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Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders. Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication. [ABSTRACT FROM AUTHOR]

Abstract Background Reliable molecular typing tools are required for a better understanding of the molecular epidemiology of Plasmodium vivax. The genes msp3a and msp1_block5 are highly polymorphic and have been used as markers in many P. vivax population studies. These markers were used to assess the genetic diversity of P. vivax strains from French Guiana (South America) and to develop a molecular typing protocol. Methods A total of 120 blood samples from 109 patients (including 10 patients suffered from more than one malaria episode, samples were collected during each episode) with P. vivax infection were genotyped. All samples were analysed by msp3a PCR-RFLP and msp1_b5 gene sequencing was performed on 57 samples. Genotyping protocol applied to distinguish between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites was based on analysing first msp3a by PCR-RFLP and secondly, only if the genotypes of the two samples are identical, on sequencing the msp1_b5 gene. Results msp3a alleles of three sizes were amplified by PCR: types A, B and C. Eleven different genotypes were identified among the 109 samples analysed by msp3a PCR-RFLP. In 13.8% of cases, a mixed genotype infection was observed. The sequence of msp1_b5 gene revealed 22 unique genotypes and 12.3% of cases with mixed infection. In the 57 samples analysed by both methods, 45 genotypes were found and 21% were mixed. Among ten patients with two or three malaria episodes, the protocol allowed to identify five new infections or relapses from heterologous hypnozoites and six treatment failures of relapses from homologous hypnozoites. Conclusion The study showed a high diversity of msp3a and msp1_b5 genetic markers among P. vivax strains in French Guiana with a low polyclonal infection rate. These results indicated that the P. vivax genotyping protocol presented has a good discrimination power and can be used in clinical drug trials or epidemiological studies.

Methionine is a key factor in modulating the cellular availability of the main biological methyl donor S-adenosylmethionine (SAM), which is required for all biological methylation reactions including DNA and histone methylation. As such, it represents a potential critical factor in nutritional programming. Here, we investigated whether early methionine restriction at first feeding could have long-term programmed metabolic consequences in rainbow trout. For this purpose, trout fry were fed with either a control diet (C) or a methionine-deficient diet (MD) for 2 weeks from the first exogenous feeding. Next, fish were subjected to a 5 month growth trial with a standard diet followed by a 2 week challenge (with theMDor C diet) to test the programming effect of the early methionine restriction. The results showed that, whatever the dietary treatment of fry, the 2 week challenge with the MD diet led to a general mitochondrial defect associated with an increase in endoplasmic reticulum stress, mitophagy and apoptosis, highlighting the existence of complex cross-talk between these different functions. Moreover, for the first time, we also observed that fish fed the MD diet at the first meal later exhibited an increase in several critical factors of mitophagy, hinting that the early nutritional stimulus with methionine deficiency resulted in long-term programming of this cell function. Together, these data extend our understanding of the role of dietary methionine and emphasize the potential for this amino acid in the application of new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish. [ABSTRACT FROM AUTHOR]

High levels of dietary lipids are incorporated in feeds for most teleost fish to promote growth and reduce nitrogen waste. However, in Senegalese sole (Solea senegalensis) previous studies revealed that increasing the level of dietary lipids above 8% negatively affect growth and nutrient utilization regardless of dietary protein content. It has been shown that glucose regulation and metabolism can be impaired by high dietary fat intake in mammals, but information in teleost fish is scarce. The aim of this study was to assess the possible effect of dietary lipids on glucose metabolism in Senegalese sole with special emphasis on the regulation of proteins involved in the muscle insulin-signalling pathway. Senegalese sole juveniles (29 g) were fed two isonitrogenous diets (53% dry matter) for 88 days. These two diets were one with a high lipid level (∼17%, HL) and a moderate starch content (∼14%, LC), and the other being devoid of fish oil (4% lipid, LL) and with high starch content (∼23%, HC). Surprisingly, feeding Senegalese sole the HL/LC diet resulted in prolonged hyperglycaemia, while fish fed on LL/HC diet restored basal glycaemia 2 h after feeding. The hyperglycaemic phenotype was associated with greater glucose-6-phosphatase activity (a key enzyme of hepatic glucose production) and lower citrate synthase activity in the liver, with significantly higher liver glycogen content. Sole fed on HL/LC diet also had significantly lower hexokinase activity in muscle, although hexokinase activity was low with both dietary treatments. The HL/LC diet was associated with significant reductions in muscle AKT, p70 ribosomal S6-K1 Kinase (S6K-1) and ribosomal protein S6 (S6) 2 h after feeding, suggesting down regulation of the AKT-mTOR nutrient signalling pathway in these fish. The results of this study show for the first time that high level of dietary lipids strongly affects glucose metabolism in Senegalese sole. [ABSTRACT FROM AUTHOR]

Background: Reliable molecular typing tools are required for a better understanding of the molecular epidemiology of Plasmodium vivax. The genes msp3a and msp1_block5 are highly polymorphic and have been used as markers in many P. vivax population studies. These markers were used to assess the genetic diversity of P. vivax strains from French Guiana (South America) and to develop a molecular typing protocol. Methods: A total of 120 blood samples from 109 patients (including 10 patients suffered from more than one malaria episode, samples were collected during each episode) with P. vivax infection were genotyped. All samples were analysed by msp3a PCR-RFLP and msp1_b5 gene sequencing was performed on 57 samples. Genotyping protocol applied to distinguish between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites was based on analysing first msp3a by PCR-RFLP and secondly, only if the genotypes of the two samples are identical, on sequencing the msp1_b5 gene. Results: msp3a alleles of three sizes were amplified by PCR: types A, B and C. Eleven different genotypes were identified among the 109 samples analysed by msp3a PCR-RFLP. In 13.8% of cases, a mixed genotype infection was observed. The sequence of msp1_b5 gene revealed 22 unique genotypes and 12.3% of cases with mixed infection. In the 57 samples analysed by both methods, 45 genotypes were found and 21% were mixed. Among ten patients with two or three malaria episodes, the protocol allowed to identify five new infections or relapses from heterologous hypnozoites and six treatment failures of relapses from homologous hypnozoites. Conclusion: The study showed a high diversity of msp3a and msp1_b5 genetic markers among P. vivax strains in French Guiana with a low polyclonal infection rate. These results indicated that the P. vivax genotyping protocol presented has a good discrimination power and can be used in clinical drug trials or epidemiological studies. [ABSTRACT FROM AUTHOR]

High levels of dietary lipids are incorporated in feeds for most teleost fish to promote growth and reduce nitrogen waste. However, in Senegalese sole (Solea senegalensis) previous studies revealed that increasing the level of dietary lipids above 8% negatively affect growth and nutrient utilization regardless of dietary protein content. It has been shown that glucose regulation and metabolism can be impaired by high dietary fat intake in mammals, but information in teleost fish is scarce. The aim of this study was to assess the possible effect of dietary lipids on glucose metabolism in Senegalese sole with special emphasis on the regulation of proteins involved in the muscle insulin-signalling pathway. Senegalese sole juveniles (29 g) were fed two isonitrogenous diets (53% dry matter) for 88 days. These two diets were one with a high lipid level (∼17%, HL) and a moderate starch content (∼14%, LC), and the other being devoid of fish oil (4% lipid, LL) and with high starch content (∼23%, HC). Surprisingly, feeding Senegalese sole the HL/LC diet resulted in prolonged hyperglycaemia, while fish fed on LL/HC diet restored basal glycaemia 2 h after feeding. The hyperglycaemic phenotype was associated with greater glucose-6-phosphatase activity (a key enzyme of hepatic glucose production) and lower citrate synthase activity in the liver, with significantly higher liver glycogen content. Sole fed on HL/LC diet also had significantly lower hexokinase activity in muscle, although hexokinase activity was low with both dietary treatments. The HL/LC diet was associated with significant reductions in muscle AKT, p70 ribosomal S6-K1 Kinase (S6K-1) and ribosomal protein S6 (S6) 2 h after feeding, suggesting down regulation of the AKT-mTOR nutrient signalling pathway in these fish. The results of this study show for the first time that high level of dietary lipids strongly affects glucose metabolism in Senegalese sole. [ABSTRACT FROM AUTHOR]