Effects of Fermentation Temperature, Drying Temperature, Caliber Size, Starter Culture, and Sodium Lactate on Listeria monocytogenes Inactivation During Salami Production.

The effect of fermentation and drying temperatures, caliber, and sodium lactate on Listeria monocytogenes inactivation was studied in salami, produced in a pilot scale, inoculated with 107 CFU/g of Listeria innocua ATCC® 33090 as a surrogate microorganism for L. monocytogenes. Fermentation temperature varied between 24 and 30°C, drying temperature between 14 and 20°C, caliber between 5.1 and 13.2 cm, and sodium lactate initial concentrations in salamis were 0 and 2%. L. innocua counts, pH and water activity were determined in salamis over time. Sodium lactate (2%) decreased pH drop and Listeria inactivation during fermentation. Baranyi & Roberts equation was used to fit the experimental data and to estimate, for each test condition, inactivation rate (k), initial (Y0), and final counts of L. innocua (YEND). Total inactivation was calculated as Y0 minus YEND (Y0-YEND). Then, using a Box Benkhen experimental design, a quadratic model for k and a two-factor interaction model (2FI) for Y0 - YEND were obtained as functions of fermentation temperature, drying temperature, and caliber size. The models predicted that maximum k and Y0 -YEND, -2.62 ± 0.14 log10 CFU/g/day and 4.5 ± 0.1 log10 CFU/g, respectively, would be obtained fermenting at 30°C and drying at 20°C regardless of caliber. Drying at 14°C allowed Listeria growth until a water activity (aw) of 0.92 was reached. Therefore, if initial Listeria contamination is high (3 log10 CFU/g), drying at low temperatures will compromise product safety.

Effect of beef long-storage under different temperatures and vacuum-packaging conditions on meat quality, oxidation processes and microbial growth.

BACKGROUND: The global beef market demands the meat industry to ensure product quality and safety in markets that are often very distant. The present study aimed to evaluate the effects of chilled (CH, 120 d) and chilled-then-frozen (CHF, 28 d + 92 d) storage conditions of beef vacuum packaged (VP) and vacuum packaged with antimicrobial (VPAM) on meat quality, oxidative status and microbial loads. Treatments resulted from the combination of storage condition and packaging type: VP + CH, VP + CHF, VPAM + CH and VPAM + CHF. RESULTS: Warner-Bratzler shear force values decreased in all treatments after 28 d of chilling. Except for VP + CH, L* values (lightness) of meat color did not differ in each treatment as the storage time increased. Meat from VP + CH had greater a* values than CHF treatments on day 120 of storage. A consumer panel did not detect differences in tenderness, flavor and overall liking between VP and VPAM beef, but they preferred CHF steaks rather than CH beef. TBARS values did not differ between VP and VPAM and between CH and CHF at any time during the storage period. At the end of storage time, all treatments except VP + CHF presented a greater concentration of thiols than at 48 h post-mortem. On day 120 of storage, VP + CH had greater catalase enzyme activity than CHF treatments while VP + CH and VP + CHF showed a greater superoxide dismutase activity than VPAM + CHF. Storage condition (CH or CHF) had a greater impact on microbial counts than the type of packaging. CONCLUSION: Freezing meat after an ageing period represents a suitable strategy to extend beef storage life without a detrimental impact on its quality. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

Microbiological Changes during Long-Storage of Beef Meat under Different Temperature and Vacuum-Packaging Conditions.

We evaluated a combination of two temperatures and two packaging materials for long-term storage of vacuum-packaged (VP) beef striploins. Microbial populations and microbiome composition were monitored during refrigerated storage (120 days between 0-1.5 °C) and refrigerated-then-frozen storage (28 days between 0-1.5 °C then 92 days at -20 °C) under low-O2 permeability VP and high-O2 permeability VP with an antimicrobial (VPAM). Pseudomonas (PSE) and Enterobacteriaceae (EB) counts in VPAM samples were significantly higher (p < 0.05) than in VP samples at 28, 45, 90, and 120 days of storage. Microbiome data showed that bacteria of the genera Serratia and Brochothrix were more abundant in VPAM samples at 120 days, while lactic acid bacteria (LAB) dominated in VP samples. Frozen temperatures inhibited microbial growth and maintained a relatively stable microbiome. Refrigerated and frozen VPAM samples showed the greatest difference in the predicted metabolic functions at the end of storage driven by the microbiome composition, dominated by PSE and LAB, respectively. Although no signs of visible meat deterioration were observed in any sample, this study suggests that VP meat refrigerated and then frozen achieved better microbiological indicators at the end of the storage period.

Production of microbial oils by the oleaginous yeast Rhodotorula graminis S1/2R in a medium based on agro-industrial by-products.

Biodiesel generated by transesterification of triglycerides from renewable sources is a clean form of energy that is currently used in many countries in blends with petrodiesel. It is mainly produced from food-grade vegetable oils obtained from oleaginous crops. High prices of these oils have made the sustainability of biodiesel production questionable. The use of nonedible feedstocks, such as intracellular triglycerides accumulated by oleaginous yeasts, appears as a feasible alternative. However, it has been demonstrated that an economically sustainable production of yeast oil could only be possible if low-cost media based on industrial subproducts, or wastes are used. In this work, we propose intracellular lipids production by a previously selected oleaginous yeast strain in a medium composed only by sugar cane vinasse and crude glycerol. Different culture strategies were studied. The highest biomass and lipid yields were obtained when the yeast R. graminis S1/2R was cultivated in batch without control of dissolved oxygen. The fatty acid methyl esters obtained under these conditions met the specification of international biodiesel standards.

Effect of UV-C Irradiation and Lactic Acid Application on the Inactivation of Listeria monocytogenes and Lactic Acid Bacteria in Vacuum-Packaged Beef.

The objective of this study was to test the effect of the combined application of lactic acid (0-5%) (LA) and UV-C light (0-330 mJ/cm2) to reduce Listeria monocytogenes and lactic acid bacteria (LAB) on beef without major meat color (L *, a *, b *) change and its impact over time. A two-factor central composite design with five central points and response surface methodology (RSM) were used to optimize LA concentration and UV-C dose using 21 meat pieces (10 g) inoculated with L. monocytogenes (LM100A1). The optimal conditions were analyzed over 8 weeks. A quadratic model was obtained that predicted the L. monocytogenes log reduction in vacuum-packed beef treated with LA and UV-C. The maximum log reduction for L. monocytogenes (1.55 ± 0.41 log CFU/g) and LAB (1.55 ± 1.15 log CFU/g) with minimal impact on meat color was achieved with 2.6% LA and 330 mJ/cm2 UV-C. These conditions impaired L. monocytogenes growth and delayed LAB growth by 2 weeks in vacuum-packed meat samples throughout 8 weeks at 4 °C. This strategy might contribute to improving the safety and shelf life of vacuum-packed beef with a low impact on meat color.

Polycyclic aromatic hydrocarbons in yerba mate (Ilex paraguariensis) infusions and probabilistic risk assessment of exposure.

The aim of this study was to assess the risk of exposure to polycyclic aromatic hydrocarbons (PAH) from yerba mate infusions in Uruguay using the margin of exposure approach (MOE) and a probabilistic method (Monte Carlo simulation). Servings/day, portion size, weekly frequency of mate consumption and body weight were the factors considered. The amount in infusions of benz[a]pyrene (B[a]P), PAH2 (sum of chrysene and B[a]P), and PAH4 (sum of benz[a]anthracene, chrysene, benz[b]fluoranthene and B[a]P) were used as markers of PAH exposure. Total content of PAH in infusions had large inter-brand variability (48-54 %) with significant differences among brands. PAH content in infusions prepared as habitually consumed was about 40 % of total content. The probability of occurrence of MOE < 10,000 varied according to the infusion preparation and the marker of exposure used, being higher for infusions prepared for total content and when B[a]P was used as marker of exposure. When the average B[a]P amount in infusion as habitually consumed was used in the simulation model, the probability of MOE < 10,000 was 9 %. The main factors contributing to B[a]P MOE variance were B[a]P amount (28.4 %), servings/day (17.3 %), and portion size (9.6 %). Heavy drinkers of yerba mate with high B[a]P content are those at risk to PAH exposure from mate infusions.

Biocontrol of Aspergillus flavus in Ensiled Sorghum by Water Kefir Microorganisms.

The capacity of microorganisms from water kefir (WK) to control Aspergillus flavus growth during the aerobic phase of ensiled sorghum grains was determined. Sorghum inoculated with A. flavus was treated with filter-sterilized and non-sterilized water kefir, ensiled, and incubated 7 days at 25 °C. A. flavus growth was quantified by qPCR after incubation. Mold growth was inhibited in the presence of water kefir while no inhibition was observed when filter-sterilized water kefir was applied, demonstrating the relevant role of the microorganisms in the kefir water in the biocontrol process. Fungal and bacterial diversity in treated sorghum mini-silos was analyzed by high-throughput sequencing. Firmicutes was the predominant bacterial phyla and Lactobacillus represented the most abundant genus, while Ascomycota was the predominant fungal phyla with Saccharomyces and Pichia as the major genera. Bacterial and yeast counts before and after incubation indicated that the microbial community obtained from WK was able to grow in the sorghum mini-silos in the presence of A. flavus. Results of the present work indicate that the use of a mixed inoculum of microorganisms present in WK may represent an alternative management practice to avoid the growth of A. flavus in ensiled sorghum grains and the concomitant contamination with aflatoxins.

Physico-chemical and antilisterial properties of nisin-incorporated chitosan/carboxymethyl chitosan films.

The objective of this work was to investigate the effect of the addition of carboxymethyl chitosan on the structural properties and antilisterial activity of nisin-incorporated chitosan films. Chitosan and carboxymethyl chitosan solutions were prepared with different mass ratios and bacteriocin nisin was added (0, 1000 and 6000 IU/ml). Filmogenic solutions were cast, dried and their physico-chemical and antimicrobial properties were investigated. For the same chitosan/carboxymethyl chitosan mass ratio, the addition of NIS at 6000 IU/ml led to changes in the macro- and microstructure, as well as in physico-chemical properties of films. On the other hand, carboxymethyl chitosan had a plasticizing effect and enhanced the distribution of the bacteriocin within the biopolymer matrix. Moreover, nisin-incorporated blend films of chitosan and carboxymethyl chitosan were more effective against Listeria monocytogenes than their pure chitosan counterparts. This study showed that different formulations of nisin-incorporated composite films of chitosan and carboxymethyl chitosan may provide options for developing bioactive packaging to improve food safety.

Evaluation of different hurdles on Penicillium crustosum growth in sponge cakes by means of a specific real time PCR.

Limited shelf life of bakery products, caused by microbial deterioration, is a concern for industries due to economic losses. Fungal spoilage of sponge cakes industrially produced in Montevideo was caused mainly by Penicillium species, in particular by Penicillium crustosum. The combination of different hurdles was studied to inhibit P. crustosum growth in sponge cakes. A full factorial design was performed to study the effect of the concentration of potassium sorbate, pH, packaging atmosphere and storage time. The results showed that packaging atmosphere and storage time were the significant factors in the ranges tested. No growth was detected in cakes stored in modified atmosphere packaging (MAP) (N2:CO2 50:50) at room temperature (25 °C) for 15 days. The effect of MAP on P. crustosum growth in cakes at room temperature was compared with the effect of air-packaging and storage at low temperature (4 °C) for 30 days. P. crustosum growth was not detected in cakes packaged in MAP, whereas it was detected after 20 days in cakes packaged in air and stored at 4 °C. This growth was quantified by a specific real time PCR developed in this work. Specific primers were designed using the sequence of ß-tubulin gene of P. crustosum as a target and PCR conditions were adjusted to ensure specificity. PCR efficiency was 107%, with a detection limit of 0.0014 ng of DNA. The qPCR method presented here, resulted specific and sensitive enough to detect the growth of P. crustosum even before biodeterioration signs were visible.

Process conditions for a rapid in situ transesterification for biodiesel production from oleaginous yeasts

BACKGROUND: Microbial oils produced by diverse microorganisms are being considered as alternative sources of triglycerides for biodiesel production. However, the standalone production of biodiesel from microorganisms is not currently economically feasible. In case of yeasts, the use of low-value nutrient sources in microbial production and the implementation of cost-efficient downstream processes could reduce costs and make microbial lipids competitive with other commodity-type oils in biodiesel production. Industrial biodiesel synthesis from oleaginous seeds is currently based on a multistep process. However, a simple process called in situ transesterification (ISTE), which takes place within the biomass without a previous lipid extraction step, is receiving increasing interest. In this work, the optimal conditions for an ISTE process to obtain biodiesel from previously selected oleaginous yeast (Rhodotorula graminis S1/S2) were defined using the response surface methodology (RSM). RESULTS: Using the RSM approach, the optimal conditions for the maximum yield with minimum reaction time included a methanol-to-biomass ratio of 60:1, 0.4 M H2SO4, and incubation at 70°C for 3 h. The optimized in situ process yield was significantly higher (123%) than that obtained with a two-step method in which fatty acids from saponifiable lipids were first extracted and then esterified with methanol. The composition of the fatty acid methyl ester mixture obtained from R. graminis S1/S2 by ISTE met Uruguayan standards for biodiesel. CONCLUSION: The characteristics achieved by the optimized method make microbial oil a potential alternative for biodiesel production from yeast at an industrial scale.

Evaluación del estado nutricional de yodo en una población de embarazadas / Assessment of iodine nutritional status in a pregnant women population

Introducción: el déficit de yodo en embarazadas puede perjudicar la salud de la madre y del recién nacido, está relacionado con diversas complicaciones obstétricas como abortos espontáneos, muertes fetales, anomalías congénitas, aumento de la mortalidad perinatal y el cretinismo. La Organización Mundial de la Salud (OMS) establece que el déficit de yodo sigue siendo la principal causa global evitable tanto de retraso mental como de parálisis cerebral, y afecta en grado variable el desarrollo y bienestar de millones de personas en el mundo. Las embarazadas constituyen una población vulnerable, con altos requerimientos de yodo. No se han realizado estudios en embarazadas en Uruguay luego de la yodación universal de la sal (1999). Objetivo principal: evaluar el estado nutricional de yodo en una población de embarazadas. Objetivo secundario: obtener una impresión cualitativa de las posibles fuentes de yodo nutricionales en esta población. Material y método: se realizó una encuesta nutricional específicamente confeccionada y se recolectaron muestras de la primera orina de la mañana para determinar yoduria en mujeres embarazadas independientemente del trimestre. Se consideró deficiencia de yodo para esta población una excreción urinaria media por debajo de 150 ?g/l (OMS, 2007). Resultados: se analizaron 96 muestras de orina. La mediana de la excreción urinaria media de yodo para esta población fue de 182 ?g/l, considerándose en rango de normalidad para las embarazadas valores entre 150 y 249 ?g/l. Conclusiones: este estudio ha confirmado que la excreción urinaria de yodo en la orina está en el rango adecuado a lo establecido por la OMS.

Abstract Introduction: iodine deficit in pregnant women may have a negative effect on the mother and the newborn, and it is associated to several obstetric complications such as spontaneous abortion, fetal death, congenital anomalies, increase of perinatal mortality and cretinism. The World Health Organization (WHO) establishes that iodine deficit is still the main global avoidable cause for mental retardation and brain palsy, and it affects the development and wellbeing of millions of people around the world at different levels. Pregnant women are a vulnerable population with high requirements. No studies in the pregnant women population have been conducted in Uruguay after the universal salt iodation in 1999. Main objective: assessment of iodine nutritional condition in a pregnant women population Secondary objective: to obtain a qualitative impression of the possible sources of nutritional iodine in this population. Methods: a specially prepared nutritional survey was conducted and first morning urine samples were collected to determine ioduria in pregnant women, regardless of the trimester. In this population, iodine deficiency was defined for this population when urine excretion was below 150 ?g/l (WHO, 2007). Results: ninety six urine samples were analysed. Median of the average urinary excretion for this population was 182 ?g/l, normal rates being between 150 and 249 ?g/l. Conclusions: the study confirmed iodine urinary excretion lies within the adequate range established by the WHO.

Resumo Introdução: a deficiência de iodo em gestantes pode prejudicar a saúde da mãe e do recém-nascido e está relacionado com diversas complicações obstétricas como abortos espontâneos, mortes fetais, anomalias congénitas, aumento da mortalidade perinatal e também com o cretinismo. A Organização Mundial da Saúde (OMS) declarou que a deficiência de iodo continua sendo a principal causa global evitável tanto de retraso mental como de paralisia cerebral, e que afeta, em diferentes graus, o desenvolvimento e o bem-estar de milhões de pessoas no mundo. As gestantes são uma população vulnerável com altos requerimentos. No Uruguay, desde 1999 quando começou a iodetação universal do sal, não são realizados estudos em gestantes. Objetivo principal: avaliar o estado nutricional de iodo em uma população de gestantes. Objetivo secundário: obter uma relação qualitativa das possíveis fontes nutricionais de iodo nesta população. Material e métodos: realizou-se um inquérito nutricional e foram coletadas amostras da primeira urina da manhã para determinar iodúria em gestantes em qualquer trimestre da gravidez. Nesta população considerou-se deficiência de iodo uma excreção urinaria média inferior a 150 ?g/l (OMS, 2007). Resultados: foram analisadas 96 amostras de urina. A mediana da excreção urinaria media de iodo nesta população foi de 182 ?g/l, considerando como normais valores entre 150 e 249 ?g/l. Conclusões: este estudo confirmou que a excreção urinaria de iodo está compreendida no intervalo considerado adequado de acordo com o estabelecido pela OMS.

Polyunsaturated fatty acid suppression of fatty acid synthase (FASN): evidence for dietary modulation of NF-Y binding to the Fasn promoter by SREBP-1c.

Dietary PUFAs (polyunsaturated fatty acids) co-ordinately suppress transcription of a group of hepatic genes encoding glycolytic and lipogenic enzymes. Suppression of Fasn (fatty acid synthase) transcription involves two PUFA-responsive regions, but the majority of PUFA sensitivity maps to a region within the proximal promoter containing binding sites for NF-Y (nuclear factor-Y), Sp1 (stimulatory protein 1), SREBP (sterol-regulatory-elementbinding protein), and USF (upstream stimulatory factor). Promoter activation assays indicate that altered NF-Y is the key component in regulation of Fasn promoter activity by PUFA. Using electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we demonstrate for the first time that PUFAs decrease in vivo binding of NF-Y and SREBP-1c to the proximal promoter of the hepatic Fasn gene and the promoters of three additional genes, spot 14, stearoyl-CoA desaturase and farnesyl diphosphate synthase that are also down-regulated by PUFA. The comparable 50% decrease in NF-Y and SREBP-1c binding to the promoters of the respective PUFA-sensitive genes occurred despite no change in nuclear NF-Y content and a 4-fold decrease in SREBP-1c. Together, these findings support a mechanism whereby PUFA reciprocally regulates the binding of NF-Y and SREBP-1c to a subset of genes which share similar contiguous arrangements of sterol regulatory elements and NF-Y response elements within their promoters. PUFA-dependent regulation of SREBP-1c and NF-Y binding to this unique configuration of response elements may represent a nutrient-sensitive motif through which PUFA selectively and co-ordinately targets subsets of hepatic genes involved in lipid metabolism.

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase.

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between -7300 and -7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between -7110 and -7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4alpha (hepatocyte nuclear factor 4alpha), and chromatin immunoprecipitation establishes that HNF-4alpha binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4alpha transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4alpha by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4alpha and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4alpha in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4alpha functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4alpha is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.

ELISA as an affordable methodology for monitoring groundwater contamination by pesticides in low-income countries.

The traditional instrumental technology for pesticide residue analysis is too expensive and labor-intense to meet the regional needs concerning environmental monitoring. ELISA methodology was used for a pilot scale study of groundwater quality in an agricultural region a few kilometers southwest of Montevideo, the capital city of Uruguay. The study spanned 2 years and examined concentrations (detection limits are given in [ppb]) of two triazine herbicides (simazine [0.3] and atrazine [0.4]) and the carbamate insecticide carbaryl [10] and its major metabolite 1-naphthol [17]. In general, pesticide concentrations were below detection limits in the samples tested and in all cases were well below the maximum contaminant levels set by the U.S. EPA. 1-Naphthol was detected frequently by ELISA, but the assay may have tended to systematically overestimate this analyte. To our knowledge, this is the first study of its type in Uruguay and perhaps the first systematic approach to monitoring for organic pesticides in groundwater water sources in the temperate region of South America.

Robust and sensitive monoclonal enzyme-linked immunosorbent assay for the herbicide molinate.

This paper reports on the generation of monoclonal antibodies and the development of a new enzyme-linked immunosorbent assay (ELISA) for the detection of molinate (S-ethyl hexahydroazepine-1-carbothioate). Hybridoma cells were generated using spleen and lymph node cells from a mouse immunized with S-2-carboxyethyl hexahydroazepine-1-carbothioate conjugated to keyhole limpet hemocyanin. After screening with a competitive ELISA, two monoclonal antibodies, mAbs 16C11 and 14D7, with IC(50) values of 82 +/- 2 and 173 +/- 8 ng/mL, respectively, were selected. MAb 16C11 can detect molinate concentrations of 1 ng/mL with no cross-reactivity to any other thiocarbamate pesticides; however, it was susceptible to the presence of organic solvents and to variation in buffer ionic strength. MAb 14D7 tolerated concentrations up to 5% of propylene glycol and 12.5% of acetonitrile in the assay buffer. The sensitivity of mAb 14D7 was further improved by decreasing the amount of coating antigen in the ELISA; the final inhibition assay showed an IC(50) of 69.2 +/- 1.4 ng/mL. In summary, mAb14D7 provided a more sensitive and robust assay, as compared with previous polyclonal antibody-based assays, with the additional advantage of being based upon a consistent and unlimited source of a defined reagent.

NF-Y involvement in the polyunsaturated fat inhibition of fatty acid synthase gene transcription.

Dietary polyunsaturated fats (PUFA) reduce the hepatic content of SREBP-1 65-75%, and this is paralleled by a comparable decrease in the expression of fatty acid synthase (FAS) gene. The close association between the nuclear content of SREBP-1 and FAS transcription has led to the conclusion that PUFA inhibit lipogenic gene transcription by suppressing SREBP-1 expression, but this conclusion is based upon correlative data. When in fact the SREBP-1/USF sites of the insulin response element of FAS were mutated, only 25% of the PUFA inhibition of FAS promoter activity was lost. On the other hand, mutating the -99/-93 NF-Y site reduced overall promoter activity 85%, and eliminated 50% of the PUFA suppression of FAS promoter activity. In addition, extended cloning and transfection-reporter assays revealed that the FAS gene contains a second PUFA response region (PUFA-RR) in the distal area of -7382/-6970. Interestingly, the distal PUFA-RR(FAS) has many similarities to the PUFA-RR of l-pyruvate kinase gene while the proximal PUFA-RR(FAS) is comparable to the PUFA-RR of the S14 and stearoyl-CoA desaturase genes.