CAMAP predictions were significantly more accurate when using original codon sequences than shuffled codon sequences which reflect amino acid usage. Furthermore, predictions were independent of mRNA expression and MAP binding affinity to MHC-I molecules and applied to several cell types and species. Combining MAP ligand scores, transcript expression level and CAMAP scores was particularly useful to increase MAP prediction accuracy. Using an in vitro assay, we showed that varying the synonymous codons in the regions flanking the MCCs (without changing the amino acid sequence) resulted in significant modulation of MAP presentation at the cell surface. Taken together, our results demonstrate the role of codon arrangement in the regulation of MAP presentation and support integration of both translational and post-translational events in predictive algorithms to ameliorate modeling of the immunopeptidome.MHC-I associated peptides (MAPs) play a central role in the elimination of virus-infected and neoplastic cells by CD8 T cells. However, accurately predicting the MAP repertoire remains difficult, because only a fraction of the transcriptome generates MAPs. In this study, we investigated whether codon arrangement (usage and placement) regulates MAP biogenesis. We developed an artificial neural network called Codon Arrangement MAP Predictor (CAMAP), predicting MAP presentation solely from mRNA sequences flanking the MAP-coding codons (MCCs), while excluding the MCC  per se, CAMAP’s novelty is to analyze the MAP-flanking mRNA sequences, thereby providing completely independent information for MAP prediction. We show on several datasets that the integration of CAMAP scores with other known factors involved in MAP presentation (i.e. MAP ligand score and mRNA expression) significantly improves MAP prediction accuracy, and further validate CAMAP learned features using an in-vitro assay. These findings may have major implications for the design of vaccines against cancers and viruses, and in times of pandemics could accelerate the identification of relevant MAPs of viral origins.MHC-I associated peptides (MAPs) are small fragments of intracellular proteins presented at the surface of cells and used by the immune system to detect and eliminate cancerous or virus-infected cells. While it is theoretically possible to predict which portions of the intracellular proteins will be naturally processed by the cells to ultimately reach the surface, current methodologies have prohibitively high false discovery rates. Here we introduce an artificial neural network called Codon Arrangement MAP Predictor (CAMAP) which integrates information from mRNA-to-protein translation to other factors regulating MAP biogenesis (e.g. MAP ligand score and transcript expression levels) to improve MAP prediction accuracy. While most MAP predictive approaches focus on MAP sequences  PLOS Computational Biology Methods paper.This is a In jawed vertebrates, virtually all nucleated cells present at their surface major histocompatibility complex class-I (MHC-I) associated peptides (MAPs), collectively referred to as the immunopeptidome ,2. MAPs The generation of the immunopeptidome can be conceptualized in two main events: (a) the generation of MAP candidates (i.e. peptides of appropriate length for MHC-I presentation) through protein degradation, and (b) a subsequent filtering step through the binding of MAP candidates to the available MHC-I molecules. Rules that regulate the second event have been well characterized using artificial neural networks (ANN) and weighted matrix approaches ,8. HowevA large body of evidence suggests that a substantial portion of MAPs are produced co-translationally –15, deriper se from our positive (hits) and negative (decoys) sequences  by a total of 33 MHC-I alleles from 18 subjects ,22. Becaequences . To faci2 > 0.95) with the amino acid distribution, compared to only 69% in the original dataset  in which original codon sequences were randomly replaced by synonymous codons according to their usage frequency in the human transcriptome . This trTo assess the importance of codon usage in MAP biogenesis, we reasoned that if codons bear important information that is operative at the translational rather than the post-translational level, then: (i) CAMAP trained to identify MCCs flanking regions should consistently perform better when trained on original codon sequences than on shuffled codon sequences (reflecting amino acid sequences), and (ii) synonymous codons should have different effects on the prediction. To test these hypotheses, CAMAP received as inputs MCCs flanking regions from hit and decoy sequences from either the original or shuffled datasets. It was then trained to predict the probability that individual input sequences were MCCs flanking regions (i.e. hit) rather than sequences from the negative dataset . We first evaluated MAPs that could originate from different transcripts within the transcriptome (i.e. transcripts with sufficient expression levels detected by RNA sequencing) as they are likely to represent conserved regions in the genome. While 79.9% of MAPs originated from unique contexts , only 2.−16, Non-source transcripts have been previously associated with higher GC content . To evalPrevious studies have shown that codon bias plays a significant role in translation efficiency (TE) by co-adaptation to the tRNA pool ,26. This-16, We evaluated whether CAMAP would still be predictive for negative examples (decoys) derived from source transcripts. While most decoys derived from source transcripts showed a CAMAP score above 0.5 and are thus classified as source, they still had a lower CAMAP score compared to hits from source transcripts  in CAMAP’s predictions. The shuffling method used above Figs  and 2 ge−4). This could be due to the limited number of synonymous codons available for swapping within a single example. This also suggests that the overall codon composition of the MAP-flanking sequences has more impact on CAMAP predictions than the codons’ position within the MAP-flanking sequences. The third nucleotide shuffling also decreased CAMAP performances (mean AUC of 0.676) but remained above that of the transcriptome shuffled dataset (mean AUC of 0.647). These results suggest that the GC content has a significant impact on CAMAP performances, as the codon swap and third nucleotide shuffling both preserves the GC content within each example, while the transcriptome shuffling does not. This is also supported by the negative correlation between GC content and CAMAP scores, as shown in As shown in Taken together, these results support a role for codon arrangements, and especially for codon usage in the regulation of MAP biogenesis which cannot be reduced to codon’s impact on gene expression levels, GC content or translation efficiency.We next validated our CAMAP trained on 9-mer MAPs derived from B-LCL using 5 datasets derived from different human and mouse cell types. All the validation datasets were described through proteogenomic analyses similarly to our B-LCL training datasets. However, all the validation datasets included MAPs of 8–11 mers, in contrast with the training dataset that contained only 9-mer MAPs. The validation datasets consisted of (i) our B-LCL dataset, this time including all peptide lengths ,22, (ii)The lower performances of CAMAP trained with shuffled sequences (representing amino acid distribution) suggests that amino acids in MAP-flanking sequences are less informative than codons regarding MAP presentation. We formally quantified this difference in information using the Kullback-Leibler (KL) divergence see . These rWe wondered whether some regions were more influential on MAP presentation than others. To address this question, we retrieved the model preferences for each codon at each position. The preferences correspond to the prediction score of our best model  when a single codon at a single position is provided as input  and mRNA transcript expression levels. We used ligand scores as predicted by NetMHCpan4.0, which was shown to possess the best predictive capacities for naturally processed peptides compared to other predictive algorithms . Becauseper se. We thus additionally assessed the value of each prediction method using a multi-layer perceptron (MLP) classifier. Results show that for all of them, adding CAMAP score to the input improves prediction accuracy  and CAMAP scores third, . As expeaccuracy . Similaraccuracy . Interesin silico, both encoding for the same OVA protein but using different synonymous codons: one predicted to enhance SIINFEKL presentation (OVA-EP), the other predicted to reduce it (OVA-RP). Accordingly, the respective CAMAP scores for OVA-RP, OVA-WT and OVA-EP were: 0.03, 0.65, and 0.96  protein containing the model MAP SIINFEKL . One conand 0.96 . All varBecause codon usage affects translation efficiency, theoretically leading to DRiP formation through premature translation arrest ,21, we eb specific T-cell activation assay  and reimplemented identically for this work using pyTorch (results presented herein derive from the pyTorch version). An Embedding layer was used to associate each label superior to 0 to a set of 2D trainable parameters; the 0 label represents a null (masking) embedding fixed at coordinates . As an output layer, we used a Softmax layer with two outputs (positive / negative). Because negative sequences are more numerous than positive ones, we used an oversampling strategy during training. At each epoch, CAMAPs were randomly presented with the same number of positive and negative sequences. All CAMAPs in this work share the same architecture  and used this encoding to transform every sequence into a vector of integers representing codons. Neural networks were first developed using the Python package Mariana  [https:/itecture , number null value.For each condition (e.g. context size), the positive and negative datasets were randomly divided into three non-redundant subsets: (i) the training subsets containing 60% of the positive and negative examples, (ii) the validation and (iii) the test subsets each containing 20% of the positive and negative examples. Examples were assigned through a sequence redundancy removal algorithm, thereby ensuring that no example was assigned to multiple subsets. We used an early stopping strategy on validation sets to prevent over-fitting and reported average performances computed on test sets. We trained 12 CAMAPs for each combination of conditions, each one using a different random split of train/validation/test sets. To mask sequences either before or after the MCCs, we masked either half with The Kullback-Leibler (KL) divergence computes how well a given distribution is approximated by another distribution. Its value can be either positive or 0, a null value indicating that the two distributions are identical. Accordingly, a higher KL divergence for codon distributions vs. amino acid distributions would indicate that codon variations are not entirely accounted for by amino acid variations. KL divergence is not a metric, as it is neither symmetric nor does it satisfy the triangle inequality. It is nevertheless an accurate and most common way of comparing two probability distributions.c at position i as a function of the number of occurrences of c at position i, divided by the total number of occurrences of that same codon:We defined the probability of having codon Q is a probability, N is a number of occurrences, c is a codon, y is a class (positive or negative), s indicates if codons have been shuffled , i is a position in sequence. For the remainder of the text we will use the following abbreviations:Here cP distributions approximate cD distributions and cPS distributions approximate cDS distributions.We then used the KL divergence to compute how well The KL divergence was defined as:We performed this calculation for both the original and the shuffled dataset, which we then compared together. If codons and amino acid distributions were equivalent, KL divergence between hits and decoys would be the same for both original and shuffled sequences, and codons would cluster along the diagonal.Preferences are a way of interpreting CAMAP networks that naturally derives from CAMAP’s mathematical formalism. The validity of the preference method for CAMAP has been demonstrated when we used it to define the EP and RP constructs. By following this method, we were able to precisely engineer codon sequences that maximized or minimized the predictions of a CAMAP network. CAMAP only has two processing layers: an embedding layer and an output layer with no latent processing in between. Moreover, the output layer has no bias. In these very specific conditions, the manifold is the embedding layer. The word embedding layer encodes the importance of codons, while the weights of the output layer encode the importance of the position.The output is thus computed as:E is the vector of embeddings for a sequence and W is the weight matrix of the output layer. When masking all codons but one, we obtain the importance (I) of that codon (c) at that position (x):Where Wx are the weights of the output layer for position x. The preference is just this quantity  normalized to a probability (using a Softmax function) for ease of interpretation.Where LogisticRegressionCV or MLPClassifier functions from the python package sklearn . In each case, the dataset containing hits and decoy sequences was split into train and test datasets with a ratio of 0.7 to 0.3, respectively. Values for CAMAP score, Ligand Score and TPM were each scaled to a range of 0–1 in the train set using MinMaxScaler from sklearn.preprocessing and the same scaling model was applied to the test set afterwards. Regression analysis was performed using LogisticRegressionCV with a 10x cross-validation using the lbfgs solver with 1000 iterations. MLP with one hidden layer of 1000 neurons was trained using MLPClassifier for 4000 iterations using the adam solver with early stopping. Classes were balanced prior to training for both models. Matthew correlation coefficients were calculated using matthews_corrcoef from sklearn.metrics. When a peptide had multiple sources (multiple transcripts or genes), only the maximum transcript expression is used.The prediction capacity of CAMAP, NetMHCpan-4.0 ligand score and transcription expression (TPM) was tested in different combinations of those parameters  using the An inducible translation reporter was generated by flanking the truncated chicken ovalbumin (OVA) cDNA (amino acids 144–386) with EGFP-P2A (in 5’) and P2A-Ametrine (in 3’) cDNA sequences. MCCs flanking contexts for the EP and RP construct were synthesized as gBlocks (purchased from Integrated DNA Technologies). The fragments were amplified by PCR and joined by Gibson assembly under a doxycycline-inducible Tet-ON promoter in a pCW backbone. Synthetic variants of the OVA coding sequence were generated in silico by varying synonymous codon usage in the MAP context regions (i.e. 162 nucleotides pre- and post-MCCs). Importantly, the amino acid sequence was preserved between the different variants; only nucleotide sequences in the MAP context (162 nucleotides on either side) differed. The sequences with the highest (EP) and the lowest (RP) prediction scores were selected for further in vitro validation and swapped into the iTR-OVA plasmid by Gibson assembly . OVA-EP Important features of our inducible translation reporter construct and T cell activation assay were: (i) No changes in amino acid sequence between the three variants: only co-translational events can differ between the three variants, post-translational events being equivalent for the three constructs; (ii) Only one start codon, at the beginning of the eGFP coding sequence: this is important for the translation reporter aspect of our construct (i.e. Ametrine/eGFP ratio), to ensure that translation can only start at the 5’-end of the whole construct, and not at the beginning of the OVA or Ametrine coding sequences; (iii) Separation of the three proteins using P2A peptide: allows the inducible synthesis of three separate proteins in a highly correlated manner .Lentiviral particles were produced from HEK293T cells by co-transfection of iTR-OVA WT, EP or RP along with pMD2-VSVG, pMDLg/pRRE and pRSV-REV plasmids. Viral supernatants were used for Raw-Kb OVA-EP, OVA-RP and OVA-WT cells were plated at a density of 250,000 cells/well in 24 well-plates 24h prior to doxycycline treatment (1 mg/ml). After the corresponding treatment duration, cells were harvested and fixed using PFA 1% for 10 minutes at room temperature and washed using DMEM 10% FBS. Raw-Kb were then co-cultured  in triplicates with the CD8 T cell hybridoma cell line B3Z cells at a 3:2 ratio for 16h (7.5 x 105 B3Z and 5 x 105 Raw-Kb) in 96 well-plates. Cells were lysed for 20 minutes at room temperature using 50 μl/well of lysis solution . 170 μl/well CPRG buffer was added , 50mM Na2HPO4•7H20, 35mM NaH2PO4•H20, 9mM KCl, 0.9mM MgSO4•7H2O). β-galactosidase activity was measured at 575 nm using SpectraMax 190 Microplate Reader (Molecular Devices). In parallel, cells were analyzed by flow cytometry using a BD FACS CantoII for eGFP and Ametrine fluorescence.Raw-KS1 Fig2) factors and (b) Kullback-Leibler (KL) divergence between positional distribution of codons and their corresponding amino acid in the shuffled (y axis) VS original (x axis) datasets. For all codons, the shuffled dataset showed greater correlations (A) and smaller KL divergence to their respective amino acid distributions than the original datasets .(A) Pearson correlation (R(TIF)Click here for additional data file.S2 Fig2 > 0.95, compared to only 69% in the original dataset (p < 5x10-5).92% of codons in the shuffled dataset reflecting the amino acids distribution with a R(TIF)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 Fig(EPS)Click here for additional data file.S8 Fig(EPS)Click here for additional data file.S9 Fig(EPS)Click here for additional data file.S10 Fig(EPS)Click here for additional data file.S11 Fig(EPS)Click here for additional data file.S12 Fig(EPS)Click here for additional data file.S13 Fig(EPS)Click here for additional data file.S14 Fig(EPS)Click here for additional data file.S15 Fig(EPS)Click here for additional data file.S16 Fig(EPS)Click here for additional data file.S17 Fig(EPS)Click here for additional data file.S18 Fig(EPS)Click here for additional data file.S19 Fig(EPS)Click here for additional data file.S20 Fig(EPS)Click here for additional data file.S21 Fig(A) Architecture of the ANN used in this work. (B) Results for the AUC on all train, validation and test subsets. Grey areas represent the 95% confidence intervals. (C) Distributions of output probabilities of CAMAPs used to calculate correlations in (TIF)Click here for additional data file.S22 FigFor each sequence in the test set we calculated the average prediction score given by CAMAPs in each condition, and calculated the Pearson correlation using the R software. Densities were calculated on all points and drawn using ggplot2. Only a random subset of the points is represented in the figures to limit their size.(TIF)Click here for additional data file.S23 Fig(TIF)Click here for additional data file.S24 Figp = 5.36 x 10−7).(A) Distribution of transcript expression levels for normal datasets (related to (TIF)Click here for additional data file.S25 Fig(TIF)Click here for additional data file.S26 Fig(TIF)Click here for additional data file.S27 Figp = 1.21 x 10−9).(A) Distribution of binding affinities for normal dataset (related to (TIF)Click here for additional data file.S28 Fig(A) Proportion of unique MAPs that can be ascribed to a single origin, 2–3, 4–10 or >10 possible origins. (B) Proportion of entries in the hit dataset that encode for MAPs with a single origin, 2–3, 4–10 or >10 possible origins(TIF)Click here for additional data file.S29 Fig(TIF)Click here for additional data file.S30 Fig(TIF)Click here for additional data file.S31 FigPearson’s R correlation score is shown on the graphs.(TIF)Click here for additional data file.S32 Figp = 1.07 x 10−4).(A) Distribution of transcripts’ GC content for normal dataset (related to (TIF)Click here for additional data file.S33 FigCAMAP was trained using hits from source transcripts and decoys from non-source transcripts only. Significance was assessed using bilateral unpaired T test.(TIF)Click here for additional data file.S34 Fig(A) Transcriptome shuffling: generates shuffled sequences by replacing each codon by one of its synonymous codons (including itself) according to the codon usage within the transcriptome, regardless of the codon used within each example. (B) Codon swap shuffling: synonymous codons present within a given example are swapped with one another. (C) Third nucleotide shuffling or N3: the third nucleotide of codons are swapped within each example to preserve amino acid sequences and GC content. Here, the global codon usage is completely different than normal codon usage in humans, as the frequency of codons is not taken into account during shuffling.(TIF)Click here for additional data file.S35 FigThe performance of CAMAP networks (n = 12) pre-trained on original (non-shuffled) datasets was evaluated on three shuffled datasets according to the methods depicted in (TIF)Click here for additional data file.S36 FigShuffled sequences represent amino acid usage, as codon-specific information are removed with synonymous codon shuffling.(TIF)Click here for additional data file.S37 FigSee (TIF)Click here for additional data file.S38 Fig(A) Comparison of the OVA-EP nucleotide sequence to the wildtype OVA sequence. The OVA-EP and OVA-WT sequences have 93.3% nucleotide identity for a total of 78 modified nucleotides. (B) Comparison of the OVA-RP nucleotide sequence to the wildtype OVA sequence. The OVA-EP and OVA-WT sequences have 92.6% nucleotide identity, for a total of 86 modified nucleotides. Mutations, shown in red, are located only in the 162 nucleotide regions flanking the SIINFEKL coding codons. Of note, the SIINFEKL coding codons (nucleotides 772–799) were not modified between the 3 constructs.(TIF)Click here for additional data file.S39 FigSingle cell eGFP and Ametrine fluorescence intensities measured at 10 hours post-induction are shown for the OVA-WT (A), OVA-EP (B) and OVA-RP (C) constructs. N.B.: only transduced cells are shown (eGFP+ cells).(TIF)Click here for additional data file.S40 Figet al. (2016) using the new versions of MAP binding affinity prediction algorithm NetMHC4.0 (A) and NetMHCpan4.0 (B).Validation of MHC-I associated peptides (MAP) dataset from Pearson H. (TIF)Click here for additional data file.S41 FigC x 2 + 27, where C is the context size in nucleotides and 27 the length of the MCCs. Related to Transcript length corresponds to (TIF)Click here for additional data file.S1 TableSIINFEKL MCCs are shown in bold, while the variant regions  are in blue and italics. Related to (DOCX)Click here for additional data file.S2 TableThe lower the number of peptides needed to capture the respective number of epitopes, the better the performance of the prediction model. This is also illustrated by the percentage of false identification  reported here. Peptides were rank ordered according to regression scores. Of note, only the maximal transcript expression was used for peptides with multiple potential origins.(DOCX)Click here for additional data file.S3 TableThe lower the number of peptides needed to capture the respective number of epitopes, the better the performance of the prediction model. This is also illustrated by the percentage of false identification  reported here. The non-linear classifier led to better predictions when using ligand score and transcript expression, with or without CAMAP scores compared to logistic regression model. Peptides were rank ordered according to regression scores. Of note, only the maximal transcript expression was used for peptides with multiple potential origins.(DOCX)Click here for additional data file.


The forensic toxicologist is challenged to provide scientific evidence to distinguish the source of ethanol  determined in the postmortem blood and to properly interpret the relevant blood alcohol concentration (BAC) results, in regard to ethanol levels at death and subsequent behavioral impairment of the person at the time of death. Higher alcohols , and 3-methyl-2-butanol ) are among the volatile compounds that are often detected in postmortem specimens and have been correlated with putrefaction and microbial activity. This brief review investigates the role of the higher alcohols as biomarkers of postmortem, microbial ethanol production, notably, regarding the modeling of postmortem ethanol production. Main conclusions of this contribution are, firstly, that the higher alcohols are qualitative and quantitative indicators of microbial ethanol production, and, secondly that the respective models of microbial ethanol production are tools offering additional data to interpret properly the origin of the ethanol concentrations measured in postmortem cases. More studies are needed to clarify current uncertainties about the origin of higher alcohols in postmortem specimens.  Blood ethanol analysis is the most frequent test performed in a forensic toxicology laboratory, as part of the investigation process of death, accident, or crime. The applied analytical procedures are rather simple and provide accurate, specific, and precise results on ethanol concentration. The relative analytical data are used to support the forensic evidence, as part of the judicial inquiry, in relation to the inter-relation between the consumption of alcoholic beverages, the measured blood ethanol concentration, and the impairment of body functions ,2,3. ParThe possible sources of ethanol detected in postmortem specimens could be either antemortem ingestion by a living person who consumed alcoholic beverages, or antemortem endogenous production due to microbial fermentation in the intestine (“auto brewery” syndrome ), or posTherefore, the discrimination of the source of ethanol, ante mortem ingestion or postmortem microbial production, is of vital importance for the proper interpretation of postmortem BAC results. It is the forensic toxicologist who is challenged to provide the relative, available scientific evidence on the ethanol levels at death and the subsequent behavioral impairment of the person at the time of death ,3,5,6.Factors, such as the putrefaction state of the cadaver at autopsy, the clinical history of the deceased, the determination of glucose levels, the identification of microbes in the analyzed sample, and the evaluation of the discrepancies between ethanol concentration from various sampling sites and from different specimens, have been used to evaluate the origin of the measured ethanol, in the effort to achieve feasible accuracy in interpreting the postmortem ethanol analysis results ,2,3,6. FThe detection of low molecular weight volatiles in human specimens has been related, for decades, with microbial activity and ethanol neoformation, while their presence in postmortem specimens has been correlated with putrefaction and microbial activity . Among tE. coli . Th. Th39]. Two more studies have reported blood ethanol and 1-propanol concentrations from postmortem cases  13,40].,40.13,40Many studies reported on the microbial ethanol generation post-sampling in postmortem samples ,3,6. On At this point, it should be mentioned that the increased temperature and the duration of storage are recognized as the premium influencing factors of the post-sampling alcohol production , besidesThe higher alcohols, 1-propanol, isobutanol, 1-butanol, and 3-methyl-1-butanol, are among the primarily targeted congeners during the alcohol congener analysis workflow and evaluation ,50. PartIn As could be concluded from As was already mentioned, the higher alcohol mostly correlated to putrefaction and microbial ethanol production is 1-propanol ,16,18,19More recently, another semi-quantitative approach, the 1-propanol concentration of 0.104 mg/dL (the “1-propanol criterion”), was suggested as an effective threshold concentration (“cut-off”) for “flagging” an autopsy blood sample as positive or “negative” for postmortem ethanol production . It was The approach of “modeling microbial (postmortem) ethanol production” has considered all the higher alcohols  as quantitative biomarkers of postmortem ethanol production ,34,35,36E. faecalis cultures, where 1-butanol preceded the other higher alcohols. This observation probably explains why 1-propanol is the most studied higher alcohol in postmortem cases, given that bacteria are the most common inhabitants of a dead body and that they, usually, invade first the different body compartments after death [The first stage of the modeling procedure was the development of bacterial, clostridial, and fungal cultures under controlled laboratory conditions, selected to approximate the conditions after death, and the recording of the concentrations of ethanol and higher alcohols produced by the microbes ,34,35,36er death ,27,37,38C. perfrigens and C. sporogenes) produced more 1-butanol than 1-propanol. This observation probably indicates that clostridia prefer the butyrate-butanol-acetate fermentation pathway, which produces 1-butanol directly from carbohydrates  predominated against 1-propanol’s levels, while 1-butanol levels were negligible. Therefore, it is reasonable to conclude that C. albicans, under the applied laboratory conditions, prefer the synthesis of branched higher alcohols than the neoformation of 1-propanol. This aspect differentiates fungal cultures [C. sporogenes). Secondly, the concentrations of 1-butanol are higher in the cultures than in postmortem cases, while some postmortem cases have 1-butanol levels as high as those determined for the C. sporogenes cultures. Thirdly, 1-propanol concentrations in cultures are within the range of 1-propanol reported usually for postmortem cases  to 100% . It became apparent, as is already known [The initial glucose levels to the BHI and SDB culture media , and isobutanol, are quantitative indicators of microbial ethanol production. Furthermore, the quantitative relationships between the microbial higher alcohols’ concentrations and the microbial ethanol’ concentrations, as expressed by the suggested models, allow the estimation of the postmortem neoformed ethanol concentrationThe applicability of the constructed models was tested in a series of autopsy blood samples where ethanol was detected simultaneously with at least one higher alcohol ,34,35,36S. aureus models [E. coli models [C. sporogenes and C. perfrigens models achieved the predefined score in 45% to 68% of the cases, respectively [E. coli models, and in 85% of the cases by the clostridia ones [E. coli, E. faecalis, C. perfrigens, and C. sporogenes models achieved the score in more than 95% of the cases [The relevant studies reported that the bacterial models succeeded the predefined score (E < ±40%) in 19% of the cases for the s models , up to 4i models ,34. The ectively . When eadia ones ,34. Whenhe cases ,34,35. Fhe cases . Althoughe cases , in posthe cases  developeSo far, the most studied and the most abundant higher alcohol in postmortem cases is 1-propanol. However, it is not the only higher alcohol which indicates microbial ethanol production. The five higher alcohols, 1-propanol, 1-butanol, isobutanol, 3-methyl-2-butanol , and 2-methyl-1-butanol , are qualitative and quantitative indicators of microbial ethanol production, as it is indicated by the respective bacterial, clostridial, and fungal ethanol production models.The models of microbial ethanol production are tools offering additional data to properly interpret BAC in postmortem cases, as well as to define the origin of ethanol in the sample. The employed, relatively simple linear models for estimating microbial ethanol production provide a noteworthy accuracy in cultures and autopsy blood.In the view of this review, the proper interpretation of postmortem ethanol analysis results should follow a step-by-step approach to estimate the suggested in the literature indicators, starting from the more easily obtainable, the detection of 1-propanol, isobutanol, methyl-butanols, and 1-butanol, during the chromatographic ethanol analysis. Then, the microbial models should be used to calculate the microbial generated ethanol and, subsequently, to evaluate the results in relation to case specific characteristics, such as putrefaction state, clinical history, and others. Finally, more selective analyses, providing more elaborate indicators of the ethanol origin , could be performed, to provide complementary data that would aim accurate interpretation of postmortem BAC.In conclusion, the evaluation of the postmortem BAC remains a complicated, multifunctional procedure. Needless to underline that more studies should be performed regarding the levels of higher alcohols in postmortem and ante mortem cases, to document the extent of applicability of the suggested models, to achieve feasible accuracy in interpreting the results of ethanol analysis, and to estimate the BAC origin in postmortem samples with increased certainty.


Traumatic myositis ossificans (MO) is an unusual complication after muscle injury and is predominantly seen in young adults and adolescents. Pediatric MO cases are even rarer. We report an 8-year-old girl who was diagnosed with a lateral humeral condyle fracture. She was treated surgically, and her elbow joint was fixed with plaster. Rehabilitation exercise was administered 1 month after the operation. Due to the wrong exercise method, a palpable bony mass appeared around the elbow 1 month later. The clinical radiological diagnosis showed MO, and conservative treatment was administered. After 3 years of follow-up, the affected limb functioned well, with no sign of recurrence. Here, we report this long-term follow-up case of MO resulting from excessive rehabilitation exercise. Myositis ossificans (MO) is a benign tumor-like lesion, which is predominantly seen in young adults and adolescents. Pediatric MO cases are even rarer . Approxivia X-ray radiography  was made directly on the lateral condyle of the humerus on the outside of the elbow joint under aseptic conditions. After separating the skin and fascia layer by layer, the fracture ends were exposed. After clearing the congestion and reducing the incarcerated soft tissue, three K-wires were used to fix the fracture. The results from intraoperative fluoroscopy showed a good reduction. The lateral collateral ligament was examined to confirm its integrity immediately. Postoperatively, a long-arm posterior splint was placed with the elbow flexed 90 degrees . The opeiography . Since tiography , 2018/12iography , 2019/3 iography , and 201iography . The resiography , and cliMO is predominantly seen in young adults and adolescents and is most commonly secondary to trauma. Although the exact etiology remains unclear, patients typically present with pain and restricted range of motion following trauma or overuse . In thisvia radiographic monitoring. Thus, the mass was not a tumor.Due to the bone hyperplasia early during the disease, such masses may be mistaken for osteosarcoma or soft-tissue tumors . Throughvia X-ray radiography, whether it is accompanied by any TRASH lesion should be determined. MRI or arthrography can be more informative than X-ray radiography in the assessment for a cartilage fracture. In clinical practice, the gap of a lateral condyle fracture is often used to determine whether surgical treatment is required. The fracture is stable when the fracture gap is <2 mm, often without osteochondral fractures, and plaster fixation is often used as the treatment modality. However, the lateral condyle of this patient was significantly displaced (>2 mm), and thus the fracture gap was deemed unstable and needed closed reduction along with K-wire fixation  often require surgical intervention . As the fixation . During An intense fibrocartilagenous callus can also develop in lateral condyle fractures, albeit rarely. Such cases are often accompanied by nonunion and lack adequate immobilization or stability to progress to the union. They are common in the conservative treatment of pediatric clavicle fractures . Studiesvia conservative treatment. Over a period of 14 months, radiographs showed increasing ossification of the mass, which required operative treatment. Kanthimathi et al. ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.HJZ, JHS, and HYZ collected the data. JC wrote the first draft of the manuscript. CG contributed to data interpretation, edited the manuscript, and approved the final version. All authors were involved in the conception of the study.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.


Bezoar is a term from Arabic “bāzahr” or ultimately from Middle Persian “p'tzhl” . It was believed to have the power of a universal antidote that works against any poison, and a glass containing a bezoar could neutralize any poison poured into it. In science, it is a mass of hair or undigested vegetable matter found in a human or animal intestines, similar to a hairball. Otherwise, the name could derive from a kind of Turkish goat whose name is just bezoar. Usually, it is found trapped in every part of the gastrointestinal system and must be distinguished by pseudobezoar, which is an nondigestible object voluntarily introduced into the digestive tract. The most common causes are a previous gastric surgery such as a gastric band (for weight loss) or gastric bypass, a reduced stomach acid (hypochlorhydria) or decreased stomach size, and a delayed gastric emptying, typically due to diabetes, autoimmune disorders, or mixed connective tissue disease. Seed bezoars are usually found in the rectum of patients without predisposing factors, causing constipation and pain. Rectal impaction is common after ingestion of seeds, while a true occlusion is rare. Although several cases of phytobezoars composed of various types of seeds are reported in the literature, bezoars of pumpkin seeds have rarely been reported. The authors report a case of fecal impaction by pumpkin seed bezoars with abdominal pain: a difficulty to void with subsequent rectal inflammation and hemorrhoid enlargement was observed. The patient underwent a successful manual disimpaction. In J.K. Rowling's Book of Harry Potter, the apprentice scientist is quizzed on bezoar during the very first Potions Class . Bezoar Usually, it is found trapped in every part of the gastrointestinal system and must be distinguished by pseudobezoar that is an nondigestible object voluntarily introduced into the digestive tract , 6.Bezoars take the name from the core substance so that we can distinguish them: phytobezoars are composed of vegetable fibers and seeds, trichobezoars are formed from hair, lactobezoars are from inspissated milk, and diospyrobezoars are from unripe persimmon fruits .The overall incidence of bezoars is felt to be low and is extremely rare in healthy individuals occurring in far less than 1% of patients in retrospective endoscopic series . Kadian Certain at-risk groups have been identified and include patients with altered upper GI anatomy after surgery and psychiatric illness or cognitive impairment. The most common causes are a previous gastric surgery such as a gastric band (for weight loss) or gastric bypass, a reduced stomach acid (hypochlorhydria) or decreased stomach size, and a delayed gastric emptying, typically due to diabetes, autoimmune disorders, or mixed connective tissue disease. Other causes are patients who cannot or do not chew their food properly, usually because they have no teeth or poorly fitting dentures and because of an excessive intake of fibers. Edentulous patients with poor mastication of food particles may also be at greater risk for bezoar development, especially if coexisting risk factors, as described above, are also present. In addition, patients with psychiatric illnesses are at an increased risk of bezoar formation due to the possible ingestion of hair and medications , 9.Many cases of bezoars have also been reported in children or adults having psychosocial problems; nevertheless, the condition can occur in normal children with no apparent psychosocial issues .Seed bezoars are usually found in the rectum of patients without predisposing factors, causing constipation and pain. Although the literature has reported several cases of phytobezoars composed of various types of seeds, bezoars formed from pumpkin seeds have rarely been reported . The diaWe report a case of a man aged 50 years with a rectal bezoar composed of pumpkin seeds ingested with their shell, necessitating extensive treatment, including manual disimpaction and rectoscopy.The description of the case follows the 2013 CARE Checklist Guidelines  with their shell. He was unemployed and showed a depressed attitude. He reported never having had problems of this nature before but having problems of constipation in the last months.The plain x-ray of the abdomen showed dilatation of the left and bowel. A proctological inspection revealed a hard bolus in the rectum  with bloUnder sedation with propofol, a disimpaction of the bezoar was accomplished with a colon washing. The patient was discharged on the same evening with a prescription of intestinal antibiotics and a large bowel toilet with polyethylene glycol and enemas.Two days after the first admission, the patient returned to the outpatient department complaining of a persistent difficulty to void with a burning sensation and blood loss. A residual hard bolus, smaller than the first, was detected in the rectum, and another disimpaction under local anesthesia was performed. A proctoscopy showed small diffuse ulcerations of the rectal mucosa and enlargement of hemorrhoids . The colThree weeks after this episode, the rectal mucosa reverted to normal, and the patient declared to move regularly without a burning sensation.Rectal seed bezoar is an uncommon cause of fecal impaction, more frequent in eastern than western countries and particularly in Middle Eastern and South Asian countries where roasted seeds are very popular . The comSeed bezoar is a subcategory of phytobezoar caused by the accumulation of indigestible vegetable or fruit seeds in the intestine lumen. They usually pass the stomach and the ileocecal valve and deposit in the colon up to the rectum, where the compound is dehydrated and forms a hard bolus impossible to evacuate . Seed beSeed bezoar occurs most frequently in the rectum both in children and adults, and symptoms are mainly constipation followed by abdominal pain and rectal burning. A true intestinal obstruction is rare, and perforation is reported only in one case , 19. FibFrom 1980 to 2018, 52 studies were reported by Manatakis  respondiPreventive therapy to avoid recurrence must be implemented when the bezoar is removed. The patient should be advised to increase the amount of water intake. Dietary habits must be investigated since inadequate chewing, swallowing whole seeds, or eating seeds with their shell may impact as bezoar , 23, 24.P = 0.02), Further investigation is needed to understand the pathogenesis of bezoar formation in this selected population  developed gastric bezoars confirmed by upper endoscopy and 94% of patients with bezoars (16 of 17) had cystic fibrosis (pulation .Seed bezoar is an uncommon cause of fecal impaction, more frequent in eastern than western countries and particularly in Middle Eastern and South Asian countries. Seed bezoar is a subcategory of phytobezoar caused by the accumulation of indigestible vegetable or fruit seeds in the intestinal lumen: it occurs most frequently in the rectum both in children and adults, and symptoms are constipation followed by abdominal pain and rectal burning. Intestinal obstruction is rare, and perforation is reported only in one case. Manual disimpaction is the commonly used procedure both in children and adults, while surgery is more frequent in adults than in children. Preventive therapy to avoid recurrence must be implemented when the bezoar is removed. An increase in the amount of water intake should be advised. Psychiatric support is mandatory in patients with recurrent episodes of seed ingestion.


The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates. There appears to be a duplicated image, with some possible signs of alteration, between the first two panels of Figure 4E.


Ceratitiscapitata flies was carried out in different locations in Tunisia.Several nematode isolates were recovered, laboratory colonies were established,and their taxonomic identities were determined based on molecular methods. Amongall the recovered nematode isolates, two of them, Oscheiustipulae TC2 and OC2, were evaluated for their capacity to controlC. capitata flies and for their ability tokill and reproduce on Galleria mellonella larvae. Our resultsshow a great potential of these two isolates as biocontrol agents as they killC. capitata eggs and pupae and interferewith the metamorphosis of C. capitata larvae.More specifically, TC2 and OC2 nematodes killed 39 and 31% ofC. capitata eggs, respectively, impaired themetamorphosis of up to 77% and up to 67% of C.capitata larvae, respectively, and killed up to 66% and upto 58% of C. capitata pupae, respectively. Theefficacy of TC2 and OC2 nematodes was particularly high on C.capitata pupae, and significant insect mortalities wereobserved even at concentrations of 1 and 5 nematodes/pupae, respectively. Wealso found that TC2 and OC2 nematodes efficiently kill and reproduce inG. mellonella larvae, suggesting thatthese insects could be used for mass-multiplication of these nematodes. Theseresults reveal the potential of O. tipulae tocomplement integrated pest management programs against C.capitata flies.A survey to collect soil nematodes with potential to control  Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), theMediterranean fruit fly or medfly, is one of the most limiting insect pests forcitrus production [Clementine,Mandarin, Navel, Maltaise,Valencia Late and Double Fine oranges areattacked by C. capitata although to differentdegrees of intensities [The citrus agro-industry is one of the most important sectors for the economy ofTunisia. Citrus production is unfortunately hampered by the occurrence of differentdiseases and pests that considerably impair tree growth and crop yields.oduction .Severalensities ,3. This ensities . The conensities ,5. The uensities ,7.Steinernema(Travassos) (Nematoda: Rhabditida) and Heterorhabditis (Poinar)(Nematoda: Rhabditida), which are associated with bacteria of the genusXenorhabdus and Photorhabdus, respectively[Oscheius (Andrássy) (Nematoda: Rhabditidae) [From the great variety of biocontrol agents, entomopathogenic nematodes (EPNs) arehighly promising due to their capacity to efficiently control different insect pests–10. The ectively–17. Seveectively–20. Aparditidae) –30.Oscheius nematodes are soil-dwelling nematodes of the Rhabditidaefamily. Several of the species of this genus are considered scavengers, orbacteriophagous free-living nematodes, while several others have been shown to killinsects. Currently, there are about 30 recognized species in the genusOscheius, and more than 13 have the ability to kill insects[Oscheiusonirici kills the larvae of Galleria mellonella(Linnaeus) , Tenebrio molitor (Linnaeus)(Coleoptera: Tenebrionidae) and Drosophila suzukii (Matsumura)(Diptera: Drosophilidae); Oscheius carolinensis has potential as abiological control agent against Pieris rapae (Linnaeus)(Lepidoptera: Papilionoidae) and T. molitor;Oscheius gingeri was efficient against G.mellonella and Conogethes punctiferalis(Guenée) (Lepidoptera: Crambidae) larvae; and Oscheius tipulae andO. rugaoensis also kill G.mellonella larvae [O. onirici isolates were shown to killdifferent insect species, while a Swiss isolate was not harmful toG. mellonella [O. saproxylicus, and O.tereticorpus, whose ability to kill insects have not beentested, however, suggesting that this trait could be much more spread in the genusthan it is currently thought [Steinernema and Heterorhabditis nematodes tocontrol agricultural pests. insects,22. For a larvae ,23–30. Ta larvae . Howeverllonella ,33. Ther thought ,32–40. COscheius nematodes as biocontrolagents and aiming at increasing their availability to be used to controlC. capitata flies, we collected soil-dwellingnematodes at several locations in Tunisia using C.capitata pupae as baits, characterized them using moleculartools to determine their taxonomic identities, and selectedOscheius nematodes to specifically evaluate their abilities tokill C. capitata at different developmental stagesand temperatures. As C. capitata pupates in thesoil, it is one of the most suitable stages for biocontrol using soil-born nematodessuch as O. tipulae. The objective of our study wasto show the great biocontrol potential of two O.tipulae isolates against C.capitata flies. Hence, our study encourages to continuestudying the biology of O. tipulae nematodes andtheir interaction with C. capitata and otherinsect pests to determine their actual biocontrol potential and to explore thepossibility of incorporating them in biocontrol programs against agriculturalpests.Given the promising potential of Citrussinensis var. Maltaise) infested by C.capitata. Twenty soil samples per location were collectedat a depth of 0-30cm using a hand shovel. Samples were placed in plastic bagsand stored at 7°C for further analyses. Soil nematodes were recovered from thesoil samples using C. capitata pupae orgreater wax moth larvae as baits according to the procedures described byBedding and Akhurst [Ceratitis capitata pupae were included as baits to obtainnematodes closely associated to this pest. For each sample, 300g of soil wereplaced in 500ml plastic containers. Then, ten C.capitata pupae, reared in artificial diets as describedbelow, were added to the plastic containers. Soil was moistened using a watersprayer. Plastic containers were incubated at 22°C in the dark for 5 days. Afterthis period, all dead insects were collected, rinsed three times with distilledwater and incubated for 24 hours at ambient temperature. Then, fiveG. mellonella larvae were added to thesame soil container. Plastic containers were incubated at 22°C in the dark for 5days. All dead insects were collected, rinsed three times with distilled water,incubated for 24 hours at ambient temperature, transferred to White traps andincubated at 22°C for 8 days in darkness [C. capitata pupae and G.mellonella larvae, or were cultured in egg yolk media . In all cases, cultures weremaintained in darkness at 25°C. Progenies were collected and cultured in freshmedia/insects. After several cycles, the nematode cultures were inspected undera light microscope  to determinepotential mixture of different nematode species.Soil samples were collected, during the winter season of 2019 and 2020, at fivedifferent locations in Tunisia: Morneg, Takilsa, Kobba, Sidi-Saad, and Ouzra.Soil samples were taken from soils of citrus crops  . Adult f5’-TTGATTACGTCCCTGCCCTTT-3’ (forward), and 26S:5’-TTTCACTCGCCGTTACTAAGG-3’ (reverse) [5’-CCTTAGTAACGGCGAGTGAAA-3’ (forward) and 536:5’-CAGCTATCCTGAGGAAAC-3’(reverse). The 18S rRNA genewas amplified using primers NEM18SF:5’-CGCGAATRGCTCATTACAACAGC-3’ (forward) and NEM18SR:5’-GGGCGGTATCTGATCGCC-3’ (reverse) [Nematode genomic DNA was extracted from about five thousand nematodes at alldevelopmental stages using the genomic DNA isolation kit from NORGEN BioTEK(Cat. 24700) following the manufacturer’s instructions. Genomic DNA was used toamplify different regions of the rRNA genes by PCR. Briefly, ITS regions  were amplified using primers 18S:reverse) . The frareverse) . PCR cycThe evolutionary histories based on the different rRNA gene sequences wereinferred by using the Maximum Likelihood method based on the General TimeReversible model (18S and D2D3) or on the Tamura 3-parameter model (ITS) ,48. BestO. tipulaenematodes to control C. capitata, we evaluatedmetamorphosis and mortality of C. capitataeggs, larvae, and pupae exposed to different concentrations of twoO. tipulae isolates, TC2 and OC2, asdescribed below.To evaluate the potential of O. tipulae TC2and OC2 nematodes on C. capitata eggs wasevaluated based on the induced mortality of these latter. Fifty eggs wereplaced in sterile Petri plates (60 mm diameter) layered with two filterpaper sheets. Then, 5000 O. tipulae TC2 or5000 OC2 nematodes suspended in 2ml of sterile water were added to eachPetri plate. Controls were treated with water only. Then, all Petri plateswere sealed with Parafilm. Six Petri plates for each treatment wereevaluated (n = 6). Egg mortalities, measured as the number of eggs that didnot hatch, were recorded three days post-treatment. The bioassay wasmaintained at 22°C.The biocontrol potential of O. tipulae TC2and OC2 nematodes on C. capitata larvae waevaluated based on successful metamorphic transition of C.capitata larvae. Twenty third instar larvae were placedin sterile Petri plates (60 mm diameter) layered with two filter papersheets. Then, either 50, 125, 250 or 500 nematodes/larva suspended in 1 mldistilled water were added to each Petri plate. Controls were treated withpure water only. Then, all Petri plates were sealed with Parafilm. FivePetri plates for each treatment were evaluated (n = 5). Due to the rapidtransition from the larval to the pupal stage, the number of healthy andunhealthy pupae was recorded three days post-treatment. The bioassay wasmaintained at 25°C.The biocontrol potential of O. tipulae TC2and OC2 nematodes on C. capitata pupae wasevaluated based on the induced mortality of these latter. Six nematode doseswere used: 1, 5, 10, 50, 100 and 500 nematodes/pupa suspended in 1 ml ofwater. Controls were treated with pure water only. For each treatment, 5Petri plates (60 mm diameter) layered with two filter paper sheets and with20 pupae each were assayed. All Petri plates were sealed with Parafilm. Thebioassays were maintained at 25°C.The biocontrol potential of O. tipulae TC2 and OC2 nematodes, themortality of C. capitata pupae exposed todifferent nematode concentrations was evaluated at different temperatures.Four different nematode concentrations were used: 50, 125, 250 and 500nematodes/pupa suspended in 1 ml of distilled water. Controls were treatedwith pure water only. Five Petri plates (60 mm diameter) layered with twofilter paper sheets and with twenty 3- to 5-day-old pupae each were assayed.All Petri plates were sealed with Parafilm. The bioassays were maintained at20, 25, and 30°C.To evaluate the impact of temperature on the biocontrol potential ofO. tipulae TC2 andOC2 nematodes to kill and reproduce on G.mellonella, the mortality of G.mellonella larvae and the number of emerging nematodes fromG. mellonella larvae exposed to thesenematodes was evaluated in three independent experiments. In the firstexperiment, Petri plates (110mm diameter) were layered with two filter papersheets, then ten first-instar G. mellonellalarvae were placed in each plate and treated with either O.tipulae TC2 or OC2 nematodes at a concentration of 500nematodes/larva suspended in 1 ml of water. Four Petri plates per nematodestrain were used. In the second experiment, Petri plates (60 mm diameter) wereeach layered with two filter paper sheets, then one first-instarG. mellonella larva was placed in eachPetri plate and treated with either O. tipulaeTC2 or OC2 nematodes at a concentration of 500 nematodes/larva suspended in 1mlof water. Ten Petri plates per nematode strain were used. In the thirdexperiment, Petri plates (60 mm diameter) were layered with two filter papersheets, then ten first-instar G. mellonellalarvae were placed in each Petri plate and treated with eitherO. tipulae TC2 or OC2 nematodes at aconcentration of either 250 or 500 nematodes/larva suspended in 1 ml of water.Three Petri plates per nematode strain and concentration were used. In allexperiments, controls were treated with pure water only. Insect mortality wasrecorded every 24h for five days. To evaluate the reproductive potential ofnematodes, all dead larvae from the third experiment were rinsed with water andindividually placed in White traps [To evaluate the potential of te traps . EmerginC. capitata pupae wasassessed by three-way ANOVA with temperature, nematode isolate and nematodeconcentration as factors. Galleria mellonella mortalities wereanalyzed by two-way repeated measures ANOVA with time and nematode isolate asfactors. Nematode reproduction was evaluated by two-way ANOVA with nematodeisolate and nematode concentration as factors. Normality and equality ofvariance were verified using Shapiro–Wilk and Levene’s tests, respectively.Holm–Sidak post hoc tests were used for multiple comparisons. All statisticalanalyses were conducted using Sigma Plot 14.5 .Differences in egg mortalities were assessed by one-way ANOVA with nematodeisolate as a factor. Differences in the number of deformed pupae and pupalmortalities were assessed by two-way ANOVA with nematode isolate and nematodeconcentration as factors. The effect of temperature on nematode biocontrolpotential against O.tipulae , Caenorhabditis elegans(TG3), and Acrobeloides spp.    andS2.equences .Howeveridi-Saad .O. tipulae TC2 and OC2show clear symptoms of infestation , andtherefore are used as biocontrol agents against many insect pests . Howeverize EPNs . Severalsothovii , O. carolinensis , O.choningensis ,63,O. mcrovilli , O.myriiophilus , O. onir onirici , O.rugagaoensis –68,O. safricana  and O.ttipulae . Some spropsophi  and O. eancensis . ApparenO. tipulaeare capable of killing C. capitata andG. mellonella, in a manner that is comparableto highly efficient entomopathogenic nematodes such asHeterorhabditis and Steinernema [C. capitata pupae. Thus, we add more evidenceon the ability of free-living, bacteriophagous nematodes to kill insects, and theirpotential to complement pest management programs based on these biological controlagents [Our results show that two populations of inernema –73. Signlagents . ClearlyO.tipulae, O. onirici andAcrobeloides spp. have been found to naturally co-occur withEPNs in certain regions of the world and have been isolated in conjunction with EPNsfrom G. mellonella cadavers retrieved from baitedtraps in the soil [Heterorhabditis and Steinernema were found insoil samples, supporting this notion, and suggesting that free-living nematodesmight out compete EPNs or that certain edaphic or climatic parameters might favorthem over the EPNs.Several studies have shown that some free-living nematodes in Rhabditida orderinteract with EPNs ,58,74,75the soil ,76,77. Uthe soil ,58. In tOscheius species, can be considered EPNs[O.tipulae nematodes, and also that less than five individuals cancause significant mortality of C. capitata pupae,but we still do not know if this effect is caused by insect-pathogenic bacteria. Themode of action is therefore uncertain and clearly deserves more attention. Hence, itremains to be determined if O. tipulae can beconsidered an EPN, based on Dillman’s definition [O. chongmingensis and O.carolinensis are considered EPNs [O.tipulae is considered a facultative kleptoparasite that competewith EPNs for insects [There is still a lot of controversy on whether or not certain species of free-livingnematodes, including red EPNs. An EPN,red EPNs. Inour finition . Other mred EPNs ,62. Howered EPNs . Similar insects ,an ento insects , and oth insects . ClearlyC.capitata and G. mellonellaare highly susceptible to these nematodes. If they rely on closely-associatedbacteria to kill remains to be investigated. The entomopathogenic action of someOscheius species has been linked to Serratiabacteria which play an essential role during the infection and presumably cause thedeath of the host insect [O. carolinensis wasconsistently associated with four bacterial species, one of which, Serratiamarcescens, appears to be carried on the cuticle of the nematodes andthrough its association provides the worms with entomopathogenic potential [Independently of their ecological classification, we show that t insect . For exaotential . Anotherotential ,82. CleaO. tipulae against C.capitata. Under which circumstances and the exact mechanismshow these free-living bacteriophagous nematodes kill insect hosts remain to beinvestigated. Likewise, their real potential to be used as a biological controlagent in the future requires further studies such as extensive field trials.In conclusion, the present study shows the high control potential of two strains ofS1 FigPhylogenetic relationships based on the nucleotide sequences of the D2-D3expansion segments of the 28S rRNA gene were inferred by using the MaximumLikelihood method based on the General Time Reversible model. The tree withthe highest log likelihood (-6470.16) is shown. The percentage of trees inwhich the associated taxa clustered together is shown next to the branches.A discrete Gamma distribution was used to model evolutionary ratedifferences among sites ). The ratevariation model allowed for some sites to be evolutionarily invariable. The tree is drawn to scale, with branch lengthsmeasured in the number of substitutions per site. NCBI accession numbers ofthe sequences used for the analyses are shown.(PDF)Click here for additional data file.S2 FigPhylogenetic relationships based on the nucleotide sequences of the internaltranscribed spacer (ITS) region of the rRNA gene were inferred by using theMaximum Likelihood method based on the Tamura 3-parameter. The tree with thehighest log likelihood (-10573.30) is shown. The percentage of trees inwhich the associated taxa clustered together is shown next to the branches.A discrete Gamma distribution was used to model evolutionary ratedifferences among sites ). The ratevariation model allowed for some sites to be evolutionarily invariable. The tree is drawn to scale, with branch lengthsmeasured in the number of substitutions per site. NCBI accession numbers ofthe sequences used for the analyses are shown.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


To evaluate systolic flow-sensitive alternating inversion recovery (FAIR) during rest and exercise stress using 2RR (two cardiac cycles) or 1RR intervals between inversion pulse and imaging.t tests.1RR and 2RR FAIR was implemented on a 3T scanner. Ten healthy subjects were scanned during rest and stress. Stress was performed using an in-bore ergometer. Heart rate, mean myocardial blood flow (MBF) and temporal signal-to-noise ratio (TSNR) were compared using paired p < 0.01) and 2RR FAIR . Mean stress MBF was higher than rest for 1RR FAIR  and 2RR FAIR . Resting mean MBF was higher for 1RR FAIR than 2RR FAIR (p < 0.05), but not during stress. TSNR was lower for stress compared to rest for 1RR FAIR  and 2RR FAIR . 2RR FAIR TSNR was higher than 1RR FAIR for rest (p < 0.05) and stress (p < 0.001).Mean heart rate during stress was higher than rest for 1RR FAIR (85.8 ± 13.7 bpm vs 63.3 ± 11.1 bpm; We have demonstrated feasibility of systolic FAIR in rest and exercise stress. 2RR delay systolic FAIR enables non-contrast perfusion assessment during stress with relatively high TSNR. Cardiovascular magnetic resonance (CMR) allows non-invasive assessment of myocardial ischemia using contrast-enhanced first-pass myocardial perfusion . AlthougIn recent years, there has been increased interest in developing CMR techniques to assess myocardial ischemia without the use of contrast agents or pharmacological stress agents. This includes T1 mapping , 6, oxygA limitation of ASL in general, and myocardial FAIR in particular, is the low signal sensitivity of this technique. This is primarily due to the intrinsically low signal variability which may be attributed to perfusion of maximum 4–8% , approxiAnother approach to increase robustness of cardiac FAIR is to time the data acquisition to the systolic rather than diastolic rest period, as this leads to an increased area of analyzable myocardium and may be preferable during high heart rates as typically encountered in stress perfusion –26. HoweAll experiments were performed on a 3 T Philips scanner  using a 24-channel torso coil. The study was approved by the institutional review board (Dnr 2016/546-32) and all subjects provided written informed consent.Ten healthy volunteers  were scanned at rest and during exercise stress. For the exercise stress test an in-bore ergometer , shown in Fig. 0 image was acquired in a separate scan without any magnetization preparation to estimate baseline magnetization for every data set. Myocardial blood flow (MBF) was calculated for both 1RR FAIR and 2RR FAIR using the formula for double-gated myocardial ASL:C and T are the control and tagged images, respectively. T1 is the longitudinal relaxation time for blood at 3 T, estimated at 1700 ms [The prosed systolic FAIR technique was ECG-triggered and used double-gating to ensure the inversion pulses were performed in the same cardiac phase but a preceding cardiac cycle as the image acquisition. In this work, cardiac FAIR was performed either with one cardiac cycle delay between inversion pulse and imaging (1RR FAIR) or with two cardiac cycle delay (2RR FAIR). Apart from this modification, the double-gated myocardial FAIR acquisition was performed as in previous studies, where tagged and control images were acquired within one breath hold of approximately ten seconds with 8 s of delay between inversion pulses. In addition, an M 1700 ms .2, FOV = 300 × 300 × 10 mm, flip angle = 50°, TR/TE = 2.2/1.1 ms, acquisition time = 110 ms, 25 ramp-up radiofrequency pulses preceded the data acquisition with linearly increasing flip angles, from 1 to 49º with 2 incrementsº. Compressed SENSitivity Encoding (SENSE) was used for image acceleration with a factor of 3. The systolic rest period was visually determined using a four-chamber cine slice with 50 cardiac phases acquired during rest and assumed to be the same for the stress scans [For the in-vivo experiments, a mid-ventricular 2D FAIR image was acquired in short-axis and each data set consisted of six pairs of tagged and control images, each pair acquired during a breath-hold and with alternating order of the control/tagged images. Imaging parameters were: spatial resolution = 2 × 2 mmss scans . The sca0) were jointly co-registered using non-linear image registration to account for differences in respiratory position within and between breath-holds [The images were processed offline to generate pixelwise maps of MBF. First, all images  was calculated based on a region of interest which was manually drawn to include the entire myocardium in the mid-ventricular slice. TSNR was defined as the ratio between the mean MBF and the standard deviation of the MBF across the 6 measurements .t test were used to statistically compare group mean differences for heart rate, mean MBF and TSNR. A threshold of P < 0.05 was used to define statistically significant differences.Two-tailed paired student’s The rest and stress 1RR FAIR and 2RR FAIR scans were successfully performed in all but one volunteer, where excessive respiratory motion during the 1RR FAIR stress scan could not be sufficiently corrected and resulted in uninterpretable MBF maps. Therefore, 1RR and 2RR FAIR data for this volunteer were not included in the statistical analysis to allow pairwise comparisons.p < 0.01) and 2RR FAIR . However, there was no statistical difference in heart-rate between 1 and 2RR FAIR for either rest and stress. The MBF of the stress scans were significantly higher compared to the rest scans for both 1RR FAIR  and 2RR FAIR . The MBF was significantly higher using 1RR FAIR during rest compared to 2RR FAIR (p < 0.05), but not during stress. The myocardial perfusion reserve (MPR) was calculated for all volunteers using 1RR FAIR and 2RR FAIR, where both rest and stress were available. The MPR for 1RR FAIR was 2.28 ± 0.72 and for 2RR FAIR 2.56 ± 1.07, which was not statistically significant (p = 0.32). The TSNR was significantly lower for the stress scans compared to rest for both 1RR FAIR  and 2RR FAIR . However, TSNR was significantly higher for 2RR FAIR compared to 1RR FAIR for both rest (p < 0.05) and stress scans (p < 0.001). The measured MBF and MPR for each volunteer using 1RR FAIR and 2RR FAIR during rest and stress are shown in Fig. Example perfusion maps for two subjects using 1RR and 2RR FAIR during rest and stress are shown in Fig. Here, we have demonstrated the ability to estimate perfusion during rest and exercise stress using systolic FAIR. We compared the use of 2RR interval delays between inversion pulse and imaging acquisition to 1RR delay and found that the former approach yielded higher TSNR at both rest and stress at the expense of producing, on average, 6% lower MBF values during rest.Similar to previous studies exploring the use of 1RR and 2RR FAIR techniques, we found a reduction in MBF for the 2RR approach . This maThe rest mean MBF was approximately 1.2–1.4 mL/g/min in this study. Others also reported a similar range (1.3–1.5 mL/g/min) , 13, butA clinical exercise test aims to reach an age-based heart rate, to induce ischemia which may be hidden at rest . In this1 mapping [The clinically most common modality for perfusion measurement is Single Photon Emission Computed Tomography (SPECT), but it provides low temporal and spatial resolution. Photon Emission Tomography (PET) is considered the clinical gold standard but requires the use of ionizing radiation, unlike MRI . There a mapping  have bee mapping  and, the1 for blood of 1700 ms was used for all subjects. However, blood T1 varies particularly with hematocrit levels, and a subject-specific blood T1 measurement may yield more accurate MBF estimation. Finally, the systolic time was determined from cine images at rest, similar to a previous perfusion publication [The study has several limitations: The study comprised of a small number of healthy subjects, and larger studies including patients with coronary artery disease are warranted to evaluate this technique, including validation against quantitative reference techniques, such as contrast-enhanced MRI. Furthermore, validation using a perfusion phantom would be desirable and will be the focus of future work , 38. Thelication , but no We have demonstrated the feasibility of systolic FAIR during rest and exercise stress. Systolic FAIR with 2RR delay between inversion and imaging enables non-contrast perfusion assessment during stress with relatively high TSNR. MBF may be slightly underestimated compared to 1RR FAIR due to partial signal saturation. Further studies are warranted to investigate the diagnostic potential of this technique in patients with coronary artery disease.


Lycorine (Lyc) is a natural alkaloid derived from medicinal plants of the Amaryllidaceae family. Lyc has been reported to inhibit the recurrence and metastasis of different kinds of tumors. However, Lyc’s effect on angiogenesis and its specific mechanism are still not clear. This study was designed to test the antiangiogenesis effect of Lyc and to explore the possible mechanisms. We performed cell experiments to confirm Lyc’s inhibitory effect on angiogenesis and employed sunitinib as a positive control. Moreover, the synergistic effect of Lyc and sunitinib was also explored. Next, we conducted bioinformatics analyses to predict the potential targets of Lyc and verified them by western blotting and immunofluorescence. Molecular docking, kinase activity assays, Biacore assays and cellular thermal shift assays (CETSAs) were applied to elucidate the mechanism by which Lyc inhibited target activity. Lyc inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Employing bioinformatics, we found that Lyc’s target was PDGFRα and that Lyc attenuated PDGFRα phosphorylation. We also found that Lyc inhibited PDGFRα activation by docking to it to restrain its activity. Additionally, Lyc significantly inhibited PDGF-AA-induced angiogenesis. This study provides new insights into the molecular functions of Lyc and indicates its potential as a therapeutic agent for tumor angiogenesis.The online version contains supplementary material available at 10.1186/s12885-022-09929-y. Malignant tumors are the main cause of death and are important obstacles to improving life expectancy worldwide .3, the vasculature on the tumor surface is disordered. This is conducive to the exogenous growth of tumors and prevents drugs from entering the tumor body, resulting in reduced drug uptake, which is the theoretical basis for the treatment of tumors with antiangiogenic drugs [Angiogenesis is one of the hallmarks of tumors , and tumic drugs . Althougic drugs . In addiic drugs , 8. TherIn recent years, natural compounds have been proven to be effective in the treatment of tumors, including in antiangiogenesis –11. LycoNetwork pharmacology is a branch of pharmacology that is based on systems biology and multiple pharmacology theories and primarily focuses on biomolecular networks . In pharIn this study, we explored the anti-angiogenic roles of Lyc in vitro and in vivo, employing sunitinib as a positive control, and then the Lyc targets were predicted by the PharmMapper database. Lyc inhibited PDGFRα activities by docking to it directly. This protocol can be considered an effective method for exploring completely new applications of natural products with exact structural formulas. This study improved the understanding of Lyc's molecular biological function and indicated that Lyc may be a promising anti-angiogenic therapy.2.Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Cell Bank of Representative Culture Preservation Committee of Chinese Academy of Sciences . HUVECs were cultured in HUV-EC-C medium and kept in an incubator at 37 °C with 5% COLyc was purchased from MedChemExpress (HY-N0288) and prepared as described before . SunitinCell viability was examined using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Cells (3000 cells/well) were seeded in a 96-well plate overnight and then exposed to different concentrations of Lyc (and/or sunitinib) and incubated for 24 and 48 h. A total of 20 µL of MTT solution  were added to each well and the cells were incubated for another 4 h at 37 °C. The supernatant was then removed and 200 µL of DMSO were added to each well to dissolve the precipitate. Absorbance was measured using a spectrophotometer  at 490 nm.Cells were seeded in six-well plates at a density of 1000 cells/well. On the next day, cells were treated with Lyc and/or sunitinib. Ten days after the treatment, cells were fixed with methanol for 15 min and then staixned with 0.1% crystal violet. Finally, stained cell colonies were photographed with a digital camera and analyzed using ImageJ , Bethesda, USA).Cells cultured in a Petri dish and treated with Lyc and/or sunitinib for a range of time points were collected and incubated with 5 µL of Annexin V and 10 µL of PI for 15 min in the dark. The samples were then evaluated using flow cytometry and the data were analyzed using FlowJo .4 cells/chamber) and different concentrations of Lyc (and/or sunitinib) were seeded in an upper Transwell chamber . HUV-EC-C medium containing 25 ng/mL PDGF-AA was added to the lower chamber. Non-migrated cells from the upper membrane were removed after incubation for 16 h at 37 °C. The migrated cells were stained with 0.1% crystal violet and photographed under a light microscope at × 200. Migrated cells were analyzed quantitatively using ImageJ.For the migration assay, 200 µL of HUV-EC-C medium containing cells (2.0 × 105 cells/well) were seeded in six-well plates. As the cells became sub-confluent, a straight cell-free wound was scratched with a 10 µL pipette tip. Cells were then washed twice with PBS and incubated in serum-free medium containing different concentration of Lyc and/or sunitinib. Cell scratches were observed and measured at different time points. Cell migration distances were analyzed quantitatively using ImageJ.Cells  pre-coating at 37 °C for 30 min. Thereafter, HUVECs were treated with different concentrations of Lyc and/or sunitinib for 4 h. After exposure, HUVECs tubes and branches were photographed under a light microscope at × 200. The formed branches were analyzed quantitatively using ImageJ.HUVECs were plated at a density of 1 × 102) and the shell membrane was removed with sterile forceps to expose the chorioallantoic membrane. A small rubber ring was placed on the chorioallantoic membrane and different concentrations of Lyc and/or sunitinib were added into the ring and incubated. At baseline and 8, 16 h later, the ring area was photographed by a scanner and the images were analyzed using Image-Pro Plus 6.0 .Five fresh 10-day-old fertile eggs were cleaned with alcohol and then incubated at 37 °C and 60–80% humidity in an egg incubator. The shell was cut to create a small window (20 × 20 mmhttp://59.78.96.61/pharmmapper/) to obtain possible Lyc targets. The target names were then corrected to the official symbol using UniProt (https://www.uniprot.org/). Gene ontology (GO) functional annotation [http://www.webgestalt.org/) [www.genecards.org/) and intersected to the predicted targets in order to get possible Lyc targets for angiogenesis-inhibiting. Finally, target names were submitted to the Cytoscape software (http://www.cytoscape.org) [Lyc’s structural formula was drawn using Chemdraw  and uploaded to PharmMapper   . Biologiape.org)  to visuaWestern blotting was conducted as we described before . BrieflyHUVECs administered with different concentrations of Lyc were grown at a 24-well plate after stimulated by PDGF-AA, fixed with 4% paraformaldehyde for 15 min and blocked with 5% bovine serum albumin for 1 h at room tempature. Sequently, primary antibody  was applied to incubate the cells overnight and incubated with Alexa Fluor 488 secondary antibody  for 2 h. Finally, cells were incubated with hoechst  for 5 min and visualized under an inverted fluorescent microscope.Molecular docking was performed using Schrödinger software, which included Protein Preparation Wizard, LigPrep, and Glide modules. PDGFRα crystal structure (PDB ID: 6JOK) was prepared from RCSB  and optiPDGFRα enzymatic assay was performed using the Kinase Activity Assay Kit  following the manufacturer's protocol. Purified human PDGFRα protein was treated with Lyc at different concentrations in a black 384-well plate. The fluorescence intensity was measured by an automatic microplate reader .Surface plasmon resonance (SPR) affinity experiment , 39 for CETSAs were conducted to detect the direct binding between Lyc and PDGFRα in celluar. Briefly, cells were pre-treated with 6 uM of Lyc for 48 h, chilled on ice, washed with PBS containing protease inhibitor and then transferred into 1.5 ml PCR tubes and heated for 3 min at appropriate temperature. Subsequently, cells were lysed, seperated and detected by western blot assays.p-values < 0.05.Results were analyzed using SPSS software . All experiments were independently repeated at least three times and data were presented as the mean ± SD. Student’ s two-tailed t-test and one-way ANOVA were performed to determine statistical significance between different groups. Differences were considered significant when 50: 24 h 9.34 µM, 48 h 4.93 µM). To further evaluate the inhibitory effect of Lyc on the growth of HUVECs, we selected sunitinib, a commonly used anti-angiogenesis agent [Tumor blood vessels provide oxygen and nutrients necessary for the metabolism of tumor cells. Thus, angiogenesis is the most basic factor in the growth and metastasis of tumors. The proliferation and migration of vascular endothelial cells toward tumor cells via a gap in the basement membrane is a key step in tumor angiogenesis. The inhibitory effect of Lyc on the proliferation and migration of HUVECs was observed to explore its impact on angiogenesis. An MTT assay was used to determine the cell viability of HUVECs treated with Lyc at different concentrations and at different time points to detect the inhibitory effect of Lyc on the proliferation of HUVECs. As shown in Fig. is agent , as the is agent , 42) hadThe structural formula for Lyc was uploaded to PharmMapper, and duplicate results were removed to obtain a total of 456 possible targets. The official symbols for the drug targets were obtained from UniProt. For a more in-depth understanding of the target protein, GO function and KEGG pathway analyses were applied in WebGestalt. Signal transduction was the most significant biological process (BP) term, cytoplasm and plasma membrane were the most significant cellular component (CC) terms, and protein binding was the most significant molecular function (MF) term . As shown in Fig. As described above, PDGFRα is dimerized and activated by the ligand PDGF. The ligand/receptor interactions proven to be important in vivo are PDGF-AA and PDGF-CC, which induce PDGFRα dimerization. Therefore, we tested whether Lyc could inhibit PDGF-AA-induced angiogenesis. As shown in Fig. Alkaloids are basic organic compounds containing nitrogen that mainly exist in plants and animals. Alkaloids have a complex ring structure, which mostly contains nitrogen. Based on this structure, alkaloids show rich physiological and pharmacological activities. Approximately 20 kinds of Lycoris spp. are widely distributed in the world. Lycoris was the first alkaloid isolated from Lycoris spp., and it is also the most common alkaloid in this family . In prevHUVECs extracted from the umbilical cord are an alternative model to study tumor angiogenesis. During proliferation and invasion, proteases secreted by tumor cells degrade the extracellular matrix (ECM), and immature vascular endothelial cells sprout into the tumor basement membrane. Then, vascular endothelial cells continue to mature and differentiate and form the vascular lumen. The capillary network further spreads and finally forms mature blood vessels . The proThe CAM assay has the advantages of simple sampling, a short experimental period and simple operation. However, the disadvantages of the CAM assay are that the local damage caused by the change in osmotic pressure and pH value affect angiogenesis, and the experimental operation process and the state of the chicken embryo chorionic membrane also have a great influence on the experimental results –53. In oThe PDGFRA gene, which maps to chromosome 4q12, encodes a highly homologous transmembrane glycoprotein,named PDGFRα,that belongs to the type III receptor tyrosine kinase family.Binding of PDGF stimulates PDGFRα dimerization, which further initiates intracellular signaling . In soliCascade signaling pathway activation induced by PDGF/PDGFRα can regulate vascular endothelial cells, enhance vascular permeability, and regulate angiogenesis . When thTyrosine kinase inhibitors (TKIs) bind to tyrosine residues in the intracellular region of membrane receptors and inhibit tyrosine phosphorylation, thereby preventing tumor-related signal activation. Molecular docking in computer-aided drug design (CADD) selects the optimal binding mode of the receptor and ligand according to the energy and spatial structure complementarity of the receptor and ligand. The reasonable relative position, molecular direction and dynamic change of receptor and ligand molecules are determined by molecular docking . In thisNatural products have unique advantages in tumor treatment, but their effective substances and mechanisms are not yet clear; thus, the specific targets of natural products have not been identified. In this study, using bioinformatics prediction, molecular docking, and in vitro and ex vivo experiments, Lyc was shown to attenuate PDGFRα activation. In addition, natural products produce pharmacological effects on different targets, but this study only examined the role of Lyc on PDGFRα. The possible Lyc impact on other targets, especially other tyrosine kinases, needs to be further investigated. Clinical trials are the most critical step in the development of drugs and determining their applicability to extended indications. We look forward to clinical trials with large sample sizes to verify the efficacy of Lyc for the treatment of tumors.Additional file 1. Additional file 2. Additional file 3.