We show here that the rate of hyperpolarized [1-13C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using 13C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-13C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo. However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic.Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1- The redox couples GSSG/GSH and NADP+/NADPH are therefore a reflection of cell redox state, which increase in the face of increased oxidative stress. Increased ROS production is often associated with chemotherapy-induced apoptosis and is an early event in tumor responses to treatment  and NADPH, which counteract the effects of relatively high levels of reactive oxygen species (ROS)eactions . The res and spectroscopic imaging (MRSI) have enabled real time measurement of metabolic fluxes in vivo by increasing the signal-to-noise ratio by more than 104-fold  to [1-13C]ascorbic acid (AA). DHA reduction can occur spontaneously by reaction with GSH or be catalyzed by the GSH-dependent thiol-disulfide oxidoreductases, glutaredoxin  and protein-disulfide isomerase and by the NADPH-dependent enzymes thioredoxin reductase  and 3α-hydroxysteroid dehydrogenase , which is an NADPH-oxidizing agent    , 27. We ne (BSO) , led to n tumors . We showa). EL4 murine lymphoma cells were incubated for 30 min with either 50 μm PMS  and by incubation with 11 mm [U-13C]glucose 30 min after oxidant treatment and then 30 s later, analyzing label incorporation into 6PG using LC-MS/MS (e–g). Both methods showed a similar and rapid increase in PPP flux with all of the oxidants.First we showed that DHA produced the same rapid increase in PPP flux as the oxidants hydrogen peroxide and PMS a. EL4 mu0 μm PMS , 1 mm hyperoxide , or 11 me . However, the increase in 6PG labeling, measured in tumor extracts in animals injected with [U-13C]glucose (b) or measured using non-invasive localized 13C MRS measurements in vivo in animals injected with hyperpolarized glucose (c and d), was not significantly different between untreated mice and mice injected with DHA. Unlike EL4 cells, the DHA-treated tumors showed no change in the GSSG/GSH ratio compared with untreated tumors; however, there was a significant increase in the activity of the PPP enzyme, glucose-6-phosphate dehydrogenase (G6PDH)  , consist (G6PDH) .13C]glutamine showed no significant increase in lactate C2 and C3 labeling following DHA treatment (C2 labeling was 0.18 ± 0.03 μmol min−1 g tumor−1 in control tumors (n = 4) versus 0.30 ± 0.09 μmol min−1 g tumor−1 in DHA-treated tumors (n = 4), p = 0.26).Flux of glutamine carbon via malic enzyme, to lactate, also produces NAPDH for glutathione reduction . HoweverThese experiments have shown that in using DHA as a probe of the capacity of a cell to resist oxidative stress, it results in an increase in the GSSG/GSH ratio, as it is reduced to AA, and a rapid increase in PPP flux. Next we asked what would happen to the rate of DHA reduction in a tumor cell that had been oxidatively prestressed. We chose to do this by depleting the glutathione pool because this would also allow us to examine how the rate of DHA reduction was related to GSH concentration.a) , which was increased a further ∼2.2-fold by the addition of DHA . 13C label incorporation into 6PG was increased by more than 2-fold at 24 h after BSO treatment , which was increased a further 4-fold by the addition of DHA  . GAPDH and G6PDH activities were unchanged; however, Grx activity increased significantly at 24 h after BSO treatment , but there was no significant change in 6PG labeling . Both GAPDH and G6PDH activities increased significantly at 24 h following BSO treatment  . Treatmea) . In botha) , indicatreatment . In Coloreatment . In summn = 5, p = 0.0013) and a 2-fold decrease in the GSSG/GSH ratio , reflecting rapid reduction of the DHA by GSH  (e), which was confirmed by increased 13C labeling of 6PG (g) at 6 and 24 h after BSO treatment. As in the experiments on the cells in vitro, this increased PPP flux can explain the lower GSSG/GSH ratio. Glycolytic flux decreased from 1.19 ± 0.09 to 0.55 ± 0.19 μmol min−1 g−1  at 6 h but increased to 0.93 ± 0.10 μmol min−1 g−1  at 24 h after BSO treatment. At 24 h, there was increased fractional labeling of the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate . GAPDH activity was unchanged, but there was an increase in G6PDH activity (e and g). Of the enzymes involved in DHA reduction, Grx activity increased 24 h after BSO treatment  and without injection of DHA . There were no changes in the activities of TrxR (69.4 ± 1.0 nmol min−1 mg tumor−1 in control (n = 6) versus 64.0 ± 1.3 nmol min−1 mg tumor−1 in BSO-treated tumors (n = 5)) and GST (40 ± 1 nmol min−1 mg tumor−1 in control (n = 6) versus 39 ± 1 nmol min−1 mg tumor−1 in BSO-treated tumors (n = 5)).Next we investigated the effects of glutathione depletion in EL4 and Colo205 tumors. BSO is cleared within 24 h of infusion in mice and causes no obvious toxicity . In EL4 SH ratio . By 24 hSH ratio . In animA by GSH . Measure 0.0051) e, which g of 6PG g at 6 anactivity , again iactivity  and consactivity , e and greatment , as was f and h). There were no significant changes in the activities of GAPDH, G6PDH, or Grx . Oxidatively prestressing EL4 tumors by BSO treatment increased the rate of hyperpolarized [1-13C]DHA reduction, which, although highly variable, was ∼3.8-fold higher in tumors treated 24 h previously with BSO than in control tumors (p = 0.044) (b–d). At this time point, there was no difference in total glutathione content and GSH concentration (g). 13C NMR spectra of extracts prepared from these tumors, where the tumors were freeze-clamped at ∼5 min after injection of the hyperpolarized [1-13C]DHA, showed that there was 70.5 ± 40.5 nmol g−1 [1-13C]DHA in 24-h BSO-treated tumors (n = 2) and 97.5 ± 34.5 nmol g−1 in control tumors (n = 2). [1-13C]AA was undetectable, although this may reflect oxidation of AA during perchloric acid extraction. Assuming that this is the concentration of [1-13C]DHA present at the time the 13C MRS measurements were made in vivo and using the [1-13C]AA/[1-13C]DHA ratio at 30 s after injection of hyperpolarized [1-13C]DHA, this equates to a [1-13C]DHA reduction rate of 0.42 ± 0.15 and 1.43 ± 0.82 nmol g−1 s−1 in control tumors and 24-h BSO-treated tumors, respectively. In Colo205 tumors, the hyperpolarized [1-13C]DHA reduction rate at 24 h after BSO treatment was also highly variable, but in this case the increase was not significant (c). Although in these BSO-treated Colo205 tumors there was no measurable decrease in the GSSG/GSH ratio, there was nevertheless a significant increase in PPP flux (f and h).Non-invasive s in vivo–20. Consr region a. Oxidat= 0.044) , b–d. Atntration  between ntration g. 13C NMnificant c. AlthouPPP flux , f and h13C]DHA led to transient respiratory arrest and cardiac depression in these mice, which was dose-dependent , as reported previously (a). However, when the cells were lysed, the rate of AA oxidation was much higher (c), 2.18 mmol liter−1 s−1versus 4.96 mmol liter−1 s−1.Because DHA has adverse, although transient, effects on mouse physiology, we also explored whether oxidative stress could be assessed using DHA into EL4 tumor-bearing mice resulted in its reduction to [1-13C]AA, as was observed previously (a). Although the data were highly variable, oxidatively prestressing EL4 tumors by BSO treatment resulted in a ∼3.8-fold increase in the rate of DHA reduction at 24 h after BSO treatment. Previous studies have suggested that the rate of DHA reduction is dependent on the levels of GSH (–−1 s−1) and tumors 24 h after BSO treatment (1.43 ± 0.82 nmol g−1 s−1) are comparable with the rate of GSH oxidation estimated previously in erythrocytes (0.28 μm s−1)  . Althoug μm s−1) . At 6 h in vitro , 52. We 50 of 320 mg kg−1, whereas we observed a transient respiratory arrest in mice at a dose of only 10 mg kg−1, suggesting that there may be species-specific differences in its effects. This toxicity might be overcome by preinjecting animals with similar doses of DHA as used for imaging, because this has been shown to improve tolerance to DHA , which is produced mainly by the mitochondria and NADPH oxidases and is found mostly intracellularly . However, the transient toxicity of DHA will limit if not prevent its potential translation into the clinic.In conclusion, we have shown that the rate of reduction of hyperpolarized glucose and measuring label incorporation into lactate using 13C NMR  and Colo205 cells (107) were incubated with 11 mm glucose for 30 min at 37 °C. Medium was collected by centrifugation and snap-frozen in liquid nitrogen. Tumor-bearing mice were injected intravenously with 0.4 ml of 200 mm glucose or 0.4 ml of 200 mm glucose and 28 mm DHA, and the animals were sacrificed and tumors were excised rapidly 4 min later. The tumors were then freeze-clamped immediately with liquid nitrogen-cooled tongs, metabolites were extracted with 7% perchloric acid, and the extracts were neutralized subsequently with KOH. Cell medium and tumor extracts were freeze-dried, and the lyophilized samples were dissolved in 20 mm phosphate buffer containing 10% 2H2O and 10 mm13C urea for 13C NMR analysis.EL4 cells (107) were incubated with 11 mm [U-13C]glucose for 30 s and then quenched in ice-cold methanol. Tumor-bearing mice were injected intravenously with 0.4 ml of 200 mm [U-13C]glucose with or without 28 mm DHA; the animals were then sacrificed, and tumors were excised rapidly at 1 min after injection and then freeze-clamped immediately. Cells were extracted at 5 × 107 ml−1, and tumors were excised at 50 mg ml−1 in ice-cold 75:25 methanol/acetonitrile containing 0.2% formic acid using metal bead-containing tubes on a Precellys24 homogenizer coupled to a Cryolys® cooler  at 4 °C. A second extraction was performed with 200 μl of water, and the organic and aqueous extracts were mixed. Solvent was removed by evaporation, and the extracts were dissolved in 0.75% octylamine in HPLC grade water. LC-MS/MS measurements of 13C-labeling of 6PG, 3PG, and PEP were based on a method published previously  at 30 °C and detected using a triple quadrupole TSQ Vantage mass spectrometer with an Accela UHPLC system  fitted with a HESI probe with a source temperature of 320 °C.EL4 and Colo205 cells , and their ions were identified from their specific mass transition in multiple reaction-monitoring mode and from their retention time, using a triple quadrupole TSQ Vantage mass spectrometer with Accela UHPLC system (Thermo Scientific) fitted with a HESI probe with a source temperature of 320 °C. Glutathione-glycine-13C2,15N was added as an internal standard.GSH and GSSG were measured as described . Cells w7 cells ml−1, and tumors were freeze-clamped and extracted at 50 mg ml−1. G6PDH (EC 1.1.1.49) activity was assayed at 22 °C, as described  in a Precellys24 homogenizer coupled to a Cryolys® cooler (Stretton Scientific) at 4 °C. Protein content was measured using Direct Detect® . Grx activity was measured using a 2-hydroxyethyl disulfide coupled assay  in a Precellys24 homogenizer coupled to a Cryolys® cooler (Stretton Scientific) at 4 °C. Protein content was measured using Bradford reagent .GAPDH (EC 1.2.1.13) activity was measured using the KDalert® kit  at 22 °C. Cells were resuspended in lysis buffer at 10escribed . Tumors ed assay . TrxR acm [3-13C]glutamine with or without 28 mm DHA. After 4 min, tumors were rapidly excised, freeze-clamped, extracted with 7% perchloric acid, and neutralized with KOH. Freeze-dried extracts were dissolved in 600 μl of 20 mm phosphate buffer, pH 7, with 10% 2H2O and 10 mm13C urea. 13C NMR spectra were acquired on a 600-MHz spectrometer  at 300 K with a TR of 3 s and 12,000 scans.EL4 tumor-bearing mice were injected intravenously with 0.4 ml of 100 mm solution of glucose containing 27 mm OX063  and 1.3 mm gadolinium chelate  was hyperpolarized using a Hypersense polarizer  at ∼1.3 K  (10 mg) was dissolved in 50 μl of DMSO-d6  with 14.8 mm trityl radical  and 1.4 mm gadolinium chelate (Dotarem). The sample was beaded in liquid nitrogen and polarized  containing 27 mm OX063  and 1.4 mm gadolinium chelate (Dotarem) was hyperpolarized as described   . Dissolu13C spectra (8-mm slice) from the tumor were acquired using an actively decoupled dual-tuned 13C/1H volume transmit coil and a 20-mm 13C receive surface coil . For experiments with glucose, spectra were acquired with a nominal flip angle of 22°, TR 100 ms, and an arrayed offset, where nine consecutive spectra were taken from the lactate region and the tenth spectrum was from the glucose region . For experiments with DHA, spectra were acquired with a nominal flip angle of 25°, TR of 1 s, spectral width 6 kHz, 1024 points. For chemical shift imaging with hyperpolarized [1-13C]DHA, 0.2 ml of the dissolution fluid was injected into the tail vein (19 s after the start of dissolution), and imaging started 15 s after injection. Chemical shift images of [1-13C]DHA and [1-13C]AA were acquired non-slice-selectively over the tumor region, using a 50-μs hard pulse, TR of 40 ms, echo time of 0.35 ms. Images were reconstructed in Matlab . At 5 min after injection of hyperpolarized [1-13C]DHA, animals were sacrificed, and the tumors were excised and immediately freeze-clamped with liquid nitrogen-cooled tongs. Labeled DHA was extracted with 7% perchloric acid, and extracts were neutralized with KOH. Freeze-dried extracts were dissolved in 20 mm phosphate buffer, pH 7, containing 10% 2H2O and 10 mm13C urea as a chemical shift standard. 13C spectra were acquired on a 600-MHz spectrometer (Bruker) at 300 K with a nominal flip angle of 30°, a TR of 3 s, and 12,000 transients. Peaks were integrated and normalized to the 13C urea standard using Topspin version 2.1 (Bruker).Mice were anesthetized with 2% isoflurane and placed inside a 7 T spectrometer , and their body temperature was maintained at 37 °C. Animals were injected via a tail vein cannula with 0.4 ml of the dissolution fluid, and data acquisition started 15 s after the start of injection. Slice-selective 13C]AA (14 mm) were injected into 2 ml of either water, RPMI medium, hydrogen peroxide solution, or an EL4 cell suspension in a 10-mm NMR tube. Spectra were acquired using a 9.4 T vertical wide bore magnet (Oxford Instruments) and a 10-mm broadband probe . A total of 180 13C spectra were acquired with an 8° flip angle pulse, TR of 500 ms, and spectral width of 16 kHz . Unpaired two-tailed in vivo data and performed all procedures involving live mice. M. W. acquired and analyzed some of the data. A. J. W. helped with the acquisition and analysis of some of the data. M. I. K. helped with the acquisition and analysis of some of the data and commented on the manuscript. B. W. C. K., T. J. L., P. D., and I. M.-R. helped with the acquisition of some of the data and commented on the manuscript. S. E. B. designed the study, acquired and analyzed some of the data, and commented on the manuscript. K. M. B. designed the study, helped interpret the data, and wrote the manuscript.K. N. T. designed the study; performed, analyzed, and interpreted most of the data; and wrote the manuscript. D.-E. H. helped with the acquisition of all


Background: Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO)-in-water emulsion in human macrophages in vitro. Materials and Methods: Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4) in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. Results: KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL) and 75% (at 25 µg/mL), whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO  also inhibited  the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. Conclusion: KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity. LPS binolecules . The keyolecules . The throlecules . In thisolecules .n-3 fatty acids are also available in other forms, such as in crude krill oil (KO), which provides EPA and DHA, mainly in the form of PLs, and as ethyl esters of pharmaceutical grade, highly-concentrated preparations [n-3 LC-PUFA in PLs, is also increasingly found on the market, and is promoted as being of “higher efficacy” [Euphausia superba Dana), has been brought onto the omega-3 market, characterized by a greater ease of absorption due to higher PL content [Applications of intravenous lipid emulsions containing fish oil reduce the length of stay in hospital , as wellarations . In thisfficacy” ,20. Addi content ,20.w/w of the fatty acids profile), with up to 95% w/w PLs and up to 45% triglycerides [KO comes from sustainable fisheries and is nearly at the beginning of a food chain, compared with fish sources that are more affected by environmental pollutants ,20. In aycerides . AccordiAs shown in We used two binding assays to evaluate the interaction of LPS with macrophage-TLR4 and the inhibitory effect of KO. As shown in p ≤ 0.05), and 5 µg/mL  when compared with the negative control (LPS (1 µg/mL) pre-incubated with KO (100 µg/mL) inhibited the binding, but not when using LPS at a concentration of 5 µg/mL . The LPS control .p ≤ 0.05) and, at 50 µg/mL LPS + KO, it was abolished (p ≤ 0.01), as shown in After co-incubation of 0.1 µg/mL LPS-EB with KO, the macrophages were treated for 3 h with different concentrations of LPS + KO: at 12.5 µg/mL LPS + KO, LPS-binding decreased by 50% (not significant); at 25 µg/mL LPS + KO LPS binding significantly decreased by 75% (p ≤ 0.01) prevented the binding of LPS-biotin and, thus, mimicked the effect of KO (p ≤ 0.01), 25 µg/mL (p ≤ 0.01), or 50 µg/mL (p ≤ 0.001) KO when compared with the control. Furthermore, LDL showed a similar inhibition pattern as KO. Additionally, the LPS-RS antagonist completely inhibited the LPS binding on the TLR4 , 25 µg/mL  or 50 µg/mL  KO in comparison with the control LPS (The TNF-α release in cell supernatants of differentiated human THP-1 macrophages treated with LPS-EB (pre-incubated with KO) was markedly reduced when 0.01 µg/mL or 0.1 µg/mL LPS was used. A significant inhibition of the TNF-α release was obtained after incubation of 0.01 µg/mL LPS with 12.5 µg/mL  or 60% (p ≤ 0.01) inhibition in comparison with control LPS  [n-3-FAs dramatically modulate the body’s response to inflammation, oxidative stress, ischemia, and immune function through several downstream bioactive mediators,  [n-3 PUFA on inflammatory gene expression via inhibition of activation of the transcription factor NF-κB [Death rates in patients caused by sepsis reach about 30%–80% , especiaand ROS) ,30. Onces, etc.) . The coms, etc.) , togethes, etc.) , may exes, etc.) . These oor NF-κB . Severalor NF-κB ,35. The or NF-κB . In thisor NF-κB , and couRhodobacter sphaeroides, which binds to the TLR4 but does not induce TLR4 signaling, we can confirm the specificity of the activation  with 100 µg/mL KO , by redu h with 1tivation . After cEscherichia coli endotoxin during a 6-h PL infusion, and attenuation of the clinical and laboratory responses were directly related to PL levels in the bloodstream [The effects we have observed with a KO emulsion are likely the result of two main mechanisms of action. First, the phopsholipids present in KO bind and neutralize LPS endotoxin. This effect has been shown previously in a study of normal human volunteers receiving an intravenous dose of odstream . Howeverodstream . The PL Finally, the KO emulsion used in this study was a crude formulation made from an unrefined natural source that is widely found in oral supplements and, thus, would be unsuitable for intravenous administration. Therefore, our study provided an indication of a proof-of-concept with this phospholipid-omega-3 combination in a cell culture model. We clearly recognize that for such a product to be a potentially viable injectable emulsion in the clinical setting, the crude KO would need to undergo refinement steps (similar to that applied to fish oil)  to conceWe conclude that KO emulsion inhibits the LPS-binding on macrophage-TLR4 and, thus, the TNF-α release induced by LPS in vitro. The addition of omega-3 fatty acids potentiates the therapeutic actions by reducing the intensity of the systemic inflammatory response. These properties may be beneficial for patients under intensive care with septicemia.Three separate batches of a 5% KO-in-water emulsion were aseptically prepared in the laboratory and sterilized prior to use. This was done to be sure the emulsions could be successfully made using a crude source of KO, since a pharmaceutical-quality grade, suitable for parenteral administration, does not exist. The mean droplet size of all the emulsions was approximately 190 nm and, thus, they were considered pharmaceutically equivalent. The composition of the final emulsions are shown in The in vitro experiments were performed using the THP-1 (human acute monocytic leukemia) cell line , cultured in 90% RPMI-1640 , 10% FBS (PAA GmbH); 100 U/mL penicillin; and 0.1 mg/mL streptomycin (PAA GmbH). All experiments were carried out in medium with 10% FBS.4) cells were seeded in 96-well plates . After differentiation with 0.1 µg/mL phorbol-12-myristate-13-acetate (PMA) Sigma-Aldrich, St. Louis, MO, USA), the medium was changed and the macrophages treated with 0.05 µg/mL-10 µg/mL ultrapure biotinylated lipopolysaccharide (LPS-EB) from E. coli O111:B4 , which is recognized only by toll-like receptor-4 (TLR4); KO 5 µg/mL to 250 µg/mL, or glycerol. After 24 h KO or 4 h LPS treatment, viability was assessed using PrestoBlue™ reagent . After 1 h the optical density (OD) was measured at 570 nm/600 nm with a SUNRISE ELISA-reader . Results are expressed as the % of viability/survival (OD570 nm/600 nm of samples × 100/OD570 nm/600 nm of control without substances).THP-1 (5 × 104) cells were seeded in 100 µL medium/well in 96-well plates . After eight days of differentiation into macrophages using 0.1 µg/mL PMA, the medium was changed and the macrophages were treated with 1 µg/mL or 5 µg/mL ultrapure LPS-EB-biotin alone or with KO. After 3 h the cells were fixed with 1% paraformaldehyde (PFA/PBS) for 20 min and incubated afterwards with streptavidin-Cy3 . Digitalized images were obtained using an inverted microscope Eclipse-TS100  and an AxioCamMRc/AxioVision digital imaging system .THP-1 (5 × 104) cells were seeded as described above and treated with 0.1 µg/mL, 1 µg/mL, or 5 µg/mL ultrapure LPS-EB-biotin. After 3 h the macrophages were fixed with 1% PFA/PBS for 20 min, then incubated with streptavidin-biotinylated horseradish peroxidase (HRP-streptavidin-biotin) complex Amersham . Subsequently, the macrophages were incubated with 50 µL peroxidase substrate Sigma Fast™ (OPD)  for 30 min at RT. The reaction was stopped with 25 µL 3N HCl, and the absorbance was measured at 490 nm/655 nm. Afterwards, the cells were stained with crystal violet solution ). The OPD absorbance was normalized against the crystal violet absorbance measured at 595 nm/660 nm.THP-1 (5 × 10PMA-differentiated THP-1 macrophages were treated for 3 h with the LPS-EB-biotin and KO mixture (both previously incubated together at 4 °C overnight), afterwards the cells were fixed with 1% PFA-PBS for 20 min, washed with PBS, incubated with streptavidin-biotin-complex-HRP , afterwards with OPD and measured as described above. The positive control was performed by incubation of macrophages with ultrapure LPS-EB-biotin alone and the negative controls incubated only with medium or with KO. In humans, low-density lipoproteins (LDLs) may bind LPS and inactivate it; we used native LDL  as a control to compare the adsorption properties of KO . The ant5 THP-1 cells were seeded in 24-well plates . After five days of PMA-induced differentiation (0.1 µg/mL), the macrophages were treated 3 h with LPS-EB and KO (previously incubated as described). After the treatment, the culture medium was harvested and centrifuged at 500× g (5 min). The cells were homogenized in RIPA buffer  for protein quantification using the bicinchoninic acid assay . Human TNF-α was determined in the supernatant using the DuoSet-ELISA kit  according to the manufacturer’s instructions; 96-well NUNC MaxiSorp™ (Thermo Fisher Scientific) were used. The amount of TNF-α was normalized with the protein content.The release of TNF-α was determined using ELISA. Therefore, 5 × 10®-12 software  was used to carry out statistical analyses by one-way analysis of variance test (ANOVA) using Dunnett’s method appropriate for multiple comparisons versus the control group. Data are shown as mean + SEM.The SigmaPlot


Meloidogyne hapla, is one of the most important nematode pathogens occurring in cold regions. It is a sedentary, biotrophic parasites of plants and overwinter in the soil or in diseased roots. This study showed that the cold tolerance for the second-stage juveniles (J2) of M. hapla was moderate with the 50% survival temperature (S50) of -2.22°C and the fatal temperature was -6°C when cooling at 0.5°C min-1. Cryoprotective dehydration significantly enhance cold tolerance of M. hapla J2 with the lowest S50 of -3.28°C after held being at -1°C for 6 h. Moreover, cold shock and cold acclimation had significant effects on the freezing survival of M. hapla J2. The lethal temperature of eggs was -18°C. Therefore, the cold tolerance of M. hapla is sufficiently favorable to withstand winters in cold temperature environments.The northern root-knot nematode,  Meloidogyne hapla, are sedentary, biotrophic parasites of plants with wide host ranges [M. hapla infect plant roots and induce root-knots, which affect water and nutrient absorption and translocation of root systems. Infection results in reduced crop yield or quality [M. hapla occurs in cold regions of crop production [M. hapla is mainly distributed in cool areas, where the mean temperature is -15°C in the coldest month and approximately 27°C in the warmest month or in high altitude mountainous areas [M. hapla is the J2, which must overcome adversely low-temperature environmental conditions before reaching plant roots. It was shown that M. hapla J2 survived at freezing temperatures [M. hapla eggs survived sub-zero temperatures in the field [M. hapla to survive at low temperatures.The northern root-knot nematode, t ranges . The sec quality , and con quality , 4. M. hoduction . Accordieratures , 7, and eratures . Meanwhihe field , 10 and he field . This inSteinernema feltiae and Heterorhabditis bacteriophora survived the low temperature of -13°C by cryoprotective dehydration, and the S50 of S. feltiae was -3.73°C [Panagrellus redivivus survived at low temperature by freezing tolerance and cryoprotective dehydration [Marshallagia marshalli survived rapid exposure to temperature below -30°C [Ditylenchus dipsaci, P. redivivus, Steinernema carpocapase, Panagrolaimus rigidus, Rhabditophanes sp. and Panagrolaimus davidi), while the S50 of P. davidi was -43.6°C lower than the others [Cold tolerance is the ability of nematodes to survive low temperatures in their living environment . There a -3.73°C . Panagreydration . Marshalow -30°C . One stue others .P. redivivus, P. davidi [S. feltiae, S. riobrave, S. carpocapsae, S. anomaly and H. bacteriophora [Escherichia coli when E. coli was subjected to cold shock [H. bacteriophora and Trichinella nativa produced proteins to respond to cold shock [M. hapla J2 are unknown, although they can survive at low temperatures.Cold acclimation is an adaptive response of organisms to low temperature that increases their capacity to tolerate freezing, and this response has been observed in . davidi , 22, S. riophora , 23. In riophora . The Cspld shock , 26. Nemld shock , 28. HowM. hapla J2 and the in vitro hatch rate of egg masses. We determined the influence of cryoprotective dehydration, cold acclimation and cold shock, on M. hapla cold tolerance.In this study, we investigated the effect of low temperature on the survival of M. hapla were generous gift by Congli Wang , and maintained on a nematode-susceptible tomato (L-402) in the greenhouse according to the described by Forge and MacGuidwin[The acGuidwin. The eggacGuidwin, 30. EggM. hapla J2 nematodes were transferred to a 0.5-ml Eppendorf tube and placed in a cooling block. The temperature of cooling block was controlled by a programmable cooling device (Temperature chamber: TEMI990). The samples were cooled from 1°C to various minimum temperatures  at 0.5°C min-1 and frozen by adding ice crystals (made by AWT) at Tmin and held for 30 min, then rewarmed to 1°C at 0.5°C min-1. The samples were then removed from the cooling block. After thawing, 300 μl AWT was added to the samples, and placed at room temperature for 24 h. Survival was determined by counting the proportion of moving nematodes after a mechanical stimulus by touching nematodes with a homemade eyelash-needle. Control samples were unfrozen at -1°C. Two runs of this regime were used with 5 replicates per run. The temperature at which 50% of the J2 were killed (S50) was determined using a Probit analysis [A 50-μl suspension containing approximately 20 analysis , 17, 19.M. hapla J2 was a cryoprotective dehydration strategy, samples were cooled from 1°C to -1°C at 0.5°C min-1, frozen by inoculating with ice crystals at -1°C and held for a specific period time  before cooling to Tmin  at 0.5°C min-1. They were kept at Tmin for 30 min and finally rewarmed to 1°C at 0.5°C min-1. Two runs of this regime were used with 5 replicates per run. Survival was determined as previously described.To test whether the cold tolerant mechanism of M. hapla J2, samples were cooled from 1°C to -1°C at 0.5°C min-1 and held for 1 h at -1°C. They were rewarmed to 1°C at 0.5°C min-1 in cool block, and then samples were taken from cool block and maintained at room temperature for 1 h [min  using similar methods as in the ‘freezing regime’. Survival was detected as before.To test the effect of cold shock on the survival of  for 1 h  before eM. hapla J2, samples were acclimated at 4°C for 12 h before cooling to Tmin  at 0.5°C min-1, the cold exposure was using the ‘freezing regime’. Survival was detected as before.To test the effect of cold acclimation on survival of M. hapla, which was exposed to low temperature, seven temperature treatments  were used. Similar sizes of fresh egg masses were chosen and sterilized by 0.4% NaOCl solution, then placed in the homemade hatching pond. Samples cooled from 1°C to Tmin at 0.5°C min-1, kept at Tmin for 30 min and then warmed to 1°C at 0.5°C min-1, finally the samples were removed from the cooling block. Control samples were kept at 25°C. All treatments were hatched in sterile distilled water at 25°C. Each treatment had 3 replicates per run, and two runs of this regime were used. The number of eggs in each egg-mass were 672 ± 5.5 (mean ± SE) on average. After 10 days, the number of hatching nematodes compared to the proportion of eggs were examined according to the formula as below:To test the hatch rate of the egg mass of 50). The minimum temperatures (Tmin) were log10 transformed to linearize the data. The relative median potency (RMP) estimated the difference of the S50 between two groups. Significant differences were defined between groups, if the 95% confidence limits (CL) of RMP estimation does not encompass the value 1. The effect of treatments on survival were tested using a factorial ANOVA [All statistical analyses were calculated by using SPSS v. 17.0 . Probit al ANOVA , 17, 19.M. hapla J2 in the freezing regime is significant. With the temperature reduced, survival decreased significantly  . Moreover, the S50 values were significantly decreased with an increased freezing time at -1°C after 2–6 h, which was lower 1.06°C after 6 h compared to the freezing regime , but there was no significant difference between 6 h and 12 h  . The S50and 12 h .M. hapla J2 subjected to cold shock at -1°C for 1 h was significantly increased , lower than the S50 in the freezing regime . Survival of M. hapla J2 significantly improved by acclimated at 4°C compared to the freezing regime , which was significantly different from the freezing regime . Moreover, the S50 values of various regimes were compared in 50 of cryoprotective dehydration for 6 h was lower than those achieved through cold shock  and acclimation , but there were no significant differences between cold shock and acclimation.Survival of ncreased . The S50g regime . And thaM. hapla declined with decreasing temperatures  (The hatching percentage for egg mass of < 0.001) .M. hapla J2 indeed survived at low temperature. Previous studies showed that the M. hapla J2 were frozen spontaneously at -8°C at the freezing rate of 1°C min-1 [-1 because the physical damage of ice formation decreased using a slow freezing rate [M. hapla J2 was modest, with the S50 of -2.22°C and fatal temperature of -6°C in the freezing regime. Comparatively, the S50 of P. davidi was -43.6°C [°C min-1 . Howevering rate . The res -43.6°C .P. davidi, S. feltiae, and H. bacteriophora [50 of M. hapla J2 was -2.22°C, and the value significantly decreased with increased time at -1°C in the cryoprotective dehydration regime. This result might explain that cryoprotective dehydration had an effect on the cold tolerance of M. hapla J2. Moreover, a previous study showed that following exposure to -15°C for 10 min, the cuticle of M. incognita J2 had been torn away from the body by freezing but not in the M. hapla J2 [Inoculating ice could make the surrounding medium freeze rapidly at high subzero temperatures and provide the basic environment of cryoprotective dehydration of nematodes . Cryoproriophora , 19. In hapla J2 . Thus, tM. hapla J2 was improved significantly by cold shock at -1°C for 1 h, and the S50 was -2.58°C, which was lower than the freezing regime. Cold shock occurs in a variety of organisms [H. bacteriophora induces the trehalose-6-phosphate synthase by cold shock [Trichinella native [P. redivivus [S. feltiae [Survival of rganisms . H. bactld shock , and Hspa native , while, edivivus  and S. f feltiae .M. hapla J2 that were acclimated at 4°C for 12 h showed a significant enhancement in survival. These were similar results to those found by Forge and MacGuidwin [P. redivivus were decreased by cold acclimation, enabling survival at lower temperatures [S. feltiae, S. anomaly and H. bacteriophora was increased after cold acclimation [S. feltiae, S. riobrave, S. carpocapsae) [P. davidi [M. hapla J2 needs to be further investigated.cGuidwin . Cold accGuidwin . The superatures . The frelimation . Moreoveocapsae)  and P. d. davidi . Meanwhi. davidi . HoweverM. hapla within 10 days was 65.30% at -6°C and 4.27% at -14°C, indicating that M. hapla survived at low temperatures, which was similar to the results by Daulton et al. [M. hapla egg masses [In nature, nematodes usually overwinter by egg masses in the plant debris or soil when the soil temperature is subzero. In this study, the percentage of egg-hatching for n et al. . HoweverM. hapla J2 was -6°C with freezing by adding ice, and cryoprotective dehydration improved the cold tolerance of M. hapla J2. The lethal temperature of M. hapla egg mass in our experiments was -18°C after freezing spontaneously. Moreover, cold acclimation and cold shock significantly improved the cold tolerance of M. hapla J2, which is advantageous for withstanding the winter in cold environments.We found that the lethal temperature of S1 FigTreatments were frozen by adding ice (open circle), and unfrozen (closed circle). The values are the mean ± SE in this figure. N = 10.(TIFF)Click here for additional data file.S2 Figmin (filled circles), and survival at freezing regime at the corresponding test temperature  without cold shock (open circles). The values are the mean ± SE in this figure. N = 10.Samples cold shocked at -1°C for 1 h and then kept at room temperature for 1 h before being cooled to T(TIFF)Click here for additional data file.


N,N′-di-Boc-N′′-triflylguanidine was applied to synthesize the guanidine derivative of indoloquinoline 1 in a gram scale for specific in vitro and in vivo biological research. Extensive studies on the antiproliferative activity against eight human tumor cell lines were completed. Compound 1 revealed the highest activity against A549 lung adenocarcinoma and MCF7 breast cancer cell lines. Thus, 1 was evaluated for the in vivo anticancer activity against 4T1 mammary gland carcinoma and KLN205 murine lung carcinoma in mouse models. The anticancer effect was observed in the KLN205 model with a 37% tumor growth inhibition at the 20 mg/kg dose. This anticancer activity of 1 was comparable to that of cyclophosphamide which inhibited murine lung tumor growth in the range of 27–43% at the dose of 100 mg/kg. The biochemistry research after 1 admission, including measurements of blood parameters like alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and urea and creatinine, were also performed.An optimization of the guanidylation process by verifying the efficacy of common guanylation reagents in order to obtain the guanidine derivatives of indoloquinoline has been performed. As a result, a high-yield procedure using  It follows that the extreme selectivity of the guanidine-DiMIQ conjugate may result from a new, unique molecular mechanism of action. Also, an important and promising feature of these compounds is the absence of hemolysis  and normal cells (human fibroblasts CCD11Lu). In addition, the impact of the compounds on tumor growth was to be investigated in an in vivo mouse model. During our previous studies, it had been experimentally verified that the guanidylation by N,N’-Bis(tert-butyloxycarbonyl)-S-methylisothiourea in a two-step synthetic sequence allows to obtain samples of guanidine derivatives for biological screening in average yields. However, this approach seemed to be problematic in the upscaling process because of the release of mephitic methyl mercaptan. For that reason, it was necessary to find a new, effective, and reproducible synthetic method which would lead us to obtain 1 and 3 guanidine analogs in a gram scale for pre-clinical tests.Thus 1 and 3.In this paper, we describe the optimization process, a new, improved synthetic strategy for the production of guanidine analogs, and biological in vitro and in vivo characteristics of compounds 1H and 13C nuclear magnetic resonance (NMR) spectra of all compounds studied were measured in CDCl3, dimethyl sulfoxide (DMSO)-D6, or D2O using Varian-NMR-vnmrs500, Varian-NMR-vnmrs600, and Varian Gemini 200 spectrometers at the temperature of 298 K. Standard experimental conditions and standard Varian programs were used. To assign the structures under consideration, the following 1D and 2D experiments were employed: the 1D selective NOESY and 2D gradient-selected COSY, 1H-13C heteronuclear single quantum correlation, and 1H-13C Heteronuclear Multiple Bond Correlation. The 1H and 13C NMR chemical shifts relate to the tetramethylsilane (for compounds dissolved in CDCl3 or DMSO-D6) and DSS (for compounds dissolved in D2O). The concentration of all solutions used for the measurements was about 10–20 mg of the compounds in 0.6–0.8 cm3 of the solvent.The 254. The chemicals and solvents were purchased from Fluka Company. Column chromatography was performed on Merck silica gel 60 (230–400 mesh).The electrospray ionization-mass spectrometry (ESI-MS) spectra were recorded on a PE Biosystems Mariner mass spectrometer. The progress of the reaction was monitored by thin layer chromatography (TLC) using Merck DC-Alufolien Kieselgel 60 F5) and water (compound 1). The injection volume was 10 µl. Ultraviolet (UV) detection at 275 nm was used.The chromatographic analysis was performed using a Shimadzu high performance liquid chromatography (HPLC) system  consisting of a Shimadzu LC-20AB pump, Shimadzu SIL-20AC autosampler, Shimadzu CTO-10AS VP column oven, and Shimadzu SPD-M20A photodiode array detector. The separation of the analyte from potential impurities was achieved using a Kromasil C8 column  placed in a thermostated column heater at 30 °C. The mobile phases consisting of A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) were used with the gradient mode at the flow rate of 1 ml/min. The samples were prepared at the concentration of about 0.2 mg/ml and they were dissolved in methanol  was added to the solution of DIPEA  and N,N-di-Boc-N'′-triflylguanidine  in DCM (40 ml). The mixture was stirred for 2 h at room temperature . Next, the mixture was washed with 2 M aqueous sodium bisulfate (50 ml) and saturated sodium bicarbonate (50 ml). Each aqueous layer was extracted with dichloromethane (2 × 30 ml). The combined organic phases were washed with brine (40 ml), dried (MgSO4), filtered, and concentrated under reduced pressure to afford crude compound 5. The compound was purified by column chromatography (chloroform: methanol 9:1 v/v) to afford a title compound 5 (2.71 g) as an orange solid with a good 94.61% yield. mp 210 °C (decomp.); 1H NMR : δ = 8.78 , 8.24–8.21 , 7.79–7.77 , 7.65 , 7.61–7.26 , 4.33 , 3.12 , 1.57 , 1.52 ; 13C NMR : δ = 163.5 2), 154.2 , 153.5 , 153.3 , 150.0 , 141.5 , 136.5 , 130.5 , 129.5 , 125.8 , 124.6 , 124.1, 123.2 , 122.1 , 121.5 , 117.8 , 116.6 , 114.7 , 83.5 , 79.2 , 33.5 , 28.2 , 28.1 , 15.1; HRESIMS m/z: 504.2611 (C28H34N5O4) [M + H]+ . The 1H NMR and 13C NMR data obtained were in good agreement with that reported in the literature  was treated with TFA (30 ml) and stirred for 24 h (TLC monitoring). The solution was evaporated to dryness, next HCl/CH3OH was added and evaporated to dryness. This procedure was repeated three times. The residue was crystallized from ethyl acetate to afford a yellow solid; yield 1.64 g (84%); mp 260 °C(decomp.); 1H NMR : δ = 8.16 , 7.95–7.87 , 7.86–7.84 , 7.66–7.58 , 7.48 , 7.39 , 3.92 , 2.76 ; 13C NMR : δ = 159.3 2), 152.7 , 148.8 , 140.8 , 137.4 , 136.7 , 132.3 , 130.2 , 129.1 , 128.8 , 125.2 , 124.1 , 123.7 , 120.9 , 118.6 , 116.3 , 38.2 , 18.2 ; The 1H NMR and 13C NMR data obtained were in good agreement with that reported in the literature  [M + H]+ ; anal. calcd. for C18H17N5 × 2HCl: C, 57.45; H, 5.09; N, 18.61; Cl, 18.84 found: C, 57.26; H, 5.18; N, 18.44; Cl, 18.67; HPLC: 97.38%.Boc-derivative 6  was added to the solution of DIPEA  and N,N-di-Boc-N′′-triflylguanidine  in DCM (10 ml). The mixture was stirred for 3 h at room temperature . Next, the mixture was washed with 2 M aqueous sodium bisulfate (15 ml) and saturated sodium bicarbonate (15 ml). Each aqueous layer was extracted with dichloromethane (2 × 20 ml). The combined organic phases were washed with brine (20 ml), dried (MgSO4), filtered, and concentrated under reduced pressure to afford crude compound 7. The compound was purified by column chromatography (chloroform:methanol 9:1 v/v) to afford title compound 7 (342 mg) as an orange solid with a good 96.9% yield; mp 220 °C (decomp.); 1H NMR : δ = 11.51 , 10.12 , 8.75 , 8.39–8.38 , 8.03–8.02 , 7.81–7.76 , 7.73–7.71 , 7.56–7.53 , 4.86 , 3.58 , 3.18 , 2.92 2), 1.55 , 1.40 ; 13C NMR : δ = 162.7 2), 162.2 , 153.5 , 152.1, 151.6 , 145.6 , 139.6 , 138.4 , 129.9 , 129.0 , 127.5 , 124.6 , 123.9 , 123.8 , 123.0 , 119.1 , 115.7 , 83.3 , 78.5 , 54.6 , 42.7 , 38.7 , 36.5 , 30.7 , 27.8 , 27.6 , 14.5 ; The 1H NMR and 13C NMR data obtained were in good agreement with that reported in the literature  [M + H]+ calc. 561.3179.The amino component 7  was treated with TFA (10 ml) and stirred for 24 h (TLC monitoring). The solution was evaporated to dryness, next HCl/CH3OH was added and evaporated to dryness. The procedure was repeated three times. The residue was crystallized from ethyl acetate to afford a yellow solid; yield 140 mg (77%); mp 200–202 °C; 1H NMR : δ = 8.22–8.21 , 8.18 , 8.07–8.05 , 7.92–7.89 , 7.76–7.71, 7.65–7.62 , 4.76 , 3.68 , 3.09 2), 2.96 ; 13C NMR : δ = 159.5 2), 150.7 , 149.6 , 141.5 , 140.7 , 135.1 , 131.8 , 129.9 , 128.2 , 127.9 , 125.4 , 124.9 , 124.3 , 124.1 , 119.6 , 113.6 , 56.5 , 46.1 2), 40.2 , 18.0 ; The 1H NMR and 13C NMR data obtained were in good agreement with that reported in the literature  [M + H]+ calcd. 360.4; anal. calcd. for C21H24N6 × 5H2O × 4HCl [596.37]: C, 42.29; H, 6.42; N, 14.09; Cl, 23.78 found: C, 42.20; H, 6.59; N, 14.00; Cl, 24.06. HPLC: 96.5%.Product 2, 95% humidity, and passaged with 0.25% trypsin-EDTA, until ~80% confluence was reached. During the course of the experiment, the microbiological purity of all cell lines was tested with the Universal Mycoplasma Detection Kit (ATCC).Cell lines were purchased from American Type Culture Collection (ATCC). The cell lines A549 and NCI-H460 were maintained in RPMI-1640 (Gibco), HepG2, ACHN and CCD-11Lu were maintained in minimum essential medium (Gibco), HCT116 and HT29 were maintained in McCoy’s Medium (Gibco), and A431 was maintained in Dulbecco’s minimum essential medium. MCF7 was maintained in Minimum Essential Medium (Gibco) with 0.01 mg/ml human insulin (Sigma). All culture media were supplemented with 10% fetal bovine serum (Gibco), streptomycin, and penicillin (Biowest). The cells were grown in standard conditions: 37 °C, 5% COThe cells were seeded in 96-well cell culture plates and on the next day treated with different concentrations of the tested compounds for the next 72 h. The cells were then incubated with 3--2,5-diphenyltetrazolium bromide at 37 °C for 3 h. Finally, the medium with the MTT solution was removed and formazan crystals were dissolved by adding DMSO. After mixing, the absorbency was measured at the 570 nm wavelength (690 nm reference filter) using a Microplate Reader EON (BioTek). The experiments were performed in triplicate.Female, 7–8-week-old Balb/c mice and 16-week-old DBA/2 mice were supplied by Mossakowski Medical Research Centre Polish Academy of Sciences . The mice were maintained in specific pathogen-free conditions. The experiments were performed according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education issued by the New York Academy of Sciences’ Ad Hoc Committee on Animal Research and approved by the Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland.2 at 37 °C). These cell lines were obtained from the ATCC  and maintained at the Hirszfeld Institute of Immunology and Experimental Therapy (IIET), Wroclaw, Poland.Murine mammary gland carcinoma 4T1 and murine lung carcinoma KLN205 cell lines were used and maintained under standard cell culture conditions , and 100 IU/ml penicillin .KLN205 cells were cultured in Eagle’s minimal essential medium  supplemented with 10% fetal bovine serum , 2 mM 4T1 cells were cultured in RPMI 1640 w/GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum , 1 mM sodium pyruvate, 3.5 g/l glucose, 100 μg/ml streptomycin , and 100 IU/ml penicillin .1 was injected intraperitoneally (i.p.) into a healthy female BALB/c mouse (three mice per dose) at the 2.5, 5, 10, and 15 mg doses of the drug per kg of the body weight, five times a week for 2–3 weeks. The mice body weight was monitored every day and body weight changes were calculated. At the end of the study, the mice were killed and subjected to autopsy.In order to determine MTD, compound orthotopically (ort.) with 50 µl PBS suspension of the 4T1 cells (2 × 105 cells/mouse) derived from an in vitro culture. On the 7th day after inoculation, when the tumors reached about 45 mm3, the mice were divided into groups (n = 10) and compound administration started. The compounds were administered intraperitoneally (i.p), compound 1 five times a week at the 5 and 10 mg/kg b.w. doses and cyclophosphamide three times a week at the dose of 25 mg/kg b.w. . The control mice received i.p. aqua pro injection (0.1 ml/10 g b.w.) in the same schedule as for compound 1.Female Balb/c mice were inoculated 3) was calculated using the formula (a2 x b)/2, where a = shortest tumor diameter in millimeters and b = longest tumor diameter in millimeters. Tumor growth inhibition for each day was calculated using the formula TGI (%) = − ([(Vt/Vctrl) × 100]−100%), where Vt is the mean tumor volume of the treated mice and Vctrl that of the untreated control animals. On the 29th day, all mice were killed and whole blood was collected for the morphological analysis using the Mythic 18 analyzer . Additionally, spleens, livers, kidneys, and lungs were dissected and weighed. Lungs were further fixed in 4% (v/v) paraformaldehyde in the PBS solution, and the metastatic nodes were counted.Tumor dimensions were measured using a caliper, and the body weight of the animals was monitored three times a week. Tumor volume (mmsubcutaneously (s.c.) with the KLN205 cells (8.5 × 105 cells/mouse) derived from an in vitro culture. On the 7th day after the inoculation, when tumors reached about 60 mm3, the mice were divided into groups (n = 7–8) and compound administration started. The compounds were administered intraperitoneally (i.p): compound 1 five times a week at the 10 and 20 mg/kg b.w. doses and cyclophosphamide once a week at the dose of 100 mg/kg b.w. . The control mice received i.p. aqua pro injection (0.1 ml/10 g b.w.) in the same schedule as for compound 1.Female DBA/2 mice were inoculated 3) was calculated using the formula described above. On day 25, all mice were killed and whole blood was collected for the morphological analysis using the Mythic 18 analyzer (Orphee). The whole blood was further centrifuged  for plasma and analyzed for biochemical parameters , aspartate aminotransferase (AST), lactate dehydrogenase (LDH), urea and creatinine (CREA)) using the Cobas c111 analyzer (Roche). Additionally, livers and kidneys were dissected and weighed.Tumor dimensions were measured using a caliper, and the body weight of the animals was monitored three times a week. Tumor volume . Compound 1 at the dose of 15 mg/kg b.w. was toxic for the Balb/c mice. Max. body weight loss reached 21% and one mice died.Compound 1) has been prepared in the following established sequence: the amidination of indoloquinoline-amine (4) using BSTU -S-methylisothiourea) as the guanylation reagent and the removal of Boc protection groups with trifluoroacetic acid  with a distinctive stench during the reaction. Thus, we have decided to search for more efficient synthetic methods to prepare compounds 1 and 3.The limitations of this approach include a relatively low yield of amidination (50–60%) and the release of gaseous methanethiol  seemed to be the most favorable, because it enables the introduction of the guanidine group in a single step and does not require the removal of any protecting group. This may increase the overall yield of the entire process and eliminate the problem of gaseous thiol released during the BSTU guanidylation. We have started to test commercially available reagents and literature procedures using PCA (1H-pyrazole-1-carboxamidine hydrochloride), DMPCA , N-Boc-1H-pyrazole-1-carboxamidine, and 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine  was applied as a guanylation reagent. The guanylation method with N,N′-di-Boc-N″-triflylguanidine seems to be the most efficient for the guanidylation of amines. Thus, it is commonly applied as the method of choice for the reaction carried out in a solution or on solid phase  with trifluoroacetic acid and then with HCl/MeOH gave N-guanyl-N--amine dihydrochloride 1 with a good 84% yield and 97.38% purity . Compound 7 was obtained in the guanidylation process with a 96.9% yield guanidine , human lung cancer (H460), human colorectal cancer (HCT 116), human colorectal cancer (HT-29), human kidney cancer (ACHN), human hepatocellular cancer (HepG2), human breast cancer (MCF7), human skin cancer (A431), and human lung fibroblasts (CCD11Lu) cell lines.The synthesized compounds 3 seemed to be significantly weaker than 1 and the DiMIQ reference compound. The most important differences in the activity against specific types of human cell lines were observed for compound 1. The MCF7 breast and A549 lung carcinoma cells turned out to be the most sensitive to 1, with IC50 = 0.5 µM and 0.19 µM, respectively. These results appeared to be 5–15 times higher in comparison to that collected for DiMIQ (IC50 = 3.4 and 2.74 µM). For two investigated cell lines, i.e., ACHN derived from human kidney cancer and HepG2 derived from human liver cancer, compound 1 exhibited the weakest cytotoxic properties among all studied compounds, while DiMIQ was the most active. These visible differences seem to be noteworthy and would present an interesting area for further scientific investigation in order to find specific biomarkers influencing sensitivity to particular cell lines. The activities of compounds 1 and DiMIQ against other investigated cell lines were comparable on the level of a few micromoles. Despite displaying high cytotoxicity against normal cells (CCD11Lu), compound 1 was selected for extended in vivo studies because of its interesting in vitro profile against breast and lung cancer cell lines.All three tested compounds showed cytotoxic properties Fig. . However1 and cyclophosphamide  were dissolved in aqua pro injection prior to use.Compound orthotopically (ort.) with the 4T1 cells (2 × 105 cells/mouse). On the 7th day after cell inoculation, compounds' administration began . Compound 1 was administered five times a week at the strength of 5 and 10 mg/kg b.w. The anticancer activity of compound 1 was not observed , but the percent of monocytes was higher and the percent of granulocytes was lower than that in the control mice . In groups that received compound ps Table . No chanps Table . After tce Table .1 was administered five times a week at the strength of 10 and 20 mg/kg b.w. starting from the 7th day after the KLN205 cells' inoculation. The anticancer activity of compound 1 was observed at the end of the experiment , and creatinine (CRE) in the blood in comparison to the control group -S-methylisothiourea allows to obtain samples of indoloquinoline guanidine derivatives for biological screening in average yields. However, this approach seemed to be problematic in the production of gram samples of compounds because of the mephitic methyl mercaptan release. In the present paper an optimization of the guanidylation process in order to obtain the guanidine derivatives of indoloquinoline has been discussed. The effectiveness of the guanylation using common guanylation reagents, such as 1H-pyrazole-1-carboxamidine hydrochloride, 3,5-dimethyl-1-pyrazolylformaminidium nitrate, N-Boc-1H-pyrazole-1-carboxamidine, and 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine , was verified. As a result of our studies, a high-yield procedure with N,N’-di-Boc-N″-triflylguanidine was applied to synthesize N-guanyl-N--amine dihydrochloride 1 which had been selected for specific in vitro and in vivo biological research. Compound 1 was obtained in a gram scale in the presence of N,N’-di-Boc-N″-triflylguanidine reagent with the overall yield of 80 and 97.38% purity according to the HPLC method. Then, a comprehensive in vitro study on the antiproliferative activity of DiMIQ, the reference compound, and two guanidine analogs 1 and 3 against eight cancer cell lines was completed. The highest activity was shown by compound 1 against A549 and MCF7 lines with the IC50 values equal to 0.19 µM and 0.5 µM, respectively. Therefore, compound 1 was selected for further investigations in the in vivo mouse models of 4T1 mammary gland carcinoma and KLN205 murine lung carcinoma. As far as the 4T1 model is concerned, we did not observe any anticancer activity of 1 at the 5 and 10 mg/kg b.w. doses, whereas the anticancer effect was observed in the KLN205 model. The tumor growth inhibition induced by compound 1 reached 37% at the 20 mg/kg dose, which was comparable to that of cyclophosphamide used as a positive control. This commonly used therapeutic agent inhibited tumor growth in mice by 27–43% at the dose of 100 mg/kg. Moreover, biochemistry research based on monitoring blood parameters such as alanine aminotransferase ALT, aspartate aminotransferase AST, lactate dehydrogenase LDH, and urea and creatinine CREA was performed. Compound 1 decreased the number of erythrocytes and the levels of hemoglobin, hematocrit, as well as creatinine in the blood in comparison to the control group. Although the total number of leukocytes was similar to that in the control group, an increase in lymphocytes and a decrease in granulocytes were observed. A similar effect was measured for cyclophosphamide.During our previous studies it had been demonstrated that the guanidylation by 1 displayed an anticancer activity in the KLN205 mouse model. This result qualified guanidine derivative 1, which is an effective inducer of apoptosis and does not induce hemolysis (Sidoryk et al. Although cancer cell lines have a prominent role in the initial stages of drug discovery, it was found that even cancer-specific drugs do not show higher efficacies in the cell lines representing respective tissues (Jaeger et al. Although the core structure of DiMIQ meets all the criteria for the DNA intercalation and TOPO2 inhibition, previous studies had shown that TOPO2 may not be the main cellular target for different DiMIQ analog compounds. Therefore, for the conjugates containing the guanidine group, a study of biological interactions with the tumor models in order to find the selectivity factors related to specific signaling pathways responsible for the cancer growth is intended.1, using N,N’-di-Boc-N″-triflylguanidine has been performed. Compound 1, produced in a gram scale, was next tested for its anticancer activity in vivo. The biological studies proved that 1 has a high anticancer activity against KLN205 murine lung carcinoma with a 37% tumor growth inhibition at the 20 mg/kg dose. These promising results of our conducted research encourage further studies using PDTX cancer models.Summarizing, an efficient of the guanidylation process for obtaining guanidine derivatives of indoloquinoline


Weissella cibaria strain CMU is used as a commercial oral care probiotic in South Korea. Here, we present the complete genome sequences of four W. cibaria strains  isolated from the saliva of an infant living in Gwangju, South Korea. Weissella cibaria is a short, rod-shaped, Gram-positive, non-spore-forming, nonmotile, heterofermentative, and catalase-negative lactic acid bacterium. This species is widely distributed in human saliva and feces (––W. cibaria strains CMU (ÖraCMU), CMS1, CMS2, and CMS3 can be used as oral care probiotics, owing to their inhibitory effect on biofilm formation and volatile sulfur compound formation . Each strain was sequenced by PacBio  technology using single-molecule real-time (SMRT) analysis version 2.3 software. The PacBio reads were assembled with the HGAP3 protocol and error corrected by Quiver version 1, performed through SMRTpipe version 2.3.0.139497. After correction, Prokka  and two circular plasmids of 18,967 bp and 3,467 bp . CMS1 and CMS2 had circular chromosomal genomes of 2,342,849 bp (G+C content of 45.3%) and 2,342,914 bp (G+C content of 45.3%), respectively. CMS3 had a circular chromosomal genome of 2,342,907 bp (G+C content of 45.24%) and one circular plasmid of 20,585 bp (G+C content of 38.3%). The chromosomal genome of CMU contains 2,038 predicted protein-coding sequences, 33 rRNAs, and 90 tRNAs, with an average gene length of 2,201 bp. The plasmids of CMU contain 21 and 2 predicted protein-coding sequences, with average gene lengths of 25 bp and 2 bp, respectively. The predicted protein-coding sequences of CMS1, CMS2, and CMS3 were 2,024 bp, 2,035 bp, and 2,035 bp, respectively, and the average gene lengths were 2,176 bp, 2,188 bp, and 2,187 bp, respectively. The plasmid of CMS3 contains 23 predicted protein-coding sequences, with an average gene length of 25 bp. The numbers of rRNAs and tRNAs—28 and 88, respectively—were the same for CMS1, CMS2, and CMS3.W. cibaria strains reported here have been deposited in GenBank under accession no. CP013936 (CMU), CP022606 (CMS1), CP013726 (CMS2), and CP013934 (CMS3).The complete genome sequences of the


Methods. The research program included 60 patients  with hyperlipidemia taking atorvastatin or rosuvastatin at a dose of 20–40 mg daily. The patients were randomly allocated in two groups. Group I (n = 30) was recommended to take the same statin at a standardized daily dose of 20 mg together with melatonin at a dose of 2 × 5 mg. Group II (n = 30) patients took statin with placebo at the same dose and time of the day. Follow-up laboratory tests  were evaluated after 2, 4, and 6 months of treatment. Results. In Group I the levels of all enzymes decreased after 6 months, particularly AST, 97,2 ± 19,1 U/L versus 52,8 ± 12,3 U/L ; ALT, 87,4 ± 15,6 U/L versus 49,8 ± 14,5 U/L ; and GGT, 84,1 ± 14,8 U/L versus 59,6 U/L . Conclusion. Melatonin exerts a hepatoprotective effect in patients taking statins.Taking statins can cause increase in the level of aspartate and alanine aminotransferase. The aim of this study was to assess the usefulness of melatonin in counteracting the adverse hepatic events from statins. Statins are widely used in the treatment and prevention of lipid metabolism disorders , 2. TheyFurthermore, statins cause hepatotoxic effect which is observed in several percent of treated patients, usually in the first weeks of the therapy –10. MostIn our study melatonin was used for this purpose because previous experimental studies had demonstrated that it protected liver against harmful effects of many toxic agents –18 as weThe liver is an organ in which intensive metabolic and detoxification processes take place. In their course large amounts of reactive oxygen species are generated and they exert a toxic effect on hepatocytes. A complex antioxidant system, in which metabolized there melatonin  is an important part, prevents that , 23.The main melatonin metabolic pathway in the liver is through hydroxylation pathway at the C-6 position by 6-hydroxylase and P450 cytochromes  –26. The An alternative metabolic pathway includes melatonin oxidation to N-acetyl-formyl-5-methoxykynuramine (AFMK) and N-acetyl-5-methoxykynuramine (AMK). The kynurenine pathway of melatonin metabolism leads to formation of a series of free radical scavengers , 28.Furthermore, melatonin decreases the production of proinflammatory cytokines , 30 and The aim of this study was to assess the usefulness of melatonin in counteracting the adverse hepatic events from statins.The research program included 60 subjects, aged 47–68 years, 41 women and 19 men.  All women were postpostmenopausal. Recruitment and diagnostic tests were conducted in the Department of Gastroenterology, Medical University of Lodz, and Outpatient Consulting Clinic “Gastro” in Lodz.The research study was performed in the years 2012–2016.Hyperlipidemia treated with statins for minimum of 6 monthsAt least 2-fold increase in the level of aspartate and alanine aminotransferase found in two consecutive testsThe persistence of increased aminotransferase levels despite the reduction in the statin doseAt the time of the inclusion of patients in the study, 38 subjects were taking atorvastatin (20 mg) and 26 rosuvastatin at the dose of 40 mg (3 patients), 20 mg (19 patients), and 15 mg (4 patients). Inclusion criteria are as follows:History of viral hepatitisCholelithiasis2Body mass index (BMI) > 30 kg/mAlcohol abuseFamilial hypercholesterolemiaEstablished hypertensionThyroid diseasesOther organic, metabolic, or mental diseasesHormone replacement therapyTaking other medications, especially analgesics and psychotropic drugs Exclusion criteria are as follows:The following biochemical parameters using standard automated technique were assessed: blood cells count and levels of bilirubin, aspartate (AST) and alanine (ALT) aminotransferase, gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), total and LDL and HDL cholesterol, triglycerides, glucose, amylase, lipase, urea, creatinine, acute phase protein, thyroid stimulating hormone (TSH), and follicle-stimulating hormone (FSH) in serum.After inclusion into the study, all patients were recommended the same balanced diet with limited animal fats and simple carbohydrates of caloric content of 1600 kcal. At the same time, they were recommended to continue the treatment with the same statin at a daily dose of 20 mg.n = 30) was recommended to take statin together with melatonin  at a dose of 2 × 5 mg, at 7:00 a.m. and 9:00 p.m. In Group II (n = 30) patients took statin with placebo  at the same dose and time of the day.The patients were randomly allocated into two groups. Group I  were evaluated after 2, 4, and 6 months of treatment.A written consent was obtained from the patients and the Bioethics Committee of the Medical University in Lodz approved the study protocol (RNN/45/12/KB).Tests were conducted in accordance with the Declaration of Helsinki and with the principles of Good Clinical Practice.U test was used for nonparametric data to perform the comparison between groups. Calculations were made using Statistical 9.1 Microsoft Co. software, and statistical significance was established at p < 0,05.All parameters were checked for normality using the Shapiro-Wilk test. Wilcoxon's rank sum test was used for the comparison of basal treatment differences between each liver enzyme level. Comparison of parameters in four time series was calculated using ANOVA Friedman test. Mann–Whitney p < 0.05) and it remained at a similar level after 4 months 66.2 ± 10.3 U/L (p < 0.01) and after 6 months 52.8 ± 13.3 U/L (p < 0.001).In Group I the initial level of aspartate aminotransferase was 97.2 ± 19.1 U/L. After introduction of melatonin to the treatment this level decreased after 2 months to 77.5 ± 10.9 U/L , 55.2 ± 11.1 U/L (p < 0.001), and 49.8 ± 14.5 U/L (p < 0.001).Alanine aminotransferase level was in Group I in the same time intervals, respectively, 87.4 ± 15.6 U/L, 65.2 ± 13.9 U/L , 62.8 ± 11.3 U/L (p > 0.05), and 59.6 ± 8.3 U/L (p < 0.01). In Group II, these values were, respectively, 75.9 ± 11.3 U/L, 72.9 ± 9.7 U/L, 69.9 ± 7.9 U/L, and 70.9 ± 9.2 U/L; the differences were statistically insignificant , ALT in 9 , and GGT in 11  on Group I patients.In Group II, after 6 months, normal results were found only in 1 patient , whereas in 6 of them AST and ALT level increased by the average of 11,3%.p < 0.05).In Group I, the level of total cholesterol decreased after 6 months from 254.2 ± 20.2 mg/dL to 212.6 ± 19.6 mg/dL and in Group II from 245.6 ± 16.4 mg/dL to 226.3 ± 20.7 mg/dL; differences between the groups were statistically significant .Triglyceride level decreased in Group I from 212,4 ± 20,3 mg/dL to 183,0 ± 14,6 mg/dL and in Group II from 193.6 ± 20,1 mg/dL to 171,4 ± 15,4 mg/dL; the differences between the groups were insignificant  immune processes are not present. In this mechanism latency period is usually longer and it may be even several months , 43.The direct drug-induced injury by molecular mechanisms is the opposite of idiosyncrasy. High concentration of a drug or its metabolite leads to the inactivation of many important cytoplasmic or mitochondrial proteins. Drugs can also disturb the processes of mitochondrial fatty acid oxidation or inhibit the activity of the respiratory chain. This results in metabolic changes such as hypertriglyceridemia, decrease in ATP production, and increased production of oxygen free radicals –46.The mechanism of statin-related hepatotoxicity is not clear. It is suggested that statins cause damage to mitochondrial membranes which leads to the leakage of aminotransferases , 48. StaStatins even at low doses may lead to hepatocellular damage; however, this happens more often at high dose , 52. An In the case of patients with nonalcoholic steatohepatitis (NASH), Gonciarz et al. , 63 admiGood tolerability and safety of melatonin result from its pharmacokinetic properties. Andersen et al.  administOur results confirm significant hepatoprotective effect of melatonin in patients treated with statins. Medications regulating lipid metabolism are taken chronically and therefore hepatoprotective factors should be used on long-term basis and should be free of side effects; melatonin satisfies such conditions.The conclusions from the obtained results support the opinion of many researchers that melatonin can be administered to protect against side effects of other drugs.


Our previous study indicated that serum 1,5-anhydroglucitol  levels slightly increased after a glucose load; therefore, this study was conducted to explore short-term changes in 1,5-AG levels after a steamed bread meal test (SBMT) and compare the agreement of 1,5-AG, glycated albumin (GA), and glycated hemoglobin A1c (HbA1c) levels between fasting and postprandial states after an SBMT.  104 participants were recruited and underwent a 100 g SBMT. Fasting, 30 min, and 120 min of 1,5-AG, GA, and HbA1c were measured. P < 0.01), and HbA1c and GA levels showed stability at 30 and 120 min. The Passing-Bablok regression linear equation showed that postprandial 1,5-AG, GA, and HbA1c levels were well fitted , and Bland-Altman difference plot showed that 100% of data points for HbA1c30 and HbA1c120 fell within the limits of agreement; 94.2%, 96.2%, 95.2%, and 95.2% of data points for 1,5-AG30, 1,5-AG120, GA30, and GA120 fell within the limits of agreement, respectively.  Levels of 1,5-AG slightly increased from 30 to 120 min after an SBMT ( Agreement analyses indicated good stability of 1,5-AG, GA, and HbA1c levels after the SBMT. HbA1c had an optimal stability, which was superior to that of GA or 1,5-AG. Fasting blood measurements for the determination of biochemical parameters have long been the norm; however, a shift in this paradigm has increasingly evolved. In a consensus statement issued in 2016, the European Atherosclerosis Society and European Federation of Clinical Chemistry and Laboratory Medicine recommended measurement of nonfasting lipid profile , 4.With regard to blood glycemic indicators, glycated hemoglobin A1c (HbA1c) is the gold standard for determining long-term glycemic control. In recent years, glycated albumin (GA) and 1,5-anhydroglucitol  have been gradually introduced into clinical practice as emerging biomarkers that reflect average glucose levels of the past 2 to 3 and 1 to 2 weeks, respectively. Therefore, assessment of these 3 indicators in the nonfasting state is considered essential in the clinical setting.Despite this, only a few studies have attempted to validate agreement between baseline and postload levels of these 3 indicators –7. Our pPrevious studies, however, have not explored the trends of 1,5-AG, GA, and HbA1c after a steamed bread meal load, and no study to date has simultaneously compared postprandial agreement of 1,5-AG, GA, and HbA1c. Therefore, based on the previous study, this study was conducted to explore short-term changes in 1,5-AG levels after an SBMT, to evaluate concordance between fasting and nonfasting levels of 1,5-AG, GA, and HbA1c in a representative Chinese population, and to simultaneously compare simultaneous agreement of these 3 indicators to specify the rationale for their clinical application. Consistency analyses were conducted with several methods to evaluate different aspects of central tendency, dispersion, and correlation.n = 104, 76 with diabetes and 28 without diabetes, age range: 47–71 years) was prospectively recruited from the Shanghai Zhabei community between May and July 2016. Each participant underwent a 100 g SBMT, and diabetes was diagnosed on the basis of the 1999 World Health Organization criteria [Polygala tenuifolia and Senega syrup therapy, acute infection or other clinically stressful conditions, thyroid disease, cancer, and mental disorders were excluded.The study population . Fasting plasma glucose (PG0), 1,5-AG0, GA0, HbA1c0, hemoglobin, albumin, and serum creatinine levels were measured in morning fasting serum samples. Each subject then underwent an SBMT. Approximately 30 and 120 min, respectively, after SBMT, 1,5-AG30, 1,5-AG120, GA30, GA120, HbA1c30, and HbA1c120 levels were determined.We developed a uniformly designed study-specific questionnaire, which included questions on history of past and present illnesses and medication. Standard measurements of body weight, height, waist circumference, and blood pressure were done, and body mass index (BMI) was calculated as BMI = weight (kg)/heightStandard laboratory measurements were performed. Plasma glucose levels were immediately obtained by the glucose oxidase method  using the 7600–120 autoanalyzer . HbA1c was detected by high-performance liquid chromatography  with an inter- and intra-assay coefficient of variability (CV) of 0.75–3.39% and 0.55–2.58%, respectively. GA was measured using an enzyme-based assay kit  on a 7600–120 autoanalyzer  with inter- and intra-assay CV of 1.95–4.73% and 1.47–3.30%, respectively. Serum 1,5-AG levels were measured by an enzymatic method  on a 7600–120 autoanalyzer  with inter- and intra-assay CV of 1.54–3.03% and 0.83–2.44%, respectively. Hemoglobin was measured using sodium dodecyl sulfate colorimetry , whereas albumin was measured by the bromocresol green method  on a 7600–120 autoanalyzer . Serum creatinine was measured by the sarcosine oxidase method  on a 7600–120 autoanalyzer .b and the intercept a to determine the probability that any difference between b and 1 and between a and 0 arose incidentally. The 95% confidence intervals (CIs) are given [y-axis against the average of baseline levels and postprandial levels along the x-axis). The 95% CIs for the difference ranges (the sample mean difference ± 1.96 standard deviation) reflected the 95% probability range wherein lies the mean difference population parameter [Intraclass correlation coefficients (ICCs), as indices of agreement, were calculated as the ratio of individual to total variability. Good consistency was considered when the ICC was higher than 0.75 . The Pasre given . The Blaarameter , 19. If t-test and Wilcoxon signed rank sum test. Intergroup comparisons of skewed data were made using the Kruskal-Wallis test. Spearman correlation analysis was performed to explore the agreement of postprandial levels of the studied indicators. The ICC model, Passing-Bablok regression, and Bland-Altman difference plots were applied to identify the bias of postprandial levels of these indicators. A two-tailed P value of <0.05 was considered to indicate statistically significant differences.All statistical analyses were conducted using SPSS 19.0 and MedCalc 12.5.0. Data are presented as mean ± standard deviation. Each variable was examined for a normal distribution, and pair analyses were carried out using paired Student's 2. As 0, PG30, and PG120 levels were 7.8 ± 2.2, 9.8 ± 2.9, and 12.1 ± 4.5 mmol/L, respectively.The 104 participants included 44 men and 60 women, with an age of 62.5 ± 6.1 years. Mean BMI was 24.6 ± 3.2 kg/m0, 1,5-AG30, and 1,5-AG120 were 16.1 ± 11.0, 17.2 ± 11.8, and 17.9 ± 12.1 μg/mL, respectively (P < 0.01). Mean GA0, GA30, and GA120 were 17.3% ± 4.2%, 17.2% ± 4.3%, and 17.2% ± 4.2%, respectively. Mean HbA1c0, HbA1c30, and HbA1c120 were 6.6% ± 1.2% (48.3 ± 12.9 mmol/mol), 6.6% ± 1.2% (48.3 ± 12.9 mmol/mol), and 6.6% ± 1.2% (48.4 ± 12.7 mmol/mol), respectively. No statistically significant differences existed in either GA or HbA1c levels between 30 and 120 min in the SBMT , and these results were replicated when separately analyzed in patients with diabetes.Mean 1,5-AGectively . 1,5-AG P < 0.01). ICCs for 1,5-AG30, 1,5-AG120, GA30, GA120, HbA1c30, and HbA1c120 were 0.997, 0.995, 0.989, 0.988, 0.999, and 0.999, respectively.Spearman correlation analysis showed that postprandial 30 and 120 min levels of the 3 indicators significantly correlated with baseline levels . The fitted linear equations, intercept (95% CI), and slope (95% CI) for postprandial 1,5-AG and GA are shown in P > 0.10). There was, further, good agreement between 1,5-AG and GA at 30 and 120 min  μg/mL. On these graphs, 94.2% and 96.2% of data points for 1,5-AG30 and 1,5-AG120 fell within the limits of agreement, respectively  ectively . Mean diμg/mL, and −0.0% (−1.8% to 1.8%). Outliers of the agreement limits only accounted for 5.8% and 4.8% of the data points, respectively  ectively . The BlaIn the present study, the change trends of 1,5-AG, GA, and HbA1c levels after SBMT in the Chinese population were investigated for the first time. The results showed that similar as the trend after a glucose load, the 1,5-AG levels were slightly elevated after SBMT, whereas levels of GA and HbA1c remained stable. Then, the agreement analyses of the 3 blood glucose-monitoring indicators after SBMT showed good stability, and in the same circumstances, HbA1c showed better stability than both GA and 1,5-AG.Previous agreement studies of postprandial glycemic indicators were predominantly conducted after an OGTT. The caloric content of 75 g glucose in the OGTT is the same as that of 100 g flour in the SBMT (1255.2 kJ) . HoweverWith the HbA1c, a standard glycemic indicator, agreement results after the SBMT were consistent with results reported from previous studies with the OGTT , 22 and The 1,5-AG accurately and rapidly reflects glycemic control across 1 to 2 weeks –27. PrevThis study was further compared with the stabilities after an SBMT and found the HbA1c to be the best indicator with 100% points falling within the boundaries of consistency and corresponding value of GA and 1,5-AG of only about 95%. In addition to the stability of the indicators themselves, another possible reason might lie in the current routine clinical assessment methods. HbA1c was measured using high-pressure liquid chromatography, and both 1,5-AG and GA were measured using enzymatic assays. High-pressure liquid chromatography is not influenced by the protein concentration of the sample or the presence of glucose . The stuThere are some limitations to this study. First, this study with its small sample did not include patients with severe hyperglycemia. Second, this study was performed among a preselected high-risk population, which might considerably affect the sensitivity and specificity of the test. Third, glycemic indicators were evaluated at only 2 time points after the SBMT. Data from more time points in a larger and different glucose tolerance status population could provide a more complete picture of variations in these indicators after a glucose load.In conclusion, using several consistency analytical methods, this study utilized steamed bread as a simulation of daily postprandial status. The results showed that the levels of 1,5-AG were slightly elevated after the SBMT, whereas those of GA and HbA1c remained stable, similar as the trend after a glucose load. Agreement analyses of the 3 glycemic indicators after the SBMT showed good stability and indicated the superiority of the HbA1c in terms of stability, better than GA and 1,5-AG. These results provide a basis for the application of these 3 glycemic indicators' nonfasting measurement in clinical practice. Finally, the present study provided a better understanding of the role of 1,5-AG in metabolism and supported further studies of its transport. Because the mechanism for this change remains unclear, further studies in different 1,5-AG transport models are needed to investigate the relevant metabolic pathways.


Screening for risk of type 2 diabetes mellitus (T2DM) is an important public health issue. Previous studies report that fasting plasma glucose (FPG) and triglyceride (TG)-related indices, such as lipid accumulation product (LAP) and the product of fasting glucose and triglyceride (TyG index), are associated with incident T2DM. We aimed to evaluate whether FPG or TG-related indices can improve the predictive ability of a diabetes risk model for middle-aged Koreans.7708 Koreans aged 40–69 years without diabetes at baseline were eligible from the Korean Genome and Epidemiology Study. The overall cumulative incidence of T2DM was 21.1% (766 cases) in men and 19.6% (797 cases) in women. Therefore, the overall cumulative incidence of T2DM was 20.3% (1563 cases). Multiple logistic regression analysis was conducted to compare the odds ratios (ORs) for incident T2DM for each index. The area under the receiver operating characteristic curve (AROC), continuous net reclassification improvement (cNRI), and integrated discrimination improvement (IDI) were calculated when each measure was added to the basic risk model for diabetes.All the TG-related indices and FPG were more strongly associated with incident T2DM than WC in our study population. The adjusted ORs for the highest quartiles of WC, TG, FPG, LAP, and TyG index compared to the lowest, were 1.64 , 2.03 (1.59–2.61), 3.85 (2.99–4.97), 2.47 (1.82–3.34), and 2.79 (2.16–3.60) in men, and 1.17 (0.83–1.65), 2.42 (1.90–3.08), 2.15 (1.71–2.71), 2.44 (1.82–3.26), and 2.85 (2.22–3.66) in women, respectively. The addition of TG-related parameters or FPG, but not WC, to the basic risk model for T2DM  significantly increased cNRI, IDI, and AROC in both sexes.Adding either TyG index or FPG into the basic risk model for T2DM increases its prediction and reclassification ability. Compared to FPG, TyG index was a more robust T2DM predictor in the stratified sex and fasting glucose level. Therefore, TyG index should be considered as a screening tool for identification of people at high risk for T2DM in practice. Type 2 diabetes mellitus (T2DM) is one of the most prevalent non-communicable diseases in the middle-aged population worldwide, largely because of recent changes in diet and lifestyle , 2. In tObesity is the most significant risk factor for incident T2DM , 16. Bod1c) was included [1c is not assessed in the routine health examination and this study did not consider reclassification ability when other blood test results apart from HbA1c were included in the T2DM risk model [Also, the increase of fasting plasma glucose (FPG) in the normal range is associated with increased incident T2DM . Of thesincluded . Howeversk model .Therefore, in the present study, we aimed to identify which of the TG-related indices that can be derived from general check-up data would improve the prediction ability of the simple T2DM risk model in middle-aged Koreans.The Korean Genome and Epidemiology Study (KoGES) consists of a gene-environment model and population-based studies . KoGES: 1c > 6.5%, or prescription for anti-diabetic medication [Height and weight were measured to the nearest 0.1 cm and 0.1 kg using a digital stadiometer and a scale, respectively. Resting blood pressure while sitting was measured by trained technicians using a standard mercury sphygmomanometer. Blood samples were collected after fasting for at least 8 h. The Friedewald formula was used to indirectly estimate low-density lipoprotein cholesterol levels in subjects with plasma TG < 400 mg/dl . Diabetedication . Subjectdication .LAP and TyG index were calculated as follows:Data are expressed as numbers and proportions for discrete variables and mean ± SD for continuous variables, and were analyzed using Chi-square and Student’s t-tests, respectively.Based on the maximized Youden index, we calculated sex-specific cut-off points of each index for T2DM. Multiple logistic regression analysis was conducted and adjusted for age, BMI, hypertensive status, family history of diabetes, current smoking and alcohol consumption status, and regular exercise. The basic model was derived from the published diabetes risk model for middle-aged Koreans and included the variables listed above , 11. We P = 0.0149). By contrast, the percentage of subjects with a family history of diabetes was higher in women than in men . There were also significant differences in anthropometric indices between sexes. Mean BMI and WC were 24.1 ± 2.9 kg/m2 and 83.3 ± 7.6 cm in men, and 24.7 ± 3.2 kg/m2 and 81.2 ± 9.5 cm in women, respectively. Mean TG, FPG, LAP, and TyG index were 170.8 ± 111.6 mg/dl, 84.5 ± 9.0 mg/dl, 38.1 ± 34.0, and 8.7 ± 0.5 in men, and 140.9 ± 75.6 mg/dl, 81.1 ± 7.7 mg/dl, 39.0 ± 30.6, and 8.5 ± 0.5 in women, respectively. TG-related indices also showed significant differences between the sexes, with the exception of LAP.Table During the 10 year follow-up, the overall cumulative incidence of T2DM was 21.1% (766 cases) in men and 19.6% (797 cases) in women. Table Table Table P < 0.0001), 48.0% (P < 0.0001), 23.6% (P < 0.0001), and 38.7% (P < 0.0001) in men, and 28.6% (P < 0.0001), 21.3% (P < 0.0001), 21.3% (P < 0.0001), and 36.0% (P < 0.0001) in women, respectively. By contrast, the addition of WC made cNRI only 5.6% (P = 0.1689), in men and − 2.3% (P = 0.5666), in women. The IDI was also higher when TG-related indices or FPG was added than when WC was added to the basic model of T2DM in both sexes.Table 1c has been recommended for diagnosis and screening of patients. Lim et al. added HbA1c to a diabetes risk model and demonstrated an increase in predictive ability for T2DM in a middle-aged Korean cohort, while Ahn et al. also demonstrated through longitudinal validation analysis that adding FPG or HbA1c to the simple diabetes risk model also improves its predictive ability [1c is better than that of the simple diabetes risk model [We showed that the predictive ability of the simple T2DM risk model was increased when TG-related indices or FPG was added. Recently, several diabetes risk models have been proposed to screen high-risk groups, some of which included blood parameters. WC was included as one of the components in the model because increased WC increases T2DM risk . However ability , 22. Thusk model , 14, 22.1c and the Oral Glucose Tolerance Test (OGTT) to the T2DM risk model, there are substantial practical constraints on screening the whole population using such measurements [1c, given that these measurements are not currently included in primary screening measurements in Korea [The advantages of the simple diabetes risk model are that it is inexpensive and patients can use it for self-assessment . Weighedurements . As an eurements , 39. Thiin Korea . To the in Korea –42. Somein Korea , 42. Thein Korea . Therefoin Korea , 44. Forin Korea . Wang etin Korea . Associain Korea . Secondlin Korea . HoweverIn conclusion, TG-related indices and FPG were more accurate than WC in the prediction of incident T2DM. In the subgroup categorized by sex and fasting glucose level, TyG index was a more robust predictor for onset T2DM than other indexes. Therefore, TyG index can be a useful screening tool for incident T2DM in middle-aged Koreans.


CD4+ T-cell counts remained unchanged regardless of cART use, or allocation to vaccine or placebo. We concluded that there was no evidence of detrimental effects of PCV7 administration on viral load or CD4+ T-cell counts six months after vaccination with PCV7.Vaccination has been associated with a transient increase in viremia in HIV-infected individuals, although contradicting evidence persist in the literature. As part of a randomized placebo-controlled efficacy trial of the PCV7 in Malawi, we collected viral load and CD4+ T-cell counts from 237 adults who received two doses of vaccine or placebo, administered 4 weeks apart. Analyses were conducted separately for cART and non-cART users. Our analysis show no difference in viral loads between vaccine and placebo groups, regardless of cART use. Viremia decreased from 4.1 to 2.9 log We hypothesised that PCV7 might have a detrimental effect on HIV control, and report a comparison of changes in VL and CD4+ T-cell counts post-vaccine between two groups: those who received cART, and those who did not. cART use in our cohort was primarily determined by date of recruitment, with public funded cART becoming available two years after recruitment started.2The methods of this study are described elsewhere t-test, and Wilcoxon Rank-Sum test were used for continuous variables. VLs and CD4+ T-cell counts were log10-transformed to approximate a normal distribution, and mean values between baseline (t0) and at 6-months (t6), and between and within vaccine and placebo groups were compared using a two-tailed paired and unpaired Student’s t-test respectively. Statistical significance was set at p < 0.05. Analyses were conducted with StataV.12.1 .For this analysis, we included 237 HIV+ cases for whom VL data at enrolment and at 6-months follow-up were available . Analyse3Of the 439 HIV+ individuals recruited for the original clinical efficacy trial, 237 provided both t0 and t6 blood samples. Seventy-nine (33.3%) were on cART at some point between samplings, of which 32 (40.5%) were on cART at enrolment: 17 on the vaccine and 15 on the placebo arm, and 47 (21 in vaccine and 26 in placebo) started within 6 months of recruitment . The meavs. 184.3 ± 9.4 non-cART), and vaccine or placebo group (184.8 ± 9.2 and 184.2 ± 9.2 respectively).For both, cART and non-cART subgroups, participants in the vaccine and placebo arms were similar in regard of age, gender, and t0 VL and CD4+ T-cell counts. The mean time between t0 and t6 sampling was 184.5 ± 9.2 days, with no differences in regard of cART use (184.7 ± 8.8 on cART users 10 copies/mL (diff 1.2 [0.89–1.51] p < 0.0001). which was consistent in vaccine (diff 1.18 [0.73–1.62] p < 0.0001 and placebo diff 1.22 [0.79–1.65] p < 0.0001) groups. This reduction was not observed among those not on cART.There were no significant differences on VL baseline values between vaccine and placebo groups at t0 nor at t6 in either those receiving cART , nor on those not on cART within the sampling period . Among those receiving cART, we observed a reduction on VL between t0 and t6, from 4.1 to 2.9 logAmong the cART group, the number of individuals with undetectable VL (VL < 400 copies/ml) increased from 16 (42.1%) at t0 to 31 (81.6%) at t6 among vaccine recipients, and from 14 (34.2%) to 31 (75.6%) in the placebo. Among the non-cART group, 4 (4.8%) and 2(2.4%) vaccine recipients had undetected VLs at t0 and t6 respectively. One patient presented a VL < 400 cell/mL at both sampling points in the placebo group.CD4+ T-cell counts were no different between vaccine and placebo groups at t0 and t6 regardless of cART treatment subgroup. Those receiving cART had an average increase of 55.7 CD4+ T-cell counts at t6 compared to t0 (p < 0.0001), which was consistent in vaccine (diff 60.6 p = 0.0004) and placebo (51.4 p = 0.0042) arms. No significant changes were observed between groups among those not on cART.410copies/mL) than those who survived, consistent with a higher risk of death independent of vaccine administration. Those who were loss to follow-up within 6 months could not be included in our analysis, as they would not have provided the second sample. The majority of these subjects were not receiving cART, and while their baseline VL and CD4+ T-cell values were not significantly different from those who remained in the study (data not shown), we cannot rule out an effect that could pretentially skew the comparison.Results show no evidence of a PCV7-induced change on either VL or CD4+ T-cell cell counts in HIV+ adults six months after receiving two doses of PCV7, consistent with published studies This study shows that the administration of two doses of PCV7 to HIV+ individuals does not have a detrimental effect on VL or CD4+ T-cell counts in HIV+ individuals, regardless of whether they receive cART therapy. Based on these findings, the initial hypothesis of the excess deaths on the vaccine group observed in the original study being the result of attrition due to higher loss to follow-up among the placebo remains a plausible explanation