Plasmodium berghei sporozoites (syrSPZ) were 3-fold less efficient in migrating to and infecting mouse liver than mosquito-inoculated sporozoites (msqSPZ). This was related to a clustered dermal distribution (2-fold-decreased median distance between syrSPZ and msqSPZ) and, more importantly, a 1.4-fold (significantly)-slower and more erratic movement pattern. These erratic movement patterns were likely caused by alteration of dermal tissue morphology (>15-μm intercellular gaps) due to injection of fluid and may critically decrease sporozoite infectivity. These results suggest that novel microvolume-based administration technologies hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.Malaria vaccine candidates based on live, attenuated sporozoites have led to high levels of protection. However, their efficacy critically depends on the sporozoites’ ability to reach and infect the host liver. Administration via mosquito inoculation is by far the most potent method for inducing immunity but highly impractical. Here, we observed that intradermal syringe-injected IMPORTANCE Malaria still causes a major burden on global health and the economy. The efficacy of live, attenuated malaria sporozoites as vaccine candidates critically depends on their ability to migrate to and infect the host liver. This work sheds light on the effect of different administration routes on sporozoite migration. We show that the delivery of sporozoites via mosquito inoculation is more efficient than syringe injection; however, this route of administration is highly impractical for vaccine purposes. Using confocal microscopy and automated imaging software, we demonstrate that syringe-injected sporozoites do cluster, move more slowly, and display more erratic movement due to alterations in tissue morphology. These findings indicate that microneedle-based engineering solutions hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines. Malaria’s high morbidity and mortality underline the pressing need for an effective vaccine to support control programs. Live, attenuated Plasmodium falciparum sporozoite (PfSPZ)-based vaccine strategies currently are in clinical development  and unsuccessful needle- and syringe-injected sporozoites (syrSPZ) is needed to boost the development of practical and efficacious attenuated sporozoite vaccines.To make live, attenuated sporozoites amenable for large-scale immunization, the U.S.-based biotech firm Sanaria has developed tools to isolate, purify, and cryopreserve sporozoites for injection. Unfortunately, the dermal or subcutaneous injection of sporozoites provided suboptimal protective efficacy . Althougin vitro and in skin  than in syrSPZ mice   . These rmsqSPZ by quantitative real-time reverse transcription-PCR (qRT-PCR) , which was at least partly caused by technical variability inherent to DNA extraction from skin lysis samples. The estimated average number of msqSPZ delivered did not significantly differ from the targeted 3,000 .Attempts to quantify the number of qRT-PCR)  showed hmsqSPZ and syrSPZ sites were imaged over an average total depth of 124 μm  by confocal microscopy in order to visualize the sporozoite distribution. In general, msqSPZ were distributed both individually and in clusters dispersed throughout the dermal tissue . Interestingly we also found hematomas and msqSPZ in the mouse peritoneum . Download Copyright © 2021 de Korne et al.2021de Korne et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00218-21.2FIG S2n = 2 per condition). ***, P < 0.001; Mann-Whitney U test. Download FIG S2, TIF file, 0.2 MB.Sporozoite-to-blood vessel distance. (A and C) Overview of the inoculation site after sporozoite delivery by mosquito (A) and the injection site after sporozoite delivery by intradermal syringe injection (C) shown as an overlay of a bright-field image, the sporozoite distribution , and the segmented blood vessels (depicted in yellow). (B) Quantification of the sporozoite-to-blood vessel distance .In contrast, dermal sites containing ion site . Zoomingd vessel   and with 5% of the msqSPZ further than 376 μm apart , with 5% of the syrSPZ further than 112 μm apart.The sporozoite distribution was quantified according to their nearest-neighbor distance (NND), confirming the dispersed nature of μm apart . In contP < 0.001, independent sample t test; Feret’s diameter, 68 ± 10 μm; P < 0.001, independent sample t test)   and B. C t test)  and B. Tpatterns .msqSPZ and 1,079 syrSPZ could be captured and analyzed. Both msqSPZ and syrSPZ were equally motile .After delivery, the majority of sporozoites displayed tortuous movement through the dermal tissue; representative examples are shown in msqSPZ and syrSPZ showed equal numbers of turns and straight paths, with the percentage of turns at frame level at 88% for both samples  . Turns were made both clockwise (CW) and counterclockwise (CCW), with a slight preference for CCW in the msqSPZ group  . In conclusion, both msqSPZ and syrSPZ traveled highly tortuous paths, although msqSPZ exhibited less-sharp turns, a more consistent turn angle, and a predominance for the well-described preferred CCW turn angle.Following both administration routes, the tracks of the sporozoites were highly curved. Traditional motility measures, such as the mean squared displacement, were thus unsuitable to accurately describe the migration behavior of both groups of sporozoites . Therefo samples , panel ire test) , panel i10.1128/mSphere.00218-21.3FIG S3FIG S3, TIF file, 0.04 MB.Mean squared displacement. (A and B) The mean squared displacement of the sporozoites after administration by mosquito inoculation or syringe injection is plotted. (A) All tracks are included. (B) The circular tracks are excluded . The mean squared displacement of the individual samples is plotted as a dotted line; a linear trendline is plotted as a solid line. Download Copyright © 2021 de Korne et al.2021de Korne et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the msqSPZ and syrSPZ, with a mean velocity of, respectively, 1.0 ± 0.4 and 0.9 ± 0.2 μm/s. The second peak, containing the highly viable and rapid msqSPZ and syrSPZ, differed between the administration routes at a mean of 2.4 ± 0.7 μm/s for msqSPZ and a mean of 1.7 ± 0.6 μm/s for syrSPZ. Thus, the rapid msqSPZ moved, on average, 1.4-fold faster than the syrSPZ.Sporozoite velocity fluctuated along tracks  and angular dispersion (AD), the sporozoites’ tracks could be divided into four typical movement patterns: short erratic tracks , short straight tracks , consistently turning tracks , and erratically turning tracks  . Represein vitro .P < 0.001, Mann-Whitney U test). Interestingly, these consistently turning rapid sporozoites were overrepresented within the msqSPZ group compared to in the syrSPZ group (2.4-fold difference), which was offset by a reciprocal increase in slower erratically turning sporozoites within the syrSPZ group (2.5-fold difference)  , whereas erratically turning sporozoites were considerably slower  .msqSPZ was larger than that of syrSPZ, as was observed by the analysis of the individual parameters , which suggested that the movement pattern or velocity of sporozoites was not dependent on their interindividual distance.Lastly, we determined whether the distribution of the sporozoites as reflected by the NND influenced the tortuosity and velocity of their tracks. In general, the average NND of rameters ; Fig. S410.1128/mSphere.00218-21.4FIG S4msqSPZ is depicted in the darker color, and the NND of the syrSPZ in depicted in the lighter color. Download FIG S4, TIF file, 0.04 MB.Relation between motility parameters and nearest-neighbor distances (NNDs). The NNDs is plotted for the tracks split into 12 subsets (S1 to S12) based on four specific movement patterns (Q1 to Q4)  and threCopyright © 2021 de Korne et al.2021de Korne et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the syrSPZ group contained more sporozoites that exhibited erratic movement at a slower speed, whereas the msqSPZ group contained more sporozoites that circled consistently at high speed.Taken together, the syrSPZ through the skin, with larger distances to blood vessels; (ii) a lack of hematomas, which are typically induced by mosquito bites; (iii) enlarged interstitial space due to syringe injection of fluid; and (iv) slower and more erratic migration patterns of syrSPZ than those of msqSPZ. Each of these parameters may impact the efficiency of sporozoite migration, blood vessel invasion, and ultimate liver infectivity and thus provide important insights into how to critically improve the delivery of sporozoite-based vaccines.Using confocal microscopy and dedicated sporozoite imaging software, we visualized sporozoites deposited by mosquito inoculation or syringe injection and assessed quantitative differences. We found that delivery by syringe injection decreases the infectivity of sporozoites by roughly 3-fold compared to mosquito inoculation, which is related to (i) a clustered distribution of msqSPZ and the fact that a substantial proportion of msqSPZ are deposited close to hematomas created by mosquito probing  tattoo device or nanoliter injector may be useful not only to create the relevant sporozoite dispersion but also to decrease injection volume to the nanoliter range , as undo 2.0 mm ), the reer range –29. In a the visualization of the sporozoite distribution throughout the inoculation and injection site (up to 100 mm2), whereas until now only one field of view (<0.5 mm2) was visualized during in vivo live imaging  and the effect of saliva inoculated by mosquitoes  techniques can provide valuable quantitative assessments of parameters affecting sporozoite migration. Our  imaging , 16, 32; imaging –35 but nimaging ––17. Furtsquitoes , 36, 37.In conclusion, detailed microscopic imaging of the dermal site appearance and migration patterns of sporozoites revealed important quantitative differences between sporozoite administration via mosquito inoculation and intradermal syringe injection. These findings open new avenues for intradermal delivery of attenuated sporozoite vaccines with enhanced efficacy.Experiments on Animals Act . All animal experiments were granted a license by the competent authority after advice on the ethical evaluation was received from the Animal Experiments Committee Leiden (protocol AVD1160020173304). All experiments were performed in accordance with the mals Act , the appmals Act  regardinPlasmodium berghei as described previously  was used. The infected mice were anesthetized, and Anopheles stephensi female mosquitoes were infected by allowing them to feed on gametocytemic mice, as described previously  msqSPZ were delivered by shaving the abdomen of the mice; 1 cm2 of skin was exposed for 15 min to around 30 infected mosquitoes (exact numbers are specified per experiment). Blood-fed mosquitoes were counted, and the presence of sporozoites in their salivary glands was confirmed using a quantitative analysis of luciferase activity after placing of the mosquitoes in a 20-μl drop of d-luciferin . (ii) syrSPZ were obtained by manual dissection of the salivary glands of infected female Anopheles stephensi mosquitoes 20 to 24 days postinfection. The salivary glands were collected and homogenized to release sporozoites in Roswell Park Memorial Institute medium  enriched with 10% fetal bovine serum (Life Technologies Inc.). The free sporozoites were counted in a Bürker counting chamber using phase-contrast microscopy to prepare the injection samples. Directly after counting of the sporozoites , a 10-μl sample containing 3,000 sporozoites was administered by injection into the abdominal skin (within the same region as defined for exposure to mosquitoes) using an insulin syringe . The number of sporozoites delivered by exposure to 30 infected mosquitoes was considered consistent with intradermal delivery of 3,000 sporozoites via syringe injection based on data in the available literature .P. berghei infection in 11 mice  was visualized, and sporozoites were quantified by measuring the luciferase activity in the liver at 44 h after the challenge with sporozoites using the IVIS Lumina II imaging system (Perkin Elmer Life Sciences). Before being imaged, the mice were shaved and anesthetized. IVIS measurements  were performed 8 min after subcutaneous injection of d-luciferin in the neck . The liver load was quantified by measuring the total flux (photons per second) of a region of interest (ROI) covering the liver . Image analysis was performed using the Living Image 4.4 software (PerkinElmer Life Sciences). Infected mice were monitored for blood-stage infections with a Giemsa-stained blood smear until day 9 postinfection. The prepatent period  was defined as the first day at which blood-stage infection with a parasitemia of >0.5% was observed.The liver stage of the 2 (25 mM), Plasmodium-specific forward and reverse primers , PhHV-specific  forward and reverse primers , a Plasmodium-specific FAM10-labeled detection probe , a PhHV-specific Cy5 double-labeled detection probe , and 5 μl of the DNA sample . Amplification consisted of 15 min at 95°C, followed by 50 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Amplification, detection, and analysis were performed with the CFX96TM real-time PCR detection system (Bio-Rad). A calibration curve to assess the sporozoite numbers in the mosquito inoculation samples was generated by analyzing skin samples injected with a dilution range of sporozoites   mosquito bites, of which 18  contained blood, the skin of 4 exposed mice was cut out, snap-frozen, and stored at −20°C until further use. Parasite burden was measured by quantitative real-time reverse transcription-PCRs (qRT-PCRs). The DNA was extracted from the frozen skin using the QIAamp DNA microkit (Qiagen) by following the manufacturer’s instruction. Amplification reactions of each DNA sample were performed in PCR plates  in a volume of 25 μl containing 12.5 μl PCR buffer , 0.5 μl MgClplicate) .msqSPZ and 4 administered syrSPZ) was excised, covered with a cover slip, and imaged using an Andor Dragonfly 500 spinning disk confocal lens on a Leica DMi8 microscope (Oxford Instruments) or a Leica true confocal scanning SP8 microscope (Leica Microsystems). The mCherry expressed by sporozoites was excited with the 561-nm laser. A 20× objective (HC PL APO 20×/0.75 IMM CORR CS2) was used, resulting in images of 617 by 617 μm. The experiments were performed within 1.5 h after tissue excision at room temperature.Immediately after sporozoite delivery, the exposed skin of 8 mice  and was converted into a binary image showing only the sporozoites. This image was further processed in two different ways: (i) the Gaussian blur filter was applied, and a pseudocolor image was created by applying the inferno-color lookup table in Fiji to visualize the location and density of the sporozoites; and (ii) the coordinates of the individual sporozoites in the msqSPZ and syrSPZ samples were determined, and the nearest-neighbor distance (the distance between the center points of neighboring sporozoites) was calculated using the ImageJ Nnd plugin. The blood vessels visible at the bright-field overview image of the dermal site were segmented using the Image Segmenter app available within MATLAB (MathWorks), and the distance from the sporozoite center to the nearest blood vessel was calculated. The number of sporozoites residing within or in close proximity to hematomas was determined using circular ROIs with a diameter of 650 μm and around the center of the hematoma. The shape of the cells visible at the zoom-in bright-field images  was described by two shape descriptors available within ImageJ, namely, roundness (4 × [area]/π × [major axis]2), with a value of 1.0 indicating a perfect circle, and Feret’s diameter .To create overview images of the dermal site, up to 570 fields of view were stitched using the Andor imaging software Fusion (Oxford Instruments). Z-slices covering an average total depth of 124 μm  were imaged. Using the Fiji package for the open-source software ImageJ , the thrmsqSPZ samples (n = 6) yielded a total of 3,400 frames. Recording syrSPZ samples (n = 8) yielded a total of 3,600 frames. Maximum-intensity projections of the recorded microscopy movies were generated using ImageJ. The motility of the sporozoites was analyzed using SMOOTmouse skin, an in-house-developed software program written in the MATLAB programming environment (MathWorks). This tool is an adapted version of the SMOOThuman skin , straightness index (<0.5 and >0.5), and velocity .First, the sporozoite tracks were characterized as motile or stationary based on their displacement, using a displacement cutoff of 21 pixels, which corresponds to the length of a sporozoite. Subsequently, the tracks of the motile sporozoites were subdivided into defined movement patterns: sharp turn, slight turn, and linear segments. Third, for the motile sporozoites, the mean squared displacement at frame level, the average velocity of their tracks, and the nearest-neighbor distance per track were calculated, and the tortuosity of the tracks was described via the straightness index  and the angular dispersion (the deviation from the mean angle of the track). Finally, the interplay between the angular dispersion, straightness index, velocity, and nearest-neighbor distance was analyzed, for which the numbers of t test and the Mann-Whitney U test. For the comparison of groups with a set value, the one-sample t test was used. For the comparison of the distribution of categorical data, the chi-square test was used, including a post hoc analysis based on residuals. A univariate general linear model was used to examine the relationship between a continuous variable and a categorical variable. P values of <0.05 were considered significant; in a case of multiple tests, the Bonferroni correction was applied to adjust the P value. All statistical tests were performed by SPSS Statistics (IBM Nederland BV). To compare the velocity distributions of both groups, the distribution was described by performing expectation-maximization-based fitting. The probability density function that could describe the sporozoite velocity distribution consisted of a mixture of 2 normal distributions. The package mixR (The average and variability of the data were summarized using the mean and standard deviation (SD) for parametric data or the median and IQR for nonparametric data. For the comparison of groups, the difference between means or medians was assessed using, respectively, the independent sample age mixR  within tage mixR  was used


Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer, with high incidence and mortality. To improve the curative effect and prolong the survival of patients, it is necessary to find new biomarkers to accurately predict the prognosis of patients and explore new strategy to treat high-risk LUAD.A comprehensive genome-wide profiling analysis was conducted using a retrospective pool of LUAD patient data from the previous datasets of Gene Expression Omnibus (GEO) including GSE18842, GSE19188, GSE40791 and GSE50081 and The Cancer Genome Atlas (TCGA). Differential gene analysis and Cox proportional hazard model were used to identify differentially expressed genes with survival significance as candidate prognostic genes. The Kaplan–Meier with log-rank test was used to assess survival difference. A risk score model was developed and validated using TCGA-LUAD and GSE50081. Additionally, The Connectivity Map (CMAP) was used to predict drugs for the treatment of LUAD. The anti-cancer effect and mechanism of its candidate drugs were studied in LUAD cell lines.We identified a 5-gene signature . Risk Score (RS) based on 5-gene signature was significantly associated with overall survival (OS). Nomogram combining RS with clinical pathology parameters could potently predict the prognosis of patients with LUAD. Moreover, gliclazide was identified as a candidate drug for the treatment of high-RS LUAD. Finally, gliclazide was shown to induce cell cycle arrest and apoptosis in LUAD cells possibly by targeting CCNB1, CCNB2, CDK1 and AURKA.This study identified a 5-gene signature that can predict the prognosis of patients with LUAD, and Gliclazide as a potential therapeutic drug for LUAD. It provides a new direction for the prognosis and treatment of patients with LUAD. Lung cancer is the most common malignant tumor with the highest morbidity and mortality worldwide, including China . Non-smaThe biological characteristics of LUAD including poor differentiation, high malignancy and more aggressiveness, are known to associate with unfavorable prognosis. However, prognostic prediction solely based on these pathological characteristics is of limited efficiency and accuracy , 8. The Moreover, due to genetic heterogeneity, appropriate treatment for patients with different genetic characteristics is also important for the prolongation of prognosis. However, most patients lose effective treatment because of primary or secondary drug resistance . Therefoin vitro for the first time. Therefore, our research provides a new strategy for the prognosis and treatment of lung adenocarcinoma.In this study, we combined multiple gene expression data sets to develop and verify a five-gene signature risk model, which can accurately evaluate individual prognosis in patients with LUAD. In addition, we combined the survival-related differentially expressed genes (DEGs) of prognostic significance with CMAP to find and verify gliclazide as a potential drug therapy for lung cancer http://ualcan.path.uab.edu/cgi-bin/ualcan-res.pl) on the 9th December, 2019. LUAD-related datasets GSE18842, GSE19188, GSE40791 and GSE50081, downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), were used as validation sets. For data cleaning, samples with missing clinical data were excluded.The mRNA expression profiles and clinical data  of LUAD were downloaded from TCGA database  was accomplished by using dplyr package in R . The sva1  ×  β1 + expression of gene2  ×  β2 +⋯+ expression of genen  ×  βn(RS) = expression of gene  provides a wealth of information about small molecular drugs, gene expression and diseases that are closely interrelated at the genomic level. Therefore, researchers can link gene expression data to disease-related drugs. In order to find new drug candidates for LUAD patients, the DEGs related to survival were uploaded to CMAP, and a corresponding analysis result could be obtained after passed the Kolmogorov–Smirnov test. The drugs with a negative score indicating their potential of anti-tumor effect were selected as the new target drug candidates for LUAD patients with high-RS.CMAPhttps://pubchem.ncbi.nlm.nih.gov/). The 3D coordinates of CCNB1  and CDK1  and AURKA  were downloaded from the PDB (http://www.rcsb.org/pdb/home/home.do).The CCNB2 of amino acids sequences were analyzed by EXpasy (http://swissmodel.expasy.org/) for lacking of its complete crystal structure. Ramachandran plots were used to assess stereo-chemical quality with the default parameters. Auto dock Vina 1.1.2 (http://autodock.scripps.edu/) and Pymol software 2.3  were used for molecular docking studies and model visualization, respectively  was dissolved with dimethyl sulfoxide (DMSO) to a 500 mM storage concentration and stored at −20°C. The concentration of DMSO was kept less than 0.4% v/v throughout each experiment. Two human LUAD cell lines A549 and H1299 were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% COCells with density of 3,000 cells/well in 96-well plates were treated with various concentrations of Gliclazide  for 24, 48 and 72 h, and then the cell-viability assay  was carried out according to the reagent’s instructions.A549 or H1299 cells (400 cells/well) were seeded into 12-well plate. Next day, cells were treated with various concentrations  of Gliclazide and continuously incubated for 10–14 days. After cells were stained with crystal violet, colonies were counted using ImageJ software .A549 or H1299 cells were treated with different concentrations of Gliclazide  for 48 h. Then, cells were harvested and fixed with 70% (v/v) cold ethanol at 4°C overnight. After 30 min-incubation with 100 μg/ml RNase A and 10 μg/ml propidium iodide (PI) staining solution in dark, cells were analyzed by FACScan flow cytometer  and evaluated using the ModFit program software.A549 or H1299 cells were treated with different concentrations of Gliclazide  for 72 h. Then, total protein was extracted for western blot as our previous studies . The fol2 test was used to evaluate the correlation of RS with clinical characteristics. Univariate and multivariate Cox proportional hazard regression analysis was also performed to access the relationship between RS and OS. Based on the multivariate Cox analysis, a nomogram was constructed with the “rms” package in R (http://www.R-project.org), SPSS 16.0 and Graph Pad Prism 7 software.Kaplan–Meier curves were drawn and the significant difference was checked by log-rank test. The Receiver Operating Characteristic (ROC) analysis was used to detect the sensitivity and specificity of the risk score in predicting survival. The area under the ROC curve (AUC) was used to evaluate the efficiency of prognosis , 24. X2 age in R , 25. A PThe flow chart of this study was shown in =(0.3013 × KIF20Aexp) + (−0.1823 × LIFRexp) + (−1.100 × RGS13exp) + (0.2402 × KLF4exp) + (0.0859 × KRT6Aexp)  of each sample in TCGA-LUAD according to the expression levels of five genes weighted by their relative coefficient using the following formula: RS To further verify the prognostic predictive value of 5-gene signature, we selected GSE50081 dataset including 128 LUAD patients as the validation set Table S2To further confirm the important role of 5-gene signature in LUAD, we analyzed the correlation of RS with clinicopathological parameters. The heatmap in To identify the significant changes of biological pathways between high- and low-RS groups, the GSEA was performed. Based on the cut-off criteria of FDR <0.25 and P <0.05, 18 significantly altered pathways were selected, including “cell cycle”, “spliceosome”, “DNA replication”, “mismatch repair” and other pathways Table S4−0.4. The analyses screened out eight drug candidates, which were not reported to play anti-LUAD effect before . Similar results were obtained by colony formation assay . Hence, these findings supported an anti-cancer effect of gliclazide by inhibiting the proliferation of LUAD cells.To experimentally validate the therapeutic efficacy of gliclazide, we assessed the effect of gliclazide on LUAD cell lines, A549 and H1299. Gliclazide treatment significantly inhibited the growth of two LUAD cells in a dose- and time-dependent manner (http://www.swisstargetprediction.ch/) to predict the key targets of gliclazide. The analyses screened out four potential target candidates including CCNB1, CCNB2, CDK1 and AURKA (https://swissmodel.expasy.org/interactive/). Ramachandran diagram and other analysis showed that the constructed CCNB2 structure is accurate and reasonable and can be used for the next step of molecular docking . Next, the binding poses and interactions of 4 drug targets with gliclazide were obtained with Auto dock Vina v.1.1.2 and the binding energy for each interaction was generated with no obstacles. The results showed that gliclazide bound to CCNB1, CCNB2, CDK1 and AURKA through visible hydrogen bonds and strong electrostatic interactions, and the hydrophobic pocket of each target was occupied successfully by gliclazide with the low binding energy of −8.9, −8.6, −8.3 and −6.9 kcal/mol, respectively  is involved in spindle formation during cell division , and KerAlthough much progress, such as target therapy and immunotherapy, has been made in lung cancer treatment, only part of patients could benefit from it . Therefoin vitro. Although the p53 status of two cells are different that wild type p53 in A549 but mutant p53 in H1299, we found that no matter the p53 status is wild type or mutant, gliclazide could induce G2M phase arrest and apoptosis in both of cell lines. Interestingly, the function of 5-gene signature is closely related to biological processes such as “Cell Cycle” and “DNA Replication”. These findings highlight the tight association of LUAD DEGs including the 5-gene signature with the processes of cell cycle and support the possible targeting of cell cycle regulate risk by gliclazide for the inhibition of high-risk LUAD patients. In our analysis, we found that the prognosis of patients with high-RS and low-RS was distinctly different, which suggested that active treatments are required for Stage I patients with high-RS rather than Stage II patients with low-RS. Based on these results, gliclazide has a great and promising therapeutic potential for NSCLC patients with high-RS.At present, EGFR-tyrosine kinase receptor inhibitor (EGFR-TKI), a widely used drug in the clinical targeted therapy, had significantly improved the prognosis of NSCLC patients. However, only 20% of them with EGFR mutant can be benefit from it and therapies for patients without EGFR mutant (EGFR wild type) are still limited. It is necessary to develop new therapies for patients with EGFR wild type. Our study also focused on anti-cancer role of Gliclazide, thus H1299 and A549lung cancer cell lines, both of which are with wild type EGFR, were selected for experimental Of note, relatively high dosage of gliclazide was required to inhibit LUAD cells, suggesting that its limitation for clinical application as a single drug. In fact, even for the anti-cancer drugs and PD-L1/PD-1 inhibitors, the effect of single drug on cancer therapy is not good enough. Two- or three-drugs combination is the common therapeutic regimens, but only part of tumor patients including lung cancer could benefit from them. Therefore, the combination of gliclazide and chemotherapy or targeted therapy, but not gliclazide alone, is more likely to achieve clinical application. On the other hand, drug modification combined with high-throughput screening is also a potential method for searching more efficient gliclazide derivatives for LUAD therapy . In thisIn summary, we identified 5-gene signature, constructed a risk score, and established nomogram based on 5-gene signature exhibiting powerful prognostic prediction effect for LUAD patients. For patients with high-RS, we found that gliclazide might be a promising anti-cancer drug by targeting cell cycle. The findings of this study provided an important reference for the prognosis and treatment of LUAD in terms of molecular biology and methodology, and thus are of great significance.The original contributions presented in the study are included in the article/YCheng and XC conducted bioinformatics analysis and wrote the main manuscript. XC and XHu designed the entire project and supervised all experiments. YCheng and KH conducted all the experiments and analyzed the data. YW, YChen, JQ, BY, XZ, ST, DS, XH, XW, and YL provided bioinformatics analysis technology, experimental technology and writing support. All authors contributed to the article and approved the submitted version.Funding was provided by the National Natural Science Foundation of China , the Key Research and Development Program of Liaoning Province (2018225060) and the Technological Special Project of Liaoning Province of China (2019020176-JH1/103).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.


Buchnera aphidicola and secondary symbiont Serratia symbiotica dominated the microbiota of Greenideinae. We simultaneously explored the relative contribution of host identity , geography and host plant to the structures of bacterial, symbiont and secondary symbiont communities. Ordination analyses and statistical tests highlighted the strongest impact of aphid species on the microbial flora in Greenideinae. Furthermore, we found a phylosymbiosis pattern in natural Greenideinae populations, in which the aphid phylogeny was positively correlated with microbial community dissimilarities. These findings will advance our knowledge of host-associated microbiota assembly across both host phylogenetic and ecological contexts.Aphids and their symbionts represent an outstanding model for studies of insect–symbiont interactions. The aphid microbiota can be shaped by aphid species, geography and host plants. However, the relative importance of phylogenetic and ecological factors in shaping microbial community structures is not well understood. Using Illumina sequencing of the V3–V4 hypervariable region of the 16S rRNA gene, we characterized the microbial compositions of 215 aphid colonies representing 53 species of the aphid subfamily Greenideinae from different regions and plants in China, Nepal, and Vietnam. The primary endosymbiont  Buchnera aphidicola within specialized bacteriocytes to compensate for the nutritional deficiency in their diet . Greenideinae is monoecious, with a holocyclic or anholocyclic life cycle. Host plants of Schoutedeniini species belong primarily to Euphorbiaceae  and secondary symbiont communities. To further understand the ecological and evolutionary processes of microbiota assemblages, we explored the presence of a phylosymbiotic pattern in Greenideinae.Based on high-throughput 16S rRNA gene sequencing, we aimed to reveal the microbial community composition associated with Greenideinae. We fully assessed the contribution of host identity , geographic distribution and host plant on bacterial, symbiont . The negative control contained an equal amount of sterile water instead of aphid DNA. To identify aphid species and eliminate parasitized samples, DNA extracts were verified using National Center for Biotechnology Information (NCBI) BLAST searches of the barcode sequence targeting the cytochrome c oxidase subunit I (COI) gene. The DNA samples were kept at −20°C.The V3–V4 hypervariable region of the 16S rRNA gene was amplified using the primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). A negative control was set up for the polymerase chain reaction (PCR), and all amplifications were conducted in triplicate. The 30-μL PCR was prepared with 15 μL of Phusion High-Fidelity PCR Master Mix , 3 μL primers and 10 ng of template DNA. The PCR thermal program was as follows: 1 min at 98°C for initial denaturation; 30 cycles of 10 s at 98°C for denaturing, 30 s at 50°C for annealing and 30 s at 72°C for elongation; and 5 min at 72°C for final extension.Amplification products were recovered using 2% agarose gel electrophoresis, and positive samples with a bright band of 400–450 bp were purified with the GeneJET Gel Extraction Kit . Sequencing libraries were constructed with the NEBNext Ultra DNA Library Prep Kit  and quantified by a Qubit 2.0 fluorometer  and an Agilent Bioanalyzer 2100 system. Finally, the libraries were sequenced on an Illumina HiSeq 2500 PE250 platform .Paired-end reads were assembled with a minimum overlap size of 10 bp and an error rate of 10% using FLASH v1.2.7 . Low-quaBuchnera and secondary symbionts)   and secombionts) , of aphige vegan . The disge vegan  using iTge vegan . We impldiversity function of vegan. Considering the non-normal distribution  of alpha diversity data, we used non-parametric Kruskal–Wallis tests to investigate the microbiota variation among groups. To simultaneously estimate the separate contributions of aphid genus (or aphid species), geography and host plant to microbial alpha diversity, three-way analysis of variance (ANOVA) was performed using the avop function in the lmPerm package  and unweighted UniFrac distance (GUniFrac function of GUniFrac)  was implemented with Bray–Curtis distance, as unweighted UniFrac distance was not suitable for this analysis. Then, the statistical significance of the microbiota variation between groups was determined by permutational multivariate analysis of variance (PERMANOVA) and analysis of similarities (ANOSIM). PERMANOVA and ANOSIM were conducted on Bray–Curtis and unweighted UniFrac distances using the adonis and anosim functions, respectively, with 10,000 permutations in vegan.The Bray–Curtis distance (UniFrac)  were calmantel function) and Procrustes analysis (procrustes and protest functions) were performed with all samples using the matrices of aphid cophenetic distances and beta diversity (Bray–Curtis and unweighted UniFrac distances) in vegan. Cophenetic distances calculate the pairwise distances between the pairs of tips from a phylogenetic tree using its branch lengths. We used the cophenetic function in the package ape , S. symbiotica (14.97%), Wolbachia (2.10%), Arsenophonus (1.80%), and Rickettsia (1.34%) were greater than 1%  were obtained. The 16S rRNA gene sequences were clustered into 162 OTUs and annotated to 35 genera, 24 families, 16 orders, 12 classes, and 6 phyla of bacteria. At the phylum level, the microbial community of Greenideinae was composed mainly of Proteobacteria, with an average relative abundance of 98.74%. Gammaproteobacteria (95.15%) and Enterobacteriales (94.72%) were the most commonly classified class and order, respectively. The most abundant family was Enterobacteriaceae (94.42%), followed by Anaplasmataceae (2.10%) and Rickettsiaceae (1.34%). At the genus level, seven symbionts were detected, among which the relative abundances of  than 1% .Buchnera was present in all the samples and predominated in most. In addition, each examined sample was simultaneously infected with 2–6 secondary symbionts. S. symbiotica and Wolbachia were the most common, with an infection frequency of 215/215, followed by Arsenophonus (171/215), Rickettsia (96/215), H. defensa (88/215), and F. symbiotica (86/215). S. symbiotica was the most abundant secondary symbiont in Greenideinae, and its relative abundance was even higher than that of Buchnera in the aphid genus Schoutedenia. The infection pattern of secondary symbionts within Greenideinae was variable among different aphid genera and species  (The primary endosymbiont  species . The mos(50/215) .Buchnera   were quite low in most samples  . The pre samples . For exaP < 0.05 for both Shannon and Simpson indices). A significant effect of geography on bacterial and symbiont communities was detected , except that there was no significant effect on the Simpson index upon using all samples (P = 0.057–0.066). However, the secondary symbiont communities did not differ significantly among geographic regions (P = 0.276–0.538).Kruskal–Wallis tests of alpha diversity revealed a significant variation in the bacterial, symbiont and secondary symbiont communities among the aphid genera, aphid species and host plants  = 2.667–4.292, P ≤ 0.013; n ≥ 3, F = 5.333–7.833, P < 0.001] using all types of data in three-way ANOVA, whereas the effect of the host plant was not significant (P = 0.127–0.636). A significant effect of geography was found only on bacterial and symbiont communities with sample size ≥ 3 compared with the effect of aphid genus and host plant . The results of three-way ANOVA also showed that the bacterial and symbiont communities were primarily structured by aphid species . Aphid species, geography and host plant did not have a significant impact on the secondary symbiont community (P = 0.174–0.924).Three-way ANOVA for alpha diversity indices highlighted the contribution of aphid identity  to shaping the microbial community structures when the effect of different factors was estimated simultaneously. We found that the microbial communities differed significantly among aphid genera [P = 0.001). Aphid species (33.4–62.3% of variance) explained more of the overall variance in the data than aphid genus (9.19–32% of variance), geographic region (13–18.1% of variance) and host plant (12.7–26.9% of variance) in all the analyses.Unconstrained NMDS plots based on all types of beta diversity matrices did not show meaningful clustering of samples structured by aphid genus, aphid species, geographic region or host plant –7. ConveR2 = 0.408–0.725, P < 0.001) and ANOSIM  corroborated the greatest effect of aphid species on structuring the microbial communities based on Bray–Curtis and unweighted UniFrac distances with sample size ≥ 3, although no statistical significance among aphid species  or aphid genera  was observed in most cases based on Bray–Curtis distances using all samples (P ≤ 0.006) and host plant (P ≤ 0.047) were revealed by PERMANOVA using all types of beta diversity data. However, their contributions  were usually limited compared to that of aphid species (R2 = 0.262–0.725). ANOSIM suggested significant dissimilarities in microbial communities among geographic regions using all samples . A significant effect of the host plant was found only in analyses of the Bray–Curtis distance .PERMANOVA ( samples . The sigP < 0.001). The results of Procrustes analysis were consistent, which showed that the microbial community structures were significantly related to aphid phylogeny .Using the Mantel test with all types of beta diversity data, we detected a positively significant correlation between aphid phylogeny and the microbial profiles of bacterial, symbiont and secondary symbiont communities  were not frequent symbiotic partners, and their relative abundances were quite low in most samples. Aphid species in Greenideinae usually move rapidly and have long siphunculi that enable them to efficiently emit alarm pheromones  can be found below: GenBank under accession numbers GQ and LJ designed the project. GQ, JC, and LJ identified voucher specimens. MQ conducted molecular experiments and all analyses and wrote the manuscript. JC assisted data analyses. All the authors contributed to revisions.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Salmonella are among the most common causes of human diseases worldwide, and infections with the serovar Infantis are becoming increasingly important. So far, diverse phenotypes and genotypes of S. Infantis have been reported. Therefore, the present study aimed to investigate the infection dynamics of two different S. Infantis strains in broilers. For this purpose, 15 birds were infected on day 2 of life with 108 CFU/ml of a pESI+ or a pESI– S. Infantis strain, respectively. Ten uninfected birds served as in-contact birds to monitor transmission. In both groups, an increase of infection was observed from 7 days of age onwards, reaching its peak at 28 days. However, the pESI+ strain proved significantly more virulent being re-isolated from most cloacal swabs and organs by direct plating. In contrast, the pESI– strain could be re-isolated from cloacal swabs and caeca only when enrichment was applied. Although the excretion of this strain was limited, the transmission level to in-contact birds was similar to the pESI+ strain. Differences in infection dynamics were also reflected in the antibody response: whereas the pESI+ strain provoked a significant increase in antibodies, antibody levels following infection with the pESI– strain remained in the range of negative control birds. The actual findings provide for the first time evidence of S. Infantis strain-specific infectivity in broilers and confirm previous observations in the field regarding differences in persistence on farms and resistance against disinfectants.Food-borne infections with  S. Infantis is actually reported as the most common isolated serovar from animal and food sources, with the majority of strains originating from broilers [S. Infantis is heterogeneous and consists of different clones and clusters [S. Infantis strains were frequently found to be associated with conjugative pESI-like megaplasmid are of global concern for the poultry industry as well as human medicine [S. Infantis strains with difficulties of elimination from farms or slaughterhouses, despite extensive cleaning and disinfection [S. Infantis strains which persist on farms were more resistant to disinfectants [Worldwide, broilers . In consbroilers , 7–9. Inclusters . These dclusters , 13, 14.medicine . An incrnfection . In thisfectants .In vivo infection experiments in mice demonstrated a higher pathogenicity and more inflammatory reactions by S. Infantis strains with multidrug resistance (MDR) [S. Infantis strain [ce (MDR) . So far,s strain . By thisS. Infantis strains in commercial broilers. For this purpose, the bacterial load in organs and faecal shedding were determined after infection altogether completed by using in-contact birds to unravel infection dynamics.Therefore, the present study focused on the colonization behaviour and antibody response of two different S. Infantis field strains, MRS-16/01939 and MRS-17/00712 derived from Austrian broiler flocks, were used. Whereas strain MRS-16/01939 proved multidrug resistant and showed growth of black colonies on xylose–lysine–deoxycholate agar , strain MRS-17/00712 was pan-susceptible presenting colourless colonies on XLD. Furthermore, strain MRS-16/01939 was reported to persist on the farm leading to recurrent infections, whereas strain MRS-17/00712 was only isolated once from the farm.Two phenotypically different S. enterica  integrated in the SeqSphere+ Software version 7.2  [Genomic DNA was isolated from overnight cultures using the MagAttract HMW DNA Kit . Paired-end sequencing was performed on a MiSeq platform . Library preparation was carried out using Nextera XT according to the instructions of the manufacturer . Raw reads were quality controlled using FastQC v0.11.9. For assembly, raw reads were de novo assembled using SPAdes version 3.11.1  . Whole-gGermany) .S. Infantis strains were first analysed through the Salmonella In Silico Typing Resource (SISTR) online tool [S. enterica serovar Infantis were downloaded from NCBI (08-04-2021) and employed for tree construction using parsnp (default parameters – correcting for recombination). Screening for the presence of pESI-like megaplasmid structures was performed by aligning the pESI plasmid from strain 119944 (accession: CP047882.1) to the assemblies of MRS-16/01939 and MRS-17/00712 using the module Whole Genome Alignment from CLC Genomics Workbench version 21.0.3 [For phylogenetic analysis, both ine tool , to deten 21.0.3 .The animal trial was approved by the institutional ethics committee and the national authority according to section 8ff of the Law for Animal Experiments, Tierversuchsgesetz (license number GZ.: 68.205/0157-V/3b/2019).™, Heartland Animal Health Inc. Fair Play, MO, USA) and each group was housed separately in isolators . Feed and water were provided ad libitum.A total of 75 one-day-old ROSS 308 broilers  were divided into 3 groups with 25 birds each. Birds were subcutaneously marked with an individual number tag  at 37°C for 24 h. Selected fresh colonies were inoculated in Luria–Bertani broth  at 37°C overnight with agitation (250 rpm). The concentrations of bacteria in the inoculums were determined by colony-forming unit (CFU) counts on MacConkey agar in serial dilutions (1:10) in duplicate. The bacterial suspensions were washed and re-suspended in phosphate-buffered saline . The inoculation dose was 6 × 108 CFU/ml for MRS-16/01939 and 3 × 108 CFU/ml for MRS-17/00712.S. Infantis strain MRS-16/01939 or MRS-17/00712, respectively. Oral application was performed by using a crop tube attached to a syringe. The remaining 10 birds in each group stayed non-infected and served as in-contact birds. Birds of group 3 remained uninfected as a negative control with 1 ml of PBS applied orally as described above.At the second day of life, 15 birds each from groups 1 and 2 were orally infected with 1 ml of V. basilica). Following experimental infection 5 birds, 3 infected, and 2 in-contacts from groups 1 and 2, together with 5 birds from the negative control group were euthanized at 7, 14, 21, 28, and 35 days of age by injecting a mixture (1:1) of Narketan®  and Sedaxylan®  intramuscularly (breast muscle) with subsequent bleeding out by cutting the V. jugularis. Necropsy was performed according to a standard protocol. Body weight, weight of liver and spleen, and gross pathological lesions were recorded. Tissue samples from liver, spleen, and caecum were collected for bacteriology.Birds were investigated daily for clinical symptoms together with the housing conditions . Before, and weekly after the experimental infection two cloacal swabs  per bird and blood samples from all birds were collected (S. Infantis was defined as: (a) negative: no growth by direct plating and enrichment, (b) low shedding: growth only by enrichment, and (c) high shedding: growth by direct plating.Weekly taken pairwise cloacal swabs were investigated to determine the shedding behaviour of both strains. For this purpose, one cloacal swab was directly streaked on XLD and brilliant-green phenol-red lactose sucrose agar . Both cultures were incubated at 37°C for 24 h. The second swab was stored in the fridge (4–8°C) until the result of direct plating was available. In case of a negative result, the second swab was investigated by an enrichment procedure according to EN ISO 6579-1:2017 . SheddinS. Infantis were expressed as CFU/g. Additionally, organ suspensions were stored in the fridge (4–8°C) until results from CFU counts were available. Negative tissue samples by direct plating were processed by applying the enrichment method as described for cloacal swabs.For bacterial quantification from organs, 1 g of liver, spleen, and caeca samples were homogenized in PBS , and plated on XLD and BPLS agars in 1:10 dilutions in duplicates. Following incubation of plates at 37°C for 24 h, colonies were counted, and the mean value was calculated per organ sample. The bacterial loads of ™ PBS Blocking Buffer, Thermo Fisher Scientific, Vienna, Austria) for 1 h. After aspiration of the blocking solution, 100 µl of test sera (diluted 1:500) were pipetted into each well in duplicates and incubated for 1 h. After washing, 100 µl of 1:5000 diluted Goat-Anti Chicken IgY (H + L)-HRP  was added to each well, and plates were again incubated for 1 h at room temperature. Following a second washing step, 100 µl of tetramethylbenzidine substrate  was added to each well, and plates were placed in the dark. After 12 min of incubation, colour reaction was stopped by adding 100 µl of 0.5 M H2SO4 to each well, and the optical density (OD) was measured with an ELISA reader  at a wavelength of 450 nm. Samples were run in duplicates on both ELISAs to calculate and analyse the mean OD values.Antibody response against MRS-16/01939 and MRS-17/00712, used as antigens, in two separate indirect ELISAs, was determined based upon an in-house protocol . Brieflyt-test was performed to determine significant differences. When normal distribution of data was not present, Wilcoxon–Mann–Whitney test and Kruskal–Wallis test were carried out. Descriptive analyses were performed for data for which the above-mentioned tests could not be applied.Data were analysed in R Core Team  with theirp2, ipf, klf, and ccdB/ccdA known to be located on this plasmid were present. Furthermore, two plasmid-encoded fimbrial operons, pef and sta, were also detected. The sequences were submitted to the NCBI database under accession numbers SAMN19328299 and SAMN19328300. Based on such differences, strain MRS-16/01939 will be referred to as pESI+ strain and MRS-17/00712 as pESI– strain throughout the manuscript.Applying whole-genome sequencing, both strains could be assigned to the sequence type 32, but they grouped into different genetic cluster types: strain MRS-16/01939 to cluster type 882, and strain MRS-17/00712 to cluster type 906. Phylogenetic analysis revealed that the strains are clearly genetically different from each other , displayp > 0.1). Furthermore, no gross pathological lesions were seen in any of the birds, except for one bird infected with pESI+ strain, which died at 11 days of age due to yolk sac infection. This bird was not included in further analysis resulting in a dataset comprising four birds only from group 1 on day 14.No clinical symptoms were recorded during the whole experimental study. No statistically significant differences were observed in body weight, liver, or spleen weight of the birds infected or the negative control .Transmission of both Salmonella throughout the experiment. Number of re-isolations from birds infected with the pESI+ strain was significantly higher compared to the pESI– strain (p = 0.011). Shedding behaviour was also different with the pESI+ strain being re-isolated throughout from all cloacal swabs whereas the pESI– strain was re-isolated only from one and two birds at 21 and 28 days of age, respectively. High shedding was only detected from birds infected with the pESI+ strain, with the highest number of positives at day 7 (80%), day 14 (79%), and day 35 (100%). Re-isolation of the pESI– strain by direct plating did not give any positive results and was only possible following enrichment  and one liver sample on day 28 with counts ranging from 1 to 7 CFU/g. No positive liver samples were obtained by direct plating at all other dates. Interestingly, spleen samples were positive by direct plating at days 14 (1/4), 21 , 28 , and 35 (2/5) with bacterial loads ranging from 1 to 300 CFU/g. The pESI+ strain was re-isolated by direct plating from the majority of caecal samples at all sampling time points: from all birds at the age of 7 (p < 0.01), 14 (p < 0.05), 21 (p < 0.01), and 28 (p < 0.01) days, and from one bird at the termination of the study. The bacterial counts ranged from 1 × 106–5 × 1010 CFU/g with a clear peak at 14 days of age , day 21 of age (p < 0.05), day 28 of age (p < 0.0001), and day 35 of age (p < 0.01) compared to birds infected with the pESI– strain and negative control group. No difference in antibody levels was found between birds infected with the pESI– strain and the negative control (No dissimilarities were found in regard to the performance of the two indirect in-house ELISAs. Infection with the pESI+ strain revealed a significant increase of antibody levels in birds at all sampling time points: day 7 of age (S. Infantis strains to persist on farms may differ. In Austria, a geographical separation was noticed with strains originating from southern parts of the country persisting heavily on farms in contrast to strains originating from northern parts [Salmonella isolates, respectively. This finding is also reflected in human isolates, where actually 41.8% of S. Infantis are MDR isolates [In vitro studies showed that this plasmid leads to superior biofilm formation, adhesion, and invasion into avian and mammalian host cells [Field observations indicate that the ability of rn parts . Thereforn parts . This isisolates . Furtherst cells , which mS. Infantis were rare, only based on a single strain and mostly performed in layer-type birds [in vivo trial was set up in which infectivity features of two different S. Infantis strains were evaluated in broilers. Infection via the oral route represents the most important way of horizontal transmission of Salmonella in chickens, in the field mainly due to environmental persistence of strains in the stables, resulting in caecal colonization, shedding of the pathogen in faeces, and consistent re-infections of birds [So far, experimental infection studies with pe birds , 37, 38.of birds .S. Enteritidis and Typhimurium, which might be caused by the acquisition of competitive microflora or the development of a specific cellular response [S. Infantis strains. An explanation for this might be the fact that different clones of one Salmonella species are able to adapt differently to the chicken intestine, as shown for S. Heidelberg in layer chickens [S. Enteritidis [In the present study, both strains successfully colonized the intestine but displayed different shedding patterns. Broilers infected with the pESI+ strain could be attributed as high shedders. This strain was already re-isolated from the majority of birds from the first sampling time point until the end of the experiment. Interestingly, the number of high shedders decreased with the course of the experiment until the age of 28 days, rising up again at 35 days of age. This is indicative of an intermittent excretion as reported for response . In contresponse , 44, 45.eritidis . In the Salmonella species in which similar results for spleens and livers were reported [S. Infantis reference strain ATCC®51741 in layer-type birds [irp2, ipf, and klf, are reported to facilitate colonization [pef and sta was elucidated, which were shown to mediate adhesion to and invasion in epithelial cells as shown for different Salmonella species [S. Infantis has been presented being less invasive compared to other Salmonella serovars [S. Typhimurium which is able to actively invade host tissues and survive intracellularly if a specific set of virulence factors encoded on the Salmonella pathogenicity island 1 and 2 are present [Also, a significant difference in the colonization ability in the caeca was recognized. The pESI+ strain was re-isolated throughout the whole experiment mostly by direct plating resulting in similar data as previously reported from infection/vaccination studies performed in layer-type birds with serovars Infantis and Enteritidis . Furtherreported , 47. In pe birds . It can nization , 48. But species , 50 So fserovars . However present .Salmonella serovars [S. Enteritidis demonstrated that antibody responses largely correlate with the degree of caecal colonization [S. Infantis infections in flocks.Until 21 days of age, the infection dynamic of the pESI+ strain was characterized by an increase and a peak in the CFU counts. Afterward, a decrease of CFU counts coincided with the rise of antibodies, underlining the development of humoral immunity, similar as reported for other serovars , 55. In nization . This fiS. Infantis strains spread rapidly to in-contact birds, and the respective proportion of positive infected and in-contact birds did not differ. This finding agrees with data from other Salmonella serovars [Despite a clear difference in shedding and colonization behavior, both serovars , 57. SimS. Infantis strain-specific virulence in commercial fast-growing broiler birds. An increased potential of S. Infantis harboring the pESI-like plasmid to colonize and infect birds was revealed, coinciding with a higher colonization rate in the caeca and the ability to invade inner organs. Awareness needs to be risen to such strains as they have a clear advantage to successfully spread within birds/farms with consequences on transmission to humans. Hence, they act as an emerging public health risk.The present study provides for the first time evidence of


Data on whether physical activity (PA) levels are related to nonalcoholic fatty liver disease (NAFLD) when considering body mass index (BMI) are scarce. We assessed whether PA affects the development or resolution of NAFLD in conjunction with BMI changes.Overall, 130,144 participants who underwent health screening during 2011–2016 were enrolled. According to the PA level in the Korean version of the validated International PA Questionnaire Short Form, participants were classified into the inactive, active, and health-enhancing PA (HEPA) groups.In participants with increased BMI, the hazard ratio (HR) and 95% confidence interval after multivariable Cox hazard model for incident NAFLD was 0.97 (0.94–1.01) in the active group and 0.94 (0.89–0.99) in the HEPA group, whereas that for NAFLD resolution was 1.03 (0.92–1.16) and 1.04 (0.88–1.23) (reference: inactive group). With increased BMI, high PA affected only new incident NAFLD. PA enhancement or maintenance of sufficient PA prevented new incident NAFLD. In participants with decreased BMI, the HRs were 0.98 (0.90–1.07) and 0.88 (0.78–0.99) for incident NAFLD and 1.07 (0.98–1.17) and 1.33 (1.18–1.49) for NAFLD resolution in the active and HEPA groups, respectively. With decreased BMI, high PA reduced incident NAFLD and increased NAFLD resolution. Maintenance of sufficient PA led to a considerable resolution of NAFLD.In this large longitudinal study, PA prevented incident NAFLD regardless of BMI changes. For NAFLD resolution, sufficient PA was essential along with BMI decrease. Maintaining sufficient PA or increasing the PA level is crucial for NAFLD prevention or resolution.The online version contains supplementary material available at 10.1186/s12889-022-13128-6. Nonalcoholic fatty liver disease (NAFLD) is a multisystem disease characterized by fat accumulation in the liver that is not triggered by excessive drinking. Lifestyle and dietary habits have resulted in a considerable increase in the prevalence of NAFLD along with obesity and diabetes . The preLifestyle modifications such as diet, exercise, and weight loss have been recommended to improve NAFLD . Most inn = 228,589) consisted of individuals who participated at least twice in a comprehensive health screening program that included assessment of PA using the validated Korean version of the International PA Questionnaire Short Form (IPAQ-SF) at Kangbuk Samsung Hospital, Seoul and Suwon, Korea, between 2011 and 2016. The aim of the health screening program was to promote health by detecting chronic diseases and associated risk factors. To examine the association of PA with the incidence or resolution of NAFLD independent of BMI changes, we excluded participants with age < 20 (n = 46), missing data on BMI (n = 216 at baseline/n = 876 at follow-up), waist circumference , fatty liver (n = 654 at baseline/n = 1044 at follow-up), and cancer history (n = 5203). Participants were also excluded if they tested positive for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) antibody (n = 8090) and if they had a daily alcohol consumption of >20 g (men) or > 10 g (women) . Individuals who were taking medications for hypertension, diabetes, or dyslipidemia were also excluded  to eliminate interference from these factors. Some participants met multiple exclusion criteria, and a total of 130,144 participants were finally included in this study . The median follow-up duration was 3.03 yr . The Kangbuk Samsung Hospital Institutional Review Board approved this study and waived the requirement for informed consent because we used only anonymized and de-identified data obtained as part of the health screening examinations (IRB No. 2018–05-050).Our study population  was performed by clinical radiologists using a 3.5-MHz probe at baseline and follow-up for all participants. The following images were obtained: (i) sagittal view of the right lobe of the liver and right kidney, (ii) transverse view of the left lateral segment of the liver and spleen, and (iii) transverse view of the liver for altered echo texture. Fatty infiltration of the liver (NAFLD) was considered present if the echogenicity of the liver was greater than that of the renal cortex, with the diaphragm and intrahepatic vessels appearing normal . The intp < 0.05. Statistical analyses were conducted using Stata .Descriptive statistics are used to summarize the characteristics of the participants in separate PA categories. The incidence rate was expressed as the number of cases of incident NAFLD or resolution of NAFLD per 100 person-yr (PY). The adjusted hazard ratio (HR) and 95% CI for incident NAFLD development and NAFLD resolution were estimated using a Cox proportional hazards model. Three regression models were generated for each outcome: model 1 was adjusted for age, sex, center (Seoul or Suwon), year of screening examination, smoking status, alcohol intake, and education level; model 2 was adjusted for variables in model 1 plus waist circumference; and model 3 was adjusted for variables in model 2 plus waist circumference changes. We used the inactive group as the reference category. The Kaplan–Meier curves were used to illustrate time-to-event outcomes in each PA group, with the results being compared using the log rank test. Statistical significance was set at A total of 95,959 individuals were identified to have no NAFLD at baseline, of whom 14,066 developed incident NAFLD during the follow-up period. 34,185 participants were identified to have NAFLD at baseline, which resolved in 3755 individuals during the follow-up period. Resolution of NAFLD was defined by absence of US criteria for NAFLD on repeat imaging. Incident NAFLD was defined as NAFLD absence at baseline and presence at follow-up by US. The baseline characteristics of the participants according to NAFLD status are shown in Supporting Table SThe HRs for NAFLD resolution or incident NAFLD development according to the PA category in different BMI change groups are shown in Table We divided the study participants into three groups to examine if there was a difference in the influence on the resolution or development of NAFLD according to their initial BMI: underweight (BMI < 18.5), normal BMI 18.5–23), and overweight (BMI > 23)  [The current NAFLD management includes dietary and PA modifications mainly aimed at weight loss . Previou1H-MRS) . A small1H-MRS) . These s1H-MRS measured intrahepatic triacyglycerol concentration (IHTAG) of 1.8%, relative reduction of 21%) and lifestyle interventions producing weight loss significantly improve liver lipid  [In our longitudinal cohort study, a higher level of PA at baseline was associated with an increase in NAFLD resolution and a decrease in subsequent NAFLD development, even after adjustment for visceral obesity (waist circumference). In our previous study, which included 36,195 US-diagnosed NAFLD patients, 19.6% had their NAFLD resolved after a mean follow-up of 4.9 ± 3.4 years . In a Sw 13–51%) . These rWith respect to temporal changes in PA, our study showed that increasing the amount of PA to an active status or maintaining an active PA or HEPA status during the median follow-up of 3 yr was associated with NAFLD resolution or prevention. Compared with the persistently inactive group, active PA or HEPA at follow-up demonstrated a higher benefit on NAFLD resolution than baseline PA level. A cross-sectional study conducted in Korea showed that an increasingly sedentary lifestyle was associated with a high prevalence of NAFLD and that the risk of developing NAFLD decreased by 6% in the minimally active group compared with the inactive group . Given tOur study had some limitations. An important limitation of this study was that the onset of new NAFLD and changes in PA levels could not be identified. Additional large-scale prospective studies are required to overcome this limitation. Present study used self-reported PA questionnaire which may be inaccurate. However, the IPAQ-SF is a widely used questionnaire in research that can determine the amount and frequency of PA relatively accurately. We diagnosed NAFLD using abdominal US. Liver biopsy is the gold standard method for the quantitative diagnosis of NAFLD. Nevertheless, US is currently the preferred method for the initial screening of NAFLD. Moreover, the sensitivity and specificity of US in diagnosing moderate to severe steatosis are rather high  . We usedIn this large longitudinal study, PA was found to be associated with the prevention of NAFLD regardless of BMI changes. PA can result in the resolution of existing NAFLD particularly when combined with BMI reduction. Maintenance or enhancement of PA even for a short period can provide benefits in terms of NAFLD prevention or resolution.Additional file 1: Supporting Fig. S1. Spline curves displaying the risk of (A) resolution and (B) incidence of NAFLD in overall and the three activity groups (Model 3).Additional file 2: Supporting Table S1. Baseline Characteristics of the Cohort Stratified Baseline NAFLD Status. Supporting Table S2. HR of Resolution and Incident NAFLD According to PA in BMI Change >0 Group (Splited by BMI Category at Baseline).


Functional tricuspid regurgitation (FTR) is common in left-sided heart pathology involving the mitral valve. The incidence, clinical impact, risk factors, and natural history of FTR in the setting of ischemic mitral regurgitation (IMR) are less known.We conducted a cohort study based on data collected from January 2012 to December 2014. Patients diagnosed with IMR were eligible for the study. The median follow-up was 5 years. The primary outcome is defined as FTR developing at any stage.2), LV internal diastolic diameter (LVIDD) 59.6 ± 3.94 (mm), RV fractional area change 22.3 ± 4.36 (%), systolic pulmonary artery pressure (SPAP) 48.4 ± 9.45 (mmHg). Independent variables associated with FTR development were age ≥ 65y [OR 1.2], failed revascularization, LA area ≥ 42.5 (mm2) [OR 17.1], LVEF ≤ 24% [OR 32.5], MR of moderate and severe grade [OR 419.4], moderate RV dysfunction [OR 91.6] and pulmonary artery pressure of a moderate or severe grade [OR 33.6]. During follow-up, FTR progressed in 39 (97.5%) patients. Covariates independently associated with FTR progression were lower LVEF, RV dysfunction, and PHT of moderate severity. LA area and LVIDD were at the margin of statistical significance .Among the 134 IMR patients eligible for the study, FTR was detected in 29.9% . In the FTR group, the average age was 60.7 ± 9.2 years , the mean LV ejection fraction (LVEF) was 37.3 ± 6.45 [%], LA area 46.4 ± 8.06 (mmIn our cohort study, FTR development and progression due to IMR was a common finding. Elderly patients with ischemic MR following unsuccessful PCI are at higher risk. FTR development and severity are directly proportional to LV ejection fraction, to the extent of mitral regurgitation, and SPAP. FTR tends to deteriorate in the majority of patients over a mean of 5-y follow-up.The online version contains supplementary material available at 10.1186/s12872-021-01982-y. Acute myocardial infarction directly affects myocardial viability and functionality and leads to structural remodeling and conduction changes seen immediately after an injury. The remodeling process results in valvular dysfunction and eventually hemodynamic changes. Ischemic mitral regurgitation (IMR) is a complication observed following the remodeling of the injured left ventricle –3.IMR is defined as mitral regurgitation following structural remodeling due to ischemic myocardial injury that is not directly related to valvular or sub-valvular pathologies such as mitral valve prolapse, endocarditis, autoimmune disease, medication, or direct radiation effect. The mechanism of IMR seems to be limited to leaflet motion, mainly due to shortening and limited relaxation of the ischemic papillary muscles. IMR is often classified as type IIIb of the Carpentier classification introduced in 1983, involving a restricted leaflet motion, most commonly the posterior leaflet . There cFunctional tricuspid regurgitation (FTR) is the consequence of various structural and functional changes involving the tricuspid annulus and the right ventricle. It is mostly secondary to left-sided mitral pathology in mitral regurgitation, mitral stenosis, and aortic stenosis , 9. PatiWe conducted an observational cohort study using Emek Medical Center's computerized database, a university-based secondary care center. Information regarding patient characteristics, demographics, and angiographic details were collected and analyzed using the Clalit health service database. Patients enrolled in the study were admitted to our heart institute for acute myocardial infarction (MI) from January 2012 to December 2014. Patients younger than 18 years and patients with primary mitral and tricuspid pathology and secondary pulmonary-related tricuspid regurgitation were excluded. The study population consisted of patients who developed mitral regurgitation following myocardial infarction. Patients were eligible for the study provided they met the inclusion criteria  was visually assessed  in multiple acoustic windows and calculated using the biplane Simpson method. Continuous-wave (CW) Doppler over TR peak gradient and the vena cava estimated central pressure using inferior vena cava (IVC) diameter and the respiratory fluctuation were used to estimate the systolic pulmonary artery pressures (SPAP) and the right atrial pressure followed by the categorization of severity grade and chamber size according to the American Society of Echocardiography (ASE) and the European Association of Cardiovascular Imaging (EACVI) guidelines , 23.The planned sample size is based on multiple variables such as IMR and FTR prevalence and a group difference of at least 15% to demonstrate a statistical difference of 80% and alpha of 5% in a two-sided test. We had to include a minimum sample size of 164 patients. Preliminary analysis revealed that one out of five patients would be eligible for the study. Following these analyses, we extended the duration of recruitment for 2 years.Our institutional Ethics Committee approved the study following the Helsinki Convention. The Emek Medical Center IRB waived informed consent due to the use of anonymous patient data and the retrospective nature of the study .Continuous variables were presented using mean ± standard deviation. Categorical variables were presented using frequencies and percentages. A chi-square test was performed to analyze the association between the study groups and categorical variables. For continuous variables, we used the t-test . All confidence intervals are at the 95% level. TR progression was analyzed using the Mantel–Cox multivariant analysis, R-squared analysis, and One-way ANOVA. Multivariable models and linear regression analysis were used to estimate the predictors of FTR. Cut-off points for continuous variables as LVIDD, LA, sPAP, and LVEF were made using the highest sensitivity and specificity point in the ROC curve. The odds ratio was calculated using descriptive analysis in SPSS and linear regression models. Some variables such as age and gender were analyzed using the Cochrane-Armitage test. Forest plot and subgroup analysis for FTR Differences was done using JMP (SAS) and considered statistically significant at the 2-sided p level of 0.05. The statistical analyses were performed using the software SPSS (IBM) and SAS 9.4 (SAS).During the study period, 960 patients were admitted to the intensive cardiac unit due to acute MI. Of these, 274 patients were excluded from the study: 236 patients were excluded due to previous known MR, non-ischemic originating mitral regurgitation, and primary mitral pathology. Thirty-eight patients were excluded due to previous TR, secondary TR, and primary tricuspid pathology. Of the 686 eligible patients, 175 (25.6%) developed ischemic mitral regurgitation. Forty-one patients were excluded due to a lack of adequate follow-up TTE exams and low imaging quality. Thus, 134 patients were in the final study cohort . Older age was directly linked to FTR severity . Multivariate linear regression analysis indicated that failed revascularization during the indexed event was an independent covariate for FTR development and severity . Door-to-balloon (D2B) time of 90 min was achieved in 94% of cases and did not influence FTR development (p = 0.56). Gender, smoking, obesity, hypertension, hyperlipidemia, diabetes mellitus, peripheral vascular disease, atrial fibrillation, chronic renal failure, prior coronary bypass surgery, or the Killip class were not found to be different between patients who developed FTR and those who did not . The two groups' demographic characteristics revealed that patients who developed FTR were older .We did not find a significant statistical correlation between the infarct-related arteries involved or the existence of multiple coronary artery diseases with the development of FTR (p < 0.0001) , LV ejection fraction (LVEF), RV function , Systolic pulmonary artery pressure (SPAP) and MR severity grade with the development of FTR  Table . We inte1) Table .Table 2E‘FTR progression’ was defined as a deterioration in TR's severity at follow-up compared to the indexed event. During follow-up, FTR progressed in 39 (97.5%) of newly developed FTR patients.p = 0.001 for moderate and severe PHT), reduced LVEF (p < 0.0001), and moderate/severe RV dysfunction (p = 0.002). LA area and LVIDD were borderline significant . Moderate or severe MR was found to be statistically linked to FTR progression  . Group 2 and group 3 had mixed echocardiographic criteria . The mean survival time was 4.58 years (95% CI: 4.11–5.04 years) in the FTR group and 5.15 years (95% CI: 4.9–5.4 years) in the no-FTR group  and the interconnection between the mitral and tricuspid apparatuses –27. Our Multivalve involvement was described in the past regarding rheumatic heart disease and congenital heart disease and not extensively in the context of ischemic heart disease –32.Myocardial infarction is still the most common cause of HFrEF, and until recently, it was thought that its primary effect was directed toward left-sided pathology through an extensive remodeling process , 34.IMR is a common complication after myocardial infarction that requires adequate follow-up regardless of the appearance of clinical symptoms –37. MildOver a five-year follow-up, we could see that FTR developed in nearly a third of IMR patients, and more surprisingly, it progressed in almost all newly developed FTR patients. Elderly patients who had unsuccessful PCI seemed to be at a higher risk than others. We identify several echocardiographic predictors for FTR development and rapid FTR deterioration and a cut-off threshold for better monitoring and future management.Our findings are consistent with prior reports of left and right chamber interconnection. Deterioration of left side chambers resulting from extensive remodeling may shift volume and pressure initially toward unadjusted right-sided chambers and result in larger LA size and systolic pulmonary artery pressure. Over time, without proper optimal heart failure medical therapy, this may result in RV dysfunction and progressive development of FTR.Bivalvular regurgitation, particularly with the tricuspid valve's involvement, dramatically influences the prognosis of heart failure, limits therapeutic options, and substantially increases the overall mortality –41.IMR should be appropriately monitored and followed up regardless of symptoms. FTR should be assessed annually to avoid significant deterioration and start prompt treatment for heart failure before the first signs of heart failure appear.Our study is designed in a retrospective fashion. The data analysis was retrospectively reviewed on computerized patient medical records. Echocardiographic images were assessed according to the ASE criteria.Quantification of the severity of mitral and tricuspid regurgitation was based on qualitative grading. These assessment methods may be confounded by several technical and hemodynamic factors, including the jets' eccentricity and atrial size. However, we cannot accurately apply quantitative or semiquantitative methods because of the nature of the study.The influence of heart failure medical therapy and its contribution to valvular dysfunction could not be evaluated here due to a lack of clinical data. Larger samples are needed to evaluate these factors.In our cohort study, FTR was a common finding in IMR patients. Third of patients with ischemic mitral regurgitation developed functional tricuspid regurgitation (FTR). Elderly patients who had unsuccessful PCI are at a higher risk. FTR progression was observed in almost all patients over a 5-years follow-up period. Rapid FTR progression was observed in patients with moderate and severe MR, LVEF ≤ 24%, LA size ≥ 42.5 mm, LVIDD ≥ 56.5 mm, and RV of moderate and severe dysfunction. FTR was found to be positively linked to poor outcomes and lower survival rates.Additional file 1. Supplementary file.


Water and electrolyte disorders commonly encountered in children post-surgery involving hypothalamus and posterior pituitary, are central diabetes insipidus, syndrome of inappropriate secretion of anti-diuretic hormone and cerebral salt wasting disease. Delayed diagnosis and inadequate management of such cases may lead to worsened neurological outcomes with a high mortality rate.Here we report the case of a 7-year-old girl who underwent surgical resection of a craniopharyngioma, following which she initially developed central diabetes insipidus. However, later on in the course of her illness she developed symptomatic hyponatremia with natriuresis which was diagnosed to be due to cerebral salt wasting disease. This combination of central diabetes insipidus and cerebral salt wasting syndrome is a rare occurrence and poses a diagnostic challenge. Diagnosis and management can be even more difficult when these conditions precede or coexist with each other.In such cases development of hyponatremia should always prompt consideration of unusual causes like cerebral salt wasting disease in addition to the classically described syndrome of inappropriate secretion of anti-diuretic hormone. Hence, a thorough knowledge of these disorders along with intensive monitoring of fluid and sodium status is critical for timely diagnosis and management of these patients. Fluid and electrolyte imbalance is commonly encountered in children secondary to central nervous system (CNS) insult. Among children undergoing surgeries in the sellar and suprasellar regions, most commonly due to craniopharyngioma, the incidence of fluid and electrolyte disorders is quite high. Central diabetes insipidus (CDI), syndrome of inappropriate antidiuretic hormone secretion (SIADH), cerebral salt wasting (CSW) disease and adipsic diabetes insipidus (ADI) can occur individually or exist simultaneously making their diagnosis and management extremely troublesome.Here, we present the case of a female child with CDI and CSW disease occurring simultaneously after surgical resection of craniopharyngioma.A 7-year-old girl presented with complaints of headache for the past 1 month and progressive right sided weakness for the past 15 days. On examination, the child had right sided hemiplegia, bilateral hemianopia and signs of optic atrophy in both eyes. MRI brain showed a 3x2x0.5 cm large sellar and supra-sellar mass with T1 weighted image showing iso to hyperintense lesion suggestive of a craniopharyngioma  6 hourly.Following surgery, she developed polyuria and her urine output was 8 ml/kg/hr. Further investigations showed elevated serum sodium (156 mEq/L), raised plasma osmolality (321 mOsm/kg) and decreased urinary osmolality (175 mOsm/kg). Her blood sugars were within normal limits (122-140 mg/dL). She was diagnosed to be suffering from CDI. Fluid losses in excess of the normal maintenance fluid rate were replaced volume for volume with 0.9% normal saline 6 hourly. She was started on IV dexamethasone  and subcutaneous vasopressin . Urine output and serum sodium levels were strictly monitored. She also complained of no light perception in both of her eyes. On the second post-operative day, she continued to have polyuria although a decrease was observed from the previous day (7 ml/kg/hr). Sodium levels increased to 160 mEq/L. The dose of vasopressin was increased further (6 IU/day) and administered when required after she had episodes of breakthrough polyuria. By the third post-operative day, her urine output had stabilized at around 3-4 ml/kg/hr. and her sodium levels were between 136 and 143 mEq/L. Her subcutaneous dose was changed to oral desmopressin (0.1 mg/day) twice daily. Oral dosage was also regulated as per her urine output with daily monitoring of serum sodium levels. On the seventh post-operative day, hormonal profile was suggestive of hypocortisolism (8 AM plasma cortisol: 1.47 μg/dL) and central hypothyroidism . She was started on levothyroxine 25 mcg. In addition, as the dexamethasone was stopped by fifth post-operative day, she was started on hydrocortisone 1.25 mg  was administered over 20-30 min followed by a continuous infusion in view of symptomatic hyponatremia. Tab fludrocortisone was started at 0.1 mg/day. Hypertonic saline infusion was tapered and stopped after 12 h as sodium levels normalized. Over the next 2 days, serum osmolality (292 mOsm/kg) and urinary osmolality (300 mOsm/kg) had normalized along with a reduction in urinary sodium level (25 mEq/L). Her sensorium had improved but polyuria persisted. Desmopressin was restarted (0.1 mg/day) once the sodium level had normalized as polyuria was due to CDI. Given the above clinical scenario, an overlap of CDI and CSW disease was considered in the present case. Figure 2/day) 8 hourly. Serum sodium and potassium levels were normal for her age. After 2 weeks, a repeat hormonal profile  was within normal limits. She is now clinically well with her permanent DI under control on 0.15 mg/day of desmopressin. As part of her neurological morbidity, she has hemiparesis with no improvement in power and has only light perception in both eyes.On follow-up after 2 weeks, her hormonal profile showed low TSH (0.52 mIU/L), normal free T4 (1.22 ng/dL) and low 8 AM cortisol (4.1 mcg/dL) requiring increase in dosages of levothyroxine to 37.5 mcg/ day and tab. Hydrocortisone to 2.5 mg  performs a crucial role in the maintenance of water and sodium homeostasis resulting from a complex interplay between hypothalamus, posterior pituitary, and adrenal glands. Factors disrupting the regulation of this normal HPA axis result in pathological conditions such as CDI, SIADH and CSW disease. Craniopharyngiomas account for 6-9% of brain tumors in children . Endocri2/kg (in 3-4 divided doses) preferably pre-operatively although a steroid sparing approach may also be used where steroids are only started when hypocortisolism is present [Post-pituitary surgery hyponatremia is commonly encountered and carries a wide spectrum of differential diagnosis such as improper fluid administration, hypocortisolism, hypothyroidism, over-correction of DI in over-dosing of desmopressin, SIADH and CSW disease. Hypocortisolism may lead to hyponatremia by causing an increase in ADH levels along with impaired renal free water excretion , 9. To p present . In caseCentral diabetes insipidus (CDI) results from a deficiency of arginine vasopressin (AVP), and is characterized clinically by polyuria, polydipsia, and dehydration –13. CDI In addition to these presentations, rarely patient might develop CSW disease due to a primary neuronal insult or as a secondary response to SIADH . CSW disThe coexistence of CDI and CSW disease is extremely rare, rather CDI is associated with SIADH especially in operated patients of sellar and supra-sellar tumors. Combined CDI and CSW disease has been reported in patients suffering from traumatic brain injury, cardiac arrest, cardiogenic and septic shock, meningitis, encephalitis, shaken baby syndrome, intracranial hemorrhage, sellar and/or supra-sellar tumors , 24–27. To conclude, in cases of CDI following surgery, development of hyponatremia should always prompt consideration of unusual causes like CSW in addition to the classically described SIADH. Early recognition and differentiation of CSW disease from SIADH is important, as management strategies for these conditions are completely different, and a misdiagnosis or even a delay in diagnosis can prove fatal. Prompt diagnosis and appropriate management of CDI and CSW disease was extremely crucial in the survival of our patient. We therefore recommend a close watch on serum sodium, and urine output in all operated cases of sellar/suprasellar tumors, with a low threshold for measuring urinary sodium excretion, urine osmolality and serum osmolality.A triphasic response in not necessarily the rule of thumb in operated cases involving the pituitary stalk.A greater emphasis is required on the importance of close monitoring after pituitary stalk injury post-surgery to prevent further endocrine morbidity and mortality.