Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Evidence for hypoxia-induced dysregulated cholesterol homeostasis in preeclampsia: Insights into the mechanisms from human placental cells and tissues.

Preeclampsia (PE) poses a considerable risk to the long-term cardiovascular health of both mothers and their offspring due to a hypoxic environment in the placenta leading to reduced fetal oxygen supply. Cholesterol is vital for fetal development by influencing placental function. Recent findings suggest an association between hypoxia, disturbed cholesterol homeostasis, and PE. This study investigates the influence of hypoxia on placental cholesterol homeostasis. Using primary human trophoblast cells and placentae from women with PE, various aspects of cholesterol homeostasis were examined under hypoxic and hypoxia/reoxygenation (H/R) conditions. Under hypoxia and H/R, intracellular total and non-esterified cholesterol levels were significantly increased. This coincided with an upregulation of HMG-CoA-reductase and HMG-CoA-synthase (key genes regulating cholesterol biosynthesis), and a decrease in acetyl-CoA-acetyltransferase-1 (ACAT1), which mediates cholesterol esterification. Hypoxia and H/R also increased the intracellular levels of reactive oxygen species and elevated the expression of hypoxia-inducible factor (HIF)-2α and sterol-regulatory-element-binding-protein (SREBP) transcription factors. Additionally, exposure of trophoblasts to hypoxia and H/R resulted in enhanced cholesterol efflux to maternal and fetal serum. This was accompanied by an increased expression of proteins involved in cholesterol transport such as the scavenger receptor class B type I (SR-BI) and the ATP-binding cassette transporter G1 (ABCG1). Despite these metabolic alterations, mitogen-activated-protein-kinase (MAPK) signaling, a key regulator of cholesterol homeostasis, was largely unaffected. Our findings indicate dysregulation of cholesterol homeostasis at multiple metabolic points in both the trophoblast hypoxia model and placentae from women with PE. The increased cholesterol efflux and intracellular accumulation of non-esterified cholesterol may have critical implications for both the mother and the fetus during pregnancy, potentially contributing to an elevated cardiovascular risk later in life.

LAT1-dependent placental methionine uptake is a key player in fetal programming of metabolic disease.

The Developmental Origins of Health and Disease hypothesis sustains that exposure to different stressors during prenatal development prepares the offspring for the challenges to be encountered after birth. We studied the gestational period as a particularly vulnerable window where different stressors can have strong implications for fetal programming of the offspring's life-long metabolic status via alterations of specific placentally expressed nutrient transporters. To study this mechanism, we used a murine prenatal stress model, human preeclampsia, early miscarriage, and healthy placental tissue samples, in addition to in vitro models of placental cells. In stressed mice, placental overexpression of L-type amino acid transporter 1 (Lat1) and subsequent global placental DNA hypermethylation was accompanied by fetal and adult hypothalamic dysregulation in global DNA methylation and gene expression as well as long-term metabolic abnormalities exclusively in female offspring. In human preeclampsia, early miscarriage, and under hypoxic conditions, placental LAT1 was significantly upregulated, leading to increased methionine uptake and global DNA hypermethylation. Remarkably, subgroups of healthy term placentas with high expression of stress-related genes presented increased levels of placental LAT1 mRNA and protein, DNA and RNA hypermethylation, increased methionine uptake capacity, one-carbon metabolic pathway disruption, higher methionine concentration in the placenta and transport to the fetus specifically in females. Since LAT1 mediates the intracellular accumulation of methionine, global DNA methylation, and one-carbon metabolism in the placenta, our findings hint at a major sex-specific global response to a variety of prenatal stressors affecting placental function, epigenetic programming, and life-long metabolic disease and provide a much-needed insight into early-life factors predisposing females/women to metabolic disorders.

Maternal Supraphysiological Hypercholesterolemia Is Accompanied by Shifts in the Composition and Anti-Atherogenic Functions of Maternal HDL along with Maternal Cardiovascular Risk Markers at Term of Pregnancy.

BACKGROUND: Maternal physiological hypercholesterolemia (MPH) occurs in pregnancy for a proper fetal development. When cholesterol increases over the physiological range, maternal supraphysiological hypercholesterolemia (MSPH) is described, a condition underdiagnosed by a lack of evidence showing its biological and clinical relevance. AIM: To determine if MSPH associates with maternal vascular dysfunction, along with changes in the composition and function of maternal HDL leading to increased cardiovascular risk. METHODS: This study included 57 women at term of pregnancy in which a lipid profile was determined. RESULTS: Maternal total cholesterol (TC) and LDL but not HDL were increased in MSPH women. The isolated HDL from a subgroup of MSPH women had a lower protein abundance and a reduced activity of the antioxidant enzyme PON1; however, an increased antioxidant capacity compared to MPH was observed, along with higher serum levels of α-tocopherol. Moreover, HDL from a subgroup of MSPH women had a lower capacity to induce NO synthesis in endothelial cells compared to MPH. In the circulation, we observed a reduced total antioxidant capacity and augmented levels of soluble VCAM, ApoB, ApoCII, ApoCIII, IL-10, and IL-12p70, as well as the cardiovascular risk ratio ApoB/ApoAI, compared to MPH women. CONCLUSION: MSPH women present dysfunctional HDL and increased atherogenic cardiovascular risk factors.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

Small extracellular vesicles from pregnant women with maternal supraphysiological hypercholesterolemia impair endothelial cell function in vitro.

Maternal physiological hypercholesterolemia MPH, maternal total cholesterol (TC) levels at term of pregnancy ≤280 mg/dL) occurs to assure fetal development. Maternal supraphysiological hypercholesterolemia (MSPH, TC levels >280 mg/dL) is a pathological condition associated with maternal, placental, and fetal endothelial dysfunction and early neonatal atherosclerosis development. Small extracellular vesicles (sEVs) are delivered to the extracellular space by different cells, where they modulate cell functions by transporting active signaling molecules, including proteins and miRNA. AIM: To determine whether sEVs from MSPH women could alter the function of endothelial cells (angiogenesis, endothelial activation and nitric oxide synthesis capacity). METHODS: This study included 24 Chilean women (12 MPH and 12 MSPH). sEVs were isolated from maternal plasma and characterized by sEV markers (CD9, Alix and HSP70), nanoparticle tracking analysis, transmission electron microscopy, and protein and cholesterol content. The endothelial cell line HMEC-1 was used to determine the uptake of labeled sEVs and the effects of sEVs on cell viability, endothelial tube formation, endothelial cell activation, and endothelial nitric oxide expression and function. RESULTS: In MSPH women, the plasma concentration of sEVs was increased compared to that in MPH women. MSPH-sEVs were highly taken up by HMEC-1 cells and reduced angiogenic capacity and the expression and activity of eNOS without changing cell viability or endothelial activation compared to MPH-sEVs. CONCLUSION: sEVs from MSPH women impair angiogenesis and nitric oxide synthesis in endothelial cells, which could contribute to MSPH-associated endothelial dysfunction.

Association between maternal obesity, essential fatty acids and biomarkers of fetal liver function.

Maternal obesity and the imbalance in linoleic acid (C18:2 n-6, LA) and alpha-linolenic acid (C18:3 n-3, ALA) levels are related with hepatic disturbances in the offspring. However, whether these alterations are present during fetal life is not well understood. Obese and normal weight pregnant women were recruited to determine fatty acids (FAs) consumption, FAs profile (in maternal erythrocytes, placenta and neonatal very low-density lipoproteins VLDL) and biomarkers of fetal liver function, such as gamma-glutamyl transferase (GGT), alpha-fetoprotein (AFP) and albumin, in umbilical cord blood. Stearic acid (C18:0, ST) was lower, and total n-3 FAs tended to be lower in umbilical cord VLDLs of obese women compared to controls. Independently of maternal obesity, GGT levels in umbilical cord blood was positively correlated with the LA content and negatively correlated with the ALA content in maternal erythrocytes. We conclude that maternal obesity and its imbalance of LA and ALA are associated with changes in biomarkers of fetal liver function.

The LDL receptor: Traffic and function in trophoblast cells under normal and pathological conditions.

During pregnancy, the developing foetus requires large amounts of cholesterol from the maternal plasma, which is mediated by proteins such as the receptor for low-density lipoproteins (LDLR). The quantity of LDLR available in the syncytiotrophoblast plasma membrane is an important factor for the uptake, metabolism, and transfer of cholesterol to foetal circulation. Because of the relevance of this receptor for cellular and systemic cholesterol metabolism in non-placental cells, the study of mechanisms associated with LDLR trafficking, such as the availability in the cell membrane, endocytosis, recycling, sorting, and degradation, have been extensively studied. Multiple protein groups are required for proper LDL/LDLR trafficking. Changes in the function of these proteins are related to hypercholesterolemia, the main risk factor associated with cardiovascular disease. It is well known that the placenta plays an essential role as a barrier between maternal lipids and the foetus and that imbalances in maternal cholesterol levels during pregnancy are frequent and associated with cardiovascular disease in the offspring. However, there is little information regarding lipoprotein trafficking in this system. In this review, we summarize the available information on LDLR trafficking, emphasizing the few reports related to receptor biology in placental cells from normal and pathological pregnancies. We conclude that extensive research on the cell biology of the placenta is required to unravel the endocytic trafficking of proteins such as LDLR in a highly specialized cell such as the syncytiotrophoblast.

[Argentine cross-cultural adaptation of the amyotrophic lateral sclerosis: functional rating scale-revised]. / Escala revisada de valoración funcional de esclerosis lateral amiotrófica: adaptación transcultural.

INTRODUCTION: Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised is a basic tool for the evaluation of people with this disease. In our country, versions are used without the necessary methodological process to adapt them culturally and to be able to certify the plausibility of the data collected. It was our goal to generate an appropriate version adapted to Argentine Spanish. METHODS: Cross-cultural adaptation of the scale was carried out, producing its Argentine version. Said production was carried out according to internationally accepted methodological procedures: sequential process of translation / synthesis / back-translation and consolidation of the version obtained by a multidisciplinary committee that solved of disagreements under Delphi methodology. The version obtained was tested on ALS people to determine of the existing syntactic - pragmatic level of difficulty, after which the final version was constituted. Difficulty level analysis was performed according to indicators: mode, median, qualitative variation index, variation ratio, accumulated frequency of positive evaluations, identification / modification of observed items (determining criterion: concordance criterion = 80%). RESULTS: After 3 rounds of consultation, the version agreed upon by the committee was obtained, obtaining agreement levels of 83.33%-100%. Its testing involved 21 functional alphabets, natives/residents, 67% men, mean age 51 years old, mean evolution 1.4 years. The general indicators supported the understanding of the version and the absence of conflicting items. DISCUSSION: The version obtained demonstrated its conceptual content validity regarding the English version, presenting not semantic nor pragmatic conflicts affecting their use in an Argentine population of ALS patients.

Introducción: La Escala Revisada de Valoración Funcional de la Esclerosis Lateral Amiotrófica es una herramienta básica para la evaluación de personas con esta enfermedad. En nuestro país se utilizan versiones sin el proceso metodológico necesario para adaptarlas culturalmente y poder certificar lo verosímil de los datos recogidos. Fue nuestro objetivo generar una apropiada versión adaptada al español argentino. Métodos: Se realizó la adaptación transcultural de la escala produciendo su versión argentina, conforme procedimientos y medidas de verificación de calidad metodológica internacionalmente aceptadas: proceso secuencial de traducción/síntesis/retrotraducción; resolución de discrepancias y consolidación de la versión obtenida por comité multidisciplinario bajo metodología Delphi; puesta a prueba de dicha versión en una población de personas con esclerosis lateral amiotrófica analizando el nivel de dificultad sintáctico-pragmático; constitución de la versión final. El análisis del nivel de dificultad se constituyó en base a los siguientes indicadores: moda, mediana, índice variación cualitativa, razón de variación, frecuencia acumulada de apreciaciones positivas, identificación / modificación de ítems observados, tomando como base un criterio de concordancia = 80%. Resultados: Tras 3 rondas de consulta se obtuvo la versión consensuada por el comité, obteniéndose niveles de concordancia del 83,33%-100%. Su puesta a prueba involucró a 21 alfabetos funcionales, nativos/residentes, 67% hombres, media edad 51 años, media evolución 1,4 años. Ausencia de dificultad a nivel comprensión y la ausencia de ítems conflictivos. Discusión: La versión obtenida demostró su validez de contenido conceptual respecto de la original, sin presentar conflictos semánticos o pragmáticos que afecten su uso en nuestra población.

Primary Human Trophoblasts Mimic the Preeclampsia Phenotype after Acute Hypoxia-Reoxygenation Insult.

Preeclampsia (PE) is a pregnancy-specific disorder that affects 3 to 5% of pregnancies worldwide and is one of the leading causes of maternal and fetal morbidity and mortality. Nevertheless, how these events occur remains unclear. We hypothesized that the induction of hypoxic conditions in vitro in primary human trophoblast cells would mimic several characteristics of PE found in vivo. We applied and characterized a model of primary cytotrophoblasts isolated from healthy pregnancies that were placed under different oxygen concentrations: ambient O2 (5% pCO2, 21%pO2, 24 h, termed "normoxia"), low O2 concentration (5% pCO2, 1.5% pO2, 24 h, termed "hypoxia"), or "hypoxia/reoxygenation" (H/R: 6 h intervals of normoxia and hypoxia for 24 h). Various established preeclamptic markers were assessed in this cell model and compared to placental tissues obtained from PE pregnancies. Seventeen PE markers were analyzed by qPCR, and the protein secretion of soluble fms-like tyrosine kinase 1 (sFlT-1) and the placenta growth factor (PlGF) was determined by ELISA. Thirteen of seventeen genes associated with angiogenesis, the renin-angiotensin system, oxidative stress, endoplasmic reticulum stress, and the inflammasome complex were susceptible to H/R and hypoxia, mimicking the expression pattern of PE tissue. In cell culture supernatants, the secretion of sFlT-1 was increased in hypoxia, while PlGF release was significantly reduced in H/R and hypoxia. In the supernatants of our cell models, the sFlT-1/PlGF ratio in hypoxia and H/R was higher than 38, which is a strong indicator for PE in clinical practice. These results suggest that our cellular models reflect important pathological processes occurring in PE and are therefore suitable as PE in vitro models.

Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of Bradyrhizobium diazoefficiens FixK2 Transcription Factor.

FixK2 is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK2 gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK2 activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK2, we replaced C183 for aspartic acid. Purified C183D FixK2 protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK2, expression of a fixNOQP'-'lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK2 encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK2 gene expression levels and FixK2 protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK2 encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK2 in vivo.

Increased Circulating Levels of PCSK9 and Pro-Atherogenic Lipoprotein Profile in Pregnant Women with Maternal Supraphysiological Hypercholesterolemia.

Maternal physiological hypercholesterolemia (MPH) occurs during pregnancy to assure fetal development. Some pregnant women develop maternal supraphysiological hypercholesterolemia (MSPH) characterized by increased levels of low-density lipoprotein (LDL). We aim to determine if proprotein convertase subtilisin/kexin type 9 (PCSK9) levels (a protein that regulate the availability of LDL receptor in the cells surface), as well as the composition and function of LDL, are modulated in MSPH women. This study included 122 pregnant women. Maternal total cholesterol (TC), LDL, triglycerides and PCSK9 increased from first (T1) to third trimester (T3) in MPH women. At T3, maternal TC, LDL, PCSK9 and placental abundances of PCSK9 were significantly higher in MPSH compared to MPH. Circulating PCSK9 levels were correlated with LDL at T3. In MSPH women, the levels of lipid peroxidation and oxidized LDL were significantly higher compared to MPH. LDL isolated from MSPH women presented significantly higher triglycerides and ApoB but lower levels of ApoAI compared to MPH. The formation of conjugated dienes was earlier in LDL from MSPH and in endothelial cells incubated with these LDLs; the levels of reactive oxygen species were significantly higher compared to LDL from MPH. We conclude that increased maternal PCSK9 would contribute to the maternal elevated levels of pro-atherogenic LDL in MSPH, which could eventually be related to maternal vascular dysfunction.

Escala revisada de valoración funcional de esclerosis lateral amiotrófica: adaptación transcultural / Argentine cross-cultural adaptation of the amyotrophic lateral sclerosis: functional rating scale-revised

Resumen Introducción: La Escala Revisada de Valoración Funcional de la Esclerosis Lateral Amiotrófica es una herramienta básica para la evaluación de personas con esta enfermedad. En nuestro país se utilizan versiones sin el proceso metodológico necesario para adaptarlas culturalmente y poder certificar lo vero símil de los datos recogidos. Fue nuestro objetivo generar una apropiada versión adaptada al español argentino. Métodos: Se realizó la adaptación transcultural de la escala produciendo su versión argentina, conforme proce dimientos y medidas de verificación de calidad metodológica internacionalmente aceptadas: proceso secuencial de traducción/síntesis/retrotraducción; resolución de discrepancias y consolidación de la versión obtenida por comité multidisciplinario bajo metodología Delphi; puesta a prueba de dicha versión en una población de perso nas con esclerosis lateral amiotrófica analizando el nivel de dificultad sintáctico-pragmático; constitución de la versión final. El análisis del nivel de dificultad se constituyó en base a los siguientes indicadores: moda, mediana, índice variación cualitativa, razón de variación, frecuencia acumulada de apreciaciones positivas, identificación/ modificación de ítems observados, tomando como base un criterio de concordancia ≥ 80%. Resultados: Tras 3 rondas de consulta se obtuvo la versión consensuada por el comité, obteniéndose niveles de concordancia del 83,33%-100%. Su puesta a prueba involucró a 21 alfabetos funcionales, nativos/residentes, 67% hombres, media edad 51 años, media evolución 1,4 años. Ausencia de dificultad a nivel comprensión y la ausencia de ítems conflictivos. Discusión: La versión obtenida demostró su validez de contenido conceptual respecto de la original, sin presentar conflictos semánticos o pragmáticos que afecten su uso en nuestra población.

Abstract Introduction: Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised is a basic tool for the evaluation of people with this disease. In our country, versions are used without the necessary methodological process to adapt them culturally and to be able to certify the plausibility of the data collected. It was our goal to generate an appropriate version adapted to Argentine Spanish. Methods: Cross-cultural adaptation of the scale was ca rried out, producing its Argentine version. Said production was carried out according to internationally accepted methodological procedures: sequential process of translation / synthesis / back-translation and consolidation of the version obtained by a multidisciplinary committee that solved of disagreements under Delphi methodology. The version obtained was tested on ALS people to determine of the existing syntactic - pragmatic level of difficulty, after which the final version was constituted. Difficulty level analysis was performed according to indicators: mode, median, qualitative variation index, variation ratio, accumulated frequency of positive evaluations, identification/ modification of observed items (determining criterion: concordance criterion ≥ 80%). Results: After 3 rounds of consultation, the version agreed upon by the committee was obtained, obtaining agreement levels of 83.33%-100%. Its testing involved 21 functional alphabets, natives/residents, 67% men, mean age 51 years old, mean evolution 1.4 years. The general indicators supported the understanding of the version and the absence of conflicting items. Discussion: The version obtained demonstrated its conceptual content validity regarding the English version, presenting not semantic nor pragmatic conflicts affecting their use in an Argentine population of ALS patients.

Human Placental Intracellular Cholesterol Transport: A Focus on Lysosomal and Mitochondrial Dysfunction and Oxidative Stress.

The placenta participates in cholesterol biosynthesis and metabolism and regulates exchange between the maternal and fetal compartments. The fetus has high cholesterol requirements, and it is taken up and synthesized at elevated rates during pregnancy. In placental cells, the major source of cholesterol is the internalization of lipoprotein particles from maternal circulation by mechanisms that are not fully understood. As in hepatocytes, syncytiotrophoblast uptake of lipoprotein cholesterol involves lipoprotein receptors such as low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI). Efflux outside the cells requires proteins such as the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. However, mechanisms associated with intracellular traffic of cholesterol in syncytiotrophoblasts are mostly unknown. In hepatocytes, uptaken cholesterol is transported to acidic late endosomes (LE) and lysosomes (LY). Proteins such as Niemann-Pick type C 1 (NPC1), NPC2, and StAR related lipid transfer domain containing 3 (STARD3) are required for cholesterol exit from the LE/LY. These proteins transfer cholesterol from the lumen of the LE/LY into the LE/LY-limiting membrane and then export it to the endoplasmic reticulum, mitochondria, or plasma membrane. Although the production, metabolism, and transport of cholesterol in placental cells are well explored, there is little information on the role of proteins related to intracellular cholesterol traffic in placental cells during physiological or pathological pregnancies. Such studies would be relevant for understanding fetal and placental cholesterol management. Oxidative stress, induced by generating excess reactive oxygen species (ROS), plays a critical role in regulating various cellular and biological functions and has emerged as a critical common mechanism after lysosomal and mitochondrial dysfunction. This review discusses the role of cholesterol, lysosomal and mitochondrial dysfunction, and ROS in the development and progression of hypercholesterolemic pregnancies.

Effect of Copper on Expression of Functional Genes and Proteins Associated with Bradyrhizobium diazoefficiens Denitrification.

Nitrous oxide (N2O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils.

Aldosterone and renin concentrations were abnormally elevated in a cohort of normotensive pregnant women.

BACKGROUND: During pregnancy, the renin-angiotensin-aldosterone system (RAAS) undergoes major changes to preserve normal blood pressure (BP) and placental blood flow and to ensure a good pregnancy outcome. Abnormal aldosterone-renin metabolism is a risk factor for arterial hypertension and cardiovascular risk, but its association with pathological conditions in pregnancy remains unknown. Moreover, potential biomarkers associated with these pathological conditions should be identified. AIM: To study a cohort of normotensive pregnant women according to their serum aldosterone and plasma renin levels and assay their small extracellular vesicles (sEVs) and a specific protein cargo (LCN2, AT1R). METHODS: A cohort of 54 normotensive pregnant women at term gestation was included. We determined the BP, serum aldosterone, and plasma renin concentrations. In a subgroup, we isolated their plasma sEVs and semiquantitated two EV proteins (AT1R and LCN2). RESULTS: We set a normal range of aldosterone and renin based on the interquartile range. We identified 5/54 (9%) pregnant women with elevated aldosterone and low renin levels and 5/54 (9%) other pregnant women with low aldosterone and elevated renin levels. No differences were found in sEV-LCN2 or sEV-AT1R. CONCLUSION: We found that 18% of normotensive pregnant women had either high aldosterone or high renin levels, suggesting a subclinical status similar to primary aldosteronism or hyperreninemia, respectively. Both could evolve to pathological conditions by affecting the maternal vascular and renal physiology and further the BP. sEVs and their specific cargo should be further studied to clarify their role as potential biomarkers of RAAS alterations in pregnant women.

Dissection of FixK2 protein-DNA interaction unveils new insights into Bradyrhizobium diazoefficiens lifestyles control.

The FixK2 protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen-fixing lifestyles in Bradyrhizobium diazoefficiens. Among the microoxic-responsive FixK2 -activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric-oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK2 is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK2 box) at the regulated promoters through three residues (L195, E196, and R200) within a C-terminal helix-turn-helix motif. Here, we mapped the determinants for discriminatory FixK2 -mediated regulation. While R200 was essential for DNA binding and activity of FixK2 , L195 was involved in protein-DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK2 box at the fixNOQP promoter impaired transcription activation by FixK2 , which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK2 -mediated activation in response to microoxia. Thus, position 11 within the FixK2 /NnrR boxes constitutes a key element that changes FixK2 targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.

Bacterial nitric oxide metabolism: Recent insights in rhizobia.

Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N2-fixing symbiosis with legumes inducing the formation of nodules in their roots. Several reports have shown NO production in the nodules where this gas acts either as a signaling molecule which regulates gene expression, or as a potent inhibitor of nitrogenase and other plant and bacteria enzymes. A better understanding of the sinks and sources of NO in rhizobia is essential to protect symbiotic nitrogen fixation from nitrosative stress. In nodules, both the plant and the microsymbiont contribute to the production of NO. From the bacterial perspective, the main source of NO reported in rhizobia is the denitrification pathway that varies significantly depending on the species. In addition to denitrification, nitrate assimilation is emerging as a new source of NO in rhizobia. To control NO accumulation in the nodules, in addition to plant haemoglobins, bacteroids also contribute to NO detoxification through the expression of a NorBC-type nitric oxide reductase as well as rhizobial haemoglobins. In the present review, updated knowledge about the NO metabolism in legume-associated endosymbiotic bacteria is summarized.