FWE < 0.05). These serum analytes were key variables in the design of novel multivariate linear mixture models. The models classified FS risk with higher accuracy than univariate approaches. The designed bi-linear classifier achieved a sensitivity/specificity of 82%/89% and was closest to the gold-standard classifier. A multivariate model assessing FS recurrence provided a difference (pFWE < 0.05) with a separating sensitivity/specificity of 72%/69%. Iron deficiency, height percentile, and age were significant FS risk factors. In addition, height percentile and hemoglobin concentration were linked to FS recurrence. Novel multivariate models utilizing blood iron status and demographic variables predicted FS risk and recurrence among infants and young children with fever.Our goal was to identify highly accurate empirical models for the prediction of the risk of febrile seizure (FS) and FS recurrence. In a prospective, three-arm, case–control study, we enrolled 162 children . Participants formed one case group (patients with FS) and two control groups . The impact of blood iron status, peak body temperature, and participants’ demographics on FS risk and recurrence was investigated with univariate and multivariate statistics. Serum iron concentration, iron saturation, and unsaturated iron-binding capacity differed between the three investigated groups (p Fever is not triggered by metabolic disorders or central nervous system (CNS) infection; both etiologies need to be excluded in the differential diagnostic workup. Children aged four months to 5 years are mostly affected with the peak incidence at 18 months of age4. Besides an increased risk of epilepsy5 and psychiatric disorder6, the recurrent FS (RFS) represent the most common long-term effect of FS6 with an estimated 14–24% of the recurrence within the same febrile illness2. Overall, the recurrence decreases with age from 50% in children younger than 12 months at the first FS to 30% afterward. After a second FS, the probability of additional FS is 50%4. The accurate prediction of FS/RFS can outline strategies to prevent FS and mitigate the burden of FS on a child’s health or avoid repeated hospital visits, which may deepen the anxiety in children and families4.Febrile seizures (FS) are the most common convulsive disorder in childhood, usually associated with a fever of 38 °C (100.4°F) or higher and an incidence of 2–11%8, thus, estimating the FS/RFS risk with higher accuracy. Iron deficiency (ID) is frequent in infants and toddlers with the concurrent peak age as FS8. The association of iron status and febrile seizures (FS) has been postulated but supported by equivocal or inconclusive reports11. This controversy may be explained by cultural and geographic differences, as iron status is closely linked to socioeconomic status, malnutrition, and weaning practices11.Multiple FS/RFS risk predictors have been proposed such as body peak temperature, iron status, electrolyte imbalance, age, sex, and genetics, but inconsistent or contradictory results across various studies were observed. Combining more than one predictor may drive the risk prediction higher12, of individual variables. Then, we designed multivariate linear mixture models sensitive and specific to FS risk and recurrence.In this work, we collected blood iron status and demographic data in prospective cohorts of children with FS, RFS, fever without seizures, and afebrile healthy controls. Next, we investigated separation ability, i.e., sensitivity (SE) and specificity (SP)This prospective case–control study was performed at the University Hospital Brno, Czechia, from April 1, 2015 to August 31, 2017 with a subsequent 5-year follow-up  under the Masaryk University Ethics Review Board approval and was conducted in accordance with the ethical principles of the Declaration of Helsinki. The informed consent form was obtained from every participant's parent/legal guardian prior to the study enrollment.4. The FS group consisted of 53 children  aged 4–70 months and formed two subgroups; non-recurrent FS  and RFS . Three children  presented complex non-recurrent FS ; all the other FS children presented with a simple FS. Fifty-three children , aged 6–70 months, had a febrile illness caused by respiratory or urinary tract infection but without seizures. The healthy control group, recruited from children coming for a regular preventive care exam, comprised 56 children  aged 6–67 months. Exclusion criteria were age below four or above 72 months, peak body temperature ≤ 37.5 °C (99.5°F) for febrile groups, psychomotor developmental delay, malnourishment, seizures lasting more than 15 min, focal signs or lateralization in a neurological exam, epilepsy, genetic epilepsy with febrile seizures plus, antiepileptic-drug usage, history of afebrile seizures, history of CNS infection or severe head trauma, electrolyte, glucose, or homeostasis imbalance. Children suffering from chronic illnesses such as cardiovascular, renal, rheumatological, or malignant diseases, hemoglobinopathies, or other blood disorders that are associated with a higher likelihood of anemia were excluded. Demographics are summarized in Table A total of 162 Caucasian children were enrolled and formed one case group (FS group) and two control groups . Inclusion criteria were age 4–72 months, electroencephalograph (EEG) without epileptiform abnormality and normal background activity corresponding to age (FS group), normal neurodevelopment, and neurological exam. The diagnostic criteria of FS followed the American Academy of Pediatrics clinical guidelinesEach participant underwent a blood draw with the analysis of red blood cell count (RBC), hemoglobin (HGB), serum iron (Fe), iron saturation (satFe), ferritin (Fer), transferrin (TF), and unsaturated iron-binding capacity (UIBC). In FS and RFS patients, electrolytes and vitamin D were also measured. Blood draw analysis results, peak body temperature, age, sex, gestational age (GA), height and weight percentiles were utilized in between-group difference testing and multivariate statistical modeling. In addition, all available screening values for all seizures were reported in participants with RFS. The precise FS duration and interval between fever onset and FS were not collected as these parent-reported outcomes tend to be inaccurate. The study participants were followed up for five years to record RFS or treatment for ID or anemia.pFWE < 0.05; FEW—family-wise error correction; non-corrected p < 0.05 was considered as a trend in the data). For variables demonstrating significant differences between case and control groups, a maximal sum of SE + SP defined the variable-specific separating threshold  investigated the presence of mutual linear relationships between variables .Pearson cross-correlation analysis  that inx0 is the constant member and the vector ϵ is Gaussian random noise. Vectors xm where index m ∈ {1, 2,…,n} represent n variables  significantly contributing (p < 0.05) to the expected signal y. Coefficients βm define magnitudes of contributions. The crucial part of linear mixture modeling is the definition of the expected signal y.The vector model1, model2, or model3) with three different expected signals  were designed and tested. In model1, y1 equals 0 at positions of healthy controls, equals 1 at positions of patients without FS, and 2 at positions of patients with FS. In the model2, only patients were considered, and y2 equals 0 at positions of patients without FS and 1 at positions of patients with FS. In model3, only patients with FS were considered, and y3 equals 1 at positions of patients with non-recurrent FS and equals 2 at positions of patients with RFS. Model-specific Wilcoxon rank-sum test, SE, SP, and the separating threshold maximizing the SE + SP sum were evaluated in the same fashion as for the univariate approach while getting closer to the gold standard classifier was the set goal.Three models  of both models into one bi-linear model y12  groups to minimize the risk of imbalanced learning14. As initial conditions were randomized, each model training was repeated 5000 times to test and guarantee model stability and reliability. Moreover, sex was also used as a binary input variable in the regression.Continuous biological factors, such as age, gestational age, height percentile, and weight percentile, were additional inputs for the linear mixture modeling via the step-wise linear regression for modely  frequency occurrence of the most common model (a priori defined by the full 100% dataset size); averages and variances of (ii) regression coefficient; (iii) explained variance; (iv) Pearson correlation coefficient between modeled and predicted signal https://github.com/umn-milab/febrile-seizure-blood-models (release r20231005).Raw input anonymized data and MATLAB language script (written in version R2018b) making statistical testing and deriving the regression models are available under the GNU General Public License version 3 at: https://www.mathworks.com/matlabcentral/fileexchange/74204-corrplotg.Tools for cross-correlation analysis are available under the same license at: The MATLAB basic programming environment, MATLAB Statistics, Machine Learning Toolbox, and Econometrics Toolbox licenses need to be available to an end-user for full program compatibility.© 2015, Dominic Siedhoff: https://www.mathworks.com/matlabcentral/fileexchange/50541-adasyn-improves-class-balance-extension-of-smote.The MATLAB implementation of the adaptive synthetic sampling is available in the ADASYN toolbox under the copyrightIron status results and demographics are summarized in Table Figure Serum electrolytes and vitamin D did not differ between FS and RFS groups. Sodium concentrations were 133 (130–137) mmol/L in FS and 133 (131–138) mmol/L in RFS. Vitamin D concentrations were 89.9 (46.9–135.1) nmol/L in FS and 77.0 (45.3–105.2) nmol/L in RFS.As expected, height and weight percentiles were linearly dependent. In addition, several blood iron status variables were mutually cross-correlated. Demographics and iron status were not significantly correlated, except for the positive correlation between age and hemoglobin. A detailed view of the cross-correlation analysis is shown in Fig. 1 or model2) or between case sub-groups  were defined.Multivariate linear mixture models with enhanced separating properties between case and control groups  between non-seizure and seizure patients with the separating threshold 0.5744 ± 0.0317. The quantitative characteristics of the estimated model1 . The quantitative characteristics of the estimated model2  of mode1 (Eq. ) and mod (2 (Eq. ) formed  , which improved predictive power to FS recurrence when compared to model1 (p = 0.0032) or model2  over the brain, heart, and skeletal muscles16, implies that ID may result in impaired neurodevelopment presenting with various degree of intellectual disability. Moreover, the elevated ferritin accompanying inflammation as an acute phase reactant is sequestered and, thus, not available for erythropoiesis and other organ systems. This defense mechanism, which aims to restrict serum iron from utilization by pathogens or tumors27, may lead or further contribute to ID, resulting in an increased risk of FS. Therefore, blood screening with an eventual iron-rich diet or iron supplementation may be warranted to prevent FS and neurodevelopmental sequelae.The previous literature on the blood iron status and FS mainly reported the association of ID and FSWe demonstrated that the bi-linear classifier consisting of two multivariate mixture models for the group separation provided high sensitivity and much improved specificity compared to univariate assessments or the models applied separately. Thus, carefully weighing the study limitations, we consider that the bi-linear classifier based on the presented models may represent a practical screening tool to determine the FS risk in febrile children. However, the robustness of the bi-linear classifier needs to be verified with a larger and more geographically and racially diverse cohort providing updated model coefficients or an extended variable list, which may result in the SE and SP at the proximity of the gold-standard classifier.11. Significant sex effects were not observed in iron status and demographics in the febrile group without seizures and FS subgroups. In the healthy control group, lower iron concentration and saturation were noted in males compared to females. In analogy, the male sex represents a risk factor for ID or ID anemia in infants and young children28. Moreover, sex may determine seizure susceptibility and type, as demonstrated in the animal model21. The sex difference or male overrepresentation in FS human studies is well documented11. In the Japanese population, the male sex was identified as one of the major predictors of FS recurrence2. Our study showed more frequent RFS in males. Sex hormones control many molecular and cellular processes in brain differentiation, including the modification of the neural response to stress or brain injury. Thus, the increased FS susceptibility in males is likely influenced by multiple factors, including iron status alteration.None of our models identified sex as a significant variable, although we observed higher FS incidence in males, which further confirms the findings of previous studiesRegarding FS recurrence, the unique trivariate model consisting of HGB concentration, body height percentile, and Fe saturation was derived. The model’s reasonable separation  and model reproducibility were suboptimal, requiring further improvement and additional variables to define a model with optimal FS recurrence predictive power.1-model2 projection may lead to an improved models’ prediction.We only utilized linear mixture modeling between investigated variables. It is possible that the proposed analysis may benefit from a non-linear transformation of some variables before the regression analysis. However, we consider that strategy of a lower potential for a marginal improvement on the current dataset as we have not observed any non-linear relationships between variables. When the dataset is enlarged, the training of a non-linear classifier in the space of the orthogonal modelA small and geographically limited Caucasian sample size represented the primary study constraint. Thus, using the full dataset for model regression with the subsequent classifier evaluation may lead to classifier overfitting in all derived and tested models. Therefore, a re-test of fixed models will be necessary at a fully independent and larger dataset that will enumerate and validate true models’ SE and SP.Body height or weight percentile tables normalized for the Czech population may differ across nations, and slightly varying regression coefficients may be derived , or, or3), oImbalanced sex distribution in case groups may bias our findings. The employed adaptive synthetic sampling was performed in an effort to minimize such a dataset effect. Future research needs to collect vitamin D samples in all investigated cases to rightfully determine its role.11 the serum Fer levels may be elevated in various inflammatory conditions as ferritin is an acute phase reactant and marker of acute and chronic inflammation. Reference26 Although the ferritin levels were not significantly different across febrile groups of children with or without seizures, the influence on overall iron status during inflammatory conditions, mainly restricted serum iron utilization26, is noteworthy and may contribute to the FS development.Similar to the previous FS studies, Refs.We confirmed the relationship between iron status and FS with a higher incidence in males. More importantly, we proposed a novel approach to evaluate the FS risk in infants and young children with fever. First, multivariate linear mixture models were derived based on blood iron status and demographic variables. The approach emphasized between-group separation properties when height percentile and age were included in the iron status observation. Next, a bi-linear classifier consisting of two multivariate mixture models provided the optimal SE + SP for FS risk. Finally, we have designed an innovative trivariate model sensitive to FS recurrence, utilizing height percentile, hemoglobin, and Fe saturation. We also hypothesize that a future extension of the novel FS recurrence model about the vitamin D variable can substantially improve its sensitivity and specificity. Future multi-center studies with a larger and more geographically and racially diverse cohort will re-test and validate the robustness of derived models to prove or disclaim them as classifiers with predictive power to FS risk or recurrence.


Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted l,d-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the wild type, as predicted for loss of an l,d-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions.Long-term survival of IMPORTANCE Water is the primary vector for transmission of L. pneumophila to humans, and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires’ disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42°C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan cross-linking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water. Legionella pneumophila is a waterborne pathogen that is the causative agent of Legionnaires’ disease, which results in pneumonia associated with high levels of lethality  or proteins particularly abundant under these conditions .To identify candidate genes that are specifically essential for long-term survival of TA sites . From thTA sites . These t8 to 4 × 107 CFU/mL). Genes specifically necessary for high fitness under starvation conditions were discovered by using the Bayesian/GUMBEL method embedded in the TRANSIT software package , 583 genes were called essential (E) after growth in bacteriological media ; membrane transport (n = 8); hypothetical proteins (n = 5); regulation (n = 6); “virulence” (n = 3); DNA replication, repair, and recombination (n = 3); intermediary metabolism (n = 5); stress (n = 1); and protein processing (n = 1) necessary for high viability under water stress conditions. Of particular note is the reidentification of rpoS sigma factor and the letAS regulatory system as being essential under water starvation conditions. Since these three genes had been previously discovered as controlling survival under water starvation, this argues that our selection procedure reproduces previously established starvation conditions in the literature , which showed a 30-fold knockdown efficiency relative to control Lp02 when assayed after growth to postexponential (PE) phase, was tested for its ability to block growth within Acanthamoeba castellanii after water starvation  or for bacteria assayed shortly after removal from broth and washing in water , an, andotO lpg2969, which showed no detectable CFU after 18 days of incubation (−7 CFU relative to nontargeting control). Water starvation conditions for 32 days resulted in decreased viability of 67% of the strains that were tested, with depletions of mip, rpoS, ccmH, and ctaD also showing no viability at this time point, with extremely poor viability of the lon, lpg1697, and mlaD depletions. After 50 days in water, depletions of the additional candidate genes lpg0011, mlaD, lon, lpg1521, ptsP, mlaE, lpg1697, and lpg1350 showed no viability on plates. Since the majority (68%) of the depletion derivates resulted in the absence of viability in water, our selection procedure identified mutants that are involved in tolerating water starvation conditions even in the absence of competition with the whole populations , 266-fold (lpg2969), 104-fold (rpoS), and 15-fold (lpg1697). In contrast, the depletion levels of the targeted transcripts from the strains showing no apparent survival defect were lower, ranging from 14-fold (cruvrD) to no effect (crnrdR)  . These dA. castellanii. After 50 days of starvation, culture aliquots of the 13 strains that showed no viability on solid medium at this time point were metabolically stimulated and cocultured with A. castellanii in an attempt to resuscitate unculturable L. pneumophila (crnrdR-harboring strains) readily responded to metabolic stimulation with increased luciferase activity and were capable of intracellular growth , electron transport (ctaD), and cytoplasmic protein turnover (lon) are critical components in supporting survival in the face of water stress  and a predicted N-terminal signal peptide . Previous studies showed the peptidoglycan layer of L. pneumophila is highly cross-linked  and tested for growth in broth culture and survival after prolonged water exposure. Growth in broth was indistinguishable for the deletion and the WT  . PE-phase mutant  to F. Suking see . This is10.1128/msphere.00454-22.2FIG S2lpg1697 gene. (A) Chromatograms of UHPLC runs. (B) Relative amounts of total cross-linkages and l,d-cross-linkages were not significantly different between the strains (two tailed unpaired t test). Download FIG S2, TIF file, 0.2 MB.Absence of detectable changes in muropeptide profiles related to loss of Copyright © 2023 Aurass et al.2023Aurass 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 lpg1697 negatively impacts the capability of L. pneumophila to persist during water stress, we were interested whether its loss also reduces intracellular growth fitness after culturing to the PE phase under permissive conditions. To this end, the WT and an isogenic type 4 secretion system mutant (Lp03) were compared to the Δlpg1697 strain by challenging both A. castellanii and murine bone marrow derived macrophages (BMDMs) at MOIs of 0.05 and 0.01, respectively  and the soil thriving species L. longbeachae encode orthologs of all 44 candidate proteins  Lsp, which was identified as contributing to fitness in cold water  and mip (lpg0791), were upregulated in ΔletS cells after experiencing water stress for 2 h (letS on these genes in water may be transient (ykuD (lpg1697), uvrB (lpg0072), and Lon protease gene (lpg1859) are activated, while two, i.e., ppsA and enhC, are repressed by RpoS. Our study suggests that the absence of either of these genes results in an attenuated capability to persist, so there may be temporal regulation of these genes that has been previously unappreciated.Previous predictions of genes important for water survival based on analyses differential transcription in response to water stress have been partially supported by the work reported here. For instance, there is a general trend of gene downregulation after introduction into water that is countered by upregulation of arvation . On the arvation   catalyzing l-d type peptidogylcan cross-links that act as alternatives to d-d type cross-links made by penicillin-binding proteins. The catalytic YkuD domain proteins use an active site cysteine and usually have a two-domain architecture with an N-terminal LysM peptidoglycan-binding domain and a C-terminal catalytic domain. With the exception of carbapenems, most β-lactam antibiotics cannot access the active site, allowing activity in the presence of most β-lactams , 63l,d-t-lactams –66. In Mnditions . Indeed,r stress  and 5.l,d-transpeptidase function, murein profiling showed no detectable compositional differences of Δlpg1697 relative to the WT. Although this failure could be due to limitations of the technique, it should be noted that there are 10 L. pneumophila genes encoding putative YkuD domain proteins that could compensate for loss of lpg1697 and mask alterations in peptidoglycan profile , 0.135 mg/mL ferric nitrate (Sigma), 0.1% (wt/vol) α-ketoglutarate, and, when appropriate, 0.1 mg/mL thymidine (Sigma), 40 μg/mL kanamycin, or 5% sucrose. Plasmids were introduced into L. pneumophila by electroporation  or on solid LB plates supplemented with 50 μg/mL ampicillin, 25 μg/mL chloramphenicol, or 50 μg/mL kanamycin when appropriate. Bacterial strains used in this study are summarized in A. castellanii (ATCC 30234) was cultured as previously described  and quantified by a SYBR green microtiter plate assay. Tagmentation and amplification of transposon-adjacent DNA for Illumina sequencing was conducted using a modified Nextera DNA Library Prep method, as described previously . SamplesA600 of 0.2, and grown overnight to an A600 of 3.8. Subsequently, cultures were washed twice in sterile tap water and inoculated to an A600 of 0.03 in 200 mL of water in 250-mL flasks. All tap water used was first passed through a 0.2-μm-pore size filter  and then autoclaved  was used for incubation to allow for assessment of the phenotype in a time frame exceeding 50 days.For confirmation of candidate genes by CRISPRi, single colonies of thyA gene by using the Gateway LR reaction. After confirmation of single clones, the final plasmids were electroporated into L. pneumophila Philadelphia-1 Lp02dCas9Lux and selected on plates without thymidine . Preprocessed reads were further analyzed by TRANSIT using the GUMBEL subroutine (Sequencing reads were processed using FASTX-toolkit and BWA (Burroughs-Wheeler Aligner), and the built-in aligner in TPP was used to map reads into the chromosome  primers were included. Transcript levels of each targeted gene were calculated by using the 2–ΔΔCT method relative to the reference strain carrying a nontargeting control plasmid.Bacterial cultures were fixed in bacterial RNA Protect reagent (Qiagen) at the time point of water culture inoculation and stored at −20°C until use. RNA was extracted by using an RNeasy kit , and cDNA was synthesized by using SuperScript VILO cDNA kit . Subsequently, cDNA was amplified with PowerUp SYBR green Master Mix  in a StepOnePlus real-time PCR system  according to the manufacturer’s instructions. Primers were designed for amplification of 150- to 200-bp fragments using the Geneious software package and are listed in L. pneumophila within A. castellanii and A/J BMDMs was monitored as described previously , and boiled for 2 h while stirring. To remove SDS from the sacculi, samples were ultracentrifuged and washed with MilliQ water several times. After the washes, sacculi were resuspended in 100 mM Tris-HCl (pH 8) buffer with proteinase K (20 μg/mL) and incubated at 37°C for 1 h. After that, the reaction was stopped by adding SDS  and boiling at 100°C for 5 min. SDS was removed using multiple described washes, and sacculi were resuspended in water. Muramidase was added to the samples, which were incubated at 37°C overnight, solubilizing muropeptides from the sacculi. Soluble muropeptides were reduced by adding NaBH4, and the pH was adjusted to 3. Muropeptides were separated using ultraperformance liquid chromatography (Waters) and later identified using a matrix-assisted laser desorption ionization-time of flight mass spectrometry system (Waters). Triplicates from the peptidoglycan profiles for each representative strain were chosen for quantification, and the area under each peak was integrated for statistical analysis.Peptidoglycan analysis was performed as previously described . In shor600] 3.5) were adjusted to an OD600 of 0.3, and 1-mL suspensions were pelleted by centrifugation at low speed . Subsequently, supernatants were removed and replaced with the indicated stress solutions and distilled water as a control. Cells were incubated under these conditions for 30 min in a shaking incubator at 37°C. Viability was determined by spot plating dilution rows on BCYEα agar plates.Osmotic challenge assays were performed as described previously with modifications . Briefly2O) or 5 μL of a 50-mg/mL carbenicillin solution, respectively. The discs were then placed on fresh BCYEα agar plates containing a fresh layer of the indicated Legionella strain. After 3 days of incubation, the zones of inhibition were determined.Disc diffusion assays were performed as described previously with modifications . BrieflyL. pneumophila strain were pregrown to the exponential growth phase in BYEα broth and back-diluted to an OD600 of 0.15. Then, 100-μL aliquots of this suspension were distributed into a 96-well plate preloaded with a concentration gradient of serial 2-fold carbenicillin dilutions in BYEα (100 μL/well). The plates were grown for 46 h in a plate reader with continuous shaking (Tecan Infinite 200 PRO), and bacterial growth was recorded as the OD. MICs were determined as the lowest antibiotic concentration for which no growth of L. pneumophila was detected after 46 h at 37°C. Wells containing BYEα broth or bacterial suspension without antibiotic were used as negative and positive growth controls, respectively.The carbenicillin MIC was determined by microtiter plate assays, as described previously with modifications . Briefly2O incubation of Tn-Seq pools were the result of one experiment performed using three independently generated pools that were incubated in parallel during the long-term experiment. These pools were subsequently analyzed as a single combined superpool during bioinformatic analysis. Confirmation of long-term efficiency of CRISPRi depletion of the dotO gene was performed once. Long-term survival analysis of CRISPRi-depleted strains in water was performed two times independently with similar results. All other presented data are from one experiment of two or three independent repetitions.The data gathered from long-term HPRJNA816157.The data for this study have been deposited in the NCBI sequence read archive under accession number


We inoculated 1.5 × 106 Ehrlich ascites carcinoma cells into female Swiss albino mice. Quercetin (50 mg/kg) was administered intraperitoneally for 15 days. Liver enzymes activity was determined using a spectrophotometric assay. The hallmarks of inflammation and fibrosis were determined using Immunohistochemistry. The effect of quercetin on tumor formation was elucidated using human breast cancer cell lines and chick chorioallantoic membrane assay. Docking study was performed to explore the binding mode of quercetin with VDR.We used Ehrlich Ascites Carcinoma (mouse mammary carcinoma) model for our in-vitro assays and chick chorioallantoic membrane assay revealed the Vitamin D mimicking effect of quercetin.In EAC tumor-bearing mice, cell numbers, tumor volume, body weight and liver weight were dramatically increased, while they significantly decreased in mice treated with quercetin. Additionally, the peritoneal neo-angiogenesis was also significantly suppressed in the quercetin-treated mice, compared to the control. In addition, quercetin treated EAC tumor bearing mice had lower levels of liver enzymes, decreased hepatic inflammation and fibrosis compared with EAC tumor bearing mice. Docking study confirmed VDR-quercetin interaction. Furthermore, via activation of VDR.Dietary flavonoid, quercetin could act as a promising therapeutic drug to suppress the breast cancer induced tumor angiogenesis, hepatic inflammation, and fibrosis possibly  Breast cancer has the highest prevalence and mortality rate in India and the world according to the 2020 Globocan cancer report . Age, favia novel signaling pathways  and human Breast cancer cell lines (MDA-MB-231 and BT-474) were kind gift from Dr. MVVST SubbaRao, CEMR laboratory. Quercetin Q4951) and vitamin D3 (C9756) were procured from Sigma-Aldrich, United States. WST-1 reagent was procured from TAKARA. Fertilized chicken eggs were procured from Ilavala poultry farm, Mysuru. The Ehrlich ascites carcinoma (EAC) model is very commonly used model to study the pathological conditions of breast cancer and associated tumor angiogenesis. These EAC cells were first discovered and isolated by the Nobel Laureate, Paul Ehrlich in the mammary gland tumor of a white mouse, therefore these tumor cells were named after him to honor his great contribution to tumor biology. Recently, we further developed this model to study the breast cancer induced liver inflammation and fibrosis . The animals were fed with a rodent normal chow diet and mineral water on an ad libitum basis.The Institutional Animal Ethics Committee (JSSAHER/CPT/IAEC/087/2021), JSS Medical College, JSS AHER, Mysore, Karnataka, India approved the animal experimental procedure. A total of 15 female Swiss albino mice were procured from M/s. Adita Biosys, Tumakuru, which were weighing 18–21grams and aged between 5 and 6 weeks and then housed for 7 days prior to all 2.3.6 EAC cells were injected into a donor mouse’s peritoneal cavity. After 10 days, ascites fluid containing EAC cells was collected from donor mice and the cells were counted using a hemocytometer. After counting, 100 μL of fluid was administered into each recipient mouse’s peritoneal cavity, which contained 1.5 million viable cells  according to the treatment pattern as follows: The non-tumor bearing mice were considered as the negative control group. The EAC group containing EAC tumor cells considered as the positive control group. The remaining EAC group was treated with quercetin every day until the 15th day . The que2.5.Elevated liver enzymes indicate the hepatic inflammation or hepatocyte damage. Mouse liver enzymes  activity was determined by adding an enzyme specific working reagent to each serum sample and incubating at 37°C for 1 min, and measuring the change in absorbance per minute during 3 min using a semi-autoanalyzer (MISPA VIVA) according to the manufacturer’s instructions .2.6.The liver tissue and peritoneum of each mouse was harvested and instantly fixed in neutral buffered formalin  and embedded in paraffin after dehydration of ethyl alcohol series for further processing. Five micrometer thick sections of liver tissue were stained with Hematoxylin and eosin staining (H&E) and Trichome Masson staining (TMS) . The int2.7.2 under humidified atmospheric conditions. 10,000 cells per well were plated in 96 well plates and treated for 24 h with various concentrations of quercetin (20–100 μM), vitamin D3 (6.25–200 μM), and a combination of both. After 24 h, 10 μL premixed WST-1 reagent was added to each experimental well, and the plates were incubated for 3 h at 37°C in 5% CO2. After incubation, absorbance was measured at 450 nm using a PerkinElmer multimode plate reader. The results were represented in percentage of inhibition by using the formula: % of Inhibition = Test/Net × 100 and the combination index (CI) was calculated by using the multiple drug-effect equation of Chou-Talalay in the CompiSyn program. Combination index (CI) represent the type of interaction between two administered drugs. The Chou-Talalay method for drug combination is based on the median-effect equation, which provides the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI < 1 indicates synergism, CI = 1 indicates additive effect and CI > 1 indicates antagonism, respectively.To measure the rate of cell proliferation of human breast cancer cell lines, the Water-soluble Tetrazolium-1 (WST-1) cell proliferation assay was performed. Cells were cultured in DMEM medium with 10% FBS and 100 U/mL streptomycin at 37°C and 5% CO2.8.ex-vivo angiogenesis. Fertilized eggs were incubated at 37°C until the 8th day. On the 9th day, a small window was made on the top of the live eggs shell by using a sterile blade. Quercetin and vitamin D3 stocks were prepared by dissolving in DMSO followed by diluted with PBS to get desired concentration and gently applied on the CAM. The eggs were then photographed after a 48-h of incubation period.The angiogenic activity was performed by using chick embryos as described by Prasanna et al. . A Chick2.9.Auto Dock Vina was used to evaluate the binding affinity of quercetin with the vitamin D receptor . The doc2.10.p values less than 0.05. The staining images were captured from stereo zoom microscope with a CCD Olympus camera attached to it (cellSens Dimension 1.12). The cell proliferation images were captured from Carl Zeiss microscope. All the data is shown in n = 5.Statistical analysis for all these experimental results were performed by using standard statistical software (Graphpad Prism version 5.0). All data were represented as means ± Standard Deviation. One-way ANOVA followed by Bonferroni’s Multiple Comparison Test was used to test the statistically significant difference among groups where * represents that 3.3.1.p < 0.0001) body weight when compared to control EAC-bearing animals (p < 0.0012) on the 15th day of tumor inoculation when compare to control EAC bearing animals (p < 0.0063) in comparison to EAC bearing animals  in liver weight when compared to control EAC bearing animals (p < 0.0002), AST (p < 0.0001), and ALT (p < 0.0001) in quercetin-treated EAC animals compared to control EAC bearing animals  which indicated that quercetin inhibited the endothelial cells proliferation. Therefore, these results collectively demonstrated that quercetin inhibits angiogenesis which is evident through reduced elevation of angiogenic markers.We noticed a significant reduction in the formation of new blood vessels in the peritoneum of quercetin treated EAC animals as compared to control EAC-bearing animals . To furt3.4.To explore the hepatoprotective action of quercetin, we performed H&E staining for all the liver tissue. Quercetin-treated EAC mice liver sections showed significantly decreased inflammation when compared to liver tissues of EAC-bearing control mice . Further3.5.p < 0.0001) and vitamin D3 (p < 0.0001) greatly suppressed the proliferation of human breast cancer cells (p < 0.0001) at desired combinations , vitamin D3 (6.25–200 μM), and quercetin (40–100 μM) combined with 50 μM and 100 μM vitamin D3 for 24 h . The WSTer cells ,B. Interinations ,D. Our r0–100 μM,inations . The phoinations .3.6.in-vitro experiments we know that quercetin has anti-tumor activity. To further elucidate the vitamin D mimicking role of quercetin, we performed CAM assay. Our CAM assay data showed that there is a noticeable reduction in the neo-angiogenesis in a dose-dependent manner  which further may activate and regulate VDR-mediated signaling pathways (via activating VDR and making it to bind Vitamin D response elements (VDRE).When we were searching for a potential target of quercetin, we came across few interesting articles which predicted that the Vitamin D receptor acts as a possible target of quercetin , 41. Thepathways , 23. A rpathways , 48. To pathways . Upon dipathways . To our via VDR and in near future we will further extend our study to validate the effect of quercetin on vitamin D target genes using breast cancer cells and liver specific cell lines.We were the first to report the potent interaction between quercetin and VDR, as well as an ameliorating effect of quercetin on breast cancer-induced hepatic inflammation and fibrosis. Limitation of our study is that we did not investigate the role of liver-specific vitamin D target genes in the prevention of breast cancer induced liver injury. Here, we tried to figure out the molecular mechanism and ameliorating effect of quercetin on breast cancer-induced liver inflammation and fibrosis 5.Through these findings, we explored the novel role of quercetin and its ameliorating effect on breast cancer-induced liver injury. Quercetin inhibited the elevation of liver enzymes, hepatic inflammation, and fibrosis in EAC-induced animals, proving its strong hepatoprotective effect. Furthermore, our findings strongly suggest that quercetin mimics vitamin D3 and our molecular docking analysis demonstrated that quercetin strongly binds to VDR. It is already known that activation of this receptor suppresses hepatic inflammation and fibrosis. Therefore, our findings in animal model clearly show that quercetin can suppress breast cancer-induced liver inflammation and fibrosis. The mechanism is possibly through the activation of VDR. Future clinical trials are warranted to investigate the effect of quercetin on liver inflammation and fibrosis in breast cancer patients and the potential to reduce the co-morbidity and improve cancer survival. In our future studies we will try to explore the role of some of the direct vitamin D target genes in mediating quercetin effects and we will also demonstrate the complete molecular mechanism behind quercetin mediated VDR activation and its therapeutic impact on breast cancer induced hepatic inflammation and fibrosis.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by JSS AHER Institutional Ethical Committee.in vivo and in vitro experiments. AS provided intellectual inputs to the manuscript. SB and SP aided in docking studies. SM and RV provided intellectual inputs to the manuscript. PS designed the project, provided overall supervision and intellectual guidance, and drafted the manuscript. All authors contributed to the article and approved the submitted version.NS performed the experiments, analyzed the data, and drafted the manuscript. VS, LP, SD, and CB helped during This work was supported by Department of Science and Technology funding through the “Promotion of University Research and Scientific Excellence” (PURSE) scheme with sanction number: SR/PURSE/2021/81 (G). NS acknowledges JSS AHER /JSSURF/29(2)/202-21) and KSTA (grant number: K.S.T.A/K/30/202-21). PS and VS (for stipend support) acknowledges Ramalingaswami Re-entry fellowship, Department of Biotechnology (DBT), Govt. of India. We also would like to acknowledge the infrastructure support provided by DST-FIST to the Center of Excellence in Molecular Biology and Regenerative Medicine (CEMR) Laboratory (CR-FST-LS-1/2018/178) and the Department of Biochemistry (SR/FST/LS-1-539/2012). SM and SD would like to thank the DBT for grant support (N0-BT/PR29598/PFN/20/1392/2018) and Senior Research Fellowship. SM would like to thank the Special Interest Group in Cancer Biology and Cancer Stem Cells (SIG-CBCSC).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Joubert syndrome (JS) is a rare genetically heterogeneous primary ciliopathy characterized by a pathognomonic cerebellar and brainstem malformation, the “molar tooth sign”, and variable organ involvement . Here, we present a case of JS in a Chinese boy.An 11-year-old Chinese boy presented with neonatal asphyxiation and hypoxia, strabismus, subsequent developmental delay, ataxia and end-stage kidney disease (ESKD). Routine blood tests showed severe anemia, increasing blood urea nitrogen and creatinine, elevated parathyroid hormone, hypocalcemia, hypokalemia and metabolic acidosis. Urine tests showed mild proteinuria. Ultrasound showed two small kidneys. Brain magnetic resonance imaging (MRI) showed dysplasia of the cerebellar vermis and extension of the upper cerebellar feet with the “molar tooth sign”. Genetic analysis showed novel compound heterozygous mutations in the RPGRIP1L gene [p.L447fs*7(p.Leu447fsTer7) and p.G908V ].In the present study, we identified novel compound heterozygous mutations in the RPGRIP1L gene in a Chinese boy. The clinical and genetic findings of this study will expand the understanding of JS. Joubert syndrome  is a rare congenital neurodevelopmental primary ciliopathy. Its population-based prevalence reaches 1.7 per 100,000 in patients ranging from 0 ~ 19 years of age . His parents were heterozygous carriers and did not present any symptoms. Gene sequencing revealed that the father carried the c.1334_c.1335insT variant, and the mother carried the c.2723G > T variant. According to the American College of Medical Genetics and Genomics (ACMG) criteria, the c.1334_c.1335insT variant of the RPGRIP1L gene was classified as a likely pathogenic variant (PVS1 + PM2) because this loss-of-function (LOF) variant could lead to a possible loss of gene function, and the c.2723G > T variant was classified as a variant of uncertain significance (PM2 + PM3 + PP3). Both conservation prediction plots and predicted three-dimensional models predicted that the substitution of amino acids (p.G908V) in the mutated protein would lead to a change in function and eventually result in disease . Brain imaging showed dysplasia of the cerebellar vermis and extension of the upper cerebellar feet with an MTS. This case was caused by a novel compound heterozygous mutation of the RPGRIP1L gene.The diagnosis of JS is based on three primary criteria: (1) the MTS on MRI, (2) hypotonia in infancy and (3) developmental delay or intellectual disability . The mosJS is classified into eight different phenotypic subgroups: pure JS, JS with ocular involvement, JS with renal involvement, JS with oculorenal involvement, JS with hepatic involvement, JS with orofaciodigital involvement, JS with acrocallosal features and JS with Jeune asphyxiating thoracic dystrophy, according to the presence of associated extra-central nervous system (CNS) features . Our patJS is characterized by extreme genetic heterogeneity, with more than 40 causative genes, all of which encode proteins responsible for the formation or functioning of the primary cilium . JS is mNPHP constitutes the most frequent genetic cause of ESKD in children and young adults. Up to 25–30% of JS individuals develop renal disease, mainly presenting as juvenile NPHP, which may remain asymptomatic for several years . Wolf MTIn this study, the patient was misdiagnosed until he presented with weight loss, severe anemia, mild proteinuria, elevated parathyroid hormone (PTH), hypocalcemia, hypokalemia, and metabolic acidosis secondary to ESKD at the age of 11. The reasons for misdiagnosis mainly included family neglect and unawareness of JS. The disease is a rare genetic developmental disorder. An estimated incidence of JS is 1/100,000 to 1/8000. Many people, even many doctors do not know or even hear of the disease. However, with the rapid development of medical technology and gene detection methods in recent years, the detection rate of JS is increasing year by year. We should pay more attention to neurological and kidney involvement, and gene detection should be suggested for those suspected patients. This kind of genotype–phenotype correlation may help to guide individualized management in JS patients.The present study identified a novel compound heterozygous mutation, p.L447fs*7 (p.Leu447fsTer7) and p.G908V , in the RPGRIP1L gene in a Chinese boy. The clinical and genetic findings of this study will expand the understanding of JS.


This study aimed to evaluate the efficacy of complete commercially available feeds with varying nutritive profiles in the diet of juvenile largemouth bass (LMB) reared in a closed tank system. LMB is a lean fish rich in health-promoting ω3 fats. It is a promising new species for aquaculture worldwide, given its lack of intramuscular bones and delicious taste. Conditions for rearing LMB in tank systems as well as its nutritive demands from the early life stage have not yet been defined. The results of this study revealed monounsaturated (MUFA) and polyunsaturated (PUFA) ω3 fatty acids to be important nutrients for juvenile LMB, with MUFA possibly outbalancing the potential negative effects of oxidative stress caused by membrane-stabilizing ω3 fats. In addition, this study suggested an interesting feature of juvenile LMB: to sacrifice the development of some parts of the skeleton  for rapid growth. Among the tested feeds, one was selected as optimal for tank-reared juvenile LMB.Micropterus salmoides). Key variations in the content of nutritive values were monounsaturated fatty acid (MUFA) and highly unsaturated FA (HUFA) ω3. Fish were fed with one of four tested diets from the 33rd to the 40th day post-hatch (DPH). Biometric indices, digestive enzyme-specific activities, thyroid hormone status, and mRNA expression of genes coding for skeleton, neuron, and muscle growth were analyzed. The lowest skeletal deformity rate and highest survival among the treatments were seen in BIP-fed fish. Dietary lipids, with an appropriate balance between MUFA and polyunsaturated FA (PUFA), alongside amino acid balance, were shown to be the main contributors to the growth of the skeleton and/or fish survival. On the other hand, fish growth is correlated with fish digestive capacity and feed moisture percent rather than feed quality. Unexpectedly, BIP-fed fish were attributed with the lowest expression of skeleton differentiation markers, which may reflect the sacrifice of scale and/or cranium growth at the expense of somatic growth. This study highlights the role of non-marine ingredients in the nutrition of post-larval LMB.This study aimed to evaluate different commercial diets , Biomar Inicio Plus (BIP)) and one experimental feed (EF) in terms of their effectiveness as post-larval diets for indoor weaned largemouth bass, LMB ( Micropterus salmoides), is a freshwater carnivorous fish species native to the American continent but introduced as a farmed species for human consumption on the Asian market at the end of the last century. LMB is an attractive new species for European aquaculture, given its delicious taste and the lack of intramuscular bones [Largemouth bass, LMB  for up to two weeks ,4. AfterAfter weaning indoors, the next phase is the on-grow phase in RAS to prepare the larvae for the grow-out diets in the pond. During traditional LMB culture, high-marine ingredient feed, Otohime C1, is commonly used for feed training after the pond-nursing phase . Then, aThree different commercial diets , and Biomar Inicio Plus (BIP)) were selected from the market and tested in this trial . ParalleProximate compositions of the diets were estimated by methods of the Hungarian Standard  ,12,13,14−1 salmon gonadotropin-releasing hormone analog . Later, fish were stocked in the spawning pond, which was equipped with 30 nests made out of artificial grass measuring 60 × 60 cm. Three days after hormonal treatments, 10 nests with fertilized eggs were removed from the spawning ponds, and eggs and newly hatched larvae were further incubated in the hatchery at a temperature of 23 ± 1 °C. Upon swim bladder inflation, 30,000 larvae were volumetrically counted and stocked in the cylindroconical larval rearing tank of water volume 1.4 m3 within the larval rearing RAS. The larval rearing system was composed of a total of 16 tanks, four of which were 1.4 m3 and 12 of which were 0.25 m3, all with the same design with a white conical bottom and black walls where water flow was maintained in an up-welling manner throughout the study. Upon stocking, larvae were fed for 10 days with enriched Artemia nauplii . Later, larvae were subjected to the weaning to dry diet of Otohime B2 feed  for 14 days in total. Upon weaning, juveniles were harvested from the larger tanks, and 60 randomly chosen juveniles were stocked in each of the 12 smaller volume tanks.The animal research was approved by the Institutional Animal Welfare Committee of the Hungarian University of Agricultural and Life Sciences, Szent István Campus (MATE-SZIC/416-1/2022). Experiments were carried out in accordance with the “Animal research: reporting of in vivo experiments (ARRIVE)” guidelines and EU Directive 2010/63/EU. Juveniles for this study were obtained from a seasonal semi-artificial reproduction of largemouth bass. Namely, in the first week of May 2023, 30 pairs of breeders were selected from the broodstock rearing pond and treated with 50 μg·kgOn the 33rd day post-hatch (DPH), weaned juveniles  were harvested from the tank, and 60 randomly chosen juveniles were stocked in each of the 12 smaller volume tanks for an 8-day-long feeding trial. Feeding treatments were assigned randomly to each tank. Each tank received 6 g of feed per tank during the first two days , then four days , 8 g per tank, and then, for the last two days (39 and 40 DPH), 9 g per tank. In the first three days, fish received a 50:50 mixture of the weaning diet, B2 and the respective treatment diet, while from the 4th day until the end of the trial, only the trial feed was supplied to the fish. Both larvae and juveniles were reared under the 16:8 LD photoperiod, and the feeding period matched the light period. Food was supplied every 5 min using the automatic belt feeder. Tanks were cleaned once a day, and mortalities were counted.−1). Fish were dipped onto a paper towel to remove water drops, and morphometric measurements were performed on a representative sample of 18 fish per tank. For molecular and biochemical analyses, six fish per tank were sampled and stored at −80 °C until further use. The experimental workflow is presented in The sampling of fish was carried out on the 41st DPH after 12 h of food restriction. Fish were anesthetized using phenoxyethanol , the upper phase was moved to another tube, and the phenol extraction step was repeated. The extracted samples were precipitated using isopropanol and 70% ethanol, then dried at RT and suspended in distilled water. RNA concentration was measured using Nanodrop 2000 .For the isolation of RNA, whole fish  were homogenized in mortar using liquid nitrogen. After powdering the samples, denaturing buffer was added to the homogenate, and the homogenate was mixed with one volume of acid phenol, 1/10 volume of Na-acetate, pH 4, and 1/5 volume of chloroform . After c® 70 µm Cell Strainer . Filtrate was additionally homogenized using Pestle B. Homogenates were freeze-thawed once to further lyze the cells and centrifuged at 4 °C. 15,500× g, for 15 min. The supernatants were transferred to another tube and stored at −20 °C until further use. Buffers used for homogenization were phosphate buffered saline (PBS) for ELISA and 50 mM Tris, pH 7, and 2 mM mannitol for digestive enzyme assays [For biochemical analyses, samples (two fish per homogenization) were homogenized after the addition of 10 volumes of homogenization buffer using a dounce tissue grinder . After 2e assays .Reverse transcription (RT) was performed using the Xpert cDNA Synthesis Kit , as suggested by the manufacturer, using 1 μg of RNA template. The reaction was performed in the SuperCycler Thermal Cycler SC3005 . Quantitative PCR (qPCR) was performed using Xpert Fast SYBR 2X Mastermix , using a five-fold dilution of complementary (c) DNA and the final 10 mM primer concentration in the mixture. Cycling conditions were:https://www.ncbi.nlm.nih.gov (accessed on 28 August 2023)) and Primer-BLAST, NCBI. The GAPDH gene was an endogenous control. One sample from the C1 group was used as the reference for the Ct calculation. NCBI Gene Identification (ID), product length in base pairs (bp), and primer efficiency slopes (obtained using cDNA from this study as a template) are provided in Primer sequences  were desTrypsin, chymotrypsin, lipase, amylase, and alkaline phosphatase (AP) activities were estimated as previously described , while t−1 ×cm−1—lipase assay)ε: the extinction coefficient of the product of the reaction  and tri-iodothyronine (T3) concentrations were estimated using BioMatic (Canada) ELISA kits, according to the manufacturer’s instructions. The concentration of proteins in the homogenates was measured using the Analyticon Biotechnologies kit. Obtained results are expressed as μg per mg of protein for trypsin, chymotrypsin, lipase, and AP; nmol ×minIn order to compare the economic performance of different feeds used in this study, a cost calculation was carried out by simulating the nutrition cost and larval nursing infrastructure operation costs  spent on rearing 1 g of juveniles. It was hypothesized that reaching a mean weight of 1 g is the ultimate aim of the indoor training phase, and the output is released for subsequent stocking in outdoor ponds. For the calculation, the following equations were used:−1 for the system of 4 m3. Given the limited experience in LMB fry rearing, a rearing density of one juvenile per liter was allowed in the calculations. The cost of juveniles weighing 0.2 g, considered to be equal among treatment groups, was not accounted for in the analysis since the ultimate aim was to compare the economic performance between feeds.The rearing time was calculated as follows, assuming an exponential growth function:https://www.statskingdom.com/kruskal-wallis-calculator.html (accessed on 28 August 2023)) .Biometric evaluation results are presented in tables as means ± standard deviations (S.D.) of the average values of three tanks . Calculations of Specific Growth Rates (SGR), allometric coefficient (b), relative condition factor (Kn), and FCR were carried out as explained previously . Survivahttps://contchart.com/outliers.aspx (accessed on 28 August)). If normality assumption and variance homogeneity could not be achieved with non-transformed and log-transformed data, the analysis was performed using the KW test with non-processed data. Pearson correlation analysis was carried out between the group average values of all evaluated variables, including feed nutritive profile. Partial correlation analysis (Pearson test), controlled for MUFA level, was used to analyze the relationship with the PUFA ω3/ω6 ratio. The same was carried out for MUFA, controlled for PUFA ω3/ω6 ratio. The correlation with the presence or absence of Bactocell® probiotic (dichotomous variable) was analyzed using the point-biserial correlation coefficient (http://www.vassarstats.net/pbcorr.html (accessed on 28 August 2023)). The results of the statistical analysis were considered significant if p < 0.05 and insignificant if a statistical trend (p < 0.1) was observed. Differences with p > 0.1 were not considered. Shapiro–Wilk and Levene’s test, Pearson correlation analysis, ANOVA, as well as table and graph drawing, were conducted using IBM SPSS Statistics 21 [corrplot R package [The results of the biochemical and molecular analyses are provided as clustered bars showing means ± S.D. of pooled samples . Statistical analysis was performed using Analysis of Variance (ANOVA), with Tukey post-hoc if the distribution of data was normal and the variance homogenous, according to the Shapiro–Wilk and Levene’s test, after the removal of single outliers (if any) using Grubbs’ test (stics 21 . A visua package .−1.The proximate compositions of the feeds are presented in p = 0.067) between total MUFA and SFA levels . The percent of FA expressed per feed weight is provided in In terms of the FA composition , as was Based on the amino acid measurements , the sump = 0.03) and higher survival (p = 0.02) percent in comparison to the EF group  and AF , as well as the lower expression of ColIα1 in comparison to EF (p = 0.04) (PSN mRNA expression in comparison to the AF group (p = 0.049). Similar to skeleton differentiation marker gene expression, the level of neurogenesis marker SOX3 was lower in BIP-fed fish in comparison to both C1 (p = 0.02) and AF groups (p = 0.01).BIP-administered fish showed lower mRNA expression levels of  = 0.04) . On the p = 0.036). The trypsin-to-chymotrypsin (T/C) activity ratio was significantly higher in the BIP group in comparison to C1 (p = 0.031) and the AF group (p = 0.031).At the level of digestive enzyme-specific activities , differeThyroid hormone levels are presented in PSN and TnnC mRNA expression. Standard length correlated positively with trypsin-specific activity and negatively with FCR. SGR showed a negative correlation with dry matter percent in the diet, as well as ColIα1, ColIα2 and PSN mRNA expression levels. FCR showed a negative correlation with standard length and the trypsin-to-chymotrypsin (T/C) activity ratio, while the allometric coefficient correlated negatively with TnnC mRNA expression, amylase specific activity, and T3 level. Fish survival correlated negatively with NFE and PUFA ω6 in the diet, while fish deformity rate showed a positive correlation with crude fiber percent in the diet. ColIα1/ColIα2 mRNA expression levels correlated positively with ColIα2/ColIα1 mRNA and T3 level and negatively (ColIα1) with trypsin specific activity. ColIα1, ColIα2, and PSN mRNA expression levels were negatively correlated with SGR. Both PSN and TnnC mRNA expression showed a positive correlation with feed dry matter percent. Alkaline phosphatase specific activity correlated negatively with lipase specific activity. Chymotrypsin specific activity showed a negative correlation with MUFA percent in the diet and a positive correlation with PLA2 specific activity. Similarly, PLA2 specific activity showed a negative correlation with MUFA percent in the diet (with or without correction for the PUFA ω3/ω6 ratio). Amylase specific activity was positively related to T3 levels. The condition factor, Kn, was not included in the correlation analysis, given the low differences in Kn values between the groups .Bivariate correlation analysis  revealedpb = −0.99, p = 0.006). In addition, probiotic presence correlated positively with the T/C activity ratio .Partial correlation analysis, controlling for the PUFA ω3/ω6 ratio, revealed a significant correlation of MUFA percent with fish survival. Likewise, partial correlation analysis, controlling for MUFA percent, demonstrated a significant correlation of the PUFA ω3/ω6 ratio with survival. Point biserial correlation analysis revealed a negative correlation between probiotic presence in the diet and FCR , previously weaned using Otohime B2. Tested feeds included three commercial diets: Otohime C1 (C1), Aller Futura (AF), Biomar Inicio Plus (BIP), and one homemade experimental feed (EF). The selection of the diets was based on two critical parameters: highly polyunsaturated fatty acid (PUFA) ω3 , and monounsaturated fatty acid (MUFA) level (high in BIP and low in C1). Namely, the high PUFA ω3 content in C1 feed most probably reflects a high marine ingredient percent in the diet . On the p = 0.07) when corrected for the PUFA ω3/ω6 ratio. A similar negative correlation trend was noticed between the PUFA ω3/ω6 ratio and the skeletal deformity rate (p = 0.09) when adjusted for the MUFA level. In line with this, the highest skeletal deformation rate was seen in fish fed EF, which showed the lowest MUFA and PUFA ω3/ω6 ratio. It appears that both factors contribute to skeletal development and that an appropriate balance between tissue MUFA and HUFA ω3 and ω6 may be critical for the adequate formation of skeletal elements. Correlation analysis additionally revealed the negative role of dietary fibers in skeleton development. Indigestible dietary fibers were shown to retard the absorption of nutrients by reducing the transit time of digestion [BIP-fed fish showed the lowest skeletal deformity rate. One of the key nutritive factors affecting skeletal development in fish is highly unsaturated FA (HUFA) level, precisely the specific balance between ω3 and ω6 HUFA in tissue, namely (the docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA))/arachidonic acid (ARA) ratio . HUFA isigestion . This miAlthough the PUFA ω3/ω6 ratio in this research has shown a negative correlation with the skeletal deformity rate, it is applicable only in the range of PUFA present in the diets in this research. This is because the results on the ARA role in skeleton growth are somewhat contradictory, given the important role of the ARA metabolite, prostaglandin E2 (PGE2), in skeleton remodeling ,35. Furtp = 0.08) and essential amino acid level (p = 0.072) (low in EF) in the diet, on the other side, were observed. This was accompanied by a negative correlation with soluble carbohydrate levels in the diet (high in EF). The above results suggest that feed quality is the main determinant of fish survival. These results, furthermore, indicate carnivorous feeding habits of post-larval LMB, supporting previous studies on the negative influence of dietary carbohydrates on the performance of LMB [Skeletal deformities are usually accompanied by high mortality, as also revealed in the present study . Similare of LMB .p = 0.064) between trypsin-specific activity and feed conversion ratio (FCR) confirmed the well-known role of trypsin as an indicator of fish digestive capacity [® in the BIP diet was negatively related to FCR and showed a positive correlation trend (p = 0.09) with trypsin-specific activity, indicating its role in improving fish digestive capacity. Bactocell® is the trade name for Pediococcus acidilactici CNCM I-4622, which was shown to improve fish skeletal development and growth rate in salmonids [Specific growth rate (SGR) and individual fish weight appeared to be positively related to the moisture percent of the diets (low in C1 and AF). High dry matter percent, although beneficial in terms of feed storage stability, may potentially increase the energy needed for food disintegration, decreasing the overall energetic value of the feed. The negative correlation trend , was observed in previous research [ColIa1 and ColIa2 mRNA seen in this research reflected both lower scale and cranial skeleton differentiation.Although the BIP group showed the lowest skeletal deformity rate, the mRNA expression of ossification markers, n type I ,46. Hencn type I . Scales  as well ,49. Sincresearch . Furtherresearch . It is tSOX3 mRNA, the marker of neural tissue growth, was also low in BIP-administered fish [p = 0.074) between SOX3 mRNA and PLA2 specific activity was observed, while overall MUFA percent in the diet appeared to suppress PLA2 activity. Although the polar and neutral lipid profiles of the diets were not investigated in this study, data from the literature suggest that neutral lipids are the preferred forms for MUFA deposition [Expression of red fish . A positposition ,54. Therposition . Therefop = 0.09) with fish length, is deemed a reliable indicator of future (one–two month) fish growth [Interestingly, C1-fed fish showed a higher chymotrypsin-specific activity in comparison to the BIP group. Chymotrypsin activity in fish was reported to suppress fish growth when growth conditions and/or food availability are suboptimal ,57, whicTnnC, was directly related to dry matter percent in the diet. In this line, TnnC mRNA showed a trend towards lower levels in the EF group in comparison to both C1 (p = 0.06) and AF (p = 0.05)-fed fish. It is possible that the low moisture percent in C1 and AF reduced the availability of the feed , imposing the need for intense swimming activity in order to reach the feed granules. This might have affected the muscle buildup, but it could also be the reason for the positive correlation trend between amylase-specific activity and the above two factors (TnnC mRNA expression (p = 0.069) and dietary dry matter percent (p = 0.078)), since carbohydrates are the most accessible substrates for rapid energy acquisition. In this line, high dry matter percent in the diet seemed to boost the overall metabolic rate, independent of TnnC expression, as evident from the positive correlation trend with T3 concentration (p = 0.061) [Expression of muscle growth marker mRNA, = 0.061) .From a biological standpoint, this research unequivocally demonstrated the superior performance of MUFA-rich BIP feed as the first juvenile feed for indoor-reared LMB, both in terms of survival and future growth potential. Although FCR in this study could not be precisely calculated, since fish were small and a common practice is to administer a large excess of feed to such small fish, feeds associated with larger SGR (BIP and EF) contributed to clearly lower per-unit rental fees owing to the shorter rearing period necessary to reach the target weight. An illustration of the key results of this study discussed above is provided in This study underlines the role of vegetable MUFA and adequate MUFA-to-PUFA balance in the prevention of skeletal deformities in post-larval LMB. It also indicates the non-overlapping roles of factors influencing fish growth  and factors influencing fish skeletal development and survival . Furthermore, this study showed the ability of LMB post-larvae to trade off the differentiation and/or growth of supporting tissues for rapid somatic growth. The consequences of this phenomenon, both beneficial in terms of improving fish growth in the long term and deleterious in terms of the adaptability of fast-growing individuals to grow-out pond environments, should be evaluated in further research. The knowledge presented here can support the selection of effective and sustainable diets for the on-growth of LMB, but it can also set the direction for the design of improved LMB diets aligned with the nutritional needs of LMB.