Evolution of a novel adrenal cell type that promotes parental care.

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

Liposomal Phytosterols as LDL-Cholesterol-Lowering Agents in Diet-Induced Hyperlipidemia.

The high blood level of low-density lipoprotein cholesterol (LDL-C) is a primary risk factor for cardiovascular disease. Plant sterols, known as phytosterols (PSs), can reduce LDL-C in a range of 8-14%. The extent of LDL-C reduction depends on its formulation. Encapsulation into liposomes is one formulation strategy to enhance the efficiency of PSs. PSs (campesterol, stigmasterol, and ß-sitosterol) have frequently been assessed alone or in combination for their LDL-C-lowering ability. However, one naturally abundant PS, brassicasterol, has not yet been tested for its efficacy. We have previously developed a novel liposomal formulation containing the PS mixture present naturally in canola that is composed of brassicasterol, campesterol, and ß-sitosterol. In this work, the efficacy of our novel liposomal PS formulation that includes brassicasterol was assessed in a hamster model. Animals were divided into five groups: (i) liposomal PS in orange juice, (ii) liposomal PS in water, (iii) marketed PS in orange juice, (iv) control orange juice, and (v) control water. The animals were fed a high-fat, cholesterol-supplemented (0.5%) diet to induce hypercholesterolemia. The treatment was administered orally once daily for 4 weeks. Fasting blood samples were collected at baseline, week 2, and week 4. The extent of the reduction of total cholesterol, LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides was compared among the groups. Liposomal PSs in both orange juice and water significantly reduced LDL-C compared to their controls. Furthermore, the liposomal PS was as effective as a marketed PS-containing product in reducing LDL-C. Liposomal PSs in both orange juice and water showed similar efficacy in LDL-C reduction, highlighting that these vehicles/food matrices do not affect the efficacy of PSs. The liposomal formulation of a natural PS mixture extracted from canola oil, with brassicasterol as a major component, exhibited a significant LDL-C reduction in a hamster model.

Determination of phytosterol oxidation products in pharmaceutical liposomal formulations and plant vegetable oil extracts using novel fast liquid chromatography - Tandem mass spectrometric methods.

Phytosterol oxidation products (POPs) formed by the auto-oxidation of phytosterols can lead to negative health consequences. New liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative and qualitative approaches were developed. For quantification, sixteen phytosterol oxidation products (POPs) in liposomal formulations; namely 7-keto, 7-hydroxy, 5,6-epoxy, and 5,6-dihydroxy derivatives of brassicasterol, campesterol, stigmasterol, and ß-sitosterol were quantified. The method has a short run time of 5 min, achieved on a poroshell C18 column, using isocratic elution. To the best of our knowledge, this is the shortest run time among reported methods for the quantitative analysis of POPs. Atmospheric pressure chemical ionization (APCI) was used, and the mobile phase was composed of acetonitrile/methanol (99:1 v/v). The quantitative method was validated as per the FDA guidelines for linearity, accuracy, precision, selectivity, sensitivity, matrix effect, dilution integrity, and stability. The method was applied for the quantification of POPs in liposomal phytosterol formulations prepared with and without tocopherols, as antioxidants. The formulation process had little impact on the formation of POPs as only 7-ketobrassicasterol was quantified in tested samples. The quantified value of POPs in liposomal samples was insignificant to impart any toxicological effects. Other degradation products such as 7-hydroxy, 5,6-epoxy and 5,6-dihydroxy derivatives of brassicasterol, campesterol and ß-sitosterol were below the lower limit of quantification. Phytosterol-containing formulations were then assessed for their oxidative stability after microwave exposure for 5 min. The incorporation of tocopherols significantly increased the stability of phytosterols in the liposomal formulations. Finally, LC-MS/MS qualitative identification of phytosterols obtained from extra virgin olive oil was performed. New POPs, namely 7-ketoavenasterol, and 7-ketomethylenecycloartenol were putatively identified, illustrating the applicability of the method to identify POPs with varying structures present in various phytosterol sources. In fact, it is the first time that 7-ketomethylenecycloartenol is reported as a POP.

Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols.

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis-mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, ß-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). ß-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.

Analytical Strategies to Analyze the Oxidation Products of Phytosterols, and Formulation-Based Approaches to Reduce Their Generation.

Phytosterols are a class of lipid molecules present in plants that are structurally similar to cholesterol and have been widely utilized as cholesterol-lowering agents. However, the susceptibility of phytosterols to oxidation has led to concerns regarding their safety and tolerability. Phytosterol oxidation products (POPs) present in a variety of enriched and non-enriched foods can show pro-atherogenic and pro-inflammatory properties. Therefore, it is crucial to screen and analyze various phytosterol-containing products for the presence of POPs and ultimately design or modify phytosterols in such a way that prevents the generation of POPs and yet maintains their pharmacological activity. The main approaches for the analysis of POPs include the use of mass spectrometry (MS) linked to a suitable separation technique, notably gas chromatography (GC). However, liquid chromatography (LC)-MS has the potential to simplify the analysis due to the elimination of any derivatization step, usually required for GC-MS. To reduce the transformation of phytosterols to their oxidized counterparts, formulation strategies can theoretically be adopted, including the use of microemulsions, microcapsules, micelles, nanoparticles, and liposomes. In addition, co-formulation with antioxidants, such as tocopherols, may prove useful in substantially preventing POP generation. The main objectives of this review article are to evaluate the various analytical strategies that have been adopted for analyzing them. In addition, formulation approaches that can prevent the generation of these oxidation products are proposed.

Labour and delivery ward register data availability, quality, and utility - Every Newborn - birth indicators research tracking in hospitals (EN-BIRTH) study baseline analysis in three countries.

BACKGROUND: Countries with the highest burden of maternal and newborn deaths and stillbirths often have little information on these deaths. Since over 81% of births worldwide now occur in facilities, using routine facility data could reduce this data gap. We assessed the availability, quality, and utility of routine labour and delivery ward register data in five hospitals in Bangladesh, Nepal, and Tanzania. This paper forms the baseline register assessment for the Every Newborn-Birth Indicators Research Tracking in Hospitals (EN-BIRTH) study. METHODS: We extracted 21 data elements from routine hospital labour ward registers, useful to calculate selected maternal and newborn health (MNH) indicators. The study sites were five public hospitals during a one-year period (2016-17). We measured 1) availability: completeness of data elements by register design, 2) data quality: implausibility, internal consistency, and heaping of birthweight and explored 3) utility by calculating selected MNH indicators using the available data. RESULTS: Data were extracted for 20,075 births. Register design was different between the five hospitals with 10-17 of the 21 selected MNH data elements available. More data were available for health outcomes than interventions. Nearly all available data elements were > 95% complete in four of the five hospitals and implausible values were rare. Data elements captured in specific columns were 85.2% highly complete compared to 25.0% captured in non-specific columns. Birthweight data were less complete for stillbirths than live births at two hospitals, and significant heaping was found in all sites, especially at 2500g and 3000g. All five hospitals recorded count data required to calculate impact indicators including; stillbirth rate, low birthweight rate, Caesarean section rate, and mortality rates. CONCLUSIONS: Data needed to calculate MNH indicators are mostly available and highly complete in EN-BIRTH study hospital routine labour ward registers in Bangladesh, Nepal and Tanzania. Register designs need to include interventions for coverage measurement. There is potential to improve data quality if Health Management Information Systems utilization with feedback loops can be strengthened. Routine health facility data could contribute to reduce the coverage and impact data gap around the time of birth.

The simultaneous quantification of phytosterols and tocopherols in liposomal formulations using validated atmospheric pressure chemical ionization- liquid chromatography -tandem mass spectrometry.

A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and ß-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05-10 µg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25-10 µg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25-10 µg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC-MS/MS method addresses three major analytical shortcomings, namely i)longer run time, ii)complex gradient elution and iii)poor baseline separation of phytosterols and tocopherols.

The Establishment of Tandem Mass Spectrometric Fingerprints of Phytosterols and Tocopherols and the Development of Targeted Profiling Strategies in Vegetable Oils.

Phytosterols and tocopherols are essential for plant biochemistry, and they possess beneficial health effects for humans. Evaluating the tandem mass spectrometric (MS/MS) behavior of phytosterols and tocopherols is needed for the development of a qualitative and quantitative method for these biologically active plant metabolites. Herein, the MS/MS dissociation behavior of phytosterols and tocopherols is elucidated to establish generalized MS/MS fingerprints. MS/MS and multistage (MS3) analysis revealed common fragmentation behavior among the four tested phytosterols, namely ß-sitosterol, stigmasterol, campesterol, and brassicasterol. Similar analysis was conducted for the tocopherols (i.e., alpha (α), beta (ß), gamma (γ), and delta (δ)). As such, a universal MS/MS fragmentation pathway for each group was successfully established for the first time. Based on the generalized MS/MS fragmentation behavior of phytosterols, diagnostic product ions were chosen for the development of profiling methods for over 20 naturally occurring phytosterols. A precursor ion scan-triggered-enhanced product ion scan (PIS-EPI) method was established. Due to enhanced chromatographic peaks, multiple ion monitoring-triggered-enhanced product ion scan (MIM-EPI) was employed for confirmation. The screening approach was applied successfully to identify blinded samples obtained from standard mixtures as well as sesame and olive oils. The oil samples contain other phytosterols, and their successful identification indicates that, the generalized MS/MS fragmentation behavior is applicable to various structures of phytosterols. A similar approach was attempted for tocopherols and was only hindered by the low concentration of these bioactive metabolites present in the oil samples.

Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives.

Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and ß-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (-9 to -14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.