In this study, expression profiling and heterosis associated genes were analyzed by RNA sequencing (RNA-Seq) in seedlings of the maize hybrid An’nong 591 and its parental lines under control and heat stress conditions.Heterosis is the superior performance of F1 hybrid and the paternal lines under both conditions, which suggested greater genetic contribution of the paternal line to heat stress tolerance. Gene Ontology (GO) enrichment analysis of the 4518 common DEGs indicated that GO terms associated with diverse stress responses and photosynthesis were highly overrepresented in the 76 significant terms of the biological process category. A total of 3970 and 7653 genes exhibited nonadditive expression under control and heat stress, respectively. Among these genes, 2253 (56.8%) genes overlapped, suggesting that nonadditive genes tend to be conserved in expression. In addition, 5400 nonadditive genes were found to be specific for heat stress condition, and further GO analysis indicated that terms associated with stress responses were significantly overrepresented, and 60 genes were assigned to the GO term response to heat. Pathway enrichment analysis indicated that 113 genes were involved in spliceosome metabolic pathways. Nineteen of the 33 overlapping genes assigned to the GO term response to heat showed significantly higher number of alternative splicing (AS) events under heat stress than under control conditions, suggesting that AS of these genes play an important role in response to heat stress.Through performing nine pairwise comparisons, the maximum number of differentially expressed genes (DEGs) was detected between the two parental lines, and the minimum number was identified between the F1 hybrid and its parental lines under control and heat stress conditions, and provides insight into the underlying molecular mechanisms of heterosis and the response to heat stress in maize.This study reveals the transcriptomic divergence of the maize FThe online version of this article (10.1186/s12870-019-1878-8) contains supplementary material, which is available to authorized users. Althoug parents . Before 1 hybrid , 5. The 1 hybrid . In addi1 hybrid , 7, 8. E1 hybrid , 9–13; hMedicago, and Arabidopsis, have also been performed based on expression profiling [Increasing evidence indicates that differential gene expression in parental lines and hybrids may be responsible for heterosis , 14, 15.rofiling –21. Generofiling . For exarofiling .1 hybrids in response to drought, and revealed that 9230 (35%) and 7185 (27%) of expressed genes exhibited nonadditive expression under control and water deficit stress in the Mo17 × B73 hybrid, respectively. Importantly, 47% of the nonadditively expressed genes overlapped between the two treatments, which suggested important roles for these genes in response to drought [Heterosis confers superior performance for a wide range of traits such as increased biomass, development rate, and grain yield, but also tolerance to environmental stresses . High teMaize is one of the most important cereal crops worldwide. In recent years, high temperature has become a serious environmental stress affecting maize production. For example, extremely high temperatures (~ 40 °C) during the flowering period resulted in severe yield losses in Huang-Huai-Hai plain of China in 2016–2018. Planting heat stress-tolerant cultivars is one of the most effective approaches to prevent stress damage. The maize hybrid An’nong 591, which is highly resistant to high temperatures, is suitable for planting in the Huang-Huai-Hai region. To investigate the molecular mechanisms in the response to heat stress, the expression profiles of An’nong 591 and its parental lines were compared in seedlings under control and 42 °C treatments by RNA-Seq, and heterosis-associated genes were analyzed to explore the underlying mechanism of heterosis. The results lay an important foundation for understanding heat-tolerance mechanisms in maize hybrids, and provide insight into the underlying molecular basis of heterosis.P < 0.01).Seedlings of the maize hybrid An’nong 591, its maternal line (CB25), and paternal line (CM1) were used to determine their phenotypic characters. When the third leaf was fully expanded, the seedlings of each genotype were treated with 42 °C/35 °C (day/night temperature) for two days to evaluate their phenotypic response to high temperature. Seedlings of CB25 showed severe leaf rolling and wilting in contrast with An’nong 591 and CM1  between the biological replicates were greater than 0.95  containing and the low quality sequences, on average, 45.81 million clean reads (6.82 Gb clean data) were obtained for each replicate. Among the 18 sequencing libraries, 72.57–75.65% of the clean reads were uniquely mapped to the maize reference genome (ZmB73_RefGen_v3). The percentages of phred scores at the Q30 level (error probability less than 0.1%) ranged from 92.36 to 93.26%, and the GC content ranged from 54.04 to 58.74%. Detailed information on the RNA-Seq data is listed in Additional file 1, CR vs. CF1, CS vs. CR, S vs. F1, R vs. F1, S vs. R, CF1 vs. F1, CR vs. R, and CS vs. S) were performed. Hierarchical cluster analysis indicated that the three biological replicates of each sample were clustered in close proximity to each other, which further reflected the highly reproducible results of RNA-Seq , photosynthesis, light reaction (GO:0019684), generation of precursor metabolites and energy (GO:0006091), photosynthesis, light harvesting (GO:0009765), response to stimulus (GO:0050896), response to light stimulus (GO:0009416), response to radiation (GO:0009314), and response to abiotic stimulus (GO:0009628) were overrepresented in all categories  were enriched for the 4518 DEGs  using three biological replicates of each sample. The majority of the 60 genes showed a low expression level under control conditions, whereas significantly up-regulated expression was detected after heat treatment  with the RNA-Seq data for each of the three genotypes  using the R package pheatmap. The average expression levels of these genes were transformed by logpes Fig. , which s1 hybrid), parental-specific expression , hybrid-specific expression (Type III: genes are expressed only in F1 hybrid), single-parental consistent expression , and co-expression of the hybrid and parental lines (Type V) [According to the differences in expression between hybrids and their parental lines, genes can be classified into five expression patterns, namely co-silence expression of the parental lines  . In the Previous studies have indicated that Types I to IV are associated with qualitative differences in gene expression, meanings presence/absence variation of these genes. Type V is associated with quantitative differences in gene expression, and the difference in expression patterns between hybrids and their parental lines is predominantly associated with this type , 26. A tA total of 3970 and 7653 nonadditive genes were identified from the total expressed genes under the control and heat conditions, respectively. We found that 56.8% (2253 of the 3970 genes) of the nonadditive genes overlapped under control and heat conditions, which suggested that nonadditive gene expression was more conserved in the hybrid under both conditions Fig. . Such pa1 hybrids in comparison with their parental lines. Three main genetic models, comprising dominance, overdominance, and epistasis hypotheses, have been proposed to explain heterosis in classical quantitative genetics [Heterosis has been widely exploited in plant breeding for decades due to the superior performance of heterozygous Fgenetics –6, 9; hogenetics . Increasgenetics , 23, 26.1 hybrid and its maternal line (11.0 and 21.3%) was significantly higher than that between the F1 hybrid and its paternal lines (9.7 and 13.6%) in both conditions. These results suggested that gene expression in the hybrid was more similar to the paternal line, especially under heat stress. We concluded that the superior heat tolerance of An’nong 591 was contributed mainly by its paternal line CM1, which was consistent with the evaluation of the phenotypic and physiological characteristics of the three genotypes.The RNA-Seq analysis showed that 56.2% of the high confidence gene models in the maize reference genome were expressed in at least one of the samples. The high transcriptomic correlation of the three biological replicates of each sample was verified by PCA and Pearson’s correlation analysis, which supported the reproducibility of the RNA-Seq data. Nine pairwise comparisons of gene expression in the three genotypes were performed, and thousands of DEGs were identified for each pair. Under both of control and heat treatment conditions, the maximum number of DEGs was detected between the two parental lines (CS vs. CR and S vs. R). Previous findings have indicated the correlation of gene expression variation and heterosis. About 70% (fold change ≥2 and FDR < 5%: 22.9%) of expressed genes were differentially expressed between maize inbred lines B73 and Mo17, and 42–57% (fold change≥2 and FDR < 5%: 7.6–10.0%) of all expressed genes were differentially expressed between one of the maize hybrids and one of its parents . In rice1 vs. F1, CR vs. R, and CS vs. S to screen for candidate genes involved in the response to heat stress, and 2096 common down-regulated and 2422 common up-regulated genes were further identified among these genes. Functional classification of the 4518 common down- and up-regulated genes was further analyzed by GO enrichment analysis. Many genes were overrepresented in biological process associated with diverse stress response. A total of 60 genes were enriched in the GO term response to heat (GO:0009408), and most of them were significantly up-regulated under heat treatment, which suggested that these common DEGs performed important functions of in response to heat stress. Among the 2096 common down-regulated genes, a total of 119 significant GO terms were enriched. We observed that GO terms associated with photosynthesis, light reaction and stress responses were overrepresented in the biological process category  exhibited nonadditive expression under the control and heat stress conditions, respectively. The proportion of nonadditive genes differed notably from that reported in previous studies. For example, 22% of the differentially regulated ESTs were significantly different from the mid-parent value in the Mo17 × B73 maize hybrid . Nonaddi crosses . In the  crosses . Togethe crosses –50. Cons crosses , 52, we  crosses –55. ThusHere, we provide a global view of the transcriptomic divergence of the maize hybrid An’nong 591 and its parental lines under heat stress using RNA-Seq, and nonadditive genes were further analyzed to explore the underlying mechanism of heterosis. Our results reveal the important roles of nonadditive genes in the response to heat stress, and provide new insight into mechanisms of heterosis in heat tolerance in maize hybrid.Zea mays L.) hybrid An’nong 591 (CB25 × CM1) and its parental lines were grown in a greenhouse at 28 °C/23 °C (day/night) with a 16-h light/8-h dark photoperiod. An’nong 591 and its parental lines were generated by Professor Qing Ma and Beijiu Cheng. For heat treatment, seedlings were incubated at 42 °C/35 °C (day/night) for two days when the third leaf was fully expanded. During the period of heat treatment, the seedlings were watered every day, and control plants were maintained under non-stress conditions. After heat treatment, the third leaf was harvested, immediately frozen in liquid nitrogen and stored at − 80 °C for RNA isolation. Before and after heat treatment, the relative water content (RWC) and relative electrolyte leakage (REL) were measured in the seedlings in accordance with methods described previously [t-test based on three biological replicates. Mid-parent heterosis (MPH) and high-parent heterosis (HPH) were determined using the following formulas: MPH (%) = (F1 − MP)/MP and HPH (%) = (F1 − HP)/HP, where MP is the average value of the two parents, and HP is the highest value of the two parents.Seedlings of the maize .Total RNA was extracted using TRIzol reagent  according to the manufacturer’s protocol. The quality and purity of RNAs was assessed by Agilent 2100 Bioanalyzer , NanoDrop spectrophotometer  and 1% agarose gel. Total RNA (1 μg) with RNA integrity number (RIN) values above 7 was used for library construction of each sample. Poly (A)-containing RNA was purified from total RNA using the NEBNext® Poly (A) mRNA Magnetic Isolation Module . The cDNA library preparations were constructed using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  according to the manufacturer’s protocol. The libraries with different indices were loaded onto an Illumina HiSeq X Ten platform for sequencing, which generated 2 × 150 bp paired-end reads. The raw reads were submitted to the GenBank GEO database under accession number GSE122866 (http://bioinformatics.psb.ugent.be/webtools/Venn/). The gene expression levels of DEGs were transformed by log10 (FPKM + 1), and used to draw a heat map with R package pheatmap.Raw reads were filtered to obtained high-quality reads by removing reads containing adapter or poly (N) sequences, low quality sequences (Q < 20) from the 5′ and 3′ ends of the reads, and sequences shorter than 75 bp. Clean reads  were subsequently aligned to the maize B73 reference genome (RefGen_v3) using HISAT software (v2.0.1) . HTSeq sGAPDH gene (accession number: NM_001111943.1) was used as an internal control for normalization, and three technical replicates of each cDNA sample were performed for qRT-PCR analysis. Data analysis was performed as reported previously [Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the gene expression levels of DEGs detected by RNA-Seq. Total RNA from the same samples as used for cDNA library construction was used for first-strand cDNA synthesis with the PrimeScript™ RT reagent kit with gDNA Eraser . Primers were designed using Primer Express 3.0 software  and are listed in Additional file eviously .http://systemsbiology.cau.edu.cn/agriGOv2/) [10 (FDR) with the R package pheatmap. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) web server (http://www.kegg.jp/) [Gene Ontology (GO) enrichment analysis was performed using agriGO (riGOv2/) . GO termegg.jp/) . PathwayTo identify nonadditive genes, the gene expression patterns that differed between the hybrid and its parental lines (CB25 and CM1) were analyzed by comparing the average expression values of all three biological replicates of the hybrids with the mid-parent values of the parental lines under the control conditions and heat stress treatment. Genes with FDR ≤ 5% were considered to be nonadditively expressed. According to the method described previously , the nonAdditional file 1:Figure S1. Comparison of expression values of the three genotypes under the control and heat treatment conditions. Expression values were transformed by log10 (FPKM). The horizontal line represents the median of each replicate of the three genotypes. (TIF 10316 kb)Additional file 2:Figure S2. Pearson’s correlation analysis of the biological replicates of each genotype. (TIF 6944 kb)Additional file 3:Figure S3. Heatmap of the differentially expressed genes under the control and heat treatment conditions. (TIF 7379 kb)Additional file 4:Figure S4. Numbers of differentially expressed genes between the hybrid and its parents. a Total number of DEGs under the control conditions. b Total number of DEGs under the heat treatment. c Total number of DEGs between the control and heat treatment conditions. d Venn diagram of common DEGs under the control conditions. e Venn diagram of common DEGs under the heat treatment. f Venn diagram of common DEGs between the control and heat treatment conditions. (TIF 12294 kb)Additional file 5:Figure S5. Hierarchical tree graphs of enriched GO terms in the biological process category for the 4518 common DEGs. Hierarchical tree graph were generated using agriGO. The GO ID , term definition, and statistical information are shown in the boxes. Significant terms (adjusted P ≤ 0.05) are in colored boxes, and GO terms with non-significant are in white boxes. Solid, dashed, and dotted lines in the graphs represent two, one and zero enriched terms at both ends connected by the line. (TIF 16606 kb)Additional file 6:Figure S6. Hierarchical tree graph of enriched GO terms in the biological process category for the 5400 nonadditive genes. (TIF 16250 kb)Additional file 7:Table S1. Gene-specific primers used for qRT-PCR analysis. (XLS 30 kb)Additional file 8:Table S2. Detailed information of the RNA-Seq data. (DOCX 22 kb)Additional file 9:Table S3. Significantly enriched GO biological process terms for 2096 common down-regulated differentially expressed genes. (DOCX 22 kb)Additional file 10:Table S4. Significantly enriched GO biological process terms for 2422 common up-regulated differentially expressed genes. (DOCX 16 kb)Additional file 11:Table S5. Comparative analysis of alternative splicing events of the 19 genes enriched in the GO term response to heat. (XLSX 14 kb)


In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes “replicative stress” that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair. Two replisomes are assembled at each origin that, upon their activation in S phase, move away from each other, establishing bidirectional replication forks. The duration of S phase is determined by the number of active origins and the velocity of replisomes, normally referred to as “fork speed” or “fork rate” (FR).e.g. red for CldU and green for IdU) to identify specific replication structures such as origins, forks, and termination events (A). Although FR is directly proportional to the length of labeled tracks and can be quantified using a conversion factor between microscopy image pixels and DNA length  and is normally counteracted by mechanisms that protect stalled forks and promote the restart of DNA synthesis –12. Exce–13–At the molecular level, the interpretation of RS phenotypes is complicated by a marked interdependency between FR and origin activity. Alterations that slow down forks trigger the activation of otherwise “dormant” origins as a compensatory mechanism , 20, whe21–i.e. lower FR) and increase in origin density (i.e. shorter IOD) in HeLa cells (B). Experimental conditions were chosen for which the primary effect could be inferred beforehand. For instance, hydroxyurea (HU) inhibits ribonucleotide reductase and reduces the pool of dNTPs, causing a decrease in FR that subsequently activates dormant origins . To confirm that the former interpretation is correct, the effect of HU on FR and origin activity was tested in the absence or presence of a CDC7 kinase inhibitor (CDC7i). CDC7 kinase activates the MCM helicase and is necessary for origin firing  and restrict origin activation, as indicated by the lower percentage of first-label origins and increased inter-origin distances . At this CDC7i concentration, the global effects on DNA synthesis and cell cycle distribution were modest .We first examined the two possible cases that lead to a concomitant slowdown of forks . If, on the other hand, the effect of HU depended on the activation of extra origins, then it would be corrected by CDC7i . The decrease in FR and concomitant activation of new origins were readily detected after HU treatment . As anticipated, when HU was combined with CDC7i, FR remained low despite a strong block in origin activity (C–E). The slight increase in FR relative to the HU-only conditions can be explained as a secondary effect of CDC7i itself following origin repression .Because HU affects fork progression independently of origin activity, the reduction in FR should be observed regardless of the presence of CDC7i . In this case, however, upon addition of CDC7i, the negative effect of UCN01 on FR was attenuated and reached similar levels as treatment with CDC7i alone (F–H). This result confirms that the change in FR is a secondary response that follows the primary influence of UCN01 on origin activation.The opposite situation, tivation , 27, 28.7i alone , F–H. Thi.e. longer IOD; Fig. 2A). In these cases, in which the use of CDC7i would be redundant with the defect in origin activity, another intervention was designed based on the DNA polymerase inhibitor aphidicolin (APH) to temporarily reduce FR. The rationale is parallel to the situation described before: if the observed reduction in origin density is the primary effect and does not depend on fork speed, then it should be resilient to APH interference. On the contrary, if it is a secondary response to faster forks, then it would be corrected by APH . Optimization experiments indicated that a short treatment with 5 μm APH was sufficient to slow down forks  without abolishing DNA synthesis or significantly altering the cell cycle .Although RS commonly refers to the slowdown of forks, there are also experimental scenarios in which forks become faster while origin activity decreases (B–D). The complementary case was tested by supplementing the cell medium with extra nucleosides to promote faster fork progression, causing a secondary decrease in origin activation (E–G). These results confirm that the primary effect of nucleoside addition was to accelerate FR.To validate the APH test, conditions were found that exerted a primary influence either on origin density or in FR. For the first case, cells were treated with CDC7i, which restricts origin activation and elicits a secondary increase in FR. As anticipated, origin density was reduced by CDC7i treatment and remained low even when FR was markedly reduced by APH , B–D. Thtivation , 24, 29.tivation , E–G. ThB). As anticipated, APH acutely decreased FR and increased origin activity (C–E), enhancing the alterations induced by the loss of PrimPol. In this context, APH does not contribute to discriminate whether PrimPol primarily affects FR or origin usage. In turn, the use of CDC7i revealed that the reduction in FR caused by PrimPol down-regulation was maintained even when extra origins could not be activated (F–H). This result indicates that the loss of PrimPol affects fork progression directly, in line with other experiments that support a role for PrimPol in facilitating fork progression through UV-damaged DNA , 60 μm CDC7i (12 h), 10 nm UCN01 (5 h), 5 μm APH (2 h), and 150 μm nucleosides (4 h).HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus penicillin–streptomycin. To down-regulate PrimPol, a stable HeLa-shPrimPol cell line generated in our laboratory was used . Short hm CldU followed by 250 μm IdU (20 min) prior to cell harvesting and lysis in 0.2 m Tris (pH 7.4), 50 mm EDTA, and 0.5% SDS. Stretched DNA fibers were prepared as described previously (Cells were pulse-labeled (20 min) with 50 μeviously . Signalseviously . For FR,Whole-cell extracts were prepared by suspension of cells in Laemmli buffer followed by sonication . SDS-PAGE, protein transfer to nitrocellulose membranes and immunoblots were performed using standard protocols. Anti-Mcm2 and anti-PrimPol have been described previously . The folm IdU for 20 min, collected, and fixed in 70% ethanol. DNA was denatured by incubation in 2 n HCl for 20 min, washed, and incubated with FITC-conjugated anti-BrdU  for 60 min at 37 °C. To monitor DNA content, cells were stained with 50 μg/ml propidium iodide (Sigma) in the presence of 10 μg/ml RNase A (Qiagen). Flow cytometry was performed in a FACSCanto II cytometer (BD Biosciences) and analyzed with FlowJo 10 (Tree Star).Cells were pulse-labeled with 250 μS. R.-A. and J. M. conceptualization; S. R.-A. and J. M. formal analysis; S. R.-A. and S. M. investigation; S. R.-A. and J. M. writing-original draft; S. R.-A. writing-review and editing; J. M. supervision; J. M. funding acquisition.


In recent years, Next Generation Sequencing (NGS) has accelerated the generation of full mitogenomes, providing abundant material for studying different aspects of molecular evolution. Some mitogenomes have been observed to harbor atypical sequences with bizarre secondary structures, which origins and significance could only be fully understood in an evolutionary framework.Parachtes and Harpactocrates, which belong to the nocturnal, ground dwelling family Dysderidae. Species of both genera have compacted mitogenomes with many overlapping genes and strikingly reduced tRNAs that are among the shortest described within metazoans. Thanks to the conservation of the gene order and the nucleotide identity across close relatives, we were able to predict the secondary structures even on arm-less tRNAs, which would be otherwise unattainable for a single species. They exhibit aberrant secondary structures with the lack of either DHU or TΨC arms and many miss-pairings in the acceptor arm but this degeneracy trend goes even further since at least four tRNAs are arm-less in the six spider species studied.Here we report and analyze the mitochondrial sequences and gene arrangements of six closely related spiders in the sister genera The conservation of at least four arm-less tRNA genes in two sister spider genera for about 30 myr suggest that these genes are still encoding fully functional tRNAs though they may be post-transcriptionally edited to be fully functional as previously described in other species. We suggest that the presence of overlapping and truncated tRNA genes may be related and explains why spider mitogenomes are smaller than those of other invertebrates.The online version of this article (10.1186/s12864-019-6026-1) contains supplementary material, which is available to authorized users. Metazoan mitogenomes are generally composed of 37 genes  plus at least one control region ) but thePholcus phalangioides (Pholcidae).Most mitogenomic studies usually focus on the use of the nucleotide and amino acid sequences as phylogenetic markers to infer fully resolved and well-supported evolutionary relationships among target taxa or test the statistical support of higher taxonomic ranks, e.g. –11. ThesGCT that lost the DHU-arm in nearly all metazoans [trnS2 of the spiders of the suborder Opisthothelae, although additional taxon sampling is needed to confirm this evolutionary pattern [Tetranychus carries three arm-less tRNA genes  except for tRNA-S1etazoans . This cletazoans . Nonetheetazoans –23. The etazoans  and was etazoans . Subsequetazoans . The traetazoans . On the  pattern . In addi pattern , 27, 28. pattern . For insand trnQ ), the huand trnT ), the orand trnV ), and thus (trnC ). The lous  , Parachtes romandiolae  , Parachtes limbarae  , Paracthes ignavus  , and Harpactocrates apennicola Simon, 1914 . The total length of the mitogenomes was around 14 kb with a coverage depth of above 200x and read length ranging from 350 to 550 bp. The coverage depth of Parachtes riberai Bosmans, 2017 was only 2-5x since nucleotide sequences were obtained by sequencing the inserts of 96 clones by the Sanger method and filling gaps by primer walking sequencing. Three mitogenomes were circularized, P. romandiolae , P. limbarae , and H. apennicola , but we failed to circularize (i.e. incomplete) P. riberai  and P. ignavus  due to the presence of an extremely long polyA run in the control region. The same issue precluded to obtain most of the control region in P. teruelis .After quality trimming, about 90% of the reads of a Roche FLX/454 run were assembled into five mitochondrial contigs, corresponding to the following species: Parachtes and Harpactocrates is a new discovery in spiders and it seems to arise from a translocation involving trnI and a large gene block  as expected for arthropods and most metazoans . Negative ATskew values were found in those genes coded on plus strand, and positive numbers on minus strand (0–0.25) with the exception of the minus strand second codon positions which displayed negative values  and ATG (9) codons, while the remaining showed non-canonical codons . However, for the tRNAs A, R, S1, and Y neither DHU nor TΨC were present or conserved across species suggesting that they could be folded as arm-less tRNAs  including three “true” arm-less tRNAs (A-S1-R). In addition, the acceptance of an arm-less secondary structure in N and E would reduce the overlapping with other genes in this tRNA block (from 73 to 53 bp). Another evidence supporting an arm-less structure for tRNA N is that there is a conserved mismatch in the first pair of DHU stem in all six species. Such mismatch implies that the canonical two bases separating acceptor and DHU arms are increased to three nucleotides and the single base separating DHU and anticodon arms are incremented to 2 bp, hence reporting an incoherence with the canonical structure of tRNAs. Regardless of the number of arms, mismatches and GU pairings were rare in the anticodon arm, i.e. mostly AU and GC bounds. In DHU and TΨC arms, if present, the mismatches in stems were also rare but they did contain GU pairings. Among those lacking both DHU and TΨC arms, trnS1 gene was the most extreme example of shortening since it was just 39 bp long in P. riberai although it overlaps 10 bp with adjacent trnA and 6 pb with trnR. Finally, most tRNAs of Parachtes and Harpactocrates usually displayed two or three mismatches in the acceptor stem, and in few extreme cases four out of the seven canonical pairs . Therefore, the predicted secondary structure of this domain highly resembled the folding predicted in other species and required minor manual corrections  was the most conserved region at the nucleotide identity level in ons Fig. . On the ons Fig.  alternatons Fig. . The secons Fig.  showed ltrnQ and trnM. Because of long polyA stretches and repetitive fragments, it could be fully sequenced only in three of the six species. The control region length was 786 and 714 bp in P. romandiolae and P. limbarae, respectively, and 747 bp in Harpactocrates. Sequences in all three species were also A + T rich , but not as high as described for most Pancrustacea species, which can reach values above 90%. The sequence of the main control region of the two Parachtes species was also conserved at nucleotide level particularly a region of 138 bp with 74% of similarity and 9% of gaps . A shorter stretch of 81 bp in the same region was also conserved in H. apennicola . The main control region of the 3 species showed several palindromic regions, which could correspond to the putative origin of replication given the presence of a characteristic long poly A stretch (~ 10–25 bp), although the ATAT (TATA) and GA(N) T motifs adjacent to 5′ and 3′ ends were missing. Some of these palindromic regions were recognized as putative origin of replication of plus strand in MITOS2, but neither their locations nor their nucleotide sequences were conserved across the 3 species.The main control region was located between genes trnL2 and trnN genes (85–94 pb in length). Nucleotide identity among the five species of the genus Parachtes ranged between 51 and 81%. The coverage and similarity of this short region relative to H. apennicola was about 50% for both features. This short non-coding region also harbors a palindromic region  of P. romandiolae is 3 bp shorter than the tRNA-R of the nematode Romanomermis culicivorax [Parachtes and Harpactocrates tRNA-S1 reduction further involves the acceptor stem, which shows multiple mismatches. This pattern has been also described in the orb-weaver spiders Neoscona doenitzi and Neoscona nautica , and hephosidae . Some ofnd trnL2 ). In fac cloning . The seqf tRNA-H .Parachtes and Harpactocrates arm-less tRNAs could only be achieved manually thanks to the highly conserved nucleotide sequences across the species, particularly the anticodon arm, which facilitated the detection of a common underlying secondary structure. The “standard” tRNAs lacking DHU or TΨC arms were always detected by MITOS2 and tRNAscan-SE in the six species with perfect pairing in their DHU or TΨC stems. In fact, the inconsistent presence or absence of DHU or TΨC arms in a particular tRNA was a crucial hint to identify both arms. The lack of covariant models searching for arm-less tRNAs and allowing multiple mismatches, could explain why three of the tRNA genes could not be found in the mite Tyrophagus longior  in tRNAscan-SE or e-values in Infernal-MiTFi within MITOS2. The accurate folding of and trnQ ), or whynR, trnP ). Finallis group , as welltonychus  and, wittonychus . FinallyParachtes and Hapactocrates to the late Oligocene [R. culicivorax are also transcribed, and include a 3′ CCA sequence as result of interacting with the Aminoacyl tRNA Synthetase, which is a key step in tRNA maturation even for arm-less tRNAs [R. culicivorax also fold into a single stable hairpin structure in a tridimensional boomerang-like form that diverges from the standard L-shape structure [Caenorhabditis elegans a gene duplication of the EF-Tu, namely EF-Tu1 and EF-Tu2, may have played a key role in the degeneration of tRNAs, since they exclusively recognize aminoacylated TΨC- and DHU-armless tRNAs, respectively; see [Drosophila melanogaster, in spite of not having arm-less tRNAs in its mitochondria, possesses a EF-Tu1 that has the ability to recognize TΨC-armless from other related arthropod species [Based on available time estimates that trace back the split between ligocene , the mulligocene . Recent ss tRNAs . In facttructure . Truncattructure . In mammely; see . A recen species . Based o species , 46. In  species . Further species , and hen species .Parachtes and Harpactocrates species  and the new location for the trnT gene  and Mesothelae  except for Argyroneta aquatica, which is approximately 16 kb. Spider mitogenomes are more compact than the average size in other arthropod groups, including myriapods (15.09 ± 0.54), insects (15.63 ± 0.85 kb) and crustaceans (15.84 ± 0.77 kb), and in other invertebrates such as annelids (15.45 ± 1.09 kb), and mollusks (16.80 ± 3.81 kb). A recent study on the short mitogenome of the isopod A. vulgare (13.9 kb), which shows overlapping genes and an incomplete and truncated set of tRNA genes, revealed the existence of novel post-transcriptional mechanisms ensuring functionality of overlapping genes such as the post-transcriptional reparation of the truncated tRNA-H explained above [A. vulgare also exhibits large overlaps between two tRNA genes transcribed in the same direction and even tRNA genes either partially or completely placed within a PCG in direct or opposite orientation [in A. vulgare, such as the one reported here between cox1 and cox2, is thought to represent residual sequence artifacts caused by rearrangement events [trnL2 gene is located between cox1 and cox2 whereas in spiders is adjacent to nad3 or nad1.The mitogenomes of ed above . The genentation . The forentation  may deveentation ). Moreovt events . InteresParachtes and Harpactocrates were A + T rich (ca 71%), which is consistent with the mean value (73.1%) recently estimated for spiders [Neoscona orb-weaver spiders [Parachtes and Harpactocrates, most of them  have been already reported in spiders [The mitogenomes of  spiders . Similar spiders ) and oth spiders ). Simila spiders , as well spiders . A close spiders , and in  spiders . Regardi spiders , mites [ spiders , amphipo spiders , and bee rich ca 1%, whichrrnL and rrnS sequences in P. romandiolae closely matched the folding predicted in other arachnids such as the whip spiders Damon diadema and Phrynus sp. (Amblypygi) [Tetranychus spiders mites (Acari) [rrnL and rrnS also resembled those inferred in other invertebrates, including insects [rrnS gene, even between close related species, some motifs had to be folded again in Mfold [RNAs with the same function show a conspicuous similarity in the arrangement of base-paired stems (i.e. topology or secondary structure) in spite of differing in the exact size and spacing of base-paired regions . This isblypygi) , and six (Acari) . The str insects , crustac insects , miriapo insects  and nema insects . We propin Mfold , and in in Mfold . The secMetacrangonyx [Parachtes and Harpactocrates mitogenomes possessed a second, shorter non-coding region, which has been suggested as the replication origin for the lagging strand in other species [Metacrangonyx longipes [trnL2 and trnN , P. teruelis , P. romandiolae , P. limbarae , P. ignavus , and Harpactocrates apennicola . Geographic coordinates, DNA extraction, and purification methods of the vouchers are detailed in the former study [Genomic DNA samples belonging to several species of the genus us study , namely er study . Sequenccox1 and rrnL (5′ to 3′), and a shorter one of about 5 kb comprising the remaining circular genome from 5′ rrnL to 3′ cox1. Polymerization of the shorter fragment was only successful if annealing temperature decreased from the optimal 68 °C to 62–60 °C probably due to high A + T richness of control region [Mitogenomes were amplified as two overlapping long PCR fragments using species-specific primers , and then DNA fragments were pooled, purified and cloned. Finally, clones were sequenced using the Sanger protocol and mitochondrial regions with no coverage were extended by primer walking (available upon request) using initial long PCR fragments as template. The mitogenomes of the remaining five species were obtained by constructing libraries from long PCR fragments and subsequently pyrosequecing them using Roche FLX/454 with a unique tag for each species-specific library.The sequence of the mitogenome of the species lsewhere , 64. Brihttp://mitos2.bioinf.uni-leipzig.de/index.py. Many tRNA genes were not identified by Infernal predictions as implemented in the MiTFi algorithm in MITOS2 or by tRNAscan-SE v1.3.1 [rrnL and rrnS was corroborated by aligning the ribosomal sequences of the six species along with the sequences of the 12 spider mitogenomes available in MetAMiGA [rrnL and rrnS from MITOS2 were inaccurate since motives from different domains were joined together. Therefore, short motifs of each domain were manually revised using both nucleotide alignments and previously known secondary structures as references. Secondary structures of short motifs were predicted in Mfold v3.6 [Artemia salina Linnaeus, 1758 and Artemia franciscana Kellogg, 1906, the amphipods Metacrangonyx “bovei” [Pseudoniphargus sorbasiensis Notenboom, 1987 [Libelloides macaronius Scopoli, 1763 [Caenorhabditis elegans Maupas, 1900 [P. romandiolae. DNA sequences were aligned in MAFFT v7.273 [Tetragnatha nitens Audouin, 1826  and Agelena silvatica Oliger, 1983 (Agelenidae), and Carrhotus xanthogramma Latreille, 1819  were not included in the analyses since they show complex gene rearrangement patterns. Finally, nucleotide and amino acid composition and codon usage profiles (RSCU) were estimated in MEGA v5.10 [Mitogenomes were annotated in the new MITOS2 version of Mitoswebserver  availablE v1.3.1  using thMetAMiGA . Secondaold v3.6  allowingas, 1900 . Since tT v7.273 . SecondaT v7.273 . AncestrT v7.273  using thGA v5.10 , and AT Additional file 1:Figure S1. A + T composition and AT and GC skews for the 6 mitogenomes reported for Parachtes and Harpactocrates. The same features are shown for each protein-coding gene and pooling by codon position and coding strand. (PDF 39 kb)Additional file 2:Figure S2. Pie plot showing amino acid frequencies for each species studied here: Parachtes teruelis (1), P. riberai (2), Harpactocrates apennicola (3), P. romandiolae (4), P. limbarae (5), and P. ignavus (6). Arrows denote those amino acid which frequencies changed greatly depending of coding strand. (PDF 385 kb)Additional file 3:Figure S3. Secondary structures predicted by Infernal-MiTFi (MITOS2) for the 22 tRNAs for the six spider species: Parachtes teruelis (mitos103), P. riberai (mitos105), P. romandiolae (mitos352), P. limbarae (mitos475), P. ignavus (mitos479), and Harpactocrates apennicola (mitos350). (PDF 397 kb)Additional file 4:Figure S4. Modified secondary structures of tRNA from those foldings predicted by mitfi (mitos2) for the 22 tRNAs for the six spider species: Parachtes teruelis (sp103), P. riberai (sp105), P. romandiolae (sp352), P. limbarae (sp475), P. ignavus (sp479), and Harpactocrates apennicola (sp350). Some tRNAs show an alternative folding either as three arms tRNA or as arm-less tRNAs. (PDF 1550 kb)Additional file 5:Table S1. Gene orders found in the 32 mitogenomes available in GenBank and MetAMiGA, that retrieved from Parachtes and Harpactocrates plus the ancestral patterns for Pancrustanea, Arthropoda and Chelicetata . (XLSX 13 kb)Additional file 6:Table S2. Primer list used to amplify long fragments 5′ cox1–3′ rrnL . Parachtes teruelis (sp103), P. riberai (sp105), P. romandiolae (sp352), P. limbarae (sp475), P. ignavus (sp479), and Harpactocrates apennicola (sp350). Note that reverse primer was identical for all species except for P. riberai (sp105). (DOC 13 kb)Additional file 7:Figure S5. Photography of long PCR fragments analyzed by agaroge gel electrophoresis after ethidium bromide staining and UV exposition. Parachtes teruelis (103), P. riberai (105), P. romandiolae (352), P. limbarae (475), P. ignavus (479), and Harpactocrates apennicola (350). S refers to short PCR fragment and L to large one. (TIF 981 kb)


The electrical activity of the crural diaphragm (Eadi), a surrogate of respiratory drive, can now be measured at the bedside in mechanically ventilated patients with a specific catheter. The expected range of Eadi values under stressed or assisted spontaneous breathing is unknown. This study explored Eadi values in healthy subjects during unstressed (baseline), stressed (with a resistance) and assisted spontaneous breathing. The relation between Eadi and inspiratory effort was analyzed.Thirteen healthy male volunteers were included in this randomized crossover study. Eadi and esophageal pressure (Peso) were recorded during unstressed and stressed spontaneous breathing and under assisted ventilation delivered in pressure support (PS) at low and high assist levels and in neurally adjusted ventilatory assist (NAVA). Overall eight different situations were assessed in each participant (randomized order). Peak, mean and integral of Eadi, breathing pattern, esophageal pressure–time product (PTPeso) and work of breathing (WOB) were calculated offline.max defined as Eadi measured at maximal inspiratory capacity reached 90 [63 to 99] μV. Median peak Eadi/Eadimax ratio was 16.8 [15.6–27.9]%. Compared to baseline, respiratory rate and minute ventilation were decreased during stressed non-assisted breathing, whereas peak Eadi and PTPeso were increased. During unstressed assisted breathing, peak Eadi decreased during high-level PS compared to unstressed non-assisted breathing and to NAVA (p = 0.047). During stressed breathing, peak Eadi was lower during all assisted ventilation modalities compared to stressed non-assisted breathing. During assisted ventilation, across the different conditions, peak Eadi changed significantly, whereas PTPeso and WOB/min were not significantly modified. Finally, Eadi signal was still present even when Peso signal was suppressed due to high assist levels.Median [interquartile range] peak Eadi at baseline was 17 [13–22] μV and was above 10 μV in 92% of the cases. EadiEadi analysis provides complementary information compared to respiratory pattern and to Peso monitoring, particularly in the presence of high assist levels.Trial registration The study was registered as NCT01818219 in clinicaltrial.gov. Registered 28 February 2013The online version of this article (10.1186/s13613-019-0564-1) contains supplementary material, which is available to authorized users. Differences between, respectively, two and more than two conditions were assessed using the Wilcoxon test (paired data) or the Friedman analysis and the corresponding post hoc tests for post hoc pairwise comparisons. Of note, when one or more breathing condition(s) could not be recorded in a subject, analysis of variance did not include this subject and the median values mentioned in the text, in the tables and in the figures summarize the values for the participants included in the comparison analysis only. Correlations were assessed using the rank Spearman correlation analysis. Statistical analyses were performed using the MedCalc software version 14.12.0 . A Fifteen healthy male volunteers with normal lung function tests were included in the study, but two of them did not tolerate nasogastric tube insertion. All the recordings in spontaneous non-assisted breathing were successfully performed and analyzed. Seventy-one of the 78 (91%) planned recordings under assisted ventilation could be recorded and stored. Due to poor comfort associated with the corresponding conditions, four of the 78 (5.1%) planned sequences under assisted ventilation could not be recorded. Due to automatic switch to the backup PS mode, NAVA could not be applied in one of the participants. For technical reasons, one NAVA recording and two esophageal pressure–time curves (during non-assisted breathing) could not be saved. Finally, in two subjects, the quality of the esophageal pressure curves recorded was not good enough for offline data analyses.The anthropometric characteristics of the participating subjects are given in Table max) and PTPeso/breath recorded during a slow inspiration to total lung capacity  are given in Table max reached 91 [64 to 104] μV.The inspiratory volumes, maximal peak Eadi (Eadimax ratio are given in Table During spontaneous unstressed non-assisted breathing (baseline), peak Eadi values were between 4 and 29 μV. Peak Eadi was above 10 μV in 92% of the recordings and the median peak Eadi was 17 [13–21] μV. Median, minimal and maximal values of peak Eadi/EadiAdding the resistance during spontaneous non-assisted breathing or applying assisted ventilation without or with the resistance affected minute ventilation (MV) by either modifying respiratory rate (RR), VT or both. Tin and Tiex were also impacted by the different conditions. Oppositely, Ti/Ttot was similar at baseline, in the presence of the resistance and under assisted ventilation without or with the resistance. Median MV, RR, VT, Tin, Tiex and Ti/Ttot are displayed in Table p = 0.03) , peak Eadi values were between 12 and 39 μV . Peak Eadi values increased compared to baseline in 10/13 subjects (77%). Overall peak Eadi increased from 16.9 [13.1–21.5] to 22.5 [15.2–28.7] µV  , but WOB/L was higher in the presence of the resistance .PTPeso/min was higher in the presence of the resistance . Of note, when the analysis was performed only for the patients who also had a legible recording of PTPeso, the difference remained significant. There were different effects of the different assisted ventilation modalities across subjects on peak Eadi  of the recordings with high-level PS. In these recordings, positive Eadi deflections were low but still visible. A representative recording illustrating this situation is displayed in Additional file p Friedman 0.10) and WOB/min (p Friedman 0.14) were not different during unstressed non-assisted breathing and under the different modalities of assisted ventilation without resistance By contrast with Eadi, PTPeso/min . Of note, when the analysis was performed only for the patients who also had a legible recording of PTPeso, the difference remained significant (p < 0.001). In the presence of the resistance, the effect of applying the different modalities of assisted ventilation on peak Eadi varied across subjects  recordings with high-level PS, in 1/10 (10%) recordings with low-level PS and never with NAVA. Eadi was still visible in all cases.p Friedman 0.17, 0.30 and 0.11, respectively).During resistive breathing, PTPeso/min Fig. d, WOB/mip < 0.001). Breath-by-breath correlation between peak Eadi and PTPeso/cycle showed a rho coefficient at 0.43 . When correlation between peak Eadi and PTPeso/cycle was assessed independently for each subject, the rho coefficients were between 0.41 and 0.83 with a median value of 0.57 [0.53–0.71]. The rho coefficient of the correlation between peak Eadi mean value and PTPeso/min was 0.43 .For all the recorded conditions and for all the subjects considered together (breath-by-breath correlation), the Spearman rho coefficients of correlations between peak Eadi, Eadi global integral, Eadi integral peak and mean Eadi were between 0.86 and 0.94  non-assisted breathing, peak Eadi increased compared to unstressed non-assisted breathing. Overall, assisted ventilation induced a decrease in peak Eadi compared to non-assisted breathing both during unstressed (without resistance) and stressed breathing. In the same conditions, changes in breathing effort assessed by Peso were often nonsignificant. In some conditions there was a paradoxical increase in peak Eadi when the level of assist was increased, underlining that Eadi could provide information not available using standard respiratory monitoring or esophageal pressure monitoring. We finally demonstrated that Eadi signal was still present when, in the presence of high assist levels, no esophageal deflections were visible on the esophageal pressure–time curve.max ratios values at baseline could be explained by differences in the subjects’ anatomical characteristics as the distance between the nasogastric tube electrodes and the crural diaphragm was previously reported as having an influence on absolute Eadi values [max ratio gave the same information.Previous data on normal baseline  peak Eadi are very sparse , 22, buti values , 30. In i values  meaning i values , 31, 32.i values . Acutelyi values  is a rel2O pressure support weaning test. Our study showed an absence of concordance between VT and Eadi variations when unstressed and stressed non-assisted spontaneous breathings were compared. Our study clearly demonstrates that respiratory drive per breath may increase even if RR decreases, which cannot be diagnosed using standard respiratory monitoring. As expected, an increase in PTPeso/min and WOB/L occurred when inspiratory load increased. An increase in peak Eadi was overall correlated with the increase in PTPeso/min and WOB/L. This is in line with the data published by Bellani et al. [During stressed (with resistance) spontaneous breathing, MV decreased compared to baseline  due to RR decrease. Tin increased, while VT remained stable. This is in line with previous data reported by Calabrese et al.  except fi et al.  who demoi et al. , 35–37.Eadi amplitude decreased when assisted breathing was compared to unstressed non-assisted breathing. In this situation, assisted ventilation induced an increase in MV due to VT increase without concomitant RR increase, in line with the data of Meric et al.  recordedEadi signal was still present even when the negative pressure drops were no longer visible on the esophageal pressure–time curve due to very low effort in presence of high assist level. The interest of Eadi monitoring in diagnosing overassist was already suggested by Sinderby et al.  and CartSeveral limitations must be mentioned. First, the respiratory profile at baseline (spontaneous non-assisted breathing without resistance) could have been influenced by the experimental setting  and the Our study demonstrated that peak Eadi normal values were within a broad range but usually above 10 µV. Our model illustrates examples of situations where Eadi amplitude does not change in parallel to PTPeso suggesting that Eadi monitoring provides additional and different information compared to respiratory pattern and to esophageal pressure monitoring. Using Eadi as advanced respiratory monitoring could thus be of interest to better integrate breathing regulation in our clinical assessment.Additional file 1: Table S1. Detailed lung function tests at baseline and in the presence of the 20 cmH2O/L/sec resistance. FVC: Forced Vital capacity, FEV1: Forced expiratory volume in one second. PEF: Peak expiratory flow. Res: Resistance. Figure S1. Effect of a resistance on peak Eadi (A) and PTPeso/min (B) during spontaneous non-assisted breathing. PTPeso/minute: esophageal pressure-time product by minute. Eadi: Electrical activity of the diaphragm. The middle line of box-and-whisker plot represents the median. The central box represents the values from the lower to upper quartile (25 to 75 percentile). The vertical line extends from the minimum to the maximum values. Figure S2. Over-assist during pressure support ventilation (PSV) indicated by absence of visible negative esophageal pressure drop. Note that peak electrical activity of the diaphragm (peak Eadi) is on average below 6 microvolts. Eso: esophageal.


Gastrointestinal stromal tumors (GISTs) account for less than 1% of all gastrointestinal tumors. The biological behaviors of GISTs vary from benign to malignant. GISTs are common in the stomach (55.6%) and small intestine (31.8%), but rarely in the rectum, colon (6%), and other sites (5.5%). Currently, the majority of published reports of primary appendiceal stromal tumors (PASTs) are case reports or case series.The PASTs described in this study were identified from a literature review (23 cases) and our center (one case). The relationship between PAST gross types and clinicopathological factors was analyzed and summarized. At the same time, the study also analyzed the related risk factors and survival of PASTs and GISTs.P < 0.001), histological type (P = 0.013), CD34 expression (P < 0.001), and DOG-1 expression (P < 0.001). Disease-free survival (DFS) analysis of 11 cases of PASTs and 227 cases of GISTs found that a comparison of 3-year and 5-year DFS was not statistically significant . In the DFS multivariate analysis, tumor mucosal ulceration, tumor size, and NIH risk classification were independent prognostic factors in 3-year and 5-year DFS.Twenty-four cases of PASTs were compared with 254 cases of GISTs from our center. The results showed that there was a significant difference between the two groups in tumor size (In this study, there was no significance in the survival of patients with appendix and gastric stromal tumors, which we hypothesized to be associated with the low sample size and incomplete follow-up records. Based on this, we conclude that the prognosis of primary appendiceal stromal tumors may be better than gastric tumors, but this needs to be confirmed in further prospective studies. Gastrointestinal stromal tumors (GISTs) account for less than 1% of all gastrointestinal tumors and are generally considered to emanate from the interstitial cells of Cajal (ICCs) . GISTs wThe biological behaviors of GISTs vary from benign to malignant. CD117, CD34, and DOG1 expression is usually positive in IHC staining, and thus these proteins are useful when confirming diagnosis . GISTs aThe cases used in this study were identified through a review of databases and from our center. Cases were retrieved from Chinese and foreign databases. The Chinese databases were China National Knowledge Infrastructure (CNKI) (seven cases), VIP (eight cases), WANFANG DATA (13 cases), while the foreign databases included PubMed 12 cases) and EMBASE (four cases). After data synthesis, 20 reports were filtered –27, whic cases anThe clinicopathological data of PASTs in this study included age, sex, tumor size, gross type, rupture, local ulceration, histological type, mitotic phase, NIH risk classification, gene mutation types, clinical symptoms, and survival data. For survival analysis, the exclusion criteria were as follows : (1) strχ2-test and Fisher exact test were applied to identify differences in clinicopathological parameters between GISTs and PASTs. Risk factors for survival were identified by univariate analysis and multivariate analysis using the Cox proportional hazards regression model. Estimations for disease-free survival (DFS: defined as the time from surgery to disease recurrence/death (months)) were obtained using the Kaplan-Meier method, and differences between Kaplan-Meier curves were investigated by log-rank test. P values of < 0.05 were considered to be statistically significant.Statistical analysis was performed using SPSS 19.0 . In this study, numerical variables were expressed as the mean ± SD. The KIT and two wild-type mutations in all of the studies. According to the modified NIH risk classification and literature reports, 11 patients were at very low risk (45.8%), two patients were low-risk (8.3%), four patients were at intermediate risk (16.7%), and seven patients were at high risk (29.2%).The clinicopathological features of the PASTs are shown in Table P > 0.05). We suspect that this may be related to the low sample size. The clinicopathological factors of 24 cases of PASTs such as age, sex, tumor size, histological type, mitotic index, CD117 expression, CD34 expression, DOG-1 expression, ulceration, and NIH risk classification were compared with 254 cases of GISTs from our center (Table P < 0.001), histological type (P = 0.013), CD34 expression (P < 0.001), and DOG-1 expression (P < 0.001).The relationship between PAST gross types and clinicopathological factors were analyzed and are summarized in Table P = 0.894 and P = 0.846, respectively) . The 3-year and 5-year DFS rates were 45.5% and 18.2%, respectively. The DFS of PAST patients was analyzed using Kaplan-Meier survival analysis and is shown in Fig. This study represented the largest number of PAST cases analyzed thus far. The clinicopathological features and prognosis of PASTs were statistically analyzed, and the survival rate of appendiceal stromal tumors was compared with that of gastric stromal tumors treated at our center; no difference was found between the two groups.PASTs are extremely rare, constituting approximately 0.1% of all cancer diagnoses . Other tGISTs are generally considered to emanate from the interstitial cells of Cajal (ICC), which are pacemaker cells that regulate gut motility . At presPASTs usually present with nonspecific or appendicitis-like symptoms and lack of corresponding hematology detection marker. Therefore, correct diagnoses of PASTs are very difficult to obtain prior to surgery. In general, CT and magnetic resonance imaging (MRI) are the first choice to study tumor location and extension . If the KIT and PDGFRA gene mutations occurred in approximately 78.5% and 5–8% of GISTs, respectively [Immunohistochemical staining is useful to confirm the diagnosis of stromal tumors –35. In Gectively . In thisComplete surgical resection with negative microscopic margins is the standard treatment for GISTs , 40.VassIt has been reported that tumor size, mitotic index, and tumor location are the best prognostic indicators for determining the malignant potential of GISTs , but theThe current study has some limitations. This is a retrospective study with a short follow-up time, so the data integrity is limited. The sample size is not large enough, and some appendix stromal tumors are less than 1 cm in diameter, which will lead to sampling errors. Because the number of stromal tumor cases identified in other locations were limited at our center , they could not be compared with appendix clinical pathology and survival characteristics.In this study, most of the PASTs were solid ; there were no cystic cases, and most of the pathological diagnosis of PASTs were spindle cells . According to the NIH classification criteria, the median risk was more than 50% . By analyzing the data of PASTs and GISTs from our center, we found that there was a significant statistical difference between tumor size, histological type, CD34 expression, DOG-1 expression, ulceration, and NIH grade. Only one patient died of postoperative lymph node metastases in all selected cases. Rutkowski et al.  reported


There was only weak evidence for geographic clustering of sequences, indicative of fluid patterns of transmission within the infected population and no evidence of any clustering by patient age such that viruses in the same lineages circulate through the entire host population. Importantly, we show that both subtypes circulated concurrently in NSW with multiple introductions into the Australian population in each year and only limited evidence for multi-year persistence.There is an ongoing global pandemic of human respiratory syncytial virus (RSV) infection that results in substantial annual morbidity and mortality. In Australia, RSV is a major cause of acute lower respiratory tract infections (ALRI). Nevertheless, little is known about the extent and origins of the genetic diversity of RSV in Australia, nor the factors that shape this diversity. We have conducted a genome-scale analysis of RSV infections in New South Wales (NSW). RSV genomes were successfully sequenced for 144 specimens collected between 2010–2016. Of these, 64 belonged to the RSVA and 80 to the RSVB subtype. Phylogenetic analysis revealed a wide diversity of RSV lineages within NSW and that both subtypes evolved rapidly in a strongly clock-like manner, with mean rates of approximately 6–8 × 10 Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections (ARTI) in humans . The burWhile the factors that contribute to the prevalence of RSV are yet to be fully defined, immunity, re-infection rates, and climate may play a role. For example, laboratory reports highlight the seasonality in temperate regions in Australia, with a peak in RSV activity typically occurring in the early winter (May/June) period and preceding the seasonal peak in influenza virus . Climate and rainfall differences in the tropical north of Australia are also likely to be important drivers of RSV disease patterns, resulting in a seasonality distinct from that observed in temperate regions, with a correlation between peak RSV and peak rainfall levels around January .Pneumoviridae) with a 15 kb genome that encodes 10 proteins [RSV is a negative-sense single-stranded RNA virus . Samples were de-identified with basic demographic information collected including age, sex and location .® easyMAG®, bioMérieux) during routine diagnostic testing was stored at −80 °C prior to the commencement of this study. This study utilized residual RSV-positive specimens collected for routine diagnostic testing at the Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW, Australia between May 2010 and December 2016. Viral nucleic acid that was previously extracted , targeting both RSVA and RSVB subtypes using previously published primers . BrieflyAll genome sequences generated in this study are available on NCBI GenBank with accession numbers MH760588 to MH760731, as shown in To place our sample set into a global context, complete and near complete genome sequences of RSVA and RSVB were obtained from GenBank. Sequences with no geographic association or sampling date were excluded. RSVA and RSVB sequences were aligned separately using the multiple-sequence alignment tool, MAFFT, using the L-INS-I algorithm followed by a visual inspection . SequencMaximum likelihood (ML) trees were estimated in RAxML ,35 emploWe used a phylogenetic approach to determine whether there was more clustering by geography and age within the NSW RSV data set than might be expected from chance alone. Specifically, Bayesian posterior trees were used for evaluating geographic and age structure in the RSVA and RSVB trees using the Bayesian Tip-association Significance (BaTS) program; which compares parsimony score (PS), association index (AI), and maximum clade size (MC) statistics . Estimatn = 10) and Mount Druitt (n = 11). Despite the wide geographic range of sampling, coverage was low. Hence, sequences were sampled only from a small number of geographic locations. We attempted whole genome sequencing on 241 archived RSV-positive viral nucleic acid extracts held and tested by ICPMR. Virus genomes were successfully sequenced for 144 specimens, of which 64 belonged to RSVA and 80 to RSVB. The number of samples was skewed with the majority collected in 2016, comprising 36 RSVA and 57 RSVB sequences, respectively A. Despitn = 628) of viral genomes from the USA, which comprised 46% of the data in this study. Other relatively well represented countries were Peru (n = 122), the Netherlands (n = 61), Kenya (n = 61), Jordan (n = 85), Viet Nam (n = 53), New Zealand (n = 92), and the sequences sampled here in NSW (n = 144). The extensive sampling biases precluded detailed phylogeographic analyses. To simplify the geographic distribution analysis, sequences were grouped according to their continent of sampling . The significant clustering for facility and electorate is also supported by the low parsimony score (PS) and association index (AI) values, i.e., 0.00 and 0.006 for facility and electorate, respectively. Surprisingly, no significant clustering was observed in the case of RSVB despite different geographic locations and age categories being well represented in the data . Finally, we also investigated the extent of phylogenetic clustering by patient age group, particularly as RSV mainly infects infants. Notably, none of the age groups showed significant clustering in RSVA or RSVB  where the patient first presented, patient electorate, and patient age were mapped across the tree . Thirteeof 0.001 , RSVA. Tthe data . Notably or RSVB . Hence, G gene had significantly higher rates in RSVB than RSVA [2 values of 0.951 and 0.927 for RSVA and RSVB, respectively, for the NSW sequences, and 0.978 and 0.990 for the global sequences (−4 substitutions per site per year (subs/site/year) for global RSVA and RSVB, respectively, and 7.29 and 7.56 × 10−4 subs/site/year for RSVA and RSVB sampled in NSW in this study, respectively  and 6.48 × 10−4  subs/site/year for RSVA and RSVB, respectively, using LSD and 7.48 × 10−4 (95% HPD 6.67–8.34 × 10−4) and 7.39 × 10−4 (95% HPD 6.34–8.45 × 10−4) subs/site/year for RSVA and RSVB, respectively, using BEAST  and 6.41 × 10−4  subs/site/year for RSVA and RSVB, respectively. These rates are within the range reported previously for paramyxoviruses [Given this strong clock-like structure, we investigated the evolutionary rates more carefully using the Bayesian Markov chain Monte Carlo (MCMC) method implemented in BEAST for the NSW data set and the least square dating (LSD) method for the global data set. In the case of the NSW data, the mean evolutionary rates were 7.06 × 10ng BEAST B. Hence,oviruses . Notablyoviruses . These goviruses ,51.We report a wide diversity of RSV lineages co-circulating in a small geographic region, reflecting a combination of continual virus entry and some sustained in situ transmission within NSW. Despite these fine-scale epidemiological insights, this study also highlighted the highly biased global sampling of RSV that hinders extensive analysis of the global distribution and transmission dynamics of RSV. We stress that increased targeted surveillance with more extensive virus sampling, particularly during suspected outbreaks, is essential to improve both our understanding of RSV ecology and evolution, and assist with vaccine design.


Anaplastic thyroid cancer (ATC) responds poorly to conventional therapies and requires a multidisciplinary approach to manage. The aim of the current study is to explore whether aggressive treatment is beneficial, especially the appropriate extent of surgery in ATC.  Patients diagnosed with ATC from 2004 to 2014 were identified from the Surveillance, Epidemiology, and End Results (SEER) database and included in our study. P = 0.177). Multivariate Cox regression analysis also showed increased mortality risk in patients with increased age , tumor extension to adjacent structures , and distant metastasis , while surgery + RT  and chemotherapy  were independently associated with improved OS. Further analysis revealed that patients undergoing total/near-total thyroidectomy (TT) had superior OS to those receiving less than TT (P < 0.001). In subgroup analysis, the benefit of TT remained significant in patients with tumors larger than 4.0 cm , with adjacent structure extension , including trachea and major vessels, but not in patients with early phase local disease such as tumor ≤ 4.0 cm or tumor within the thyroid or with minimal extrathyroidal extension. Patients with very locally advanced disease or distant metastasis could not benefit from TT as well.  A total of 735 ATC patients were identified. The two-year overall survival (OS) rates for stage IVA, IVB, and IVC patients were 36.5%, 15.6%, and 1.4%, respectively. By directly comparing eight treatment modalities, we found that surgery + radiotherapy (RT) ± chemotherapy was the most effective treatment strategy. surgery + chemotherapy and RT + chemotherapy had comparable results (hazard ratio (HR) = 1.461, 95% confidential interval (CI): 0.843-2.531,  In operable cases, surgery + RT ± chemotherapy was the optimal treatment modality. Otherwise, RT + chemotherapy was the appropriate strategy. However, TT was not beneficial for very early stage or metastatic ATC. Anaplastic thyroid cancer (ATC), although accounting for less than 0.2% of thyroid cancer, is one of the most lethal malignancies and accounts for up to 14-39% of total mortality in thyroid cancer . The cliATC responds poorly to conventional therapies of thyroid cancer and requires a multidisciplinary approach under many circumstances. The combination of surgery, radiation therapy (RT), and chemotherapy is recommended to improve the locoregional and distant control of the disease . PreviouData from the population-based Surveillance, Epidemiology, and End Results (SEER) database, on the other hand, provides population-based data in the current status of ATC treatment. There were two previous studies based on the SEER database including 516  and 261 Based on the latest version of SEER database, we aimed to explore whether aggressive treatment  is beneficial, especially the appropriate extent of surgery in ATC patients on a population-based level. Up to our knowledge, it is the first time to directly compare the results of eight treatment approaches and to reevaluate the role of total thyroidectomy in different clinical scenarios in ATC.The SEER database of the US National Cancer Institute with anaplastic thyroid carcinoma  was used to identify ATC patients. We limited the cohort: (1) from 2004 to 2014 because of the lack of complete patient profiles in earlier cases, (2) patients with active follow-up during the study period until the cutoff follow-up date or the occurrence of endpoint event, and (3) patients with a known cause of death. Patients whose diagnoses were only based on autopsy reports or death certificate were excluded. The selection paradigm was presented in Supplementary Patient baseline information , disease characteristics , and treatment  were included into analyses. The 7th edition of TNM staging system was used to stage the patients. Tumor size was recorded by “CS tumor size (2004+)”, and extension of primary tumor was divided into three categories according to “CS extension (2004+)” as (1) confined to thyroid, (2) minimal extension , and (3) adjuvant structures/organs . Surgery was defined as lobectomy with/without resection of the contralateral lobe while tracheotomy and excision biopsy were excluded. We further divided the extent of surgery by “RX Summ-Surg Prim Site (1988+)” into two parts: (1) less than total thyroidectomy  and (2) total/near-total thyroidectomy (TT). Information of cervical lymph node management was extracted from the “RX Summ-Scope Reg LN Sur (2003+),” “regional nodes examined (1988+),” and “regional nodes positive (1988+).” The primary outcome of the present study was overall survival (OS), the duration of which was defined as the time from diagnosis to death from any cause.P value < 0.05 was considered to be statistically significant.Cox regression models were used to identify risk factors contributed to OS and to compare the hazard ratios (HRs) and 95% confidential intervals (CIs) of different treatment modalities. Survival rates were estimated using the Kaplan-Meier method, and the significance of comparisons was calculated by the log-rank test. All statistical analyses were conducted with IBM SPSS version 24 . A two-sided P < 0.001).A total of 735 ATC patients diagnosed between 2004 and 2014 were identified from the SEER database and included into the analysis. The median age at diagnosis was 70 years old (range: 26-98). The majority of patients were female . The median tumor size was 6.4 cm. Of all patients, 362 had lymph node metastasis and 335 had distant metastasis . The OS According to the treatment strategies, we categorize the patients into eight treatment groups: 159 patients did not receive surgery, RT, or chemotherapy (T1); 27 had only chemotherapy (T2); 188 had only surgery (T3); 19 had surgery + chemotherapy (T4); 80 had only RT (T5); 125 had RT + chemotherapy (T6); 56 had surgery + RT (T7); 141 had surgery + RT + chemotherapy (T8).P = 0.691; T5 vs. T2: HR = 1.310, 95% CI: 0.822-2.090, P = 0.256; T5 vs. T3: HR = 1.163, 95% CI: 0.866-1.561, P = 0.316). Adding chemotherapy to RT or surgery was more effective than RT alone or surgery alone, while surgery + chemotherapy (T4) and RT + chemotherapy (T6) had comparable results . surgery + RT (T7) revealed significantly decreased HRs compared with other treatments (T1-6); however, adding chemotherapy to surgery + RT did not seem to further improve the OS .Subsequently, we directly compared OS rates in eight treatment groups . ChemothP < 0.001) and 3.0% in the RT-alone group (P < 0.001) (P = 0.663). Using the multivariate Cox regression model (P < 0.001), tumor extension to adjacent structures , and distant metastasis . surgery + RT could significantly improve OS compared with surgery alone . Chemotherapy was also an independent prognostic factor for improved OS .Next, we limited the analysis into 316 patients who had locoregional treatments (surgery or RT). Patients in the surgery + RT group showed a significant survival benefit than other groups. The two-year OS in the surgery + RT group was 28.8% compared with 7.7% in the surgery-alone group (< 0.001) . Howeveron model , we obseIn 197 patients who had both surgery and RT, only seven had RT prior to surgery, 187 had RT after surgery, two had RT both before and after surgery, and one patient had both RT and surgery, but the sequence was unknown.Since the detailed information regarding the regimens of chemotherapy and doses of RT was not accessible in the SEER database, we put emphasis on the exploration of the optimal surgical strategies in ATC in the setting of multidisciplinary treatment.N = 195) and less than TT group (N = 130). These two groups were comparable in all baseline characteristics, such as gender, age, tumor extension, regional lymph node, and distant metastasis status. However, more patients had a tumor more than 6.4 cm in the less than TT group . The benefit of TT remained significant in all gender and age groups and patients with tumor size > 4.0 cm , with adjacent structure extension , including trachea and major vessels. Nevertheless, TT was not beneficial for patients with early phase local disease such as tumor ≤ 4.0 cm (P = 0.437), tumor within the thyroid (P = 0.382), or minimal extension (P = 0.681). Patients with very locally advanced disease  or distant metastasis (P = 0.122) could not benefit from TT as well . Pathology revealed that 54 patients had no lymph node metastasis, and the median of positive lymph nodes was 2 (range: 1-53). The median percentage of lymph node positivity was 23% (range: 0%-100%).P < 0.001). Then we divided the patients into two groups according to whether they had thyroid surgery. Interestingly, although lymph node dissection could improve OS in patients who had thyroid surgery  should be applied to ATC patients whenever applicable. As previous studies indicated, extensive surgery with adjuvant RT was the optimal local regional treatment strategy in ATC, and long-term survival might be acquired in these cases, especially when the tumor was less invasive –16. A reIn cases with adequate locoregional therapy (surgery + RT), our analysis showed that adding chemotherapy could not further improve OS. Nevertheless, Rao et al. reported that up to 74% of patients had distant disease progression during treatment and there was a potential opportunity to improve outcomes with earlier initiation of systemic therapy . One of Another highlight of the current study was that we reevaluated the role of TT and found that TT was not beneficial for all patients. For patients with locally restrained disease, the TT group showed nonsuperior OS than the control group. These results were in accordance with previous studies that complete resection (R0/R1) was associated with improved survival in ATC patients –24. SincOn the other hand, in patients with larger tumor size and adjacent structures extension, TT had a survival advantage due to the complete resection of the tumor which could not be achieved by less than TT. However, for patients with very advanced disease (such as distant metastasis), our data suggested that TT was not correlated with OS due to the extreme fatal nature of ATC. These were correlated with previous studies that there was no benefit of surgery in IVC patients .Up to 49.3% of ATC patients presented with cervical lymph node metastases, still, the surgery of reginal lymph nodes was seldom discussed. Our findings suggested that cervical lymph node dissection could improve OS in patients with thyroid surgery. This result was consistent with multivariate Cox analysis of the current study and previous studies that lymph node metastasis was not an independent prognostic factor for OS in ATC patients.The strengths of the current study were as follows: (1) unlike previous SEER-based studies, we were able to include more variables into our analyses owning to the update of the database since 2004; (2) we analyzed the scope of surgery of the thyroid and regional lymph nodes in ATC; and (3) for the first time, we directly compare all the treatment strategies of ATC. However, our research was limited by its retrospective nature, and the lack of more detailed information of RT and chemotherapy made it impossible to explore the optimal RT doses and chemotherapy regimens.By analyzing 735 patients treated during 2004-2014, we identified that aggressive treatment could improve OS in ATC. In operable cases, surgery + RT ± chemotherapy was the optimal treatment option; otherwise, RT + chemotherapy was the appropriate strategy. However, the scope of thyroid surgery should depend on the scope of local disease. TT was not beneficial for very early stage or metastatic ATC compared with less than TT.


LPL, APOA5, APOC2, GPIHBP1 and LMF1, have been frequently reported to cause or predispose to HTG.Hypertriglyceridemia (HTG) is one of the most common etiologies of acute pancreatitis (AP). Variants in five genes involved in the regulation of plasma lipid metabolism, namely A Han Chinese patient with HTG-induced AP was assessed for genetic variants by Sanger sequencing of the entire coding and flanking sequences of the above five genes.LPL, APOC2, APOA5, GBIHBP1 and LMF1 genes in the patient. We found no putative deleterious variants, with the exception of a novel and heterozygous nonsense variant, c.1024C > T , in exon 7 of the LMF1 gene.The patient was a 32-year-old man with severe obesity (Body Mass Index = 35) and heavy smoking (ten cigarettes per day for more than ten years). At the onset of AP, his serum triglyceride concentration was elevated to 1450.52 mg/dL. We sequenced the entire coding and flanking sequences of the LMF1 nonsense variant was found in a HTG-AP patient with severe obesity and heavy smoking, highlighting an important interplay between genetic and lifestyle factors in the etiology of HTG.This is the first time that a heterozygous The online version of this article (10.1186/s12944-019-1012-9) contains supplementary material, which is available to authorized users. Hypertriglyceridemia-induced acute pancreatitis (HTG-AP) usually refers to acute pancreatitis (AP) occurred in subjects with fasting serum triglyceride (TG) levels of > 1000 mg/dL (> 11.3 mmol/L) or > 500 mg/dL (> 5.65 mmol/L) with coincidentally detected milky serum and in the absence of other etiologic factors. Hypertriglyceridemia (HTG) is responsible for 2–5% of AP patients in Western countries , demonstLPL , APOA5  complex ), APOC2 ctivator ), GPIHBPg of LPL ), and LMn of LPL ), have bn of LPL .Herein, we describe a case with HTG-AP that appeared to be predisposed by a combination of genetic susceptibility and exposure to harmful lifestyle factors.The patient was a 32-year-old man with severe obesity (Body Mass Index (BMI) = 35) and heavy smoking (ten cigarettes per day for more than ten years). He was diagnosed to have moderate HTG ) three years ago (at the age of 29) but had not taken lipid-lowering drugs. His father had diabetes mellitus, his mother was healthy, and there was no known history of HTG in the family.He was hospitalized elsewhere due to severe abdominal pain associated with nausea and vomiting on June 5, 2017. He was transferred to our Surgical Intensive Care Unit for further treatment on June 11, 2017. The study was performed in accordance with the Helsinki Declaration and was approved by the Ethics Committee of Jingling Hospital, Nanjing University. Bio-samples were obtained after written informed consent.Blood samples were collected after fasting for 12 h. Serum glucose (GLU), TG, total cholesterol (TC), high-density lipoprotein (HDL), low density lipoprotein (LDL), apolipoprotein A1 (APOA1), and apolipoprotein B (APOB) levels were measured enzymatically on an automatic analyzer .LPL, APOA5, APOC2, LMF1, and GPIHBP1 were amplified by polymerase chain reaction (PCR) and subsequently sequenced using both forward and reverse primers (primer sequences available upon email request). Identified mutations were subjected to confirmation by independent PCR amplification and sequencing. Nomenclature for the description of the reported LMF1 mutation followed the Human Genome Variation Society (HGVS) recommendations [LMF1 mRNA reference sequence.Genomic DNA was extracted from the peripheral blood cells using a Gentra Puregene Blood kit  according to the manufacturer’s instructions. The entire coding and flanking sequences of ndations . GenBank9/L) as well as inflammation markers C-reactive protein  and procalcitonin . Arterial blood gas analysis showed that pondus hydrogenii (PH) was 7.382 mmol/L, partial pressure of oxygen (PaO2) was 86.8 mmol/L, and fraction of inspiration oxygen (FiO2) was 33%. Renal and cardiovascular functions were normal.When the patient was initially hospitalized elsewhere, his blood sample was milky, with the serum TG concentration being elevated to 1450.52 mg/dL (16.39 mmol/L). Moreover, his serum TC and GLU levels were elevated to 6.55 mmol/L and 20.35 mmol/L, respectively. The patient was diagnosed with severe AP with the complication of acute necrotic collection , in a Han Chinese patient with HTG-AP. To our best knowledge, this variant has not been previously reported to be associated with HTG in the literature. Moreover, this variant was also absent in the ClinVar database . Further, survey of the Genome Aggregation Database  revealed that the c.1024 C > T variant was present at an average allele frequency of 0.00002857  in all populations combined; and its allele frequency in the East Asian population was 0.0002238 . In short, the LMF1 c.1024 C > T variant is extremely rare in normal populations.In this study, we identified a heterozygous LMF1 gene encodes a 567 amino acid protein. Recently, Serveaux Dancer and colleagues assessed the functionality of two LMF1 nonsense variants, p.Tyr439* and p.Trp464*, by means of an in vitro assay. Specifically, they co-transfected HEK293T cells with the wild-type LPL expression vector and the LMF1 mutant expression vector of interest; and then measured the LPL activity in the culture media of the co-transfected HEK-293 T cells. As such, Serveaux Dancer and colleagues found that both the LMF1 p.Tyr439* and p.Trp464* nonsense variants caused an almost complete loss of the LPL activity [LMF1 c.1024C > T (p.Arg342*) variant reported here, which removes ~ 100 more residues from the C terminus of the protein as compared to p.Tyr439* and p.Trp464*, can be reasonably concluded to cause a complete functional loss of the affected LMF1 allele.The human activity . The LMFLPL, APOC2, APOA5, GBIHBP1 and LMF1 genes identified only a single and heterozygous deleterious variant, LMF1 p.Trp464* (N.B. one of the aforementioned loss-of-function LMF1 nonsense mutations) [LMF1 variants had been described to have either normal or at most borderline HTG. Thus, our patient represents the second heterozygous carrier of a LMF1 nonsense variant who had severe HTG. This notwithstanding, it is pertinent to emphasize an important difference between the two relevant severe HTG patients in terms of their underlying etiological factors. In the Serveaux Dancer study, the two other heterozygous carriers of the LMF1 p.Trp464* variant had normal TG concentration; and the 8-month-old proband with the heterozygous LMF1 p.Trp464* variant exhibited severe HTG (1860 mg/dL (21 mmol/L)) on an episode of severe acute gastroenteritis (a condition known to be associated with moderate HTG) without AP. In our study, we were not able to analyze the patient’s parents but neither of them was known to have HTG. Notably, our patient had two harmful lifestyle factors, severe obesity and heavy tobacco abuse. Obesity, producing more TG, is not only associated with primary HTG [LMF1 variants of similar functional effects, they had quite different secondary HTG-predisposing factors.A particularly interesting finding of the Serveaux Dancer study is that in one of their subjects with severe HTG, their mutational analysis of the tations) . Prior tmary HTG  but alsomary HTG . Tobaccomary HTG . In addimary HTG . In shorLMF1 nonsense variant, c.1024C > T (p.Arg342*), is assumed to be half of that in health controls. We were not able to confirm this assumption due to the unavailability of pathophysiologically relevant tissue(s) from the patient. Second, “LPL is also expressed at high levels in certain regions of the brain. Why LPL is produced in neurons of the brain remains an enigma ….” [https://www.proteinatlas.org/search/lmf1).Finally, we would like to make two points. First, the LMF1 protein expression level in the patient carrying the heterozygous igma ….” . IntriguLMF1 nonsense variant in a HTG-AP patient with severe obesity and heavy smoking, highlighting an important interplay between genetic and lifestyle factors in the etiology of HTG.In conclusion, we have for the first time discovered a novel and heterozygous Additional file 1:Sequencing data. (DOCX 22 kb)