Dietary docosahexaenoic acid (DHA) downregulates liver DHA synthesis by inhibiting eicosapentaenoic acid elongation.

DHA is abundant in the brain where it regulates cell survival, neurogenesis, and neuroinflammation. DHA can be obtained from the diet or synthesized from alpha-linolenic acid (ALA; 18:3n-3) via a series of desaturation and elongation reactions occurring in the liver. Tracer studies suggest that dietary DHA can downregulate its own synthesis, but the mechanism remains undetermined and is the primary objective of this manuscript. First, we show by tracing 13C content (δ13C) of DHA via compound-specific isotope analysis, that following low dietary DHA, the brain receives DHA synthesized from ALA. We then show that dietary DHA increases mouse liver and serum EPA, which is dependant on ALA. Furthermore, by compound-specific isotope analysis we demonstrate that the source of increased EPA is slowed EPA metabolism, not increased DHA retroconversion as previously assumed. DHA feeding alone or with ALA lowered liver elongation of very long chain (ELOVL2, EPA elongation) enzyme activity despite no change in protein content. To further evaluate the role of ELOVL2, a liver-specific Elovl2 KO was generated showing that DHA feeding in the presence or absence of a functional liver ELOVL2 yields similar results. An enzyme competition assay for EPA elongation suggests both uncompetitive and noncompetitive inhibition by DHA depending on DHA levels. To translate our findings, we show that DHA supplementation in men and women increases EPA levels in a manner dependent on a SNP (rs953413) in the ELOVL2 gene. In conclusion, we identify a novel feedback inhibition pathway where dietary DHA downregulates its liver synthesis by inhibiting EPA elongation.

Optimization of ionizable lipids for aerosolizable mRNA lipid nanoparticles.

Although mRNA lipid nanoparticles (LNPs) are highly effective as vaccines, their efficacy for pulmonary delivery has not yet fully been established. A major barrier to this therapeutic goal is their instability during aerosolization for local delivery. This imparts a shear force that degrades the mRNA cargo and therefore reduces cell transfection. In addition to remaining stable upon aerosolization, mRNA LNPs must also possess the aerodynamic properties to achieve deposition in clinically relevant areas of the lungs. We addressed these challenges by formulating mRNA LNPs with SM-102, the clinically approved ionizable lipid in the Spikevax COVID-19 vaccine. Our lead candidate, B-1, had the highest mRNA expression in both a physiologically relevant air-liquid interface (ALI) human lung cell model and in healthy mice lungs upon aerosolization. Further, B-1 showed selective transfection in vivo of lung epithelial cells compared to immune cells and endothelial cells. These results show that the formulation can target therapeutically relevant cells in pulmonary diseases such as cystic fibrosis. Morphological studies of B-1 revealed differences in the surface structure compared to LNPs with lower transfection efficiency. Importantly, the formulation maintained critical aerodynamic properties in simulated human airways upon next generation impaction. Finally, structure-function analysis of SM-102 revealed that small changes in the number of carbons can improve upon mRNA delivery in ALI human lung cells. Overall, our study expands the application of SM-102 and its analogs to aerosolized pulmonary delivery and identifies a potent lead candidate for future therapeutically active mRNA therapies.

A moderate-fat diet containing soy protein does not differentially impact energy balance in male and female mice compared to dairy protein.

Dairy and nondairy plant-based alternative proteins are reported to differentially influence body weight; however, most research has compared plant-based alternatives with isolated dairy proteins rather than a complete milk protein (containing casein and whey). This is notable given that people do not generally consume isolated dairy proteins. Therefore, the present study aimed to investigate the impact of a soy protein isolate (SPI) on factors influencing body weight gain in male and female mice in comparison to skim milk powder (SMP). Based on current knowledge in rodents, we hypothesized that SPI would promote body weight gain compared with SMP. Mice (n = 8 per sex per diet) consumed a moderate-fat diet (35% kcal from fat) containing either SPI or SMP for 8 weeks. Body weight and food intake were measured weekly. Energy expenditure, physical activity, and substrate use were measured using metabolic cages. Fecal energy content was measured with bomb calorimetry. Body weight gain and food intake during the 8-week feeding study was not different in mice consuming either SPI or SMP; however, males had a higher body weight, adiposity, and feed efficiency compared with females (all P < .05). Fecal energy content was approximately 7% higher in both male and female mice fed the SPI diet compared with the SMP diet. Neither protein source affected substrate utilization, physical activity, or energy expenditure. Physical activity in the dark phase trended higher in females compared with males (P = .0732). The present study suggests that the consumption of SPI in the context of a moderate-fat diet has little impact on numerous factors influencing body weight regulation in male and female mice compared with a complete milk protein.

Tactile sensitivity and motor coordination in infancy: Effect of age, prior surgery, anaesthesia & critical illness.

BACKGROUND: Tactile sensitivity in the infant period is poorly characterized, particularly among children with prior surgery, anaesthesia or critical illness. The study aims were to investigate tactile sensitivity of the foot and the associated coordination of lower limb motor movement in typically developing infants with and without prior hospital experience, and to develop feasible bedside sensory testing protocols. MATERIALS AND METHODS: A prospective, longitudinal study in 69 infants at 2 and 4 months-old, with and without prior hospital admission. Mechanical stimuli were applied to the foot at graded innocuous and noxious intensities. Primary outcome measures were tactile and nociceptive threshold (lowest force required to evoke any leg movement, or brisk leg withdrawal, respectively), and specific motor flexion threshold (ankle-, knee-, hip-flexion). Secondary analysis investigated (i) single vs multiple trials reliability, and (ii) the effect of age and prior surgery, anaesthesia, or critical illness on mechanical threshold. RESULTS: Magnitude of evoked motor activity increased with stimulus intensity. Single trials had excellent reliability for knee and hip flexion at age 1-3m and 4-7m (ICC range: 0.8 to 0.98, p >0.05). Nociceptive threshold varied as a function of age. Tactile sensitivity was independent of age, number of surgeries, general anaesthesia and ICU stay. CONCLUSIONS: This brief sensory testing protocol may reliably measure tactile and nociceptive reactivity in human infants. Age predicts nociceptive threshold which likely reflects ongoing maturation of spinal and supraspinal circuits. Prior hospital experience has a negligible global effect on sensory processing demonstrating the resilience of the CNS in adverse environments.

Passivated Impedimetric Sensors for Immobilization-Free Pathogen Detection by Isothermal Amplification and Melt Curve Analysis.

The ongoing SARS-CoV-2 pandemic demonstrates that the capacity of centralized clinical diagnosis laboratories represents a significant limiting factor in the global fight against the newly emerged virus. Scaling up these capacities also requires simple and robust methods for virus diagnosis to be easily driven by untrained personnel in a point-of-care (POC) environment. The use of impedance sensors reduces the complexity and costs of diagnostic instruments and increases automation of diagnosis processes. We present an impedance point-of-care system (IMP-POCS) that uses interdigitated electrodes surrounded by an integrated heating meander to monitor loop-mediated isothermal amplification (LAMP) and melt curve analysis (MCA) consecutively in a short time. MCA permits distinguishing false- from true-positive results and significantly raises the validity of pathogen detection. Conclusively, the herein-developed miniaturized total analysis system (µTAS) represents a powerful and promising tool for providing reliable, low-cost alternatives to standard clinical diagnosis.

Plant and marine N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis varies according to nutritional state.

Marine sourced N3-PUFA regulate lipid metabolism in adipose tissue and liver; however, less is known about plant sourced N3-PUFA. The goal of this study was to investigate plant and marine N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis according to nutritional state. Mice were fed low-fat diets (7% w/w) containing either lard, flaxseed, or menhaden oils for 8 weeks, and were euthanized in either fed or fasted states. Substrate utilization and physical activity were assessed during the transition from a fed to fasted state. Plasma biomarkers (triacylglycerol [TAG], non-esterified fatty acids [NEFA]), as well as liver and epididymal adipose tissue (eWAT) lipogenic and lipolytic markers, were measured. Neither plant nor marine N3-PUFA influenced substrate utilization or activity during the transition from a fed to fasted state. In the fed state, marine N3-PUFA reduced plasma TAG levels compared to the other diets, with no further reduction seen in fasted mice. Hepatic lipogenic markers (Fasn, Acc, Scd1, and Elovl6) were reduced in the fed state with marine N3-PUFA, but not plant N3-PUFA. In the fasted state, mice fed either N3-PUFA accumulated less liver TAG, had lower plasma NEFA, and suppressed eWAT HSL activity compared to lard. Marine N3-PUFA are more potent regulators of lipogenesis than plant N3-PUFA in the fed state, whereas both N3-PUFA influence eWAT lipolysis and plasma NEFA in the fasted state. This work provides novel insights regarding N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis according to nutritional state.

Manufacturing Stable Bacteriophage Powders by Including Buffer System in Formulations and Using Thin Film Freeze-drying Technology.

PURPOSE: Bacteriophage (phage) therapy has re-gained attention lately given the ever-increasing prevalence of multi-drug resistance 'super-bugs'. To develop therapeutic phage into clinically usable drug products, the strategy of solidifying phage formulations has been implemented to diversify the dosage forms and to overcome the storage condition limitations for liquid phage formulations. METHOD: In our work, we hypothesize and tested that an advanced technology, thin film freeze-drying (TFFD), can be used to produce phage containing dry powders without significantly losing phage viability. Here we selected T7 phage as our model phage in a preliminary screening study. RESULTS: We found that a binary excipient matrix of sucrose and leucine at ratios of 90:10 or 75:25 by weight, protected phage from the stresses encountered during the TFFD process. In addition, we confirmed that incorporating a buffer system in the formulation significantly improved the survival of phage during the initial freezing step and subsequent sublimation step in the solidifying processes. The titer loss of phage in SM buffer (Tris/NaCl/MgSO4) containing formulation was as low as 0.19 log plaque forming units, which indicated that phage function was well preserved after the TFFD process. The presence of buffers markedly reduced the geometric particle sizes as determined by a dry dispersion method using laser diffraction, which indicated that the TFFD phage powder formulations were easily sheared into smaller powder aggregates, an ideal property for facilitating a variety of topical drug delivery routes including pulmonary delivery through dry powder inhalers, nebulization after reconstitution, and intranasal or wound therapy, etc. CONCLUSION: From these findings, we show that introducing buffer system can stabilize phage during dehydration processes, and TFFD, as a novel particle engineering method, can successfully produce phage containing powders that possess the desired properties for bioactivity and potentially for inhalation therapy.

Soy Consumption, but Not Dairy Consumption, Is Inversely Associated with Fatty Acid Desaturase Activity in Young Adults.

Past research using hepatic rat microsomes showed that soy protein suppressed delta-6 desaturase activity (D6D) compared to casein (a dairy protein). The effects of soy and dairy on desaturase pathway activity in humans remain poorly investigated. The objective of this analysis was to investigate the association between soy and dairy consumption with plasma fatty acids and estimate the desaturase pathway activity in a multiethnic Canadian population of young adults. We analyzed data from men (n = 319) and women (n = 764) previously collected for the Toronto Nutrigenomics and Health Study. Food frequency questionnaires and plasma fatty acids were assessed. Relationships between soy and dairy beverages and food consumption with estimated desaturase activities were assessed by regression models and by grouping participants according to beverage and food intake data. Weak inverse associations (p ≤ 0.05) were found between soy consumption and the overall desaturation pathway activity, specifically D6D activity. When participants were grouped based on soy and dairy consumption habits, omega-6 LC-PUFAs, as well as various estimates of the desaturase pathway activity, were significantly lower in individuals consuming soy (with or without dairy) compared to individuals consuming only fluid milk and dairy products. In conclusion, soy consumption, not dairy consumption, appears to suppress desaturase pathway activity.

Diet Regulation of Long-Chain PUFA Synthesis: Role of Macronutrients, Micronutrients, and Polyphenols on Δ-5/Δ-6 Desaturases and Elongases 2/5.

Deficiencies in the n-3 (ω-3) long-chain PUFAs (LC-PUFAs) EPA and DHA are associated with increased risk for the development of numerous diseases. Although n-3 LC-PUFAs can be obtained by consuming marine products, they are also synthesized endogenously through a biochemical pathway regulated by the Δ-5/Δ-6 desaturase and elongase 2/5 enzymes. This narrative review collates evidence from the past 40 y demonstrating that mRNA expression and activity of desaturase and elongase enzymes are influenced by numerous dietary components, including macronutrients, micronutrients, and polyphenols. Specifically, we highlight that both the quantity and the composition of dietary fats, carbohydrates, and proteins can differentially regulate desaturase pathway activity. Furthermore, desaturase and elongase mRNA levels and enzyme activities are also influenced by micronutrients (folate, vitamin B-12, vitamin A), trace minerals (iron, zinc), and polyphenols (resveratrol, isoflavones). Understanding how these various dietary components influence LC-PUFA synthesis will help further advance our understanding of how dietary patterns, ranging from caloric excesses to micronutrient deficiencies, influence disease risks.

Aerosolizable Lipid Nanoparticles for Pulmonary Delivery of mRNA through Design of Experiments.

Messenger RNA is a class of promising nucleic acid therapeutics to treat a variety of diseases, including genetic diseases. The development of a stable and efficacious mRNA pulmonary delivery system would enable high therapeutic concentrations locally in the lungs to improve efficacy and limit potential toxicities. In this study, we employed a Design of Experiments (DOE) strategy to screen a library of lipid nanoparticle compositions to identify formulations possessing high potency both before and after aerosolization. Lipid nanoparticles (LNPs) showed stable physicochemical properties for at least 14 days of storage at 4 °C, and most formulations exhibited high encapsulation efficiencies greater than 80%. Generally, upon nebulization, LNP formulations showed increased particle size and decreased encapsulation efficiencies. An increasing molar ratio of poly-(ethylene) glycol (PEG)-lipid significantly decreased size but also intracellular protein expression of mRNA. We identified four formulations possessing higher intracellular protein expression ability in vitro even after aerosolization which were then assessed in in vivo studies. It was found that luciferase protein was predominately expressed in the mouse lung for the four lead formulations before and after nebulization. This study demonstrated that LNPs hold promise to be applied for aerosolization-mediated pulmonary mRNA delivery.

Effects of Cocoa-Derived Polyphenols on Cognitive Function in Humans. Systematic Review and Analysis of Methodological Aspects.

The effects of cocoa-derived polyphenols on cognitive functions have been analyzed through numerous studies using different interventions (doses, vehicles, time frame, cognition tests, and characteristics of participants) which may hamper the interpretation and comparison of findings across investigations. Thus, a systematic review was conducted to analyze the effects of cocoa-derived polyphenols intake on human cognition and discuss the methodological aspects that may contribute to the heterogeneity of findings. Randomized clinical trials evaluating the effect of cocoa polyphenols on cognitive function in healthy subjects were selected according to selection criteria. Twelve studies were selected. Quality was assessed according to the Cochrane risk for bias tool. The most common risk for bias was the lack of information about the sequence generation process. Effects on cognitive function were observed after consumption of 50 mg/day of (-)-epicatechin and in studies using a component-matched placebo and cocoa as the polyphenol vehicle given to healthy adults (18-50 years). Memory (n = 5) and executive function (n = 4) showed the most significant effects with medium and large effect sizes after intake of intermediate doses of cocoa flavanols (500-750 mg/day). Overall, this set of studies suggest a positive effect of cocoa polyphenols on memory and executive function. However, the available evidence is very diverse and future studies may address the identified sources of variation to strengthen current evidence on this promising field.

Quantitative PCR of T7 Bacteriophage from Biopanning.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., "biopanning"). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2-4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g., immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

Evaluación del impacto ambiental del proceso de modificación química de almidones nativos como potenciales excipientes en la industria cosmética / Evaluation of the environmental impact of the process of chemical modification of native starches as potential excipients in the cosmetic industry

RESUMEN El almidón por ser un polímero natural biodegradable es utilizado en la industria cosmética, gracias a que posee un bajo impacto ambiental. Para mejorar las propiedades anfifílicas de este polímero, se introduce una cadena alifática en la estructura, que permita interactuar con las cadenas apolares del almidón, generando así un aumento de su capacidad lipofílica. Por tanto, en este trabajo se usaron almidones de ñame, yuca, batata y maíz USP, este último como referencia, y fueron sometidos a tres metodologías de modificación: 1. Esterificación directa, 2. Hidrólisis y posterior esterificación y 3. Esterificación mediante microondas, empleando anhídrido dodecenil succínico (DDSA). En todos los casos, se incrementó significativamente la capacidad captadora de aceite y la emulsificante (tensoactiva) de los almidones; sin embargo, el "método en microondas" presentó algunas ventajas en comparación con otros métodos, de las cuales destacamos: aumento de la eficiencia de esterificación, disminución del tiempo de reacción de esterificación y menor impacto ambiental.

SUMMARY Starch, as a biodegradable natural polymer, is used in the cosmetic industry, exhibiting a low environmental impact. To improve the amphiphilic properties of this polymer, an aliphatic chain is introduced in the structure, which allows interacting with the polar chains of the starch, generating an increase of its lipophilic capacity. Therefore, in this paper we evaluated starches of yam, cassava, sweet potato and corn USP (as reference), which were subjected to three chemical modification methodologies: 1. Direct esterification; 2. Hydrolysis and esterification; 3. Esterification by microwaves, by using dodecenyl succinic anhydride (DDSA). In all cases significant increasing of the oil capturing capacities and emulsifier (active) of the starches. However, the "microwave method" presented some advantages compared to other methods, in particular, an increase in esterification efficiency, a decrease in the esterification reaction time and a lower environmental impact.

Evaluación de la actividad antioxidante de las pulpas de Rubus glaucus B, Vaccinium floribundum K y Beta vulgaris L / Evaluation of the antioxidant activity of pulps from Rubus glaucus B., Vaccinium floribundum K. and Beta vulgaris L

Introducción: actualmente se ha demostrado una relación entre el consumo de alimentos con altos niveles de compuestos antioxidantes, los cuales están directamente asociados con la prevención de enfermedades. Objetivos: evaluar la actividad antioxidante de las pulpas Rubus glaucus B, Vaccinium floribundum K y Beta vulgaris L. Métodos: Rubus glaucus Benth variedad Castilla, Vaccinium floribundum K y Beta vulgaris L fueron adquiridas en un mercado local de la ciudad de Cartagena, Bolívar (10°25'25″N 75°31'31″O). La pulpa se obtuvo a partir del fruto (mora y agraz) y raíz (remolacha); y le se determinó pH, Brix, acidez titulable, índice de madurez, humedad, ceniza, grasa, proteínas, fibra cruda, carbohidratos y la actividad antioxidante fue determinada mediante la técnica de actividad antiradicalaria por el método DPPH, asimismo el contenido de fenoles totales se realizó por el método colorimétrico Folin-Ciocalteu. Resultados: los sólidos solubles totales oscilaron entre 5,94 - 10,91 Brix, pH 2,9 - 5,96, ceniza 0,407 - 1,05 g/100 g, humedad 87,263 - 83,95 g/100 g, proteína 0,624 - 1,59 g/100 g, grasa 0,12 - 0,62 g/100 g, fibra cruda 2,107 - 3,637 g/100 g, carbohidratos 10,01 - 14,446 g/100 g para las pulpas de mora, agraz y remolacha. Los resultados de la prueba de actividad antioxidante presentaron valores de IC50 mediante la técnica de DPPH• en el rango de 53,33 - 141,88 µg/mL, lo cual está directamente relacionado con el contenido en fenoles. Conclusiones: por lo tanto, la pulpa de Rubus glaucus Benth variedad Castilla, Vaccinium floribundum K y Beta vulgaris L, son considerados como promisorios para diseñar productos nutracéuticos por su elevada actividad antioxidante(AU)

Introduction: A direct relationship has been proved to exist between consumption of foodstuffs with a high content of antioxidant compounds and disease prevention. Objectives: Evaluate the antioxidant activity of pulps from Rubus glaucus B., Vaccinium floribundum K. and Beta vulgaris L. Methods: Rubus glaucus Benth variety Castile, Vaccinium floribundum K. and Beta vulgaris L. were acquired at a local marketplace in the city of Cartagena, Bolívar (10°25?25?N 75°31?31?W). The pulp was obtained from fruits (Andean raspberry and Andean blueberry) and roots (beet). Determination was conducted of its pH, Brix, titratable acidity, maturation index, humidity, ash, fat, proteins, crude fiber, carbohydrates, and antioxidant activity using the technique of anti-radical activity by the DPPH method. Content of total phenols was determined by the Folin-Ciocaltey colorimetric method. Results: Total soluble solids ranged between 5.94 - 10.91 Brix, pH 2.9 - 5.96, ash 0.407 - 1.05 g / 100 g, humidity 87.263 - 83.95 g / 100 g, protein 0.624 - 1.59 g / 100 g, fat 0.12 - 0.62 g / 100 g, crude fiber 2.107 - 3.637 g / 100 g, carbohydrates 10.01 - 14.446 g / 100 g for pulps from Andean raspberry, Andean blueberry and beet. Antioxidant activity testing using the DPPHo technique obtained CI50 values of 53.33 - 141.88 ?g/ml, directly proportional to phenol content. Conclusions: It is therefore concluded that pulp from Rubus glaucus Benth variety Castile, Vaccinium floribundum K. and Beta vulgaris L., is considered to be promising for the design of nutraceutical products due to its antioxidant activity(AU)