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Development of tomato varieties and hybrids with resistance to the pathogen Fusarium oxysporum f sp. Lycopersici is a priority in breeding of vegetable crops. Evaluation and selection of the initial material is important in effective breeding for resistance to a pathogen. To do this, along with classical methods in tomato breeding, the method of molecular marking is widely used. The purpose of this study was to screen the existing collection of tomatoes with molecular markers I-2/5, I2OH and At-2, which allow identification of resistance genes to the first and second races of the fusarium pathogen. As a result of the work, genotypes were identified that can be used as donors of resistance genes for further breeding work.

Abstract Background Mercury is a prominent environmental contaminant that causes detrimental effects to human health. Although the liver has been known to be a main target organ, there is limited information on in vivo molecular mechanism of mercury-induced toxicity in the liver. By using transcriptome analysis, phenotypic anchoring and validation of targeted gene expression in zebrafish, mercury-induced hepatotoxicity was investigated and a number of perturbed cellular processes were identified and compared with those captured in the in vitro human cell line studies. Results Hepato-transcriptome analysis of mercury-exposed zebrafish revealed that the earliest deregulated genes were associated with electron transport chain, mitochondrial fatty acid beta-oxidation, nuclear receptor signaling and apoptotic pathway, followed by complement system and proteasome pathway, and thereafter DNA damage, hypoxia, Wnt signaling, fatty acid synthesis, gluconeogenesis, cell cycle and motility. Comparative meta-analysis of microarray data between zebrafish liver and human HepG2 cells exposed to mercury identified some common toxicological effects of mercury-induced hepatotoxicity in both models. Histological analyses of liver from mercury-exposed fish revealed morphological changes of liver parenchyma, decreased nucleated cell count, increased lipid vesicles, glycogen and apoptotic bodies, thus providing phenotypic evidence for anchoring of the transcriptome analysis. Validation of targeted gene expression confirmed deregulated gene-pathways from enrichment analysis. Some of these genes responding to low concentrations of mercury may serve as toxicogenomic-based markers for detection and health risk assessment of environmental mercury contaminations. Conclusion Mercury-induced hepatotoxicity was triggered by oxidative stresses, intrinsic apoptotic pathway, deregulation of nuclear receptor and kinase activities including Gsk3 that deregulates Wnt signaling pathway, gluconeogenesis, and adipogenesis, leading to mitochondrial dysfunction, endocrine disruption and metabolic disorders. This study provides important mechanistic insights into mercury-induced liver toxicity in a whole-animal physiology context, which will help in understanding the syndromes caused by mercury poisoning. The molecular conservation of mercury-induced hepatotoxicity between zebrafish and human cell line reveals the feasibility of using zebrafish to model molecular toxicity in human for toxicant risk assessments.

Glucose is the main energy source of the brain, yet recent studies demonstrate that fatty acid oxidation (FAO) plays a relevant role in the pathogenesis of central nervous system disorders. We evaluated FAO in brain mitochondria under physiological conditions, in the aging brain, and after stroke. Using high-resolution respirometry we compared medium-chain (MC, octanoylcarnitine) and long-chain (LC, palmitoylcarnitine) acylcarnitines as substrates of β-oxidation in the brain. The protocols developed avoid FAO overestimation by malate-linked anaplerotic activity in brain mitochondria. The capacity of FA oxidative phosphorylation (F-OXPHOS) with palmitoylcarnitine was up to 4 times higher than respiration with octanoylcarnitine. The optimal concentration of palmitoylcarnitine was 10 μM which corresponds to the total concentration of LC acylcarnitines in the brain. Maximal respiration with octanoylcarnitine was reached at 20 μM, however, this concentration exceeds MC acylcarnitine concentrations in the brain 15 times. F-OXPHOS capacity was highest in mouse cerebellum, intermediate in cortex, prefrontal cortex, and hypothalamus, and hardly detectable in hippocampus. F-OXPHOS capacity was 2-fold lower and concentrations of LC acylcarnitines were 2-fold higher in brain of aged rats. A similar trend was observed in the rat model of endothelin-1-induced stroke, but reduction of OXPHOS capacity was not limited to FAO. In conclusion, although FAO is not a dominant pathway in brain bioenergetics, it deserves specific attention in studies of brain metabolism., Competing Interests: Declaration of competing interest Luiza Helena Daltro Cardoso and Cristiane Cecatto are employees and Erich Gnaiger is the founder and CEO of Oroboros Instruments, Innsbruck, Austria. The other authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background and Purpose: Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), and exert adverse effects on mitochondrial function, although the mechanisms underlying these effects remain unclear. We used a tamoxifen-induced Hmgcr-knockout (KO) mouse model, a multi-omics approach and mitochondrial function assessments to investigate whether decreased HMGCR activity impacts key liver energy metabolism pathways., Experimental Approach: We established a new mouse strain using the Cre/loxP system, which enabled whole-body deletion of Hmgcr expression. These mice were crossed with Rosa26 Cre mice and treated with tamoxifen to delete Hmgcr in all cells. We performed transcriptomic and metabolomic analyses and thus evaluated time-dependent changes in metabolic functions to identify the pathways leading to cell death in Hmgcr-KO mice., Key Results: Lack of Hmgcr expression resulted in lethality, due to acute liver damage caused by rapid disruption of mitochondrial fatty acid β-oxidation and very high accumulation of long-chain (LC) acylcarnitines in both male and female mice. Gene expression and KO-related phenotype changes were not observed in other tissues. The progression to liver failure was driven by diminished peroxisome formation, which resulted in impaired mitochondrial and peroxisomal fatty acid metabolism, enhanced glucose utilization and whole-body hypoglycaemia., Conclusion and Implications: Our findings suggest that HMGCR is crucial for maintaining energy metabolism balance, and its activity is necessary for functional mitochondrial β-oxidation. Moreover, statin-induced adverse reactions might be rescued by the prevention of LC acylcarnitine accumulation., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Previously, we showed that fluvastatin treatment induces myofibrillar damage and mitochondrial phenotypes in the skeletal muscles of Drosophila. However, the sequential occurrence of mitochondrial phenotypes and myofibril damage remains elusive. To address this, we treated flies with fluvastatin for two and five days and examined their thorax flight muscles using confocal microscopy. In the two-day fluvastatin group, compared to the control, thorax flight muscles exhibited mitochondrial morphological changes, including fragmentation, rounding up and reduced content, while myofibrils remained organized in parallel. In the five-day fluvastatin treatment, not only did mitochondrial morphological changes become more pronounced, but myofibrils became severely disorganized with significantly increased thickness and spacing, along with myofilament abnormalities, suggesting myofibril damage. These findings suggest that fluvastatin-induced mitochondrial changes precede myofibril damage. Moreover, in the five-day fluvastatin group, the mitochondria demonstrated elevated H 2 O 2 and impaired fatty acid oxidation compared to the control group, indicating potential mitochondrial dysfunction. Surprisingly, knocking down Hmgcr (Drosophila homolog of HMGCR) showed normal mitochondrial respiration in all parameters compared to controls or five-day fluvastatin treatment, which suggests that fluvastatin-induced mitochondrial dysfunction might be independent of Hmgcr inhibition. These results provide insights into the sequential occurrence of mitochondria and myofibril damage in statin-induced myopathy for future studies., (© 2024. The Author(s).)

Right ventricular (RV) and left ventricular (LV) dysfunction is common in a significant number of hospitalized coronavirus disease 2019 (COVID-19) patients. This study was conducted to assess whether the improved mitochondrial bioenergetics by cardiometabolic drug meldonium can attenuate the development of ventricular dysfunction in experimental RV and LV dysfunction models, which resemble ventricular dysfunction in COVID-19 patients. Effects of meldonium were assessed in rats with pulmonary hypertension-induced RV failure and in mice with inflammation-induced LV dysfunction. Rats with RV failure showed decreased RV fractional area change (RVFAC) and hypertrophy. Treatment with meldonium attenuated the development of RV hypertrophy and increased RVFAC by 50%. Mice with inflammation-induced LV dysfunction had decreased LV ejection fraction (LVEF) by 30%. Treatment with meldonium prevented the decrease in LVEF. A decrease in the mitochondrial fatty acid oxidation with a concomitant increase in pyruvate metabolism was noted in the cardiac fibers of the rats and mice with RV and LV failure, respectively. Meldonium treatment in both models restored mitochondrial bioenergetics. The results show that meldonium treatment prevents the development of RV and LV systolic dysfunction by enhancing mitochondrial function in experimental models of ventricular dysfunction that resembles cardiovascular complications in COVID-19 patients.

The pancreas development depends on complex regulation of several signaling pathways, including the Hedgehog (Hh) signaling via a receptor complex component, Smoothened, which deficiency blocks the Hh signaling. Such a defect in birds and mammals results in an annular pancreas. We showed that in developing zebrafish, the mutation of Smoothened or inhibition of Hh signaling by its antagonist cyclopamine caused developmental defects of internal organs, liver, pancreas, and gut. In particular, the pancreatic primordium was duplicated. The two exocrine pancreatic primordia surround the gut. This phenomenon correlates with a significant reduction of the gut's diameter, causing the annular pancreas phenotype., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Long-chain ω -3 polyunsaturated fatty acids (PUFAs) are known to induce cardiometabolic benefits, but the metabolic pathways of their biosynthesis ensuring sufficient bioavailability require further investigation. Here, we show that a pharmacological decrease in overall fatty acid utilization promotes an increase in the levels of PUFAs and attenuates cardiometabolic disturbances in a Zucker rat metabolic syndrome model. Metabolome analysis showed that inhibition of fatty acid utilization by methyl-GBB increased the concentration of PUFAs but not the total fatty acid levels in plasma. Insulin sensitivity was improved, and the plasma insulin concentration was decreased. Overall, pharmacological modulation of fatty acid handling preserved cardiac glucose and pyruvate oxidation, protected mitochondrial functionality by decreasing long-chain acylcarnitine levels, and decreased myocardial infarct size twofold. Our work shows that partial pharmacological inhibition of fatty acid oxidation is a novel approach to selectively increase the levels of PUFAs and modulate lipid handling to prevent cardiometabolic disturbances., Competing Interests: JK, MMK, HC, SG, MD, and EL are listed as inventors in several international patent applications regarding the use of methyl-GBB for the treatment of cardiovascular and cardiometabolic diseases. None of the authors currently own any of these patents., (Copyright © 2021 Janis Kuka et al.)

Aim: Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite synthesized in host organisms from specific food constituents, such as choline, carnitine and betaine. During the last decade, elevated TMAO levels have been proposed as biomarkers to estimate the risk of cardiometabolic diseases. However, there is still no consensus about the role of TMAO in the pathogenesis of cardiovascular disease since regular consumption of TMAO-rich seafood (i.e., a Mediterranean diet) is considered to be beneficial for the primary prevention of cardiovascular events. Therefore, the aim of this study was to investigate the effects of long-term TMAO administration on mitochondrial energy metabolism in an experimental model of right ventricle heart failure. Methods: TMAO was administered to rats at a dose of 120 mg/kg in their drinking water for 10 weeks. Then, a single subcutaneous injection of monocrotaline (MCT) (60 mg/kg) was administered to induce right ventricular dysfunction, and treatment with TMAO was continued (experimental groups: Control; TMAO; MCT; TMAO+MCT). After 4 weeks, right ventricle functionality was assessed by echocardiography, mitochondrial function and heart failure-related gene and protein expression was determined. Results: Compared to the control treatment, the administration of TMAO (120 mg/kg) for 14 weeks increased the TMAO concentration in cardiac tissues up to 14 times. MCT treatment led to impaired mitochondrial function and decreased right ventricular functional parameters. Although TMAO treatment itself decreased mitochondrial fatty acid oxidation-dependent respiration, no effect on cardiac functionality was observed. Long-term TMAO administration prevented MCT-impaired mitochondrial energy metabolism by preserving fatty acid oxidation and subsequently decreasing pyruvate metabolism. In the experimental model of right ventricle heart failure, the impact of TMAO on energy metabolism resulted in a tendency to restore right ventricular function, as indicated by echocardiographic parameters and normalized organ-to-body weight indexes. Similarly, the expression of a marker of heart failure severity, brain natriuretic peptide, was substantially increased in the MCT group but tended to be restored to control levels in the TMAO+MCT group. Conclusion: Elevated TMAO levels preserve mitochondrial energy metabolism and cardiac functionality in an experimental model of right ventricular heart failure, suggesting that under specific conditions TMAO promotes metabolic preconditioning-like effects., Competing Interests: 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., (Copyright © 2021 Videja, Vilskersts, Korzh, Cirule, Sevostjanovs, Dambrova and Makrecka-Kuka.)

Purpose: Sodium-glucose cotransporter 2 (SGLT2) inhibitors prevent heart failure and decrease cardiovascular mortality in patients with type 2 diabetes. Heart failure is associated with detrimental changes in energy metabolism, and the preservation of cardiac mitochondrial function is crucial for the failing heart. However, to date, there are no data to support the hypothesis that treatment with a SGLT2 inhibitor might alter mitochondrial bioenergetics in diabetic failing hearts. Thus, the aim of this study was to investigate the protective effects of empagliflozin on mitochondrial fatty acid metabolism., Methods: Mitochondrial dysfunction was induced by 18 weeks of high-fat diet (HFD)-induced lipid overload. Empagliflozin was administered at a dose of 10 mg/kg in a chow for 18 weeks. Palmitate metabolism in vivo, cardiac mitochondrial functionality and biochemical parameters were measured., Results: In HFD-fed mice, palmitate uptake was 1.7, 2.3, and 1.9 times lower in the heart, liver, and kidneys, respectively, compared with that of the normal chow control group. Treatment with empagliflozin increased palmitate uptake and decreased the accumulation of metabolites of incomplete fatty acid oxidation in cardiac tissues, but not other tissues, compared with those of the HFD control group. Moreover, empagliflozin treatment resulted in fully restored fatty acid oxidation pathway-dependent respiration in permeabilized cardiac fibers. Treatment with empagliflozin did not affect the biochemical parameters related to hyperglycemia or hyperlipidemia., Conclusion: Empagliflozin treatment preserves mitochondrial fatty acid oxidation in the heart under conditions of chronic lipid overload.

The suppression of energy metabolism is one of cornerstones of cardiac dysfunction in sepsis/endotoxaemia. To investigate the role of fatty acid oxidation (FAO) in the progression of inflammation-induced cardiac dysfunction, we compared the effects of FAO-targeting compounds on mitochondrial and cardiac function in an experimental model of lipopolysaccharide (LPS)-induced endotoxaemia. In LPS-treated mice, endotoxaemia-induced inflammation significantly decreased cardiac FAO and increased pyruvate metabolism, while cardiac mechanical function was decreased. AMP-activated protein kinase activation by A769662 improved mitochondrial FAO without affecting cardiac function and inflammation-related gene expression during endotoxaemia. Fatty acid synthase inhibition by C75 restored both cardiac and mitochondrial FAO; however, no effects on inflammation-related gene expression and cardiac function were observed. In addition, the inhibition of carnitine palmitoyltransferase 2 (CPT2)-dependent FAO by aminocarnitine resulted in the accumulation of FAO intermediates, long-chain acylcarnitines, in the heart. As a result, cardiac pyruvate metabolism was inhibited, which further exacerbated inflammation-induced cardiac dysfunction. In conclusion, although inhibition of CPT2-dependent FAO is detrimental to cardiac function during endotoxaemia, present findings show that the restoration of cardiac FAO alone is not sufficient to recover cardiac function. Rescue of cardiac FAO should be combined with anti-inflammatory therapy to ameliorate cardiac dysfunction in endotoxaemia., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Significant impairments in mitochondrial function are associated with the development of multi-organ failure in sepsis/endotoxaemia, but the data on the dynamics of simultaneous mitochondrial impairment in multiple organs are limited. The aim of this study was to evaluate the changes in heart, brain and kidney mitochondrial function in an experimental model of lipopolysaccharide (LPS)-induced endotoxaemia.Samples were collected 4 and 24 h after single injection of LPS (10 mg/kg) in mice. Marked increases in inflammation-related gene expression were observed in all studied tissues 4 h after LPS administration. At 24 h post LPS administration, this expression of inflammation-related genes remained upregulated only in kidneys. Significantly increased concentrations of kidney function markers confirmed that kidneys were severely damaged. Echocardiographic measurements showed that the ejection fraction and fractional shortening were significantly reduced 4 h after LPS administration, whereas 24 h after LPS administration, the cardiac function was restored to baseline. A two-fold decrease in mitochondrial oxidative phosphorylation (OXPHOS) capacity in the kidney was observed 4 and 24 h after LPS administration. Significant decrease in mitochondrial fatty acid oxidation was observed in heart 4 h after LPS administration. Furthermore, 24 h after LPS administration, the respiration rates in cardiac fibers at OXPHOS and electron transport (ET) states were significantly increased, which resulted in increased ET coupling efficiency in the LPS-treated group, whereas four-fold increases in the H2O2 production rate and H2O2/O ratio were observed. The brain mitochondria demonstrated a slightly impaired mitochondrial functionality just 24 h after the induction of endotoxaemia.In conclusion, among studied tissues kidney mitochondria are the most sensitive to endotoxaemia and do not recover from LPS-induced damage, whereas in brain, mitochondrial function was not significantly altered. In heart, endotoxaemia induces a decrease in the mitochondrial fatty acid oxidation capacity, but during the phase of suppressed inflammatory response, the ET efficiency is improved despite the marked increase in reactive oxygen species production.

We have developed a multi-channel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo. The Fish and Chips was micro-fabricated in silicon and glass for reproducibility and accuracy of the microfluidic structure. The microfluidic platform consists of three parts: a microfluidic gradient generator, a row of eight fish tanks, in which the fish embryos are individually placed, and eight output channels. The fluidic gradient generator supports dose-dependent drug and chemical studies. A unique perfusion system ensures a uniform and constant flow of media to the fish tank while the wastes are efficiently removed. The fish tanks restrict the embryo movements, except rotationally, for live imaging of internal tissues and organs. The embryos showed developmental abnormalities under the influence of the drug valproic acid (VPA).

The early and long-term results of 225 gastrectomies, performed on the reason of gastric cancer, were analyzed. The comparative analysis of early and long-term results showed the statistically significant differences in complication and lethality rates between apparate and manual esophagoenteroanastomoses, especially considering the rate of reflux-esophagitis.

Betaine homocysteine S-methyltransferase (BHMT, EC 2.1.1.5) is a key enzyme in the methionine cycle and is highly expressed in the liver. Despite its important biochemical function, it is not known whether BHMT plays a role during organ development. In this report, we showed that early in development of zebrafish before endoderm organogenesis, bhmt is first expressed in the yolk syncytial layer and then after liver formation becomes a liver-enriched gene. By using the anti-bhmt morpholinos that deplete the Bhmt, we found that in morphant embryos, several endoderm-derived organs, including liver, exocrine pancreas, and intestine are hypoplastic. Strikingly, the number of β-cells in the pancreatic islet was increased rather than reduced in the morphant. Additional studies showed that Bhmt depletion elevates the sonic hedgehog (shh) transcript level in the morphant, whereas Bhmt-depletion in the Shh-deficient mutant syu failed to rescue the isletless phenotype. These molecular and genetic data strongly suggest that Shh functions downstream of Bhmt to promote β-cell development. Therefore, although there are still many intriguing questions to be answered, our finding may identify a novel function for Bhmt involving modulation of Shh signaling to control β-cell development.

Developmental patterning and growth of the vertebrate digestive and respiratory tracts requires interactions between the epithelial endoderm and adjacent mesoderm. The esophagus is a specialized structure that connects the digestive and respiratory systems and its normal development is critical for both. Shh signaling from the epithelium regulates related aspects of mammalian and zebrafish digestive organ development and has a prominent effect on esophageal morphogenesis. The mechanisms underlying esophageal malformations, however, are poorly understood. Here, we show that zebrafish Ihha signaling from the epithelium acting in parallel, but independently of Shh, controls epithelial and mesenchymal cell proliferation and differentiation of smooth muscles and neurons in the gut and swimbladder. In zebrafish ihha mutants, the esophageal and swimbladder epithelium is dysmorphic, and expression of fgf10 in adjacent mesenchymal cells is affected. Analysis of the development of the esophagus and swimbladder in fgf10 mutant daedalus (dae) and compound dae/ihha mutants shows that the Ihha-Fgf10 regulatory interaction is realized through a signaling feedback loop between the Ihha-expressing epithelium and Fgf10-expressing mesenchyme. Disruption of this loop further affects the esophageal and swimbladder epithelium in ihha mutants, and Ihha acts in parallel to but independently of Shha in this process. These findings contribute to the understanding of epithelial-mesenchymal interactions and highlight an interaction between Hh and Fgf signaling pathways during esophagus and swimbladder development., (Copyright © 2011. Published by Elsevier Inc.)

Background: Wnt signaling plays critical roles in mammalian lung development. However, Wnt signaling in the development of the zebrafish swimbladder, which is considered as a counterpart of mammalian lungs, have not been explored. To investigate the potential conservation of signaling events in early development of the lung and swimbladder, we wish to address the question whether Wnt signaling plays a role in swimbladder development., Methodology/principal Findings: For analysis of zebrafish swimbladder development, we first identified, by whole-mount in situ hybridization (WISH), has2 as a mesenchymal marker, sox2 as the earliest epithelial marker, as well as hprt1l and elovl1a as the earliest mesothelial markers. We also demonstrated that genes encoding Wnt signaling members Wnt5b, Fz2, Fz7b, Lef1, Tcf3 were expressed in different layers of swimbladder. Then we utilized the heat-shock inducible transgenic lines hs:Dkk1-GFP and hs:ΔTcf-GFP to temporarily block canonical Wnt signaling. Inhibition of canonical Wnt signaling at various time points disturbed precursor cells specification, organization, anterioposterior patterning, and smooth muscle differentiation in all three tissue layers of swimbladder. These observations were also confirmed by using a chemical inhibitor (IWR-1) of Wnt signaling. In addition, we found that Hedgehog (Hh) signaling was activated by canonical Wnt signaling and imposed a negative feedback on the latter., Significance/conclusion: We first provided a new set of gene markers for the three tissue layers of swimbladder in zebrafish and demonstrated the expression of several key genes of Wnt signaling pathway in developing swimbladder. Our functional analysis data indicated that Wnt/β-catenin signaling is required for swimbladder early development and we also provided evidence for the crosstalk between Wnt and Hh signaling in early swimbladder development.

Although Wnt and Hedgehog (Hh) signaling pathways play important roles in mouse lung development, these have not been explored in the development of Xenopus lung. This may be due to the lack of specific molecular markers for different layers of tissue in Xenopus lung and/or insufficient knowledge on expression patterns of Wnt and Hh signaling components in Xenopus lung. In this study, we first described the early morphogenesis of Xenopus laevis lung by using surfactant protein C (sftpc) as a marker of lung epithelium and compared it with the expression patterns of several genes of Wnt and Hh pathways in Xenopus lungs. Our data showed that wnt7b was expressed in the entire lung epithelium from stage 37 to stage 45, while two other Wnt signaling components, wnt5a and wif1 (wnt inhibitory factor 1), were expressed in the mesenchyme layer of the entire lungs through stages 39-41. We also found that sonic hedgehog (shh) was expressed at stage 41 only in the anterior, but not in the posterior part of the lungs. These results show the expression of wnt5a, wnt7b, wif1 and shh in different layers of tissue of Xenopus lungs at early developmental stages, which implies different roles of these genes in the early development of Xenopus lungs. Our study for the first time defined specific molecular markers for description of early lung development in Xenopus, as well as provided information about expression of components of Wnt and Hh pathways in early Xenopus lungs, which should be useful for future functional studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Mercury is a prominent environmental contaminant that causes detrimental effects to human health. Although the liver has been known to be a main target organ, there is limited information on in vivo molecular mechanism of mercury-induced toxicity in the liver. By using transcriptome analysis, phenotypic anchoring and validation of targeted gene expression in zebrafish, mercury-induced hepatotoxicity was investigated and a number of perturbed cellular processes were identified and compared with those captured in the in vitro human cell line studies., Results: Hepato-transcriptome analysis of mercury-exposed zebrafish revealed that the earliest deregulated genes were associated with electron transport chain, mitochondrial fatty acid beta-oxidation, nuclear receptor signaling and apoptotic pathway, followed by complement system and proteasome pathway, and thereafter DNA damage, hypoxia, Wnt signaling, fatty acid synthesis, gluconeogenesis, cell cycle and motility. Comparative meta-analysis of microarray data between zebrafish liver and human HepG2 cells exposed to mercury identified some common toxicological effects of mercury-induced hepatotoxicity in both models. Histological analyses of liver from mercury-exposed fish revealed morphological changes of liver parenchyma, decreased nucleated cell count, increased lipid vesicles, glycogen and apoptotic bodies, thus providing phenotypic evidence for anchoring of the transcriptome analysis. Validation of targeted gene expression confirmed deregulated gene-pathways from enrichment analysis. Some of these genes responding to low concentrations of mercury may serve as toxicogenomic-based markers for detection and health risk assessment of environmental mercury contaminations., Conclusion: Mercury-induced hepatotoxicity was triggered by oxidative stresses, intrinsic apoptotic pathway, deregulation of nuclear receptor and kinase activities including Gsk3 that deregulates Wnt signaling pathway, gluconeogenesis, and adipogenesis, leading to mitochondrial dysfunction, endocrine disruption and metabolic disorders. This study provides important mechanistic insights into mercury-induced liver toxicity in a whole-animal physiology context, which will help in understanding the syndromes caused by mercury poisoning. The molecular conservation of mercury-induced hepatotoxicity between zebrafish and human cell line reveals the feasibility of using zebrafish to model molecular toxicity in human for toxicant risk assessments.

Background: Recently we have performed a detailed analysis of early development of zebrafish swimbladder, a homologous organ of tetrapod lung; however, the events of swimbladder development are still poorly characterized. Many studies have implicated the role of vascular system in development of many organs in vertebrates. As the swimbladder is lined with an intricate network of blood capillaries, it is of interest to investigate the role of the vascular system during early development of swimbladder., Results: To investigate the role of endothelial cells (ECs) and blood circulation during development of the swimbladder, phenotypes of swimbladder were analysed at three different stages (approximately 2, 3 and 5 dpf [day postfertilization]) in cloche (clo) mutant and Tnnt2 morphants, in the background of transgenic lines Et(krt4:EGFP)sq33-2 and Et(krt4:EGFP)sqet3 which express EGFP in the swimbladder epithelium and outer mesothelium respectively. Analyses of the three tissue layers of the swimbladder were performed using molecular markers hb9, fgf10a, acta2, and anxa5 to distinguish epithelium, mesenchyme, and outer mesothelium. We showed that the budding stage was independent of ECs and blood flow, while early epithelial growth, mesenchymal organization and its differentiation into smooth muscle, as well as outer mesothelial organization, were dependent on ECs. Blood circulation contributed to later stage of epithelial growth, smooth muscle differentiation, and organization of the outer mesothelium. Inflation of the swimbladder was also affected as a result of absence of ECs and blood flow., Conclusion: Our data demonstrated that the vascular system, though not essential in swimbladder budding, plays an important role in the development of the swimbladder starting from the early growth stage, including mesenchyme organization and smooth muscle differentiation, and outer mesothelial organization, which in turn may be essential for the function of the swimbladder as reflected in its eventual inflation.

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)(sq33-2) and Et(krt4:EGFP)(sqet3) that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.

Background: Early events in vertebrate liver development have been the major focus in previous studies, however, late events of liver organogenesis remain poorly understood. Liver vasculogenesis in vertebrates occurs through the interaction of endoderm-derived liver epithelium and mesoderm-derived endothelial cells (ECs). In zebrafish, although it has been found that ECs are not required for liver budding, how and when the spatio-temporal pattern of liver growth is coordinated with ECs remains to be elucidated., Results: To study the process of liver development and vasculogenesis in vivo, a two-color transgenic zebrafish line Tg(lfabf:dsRed; elaA:EGFP) was generated and named LiPan for liver-specific expression of DsRed RFP and exocrine pancreas-specific expression of GFP. Using the LiPan line, we first followed the dynamic development of liver from live embryos to adult and showed the formation of three distinct yet connected liver lobes during development. The LiPan line was then crossed with Tg(fli1:EGFP)y1 and vascular development in the liver was traced in vivo. Liver vasculogenesis started at 55-58 hpf when ECs first surrounded hepatocytes from the liver bud surface and then invaded the liver to form sinusoids and later the vascular network. Using a novel non-invasive and label-free fluorescence correction spectroscopy, we detected blood circulation in the liver starting at approximately 72 hpf. To analyze the roles of ECs and blood circulation in liver development, both cloche mutants (lacking ECs) and Tnnt2 morphants (no blood circulation) were employed. We found that until 70 hpf liver growth and morphogenesis depended on ECs and nascent sinusoids. After 72 hpf, a functional sinusoidal network was essential for continued liver growth. An absence of blood circulation in Tnnt2 morphants caused defects in liver vasculature and small liver., Conclusion: There are two phases of liver development in zebrafish, budding and growth. In the growth phase, there are three distinct stages: avascular growth between 50-55 hpf, where ECs are not required; endothelium-dependent growth, where ECs or sinusoids are required for liver growth between 55-72 hpf before blood circulation in liver sinusoids; and circulation-dependent growth, where the circulation is essential to maintain vascular network and to support continued liver growth after 72 hpf.

We identified a zebrafish caudal-related homeobox (cdx1b) gene, which shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs.

Arsenic is a prominent environmental toxicant and carcinogen; however, its molecular mechanism of toxicity and carcinogenicity remains poorly understood. In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 15 parts/million (ppm) arsenic [As(V)] for 8-96 h to identify global transcriptional changes and biological networks involved in arsenic-induced adaptive responses in vivo. We found that there was an increase of transcriptional activity associated with metabolism, especially for biosyntheses, membrane transporter activities, cytoplasm, and endoplasmic reticulum in the 96 h of arsenic treatment, while transcriptional programs for proteins in catabolism, energy derivation, and stress response remained active throughout the arsenic treatment. Many differentially expressed genes encoding proteins involved in heat shock proteins, DNA damage/repair, antioxidant activity, hypoxia induction, iron homeostasis, arsenic metabolism, and ubiquitin-dependent protein degradation were identified, suggesting strongly that DNA and protein damage as a result of arsenic metabolism and oxidative stress caused major cellular injury. These findings were comparable with those reported in mammalian systems, suggesting that the zebrafish liver coupled with the available microarray technology present an excellent in vivo toxicogenomic model for investigating arsenic toxicity. We proposed an in vivo, acute arsenic-induced adaptive response model of the zebrafish liver illustrating the relevance of many transcriptional activities that provide both global and specific information of a coordinated adaptive response to arsenic in the liver.

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

Epstein-Barr virus-immortalized lymphoblastoid cell lines were derived from five patients with late radionecrosis. Two of these cell lines exhibited postradiation viability levels intermediate between normal cell lines and that from an individual with ataxia telangiectasia. Compared with controls, these two cell lines exhibited impaired ability to rejoin DNA double-strand breaks on pulsed-field gel electrophoresis and 6-10-fold reduced DNA-dependent protein kinase (DNA-PK) activity in vitro in cell-free extracts. Immunoblotting showed normal levels of Ku70, Ku80 and XRCC4 and the presence of DNA-PKcs in both cell lines. These findings suggest that DNA-PK might be an important factor affecting the predisposition of radiotherapy patients to late radionecrosis.

The aim of the work was to examine immunotropic features of celluroplasmine++ (Cp) and estimate its capacity as radioprotector. The pulse radiolysis method was used to set the mechanism of elementary reactions, which were responsible for antioxidant activity, and to demonstrate a particular role of reversible oxycomplexis, Cp...O2. Within in vitro test-systems the effects of Cp were examined on the model of interaction between lymphocytes from Shelter staff which constantly contacts with ionizing irradiation, and autologous erythrocytes in rosset-forming phenomenon (223 Shelter employees in comparison with 253 donors of Kyiv blood transfucion station). Expression of this Index in the presence of antigens with known poly-specificity was determined (antigens were taken from cortical and pyramid sections of kidney, liver, lungs, myocard, pancreatic gland, grey matter and aorta). Simultaneously the presence of analogous autoantibodies was determined (ELISA). It has been shown that Cp can reprogram the level of expression of immunovaluable receptors towards to norm. It has been also defined that Cp presence in blood irradiated in vitro (1-15 rem) promotes the masking of active centers of autoantibodies of different tissue specificity.