Improved SARS-CoV-2 RNA recovery in wastewater matrices using a CTAB-based extraction method.

Wastewater-based epidemiology has allowed tracking the magnitude and distribution of SARS-CoV-2 in communities, allowing public health officials to prepare for impending outbreaks. While many factors influence recovery of SARS-CoV-2 from wastewater, proper extraction, concentration, and purification of RNA are key steps to ensure accurate detection of viral particles. The aim of this study was to compare the efficiency of four commonly used RNA extraction methods for detection of the SARS-CoV-2 RNA genome in sewage samples artificially inoculated with the virus, in order to identify a protocol that improves viral recovery. These methods included CTAB-based, TRIzol-based, and guanidinium thiocyanate (GTC)-based extraction procedures coupled with silica spin column-based purification, and an automated extraction/purification protocol using paramagnetic particles. Following RNA extraction, virus recovery rates were compared using RT-qPCR-based detection. The CTAB-based approach yielded the highest recovery rates and was the only method to consistently demonstrate stable virus recovery percentages regardless of the specific physicochemical characteristics of the samples tested. The TRIzol method proved to be the second most effective, yielding significantly higher recovery rates compared to both the GTC-based and the automated extraction methods. These results suggest that the CTAB-based approach could be a useful tool for the recovery of viral RNA from complex wastewater matrices.

Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms.

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the ß-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region. IMPORTANCE: Little is known about the ecology of critically important antibiotic resistance among bacteria with the potential to be opportunistic human pathogens (e.g., Escherichia coli) on South American farms. By studying 70 pig and dairy cattle farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli and 3GC-R plasmids were diverse, suggestive of long-term circulation in this region. We identified the de novo mobilization of the resistance gene blaROB from pig pathogens into E. coli on a novel mobile genetic element, which shows the importance of surveying poorly studied regions for antibiotic resistance that might impact human health.

A simplified scoring system for the diagnosis of diarrhea and respiratory diseases in dairy calves.

Objective: To compare the capacity of a simplified calf health scoring chart (SIM score) with the University of Wisconsin's calf health scoring chart (WIN score) for the diagnosis of calf diarrhea and calf respiratory disease (RD). Animals and procedures: Holstein calves (N = 222) were clinically evaluated for diarrhea and RD diagnosis using the WIN and SIM scores. The WIN score was based on fecal consistency for diagnosis of diarrhea (0 = feces of normal consistency to 3 = watery feces; score ≥ 2 = positive diagnosis); and on nasal discharge, ocular discharge, coughing, ear position, and rectal temperature for diagnosis of RD (each clinical sign receives a score of 0 to 3; aggregate score ≥ 5 = positive diagnosis). The SIM score was based on a hide cleanliness score for diagnosis of diarrhea [0 = negative (calf was clean) and 1 = positive (tail head region, thighs, and/or legs were soiled)]; and on nasal discharge, ocular discharge, coughing, and ear position for diagnosis of RD (rectal temperature measurement was not required and each clinical sign had 2 levels of severity; aggregate score ≥ 5 = positive diagnosis). Results: In the RD diagnosis, the SIM score had a sensitivity of 88.24%, a specificity of 95.01%, a positive predictive value (PPV) of 55.56%, and a negative predictive value (NPV) of 99.13%. In the diarrhea diagnosis, the SIM score had a sensitivity of 94.62%, a specificity of 49.64%, a PPV of 18.22%, and an NPV of 98.73%. Conclusion: Compared with the WIN score, the SIM score is a reliable test for diagnosing RD but not for diagnosing diarrhea.

Un système de notation simplifié pour le diagnostic de la diarrhée et des maladies respiratoires chez les veaux laitiers. Objectif: Comparer la capacité d'un tableau de notation simplifié de la santé du veau (score SIM) avec le tableau de notation de la santé du veau (score WIN) de l'University of Wisconsin pour le diagnostic de la diarrhée du veau et de la maladie respiratoire du veau (RD). Animaux et procédures: Des veaux Holstein (N = 222) ont été évalués cliniquement pour le diagnostic de diarrhée et de RD à l'aide des scores WIN et SIM. Le score WIN était basé sur la consistance fécale pour le diagnostic de diarrhée (0 = fèces de consistance normale à 3 = fèces aqueuses; score ≥ 2 = diagnostic positif ); et sur l'écoulement nasal, l'écoulement oculaire, la toux, la position des oreilles et la température rectale pour le diagnostic de RD (chaque signe clinique reçoit un score de 0 à 3; score global ≥ 5 = diagnostic positif ). Le score SIM était basé sur un score de propreté de la peau pour le diagnostic de diarrhée [0 = négatif (le mollet était propre) et 1 = positif (la région de la tête de la queue, les cuisses et/ou les pattes étaient souillées); et sur l'écoulement nasal, l'écoulement oculaire, la toux et la position des oreilles pour le diagnostic de RD (la mesure de la température rectale n'était pas requise et chaque signe clinique avait 2 niveaux de gravité; score global ≥ 5 = diagnostic positif ). Résultats: Dans le diagnostic de RD, le score SIM avait une sensibilité de 88,24 %, une spécificité de 95,01 %, une valeur prédictive positive (VPP) de 55,56 % et une valeur prédictive négative (VPN) de 99,13 %. Dans le diagnostic de diarrhée, le score SIM avait une sensibilité de 94,62 %, une spécificité de 49,64 %, une VPP de 18,22 % et une VPN de 98,73 %. Conclusion: Comparé au score WIN, le score SIM est un test fiable pour diagnostiquer le RD mais pas pour diagnostiquer la diarrhée.(Traduit par Dr Serge Messier).

Occurrence of Norovirus, Rotavirus, Hepatitis a Virus, and Enterovirus in Berries in Argentina.

Berries have been implicated as the probable vehicle of infection in multiple outbreaks of norovirus and hepatitis A virus (HAV). These foods often receive minimal or no processing and may be exposed to virus contamination at each stage of production. In an increasingly globalized world, berries have a wide distribution and can give rise to the spread of diseases in distant parts of the world. With the aim of describing the virological quality of the berries cultivated in Argentina, a total of 184 soft fruits of different varieties (strawberries, blueberries, raspberries, blackberries, currants, pomegranate arils, cassis, and elder) were collected during the periods 2016-2018 and 2020. Viral particles were eluted and concentrated by polyethylene glycol precipitation according to ISO 15216-2:2019 guidelines. Genome detection of norovirus (NoV) genogroups I (GI) and II (GII), HAV, rotavirus, and enterovirus was performed by real-time RT-PCR with TaqMan probes. Positive samples were amplified by conventional RT-PCR and the amplicons were purified and sequenced in both directions. Phylogenetic analysis was performed using the Neighbor-Joining method based on the evolutionary model Kimura-2-parameters. NoV GII.6 was detected in 1/184 (0.5%) of the soft fruits, corresponding to a raspberry sample obtained during the fall of 2017. No presence of other human enteric viruses was found in the other berries analyzed. The collected data are the first in Argentina in relation to the prevalence of enteric viruses in berries and is useful as reference data for a risk assessment of soft fruits as vehicles of foodborne pathogenic viruses.

Nutritional and environmental contributions to autism spectrum disorders: Focus on nutrigenomics as complementary therapy.

The prevalence of autism spectrum disorders (ASD) has risen sharply in the last 30 years, posing a major public health concern and a big emotional and financial challenge for families. While the underlying causes remain to be fully elucidated, evidence shows moderate genetic heritability contribution, but heavy environmental influence. Over the last decades, modern lifestyle has deeply changed our eating, rest, and exercise habits, while exposure to air, water, and food chemical pollution has increased due to indiscriminate use of pesticides, food additives, adjuvants, and antibiotics. The result is a drastic change in the quality of our energy source input, and an overload for antioxidant and detoxification pathways that compromises normal metabolism and homeostasis. Current research shows high prevalence of food selectivity and/or food allergy among children with autism, resulting in essential micronutrient deficits that may trigger or aggravate physical and cognitive symptoms. Nutrigenomics is an emerging discipline that focuses on genotype-micronutrient interaction, and a useful approach to tailor low risk, personalized interventions through diet and micronutrient supplementation. Here, we review available literature addressing the role of micronutrients in the symptomatology of ASD, the metabolic pathways involved, and their therapeutic relevance. Personalized and supervised supplementation according to individual needs is suggested as a complement of traditional therapies to improve outcome both for children with autism and their families.

Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters.

Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2% and 45.0%, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 102 GC/mL) than PEG precipitation (4.3 × 103 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.

Prevención de desnutrición aguda moderada con un suplemento alimenticio listo para consumir en niños preescolares de comunidades rurales / Moderate acute malnutrition prevention with a ready-to-use therapeutic food in pre-school children from rural communities

Resumen Introducción: La desnutrición infantil en México alcanza prevalencias de 27.5 % en zonas rurales. Objetivo: Evaluar la efectividad de un suplemento alimenticio listo para consumir (SALC) para corregir desnutrición aguda leve y prevenir desnutrición aguda moderada en preescolares de comunidades rurales. Método: Ensayo clínico aleatorizado por grupos: con y sin SALC (g-SALC y g-S/SALC); se incluyeron niños de dos a cinco años, con puntuaciones-Z de peso para la talla (pZ-P/T) mayor de −2 y menor de −1 y nivel socioeconómico bajo. Todos recibieron educación sobre nutrición, salud e higiene dos veces al mes; los niños del g-SALC debieron consumir diariamente una porción del suplemento. Se evaluó pZ-P/T al inicio y a los cuatro, seis y 12 meses. La comparación entre grupos se realizó con el modelo de riesgos proporcionales de Cox. Resultados: Respecto a la recuperación de desnutrición aguda leve, en g-SALC se observó 68.7 versus 52.1% en el grupo control en el análisis de intención para tratar, con una razón de riesgo (HR) = 1.25; en el análisis por protocolo del primer semestre se observó una HR = 1.48 y en el segundo semestre, HR = 1.56. Un paciente progresó a desnutrición aguda moderada. Conclusiones: El g-SALC mostró resolución significativamente mayor de desnutrición aguda leve.

Abstract Introduction: Child malnutrition in Mexico reaches a prevalence as high as 27.5 % in rural areas. Objective: To assess the effectiveness of a ready-to-use supplementary food (RUSF) to correct mild acute malnutrition and prevent moderate acute malnutrition in preschool children from rural communities. Method: Randomized clinical trial, with assignment to two groups: group with RUSF (RUSF-g) or group without it (non-RUSF-g); children aged from two to five years, with weight-for-height Z-scores (WHZ) between -2 and -1 and low socioeconomic status were included. All received education on nutrition, health and hygiene twice monthly; the RUSF-g children had to consume four biscuits of the supplement every day. WHZ was assessed at baseline and at four, six, and 12 months. The comparison between groups was carried out with Cox proportional hazards model. Results: With regard to mild acute malnutrition correction in the RUSF-g, 68.7 versus 52.1 % in the control group was observed in the intent-to-treat analysis, with a hazard ratio (HR) = 1.25; in the per-protocol analysis of first semester, a HR = 1.48 was observed, and in the second semester, HR = 1.56. One patient progressed to moderate acute malnutrition. Conclusions: The RUSF-g showed a significantly higher resolution of mild acute malnutrition.

Endometrial expression of key genes related to fertility in repeat breeder and non-repeat breeder cows.

The aim of the present study was to compare the endometrial gene expression of epidermal growth factor receptor (EGFR), nodal growth differentiation factor (NODAL), prostaglandin-endoperoxide synthase 2 (PTGS2), oestrogen receptor 1 (ESR1) and progesterone receptor (PGR) in repeat breeder cows (RBC) and non-RBC during diestrus. Endometrial samples were collected by cytobrush technique and stored in RNA stabilizing solution at -20°C until RT-qPCR analysis. Differences in endometrial mRNA expression of selected genes were assessed by ANOVA and simple (r) and the partial correlations (rp) among selected genes were performed. Results demonstrated that mRNA expression of EGFR and NODAL were higher in RBC than in non-RBC (3 and 25-fold change, p < .01 and p < .01, respectively), while the mRNA expression of PTGS2 was lower (1.56-fold change, p < .01). Although there were no differences detected in the mRNA expression of ESR1 and PGR, there was a positive correlation between the expression of ESR1 and EGFR (0.84, p < .05) and a negative correlation between PGR and PTGS2 (-0.49, p < .05). In conclusion, the difference on the endometrial mRNA expression of the genes included in the study between RBC and non-RBC indicates a deregulation of important mechanisms that are vital to establish a successful pregnancy. Thus, the present study provides useful insight as a base for future studies to elucidate the causes of RBC.

Analysis of puerperal metritis treatment records in a grazing dairy farm in Argentina.

OBJECTIVE: To assess the efficacy of antibiotic usage for the treatment of puerperal metritis (PM) and its association with reproductive performance, a retrospective cohort study including a total of 9168 records of cows from a dairy farm in Argentina was run. MATERIAL AND METHODS: Cows having a PM3 (metricheck, scale 0-3) and treated with ceftiofur (ceftiofur crystalline free acid, 6.6 mg/kg) at 0-21 days postpartum (p. p.) (n = 2688), and cows having a PM 1-2 and not treated with an antibiotic at 0-21 days p. p. (n = 6480) were included in the study. All cows were reexamined with metricheck to assess the clinical cure (vaginal discharge [VD] score 0), partial cure (VD score similar or lower than previous), no cure (VD score higher than previous). Cows with a metricheck VD1-3 after 0-21 days p. p. were diagnosed as clinical endometritis (CE) 1-3. The occurrence of PM1-3, cure rate, calving to conception interval, the hazard of pregnancy, odds for non-pregnancy, and odds for CE were analyzed using SAS software. RESULTS: A total of 8876 PM1-3 records were included, 2435 records of PM3 treatments with ceftiofur (27.43 %), and 6441 records of PM1-2 (72.57 %) with no treatment. Cows having PM1 and PM2 became pregnant 14 and 12 days earlier than cows with PM3 (p < 0.001). The PM3 ceftiofur treated cows had a clinical cure of 24.85 % (PM0); 53.63 % had a partially cure; and 18.52 % no cure. Conversely, cows with PM1-2 had a 51.96 %, 20.70 %, and 24.53 % cure rate, respectively (p < 0.001). Cows having complete cure became pregnant 13 and 11 days earlier than cows having partial cure and no cure (p < 0.001). Cows that had PM3 during the first 21 days p. p. had twice the chances of developing CE compared to cows having PM1-2 (41.28 % vs. 24.14 %, p < 0.001). After 21 days p. p., less than 1 % of cows with clinical cure developed CE compared to 63.32 % that developed CE with partial cure, and 38.21 % with no cure (p < 0.001). CONCLUSION AND CLINICAL RELEVANCE: After ceftiofur treatment, 78 % of cows were cured when measured by disappearance of fetid VD but only 25 % of cows had clinical cure when measured by appearance of a clear VD. The cows that remained with clinical metritis had more chances of having CE after 21 days p. p. and had more days open than cows with clear normal VD.

Moderate acute malnutrition prevention with a ready-to-use supplementary food in pre-school children from rural communities.

INTRODUCTION: Child malnutrition in Mexico reaches a prevalence as high as 27.5 % in rural areas. OBJECTIVE: To assess the effectiveness of a ready-to-use supplementary food (RUSF) to correct mild acute malnutrition and prevent moderate acute malnutrition in preschool children from rural communities. METHOD: Randomized clinical trial, with assignment to two groups: group with RUSF (RUSF-g) or group without it (non-RUSF-g); children aged from two to five years, with weight-for-height Z-scores (WHZ) between -2 and -1 and low socioeconomic status were included. All received education on nutrition, health and hygiene twice monthly; the RUSF-g children had to consume one portion of the supplement every day. WHZ was assessed at baseline and at four, six, and 12 months. The comparison between groups was carried out with Cox proportional hazards model. RESULTS: With regard to mild acute malnutrition correction in the RUSF-g, 68.7 versus 52.1 % in the control group was observed in the intent-to-treat analysis, with a hazard ratio (HR) = 1.25; in the per-protocol analysis of first semester, a HR = 1.48 was observed, and in the second semester, HR = 1.56. One patient progressed to moderate acute malnutrition. CONCLUSIONS: The RUSF-g showed a significantly higher resolution of mild acute malnutrition. INTRODUCCIÓN: La desnutrición infantil en México alcanza prevalencias de 27.5 % en zonas rurales. OBJETIVO: Evaluar la efectividad de un suplemento alimenticio listo para consumir (SALC) para corregir desnutrición aguda leve y prevenir desnutrición aguda moderada en preescolares de comunidades rurales. MÉTODO: Ensayo clínico aleatorizado por grupos: con y sin SALC (g-SALC y g-S/SALC); se incluyeron niños de dos a cinco años, con puntuaciones-Z de peso para la talla (pZ-P/T) mayor de −2 y menor de −1 y nivel socioeconómico bajo. Todos recibieron educación sobre nutrición, salud e higiene dos veces al mes; los niños del g-SALC debieron consumir diariamente una porción del suplemento. Se evaluó pZ-P/T al inicio y a los cuatro, seis y 12 meses. La comparación entre grupos se realizó con el modelo de riesgos proporcionales de Cox. RESULTADOS: Respecto a la recuperación de desnutrición aguda leve, en g-SALC se observó 68.7 versus 52.1 % en el grupo control en el análisis de intención para tratar, con una razón de riesgo (HR) = 1.25; en el análisis por protocolo del primer semestre se observó una HR = 1.48 y en el segundo semestre, HR = 1.56. Un paciente progresó a desnutrición aguda moderada. CONCLUSIONES: El g-SALC mostró resolución significativamente mayor de desnutrición aguda leve.

Identification of Escherichia coli and Trueperella pyogenes isolated from the uterus of dairy cows using routine bacteriological testing and Fourier transform infrared spectroscopy.

BACKGROUND: Uterine disorders are common postpartum diseases in dairy cows. In practice, uterine treatment is often based on systemic or locally applied antimicrobials with no previous identification of pathogens. Accurate on-farm diagnostics are not available, and routine testing is time-consuming and cost intensive. An accurate method that could simplify the identification of uterine pathogenic bacteria and improve pathogen-specific treatments could be an important advance to practitioners. The objective of the present study was to evaluate whether a database built with uterine bacteria from European dairy cows could be used to identify bacteria from Argentinean cows by Fourier transformed infrared (FTIR) spectroscopy. Uterine samples from 64 multiparous dairy cows with different types of vaginal discharge (VD) were collected between 5 and 60 days postpartum, analyzed by routine bacteriological testing methods and then re-evaluated by FTIR spectroscopy (n = 27). RESULTS: FTIR spectroscopy identified Escherichia coli in 12 out of 14 samples and Trueperella pyogenes in 8 out of 10 samples. The agreement between the two methods was good with a Kappa coefficient of 0.73. In addition, the likelihood for bacterial growth of common uterine pathogens such as E. coli and T. pyogenes tended to increase with VD score. The odds for a positive result to E. coli or T. pyogenes was 1.88 times higher in cows with fetid VD than in herdmates with clear normal VD. CONCLUSIONS: We conclude that the presence of E. coli and T. pyogenes in uterine samples from Argentinean dairy cows can be detected with FTIR with the use of a database built with uterine bacteria from European dairy cows. Future studies are needed to determine if FTIR can be used as an alternative to routine bacteriological testing methods.

Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases.

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis.

Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis.

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.

Membrane organization and regulation of cellular cholesterol homeostasis.

An excess of intracellular free cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor because it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. We hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line overexpressing stearoyl coenzyme A (CoA) desaturase (SCD cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty acids, and determined this effect on membrane properties, cell viability, and Chol homeostasis. PM in SCD cells has a higher ratio of phospholipids to sphingomyelin and is slightly enriched in Chol. These cells showed an increase in the ratio of cholesteryl esters to free Chol; they were more resistant to Chol toxicity, and they exported more caveolin than control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

Endothelin 1 and 3 enhance neuronal nitric oxide synthase activity through ETB receptors involving multiple signaling pathways in the rat anterior hypothalamus.

We have previously reported that endothelin 1 and 3 (ET-1, ET-3) through the ETB receptor decrease norepinephrine release in the anterior hypothalamus and activate the nitric oxide (NO) pathway. In the present work we sought to establish the receptors and intracellular mechanisms underlying the increase in nitric oxide synthase (NOS) activity stimulated by ET-1 and ET-3 in the rat anterior hypothalamus. Results showed that ETs-stimulated NOS activity was inhibited by a selective ETB antagonist (BQ-788), but not by a selective ETA antagonist (BQ-610). In addition, NOS activity was not altered in the presence of an ETA agonist (sarafotoxin 6b), but it was enhanced in the presence of a ETB agonist (IRL-1620). Both Nomega-nitro-L-arginine methyl ester (NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) diminished ETs-stimulated NOS activity. The stimulatory effect of ETs on NOS activity was inhibited in the presence of PLC, PKC, PKA and CaMK-II inhibitors (U-73122, GF-109203X, H-89 and KN-62, respectively), and the IP3 receptor selective antagonist, 2-APB. Our results showed that both ET-1 and ET-3 modulate neuronal NOS activity through the ETB receptor in the rat anterior hypothalamus involving the participation of the PLC-PKC/IP3 pathway as well as PKA and CaMK-II.

Endothelin-1 and -3 diminish neuronal NE release through an NO mechanism in rat anterior hypothalamus.

The existence of endothelin binding sites on the catecholaminergic neurons of the hypothalamus suggests that endothelins (ETs) participate in the regulation of noradrenergic transmission modulating various hypothalamic-controlled processes such as blood pressure, cardiovascular activity, etc. The effects of ET-1 and ET-3 on the neuronal release of norepinephrine (NE) as well as the receptors and intracellular pathway involved were studied in the rat anterior hypothalamus. ET-1 (10 nM) and ET-3 (10 nM) diminished neuronal NE release and the effect blocked by the selective ET type B receptor antagonist BQ-788 (100 nM). N(omega)-nitro-L-arginine methyl ester (10 microM), methylene blue (10 microM), and KT5823 (2 microM), inhibitors of nitric oxide synthase activity, guanylate cyclase, and protein kinase G, respectively, prevented the inhibitory effects of both ETs on neuronal NE release. In addition, both ETs increased nitric oxide synthase activity. Furthermore, 100 microM picrotoxin, a GABA(A)-receptor antagonist, inhibited ET-1 and ET-3 response. Our results show that ET-1 as well as ET-3 has an inhibitory neuromodulatory effect on NE release in the anterior hypothalamus mediated by the ET type B receptor and the involvement of a nitric oxide-dependent pathway and GABA(A) receptors. ET-1 and ET-3 may thus diminish available NE in the synaptic gap leading to decreased noradrenergic activity.