Prospective migrants to countries where the incidence of tuberculosis (TB) is low (low-incidence countries) receive TB screening; however, screening for latent TB infection (LTBI) before immigration is rare. We evaluated the cost-effectiveness of mandated and sponsored preimmigration LTBI screening for migrants to low-incidence countries. We used discrete event simulation to model preimmigration LTBI screening coupled with postarrival follow-up and treatment for those who test positive. Preimmigration interferon-gamma release assay screening and postarrival rifampin treatment was preferred in deterministic analysis. We calculated cost per quality-adjusted life-year gained for migrants from countries with different TB incidences. Our analysis provides evidence of the cost-effectiveness of preimmigration LTBI screening for migrants to low-incidence countries. Coupled with research on sustainability, acceptability, and program implementation, these results can inform policy decisions.  The World Health Organization (WHO) has continued working toward tuberculosis (TB) elimination, aiming to reduce the overall TB burden by ≈90% to <1 case/1 million persons in countries where TB incidence is low (low-incidence countries)  has been suggested as a method to accelerate the decline of TB  suggested mandatory LTBI screening and treatment as part of routine preimmigration medical exams  vaccination status, chest radiograph results, and LTBI prevalence. Patient age was defined based on an age distribution of a reference cohort of permanent residents to Canada in 2014 , IGRA, or sequential screening, in which persons testing positive by TST were given a confirmatory IGRA. We defined a positive TST result as an induration measuring The model took a healthcare system perspective for the fully sponsored and mandated preimmigration LTBI screening: all LTBI screening costs preimmigration, along with typical postarrival costs, were the responsibility of the receiving country’s healthcare system. We used a 3% annual discount rate for costs and outcomes .A simplified model structure is displayed in https://www.simio.com).We made the following assumptions in the model. Those with previous TB or an abnormal chest radiograph result identified during the preimmigration medical exam were also referred for postarrival follow-up. With the intervention, all those who began screening completed it, eliminating dropout during this stage of the LTBI cascade of care. Drug-resistant TB and self-cure of LTBI were not modeled. It was assumed that all those who tested positive were offered LTBI treatment to limit extrapolation of care provider decisions. All reactivation TB cases had a 17.6% chance of causing a secondary case; further transmission was not modeled . Modelin–We derived model estimates from the literature or expert opinion . A meta-We derived all costs from Canada sources and assumed that the costs of screening abroad were equal to screening costs in Canada. We derived costs for LTBI treatment and screening, including drugs, screening tests, routine monitoring, and clinician time, from the British Columbia Centre for Disease Control. Adverse event costs, including hospitalization rates and time, and the cost of TB disease were as reported in the literature .We derived health utility data from a study  to capture uncertainty of model estimates using an outer sample size of 1,000 and inner sample size of 50,000 , 3. To gAmong migrants from moderate- to very high–incidence countries, IGRA screening coupled with postarrival rifampin treatment was the optimal intervention strategy in deterministic analysis. Sequential screening coupled with postarrival rifampin treatment was the optimal intervention strategy among migrants from low-incidence countries. Intervention strategies involving TST identified the most migrants for postarrival follow-up, whereas strategies involving sequential screening identified the fewest. Intervention strategies involving rifampin resulted in the fewest TB cases (46% reduction compared with the base case) .For migrants from low-incidence countries, screening with TST alone resulted in a net loss in population QALYs because of poor specificity of the TST. Sequential screening, the most specific screening method, coupled with postarrival rifampin treatment yielded the lowest cost per QALY gained at $191,889. IGRA screening, the most sensitive screening method, coupled with rifampin treatment resulted in the fewest TB cases (46.2% reduction) but had a higher cost per QALY gained  because of its lower specificity compared with that of sequential screening.For migrants from moderate-incidence countries, the optimal intervention strategy was IGRA screening coupled with postarrival rifampin treatment for those from moderate-incidence countries with a cost per QALY gained of $43,343. Sequential screening coupled with postarrival rifampin treatment was cheaper overall but had a cost per QALY gained of $47,561.Among migrants from high-incidence countries, IGRA screening coupled with postarrival rifampin treatment was the optimal intervention strategy, at a cost per QALY gained of $26,350. Sequential screening coupled with rifampin treatment was less expensive, but also less efficient, with a cost per QALY gained of $29,997.Among migrants from very high–incidence countries, IGRA screening coupled with postarrival rifampin treatment had a cost per QALY gained of $16,291 compared with the base case. Sequential screening with rifampin treatment again was slightly cheaper, resulting in a cost per QALY gained of $20,165.Among migrants from low-incidence countries, sequential screening coupled with postarrival rifampin treatment was the most cost-effective option in deterministic analysis. In PSA, this intervention had a probability of cost-effectiveness of 49.1% at a WTP threshold of $50,000/QALY and 50.7% at a WTP threshold of $100,000/QALY. This probability did not substantially increase past these thresholds, however, resulting in a probability of cost-effectiveness of 52% at a WTP threshold of $200,000/QALY , panel AAmong migrants from moderate-, high-, and very high–incidence countries, IGRA screening coupled with postarrival rifampin treatment was the most cost-effective option in deterministic analysis. This intervention strategy at WTP thresholds of $50,000/QALY gained had probabilities of cost-effectiveness of 57.5% among migrants from moderate-incidence countries , panel B>30 cases/100,000 persons appears to be an effective method for reducing TB incidence post-arrival. The use of IGRA screening coupled with postarrival rifampin treatment provided the lowest cost-effectiveness ratio in migrants from these countries. This intervention strategy reduced TB incidence by >45% and yielded costs <$50,000/QALY gained.The intervention of preimmigration LTBI screening followed by postarrival treatment among new migrants from countries with a TB incidence Because prevalence of LTBI was low among migrants from countries with a TB incidence <30 cases/100,000 persons and specificities of LTBI diagnostic tests are imperfect, this intervention may result in a high number of uninfected persons receiving treatment unnecessarily. This finding suggests that with some strategies, the QALYs lost due to treatment side effects among those with false-positive diagnostic results may be greater than the QALYs gained by averted TB in those with true-positive diagnostic results. If screening and treatment must be performed in these low LTBI prevalence populations, more specific screening methods  are preferred to avoid inappropriate treatment.Probabilistic sensitivity analysis suggests a certain degree of uncertainty in results. The behavior of CEACs as WTP thresholds increase suggests that the intervention offers small increases in population QALYs or large increases in cost in many replications. It is important to understand how well the model parameters represent the local setting when using the results of this analysis to inform evidence-based policy. These results suggest that intervention offers domestic benefits to the receiving country, but several factors need to be carefully examined. IGRA use in high-resource settings suffers from variability, in part related to several operational issues (This model considered only the costs of persons who became permanent residents. The data from Canada indicated that ≈50%–60% of those who begin the process of becoming a permanent resident successfully complete it (Regardless of how preimmigration LTBI screening is implemented, investment in LTBI infrastructure in high TB incidence settings will be essential for global TB elimination. Evidence suggests that introduction of routine preimmigration TB screening in many high-income, low-incidence countries has played a role in improving infrastructure for TB programs in low-resource areas (The cost-effectiveness of preimmigration LTBI screening and postarrival treatment has not been evaluated since 2003. Previously, Schwartzman and Menzies (Our analysis has several strengths. Use of discrete event simulation enabled realistic modeling of time spent in various health states, which is difficult to implement in Markov models. This type of model also allowed age-representative modeling of new migrants for application of age-adjusted LTBI prevalence. The source of most of the cost data was the British Columbia Center for Disease Control, which handles most TB cases in the province of British Columbia. This analysis estimated LTBI prevalence and abnormal chest radiograph prevalence using several years of immigration and TB data from Ontario. The data are likely to be generalizable, because Ontario accepts 40% of new permanent residents (In this study, we assumed that all migrants were recommended postarrival LTBI treatment when they had a positive LTBI diagnostic test, which is not necessarily true; for some persons, the risk for serious adverse events may outweigh the benefit of treatment. Social factors and concurrent conditions may increase the risk for reactivation of LTBI. We have shown that the benefits of rifampin treatment for migrants from moderate- to very high–incidence countries who test positive by IGRA preimmigration outweigh the potential risks of adverse events. However, in practice, individual adverse-event risk is considered, and treatment may not be offered to all migrants. Further research designed to identify the specific populations who should be offered treatment would help inform future analyses.We derived the reactivation rate of LTBI from the literature, but because many of those studies were based on TB incidence in those who were positive by TST, it is possible that the predictive value of the TST caused underestimation of true reactivation rates. Our analysis did not consider 3 months of once-weekly isoniazid and rifapentine as an LTBI treatment modality because it was not universally available. Literature data, however, suggest this modality may yield similar results to rifampin treatment (Our analysis used a healthcare system perspective, which does not consider costs incurred by persons experiencing the intervention (In conclusion, preimmigration IGRA screening coupled with postarrival rifampin treatment among migrants from countries with moderate to very high incidence of TB resulted in the lowest cost-effectiveness ratios. This evidence can be used to support policy decisions surrounding preimmigration LTBI screening in high-income, immigrant-receiving countries, when coupled with evaluations on program implementation, acceptability, and sustainability. Next steps in research should be to identify subgroups at highest risk for progression to TB disease to limit individual risk associated with LTBI treatment and improve the likelihood of feasibility and sustainability.Additional information about cost-effectiveness of latent tuberculosis infection screening before immigration to low-incidence countries.


An aberrant right subclavian artery (ARSA) is a relatively prevalent vascular anomaly. What is the most appropriate treatment for thoracic aortic aneurysm combined a non-aneurysmal change ARSA?A 52-year-old man was admitted to our institute due to a history of chronic cough, dysphagia and an abnormal chest radiographic finding. Because of his progressive symptoms and large fusiform thoracic aneurysm, we performed the hybrid repair for simultaneous relief of an ARSA causing dysphagia and thoracic aneurysm.In case without aneurysm of ARSA, especially in conjunction with approximate thoracic aneurysm, our approach is suitable because the revascularization using the right carotid to subclavian artery re-routing prior to endograft deployment is justified in order to preserve circulation of posterior brain, spinal cord, internal mammary artery and upper limb and to prevent large retrograde type II endoleaks, as well as simplicity and durability. An aberrant right subclavian artery (ARSA) is a relatively prevalent vascular anomaly . We consA 52-year-old man was admitted to our institute due to a history of chronic cough, dysphagia and an abnormal chest radiographic finding. A chest x-ray and computed tomographic angiography scan (CTA) revealed an ARSA behind the esophagus with about 5.6-cm sized proximal descending aortic aneurysm Fig. a. The esAn ARSA is the most common congenital abnormality of the aortic arch . An ARSAConventional open repair for ARSA combined thoracic aneurysm is still associated with high mortality and morbidity, as well as neurological events , 2. Ther


The dynamics of the tripartite relationship between the host, gut bacteria and diet in the gut is relatively unknown. An imbalance between harmful and protective gut bacteria, termed dysbiosis, has been linked to many diseases and has most often been attributed to high-fat dietary intake. However, we recently clarified that the type of fat, not calories, were important in the development of murine colitis. To further understand the host-microbe dynamic in response to dietary lipids, we fed mice isocaloric high-fat diets containing either milk fat, corn oil or olive oil and performed 16S rRNA gene sequencing of the colon microbiome and mass spectrometry-based relative quantification of the colonic metaproteome. The corn oil diet, rich in omega-6 polyunsaturated fatty acids, increased the potential for pathobiont survival and invasion in an inflamed, oxidized and damaged gut while saturated fatty acids promoted compensatory inflammatory responses involved in tissue healing. We conclude that various lipids uniquely alter the host-microbe interaction in the gut. While high-fat consumption has a distinct impact on the gut microbiota, the type of fatty acids alters the relative microbial abundances and predicted functions. These results support that the type of fat are key to understanding the biological effects of high-fat diets on gut health. The mammalian gut has co-evolved with thousands of bacterial species and together they form a complex dynamic relationship of which the physiological consequences are largely undiscovered. The gut microbial ecosystem has the potential to influence the overall health status of the mammalian host by forming an interface between the gut mucosal surface and the luminal food ingested into the body. Molecular crosstalk between the microbiome and the host epithelium influences intestinal barrier function, in part through the release of microbial metabolites like short-chain fatty acids (SCFA) . Increas-/- mice via conjugation of hepatic bile acids which promoted growth of Bilophila wadsworthia , Aldercretzia spp. [Actinobacteria (P = 0.03)], family S24_7 [Bacteroidetes (P = 0.002)], and Ruminococcus spp. [Firmicutes (P = 0.005)]. Uniquely, olive oil resulted in an increased abundance of several Firmicutes including Clostridiaceae (P = 0.003), Peptostreptococcaceae (P = 0.01), Ruminococcaceae (P = 0.005), and Dorea spp (P = 0.003). In contrast, milk fat promoted different families of Firmicutes including Erysipelotrichales (P = 0.008) and several genera from Ruminicoccus (P = 0.003). Corn oil enhanced the abundance of Firmicutes family members from Turicibacteraceae (P = 0.008) in addition to Coprococcus spp. (P = 0.002). Overall, high-fat diets resulted in analogous modulation of the gut microbiota at higher taxonomic levels, but the type of fatty acid present in the dietary lipid uniquely altered the intestinal microbes at lower taxonomic levels.The differences in bacterial communities between the dietary cohorts were further evident when samples were ordered according to their Firmicutes to Bacteroidetes ratio. In agreement with previous literature ,4, our rSince microbial compositions change according to type of lipid diets, we next investigated how lipids affect the functionality of the microbiota using amplicon sequencing predictions and comparing SCFA metabolites. Amplicon sequencing functional content was predicted from marker genes (16S rRNA) and LDA was performed . The lowTo determine if virulence attributes of the gut microbiome were modulated by high-fat diets, we used 16S OTUs to categorize functionality. We found that all high-fat diets predicted an increase in the abundance of mobile genetic elements A; howeveP value ≤0.006 in the submucosa α/ retinoid X receptor (RXR)α pathways compared to the low-fat control. IPA selected proteins used to determine PPAR activation included: acyl-CoA oxidase 1, apolipoprotein A1, cytochrome P450 family 2 subfamily C member 18, fatty acid synthase, glycerol-3-phosphate dehydrogenase 1, heat shock protein 90 beta family member 1, mitogen-activated protein kinase 1, and protein disulfide isomerase family A member 3. Overall, increased consumption of fat regardless of the saturation index, results in decreased cell death and tRNA charging signaling and increased PPARα/RXRα activation signaling.Two of the most highly affected host pathways in the corn oil group were glycolysis I and oxidative phosphorylation, indicating increased energy demand in the mucosal epithelial cells of corn oil fed mice A. These The milk fat dietary group had upregulated acute phase response A. Acute Similar to the low-fat group, the olive oil group had increased proteins involved in actin cytoskeleton signaling and epithelial integrity A due to To understand potential interactions between the bacteriome and host mucosal proteins, we evaluated Spearman correlations between mean phyla abundance and the selected mucosal proteins contributing to IPA pathways and networks . The heaThe mammalian gut has co-evolved with trillions of microorganisms, the collection of which is referred to as the gut microbiome. We have yet to understand how microbes succeed in the gut as a consortium and then co-exist in a community and affect the host responses. It has been hypothesized that several external factors, including diet, play a role in the host-microbe interaction in the gut. Several studies over the past few years have shown that a high-fat diet can lead to different gut microbial profiles, yet the effects on bacterial taxa and their functional responses caused by distinct types of fatty acids are not well understood. To define the specific changes in bacterial taxa as well as functional outputs, we analyzed the effect of commonly consumed dietary lipids on the colonic microbiome.Citrobacter rodentium [While there were differences between the high-fat and low-fat diets suggesting calories may play a role, these diets are not directly comparable since the macronutrients and micronutrients are different in the low-fat diet. By changing the amount of dietary fat, the proportion of carbohydrates and proteins automatically changes making it difficult to disentangle lipid driven changes. For example, the availability of dietary carbohydrates has been shown to modulate biofilm development  and acquodentium , whereasodentium  despite The data presented in this work show that diet induced changes to the microbiome mirrors diet induced responses from the host. For instance, the predicted increase in invasive potential observed in the corn oil bacteriome parallels the predicted decrease in pathogen resistance and barrier function observed in the host. Specifically, the corn oil diet increased the microbial diversity in the gut that was predicted to increase microbial virulence traits such as increased microbial motility and bacterial signal transduction by two component regulatory systems. The host proteome indicated the protective barrier protein MUC2 was decreased alongside proteins important for tight and adherins junctions, and endothelial integrity (bleeding). Furthermore, decreased peristalsis (contractility of muscle), and increased oxidative stress response predicted in the corn oil group may be in response to increased microbial invaders. This supports the phenotype observed in previous studies using a similar diet which showed n-6 PUFA results in increased oxidative stress and tissue damage ,52, incrSimilar parallels between the microbiome and host responses were observed in the milk fat group. Specifically, the milk fat diet increased microbes in the gut whose functions are involved in carbohydrate and lipid metabolism. This was reflected in the host by increased proteins involved in fatty acid β-oxidation. Mounting evidence supports that sirtuins link metabolism and host inflammation. While inflammation is required to defend against invading organisms, compensatory mechanisms are required to prevent chronic inflammation. Host sirtuins, increased in the milk fat group, improve outcomes in chronic inflammatory diseases and sepsis by ‘mending’ the host or promoting restitution through immune repression and restoring homeostasis following stress responses . MoreoveThis relationship between the microbiome, host and dietary lipids is not limited to the bacteriome and there is evidence that the virome may similarly be involved. In support of this, sequencing data predicted upregulated RIG-I-like receptor signaling pathways in the olive oil dietary group. The RIG-I-like family of pattern recognition receptors identify viral RNA , and areOverall, we conclude that the type of dietary lipids distinctly impacts the gut microbiome. While high fat consumption has a distinct impact on the gut microbiota as compared to a normal chow diet, the type of fatty acids alters the relative microbial abundances where olive oil was most distinct from the corn oil and milk fat. The corn oil and milk fat diets shared similarities in diversity but had different functional characteristics. We show that the corn oil diet, rich in n-6 PUFA, resulted in a microbiome with enhanced predicted virulence and pathogenicity associated with increased host inflammation, oxidative stress and increased barrier dysfunction. While the milk fat diet, rich in SFA, resulted in a host-microbe relationship that promoted inflammation which could result in inflammatory induced intestinal damage, there was a compensatory protective response evident by the production of sirtuins and SCFAs. In marked contrast to both corn oil and milk fat, the olive oil diet, rich in MUFA, resulted in a host-microbe dynamic suggesting the involvement of the less-explored virome. These results suggest that fat type is an important consideration for gut health and not all high-fat diets are detrimental. However, given this study is descriptive in nature, future studies should focus on well-designed experiments unraveling the mechanisms of each lipid on gut health. These results have the potential to guide evidence-based nutrition recommendations for IBD patients who can suffer from nutrient deficiencies from overly restrictive dietary regimes including low-fat diets.


Carcinogen-induced mouse models of liver cancer are used extensively to study the pathogenesis of the disease and are critical for validating candidate therapeutics. These models can recapitulate molecular and histological features of human disease. However, it is not known if the genomic alterations driving these mouse tumour genomes are comparable to those found in human tumours. Herein, we provide a detailed genomic characterisation of tumours from a commonly used mouse model of hepatocellular carcinoma (HCC).We analysed whole exome sequences of liver tumours arising in mice exposed to diethylnitrosamine (DEN). Mutational signatures were compared between liver tumours from DEN-treated and untreated mice, and human HCCs.Hras, in codon 584 of Braf or in codon 254 of Egfr. Truncating mutations of Apc occurred in 21% of neoplasms, which were exclusively carcinomas supporting a role for deregulation of Wnt/β-catenin signalling in cancer progression.DEN-initiated tumours had a high, uniform number of somatic single nucleotide variants (SNVs), with few insertions, deletions or copy number alterations, consistent with the known genotoxic action of DEN. Exposure of hepatocytes to DEN left a reproducible mutational imprint in resulting tumour exomes which we could computationally reconstruct using six known COSMIC mutational signatures. The tumours carried a high diversity of low-incidence, non-synonymous point mutations in many oncogenes and tumour suppressors, reflecting the stochastic introduction of SNVs into the hepatocyte genome by the carcinogen. We identified four recurrently mutated genes that were putative oncogenic drivers of HCC in this model. Every neoplasm carried activating hotspot mutations either in codon 61 of Our study provides detailed insight into the mutational landscape of tumours arising in a commonly used carcinogen model of HCC, facilitating the future use of this model to better understand the human disease.Mouse models are widely used to study the biology of cancer and to test potential therapies. Herein, we have described the mutational landscape of tumours arising in a carcinogen-induced mouse model of liver cancer. Since cancer is a disease caused by genomic alterations, information about the patterns and types of mutations in the tumours in this mouse model should facilitate its use to study human liver cancer. Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer, which is currently the sixth most frequently diagnosed human cancer. Liver cancer is the second most common cause of cancer death globally and its incidence is increasing in countries with historically low rates.[9]Hras or Braf proto-oncogenes.There are an increasing number of experimental mouse models used in HCC research to study the disease pathogenesis and to assess novel therapeutics.No single mouse model can capture all aspects of human HCC, although each can recapitulate at least some of the genetic and/or cellular features of the human disease. For example, a comparison of global gene expression profiles showed that HCC from DEN-treated mice resembles a subclass of human HCC associated with poor prognosis.Male C3H/HeOuJ mice were administered a single intraperitoneal injection of DEN (20 mg/kg body weight) aged 14–16 days. Liver samples were collected during the first 24 h following DEN administration; tumour samples were collected up to 40 weeks after treatment. Untreated C3H male mice were used for reference tissue samples or aged up to 76 weeks for spontaneous liver tumour samples. Tissue samples were snap frozen for DNA extraction and/or fixed in neutral buffered formalin for histological analyses.6-ethyl-2-deoxyguanosine (Squarix Biotechnology); and Ki67 (Bethyl Laboratories). Quantification of nuclear staining for O6-ethyl-2-deoxyguanosine and phospho-histone H2AX was done using ImageScope software (Leica Biosystems). Tumours were classified according to the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) guidelines.Histochemical staining with haematoxylin and eosin (H&E) or using the Gomori’s method was carried out on formalin-fixed paraffin-embedded tissue sections. Immunohistochemistry was performed using antibodies against β-catenin (BD Biosciences); phospho-histone H2AX (Merck Millipore); OGenomic DNA was isolated from liver tumours and from ear/tail samples using the AllPrep DNA/RNA mini kit or the DNeasy blood & tissue kit (Qiagen), according to the manufacturer’s instructions. Exome capture libraries were prepared following the instructions of the SureSelectXT mouse all exon target enrichment system (Agilent Technologies). Exome libraries were sequenced using a 125 base pair paired-end read protocol on an Illumina HiSeq 2500.Sequencing reads were aligned to the C3H_HeJ_v1 mouse genome assembly  C3H/HeOuJ male mice were administered a single intraperitoneal injection (20 mg/kg body weight) of DEN.DEN primarily targets the liver, in which it is metabolically activated by cytochrome P450 enzymes in hepatocytes.All DEN-treated mice developed multiple, macroscopically identifiable tumours by 25 weeks after administration, concordant with previous studies.The development of spontaneous tumours in untreated C3H showed greater histological and temporal variability (37–76 weeks). Importantly, DN and HCC tumours arising in these untreated mice were histologically indistinguishable from those treated with DEN C. FurtheWhole exome sequencing was performed on DNA isolated from 50 discrete neoplasms excised from the livers of 33 individual C3H male mice given a single intraperitoneal administration of DEN as juveniles. 34 of the DEN-induced neoplasms were of sufficient size to provide additional tissue for histopathological examination; of these, 16 were classified as DNs and 18 as HCCs. The whole exome sequences of the remaining 16 DEN-induced neoplasms were used only for the phylogenetic analysis . Spontaneous neoplasms carried many low abundance SNVs, and non-synonymous variants appear to be preferentially selected as a subset of these SNVs had increased VAFs  was preceded by a C (or A) and followed by a T (or G). Hierarchical clustering on the 96 possible trinucleotide substitution contexts showed a consistent mutational pattern shared among both DNs and HCCs arising in C3H inbred mice exposed to DEN B. We alsMathematical modelling of mutational processes in human cancer has defined over 30 mutational signatures, several of which are associated with exposure to specific environmental mutagens.The neoplasms which arose following carcinogen exposure carried a high mutational load in their exomes. For example, each DEN-initiated DN had an average of 583 somatic SNVs in its protein-coding sequence, compared with 26 SNVs in an average spontaneous neoplasm. As expected, 72% of these point mutations are predicted to be non-synonymous, with no detectable bias in the distribution of missense, nonsense and splice site mutations A. WithinHras is the predominant, although not obligatory, oncogenic driver of HCC in juvenile male C3H mice that have been administered a single dose of DEN. Over half of the DEN-initiated tumour samples harboured a non-synonymous mutation in the Hras proto-oncogene, almost exclusively an activating hotspot mutation in codon 61 , although the mutation spectrum was different. Almost half of the point mutations were identical G:C to T:A transversions in codon 117   . This coNs Figs. . The can-catenin C, suggesBy comparison, spontaneous tumours from untreated mice contained few detectable point mutations in cancer genes . As prevWe used a pathway analysis approach to assess how well the DEN mouse model recapitulates human HCC, as defined by cancer-associated gene mutations. Tumour samples were annotated with a list of Reactome pathways that contained a mutated cancer-associated gene(s). Based on these mutated pathways murine liver tumours, both DEN-induced and those arising in untreated C3H mice, clearly clustered separately from human HCCs .Chemically-induced mouse models of liver cancer are important tools widely used to study the molecular pathogenesis of human HCC.Our strategy comparing spontaneously occurring liver tumours with those initiated by exposure to DEN allowed the direct comparison of the histopathology and genomic impact of carcinogen exposure. By controlling the initiating carcinogenic event the liver lesions in DEN-treated mice developed from early DNs to carcinoma within a short, relatively consistent timeframe. The liver lesions arising in untreated mice arose at a much lower incidence and with a longer, more variable latency. Based on their histological appearance, liver tumours resulting from exposure to DEN were indistinguishable from those that arose spontaneously in untreated mice. This result parallels that found in human liver tumours, where heterogeneous molecular phenotypes can underlie HCC samples that are histologically similar. Indeed, these murine dysplastic lesions and carcinomas mimicked the histological features of their corresponding human tumours. In sharp contrast, however, this similarity was not seen in mutational landscapes: the exomes of DEN-induced neoplasms clearly reflected the DNA damage caused by chemical carcinogenesis.DEN-induced tumours carried a notably high burden of somatic mutations, which allowed us to demonstrate that multiple tumours can evolve independently within an individual liver. We did not identify evidence of metastatic clones within a liver, although this may reflect the small sample size. The mutational frequencies were much higher than those seen in most human solid tumours, including HCC. Perhaps not surprisingly, human lung and skin cancers that result from environmental exposure to potent mutagens are among the few human tumour types with mutational burdens similar to those we report here.Hras, Braf, Egfr and Apc.Exposure to DEN left a common mutational imprint in the tumour exomes of treated mice. Indeed, the same small subset of reported signatures of mutational processes was readily identified computationally in every DEN-induced tumour. Notwithstanding this common imprint, each individual exome, including those of neoplasms arising within the same liver, carried a unique combination of somatic base substitutions. The majority of these SNVs are likely to be passenger mutations. However, we could identify four recurrently mutated genes that are putative oncogenic drivers of HCC in DEN-treated C3H male mice: Hras or Braf. The remaining ∼20% of samples carried a potentially activating hotspot mutation in Egfr, one of the upstream receptor tyrosine kinases that can regulate the Ras signalling pathway. This suggests that constitutive activation of the Ras/Raf/MEK/ERK signal transduction pathway is a hallmark feature in this mouse model of liver cancer.The main genetic trait of DEN-initiated tumours is acquisition of mutations which deregulate signalling cascades involved in cell proliferation and survival. Over 80% of DEN-initiated tumour samples carried an activating hotspot driver mutation in either Hras proto-oncogene is frequently reported in both spontaneous and chemically-induced liver tumours in mice.Hras mutations is strongly influenced both by the mouse strain used, as well as by the type and dose of chemical and experimental induction protocol employed. Indeed, even between spontaneous and treatment-induced tumours in C3H mice, we observed a difference in the location of the hotspot activating mutation in Hras . The mutational activation of Braf is also reported to be influenced by the mouse strain and appears to be related to the strain’s susceptibility to hepatocarcinogenesis.Braf in the DEN-initiated liver tumours in the highly susceptible C3H mouse strain compared to those arising in the more resistant C57BL/6J strain. Changing the strain in the DEN model can therefore be used to increase the probability of a specific driver mutation in the resulting tumour, although the same signalling pathway will likely be affected. This study and others have shown that dysregulation of the Ras/Raf/MEK/ERK pathway is a common route to hepatocarcinogenesis in mice, especially in genotoxic models.[31]Activation of the Apc is a putative, although not obligatory, gatekeeper of malignant transformation in the DEN liver cancer model; we detected a significant recurrence of Apc-truncating mutations exclusively in carcinoma samples and absent in the DNs arising in DEN-treated C3H mice. Activating β-catenin (Ctnnb1) mutations have previously been implicated in progression to carcinoma, but in a two-stage model where DEN is given as the initiator followed by treatment with phenobarbital as a tumour promoter.Apc and consequent aberrant nuclear expression of β-catenin.bona fide cancer genes that were recurrently mutated with significance in the set of DEN-initiated tumour samples from this study. Instead, we saw a diversity of low-incidence, non-synonymous point mutations in numerous oncogenes and tumour suppressors, consistent with the known mechanism of mutagenesis by DEN. Specifically, the introduction of a large mutagenic SNV burden stochastically across the genome resulted in heterogeneity at the level of driver gene mutations in the resulting individual tumours. However, it is also possible that common driver genes could have been dysregulated by alternative genetic or epigenetic processes during tumorigenesis.Aside from these four driver genes, there were no other Hras, Braf or Egfr, which recurrently occur at a high frequency in the DEN-induced liver tumours, are rarely observed in cases of human HCC. The most common cellular processes and pathways implicated in the pathogenesis of human HCC are telomere maintenance, WNT/β-catenin signalling and p53 cell cycle control.RAS family members are rare, a subset of HCC cases have been reported to have aberrantly activated RAS-MAPK signalling which correlates with a poor prognosis.CTNNB1 mutations are frequently observed in human HCC samples, while loss-of function mutations in Apc were found in mouse DEN carcinomas.There are currently several mouse models of liver cancer, each of which recapitulates specific genetic, molecular, and/or histological features of the human disease.in vivo models and thereby inform the selection of the most appropriate models to study human liver cancer.Our study demonstrates how the application of exome sequencing on carefully designed cohorts can reveal novel insights into widely used mouse models of liver cancer. Such oncogenomic descriptions will deepen our understanding of the advantages and limitations of preclinical Cancer Research UK , the Wellcome Trust  and the European Research Council .This research was supported by The authors declare no conflicts of interest that pertain to this work.Please refer to the accompanying Study design: FC, TFR, SJA, CF, ML, DTO. Experiments: FC, SJA, CF. Data curation: ML, TFR. Computational analyses: TFR. Interpretation of data: FC, TFR, SJA, CF, DTO. Study concept and writing the draft manuscript: FC. Data visualisation for the manuscript: TFR, SJA, CF. Critical revision of the manuscript: FC, TFR, SJA, DTO. Provision of whole genome sequencing: JSL. Acquisition of funding: DTO.


There are few cases of multiple bronchial stenoses reported in the literature and none of the severity described here. The case is relevant due to its rareness, the pathophysiological insights derived, the successful interventional pulmonology strategies demonstrated, and as an example of a rare indication for high-risk lung transplantation.A 47-year-old man developed multiple recurrent bronchial web-like stenoses five weeks after an episode of severe tracheo-bronchitis presumed secondary to a chemical inhalation injury which initially caused complete bilateral lung collapse necessitating veno-venous extracorporeal membrane oxygenation. The stenoses completely effaced bronchi in many locations causing severe type II respiratory failure requiring mechanical ventilation and bronchoscopic puncture / dilatation then ultimately bilateral lung transplantation.This very rare case highlights the morbid sequelae that can arise after catastrophic tracheobronchitis which now, in the era of extracorporeal membrane oxygenation, may be survivable in the short-term. Bronchial stenoses are commonly caused by malignancy, trauma, infection (e.g. tuberculosis) and autoimmune conditions however they are generally singular or few –3. ThereA 47-year-old previously well male electrician from rural Australia presented with five days of worsening dyspnoea, productive cough and scant haemoptysis unresponsive to oral antibiotics and corticosteroids. He had a history of depression (desvenlafaxine) and active tobacco smoking (25 pack years). Initial chest x-ray was normal. He rapidly progressed to severe type I respiratory failure over the ensuing day requiring intubation and mechanical ventilation. Marked inspiratory and expiratory airflow limitation precluded adequate gas exchange and therefore veno-venous extra-corporeal membrane oxygenation (ECMO) was emergently instituted. Complete bilateral lung collapse developed over the next day Figs.  and 2.Rhinovirus identified by polymerase chain reaction. Vasculitis screen including antineutrophil cytoplasmic antibody (ANCA) was negative. The presumptive diagnosis was an inhalational injury.The lack of airspace opacity on initial chest x-ray excluded acute respiratory distress syndrome and suggested the severe airflow limitation may be due to airway obstruction. Bronchoscopy confirmed severe diffuse airway inflammation characterised by purulent exudate, ulcers and nodules throughout the airways Fig. . EndobroManagement consisted of broad-spectrum antimicrobial therapy , repeat bronchoscopic toilet and supportive care. The airway inflammation improved and transition to mechanical ventilation occurred after 13 days when only mild expiratory airflow obstruction was observed. A tracheostomy tube was sited two days after cessation of ECMO and ventilatory support was gradually weaned, ceasing seven days later. The patient was transferred to the ward on day 24 of admission. The only complication was critical-illness myopathy. He was discharged three days later for outpatient follow-up. He could mobilise 50 m and had no symptoms at rest.The patient re-presented six days post-discharge with progressive dyspnoea, wheeze and a mild cough productive of yellow sputum. Examination revealed increased work of breathing and a diffuse wheeze throughout the respiratory cycle. Fibre-optic nasoendoscopy to mid-trachea did not reveal paradoxical vocal cord motion or obstruction. Computed tomography (CT) imaging demonstrated widespread bronchial wall thickening from large to medium airways and mild ground-glass opacity in the peripheral upper lobes bilaterally Fig. . Broncho2 0.27 revealed pH 7.33, PaO2 99 mmHg, PaCO2 74 mmHg, bicarbonate 34 mmol/l).The patient was treated empirically with high-dose prednisolone, inhaled bronchodilators and antibiotics. He had significant anxiety and was managed with sertraline and olanzapine plus pro re nata clonazepam. His dyspnoea worsened and over the ensuing 17 days he developed increasing headache and drowsiness. He was found to be in severe but partially compensated type II respiratory failure with a normal alveolar arterial oxygen (Aa) gradient  and requiring intubation and mechanical ventilation. There was difficulty with mechanical ventilation with peak inspiratory pressures of 69 cmH2O required to achieve tidal volumes of 3 ml/kg. Expiratory airflow limitation plus plateau airway pressures < 20 cmH2O indicated a predominantly obstructive ventilatory defect and intermittent circuit disconnection was required to relieve gas trapping.The patient deteriorated despite a trial of non-invasive ventilation, becoming obtunded . Claustrophobia prevented plethysmography.The patient re-presented after 6 days with increasing dyspnoea, wheeze and mild type I respiratory failure. Bronchoscopy revealed aggressive recurrence of the webbing with occlusion of various segmental bronchi, some of which were re-canalised with puncture and dilatation. Mucosa in some areas sheared away from the underlying bronchial cartilage, precluding further intervention. The trachea appeared largely spared and the proximal main bronchi only minimally affected. An extensive history was conducted including from collateral sources regarding possible inhaled exposures in his home due to the repeated deterioration early after discharge. No causative agent could be identified except for malathion which the patient had used to spray ants a few days prior to his first presentation and not since. As the exposure was only identified at this later stage, cholinesterase levels could not be performed on blood samples from the original admission.Due to the severe and rapidly recurring webbing plus the increasing danger of endobronchial intervention, the patient was rapidly worked-up for lung transplantation. There were concerns regarding transplanting for a respiratory disease of unknown origin, that the underlying disease may be systemic, that it may affect the anastomoses and native large airways or that it may recur in the graft. Furthermore the patient was deconditioned and could not complete standard lung transplantation work-up. Surgical options included a standard bilateral sequential lung transplant versus a domino heart-lung transplant with a tracheal anastomosis. The patient underwent the former one month later at the quaternary referral centre with pathology of the explant revealing very severe necrotising bronchitis extending deep into the wall with associated granulation and fibrosis, most severe in the central bronchi, with some areas of histiocytic inflammation particularly in the distal trachea; however no specific diagnosis could be reached. Twelve months post-transplant, the patient is progressing well with normal graft function and no evidence of disease recurrence or systemic illness.This case warrants discussion for the diagnostic lessons arising from the re-presentation with type II respiratory failure, as an example of interventional pulmonology strategies, as a very rare case of severe diffuse bronchial stenoses after presumed inhalational airway injury and as an extremely rare indication for high-risk lung transplantation.The cause of the severe type II respiratory failure upon re-presentation was initially uncertain. Differential diagnoses included parenchymal lung disease  and other possibilities that were excluded as above . Given the homogenous gas trapping on imaging, it was theorised that an initial inhalational chemical injury had caused bronchiolitis obliterans.The discovery of severe diffuse bronchial stenoses on repeat bronchoscopy only 17 days later provided a superior explanation for the type II respiratory failure and difficulty in ventilation. This was confirmed by the dramatic improvement after bronchoscopic dilatation. We deduce collateral ventilation allowed segments distal to complete membranous bronchial occlusion to remain aerated. Fixed large airway obstruction and attenuated airflow through pathways of collateral ventilation would have retarded both inspiration, manifesting as high peak inspiratory pressure, as well as expiration, promoting the gas trapping and hypoventilation that characterised this case.There are many potential causes of one or a few bronchial stenoses. Reported aetiologies include congenital lesions, infection , autoimmune disorders , inhalational injury , benign and malignant tumours, malignancy treatment , trauma  and miscellaneous  –6.Far fewer causes of multiple bronchial stenoses have been identified and reports in the literature are rare. Mailloux and colleagues report a case of multiple web-like bronchial stenoses arising after severe illness secondary to infection with H1N1 influenza A which has some notable similarities with our case, including the delayed presentation after the initial airway injury . KeatingMalathion is an organophosphate pesticide considered to have low acute toxicity. Its effects on humans are mediated by inhibition of acetylcholinesterase, accumulation of acetylcholine and cholinergic toxicity. Malathion was the only inhaled toxin identified in our patient but the clinical features we observed are not typical of those described in historical studies of malathion toxicity in humans . AlthougWe hypothesise this case is rare as this injury would have been lethal in the pre-ECMO era, and that the multiplicity of stenoses here reflects the severity of the original airway injury. In one sense our case highlights what can be achieved with ECMO but, in another, it is a cautionary tale of the morbid sequelae that may arise after ‘surviving the unsurvivable’.


The imaging findings of hypoglycemic encephalopathy can be considerably similar to those of ischemic infarction or toxic leukoencephalopathy. We demonstrated unusual magnetic resonance (MR) imaging features of hypoglycemic encephalopathy which can be confused with other pathology both on imaging and acute clinical presentation. The diffusion-weighted imaging (DWI) and apparent diffusion coefficients (ADC) map findings in our case further supports the hypothesis of hypoglycemia-induced “excitotoxic injury” of glial cells and myelin sheath that might protect neuron axons from intracellular edema and irreversible damage.A 72-year-old woman presented with poor appetite and was initially drowsy at home; the symptoms progressed to loss of consciousness accompanied by mild incontinence. The initial glucose level was 44 mg/dL, but no nausea, vomiting, fever, or cold sweating was reported. Physical examination after intravenous glucose supplementation revealed the absence of focal neurological signs, facial palsy, and tongue or eye deviations. The images obtained 24 h after symptoms onset revealed symmetrical hyperintensities on DWI  associated with hypointensities on ADC map along the corticospinal tract, from the levels of the cerebral peduncle and the posterior limbs of the internal capsule to the level of the corona radiata, which may mimic the imaging findings of acute ischemic infarction or amyotrophic lateral sclerosis. The patient received sliding-scale insulin therapy and rehabilitation, and she recovered consciousness without motor function deficits after 1 month. Moreover, repeat DWI and ADC map showed the complete disappearance of the lesions.In the phenomenon of excitotoxic injury, axons could be protected from intracellular edema and irreversible damage, which may explain the reversible clinical symptoms and imaging abnormality after controlling for blood glucose because of the preserved motor axon. The diagnosis of acute symptomatic hypoglycemic encephalopathy through clinical and imaging features can be challenging. It is crucial to differentiate it from ischemic encephalopathy since the management and clinical outcome are different.The online version of this article (10.1186/s12880-019-0310-z) contains supplementary material, which is available to authorized users. The diagnosis of hypoglycemia-induced encephalopathy can be challenging clinically and can even be confounded by unusual neuroimaging findings. Hypoglycemia can be caused by a spectrum of medical conditions but is most commonly a result of underlying diabetes mellitus (DM). In patients with type 1 DM, hypoglycemia is caused by reduced sympathoadrenal responses, causing “hypoglycemia unawareness” . FurtherA 72-year-old woman presented with poor appetite and was initially drowsy at home; the symptoms progressed to loss of consciousness accompanied by mild incontinence. Therefore, the patient was admitted to the emergency department with an initial glucose level of 44 mg/dL, and no nausea, vomiting, fever, or cold sweating was reported. After intravenous glucose supplementation, she partially recovered consciousness , and her serum glucose level increased to 242 mg/dL. Physical examination revealed the absence of focal neurological signs, facial palsy, and tongue or eye deviations; however, mildly increased deep tendon reflexes were noted at the bilateral lower limbs. The images obtained 24 h after symptoms onset revealed symmetrical hyperintensities on DWI b-value: 1000) associated with hypointensities on ADC map along the corticospinal tract, from the levels of the cerebral peduncle and the posterior limbs of the internal capsule to the level of the corona radiata, but there was no abnormal signal on T2-fluid attenuated inversion recovery (FLAIR) images


A novel splicing isoform of MDV UL15 could also be confirmed by mass spectrometry and RT-PCR. In addition, we could demonstrate that the associated transcriptional motifs are highly conserved and closely resembled those of the host transcriptional machinery. Taken together, our data allow a comprehensive re-annotation of the MDV genome with novel genes and splice variants that could be targeted in further research on MDV replication and tumorigenesis.Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and poses a serious threat to poultry health. In infected animals, MDV efficiently replicates in B cells in various lymphoid organs. Despite many years of research, the viral transcriptome in primary target cells of MDV remained unknown. In this study, we uncovered the transcriptional landscape of the very virulent RB1B strain and the attenuated CVI988/Rispens vaccine strain in primary chicken B cells using high-throughput RNA-sequencing. Our data confirmed the expression of known genes, but also identified a novel spliced MDV gene in the unique short region of the genome. Furthermore,  Marek’s disease virus (MDV), also known as Gallid alphaherpesvirus 2, causes a deadly lymphoproliferative disease in chickens. Typical clinical symptoms include immunosuppression, paralysis and polyneuritis, acute brain edema, and lymphoma that develop as early as 3 weeks post infection ,2. MDV hMDV infection is initiated by the inhalation of the virus from a contaminated environment. Macrophages and other phagocytic cells are thought to take up the virus and transfer it to lymphatic tissues, where B and T cells are infected . B cellsL) and a unique short (US) sequence that are flanked by terminal (TRL and TRS) and internal (IRL and IRS) inverted repeat regions . Th. Th56]. g events , contradSome identified splice variants would result in proteins with an altered membrane topology . This is for example the case for splice variants of pp24 and of pp38, which results in changes of the previously assessed hydrophobic anchor domains of both proteins . While pS region contains many genes that play important roles in the viral life cycle. Intriguingly, we detected splice variants of several envelope glycoproteins as described above. In addition, we identified a hitherto uncharacterized spliced transcript of a gene located downstream of SORF2A, termed SORF6  and vIL8 splicing was detected in infected primary B cells 16 hpi although more extensive splicing activity has been observed in this region of the MDV genome in vitro and in vivo ,73,74. TIn the RNA-seq data, we did not detect any reads complementary to vTR. However, this region is annotated as a hypothetical MDV gene termed RLORF1 (an arginine-rich protein/ICP0-like protein) . RLORF1 Furthermore, we detected several poly(A) cleavage sites in combination with appropriate motifs within the MDV repeat regions, that are to our knowledge undescribed. The presence of these transcriptional signals in combination with sufficient read coverage suggest the existence of hitherto hypothetical protein coding regions, including RLORF11 and MDV082.Although we enriched for poly(A) mRNA, some newly identified introns do not lie in annotated MDV coding sequences and some splice donor and acceptor sites do not give rise to conclusive protein-encoding mRNAs. Such sequences could be easily regarded as ‘nonsense’ transcripts that are rapidly degraded. However, the importance of noncoding RNAs (ncRNAs) in MDV infections is expanding. Several viral ncRNAs were found to be expressed by MDV ,76,77,78Taken together, our MDV RNA-seq data provide novel insights into the transcriptional profile of the RB1B and CVI988 strains. Despite stark differences in their pathogenicity, the two viruses show a similar transcriptomic profile in primary chicken B cells.B cells are a major target for lytic MDV replication in vivo ,10; howeS region of the MDV genome that we will characterize in future studies. While some of the detected splice sites were previously published, we identified several novel splice variants and confirmed some of them by RT-PCR and/or MS. However, more work is certainly required to dissect their relevance in the MDV life cycle. The RNA-seq revealed the expression of 94 MDV transcripts and the presence of 71 introns that lead to mostly novel splice forms and antisense transcripts. In addition, we could identify a novel gene in the UWe found that MDV produces bicistronic and polycistronic transcripts as a mechanism to maximize its coding capacities. Poly(A) cleavage after the upstream AATAAA motif seems to be the most frequent and functional polyadenylation signal in MDV. The identification of possible alternative transcript termination (ATT) needs further experimental evidence . ATT is The comparison of the transcriptome between the very virulent RB1B strain and the CVI988 vaccine revealed differences in only a few transcripts . HoweverIn summary, our data demonstrate that the MDV genome is more complex than previously assumed. It provides a source of reference for MDV transcripts expressed in primary chicken B cells and lays the foundation for future research on MDV-encoded gene products and splice variants.


Escherichia coli (ExPEC) strain PPECC42 under hyper-osmotic conditions. In this study we investigated the role of OmpX in biofilm formation and curli production of ExPEC PPECC42. Our data showed that OmpX disruption or overexpression didn't significantly affect the biofilm formation and curli production of the wild-type strain. However, in the tolC-deleted mutant, overexpressing OmpX suppressed the effect of TolC inactivation on ExPEC biofilm formation and curli production under hyper-osmotic growth conditions. Real-time qRT-PCR confirmed that OmpX overexpression affected curli production by regulating the transcription of the curli biosynthesis-related genes in the ΔtolC strain. Our findings suggest that OmpX is involved in biofilm formation and curli production.Our previous study showed that the inactivation of the efflux pump TolC could abolish biofilm formation and curli production of extraintestinal pathogenic  Escherichia coli (ExPEC) are a sub group of pathogenic E. coli strains causing a variety of infections and diseases at extraintestinal sites in humans and animals, which are typically characterized by multi-organ infections including urinary tract infections, meningitis, polyserositis, and septicemia .Our previous study showed that inactivation of TolC compromised the ability of biofilm formation and curli production in porcine ExPEC strain PPECC42 in response to hyper-osmotic conditions      . An isogenic ompX-deleted strain of ExPEC PPECC42, in which a 345 bp fragment was deleted within the ompX open reading frame (ORF), was constructed as described previously  using a BioPhotometer . The data were obtained from three independent experiments, with each having three biological replicates.Fresh LB medium, M9 or 1/2 M9 minimal medium was inoculated with a 1:100 dilution of overnight cultures and incubated at 37°C under shaking at 200 630) using a Universal Microplate Reader . All assays were performed with 12 replicates. The cutoff OD (ODc) was defined as three times standard deviation above the mean OD of the negative control.Biofilm formation was evaluated using crystal violet assay as described previously  and 20 μg/mL Coomassie brilliant blue  as described previously  method  from the cells of each strain cultured in different conditions. cDNA was reverse transcribed from 2 μg of RNA using HiScript TM first strand cDNA synthesis kit , and used as the template for qRT-PCR with AceQ™ qPCR SYBR Green Master Mix Kit  using a Bio-Rad detection system . The expression of the target genes in each strain was normalized to that of the house-keeping gene p < 0.05.Statistical analyses were performed using GraphPad prism version 5.0 software . Statistical difference was calculated using the One-way ANOVA test and considered significant at a ::ompX into each strain. The expression level of OmpX in each strain was analyzed by using real-time qRT-PCR. As shown in Figure ompX mutant, but was detected in the ΔtolC::ompX and WT::ompX strains, suggesting that the strains construction was successful. The growth curves of each strain were determined in LB medium, M9 minimal medium, and 1/2 M9 minimal medium, and the results showed that all the strains presented a similar growth pattern to the WT strain  or deleted ompX (indicated as ompX) showed a comparable level of biofilm formation to the wild-type strain (WT), indicating the deletion or overexpression of OmpX alone does not affect the biofilm formation of the wild-type ExPEC.We next investigated the effect of OmpX deletion and overexpression on biofilm formation of ExPEC. Cells were grown in 1/2 M9 or M9 medium at 28°C for 120 h and the biofilm formation was measured using crystal violet biofilm assay. As shown in Figures tolC led to decreased biofilm formation when the cells were grown in M9 medium  restored the ability to form biofilm to a similar level of the wild-type strain  assay was used to test the curli production. As shown in Figure tolC, ΔtolC::ompX, and WT::ompX, presented a rdar  morphology on 1/2 M9-CR agar plates, indicating normal curli production. However, in M9 medium and under 0.06 M NaCl- or 0.8% sucrose-induced hyper-osmotic conditions, in contrast with the wild-type strain that still presented a rdar morphology, the ΔtolC strain showed a significantly whiter morphology, which was consistent with our previous study that the disruption of tolC reduced the curli production. Intriguingly, overproducing OmpX restored the morphology of the cells of ΔtolC strain to a similar level to the WT strain. This result suggests that OmpX overexpression suppresses the reduced curli production resulted from tolC deletion under hyper-osmotic stresses.Our previous study showed that the inactivation of csgD and csgB, were further tested in the four strains grown in the M9 medium. As shown in Figure tolC mutant showed a significantly down-regulated expression of both of csgD and csgB genes. In contrast, the ΔtolC::ompX strain exhibited a much higher expression level of sboth genes than the ΔtolC strain. The results further suggested that the regulation of OmpX overexpression on curli production is due to its effect on the transcription of the curli biosynthesis-related genes.The transcription levels of two separate curli biosynthesis-related genes, tolC mutant, indicating potential interplays between TolC and OmpX. Hence, this study was designed to explore the role of OmpX in ExPEC biofilm formation and curli production, especially under hyper-osmotic conditions, and to determine whether OmpX mediated the effect of TolC inactivation on ExPEC biofilm formation.Biofilms can increase bacterial resistance to external environmental stresses, and help the bacteria survive in different hostile environments (Steenackers et al., tolC background OmpX overexpression significantly suppressed the inhibited biofilm formation and curli production under hyper-osmotic conditions. TolC is able to regulate many proteins and interact with other outer membrane proteins in E. coli under different concentrations of glucose (Yang et al., tolC increased the transcription of ompC and micF under high osmotic conditions, whereafter reduced the amount of OmpF (Misra and Reeves, E. aerogenes (Dupont et al., E. coli (Otto and Hermansson, Our data showed that the disruption or overexpression of OmpX alone did not have any effect on biofilm formation; however, in the ΔTherefore, this study suggests that TolC and OmpX play important roles in responding to and resisting osmotic stresses. When confronting hyper-osmotic conditions, the bacterial cells lacking TolC became unable to carry out proper osmolarity regulations, therefore were sensitive to these stresses. Under this circumstance, OmpX overexpression may modify the expression of NaCl or sucrose transportation related proteins or signal pathways, increase the efflux or decrease the influx of NaCl or sucrose across the outer membrane, and thus relieve the hyper-osmolarity stresses and regulate curli production and biofilm formation. The current study provides a new insight into the role of OmpX in bacterial biofilm formation and environmental osmotic adaptation.BL, QH, AC, XL, BH, and LZ performed research. QH, BL, ML, XM, and SL designed research and analyzed the data. BL, QH, and SL wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Danio rerio) have emerged as a valuable vertebrate model to study cardiovascular development over the last few decades. The facile genetic manipulation via forward and reverse genetic approaches combined with noninvasive, high-resolution imaging and phenotype-based screening has provided new insights to molecular pathways that orchestrate cardiac development. Zebrafish can recapitulate human cardiac pathophysiology due to gene and regulatory pathways conservation, similar heart rate and cardiac morphology and function. Thus, generations of zebrafish models utilize the functional analysis of genes involved in CAD, which are derived from large-scale human population analysis. Here, we highlight recent studies conducted on cardiovascular research focusing on the benefits of the combination of genome-wide association studies (GWAS) with functional genomic analysis in zebrafish. We further summarize the knowledge obtained from zebrafish studies that have demonstrated the architecture of the fundamental mechanisms underlying heart development, homeostasis and regeneration at the cellular and molecular levels.Coronary artery disease (CAD) is the leading form of cardiovascular disease (CVD), which is the primary cause of mortality worldwide. It is a complex disease with genetic and environmental risk factor contributions. Reports in human and mammalian models elucidate age-associated changes in cardiac function. The diverse mechanisms involved in cardiac diseases remain at the center of the research interest to identify novel strategies for prevention and therapy. Zebrafish ( According to the World Health Organization (WHO) global observatory data, 17.9 million people die each year from cardiovascular diseases (CVDs) which is estimated to be 31% of global deaths .Nevertheless, population studies are revealing the genetic basis of CAD, highlighting also the complexity of its genetic causality . The devDanio rerio) have provided significant advantages regarding our understanding of cardiovascular disease causality. The biological characteristics, ease of genetic manipulation, facilitation of chemical screening and genetic similarity to humans are only some of the features that contributed to the emergence of this animal model as a useful and valuable tool for the cardiovascular research field. A set of zebrafish models has been generated to study lipid metabolism and hypercholesteremia, which are known major risk factors for development of cardiovascular disease. Zebrafish ldlr mutants were validated as a good model to study vascular lipid accumulation, a hallmark of human atherosclerosis [Danio rerio could be also used to study age-associated cardiac disease.Although technological advantages provide cumulative information of genetic causality, the prediction of pathogenicity and the interpretation of clinical consequences of a genetic variant remain challenging. There are several strategies to validate the candidate variants derived from sequencing approaches. Bioinformatic tools provide a powerful advantage to prioritize the candidates by using computational methodologies and algorithms. However, this is largely limited to common variants, as they demand accurate differences in allele frequencies. As a consequence, rare and de novo variants considered as of uncertain significance are categorized between benign and pathogenic ,7. Anothclerosis . Zebraficlerosis ,10,11. AIn this review, we highlight the importance of zebrafish models for the functional analysis of genes involved in CVDs derived from large-scale human population analysis. We summarize the experimental studies that have been conducted in zebrafish models and how these have augmented the understanding of genes relevant to CAD physiology.Danio rerio) are a small tropical fish belonging to the minnow family Cyprinidae and are native to Southeast Asia. In recent years, zebrafish have emerged as an excellent model system in biomedical research due to its advantageous characteristics. The experimental value of this organism lies in the general biological and physiological features it possesses as well as in the technical and genetic manipulations that can be conducted. Zebrafish are small fish and each breeding pair can produce a large number of offspring weekly. The embryos develop rapidly, exhibiting optical transparency during the first hours post fertilization (hpf) that allow direct observation using light microscopy. Another advantage over mammalian models is the external fertilization and embryonic development, which allows non-invasive techniques to be applied in order to monitor the early developmental stages. Furthermore, zebrafish embryos have the ability to fully function without blood circulation for 4–5 days post fertilization (dpf) by obtaining adequate oxygen through passive diffusion due to their small size. This characteristic gives the advantage to generate and study models of severe developmental cardiovascular disorders that are embryonic lethal in mice [Zebrafish  that revealed many interesting features when compared to the human genome . It was ralogues . At thisaralogue . Accumulegfl7  mutants have no phenotype, while knockdown of egfl7 leads to severe vascular defects. It was demonstrated that upregulation of other extracellular matrix (ECM) genes  have occurred in egfl7 mutants but not in knockdown embryos. Despite its fundamental role in whole organism robustness, the mechanisms that drive genetic compensation remain poorly understood. In a recent review, two models were proposed: (a) the DNA damage response induces chromatin reorganization so as to increase chromatin accessibility to compensatory gene regulators, and (b) the mutated regions produce transcripts that are targeted for degradation and subsequently, the RNA fragments induce a compensatory response either by triggering chromatin remodeling or guiding common (for mutated and compensated genes) RNA binding proteins (RBP) and/or miRNAs to act instead of compensating gene mRNA stabilization [Genetic compensation has been documented in a number of animal models as a mechanism to fine-tune their transcriptome in order to adapt their fitness and maintain their viability caused by genetic changes. There are a lot of studies focusing on the functional and genetic compensation established in model systems, like Arabidopsis ,26, yeaslization .N-ethyl-N-nitrosourea (ENU) to induce random mutation, is widely used in zebrafish as it can overcome the limitations of the other vertebrate models like suitable breeding scheme, genomic architecture, large-scale husbandry and ease in monitoring early developmental phenotypes [silent heart (sih) which causes a non-contractile heart phenotype [sih embryos survive until 7 dpf due to their ability to uptake adequate oxygen though diffusion and so this line serves as an excellent model to study the structural changes and molecular pathways involved in cardiac disorders while this phenotype would be lethal in other vertebrates. The combination of ENU mutagenesis with transgenic lines harboring reporter genes such as Tg(flk:EGFP) and screens on specific cardiac development and morphogenesis have also been conducted [Despite the overall biological limitations, there is a wide toolbox for genetic engineering that facilitates the modeling of human diseases and we will summarize the most commonly used tools to manipulate the zebrafish genome. Forward and reverse approaches have been successfully applied in zebrafish to identify molecular pathways and to elucidate the role of known or novel genes related to human diseases such as CAD. Forward genetics, i.e. by using chemical mutagens like enotypes . Numerouenotypes ,39,40,41enotypes ,43. Althonducted ,44. Anotonducted . This vaonducted ,47,48.In recent years, zebrafish have become an attractive model system due to the successful application of reverse genetic approaches as well. The most commonly applied technique is gene knockdown by injecting morpholino oligomer molecules (or simply “morpholino”) at the one-cell stage zebrafish embryos to investigate if knocking down a gene causes a phenotype. Morpholinos (MO) are chemically modified oligonucleotides with base-pairing ability similar to the natural oligomers and are effective in a dose-dependent manner . The MOsabcc9, kcnj8 and pln) encode for subunits of an ATP-sensitive potassium channel (KATP) linked to Cantú syndrome, a rare genetic syndrome included in cardiovascular disorders. As CRISPR/Cas9 methodology evolves, the capacity of this system to generate specific patient-derived mutations in animal models promises a broad application paving the way for new therapeutic strategies [Fortunately, recent advances in reverse genetic approaches also revolutionized the toolbox of zebrafish genome engineering. Genome editing with zing finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered, regularly interspaced, short, palindromic repeats/Cas (CRISPR-associated) (CRISPR/Cas) systems were applied for targeted genome modifications ,57,58,59rategies ,63.tomo-seq technique [tomo-seq in combination with high-throughput RNA sequencing generated a high-resolution genome-wide atlas of gene expression in the regenerating zebrafish heart [To improve our understanding on heart formation during development, specific pathways and regulators must be identified as well as their role under diseased conditions. A recently developed strategy focusing on unraveling genes restricted to well-defined regions of the heart to ensure proper function, morphology and shape is the echnique ,65. Thissh heart ,67. ThesSince the zebrafish has proven to be an excellent model for human cardiac research, another approach to study cardiovascular diseases is the structure of in vitro three-dimensional (3D) heart-like cell aggregates, consisting of myocardial tissue formed spontaneously from enzymatically digested whole embryonic zebrafish larvae (zebrafish heart aggregate(s)—ZFHA(s)) ,68. The Genetic disposition, jointly with traditional risk factors, is considered to manifest in cardiovascular diseases. For many decades, epidemiological studies focused on the effort of unraveling the causes and specific predictors for CVD. Up to now, modifiable and non-modifiable risk factors such as obesity, hypertension, smoking, diabetes, blood pressure, and sex have been studied ,72,73,74KCNIP1) which associated to AF susceptibility. In a recent study, it was demonstrated that KCNIP1-knockdown and overexpression in zebrafish hearts modulates atrial rates [KCNIP1 gene determined the mRNA level of KCNIP1, and KCNIP1-encoded protein KCHIP1 was linked to the mechanism of AF, maintaining high atrial rates which indicates a possible target for AF treatment [KCNH2 mutation (potassium voltage-gated channel subfamily H member 2), which has been identified in long QT syndrome type 2 (LQT2) patients, was evaluated in larval zebrafish [KCNH2 mutation using microscopy and electrophysiology. Another study focused on the transcription factor PRRX1 as it is a strong candidate gene based on previous genome-wide association studies on AF [PRRX1 locus (~158 kb region in AF cases) to identify common and rare SNPs variants, they confirmed that the causative variant resides in an upstream enhancer of PRRX1 modulated by SNP rs577676, which reduces PRRX1 expression. CRISPR-Cas9-mediated knockout of PRRX1 in human embryonic stem cells (hESCs) and morpholino-mediated knockdown of the putative PRRX1 orthologues in the zebrafish (prrx1a and prrx1b) showed that even a small modulation of PRRX1 expression may be sufficient to modulate the atrial action potential duration (APD), a hallmark of AF.Atrial fibrillation (AF) is the most common sustained arrhythmia and can lead to heart failure and cardiovascular death. Due to the similarity of electrical properties of zebrafish heart to those of the human heart, zebrafish have been established as a powerful animal model to study cardiac arrhythmias ,18,20,79al rates . Intronireatment . Later oebrafish . By usines on AF . In thishspb7 mutants (TALEN-generated) display mild focal cardiac fibrosis, cardiomegaly and sarcomeric abnormalities. They also identified large cytoskeletal proteins Filamin C (FLNC) and TITIN as HSPB7 binding partners and proposed that hspb7 functions through a damage-induced network as loss of hspb7 stimulated autophagic pathways and inhibition of autophagy (treatment with bafilomycin A (BafA) and chloroquine (CQ) inhibitors) in hspb7 mutants, resulting in increased sensitivity and more severe cardiac defects. Another group used single-stranded oligodeoxynucleotides to precisely introduce the human PBX3 p.A136V variant in the homologous zebrafish pbx4 gene through CRISPR-Cas9 genome editing in order to test whether this Pbx gene variant  patient cohort) acts as a genetic modifier in zebrafish heart development [pbx4 p.A131V variant enhances myocardial morphogenesis defects caused by loss of the CHD gene, cardiac specification factor, hand2. The study provides an example of precision genome editing in zebrafish to demonstrate a function for a human disease-associated variant.GWAS studies on idiopathic cardiomyopathies have implicated polymorphisms in small heat shock protein, beta 7 (HSPB7) as potential contributors and a recently published work highlighted its cardioprotective role . The reselopment . It was tensin1 (TNS1) after functional analysis using zebrafish embryos [lmcd1 morphants exhibit significantly increased atrioventricular regurgitation and moderate reduction in cardiac looping while a similar phenotype was observed for tensin1, thus supporting their role as candidate genes for MVP pathogenesis. Atrioventricular septal defects (AVSD) also represent abnormalities in atrioventricular valves, and atrial and ventricular septa. The study by Ferese et al., identified NFATC1 rare variants in a small but significant proportion of cases from two cohorts of AVSD patients [nfatc1 zebrafish mutants, providing evidence of their functional relevance in vivo and supporting a role of defective NFATC1 function in the etiology of AVSD. Another recent study aimed to functionally characterize the zebrafish orthologues of six human candidate genes  in GWAS-derived loci for heart rate variability using a large-scale, image-based screen in zebrafish embryos and larvae [HCN4 as a druggable gene using the Drug Gene Interaction Database (DGIdb) and revealed several interaction partners for all the tested causal genes that can be drug-treated based on the current pharmaceutical standards.Valvulopathies are diseases of the cardiac valves  and non-syndromic mitral valve prolapse (MVP) is a common degenerative valvulopathy that can cause heart failure and sudden death. Studies in zebrafish revealed the effect of intracardiac flow dynamics on their development . A meta- embryos . The zebpatients . The autd larvae . The autLDLR, APOB and PCSK9, as well as SNPs at numerous loci) have been reported, only approximately 20% of the variation in low-density lipoprotein cholesterol (LDL-C) levels is explained [APOOP1, increased the LDL-C levels on both the control and those supplemented with a 4% w/w cholesterol (HCD) larval diet and upregulated the expression of genes involved in cholesterol synthesis. In addition, overexpression of APOOP1 resulted in an approximately 20-fold increase in the average number of vascular plaques and suppression of its parent gene, apO, increased LDL-C and plaques suggesting a regulatory interaction between these two genes. Another factor that could eventually increase the risk for CVD is the excessive lipid deposition within adipose tissue (AT). A large-scale meta-analyses of GWAS has identified several loci associated with this trait including Plexin D1 (PLXND1)—a gene known to modulate angiogenesis—and Minchin et al., performed a functional analysis to elucidate its role in body fat distribution in a zebrafish system [plxnd1 had a reduced capacity to store lipids in visceral AT (VAT) and that type V collagens were upregulated, suggesting that they mediated the inhibitory effect of Plxnd1 on VAT growth. One more study that used the zebrafish model to validate a rare causal gene involved in congenital cardiomyopathy resulting in a lethal restrictive phenotype was performed by Louw et al. [KIF20A gene and demonstrated that MO-mediated kif20a knockdown zebrafish embryos develop a progressive cardiac phenotype , suggesting that kif20a is evolutionary conserved in heart development and required for proper heart function. These last studies are examples of how the zebrafish model system facilitates the validation and functional analysis of genes not only derived from large-scale gene association studies, but also can be a platform of studying novel and rare genetic variants that contribute to multifactorial complex diseases  and heart failure (HF), cellular and molecular mechanisms underlying their pathophysiology still remain unclear. During HF, there is a progressive loss of cardiomyocytes (CMs), which eventually leads to the formation of fibrotic, non-functional scar development. In order to gain deeper knowledge into human cardiac repair, various injury models have been established. Although newborn mice exhibit a heart regenerative capacity, it is lost by seven days after birth and the adult mammalian hearts retain a low capacity for regeneration, mainly due to the cell-cycle arrest of CMs ,102,103.col1a2-expressing fibroblasts impaired CM proliferation and scar resolution [The role of cell-specific contributions and immune responses during heart repair has been extensively studied ,105. Amosolution . Thus, treg) which infiltrate the damaged heart and produce tissue-specific mitogens essential for robust regeneration [In addition to cardiac tissue activation, immune cell populations respond to heart injury by promoting inflammation. A transient delay of macrophage recruitment, using clodronate liposome (CL) injections one day prior to injury was sufficient to disrupt neovascularization, neutrophil clearance and heart regeneration following cardiac injury in zebrafish . Neutropneration . Finallyneration . Therefoneration ). ParallIn summary, zebrafish have emerged as a valuable tool in biomedical research and contributed to the deeper understanding of cardiac development and pathophysiology. Due to the efforts of forward genetic screens and manipulation, zebrafish research has been pivotal in unraveling major factors of cardiac function as well as the molecular pathways in which they are involved. The efforts that had been made out of the combination of population-based GWAS and functional analysis in zebrafish models shed light on uncharacterized mechanisms that give the knowledge to design new prognostic and therapeutic strategies. The growing toolbox of transgenic zebrafish lines, tissue-specific genetic manipulation, high-resolution imaging and high-throughput chemical screens will provide an excellent model in clinical and basic research to dissect the architecture of cardiac diseases.