Additionally, we determined the influence of metformin on mice adipose tissue metabolism. This study has shown for the first time that metformin inhibits the proliferative potential of ASCs in vitro in a dose- and time-dependent manner. In addition, we have found a significant correlation between the activity of ASCs and osteopontin at the mRNA and protein level. Furthermore, we have demonstrated that 5 mM and 10 mM metformin have cytotoxic effect on ASCs, causing severe morphological, ultrastructural, and apoptotic changes. The reduced level of OPN in the adipose tissue of metformin-treated animals strongly correlated with the lower expression of Ki67 and CD105 and increased caspase-3. The metformin influenced also circulating levels of OPN, which is what was found with systemic and local action of metformin. The results are a valuable source of information regarding the in vitro effect of metformin on adipose-derived stem cells.Metformin is applied not only as antidiabetic drug, but also in the treatment of obesity or as antiaging drug. The first part of the research discussed the effect of metformin at concentrations of 1 mM, 5 mM, and 10 mM on the morphology, ultrastructure, and proliferation potential of mice adipose-derived multipotent mesenchymal stromal cells (ASCs)  Metformin  is a widely used drug for the treatment of type 2 diabetes mellitus, condition associated with insulin sensitivity and obesity. Additionally, it has been shown that metformin may inhibit growth of tumor cells and thus potentially may find application in therapy of various types of cancer –6. MetfoThe progressive aging of the human population together with increasing obesity are the main reasons of metabolic diseases . The WorProgenitor cells in the uncultured stroma-vascular fraction (SVF) derived from adipose tissue usually amount to up to 3% of the whole cells. This unique population is defined as adipose-derived multipotent stromal cells, ASCs , 16. AltGiven the fact that metformin exerts an unquestionable influence on adipose tissue, which becomes a source of a unique stem cell population, it was reasonable to investigate the effects of this compound on the proliferation potential, morphology, and ultrastructure of the ASCs.1, S, and G2 phases of the cell cycle [Markers that can be associated with cells proliferation are Ki-67 and osteopontin (OPN). Additionally, expression of another marker, for example, CD105/endoglin, should be taken into consideration, because it defines MSCs provenance and is associated with preadipocytes proliferation and differentiation potential , 21. Ki-ll cycle , 23 and ll cycle . In turnll cycle . It is ell cycle , 27. Morll cycle . The ostll cycle . in vitro and in vivo studies. Analysis of cytotoxic effect of metformin was performed on ASCs isolated from mice. We evaluated not only proliferative activity of ASCs exposed to metformin, but also their morphology and ultrastructure. Additionally we investigated the OPN gene expression and protein secretion.Bearing in mind all mentioned facts, determination of expressions of Ki-67 and osteopontin in adipose tissue of mice treated with metformin seems to be crucial regarding its influence on ASCs proliferation activity. To the best of our knowledge, this issue has never been investigated previously. Therefore, to obtain complex information concerning effect of metformin of ASCs and adipose tissue, we decided to approach this subject bidirectionally, performingMetformin accumulates in tissues of diabetic mice in concentrations several times higher than those in blood , 31, and in vivo study was to evaluate the effect of metformin on histological structure of adipose tissue and to determine expression of proliferation markers. We believed that the data presented in this study will provide meaningful information of clinical significance, especially due to the fact that substantial part of our research was dedicated to cells morphology investigation, important in diagnosis of pathology.The aim ofAll reagents used in this experiment were purchased from Sigma-Aldrich (Poland), unless indicated otherwise. (Dec. No. 177/2010 of 11.15.2010).All experimental procedures were approved by the II Local Ethics Committee of Environmental and Life Science University2 and 95% air. The cells were passaged after reaching 80–90% confluence. Subsequent cultures were propagated in DMEM containing 4500 mg/L of glucose. Culture media were supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin). The medium was changed every two days. Before experiment, the cells were passaged three times, using trypsin solution  according to manufacturers' instruction.The cells were isolated from subcutaneous adipose tissue of six C57BL/6 mice using protocol established previously , 33. Mat in vitro tests. Analyzed cells were adjusted in terms of subculture . For staining, cultures were maintained in 24-well dishes designated for immunofluorescence preparations (Sarstedt). For each staining, cultures were run in triplicate. To determine the phenotype of cells, the expression of following markers was investigated: CD29, CD44, CD45, CD73, and CD105. The antibodies used for the analysis were purchased from Sigma (rabbit anti-mouse integrin-b-1 (CD29), dilution 1 : 100; rabbit anti-mouse NT5E (CD73), dilution 1 : 200; rabbit anti-mouse endoglin (CD105), dilution 1 : 200; rabbit anti-mouse CD45, dilution 1 : 100), except for anti-CD44 which was obtained from R&D Systems . Secondary antibody conjugated with atto-488 fluorescence label was purchased from Sigma . Immunocytochemistry was performed using the general protocol described by the manufacturer. Incubation with primary antibodies was carried out overnight, while the reaction with secondary antibodies was performed at 37°C for 1 hour in the dark. In order to determine background fluorescence and exclude nonspecific staining, negative controls were included to assess the binding specificity of secondary antibodies. Negative controls were incubated with secondary antibody for 1 hour at 37°C in the dark. Additionally, specimens were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. The samples were imaged using an inverted fluorescence microscope . Images were captured with Cannon PowerShot camera and merged using AxioVision 4.8 software .Phenotypic characterization of cells was performed using ASCs cultures derived from animals assigned forOsteogenic and adipogenic differentiation of ASCs were induced using commercial kits . Cultures under standard and osteo- and adipogenic conditions were maintained in 24-well dishes. Experiments were carried out simultaneously, each in triplicate according to the previous findings . Osteoge4. Cells were suspended in 0.5 mL of culture medium per well. Metformin hydrochloride  was crushed in a mortar and dissolved in the culture medium at the following concentrations: 1 mM, 5 mM, and 10 mM. Nontreated cells served as a control for comparison with the test culture. In order to performe further analysis , each culture was performed in six replications. Proper number of culture plates was performed to investigate cells in the chosen time-points. The first dosage of metformin was added to the culture after 24 hours. During the cell treatment medium was changed every day. Culture media were collected for determination of protein level. The viability of the cells was evaluated after 24, 48, and 72 hours using a resazurin-resorufin system (Alamar Blue). To perform the test, culture media were removed and replaced with a medium containing 10% of the dye. Cells were incubated with the dye in the CO2 incubator for 2 hours. The supernatants were collected and transferred to a 96-well microplate reader . The absorbance of the supernatants was measured spectrophotometrically at a wavelength of 600 nm for resazurin and 690 nm as a reference wavelength. Each measurement included a blank sample, containing complete medium without cells. Proliferation activity was described using ΔΔA value, expressing difference of absorbance of supernatants at 600 and 690 nm and including absorbance of blank samples.For assays, cells were seeded in 24-well plates at a concentration equal to 3 × 10ASC morphology was evaluated under epifluorescent microscope  and scanning electron microscope . The analysis of morphology was performed after 24 h and 72 h of culture in 24-well plates. The preparation of ASCs for fluorescent microscopy was performed according to established procedure , 35 and For SEM analysis, (i) cells were fixed in 2.5% glutaraldehyde in DMEM, (ii) rinsed with HBSS, (iii) dehydrated in a graded ethanol series , (iv) air-dried for 30 min at room temperature, and (v) coated with gold using 300-second program . Prepared samples were captured using a SE1 detector at 10 kV filament tension  under 500x and 5000x magnification.For TEM analysis, we used procedure described previously . Brieflyβ2M) was used as housekeeping gene; sequences of primers were as follows: (i) forward 5′-CATACGCCTGCAGAGTTAAGCA-3′ and (ii) reverse 5′-GATCACATGTCTCGATCCCAGTAG-3′ (NCBI accession number: NM_009735.3). Quantitative RT-PCR was carried out in a total volume of 20 μL using SensiFast SYBR and Fluorescein Kit (Bioline). The concentration of primers in each reaction was 500 nM. The following cycling conditions were applied: 95°C for 2 min, followed by 45 cycles at 95°C for 5 sec, annealing 58°C for 10 sec, and 72°C step for 5 sec with a single fluorescence measurement. Analysis of the dissociation curve of amplicons was performed to determine the specificity of the PCR products. Melting curve was determined with a gradient program of the range from 65 to 95°C at a heating rate of 0.2°C/s and continuous measurement of the fluorescence. Reactions were performed in three repetitions. The values of the threshold cycle (Ct) obtained in each experiment were used to calculate fold change in relation to the expression of housekeeping gene. Real-time PCR was performed using CFX Connect Real-Time PCR Detection System (BioRad).After 24 and 72 hours of culture, cells were rinsed twice using HBSS and then homogenized using 0.8 mL of TRI Reagent. Procedure of cells' homogenization was performed directly in the culture dish. Preparations were obtained from samples prepared in duplicate. Total RNA was isolated using a single-step method described by Chomczynski and Sacchi . The resThe osteopontin level was determined in supernatants collected after experimental cultures. For analysis, each culture medium was prepared in duplicate. The concentration of OPN was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA kit . ELISA was performed according to the manufacturer's instructions. For the analysis, supernatants were diluted 5 times. All tested samples and standards were measured in two replicates. ad libitum during the 5-week experiment. The animals were purchased from Animal Laboratory House, Wroclaw Medical School, and housed in the Animal Experimental Laboratory . Mice received a standard diet with 4,2% fat  and were maintained on a 12-hour light-dark cycle at 22 ± 0.2°C.Eighteen C57BL/6 mice  were housed three per cage in an ultraclean facility on ventilated racks and were provided food and watern = 9) receiving drinking water only and (ii) the group receiving metformin in drinking water at a concentration equal to 2,8 mg/day  (n = 9). Water was changed every two days.The animals used in the study were divided into two groups: (i) control . Blood was collected from animals using cardiac puncture method. Biochemical analysis of blood samples, such as glucose and lipids measurements, was performed with Erba XL 300 platform . After euthanasia of animals, adipose tissue was collected from abdominal subcutaneous layer of all animals.μm thickness and subsequently stained using Mayer hematoxylin (Sigma-Aldrich). Sections were prepared in six replicates and deposited on Superfrost Ultra Plus Adhesion Slides (Thermo Scientific).Subcutaneous adipose tissue (0.5 g) was fixed in a cold 4% paraformaldehyde (Sigma-Aldrich). Next, samples were transferred to 0.1 M phosphate buffer and incubated for 24 hours. The samples were then treated with 18% sucrose for three days. The samples were applied on a specimen holder using tissue freezing medium and transferred to a freezer (−20°C). The samples were cut into sections of 0.5 Morphometric analysis of adipose tissue was performed with Axio Imager light microscope (Zeiss) using 10 fields of view for each individual (each slide) based on our previous experience . Histolohttp://rsbweb.nih.gov/ij/) was used to measure size and area of adipocytes. Adipocytes that were damaged or overly distorted owing to processing were not included in the measurements. Moreover, number of crown-like structures (CLS) was determined from six random microscopic fields magnified 100x.The Image J software (μm frozen sections were placed on Superfrost Ultra Plus Adhesion Slides (Thermo Scientific). The samples were stored at −20°C and before preparations were rehydrated. For each staining, two specimens were prepared. Antibodies used in the experiment were raised against mouse OPN, Ki67 (1 : 200) (both purchased from Abcam), and caspase-3 markers (1 : 100) (Sigma) as well as CD44 (1 : 500) and CD105 (1 : 100) antigens (both from Abcam). Obtained samples were incubated with specific antibodies over night at 4°C. After rinsing with HBSS, sections were incubated for 1 hour with goat anti-rabbit IgG antibody conjugated with atto-488 (dilution 1 : 200), avoiding direct light. Nuclei were counterstained using 4′,6-Diamidino-2-Phenylindole . Incubation of specimens with DAPI (5 min) was followed by HBSS washing. Slides were mounted using Fluoromount to image the sections. Specimens were visualized using epifluorescence microscopy  with a 100-fold magnification. Images were saved and processed using Axio Imager 4.7 software. Nonspecific staining of the samples was reduced by incubation with a blocking buffer for 30 minutes. The incubation buffer contained goat serum (1 : 100), bovine albumin (1 : 10), and Triton-X (0.3%).For immunohistochemistry, 4 The OPN concentration was measured using ELISA kit . Serum was obtained from all control and experimental animals. The test was performed similarly as it was described in paragraph 1.7, with the difference that serum samples required 100-fold dilution. All samples and standards were measured in duplicate.P < 0.05 were considered significant.The normality of the population data was determined using Shapiro-Wilk test, while equality of variances was assessed by Levene's test. Differences between groups were determined using one- or two-way analysis of variance (ANOVA). Statistical analysis was performed with STATISTICA 10.0 software . Differences with a probability of To confirm multipotent and mesenchymal character of isolated stromal cells, immunohistochemical staining and multipotency assay were performed. Analyses were conducted with regard to the recommendations of International Society of Cellular Therapy , 40. Imm in vitro, after exposition to the investigated concentrations of metformin, was evaluated after 24, 48, and 72 hours of culture  shedding . The ultAnalysis of OPN gene expression was investigated in cells derived from experimental and control cultures, after 24 h and 72 h of propagation . QuantitThe level of secreted metformin in experimental cultures decreased in time and in dose-dependent manner, while in control cultures highest concentration of OPN protein was noted after 72 h of culture. The concentration of OPN decreased significantly in all experimental cultures after 48 h of propagation .P < 0.001).In our experiment, administration of metformin caused significant weight loss in C57BL/6 mice. Additionally, mice treated with metformin had lowered glucose and triglyceride levels, whereas cholesterol level increased . All difμm) than adipocytes of experimental mice (13.97 ± 2.01 μm). The areas of adipocytes derived from experimental animals were also smaller than in control group. Additionally, population of adipocytes in tissue of control mice was more homogenous both in size (within a range of 15.46–18.67 μm) and in shape , while adipocytes forming adipose tissue of experimental mouse had divergent size (within a range of 11.02–18.21 μm) and morphology. The formation of crown-like structures in adipose tissue of experimental animals was more evident; however comparative analysis of CLS number showed that difference between groups is not relevant.Evaluation of the adipocytes morphology and results of morphometric analyses are shown in + and CD105+. However, substantial differences in the quality and intensity of reaction between investigated groups were observed; results were presented in +Figures , resp. i+Figures . The exp+Figures  when com+Figures . The rea+Figures  than in +Figures . In turn+Figures , when co+Figures .Evaluation of OPN serum concentration measured with ELISA showed that mice treated with metformin exhibit significantly lower OPN level than mouse in control group. The results of statistical analysis are presented in  in vitro and in vivo studies. Metformin is known mainly for its antidiabetic properties, but it may also produce physiological effects comparable to calorie restriction. For instant, it may decrease fat mass and reduce size of adipocytes, as it was shown in depots of subcutaneous tissue of patients with diabetes [ in vitro studies that metformin may increase glucose uptake of adipocytes and embryonic tissues [In the present study we investigated the effects of metformin on adipose-derived mesenchymal multipotent stromal cells (ASCs) and adipocytes, especially in relation to their proliferative potential. We have also focused on the evaluation of the influence of metformin on the morphology of both cells population of adipose tissue, and thus the results presented here includediabetes , 42. Add tissues , 44. Bec tissues .As it was previously shown, metformin may inhibit proliferation of stromal cells—fibroblasts  and canc in vitro in experimental and control cultures with the supernatant concentration of OPN measured with ELISA, we have found similarity in the pattern of curves' course. In the first 24 hours of cultures the proliferation activity of cells and OPN concentration in all investigated groups were at the same level. Both proliferation and OPN concentration significantly decreased after 48 h in cultures exposed to the metformin. Inhibition of proliferation activity of cells was also correlated with OPN decrease at mRNA level; the transcript level depends on dose used in culture, and the lowest expression of OPN mRNA was noted in cultures treated with 10 mM of metformin.When comparing proliferation activity of ASCsInhibited expression of OPN gene along with other osteogenic markers after treatment with metformin was noted previously in primary osteoblasts and mouse osteoblastic cell line (MC3T3-E1) . In this in vitro [μg/mL inhibits fibroblast proliferation, at both the normoxia and hypoxia conditions. Gao et al. [ in vitro was established for doses from 5 to 30 mM [ in vivo [The metformin action in time- and dose-dependent manner was also noticed by the other researcher groups in various cell cultures. The concentrations of metformin which was used in this study were also tested in cultures of human oral squamous cell carcinoma and significantly reduced the colony formation of those cellsin vitro . Dose rein vitro  has showo et al.  showed ao et al. , 52, 53.to 30 mM , 4–6, 54 in vivo . But ano in vivo . It was  in vitro studies, possess a cytotoxic effect on the cells and may cause (i) disintegration of cells, (ii) nucleus fragmentation and production of apoptotic bodies, (iii) enlargement of mitochondria, (iv) inhibition of MVs shedding, and (v) limited development of cellular projections. To the best of our knowledge, this is the first report describing morphological and ultrastructural changes of ASCs under the influence of metformin. However the observations concerning ultrastructural alterations resulting from the metformin action are consistent with the descriptions presented by Pounaghi et al. [Additionally, results of microscopic evaluation of ASCs morphology and ultrastructure have shown that high doses of metformin (5 mM and 10 mM), which were used fori et al.  who havei et al. , 57.Kiefer et al.  have notAdditionally, in our model, the decrease of tissue OPN expression after metformin treatment correlated also with decreased levels of this protein in mice serum. Number of human studies reported elevated circulating OPN levels in obese individuals compared with lean subjects . SurprisOur experiment also showed the effect of metformin on an adipocytes size. Morphometric correlation of adipocytes size from adipose tissue of control and experimental group revealed that metformin treatment significantly decreased diameter and area of adipocytes. This observation is in a good agreement with Ciaraldi et al.  and StumQuantitative evaluation of crown-like structures occurring around adipocytes revealed that macrophage infiltration into adipose tissue is similar in both investigated groups of animals. The fact that metformin may regulate body weight , 12 was In conclusion, metformin affects morphology of adipose-derived multipotent mesenchymal stem cells and adipocytes. Moreover, this compound highly influences proliferation of cells derived from adipose tissue. Targeting OPN expression and synthesis by the metformin action may significantly inhibit proliferation and differentiation of adipocytes and adipocytes precursors. Because obesity, cancer, and aging have numerous interrelationships, the recognition of metformin influence on OPN expression may bring a key that will help to tackle epidemics of obesity, diabetes, and cancer. Additionally, dose titration of metformin appears to be crucial, as effects of metformin action strongly relates to its concentration. An important part of future studies, related to metformin effects on stromal cells, should be a detailed analysis of the mechanisms of its action. As it was previously noted, the inhibitory effect of metformin on cells proliferation and differentiation may vary among cells. It seems that a strong clue that will allow for clarification of this issue might be elucidation of AMPK role in these cellular processes.


Hucho hucho) from: Poland, Germany, Slovakia, and Ukraine were investigated using ten microsatellite DNA loci. Microsatellite DNA analysis was successfully applied for the first time in the Polish broodstock of this fish species. The genetic variation and genetic distance between these broodstocks were evaluated. In addition, we examined the potential effects of a genetic bottleneck on the genetic variation of the broodstocks. The European huchen broodstocks exhibited moderate genetic diversity (PIC = 0.405–0.496 and I = 0.831–1.047) with the exception of German broodstock which presented higher genetic diversity (PIC = 0.590 and I = 1.254). Observed (Ho) and expected (He) heterozygosity across the investigated loci in all broodstocks ranged from 0.434 to 0.686 and from 0.452 to 0.650, respectively. Overall, the studied broodstocks were in Hardy-Weinberg equilibrium (HWE); however, from 8 to 42 % of the loci deviated from HWE in each stock. The Garza-Williamson index (M = 0.146–0.279) and values of the heterozygosity excess revealed a reduction of genetic variation in all studied broodstocks because of the founder or bottleneck effect. The analysis of genetic differentiation (Fst) and Nei’s genetic distance between pairs of broodstocks revealed that Polish and Ukrainian broodstocks of European huchen were characterized by the closest genetic distance. In contrast, the highest genetic divergence parameters (Fst and Nei’s distance) were observed among German, Slovak, and Ukrainian broodstocks.Four broodstocks of European huchen ( Hucho hucho), also called Danube salmon, is the largest salmonid species and one of the most endangered members of the Salmonidae family (Holcik Hucho taimen) are exclusively freshwater residents. Furthermore, this fish species can live for more than 20 years, and adults are large piscivorous predators of up to 1.65 m standard length (SL) and with a maximum mass of 60 kg (Kottelat and Freyhof European huchen (y Holcik . It is ey Holcik . EuropeaBrachymystax lenok)   pelvic or pectoral fin clips were placed in Eppendorf tubes and kept in 96 % ethanol at a temperature of 4 °C until DNA extraction. DNA was isolated from collected fin clips using standard Chelex 100 procedure , Germany , Slovakia  and Ukraine  during the years 2011-2013  and 0.6 unit GoTaq flexi DNA polymerase . Re-distilled water was used to bring the reaction mixture to the desired final volume. Amplification was performed with a Mastercycler gradient thermocycler  under the following conditions: an initial denaturation at 94 °C for 3 min, followed by 34 cycles at 94 °C for 30 s, annealing at 53–61 °C  (Table Ar) were computed by GenePop software (version 4.2.1) (Rousset Ho) and expected heterozygosity (He), the exact Hardy-Weinberg (H-W) equilibrium test, as well as linkage disequilibrium (LD) were calculated using Arlequin software (version 3.5) (Excoffier and Lischer Fis) were calculated using Fstat software (version 2.9.3)  was also calculated employing PowerMarker software (version 3.25) (Liu and Muse Fst) and Nei’s genetic distance. Shannon’s index (I) for each loci within tested broodstocks was calculated using PopGene software (version 1.3.2) (Yeh and Boylet M), which was computed applying Arlequin software 3.5. A test for bottleneck assessment was also conducted using the Bottleneck software (version 1.9)  and infinite allele model (IAM) for four broodstocks of European huchen were tested. This method is based on the assumption that in non-bottlenecked broodstock (close to mutation drift equilibrium) the value of expected heterozygosity (He) is equal to Heq (heterozygosity expected in a mutation-drift equilibrium). The excess of He over Heq is the evidence of severe reduction in broodstock effective size that may occur because of a bottleneck event. In order to accommodate the obtained genotypic data to the requirements of employed software, every tetrasomic locus was examined as two disomic loci and as result the mean values was considered for estimation of genetic parameters.The Micro-Checker software (version 2.2.3) was used to check microsatellite null alleles, scoring errors due to stuttering and large allele drop-out in samples  Goudet . The polStr-12, Str-15, Str-60, Str-73, Str-543, Ssa-85, Ssa-171, BleTet-2, Sfo-292, Omm-1016, Omm-5017) were monomorphic in all broodstocks, one (Str-85) did not produce any PCR product and seven loci  produced non specified bands (stutter bands). Ten microsatellite loci  were selected for further analyses because they were characterized by good quality products and were polymorphic  and 467 bp. The number of amplified alleles per locus ranged from one (Sfo-262) to 26 (Omm-1077) with an average of 8.1 alleles per locus  of the four broodstocks of European huchen are shown in Tables PIC) in fish from Poland, Germany, Slovakia, and Ukraine were 0.430, 0.590, 0.496, and 0.405, respectively. The mean allelic richness varied from 3.944 to 5.200 in studied broodstocks of European huchen  in the investigated broodstocks ranged from 0.434 (Ukraine) to 0.686 (Germany) and were close to the mean values expected under H-W equilibrium (He) (Table Heq). In all broodstocks, under the infinite allele model (IAM) and stepwise mutation (SMM) models, heterozygosity excess were detected in most of analyzed loci; however, observed He>Heq differences were significant in only Polish  and Ukrainian  broodstocks. All investigated loci differed in terms of the Garza-Williamson index (M). In all samples M values were the lowest for the most polymorphic loci (such as BleTet-9 and Omm-1077) and the highest for the least polymorphic loci . The mean observed M values in the examined broodstocks varied from 0.146 to 0.279. The mean fixation index (Fis) in fish from Poland, Germany, Slovakia, and Ukraine were −0.071, −0.082, −0.047, and 0.053, respectively (Table The mean observed heterozygosity (Fst) of the four broodstocks of European huchen was 0.1139. Analysis of the genetic structure of the studied broodstocks with AMOVA method revealed that 13.20 % of the genetic diversity was distributed among broodstocks and 86.80 % occurred among individuals within the broodstocks. The Nei’s genetic distances among the four broodstocks and the Fst matrix are shown in Table Fst values, occurred between Ukraine and Slovakia (0.1731). The lowest genetic divergence and genetic distance were observed between Poland and Ukraine , while the highest genetic distances were observed among German, Slovak, and Ukrainian broodstocks  with the exception of German broodstock which presented high genetic diversity (the mean He and PIC were close to 0.6). Similar moderate indices of genetic diversity to those observed in the present study were reported for some populations of taimen from Heilongjiang River Basin (China)  consistently ranked the broodstocks in order of decreasing diversity (Germany > Slovakia > Poland > Ukraine). However, there were no significant differences between these parameters among the four studied broodstocks (P > 0.05).The comparison of the overall number of private alleles, allelic ranges and its frequency clearly demonstrates genetic differences between studied broodstocks of European huchen. This confirms the hypothesis that the broodstocks studied do not share a common gene pool at the population genetic level. Genetic diversity, which can be evaluated as the allele number by locus and the mean number across loci, differed among the studied groups of fish. Examined broodstocks of European huchen exhibited moderate genetic diversity  indicating a small overall deficiency of heterozygotes, while the rest of the analyzed broodstocks of European huchen were characterized by negative Fis indicators exhibiting small overall excess of heterozygotes against Hardy-Weinberg expectations. In contrast, in domesticated stocks of fish the observed heterozygosity (Ho) sometimes exceeds the expected heterozygosity (He). For example, this tendency was observed in Chinook salmon (Oncorhynchus tshawytscha) stocks   and stepwise mutation models (SMM) in Polish and Ukrainian broodstocks. The heterozygosity excess at observed loci in mentioned broodstocks might be indicative of a small founding population size or bottleneck events as was hypothesized in the case of the hatchery stocks of brook trout   from China ; however, the majority of diversity were observed between individuals within a broodstocks (86.80 %). Similar genetic diversity (13.10 %) was observed among two hatchery stocks of barfin flounder (A continuous genetic monitoring of hatchery stocks is an important tool for development of sustainable conservation management programs. Management based on supportive and captive breeding carries the risk of adverse modifications to a gene pool, resulting in loss of genetic diversity, decrease of heterozygosity, or inbreeding depression. The consequence of inbreeding depression in hatchery stocks can be the negative effects on: hatching rate, fry survival, growth rate, and spawning performance, as well as inefficiency of feed conversion, occurrence of deformations and presence of short-lived albino fish (Guo-Sheng et al. The potential for evolution and adaptation to new environments is limited by the genetic diversity of the population. Natural selection favors individuals that are better adapted to live in natural environment than in captivity, thus research programs aimed at retaining the evolutionary genetic adaptive potential of European huchen stocks are very important. Therefore the optimum degree of genetic differentiation and a high genetic diversity should be permanently controlled. Moreover, European huchen habitat continues to be threatened by further anthropogenic factors influence and particularly unfavorable conditions may adversely impact on the populations characterized by low genetic variation level. The lack of information on the genetic structure of European huchen broodstock in Poland essentially limits the sustainable conservation of this species. Therefore, baseline genetic data are crucial to guide future population specific conservation programs and research efforts on European huchen in Poland.


Objective. TRAIL is a potential biomarker of cardiovascular (CV) disease. Ankylosing spondylitis (AS) is a chronic inflammatory disease associated with metabolic syndrome (MeS) and accelerated atherosclerosis. We assessed whether disease activity, systemic inflammation, and MeS features were associated with circulating TRAIL levels in AS patients undergoing TNF-α antagonist infliximab therapy and if infliximab infusion modified TRAIL levels. Methods. We measured TRAIL serum levels in 30 nondiabetic AS patients without CV disease undergoing anti-TNF-α therapy, immediately before and after an infliximab infusion, and in 48 matched controls. Correlations of TRAIL levels with disease activity, systemic inflammation and MeS features, adipokines, and biomarkers of endothelial activation were evaluated. Changes in TRAIL levels following anti-TNF-α infusion were analyzed. Results. TRAIL levels were higher in AS patients than controls. TRAIL levels displayed an inverse correlation with total and LDL cholesterol. We observed an inverse correlation with QUICKI and a marginal association with HOMA-IR. We also found an inverse correlation with resistin and a marginal association with apelin and OPN. Anti-TNF-α infusion did not change TRAIL levels after 120′. Conclusion. Elevated TRAIL levels in AS patients may be the result of a compensatory mechanism to reduce CV risk in these patients. In addint of AS . Interespatients . Additiond in AS –12. Sincpatients , the anaTNF-related apoptosis-inducing ligand (TRAIL) belongs to the TNF superfamily and was proposed as a potential biomarker of CV disease . This prα monoclonal antibody infliximab infusion modified TRAIL levels.Taking all these considerations into account, in the present study, we aimed to study if TRAIL serum levels were altered in AS patients undergoing infliximab therapy when compared to controls. We also aimed to assess potential associations between disease activity, systemic inflammation, adipokines, biomarkers of endothelial activation, and MeS features with circulating TRAIL levels in these patients. Finally, we also aimed to determine whether an anti-TNF-We assessed a series of 30 patients with AS attending hospital outpatient clinics seen over 14 months (January 2009 to March 2010), who fulfilled the modified New York diagnostic criteria for AS . They weFor ethical reasons, patients included in the present study were not randomized to a placebo group. The same procedure has been found acceptable and followed in studies on the short-term effect of infliximab therapy on adipokines and biomarkers of endothelial cell activation in patients with rheumatoid arthritis (RA) –22.Patients on treatment with infliximab seen during the period of recruitment with diabetes mellitus or with plasma glucose levels greater than 110 mg/dL were excluded. None of the patients included in the study had hyperthyroidism or renal insufficiency. Also, patients seen during the recruitment period who had experienced CV events, including ischemic heart disease, heart failure, cerebrovascular accidents, or peripheral arterial disease, were excluded. Patients were diagnosed as having hypertension if blood pressure was ≥140/90 mmHg or they were taking antihypertensive agents. Patients were considered to have dyslipidemia if they had hypercholesterolemia and/or hypertriglyceridemia . Obesity was defined if body mass index (BMI)  was greater than 30.α monoclonal antibody infliximab was started because of active disease. All patients included in the current study had begun treatment with NSAIDs immediately after the disease diagnosis. All of them were still being treated with these drugs at the time of the study. At the time of this study, most patients were on treatment with naproxen 500–1000 mg/d. Although the 2010 updated recommendations facilitate initiation of TNF-α blockers in AS and only ask for 2 NSAIDs with a minimum total treatment period of 4 weeks [α therapy in these series of patients recruited between January 2009 and March 2010, they had to be treated with at least 3 NSAIDs prior to the onset of infliximab therapy.In all cases, treatment with the anti-TNF- 4 weeks , for theA clinical index of disease activity (Bath Ankylosing Spondylitis Disease Activity Index—BASDAI—range of 0 to 10)  was evalα monoclonal antibody infliximab , the mean BASDAI ± standard deviation (SD) was only 2.94 ± 2.11.The main demographic, clinical, and laboratory data of this series of 30 AS patients at the time of the study are shown in α therapy. Also, patients gave informed consent to participate in this study. Neither this study nor the former studies on the short-term effect of infliximab therapy on insulin resistance, adipokines, and biomarkers of endothelial cell activation in AS were supported by any pharmaceutical drug company.The local institutional committee approved anti-TNF-μU/mL) × glucose  (mmol/l) ÷ 22.57) [In all cases, the drug was given to patients as an intravenous infusion in a saline solution over 120 minutes. All measurements were made in the fasting state. Blood samples were taken at 0800 hours for determination of the erythrocyte sedimentation rate (ESR) (Westergren), C-reactive protein (CRP) (latex immunoturbidimetry), lipids (enzymatic colorimetry), plasma glucose, and serum insulin . As previously described, insulin resistance was estimated by the homeostasis model assessment of insulin resistance (HOMA-IR) using the formula (insulin  ÷ 22.57) . A comme7 . A comr).Variables were expressed as mean ± SD, median, interquartile range (IQR), or percentages. Correlation between basal TRAIL at time 0 and selected continuous variables was performed, adjusting for age at the time of the study, sex, and classic CV risk factors via estimation of the Pearson partial correlation coefficient (t-test. Differences in TRAIL levels between men and women and patients with hypertension or without were assessed by the Mann-Whitney U test.The associations between baseline characteristics and serum TRAIL concentrations were assessed by Student's paired t-test.TRAIL serum levels before (at time 0) and after infusion (at time 120 minutes) were compared using the paired Student's P  values ≤ 0.05 were considered to indicate statistical significance. Analyses were performed using Stata 12/SE .Two-sided P < 0.0003) : 160.72 (128.69–195.99)) than in healthy controls : 107.49 (85.32–137.31)) ( 0.0003) .Circulating TRAIL levels did not correlate with disease duration, BASDAI, or VAS spinal pain at the time of the study . Likewisr = −0.427; P = 0.05) and a marginal association with apelin . No association with the rest of adipokines was observed  and LDL cholesterol . In addition, we observed a negative correlation of TRAIL with insulin sensitivity index  and a marginal association with IR   . However = 0.06) . Likewis = 0.06) . Besidesr = −0.372; P = 0.06)  .P = 0.772).TRAIL serum levels did not change following an infliximab infusion. In this regard, the mean ± SD values of TRAIL were 160.57 ± 47.48 pg/mL immediately prior to infliximab infusion (time 0) and 157.06 ± 65.90 pg/mL at the end of the infusion (time 120 minutes)  due to prolonged biologic therapy (median duration: 23 months). Interestingly, TRAIL levels were higher in AS patients than in controls. In keeping with our results, increased levels of TRAIL were found in rheumatoid arthritis patients undergoing disease-modifying antirheumatic drug therapy for 1 year when compared to controls . LikewisNext, we investigated the potential association of TRAIL with metabolic indices, since AS patients often display MeS features . In thisFurthermore, we also disclosed an inverse correlation of TRAIL with the proinflammatory adipokine resistin and a marginally negative association with the proinflammatory biomarker of endothelial activation OPN. In line with these results, a marginally positive correlation between TRAIL and apelin, an anti-inflammatory adipokine, was also observed. These results further support the previously described anti-inflammatory role of TRAIL .α therapy, a chronic inflammatory state is plausible to exist in our patients (CRP 24 at disease diagnosis and 6.24 mg/L at the time of the study). In this regard, it was reported that CRP values greater than 3 mg/L were associated with high risk of future CV events [Unlike patients with diabetes or obese individuals , 28, in V events . Therefoα blockade on TRAIL and other molecules implicated in the mechanisms leading to CV disease in AS.Finally, although the aim of the study was to evaluate the immediate direct effect of an infliximab infusion on TRAIL levels, adipokines, biomarkers of endothelial activation, and metabolic syndrome, it may constitute by itself a potential limitation since it is possible that the influence of infliximab on TRAIL levels may require several steps before directly reducing TRAIL levels. Therefore, further studies aimed at determining the effect of infliximab on TRAIL levels at different times, especially at later times, are needed to fully establish the effect of anti-TNF-In conclusion, our results suggest that elevated TRAIL levels in AS patients may be the result of a compensatory mechanism to reduce CV risk as a result of IR and chronic inflammatory state in these patients.


Salvadora persica (miswak) on oral microorganisms, but information concerning its antibacterial activity against other human pathogens, particularly multidrug resistant (MDR) isolates, is scarce. Therefore, this study aimed to assess the in vitro antibacterial activities of Salvadora persica L. extracts against 10 MDR bacterial clinical isolates other than oral pathogens. The antibacterial activity of aqueous and methanol miswak extracts was assessed using the agar dilution and minimum inhibitory concentration (MIC) methods. Overall, the 400 mg/mL of miswak extract was the most effective on all strains. The methanol extract exhibited a stronger antibacterial activity against Gram-negative (3.3–13.6 mm) than Gram-positive (1.8–8.3 mm) bacteria. The lowest MIC value was seen for E. coli , followed by Streptococcus pyogenes (1.56 µg/mL). The highest MIC value  was recorded for methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, and Stenotrophomonas maltophilia. This study demonstrates, for the first time, the moderate to strong antibacterial activity of miswak extracts against all tested MDR-pathogens. Methanol extract appears to be a potent antimicrobial agent that could be considered as complementary and alternative medicine against resistant pathogens. Further studies on a large number of MDR organisms are necessary to investigate and standardize the inhibitory effect of miswak extracts against these emerging pathogens.Much effort has focused on examining the inhibitory effect of Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and carbapenem-resistant Gram-negative bacilli clinical isolates poses a serious therapeutic problem because no new antimicrobial agents are currently available for treatment of infected patients [The emergence of multidrug resistant (MDR) human bacterial pathogens during 1990s and more recently the extensively resistant clinical isolates is hampering efforts to control and manage human infections by these organisms . Moreovepatients –4.Plants are important in human's life and fulfill his every day's needs. They are used as cosmetics, food, flavors, ornamental, and medicine. Medicinal plants have become part of complementary medicine worldwide, because of their potential health benefits. Various plant extract has great potential against infectious agents and can be used for therapeutic purposes , 6. Salvadora persica L, also called miswak, belonging to the Salvadoraceae family, is one of the most important ones among 182 species of plants being used as chewing sticks. It has been widely used in many Asian, African, and Middle Eastern countries. The roots, twigs, and stems of this plant have been used for oral hygiene and small miswak sticks have been used as toothpicks for maintaining oral hygiene [The toothbrush tree, hygiene , 8. It h hygiene . Staphylococcus aureus, Streptococcus mutans, Streptococcus faecalis, Streptococcus pyogenes, Lactobacillus acidophilus, Pseudomonas aeruginosa, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Haemophilus influenzae, and Candida albicans [ Salvadora persica extract is also an effective antimicrobial agent when utilized clinically as an irrigant in the endodontic treatment of teeth with necrotic pulps [Previous in vitro studies have reported the antibacterial and antifungal effects of miswak on cariogenic bacteria and periodontal pathogens includingalbicans –16. Moreic pulps –20. Salvadora persica on oral organisms, but information concerning the antibacterial activity of Salvadora persica against other human pathogens, particularly MDR isolates, is scarce [ Salvadora persica L. extracts against 10 MDR bacterial clinical isolates  including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), penicillin-resistant Streptococcus pyogenes, Enterococcus faecalis, and 6 carbapenem-resistant Gram-negative bacilli: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Acinetobacter baumannii, and Stenotrophomonas maltophilia.Much effort has focused on examining the inhibitory effect ofs scarce , 15. TheThis in vitro descriptive study was conducted during the period from March to August 2015. A total of 10 pathogenic MDR bacterial strains other than oral pathogens were included in this study. Streptococcus pyogenes, Enterococcus faecalis, and 6 carbapenem-resistant Gram-negative bacilli: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Acinetobacter baumannii, and Stenotrophomonas maltophilia. All isolates were identified to the species level using standard methods and verified using the VITEK-2 system  according to the manufacturer's instructions. Seven nosocomial strains were isolated from urinary tract infection, two strains were isolated from blood stream infection, and one strain was isolated from ventilator-associated pneumonia. Antimicrobial resistance patterns of all isolates were determined using the reference broth microdilution method following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [ S. aureus and S. epidermidis strains was identified by PCR amplification of the mecA gene as previously described [All bacterial strains were isolated from clinical specimens of hospitalized patients with nosocomial infections identified according to the Centers for Disease Control and Prevention/National Healthcare Safety Network (CDC/NHSN) criteria . The sele (CLSI) . Methiciescribed . Carbapeescribed . Multidrescribed .Salvadora persica chewing sticks were purchased from the local market of Jazan Province in Saudi Arabia during spring 2015 when plants were flowering. The sticks were washed with distilled water, cut into small species, and allowed to dry at room temperature for 2 weeks. Then, they were grounded to powder using electrical blinder. Salvadora persica powder with 1 L of distilled water (for aqueous extract) and 95% methanol , with methanol extract for 24 h. The mixture was then filtered using Whatman number 1 filter paper, and the filtrate was then evaporated in vacuum evaporator at 60°C (for aqueous) and 40°C (for methanol). The extracts were stored in sterile bottles and kept frozen at −20°C until further use [Preparation of aqueous and methanol extracts was carried out by mixing 100 g of620). Growth was checked every 30 minutes, and the exponential phase of bacterial growth was identified by the increased OD620 reading. Then, the inoculum density of each bacterial suspension was adjusted to a final density equivalent to 0.5 McFarland Standard (1.5 × 108 CFU/mL) in sterile saline (0.84% NaCl).All bacterial isolates were grown to the exponential phase in tryptic soy broth (TSB)  at 37°C for 18 h. The bacterial growth was estimated as turbidity using spectrophotometer to measure the light absorption of the microbial mass as determined by the optical density readings at 620 nm  methods.µL of bacterial suspension was spread smoothly on the agar plates. The required numbers of wells, each 3 mm in diameter, were cut out of the agar using a sterile glass capillary ensuring proper distribution of holes in the periphery and one in the center for each agar plate. Then, wells were filled with 50 µL of sterile extract (aqueous or methanol) made from Salvadora persica stock solution . This was followed by 2 h preincubation at room temperature for proper diffusion of the plant extract into the media. Then, the plates were incubated at 37°C for 24 h [µg vancomycin for Gram-positive isolates and 10 µg tobramycin for Gram-negative strains, were used as positive controls, and water (for aqueous extract) or methanol (for methanol extract) was used as negative control. The test for each microorganism was repeated three times to ensure reproducibility. The average zones diameter values from three repeats were taken in determination of the final inhibition zones. This was done to ensure that all inhibition zones within each experiment were obtained under the same experimental conditions.The antimicrobial testing was performed on Mueller Hinton agar plates (Difco Laboratories) using the agar diffusion method. Briefly, 100 for 24 h . The mea Salvadora persica extracts was determined using the standard microdilution method in 96 multi-well microtiter plates, as previously described [µL from each of the aqueous and methanol extracts was pipetted into the first well of each microtiter plate row, and 50 µL of TSB was distributed from the 1st to the 12th well of each row. Twofold serial dilution was achieved by transferring 50 µL of scalar dilution from the first to the subsequent wells of each row. The final concentration of the extracts adopted to evaluate antibacterial activity was included from 25 mg/mL to 0.003 mg/mL. Finally, 10 µL of each bacterial suspension was added to each well. Two row lines in each plate were used as controls: one row line with vancomycin as a positive control for Gram-positive isolates and another row line with tobramycin for Gram-negatives strains . Plates were incubated at 37°C for 18–24 h. The lowest concentration at which no turbidity occurred was taken as the MIC value. Plates were analyzed individually to determine MIC and the average MIC values from three repeats were taken in determination of the final MIC values for each extract to ensure accuracy and reproducibility.The MIC of theescribed , with sl Salvadora persica against 10 MDR pathogenic organisms are listed in  E. coli (4.8–13.6 mm), followed by K. pneumoniae (4.6–12.7 mm) and Serratia marcescens (4.5–12.5 mm). Streptococcus pyogenes was the most susceptible one to methanol extract among all Gram-positive pathogens with inhibition zone diameter of 2.2–7.4 mm.The antibacterial activities of aqueous and methanol extracts of E. coli (0.39 mg/mL for methanol extract and 1.56 mg/mL for aqueous extract), followed by Streptococcus pyogenes (1.56 mg/mL for both extracts). The highest MIC value (12.5 mg/mL for aqueous extract and 6.25 mg/mL for methanol extract) was recorded for MRSA, Acinetobacter baumannii, and Stenotrophomonas maltophilia.The MIC values of aqueous and methanol extracts are presented in  Salvadora persica.Antimicrobial resistance has always been a global health concern challenging treatment of human infections caused by MDR-bacterial pathogens . This pr Salvadora persica for antimicrobial activities against seven oral pathogens [ Salvadora persica extracts is attributed to the different phytochemical constituents. Mohammed investigated the phytochemical constituents of Salvadora persica extracts and revealed the presence of flavonoids, sterols, saponins, tannins, basic alkaloids, and reducing components in methanol extract and saponins, tannins, and reducing components in aqueous extract which could be responsible for the observed antimicrobial property of methanol extract compared with aqueous extract [ Salvadora persica extracts [In this study, the 400 mg/mL of miswak extracts was the most effective one on all pathogens and the methanol extract exhibited a stronger antibacterial activity against Gram-negative than Gram-positive bacteria. These results are in agreement with previous findings from other studies , 16, 17.athogens . In theiextracts . In theiextracts . E. coli, K. pneumoniae, P. aeruginosa, and S. marcescens among Gram-negative pathogens and Streptococcus pyogenes among Gram-positive bacteria. Previous studies have reported that Salvadora persica extracts were effective against S. aureus, Streptococcus mutans, Streptococcus pyogenes, E. faecalis, and P. aeruginosa [ A. baumannii, and S. maltophilia. To the best of our knowledge, this is the first study investigating the antibacterial activity of miswak extracts against these MDR-pathogens. Our finding is of considerable concern. These emerging pathogens become a significant health problem because of their remarkable ability to innate and acquire resistance to multiple antimicrobial classes and to survive in nosocomial environments [In this study, the methanol extract had promising MIC values againstruginosa , 16, 17.ronments , 4. A. baumannii, and S. maltophilia are necessary to further investigate and standardize the inhibitory effect of miswak extracts against these emerging pathogens.This study demonstrates, for the first time, the strong to moderate antibacterial activity of methanol and aqueous miswak extracts against all tested MDR-pathogens. However, methanol extract has stronger antibacterial effect, and it appears to be a potent antimicrobial agent that could be considered as complementary and alternative medicines against resistant pathogens. Further in vitro and in vivo studies on a large number of clinical isolates of MRSA,


Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense.Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Mgfnr not only resulted in decreased N2 production due to reduced N2O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr.Deletion of 2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization.We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O Magnetospirillum gryphiswaldense (in the following referred to as MSR-1) and other MTB, magnetosomes are membrane-enveloped magnetic crystals of magnetite (Fe3O4) that are aligned in chains [2O4] occurs by coprecipitation of ferrous and ferric iron in supersaturating concentrations, which requires a balanced ratio of ferrous and ferric iron 2+ or a [2Fe-2S]2+ cluster [2+ cluster per monomer. The Fnr dimers are able to bind target promoters and regulate transcription. Exposure of the [4Fe-4S]2+ clusters to oxygen results in its conversion to a [2Fe-2S]2+ oxidized form, which triggers conformational changes and further induces the protein monomerization and prevents its binding to DNA 2+ cluster in regulating EcFnr activity, and some single amino acid substitutions at positions not conserved in the Fnr family led to increased stability of Fnr to oxygen and activated transcription of nitrate reductase genes under aerobic growing conditions 2+ cluster. However, MgFnr was able to complement ΔEcfnr mutant back to WT-like growth, which indicates that MgFnr also has the universal properties of EcFnr. Nonetheless, ΔEcfnr mutant and ΔMgfnr mutant displayed significantly different phenotypes during anaerobic growth, such as a largely decreased growth yield in ΔEcfnr mutant, but no defective growth in ΔMgfnr mutant. These differences might be explained by different media used for cultivation because in E. coli deletion of Ecfnr only resulted in growth defect in some minimal media [Mgfnr but also overexpression of Mgfnr in WT affected anaerobic and microaerobic magnetite biomineralization in the presence of nitrate and caused the synthesis of smaller magnetosome particles, which indicates that the balanced expression of MgFnr is crucial for WT-like magnetosome synthesis and the expression level is under precise control, be regulated by oxygen. Therefore, MgFnr might play an important role in maintaining redox balance for magnetite synthesis by controlling the expression of denitrification genes, and thus the expression of MgFnr is required to be strictly regulated. On the other hand, since MgFnr serves as an activator for expression of denitrification genes (nor and nosZ) under microaerobic conditions while as a repressor on the same genes under aerobic conditions, it is proposed that other oxygen sensors involved in expression of nor and nosZ are regulated by MgFnr. For example, a NosR protein has been shown to be required to activate the transcription of nos gene in Pseudomonas stutzeri[Since deletion of nditions ,30,32,36 cluster ,38. An Eal media  while th stutzeri. However2 and further displays additional functions for aerobic conditions, which might result from some non-conserved amino acids.We demonstrated for the first time that MgFnr is a genuine oxygen regulator in a magnetotactic bacterium and mediates anaerobic respiration. The expression of MgFnr is required to be precisely controlled, which is regulated by oxygen. In addition, MgFnr is also involved in regulation of magnetite biomineralization during denitrification, likely by controlling proper expression of denitrification genes. This allows the transcription to be adapted to changes in oxygen availability, and thus maintaining proper redox conditions for magnetite synthesis. Despite of general similarities with Fnr proteins from other bacteria, MgFnr is more insensitive to O2 respiration enzymes, magnetite biomineralization is also probably regulated by other unknown O2 sensors. Therefore, further research on respiratory pathways in MTB is likely to gain more insights into the mechanism of oxygen-dependent regulation of biomineralization.Although oxygen is known to be a major factor affecting magnetite biomineralization for decades, the mechanism of this effect in MTB is still unknown. The common observation that magnetosomes are only synthesized under oxygen-limited conditions raised the possibility of protein-mediated regulation of the biomineralization process. However, although MgFnr mediates oxygen-dependent regulation, its relatively subtle and indirect effects on magnetite biomineralization cannot account for the observed complete inhibition of magnetite biosynthesis under aerobic conditions. In addition to a possible effect caused by directly perturbing the redox balance of iron ions required for magnetite synthesis, another level of genetic regulation may exist in MSR-1. Since MgFnr only affects expression of denitrification genes but not genes encoding OE. coli strains were grown in lysogeny broth (LB) at 37°C, and MSR-1 strains were cultivated at 30°C in nitrate medium as described before [E. coli: tetracycline (Tc), 12 μg/ml, kanamycin (Km), 25 μg/ml, and gentamicin (Gm), 15 μg/ml; MSR-1: Tc, 5 μg/ml, Km, 5 μg/ml, and Gm, 30 μg/ml. When E. coli strain BW29427 was used as donor in conjugation, 300 μM diaminopimelic acid (DAP) was added.Bacteria strains and plasmids used in this study are shown in Additional file d before . In ammomag) were monitored under microaerobic and anaerobic conditions in 250 ml flasks containing 100 ml media. For microaerobic conditions, flasks were sealed with butyl-rubber stoppers under a microaerobic gas mixture containing 2% O2 and 98% N2 before autoclaving. Anaerobic conditions were achieved by removing oxygen from gas mixture. For aerobic conditions, strains were cultured in free gas exchange with air in 300 ml flasks containing 20 ml medium agitated at 200 rpm. Optical density (OD) and magnetic response (Cmag) were measured photometrically at 565 nm as previously described [Experiments for growth and magnetic response . Enzymes, including restriction enzymes and T4 DNA ligase, were purchased from Fermentas. To generate the unmarked Δescribed . An abouMgfnr mutant, the Mgfnr gene with its own promoter region was ligated into Acc65I/SacII-digested pBBR1MCS-2, yielding pLYJ110. Subsequently, pLYJ110 was transformed into MSR-1 WT and ΔMgfnr mutant by conjugation. The Ecfnr gene from E. coli K-12 was also hetero-complemented into ΔMgfnr mutant and WT. The PCR fragment of Ecfnr from E. coli was digested with HindIII and XbaI and ligated into pLYJ36 to yield pLYJ153.For genetic complementation of ΔEcfnr mutant with kanamycin marker was excised with the E. coli Quick and Easy gene deletion kit (Gene Bridges) and the Bac modification kit (Gene Bridges), as reported in [Ecfnr mutant. To express MgFnr protein from MSR-1, Mgfnr was ligated into SmaI/XbaI-digested pBBR1MCS-2 to yield pLYJ132. Plasmid pLYJ132 was then transformed into ΔEcfnr mutant. For transcomplementation analysis, strains were anaerobically grown in glucose minimal medium and lactate minimal medium [First, Δorted in . This unl medium .Mgfnr-N27D), pLYJ142 (Mgfnr-I34L), pLYJ143 (Mgfnr-D153E), and pLYJ144 (Mgfnr-L98H), respectively. The different MgFnr mutants were subsequently obtained by a two-step homologous recombination technique in the same manner as described previously [Mgfnr variants were confirmed by PCR and sequencing.Substitutions at amino acid positions 27, 34, 98, and 153 were created by site-directed mutagenesis. First, PstI-SpeI digested fragment for each of substitutions was cloned into pOR093 to create pLYJ141  to remove absorbed iron. After resuspended with 100 μl 65% HNOd before . BrieflyMgfnr strains were grown under microaerobic conditions in the presence of nitrate. 1 ml culture at different time points was taken to detect nitrate and nitrite concentration as described in [WT and Δribed in . NitrateMgfnr strains were grown under microaerobic conditions in the presence or absence of nitrate. The cells were centrifuged and resuspended in fresh ammonium medium. Then the suspension was placed in the measuring chamber (1.5 ml) of a mass spectrometer . The bottom of the chamber (Hansatech electrode type) was sealed by a Teflon membrane, allowing dissolved gases to be directly introduced through a vacuum line into the ion source of the mass spectrometer. The chamber was thermostated at 28°C, and the cell suspension was stirred continuously by a magnetic stirrer. For O2 respiration measurement, air was sparged into the suspension before chamber closing. The consumption of oxygen by the cells was followed at m/e = 32. For denitrification, the cells were sparged with Argon and nitrate reduction was measured using 2 mM K15NO3 (CEA 97.4% 15 N). NO, N2O and N2 concentrations were followed as a function of time.WT and ΔIf not specified, MSR-1 WT and mutants were grown at 30°C under anaerobic or microaerobic conditions for 20 h, concentrated and adsorbed onto carbon-coated copper grids. Samples were viewed and recorded with a Morgagni 268 microscope  at 80 kV. For magnetosome analysis, more than 300 crystals were characterized for each strain.fnr genes were identified by BLASTP (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) homology searching in the genomes of MSR-1 (GenBank: CU459003.1), M. magneticum (GenBank AP007255.1), M. magnetotacticum (NCBI reference sequence NZ_AAAP00000000.1), Mc. marinus (GenBank accession number CP000471.1), and D. magneticus strain RS-1 (GenBank accession number AP010904.1). ClustalW was used for sequence alignment. The identification of Fnr binding sites in the promoter regions of the different operons encoding denitrification enzymes were performed with the virtual footprint software .The authors declare that they have no competing interests.YL and DS conceived and designed the research. YL, MS, SB, KS, and DP performed the experiments and analyzed the data. YL and DS wrote the manuscript. All authors read and approved the final manuscript.Magnetosome formation in WT overexpressing MgFnr. Plasmid pLYJ110 and pLYJ153 contains fnr gene from MSR-1 and E. coli, respectively. Cells were grown in anaerobic nitrate medium. Bar, 100 nm.Click here for fileDetection of Fnr binding sites in the upstream regions ofnap, nirS, nor, and nosZ. The putative Fnr binding sites in the promoter regions are indicated in yellow.Click here for fileTranscription ofnosZfused togusAinMgfnrvariant strains under microaerobic in the presence of nitrate. Expression was measured by β-glucuronidase activity.Click here for fileMagnetosome formation in differentMgfnrvariant strains. Cells were grown in microaerobic nitrate medium. Bar, 100 nm. Irregular shaped particles are indicated by black arrows.Click here for fileBacterial strains and plasmids used in this work.Click here for file


Cancer creates a tremendous financial burden. Cancer-related costs are categorized into direct, indirect, and psychosocial costs. Although there have been many reports on medical care costs, which are direct, those on other costs are extremely scarce. We estimated travel time and costs required for cancer patients to receive outpatient treatment. We studied 521 cancer patients receiving anti-cancer treatment between February 2009 and December 2012 at the Outpatient Chemotherapy Center of Teikyo University Chiba Medical Center. Address data were extracted from Data Warehouse electronic medical records, and travel distance and time required for outpatient treatment were calculated via MapInfo and ACT Distance Calculator Package. Transportation costs were estimated on the basis of ¥274 (=$3.00) per kilometer. The study design was approved by an ethics review board of Teikyo University (12–851). Average round-trip travel distance, time, and cost for all patients were 26.7 km, 72.5 min, and ¥7,303 ($79.99), respectively. Cancer patients incurred a travel cost of ¥4000–¥9000 ($40.00 to $100.00) for each outpatient treatment. With population aging, seniors living alone and senior households are increasing, and outpatient visits are becoming a common burden. Cancer creates a tremendous financial burden . Cancer-http://www.civil.eng.osaka-u.ac.jp/plan/staff/inoi/keikaku08_3.pdf). The study design was approved by an ethics review board of Teikyo University (12–851). Address data were anonymized and de-identified prior to analysis.We studied 521 cancer patients receiving anti-cancer treatment between February 2009 and December 2012 at the Outpatient Chemotherapy Center of Teikyo University Chiba Medical Center. Address data were extracted from Data Warehouse electronic medical records. Travel distance and time required for outpatient treatment were calculated via software MapInfo®  and ACT Distance Calculator Package® . Transportation costs were estimated on the basis of ¥274 (=$3.00) per kilometer  about $360 per month [The Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) altered the guidelines for reimbursements for outpatient chemotherapy drugs and drug administration services in the United States. Friedman et al. reported that access to care between pre-MMA and post-MMA patients has no statistically significant difference . Shea etAs this study was performed only in a single facility, a meaningful tendency in travel time and costs was not seen in relation to the number of patients and specialists for each cancer type. A study in multiple facilities may show a notable trend.Cancer patients incurred a travel time of 45–90 min and a cost of $40–$100 for each outpatient treatment. Cancer patients carry rising burdens of health care-related out-of-pocket expenses. With population aging, seniors living alone, and increasing number of senior households, outpatient visits are becoming a common burden.


P < 0.05). Furthermore, the results showed that only high-dose SA significantly affected the transcription of hepatic and adipose tissue insulin signaling genes. The data suggested that SA prevented HFD-induced insulin resistance in rats after 12 weeks of administration through nontranscriptionally mediated biochemical changes that may have differentially sialylated glycoprotein structures at a low dose. At higher doses, SA induced transcriptional regulation of insulin signaling genes. These effects suggest that low and high doses of SA may produce similar metabolic outcomes in relation to insulin sensitivity through multiple mechanisms. These findings are worth studying further. N-Acetylneuraminic acid (Neu5Ac) is a biomarker of cardiometabolic diseases. In the present study, we tested the hypothesis that dietary Neu5Ac may improve cardiometabolic indices. A high fat diet (HFD) + Neu5Ac (50 or 400 mg/kg BW/day) was fed to rats and compared with HFD + simvastatin (10 mg/kg BW/day) or HFD alone for 12 weeks. Weights and serum biochemicals  were measured, and mRNA levels of insulin signaling genes were determined. The results indicated that low and high doses of sialic acid (SA) improved metabolic indices, although only the oral glucose tolerance test, serum triglycerides, leptin, and adiponectin were significantly better than those in the HFD and HFD + simvastatin groups ( N-acetylated derivatives of neuraminic acid that occur naturally in glycoproteins and gangliosides. SA is a biomarker of cardiometabolic diseases, where it is thought to be a consequence of long-term inflammation [Sialic acids (SAs) areammation . This clammation . Additioammation , 4, whilammation , 6. Whatammation .There are numerous pharmacological and alternative therapies for cardiovascular diseases, but most of them have proven ineffective in curbing the rising burden of the diseases. This is driving the search for alternatives to currently available therapies . MoreoveNeu5Ac was purchased from Carbosynth Limited , while analytical grade ethanol was purchased from Merck . Lipid profile kits were purchased from Randox Laboratories Ltd. , while ELISA kits  were purchased from Elabscience Biotechnology Co., Ltd. , and Millipore , respectively. An RNA extraction kit was purchased from RBC Bioscience Corp. , and a GenomeLab GeXP Start Kit was purchased from Beckman Coulter, Inc. . Simvastatin was purchased from Pfizer , and RCL-2 solution was purchased from Alphelys . Cholesterol and cholic acid were purchased from Amresco  and Santa Cruz Biotechnology , respectively. Standard rat pellet was purchased from Specialty Feeds , while palm oil was supplied by Yee Lee Edible Oils Sdn. Bhd. .n = 30) were housed in individual cages at 25 ± 2°C, with 12/12 h light/dark cycle, and allowed to acclimatize for 2 weeks with free access to normal pellet and water. The rats were then assigned to one of five groups  of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, approved the use of animals in this study , and the animals were handled as stipulated by the guidelines for the use of animals. Sprague Dawley rats , insulin (R2 = 1), and leptin (R2 = 0.9996).Serum from blood collected in plain tubes was used for measurements of adiponectin, leptin, and insulin using the respective ELISA kits according to the manufacturer's instructions. Absorbances were read on a BioTeK Synergy H1 Hybrid Reader  at the appropriate wavelengths . The results were analyzed on μU/mL))/2430 [Lipid profile analyses were performed using serum from blood collected at the end of the study by cardiac puncture after an overnight fast. Samples were analyzed using Randox analytical kits according to the manufacturer's instructions with a Selectra XL instrument . After an overnight fast, the oral glucose tolerance test (OGTT) was performed by oral gavage of 2 g/kg BW D-glucose to every rat, and the blood glucose levels were measured at 0, 30, 60, and 120 minutes via tail vein puncture, using a glucometer . The area under the curve for glucose in the OGTT was calculated as reported previously . Then, hL))/2430 . Rattus norvegicus gene sequences from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/nucleotide/) and tagged with an 18-nucleotide universal forward and 19-nucleotide universal reverse sequence, respectively. Primers (μL) were analyzed on a GeXP genomelab genetic analysis system  after mixing with sample loading solution and DNA size standard 400 as recommended by the manufacturer. Results were analyzed with the Fragment Analysis module of the GeXP system software and normalized on the eXpress Profiler software. Fold changes were calculated by dividing the expression value for the different treatment groups by the expression value for the normal group.Primers for the gene expression study were designed using the Primers  were supn = 6) of the groups was used for the analyses. One-way analysis of variance (ANOVA) with Tukey's post hoc test was performed using SPSS 17.0 software  to assess the level of significance of the differences between means with a cutoff of P < 0.05.The means ± standard deviation  in comparison with the HFD group (P < 0.05).The OGTT, insulin, HOMA-IR, and lipid profile results are shown in FD group . MoreoveFD group ; the HFDP < 0.05). Adiponectin level in the HFD + SIM group was also lower than that of the HFD group, while the HFD + SAH and HFD + SAL groups had higher levels, despite being not significantly different from the HFD group.κbkβ), and mitogen-activated protein kinase (Mapk1) in the liver, while it upregulated pyruvate kinase (Pk) in the adipose tissue. Other genes involved in the insulin signaling pathway were not changed by SA , a glycoprotein, is an established marker for fat catabolism [As can be recalled, the HFD + SIM, HFD + SAH, and HFD + SAL groups had lower weight gains in comparison with the HFD group. Simvastatin has weight-reducing properties  and henctabolism , and dietabolism , 15. Addtabolism . Elevatetabolism , and in tabolism .κbkβ [The gene expression data from this study showed that the HFD + SIM and HFD groups had transcriptional changes of insulin signaling genes that tended towards insulin resistance. Furthermore, the data showed that the HFD + SAH group attenuated the HFD-induced transcriptional changes, which tended towards improved insulin signaling. Specifically, it upregulated the expression of a central mediator of the intracellular signal transduction of insulin sensing (Pi3k), whose transcriptional downregulation has been linked with obesity-induced insulin resistance , 18. Simκbkβ  especialκbkβ , 23. Theκbkβ . Elevateκbkβ .Taken together, the data showed that although simvastatin improved lipid profiles, it increased insulin resistance, as reported previously . SA, on We demonstrated that SA prevents HFD-induced insulin resistance through transcriptional and nontranscriptional mechanisms. At lower doses, sialylation of glycoprotein targets may be responsible for the preventive effects of SA against insulin resistance. In addition, at higher doses, transcriptional regulation of insulin signaling genes may provide longer-lasting effects. These findings are worth evaluating further.


Background: Acute kidney injury (AKI) is a common complication after liver transplantation (LT), and considerably increases the morbidity and mortality of the procedure. The gold standard of measuring the kidney function, the serum creatinine level (sCr), has poor specificity and sensitivity for the early diagnosis of AKI. Novel biomarkers for the prediction or early diagnosis of AKI, would potentially increase the opportunities for therapeutic interventions.Objective: To compare the diagnostic value of the standard renal marker, sCr and neutrophil gelatinase-associated lipocalin (NGAL) to predict AKI within 48 hours of LT.Methods: During a 9-month period from 2010 to 2011, NGAL was measured two times in 90 patients who underwent LT from deceased donors—after induction of anesthesia (NGAL1) and 2 hours after reperfusion of the liver graft (NGAL2). Patients were grouped according to the presence of risk factors for developing AKI according to the Acute Kidney Injury Network criteria .) curve and area under curves (AUCs) of ΔNGAL and sCr levels during the first 48 hours of LT were similar in predicting AKI. The AUCs of the ΔNGAL to predict AKI was 0.64 (95% CI: 0.52–0.76). The development of AKI was significantly correlated with the number of units of fresh frozen plasma transfused intra-operatively (p=0.017) and cold ischemic time (p=0.042).Results: 60 men and 30 women with mean±SD age of 40.2±14.2 years were included in this study. The incidence of AKI was 34% (95% CI: 24%–44%). The difference between the NGAL1 and NGAL2 (ΔNGAL) and the baseline NGAL concentration was predictive of AKI in all patients. Receiver operating characteristic (ROCConclusion: Serum NGAL concentrations obtained during surgery is a predictor of AKI in patients undergoing LT. According to the AKIN criteria, AKI is defined as either an absolute increase in plasma creatinine of at least 0.3 mg/dL (26.4 mmol/L) above the baseline value within 48 hours of OLT or a at least 50% increase in plasma creatinine during the prior seven days . Although the role of NGAL in non-transplant AKI is well recognized, its role in clinical transplantation is not still clear.Orthotopic liver transplantation (OLT) is the definitive treatment for end-stage liver disease (ESLD). Acute kidney injury (AKI) is one of the most significant complications of OLT with high rates of morbidity and mortality. During the post-operative period, the rate of AKI varies between 12% and 70%; 71% of patients who developed AKI will require renal replacement therapy [mg/dL 26. mmol/L aWe conducted this study to re-evaluate the predictive value of serum NGAL for intra-operative AKI in patients undergoing OLT and investigated peri-operative variables that could have been associated with the development of AKI.All adult patients who underwent OLT from deceased donors in Organ Transplant Center, Shiraz University of Medical Sciences, from September 2010 to January 2011, were included in this study. Patients who aged less than 18 years or had chronic hypertension, systemic infections, any systemic renal inflammatory conditions such as lupus erythematosis, anemia, hypoxia, malignancies, or pre-operative creatinine level >2 mg/dL, were excluded from the study. Also, those who were candidate for combined kidney-liver transplantation and those who received sirolimus or cyclosporine as their maintenance immunosuppressive regimen instead of tacrolimus were excluded from this study.i.e., the baseline NGAL; NGAL1), and two hours after reperfusion (NGAL2). Plasma NGAL was measured using a commercially available ELISA kit  according to the manufacturer’s instructions.Blood samples for the determination of NGAL were drawn at two different times—immediately after the induction of anesthesia  was administered according to serial thromboelastography results. All patients received 1000 mg intraoperative methylprednisolone for the induction of immunosuppressive regimen. All donor organ allografts were implanted either by standard caval replacement with bicaval anastomoses or by piggyback techniques. Veno-venous bypass was used in none of the patients.The anesthesia included the standard induction with thiopental (2–3 mg/kg), fentanyl (2–3 µg/kg), and a non-polarizing muscle relaxant or, in selected cases, succinylcholine. The anesthesia was maintained by isoflurane (1%–1.5%). Fentanyl was given either as a bolus or as continuous infusion as determined by the attending anesthesiologist. Patients were ventilated with a fraction of inspired oxygen of 0.5–1 L/min, tidal volume of 7–10 mL/kg, and a positive end-expiratory pressure of 0–5 cm HSerum creatinine (Cr) levels were determined preoperatively (baseline) and then daily up to two days after the surgery. Serum Cr levels were used as a surrogate for renal function. AKI was defined as an absolute increase in plasma creatinine at least 0.3 mg/dL (26.4 mmol/L) above the baseline value within 48 hours of OLT.Data analysis ® ver 16.0 for Windows® . All continuous data were expressed as mean±SD (range). For univariate analysis of risk factors for AKI, differences between the two groups were analyzed with Mann-Whitney U test for continuous variables and with χ2 test for categorical variables. Receiver operating characteristic (ROC) curve was used to determine the cutoff point for plasma NGAL level to predict AKI. Based on the cutoff value, we then categorized patients into two groups of NGAL–  and NGAL+ . The patients were also stratified by their serum Cr according to the consensus diagnostic increases in serum creatinine defining AKI into sCREA– and sCREA+. All tests were two-sided. P≤0.05 was considered statistically significant.All data were analyzed by SPSSA total of 90  patients were enrolled in this study. The mean±SD age of recipients was 40.2±14.3 years. They had a mean±SD body weight of 68.3±13.3 kg, and a MELD score of 22.0±6.2. Demographics of patients are shown in The number of units of FFP transfused intra-operatively (p=0.017) and cold ischemic time (CIT) (p=0.042) were risk factors for AKI in OLT recipients . There wThe area under the curve (AUC) of the ROC curve of the difference NGAL1 from NGAL2 (ΔNGAL) to predict AKI was 0.64 (95% CI: 0.52–0.76); the best cutoff value (the point closest to sensitivity = specificity = 1) was 0.57 with a sensitivity of 71% and a specificity of 59% . The AUC for ROC curve of serum NGAL2 to predict AKI was 0.54 . ΔNGAL was predictive for AKI; serum NGAL2 concentration was not.–/NGAL–, 42 (47%) were sCRE–/NGAL+, 13 (14%) were sCREA+/NGAL– and 18 (20%) were sCREA+/NGAL+. Although, the incidence of AKI was 34.4%, six patients ultimately needed renal replacement therapy—four were sCREA+/NGAL+, and two were sCREA+/NGAL–.In our study, 17 (19%) patients were sCREANGAL1 was more than normal in nine (10%) of patients—in four NGAL2 was less than NGAL1 .The need for a simple, accurate, and minimally invasive marker for early diagnosis of AKI has been a limiting factor in clinical nephrology research and practice. The reference standard test for the diagnosis of AKI in current clinical practice is serum Cr level. Furthermore, prognostic scoring systems such as MELD, have shown that serum Cr is an important prognostic biomarker in cirrhotic patients, but its sensitivity and specificity is poor because it overestimates the renal function in patients with low muscle mass. The decreased hepatic production of Cr results in lower serum Cr levels , 10. RecParikh demonstrated that NGAL, an early biomarker of AKI after cardio-pulmonary bypass surgery, is markedly induced within 2–6 hour after the initiation of the operation in patients destined for AKI but it declines after 12 hour, when IL-18 and KIM-1 are easily detectable [Previous studies have shown that NGAL levels reach the peak approximately 2 hours post-AKI . Nonetheet al, demonstrated that the cutoff value of NGAL concentration for prediction of AKI across all settings ranged from 100 to 270 ng/mL [In different clinical settings of AKI, Haase, 70 ng/mL . Recentl70 ng/mL . In this70 ng/mL .While different definitions of AKI , variouset al, of cohort studies of patients with cardio-renal disease, demonstrated that an increase in plasma NGAL without an increase in plasma Cr can predict an increase in dialysis and mortality rate of the patients [A recent analysis by Haase, patients .et al, confirmed that the intensive care unit patients with increased serum Cr at admission but negative NGAL (<130 ng/mL), rarely develop AKI and might not be planned for prolonged hospitalization [Nickolas, lization . This suet al, measured urinary biomarkers of injury, i.e., cystatin C, NGAL, c-glutamyl transpeptidase, IL-18, and kidney injury molecule 1 (KIM-1) at 0, 12, and 24 hours following ICU admission; it was shown that while the median concentrations of KIM-1, cystatin C, and IL-18 were significantly greater in those with pre-renal AKI compared to those without, NGAL and c-glutamyl transpeptidase concentrations were not significantly different. The results suggest that pre-renal AKI represents a milder form of injury. The reason why some biomarkers are increased requires further study [Pre-renal AKI has recently been considered to represent a physiological response to under-perfusion. Its diagnosis is retrospective after a transient rise in plasma Cr, usually associated with evidence of altered tubular transport, particularly that of sodium. To test the reversibility of pre-renal AKI, Nejat, er study . Furtheret al, found that chronic renovascular hypertension (RVH) is associated with elevated NGAL levels, likely due to ongoing kidney and systemic inflammation and ischemia [Our study has many limitations. First, the overall sample size was small and the incidence of post-operative AKI was significantly lower than that reported earlier, leaving a small pool of patients for analysis. This shortcoming diminished the statistical power of our study and precluded a more robust calculation of our biomarker’s predictive values. Second, serial NGAL levels may be more useful in the early diagnosis of AKI. Plasma NGAL measurements may be influenced by a number of coexisting variables including chronic kidney disease, chronic hypertension, systemic infections, inflammatory conditions, anemia, hypoxia and malignancies . Patientischemia .Previous studies on AKI revealed that urine IL-18 and NGAL reach their peak almost six hours post-cardiac surgery . Future In conclusion, we found that ΔNGAL is a good marker for early detection of AKI after OLT. More studies on larger groups of patients, and combination of novel biomarkers of renal injury and function are needed to further elucidate the diagnostic values of these markers.