Mucosal epithelia encounter both physicochemical and biological stress during their life and have evolved several mechanisms to deal with them, including regulation of immune cell functions. Stressed and damaged cells need to be cleared to control local inflammation and trigger tissue healing. Engagement of the activating NKG2D receptor is one of the most direct mechanisms involved in the recognition of stressed cells by the immune system. Indeed, injured cells promptly express NKG2D ligands that in turn mediate the activation of lymphocytes of both innate and adaptive arms of the immune system. This review focuses on different conditions that are able to modulate NKG2D ligand expression on the epithelia. Special attention is given to the mechanisms of immunosurveillance mediated by natural killer cells, which are finely tuned by NKG2D. Different types of stress, including viral and bacterial infections, chronic inflammation, and cigarette smoke exposure, are discussed as paradigmatic conditions for NKG2D ligand modulation, and the implications for tissue homeostasis are discussed. Mucosal epithelia represent the frontline of multicellular organisms, and they are continuously exposed to several types of stress. Pathogens and environmental stress  can result in cell damage and loss of tissue function. To control inflammation and promote tissue repair, different mechanisms for the detection and elimination of stressed cells have evolved, including activation of the immune system.+ αβ T cells, γδ T cells, and iNKT cells. Engagement of NKG2D triggers the cytolytic function of effector CD8+ T cells independently of TCR recognition in some circumstances  cells, CD8mstances –4 while mstances –7. In humstances , 10 but mstances –13. Evenr stress , only rer stress .Expression of NKG2D ligands has been shown in tissues from patients with chronic inflammatory diseases, including rheumatoid arthritis , 17, typThis review focuses on different conditions that are able to modulate NKG2D ligands on epithelial cells, and, in particular, on the role of NKG2D/NKG2D ligands in controlling the homeostasis of the gut and lung epithelia during inflammation. Recent findings link toll-like receptor (TLR) signaling to NKG2D ligand expression, and we can now think of epithelial and immune cells as an integrated system able to deal promptly with environmental stress.Among the pattern recognition receptors, TLRs play a key role in innate immunity, serving as first line sensors of structurally conserved bacterial and viral components, the so-called pathogen-associated molecular patterns. It is clear that TLRs can be triggered also by endogenous ligands, such as nucleic acids released by necrotic cells and matrix components generated during tissue injury, collectively called damage-associated molecular patterns. As TLR engagement leads to the production of inflammatory cytokines and chemokines, which contribute to local inflammation and leukocyte recruitment, the recent findings showing that TLR signaling is able to modulate the NKG2D axis and thus lymphocyte activation are of great interest Table .via TLR3 activation  patients  in active Crohn’s disease have reported no major improvement, but further investigations regarding dose ranging and dose regimen are needed  stress  and innate-like T cells (such as CD8αα+ T cells), which contribute to mucosal inflammation in an NKG2D-mediated manner  stress . A recend manner . Expressd manner –45. Inded manner , 46, 47.phocytes . This inAn interesting issue to be considered for both Crohn’s disease and celiac disease regards NKG2D ligand polymorphism. MICA gene is highly polymorphic with more than one hundred alleles, which affect both RNA and protein expression levels . Some stAkkermansia muciniphila decrease NKG2D ligand expression. An interesting association between the above conditions and the intestinal levels of IL-15 has been postulated, once again linking the inflammatory milieu to NKG2D ligands  is associated with increased bacterial clearance . COPD is a severe form of airway epithelium inflammation largely due to cigarette smoke exposure. Cigarette smoke is known to induce necrotic and apoptotic cell death with concomitant nucleic acid release . This lel injury . The strl injury .Which ligand is displayed? Is this choice stress type dependent?Do different ligands engage the receptor in different ways?What is the contribution of NKG2D ligands to the pathogenesis of gut and lung inflammatory diseases? Does NKG2D ligand polymorphism play a role in IBD?Expression of NKG2D ligands can be achieved in several circumstances, ranging from viral and bacterial infections to physiochemical stress Table . This veIndeed, both NKG2D and NKG2D ligand genes are highly polymorphic. This polymorphism has been associated with host–pathogen coevolution, with great relevance for viral infections, but functional consequences have also been described during hematopoietic transplantation and cancer immunosurveillance , 66, 67.via a glycosylphosphatidylinositol molecule and thus are supposed to be trapped in the lipid raft-enriched apical side of epithelial cells (Figure Another important point to be considered is the strict polarity of epithelial tissues. In the gut, the apical side of the epithelial layer displays different functions from the basolateral side, where IELs are located. Human ULBP1–3 and murine RAE-1 are anchored to the cell membrane s Figure . Hence, s Figure . Remarkas Figure , 69. Furs Figure , 71.Great interest is growing regarding the role of the microbiome in human pathophysiology, as commensal bacteria can establish an immune regulatory milieu in the intestine , 73. TheFA searched for literature articles; conceived and wrote the manuscript. ANS conceived and wrote the manuscript. ALS, CC, and GS revised and critically contributed to the manuscript drafting. FA, ALS, CC, GS, and ANS approved the final version of the manuscript.The authors declare that research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.


Ovine SNP 600 K BeadChip array for genome-wide detection of CNVs in 48 Chinese Tan sheep. A total of 1,296 CNV regions (CNVRs), ranging from 1.2 kb to 2.3 Mb in length, were detected, representing approximately 4.7% of the entire ovine genome (Oar_v3.1). We combined our findings with five existing CNVR reports to generate a composite genome-wide dataset of 4,321 CNVRs, which revealed 556 (43%) novel CNVRs. Subsequently, ten novel CNVRs were randomly chosen for further quantitative real-time PCR (qPCR) confirmation, and eight were successfully validated. Gene functional enrichment revealed that these CNVRs cluster into Gene Ontology (GO) categories of homeobox and embryonic skeletal system morphogenesis. One CNVR overlapping with the homeobox transcription factor DLX3 and previously shown to be associated with curly hair in sheep was identified as the candidate CNV for the special curly fleece phenotype in Tan sheep. We constructed a Chinese indigenous sheep genomic CNV map based on the Illumina Ovine SNP 600 K BeadChip array, providing an important addition to published sheep CNVs, which will be helpful for future investigations of the genomic structural variations underlying traits of interest in sheep.Copy number variants (CNVs) represent a form of genomic structural variation underlying phenotypic diversity. In this study, we used the Illumina  Previous studies have shown that CNVs influencing genes or gene regions are associated with important phenotypic traits in livestock. For example, duplications of the KIT gene constitute the Dominant white locus in pigs3. In chicken, the pea-comb phenotype is caused by a CNV in intron 1 of the SOX5 gene4, and the late feathering locus comprises a partial duplication of the PRLR and SPEF2 genes5. A 1.6-kb deletion in TBX3 disrupts the asymmetric hair pigmentation that underlies Dun camouflage coloring in horses6. As one of the first domesticated animals, sheep (Ovis aries) have played an important role in human society7. Although increasing attention has been paid at identifying ovine CNVs13, the total number of CNVs, particularly in Chinese indigenous sheep, has been limited. None of the previously described ovine CNVs except for the agouti duplication, which affects the ASIP locus in sheep and contributes to coat color variability, have been determined to have a direct effect on a sheep trait15. Therefore, more efforts are needed to identify CNVs, one of the most important types of genomic variation, in the sheep genome.Copy number variations (CNVs), which represent a type of genomic structural variation, are DNA segments ranging in size from 1 kilobase (kb) to several megabases (Mbs) in which duplication or deletion events have occurred16. Compared to the other two technological platforms, SNP array is more cost-effective, allowing users to increase the number of samples on a limited budget and, as a result, achieve a more desirable performance in large-scale CNV detections, particularly at the genome-wide scale17. SNP arrays are also advantageous due to their high signal-to-noise ratios and the use of B-allele frequency, which facilitates the interpretation of results18. Furthermore, less sample per experiment is required for SNP arrays compared to that required for array-based comparative genomic hybridization (aCGH)19. However, the main bias of SNP arrays on CNV detection is the low SNP coverage of the genomic regions that often harbor CNVs20. This bias may be minimized to some extent by increasing the genome coverage using commercial high-density SNP arrays. Therefore, the detection of CNVs by high-density SNP arrays has become in increasingly common and performed successfully in various species.In recent years, advances in high-throughput genome scanning technologies, especially SNP arrays, DNA hybridization on array platforms, and next-generation sequencing methods, have allowed the identification of genome-wide structural variants that, due to their small size, are undetectable using microscopic techniquesKRT1.2 (keratin 1.2)21 and KAP1.3 (keratin associated protein 1.3)22 genes, which are related to wool curvature). The latest skin transcriptome profiling of Tan sheep at ages 1 and 48 months determined that the keratin genes  and their associated pathways, which were previously shown to be associated with hair/fleece development and function, are expressed differentially between 1 and 48 months of age23. However, the genetic components behind the white curly fleece phenomenon and the adaptation to harsh environments in Tan sheep are still unclear.Tan sheep, one of the most important sheep breeds indigenous to China, are reared in Ningxia Province and are renowned for their production of high-quality pelts and long-term adaptation to the dry, cold and windy climate of northwestern China. The famous lamb pelts from Tan sheep are the result of long-term artificial selection and thus exhibit a lustrous white curly fleece that disappears gradually with age. Earlier studies have focused on various candidate genes to investigate the genetic mechanism of the distinct curly fleece trait .The primary aim of this study was to conduct a genome-wide survey of sheep CNV regions (CNVRs) using the Illumina After employing a stringent CNV calling pipeline, we identified 5,190 autosomal CNVs  in 48 Chinese Tan sheep . The average distance between the CNVRs on each chromosome ranges from 467.5 kb on OAR6 to 544.9 kb on OAR11. The closest CNVRs are 4.5 kb apart on OAR3, whereas the largest inter-CNV distance is 34.4 Mb on OAR23.We generated a genome-wide map of CNVRs in Tan sheep Fig. . The chrRemarkably, compared with the number of gain events, we observed almost ten times more loss events and a longer loss length, with the number of regions with CNV losses and gains being 1,173 and 119, respectively. Both types were found in only five regions. For each CNVR, the relative frequency of animals with an overlapping CNV ranged from 2.1% to 37.5%  overlapping regions  CNVRs identified in our study are novel Fig. . To veri16. As stated earlier, we identified 5,190 CNVs, which accounted for 4.7% of the entire ovine genome. Among these, 463 (8.9%) overlapped with 1,255 gaps, indicating that the majority (90%) of the CNVs that we identified are located within the gap-free genomic region. Among the 463 CNVs that overlap with gaps, 140 (>30%) were confirmed by other studies. Of the eight randomly selected CNVs for qPCR validation, four overlapped with gaps and were verified by qPCR, suggesting that these gap-overlapping CNVs are also high-confidence CNVs.The reference genome used in this study, Oar_v3.1, has 21,585 gaps larger than 1 kbhttp://www.biomart.org/) was used to retrieve the gene content in the 1,296 CNVRs . The 81% of our CNVRs overlap with 4,541 genomic genes , RXRA (retinoic X receptor A), KLF11 (Kruppel-like factor 11), PPP1CA , and PDGFA , that were previously reported to overlap with CNVs in fat-tailed sheep9. We found a total of 1,094 CNVRs (84.4%) that overlap with at least one base with QTLs from the sheep Animal QTLdb  and GTPase activity (P = 4.63E-07). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the Notch signaling pathway, the MAPK signaling pathway and the VEGF signaling pathway to be significantly enriched , two well-known genes associated with hair development, belong to the most significantly enriched GO category of homeobox. Moreover, HOXD12 (homeobox protein Hox-D12) and TBX6 (T-box transcription factor 6), as well as HOXB3 (homeobox protein Hox-B3), which is involved in embryonic skeletal system morphogenesis, overlap with the CNVRs potentially specific to Tan sheep.Using the same 600 K ovine BeadChip platform, Zhu ies Fig. . GO analet al.11 and Liu et al.10 used low-density SNP arrays with 50 K probes. Thus, more than 10 times better resolution was achieved by this study than the earlier two SNP array studies, which also resulted in the smaller mean/median length of CNVRs  for CNV detection. As a result, Zhu et al.9 identified half as many CNVRs as the present study, and the median size of the CNVRs they detected was twice as large as that of our study. As expected, when we applied the same filtration process used by Zhu et al.9, a CNVR overlap of nearly 60% was reached, which is much higher compared to that found in the previous four studies . The estimate is still higher, even when compared with a comprehensive CGH array-based CNV study that covered 2.7% of the ovine genome13. This difference could be due to the underestimation of sheep CNVs by Jenkins et al.13, in which a cattle reference genome was used for the probe design and, thus, sheep CNVs in regions that were deleted in or of low homology with the reference genome were likely ignored. The difference could also be due to the overestimation by the CNV calling algorithm, PennCNV, in our study. The current SNP array-based detection of CNVs remains prone to false positives and shows low concordance between multiple calling algorithms26. Although PennCNV software is widely used for Illumina SNP arrays27, particularly for high-density SNP arrays9, a certain proportion of false positives exists in our findings. SNP arrays may also miss CNVs because their SNP chip coverage shows inherent bias against the genomic regions harboring CNVs20. For example, segmental duplications (SDs), one of the catalysts and hotspots for CNV formation, are often affected by low probe density due to the difficulties of array design28. Furthermore, common CNVs may cause SNPs to be rejected when the SNPs deviate from Mendelian inheritance and the Hardy-Weinberg equilibrium20.The 1,296 autosomal CNVRs reported in this study account for approximately 4.7% of the entire ovine genome. This estimate is similar to the range reported in horseset al.9 and Liu et al.10  and large Chinese sheep populations are needed to investigate the high loss/gain ratio in future studies.Of the 1,296 identified CNVRs, more losses than gains were observed. This imbalance was also observed in reports by Zhu 13 and 80% of the randomly selected novel CNVRs were further confirmed by qPCR, indicating the accuracy of our CNV detection using a high-density SNP chip in the sheep genome. Currently, three algorithms for CNV detection based on SNP arrays have been developed, which are available in different programs, including PennCNV30, cnvPartition  and QuantiSNP31. According to a previous comprehensive assessment of multiple CNV calling algorithms for array-based detection, the concordance of these algorithms is low26. The common set of CNVs detected by multiple algorithms may avoid bias to some extent but may also miss many false negatives, whereas the union set may contain a large number of false positives. This makes it difficult to determine the appropriate number of CNVs, as summarized by Winchester et al.20. According to the number of citations in PubMed, PennCNV software is currently the most widely used for Illumina chips 27, particularly for high-density SNP data9. Compared to other algorithms such as CNVPartition and QuantiSNP, PennCNV is more reliable for assessing the number of copies when using Illumina high-density arrays because it incorporates the allelic intensity ratio at each SNP marker and the total signal intensity, the allele frequency of SNPs, the distance between neighboring SNPs, and the GC content to overcome biases32. In this study, we used high-density SNP arrays and stricter filtering criteria (SD of LRR < 0.30 and BAF = 0.01) to reduce the rate of false-positive results, resulting in a qPCR confirmation percentage of 80%. We also found a good correlation between our CNV prediction and qPCR validation . Previous studies have shown that SNPs in the 3′UTR and promoter regions of DLX3 have a significant effect on wool curvature in Chinese Merino sheep38. Therefore, this candidate gene is worthy of validation for its functional relevance to the special curly fleece in Tan sheep. In addition to the unique trait of curly fleece, Tan sheep exhibits the common fat-tail phenotype, as do other sheep indigenous to China, for adaptation to the dry  and cold  climate. Thus, by comparison with the previous CNV study of the two typical Chinese fat-tail breed9, we found that the same set of CNVR-harboring genes involved in lipid metabolism and the same GO category of lipid metabolism were significantly enriched (P = 0.001), indicating that the identified CNVRs are also likely associated with the fat-tail phenotype in Tan sheep.More than 80% of the CNVRs identified in this article span 1,094 QTLs Table  belongined Table . GO analBlood samples were randomly collected from 48 unrelated Tan sheep (6 rams and 42 ewes) from multiple flocks in Ningxia province. Each sheep was carefully confirmed to match the phenotypic characteristics of the Tan sheep breed. Genomic DNA was extracted from blood using the Promega Wizard Genomic DNA Purification Kit  according to the standard protocol provided by the manufacturer. A NanoDrop 2000 was used to measure the purity and concentration of the genomic DNA.Procurement of peripheral blood was performed according to the guidelines for the care and use of experimental animals established by the ethics committees of the Ministry of Agriculture of People’s Republic of China. All of the animal experiments were approved by the Chinese Academy of Agricultural Sciences (CAAS) .Ovine SNP600K BeadChip array, which contains oligo probes for 685,734 SNPs, with the majority (80%) equally spanning the ovine genome, 10% reported in the literature as functional variants, 7% overlapping with the Illumina Ovine SNP50K array, and 3% accessible using a genotyping-by-sequencing protocol39. The raw data were extracted using GenomeStudio (Illumina) and strict quality control was used for SNP filtering to increase the accuracy of the CNV detection. First, we removed individuals in which the call rate was <90%. Second, we discarded SNPs with a >10% missing genotype and a minor allele frequency (MAF) <0.05. Third, we removed SNPs that severely deviated from Hardy-Weinberg equilibrium (multiple test-adjusted P < 10−5) within each population. In addition, identical-by-descent (IBD) was conducted using Plink software. Finally, 495,786 autosomal SNPs were subjected to the subsequent CNV detection and analysis. The X and Y chromosomes were excluded40.According to the manufacturer’s protocols , all genomic DNA samples from 48 sheep were genotyped using the Illumina http://www.openbioinformatics.org/penncnv/), which allows for the detection of CNVs based on Illumina or Affymetrix SNP chip data. Illumina GenomeStudio software can export the signal intensity data of the log R ratio of R (LRR) and B allele frequency (BAF) for each SNP. The population frequency of B allele (PFB) file was calculated based on the average BAF of each marker in the population. The PennCNV algorithm30 was only applied to autosomes (command: -lastchr 26) to identify individual-based CNVs. To increase the confidence of the detected CNVs, quality control was performed by employing standard exclusions of the LRR (standard deviation of LRR) <0.3, a BAF drift <0.01 and a waviness factor <0.05. We classified the status of these CNV into two categories: “loss” (CNV containing a deletion) and “gain” (CNV containing a duplication).CNVs were detected using a hidden Markov Model  that were identified across all of the samples, according to previously reported methodshttp://www.biomart.org/) in the Ensembl database was employed to identify genes located within or partially overlapping with the identified CNVRs. CNVRs that overlapped with the gene’s coding region by at least 1 bp were used to calculate the proportion of CNVR overlapping genes. Functional annotation was performed in DAVID (http://david.abcc.ncifcrf.gov/) for GO terms and KEGG pathway analyses. Because the sheep genome annotation is limited, the ovine Ensembl gene IDs were converted into orthologous human Ensembl gene IDs for the functional enrichment analysis. Furthermore, these CNVRs were mapped to the sheep QTLs from the Animal QTL database .BioMart , we removed all CNVs less than 100 kb according to the technique described by Zhu et al.9.To evaluate the reliability of the CNVRs detected, we compared our results to five existing studies of sheep CNVs detected using various platforms and involving different breedsDGAT2 in the ovine genome were used as reference genes according to our own study and a previous study10. Four samples in which a normal copy number was identified in the target regions were used as reference samples. PCR experiments were conducted using Power SYBR Green PCR Reagent Kit (Applied Biosystems). The qPCR conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Three replications were performed for each sample. Fold changes were determined using a standard 2−ΔΔCT method that compares the ΔCT value of a reference sample with the sample of interest for the ΔΔCT calculation and compares the CT (cycle threshold) values of a reference gene to the gene of interest for the ΔCT calculation. Fold changes were normalized to a diploid number for a better comparison of copy number in all qPCR plots.We performed qPCR analysis on ten random selected genomic regions harboring CNVs identified in this study on an ABI7500  sequence detection system. The primers (Additional file: Table dataset1


After implantation of the chondrocyte-treated scaffold into the subcutaneous tissue of nude mice, the TET presented properties of mature cartilage tissue. To further evaluate the feasibility of repairing whole segment tracheal defects, replacement surgery of rabbits’ native trachea by TET was performed. Following postoperative care, mean survival time in Group I was 14.38 ± 5.42 days, and in Group II was 22.58 ± 16.10 days, with the longest survival time being 10 weeks in Group II. In conclusion, we demonstrate the feasibility of repairing whole segment tracheal defects with 3D printed TET.Long segmental repair of trachea stenosis is an intractable condition in the clinic. The reconstruction of an artificial substitute by tissue engineering is a promising approach to solve this unmet clinical need. 3D printing technology provides an infinite possibility for engineering a trachea. Here, we 3D printed a biodegradable reticular polycaprolactone (PCL) scaffold with similar morphology to the whole segment of rabbits’ native trachea. The 3D-printed scaffold was suspended in culture with chondrocytes for 2 (Group I) or 4 (Group II) weeks, respectively. This  In children and apart from the aforementioned causes listed above, trachea stenosis may also be a congenital abnormality. For several decades, many surgeons have made great effort in seeking novel treatments for this disease. Tracheal resection and re-anastomosis existed as a clinical solution as early as the late nineteenth century, but it was contraindicated for stenotic segments longer than 2 cm in children and 5 cm in adults due to the risk of excess tension2. Recently, slide tracheoplasty has improved outcomes significantly for preciously inoperable patients4. However, performing such a procedure comes at a sacrifice of tracheal length for tracheal inner diameter. In our center alone we have nearly ten-years’ experience with the surgical management of congenital tracheal stenosis; still we find current surgical strategies have major limitations regarding the location and length of trachea stenosis, as well as postoperative problems with tracheomalacia and granulation formation3. One novel solution to overcoming these obstacles is the use of artificial substitutes to replace long-segment narrowed trachea.Trachea stenosis is a rare but life-threatening condition in patients. In adults, it is usually caused by prolonged endotracheal intubation, tracheostomy, trauma, trachea cancer and inflammation6, and synthetic materials are limited in biocompatibility and granulation formation8. In contrast, because of the proper autologous availability, Tissue-Engineered Trachea (TET) seems to be an ideal solution for trachea replacement9. By choosing suitable materials for scaffolds and seeding these scaffolds with homogenous cells, TET can emulate similar biological structures and functions to that of native trachea. Furthermore, with the progress of 3D printing techniques, it is possible to design an individualized tracheal model that is suitable for the host in morphology, as well as properly support force to maintain the shape of the TET11.Many studies have turned to grafting technologies to overcome the unmet clinical needs facing tracheal repair. However, allografts and autografts are limited due to a severe lack of healthy donors and a heightened risk of immune-rejection16. However, these studies only aimed to fix the anterior-end defects of tracheas and further studies are needed in order to demonstrate similar technologies could be employed for whole segment tracheal replacement. A recent study successfully reported construction of whole TET with mechanical properties similar to that of the native trachea by a 3D printing scaffold17. Still, a major caveat in this study was the complexity of the manufacturing of the TET. Additionally, the study lacks any in vivo evidence that the TET could be used in experimental replacement surgery.In recent years, several authors have attempted to make trachea scaffolds through the combination of Tissue-Engineering and 3D-Printing techniquesin vivo, to demonstrate the feasibility and potential problems associated with these TETs.In this study, we 3D-printed a scaffold of the whole segment of rabbit trachea, which had similar morphology to that of the native trachea as well as the proper support force to maintain the lumen. A biodegradable material, PCL, was used to build this scaffold due to its appropriate melting point and malleability for 3D printing. We also presented a convenient method to achieve cartilage tissue regeneration and TET reconstruction, and used these TETs to replace the native rabbit trachea, 18. The inner layer of the scaffold consists of vertical and horizontal PCL cross bars. The PCL cross bars have a diameter of 0.12 mm, which simultaneously acts as a support system to strengthen the scaffold while providing enough space to coat those areas with chondrocytes. The grids of the PCL bars are 0.24 * 0.12 mm. Figure We carefully designed a PCL scaffold that closely resembled the dimensions to that of the native trachea of a 4-months old New Zealand white rabbit Fig. . The lonIn order to ascertain if any structural changes incurred to the chemistry of the PCL during the 3D-printing process of the TET, we carried out Fourier Transform Infrared Spectroscopy (FT-IR) analysis. We analyzed absorption spectra at several wavelengths, which correspond to different carbon compounds found in the PCL material. Analysis revealed absorption spectra patterns were not altered between the three groups of PCL raw material, 3D-printed PCL scaffolds, and sterilized 3D-printed samples Fig. .Additionally, to verify the thermoanalytical properties were also similar between the raw PCL material, 3D-printed PCL scaffolds and the sterilized 3D-printed PCL, we performed Differential Scanning Calorimetry (DSC) assays. We found sharp endothermic peaks at around 60 °C in all three groups, which correlates to the melting points of the PCL materials Fig. . During in vivo engraftment.Next, to get a practical perspective on the mechanical properties of the 3D-printed scaffold we performed radial compressive force-deformation analysis Fig. . Accordiin vitro with seeded chondrocytes. Favorable adhesion of the chondrocytes to the PCL scaffold was observed by microscopy , photo-macrograph analysis and histological analysis . Due to the poor survival rates associated with bronchoscopy examination, along with the technical hurtle of passing the bronchoscope through the scaffold segment, this test became obsolete on the vast majority of animals undergoing TET surgery.Next we sought to compare mean survival rates of animals receiving TET replacement surgery, between the two groups of chondrocyte treated scaffolds. Analyses of the post-operative survival rates are shown below Fig. . Group IA detailed autopsy examination was performed at time of death. According to the results from the autopsy, our TETs provided strong functional support to the native operated tracheas. This was made evident by the lack of observed tracheal collapse or malacia at the time of autopsy. The whole segment of TETs displayed some stenosis where the middle sections were the narrowest Fig. . We obseWe did, however, observe deposits of cartilage-like tissue in histological stainings at time of autopsy Fig. . We specFurthermore, the autopsies revealed other causes of death, which included pneumonia, infection in scaffold segment and anastomotic stenosis Table . Both in19. In this study, we designed and 3D-printed an intact trachea scaffold with similar morphology to that of a native rabbits’ trachea, which presented good cytocompatibility, as well as provided solid functional support in the early stages after replacement operation. This study highlights for the first time, the possibility of repairing whole tracheal defects with TET in an animal model.Recently, the rise of 3D printing technology provides infinite possibilities for tissue engineering15. The latter of these two studies reported neocartilage formation at 8–12 weeks following surgical implantation. However, their scaffolds were less than 1 cm in length and only fit partial tracheal defects less than the size of one semicircle of annulus tracheal. Although these studies identified significant breakthroughs of TET, the length and shape of the scaffolds limited the application of the TET.In contrast to increasing advances in tissue engineering and grafting technologies, still an unmet clinical need persists in regards to repairing partial tracheal defects using 3D printed scaffolds. To date, several studies have tried to utilize 3D-printing scaffolds, with and without cell-coating pre-treatment steps, in order to repair partial tracheal defectset al., the etteam reported an intact tracheal scaffold with similar morphology to the native trachea and appropriate mechanical behavior similar to native trachea17. The reconstructed TET contained a gelatin portion that had been seeded with chondrocytes, allowing the formation of neocartilage tissue following transplantation into the dorsal subcutaneous spaces of nude mice. However, the procedures used here to make such a scaffold are fairly complicated. Furthermore, the most objective criteria of TET replacement surgery are the in vivo evaluations of the TET, which the authors did not include in their study.In order to treat long segments of tracheal stenosis, whole tracheal scaffolds display a more promising artificial substitute. In a current study by Park in vitro and in vivo. This is one of the first papers to utilize a 3D-printed TET for rabbit tracheal transplantation and nicely demonstrates the feasibility of repairing whole segment tracheal defects with such a procedure.Here, we presented a novel design for a 3D-printed whole tracheal scaffold by employing a rather simple method. The TET construct presented good cartilaginous properties both Our chondrocyte treated TET scaffold has several advantages worth highlighting: 1) The ployporous structure is conducive to cell adhesion and proliferation, which makes the method of tissue reconstruction simple; 2) The morphology of the scaffold is very similar to that of native trachea and can meet physiological demand. In addition, with 3D-printing technology, it is easy to change the morphology of the scaffold to achieve individualized treatment. 3) As a biodegradable material, PCL has both appropriate support strength and favorable cytocompatibility. The long-term goal would be to achieve new anastomosis through a PCL-decayed, reconstructed cartilage formation enhanced by host epithelization.One major caveat of our TET design was that our scaffold lacks contractility properties supporting that of a native trachea, and the chondrocyte treated TET needs major modifications in order to tolerate secretion or discharge. Additionally, with 3D-printing technologies, always some design/printing errors may occur, which can render the shape of the scaffold and set it slightly apart to those dimensions of the native trachea.21. This progress, mediated by a wide range of coordinated cellular reactions, has a close relation with infection, inflammation, tissue necrosis and immunological rejection23. In our opinion, the stenosis seen in the scaffold segment and granulation formation after transplantation surgery is related to the lack of any protective epithelial layer along with an accompanying inflammatory reaction.Irregardless of such pros and cons presented with our TET, repairing whole segment tracheal defects by TET is a complex procedure, especially due to diminishing epithelial tissue within the segment, which can easily cause obstructed secretion, damage of respiratory function and/or infection. The primary postoperative causes of death in our study are immediately relevant with the reasons mentioned above. Granulation formation is a common complication of trachea surgeryIn this study, we report the successful formation of cartilage tissue in the dorsal subcutaneous spaces of nude mice, but such a response failed in the rabbits’ airway. The nude mouse had an inhibited immune system, and subcutaneous space is an aseptic condition. In contrast, in the rabbit, the environmental conditions in the airway are not of a sterile condition, bacterial colonization from the respiratory tract may cause local inflammation, which is harmful for the maturation of cartilage tissue.Another common complication of tracheal surgery is tracheomalacia, which was not observed in our experiment, indicating a favorable mechanical support of our TET. The mechanical properties of the scaffold is inextricably linked to the strength and diameter of material. Increasing the thickness of the diameter of the scaffold often equates to an increased strength of the scaffold, both of which prolongs degradation time. Here, due to post-transplantation survival rates not exceeding 10 weeks, it is difficult to ascertain the long-term functional duration of our TET. In particular, more information regarding how degradation will alter the reparative processes needs further evaluation. Additional experiments are also needed to ensure scaffold diameters are functionally optimized.29. Angiogenesis and neovascularization provides nutritional support to the implanted TET and have previously been shown to be key factors influencing ingrowth of epithelium24. Epithelization provides critical functions such as the prevention of bacteria, as well as ciliary beating which helps enable discharge of secretion. In order to induce vascularization and epithelization which could aid in the long-term survival of TET transplants, several approaches exist, including pre-vascularization in vivo25, pretreating the scaffold or cell-scaffold interface with growth factors (such as VEGF)23, and/or using human turbinate mesenchymal stromal cell sheets as an inner surface layer in TET for epithelial regeneration13.Lastly, in order to maintain function and improve long-term survival and grafting rates, vascularization and epithelization are two important aspects when dealing with TET transplants22. However, to perform such studies could enhance a risk of serious infection, requiring more strict conditions for animal housing and postoperative care. At least according to our results, the 4-weeks cultured TET group presented a longer survival time than the 2-weeks cultured TET. Prolonged culture times or culturing scaffolds with more cells may be an additionally helpful way to prolong the maturation of the transplanted TET.Another way to reduce inflammation and granulation formulation following transplantation of TET is through inhibiting the immune system. This is easily attainable by using dexamethasone or IL-1 receptor antagonistsAt present, the concept of utilizing a functionally sustained TET for intrathoracic tracheal replacement surgery shows much promise. With the help of 3D-printing, we establish a simple solution for the adhesion of cells on a 3D-printed scaffold and proved the feasibility of cartilage tissue formation. Although there are some experimental setbacks in regards to the animal replacement experiments, we envision the ideas and methods of the scaffold design and tissue reconstruction presented in this study can be a meaningful early-stage determinant, and help to inspire more practicable approaches to repair long-segment trachea stenosis. In order to move forward with clinical application, information within the parameters of morphology, function and degree of comfort are severely required.in vitro and in vivo. Such technology could one day be considered for tracheal replacement therapies, as we displayed evidence that the TETs can endure long-term implantations, up to 10 weeks survival time in vivo. In conclusion we demonstrate the feasibility of repairing whole segment tracheal defects with the use of 3D printed TET.With the combination of 3D-printing and tissue engineering, we 3D-printed an intact tracheal scaffold with biodegradable material and successfully reconstructed TET with a chondrocyte suspension. The TET presented good cartilaginous properties both A Total of twenty-one New Zealand white rabbits (4-months old) were purchased from Shanghai Chedun Experimental Animal Raising Farm . Four male nude mice (7-weeks old) were purchased from Shanghai Slaccas Experimental Animal Ltd. . All experimental protocols were complied with the relevant guidelines and regulations and approved by the Animal Care and Experiment Committee of Shanghai Jiao Tong University School of Medicine.The 3D printing PCL scaffold was designed exactly as native trachea of a 4-months old New Zealand white rabbit. A widely used biodegradable material, PCL, was used to print the scaffold. All scaffolds were 3D printed by Beijing Advanced Medical Technologies Ltd. Inc. . Rapid Stent Fabrication System was used as a preparation system of the scaffold.To further detect whether there are some changes of chemical/material properties, PCL raw material, 3D printed PCL sample and irradiation sterilized 3D printed PCL sample were tested by Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC).Three scaffolds were compressed on a computer stretch and compression tester . The presser foot controlled compression of the scaffold with the speed of 2 mm/min. The radial compressive force and the percentage of deformation was recorded for the Radial compressive force-deformation curve. All compressing tests were conducted under standard environment conditions .30. Briefly, primary cartilage tissue with a size of about 2.0 * 3.0 cm was obtained from auricle of rabbit and separated under sterile conditions. The cartilage slice was washed with phosphate buffered solution  and digested with 0.25% trypsin plus 0.02% Ethylene Diamine Tetraacetie Acid (EDTA)  at 37 °C for 30 minutes, minced into approximately 1 mm3 pieces, and then digested with 0.1% collagenase NB4  in serum-free Dulbecco’s Modified Eagle’s Medium  at 37 °C for about 12 hours. Then, the cells were harvested, cultured, and expanded according to reported methods30. The chondrocytes in passage 2 were harvested for the construction of tubular cartilage  with 7.5 ml DMEM containing 10% FBS, 50 μg/ml of vitamin C , 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 0.1 mM nonessential amino acids . After 3 days culture, all clusters were put on a shaking table at 100 rpm with 5% CO2 and 37 °C for 2 or 4 weeks.The harvested chondrocytes were resuspended in DMEM containing 10% fetal bovine serum  to a final concentration of 5.0 *10To detect the cytocompatibility of the scaffold, the blank scaffold, 2-weeks cultured TET and 4-weeks cultured TET was observed by SEM in Shanghai Jiao Tong University, Bio-X Institutes. All samples were fixed in 4% paraformaldehyde for 24 h and dehydrated with graded concentrations  of ethanol. Subsequently, the samples were coated with gold/palladium and observed by SEM Quanta 250 .in vitro for 2 weeks or Group II, TET cultured in vitro for 4 weeks. Each sample was implanted into the dorsal subcutaneous spaces of each nude mouse. Animal care and housing was provided by Experimental Animals Department of Shanghai Children’s Medical Center. After 6 weeks, all implants were retrieved from each mouse for macrograph and histological analysis.Four nude mice (7-weeks old) were distributed into two experimental groups: Group I, TET cultured in vitro for 2 weeks (n = 8), group II, TET cultured in vitro for 4 weeks (n = 12). All rabbits were anesthetized with intravenous injection of pentobarbital sodium (30 mg/kg). Under sterile conditions, a midline incision was made in the anterior neck. Surrounding tissue was separated carefully to expose the trachea. An approximate 1.6 cm segment of trachea was cut off at 1.0 cm below the cricoid cartilage. A tissue-engineering trachea was placed into the defect trachea and performed end-to-end continuous anastomosis with 6–0 absorbable PDS II sutures  in both proximal and distal native trachea. Then incision was closed in layers and the animal was allowed to breathe spontaneously and recover from anesthesia.20 rabbits were divided randomly into two groups: Group I, TET cultured Postoperatively, all rabbits were given intramuscular injection of cefuroxime for 5 days to prevent infection. Animal care and monitoring was provided by Experimental Animals Department of Shanghai Children’s Medical Center. Once the animals died, the survival period was recorded, autopsy was performed to analyze the causes of death. The implanted trachea including part of native trachea was dissected from surrounding tissues and taken out. To observe the stenosis of the inner lumen and granulation formation, the implanted trachea was cut off ring by ring for gross view and histology analysis.30. The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned into 8 μm sections. The sections were stained with hematoxylin and eosin (H&E) to assess tissue structure and Toluidine blue and Safranin O to locate the Glycosi Aminoglyca Good deposits.All histology staining were performed as previously describedAll the quantitative data were recorded as mean ± standard deviation. SPSS 23.0 statistical software  was used for statistical analysis. Survival rates were estimated with the Kanlan-Meier method and Breslow test. A p-value less than 0.05 were considered as statistically significant.


The Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study 2015 provides an up-to-date analysis of the burden of lower respiratory tract infections (LRIs) in 195 countries. This study assesses cases, deaths, and aetiologies spanning the past 25 years and shows how the burden of LRI has changed in people of all ages.We estimated LRI mortality by age, sex, geography, and year using a modelling platform shared across most causes of death in the GBD 2015 study called the Cause of Death Ensemble model. We modelled LRI morbidity, including incidence and prevalence, using a meta-regression platform called DisMod-MR. We estimated aetiologies for LRI using two different counterfactual approaches, the first for viral pathogens, which incorporates the aetiology-specific risk of LRI and the prevalence of the aetiology in LRI episodes, and the second for bacterial pathogens, which uses a vaccine-probe approach. We used the Socio-demographic Index, which is a summary indicator derived from measures of income per capita, educational attainment, and fertility, to assess trends in LRI-related mortality. The two leading risk factors for LRI disability-adjusted life-years (DALYs), childhood undernutrition and air pollution, were used in a decomposition analysis to establish the relative contribution of changes in LRI DALYs.In 2015, we estimated that LRIs caused 2·74 million deaths  and 103·0 million DALYs (95% UI 96·1 million to 109·1 million). LRIs have a disproportionate effect on children younger than 5 years, responsible for 704 000 deaths (95% UI 651 000–763 000) and 60.6 million DALYs (95ÙI 56·0–65·6). Between 2005 and 2015, the number of deaths due to LRI decreased by 36·9% (95% UI 31·6 to 42·0) in children younger than 5 years, and by 3·2% (95% UI −0·4 to 6·9) in all ages. Pneumococcal pneumonia caused 55·4% of LRI deaths in all ages, totalling 1 517 388 deaths (95% UI 857 940–2 183 791). Between 2005 and 2015, improvements in air pollution exposure were responsible for a 4·3% reduction in LRI DALYs and improvements in childhood undernutrition were responsible for an 8·9% reduction.LRIs are the leading infectious cause of death and the fifth-leading cause of death overall; they are the second-leading cause of DALYs. At the global level, the burden of LRIs has decreased dramatically in the last 10 years in children younger than 5 years, although the burden in people older than 70 years has increased in many regions. LRI remains a largely preventable disease and cause of death, and continued efforts to decrease indoor and ambient air pollution, improve childhood nutrition, and scale up the use of the pneumococcal conjugate vaccine in children and adults will be essential in reducing the global burden of LRI.Bill & Melinda Gates Foundation. Lower respiratory tract infections (LRIs) are a substantial public health problem and a leading cause of illness and death in people of all ages. Previous estimates found that in 2013, LRIs caused more than 2·6 million deaths worldwide, making them the fifth leading cause of death overall and the leading infectious cause of death in children younger than 5 years.Estimates of the burden of LRIs and their aetiologies are being produced annually as part of the Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study, which provides a unique source for tracking trends in LRI-related morbidity and mortality. Results from the GBD study that quantify the burden of LRI will help to measure progress towards the Sustainable Development Goals, including Goal 3, which is to ensure healthy lives and wellbeing for people of all ages.Evidence before this studyLower respiratory tract infections are a leading cause of morbidity and mortality, particularly in children younger than 5 years, and the global burden has been estimated by several groups, including the Global Burden of Disease study (GBD). We build on previous GBD studies with updated data and methods. Updated cause-of-death data came from additional years of vital registration systems  and by searching PubMed and Google Scholar for “verbal autopsy” on March 3, 2015. Updated non-fatal and aetiology data came from a PubMed search on June 1, 2015, for “lower respiratory infections”, “bronchiolitis”, and “pneumonia”. Articles published between Jan 1, 2012, and Dec 31, 2015, were selected without language restrictions.Added value of this studyThis manuscript provides a comprehensive assessment of LRI burden based on GBD 2015, including newer and more robust evidence on the mortality, morbidity, and risk factors associated with LRIs, including four aetiologies, and is the first cause-specific description of LRI in a GBD study. In addition to descriptions of trends in morbidity and mortality, we use the Socio-demographic Index to relate changes in LRI burden to demographic transitions and assess the effect of changing population characteristics and risk factor exposure to decompose the trends in LRI burden.Implications of all the available evidenceWe show a decreasing burden of LRI in children younger than 5 years, but an increase in the burden in adults. Furthermore, we show the change in risk factor exposure globally, providing health professionals with valuable information needed to design and implement effective programmes and policies to reduce the burden of LRI. We also identify high-burden LRI regions that need more attention. Expanded use of the pneumococcal conjugate vaccine, interventions to improve under-5 nutrition, and a focus on appropriate case management could reduce the burden of LRI. Comprehensive and reliable data on LRI morbidity and mortality globally are still needed.Haemophilus influenzae type B [Hib], Streptococcus pneumoniae [pneumococcal pneumonia], influenza, and respiratory syncytial virus [RSV]), covering deaths, episodes, disability-adjusted life-years (DALYs), risk factors contributing to the burden of LRIs, and the relationship between LRIs and social development for 195 countries from 1990 to 2015 for both sexes and by age.Here, we present results from the GBD study 2015, describing the burden of LRIs and four aetiologies  showing the 2·5 and 97·5 percentiles of the distribution.The GBD Cause of Death database contains all available data from vital registration systems, surveillance systems, and verbal autopsies  framework.The sum of all cause-specific mortality models must be equal to the all-cause mortality estimate.LRIs were defined as clinician-confirmed or radiologically confirmed pneumonia or bronchiolitis and were divided into moderate and severe or very severe episodes on the basis of WHO case definitions for pneumonia.LRI morbidity  was modelled using DisMod-MR version 2.1 (DisMod), a Bayesian, hierarchical, mixed-effects meta-regression platform.DALYs are the sum of YLLs and YLDs and represent the cumulative burden of disease due to LRI.We estimated LRI aetiologies separately from overall LRI mortality and morbidity using two distinct counterfactual modelling strategies to calculate population attributable fractions (PAFs) for influenza, RSV, Hib, and pneumococcal pneumonia. The PAF is the proportional reduction in LRI morbidity or mortality that would be observed if the exposure to the pathogen were zero. We did not attribute aetiologies to neonatal pneumonia cases or deaths because of an absence of reliable data in this age group, and we did not consider Hib in age groups older than 5 years for the same reason.We used a vaccine probe design to estimate the PAF for pneumococcal pneumonia and Hib by first calculating the ratio of vaccine effectiveness against non-specific pneumonia to pathogen-specific pneumonia at the study level.For Hib, we assumed that the vaccine efficacy against invasive Hib disease is the same as against Hib pneumonia. However, we did not make the same assumption for pneumococcal pneumonia because a study of pneumococcal conjugate vaccine (PCV) found that the vaccine efficacy against invasive pneumococcal disease might be significantly higher than against pneumococcal pneumonia.Influenza and RSV were estimated by calculating an attributable fraction that relates the odds ratio (OR) of LRI given pathogen detectionWe conducted a systematic literature review of the proportion of LRI cases that test positive for influenza and RSV and used the meta-regression tool DisMod to estimate the proportion of people with LRI who are positive for influenza and RSV, separately, by location, year, age, and sex.Different PAFs were measured for non-fatal and fatal LRI episodes. Fatal PAFs were adjusted using a scalar from the DisMod proportion models that represents the relative frequency of detection in inpatient versus non-inpatient sample populations. In the absence of aetiological data from fatal cases of LRI after death, we assumed that episodes of LRI requiring hospital admission were a reasonable proxy of severe and fatal episodes. Mortality is lower in patients with viral pneumonia than in those with pneumonia with bacterial causes. Therefore, we adjusted the fatal PAF estimates by establishing the ratio of case fatality in viral to bacterial causes of pneumonia from hospital data coded specifically to these causes, representing the relative fatality in people who were treated .On the basis of methods used to construct the Human Development Index, GBD 2015 used the Socio-demographic Index (SDI), a summary measure of a country's development based on lag-distributed income per capita, average educational attainment, and total fertility rate.Methods for risk factor attribution to LRI are described in detail elsewhere.27The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.At the global level, under-5 LRI mortality occurred in 104·8 children per 100 000 (95% UI 97·0–113·6) and varied by region and country . AccordiWe calculated that, in 2015, LRIs caused 103·0 million DALYs (95% UI 96·1 million to 109·1 million) in all ages and 60·6 million DALYs (95% UI 56·0 million to 65·6 million) in children younger than 5 years , the USA (235·2 per 100 000 [224·0–247·0] vs 2·7 per 100 000 [2·4–3·0]), and Japan (613·7 per 100 000 [588·4–639·2] vs 2·8 per 100 000 [2·4–3·2]).Although nearly 60% of LRI DALYs were from children younger than 5 years, our findings suggest that LRI mortality was substantial across all ages, and in elderly people in particular. In adults aged 70 years or older, 1·27 million deaths (95% UI 1·15–1·34 million) were estimated to be caused by LRIs in 2015. In some countries, we estimated a much larger number of deaths due to LRIs in older adults (≥70 years) than in children younger than 5 years—eg, in China , and influenza was responsible for 10 151 (5 731–16 790) in children younger than 5 years, together accounting for 6·6% of LRI deaths in this age group . The burWe estimated that the leading risk factors for LRI DALYs in 2015 were childhood wasting (responsible for 44·6% [95% UI 31·7–52·8] of DALYs worldwide), household air pollution , and ambient particulate matter . Suboptimal breastfeeding was the third-leading risk factor for under-5 DALYs globally and the leading risk factor for under-5 DALYs in high SDI locations. Other risk factors for LRI, such as smoking, alcohol use, and zinc deficiency, were responsible for less than 10% of LRI DALYs globally.Health Metrics and Evaluation visualisations.A decomposition of the change in attributable DALYs between 2005 and 2015 by country is shown in The GBD 2015 study estimated that LRIs were the fifth-leading cause of death (of 249 causes) and the leading infectious cause, responsible for 2·74 million deaths (95% UI 2·50 million to 2·86 million). LRIs were the third-leading cause of under-5 mortality behind preterm birth and neonatal encephalopathy, accounting for 12·1% of deaths in this age group. Our analysis suggests that the number of deaths due to LRI in children younger than 5 years decreased by 37% between 2005 and 2015. We found that, although all-age LRI mortality rate decreased, growing and ageing populations have contributed to no significant decline in total LRI deaths between 2005 and 2015. Despite dramatic improvement in the under-5 LRI mortality rate, LRI remains a preventable cause of death in young children and elderly adults, particularly in south Asia and sub-Saharan Africa, and was the second-leading cause of DALYs in 2015. The findings call for renewed efforts to control and prevent LRIs across all age groups.Some solutions to prevent LRI deaths do not require major advances in technology. Measures to protect, prevent, and treat LRIs are highlighted in the Global Action Plan for Pneumonia and Diarrhoea.Although such interventions were not estimated in GBD 2015, improved access to health care and emphasis on appropriate treatment have probably played a crucial role in reducing LRI mortality, with proper treatment reducing mortality by 20–42%.In 2015, approximately 65% of children younger than 5 years received the Hib vaccine and 40% received the PCV.The expanded use of PCV might have several indirect effects on LRI burden. PCV might prevent influenza and RSV mortality, because up to half of severe viral infections are complicated by pneumococcal pneumonia.Our results suggest that LRIs were the second-leading cause of DALYs globally in 2015 after ischaemic heart disease.Household solid fuel use as a risk factor for LRI has decreased since 2005, particularly in Latin America and southeast Asia, which are undergoing rapid urbanisation and economic development. Economic development that shifts energy requirements away from household burning of biomass might reduce exposure to indoor air pollution at the expense of outdoor and ambient particulate matter from large-scale energy production facilities like coal-burning power plants.Our estimates of LRI mortality, morbidity, and aetiology attribution are limited by data availability and especially the sparsity of data in sub-Saharan Africa, the region with the greatest LRI burden and need for high-quality data . Only exStaphylococcus aureus, or intracellular pathogens like Mycoplasma pneumoniae, might be important aetiologies for LRIs and are not included in GBD 2015.Even with the application and expanded use of PCR diagnostic techniques, data on the aetiology of pneumonia remain sparse, particularly in areas with high disease burden. This scarcity of data is largely due to the difficulty of obtaining appropriate samples for testing, particularly in children, the relatively high cost of PCR, and challenges in culturing and diagnosing many pathogens that cause respiratory infections.The attributable fraction strategy for Hib and pneumococcal pneumonia assumes that the vaccine efficacy against invasive disease is the same as for pneumonia. A study by Bonten and colleaguesOnly four randomised controlled trials on Hib vaccine efficacy have been done in children younger than 5 years. Despite a plausible disease burden in older children and adults, we decided to apply the attributable fraction of LRI episodes and deaths due to Hib pneumonia to the under-5 age group only. The lower bound of the Hib PAF estimates is below zero  at the global level, reflecting in part the scarcity of reliable data on Hib vaccine efficacy.The attribution of the viral pathogens to LRI mortality was based on the relative case fatality of bacterial to viral aetiologies, and cases of LRI admitted to hospital might not be representative of cases not admitted to hospital. Efforts to improve surveillance, such as the African Network for Influenza Surveillance and Epidemiology,The GBD 2015 estimates of LRI mortality and burden are generally similar to the GBD 2013 estimates.Our estimates of pneumonia mortality in children younger than 5 years differ from those produced by the WHO Department of Evidence, Information and Research and the Maternal and Child Epidemiology Estimation (MCEE) group. The GBD 2015 estimates for under-5 mortality due to LRI in 2015  were much lower than those from the MCEE (920 000 deaths).Despite substantial reductions in under-5 LRI mortality in many countries, the burden remains high, particularly in areas of low sociodemographic development, and has increased in some populations, particularly elderly people. Estimates of the global burden of LRI will be improved by more high-quality data on mortality, morbidity, and aetiologies, especially in sub-Saharan Africa where the burden is highest and data are most scarce. The creation and expansion of civil registration systems in Africa and south Asia are gaining momentum, and such data will not only improve global comparative mortality assessments such as the GBD study, but also increase the evidence for guiding decision about local policy.mokdaa@uw.eduCorrespondence to: Dr Ali H Mokdad, Institute for Health Metrics and Evaluation, University of Washington, Seattle, WA 98121, USA


NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the  Neurofibromatosis 1  is an autosomal dominant disorder with a complete penetrance and highly variable expressivity. It is one of the most common genetic disorders with an incidence of 1 case in 2000 births [NF1 gene, which is a tumor suppressor gene located at chromosome 17q11.2. It spans 282 kb and comprises 60 exons , being one of the largest human genes [NF1 gene presents one of the highest frequencies of spontaneous mutations in the human genome. Therefore, half of individuals with NF1 present no family history of the disorder [NF1 is caused by a spectrum of mutations that affect the disorder .NF1 mutations dispersed throughout the gene have been reported and include missense/nonsense, splicing, small insertions or deletions (indels), and also gross chromosomal alterations. Although no true hotspots have been identified in the NF1 gene, recurrent mutations are not uncommon in individuals with NF1 [To date, according to the online Human Gene Mutation Database (data accessed on November 2016), more than 2600 different with NF1 .NF1 gene mutations represent a considerable research and diagnostic challenge because of its large size, existence of a number of highly homologous pseudogenes located throughout the human genome , absence of mutational hotspots as well as the diversity of mutation types, including deep intronic splicing mutations [NF1 gene mutation detection rate in NF1 patients who meet the clinical diagnostic criteria is reported to be 50%–95% , microsatellite marker analysis, and fluorescence in situ hybridization (FISH)).Identification and characterization of utations ,7. The NNF1 mutation detected in the genetic tests, are related to mutations located in NF1 gene regions that are not commonly screened ) [Therefore, current NF1 molecular diagnostic testing based on Sanger sequencing is labor-intensive, time-consuming , and expensive . Moreove (UTRs)) . It is a (UTRs)) , which aNF1 gene [NF1 regions.In the last few years, DNA sequencing has been passing through a revolution with the use of next-generation sequencing (NGS). It permits a fast and simple sequencing, making possible the analysis of large genomes. With NGS, rather than just sequence the coding exons and conserved splice sites, it is possible to screen the entire sequence of a target large gene from a gDNA sample, including the intronic regions. Therefore, NGS seems to be a very useful tool to sequence the NF1 gene . In thisThis study was approved by the ethical committee of Antônio Pedro University Hospital of Universidade Federal Fluminense, Niterói, Brazil (#121/11).Some clinical features of the participants of this study are shown in ® fluorometer (Thermo Fisher Scientific). Integrity of DNA was evaluated by agarose-gel electrophoresis.Extraction of DNA from leukocytes and purification were performed with QIAamp DNA Blood Mini kit , according to the manufacturer’s instructions. Purity of DNA was assessed by spectrophotometry using NanoDrop 2000 . DNA yields were determined using a double stranded DNA (dsDNA) broad range (BR) kit on a QubitNF1 gene. The designed nonoverlapping probe set covered 90.5% of the targeted region (chr17: 29421944-29704695).Capture probe set was designed and synthesized by Thermo Fisher Scientific for the Ion TargetSeq Custom Enrichment kit (Thermo Fisher Scientific) to target all the coding exons, UTRs, as well as the intronic regions of the ® sonicator , using two cycles of 15 min. Then, DNA fragments were end-repaired, nick repaired, and the barcodes and adaptors were ligated to fragmented DNA. After, we size-selected and amplified the library using the IonXpress Plus gDNA Fragment Library kit, Ion Xpress Barcode Adapters and E-Gel Size Select 2% agarose gels (Thermo Fisher Scientific), according to the manufacturer’s instructions. The amplified library was then hybridized to the NF1 capture probe set for two days (two hybridizations). Libraries were quantified in Qubit® fluorometer (Thermo Fisher Scientific) with dsDNA high sensitivity (HS) kit. The quantity and quality of the library were also verified with Agilent Bioanalyzer DNA High Sensitivity kit . Libraries were then pooled in equimolar concentrations and diluted to 26 pM, prior to clonal amplification onto Ion Sphere Particles using the Ion One Touch 2 System and the Ion OneTouch 200 Template kit v2 (Thermo Fisher Scientific), following the manufacturer’s instructions. Coated spheres were enriched on the Ion Torrent ES (Thermo Fisher Scientific) before loading onto Ion 316 or 318 sequencing chips (Thermo Fisher Scientific). NGS was performed on Ion Torrent Personal Genome Machine—Ion Torrent PGM™ (Thermo Fisher Scientific).Libraries were constructed from 2–3 µg of DNA. DNA was sheared into 200 bp fragments on a BioruptorPrimary analyses of NGS data were performed using Torrent Suite software on the Ion Torrent Browser (Thermo Fisher Scientific): base calling, quality score assignment, adapter trimming, and demultiplexing. Reads were generated using Ion Torrent platform pipeline software, which includes standard control of the read quality. Using the same software, reads were aligned against human whole genome (hg19). Only high-quality reads (Q ≥ 20) were aligned to the reference sequence using TMAP (Torrent Mapping Alignment Program).® Variant Analysis™ software (Qiagen) and SNPnexus web server were used. Variants were also viewed manually on Integrative Genomics Viewer  and on Alamut® Visual  to eliminate strand bias and reduce false positive calls.Germline variants in the targeted regions were detected using the variant caller plug-in of the Ion Torrent Suite software. Variant caller was run with default germline low stringency settings. For each sample, all variants were exported through a vcf  file. Variants were filtered following the criteria of a minimum coverage of 30×, and a sample variant frequency between 30%–70%. For variant interpretation, IngenuityNF1 gene (neurofibromin), PolyPhen2 and SIFT were applied through Ingenuity® Variant Analysis™ software and SNPnexus web server. In silico splicing predictions were investigated with Alamut® Visual, which uses the following prediction methods: SpliceSiteFinder-like; MaxEntScan; NNSPLICE; GeneSplicer; Human Splicing Finder; Known Constitutive Signals; ESEFinder; RESCUE-ESE. Because Alamut® Visual integrates only Human Splicing Finder’s prediction capabilities on donor and acceptor splice sites, and does not investigate the mechanisms of the splicing abnormalities caused by mutations at the nonconsensus splice sites, we also used the online Human Splicing Finder (http://www.umd.be/HSF/).To predict the potential effect of the mutation on the structure and function of the protein codified by the All variants were confirmed by Sanger sequencing in an AB3130 Genetic Analyzer . Primers were designed using NCBI’s Primer BLAST tool  and the TM runs (12 samples) was 96.9 Mbp, constituting 83.3% of total output. Overall average depth was 177, ranging from 52 to 470. The alignment parameters of the reads for each sample and for each run are shown in NF1 exons and introns is shown in NF1 exons were highly covered, with an average coverage depth ranging from 68.5× to 280.4×. The average coverage depth of the intronic regions ranged from 46.1× to 228.4×. The distribution of reads into different chromosomes is shown in NF1 gene.The average number of bases with Q ≥ 20 across the three Ion Torrent PGMTo detect the pathogenic variants, we blindly inspected all mapped sequence reads from NGS from the 12 samples (11 unrelated patients). Following variant filtering, a total of 3554 variants were identified . We identified eight recurrent and two novel mutations. A summary of the mutations found in this study is shown in NF1 pathogenic variants identified in this study. Splicing alterations  accounted for 50% (5 out of 10) of the mutations. All were recurrent, with the exception of the mutation c.288 + 5G > A; the splicing effect was predicted by Alamut® Visual. For the mutation c.6483C > G, Alamut® Visual predicted that the reading frame was interrupted by a premature stop codon. As demonstrated by Ars et al. [Point mutations accounted for 100% of the 10 ® Visual, creates a frame shift starting at codon A2623. The new reading frame ends in a STOP codon 34 nucleotides downstream (p.A2623Qfs*35). Missense mutations  accounted for 20% (2 out of 10) of the pathogenic variants and were all recurrent.Truncating mutations  corresponded to 30% (3 out of 10) of the pathogenic variants. The mutation c.185delT was already described by Forzan et al. . This isNF1 gene, including the exonic and intronic regions by hybridization capture-based NGS. Although some previous studies had evaluated the use of NGS technology to investigate mutations in the NF1 gene [We developed an approach to analyze the entire NF1 gene ,19,20,21NF1 region for sequencing, was based on its ability to capture a large target region in a single experiment, more conveniently, with less hands-on time [NF1 gene. Most (99.3%) of the bases of the NF1 gene not covered by the designed probe set were located outside the exonic regions. Therefore, there was a small percentage of the exonic regions that was not covered by the capture probe set.In our study, the choice of using a hybridization-based targeted selection to prepare the gDNA library, instead of using PCR-based enrichment to extract the -on time . We coulThe average of the percentage of the target covered at least once and ≥30× was 97.7% and 96.0%, respectively, showing an excellent capture performance. Also, we were able to reach reads in the 150 bp range with total sequence throughput that guaranteed high coverage depth, which is important to achieve high sensitivity, preventing false-positive variant calls . We idenNF1 gene had a high depth of coverage with exception of exon 1. As it occurs in other genes, the first exon of the NF1 gene presents higher GC content (71%), compared to the other exons (42%) [NF1 gene had been previously reported in other studies with NGS, using both PCR-based enrichment and hybridization-based targeted selection of the NF1 gene for library preparation [NF1 gene is also a challenge in cDNA-based protocols with Sanger methodology [All exons and introns of the  content 1%, compans (42%) ,26, and  content 1%, compaparation . Regardihodology .NF1 gene not covered by the designed probe set or in regions with <30× of coverage depth. As suggested in the literature, in cases of patients with NF1 with no NF1 gene mutation detected, it is possible that the disease is caused by increased methylation of the NF1 promoter region, which would downregulate gene expression [NF1 gene, which are not easily detectable by our approach. Literature reports that ≈5% of NF1 mutations are microdeletions, which have been detected by FISH, MLPA analysis, and/or array comparative genomic hybridization (array-CGH) [NF1 gene are small copy-number changes involving one to multiple exons detectable by long-range reverse transcription polymerase chain reaction (RT-PCR) and/or MLPA [NF1 gene.In the only case in which no mutation was found using NGS, we sequenced the coding region of exon 1 by Sanger method. Nevertheless, no pathogenic variation was found. It is possible that the disease-causing mutation was located outside exon 1, in regions of the pression . There iray-CGH) . Moreove/or MLPA . Althoug/or MLPA ,30. We bNF1 gene is the high number of nonfunctional pseudogenes throughout the human genome. Because pseudogenes are not subject to selective pressure, their mutation rate is much higher than that of the functional genes from which they originate, and therefore their unintended sequencing can lead to false positive variant calls [NF1 sequence, reads were aligned against the whole human genome and only high-quality reads (Q ≥ 20) were aligned to the reference sequence. Sanger sequencing confirmed all the mutations identified by NGS, showing that there were no false positive variants from pseudogenes reads.Another challenge of sequencing the nt calls . Target nt calls . In our Naturally, with our approach, the number of total variants that were identified was high (average of 238 variants/patient) compared to other studies that have focused on the exonic regions (average of 30 variants/patient) [TM data. By manually checking all possible pathogenic variants calls with the IGV software, we were able to identify some as homopolymer artifacts and thus they did not lead to false positive results.Homopolymer errors are the dominant error type in Ion Torrent PGMNF1 gene cause aberrant splicing and over 57% of them reside outside the canonically conserved GT-AG dinucleotides at the splice donor and acceptor sites [NF1 gene belong to the class of deep intronic mutations (located >500 bp away from exon boundaries), representing 1%–3% of all NF1 mutations [Most (80%) of the mutations identified in this study were recurrent and two (20%) were new. Splicing defects accounted for 40% of the mutations in our sample series. In the literature, approximately 29% of all reported mutations in the or sites . Moreoveutations ,32,33,34utations .NF1 gene, some authors propose the use of mRNA/cDNA for mutation detection, which also identifies deep intronic splicing mutations [Based on the high prevalence of splicing mutations in the utations . Over thutations . Neverthutations ,10,24. FNF1 gene based on gDNA only sequences the exons and conserved splice sites, which span ≈5 kb, and therefore does not identify deep intronic mutations. Of course, it would be impracticable in clinical settings to use Sanger methodology to sequence the whole NF1 gene, including intronic and exonic regions, which spans ≈282 kb. One great advantage of the NGS technology associated with the in silico prediction tools is the capacity to analyze the entire intronic sequence of a target gene, making possible the identification of deep intronic mutations from a gDNA sample. Nowadays, there are a number of in silico tools that have been developed to assess the effect of DNA sequence variations on splicing, including deep intronic mutations. The online Human Splicing Finder correctly predicted the activation of an exonic cryptic acceptor site in one of our samples (c.1260 + 1604A > G). This deep intronic mutation is recurrent and was reported in three previous studies [The use of gDNA is much easier in clinical settings in comparison to cDNA. Nevertheless, the approach that has been used in the literature to analyze the  studies ,24,27. I® Visual, was classified as a frameshift mutation with a truncating effect.Currently, on a routine basis, there is a need to confirm the in silico-predicted splicing alterations . Follow-NF1 mutation provides a presymptomatic/prenatal test in the offspring of sporadic patients [NF1 gene, therefore facilitating the study of the effect of specific mutations on structure and function of the gene product, and leading to a better understanding of the correlation between genotype and phenotype.Developing a 100% sensitive genetic test for NF1 is necessary to help to establish the diagnosis of patients who do not fulfill the NIH clinical diagnostic criteria , which ipatients . In the NF1 gene not covered by the designed probe set. Nevertheless, our results, together with the results of others that used NGS to sequence the NF1 gene [NF1 mutation screening. In this study, we show the results of a simple and fast approach, using hybridization capture-based NGS with gDNA as starting material, to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations. More studies with young patients with sporadic disease and patients with atypical features would be important to demonstrate the clinical utility of our approach.Limitations of this study include the small sample size and also the existence of small regions of the NF1 gene ,18,19,20


The outcome was in general comparable with the results obtained using two reference methods, i.e., visual analysis of liver morphology by fluorescence microscopy and size analysis of fluorescent 2D liver images. In addition, our study shows that administering compounds into the yolk is relevant in the framework of hepatotoxicity testing. Taken together, cell imaging counting provides a novel and rapid tool for screening hepatotoxicants in early stages of drug development. This method is also suitable for testing of other organ-related toxicities subject to the organs and tissues expressing fluorescent proteins in transgenic zebrafish lines.Drug-induced liver injury (DILI) is the most common reason for failures during the drug development process and for safety-related withdrawal of drugs from the pharmaceutical market. Therefore, having tools and techniques that can detect hepatotoxic properties in drug candidates at an early discovery stage is highly desirable. In this study, cell imaging counting was used to measure in a fast, straightforward, and unbiased way the effect of paracetamol and tetracycline, (compounds known to cause hepatotoxicity in humans) on the amount of DsRed-labeled hepatocytes recovered by protease digestion from Tg( Drug-induced liver injury (DILI) is the most common cause of liver injury affecting approximately 14–19 cases per 100,000 patient-years in Western patients. Among the patients diagnosed with DILI in a French study, 6% died and 12% required hospitalization . TherefoDanio rerio) are a promising small vertebrate animal model for use in biomedical research and represent a powerful alternative model to implement the 3R principle of humane animal research  [Zebrafish (inement) ,5,6. In inement)  and dispinement) . In addiinement) , so hepalfabp10a) in hepatocytes, and liver morphology using a Tg(fabp10a:DsRed) zebrafish transgenic line that specifically expresses fluorescent DsRed in hepatocytes [Current hepatotoxicity testing using zebrafish includes endpoints such as liver degeneration and yolk retention , expressatocytes . UnfortuAs hepatocytes account for 70%–75% of hepatic cells and carry out most of the liver functions , it is afabp10a:DsRed) transgenic zebrafish in a fast and straightforward way. Two compounds known to cause hepatotoxicity in humans, i.e., paracetamol and tetracycline [In this study cell imaging counting (CIC) was used to quantify the amount of DsRed-labeled hepatocytes recovered from Tg or dose , i.e., the highest concentration/dose of compound causing insignificant larval death  caused larval death in several of the conditions tested , whereasfabp10a:DsRed) zebrafish larvae with the hepatotoxicants at their MC/MD (and dilutions) at 3 dpf and assessed their hepatotoxic effects after three days of treatment (immersion) or three days after treatment (injection) by visual analysis of liver morphology using fluorescence microscopy. We observed a range of abnormal liver phenotypes including reduced and enlarged livers  expressing DsRed in hepatocytes under the fabp10a promoter. To dissociate and recover hepatocytes from the larvae we investigated the effect of different common proteases  and mechanical forces . We found that dispase, in combination with a gentle up and down pipetting of the digested larvae, resulted in a high recovery of well-separated and viable hepatocytes  incubated between 3 and 6 dpf with 1% dimethyl sulfoxide (DMSO) (vehicle control). The procedure was independently repeated three times, and the data were pooled. The average number of hepatocytes recovered amounted to 1509 ± 328 (average ± standard deviation (SD)) per larva (n = 240).To demonstrate the reproducibility of the method we quantified hepatocytes isolated from 6 dpf larvae  but also in case of the pericardial injection (only at MD) .The non-hepatotoxic compounds  did not induce any change in the amount of recovered hepatocytes as compared to the amount coming from livers of non-treated animals by any of the administration routes (results not shown).In order to investigate the pharmacokinetics of paracetamol and tetracycline after immersion or injection at their MC/MT, we quantified the compounds in larval extracts as a function of time (0–24 h post treatment) using ultra-high performance liquid chromatography with ultraviolet detection (UHPLC-UV). The validation data of the extraction and chromatography method can be found in the The first objective of this study was to evaluate cell imaging counting (CIC) as a new and efficient approach for ex vivo hepatotoxicity assessment in zebrafish larvae. CIC is a high-content imaging and quantification tool that is used for detecting cells and even changes in cells at a subcellular level ,14. UsinTo evaluate the sensitivity of CIC to detect drug-induced hepatotoxicity, visual analysis of liver morphology by fluorescence microscopy and size analysis of fluorescent 2D liver images were employed as reference methods. The data show that all three methods were able to identify the hepatotoxic effects of paracetamol and tetracycline, at least when larvae were immersed in the non-lethal maximal concentration (MC) . As compThe reason underlying the differences in sensitivity of the three methodological approaches is presently not clear. Obviously, further studies including detailed microscopic analyses of liver sections are needed that would allow us to correlate the outcome, as observed in this study, with histological changes and cellular viability.Herein we also explored the applicability of using injections for the delivery of compounds investigated for their hepatotoxic effects. In case of paracetamol a clear effect was detected after yolk injection at MD, as identified by all three methods. Thus hepatotoxicity can also be induced by a single overdose of paracetamol in zebrafish larvae, as observed for humans . TetracyOverall, the outcome therefore shows that also injections, especially in case of administering compounds into the yolk, can produce significant results in the framework of hepatotoxicity testing. However, as the pharmacokinetic data indicate that both paracetamol and tetracycline are absorbed by immersion, leading to obvious liver effects, it is not clear from the present data that yolk injections could be advantageous over simple bathing of larvae in compound solutions. Obviously, the yolk administration route that can also be performed automatically  is partiN-acetyl-p-benzoquinone imine (NAPQI) are formed (not tracked down in this study) that exert their effect over time. Of interest, NAPQI-GSH adduct (GSH is the reduced form of glutathion) has been found when paracetamol was incubated with liver microsomes of adult zebrafish, possibly as the result of the expression of cytochromes P450 (CYPs), CYP3A65 with contributions of CYP3C1 [The pharmacokinetic profile of paracetamol shows that almost no compound could be recovered from the larvae 24 h after yolk injection, although the hepatotoxic effects detected at 6 dpf are similar to the immersion results. This seems to indicate that the injurious effects take place particularly during the first 24 h after administration (3–4 dpf). Alternatively, it is possible that metabolites like f CYP3C1 . Of intef CYP3C1 , althougInterestingly, the pharmacokinetic profiles after yolk or pericardial injection of paracetamol are somewhat similar, although the hepatotoxic effects induced were different. Likely, both administration routes result in a dissimilar overall distribution of the compound in organs and tissue, affecting the final hepatotoxic effect.In case of injected tetracycline, different pharmacokinetic profiles were observed as compared to the ones seen after injection of paracetamol. The concentrations remained somewhat constant over the first 24 h after administration, and it looks like the compound is not readily excreted from the larval body. It is possible that tetracycline binds to a large extent and with high affinity to local tissue without being distributed over the rest of the larvae. Anyhow, the final hepatotoxicity induced by the different administration routes as detected by liver morphology analysis is surprisingly quite similar. In contrast, when using CIC to assess hepatotoxicity, immersion led to pronounced effects , whereas no impact was observed after injection. Again, the overall outcome regarding tetracycline is likely the consequence of a complex interplay between distribution, metabolism and detection of hepatotoxicity. These first intriguing results regarding pharmacokinetics and organ toxicity in zebrafish larvae definitely demand further investigation, and it looks like detailed metabolism data are required to fully understand the results. However, this experimental work was beyond the scope of the present study.Paracetamol, tetracycline, sucrose and rutin were purchased from Sigma-Aldrich . Protease inhibitor cocktail (1.5× solution) was made by dissolving 1 tablet  in 6.5 mL Dulbecco’s phosphate-buffered saline (DPBS)  without calcium and magnesium.Dispase II (Sigma-Aldrich) was dissolved in DPBS  without calcium and magnesium to make a 20 mg/mL stock solution.UHPLC experiments were performed using ammonium formate purchased from Fluka . Formic acid was obtained from Acros Organics , acetonitrile and methanol was purchased from Fischer Scientific . All reagents were of analytical grade or better. Ultrapure water was produced using a Millipore Milli-Q Gradient system . All pH modifications were done with a Metrohm 691 pH meter .All zebrafish experiments carried out were approved by the Ethics Committee of the University of Leuven  and by the Belgian Federal Department of Public Health, Food Safety & Environment .fabp10a promoter (herein referred to as Tg(fabp10a:DsRed)) was created and raised in our laboratory [fabp10a:DsRed) zebrafish (larvae and adults) were kept in standard conditions for zebrafish . Tg(fabp10a:DsRed) adults from the third and fourth generation were mated . Only embryos of good quality  were selected for experiments and kept in petri dishes containing Danieau’s solution until compound exposure. All larvae were derived from the same spawns of eggs for statistical comparison between the control and treated groups.A transgenic zebrafish line expressing DsRed in hepatocytes under the boratory . Tg(fabpfabp10a:DsRed) larvae were transferred to 24-well plates  with each well containing 10 larvae in 1 mL Danieau’s solution and the dissolved drug or the vehicle control for immersion. For yolk injections, and pericardial injections into the bloodstream via the common cardinal veins [Compounds tested were dissolved in DMSO (stock solution) and then further diluted in Danieau’s solution with a final concentration of 1% DMSO or diluted with 0.9% NaCl solution (1:1 ratio) to obtain a working solution for immersion and yolk/pericardial injections, respectively. At 3 dpf, hatched Tg and arranged so that they were lying on their right sides with their heads facing left. This enabled us to view the left lateral side of the larvae and the larger lobe of the liver that was scored in a blinded manner under a fluorescence microscope . Livers were considered abnormal if their typical globular structure underwent gross morphological changes. Images of the livers  were imported to ImageJ software  for determining the liver size. To that end, the picture was set to scale , the liver area extracted  and the area calculated . At 6 dpf treated larvae were anesthetized by 0.5 mM tricaine in a 6 well-plate  with one larva per well and incubated with 200 µL 2.5 mg dispase/mL DPBS for 4 h. The proteolytic activity was stopped by adding 100 µL of 1.5× protease inhibitors cocktail solution. The hepatocytes were dispersed by pipetting up and down 20 times with a 1 mL automatic pipette. After digestion, hepatocytes were positioned at the bottom of the well by centrifugation at 200× g  for 15 min.In order to digest the larvae and separate hepatocytes from the liver, larvae were transferred to 96 well-plates , with a 2×/0.1 NA lens, DsRed fluorescence channel, a 0.1-s exposure time, and the intensity segmentation (172-4095) set to ensure hepatocytes were recognized selectively. ® tubes pre-filled with acid washed glass beads  and 270 µL of extraction medium  was added (10 larvae per Eppendorf® tube). Samples were centrifuged for 15 s at 14,100× g  to insure high homogenization efficiency. Subsequently, the samples were homogenized with ultrasonication  at +4 °C and high energy input setting to ensure the highest possible disruption of the cells. The overall treatment time was 15 min spread over 30 cycles of 30 s treatment followed by pauses of 30 s in-between. Six samples were homogenized during each treatment cycle. Subsequently, treated samples were centrifuged at 14,100× g for 15 min and 200 µL of supernatant was transferred to an empty Eppendorf tube (1.5 mL) and stored on ice until all samples were processed. All samples were then placed into a vacuum oven  and evaporated until dry at +45 °C during 45 min. The samples were then reconstituted with a mobile phase-like solution (6% acetonitrile in water), vortexed for 5 s, resuspended at least three times with a micropipette and sonicated  for 15 min. The reconstitution volume was chosen such that the linearity range of the method was not exceeded and is given in At given time points , treated larvae were collected and transferred to 1.5 mL EppendorfAn Agilent 1290 UHPLC system  consisting of an autosampler, quaternary pump, degasser and DAD-detector (diode array detector) was used for all uptake measurements. Data acquisition and peak processing was performed using OpenLAB CDS Chemstation Edition 01.04 software (Agilent). All calculations concerning the evaluation of recorded data were made in MS Excel .p (particle diameter) = 1.7 µm) from Waters Inc.  at a flow rate of 0.4 mL/min. Gradient elution was performed starting at 97:3 (v/v) 10 mM ammonium formate (pH 2.8 modified with formic acid):acetonitrile, and changed to 18:82 (v/v) 10 mM ammonium formate (pH 2.8):acetonitrile in 10.5 min. The injection volume (Vinj) of each sample was 2 µL. After elution of the compounds, a cleaning step at 100% acetonitrile was applied for 1.3 min after which the column was re-equilibrated for 7.0 min at initial conditions. Paracetamol was detected at a wavelength of 254 nm, while a wavelength of 274 nm was used for the measurement of tetracycline.Compounds of interest were separated chromatographically on an Acquity BEH C18 column  zebrafish larva to be 0.25 mg.The larval amount of hepatotoxicant (mg/kg) was calculated assuming the weight of a 3 or 4 dpf Tg regarding numbers of hepatocytes and liver sizes were analyzed for statistical significance using an ANOVA test. Regarding the liver morphology, Fisher’s exact test was used for the analysis. ex vivo to detect hepatotoxicity induced by paracetamol and tetracycline in our transgenic zebrafish model. The outcome was in general comparable with the results obtained using two reference methods, i.e., visual analysis of liver morphology by fluorescence microscopy and size analysis of fluorescent 2D liver images, although clear differences exist depending on the administration routes investigated, i.e., immersion, yolk and pericardial injections. In general, immersion of larvae resulted in more consistent results among the different hepatotoxicity tests.We successfully applied CIC Unlike the other methods, our CIC-dependent approach measures directly and in an unbiased way the effect of compounds on the number of hepatocytes in the liver, is quick and reliable, and therefore provides a novel and straightforward tool for screening hepatotoxicants in early stages of drug development. As CIC allows for high-content imaging, the system is also amenable to more detailed analysis of recovered cells, e.g., multiparametric cytotoxicity testing. In addition, the method is also suitable for testing of other organ-related toxicities subject to the organs and tissue expressing fluorescent proteins in transgenic zebrafish lines.