Adenosine is a nucleoside that is particularly interesting to many scientific and clinical commun... more Adenosine is a nucleoside that is particularly interesting to many scientific and clinical communities as it has important physiological and pathophysiological roles in the kidney. The distribution of adenosine receptors has only recently been elucidated; therefore it is likely that more biological roles of this nucleoside will be unveiled in the near future. Since the discovery of the involvement of adenosine in renal vasoconstriction and regulation of local renin production, further evidence has shown that adenosine signaling is also involved in the tubuloglomerular feedback mechanism, sodium reabsorption and the adaptive response to acute insults, such as ischemia. However, the most interesting finding was the increased adenosine levels in chronic kidney diseases such as diabetic nephropathy and also in non-diabetic animal models of renal fibrosis. When adenosine is chronically increased its signaling via the adenosine receptors may change, switching to a state that induces renal...
Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (Na... more Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8 and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8 and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10 and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair ...
Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the ... more Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein–protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein–protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, ...
Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzym... more Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our findings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immunofluorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of different localization signals on FBPase for diverse metabolic functions.
Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemi... more Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemia generates reactive oxygen species (ROS), contributing to diabetic complications, especially in DN. Sodium Tungstate (NaW) is an effective antidiabetic agent for short and long-term treatments of both type 1 and type 2 diabetes models. In this study, we evaluated the effect of NaW on ROS production in bovine neutrophils incubated with platelet-activating factor (PAF) and in HK-2 cells induced by high glucose or H2O2. In addition, we evaluated the effect on iNOS expression in the type 1 diabetic rat model induced with streptozotocin (STZ). NaW inhibited ROS production in PAF-induced bovine neutrophils, and human tubular cells (HK-2) were incubated in high glucose or H2O2. In addition, NaW inhibited iNOS expression in glomeruli and tubular cells in the type 1 diabetic rat. This study demonstrates a new role for NaW as an active antioxidant and its potential use in treating DN.
The development of fish oral vaccines is of great interest to the aquaculture industry due to the... more The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 μm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering prote...
An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for s... more An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1β and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only th...
Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosi... more Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosis in salmonids farms in Chile. It was recently reported that P. salmonis produces exotoxins that play a role in the pathogenesis. However a delivery system has not yet been identified. Outer membrane vesicles (OMVs) are 10-300 nm spherical-bilayer structures discharged from the surface of many Gram-negative bacteria, which are able to deliver toxins and virulence factors. Recently, we described the production of OMVs by P. salmonis in both, in normal growth in broth and inside the host cells after infection. However, pro-immflamatory cytokines expression induced by P. salmonis OMVs has not yet been characterized. Thus, the aim of this study was to investigate if OMVs purified from P. salmonis are able to generate an immflamatory immune response in fish macrophages. P. salmonis was grown in basal broth supplemented with Cysteine (3.18 mM) and ferric chloride (0.05 mM) at 18°C until early ...
Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the et... more Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the etiologic agent of Piscirickettsiosis, a systemic infection of multiple organs and tissues among as kidney, liver, spleen, brain, intestine, ovaries, and gills in several salmonids species such as Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch), causing high mortality and serious economic losses for fish farming in the south of Chile. P. salmonis is able to infect, evade the immune response and replicate inside of several fish cell lines such as RTS-11, SHK-1 and CHSE-214. However, there are no reports concerning the infection and lifestyle of P. salmonis inside SHK-1 cell line. Thus, the aim of this study was to characterize, in a time-course, the infection and intracellular lifestyle of P. salmonis in SHK-1 fish cell line. SHK-1 cells were cultured at 20oC in 75 cm flask, in Leibovitz’s L-15 medium supplemented with 10% FBS. P. sa...
Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with... more Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and ses...
Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-... more Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of ...
Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish ... more Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by...
Adenosine is a nucleoside that is particularly interesting to many scientific and clinical commun... more Adenosine is a nucleoside that is particularly interesting to many scientific and clinical communities as it has important physiological and pathophysiological roles in the kidney. The distribution of adenosine receptors has only recently been elucidated; therefore it is likely that more biological roles of this nucleoside will be unveiled in the near future. Since the discovery of the involvement of adenosine in renal vasoconstriction and regulation of local renin production, further evidence has shown that adenosine signaling is also involved in the tubuloglomerular feedback mechanism, sodium reabsorption and the adaptive response to acute insults, such as ischemia. However, the most interesting finding was the increased adenosine levels in chronic kidney diseases such as diabetic nephropathy and also in non-diabetic animal models of renal fibrosis. When adenosine is chronically increased its signaling via the adenosine receptors may change, switching to a state that induces renal...
Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (Na... more Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8 and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8 and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10 and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair ...
Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the ... more Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein–protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein–protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, ...
Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzym... more Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our findings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immunofluorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of different localization signals on FBPase for diverse metabolic functions.
Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemi... more Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemia generates reactive oxygen species (ROS), contributing to diabetic complications, especially in DN. Sodium Tungstate (NaW) is an effective antidiabetic agent for short and long-term treatments of both type 1 and type 2 diabetes models. In this study, we evaluated the effect of NaW on ROS production in bovine neutrophils incubated with platelet-activating factor (PAF) and in HK-2 cells induced by high glucose or H2O2. In addition, we evaluated the effect on iNOS expression in the type 1 diabetic rat model induced with streptozotocin (STZ). NaW inhibited ROS production in PAF-induced bovine neutrophils, and human tubular cells (HK-2) were incubated in high glucose or H2O2. In addition, NaW inhibited iNOS expression in glomeruli and tubular cells in the type 1 diabetic rat. This study demonstrates a new role for NaW as an active antioxidant and its potential use in treating DN.
The development of fish oral vaccines is of great interest to the aquaculture industry due to the... more The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 μm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering prote...
An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for s... more An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1β and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only th...
Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosi... more Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosis in salmonids farms in Chile. It was recently reported that P. salmonis produces exotoxins that play a role in the pathogenesis. However a delivery system has not yet been identified. Outer membrane vesicles (OMVs) are 10-300 nm spherical-bilayer structures discharged from the surface of many Gram-negative bacteria, which are able to deliver toxins and virulence factors. Recently, we described the production of OMVs by P. salmonis in both, in normal growth in broth and inside the host cells after infection. However, pro-immflamatory cytokines expression induced by P. salmonis OMVs has not yet been characterized. Thus, the aim of this study was to investigate if OMVs purified from P. salmonis are able to generate an immflamatory immune response in fish macrophages. P. salmonis was grown in basal broth supplemented with Cysteine (3.18 mM) and ferric chloride (0.05 mM) at 18°C until early ...
Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the et... more Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the etiologic agent of Piscirickettsiosis, a systemic infection of multiple organs and tissues among as kidney, liver, spleen, brain, intestine, ovaries, and gills in several salmonids species such as Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch), causing high mortality and serious economic losses for fish farming in the south of Chile. P. salmonis is able to infect, evade the immune response and replicate inside of several fish cell lines such as RTS-11, SHK-1 and CHSE-214. However, there are no reports concerning the infection and lifestyle of P. salmonis inside SHK-1 cell line. Thus, the aim of this study was to characterize, in a time-course, the infection and intracellular lifestyle of P. salmonis in SHK-1 fish cell line. SHK-1 cells were cultured at 20oC in 75 cm flask, in Leibovitz’s L-15 medium supplemented with 10% FBS. P. sa...
Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with... more Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and ses...
Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-... more Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of ...
Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish ... more Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by...
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Papers by Alejandro Yañez