Background and objectives: Kidneys from donation after circulatory death (DCD) are more likely to be declined for transplantation compared with kidneys from donation after brain death (DBD). The aim of this study was to evaluate characteristics in the biopsies of human DCD and DBD kidneys that were declined for transplantation in order to rescue more DCD kidneys. Materials and Methods: Sixty kidney donors  were recruited into the study and assessed using donor demographics. Kidney biopsies taken post cold storage were also evaluated for histological damage, inflammation , von Willebrand factor (vWF) expression, complement 4d (C4d) deposition and complement 3 (C3) activation using H&E and immunohistochemistry staining, and Western blotting. Results: More DBD donors (16/24) had a history of hypertension compared with DCDs . The mean warm ischemic time in the DCD kidneys was 12.9 ± 3.9 min. The mean cold ischemic time was not significantly different between the two groups of kidney donors . The score of histological damage and MPO, as well as the reactivity of vWF, C4d and C3, varied between kidneys, but there was no significant difference between the two donor types (p > 0.05). However, vWF reactivity might be an early indicator for loss of tissue integrity, while C4d deposition and activated C3 might be better predictors for histological damage. Conclusions: Similar characteristics of DCD were shown in comparison with DBD kidneys. Importantly, the additional warm ischemic time in DCD appeared to have no further detectable adverse effects on tissue injury, inflammation and complement activation. vWF, C4d and C3 might be potential biomarkers facilitating the evaluation of donor kidneys. In the UK, approximately 12 to 15% of kidneys from deceased donors are retrieved, but then declined for transplantation . There iOne of the major risk factors in DCD donor kidneys is the impact of warm ischemic injury. DCD kidneys have higher rates of delayed graft function (DGF) and a higher risk of primary non-function (PNF) compared with DBD kidneys ,5,6,7. TThere are also other factors in injury and inflammation that may affect long-term graft survival. Myeloperoxidase (MPO), a marker mainly for neutrophil granulocytes, catalyses the formation of reactive nitrogen species and is elevated in patients with chronic kidney disease and associated with its progression . HypotheThe von Willebrand factor (vWF) binds to the surface of endothelium when disrupting the endothelium-exposed sub-endothelial matrix. Under arterial shear conditions, platelets are tethered onto the immobilized vWF along the surface of the injured vessel wall, leading to platelet adhesion and thrombus formation at the site of injury. Platelet–vWF interactions are markedly abnormal in patients with end-stage renal disease, which was normalised by a successful kidney transplantation .The complement system is an important mediator of IRI and allograft rejection . Renal pThe aim of this study was to reduce unnecessarily discarding DCD kidneys by systematically evaluating donor characteristics and assessing the level of injury, inflammation and complement activation in biopsy samples from DBD and DCD kidneys that were retrieved but then declined for transplantation. Certain injury parameters such as vWF reaction, C4d deposition and C3 activation might be potential biomarkers to be used as the judging criteria of donor kidney quality and facilitate clinical decision-making.From October 2012 to October 2013, 64 kidneys were deemed unsuitable for transplantation by the National Organ Allocation Scheme and were then recruited to this laboratory research project. The consent from the donor family for the use of the organs for transplant and research was obtained by the transplant coordinators before organ retrieval. Ethical approval was granted for the study by the National Research Ethics Commission in the UK . The definition of expanded criteria donor (ECD) included any donor aged ≥ 60 years or a donor aged ≥ 50 years plus two out of three of the following features: a history of hypertension, a raised terminal serum creatinine (>132 µmol/L) or death from a cerebrovascular accident.A single wedge biopsy was taken on arrival at the laboratory after a period of static cold storage. The tissue was divided and either fixed in 10% formal saline then embedded in paraffin wax, or frozen in liquid nitrogen and stored at −80 °C. Four µm paraffin sections were stained with haematoxylin and eosin (H&E) for histopathological scoring or anti MPO, vWF and C4d antibodies for immunohistochemical analysis.H&E-stained sections were assessed using the Remuzzi score by a consultant pathologist who was blinded to the donor types. Four different parameters, glomerular global sclerosis, tubular atrophy, interstitial fibrosis and vascular lesions, were used in the scoring system . The scoImmunohistochemical staining of myeloperoxidase activity (MPO), a marker mainly for neutrophil granulocytes, was undertaken on paraffin sections using a DAKO ChemMate EnVisionTM Detection Kit . The sections were digested by 40 µg/mL proteinase K for 15 min at 37 °C, then blocked by peroxidase-blocking reagent. The sections were labelled by an anti-MPO antibody  at 4 °C overnight. The antibody binding was revealed by a secondary antibody and 3′-amino-9-ethylcarbazole . MPO + cells in tubular, interstitial and glomerular areas were semi-quantitatively scored by counting the number of positive cells in 20 fields at 400× magnification.vWF reactivity detected by immunohistochemical staining in the glomerular areas of the declined kidneys was assessed under light microscope at 100× and 400× magnification and classified according to its intensity . Normal glomeruli were not stained by vWF . Two indImmunohistochemical analysis was performed using a monoclonal antihuman C4d antibody as the primary antibody. The staining of C4d was examined under light microscope at 100× and 400× magnification. Two to three independent observers blinded to the sample groups evaluated the staining pattern and density . The extent of tubular involvement for C4d deposition was qualitatively scored and was reproducible between observers.Tissue samples obtained from kidney donors were processed for Western blotting analysis using a polyclonal anti C3 antibody, which reacts with native and activated C3. A total of 58 samples were analysed, so conditions  were initially standardised. The membranes were probed with β-actin as a loading control to normalise C3 reactivity.t-test for parametric or Mann–Whitney U test for nonparametric variables. Categorical variables were analysed by Fisher’s exact test. A value of p < 0.05 was considered statistically significant. GraphPad Prism 6 was used for the statistical analysis .Continuous data were presented as mean ± SD, and range, where appropriate. Data were compared using the student’s p = 0.174). There was no significant difference in the donor age (p = 0.878). In the DBD kidneys, death was caused by an intracranial haemorrhage (ICH) in 88% of donors compared with 33% in the DCD group (p < 0.0001). There were significantly more DBD donors (67%) who had a history of hypertension compared with the DCD group . The period of ventilation was significantly longer in the DCD donors (p = 0.008).Sixty-four kidneys were recruited into this research study. Four kidneys were excluded: one for damage to the renal vessels and three for not being chilled in transit. Therefore, 60 kidneys (24 DBD and 36 DCD kidneys) were analysed. The donor demographics are listed in p > 0.05). At retrieval, the level of SCr was increased >132 µmol/L in 6 DBD and 11 DCD donors (p = 0.575). The cold ischemic time , which exceeded 30 h in 14 of the DBD and 14 of the DCD kidneys (p = 0.296). The mean warm ischemic time (WIT) in the DCD kidneys was 12.8 ± 3.9 min.There was no significant difference in the serum creatinine SCr,  level atime CIT,  was longKidneys were declined for transplantation for a variety of reasons . The mosp > 0.05; Six biopsies that had fewer than 25 glomeruli were excluded from the analysis. There was no significant difference in the Remuzzi scores between the DBD and DCD kidneys . Seven DBD kidneys were scored mild and another three were moderate ATI, while the DCD kidneys were scored 9 mild, 10 moderate and 1 severe ATI. In addition, the mean number of MPO positive cells was similar between groups .All kidneys were also graded for acute tubular injury (ATI), and there was no statistical difference between the DBD and DCD kidneys , moderate (++), mild (+) and negative staining of vWF were found in DBD kidneys and DCD kidneys, without significant differences between the two groups , with which the level of moderate vWF was reversely related  and 88.6% of DCD kidneys with four negative for C3 (n = 34). The reactivity with the polyclonal anti C3 antibody of bands less than 68 kDa, representing 43 kDa iC3b, 41 kDa C3dg and 27 kDa C3c, were recorded as C3 active products  showed evidence of complement activation compared with the DCD kidneys 46.9%, . InteresAlthough DCD kidneys are more likely to be rejected for transplantation, this study showed similar levels of histologic damage, inflammation, cellular injury and complement activation in DBD and DCD donor kidneys that were declined for transplantation. The significant differences in clinical risk factors such as higher percentage of hypertension and intracranial haemorrhage in DBD donors, but additional warm ischemic time and prolonged ventilation in DCD donors, may be complementarily balanced in the outcome of detected injury parameters. In addition, vWF reactivity as an endothelial cell activation marker and complement activation might mirror the ischemic insult up to a certain degree and then decline when damage becomes severe, while C4d deposition and activated C3 might be predictors for concomitant histological damage.In the UK, the number of DCD kidney transplants has risen over the years . HoweverKidneys recruited in this study were discarded for a variety of reasons. Past medical history was the commonest cause of decline. Fourteen of these were from donors with suspected malignancies, which would not be considered for transplantation, but were included in the analysis. Inadequate in situ perfusion was also a common cause of decline particularly in the DCD kidneys (40%). This could result from improper placement of the aortic cannula, renal vasospasm or intravascular thrombosis ,24. A laWe used a range of histological parameters to assess injury, inflammation and complement activation. The Remuzzi score was used to assess and grade the histological damage of the samples. This scoring system was originally developed to assess whether kidneys from marginal donors had enough viable nephrons to be transplanted . Their rThe expression of vWF in renal glomeruli has been reported in patients with hypertension or vascular disease . A signiCold ischemia is another major risk factor for DGF and reduced graft survival ,28,29,30Brain death is viewed by some as a risk factor for poor allograft survival causing variations in the catecholamine level that could lead to hypoxia and inflammation in the donor organ. In this series, the majority of the DBD donors suffered intracranial haemorrhage (88%) and hypertension (67%) compared with 33% and 22% of the DCD donors, respectively. Conversely, DCD donors were subjected to significantly prolonged ventilation (3.6 times) compared with DBD donors. Damman et al. found an increased C3 mRNA expression in kidney biopsies from DBD compared with DCD donors [This study aimed to distinguish characteristics of biopsy samples taken from DBD and DCD donor kidneys that had been declined for transplantation. Clinically, the distinct difference between DBD and DCD kidneys is the higher rate of DGF in DCD kidneys, yet this argument does not affect graft survival. The pathogenesis of DGF in DCD kidneys was triggered by the additional warm ischemic insult. However, our findings suggest that the DCD kidneys declined for transplantation showed a similar level of histological injury , inflammThe outcome from the discarded donor kidneys in this study is in agreement with a report by Damman et al. that showed similar hypoxic, complement and coagulation pathways in DBD and DCD donors . AlthougThis study has shown a great deal of variability in the quality of DBD and DCD kidneys declined for transplantation. There were no significant differences in the histologic damage, cellular injury, inflammation and complement activation renal biopsies of the DBD and DCD kidneys. vWF reactivity and complement activation might be early indicators for a certain degree insult, while C4d deposition and C3 activation might be also good predictors for histological damage. These potential biomarkers in conjunction with NMP could be applied to better assess and rescue viable DCD donor kidneys.


Regarding gastric atrophic and nonatrophic mucosae, TXI mode 1 had a significantly greater color difference than WLI . TXI may be a useful observation modality in the endoscopic screening of the upper gastrointestinal tract.In 2020, Olympus Medical Systems Corporation introduced the Texture and Color Enhancement Imaging (TXI) as a new image-enhanced endoscopy. This study aimed to evaluate the visibility of neoplasms and mucosal atrophy in the upper gastrointestinal tract through TXI. We evaluated 72 and 60 images of 12 gastric neoplasms and 20 gastric atrophic/nonatrophic mucosa, respectively. The visibility of gastric mucosal atrophy and gastric neoplasm was assessed by six endoscopists using a previously reported visibility scale (1 = poor to 4 = excellent). Color differences between gastric mucosal atrophy and nonatrophic mucosa and between gastric neoplasm and adjacent areas were assessed using the International Commission on Illumination L*a*b* color space system. The visibility of mucosal atrophy and gastric neoplasm was significantly improved in TXI mode 1 compared with that in white-light imaging (WLI) (visibility score: 3.8 ± 0.5 vs. 2.8 ± 0.9,  Despite the decreasing trend because of the widespread use of eradication therapy for Helicobacter pylori (H. pylori), gastric cancer remains the third leading cause of death worldwide3. In Japan, the incidence of gastric cancer ranks second among men and fourth among women5. Early detection of gastric cancer leads to early treatment, commonly endoscopic treatment. Its early detection promotes reduction in tumor invasion and prevention of cancer progression. The diagnosis of atrophic gastritis, which is a risk factor for gastric cancer, via upper gastrointestinal endoscopy is also vital. In upper gastrointestinal endoscopy, image-enhanced endoscopy (IEE) contributes to the detection of early gastric cancer and the diagnosis of atrophic gastritis; the most common IEEs are narrow-band imaging (NBI), blue-laser imaging (BLI), and linked color imaging (LCI)13. In particular, NBI facilitates the diagnosis and identification of early-stage gastric cancer by illuminating the mucosal surface structures at two narrow wavelengths (blue light at 390–445 nm or green light at 530–550 nm) that are easily absorbed by blood hemoglobin and highlighting the capillaries in the mucosal surface layer14. Similarly, BLI irradiates a narrow wavelength laser beam (450 ± 10 nm for white-light observation and 410 ± 10 nm for narrow-band observation) to the mucosa to highlight capillaries and fine patterns in the mucosa. In addition, the wavelength of the laser light is narrower than that of the narrow-band light of NBI, so the mucosa can be observed with higher specificity. Meanwhile, LCI increases the light output of the phosphor excited by enhancing the light output at 450 nm, making the screen brighter, and the image processing makes the color separation in the red region clearer. LCI also highlights the subtle color differences in the mucosa through imaging that further extends saturation and hue differences, thereby contributing to the diagnosis of inflammation and neoplasia12.Although the mortality rate of gastric cancer is decreasing worldwide, it still exceeds 800,000In 2020, Olympus Medical Systems Corporation  introduced a new IEE called Texture and Color Enhancement Imaging (TXI). In TXI, an image usually obtained by white-light irradiation is divided into texture image and base image, which are recombined after enhancing the texture and correcting the color tone and brightness. TXI optimizes three elements, namely, “structure,” “color tone,” and “brightness,” of the mucosal surface in ordinary white-light observation. Although TXI is currently expected to be useful, the conditions and lesions that can benefit from TXI in actual clinical practice are still poorly known. In addition, the efficacy of TXI remains unreported. TXI is expected to reveal subtle changes in mucosal irregularities, color tone, and vascular permeability, which are hardly detectable by white-light imaging (WLI), by emphasizing and correcting the color and contour of the mucosa. Therefore, this study aimed to assess the efficacy of TXI in visualizing gastric mucosal atrophy and gastric neoplasms.This study employed the CV-1500 light source equipped with a TXI system, as well as the GIF-EZ1500 and GIF-H290Z endoscopes, . TXI has two types: mode 1 and mode 2. In TXI mode 1, the overall image is slightly blue-white and the color tone changes are more coordinated than those in TXI mode 2, which has an orange color tone and provides an image similar to WLI. We also compared TXI mode 1 and mode 2 to contribute to appropriate mode selection. For the structure-enhanced mode, “A7” was selected for NBI and WLI, while “strong” was selected for TXI.The following is a description of the TXI scans: First, the image obtained from WLI is divided into a base image and a texture image. Then, texture enhancement is applied to the texture image and brightness adjustment is applied to the base image. These two images are combined to produce a TXI mode 2 image. Then, a TXI mode 1 image can be obtained by color enhancement of the TXI mode 2 image. Figure H. pylori infection. In addition, 11 consecutive patients with 12 gastric neoplasms, who were examined during the same period, were evaluated. Although 15 gastric neoplasms could be imaged, three were excluded because appropriate images could not be successfully captured. Gastric mucosal atrophy was diagnosed endoscopically by two experts (T.I. and T.M.) using WLI, whereas gastric neoplasms were diagnosed by biopsy or endoscopic submucosal dissection. For both gastric mucosal atrophy and gastric neoplasms, images were taken continuously with WLI, TXI mode 1, and TXI mode 2 in the same composition. For gastric neoplasms specifically, images were also taken continuously after indigo carmine spraying, again with the same composition and imaging modes. The infection and eradication statuses of Helicobacter pylori were obtained from the patients’ medical records. Infection with H. pylori was regarded as positive if the record included at least one positive result from the urease breath test  or rapid urease test  or indicated detection of H. pylori using serum IgG antibody 17. All the patients were confirmed to have a current or history of H. pylori infection. Endoscopic findings of gastric mucosal atrophy were evaluated in accordance with the Kimura–Takemoto classification18 The atrophic pattern was differentiated on the basis of the following features: C1, atrophic mucosa is only found in the antrum; C2, atrophic mucosa is found at the gastric angle or in the lower corpus; C3, atrophic mucosa is also found in the upper corpus; O1, atrophic mucosa surrounds the gastric cardia, but the folds of the great curvature are relatively maintained; O3, the entire gastric mucosa is atrophic, and the folds of the greater curvature as a whole disappeared; and O2, an intermediate condition exists between O1 and O313.This study enrolled consecutive patients who underwent esophagogastroduodenoscopy in July and August 2020 for gastric mucosal atrophy or gastric neoplasms, including early gastric cancer and adenomas, at Chiba University Hospital in Japan. A total of 23 patients were enrolled in the study after providing an informed consent; however, four patients were excluded because they could not obtain an adequate imaging set. Thus, we evaluated 19 patients with gastric mucosal atrophy and gastric neoplasms, including gastric adenomas or early-stage gastric cancer. All the patients were confirmed to have a current or history of This study was reviewed and approved by the Institutional Review Board of Chiba University School of Medicine and registered in the University Hospital Medical Information Network . All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from each participant.20. The visibility scores for gastric mucosal atrophy were rated on a 4-point scale as follows: 4, excellent (can be easily determined); 3, good (can be detected with careful observation); 2, fair (can barely be detected); and 1, poor (cannot be detected with repeated observation). Figure In assessing the visibility score of the atrophic mucosa, first, images were taken in a composition that allowed single-image observation of the atrophic and nonatrophic mucosae. Images were acquired from the middle or distant view, with WLI, TXI mode 1, and TXI mode 2 in the same composition ; 3, good (detectable with careful observation); 2, fair ; 1, poor (undetectable with repeated observation). Figure In assessing the visibility score of gastric neoplasms, first, imaging was performed from the middle or distant view with the same composition for each lesion by WLI, TXI mode 1, and TXI mode 2. We assessed 36 images. Next, after spraying indigo carmine, we imaged the lesions in mid- or distant view using WLI, TXI mode 1, and TXI mode 2. Likewise, 36 images were obtained in this imaging , a red-green axis , and a yellow-blue axis , is designed to approximate human perception. The Euclidean distance between two points in the CIELAB color space is proportional to the difference in color perception. Here, we evaluated the perceived color difference in endoscopic images, with ΔE values calculated according to the CIELAB color space system24. We evaluated the color difference between gastric atrophic and nonatrophic mucosae by comparing the WLI scans with TXI mode 1 and TXI mode 2 images of the same composition taken consecutively 2 + (Δa*)2 + (Δb*)2]1/2. Furthermore, we excluded pixels affected by halation.Color differences (ΔE) were calculated using the International Commission on Illumination L*a*b* (CIELAB) color space systemSimilar to how the color difference between gastric atrophic and nonatrophic mucosae was evaluated, we compared gastric neoplasm and non-neoplasm images taken consecutively in the same composition with WLI, TXI mode 1, and TXI mode 2. The WLI, TXI mode 1, and TXI mode 2 images were also obtained after indigo carmine spraying .Clinical data were expressed as percentages, averages, and ranges. We calculated the mean and standard deviation (SD) of the visibility scores and color differences (ΔE). The mean visibility scores rated by all endoscopists (experts and trainees) were also analyzed. Scores between modalities were compared using the Wilcoxon signed-rank test. Moreover, Nineteen patients with gastric mucosal atrophy and/or gastric neoplasm were evaluated . On the basis of the Kimura–Takemoto classification, mucosal atrophy was classified as follows: C1 in four patients (5.3%), C3 in one (5.3%), O1 in seven (36.8%), O2 in five (26.3%), and O3 in five (26.3%). Seven gastric neoplasms (46.7%) were found in the middle stomach and eight (53.3%), in the lower stomach. Regarding their morphology, nine cases (60.0%) were 0-IIa; four (26.7%), 0-IIc; and two (13.3%), 0-IIa + IIc. The gastric neoplasms consisted of differentiated adenocarcinoma in 10 cases (76.9%) and adenoma in 3 cases (23.1%).p < 0.01 on both comparisons). However, TXI mode 1 was significantly better than TXI mode 2 (p < 0.01). Table Gastric atrophic mucosa was more visible in TXI mode 1 and in TXI mode 2 than in WLI (p < 0.01). Similarly, TXI mode 2 was better than WLI (p < 0.01). However, TXI mode 1 was significantly better than TXI mode 2 (p < 0.01). Table Gastric neoplasm lesions were more visible in TXI mode 1 than in WLI (p < 0.01). TXI mode 1 also had a significantly higher color difference than TXI mode 2 . Meanwhile, the color difference between WLI and TXI mode 2 was not significant . These results are summarized in Table The color difference (ΔE) between gastric atrophic and nonatrophic mucosae was significantly higher for TXI mode 1 than for WLI . Table The color difference (ΔE) between neoplastic and non-neoplastic areas was significantly higher for TXI mode 1 than for WLI [8.000 ± 4.263 vs. 18.728 ± 16.046, The results of this study showed that screening endoscopy using TXI mode 1 may provide more visible gastric neoplasms, including early gastric cancer and gastric adenomas, and gastric mucosal atrophy than screening endoscopy using WLI. TXI mode 1 also seemed to be a better modality for visualizing lesions and gastric mucosal atrophy than the newly introduced TXI mode 2. To our knowledge, this study is the first to compare color assessment and visibility of gastric neoplasms and gastric mucosal atrophy using the CIELAB color space system and visibility score in TXI.This study also examined the visibility and color differences of gastric neoplasms and gastric mucosal atrophy individually according to the assessments made by six endoscopists. Typically, endoscopists find lesions by using the unevenness of the mucous membranes and changes in color shades when examining the inside of the stomach. Hence, gastrointestinal endoscopy requires techniques that can detect slight differences in color shades. The same can be said for atrophic mucosa.WLI is known to detect lesions that show clear differences in color shading or mucosal surface irregularities, but has difficultly recognizing lesions with little mucosal unevenness or only a slight change in color tone. To recognize such lesions, we need to detect the color difference between gastric neoplasm and non-neoplastic areas. TXI is a new technology that is expected to improve the visibility of these differences by emphasizing the color tone and structure, such as the outline, of the target lesion. The current study evaluated the objective color tone by using the CIELAB color space and visibility score for each imaging mode to assess the visibility of gastric mucosal atrophy and gastric neoplasm. Results showed that TXI mode 1 had a better contrast than WLI for the color difference between gastric neoplasm and non-neoplastic areas. Likewise, the contrast for the visibility of gastric mucosal atrophy was higher in TXI than in WLI. As mentioned, TXI has two modes, namely, mode 1 and mode 2, and TXI mode 1 showed a better contrast than TXI mode 2 in the visibility of gastric neoplasm and gastric mucosal atrophy. Even after the spraying of indigo carmine, which we frequently use for visualizing slight changes in gastric mucosa, TXI mode 1 also showed a better contrast than WLI or TXI mode 2 in the color difference. Moreover, the visibility score of TXI mode 1 was significantly higher than that of WLI and TXI mode 2. Therefore, TXI mode 1 is a better imaging method than WLI and TXI mode 2 for visualizing early-stage gastric cancer, adenomas, and gastric mucosal atrophy. The visibility scores of experts and trainees revealed basically similar trends. However, after indigo carmine spraying, the visibility ability of TXI mode 1 and TXI mode 2 was significantly increased compared with WLI when judged by experts. When judged by trainees, TXI mode 2 did not show a significant increase in visibility compared with WLI, possibly because of their lack of experience in observing indigo carmine sprays. TXI mode 1 is an image mode with a strong blue-white impression, and the blue color of indigo carmine and other substances are emphasized. Thus, TXI mode 1 may aid in detecting lesions when combined with indigo carmine spraying. However, TXI mode 1 requires some familiarity with the color tone of the images compared with WLI, which is more familiar among endoscopists. Meanwhile, TXI mode 2 is an orange-tinged image mode that is relatively close to WLI. The selection of imaging mode for screening upper gastrointestinal endoscopy depends on the decision of each endoscopist.This study, however, has several limitations. First, it was a small, single-center study. Second, the number of images evaluated was small. Third, in terms of visibility, the decision was made according to still images rather than video. Fourth, endoscopy experts were clearly expecting that imaging modes other than WLI would have better visibility because of the nature of these modes. Fifth, we examined visibility, not detectability. However, considering our results that the use of TXI improved the visibility and color difference of gastric neoplasms, we believe that TXI has the potential to increase the detectability of these neoplasms. For future research, a larger sample size is required, and the detection rate of gastric neoplasms, including early gastric cancer and gastric adenomas, will need to be studied in a multicenter, prospective, randomized, controlled trial. Sixth, we did not compare TXI with other modalities such as NBI, BLI, and LCI. In this study, we focused on the evaluation of gastric mucosal atrophy and detection of gastric neoplasms in screening. For these reasons, we did not collect data that could be directly compared with NBI, which is commonly used for scrutiny. BLI and LCI are similar observation methods, but because we used Olympus endoscopes in this study, we could not compare LCI and TXI. The seventh limitation is the identities of the images themselves. TXI scans are outputted by processing images obtained from WLI scans. The images used in this study for color difference and visibility scoring were taken using TXI and WLI in the same composition and object in succession. The WLI and TXI scans used in this study were not created from the exact same WLI scan, but were images taken in succession.In conclusion, TXI mode 1 is recommended for the endoscopic screening of the upper gastrointestinal tract. In addition, TXI mode 1 observation after indigo carmine spraying is useful for lesion visibility because of the enhanced blue coloration.


All subjects also underwent an MRI scan where multiple inversion time (MTI) pulsed arterial spin labeling (PASL) was used to quantify resting CBF and CVR to 5% CO2. Region of interest analysis showed a non-significant inverse correlation between whole-brain gray matter CBF and 2peak; r = −0.4, p = 0.08, corrected p (p′) = 0.16 and a significant positive correlation between 2peak and whole-brain averaged gray matter CVR; r = 0.62, p = 0.003, p′ = 0.006. Voxel-wise analysis revealed a significant inverse association between 2peak and resting CBF in the left and right thalamus, brainstem, right lateral occipital cortex, left intra-calcarine cortex and cerebellum. The results of this study suggest that aerobic fitness is associated with lower baseline CBF and greater CVR in young adults.This cross-sectional study investigated the effects of aerobic fitness on cerebrovascular function in the healthy brain. Gray matter cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) were quantified in a sample of young adults within a normal fitness range. Based on existing Transcranial Doppler ultrasound and fMRI evidence, we predicted a positive relationship between fitness and resting gray matter CBF and CVR. Exploratory hypotheses that higher  Aerobic fitness has emerged as a modifiable lifestyle factor which reduces the risk of all-cause mortality , cardiovmax in younger adults despite observing the same trend as 2max in frontal regions but positively correlated in periventricular white matter and portions of the somatosensory cortex. These conflicting CVR data represent an issue of inconsistent findings regarding aerobic fitness and its effects on cerebrovascular function across the lifespan.Neuroimaging studies have provided evidence of a neuroprotective effect of aerobic fitness in older adults, linking higher aerobic fitness with greater brain volume  and cereRecent findings from 2 and 2peak, a measure of aerobic fitness, in line with previous findings  MRI at multiple post-labeling delay times, variations in resting CBF and CVR, two widely used indices of cerebrovascular function, in a sample of young, healthy adults within a normal range of fitness levels . Based on the extant TCD and fMRI evidence, we predicted a positive relationship between fitness and resting gray matter CBF. We also expected to see a positive correlation between CVR to COfindings . Tests cTwenty healthy adults  were recruited from Cardiff University. The study was approved by the Cardiff University School of Psychology Research Ethics Committee and performed in accordance with the guidelines stated in the Cardiff University Research Framework . All participants were non-smokers and educated to university level. Informed written consent was obtained from all subjects.2 inhalation can cause sensations of breathlessness, light headedness and anxiety in some individuals. For this reason, all volunteers took part in a gas modulation session in a MR scanner simulator. A stepwise protocol was employed to allow participants time to become accustomed to CO2 inhalation  and work rate were also recorded at baseline and at 2 min intervals and electrocardiography (ECG) was monitored for the duration of the test. Following termination of the 2peak test participants were monitored for 10 min. In this 10 min recovery period blood pressure, HR and blood lactate were sampled every 2 min before subjects left the lab.Blood lactate concentration was sampled from the earlobe using Unistik 3 1.8 mm lancets  and tested using the Lactate Plus system  at baseline, 2 min intervals and at exhaustion. Blood pressure (BP) was recorded at baseline, HR at baseline and 2 min intervals during the test. Respiratory gas exchanges were continuously monitored using breath by breath analysis with the Cortex Metalyser 3b . 2 (PETCO2) and O2 (PETO2) via the Biopac < cps:sup > ® < /cps:sup > system . The MEDRAD system  was used to monitor O2 saturation throughout the experiment.In Visit 3, volunteers underwent the MR scan. Images were acquired on a 3T whole body MRI system  using an eight-channel receive-only head coil. Heart rate was recorded using a pulse oximeter and a respiratory bellows monitored breathing. A sampling line connected to the face mask of the breathing circuit  was used3, matrix size 64 × 64 mm, FOV = 19.8 cm, flip angle = 90°, inter-slice time 55 ms, 15 slices, slice gap 1.5 mm for maximum brain coverage. Label thickness was 200 mm with 10 mm gap between the end of the label and the most proximal imaging slice.PASL data were acquired using a single subtraction PICORE QUIPSS II  with a d0, CSF), needed for the quantification of CBF. The acquisition parameters for this CSF were identical to the PASL acquisition, except that no labeling was applied so that the image was acquired with fully relaxed magnetization. A minimal contrast image was also acquired to correct for coil inhomogeneities with TE = 11 ms, TR = 2 s. The PASL acquisition was performed twice, first during rest, then while volunteers were hypercapnic. The hypercapnic modulation was carried out using the prospective control method described by 2, and medical oxygen, the latter incorporated as a safety backup but not used during experimentation, the circuit is described in detail by ETCO2 elevation of 7mmHg was targeted using 5% CO2  and the scan commenced once participants reached the target CO2 level. ASL data acquisition lasted ∼6 min during rest, followed by a delay to allow the onset of hypercapnia. Once a PETCO2 elevation of approximately 7mmHg was achieved, the ∼6 min ASL acquisition was repeated.A calibration image without any labeling was acquired before the perfusion mages using the same acquisition parameters, except a long TR; this was used to obtain the equilibrium magnetization of cerebrospinal fluid  scan was also acquired for registration of functional data and voxel-based morphometry (VBM) analysis; TR/TE = 7.8/2.9 ms, resolution = 1 mm isotropic.2peak was calculated from the respiratory gas exchanges which were continuously monitored using breath-by-breath analysis and averaged over 10 s blocks. 2peak was defined as the 30 s averaged value at maximum recorded work rate. HR, respiratory exchange ratio (RER) and blood lactate were recorded every 2 min and at termination of the fitness test to validate 2peak criteria being reached. The test result was considered valid if heart rate exceeded 90% of age predicted maximum, RER > 1 and blood lactate concentration was greater than 8 mM at the end of the test . The CBF timeseries for the normocapnia and hypercapnia scans were corrected for coil sensitivity inhomogeneities using the minimal contrast images  in Matlab . Permutation testing was used as an alternative to Bonferroni correction for multiple comparisons which is too conservative when potentially related variables are tested. This approach was also applied to examine the associations between gray matter CBF and CVR, and cognitive test scores.Individual subject gray matter masks were created using FMRIB’s Automated Segmentation Tool (FAST) and thresholded to include voxels with >50% gray matter probability. ROI analysis of the relationship between gray matter CBF at rest, CVR and 3 (2peak and (ii) CVR and 2peak. The individual subject masks described in the ROI Analysis section were used to confine this analysis to global gray matter only. Randomize is a permutation testing method that uses threshold free cluster enhancement (TFCE) (p < 0.05 (FWE corrected).Follow up voxelwise analysis was conducted using FSL’s Randomize tool3  to examit (TFCE)  to corre2peak test all subjects satisfied test criteria by working to volitional exhaustion. Based on typical 2max criteria, 17 subjects fulfilled criteria for a maximal exercise test  between baseline PETCO2 and CBF, but no strong associations between 2peak and baseline PETCO2  or the ΔPETCO2 during hypercapnia  of blood. Brain capillary [Hb] cannot be measured directly in vivo, however, peripheral capillary samples may provide an indication of Hb differences between subjects or groups which could help to explain biological mechanisms driving CBF differences.Hemoglobin (Hb) levels were not measured. Hb is responsible for transport of Os oxygen  and therSecond, investigations were restricted to gray matter. Due to the limited SNR of ASL, reliable quantification of CBF and CVR in white matter is difficult as CBF is much lower than in gray matter. However, adequate blood supply and energy metabolism is necessary for all round brain health, and aging is associated with damage to white matter microstructure and reductions in myelination . Therefo2peak. An alternative possibility is that CBF is more heterogeneous throughout the brain than CVR which is more affected by low SNR; therefore greater averaging of data in the ROI analysis allowed CVR differences to be detected.The voxelwise and ROI analyses did not show the same statistical significances. While the voxelwise analysis of CBF revealed regions of significantly lower CBF with fitness, the ROI analysis showed a non-significant trend in the same direction for global gray matter. The ROI analysis of CVR, however, showed a moderate (0.6) significant correlation with fitness while the voxelwise analysis did not reach statistical significance. ASL is an intrinsically low SNR technique compared to BOLD fMRI. Although ASL offers significant benefits in terms of quantification of physiological parameters, the SNR may have prevented detection of greater voxelwise associations with t(18) = −0.119, p = 0.734 but controlling for menstrual cycle phase, which was not done here, could reduce variability within cohorts. This is a preferable option to limiting studies to males only as hormonal differences may play a role in the acute response to exercise and possibly in mediating the effects of fitness on brain health.Lastly, there are known hormonal effects on exercise test performance  and CBF The results of this study suggest that aerobic fitness is associated with lower CBF and greater CVR in young, healthy adults, however, the modest effects observed need replication in larger samples. We currently know very little about the functional relevance of CBF or CVR differences in young adults, and how the observed neurophysiological effects of physical training differ from those observed in older adults. With recent advances in quantitative MRI techniques, non-invasive mapping of multiple indices of cerebrovascular function, e.g., CBF, CVR, CBV OEF and the cerebral metabolic rate of oxygen consumption is possible within a single scan session . NotablyApplication of these techniques to study brain function in both young and older trained and sedentary adults will provide information necessary to move forward in developing exercise training protocols to increase the adoption of fitness training as a preventative health tool.Research over the next decade should also work to establish whether regular exercise regardless of intensity level delivers brain benefits, or whether there is an aerobic fitness threshold, above which benefits such as maintained CBF with aging, are observed. Answering this question will guide optimal exercise dose recommendations and interventional studies.https://www.biorxiv.org/content/10.1101/539072v1. This work first appeared in the lead author’s thesis . Informed written consent was obtained from all subjects.CF conceived and designed the study with input from RW. CF and JS coordinated the project and collected the data with assistance from DH. CF and RW analyzed the data. CF prepared the manuscript with RW providing input and interpretation of results as well as reviewing and editing the final manuscript. VT supervised CF throughout the project. All authors approved the manuscript before submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Serum microbial and mitochondrial metabolites were measured by GC-MS and compared with the biomarkers and clinical neurological scores. The identified associations between specific bacterial taxa in fecal samples, neurological status and serum levels of metabolites suggest that impacts on specific members of the gut microbiota and their metabolism might be a promising tool for regulating brain function in future.The community structure and metabolic potential of gut microbiome is not well investigated, especially in chronically critically ill patients with prolonged dependence on support systems after severe brain disorders. Microbial phenolic metabolites can target the brain function by the direct and indirect modulation of inflammation. The aim of this study was to investigate the features of the gut microbiota and profile of certain metabolites in the progression and reversibility of neurological disorders in chronically critically ill patients. Fecal samples were collected in dynamics from such patients ( Over the past decades, advances in therapeutic techniques have substantially improved the survival of patients after acute events, and simultaneously expanded the population of patients with prolonged dependence on life support systems. This gave rise to the concept of chronic critical illness (CCI) . CCI criMost surviving patients after severe brain injury meet CCI criteria. They are compromised by the risk of the CCI-attributed neurological dysfunction aggravated by their condition. It may be caused by the functional or metabolic impairment mediated by pro- or noninflammatory factors, including microglia hyperactivation, blood brain barrier disruption and altered neurotransmission .Taxonomic changes and dysfunction of the gut microbiota may be important factors in CCI. The gut microbiota has the ability to affect the central nervous system (CNS) function ,5, makinStaphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumanii, Escherichia coli and Pseudomonas aeruginosa.  dolcium and Ruminococcus bromii/unclassified were significantly enriched in the microbiome of the patients with the nasogastric tube or gastrostomy compared to those with oral enteral nutrition (FDR < 0.05), but this finding requires further confirmation in clinical studies with larger group sizes. We evaluated the effect of antibiotics use, nutrition type, diagnosis and infectious complications  on the gIn this study, we found that the abundance of the specific gut microbial taxa along with the serum levels of certain microbial and mitochondrial metabolites in the CCI patients were significantly different from the ones of the healthy subjects, independent oftheir neurological dynamics. The features of the clinical course allowed us to include heterogeneous patients in the studied groups, regardless of their primary nosological form of severe brain damage.Proteobacteria and Gram-positive opportunistic bacteria, such as Enteroccocus and Streptococcus coupled with the reduced abundance of commensal microorganisms. The balance-based approach showed that the main predictors for the CCI group were Eggerthella lenta (58% reproducibility), while for the healthy group there were unclassified species from Coprococcus/unclassified genus, similarly to the analysis of individual taxa. These facts are consistent with the findings of the previous studies of the gut microbiota in critically ill patients with brain injury [Akkermansia, was correlated with their clinical outcomes [Prevotella, Klebsiella, Streptococcus and Clostridium XI) were found to be associated with some neuropsychiatric disorders [Eggerthella lenta is an emerging and uncommon human anaerobic opportunistic pathogen under-recognized due to the limitations of phenotypic identification that can lead to the severe bloodstream infection [Eggerthella lenta in the gut [Gut microbiota dysbiosis in patients with CCI is characterized by prevalence of n injury ,26. The outcomes . Howeverisorders ,29. The nfection . Notewor the gut , which mRuminococcus, Roseburia and Clostridiaceae were positively correlated with the improvement of the neurological status-specifically, by manifesting positive association with the Rivermead Mobility Index and negative association-with the Rankin scale. Among them, Roseburia known for its metabolic capacity of producing short-chain fatty acids has been reported to be depleted in fecal samples of sarcopenic subjects [Clostridiaceae in the gut was associated with the positive neurological dynamics in the CCI patients. Previously, it has been shown that the arthritis phenotype is also characterized by an increase in the number of Clostridiaceae [Clostridium bacteria are among the phylotypes able to metabolize aromatic amino acids, such as tyrosine [Clostridium among the patients should be further investigated on larger cohorts. Another interesting finding, A. muciniphila is best known for its potential anti-inflammatory properties [A. muciniphila was abundant in a number of patients with positive dynamics, presumably indicating its important role in the neurorehabilitation. Overall, the described results provide additional support for the “gut-brain axis” concept. We identified the associations between several gut microbial taxa and neurological scales . The Rumsubjects . In addiidiaceae . The Clotyrosine . The reloperties ; neverthoperties . As showoperties . HoweverBifidobacterium, which is relatively less abundant in the CCI patients, in turn, suggesting the importance of this finding as a reflection of the gut microbiota dysfunction. Three phenolic metabolites-PhLA, p-HBA and HVA-had higher levels in CCI patients, while only one (PhPA) had higher values in healthy controls, which resonates with the results of our pilot study . The levCoprococcus_u  and the disease-associated dysbiosis was characterized by depletion of commensal microbes, contrarily to the very few taxa significantly enriched in the patients . We obseTo summarize, the gut microbiota of the CCI patients with brain dysfunction undergoes certain transformations and can be considered as a “damaged organ”, which is schematically shown in This prospective observational study was performed in the Department of Intensive Care at Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, Moscow, Russian Federation. The local Ethics Committee approved the study  which was conducted in accordance with the ethical standards of the Declaration of Helsinki and formal consent for participation in this study was obtained from each patient or his/her legal representative.Study Participants. We included adult (>18 years) patients admitted to the Department between June 2019 and December 2019. The following inclusion criteria were applied: patients with brain injury of various etiology due to the CCI criteria  and admitted to ICU for the intensive neurological care. Exclusion criteria: chemotherapy/hormonal therapy with steroids; suspected neurological infection, acute neurological disorders. n = 44) was from 23 to 75 years (median: 50 (IQR: 31–64) years). The study group was balanced by gender: 23 (52%) females and 21 (48%) males. The pathologies recorded during the study included an acute cerebrovascular event (n = 16), severe traumatic brain injury (TBI) (n = 13), neurosurgery complications in the postoperative period (n = 12), anoxic brain injury after a successful cardiopulmonary resuscitation effort to reverse clinical death (n = 3). Some patients were on mechanical ventilation (n = 14) and some had a tracheostomy tube (n = 9). All patients were on enteral feeding; among them, over a half (n = 23) were fed through a nasogastric or gastrostomy tube. Antibacterial therapy was administered to patients (n = 13) who had urinary and/or lower respiratory tract infection. The age range of the CCI patients , the National Institutes of Health Stroke Scale (NIHSS), the Rivermead Mobility Index Scale and the Rankin Scale. The Rankin Scale is used to measure the degree of neurological disability. It is widely used for the clinical outcome measure in the stroke clinical trials. The scale ranges from 1 to 5 where score 1 shows no significant disability and score 5 indicates severe disability, which requires constant nursing care and attention The Rivermead Mobility Index (RMI) is a hierarchical mobility scale used in neurological rehabilitation. It reflects to different aspects of mobility, with the sum scores ranging from 0 to 15, with higher scores indicating better mobility.The Glasgow Coma Scale (GCS) is a clinical scale used to reliably measure a person’s level of consciousness after a brain injury. A person’s GCS score can range from 3 (completely unresponsive) to 15 (responsive).The National Institutes of Health Stroke Scale, or NIH Stroke Scale (NIHSS) is a tool which is used to objectively quantify the impairment caused by a stroke. The maximum possible score is 42, which reflects no stroke symptoms; the minimum score is 0 and reflects a severe stroke.A positive dynamic was defined as a decrease in the number of points by 1 or more on the NIHSS scale; a negative dynamic was defined as an increase in the number of points by one or more on the NIHSS scale or a lethal outcome. Characteristics of the two groups of CCI patients is demonstrated in Blood  and gut microbiome samples (feces) were collected simultaneously in the morning at regular intervals (once a week). Fecal samples were obtained by collecting a small amount of feces as a rectal swab and dissolved in 1 mL of sterile saline solution; after thorough mixing, it was divided into two Eppendorf tubes. All samples (serum and feces) were frozen and stored at −30 °C prior to analysis that included GC-MS, biomarkers and general clinical analyses, laboratory tests and sequencing. The total number of simultaneously taken fecal and blood samples was 83. In 18 patients, 1 sample was taken; in 16 patients—2 samples; in 10 patients—3 samples or more.As a control group, we used previously obtained data on serum metabolites of 20 healthy volunteers without any signs of neurological or somatic pathology . The ageThe 2,3,4,5,6-D5-benzoic acid , benzoic acid , phenylpropionic acid , phenyllactic acid , 4-hydroxyphenylacetic acid , 4-hydroxyphenylpropionic acid , homovanillic acid , 4-hydroxyphenyllactic acid , 3,4-dihydroxybenzoic acid , succinic acid (≥99%), fumaric acid (≥99%), N,O-bis(trimethylsilyl)trifluoroacetamide trifluoroacetamide), hexane (≥97.0%) were obtained from Merck ; sulfuric acid, acetone, diethyl ether, sodium chloride were laboratory reagent-grade and obtained from Khimreactiv .m/z range of 50–480 in the electron-impact mode at 70 eV, using Xcalibur 2.2 software. The MS source was 200 °C and the GC-MS interface was kept at 250 °C. Scan rate was 3 scans/s; cathode delay time 4 min.The blood serum samples were thawed at a room temperature prior to analysis. The GC-MS analyses were performed on a Trace GC 1310 gas chromatograph equipped with an ISQ LT mass spectrometer and AI 1310 autosampler using the capillary column TR-5ms (95% poly(dimethylsiloxane) + 5% phenyl polysilphenylene-siloxane phase, 30 m × 0.25 mm, df = 0.25 µm) obtained from Thermo Scientific . The column flow was constant at 1.5 mL/min with helium as the carrier gas, split 1:10. The GC analysis was performed in 25 min with a starting oven temperature of 80 °C (hold time 4 min) and a single ramp of 10 °C/min to 250 °C (hold time 4 min). The injector temperature was 200 °C. Full-scan mass spectra were recorded with an The conditions of liquid–liquid extraction of the microbial and mitochondrial metabolites extraction were previously described . Brieflym/z values and their concentrations were calculated using the equations of linear functions which were previously described [Trimethylsilyl derivatives of the microbial and mitochondrial metabolites were identified using retention times and characteristic escribed ,23.g for 5 min. Aqueous phase was transferred to new sterile tube and used for PCR dilutions. The obtained DNA solution was stored at −20 °C.Defrozen fecal solution (200 mL) was placed in the 2.0 mL tube containing 3:1 mix of 0.1 mm and 0.5 mm pre-sterilized glass beads . Then 1 mL of a warm 60 °C lysis buffer  was added. The mixture was vortexed and homogenized with MiniLys  for 3 min. The lysate was incubated at 70 °C for 15 min and centrifuged for 20 min at 14,000 rpm. The supernatant (1 mL) was transferred to the sterile tube and put on the ice. The pellet was added to a 1 mL of lysis buffer and the homogenization process was repeated. The supernatants were combined in the 15 mL tubes with the addition of 4 mL of 96% ethanol and 200 μL of 3 M sodium acetate. The mixture was incubated at −20 °C for not less than 1 h. The mixture was centrifuged for 15 min at 14,000 rpm at +4 °C, the supernatant was discarded, the DNA pellet was washed twice with 80% ethanol. The pellet was dried at 53 °C for 30–60 min and resuspended in 200 μL of sterilized milliQ water. The mixture was centrifuged and transferred into new tubes. Resulting DNA solution was treated with 10 μL of RNAse A (5 mg/mL) for 1 h at 37 °C, followed by additional round of chloroform purification. Chloroform was added to the solution in 1:1 ratio, tube was vortexed for 1 min and centrifuged at 5000× Amplicon sequencing of V4 variable region of microbial 16S rRNA gene was performed on a MiSeq sequencer  as described before .Raw microbiome data are available in the Sequence Read Archive (SRA) by the accession number PRJNA688839. The reads were processed using the Knomics-Biota system   were clr transformed; zero values were preliminarily imputed using Bayesian-multiplicative treatment implemented in the cmultRepl function in package zCompositions . The metMetabolic and gut microbial taxonomic profiles of samples from the CCI patients and healthy subjects were compared using the 1st time point per subject for the CCI patients with linear models. Taxonomic profiles were analyzed at different ranks—from order down to species. Adjustment for multiple comparison was applied separately for taxonomic and metabolic analysis results using Benjamini-Hochberg method. Patients with certain diagnosis were compared to all other patients during the analysis of diagnoses. The analysis was performed for all diagnoses except anoxic brain injury effort due to the small sample size in this group.The associations of metabolic and taxonomic profiles with dynamics, neurological scales, diagnosis, infectious complication, therapy  as well as between each other were assessed using mixed effect linear models. Measurements from all time points were used and patients ID was treated as a random effect in a model. Similar to the health status analysis, the taxonomic ranks from species to order were analyzed. Adjustments for multiple comparisons were applied separately within each metadata group  and data type (taxonomic and metabolic) using Benjamini-Hochberg method. p-value was observed. For health status analysis, the first time point measurements per patient were used and for the other analyses—the last point. We use threshold for the reproducibility of bacterial taxa > 50% and final model R2 > 0.2 to identify reliable associations using balances-based approach.The taxonomic data associations with health status , dynamics and metabolic profiles were calculated with balances-based approach  as descrDifferences in changes of microbial abundances and metabolite levels between the patients with different dynamics were assessed by ANCOVA approach, where dynamics were treated as an intercept. Alpha diversity associations with dynamics were assessed using mixed-effect linear models, where patient ID was treated as a random effect.Clostridiaceae, Coproccocus and one of its metabolites (PhPA) found in patients with positive dynamics should be further confirmed in neurorehabilitation. It is possible that disrupted profile of the microbial metabolites of phenolic and other chemical classes plays an important role in neurological deficits. We are confident that CCI patients need microbiota-targeted therapies that can improve the efficacy of neurorehabilitation even in patients with severe brain damage. The CCI patients have a significant and patient-specific imbalance in the taxonomic composition of the gut microbiota. Pathophysiological role of an increased abundance of taxon associated with the biodegradation of tyrosine such as


We characterized copper (Cu) concentrations in the water, sediments, and shoreline plants of stormwater ponds in the urban Tampa, Florida area. We selected 6 urban residential stormwater ponds that receive summer wet season (May to September) Cu sulfate applications at least twice a month. We collected triplicate water and sediment samples from each pond and analyzed for Cu, as well as nutrient pools (inorganic N and P) and a suite of other physicochemical properties . We analyzed shoreline plant tissue samples for Cu. The raw dataset provides values for Cu concentrations in water, sediments and plant tissue, and other measured parameters in water and sediments.This dataset is important for characterizing the fate and potential mobility of Cu in freshwater ponds treated with Cu sulfate algaecides. This applied research data will provide baseline understanding of Cu concentrations in water, sediments, and select plant tissue samples, providing insights on potential toxicity of Cu and any threats that Cu sulfate algaecides may pose to aquatic organisms and downstream waters. This dataset can also inform future research designs aimed at elucidating the effects of Cu on denitrifying bacteria and N removal in stormwater pond ecosystems. Finally, the plant tissue data shows variable Cu concentrations among plant species, and this data can inform future phytoremediation experiments. Specifications TableValue of the data•The data provide information about Cu concentrations in environmental pools  of stormwater ponds, a type of waterbody that numbers in the tens of thousands in Florida alone •Aquatic biologists, stormwater scientists and engineers, and researchers can benefit from the data. The data can be used by those interested in heavy metal (Cu) contamination in freshwater bodies, in phytoremediation of Cu contamination, and in how we can manage copper sulfate algaecide applications in urban waters.•These data may be used to inform future experiments related to phytoremediation of Cu in soils and sediments.•These data may be used to inform future experimental designs aimed at learning the effects of Cu on aquatic organisms, including the microbial community responsible for N transformations and removal, thus providing insights on the connections between algal management strategies and nutrient cycling in the ponds.1Stormwater ponds are a type of stormwater control measure used in urban environments to primarily reduce flooding associated with urban runoff, though they are often expected to perform some level of pollutant removal as well , 3. They4 • 5H2O) is the active ingredient in several trademarked algaecides used to control algae in stormwater ponds and other freshwater systems [2+) are often applied to stormwater ponds on a weekly or biweekly basis to control rapid proliferation of algae Copper sulfate , accumulating up to 1596 mg/Kg Cu  and nutrient pools (N and P) for water and sediments. Copper concentrations were consistently higher in sediments than in water samples. With the exception of pond L237, Cu in water seldom exceeded 0.02 mg/L and exchangeable Cu in sediment was generally below 2 mg/Kg  and 2. Pmg/Kg Cu .Fig. 1Co4—N, ortho-P, and total dissolved P concentrations in water and with sediment Cu and P concentrations. Copper concentrations in sediment were positively and strongly correlated with water pH; Cu, ortho-P and total dissolved P concentrations in water; and with the sediment P concentration.A Pearson correlation matrix of water and sediment parameters is in Supplementary File 1 in the worksheet titled “Correlation Matrix.” Copper concentrations in water were positively and strongly correlated (correlation coefficient >0.50) with NH22.12+) at least twice a month during the summer rainy season (May to September).We selected six residential stormwater ponds in Lakewood Ranch, a medium-density neighborhood in Brandenton, Florida, which is part of the Tampa Bay watershed. The six ponds receive stormwater runoff from neighborhood streets, roofs, and sidewalks and are frequently impaired by excess aquatic vegetation and planktonic algae. Neighborhood demographics and stormwater parameters in the neighborhood have been characterized in previous studies 2.2in situ measurement of water temperature, surface dissolved oxygen, pH, and conductivity. We then collected 250 ml of pond water from each of the 3 points in all ponds. Water samples were placed into acid-washed HDPE bottles and then placed in a cooler on ice for transport back to the lab. Triplicate sediment samples were collected from the top 15 cm of each sampling point, placed in plastic bags and transported to the lab in a cooler. On the day of sampling we also walked the shoreline of each pond and made an inventory of non-turfgrass vegetation growing within 0.5 m of the shoreline, both inside and outside the ponds . For each plant species, we collected leaf, flower, stem, or shoot samples  at the University of Florida IFAS Analytical Research Laboratory using air-segmented continuous autoflow analyzers via EPA methods 353.2, 350.1, and 365.1, respectively. Water samples were analyzed for total alkalinity using LaMotte's KH/Alkalinity Test Kit 4491-DR-01 and direct reading titrator method. Sediment samples were air dried and passed through a #10 sieve prior to extraction and analysis. For sediment exchangeable Cu 1.0 g of oven dried (105 °C for 12 h) of sediment was extracted with 25 ml of 1 M NH4NO3. Extracts were then analyzed for Cu via ICP spectrophotometry. Sediment nutrient pools (inorganic N and P) were determined after extracting 20 g of air dry sediment with 150 ml of 2 N KCl and analyzing extracts in the same manner as water samples. Plant tissue samples were rinsed with deionized water, placed on a lab bench to air dry and then ground with a plant tissue grinder. They were analyzed for Cu via ICP spectrophotometry.Water samples were analyzed in the lab for Cu2.3We used Microsoft Excel to create a Pearson correlation matrix for measured water and sediment parameters.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Salmonella serovars (NTS) among food animals is of special concern as this may transmit resistant pathogens to humans during handling or consumption of animal products. In Nepal, the possibility of AMR Salmonella serovars among food animals is an important area of research, particularly in light of the rapidly growing poultry industry, lack of surveillance and proper biosecurity measures; and paucity of relevant data. This study was conducted with the aim to estimate the burden of NTS and associated antimicrobial resistance in the environments of commercial poultry farms and the poultry carcasses in slaughter house. This study also intends to find some basic knowledge of the poultry farmers and their practice relating to the use of antimicrobials, vaccination and biosecurity measures.Antimicrobial resistance (AMR) among bacterial pathogens is a fast-growing public health concern. AMR in non-typhoidal Taking one health approach, a cross-sectional study was carried out in Chitwan district of Nepal between May and October 2017. Various environmental samples viz. farm litter, feed, water, poultry faeces, vehicle swabs, farm swabs from 12 broiler poultry farms and various sections of poultry carcasses from 21 slaughter houses were aseptically collected. These were microbiologically assessed for the presence of NTS serovars and their phenotypic and genotypic indicators of antimicrobial resistance. The poultry farmers were also briefly interviewed regarding their basic biosecurity related knowledge and practices before collecting the environmental samples.Overall, of total environmental samples collected, 50% (31/62) tested positive for NTS serovars with environmental swabs  being the most culture positive sample types. Similarly, of 159 tissue samples collected from 24 carcasses, 79% (126/159) were culture positive for NTS serovars. Nearly 97% (153/157) of isolates showed antimicrobial resistance to tetracycline, while 11% (17/157) to ciprofloxacin and 5% (8/157) of isolates were resistant against azithromycin. All 157 isolates were sensitive to meropenem. In terms of AMR genes, tetA , QrnS , mefA  and VIM-1  were detected in the isolates that corresponded to the AMR to tetracycline, ciprofloxacin, azithromycin and meropenem respectively. In farmers interview, only 42% (5/12) of farmers mentioned of using basic biosecurity measures such as applying lime powder around the farm; 84% (10/12) of farmers reported vaccinating their birds with some vaccine and 75% (9/12) of farmers used various antimicrobials prophylactically such as neomycin , colistin , furaltadone , doxycycline , sulfatrimethoprim  and tylosin .This study revealed gross contamination of farm environment and subsequent poultry meat samples with NTS serovars that were resistant to several clinically important antimicrobials. Further, inadequacy of even basic biosecurity measures and frequent prophylactic use of antimicrobials in the commercial poultry farms was observed. This reinforces an urgent need to raise awareness and implement proper biosecurity approaches from farms to slaughter houses in order to reduce the burden of NTS contamination of surrounding environment and poultry products. Further, high prevalence AMR among NTS isolates also underscores the need to strengthen the policies to prevent the rampant use of clinically used human antimicrobials in poultry sector. Salmonella enterica (NTS) caused over 153 million illnesses and 122,000 deaths around the world, with children under 5 bearing a disproportionate burden [Salmonella contaminated, poultry is among the most frequent, resulting in international efforts to control and reduce the burden of Salmonella among poultry [In 2010, non-typhoidal  poultry .Salmonella tend to be more severe and lead to higher hospitalizations and deaths [Salmonella globally [In addition to the significant public health impact due to large burden of NTS illnesses, the increase in antimicrobial resistance (AMR) is a great concern . Infectid deaths . While gglobally , 8, systSalmonella in the region.In Nepal, poultry production is a significant economic activity and represented 4% of the country’s gross domestic product in 2017 . AccordiSalmonella enterica Typhi isolated from febrile humans [Salmonella enterica in poultry sector of Nepal. Still, the limited data available highlight the data gaps that must be addressed. Therefore, we used a one-health approach to conduct a preliminary study on prevalence of non-typhoidal Salmonella and associated AMR in poultry sector in Chitwan region of Nepal using both phenotypic and molecular characterization methods.Most surveillance efforts conducted in Nepal have focused on e humans , 14, andThe study was conducted in the Chitwan district in southern Nepal between May and October 2017; this time period coincides with summer monsoons and typically expected to have the highest rates of human salmonellosis. Chitwan is one of the largest poultry egg and broiler producing districts in the country , with a Twelve commercial poultry farms of Chitwan district registered with National Poultry Federation were selected to represent a range of production volume, housing types and hygiene. Upon receiving an informed consent from the farm owner, an informal interview was conducted by the research team with the farm owner to obtain information on volume of production and usual practices, such as the use of vaccines, antimicrobials, and hygiene and basic biosecurity measures. The GPS location of each farm was recorded using a mobile app. Environmental samples were collected by taking the following from each farm: approximately 20 g of farm litter; 25 g of poultry feed collected directly from the feed sack in use; and a spatula full of moist poultry feces from the chicken farm. Each sample was collected in a sterile container. Approximately 100 ml of drinking water used for poultry was collected in a sterile screw capped container by direct decanting. Further, a sterile cotton swab moistened with sterile skim milk was dragged along the inner walls of the poultry farm and immersed in vial containing 10 ml of sterile skimmed milk. A similar strategy was used with a sterile cotton swab to sample the wheels, side bars, and bird cages of the vehicle used for transporting birds from the farm to the slaughter facility. All samples were stored on ice and transported to the laboratory within 6 h.Twenty-one slaughter houses in Chitwan district were selected such that they received the birds that were reared at the farms that were included and sampled in this study. The GPS points for each establishment were recorded using smart phone app. In each establishment, a chicken carcass was randomly selected. From each carcass, roughly 50 g of each seven different types of meat samples were collected using a sterile scalpel blade and were aseptically transferred into a sterile container. The sample types were gizzard, liver, heart, intestine, skin, spleen and muscle. These were stored and transported on ice to the laboratory within 6 hours of sample collection.All samples except swabs were pre-enriched in Buffered peptone water  and incubated  at 35 ± 2 °C for 18 h. Swab samples in skimmed milk were directly incubated  at 35 ± 2 °C for 18 h. Samples were selectively enriched by transferring 100 μl of pre-enriched content into 9.9 ml of Rappaport Vassiliadis Broth  and incubated  at 40 ± 2 °C for 18 h. A loop full of content was sub-cultured on Xylose Lysine Deoxycholate (XLD) agar  and incubated  at 35 ± 2 °C for 18 h.Salmonellaenterica serovars. For further confirmation of Salmonella enterica genus, the biochemical IMVic tests were performed for Salmonella enterica like isolated bacterial colonies. In IMViC biochemical tests, the isolates giving indole negative, methyl red positive, voges-proskauer negative, citrate utilization positive, alkaline slant by acidic butt with or without H2S in TSI (triple sugar iron) test, motile and urease negative results were confirmed as Salmonella enterica isolates. The typhoidal Salmonella enterica serovars viz., Salmonella enterica Typhi, Paratyphi A, B and C are highly adapted to humans and have no known animal or environmental reservoirs [Salmonella enterica serovars isolated from poultry and environmental samples in this study were assumed to be non-typhoidal Salmonella enterica serovars (NTS).The inoculated XLD plates were examined for transparent red colonies with black centers, the characteristic feature of servoirs . Thus, aThe antimicrobial susceptibility test was performed by modified Kirby Bauer disk diffusion method for azithromycin (15 μg), tetracycline (35 μg), ciprofloxacin (10 μg) and meropenem (10 μg). The disc diffusion breakpoint interpretation was carried out as per the CLSI  2017 guidelines [Salmonella enterica isolates were sub-cultured on XLD agar plate from the stored glycerol stock. Upon incubation, the plates were first examined for colony purity. Two to three well isolated colonies of Salmonella enterica were randomly selected from an overnight culture and re-suspended in 200 μl of sterile distilled water. Bacterial DNA was extracted using Quick-DNATM Fungal Bacterial Miniprep Kit  following manufacturer instructions. Briefly, the bacterial cells in the suspension were physically disrupted by vigorously vortexing in BashingBead buffer (provided in the kit) followed by centrifugation  at 10,000 x g for 1 min. The disrupted bacterial cells in the lysate were treated with Genome lysis buffer. The released crude bacterial DNA was collected on the Zymo-Spin™ IC Column followed by centrifugation  at 10,000 x g for 1 min. Upon discarding the flow through, the DNA adsorbed on the column was washed twice with 200 μl of DNA Pre-Wash Buffer and 500 μl g-DNA Wash Buffer followed by centrifugation  at 10,000 x g for 1 min. Finally, bacterial DNA was eluted from the column in 20 μl of DNA Elution Buffer and stored at 2–8 °C.The biochemically confirmed A Taq-man chemistry based real time PCR detection of antimicrobial resistance genes was carried out using a commercial kit Qiagen Microbial DNA qPCR Assay . The following antimicrobial resistance genes for specific antimicrobials were tested: QnrS  for fluoroquinolones; OXA-62 , NDM , IMP-1group  and VIM-1group  for beta-lactam antimicrobials; aacC1 for aminoglycosides; tetA  for tetracyclines; ermA  and mefA  for macrolides/lincosamides. A 25 μl of PCR reaction volume for each sample was set up containing 12.5 μl of hotstart qPCR mastermix, 9.5 μl of distilled water, 1 μl of qPCR assay constituting a mixture of AMR gene specific forward primer, reverse primer and specific hydrolysis probe and 2 μl of extracted DNA. In each batch of PCR run, a PCR positive control tube (added with the PCR positive control as provided by the manufacturer) and a no template control (NTC) tube (added with sterile distilled water) were included as internal controls. The PCR reaction for each AMR marker was prepared per sample. The real time detection of AMR genes was carried out in Sa Cycler-96 thermocycler  by using the following thermal-cycle protocol; initial activation at 95 °C/10 min and 40 cycles of denaturation at 95 °C/15 s followed by annealing, extension and detection (in FAM channel) at 60 °C/2 min. The ramp rate was set to 1 °C/second. After the completion of PCR program, the threshold cycle (CT) value for each well was calculated. The baseline and threshold were manually adjusted by normalizing the background signal from the NTC well. Finally, for data analysis, the threshold cycle values for all wells were exported to an Excel spreadsheet for scoring positive or negative results for each AMR marker for all samples.A statistical analysis was carried out using STATA software. The descriptive statistics of qualitative variables were expressed in absolute frequency (N) with percentage (%). That of quantitative variables was calculated in mean ± standard deviation (SD) or median with interquartile range (IQR). For the purposes of analysis in this study, the isolates showing intermediate antibiotic sensitivity were classified as sensitive.Interviews were conducted with the farm owners from each of the 12 poultry farms visited. Broiler operations ranged in size from 70 birds to 1000 birds . All farms used commercial pellet feed and raised birds of the same age in deep litter. At the end of each harvest, 75% (9/12) of the farms disposed of the farm litter through a contractor that is to be used as a fertilizer, while the rest disposed it by themselves in their own farms. Regarding employing biosecurity measures, approximately 42% (5/12) of farmers reported using lime powder around their farm, 16.6% (2/12) failed to maintain any biosecurity measures and remaining 41.6% (5/12) had no knowledge about biosecurity measures. Nearly 84% (10/12) of farmers reported vaccinating their birds with some vaccine. Seventy five percent (9/12) of farmers used antimicrobials, of which eight farms reported of obtaining the antibiotics from the feed supplier. The classes of antimicrobials used prophylactically in farms varied viz., neomycin , colistin , furaltadone , doxycycline , sulpha-trimethoprim  and tylosin .Salmonella enterica culture positive sample. All collected poultry drinking water samples were culture negative. Table Salmonella enterica serovars.Twelve broiler farms were interviewed, but environmental samples could be collected from 11 farms only. A total of 62 environmental samples were collected from the farms, of which 50% (31/62) tested positive for NTS serovars. Almost 67% (8/12) of the farm and vehicle swab samples and 50% (6/12) of faecal samples were culture positive for NTS serovars. With exception of one farm, all farms had at least one In total, 159 poultry tissue samples including skin, muscle, heart, intestine, liver, gizzard, and spleen were collected from 24 chicken carcasses obtained from slaughter houses. Of these, 79% (126/159) were culture positive for NTS serovars. At least 70% of each tissue types were culture positive for NTS serovars; all chicken carcasses had at least four NTS culture positive samples  and environmental samples . The gastrointestinal tract of poultry is one of most common natural niche for several serovars of NTS. The high prevalence of NTS in poultry carcasses indicated possible high level of visceral contamination of the meat products. One study conducted in retail meat shops of Kathmandu reported the detection of Salmonella among 141 retail markets [Similar studies conducted in other low- and middle-income Asian countries have reported a wide range of prevalence of NTS in poultry and environmental samples. Studies in India found a range of 0 to 10% NTS prevalence among poultry meat and tissue , 19. Ano markets .Salmonella isolates from the previous study of retail meat in Chitwan, 85% (n = 23) were multi-drug resistant. The most common antibiotics for Salmonella resistance were ampicillin, nitrofurantoin, and doxycycline. Similar to our findings, the Vietnamese studies detected high percentages of NTS isolates resistant to tetracycline varying from 40% [Among from 40%  to 78% [from 40%  and low from 40% , 19; andfrom 40% .QnrS gene was detected in 16% of samples overall. However, none of the isolates that were resistant to ciprofloxacin had the QnrS marker; the majority specimens with the marker tested sensitive to ciprofloxacin indicating possible alternative genetic mechanism for observed ciprofloxacin resistance. The burden of antimicrobial resistance genes in bacterial isolates may depend on the extent of specific antibiotic agent used in that setting. The prolong use of antibiotics, particularly those of broad spectrum, may exert a selective pressure on the microbial community in the setting, which may facilitate rapid emergence and selection of antimicrobial resistant isolates and increased horizontal transmission of antimicrobial resistance genes. As antibiotics are extensively used in poultry in an unregulated way, mostly in developing countries, AMR among poultry should be closely monitored by the surveillance of AMR genes in poultry isolates.In this study, the Salmonella enterica serotypes based on the use of Salmonella specific sample enrichment method, the colony characteristics on selective media and biochemical characteristics. Further, it was assumed that all Salmonella enterica serovars isolated in this study were non-typhoidal Salmonella serovars based on the fact that the typhoidal Salmonella serovars have only the humans as reservoirs. However, this study cannot guarantee that all Salmonella enterica isolates from samples were NTS. For example, it could not be ruled out if there were any prior human contamination of the samples, such as resulting from the handling of poultry samples by a person infected or colonized with typhoidal Salmonella serovars .One of the limitations of this study is the lack of bacterial serotyping method for specific identification of NTS serotypes. The selected bacterial isolates were considered to be one of the Salmonella in poultry meat samples that were meant for human consumption and in the surrounding where the commercial chicken are raised. Such findings are important to reinforce and educate about biosecurity measures to reduce the burden of Salmonella contamination of animal derived food.However, the main aim of this study was to assess the burden of AMR Salmonella contamination of animal derived foods. Additionally, this study emphasises on the need to conduct a routine surveillance of AMR on potential human pathogens contaminating the human foods of animal origin so as to assess the burden and diversity of AMR genes that may have direct impact on human health. In this study, it was found that the use of antibiotics as prophylactics in poultry was a common finding. Given a high burden of AMR genes among NTS isolates in this study, it also underscores the urgency of proper regulation of antimicrobials in poultry industry to prevent an inappropriate use, particularly of last resort clinical antimicrobials like colistin. Additionally, awareness programs at local farm and community levels on antimicrobial stewardship and impact of poultry linked AMR on human health may be more effective and sustainable to prevent the misuse of antimicrobials in poultry sector.In this study, a high prevalence of antimicrobial resistance NTS was observed in the poultry samples and in the farm environments. Further, a high prevalence of AMR NTS isolates was observed in specimens taken from transporting vehicles which could implicate in an easy spread of resistant NTS beyond farm. Additionally, low levels of knowledge and practice regarding biosecurity amongst farmers found this study could further exacerbate the existing problem of high burden of antimicrobial resistant NTS in poultry industry. The findings from this study thus emphasizes on the need to educate and enforce optimal biosecurity measures at all levels of food supply value chain from the farms, slaughter houses to the retail shops so as to reduce the burden of