Listeria monocytogenes is an opportunistic foodborne pathogen that causes human Listeriosis and high mortality particularly in immunocompromised individuals. Pregnant women are more prone to L. monocytogenes infection resulting in abortions.Lactobacillus brevis (LB) MF179529, a probiotic bacterial strain, was investigated in a murine model.In the present study, antilisterial activity of L. monocytogenes required to cause symptomatic listeriosis. In the main trial, mice were divided into 4 groups. Group I was kept as negative control, group II was exposed to L. monocytogenes and maintained as positive control. Group III was fed with L. brevis only, while group IV received L. brevis for 3 days prior to L. monocytogenes infection. A volume of 200 μl of L. monocytogenes ATCC 19115 and L. brevis MF179529 bacterial suspension corresponding to cell density of 109CFU/ml were given to respective groups by intragastric route. Progress of infection was monitored for 7 days including general health scoring, listeria dispersion in organs, bacterial load in intestine and blood biochemistry were recorded on 3rd, 5th and 7th days post infection (dpi).Initially a pilot study was conducted to determine the dose of 9CFU/ml of L. monocytogenes ATCC 19115 in mice.Clinical listeriosis was induced by 10L. brevis supplementation resulted in significant reduction in dispersion and propagation of L. monocytogenes in liver, spleen and intestine. L. brevis MF179529 consumption led to a significant elevation of number of lactic acid bacteria and reduction of total plate count, anaerobic count and coliform population in intestine. Moreover, total leukocyte and neutrophil counts of treated animals were similar to the negative control while positive control group displayed higher number. Safety evaluation of L. brevis was performed by monitoring general health, hematological and serological parameters of L. brevis fed and negative control group (group III and I). No significant difference in feed intake, body temperature, body weight and blood picture could be detected in L. brevis supplemented and control groups.Animals of group IV displayed minor signs of infection. L. brevis in L. monocytogenes infection and suggest that L. brevis could be used for prophylactic measure.Our results indicate ameliorative role of The online version of this article (10.1186/s12906-019-2444-5) contains supplementary material, which is available to authorized users. Listeria monocytogenes (LM) is a facultative intracellular food borne pathogen which is causative agent of human listeriosis, gastrointritous, abortion, meningoencephalits and septicemia [Pathogenic microbes are important due to their role in health complications and food spoilage. pticemia . Immuno-pticemia . Ingestipticemia . Listeripticemia .Lactobacillus and Bifidobacterium genera are used commercially [Probiotics are defined as live nonpathogenic microorgansims, when used in adequate amount give health benefits to the host . They plercially , 8. Probercially . The antercially . Probiotercially . Probiotercially .. LAB are known to produce fermented products as well as bacteriocins that have the inhibitory effect against clinical and foodborne pathogens including LM [. Thus LAB are recommended to be used as food supplement and therapeutic agent against infectious diseases [Can be used as an alternative to chemical growth promoter, and growth performance of the host. LAB are reported to produce antimicrobial compound such as lactic acid, hydrogen peroxide, reuterin, diacetyl, acetoin reutricyclin, antifungal peptides and bacteriocins . Bacteriuding LM , 15. Thudiseases .L. brevis (LB) MF179529 against LM using a murine infection model.To the best of our knowledge, this is the first report on antimicrobial efficacy of L. brevis (LB) MF179529, a strain previously isolated from fresh cow fecal samples and identified by 16S rRNA sequencing in our laboratory. The bacterial strain was refreshed by plating on de Man Rogosa Sharpe (MRS) agar following incubation at 35 °C for 48 h anaerobically. This strain can be obtained from First Fungal Culture Bank of Pakistan (FCBP) with accession number FCBP-692, a registered World Federation of Culture Collections WFCC) in Pakistan. Listeria monocytogenes (LM) ATCC 19115 was obtained from the American Type Culture Collection. LM ATCC 19115 strain was cultivated in Tryptic Soya Broth (TSB) supplemented with 0.6% (w/v) yeast extract at 37 °C for 24 h. Both strains were preserved in 20% glycerol stocks at − 80 °C and were revived by sub culturing from glycerol stock whenever required.5,107 and 109 CFU/ml and bacterial density of LM corresponding to 109 CFU/ml was selected as infectious dose to be used in the main experiment .LB and LM were grown in de Man Rogosa Sharpe (MRS) and Tryptic Soya Broth (TSB) respectively for specified time period. In order to select the infectious bacterial density, overnight culture of LM was centrifuged and pellet was washed thrice with 0.01 M sterile Phosphate Buffered Saline (PBS) buffer (pH 7). Pellet was re-suspended in sterile 0.01 M PBS buffer, OD was adjusted to 1 ± 0.05 at 600 nm. Three doses were used in pilot study viz, 10Mus musculus, 6–8 week old) were purchased from University of Veterinary and Animal Sciences, Lahore and acclimatized in animal house of Department of Zoology, University of the Punjab, Lahore, for one week prior to experimentation. Mice were kept in temperature controlled environment at 28 ± 2 °C with photoperiod 12 h light/dark cycles, humidity 30–35%. They were housed in standard sized cages (12 ″ × 18″ inches) whereas basal diet and fresh water were provided ad libitum.Healthy albino male mice (n = 24) were divided into 4 groups at random, group A, B and C were inoculated once with 200 μl of LM suspension corresponding to cell density of 105, 107 and 109 CFU/ml respectively through intragastric gavage. Group D was maintained as negative control and treated similarly with equal volume of 0.01 M sterile PBS. Re-isolation of LM from visceral organs  was used as a criterion of induction of listeriosis. The bacterial density which led to dispersion of LM in liver, spleen and intestine of all animals was used to induce infection in the main study.The bacterial density of LM required to induce infection was determined in a pilot study. Mice (L. brevis while group IV received both LB and LM. Mice were fed with L. brevis (109 CFU/ml/ mouse) orally by intragastric gavage for 3 consecutive days before oral infection with L. monocytogene. Sampling was performed at 3rd, 5th and 7th day of post infection.A total of 60 mice were divided in four groups I-IV. Groups I and II were used as negative and positive control respectively, group III received only Mice were observed daily for clinical signs and symptoms of infection, including animal movement, fur condition, structure of backbone, dehydration and feeding activity and symptoms were scored from 0 to 4. The scores represent following physical attributes (0) Active, alert showing no sign of discomfort; (1) Active, ruffled fur beginning; (2) Slow, ruffled fur with pronounced circumflexed back; (3) Sluggish, ruffled fur, circumflexed back, skin lesion; (4) Sluggish, ruffled fur, circumflexed back, closed and sunken eyes, difficulty in feeding. Animals were scored daily for their health conditions upto 7 days post infection. The GHS of infected animals (II) group were compared with LB treated group (IV).The body temperature and body weight of each animal was monitored daily throughout the experiment using rectal thermometer and balance (SHIMADZU -ELB300). Similarly, feed intake of each animal was monitored and measured by subtracting feed left behind from the feed given and dividing it by number of the animals in the group.Total 5 mice from each group were sacrificed following ULAM guide lines of euthanization by intra peritoneal injection of ketamine (200 mg/kg of body weight) on 3rd, 5th and 7th day post infection (dpi). Quantification of LM in tissues was performed by removing liver, spleen and intestine aseptically from slaughtered mice. Dilutions of weighed amount of tissue homogenate were prepared in sterile 0.01 M PBS and plated on lithium chloridephenylethanol-moxalactam (LPM) agar for the determination of LM count per gram of the tissue.Assessment of intestinal microbial composition of all groups was done by enumerating coliform, Lactic Acid Bacteria (LAB), Total Plate Count (TPC) and Anaerobic Plate Count (APC) on selective media; MacConkey, MRS, nutrient agar under aerobic condition and nutrient agar under anaerobic condition respectively.Blood was collected by cardiac puncture and kept in EDTA coated and uncoated vails for hematological and serological analysis respectively. For differential leucocytes counting blood smear was prepared on glass slide and fixed with 100% methanol for 5 min. Later on, it was stained with Writght-Giemsa stain for 10 min. Slides were analyzed for lymphocytes, neutrophils, monocytes and eosinophils counts under oil immersion. Serum analysis was performed through fully automated analyzers  using chemical reagents of Beckmann coulter, serum parameters included ALP, ALT/SGPT, AST/SGOT, TBil, Urea, Creatinine, CHOl, TG, HDL and LDL.p value < 0.05 was considered significant.Statistical analysis was performed using IBM SPSS software  version 21 Chicago, IL, USA. Experiments were performed in triplicate from each sample. All results are presented as group means ± S.E. One Way ANOVA followed by Tukey’s test, independent sample t test and Mann-Whitney U tests were performed as and where appropriate. The Lactobacillus brevis on the basis of 16S rRNA sequencing and assigned accession number MF179529 by National Center for Biotechnology Information (NCBI). In pilot study, LM could not be re-isolated from tissues including liver, spleen and intestine of animals exposed to lower bacterial density (105 CFU/ml). Animal exposed to a dose of 107 CFU/ml showed presence of LM in few tissues. It was reisolated from liver and intestine of 2/6 and spleen of 1/6 animals. A single intragastric dose of 109CFU/ml of LM resulted in translocation of pathogen in all tissues of sacrificed animals  against Listeria infection (109 CFU/ml) was monitored. General health conditions of LB fed group were better than positive control group (P ≤ 0.05). The comparison of GSH scores in LB treated and infected group (IV and II) is presented in Table In the main study, protective effect of orally administered LB MF179529  and LB fed (III) group remained unaltered, while continuous decrease was observed in positive control (II) group where it reduced from 34 to 28 g. LM + LB treated group (IV) showed initial decrease in body weight at 3rd dpi but later on it increased to level of negative control  group as compared to positive control (II) group. LM count of group IV was 4.45 CFU/g, 4.15 CFU/g in liver and 4.45 CFU/g and 4.15 CFU/g in spleen at 3rd and 5th dpi respectively. However, it could not be re-isolated at 7th dpi LM from both tissues. On the other hand, its value was 5.3, 5.1 and 4.8 log CFU/g at 3rd, 5th and 7th dpi respectively in intestine. The LM count in group II remained high in all tissues  during course of experiment Fig. .Fig. 2CoInfluence of LB treatment on the microbial composition of intestine of mice was analyzed by comparing coliform, LAB, TPC and APC count of group I-IV. LB supplementation lead to significant reduction in TPC and coliform count in group III and IV Fig. a and d. p > 0.05)  and elevation in TLC was noticed at 3rd dpi in positive control (II) group. Platelets count of group II also decreased significantly as compared to negative control group. But LB treated groups (III and IV) showed higher platelets count as compared to negative control group. A significant increase in neutrophils and decrease in lymphocytes was also observed in positive control group (II) while no such alteration was observed in LB treated groups (III and IV). The variation in eosinophils and monocytes levels was not significant among groups  and monitored for morbidity as well as mortality up to 7 dpi. Mice receiving 109CFU/ml displayed clinical signs of infection including circumflex back, ruffled fur and sluggish movement. Additionally, LM was successfully re-isolated from liver, spleen and intestine indicating successful establishment of clinical infections and dissemination of pathogens. On the other hand, in animals exposed to dose of 107 CFU/ml, re-isolation frequency was very low indicating slight infection. In light of these results bacterial density of 109 CFU/ml was decided for main study.pathogen . Higher [9 of LM . Therefo[9 of LM . Mice weL. brevis MF179529 against LM ATCC 19115 infection in mice, main study was conducted. Previously, most of the literature reports the inhibitory effects of Pediococcus acidilactici, Lactobacillus casei, Lactobacillus brevis and Lactobacillus paracasei on LM. L. brevis MF179529 was one of our lab isolates with significant antimicrobial activity against both Gram positive and Gram negative pathogenic isolates (data not shown). To the best of our knowledge, no data exists on antilisterial effect of LB.In an effort to analyze the protective effect of local strain 9 CFU/ml bacterial density of LB MF179529 was offered to mice three days prior to LM infection. [On the basis of previous literature on LAB species, 200 μl of 10fection. , 24. A sLactobacillus casei [To further strengthen the role of LB in decreasing LM infection, the pathogen load in different organs was quantified. LB treated group displayed low tissue invasion in liver, spleen and intestine relative to positive control group. Interestingly, complete elimination of pathogen was noticed in liver and spleen at 7th dpi. Consistently, LM counts in intestinal tract also reduced gradually from 3rd to 7th dpi, although it was not totaly eliminated. It may be due to secretions of antimicrobial compounds, competition among LB and LM for colonization in GI tract etc. Our findings are consistent with Becattini et al.  who repous casei –29.Of note, another important contribution of LB MF179529 consumption on GI tract was noticed by alteration in intestinal microbial equilibrium. A significant increase in lactic acid bacteria along with reduction of other intestinal bacteria including LM proposed antilisterial activity of LB. It may be due to competitive survival of LB in GI tract . RecoverHematological studies provide important information about multiple disorders including bacterial infections. Phagocytes especially macrophages and neutrophils plays an important role against bacterial infections. To further confirm the remodeling role of LB, the hematological picture of all groups was compared. Our results indicated that Hb level decreased in infection group making it a high risk factor to the health . It is kIn vivo safety assessment of LB was performed by comparing hematological and serum biochemical profiles in control and LB fed group. No significant variation in any serological parameter could be recorded indicating safe nature of LB MF179529. In culmination, our results suggest that LB MF179529 from cow intestine is useful for prevention of invasion and facilitates suppression of LM ATCC19115 infection in mice.Listeria infected mice. LB reduces LM load in liver, spleen and intestine and favorably perturbates intestinal microbial equilibrium. We suggest that LB MF179529 ingestion has health promoting capacities and a good candidate to be employed as a probiotic against listeria infection. Further studies are needed to best understand the mechanisms by which this microorganism promotes resistance against infectious diseases.In the present study, our results indicated that oral administration of LB MF179529 provides protective effect to the Additional file 1:Figure S1. Variations in body weight due to LM infection and its amelioration with LB. Body weight at day 1st among all groups statistically similar (p ≥ 0.05). (DOCX 19 kb)Additional file 2:Figure S2. Comparison of feed intake among 4 groups days post infection. Comparison was made using One way ANOVA followed by DMRT. Group II mice received only LM and showed significant reduction in feed intake as compared with the control group. In other groups no significant differences in feed intake was observed. Asterics show significant difference at P < 0.05. (DOCX 20 kb)


To investigate the value of dynamic monitoring peripheral blood lymphocyte-to-monocyte (LMR) ratio in evaluating the treatment response and prognosis of patients with extranodal NK/T cell lymphoma (ENKTL).A total of 148 patients with ENKTL were retrospectively analyzed in the Affiliated Tumor Hospital of Zhengzhou University between March 2012 and March 2018. The optimal cut-off value of LMR was determined using the receiver operating characteristic curve (ROC) method, then patients were divided into low LMR group and high LMR group. The LMR level was dynamically measured at various time points, and the relationships between LMR and therapeutic response, and survival were analyzed.P = 0.009). The 5-year overall survival (OS) and progression-free survival (PFS) were 49.28% and 44.89% in the low LMR group, respectively; 5-year OS and PFS in the high LMR group were 84.50% and 67.12%, respectively, significantly longer . The OS and PFS of patients with elevated LMR after treatment were longer than those with decreased LMR after treatment . The LMRs at relapse were significantly lower in both high and low LMR groups than those of the last follow-up . Univariate and multivariate analysis demonstrated that low LMR was an independent risk factor for poor prognosis in ENKTL patients .The complete remission rate (CR) was 85.7% in patients with high LMR at diagnosis, which was remarkably higher than that of patients with low LMR at diagnosis (64.9%) (Lymphocyte to monocyte ratio can be used as an indicator of treatment response, prognosis and recurrence in patients with ENKTL. Low LMR before and after treatment is a poor prognostic factor. Extranodal NK/T cell lymphoma (ENKTL) is a unique histopathologic subtype of non-Hodgkin’s lymphoma (NHL), characterized by vascular damage and destruction, necrosis, cytotoxic phenotype, and Epstein–Barr virus (EBV) infection . ENKTL, Serum lactate dehydrogenase (LDH) reflects cell proliferation activity, and the elevated LDH is a parameter reflecting poor prognosis. The prognostic value of LDH has been confirmed in non-Hodgkin’s lymphoma (NHL), and included in the International Prognostic Index (IPI) . BesidesTherefore, the purpose of this study was to investigate the predictive value of dynamic monitoring of LMR in peripheral blood at various time points before and after therapy in patients with ENKTL.This study was a retrospective analysis of a cohort of 148 newly-diagnosed ENKTL patients treated in the Affiliated Tumor Hospital of Zhengzhou University between March 2012 to March 2018. The inclusion criteria are as follows: (1) pathologically and immunohistochemically confirmed diagnosis of ENKTL according to the WHO 2016 classification of the tumors and hematopoietic and lymphoid tissues [Data collected included Eastern Cooperative Oncology Group (ECOG), performance status (PS), age, gender, involved sites, B symptoms, serum LDH level, serum β2 microglobulin (β2 M), bone marrow involvement, complete blood count (CBC), absolute lymphocyte count (ALC), absolute monocyte count (AMC), liver and renal function, serum potassium, sodium, chloride and calcium levels, International Prognostic Index (IPI), Korean Prognosis Index (KPI) indices, and stage (Lugano staging system). This study was approved by the Medical Ethical Committee of the Affiliated Tumor Hospital of Zhengzhou University. All data of the recruited patients were obtained after informed consent in accordance with the Declaration of Helsinki.Complete blood count and serum LDH level were measured and recorded at various time points . The LMR was calculated through ALC divided by AMC. Similarly, the LMR/LDH was calculated through LMR value divided by serum LDH value. Namely, the LMR and LMR/LDH at diagnosis were obtained from data within a week before the first course of chemotherapy; the LMR during treatment was from data before every other course of chemotherapy; the LMR during follow-up was from data every 3 months in the 1st year, every 6 months in the 2nd year, and then once yearly. The optimal cut-off values of both LMR and LMR/LDH were determined using Cutoff Finder via the receiver operating characteristic curve (ROC) method (Euclidean distance).Patients with early stage disease treated with 4–6 cycles of chemotherapy followed by involved-field radiotherapy (IFRT). Patients with advanced disease treated with 6–8 cycles of chemotherapy as first-line treatment; subsequently, IFRT could be delivered as consolidation, palliative, or salvage therapy depending on physician condition of patients. Of the 148 patients, 5 (3.38%) received radiotherapy alone, and 143 (96.62%) received chemotherapy, of which 94 (63.5%) received chemotherapy plus radiotherapy, and 39 (26.4%) received chemotherapy alone. The chemotherapy regimens were shown in Table Overall survival (OS) is defined as the duration from disease diagnosis to the last follow-up or death. Progression-free survival (PFS) is defined as the duration from disease diagnosis to disease progression or disease-related death. These treatment responses were evaluated every two courses of chemotherapy through clinical manifestations, B-ultrasound, Computed Tomography (CT), Magnetic Resonance Imaging (MRI), or Positron Emission Computed Tomography (PET-CT). According to standard response criteria for NHL , the resIn the follow-up analysis, CBC, blood chemistries  and imaging  were performed every 3 months in the 1st year, every 6 months in the 2nd year, and then once yearly. Morphological examination of bone marrow cells was performed in patients with bone marrow infiltration at the beginning of disease. The follow-up deadline was September 2018. The survival time of lost patients was calculated to the last follow-up date.P < 0.05 was defined as significance. All statistical analyses were conducted using SPSS 21.0 and Graphpad Prism 7.0 software.Categorical characteristics and ratio were compared using the Chi-square or Fisher’s exact test. The Kaplan–Meier method was used to calculate the probability of survival, and difference between groups were compared by log-rank test. Cox proportional-hazards regression model was applied to perform Univariate and multivariate. A two-sided 2 was dominant. Among 148 patients, 42 (28.4%) were positive for serum EBV-DNA, 83 (56.1%) were negative for serum EBV-DNA, and 23 (15.5%) were undetected. The median ALCs were 1.4 × 109/L (range 0.23–3.35), and the median AMCs were 0.35 × 109/L (range 0.02–0.84). 148 eligible patients were categorized into the high and the low LMR groups according to the optimal cut-off value of the LMR or LMR/LDH, respectively. The characteristics were shown in Table A total of 148 cases of newly diagnosed ENKTL patients, with median age was 42 years (range 11–72 years), were eligible to be evaluated. 37 patients (25.0%) experienced recurrence or disease progression, and 24 patients (16.2%) died. The median follow-up time was 32 months (range 6–74 months). The majority of patients were < 60 years, Lugano stage I/IIP = 0.01  was 0.738 (95% CI 0.619–0.856), P = 0.009). The LMR significantly reduced when the high LMR group reached the good response (CR + PR) (P = 0.032); however, there was no statistical change among the non-response patients (P = 0.098). There was no statistical change in the low LMR group regardless of whether the response was achieved or not .The CR rate in the high LMR group at diagnosis (85.7%) was remarkably higher than that in the low LMR group (64.9%) (P < 0.001) and PFS  in the low LMR group at diagnosis were significantly lower than those in the high LMR group. The OS  and PFS  in the low LMR/LDH group at diagnosis were significantly lower than those in the high LMR/LDH group.The OS  and PFS  than those in Group 2. Patients with low LMR pretreatment were stratified into two subgroups: Group 3, those with increased LMR at the end of therapy (≥ the LMR level at diagnosis) (n = 15), and Group 4, those with no increase in LMR (< the LMR level at diagnosis) (n = 17). Similarly, patients in Group 3 had longer OS  and PFS  than those in Group 4.According to the change of LMR level during treatment, the patients with high LMR pretreatment were stratified into two subgroups: Group 1, those with increased LMR at the end of therapy (≥ the LMR level at diagnosis) (n = 80), and Group 2, those with no increase in LMR after treatment (< the LMR level at diagnosis) (n = 24). Patients in Group 1 had longer OS . Nevertheless, the OS  and PFS  of patients who received chemotherapy plus radiotherapy were significantly longer than those of patients who received chemotherapy alone. After treatment, there was no statistical difference in LMR levels among patients treated with various chemotherapy regimens (P = 0.148), however, the LMR of patients with chemotherapy plus radiotherapy was higher than that of patients with chemotherapy alone in both high and low LMR groups .There was no statistical difference in survival among patients with various chemotherapy regimens .The LMR was dynamically monitored at the follow-up of 3, 6, 9 and 12 months after treatment. The level of LMR increased over time, and eventually tended to a certain level in both groups. Nevertheless, compared with the LMR level at the last follow-up, the LMR level at relapse was significantly lower in both high and low LMR groups , and chemotherapy or radiotherapy alone could be significantly associated with shorten OS and PFS. Clinical factors with statistically significance for OS and PFS (P < 0.05) were included in the multivariate analysis (Table P = 0.009) and PFS (P = 0.025).Univariate analysis was shown in Table is Table . Due to Two clinical prognostic models for ENKTL, IPI and KPI, are limited in terms of prognostic capability, especially in the era of asparaginase-based chemotherapy . In thisPrevious studies already demonstrated the involvement and prognostic values of EBV infection in ENKTL . More reIn the present study, LMRs were dynamically monitored at various time points , and the relationships between LMR and therapeutic response, and survival were analyzed, respectively. As reported in the literatures , 20 and ENKTL is a highly aggressive lymphoma. Fortunately, asparaginase-based regimens significantly improve the prognosis . The ratHowever, there are still some limitations in this study e.g., this was a single-center and retrospective study. Thus, it is necessary to conduct a multi-center and prospective study, and to expand the sample size to verify these results in the future.In conclusion, this study, for the first time, retrospectively analyzed the correlation between the dynamic changes of LMR at various time points and the treatment responses, prognosis, and recurrence in ENKTL patients. We found that low LMR at diagnosis was a simple and effective indicator of poor response to treatment and poor prognosis. Additionally, further reduction of LMR during follow-up was associated with disease recurrence.


The PANI/ZnO composite photocatalysts were synthesized to efficiently remove organic dye  from model wastewater. The PANI/ZnO composite photocatalysts were studied with the intention of efficient removal of organic dye  from wastewater to obtain low-cost heterogeneous catalysts that offer high catalytic activity and stability. The conductive PANI polymer, which absorbs Vis irradiation, was used in this work as ZnO absorbs only ultraviolet (UV) irradiation; thus, the composite photocatalysts’ activity was broadened into the Vis region. Characterization of the composite photocatalysts was done by Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy, electric conductivity, UV-Vis spectroscopy, and by specific surface area (SBET) measurements. The composites’ photocatalytic activity under solar irradiation was validated by monitoring degradation of the AB25 dye. This study presented that it was possible both to prepare PANI and to prevent ZnO dissolution if in situ polymerization starts from neutral media with the addition of DEG. Additionally, efficient removal of AB25 dye, about 90% in 60 min, was achieved. The first-order rate constants of the photodegradation of AB25 by PANI/ZnO 0.02/0.024/0.04 DEG (and pure ZnO)) were computed to be 0.0272/0.0281/0.0325 (and 0.0062) min Increased awareness regarding a material’s performance and increased competition in the global market for polymer composites have fueled their growth. Composites  are becoming an essential part of today’s materials due to the construction of more complex hybrid materials. Nanoarchitectonics is an emerging field that refers to the rational design and construction of nanostructures into functional materials with control at the nanoscale ,2,3.It is now recognized that materials are often the limiting factor in bringing a new technical concept to fruition; thus, polymers are often materials of choice in these demanding applications. Furthermore, composites can be defined as materials consisting of two or more chemically and physically different phases that are separated by a distinct interface. Therefore, different materials are combined judiciously to achieve a system  with more useful structural or functional properties that are unattainable by any of the constituents alone.2) photocatalyst.To obtain superior composites properties the interfacial adhesion should be strong. This can be achieved on the molecular level by a chemical reaction or adsorption . In situ2 during photocatalytic reactions. To overcome the aforementioned obstacles and to enhance photocatalysis under ambient conditions, several authors modified the surface and structure of ZnO [The main barriers for extensive ZnO applicability are the narrow light-absorption range, the charge-carrier recombination, the photoinduced corrosion–dissolution at extreme pH conditions, and the formation of inert Zn(OH)e of ZnO ,6,7,8,9.e of ZnO , metal oe of ZnO ,12, and e of ZnO ,14.PANI was chosen as one of the promising conductive polymers to tune composites’ optical, electrical, and photocatalyst properties ,16. The However, the challenge is to find experimental conditions that result in encapsulation of ZnO nanoparticles , which cCB− (obtained by absorption of a photon from Vis irradiation) into the ZnO conduction band (CB), and at the same time it can accept hVB+ from the ZnO valence band (VB). Thus, the obtained iterations yield a PANI-ZnO synergistic effect, which hinders the recombination process in ZnO and increases activity under Vis radiation [Furthermore, the charge transfer is governed by radical cations (polaron) or dications (bipolaron) that are delocalized over several units of the polymers ,22. The adiation .CB− and hVB+ transform H2O and O2 into OH−1 radicals which in turn degrade the organic pollutants [2 (3.2 eV) absorption to the visible light region, several authors have prepared carbon-doped TiO2, which has a lower bandgap (≈3.01 eV) [2 was not solved. As an alternative to TiO2, ZnO has also been studied as a potential candidate for wastewater treatment due to, e.g., its similarity in charge carrier dynamics upon bandgap excitation.Afterward, the typical photocatalysis mechanism for metal-oxides proceeds, i.e., ellutants ,25. Furtllutants ,27,28. Sllutants ,30. To i3.01 eV) ,32,33. HThe purpose of this work is to prevent the dissolution of ZnO particles during the in situ chemical oxidation of aniline and to prepare a PANI/ZnO composite photocatalyst. Consequently, the photocatalytic activity of the pure ZnO is extended to the region of solar radiation. The properties of the photocatalyst were studied in detail and the photodegradation performance of PANI/ZnO catalysts was validated by degradation of acid blue dye (AB25) in water.4)2S2O8, Morris Plains, NJ, USA) Acros Organics; and sulfuric acid  and diethylene glycol  were used to synthesize composite samples. Commercial organic dye C.I. Acid Blue 25 (AB25) Ciba-Geigy was used as the water pollutant. The chemicals were used as received, without any additional pre-treatment. All aqueous solutions were prepared with ultrapure water from the Merc Millipore Direct-Q3 UV water purification system .Zinc oxide (ZnO), nanopowder, particle size <100 nm, Sigma-Aldrich ; aniline  and ammonium persulfate  light, which can be presented by Equation (1) ,35:(1)ZnHowever, intense ZnO solubility in aqueous solutions also prevents its wider application.In strong acid media:In an alkaline medium:Upon UV irradiation:Thus, both strongly acidic and alkaline conditions do not favor both the composite’s synthesis and photocatalytic process.2O) several times, separated by the centrifuge, and dried at 60 °C for 24 h.Polyaniline/zinc oxide (PANI/ZnO) composites preparation was conducted by in situ chemical oxidation polymerization at room temperature whilst the starting pH media of a water and diethylene glycol (DEG) mixture was neutral. The aniline monomer was oxidized by APS in the presence of ZnO nanoparticles. The constant mole ratio of monomer and oxidant was n(An):n(APS) = 1:0.25, the weight ratio of m(PANI):m(ZnO) was 15:85 for all prepared samples, and the concentration of DEG was 0.02, 0.024, and 0.04 M. Samples are denoted as: PANI/ZnO 0.02 DEG, PANI/ZnO 0.024 DEG, and PANI/ZnO 0.04 DEG. The procedure for the PANI/ZnO 0.02 DEG sample was as follows: solution A was prepared using 0.8 g ZnO nanoparticles, 0.386 mL of DEG, and 0.588 mL of aniline in 25 mL distilled water and sonicated for 15 min to obtain a stable suspension. Solution B consisted of 0.367 g APS in 25 mL distilled water . An An-Z−1 with a resolution of 4 cm−1.Fourier transform infrared (FTIR) spectroscopic data were obtained with a Perkin-Elmer Spectrum One FT-IR spectrometer  equipped with an attenuated total reflection accessory  in reflection mode. The spectra were collected in the range 650–4000 cmUV-Vis spectra of powdery samples of PPy/ZnO composites were recorded by a Shimadzu UV-Vis-near infrared (NIR) (UV-3600) spectrometer  with the integrated sphere. Barium sulfate was used as the reference material.The morphology of the studied samples was analyzed using scanning electron microscope (SEM) images obtained by a thermal field emission scanning microscope   operating at 3 kV.Thermal properties of the composites were analyzed by thermogravimetric analysis (TGA) using a TA Instruments Q500 analyzer . TGA curves were obtained under a nitrogen atmosphere (40 mL/min) and a heating rate of 10 °C/min from room temperature to 800 °C. The derivative thermogravimetric (DTG) curves were directly derived from the original TGA thermogram using the product software. ρ is the specific electrical resistance (Ω cm), R is the electrical resistance (Ω), and d is the thickness of pastilles (cm). The electric conductivity (κ/S cm−1) was calculated as a reciprocal value of the electrical resistance (Equation (7)):Conductivity of PANI/ZnO composites was determined by measuring their electrical resistance with the four-point probe method . Electrical resistance is calculated according to Equation (6):For each sample, at least 10 measurements were made and results are given as their average value . Note thθ with steps of 0.02° 2θ and a counting time of 0.6 s. The fits were carried out by means of a split-type pseudo-Voigt profile function and the polynomial background model. The crystallite size was determined using Scherrer’s expression. Isotropic vibration modes were assumed for all atoms. The instrumental line broadening was determined using LaB6 powder.Prepared samples were characterized by powder X-ray diffraction (PXRD) at room temperature (RT) using a Shimadzu XRD6000 diffractometer device  adsorption isotherms were obtained at −196 °C using Gemini 2380 . The Brunauer-Emmett-Teller (BET) specific surface area (SBET) of composites was calculated using N2 adsorption isotherms. All the samples were dried at 50 °C for 1 h and degassed under vacuum at 400 °C for 24 h prior to analysis.Nitrogen  . In a typical procedure, 50 mg/L of the PANI/ZnO composite was added into 50 mL aqueous solution of AB25 (30 mg/L), and the pH media was adjusted to 7. Then, the mixture was kept in the dark with constant shaking for 30 min to reach the adsorption-desorption equilibrium. Furthermore, the irradiation was carried out at a horizontal lamp-sample distance of 25 cm and the photocatalytic process was carried out for 60 min. At given intervals (15 min), an aliquot of photoreacted AB25 solutions was taken and was filtered  filters ) and then subjected to UV-Vis spectroscopic measurements to determine the AB25 concentration. The degradation of AB25 was determined by the intensity of the absorption peak at 622 nm using a Perkin Elmer Lambda EZ 201 spectrophotometer . The results were presented as the concentration of remaining dye in the wastewater after treatment according to the equation:−1 and they can be attributed to N–H, C=N, C=C, and C–N stretching, respectively. The absorption intensity ratio of PANI bands (1583 and 1494 cm−1) typical for quinoid (Q) and benzoid (B) units is 1:3. This ratio is in accordance to the Q versus B units ratio (1:3) in FTIR spectra of pure PANI, ZnO and PANI/ZnO composites are given in −1, which correspond to C–H stretching [−1, respectively. Additionally, diethylene glycol (DEG) induced bands at 1026 and 963 cm−1  and at 778 cm−1 (–CH2), which confirms its presence in composites even after washing.Furthermore, the pure PANI shows bands at 1139 and 816 cmretching . In the −1, which represents the stretching vibrations of water molecules [−1 characterize Zn–O–Zn and Zn–O stretching. The shift of characteristic ZnO bands in PANI/ZnO composites is due to interactions between PANI and ZnO nanoparticles. Also, a disappearance of the absorption band at 872 cm−1, which is typical for ZnO composites, suggests that PANI encapsulated ZnO and/or it was immobilized on the ZnO surface.The FTIR spectrum of ZnO shows an intensive band at 3488 cmolecules . The abs−1) was computed by using Equations (6) and (7) and the corresponding values are given in −7 S cm−1) than the composite samples (≈10−6 S/cm−1). Increased conductivity confirms the presence of a relatively well-conducting PANI polymer, which was prepared from a neutral aqueous medium with the addition of DEG. This result agrees with the literature as it is known that PANI synthesized in neutral media has poor conductivity [The conductivity of PANI/ZnO composites synthesized with a different concentration of DEG was determined by the four-point method. The conductivity .Furthermore, it is expected that increased PANI conductivity in composites will enhance interactions between PANI and ZnO. The PANI conductivity in composites shows slight variations due to the same composition (see thermogravimetric (TG) results in ortho-position) [TGA was conducted to determine the thermal stability and the composition of the prepared PANI/ZnO composites. The PANI/ZnO weight loss was monitored in the range of 40–800 °C and the results are given in osition) . The thiosition) .maxT2,3,4) obtained from the DTG profiles  show some variation  in PANI/ZnO. Therefore, this DEG-induced transformation should additionally boost photocatalysts’ properties, i.e., it should increase the efficiency of PANI/ZnO composites.The specific surface area (BET) values were computed using nitrogen adsorption isotherms of PANI/ZnO composites and they are presented in X-ray diffractograms show that the PANI/ZnO samples consist of crystalline phases, among which dominates the zincite (ZnO) pattern (ICDD PDF#36-1451) in a hexagonal P63 mc wurtzite type of structure . The othθ), which corresponds to interplanar distances of 1.39 nm. However, there are only minute quantitative and broadening differences between composites and no differences in shifting. This indicates that in situ polymerization has not induced the ZnO crystal structural changes. The same cannot be said for PANI, as it clearly undergoes structural changes at a higher-order level due to interaction with ZnO.The composite constituents were also separately recorded a–c. ZnO θ values. Interestingly, PANI/ZnO composites retain the effect characteristic for a higher level of mutual microstructural ordering, which is visible from the presence of mesostructural peaks at ≈5° 2θ. The composites’ peak positions correspond to interplanar distances of about 1.74 ± 0.01 nm, which is a higher value when compared to pure PANI (1.39 nm).Furthermore, the rod-like configuration of polymeric chains is obviously infiltrated by ZnO particles that extend to the interplanar distances, giving rise to a mesostructural peak at lower 2The retained intensity ratio of the ZnO reflection indicates that the preferred orientation is excluded. One can assume that ZnO crystallites are predominately isotropic. PANI reflections seem a bit broader and retain less of the intensity ratio, which points to smaller, less isotropic crystallites. Again, minute changes in shift and intensity of the mesostructured reflection point to the fact that initial compositional changes in composites (stoichiometry) can hardly contribute to the extent of mesostructural ordering.Insight into lattice strain parameters may be additionally offered. We can conclude that the PANI/ZnO composite yields a synergetic effect with respect to mesostructural ordering, which may be well-utilized in sorption or ion-exchange performance in catalysis. The investigation of morphology by means of FE SEM gave additional insight. It seems that the composite poses on large micron-seized plate-like  particles of PANI separated, or better to say intercalated, with relatively isotropic, partially agglomerated ZnO nanoparticles about 200 nm in size. PANI size and ZnO agglomeration varies for samples prepared with different DEG amounts; however, it seems that a lower DEG amount strongly favors a ZnO-nanoparticles-intercalated PANI-plate-like morphology, which is in concordance with the XRD results.The oxidation state of PANI in composites and the photocatalysts’ energy bandgap were studied by using UV-Vis spectroscopy. UV-Vis diffuse reflectance spectra  of PANI/Furthermore, optical ZnO and PANI bandgaps were estimated from the insert plots presented in According to the literature ,52,53, dThe prepared PANI/ZnO composite photocatalysts were validated by monitoring the degradation of AB25 dye in water (30 mg/L) under simulated solar irradiation using 1 g/L of catalysts. The pH of suspension during photocatalysis was adjusted to 7 due to the sensitivity of ZnO on the pH media. 2 [During the adsorption process, PANI/ZnO composite samples removed ≈20%, whereas the pure ZnO removed ≈40%, of AB25 dye a. Next, 2 .The photocatalytic mechanism of dye degradation is presented in 2 and H2O [The most important species for the photocatalytic process are hydroxyl radicals (2.80 eV) . Thus, t and H2O ,56,57.appk is the apparent rate constant, and c0 and ct are the concentration of dye after 30 min of adsorption (in the dark) and the concentration of dye at time t, respectively.The results in c0/ct) versus t data) suggests a pseudo-first-order reaction for all studied composite catalysts. By comparing the obtained appk values , which increased the number of common spots between PANI and ZnO .This presented approach showed significant progress in the prevention of ZnO’s dissolution during in situ PANI/ZnO photocatalyst synthesis. Thus, the same approach should be used when preparing PANI composites with metal-oxides, such as ZnO, that are sensitive to low pH.


Innovative treatment modalities have not yet shown a clinical benefit in patients with septic shock. To reduce severe cytokinaemia, CytoSorb as an add-on to continuous renal replacement therapy (CRRT) showed promising results in case reports. However, there are no clinical trials investigating outcomes.n = 67) or CRRT alone (n = 49). The primary outcome was the 28-day all-cause mortality rate. Patients were weighted by stabilized inverse probability of treatment weights (sIPTW) to overcome differences in baseline characteristics.In this investigator-initiated retrospective study, patients with septic shock were treated with CRRT + CytoSorb (p < 0.001), lower mean arterial pressure (p = 0.007) and higher levels of noradrenaline (p < 0.001) compared to the CRRT group. For CytoSorb, the mean predicted mortality rate based on a SOFA of 13.8 (n = 67) was 75% (95%CI 71–79%), while the actual 28-day mortality rate was 48% . For CRRT, based on a SOFA of 12.8 (n = 49), the mean predicted versus observed mortality was 68% versus 51% . By sIPTW analysis, patients treated with CytoSorb had a significantly lower 28-day mortality rate compared to CRRT alone . Independent predictors of 28-day mortality in the CytoSorb group were the presence of pneumosepsis , higher levels of lactate at the start of CytoSorb  and older age .At the start of therapy, CytoSorb-treated patients had higher lactate levels  contains supplementary material, which is available to authorized users. Sepsis and septic shock are a major health burden worldwide leading to approximately 5 million deaths annually –3. AlthoRecently, CytoSorb has been developed and approved in Europe since 2011 for use in patients with severe cytokinaemia –11. CytoHence, the aim of the current study is to investigate whether the application of CytoSorb in addition to CRRT leads to a reduction in 28-day mortality compared to CRRT alone in patients with septic shock in the ICU, by using the inversed probability of treatment weights method.In this retrospective investigator-initiated study, patients admitted to the ICU of the Maasstad Hospital with septic shock  treated Septic shock was defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection, identified by persisting hypotension requiring vasopressive medication to maintain mean arterial pressure (MAP) ≥ 65 mmHg and having a serum lactate level > 2 mmol/L despite adequate volume resuscitation .The primary endpoint was the 28-day all-cause mortality compared for CytoSorb versus CRRT alone. Secondary endpoints included the comparison between the observed 28-day mortality rate in the CytoSorb treatment group versus the predicted mortality according to the SOFA score , 22, andT-test.For the first part, the predicted probability according to the SOFA score at the start of therapy , 22 was p value < 0.1 in univariate analysis were considered for multivariable regression analyses. First, a full multivariable model was constructed where all possible important variables were forced into the model. Second, using the same variables from the full model, the final model was constructed with the backstep likelihood ratio method. SPSS version 22.0  was used to perform statistical analyses. All statistical tests were two-sided and evaluated at the 0.05 level of significance.For the second part, all evaluations were carried out using the inverse probability of treatment weights (IPTW), including a stabilizing method to avoid bias from extreme weights , 24. Stap = 0.027) and at the start of therapy (p < 0.001), were administered higher levels of noradrenaline (p < 0.001) and had lower mean arterial pressure (p = 0.007). Patients were treated with CRRT for a mean duration of 4.96 (SE 0.63) days in the CRRT group and 4.97 (SE 0.55) days in the CytoSorb group (p = 0.990). In the CytoSorb group, patients were treated with CytoSorb added to CRRT for a mean duration of 2.34 (SE 0.16) days and with CRRT only for a mean 2.66 (SE 0.52) days. The mean duration from ICU admission to the start of treatment was 2.1 (SE 0.36) versus 1.66 (SE 0.38) days (p = 0.416) for CRRT versus CytoSorb, respectively. In total, 88% (n = 59) patients commenced CytoSorb directly together with CRRT because of septic shock (i.e. no delay between CRRT and CytoSorb), 4 patients had a delay in the start of CytoSorb after CRRT of 1 day and another 4 had a delay of > 1 day. Moreover, the mean duration from hospital admission to the start of treatment did not differ between the groups . After adjustment with sIPTW, the CRRT and CytoSorb groups were largely comparable . For the CRRT only group, the mean SOFA score was 12.8 (SE 3.2), corresponding to a mean predicted mortality rate of 67.9% (95%CI 60.7–75.2%), while the observed mortality rate was 51.0% (95%CI 36.9–65.2%) with a mean difference − 16.9% .When no correction for baseline variables was applied, it was shown that the 28-day all-cause mortality rate was similar for CytoSorb versus CRRT . In this analysis, CKD was not associated with 28-day mortality , and a higher MAP was associated with a lower chance of mortality .By sIPTW chi-square analysis, the 28-day mortality significantly differed for CytoSorb versus CRRT: 53.0% versus 72.3%, respectively (p = 0.014), lactate levels , noradrenaline levels , and older age . To investigate whether CytoSorb treatment may have been initiated earlier in patients with worse septic shock, interactions between therapy timing and lactate levels or SOFA score at the start of therapy were applied, which were found non-significant . By multivariable analysis, independent predictors of 28-day mortality in the CytoSorb group were older age , higher levels of lactate at the start of therapy  and pneumosepsis . Other factors were not independently associated with mortality  patients received CytoSorb at least 1 day after CRRT was already started. We performed a sensitivity analysis in which we excluded these patients and found no deviations from the main results.In our cohort, it was found that CytoSorb therapy did not seem to provide with a survival benefit for patients with pneumosepsis. This may in part be due to combined difficulties in adequate ventilation and/or presence of ALI or ARDS in these severely ill patients. These findings are underlined by a recent randomized trial which assessed IL-6 levels as a primary outcome in patients with ALI and ARDS and found no survival benefit in these patients as a secondary (but not powered for) outcome measure .Since the current study is a retrospective data analysis, there is inherent bias to take into account. Importantly, by applying IPTW, confounding by indication was as much as possible eradicated . Still, Albeit patients had a severe refractory septic shock and were treated with CytoSorb as a last -resort, one may argue that this treatment modality could have potential detrimental effects. One of these effects may be that CytoSorb could filter out antibiotics leaving patients exposed to levels below the therapeutic range . In the We have shown, to our knowledge, in the largest cohort of septic shock patients to date, that CytoSorb treatment may lead to an improved 28-day survival compared to CRRT alone, both on basis of observed versus predicted mortality rates as well as by IPTW. The current data should be further corroborated by randomized clinical trials.Additional file 1:Figure S1. Absolute standardized differences between CRRT and CytoSorb for variables before and after weighting. (PDF 403 kb)


Idebenone (IDE), a strong antioxidant widely investigated for the treatment of neurodegenerative diseases and skin disorders, shows low oral and topical bioavailability due to its unfavorable physico-chemical properties. In this work, to improve IDE topical effectiveness, we explored a two-steps approach: (1) we synthesized an IDE ester (IDEPCA) with pyroglutamic acid, a molecule whose hydrating effects are well known; (2) we loaded IDEPCA into solid lipid nanocarriers (SLN). We evaluated in vitro antioxidant and anti-glycation activity and in vivo hydrating effects after topical application in human volunteers from gel vehicles of IDEPCA SLN in comparison to IDE SLN. All SLN showed good technological properties . The oxygen radical absorbance capacity assay showed that IDEPCA SLN and IDE SLN had similar antioxidant activity while IDEPCA SLN were more effective in the in vitro NO scavenging assay. Both IDEPCA and IDE SLN showed the same effectiveness in inhibiting the formation of advanced glycation end products. In vivo experiments pointed out a better hydrating effect of IDEPCA SLN in comparison to IDE SLN. These results suggest that the investigated approach could be a promising strategy to obtain topical formulations with increased hydrating effects. Hdiseases ,12,13 andiseases . Owing tdiseases ,16. IDE diseases . In addidiseases ,18,19,20diseases ,22, maindiseases  pointed diseases ,25,26. Tdiseases ,28. Prevdiseases ,30. The ®, GO), Cetyl palmitate (CP), Methylisothiazolinone and Methylchloroisothiazolinone (Acnibio AC®) and triethanolamine (TEA) were bought from ACEF . Idebenone (IDE) was obtained from Carbosynth . Polyoxyethylene-20-oleyl ether , 2,2’-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), fluorescin (FL) Trolox (TRL), aminoguanidine carbonate (AMG), bovine serum albumin (BSA), D-fructose, sodium nitroprusside and Griess reagent (1% dihydrochloride sulphanylamide and 0.1% naphthylethylenediamine (NED) dihydrochloride in 5% of hydrochloric acid) and sodium azide were purchased from Sigma-Aldrich srl . Curcumin was obtained from MP Biomedicals, . Imidazolidinyl urea (Kemipur 100) was obtained from Farmalabor .Glyceryl oleate , dry acetonitrile (CH3CN), 4-dimethylaminopyridine (DMAP), ethyl acetate (EtOAc), and hydrochloric acid (HCl), used to synthesize IDE ester (IDEPCA), were reagent grade and were bought from Sigma-Aldrich srl . DMSO-d6 used for NMR analysis was purchased from Sigma-Aldrich srl .The following chemicals 2-Pyrrolidone-5-carboxylic acid (PCA), The synthesis of the ester IDEPCA was reported in 1H NMR spectrum was performed on a Varian Inova Unity 200 spectrometer (200 MHz)  using DMSO-d6 as solvent. Chemical shifts are given in δ values (ppm), using tetramethylsilane as the internal standard; coupling constants (J) are given in Hz. Signal multiplicities are characterized as s (singlet), d (doublet), t (triplet), and m (multiplet). Reaction progress and purity of the synthesized compound were checked on TLC  using EtOAc as eluent and spots were visualized by UV light at 254 and 366 nm as the wavelength. IDEPCA was purified by flash column chromatography using Merck silica gel (0.040–0.063 mm).Infrared spectrum was recorded on a Perkin Elmer series FTIR 1600 spectrometer . A sample droplet was placed between two disks of pure NaCl (neat sample). Signal intensity was characterized as s (strong), m (medium), w (weak). 3CN (10 mL) was added dropwise to a mixture of IDE , PCA , and DMAP  in dry CH3CN (25 mL), at 0 °C. Then, the mixture was stirred under nitrogen at room temperature for 3.5 h. The obtained precipitate of dicyclohexylurea was eliminated by filtration and the solvent was evaporated in vacuum to dryness. The residue was dissolved in EtOAc, washed with 0.5 N HCl (25 mL × 2), H2O (25 mL), and brine (25 mL). The organic phase was dried on anhydrous sodium sulphate, filtered, and evaporated in vacuum to dryness. The obtained oil was purified by flash column chromatography using EtOAc as eluent to give IDEPCA as a pure orange oil .A suspension of DCC  in dry CH−1 2927 (s), 2854 (m), 1742 (s), 1704 (s), 1651 (s), 1610 (s), 1456 (m), 1266 (s), 1204 (s), 1156 (w). 1H NMR (DMSO-d6) δ 1.20–1.36 , 1.50–1.65 , 1.92–2.05 , 2.11 , 2.22–2.42 , 3.88 , 4.07 , 4.16 , 8.01 . C24H35NO7.IR  cmA set of structural properties and molecular descriptors of IDE, PCA and IDEPCA were calculated using MarvinSketch, and reported in IDE and IDEPCA water solubility was determined in triplicate by adding an excess of each compound to deionized water and stirring with a magnetic stirrer for 24 h at room temperature, while avoiding light exposure.The resulting suspensions were filtered and the concentration of the compound in its saturated solution was determined spectrophotometrically  at 278 nm for both IDE and IDEPCA. Prior to the solubility determination, a standard working curve was constructed from a known concentration of each compound in deionized water. The sensitivity of the assay was 0.5 μg/mL for IDE and IDEPCA.Three aliquots of IDEPCA water saturated solution were stored at room temperature for 15 days to assess IDEPCA stability in aqueous medium. At intervals , samples of these solutions were withdrawn and IDEPCA content was assayed spectrophotometrically as reported above.w/w) of each compound used to prepare such SLN are reported in w/w) and its derivative IDEPCA was used in an equimolar amount (1.33% w/w). The aqueous phase consisted of deionized water containing Acnibio AC® 0.05% w/w and Kemipur 100 0.35% w/w as preservatives. Unloaded (SLN A) and IDE or IDEPCA loaded SLN were prepared using the same procedure. Briefly, the oil phase and the aqueous phase were separately heated and when both the oil and the water phase were at 90 °C, the aqueous phase was slowly added to the oil phase under stirring. Then, the mixture was allowed to cool to room temperature under continuous mixing and the phase inversion temperature (PIT) was recorded using a conductivity meter  when the turbid mixture turned into clear. Thin layer chromatography (TLC) analyses were performed to assess IDE and its derivative stability under these conditions, as previously reported [Solid lipid nanoparticles (SLN) were prepared by the phase inversion temperature (PIT) method, according to a procedure previously described . For theSLN samples were stored in airtight jars at room temperature and in the dark until used.w/w) aqueous solution of uranyl acetate was added over 2 min. Before starting the analysis with a transmission electron microscope  operating at an acceleration voltage of 200 KV, the surplus was removed and the sample was allowed to dry at room temperature.For transmission electron microscopy (TEM) analysis, 5 μL of SLN samples were placed on a Formvar (200-mesh) copper grid . When all of the sample was absorbed, the excess was removed by filter paper and a drop of 2%  were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 , using a 4 mW laser diode at 670 nm and scattering light at 90°.All SLN were analyzed after dilution  and sample were adapted to 25 °C for 2 min prior to the analysis. The measurement were carried out in triplicate and the results were expressed as mean ± SD. ζ-potential was determined by laser Doppler velocimetry using the same Zetasizer reported above, after diluting each sample with KCl 1 mM (pH 7.0), according to a procedure previously reported .Particle sizes, PDI and ζ potential values of SLN samples were measured at intervals . During storage, samples were maintained at room temperature and protected from light exposure.S is the area under the fluorescence decay curve of the sample, trolox, or blank calculated with Origin®7 .The antioxidant activity of IDE and IDEPCA, free or loaded into SLN, was assessed in vitro using the oxygen radical absorbance capacity (ORAC) assay, according to the procedure previously reported by Cao et al. ,34. DataA0 is the absorbance (nm) of untreated sample, A1 (nm) is the absorbance (nm) of treated samples .The ability of IDE or IDEPCA, free or loaded into SLN (1 mg/mL), to inhibit the spontaneous NO production from an aqueous solution of sodium nitroprusside (20 mM) at 25 °C for 3 h was evaluated using Griess reagent . Curcumi3 0.02% w/v as positive controls. BSA alone was used as negative control as it did not provide any formation of fluorescent AGEs. Aminoguanidine (AMG) (3 mM) was used as the reference compound [Anti-glycation ability was evaluated determining the inhibition of fluorescence produced by advanced glycation end products (AGEs) formation through Maillard reaction, using the method of Derbre et al. . Proteincompound . Final gw/w) was used as rheological additive and TEA (1.00% w/w) for neutralization. Carbopol Ultrez 21 was dispersed in the aqueous phase consisted of deionized water containing 0.35% w/w Kemipur 100 and 0.05% Acnibio AC (gel C), unloaded SLN (gel SLN), 0.32% w/w PCA in deionized water (gel PCA), IDE loaded SLN (gel IDE), IDEPCA loaded SLN (gel IDEPCA), IDE loaded SLN and 0.32% w/w PCA (gel IDE/PCA). Owing to IDE poor water solubility, we could not prepare a gel containing only free IDE as control, even when adding low amounts of solubilizing agents to the vehicle. Full polymer hydration was obtained by maintaining the mixture in the dark at room temperature for 24 h. Formulations were made viscous by adding TEA (1.00% w/w) drop by drop, under slow mixing to avoid air bubbles formation. All gels were stored in airtight jars at room temperature and in the dark to protect from photo-degradation until use.The composition of gel formulations is reported in To determine the in vivo efficacy of SLN containing IDE or IDEPCA from gel formulations, twelve female subjects with average photo aging were selected (age range 40–60 years). Investigations were performed according to the rules of the Declaration of Helsinki of 1975, revised in 2008. The local ethics committee declared that no approval was needed for this type of study, due to its nature and the safety of the formulations under investigation.After receiving the required information about the nature of the study, all volunteers provided their written informed consent. Four different gels, randomly chosen among the six prepared gels, were supplied to each volunteer in containers labeled with color codes (double blind study).2) into different areas over the back of the hands, twice a day, morning and evening, for one week, without applying any other product.The volunteers were instructed to apply each gel (about 2 mg/cmp < 0.05).Measurements of skin hydration have been determined before the application of the products  and after one week of treatment, using the specific probes of the instrument Soft Plus . Using these probes, skin hydration was assessed by capacity measurements in the range 0–100 u.c. (arbitrary units) with a resolution of 1 u.c. and a 5% precision. All data were acquired under controlled temperature (22 ± 1 °C) and humidity (35% ± 5%) conditions. Each measurement was performed in triplicate. Skin hydration values were reported as difference between the values obtained before and after applying the samples under investigation (Δ hydration). Data were expressed as mean ± S.D. Statistical analysis of the results was performed using Students’ test , which were not affected by IDE or IDEPCA loading see . PDI valMorphological analyses pointed out the presence of spherical particles whose sizes roughly matched those determined by DLS . All theThe PIT or HLB  temperature has been reported to be an indicator of the stability of colloidal systems as the higher the PIT value, the higher the formulation stability . As shoLoaded SLN were prepared using equimolar amounts of IDE and IDEPCA. According to the data reported in IDEPCA proved stable in water during storage at room temperature for 15 days as no significant change of IDEPCA content was observed during this period (data not shown).Scientific evidence shows that glycation along with oxidative processes are major responsible for both several diseases and related risk of complications, especially cardiovascular, and aging processes . GlycatiThe ORAC assay has emerged as a valuable analytical method to determine the antioxidant potential of a range of substances ,50, reprAs illustrated in Nitric oxide (NO) is an important chemical mediator involved in the regulation of various physiological processes whose concentration increase is associated with several diseases . In thisAGEs are a group of highly complex oxidant compounds produced by metabolic reactions including the Maillard reaction, generated in vivo as response to an increased oxidative stress. The results of the in vitro anti-glycation assay pointed out that both IDE and IDEPCA showed a capacity to inhibit AGEs formation significantly higher than Trolox and their incorporation into SLN provided a two-fold increase of this activity, with percentages of inhibition higher than 90%. The lower ability to inhibit AGEs formation of free IDE in comparison to IDE loaded into SLN could be due to the high protein binding of this antioxidant , resultiw/w IDE into SLN as this is the highest concentration used in marketed products and has already been tested in lipid nanocarriers for topical delivery of IDE [Gels and emulsions are the most frequently used vehicles for topical application of lipid nanoparticles . Souto ey of IDE . The valp < 0.05) was observed with respect to all other gels. To evaluate the influence of IDE esterification with PCA, a gel containing a physical mixture of IDE loaded SLN and PCA (in the same molar ratio of IDEPCA) was assessed for its ability to improve skin hydration. This formulation provided an effect lower than IDEPCA loaded SLN but similar to that observed for gels incorporating unloaded or IDE loaded SLN or containing only PCA.Topical application of gel PCA and gel SLN (containing unloaded SLN) resulted in an improvement of skin hydration and no significant difference was observed between these two formulations. As shown in The highest hydrating ability of gel IDEPCA could be attributed to a better capacity of IDEPCA to penetrate into the skin layers in comparison to IDE. The better hydrating effect of IDEPCA loaded SLN is supported by previous patents on PCA esters designed to improve skin hydration ,59. FurtIDE esterification with pyroglutamic acid provided an IDE derivative (IDEPCA) with improved in vitro antioxidant activity in comparison to IDE. IDE or IDEPCA loading into SLN resulted in a decrease of antioxidant activity while the ability to inhibit AGEs formation notably increased. In vivo studies performed on human volunteers utilizing IDE or IDEPCA SLN incorporated in gel vehicles pointed out a greater hydrating effect of IDEPCA SLN in comparison to IDE SLN. These results suggest that the synthesis of ester derivatives with pyroglutamic acid and their loading into SLN could be a valuable approach to develop topical formulations with increased hydrating effects.


Growth hormone (GH) is used to treat short stature and growth failure associated with growth disorders. Birth size and GH status variably modulate response to GH therapy. The aim of this study was to determine the effect of birth size on response to GH therapy, and to determine the impact of GH status in patients born small for gestational age (SGA) on response to GH therapy. Data from the prospective, non-interventional American Norditropin Studies: Web-Enabled Research (ANSWER) Program was analyzed for several growth outcomes in response to GH therapy over 3 years. GH-naïve children from the ANSWER Program were included in this analysis: SGA with peak GH ≥10 ng/mL (20 mIU/L), SGA with peak GH <10 ng/mL (20 mIU/L), isolated growth hormone deficiency (IGHD) born SGA, IGHD not born SGA and idiopathic short stature. For patients with IGHD, those who did not meet criteria for SGA at birth showed greater improvements in height SDS and BMI SDS than patients with IGHD who met criteria for SGA at birth. For patients born SGA, response to GH therapy varied with GH status. Therefore, unlike previous guidelines, we recommend that GH status be established in patients born SGA to optimize GH therapy. Treatment with recombinant human growth hormone (GH) is widely utilized for improving height in children with growth failure and conditions in which it is efficacious, including isolated growth hormone deficiency (IGHD), idiopathic short stature (ISS) and small for gestational age (SGA) , 2. AlthThe effectiveness of GH therapy for increasing short-term growth rates and adult height in patients with various growth disorders is well documented , 10, 11.The non-interventional American Norditropin Studies: Web-Enabled Research (ANSWER) Program was launched in 2002 and designed to assess the real-life clinical outcomes of pediatric and adult patients treated with Norditropin as prescribed by physicians according to standard clinical practice . ProspecData were extracted from the prospective, non-interventional ANSWER Program, which was launched in 2002 and aimed to evaluate long-term safety and effectiveness outcomes of US pediatric and adult patients treated with Norditropin (somatropin (rDNA origin) injection; Novo Nordisk A/S) . The ANSThe analyses of data from the ANSWER Program described in this report evaluated changes in growth-related clinical end points in response to 3 years of treatment with Norditropin. The analysis population included GH treatment-naïve children (aged <18 years at the end of 3 years of data collection) diagnosed with ISS, IGHD or SGA . ISS was defined as idiopathic short stature when excluding all other potential causes of growth failure. Diagnostic classification was determined by the participating physician who evaluated the patient. The IGHD population was subdivided based on fulfillment of SGA criteria according to historical auxological data, and the SGA population was subdivided by peak GH levels at baseline  as determined by GH stimulation test. This cutoff point of 10 ng/mL (20 mIU/L) is commonly considered a threshold indicating normal or below normal GH levels  at baseline were included. Patients with prior GH treatment who started more than 1 week prior to enrollment were excluded. We also excluded patients with missing birth weight and/or length information at baseline (enrollment visit). Participants with missing, inconsistent or improbable baseline values of key variables  or any of the GH response end points above were also excluded. Since many patients were missing baseline GH peak values and/or IGF-I values and/or BA imaging values at baseline, we used in lieu of missing any of these three baseline values the values from the closest visit date to enrollment date within the interval from 18 months before to 12 months after the enrollment date.z-scores) were calculated from actual height and weight using standard references provided by the US Centers for Disease Control and Prevention , insulin-like growth factor 1 (IGF-I) SDS and GH dose. HSDS and BMI SDS ) unless otherwise noted. Differences in ΔHSDS and ΔIGF-I SDS at each year of GH treatment were compared by analysis of covariance (ANCOVA), with diagnostic population, gender and baseline age as fixed effect covariates. Least-squares (LS) means for ΔHSDS and ΔIGF-I SDS at year 3 were estimated using the Tukey–Kramer test. All analyses were performed using SAS version 9.3 (SAS Institute).All statistical tests were two-sided and n = 709); the second largest subpopulation was those diagnosed with IGHD who were also born SGA (n = 86) (s.d.) peak GH (ng/mL) levels were higher for children born SGA who were GH sufficient (17.04 (5.08)) compared to those born SGA who were GH deficient. For children with IGHD, those who met criteria for SGA had lower mean (s.d.) peak GH (ng/mL) levels compared to those with IGHD who did not meet criteria for SGA (6.82 (5.30) vs 7.44 (7.11)). Mean (s.d.) birth length SDS (BL SDS) was lower for children born SGA who were GH sufficient (−2.75 (1.97)) compared to those born SGA who were GH deficient (−2.08 (1.35)). Mean (s.d.) birth weight SDS (BW SDS) was lower for children born SGA who were GH sufficient (−2.50 (0.72)) compared to children born SGA with GH deficiency (−1.89 (1.12)).Of the 14,680 GH-naïve children who enrolled in ANSWER by the end of July 2016, 882 met all inclusion criteria and were included in this analysis. The subpopulations in this analysis were SGA and GH sufficient , SGA with GH deficiency , IGHD born SGA, IGHD not born SGA and ISS. The majority of these patients were diagnosed with IGHD and not born SGA ((n = 86) . Mean BAs.d.) BMI SDS from year 1 to year 3 increased more for those with GH deficiency (−0.85 (2.28) to −0.21 (1.11)) compared with those who were GH sufficient ; year 3, −0.57 (1.06)). However, differences in mean (s.d.) height SDS from year 1 to year 3 were greater for patients born SGA who were GH sufficient ; year 3, −1.63 (0.73)) compared to patients born SGA who were GH deficient ; year 3, −1.18 (0.79)). Increase in mean (s.d.) IGF-I SDS was also greater for patients born SGA with GH deficiency ; year 3, 1.19 (2.29)) compared to patients born SGA who were GH sufficient ; year 3, 1.40 (1.14)). Improvements in growth-related outcomes were greater for IGHD patients who were not born SGA compared to those with IGHD who were born SGA. Increases in mean (s.d.) HSDS from year 1 to year 3 were slightly greater for patients with IGHD not born SGA (−1.86 (0.78) to −1.04 (0.80)) than for patients with IGHD born SGA (−2.08 (0.69) to −1.34 (0.72)). Similarly, increase in mean (s.d.) BMI SDS was greater for IGHD patients not born SGA ; year 3, 0.02 (1.07)) compared to IGHD patients born SGA ; year 3, −0.28 (1.03)). Of note, mean (s.d.) IGF-I SDS increased from 0.19 (1.69) to 1.00 (1.54) from year 1 to year 3 for IGHD patients not born SGA compared to an increase of 0.96 (1.54) to 1.03 (1.42) for IGHD patients born SGA.In general, the growth-related outcomes, IGF-I SDS, BMI SDS and HSDS improved over the 3 years of follow-up, as shown by mean SDS for these outcomes at each year after enrollment . For allConsistent with these response data, mean ΔHSDS relative to baseline levels progressively increased over each year of follow-up for each patient subpopulation . Increass.d. in year 3 over year 1. A significant decline in ∆HSDS by approximately 0.059 was estimated per each year of delay in start of treatment. For each unit of increase in baseline HSDS, improvement from GH treatment declined by an estimated 0.1275 ΔHSDS.To control for potential covariates influencing ΔHSDS results, differences were also compared by ANCOVA and adjusted for year of follow-up, gender, baseline age and baseline HSDS, with an estimated difference from baseline of 0.5855 . SignifiANCOVA was similarly used to estimate differences in ΔIGF-I SDS while adjusting for year of follow-up, gender, baseline age and baseline HSDS. After controlling for each of these variables, significant differences were observed between subpopulations in ∆IGF-I SDS . The GH-Estimates of ∆HSDS and ΔIGF-I SDS at year 3 were generated as LS means by assigning mean values for all other covariates  . The estThis analysis of data from the ANSWER Program demonstrates that treatment with GH resulted in improved growth outcomes for children with IGHD or SGA (GH sufficient or deficient) and ISS. With few exceptions, improvements were shown in all outcomes  for all population groups over 3 years of treatment. Increases in ∆HSDS over time are consistent with results reported in other ANSWER publications , 13, 26.Improvements in mean HSDS were observed for all patient subpopulations, indicating the benefits of GH treatment. In the analysis presented here, two subpopulations of patients with GHD who were born SGA were compared: those who were primarily diagnosed with SGA and found to be GH insufficient and those who were diagnosed with IGHD and whose historical auxological data fulfilled criteria for SGA. The SGA group who were GH insufficient showed a greater improvement in HSDS than patients with IGHD born SGA. Previous reports of data from the ANSWER Program indicated that increases in HSDS were similar between patients born SGA and those with IGHD , 7. The In addition to HSDS, mean IGF-I SDS also increased in most patient groups while GH dose remained constant over the 3-year period in all groups. Although the IGHD born SGA subpopulation demonstrated similar changes in HSDS to the other groups, mean IGF-I SDS only increased from 0.96 to 1.03. In comparison, the other groups with GH insufficiency demonstrated greater increases in IGF-I SDS; the IGHD not born SGA group increased from 0.19 in year 1 to 1.00 in year 3, and SGA with GH deficiency group increased from −0.11 to 1.19; the SGA and GH sufficient group also showed a greater increase in IGF-I SDS, from 0.68 in year 1 to 1.40 in year 3.The repeated measurements model for ∆HSDS derived from this analysis indicates that older baseline age and having a lower difference in HSDS at baseline may reduce the effect of GH treatment on HSDS. The model is consistent with findings from other analyses on data from the ANSWER Program that included patients with GHD, SGA and ISS, which demonstrated that younger age at baseline and lower baseline HSDS were factors that were associated with higher ∆HSDS after approximately 4 years of GH treatment , 13. ThiThere are several potential limitations of this study. Given that the ANSWER Program is an observational study, there may be variations in data collection due to the large number of investigators who participated. In addition, it was at the discretion of the reporting physician to provide their patient’s diagnosis, and thus, variability among physicians’ diagnoses contribute to a certain lack of uniformity. There is also potential for information bias due to missing or erroneous data points resulting from misdiagnosis or failure to report confounding variables. Differences in laboratory assays  and diagnostic procedures among sites reporting information may affect outcomes, and considering the variability in IGF-I assays, this especially limits interpretation of IGF-I data. Another potential limitation is the differences in availability of diagnostic technologies among clinics; for this reason, some patients may be missing results for some tests, such as the GH stimulation test. Further, as noted earlier, there are situations of GH resistance that may occur within the categorical diagnoses of both ISS and SGA that are difficult to diagnose or that are yet to be described.In conclusion, results of this analysis demonstrate that the greatest benefits of GH treatment were shown for patients diagnosed with IGHD who were not born SGA, those diagnosed with IGHD who were born SGA and those born SGA who were GH deficient. Of note, among patients with IGHD, birth size appears to affect response to GH therapy, as IGHD patients who were not born SGA showed greater improvements in growth-related outcomes compared to IGHD patients who were born SGA. Regarding patients born SGA, GH responses were variable between those who were GH sufficient and those who were GH deficient. While the assessment of GH status in patients born SGA has not been recommended in the literature or by existing guidelines, the results reported here suggest that determining GH status may help explain some of the variation in response to GH therapy in children born SGA. Therefore, GH status may be yet another consideration for GH therapy optimization in these patients. Further studies, including genetic analyses, are needed to elucidate additional aspects of SGA that impact growth.R Rapaport has served as a consultant for Novo Nordisk Inc. and Sandoz. P Lee has served on advisory boards for Abbvie and Novo Nordisk, speakers bureau for AbbVie and is on the safety monitoring committee for Versartis. J Ross has served as a consultant for Novo Nordisk and Opko and has research supported by Versartis and Novo Nordisk. P Saenger has nothing to declare. V Ostrow is an employee of Novo Nordisk Inc. G Piccoli is an employee of Novo Nordisk Inc.


Most studies on the human microbiota have analyzed stool samples, although a large proportion of the absorption of nutrients takes place in upper gut tract. We collected samples from different locations along the entire gastrointestinal tract from six patients who had simultaneously undergone upper endoscopy and colonoscopy, to perform a comprehensive analysis using culturomics with matrix assisted laser desorption ionisation - time of flight (MALDI-TOF) identification and by metagenomics targeting the 16S ribosomal ribonucleic acid (rRNA) gene.p < 0.02). We isolated fewer aero-intolerant species in the upper gut: 37 from the stomach and 150 from the left colon (p < 0.004). Using metagenomics, 1,021 species were identified. The upper gut microbiota was revealed to be less rich than the lower gut microbiota, with 37,622 reads from the stomach, 28,390 from the duodenum, and 79,047 from the left colon (p < 0.009). There were fewer reads for aero-intolerant species in the upper gut .Using culturomics, we isolated 368 different bacterial species, including 37 new species. Fewer species were isolated in the upper gut: 110 in the stomach and 106 in the duodenum, while 235 were isolated from the left colon (p < 0.001).Patients taking proton pump inhibitors (PPI) were then revealed to have a higher stomach pH and a greater diversity of species in the upper digestive tract than patients not receiving treatment  contains supplementary material, which is available to authorized users. Previous studies have shown that the absorption of fat, simple carbohydrates and proteins begins in the duodenum and is completed in the jejunum . The dis CFU/ml) , which m CFU/ml) . In the um 104–10 CFU/ml thydrates . Studieshydrates , whereasThe composition of the human gut microbiota was significantly revived by metagenomic studies. However, 16S rRNA sequencing-based methods are biased by the different deoxyribonucleic acid (DNA) extraction methods used  and can We believe that the human-gut functional subdivision corresponds an equally specific microbiota functional compartmentalization that is missed when working exclusively on fecal samples. To date, few human gut microbiota studies have involved samples from the entire gut tract, in comparison to studies using stool samples . To reac: Table S2). The mean ± SD pH was significantly lower in the stomach and duodenal samples for untreated patients compared to patients receiving PPI  . In total, 219 (60%) isolates were strictly aero-intolerant. The bacterial species belonged to Firmicutes , followed by Bacteroidetes , Actinobacteria , Proteobacteria , Fusobacteria , Synergistetes , Verrucomicrobia  and Lentisphaerae  . These isolates belonged to 141 different genera: Clostridium was the most common  followed by Bacteroides , Streptococcus  and Lactobacillus . In total, 304 (83%) bacterial species were bacteria previously described in the human gut  per patient and 368 different bacterial species were identified  were isolated in at least 50% of the samples tested.We looked for the presence of a bacterial core on the basis of culturomics but we did identify any common bacterial species in all samples tested. However, eight different species . Most of the Operational Taxonomic Unit (OTUs) assigned to known species belonged to Firmicutes , followed by Proteobacteria , Bacteroidetes , Actinobacteria , Fusobacteria , Spirochaetes , Synergistetes , Verrucomicrobia , Cyanobacteria , Deinococcus-Thermus , Tenericutes , Acidobacteria , Aquificae , Lentisphaerae , Negibacteria  and Saccharibacteria  . Throughout the study 1,021 bacterial species were identified by this approach , with a mean value of 434 ± 75 species per patient (ranging from 315 to 518). In particular, Methanobrevibacter smithii was identified in the ileum, right and left colon of patient 1 and in the stomach and left colon of patient 3.Metagenomics enabled the identification of 16 different bacterial phyla  and three different species  were detected in all the samples. Moreover, we found 64 (6%) different genera and 103 (10%) different species in at least 50% of the samples tested.The existence of a common bacterial core among the different parts of the digestive tract was then examined. We found that five different genera  were only identified by culturomics, 827 (69%) only by metagenomics, and 194 (16%) species by both Fig. .p < 0.001).Significantly the proportion of aero-intolerant species was found to be higher using culturomics  than metagenomics  (: Table S4). All these species were also detected by metagenomics, corresponding to 11,990 reads  compared to the duodenum  but significantly fewer when compared to the ileum , and the right and left colon  . In addition, we found significantly fewer aero-intolerant species in the stomach  and duodenum  than in the ileum , and the right and left colon  .Using culturomics, no differences were found between the number of species isolated in the stomach  and the duodenum  than in the left colon  . The microbiota of the stomach and the duodenum presented significant less richness (estimated by the Chao1 index) and biodiversity (assessed by a nonparametric Shannon index) than the ileum (p = 0.009 and p = 0.01 respectively), the right (p = 0.007 and p = 0.009 respectively) and the left colon (p = 0.004 and p = 0.007 respectively) . In addition, we observed significantly fewer reads for aero-intolerant species in the stomach  and the duodenum  than in the ileum  (p = 0.02 each), right colon  and left colon  . It is interesting to note that some phyla were identified only in the upper gastrointestinal tract and not in the lower digestive tract such as Spirochaetes, Deinococcus-Thermus, Tenericutes, Acidobacteria, Aquificae and Negibacteria .Metagenomics revealed significantly fewer reads in the stomach  revealed a relationship between the stomach microbiota and low pH Fig. . Stomach methods . At thisp < 0.001). Patients treated with PPIs had a higher recovery percentage revealed by both techniques than the untreated group  and in the group treated with PPIs, we identified 960 species including 462 aero-intolerant species (48%) using the two techniques. Patients taking PPIs had a greater diversity of bacterial species than patients not receiving treatment  of 46.3 ± 15.4 in the stomach compared to 29.3 ± 11 in the colon [We observed an increasing gradient in the number of reads and the number of isolates when comparing the upper gastrointestinal tract with the lower. The increasing degree of bacterial diversity along the gut had already been previously reported. The acidic environment in the stomach, the oxygen gradient from the mouth to the rectum, the transit speed, the biliary salts, the mucosal secretion of IgA, and the secretion of antimicrobials by Paneth cells in the small intestine may explain the lower bacterial diversity in the upper gastrointestinal parts , 27. In er tract . Previouer tract , with sper tract . Other she colon , 32. To Firmicutes population associated with a decrease in Bacteroidetes. Similarly, previous studies have revealed important microbiota changes in the terminal ileum with PPI administration, which can be summarized as an increase in Firmicutes (Lactobacillus or Clostridium) and/or a decrease in Bacteroidetes [Akkermansia and Coprococcus and an increase in Bacteroidales) and weight loss after the administration of omeprazole in C57BL/6 J mice has been reported [Our results are astonishing compared to the literature  but we froidetes , 35. Furreported . Moreovereported , 38, whireported , but thireported , 41.This work revealed that the gut microbiota is much different from its upper part, where foods are absorbed, and its lower part, the colon, which can be likened to waste, with bacterial growth due to fermentation . This ex2. They included one obese patient (patient 3) and one patient with a Crohn’s disease (patient 4). Three patients were currently receiving long-term treatment with PPI: patient 1 because of a gastro-esophageal reflux, patient 2 because of a gastric ulcer and patient 6 because of an esophagitis . Patients were excluded if, in the previous 6 months, they had received antibiotherapy or had taken a trip outside Europe. Patients gave their signed informed consent and completed a clinical questionnaire. Overall, six adult volunteers  were include  previously supplemented with 5 ml of sheep blood and 5 ml of 0.2 μm filtered rumen . SamplesAll samples were cultured in eight different agar media  to quickly identify the colonies . Each isTotal genomic DNA was extracted from colonies using a QIAamp tissue kit  and stored at 4 °C until use for PCR amplification and sequencing of the 16S rRNA gene, using previously described methods . All seq2 cluster density with a cluster passing quality control filters of 94.8% . The raw data were configured in fastq files for R1 and R2 reads.Thirty samples were extracted from six patients in both protocols as previously described in the literature. This concomitant extraction by two protocols (named protocol 1 and protocol 5) including one protocol with a deglycosylation step . Indeed,http://www.ebi.ac.uk/).The paired end sequences of corresponding raw fastq files were assembled into longer joined sequences by FLASH  with a qFrom the Silva website, the Silva small and large subunit (SSU and LSU) database  of releaFinally, our local reference database was used, containing a total of 456,714 well-annotated sequences.Blast were then used to search the OTUs against the Silva reference database . The beshttp://www.mediterranee-infection.com/article.php?laref=374). According to oxygen tolerance, each phylotype was assigned as ‘aero-intolerant’ or ‘aerotolerant’.A bacterial oxygen tolerance database was set up based on the literature Additional file 2:Figure S1. Comparison of the pH samples along the digestive tract. (JPG 74 kb)Additional file 3:Figure S2. Phyla identified along the gastrointestinal tract by culturomics (number of identified species): Proteobacteria (a), Bacteroidetes (b), Firmicutes (c) and metagenomics (number of generated reads): Proteobacteria (d), Bacteroidetes (e), Firmicutes (f), * p < 0.05. (TIFF 184 kb)Additional file 4:Table S3. Number of species, ranged by phyla, along the digestive tract identified by culturomics, metagenomics and both assays. (DOCX 15 kb)Additional file 5:Table S4. New bacterial species isolates by culturomics. (DOCX 19 kb)Additional file 6:Figure S3. Number of identified species by culturomics (a), with aerointolerant (b) and aerotolerant (c) species identified along the digestive tract, and number of generated reads by metagenomics (d) with number of reads from aerointolerant (e) and aerotolerant species (f) along the digestive tract, * p < 0.05. (TIFF 197 kb)Additional file 7:Figure S4. Shannon index along the digestive tract, * p < 0.05. (TIFF 62 kb)Additional file 8:Table S1. Culture conditions used for culturomics in this study. (DOCX 13 kb)