemius muscle and heart tissue of male C57BL/6 mice.Previous studies have revealed the inductive effect of branched-chain amino acids (BCAAs) catabolism on fatty acid oxidation and metabolism, especially in muscle cells. In the present investigation, we have attempted to address whether a combination of BCAAs supplement consumption with aerobic exercise could elaborate the expression of Thirty-six young male mice with an average weight of 18 ± 2 g were selected. Mice were randomly assigned to 6 groups: 20 mg/mL of BCAAs consumption with simultaneous exercise-training, 60 mg/mL of BCAAs consumption with simultaneous exercise-training, exercise-trained with no BCAAs consumption group, 20 mg/mL BCAAs without exercise-training, 60 mg/mL BCAAs without exercise-training, and untrained mice without BCAAs consumption.Fndc5, PPARγ, Pgc-1α gene expression in skeletal muscles although, circulating Irisin levels remained unchanged (p < 0.05). Interestingly, plasma urea and lactate levels were significantly increased in 60 mg/mL BCAAs administrated mice which performed exercised (p < 0.05). Two-way analysis of variance (ANOVA) was used to examine significant difference between groups and sedentary group.The findings showed a combination of 20 mg/mL BCAAs with aerobic exercise significantly increased Fndc5, PPARγ, Pgc-1α in gastrocnemius muscle similar with counterparts in heart tissue. Of note, higher serum irisin levels were detected after 20 mg/mL BCAAs supplementation coincided with the exercise.Results showed inductive effect of 20 mg/mL BCAAs on expression levels of An Overview on supplemantaion of branched chain amoinoacids on metablism of skeletal muscle and heartThe online version of this article (10.1186/s12986-018-0298-3) contains supplementary material, which is available to authorized users. Sirtuin 1 (Sirt1) expression besides a reduction in oxidative damage  was implemented  was more than exercise (Ex)   Fig. . Importated Fig. . Data reEx) Fig. . Notablycle Fig. . To our Ex) Fig. . InteresARγ Fig. . HoweverARγ Fig. . MoreoveARγ Fig. .Fig. 7Mo. Correlation of irisin levels with Fndc5 revealed that circulating irisin had positive associations (r = 0.65 and 0.7 for muscle and heart respectably) with the expression of this gene  supplementation (20BCAA) was beneficial to increase the transcript level of Fndc5. However, higher amount of BCAAs (60 mg/mL) did not change Fndc5 transcript level even though the expression of mitochondrial genes decreased. This is possibly due to triggering an increase in energy expenditure [Fndc5 gene expression in the skeletal and heart muscles remains to be obscure.Physical exercise is a well-recognized factor that could increase pression , 21, 30.enditure , 31. DetFndc5 transcription was evident in our data. We found this interaction had positive effect on Fndc5 expression and metabolism. Also, we indicated protein content of FNDC5 decreased in 60BCAA as compared with sedentary group. Interestingly, we observed the protein level of FNDC5 in the skeletal muscle was higher in exercised mice in contrast with 20BCAA group. Presumably, this incompatibility between mRNA and protein levels of FNDC5 in the mice is related to the activity of some regulators which affect protein translation [Additionally, the synergetic effect of exercise interaction with 20 mg/mL BCAAs supplementation (20BCAA/Ex) on induction of nslation , 33.Fndc5 gene expression [Fndc5 of skeletal muscle in trained individuals [Fndc5 is irisin, which affects total body energy expenditure, as well as browning of WAT (white adipose tissue) [Fndc5 and irisin levels in plasma [Our findings were in good agreement with Aydin et al. which reported a positive association with metabolism and pression . Unlike ividuals . This di tissue) . There an plasma , 21, 35.Pgc-1α, is a co-activator of various nuclear receptors as well as transcription factors. Previous study has indicated that Pgc-1α is involved in regulation of energy metabolism, as well as thermogenesis, in skeletal muscle. In skeletal muscle, Pgc-1α regulates not only the mitochondrial function but also metabolic activity of the cell [Pgc-1α [the cell , 37. Tad [Pgc-1α . However [Pgc-1α .Sirt 1 PPARγ, Pgc-1α, Tfam, Cox4i1, ATP5a1, ATP5b and Fndc5 involved in mitochondrial biogenesis and for the first time we have shown that endurance exercise cold increase ATP5a and ATP5b mRNA expression. In present study, we have demonstrated that Sirt 1 and Fndc5 mRNA had a similar trend and they had a positive correlation with each other. PPARγ expression was observed in trained mice, emphasizing that exercise could enhance PPARγ gene expression [Sirt1 up-regulation and life span extension, and increasing in mitochondrial biogenesis, and ROS production decrease in the skeletal muscle of male mice [In this study we have examined the effect of exercise and BCAAs supplementation on transcript levels of pression . Our resale mice . One of Pgc-1α and PPARγ expression [Pgc-1α, Tfam and Sirt1 expression in skeletal muscle. Furthermore, BCAAs supplementation is responsible for antioxidant defense and reduction of ROS [Pgc-1α expression triggers the interaction of Pgc-1α with PPARγ, which elaborates the oxidative capacity of mitochondria. On the other hand, Pgc-1α mediates BCAAs metabolism in skeletal muscle through increasing the transcript levels of branched chain amino acid transaminase 1(Bcat1), Bcat2 and branched-chain alpha-keto acid dehydrogenase (Bckdh) [Fndc5 through unidentified cascade.According to our knowledge endurance exercise increases the capacity of skeletal muscle for aerobic metabolism through an increase in mitochondrial capacity to generate ATP via oxidative phosphorylation , presumapression . Furthern of ROS . Enhance (Bckdh) . Increme (Bckdh) , 39. FinFndc5, PPARγ, Pgc-1α similar with heart tissue. In addition, Irisin levels were increased at most level after 20 mg/mL BCAAs supplementation which coincided with the exercise (20BCAA/Ex). However, overloading of higher amount of BCAA may have adverse these effects on transcript levels of Fndc5. However, physiological aspects of this finding needs to be confirmed in further studies.Our present study suggests that consuming 20 mg/mL BCAAs with exercise (20BCAA/Ex) had positive effect on expression level of gastrocnemius muscle Additional file 1:Figure S1. Comparative study on amino acid content of proteins in human and mouse. As shown, the assessed proteins were the same as were obtained by STRING analysis (Fig. sis Fig. . Dark co


To examine atypical manifestations of Kawasaki disease (KD) in children.Cardiovascular complications during acute KD are a major contributor to its mortality rate. It can involve the pericardium, the myocardium, the endocardium, and/or the coronary arteries; however, cardiomegaly and nephrotic syndrome (NS) during the acute stage of KD have seldom been reported.Two children, each with a fever lasting more than 5 days, were diagnosed with cardiomegaly using echocardiography in the early phase of Kawasaki disease (within 2 weeks). Case 1 was misdiagnosed with NS because of the proteinuria, hypoalbuminemia, and edema present at the onset of the disease.A diagnosis of incomplete KD was based on a constellation of clinical manifestations and symptoms and was supported by laboratory results.Intravenous immunoglobulin (IVIG) and aspirin were administered, supplemented with and without supplemental steroid therapy (case dependent).The clinical manifestations and syndromes of the two cases were completely resolved and their heart size restored to normal within 2 weeks, with no evidence of coronary artery lesions (CAL).Physical findings and manifestations are atypical in incomplete KD. Cardiomegaly and nephrotic syndrome can be an early manifestation of KD; cardiomegaly, especially, should be recognized as a possible manifestation of the acute stage of KD. Furthermore, these symptoms can be rapidly relieved by treatment with IVIG, with or without supplemental steroid therapy. The average annual incidence rate of KD in Japan is 264.8 per 100,000 children under 5 years of age, compared to only 11.4, 5.4, and 7.4 per 100,000 in Finland, Norway, Sweden, respectively, and 20.8 in USA.–4 KD can damage multiple organs; induce coronary artery lesions (CAL); and/or cause carditis, hepatitis, arthritis, central nervous system disease, KD shock syndrome, muscle and kidney damage, and hyponatremia. Cardiovascular complications during acute KD are a major contributor to its mortality rate. It can involve the pericardium, the myocardium, the endocardium, and/or the coronary arteries; however, cardiomegaly and nephrotic syndrome (NS) during the acute stage of KD have seldom been reported. Here, we report cases of two Chinese children with cardiomegaly in the acute stage of KD, one of whom experienced concomitant NS at the onset of the disease.Kawasaki disease (KD) is an acute, febrile, systemic vasculitis of unknown etiology that predominantly affects children <5 years of age.This study retrospectively analyzed the clinical data of 2 children with KD who were admitted between 2016 and 2017 in the West China Second University Hospital and followed up for 6 months. This study was approved by the Ethics Committee of the West China Second University Hospital. Patients’ parents have provided informed consent for publication of the case.A total of 2 children with cardiomegaly in early phase of Kawasaki disease are included in this series.29/L (3.6–9.7 × 109/L), percent of neutrophil granulocyte (N%) 66% (23.6–75%), hemoglobin (Hb) 104 g/dL (110–146 g/L), platelet count (PLT) 187 × 109/L (100–450 × 109/L), C-reactive protein (CRP) 62.54 mg/L (0–8 mg/L), triglyceride (TG) 3.81 mmol/L (<2.83 mmol/L), serum potassium 2.93 mmol/L (3.5–5.5 mmol/L), serum sodium 138.7 mmol/L (135–145 mmol/L). Urine test: protein was 2+, red blood cell (RBC) and pathocast were negative. Urine and blood cultures were sterile. The physical examination on admission revealed a temperature of 38.6 °C, a respiration rate of 25 beats/min, a heart rate of 114 beats/min, a blood pressure of 99/68 mm Hg, and mildly irritated, red skin rashes over the body with normal skin between. The eyelids and toes were edematous. The bilateral conjunctiva had hyperaemia without tumefaction of the lymph node. Additionally, the patient displayed cracked lips, a strawberry tongue, perianal desquamation, and abnormalities at the Bacilli Calmette-Guerin inoculation site. The abdomen was distended  and positive for shifting dullness. The physical examination of the lung, heart, and nervous systems was normal. The laboratory evaluation of our hospital on admission was as follows: WBC 5.4 × 109/L, N% 69.3%, Hb 101 g/L, PLT 152 × 109/L, CRP 40.0 mg/L (0–8 mg/L), erythrocyte sedimentation rate (ESR) 2 mm/hour (<21 mm/hour), alanine aminotransferase (ALT) 55 U/L (21–72 U/L), aspartate aminotransferase (AST) 67 U/L (17–59 U/L), lactic dehydrogenase (LDH) 994 U/L (313–618 U/L), albumin 24.7 g/L (35–50 g/L), serum sodium 131.9 mmol/L (137–145 mmol/L), serum potassium 3.5 mmol/L (3.5–5.1 mmol/L), and serum calcium 2.01 mmol/L (2.1–2.55 mmol/L). Routine urine: protein 2+, RBC 0–3/hp, WBC 0–3/hp, pathocast 1+. 24-hours proteinuria: 0.5 g/24 h (<0.14 g/24 h). Urine and blood cultures were sterile. Coagulation function test: International Normalized Ratio (INR) 1.16 (0.8–1.5), prothrombin time (PT) 14.3 seconds (9.4–15.4 seconds), activated partial thromboplastin time (APTT) 60.0 seconds (20.6–40.6 seconds), fibrinogen (Fg) 166 mg/dL (200–400 mg/dL), D-dimer 6.11 mg/L (<0.55 mg/L), fibrinogen degradation product (FDP) 20.9 μg/mL (<5 μg/mL). The assessment of immune function: IgG 23.90 g/L (3.41–19.6 g/L), IgA 0.64 g/L (0.19–2.20 g/L), IgM 0.94 g/L (0.43–1.63 g/L), complement C3 0.42 g/L (0.70–2.06 g/L), complement C4 0.27 g/L (0.11–0.61 g/L), CD3 73.9 (39–73%), CD4 46.7 (25–50%), CD8 24.1 (11–32%), CD4/CD8 1.9 (1.5–2.0). Echocardiography showed that the left heart was slightly enlarged (left ventricle (LV) = 36 mm, left atrium (LA) = 22 mm) with normal heart function (ejection fraction (EF) = 62%, patent foramen ovale, normal size of the coronary arteries (CA), (left coronary artery (LCA) = 2.3 mm, Z-score <2; left anterior descending coronary artery (LAD) = 2.4 mm, Z-score <2; left circumflex coronary artery (LCX) = 1.2 mm, Z-score <2; right coronary artery (RCA) = 2.0 mm, Z-score <2), and a small amount of pericardial effusions (PEs)  1.85 × 109/L, the Hb rose to 108 g/L, and the PLT increased to 543 × 109/L. CRP levels returned to normal. Two weeks after discharge, echocardiography revealed that the size of the heart returned to normal without evidence of CAL. The child underwent routine echocardiography, blood tests, and urine tests for 1 year; all follow-up tests were normal.After admission, he was diagnosed with incomplete KD and treated with immunoglobulin (2 g/kg). His abdominal distension and limb edema abated, and his temperature normalized. One day later , his fever returned. After immunoglobulin (1 g/kg) was administered, followed by aspirin (50 mg/kg/day), his temperature returned to normal. On the next day , the echocardiograph indicated that the left heart was less enlarged than before  and without CAL. Routine urine and 24-hours proteinuria tests returned to normal 3 days later , and the aspirin dosage was reduced to 5 mg/kg/day. Within 4 days, his temperature normalized, and the patient was discharged . One week after discharge, the WBC was 5.1 × 1039/L, N% 82%, Hb 98 g/L, PLT 387 × 109/L, CRP 103 mg/L, ESR 73 mm/h, ALT 194 U/L, AST 314 U/L, albumin 29.2 g/L, LDH 1130 U/L, troponin 0.156 μg/L and CK-Mb was normal. Coagulation function test: INR 1.3, PT 16.0 seconds, APTT 36.1 seconds, Fg 469 mg/dL, TT 16.4 seconds. The test of urine routine was normal. Thyroid function: T3 1.00 nmol/L (1.4–3.7 nmol/L), T4 87.7 nmol/L (82–171 nmol/L), TSH 3.615 mIU/L (0.64–6.27 mIU/L), anti-thyroglobulin antibody 104.6 μ/mL (<60 U/mL) and anti-thyroperoxidase antibody 206.7 U/mL (<60 U/mL). Autoantibodies tests showed antinuclear antibodies (ANA) were positive (1:100). Echocardiography showed a slightly enlarged heart   and in tricuspid valve (Vmax = 2.4 m/second), normal heart function (EF = 62%), and normal sized coronary arteries (Z-score <2). Enhanced chest CT test showed a little shadow on the upper lobe of the lungs and stripes on the lower lobe of the left lung, a few pleural effusions, and a slightly enlarged heart shadow.A 10 years and 5-month-old girl was admitted to our hospital for fever for the last 8 days with recent discovery of cardiomegaly. Her highest temperature was 39.2 °C and was accompanied by right submandibular neck lymph node enlargement, abdominal discomfort, and rash. Although she was treated with antibacterial and antiviral drugs for several days in the previous hospital, she still had a high temperature, vomiting, and sickness. In addition, chest radiography found a heart shadow enlargement  for the diagnosis of incomplete KD. Her temperature returned to normal and the erythema disappeared after 2 days. We added a small dose of prednisone (0.5 mg/kg day) and aspirin (3 mg/kg day) for successive treatment. Three days after intravenous immunoglobulin (IVIG), we rechecked the echocardiography, which revealed that the cardiac size was slightly recovered , and the heart function was still normal. One week after IVIG, echocardiography showed a normal cardiac size  without CAL. A reexamination of the blood revealed WBC 7.6 × 104 The patient in case 1 had a fever for 7 days; 3 principal clinical features ; and 4 laboratory findings . The patient in case 2 had fever for 10 days; only 2 principal clinical features ; but 5 laboratory findings . Echocardiography in both patients revealed early phase cardiomegaly, which was completely relieved after IVIG was administered with or without steroid therapy.These two cases were diagnosed with incomplete KD in accordance with the 2017 guideline of KD from the American Heart Association.,6 Young infants  and older children are more likely to experience incomplete KD.,8 Although the prognosis of children with incomplete KD is comparable to those with complete KD, prolonged fever is one of the strongest predictors of CAL. Compared with typical KD, the physical findings of incomplete KD usually lack 2 of the 6 basic symptoms, most frequently cervical lymphadenopathy and polymorphous exanthema; however, characteristic mucous membrane changes are present in both typical and incomplete disease. On the other hand, incomplete KD can present with atypical manifestations, such as pneumonia, nephritis, arthritis, uveitis, myositis, retinal vasculitis, and central nervous system involvement, and NS and cardiomegaly are both rare clinical manifestations that can appear during the acute stage of KD. One case in our article was diagnosed with NS because of proteinuria, hypoalbuminemia, and edema; cardiomegaly was apparent in both patients in this report.An estimated 15–20% of KD patients are diagnosed with incomplete KD, and the incidence rate is thought to be rising over the years.,13; however, cardiomegaly has rarely been reported and the pathogenic mechanism is still unclear. The patient in case 1 had PE with normal levels of CK-Mb and troponin and lacked evidence of myocarditis, but vasculitis was evident because of the kidney involvement. As Okada reported, vasculitis and microvascular hyperpermeability are important events in the pathophysiology of pericardial effusion in KD. The patient in case 2 had mitral regurgitation combined with an increased level of both CK-Mb and troponin, which suggested that the cardiomegaly might be caused by myocarditis. Suzuki also reported that tricuspid and mitral regurgitation are often transient in the acute phase of KD and could be attributed to myocarditis. Crystal suggested that ventricular dilation may have subclinical myocardial involvement; it may be increased in patients on the initial echocardiography but remain within the normal range. Hence, cardiomegaly is a previously unrealized, type of cardiac damage involved in the acute phase of KD, whose etiology may be multi-factorial, involving vasculitis and myocarditis, and should be further studied. Cardiomegaly is not in the current guideline as a diagnostic manifestation; however, we need to be aware of the potential for this type of noncoronary damage when a diagnosis of incomplete KD is being considered.Noncoronary cardiac abnormalities, including PE, valvar regurgitation, left ventricular dysfunction, and aortic root dilation are common in the acute stage of KD Only three articles, including 5 children, have reported NS in the acute phase of KD.–20 In 1985, Lee reported a 3-month-old infant who presented with NS during the acute phase of KD and improved after steroid therapy. Unfortunately, the child died because of a massive myocardial infarction due to coronary aneurysm. Four other patients  recovered completely without any sequelae, and the proteinuria in all of the patients disappeared within 2 weeks with administration of IVIG without steroids, similar to our patient in case 1. The pathogenic mechanism of NS in KD is still unclear, but activation of the immune system (such as T-cell immune-complex mediated kidney injury) is postulated as a possible mechanism.,21,22KD is a systemic vasculitis that can result in kidney injures, including sterile pyuria, acute nephritis syndrome, renal tubular abnormalities, NS, and renal artery lesions (aneurysms and stenosis) that are seldom seen in patients.−1 day−1) was administered for 1 week and then tapered down in a second week. Although the patient in case 1 had PE and cardiomegaly, IVIG was administered without corticosteroids because his inflammation biomarkers were not very high and his ESR and WBC were normal. Echocardiography revealed that both patients’ hearts had recovered within 1 month. According to the 2017 guideline of KD, corticosteroids were recommended in children at high risk for the development of coronary artery aneurysms.,23 Whether PE is a risk factor for the development of CAL is debatable; so the indications for steroid utilization in KD with noncoronary cardiac abnormalities are undefined.The patients’ clinical manifestations resolved and their hearts returned to a normal size after treatment with IVIG and aspirin, with or without steroid therapy. The patient in case 2 experienced a strong inflammatory response evidenced by high levels of CRP, ESR, WBC, and multiple positive antibodies; so low dose prednisone (1 mg kg5Because of the increased rate of cases with incomplete KD, we are seeing more evidence of atypical manifestations. Cardiomegaly and nephrotic syndrome can be an early manifestation of KD; cardiomegaly, especially, should be recognized as a possible heart manifestation in the acute stage of KD. Furthermore, these symptoms are rapidly relieved by treatment with IVIG, with or without supplemental steroid therapy.The authors thank the patient's parents for providing permission to use the information of their children.Conceptualization: Xiaotang Cai.Data curation: Liyuan Wang, Shunli Liu.Investigation: Yongmei Xie.Methodology: Zhiling Wang.Project administration: Xiaotang Cai.Resources: Xiaomei Sun.Supervision: Zhiling Wang.Writing – original draft: Liyuan Wang.Writing – review & editing: Liyuan Wang, Xiaomei Sun, Xiaotang Cai.


The antioxidant enzyme, 3-mercaptopyruvate sulfurtransferase  is localized in the cytosol and mitochondria, while the evolutionarily-related enzyme, rhodanese  is localized in the mitochondria. Recently, both enzymes have been shown to produce hydrogen sulfide and polysulfide. Subcellular fractionation of liver mitochondria revealed that the TST activity ratio of MST-knockout (KO)/wild-type mice was approximately 2.5; MST activity was detected only in wild-type mice, as expected. The ratio of TST mRNA expression of KO/wild-type mice, as measured by real-time quantitative polymerase chain reaction analysis, was approximately 3.3. It is concluded that TST is overexpressed in MST-KO mice. Rhodanese  and 3-Mercaptopyruvate sulfurtransferase MST, EC 2.8.1.2) and are ubiquitous, evolutionarily related enzymes distributed in both prokaryotes and eukaryotes .8.1.2 an. The 5′ MST performs antioxidative function ,15,16,17The study was conducted in accordance with the Declaration of Helsinki, and the all experiments were performed in accordance with the guidelines and regulations for the Care and Use of Laboratory Animals, Nippon Medical School (#28-008). Littermate wild-type and KO mice  were deeg for 10 min at 4 °C, the supernatant was used for TST and MST activity assays and western blot analyses for both enzymes.Livers were excised from three littermate wild-type (#1–3) and three KO mice (#1–3) sacrificed according to the method described above. Mitochondrial and cytosolic fractionations were prepared using a Mitochondria Isolation Kit for Tissue . After an approximately 100 mg portion of each liver was washed with 4 mL of 20 mM potassium phosphate buffer (pH 7.2 at 4 °C) twice, it was cut into small pieces in 800 µL of the same buffer. Each homogenate was prepared using a Potter homogenizer  with a loose-fitted pestle with three strokes, then each fractionation was performed according to protocol 1 described in the kit. Mitochondrial fractions were added to 50 µL of the same buffer, incubated on ice for 30 min, and vortexed. After being frozen at −80 °C for 30 min, they were incubated on ice for 30 min and vortexed. They were then frozen again at −80 °C for 30 min, added to 5 µL of 10% triton X , and incubated on ice for 30 min. After centrifuging at 20,000× MST activity catalyzes the trans-sulfuration from 3-mercaptopyruvate to 2-mercaptoethanol and TST activity from thiosulfate to cyanide. The activities of both enzymes were measured for each mitochondrial fraction obtained according to the method described above. MST and TST activities were measured by a modification of the methods of Vachek and Wood  and SörbMouse livers were excised from littermate wild-type and KO mice, which were sacrificed by the method described above. Each mRNA was isolated using an mRNA Isolation Kit . The mRNA concentrations were measured using a NanoDrop Lite (Thermo Fisher Scientific). The cDNA samples were synthesized using a QuantiTect Reverse Transcription kit : Each mixture contained 1 μg of each RNA template, 2 μL of gDNA Wipeout Buffer, and RNase-free water for a final volume of 14 μL. Synthetic reactions were performed at 42 °C for 2 min. Further reactions were performed on the obtained 14-μL mixture, 1 μL of Quantiscript Reverse Transcriptase (RT), 4 μL of Quantiscript RT buffer, and 14 μL of RT Primer Mix at 42 °C for 15 min, and then 95 °C for 3 min.Mouse TST primers were designed based on the GenBank nucleotide database  of mouseMitochondrial fractions  were used for western blot analysis—each 30-mg sample was loaded into a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis  chamber with a stacking gel. Each sample was added to 2% SDS, 100 mM Dithiothreitol (DTT), 0.1% bromophenol blue, and 10% glycerol in 50 mM Tris HCl buffer , and then heated at 100 °C for 10 min. All reagents were purchased from Wako Pure Chemicals Industries, Ltd. . Separated proteins were transferred to Immuno-Blot polyvinylidene difluoride (PVDF)  with an electrotransfer apparatus .Anti-rat MST rabbit polyclonal antibody was prepared and partially purified as described previously , and shoWestern blot analyses with anti-MST and anti-TST polyclonal antibodies  were performed. Alkaline phosphatase-conjugated goat anti-rabbit IgG  was used as a secondary antibody. A 5-Bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) Color Development Substrate  was used for staining alkaline phosphatase-conjugated probes.Protein concentrations were determined using a Coomassie protein assay kit  with crystalline bovine serum albumin  as the standard.t-test, and p values less than 0.05 were deemed significant. All results have been rounded to no more than three significant figures.The significance of difference between values was estimated with the Student’s −1 and 6.61 ± 0.563 × 10−1 unit/mg, respectively  were 2.65 ± 0.0331 × 102 ng/μL (A260/A280 = 2.02) and 6.11 × 102 ng/μL (A260/A280 = 1.99), respectively. The cDNA synthesis yields for wild-type and KO mice were 1.13 × 103 μg/μL (A260/A280 = 1.85) and 1.15 × 103 μg/μL (A260/A280 = 1.85), respectively.Isolated mRNAs of wild-type and KO mice were measured to be 5.36 × 101 and 1.03 ± 0.158 × 102 bp, respectively . Internal control genes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 1.82 ± 0.119 × 102 and 1.28 ± 0.150 × 102 bp, respectively. Corrected values of TST mRNAs GAPDH  for wild-type and KO mice were 2.43 ± 0.911 × 10−1 and 8.01 ± 1.23 × 10−1, respectively. The results of the revaluation were 3.03 ± 1.14 × 10−1 and 1.00 ± 0.154, respectively  for wild-type and KO mouse were 8.75 ± 3.28 × 10−2 and 7.94 ± 0.122 × 10−1, respectively. The results of the revaluation were 1.10 ± 0.413 × 10−1 and 1.00 ± 0.0153, respectively   and tran− or Cys S-S−), reduction by thioredoxin produced hydrogen sulfide and polysulfide [−), Cys-thiosulfinate (Cys-Sγ-SO2−), and Cys-thiosulfonate (Cys-Sγ-SO3−), reduction of these molecules by thioredoxin was proposed to produce sulfur dioxides [MST functions as an antioxidant ,15,16,17ysulfide . On the dioxides . Hydrogedioxides ; hydrogedioxides . Polysuldioxides ; polysulcis-element of TST gene has not been clarified. Further, TST and MST are family enzymes and both enzymes catalyze the same substrates, although Km and Kcat are different [On the other hand, decrease in hydrogen sulfide, polysulfides, and sulfur oxide or increase in thiosulfate could directly or indirectly regulate a functional protein; TST, a family enzyme of MST could be up-regulated , howeverifferent ,2. Thiosifferent ,2, was a2S and H2Sn in mitochondria.The ratio of TST activity in MST-KO to wild-type mice was approximately 2.5. Real-time quantitative polymerase chain reaction analysis revealed the ratio was approximately 3.3. These facts led us to conclude that TST was overexpressed in the KO mice. Double (MST and TST)-KO mice, therefore, should be produced to investigate the pathophysiology of defects in H