Deceased donor kidneys with acute kidney injury (AKI) are often discarded because of concerns about inferior transplant outcomes. A means of grading the quality of such kidneys is the performance of procurement biopsies.This is a retrospective study of 221 brain death donors with marginal kidneys transplanted in 223 recipients in Germany. Marginal kidneys were defined as kidneys with procurement biopsies done exceptionally to assess suitability for transplantation in otherwise potentially discarded organs. The impact of deceased donor AKI on patient survival and death-censored graft survival at 1, 3 and 5 years and graft function at 1 and 3 years after transplantation was investigated.Stage 1: 1.435 (95% CI 0.438–0.702), ORStage 2: 2.463 (95% CI 0.656–9.245), ORStage 3: 4.784 (95% CI 1.421–16.101)] but a similar graft and patient survival compared to recipients of donors without AKI and with AKI stage 1 and 2 as well. The coexistence of recipient DGF and donor AKI was associated with the lowest graft survival and function rates.Recipients of kidneys with stage 3 AKI had a greater incidence of delayed graft function [DGF; ORThe transplantation of deceased donor marginal kidneys with AKI confers a higher risk for DGF but is associated with acceptable graft and patient outcomes, which do not differ in comparison with marginal donor kidneys without AKI. Graft prognosis is especially poor if donor AKI and recipient DGF concur. Donor AKI was a risk factor independent of the histological lesions of procurement biopsies.The online version contains supplementary material available at 10.1007/s11255-022-03277-3. Kidney transplantation (KT) continues to remain the best available renal replacement therapy for most patients with end-stage renal disease. The shortage of organs and the continuously increasing number of patients on the waiting list led to the increased usage of organs from marginal donors . The chaAcute kidney injury (AKI) is very common in kidney donors and is strongly correlated with DGF –8. PreviGiven these open questions, we aimed to investigate AKI in kidneys from marginal donors with post-explantation biopsies. We evaluated the impact of clinical donor characteristics and histological findings of their biopsies on short-term patient and graft survival and short-term graft function.We extracted data from the Deutsche Stiftung Organtransplantation (DSO) Region Nord and from the German transplant centers of kidneys allocated between January 2003 and March 2012. We included adult recipients of deceased-donor kidney-only transplants of marginal organ quality in Germany. Recipients were excluded if they were < 18 years old at the time of transplantation, if they received multiple types of organs, or if their donors were from outside of Germany. According to German regulations, only brain-dead donors were included in the study. For the same reason, normothermic ex vivo kidney perfusion systems for organ preservation were also not used.Donor variables, procurement biopsy results, recipient variables and transplant factors included in the analysis are listed in Tables Marginal kidneys were defined according to the current clinical practice in Germany, i.e., kidneys with procurement biopsies done exceptionally to assess suitability for transplantation in otherwise potentially discarded organs. Such kidneys had, for example, proteinuria or presumed chronic kidney disease, were of poor macroscopic or perfusion quality, had heavy aortic patch and/or renal artery atherosclerosis, had multiple accessory renal arteries or were recovered from donors with long ICU stay, diabetes and multiorgan failure. Macroscopic grading of the external aspect of the donor kidney was provided by the explanting surgical team as good, medium, or poor; likewise, atherosclerosis was characterized as no, mild or severe and perfusion quality as good, medium or poor. Extended criteria donors (ECD) were classified as brain death donors 60 years of age or donors 50 to 59 years of age with at least 2 of the following features: history of hypertension, terminal serum creatinine > 1.5 mg/dl or cerebrovascular cause of death .The original Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to calculate eGFR . AKI wasOverall graft loss was defined as time from transplantation to return to dialysis, or death with a functioning graft. Death-censored graft failure was the same apart from censoring those who died with a functioning graft. Patient death was defined as the time from date of transplantation to patient death, not censored at graft failure. All survival times were censored at the end of follow-up or loss to follow-up.Four different outcomes were analyzed: (1) primary non-function (PNF), (2) DGF, (3) recipient eGFR/creatinine at 3, 12 and 36 months, and (4) graft loss, death-censored graft failure and patient survival at 1 and 3 years.All biopsies were processed in paraffin according to the routine protocol at the Institute of Pathology, Hannover Medical School, which involves multiple level sections stained with hematoxylin and eosin, periodic-acid Schiff, Jones silver, trichrome elastica. Histopathological parameters were retrospectively determined by an experienced nephropathologist and included type of biopsy, total number of glomeruli and ratio of globally sclerosed glomeruli, focal and segmental glomerulosclerosis (FSGS), number of arteries (media ≥ 2 smooth muscle cell layers), presence of FSGS, Banff Lesion Scores i, t, v, g, ptc, ci, ct, cv, cg, ah according to Banff 2011 –18, arteP < 0.05. Variables were also considered confounders if they changed the coefficient of the explanatory variable by > 10%. The different exposure variables were inserted into the model. We compared the models using F test, adjusted R2 and the Hosmer–Lemeshow goodness of fit and the C statistic.The baseline characteristics of the study cohort were expressed as mean (SD). We created logistic regression models for the outcome of DGF, adjusting for covariates. The linearity assumption was assessed through categorization of continuous variables. We checked for interaction terms using forward elimination. Nonsignificant variables were removed from the model using backward elimination with a cutoff of F test and adjusted R2.Three multilinear regression models for the outcomes of recipient eGFR at 3, 12 and 36 months were created, adjusting for covariates. Collinearity of different variables was assessed using the variance inflation factor. The linearity assumption was assessed using scatter plots of residual values for each continuous variable. Effect modification was assessed for using the forward elimination method. Nonsignificant variables were removed from the model using backward elimination. The different exposure variables were then assessed in the different models. The Wald test was used to assess the significance of exposure variable plus any interaction terms. We then compared the variables of interest for the different models using the C statistic and Akaike Information Criterion (AIC).Three separate multivariable Cox proportional hazards models were created to assess the outcomes of death-censored graft failure, and patient death. Nonlinear continuous variables were made categorical. The nonsignificant variables were removed from the model using backward elimination. Wald statistics were used to assess the significance of exposure variables. The models were assessed using the Harrell The variables for which the various models were adjusted for in the multivariate analyses are summarized in Supplementary File 1.P value below of 0.05 was considered significant in all two-sided tests. Statistical analysis was performed with SPSS software, v24  and IBM SPSS Statistics Essentials for R.A The study protocol was conducted in accordance with the Declarations of Helsinki and Istanbul on organ trafficking and transplant tourism and approved by the Ethics Committee of Hannover Medical School (No. 1519-2012).From 442 kidneys of marginal quality considered for transplantation and with procurement biopsies, 149 were not transplanted. For the remaining 293 transplanted kidneys, follow-up data were available for 223 organs . Furthermore, the KDRI was 1.553 ± 0.506 and 1.426 ± 0.516 in the non-AKI and AKI donor groups. The mean donor BMI and the prevalence of diabetes were higher, whereas less numbers of ECDs were observed in the AKI group. No differences in donors’ gender, hypertension, smoking rate, Hepatitis B and C and CMV serology, causes of brain death and distribution of KDPI were observed between the non-AKI and AKI donor groups. Traumatic brain injury was more common in donors without AKI, but duration of brain death was comparable between groups. Donors with AKI remained for a longer time in the ICU and received less often volume expanders and steroids.Donors’ characteristics are summarized in Table In the AKI donor group serum creatinine at admission, peak serum creatinine and creatinine and recovery were higher. Furthermore, AKI donors had more proteinuria and reduced diuresis in the last 24 h before cross-clamp.Regarding perfusion and organ quality, there were no differences between kidneys with and without AKI Table . HoweverTable P = 0.002), patient and death-censored graft survival at 1, 3 and 5 years graft function at 3 months, 1 and 3 years, proteinuria and number of rejections were similar in both groups.Clinical outcomes are presented in Tables C statistics: AKI = 0.772 (CI 0.703–0.842), AKIN classification = 0.784 (CI 0.714–0.853))]. Increasing stage of donor AKI was associated with a higher rate of DGF  showed that there was no significant association between AKI stage and patient survival, death-censored and non-death-censored graft failure Table . SimilarR2 values were worse .Interestingly, there was weak evidence of an association between 3- and 12-month recipient eGFR and increasing stage of donor AKI Table . The modResults on graft survival are illustrated in Fig. The deceased donor pool is limited and living kidney donation does not suffice to close the gap in organ shortage. For donors with AKI, several aspects have to be taken into account, such as surgical issues, hemodynamic compromise, immunological issues and ischemia reperfusion injury . UnfortuShort-term patient and allograft survival and graft function appear acceptable after transplantation of marginal kidneys with and without AKI to expand the donor pool.The incidence of DGF was significantly higher only in recipients of kidneys from marginal donor kidneys with AKIN stage 3, but not in recipients of donor kidneys with AKIN stage 1 and 2.Donor AKI and recipient DGF in combination were considerably associated with graft loss.Histopathological assessment of donor kidneys was not helpful in predicting outcomes;The rate of cumulative rejections or the level of proteinuria at follow-up was not higher in recipients of marginal donor kidneys with AKI.The main findings of our study were:Similar to others, we confirmed classical risk factors for AKI, such as diabetes mellitus, BMI and preexisting chronic kidney disease –23. MoreThe discrepancy between macroscopic and microscopic findings in the no-AKI group  implicates that macroscopic assessment of a recovered organ from transplant team is a subjective and probably not accurate parameter of organ quality. Furthermore, it underpins the pitfalls of procurement biopsies in marginal kidneys and corroborates the allocation policy of the European senior program (ESP) of Euro Transplant (ET), where biopsy is not a prerequisite and indeed is not performed in the great majority of the recovered organs. Regarding histopathology, we suppose that the opposite as expected patterns in donor kidneys with AKI are due to selection bias, since only post-explantation biopsies of marginal kidneys were assessed. In that cases, histological findings are unfavorable in general and differences between marginal donor kidneys with and without AKI would not be anticipated. This was also probably the reason why, except for DGF, histological findings failed to predict clinical outcomes, in the multivariate analysis. Perhaps, the statistical analysis did not confirm the significance of these lesions, although their clinical relevance was evident, i.e., that AKI kidneys could be transplanted with satisfactory outcomes. Hence, our results do not support the routinely performance of procurement biopsies in deceased donor kidneys with AKI.The occurrence of DGF in presence of donor AKI is plausible but the extremely poor outcomes after concurrence of both unfavorable conditions are probably due to the superimposed damage in the transplanted organ before recovery from AKI. The development and severity of AKI are known risk factors for the transition to chronic- or end-stage renal disease . What’s The clinical implication of our findings is that patients at high risk of developing DGF should be cautiously selected if kidneys with AKI are offered and in the case of transplantation, careful observation is required during the first three months of follow-up. Such a time frame is of relevance for the allocation policy of ET since patients can be relisted without losing previous accrued waiting-list points in case of graft failure. Insisting on rescuing a graft deemed to get lost could finally result in much longer waiting times for a second transplant. Giving-up the graft and re-initiating dialysis timely is therefore crucial for those patients.Strengths of the study are the analysis of detailed donor items, concerning the treatment during the ICU stay and the explantation procedure, with data on kidney function from admission to the ICU until recovery, elaborate hemodynamic parameters, concomitant medications and histopathological scoring of the procurement biopsies in a center with experienced nephropathology.Limitations of our study were the small sample size, the retrospective design, the amount of missing data and the bias toward marginal organs. Lastly, quality of life was not investigated. This is a fundamental item considering the significantly inferior outcomes in graft survival.In conclusion, our results suggest that transplantation of marginal kidneys with AKI puts recipients in disadvantage only regarding transplanted organ survival and function but not patient survival and may be recommended, always considering an unfavorable risk-to-benefit ratio. Since functioning grafts in the long run outperform dialysis, an individualized approach in the context of a personalized medicine strategy is essential. The non-acceptance of an organ leads in the end to an increase in patients’ mortality due to their longer time on the waiting list .Supplementary file1 (DOCX 18 kb)Supplementary file2 (DOCX 25 kb)Below is the link to the electronic supplementary material.


E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human β-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalismprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1 ΔmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF.The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While  Enterococcus faecalis is a Gram-positive commensal bacterium that naturally inhabits the harsh environment of the human gastrointestinal tract. The enterococci are among the most clinically significant nosocomial pathogens and cause a variety of opportunistic infections in susceptible individuals, including endocarditis, urinary tract infections, bacteremia, and wound infections , amphipathic molecules that are part of the innate immune response against microbes during infection . CAMPs aE. faecalis, CAMPs are thought to target phosphatidylglycerol (PG), an anionic phospholipid which constitutes a major portion of the cell membrane , and a flippase domain responsible for transferring modified PG from the inner leaflet to the outer leaflet of the cytoplasmic membrane  were deleted, exogenous fatty acid supplementation resulted in tolerance to the cationic antibiotic daptomycin and changes in lipid composition  in in OG1RF . Our finymyxin B . In OG1Rposition . Mutatiofaecalis . Such adfaecalis  and phosfaecalis .mprF genetic perturbation with cationic antimicrobial resistance and lipidomic alterations suggest a gap in knowledge about how mprF brings about such adaptive lipid remodeling. Thus, we sought to more thoroughly investigate the mechanism by which mprF affects lipid homeostasis in E. faecalis. Using lipidomic methods that we had previously established , and assays for membrane fluidity, secretion, biofilm formation, and CAMP susceptibility, we have discovered a previously unappreciated role for mprF in global lipid regulation and cellular physiology.Our previous work and the above literature linking ablished , we quanE. faecalis more strongly at foci situated at or near the division septum in the ΔmprF2 mutant than in the wild type, and that only MprF2, and not MprF1, protects against hBD-2-mediated killing  at 50 μg/mL  to quantify phosphatidylglycerol (PG) and lysyl-PG (L-PG) molecules in this strain and in two daptomycin-resistant strains  studies which lacked the sensitivity to discriminate between individual lipid molecules and quantify their levels, using LC-MS/MS, we were able to observe that MprF1 also contributes to L-PG synthesis in E. faecalis OG1RF under these laboratory conditions.We previously observed that human β-defensin 2 (hBD-2) binds  killing . The Δmp strains . In the ble L-PG , as prevble L-PG , whereas10.1128/mbio.03073-22.1FIG S1mprF mutants. Deletion of mprF paralogs increases susceptibility to CAMPs to different degrees. CFU enumerated after 2-hour exposure to increasing concentrations of (A) hBD2 or (B) LL-37. Error bars represent the standard error of mean from 2 biological replicates averaged from 6 technical replicates each. *, 0.05 > P ≥ 0.01; ****, P < 0.0001; Fisher’s least significant difference (LSD) test for ANOVA. Loss of mprF contributes to changes in individual species of L-PG and PG. Normalized quantities of (C) 8 native lysyl-PG species, (D) 18 native PG species. Error bars represent the standard error of mean from 5 biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Dunnett’s test for ANOVA. Inactive MprF2 does not complement L-PG and PG levels. Normalized amounts of total lysyl-phosphatidylglycerol (L-PG) (E) and total phosphatidylglycerol (PG) quantities (F) in E. faecalis wild type and mprF mutants are shown. Each bar represents the mean ± standard error of measurement calculated from 5 biological replicates, each represented by an open circle. **, P < 0.001; ****, P < 0.0001; Fisher’s least significant difference (LSD) test for ANOVA. Inactive MprF2  does not complement L-PG and PG levels at the individual species level. Normalized quantities of (G) 8 native lysyl-PG (L-PG) species, and (H) 18 native phosphatidylglycerol (PG) species. Each bar represents the mean ± standard error of measurement calculated from 5 biological replicates, each represented by an open circle. No difference in lipoteichoic acid (LTA) levels between wild type and mprF mutants. (I) Western blots for LTA in whole cell lysates and supernatants. SecA used as a loading control. Loss of mprF contributes to changes in individual species of DGDAG. Semi-quantitative analysis of (J) 15 native DGDAG species in the mprF mutants. Error bars represent the standard error of the mean from 5 biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Dunnett’s test for ANOVA. Download FIG S1, PDF file, 1.0 MB.CAMP susceptibility assessment and analysis of individual species of lysyl-PG (L-PG), phosphatidylglycerol (PG) and, diglucosyl-diacylglycerol (DGDAG) and lipoteichoic acid (LTA) in the Copyright © 2023 Rashid et al.2023Rashid 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 mprF gene expression, we performed quantitative reverse transcription PCR (RT-qPCR) of the mprF paralogs in the wild type. RT-qPCR revealed that mprF2 expression is 200-fold higher than mprF1 expression  Absolute quantitation of mprF1 and mprF2 transcripts using reverse transcription quantitative PCR (RT-qPCR) of mid-log phase cultures of the wild type displaying fold change of mprF2 relative to mprF1 copy number. (B) Relative quantification of mprF1 and mprF2 transcripts using RT-qPCR of mid-log phase cultures of WT, ΔmprF1, ΔmprF2 and ΔmprF1 ΔmprF2. Data displays fold change of transcripts relative to the wild type. Each bar represents the mean ± standard error of measurement calculated from 3 biological replicates averaged from 3 technical replicates each. ***, P < 0.001; Unpaired t-test for (A), Fisher’s least significant difference (LSD) test for ANOVA for (B). No difference in growth rates or cell viability between wild type and mprF mutants. (C) Optical density measured at 600 nm for the wild type and mprF mutants across 9 time points is shown. Error bars represent the standard error of the mean from 2 biological replicates averaged from 2 technical replicates each. (D) CFU enumeration results of the wild type and mprF mutants across 9 time points are shown. Error bars represent the standard error of the mean from 2 biological replicates averaged from 5 technical replicates each. (E) Percentage of live and dead cells for OG1RF WT and mprF mutants across 4 bacterial growth phases. Enumerated from at least 100 cells per strain. Download FIG S2, PDF file, 0.2 MB.Expression of the Copyright © 2023 Rashid et al.2023Rashid 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 mprF2 and ΔmprF1 ΔmprF2 mutants had significantly less PG than wild type. Moreover, like our observation for L-PG, ΔmprF1 also had an intermediate amount of PG compared to the wild type and the other two mprF mutants (pgsA) is thought to be essential in E. faecalis, based on the fact that this gene is essential in many other bacterial species , as previously described in B. licheniformis  reveals that full length MprF is required for restoration of PG and L-PG levels in ΔmprF1ΔmprF2. Normalized quantities of PG and L-PG from the ΔmprF1ΔmprF2 with plasmid-based expression using the PsrtA promoter of full length mprF1, mprF2 or the N-terminal, C-terminal domains of either mprF. Total normalized quantities of (A) L-PG and (B) PG in the strains tested as well as individual normalized quantities of (C) L-PG and (D) PG. Error bars represent the standard error of mean from 5 biological replicate. Plasmid backbone used here for all strains are pGCP123-PsrtA, except for pEmpty which are pGCP123 instead. Download FIG S3, PDF file, 0.5 MB.Expression of individual Copyright © 2023 Rashid et al.2023Rashid 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 mprF2 is required for L-PG production  Text detailing how the identities of the TLC spots in Copyright © 2023 Rashid et al.2023Rashid 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/mbio.03073-22.4FIG S4mprF mutants. (A) Iodine staining, (B) ninhydrin staining and (C) 32P-radiolabeling staining of 1D-TLCs of empty vector controls of WT, ΔmprF1, ΔmprF2, ΔmprF1 ΔmprF2 and pmprF2-HA complemented ΔmprF2, ΔmprF1ΔmprF2 together with PG, CL and lysyl-PG standards under chloroform: methanol: water (65:25:4) solvent system. Iodine stains most lipids while ninhydrin stains amino modified lipids. 6 major spots were observed. (D) These spots were scraped off iodine strained TLC plates of WT and ΔmprF1 ΔmprF2 and placed through lipid extraction before analysis using LC-MS. Spots were identified and labelled accordingly. (E) 1D- and 2D-TLCs of 14C and 32P labelled lipid extracts of wild type and ΔmprF1 ΔmprF2. chloroform: methanol: water (65:25:4) solvent system was used in the first dimension while chloroform: hexane, methanol, acetic acid (50:30:10:5) was used in the second dimension. Comparing positions of the spots in the first and second dimension, we can resolve the positions of PG and CL. In the solvent system used in the second dimension, CL migrates ahead of PG. Download FIG S4, PDF file, 0.4 MB.Identification of lipid spots in 1D- and 2D-TLCs of lipid extracts from WT and the Copyright © 2023 Rashid et al.2023Rashid 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/mbio.03073-22.8TABLE S1Table S1, DOCX file, 0.05 MB.Supplementary tables for methods. (A) Bacteria strains and culture conditions. (B) Lipid standards used. Procured from Avanti Polar Lipids. (C) Primers used for RT-qPCR. (D) Components in chemically defined media (CDM). (E) Antibody and developing solution concentrations for Western blot. (F) PCR Primers. (G) Antimicrobial peptides used. Download Copyright © 2023 Rashid et al.2023Rashid 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/mbio.03073-22.10TABLE S2E. faecalis from untargeted MS. (B) MRM transition list for DGDAG. (C) Lipid species observed from untargeted MS analysis of TLC spots. (D) GC-FAME analysis of BHI. (E) Fatty acids detected in wild type and ΔmprF1 ΔmprF2 grown in BHI or CDM. (F) RNAseq analysis of ΔmprF1 vs wild type. (G) RNAseq analysis of ΔmprF2 vs wild type. (H) RNAseq analysis of ΔmprF1 ΔmprF2 vs wild type. Download Table S2, XLSX file, 0.2 MB.(A) DGDAG species detected in Copyright © 2023 Rashid et al.2023Rashid 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 14C radiolabeled and iodine-stained TLC plates indicate that L-PG is lower only in ΔmprF2 and ΔmprF1 ΔmprF2 and that reduction was restored by complementation with pmprF2-HA (mprF2 and ΔmprF1 ΔmprF2 (32P radiolabeling) but no amino groups (negative ninhydrin staining)  that were consistent across all species, suggesting that these species belong to the same class   to E. Bame class . Compleme levels .14C-radiolabeled TLC revealed faint spots for GPDGDAG. Since it was not possible to discern any differences in GPDGDAG levels via TLC  (which is polyglycerophosphate polymerized onto a GPDGDAG membrane anchor) might be affected as well  surrogate standard . ΔmprF1 pe level . The sampe level .mprF1 ΔmprF2 while CL appeared unchanged   , F. All mprF deletion resulted in alterations in phospholipid and glycolipid levels, we hypothesized that these altered levels might be due to altered expression of genes involved in lipid metabolism. To test this hypothesis, we performed RNA sequencing to compare the gene expression profiles of the wild type and the mprF mutants. In total, 301 genes (14% of the genome) were differentially expressed between the wild type and ΔmprF1 ΔmprF2  were downregulated in the double mutant  Global gene expression profile comparing E. faecalis OG1RF with ΔmprF1 ΔmprF2 . Gene functional categories are based on KEGG pathway membership and manually curated for category assignment. The majority of the differentially expressed genes were categorized under metabolism and membrane transport. A vertical black line represents the median value for each category. (B) Principal component analysis of global gene expression profile comparing E. faecalis wild type (orange circle), ΔmprF1 (green triangle), ΔmprF2 (blue square), and ΔmprF1 ΔmprF2 (purple cross). Smaller symbols represent the spread of each biological replicate and larger symbols represent the centroid of their respective clusters. The two dimensions measured account for 70.6% of the variability of all differentially expressed genes. (C) Venn diagram represents differentially expressed E. faecalis genes in the mprF mutants relative to wild type. The number of genes (FDR < 0.05) in each section was calculated from mprF1 ΔmprF2 showing a slight increase in pgsA and decrease in fatty acid biosynthesis gene expression levels. Each bar represents the mean ± standard error of measurement calculated from 4 biological replicates averaged from 3 technical replicates each. **, P < 0.01; ****, P < 0.0001; Fisher’s least significant difference (LSD) test for ANOVA. Download FIG S5, PDF file, 0.2 MB.ΔCopyright © 2023 Rashid et al.2023Rashid 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 de novo fatty acid biosynthesis is downregulated in ΔmprF1 ΔmprF2, we speculated that this mutant might require exogenous fatty acids for growth and survival. E. faecalis can incorporate exogenous fatty acids into its membrane when grown in bovine heart infusion (BHI) culture medium (16:0] and stearic acid [C18:0])  lacking fatty acids, while growth of the single mutants was less severely impaired  . We thusimpaired . Supplemimpaired  or stearimpaired  promotedimpaired  promotedimpaired  and D. Uimpaired  and F. Oimpaired , H and I10.1128/mbio.03073-22.6FIG S6mprF mutants cultured in chemically defined media (CDM) while high concentrations of unsaturated fatty acids, myristic acid, and lauric acid fail to promote growth. Growth curves of wild type and ΔmprF1 ΔmprF2 grown under fatty acid supplementation of (A) palmitic acid (C16:0) or (B) stearic acid (C18:0) at lower concentrations of 62.5, 31.25 or 15.63 ng/mL.  Growth curves of combinations of palmitic and stearic acid in a 3:1 ratio at varying concentrations. Growth curves of wild type and ΔmprF1 ΔmprF2 grown under higher concentrations of unsaturated fatty acid supplementation of (E) palmitoleic acid (C16:1 cis-9) or oleic acid (C18:1 cis-9) and (F) cis-vaccenic acid (C18:1 cis-7) or linoleic acid , as well as (G) lauric acid (C12:0), myristic acid (C14:0) or palmitic acid (C16:0) and (H) stearic acid (C18:0) or arachidic acid (C18:0). Fatty acids were supplemented at 5 μg/mL. (I) Growth curves of 1, 5, and 10 μg/mL of arachidic acid. Equal volumes of ethanol (EtOH) were used as the solvent control for comparison. Total fatty acid concentrations are shown. Data points for all graphs represents the mean ± standard error of measurement calculated from 3 biological replicates averaged from 3 technical replicates each. Fatty acid abbreviations:  and . For curves that overlap the blank, arrows corresponding to their respective colors are shown next to the blank curve. Download FIG S6, PDF file, 0.8 MB.Saturated fatty acid supplementation at low concentrations promotes growth of Copyright © 2023 Rashid et al.2023Rashid 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/mbio.03073-22.7FIG S7mprF1 ΔmprF2. (A) Proportion of acyl-ACPs within WT and ΔmprF1 ΔmprF1. Values obtained through mass spectrometry were normalized against internal standards and total protein concentration before conversion to proportions. Error bars represent the standard error of mean from 4 biological replicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; Fisher’s LSD test for ANOVA. Control experiments show that the Laurdan assay can sensitively and accurately measure differences in membrane fluidity. Wild type cells grown at a range of temperatures that result in different membrane fluidities (B) and exposure to the membrane fluidizer, benzyl alcohol (C). Benzyl alcohol was spiked in just after the initial reading at time, t = 0 minutes. Experiments were done in a microtiter plate set-up with measurements taken using a plate reader instead due to the technical constraints in the microscope set up in FIG S7, PDF file, 0.1 MB.Acyl-ACP analysis and control experiments testing sensitivity of the Laurdan dye. Analysis of individual species of acyl-ACPs in WT and ΔCopyright © 2023 Rashid et al.2023Rashid 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 Lactococcus lactis uses the multiple antibiotic resistance repressor (MarR) family repressor FabT to regulate expression of the fab gene operon  species that have long-chain acyl moieties  amino acid sequence present at the acyl attachment site, leaving behind a DSL peptide connected to 4’-phosphopantetheine and an acyl-group, which was then detected using mass spectrometry. The conserved nature of the Asp-N cleavage site and consistent structure of the digestion products makes quantification of acyl-ACP species possible  possesses the conserved DSLD sequence, while AcpB (the ACP involved in uptake of exogenous fatty acids) does not . Gas chromatography analysis of total fatty acid methyl esters (GC-FAME) was performed on lyophilized cell pellets of the wild type and ΔmprF1 ΔmprF2 grown in either BHI or CDM. The fatty acid profiles of the wild type and ΔmprF1 ΔmprF2 grown in BHI were very similar , ~11% less cis-vaccenic acid (C18:1 ω7 cis), and ~2.5% less C19 cyclo ω7 acid relative to the wild type, whereas the wild type showed a decrease of 2% and 3% in palmitic acid (C16:0) and stearic acid (C18:0), respectively, and an ~6% increase in cis-vaccenic acid (C18:1 ω7 cis) compared to growth in BHI , an ~7% decrease in cis-vaccenic acid (C18:1 ω7 cis), and an ~3.5% decrease in C19 cyclo ω7 relative to growth in BHI  Secretion. Anionic membrane phospholipids promote efficient secretion via the generalized Sec pathway  Membrane fluidity. The fluidity of the cell membrane is influenced by the amount of unsaturated lipids present, where a higher degree of unsaturation results in more fluid membranes (mprF mutants (o) or disordered (Ld) the local lipid environment is, its emission spectra will blue-shift (in Lo regions) or red-shift (in Ld regions), respectively  Biofilm formation. In E. faecalis strain 12030, the loss of mprF enhances biofilm formation  across all daily time points over a 5-day period  leads to unexpected lipidomic, transcriptomic, and functional changes in E. faecalis OG1RF, ΔmprF1, ΔmprF2, and ΔmprF1 ΔmprF2. One advantage of such methods is that they are much more sensitive than traditional thin-layer chromatography (TLC), enabling a more comprehensive understanding of lipid homeostatic shifts that are dependent upon mprF  than ΔmprF1 , L-PG is completely absent in ΔmprF2 (mprF1 present). The unexpected observation that MprF1 alone does not synthesize L-PG suggests that MprF2 expression is necessary for MprF1 to function. This possibility warrants further investigation.In the current study, we used previously established mass spectrometry-based methods  to analypon mprF . When wen ΔmprF1 . Unlike mprF2 and ΔmprF1ΔmprF2 have significantly lower PG levels than wild type and ΔmprF1. This is a novel and unexpected finding because mprF acts downstream of pgsA, one of two genes  involved in PG synthesis . Thus, only a fraction of the lipidome was involved in adaptive remodeling. Different sets of lipidomic features  were involved in responses to changes in temperature, high salt concentrations, and stationary growth phase. One of the more striking observations was that varying the degree of acyl chain saturation in PE, PG, or phosphatidylcholine (PC) had varying effects on lipid packing, a property that is correlated with other physical properties, including viscosity , as previously described . For metE. faecalis were quantified by LC-MS/MS using multiple reaction monitoring (MRM) using a previously described methodology  . The QToF instrument was set to positive ion mode, at an electrospray voltage of −3,500 V (Vcap), a temperature of 200°C, a drying gas rate of 14 L/min. Spectra were acquired in auto-MS2 mode with MS1 acquisition rate at 4 spectra/s and the MS2 acquisition rate at 20 spectra/s with fixed collision energy at 40 eV. The list of detected species of DGDAG in the samples can be found in To determine the species of DGDAG present in m/z, 1,071.6; MS2 m/z, 153.0. Integrated peak areas were normalized against cell weight.Due to the absence of suitable internal standards, semiquantitative analysis of DGDAG was carried out instead. Lipid extraction was performed as described above without addition of internal standards. Analysis of DGDAG lipid species was performed by LC-MS/MS via MRMs using monoglucosyl-diacylglycerol (MGDAG) 34:1 as a surrogate standard  for extemprF1, ΔmprF2, and ΔmprF1 ΔmprF2 strains. Detailed methods are described in the supplementary information file.Sequencing of RNA was done from OG1RF, Δ15N acyl-ACP standards  were then spiked into the sample at 5-μM equimolar concentrations. Proteins were precipitated from standard spiked lysates via TCA-precipitation and resuspended in 50 mM MOPS buffer, pH 7.5. Resuspended proteins were then treated with the Asp-N protease at 20:1 ratio (protein:enzyme) at 37°C overnight. Reaction was then stopped by addition of methanol to a final concentration of 50%. These samples were then analyzed using LC-MS with MRMs using previously described solvent gradients and MRM parameters  or CDM (72 h) were lyophilized and sent together with powdered BHI for GC-FAME analysis at the Identification Service of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. Cellular fatty acids were converted into fatty acid methyl esters (FAME) and analyzed by GC-MS using used C21:0 FAME in a defined amount per biomass as internal standard for normalization.Late stationary phase cultures of wild type and Δ14C]-acetate or [32P]-disodium phosphate  was added into 5 mL of media at 0.2 μCi/mL or 1 μCi/mL, respectively, before culturing strains overnight at 37°C for 16 to 18 h at static conditions. Lipids were then extracted as previously described and resuspended in 50 μL of chloroform-methanol solution (1:1 vol/vol) (Radiolabeling of lipids was performed as previously described with the following modifications : [14C]-avol/vol) . Next, 1SDS-PAGE and Western blot were performed as described in a previous study . 4–12% o600) readings of the cultures were measured. Cell-free supernatants were obtained by centrifugation at 6,000 rcf for 5 min at 4°C and filtering the supernatants into fresh Eppendorf tubes using 0.2-μm syringe filters. Next, 1.6 mL of filtered supernatant was mixed with 400 μL of 100% wt/vol tricholoroacetic acid (TCA) solution (1:4 ratio of TCA to sample) and incubated at 4°C for 10 min. Tubes were centrifuged at 20,000 rcf for 15 min at 4°C. The precipitated protein pellet was washed once with 2 mL of 100% ice-cold acetone and placed on a 98°C heat block to evaporate residual acetone. The pellets were resuspended in 500 μL of PBS. Twenty-five μL of these protein solutions were used for estimation of protein content using the Pierce BCA protein assay kit  in a microtiter plate format according to the manufacturer’s protocol. Protein concentrations of the samples were then normalized to OD600 of 1.0 based on the respective OD600 readings of the individual cultures.Late stationary phase cultures were prepared by growing cultures in BHI broth overnight for 16 to 18 h at 37°C, static conditions. Optical density at 600 nm . PhoZF secretion was monitored by its ability to convert para-nitrophenyl phosphate (pNPP) into a colored product that can be measured by absorbance at 405 nm. Mid-log phase cultures of strains harboring the pABG5 plasmid containing the chimeric alkaline phosphatase enzyme (PhoZF) were normalized to an OD600 of 0.5. Cell-free supernatants were obtained by centrifuging samples at 6,000 rcf for 5 min at 4°C and filtering the supernatant through 0.2-μm syringe filters. Next, 25 μL of supernatant was added to 200 μL of 1M Tris-HCl, pH 8.0, in a 96-well microtiter plate. Then, 25 μL of 4 mg/mL para-nitrophenyl phosphate (pNPP)  was added to each well to start the reaction. The plate was placed into a Tecan Infinite M200 Pro spectrophotometer and incubated at 37°C with the absorbance read at 405 nm every 10 min for 18 h.Secretion of the strains were monitored by their ability to secrete a chimeric alkaline phosphatase, PhoZF (600 of 0.7 in PBS and incubated with 100 μM Laurdan for 10 min at 37°C. Cells were washed twice with PBS and 10 μL of the cell suspension was spotted onto PBS-agarose pads (1% wt/vol) mounted on glass slides. Coverslips were placed over the agarose pads and sealed using paraffin wax.Late stationary and mid-log phase cultures were normalized to an ODSlides were imaged using a Zeiss LSM 880 laser scanning microscope with Airyscan, using a Plan-Apochromat 63x/1.4 Oil DIC objective with an incubation chamber set to 37°C. The slides were equilibrated for 10 min within the chamber before imaging and excited using a 405-nm laser with emission collected between 419 and 455 nm (blue) and between 480 and 520 nm (green) simultaneously. Digital images were acquired using the Zen (Zeiss) software and analyzed using ImageJ. Using ImageJ, regions of interests (ROIs) of individual cells or cell clusters were selected and mean fluorescence intensities (MFIs) of each ROI for each channel were measured and tabulated in Microsoft Excel. Using the following formula, the average GP values for each ROI were calculated and then plotted using GraphPad Prism software:Laurdan was validated to be responsive to changes in membrane fluidity via control experiments subjecting stained cells to a gradient of temperatures, and membrane fluidizer, benzyl alcohol  and C.Mid-log phase cultures of the strains were tested for their daptomycin MIC using the microplate broth dilution methods, as previously described .mprF1, ΔmprF2, and ΔmprF1 ΔmprF2 across a 5-day period . These bacterial cultures were then normalized to an OD600 of 0.5 and harvested by centrifugation at 6,000 rcf for 5 min at 4°C. The supernatant was discarded, and the remaining cell pellet was washed and resuspended in 1 mL of 0.01 M low-salt phosphate buffer (PB). Bacterial resuspensions were then serially diluted 500-fold and 25 μL of each sample was added twice into a 96-well microtiter plate, for a total of 2 technical replicates. An equal volume of human β-defensin 2 (hBD2) or LL-37  was added to the samples and incubated statically for 2 h at 37°C . Then, 5 μL of bacterial suspension from each well was then spotted 3 times onto a BHI agar plate, for a total of 6 technical replicates per sample. The spotted agar plates were then incubated statically overnight at 37°C and surviving bacteria were determined by CFU enumeration.Overnight OG1RF cultures were subcultured in BHI liquid broth at a 1:10 dilution and grown to mid-log phase .RNAseq files are available on NCBI, Sequence Read Archive (SRA accession no.


Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events  than the R group . The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder ( Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae [Nidovirales and the family Coronaviridae. The SARS-CoV-2 virus triggered the breakout of the coronavirus disease 2019 (COVID-19) pandemic, which is yet ongoing. There have been few reports of nidoviruses in aquatic species, which are mostly confined to the subfamily Piscanivirinae and the family Tobaniviridae [Piscanivirinae subfamily is comprised of two genera, namely Bafinivirus and Oncotshavirus. White bream virus isolate DF24/00 (WBV DF24/00), the first fish nidovirus, was isolated in 2001 from white bream (Blicca bjoerkna) in Germany [Piscanivirinae include fathead minnow nidovirus (FHMNV) [The International Committee on Taxonomy of Viruses (ICTV) formally recognized the order Nidovirales in 1996, with four families: iviridae , there aiviridae . The Pis (FHMNV) , chinook (FHMNV) , crucian (FHMNV) , Atlanti (FHMNV) , and chi (FHMNV) . These nStudies on virus–host interactions at various levels are critical for gaining a full understanding of virus properties. To understand the molecular processes of virus infection and possible locations of intervention, a thorough investigation of gene expression following virus infection at the entire genome scale is required. High-throughput sequencing is a precise method for investigating transcriptome data on fish immune to infection with bacteria, viruses, or parasites ,10,11. HParalichthys olivaceus) is widely distributed along the Korean Peninsula, Japan, and China. In China, it is a commercially significant marine culture fish. The recent expansion of the fish culture business has resulted in several illnesses that have resulted in significant losses in the Japanese flounder population. The diseases induced by viral infection are becoming a major threat to the Japanese flounder culture [Japanese flounder , Chinook salmon (Oncorhynchus tshawytscha) embryonic tissue (CHSE), or Japanese flounder brain (JFB) cells in 6 well plates at 1:10 dilutions and was incubated at 17 °C in L15 with 5% fetal bovine serum for 20 days. The infected JFB cells and supernatant mixture were lysed for 3 cycles of freeze-thaw at −80 °C. Cell debris and contaminants were removed by centrifuging at 1807× g for 20 min at 4 °C. The purification of virions was performed using ultracentrifuge (himac CP70MX) according to the protocol described by Gao et al. [The pooled tissues were homogenized in sterile PBS (1:10 weight–volume ratio) and centrifuged at 1000× GenBank Virus RefSeq database using Bowtie software and then assembled into contigs by the SPAdes and Velvet software. After assembled contigs, annotation was conducted with five databases .To narrow down the range of candidate viruses, the RNA of virus-infected JFB cells was extracted using RNA simple total RNA kit , and cDNA was synthesized by PrimeScript RT reagent Kit with gDNA Eraser . The RNA was then used for small RNA library construction, siRNA sequencing, and analysis according to the protocol described by Wu et al. . In partTo obtain the whole genome sequence of the isolated virus, the RNA of purified virions was extracted, and cDNA was synthesized. After quality checking, the sequencing library was constructed and clustered following the manufacturer’s recommended protocols. The library was sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated. The raw reads were trimmed using Trimmomatic , and theThe full genome sequence of the isolated virus in this work, as well as additional ten nidovirus sequences retrieved from NCBI, were utilized for phylogenetic analysis. The sequences were aligned, and phylogenetic relationships were inferred using the neighbor-joining method. The phylogenetic tree was constructed with 1000 bootstrap replications of the Tamura–Nei model using MEGA X .2O. The cycling conditions were as follows: 98 °C for 3 min; followed by 35 cycles of 10 s at 98 °C, 5 s at 55 °C, and 5 s at 72 °C on a GeneAmp PCR system 9700 . The primer sequences were listed in For the detection of CSBV Bces-Po19, each reaction volume (50 μL) contained 200 ng of cDNA, 25 μL of 2×PrimeSTAR Max Premix , 300 nM of each primer, and ddHThe gills, liver, spleen, kidney, heart, and stomach of diseased fish were sampled and fixed in Bouin’s solution for 24 h before being rinsed with 70% alcohol and kept at room temperature. The samples were dehydrated in ethanol serial dilutions, sectioned (5 μm thickness), and then stained with Harris’s hematoxylin and eosin. The liver, kidney, ovary, and spleen of diseased fish as well as inoculated JFB cells containing 70% cytopathic effects (CPEs) were processed for TEM  using the previously reported methodology ; the fixWe randomly selected 3 moribund fish that displayed symptoms (Sensitive (S)) and 3 fish that had no symptoms (Resistant (R)). Each fish’s head kidney was immediately frozen in liquid nitrogen for 48 h and kept at −80 °C until use. The TRIZOL Kit was used to extract RNA . To remove genomic DNA from extracted RNA, RNase-free DNase I was used. The quality of the extracted RNA was evaluated using an Agilent 2100 Bioanalyzer System . The presence of virus in all S and R samples was confirmed by PCR using specifically designed primers  in the iThe sequence data obtained were processed with the SMRTlink 5.0 software. Circular Consensus Sequence (CCS) was created from subread BAM files with the following parameters: min_length 200, max_drop_fraction 0.8, no_polish TRUE, min_zscore −9999, min_passes 1, min_predicted_accuracy 0.8, and max_length 18,000. The output of CCS BAM files output was acquired as full-length reads and non-full-length reads using pbclassify.py script, ignore polyA false, and minSeqLength 200. The non-full-length and full-length fasta files were sent into the cluster step, which involved isoform-level clustering (ICE), followed by final arrow polishing, hq_quiver_min_accuracy 0.99; bin_by_primer, false; bin_size_kb, 1; qv_trim_5p, 100; and qv_trim_3p, 30. Using the Illumina RNA-Seq data and the LoRDEC program, further nucleotide mistakes in consensus reads were repaired . FollowiUnmapped transcripts and novel gene transcripts obtained from gene structure analysis were annotated as NT using BLAST ; the software Diamond BLASTX  was used to annotate to NR, KOG/COG, Swiss-Prot and KEGG; and the software of Hmmscan  was used in Pfam database analysis. p-value of 0.05 were set as the threshold for significantly differential alternative splice. The full-length transcript that was mapped on two or more loci in the reference genome with more than 99% alignment coverage and having the locus 100 kb apart from each other on the reference genome and a 10% alignment of each gene locus to the corresponding transcript was predicted as a fusion transcript [TAPIS was used to analyze the gene structure of polished isoforms. The exon-intron structure of each transcript was predicted. Newly found loci and isoforms were identified by comparing them to the reference genome annotation, using the same criteria as for loci and isoform identification . Alternaanscript . The animal TFDB 2.0 database was used for transcript factor (TF) analysis . The trahttps://bigd.big.ac.cn/gsa (Unpublished Data).Total RNA from S1 to S3 and R1 to R3 were prepared separately for RNA-Seq. Briefly, the sequencing libraries were created with the NEBNext UltraTM RNA Library Prep Kit for Illumina  and then tested for quality with an Agilent Bioanalyzer 2100. Following library preparation, index-coded samples were clustered, and libraries were sequenced using an Illumina Hiseq XTen platform, yielding paired-end reads. The raw sequencing data presented in this paper were deposited in the Genome Sequence Archive in National Genomics Data Center, Beijing Institute of Genomics , Chinese Academy of Sciences, under accession number CRA003368 that are publicly accessible at p < 0.05 were differentially expressed. Raw reads were cleaned before being mapped to the Japanese flounder reference genome  using Hip < 0.05 were deemed significantly enriched. The statistical enrichment of DEGs in KEGG pathways was tested using KOBAS (v3.0) [GOseq (v1.10.0) was used to perform a gene ontology (GO) enrichment study of differentially expressed genes (DEGs) . GO keywS (v3.0) , which w−ΔΔCt method was used to analyze gene expression levels. qRT-PCR was used to confirm the RNA-Seq data. Ten DEGs with high levels of significance were selected for qRT-PCR analysis, with the 18S gene serving as a control . The qRTThe moribund fish exhibited bleeding on their fins, muscles, liver, gonads, etc. . The infThe infected EPC, CHSE, and JFB cells all demonstrated severe CPEs that resulted in large plaques of detached cells and the loss of the monolayer . The infThe isolated virus was named as CSBV Bces-Po19, and its full-length genome sequence (NCBI accession no.: OM830952) was 26,597 nt in length, including a 5′ UTR (959 nt) and five open reading frames (ORFs) that encode ORF1a , ORF1b , spike glycoprotein , membrane protein , nucleocapsid protein , and a 3′ UTR (215 nt) A. Oncotshavirus, both nucleotide and amino acid homology analysis were performed. Phylogenetic analysis of the viral genome placed the CSBV Bces-Po19 in the Oncotshavirus genus, within the Tobaniviridae family of Nidovirales, forming a clade with CSBV WHQSR4345 (NCBI accession No. MG600027) and CSBV HB93 (NCBI accession No. MH171482) (To identify the genetic relationship between CSBV Bces-Po19 and other viruses belonging to the genus H171482) B. The comparison of complete genome sequences in GenBank Blast searches showed that the CSBV Bces-Po19 isolate had a similarity of 93.17–98.62% (in nucleotides) to other aquatic nidoviruses isolates, with the highest percent identity to CSBV WHQSR4345. Nucleotide and amino acid sequence analyses of five ORFs are shown in Using the designed CSBV Bces-Po19-specific primers, bands with 757 bp were detected in CSBV Bces-Po19-infected JFB cells, while no band was detected for the control cells. However, the internal reference gene β-actin was found in all samples in both control and infected cells . This reThe primers used in this experiment were designed to amplify ORF1a of CSBV Bces-Po19. However, due to the close species relatedness, CSBV Bces-Po19 primer pairs could cross-react with CSBV WHQSR4345, CSBV HB93, and CSBV Cefas-W054. As such, the viral species should be initially screened by PCR and then further validated through whole-genome sequencing.The ultrastructural examination was performed on the liver, kidney, ovary, and spleen of Japanese flounder infected with CSBV Bces-Po19. Cell necrosis was the most common pathogenic alteration in all four tissues. The spherical virions were visible, measuring 55–110 nm in diameter, and featured surface spikes that protruded 14–21 nm from the virion wrap A,B. The Pathological alterations in CSBV Bces-Po19-infected JFB cells included necrosis and extensively disrupted spherical and bacilliform virions A–C. In tFrom the head kidney samples of CSBV Bces-Po19-sensitive and -resistant Japanese flounder, Illumina sequencing yielded 41,175,958 to 55,892,426 clean reads. The Japanese flounder reference genome was uniquely mapped to an average of 90.13% of clean reads . FPKM vaThe Iso-Seq on PacBio Sequel machine produced 23.31 and 24.30 G polymerase read bases for CSBV Bces-Po19 R and S samples, respectively . After eThe polished consensus reads were adjusted with Illumina RNA-Seq data to reduce further nucleotide mistakes in full-length sequencing reads. After correction, 463,495,705 (R) and 535,777,939 (S) nucleotides were retrieved, representing 99.86% and 99.84% of the total nucleotides before correction, respectively . The N50The polished consensus reads, after correction, were mapped to the reference genome of Japanese flounder, yielding a mapping rate of 86.84% (R) and 84.60% (S), respectively . There wDEG analysis was used to analyze the transcriptome profile of Japanese flounder in response to CSBV Bces-Po19. A comparison of the CSBV Bces-Po19 S and R transcriptomes revealed 1444 DEGs, 931 of which were highly up-regulated and 513 of which were significantly down-regulated A. A heatA KEGG pathway analysis found that 129 pathways were implicated in the Japanese flounder’s reaction to CSBV Bces-Po19. Ribosome biogenesis in eukaryotes, metabolic pathways, RNA transport, spliceosomes, and pyrimidine metabolism are the top five enriched pathways B. IntereSUPPA software was used to identify seven types of AS events: skipped exon (SE), mutually exclusive exon (MX), alternative 5′ splice site (A5), alternative 3′ splice site (A3), retained intron (RI), alternative first exon (AF), and alternative last exon (AF) (AL). There were 12,352 AS events in 10,743 of the total S group genes and 11,452 AS events in 12,989 of the total R group genes . This sutead3 gene had the most AS in the R group, with 52 AS, which is 25 higher than the S group. The highest number of AS was found in the S group in the slco4a1 gene, which contained 57 AS, which is 21 more than the R group  were found in three immune-related pathways, namely “phagosome”, “p53 signaling pathway”, and “NOD-like receptor signaling pathway”. Two AS isoforms were found in both the “phagosome” and “p53 signaling pathways”. Six AS isoforms were discovered in the RNA-related pathways: “RNA transport”, “RNA degradation”, “ribosome biogenesis in eukaryotes”, “spliceosome”, and “mRNA surveillance pathway” were found to have six AS isoforms . The AS isoforms eif4g1_novel05, rmb8a_novel01, eif2s2_novel01, and ran_novel05 were involved in the “RNA transport” pathway. All the AS isoforms had similar expression patterns with their related genes. A total of twelve isoforms were up-regulated, and four isoforms were down-regulated in the S group in comparison with the R group  splicing, resulting in multiple mature mRNAs from a single pre-mRNA. The AS increases transcriptome complexity and thereby the proteome diversity of a cell, and it also modulates the abundance of functional mRNAs . It is ccdk4/6, necessary for cells to enter the G1 phase from the G0 phase [A variety of viruses have been reported to cause G0/G1, S, or G2/M arrest in the host cells, hence promoting replication of progeny viruses after viral infection . InfectiG0 phase , was sig


Classification and phenotype identification of lettuce leaves urgently require fine quantification of their multi-semantic traits. Different components of lettuce leaves undertake specific physiological functions and can be quantitatively described and interpreted using their observable properties. In particular, petiole and veins determine mechanical support and material transport performance of leaves, while other components may be closely related to photosynthesis. Currently, lettuce leaf phenotyping does not accurately differentiate leaf components, and there is no comparative evaluation for positive-back of the same lettuce leaf. In addition, a few traits of leaf components can be measured manually, but it is time-consuming, laborious, and inaccurate. Although several studies have been on image-based phenotyping of leaves, there is still a lack of robust methods to extract and validate multi-semantic traits of large-scale lettuce leaves automatically.In this study, we developed an automated phenotyping pipeline to recognize the components of detached lettuce leaves and calculate multi-semantic traits for phenotype identification. Six semantic segmentation models were constructed to extract leaf components from visible images of lettuce leaves. And then, the leaf normalization technique was used to rotate and scale different leaf sizes to the “size-free” space for consistent leaf phenotyping. A novel lamina-based approach was also utilized to determine the petiole, first-order vein, and second-order veins. The proposed pipeline contributed 30 geometry-, 20 venation-, and 216 color-based traits to characterize each lettuce leaf. Eleven manually measured traits were evaluated and demonstrated high correlations with computation results. Further, positive-back images of leaves were used to verify the accuracy of the proposed method and evaluate the trait differences.The proposed method lays an effective strategy for quantitative analysis of detached lettuce leaves' fine structure and components. Geometry, color, and vein traits of lettuce leaf and its components can be comprehensively utilized for phenotype identification and breeding of lettuce. This study provides valuable perspectives for developing automated high-throughput phenotyping application of lettuce leaves and the improvement of agronomic traits such as effective photosynthetic area and vein configuration.The online version contains supplementary material available at 10.1186/s13007-022-00890-2. The morphogenesis and growth of lettuce leaves have complex regulation mechanisms. In addition to genetics, habitat can also influence the variation in their leaf structure, color, and venation. Explainable and highly distinguishable features for large-scale lettuce varieties are very important for the applications of phenotype identification and phenotype–genotype association analysis. It is impossible to find two identical leaves worldwide , but theThe basic structure of leaves is established in the early stage of leaf development. Leaf morphogenesis includes the formation of the petiole, mid-rid, lamina, and marginal structures . These iFrom an image processing perspective, data-driven image techniques such as convolutional neural networks (CNNs) and their derived models can be used for the segmentation and classification of vegetable plants/leaves , 16. CNNThis study aimed to develop an automated phenotyping pipeline to quantify detached lettuce leaves and evaluate the multi-semantic traits for large-scale lettuce cultivars. The study highlights the efficacy of an automated phenotyping pipeline in recognizing multiple semantic components of lettuce leaves and evaluating traits differences not only for leaf components but between positive and back leaves.More than 400 lettuce varieties were grown in a terraced greenhouse at the Beijing Academy of Agriculture and Forestry Sciences (BAAFS) from January 1 to February 25, 2020. Lettuce varieties included Butterhead, Celtuce love, Italian, Red Coral, Red Lettuce, Red Oakleaf, and Salad Grand Rapid (SGR) . A studiAs shown in Fig. The classical U-NET  was usedLettuce leaves could be represented using size-related and size-free traits. The size-related traits of lettuce leaf consist of common geometry indices, such as leaf area, perimeter, length, and width. These traits were calculated based on the resulted semantic components from the end-to-end semantic segmentation. However, leaves of different lettuce varieties had significant differences in size and leaf area at the same growth period. Thus, these leaves were rotated to the same orientation and scaled to the same size (width) to eliminate the effect of the size difference and improve the efficiency of the phenotyping process.Leaf normalization was performed to obtain standard “size-free” images of lettuce leaves via rotation and scaling procedures. The leaf was rotated to an upright position according to its mid-rid  Fig. A. It couFour semantic components  of positive and back leaves could be skeletonized Fig. A–D. The Geometry and color traits of each semantic component could be calculated directly using semantic segmentation results. However, more valuable descriptions could be extracted from these semantic components, such as the petiole and vein architecture. The phenotyping pipeline of lettuce leaves was proposed to calculate the petiole, vein architecture, and related statistical traits, as shown in Fig. The petiole was defined as the region along the mid-rid from the leaf base to the first lamina; thus, the mid-rid could be divided into the petiole and first-order veins. The laminas and mid-rid could be combined into a new object by a dilate operation of the mid-rid region. The max contour of the new object could contain all adjacent lamina regions, and the lamina closest to the leaf base was used to determine the petiole. Moreover, the mid-rid could divide the whole leaf into the left and right parts. Thus, the skeleton line of the mid-rid was utilized to divide semantic components  into two parts (the left and right parts).The vein architecture mainly contained the angles and distribution of the second-order veins. In this study, the lamina-based approach was used to analyze vein architecture Fig. A. All laVein angles could be calculated based on the first-order laminas (LM_L and LM_R). The contour of each lamina was simplified as a polygonal chain using fewer points based on the Douglas–Peucker algorithm. The approximated polygon retained the original contour shape (controlled by the specified tolerance), and fewer nodes were used to eliminate the ambiguity in angle calculation. The simplified polygon had a subset of the points of the original contour. Due to the normalized leaves, a default tolerance, such as five, could be applied to almost all the laminas. Among the polygon nodes, the closest point to the leaf base and mid-rid was selected as the appropriate attachment point. The corresponding vein angle of each attachment point was calculated using its two adjacent line segments of the polygon.The complex shape and structure of the lettuce leaves, such as leaf folds, twisted veins, multiple main veins, etc., presented challenges to the leaf phenotyping. For these particular leaf cases, it was difficult to extract leaf semantic components accurately by image techniques. The phenotyping pipeline could only be applied for the leaf types that conformed to data annotation specification Fig. D, not foSince data annotation was highly time-consuming and labor-intensive, only 41 representative lettuce leaves were annotated based on the specification in Fig. Leaf normalization operation was an important procedure to ensure the consistency of phenotyping analysis. Each leaf image was rotated in an angle sequence  to construct test image sets which were used to evaluate the robustness and reliability of the proposed leaf normalization method. The data normalization results for the positive and back leaves are shown in Fig. The rotation invariance of data normalization could be assessed by the scaling factors. The mean and std of scaling factors were 1.949 and eaf Fig. . The Smaeaf Fig. A. The noeaf Fig. B–G. ThisLeaf area rapidly increased during leaf vegetative growth, while the vein architecture (the mid-rid and second-order veins) slightly increased. Scaling factors could be used as an individual trait of lettuce leaves since they directly reflected leaf growth. Data normalization was valuable to extract size-related traits for evaluating lettuce leaves with large size differences. Moreover, data normalization greatly reduced the image size, thus improving the computation performance of the time-consuming techniques.Eleven traits of 112 images from 56 lettuce leaves (each leaf contained positive and back images) were manually measured to validate the computation accuracy of the phenotyping pipeline. Manually measured traits are listed in Table The comparison analysis of the 11 traits was conducted to identify the relationship between manual and computation measurements. MR_L had a higher correlation coefficient than BD_L. PE_L was determined by the position judgment of the first lamina in manual measurement. PE_L had the least correlation between manual and computation methods  were utilized to verify the accuracy of the proposed method and evaluate the trait differences between positive and back images.The common geometry traits of lettuce leaves, such as length, width, pixel area, convex hull area, were calculated based on each semantic component after data normalization  were used to represent the color traits of each semantic component of leaves. Each color space had three channels (18 channels). The mean and standard deviation of each channel was calculated. A feature set was constructed for each semantic component (each feature vector contained 18 mean color features) to identify the most important color feature of each semantic component. The orientation type (positive or back leaves) was used as a classification variable.A classification decision tree was used to determine the most important color features based on the classification variable Fig. . All colColor features of six semantic components were integrated into a feature set containing 216 color features to quantify the whole leaf color. The orientation (positive-back leaf) and semantic components were respectively used as classification variables to investigate the important scores of color features Fig. A and B. These semantic components of lettuce leaves contributed lots of accurate and quantitative traits that were difficult to be measured manually. Some traits had clear physiological and ecological significance, or are related to lettuce quality and yield, such as color differences of positive-back leaves, and area ratio of leaf veins, etc. Thus, these image-based traits were not only valuable for gene function analysis and mapping of lettuce varieties, but also could be used for classification and identification of lettuce leaves. The selection of important features were valuable for refining the description of lettuce leaf and its components. Principal component analysis (PCA) could be used for dimensionality reduction via fusion to reduce the number of features greatly. Both the single important feature or principal components could be used for leaf classification and grading. Herein, the feature sets were classified into four types , then used for PCA analysis (400 lettuce varieties). The top 10 principal components of each feature set could explain 98.415%, 97.493%, 95.138%, and 89.004% of the overall variance, respectively  of 0.77. CLR_PC4 was negatively correlated with GEO_PC1 and VEN_PC2 . The first three PCs of COM and CLR were highly positively correlated. The detailed correlation coefficients among different PCs are shown in Fig. Lettuce leaves could be clustered into different categories based on their feature set by minimizing the intra-class gap and maximizing the inter-class gap. Each type of lettuce leaves was assigned an individual label for further analysis. The hierarchical clustering based on Euclidean distance was conducted using four types of PCs Fig. . For posHere we developed a phenotyping pipeline to extract and quantify multiple semantic components of leaves for different lettuce varieties. By fine definition and image annotation, semantic segmentation models were employed to automatically decompose lettuce leaves into six types of semantic components. Through the normalization operations, leaves of great differences in size and orientation were rotated and scaled to the same level for trait quantification and comparison. The positive-back lettuce leaves were also for the first time used to evaluate the accuracy of semantic segmentation and their essential difference from the geometry, color, and venation perspectives. These semantic components contribute a large number of traits that are difficult to be measured manually and are helpful to discover the statistical differences of lettuce leaves for leaf classification and identification. The proposed method can be improved in segmentation accuracy by supplementing richer annotated leaves and be extended for leaf phenotyping of other crops.Additional file 1:Table S1. Model evaluation for six semantic components of lettuce leaves. Table S2. Geometry traits of lettuce leave. Table S3. Vein architecture traits of lettuce leaves.


The application of dendrimeric constructs in medical diagnostics and therapeutics is increasing. Dendrimers have attracted attention due to their compact, spherical three-dimensional structures with surfaces that can be modified by the attachment of various drugs, hydrophilic or hydrophobic groups, or reporter molecules. In the literature, many modified dendrimer systems with various applications have been reported, including drug and gene delivery systems, biosensors, bioimaging contrast agents, tissue engineering, and therapeutic agents. Dendrimers are used for the delivery of macromolecules, miRNAs, siRNAs, and many other various biomedical applications, and they are ideal carriers for bioactive molecules. In addition, the conjugation of dendrimers with antibodies, proteins, and peptides allows for the design of vaccines with highly specific and predictable properties, and the role of dendrimers as carrier systems for vaccine antigens is increasing. In this work, we will focus on a review of the use of dendrimers in cancer diagnostics and therapy. Dendrimer-based nanosystems for drug delivery are commonly based on polyamidoamine dendrimers (PAMAM) that can be modified with drugs and contrast agents. Moreover, dendrimers can be successfully used as conjugates that deliver several substances simultaneously. The potential to develop dendrimers with multifunctional abilities has served as an impetus for the design of new molecular platforms for medical diagnostics and therapeutics. It showed great relaxation, which confirms that Mn (II) is a potential alternative to Gd-based measures [Contrast agents used in magnetic resonance imaging (MRI) allow for the obtention of more sensitive images. The two common types of images are hyperintense images  and the MDA2 antibody targeting malondialdehyde (MDA) -lysine epitopes to significantly enhance atherosclerotic lesions . In theiThe development of nanotechnology is an innovative approach to replace the classic regimens of anti-cancer therapies and classic diagnostics. The modification of drugs and contrast agents that already exist, as well as the search for new ones, is the future and a challenge of modern medicine. The use of dendrimer-coated magnetic nanoparticles allows for the development of a drug delivery system and targeted therapy that improves the effectiveness of therapy and reduces the risk of toxicity by using a lower drug dose. In addition, implementing treatment regimens using pH-sensitive pharmaceuticals would help to reduce drug resistance during anti-cancer therapy. Therefore, it is essential to characterize dendrimers and pay attention to analytical techniques in the process of their manufacture. Depending on the dendrimer group and the surface group, it is possible to create conjugates of interest. The use of nanoparticles allows for the minimization of side effects and the adjustment of the appropriate dose of the drug, which will bring the desired therapeutic effect. Due to their properties and the ability to adjust the size of molecules, PAMAM dendrimers, used as drug carriers, can be successfully used in anti-cancer therapy. It is possible to attach specific ligands to the surface of the dendrimer, which will be recognized by receptors overexpressing cancer cells. In addition, research into new MRI contrast agents is proving to be important. The currently used contrast agents cause a number of side effects due to the high dose administered. The research focuses on the development of contrast agents based on functionalized Gd dendrimers that could be administered in clinical trials at a lower dose while maintaining the same sensitivity and diagnostics in clinical trials as traditional contrast agents. This review presents advancements in dendrimer-based research in various fields. It should be emphasized that the spectrum of the use of dendrimers is very wide. The applications of dendrimers include, but are not limited to, chemotherapy, radiation therapy, photothermia, and photodynamic therapy. In addition, they are also used in gene therapies because they allow for the condensation of nucleic acid materials. Due to the unknown metabolism of the therapies used, there is a need for further research into the use of dendrimers, despite the fact that they are characterized by higher biocompatibility and the improved effectiveness of the therapies used. Despite numerous studies, there is a need for continued research and an attempt to transfer them to in vivo research. There is a need to improve the effectiveness of the applied therapies and to understand metabolic mechanisms. In summary, the use of dendrimers in oncology is a modern approach that can overcome the limitations of standard diagnostics and therapy by improving the time required to detect neoplastic lesions and monitoring the effectiveness of the therapy. The use of dendrimers for the synthesis of new drugs or the modifications of existing drugs is a challenge of modern pharmacology. In addition, the development of effective drug delivery methods to cancer cells would allow for better therapy outcomes. The development of targeted therapies would make it possible to improve the effectiveness of treatment and minimize the negative side effects of the used therapies. It will also make it possible to bypass the disadvantages of classical diagnostic and therapeutic methods and improve the effectiveness of the used therapies.


The prevalence of chronic kidney disease (CKD) risk factors such as diabetes mellitus, hypertension, and obesity among the young Malaysians are increasing. Understanding the factors associated with CKD knowledge could assists healthcare providers to design health education programmes. There are scarce local studies on CKD knowledge and its associated factors among university students. This subpopulation comprises of young people with diverse background and characteristics. This study was aimed to assess the CKD knowledge and its associated factors among university students. A cross-sectional study was conducted among Universiti Kebangsaan Malaysia students from July 2020 to August 2020. A convenience sampling method was applied. All students were invited to complete an online survey using Google Forms that were sent to their email. The survey consisted of questions related to their sociodemographic, socioeconomics, university programme enrolled, medical history, lifestyle characteristics and CKD knowledge. The data were analysed using SPSS Statistics 26.0. Multiple logistic regression analysis was performed to identify the final associated factors after controlling for confounders. A total of 3074 students participated and 32.6% of them had below average CKD knowledge. Students of male gender, enrolment in undergraduate programmes and non-health-related faculties/institutes were more likely to have below average CKD knowledge. Students who are Chinese, from high monthly household income family and with family history of CKD were less likely to have below average CKD knowledge. Almost one-third of the students had below average CKD knowledge. The six associated factors with CKD knowledge were non-modifiable. Of the six factors, three were associated with students being more likely to have below average CKD knowledge; another three were associated with students being less likely to have below average CKD knowledge. Future health education programmes to enhance CKD knowledge should be designed focusing on students who are more likely to have below average CKD knowledge. CKD is an important global health problem as its prevalence and global burden are rising rapidly. Worldwide, approximately 200 million people have CKD [Chronic kidney disease (CKD) is defined as abnormalities of kidney structure or function, that are present for > 3 months, and present health implications. CKD is classified based on the cause, estimated glomerular filtration rate (eGFR), and albuminuria category . CKD canIn Malaysia, CKD prevalence increased from 9.1% in 2011  to 15.5%A person who is knowledgeable and aware of CKD would tend to adopt a healthy lifestyle, which could lower the risk of CKD . Early-sCKD can affect any age group, including the young. The prevalence of CKD risk factors, especially diabetes mellitus and hypertension among young adults in Malaysia has increased throughout the years . Among tUniversity students are a segment of the general population wherein the majority are young adults. This subpopulation comprises people with diverse sociodemographic, and socioeconomic characteristics and university programme enrolment. Some university students join health- or non-health related programmes that might or might not provide detail information on CKD. Regardless, university students need to be equipped with such knowledge, to understand the seriousness of CKD and its implications to not only for themselves but also their family members. The adaptation of a healthy lifestyle in early years would likely be maintained throughout a person’s lifetime  and woulUniversity students in Rwanda, East Africa, showed poor knowledge on CKD risk factors and preventive measures . FurtherMore studies should be conducted as Malaysia is a multi-ethnic country with highly diverse sociocultural characteristics. Such studies could aid recognition of the knowledge gap in certain sub-population, who could be disproportionately affected by CKD, based on factors such as age and ethnicity . TherefoA cross-sectional study was conducted among undergraduate and postgraduate students of Universiti Kebangsaan Malaysia (UKM) from July to August 2020 to assess CKD knowledge and its associated factors. UKM is a public university which was established in 1970 and has since become a reputable research public university . The maiA convenience sampling method was applied in which students were invited to participate in the study via email; their email addresses were provided by the UKM Centre of Academic Management. The current study adhered to the tenets of the Declaration of Helsinki. Study approval was obtained from the UKM Research Ethics Committee (research code: FF-2020-271). The inclusion criteria were Malaysian citizens, aged ≥18 year and full-time students. The exclusion criterion was self-reported history of CKD. The sample size was calculated using PS software  at 5% siThe students were invited to participate in July–August 2020. A set of questionnaire prepared in Google Forms was distributed via the students’ email addresses. Informed consent was provided in the first section of the online survey form where they are fully informed as to the intent and purpose of the study. The written informed consent has been waived by the UKM Research Ethics Committee. No minors were involved in this study. Students who agree to have their data used for the study, can click an “I Agree” button and have their data submitted online. Students who do not agree to have their data used in the study, can click an “I Do Not Agree” button and their data is not submitted and collected online. We opted to use this method because the study was conducted when Malaysia was under a Movement Control Order following the coronavirus disease 2019 (COVID-19) pandemic outbreak. Students were prohibited from being on-campus to curb the pandemic, and all teaching and learning sessions were conducted online. The questionnaire consisted of two sections: Section A, which assessed the student’s sociodemographic, socioeconomics, university programme enrolled, medical history and smoking status.Section B consisted of a validated CKD knowledge questionnaire from a study from Singapore . ApprovaAnatomy: How many healthy kidney(s) does a person need to lead a normal life? (correct answer: one)Physiology: What is the function of a kidney in the human body? (correct answer: to filter waste products in the blood)Aetiology: What can cause kidney disease? Presentation: What are the symptoms of early kidney disease that might progress to kidney failure? (correct answer: can present without any symptoms/ complaints)Progression: Which of the following statement(s) about kidney disease is incorrect? (correct answer: identified that CKD cannot be cured with medication)Resources available: Where can dialysis treatment be carried out? Treatment: What is the best medical treatment for end-stage kidney failure? (correct answer: kidney transplant)Each correct answer was awarded 1 point; incorrect answers or ‘I don’t know’ were awarded 0 points, yielding a maximum score of 7 points and a minimum score of 0 points.The study outcome was below average knowledge, which was defined as a score for <4/7 correct answers. Average CKD knowledge was considered a score for ≥4/7 correct answers . The socUniversity programme enrolled comprised programme level (undergraduate or postgraduate), faculty/institute  and years of study. Medical history included personal medical history  and family history of chronic illness . Smoking status was assessed by asking ‘Have you ever smoked ’ and was categorized as yes or no.n) and percentage (%). Normally distributed data were described using the mean and standard deviation (SD). The crude odds ratio (COR) and its corresponding 95% confidence interval (CI) were determined using simple logistic regression. Adjusted analysis of multiple logistic regression analysis (forward likelihood ratio) was performed to identify the adjusted OR (AOR) of the final factors associated with below average CKD knowledge after controlling for possible confounders. The final model was tested for all possible two-way interactions (multiplicative interaction) between the independent variables, and its fitness was assessed. Statistical significance was set at p < 0.05.The data were analysed using SPSS Statistics 26.0 . Categorical data were described as the frequency  years. The majority of the students were young adults . The majority were female (76.4%) and Malay (72.5%). Most of the respondents (70.5%) were not working and 55.2% were in the B40 monthly household income group. A total of 80.6% and 85.3% were single and did not have children, respectively. Almost two-thirds of the students (62.5%) were undergraduates at non-health-related faculties/institutes (69.9%). Most of the students (86.7%) did not have any personal medical history, family members with chronic illnesses (73.8%) and non-smokers (91.5%). Out of the 3074 students, 32.6% (95% CI: 31%-34.3%) had below average CKD knowledge. Approximately half of the respondents answered correctly for knowledge on anatomy of the kidney (43.5%). Most of the students answered correctly for knowledge on kidney physiology or function (96.5%) and CKD aetiology (67.5%). However, approximately one-third of the students answered correctly for CKD symptom presentation (38.4%) and knowledge on CKD progression (34.7%). More than half of the students (53.8%) answered correctly for knowledge of resources of dialysis treatment centres. Most of the students correctly answer the best treatment for end-stage renal failure (81.7%). Despite the low response rate in this study, the sample size was relatively larger than that of a previous local study  involvinCKD is a common and growing public health problem that requires everyone to have adequate knowledge to reduce its development and progression. Our results indicate that almost one-third of UKM students have below average knowledge of CKD. This is lower than that of a local study that showed that 43.5% of respondents had below average knowledge . In thatOur study reported a high proportion of correct answers for physiology as compared to a Greek study involving primary school pupils . In thatA greater proportion of students in our study correctly identified the risk factors (aetiology) and best medical treatment for end-stage kidney failure in our student’s population as compared to the general populations , 35. ThiThe low knowledge could be due to health education on disease progression commonly being designed for high-risk groups or patients with CKD . It is cThe present study showed that male students were more likely to have below average CKD knowledge. The below average CKD knowledge among male students is possibly due to sex differences in health information-seeking behaviour, which requires further evaluation. A study among college students in China suggested that male students are less likely than female students to seek health information on self-care, disease prevention and self-medication . Female Here, we found that Chinese students and students from high monthly household income families were less likely to have below average CKD knowledge. The association of ethnicity with CKD knowledge is inconsistent with that from a local study  and fromThere is limited information on the association of household family income and CKD knowledge among students for further comparison. However, a review has suggested that a higher household income enables parents to acquire better goods such as healthy diet, books and learning materials for their children to thrive better, including cognitive development and health . MoreoveThe present study indicates that undergraduate students and students from non-health-related faculties/institutes were more likely to have below average CKD knowledge compared to postgraduate students and those at health-related faculties/institutes. A descriptive study of university students in the United States showed that CKD knowledge was low in this population, particularly among the younger students . We postApart from the level of educational attainment, the students’ health literacy might have been influenced by their younger age. A Malaysian Health Literacy Survey showed that the Malaysian aged 18–24 years had a higher proportion of limited health literacy compared to Malaysian aged 25–39 years . A GermaThere is a scarcity of information measuring the association between faculty/institute type with CKD knowledge. Most recent studies have involved students either in health-related , 46 or nThe strength of our study included the participation of Malaysian students of varying ethnicities and backgrounds. To our knowledge, this is among the first local studies on CKD knowledge and its associated factors that include both undergraduate and postgraduate students across all courses and programmes. The findings presented were also adjusted for confounders. Furthermore, our findings can be used for formulating future studies. Our study has several limitations. This was a single-centre, cross-sectional study that used non-probability sampling. Causal inferences may not be drawn, and the findings cannot be inferred to the whole student population. Selection bias may be present, where the participating students could be more health conscious and more inclined to answer the questions. There is the possibility of information bias, as the students could have sought the correct answers prior to answering the survey, resulting in overestimation of CKD knowledge in this study.Despite the involvement of content experts (nephrologists and primary care physicians) in the questionnaire development, the authors suggested that further studies should be performed with a more refined questionnaire . We postFurthermore, the questionnaire validation was not clearly explained. A questionnaire is one of the most common techniques used to measure the knowledge level in a community . A validFactors that we hypothesized earlier that would influence CKD knowledge, such as health information-seeking behaviour and health literacy, should be examined further. More studies are needed to explore the types of knowledge , which influence motivational factors (perceived health threat, attitude towards health action, attitude towards health outcome and subjective norms that lead to decisions .Stratified analysis based on programme level and of faculty/institute type should also be investigated as the students have different roles and obligations that might influence their knowledge level. Our study could provide a foundation for intervention strategies for improving the CKD knowledge of this young population. Although CKD prevalence in childhood is uncommon and children were not our focus study population, we hope that our findings highlight the need to design a school education programme on kidney function and the CKD risk factors as early as the primary school level. Such a programme may ensure a healthy and health-literate population starting from a young age . In addiAlmost one-third of the students in this study had below average CKD knowledge. The associated factors were non-modifiable and included male gender, Chinese ethnicity, high monthly household income family, undergraduate students, enrolment in non-health-related faculties, and family history of CKD. Future health education programmes or training to enhance CKD knowledge should be designed focusing on students who are likely to have below average CKD knowledge.