Oncostatin M and STAT3 Signaling Pathways Support Human Trophoblast Differentiation by Inhibiting Inflammatory Stress in Response to IFNγ and GM-CSF.

Interleukin-6 (IL-6) superfamily cytokines play critical roles during human pregnancy by promoting trophoblast differentiation, invasion, and endocrine function, and maintaining embryo immunotolerance and protection. In contrast, the unbalanced activity of pro-inflammatory factors such as interferon gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at the maternal-fetal interface have detrimental effects on trophoblast function and differentiation. This study demonstrates how the IL-6 cytokine family member oncostatin M (OSM) and STAT3 activation regulate trophoblast fusion and endocrine function in response to pro-inflammatory stress induced by IFNγ and GM-CSF. Using human cytotrophoblast-like BeWo (CT/BW) cells, differentiated in villous syncytiotrophoblast (VST/BW) cells, we show that beta-human chorionic gonadotrophin (ßhCG) production and cell fusion process are affected in response to IFNγ or GM-CSF. However, those effects are abrogated with OSM by modulating the activation of IFNγ-STAT1 and GM-CSF-STAT5 signaling pathways. OSM stimulation enhances the expression of STAT3, the phosphorylation of STAT3 and SMAD2, and the induction of negative regulators of inflammation (e.g., IL-10 and TGFß1) and cytokine signaling (e.g., SOCS1 and SOCS3). Using STAT3-deficient VST/BW cells, we show that STAT3 expression is required for OSM to regulate the effects of IFNγ in ßhCG and E-cadherin expression. In contrast, OSM retains its modulatory effect on GM-CSF-STAT5 pathway activation even in STAT3-deficient VST/BW cells, suggesting that OSM uses STAT3-dependent and -independent mechanisms to modulate the activation of pro-inflammatory pathways IFNγ-STAT1 and GM-CSF-STAT5. Moreover, STAT3 deficiency in VST/BW cells leads to the production of both a large amount of ßhCG and an enhanced expression of activated STAT5 induced by GM-CSF, independently of OSM, suggesting a key role for STAT3 in ßhCG production and trophoblast differentiation through STAT5 modulation. In conclusion, our study describes for the first time the critical role played by OSM and STAT3 signaling pathways to preserve and regulate trophoblast biological functions during inflammatory stress.

Chromatic and spatial image statistics predict infants' visual preferences and adults' aesthetic preferences for art.

Aesthetics has been characterized as a triadic interaction of perceptual, emotional, and conceptual neural systems (e.g., Chatterjee & Vartanian, 2014). There has been much empirical effort to identify the visual features that contribute to the perceptual component of this triad (e.g., Mather, 2020). Here, we measured infants' visual preferences and adults' aesthetic preferences for 40 of van Gogh's landscape paintings and investigated the contribution of the chromatic and spatial image statistics of the art to infants' and adults' responses. We found that infants' and adults' responses were significantly related: infants looked longer at the art that the adults found more pleasant. We also found that our combination of chromatic and spatial image statistics could account for around two thirds of the variance in infant looking and adult pleasantness ratings. The amount of variation in the luminance and saturation of the art's pixels contributed to both infants' visual preferences and adults' aesthetic preferences, potentially identifying two "perceptual primitives" of aesthetics that can be traced back to early sensory biases in infancy. We also identified important differences in the types of image statistics that predict infants' and adults' responses. We discuss the findings in relation to theories of aesthetics, natural scene statistics, and infant vision and perception.

DART-MS Facilitated Quantification of Cannabinoids in Complex Edible Matrices-Focus on Chocolates and Gelatin-Based Fruit Candies.

Traditional methods for detecting and quantifying cannabinoids in Cannabis sativa materials are most often chromatography-based, and they generally require extensive sample preparation protocols to render materials into a form that can be injected into the systems without the risk of contaminating or damaging the equipment. This challenge is amplified when interrogating the increasingly broad range of matrix types that cannabinoids are infused within, such as edibles that also contain sugars, fats, lipids, and carbohydrates. The requisite application of highly nuanced methods that must be developed for each matrix type is, in addition to being resource-intensive and time-consuming, highly impractical and unsustainable for crime laboratories endeavoring to perform such analyses in a routine manner, since they are often under-resourced while typically also confronting sample testing backlogs. A key to resolving this issue is to identify an analysis approach that avoids the requirement for nuanced method development by being applicable to a broader range of matrix types. Ambient ionization mass spectrometry (AIMS) methods have shown great promise in their ability to rapidly interrogate samples. Therefore, this study focused on developing validated protocols using AIMS (specifically, direct analysis in real time-high-resolution mass spectrometry, or DART-HRMS) to detect and quantify Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in edible matrices. Calibration curves were developed using deuterated counterparts of THC and CBD as internal standards. Following the use of high cannabinoid recovery rate extraction protocols for chocolates and gelatin-based fruit candies or "gummies", the DART-HRMS approach was applied to quantify cannabinoid levels in commercially available cannabinoid-infused candies, yielding results similar to those reported on the product labels. Importantly, the developed method circumvented challenges encountered using traditional approaches. As the Cannabis field continues to evolve and new matrix types emerge on the market, the DART-HRMS detection and quantification protocols can be readily applied without the need for major procedural adaptations.

Combined ambient ionization mass spectrometric and chemometric approach for the differentiation of hemp and marijuana varieties of Cannabis sativa.

BACKGROUND: Hemp and marijuana are the two major varieties of Cannabis sativa. While both contain Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of C. sativa, they differ in the amount of THC that they contain. Presently, U.S. federal laws stipulate that C. sativa containing greater than 0.3% THC is classified as marijuana, while plant material that contains less than or equal to 0.3% THC is hemp. Current methods to determine THC content are chromatography-based, which requires extensive sample preparation to render the materials into extracts suitable for sample injection, for complete separation and differentiation of THC from all other analytes present. This can create problems for forensic laboratories due to the increased workload associated with the need to analyze and quantify THC in all C. sativa materials. METHOD: The work presented herein combines direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) and advanced chemometrics to differentiate hemp and marijuana plant materials. Samples were obtained from several sources (e.g., commercial vendors, DEA-registered suppliers, and the recreational Cannabis market). DART-HRMS enabled the interrogation of plant materials with no sample pretreatment. Advanced multivariate data analysis approaches, including random forest and principal component analysis (PCA), were used to optimally differentiate these two varieties with a high level of accuracy. RESULTS: When PCA was applied to the hemp and marijuana data, distinct clustering that enabled their differentiation was observed. Furthermore, within the marijuana class, subclusters between recreational and DEA-supplied marijuana samples were observed. A separate investigation using the silhouette width index to determine the optimal number of clusters for the marijuana and hemp data revealed this number to be two. Internal validation of the model using random forest demonstrated an accuracy of 98%, while external validation samples were classified with 100% accuracy. DISCUSSION: The results show that the developed approach would significantly aid in the analysis and differentiation of C. sativa plant materials prior to launching painstaking confirmatory testing using chromatography. However, to maintain and/or enhance the accuracy of the prediction model and keep it from becoming outdated, it will be necessary to continue to expand it to include mass spectral data representative of emerging hemp and marijuana strains/cultivars.

Rapid Detection and Quantification of Hallucinogenic Salvinorin A in Commercial Salvia divinorum Products by DART-HRMS.

In recent years, national laboratories have identified several plant-derived materials as concerns to public health because of their psychoactive effects, potential for abuse, and the lack of federal regulation of their use. One of these is Salvia divinorum (aka Salvia), which has received focused attention due to its increasing recreational use and the ease by which it can be acquired. Traditional chromatographic approaches for the detection of the major psychoactive component of Salvia (i.e., salvinorin A) typically require time-consuming sample pretreatment prior to identifying the presence of salvinorin A in plant material unknowns. In this study, direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) was used to rapidly screen for Salvia plant material. This approach facilitated the analysis of bulk material in its native form, thereby bypassing sample pretreatment steps. In addition, a validated DART-HRMS method was developed for the quantification of salvinorin A in commercial Salvia products (e.g., raw plant materials, enhanced leaf extracts). In this regard, cholesterol was found to be a suitable internal standard. The average salvinorin A content in raw Salvia leaves was determined to be 1.54 mg/g, while the salvinorin A quantified in enhanced Salvia leaf extracts was between 13.0 and 53.2 mg/g.

Introducing "DoPP": A Graphical User-Friendly Application for the Rapid Species Identification of Psychoactive Plant Materials and Quantification of Psychoactive Small Molecules Using DART-MS Data.

The widespread abuse of "legal high" psychoactive plants continues to be of global concern because of their negative impacts on public health and safety. In forensic science, a major challenge in controlling these substances is the paucity of methods to rapidly identify them. We report the development of the Database of Psychoactive Plants (DoPP), a new user-friendly tool featuring an architecture for the identification of plant unknowns, and the necessary regression statistics for the development and validation of psychoactive compound quantification. The application relies on the knowledge that terrestrial plants exhibit species-specific chemical signatures that can be revealed by direct analysis in real time─high-resolution mass spectrometry (DART-HRMS). Subsequent automated machine learning processing of libraries of these spectra enables rapid discrimination and species identification. The chemical signature database includes 57 available plant species. The rapid acquisition of mass spectra and the ability to sample the materials in their native form enabled the generation of the vast amounts of spectral replicates required for database construction. For the identification of sample unknowns, a data analysis workflow was developed and implemented using the DoPP tool. It utilizes a hierarchical classification tree that integrates three machine learning methods, namely, random forest, k-nearest neighbors, and support vector machine, all of which were fused using posterior probabilities. The results show accuracies of 98 and 99% for 10-fold cross-validation and external validation, respectively, which make the classification model suitable for identity prediction of real samples.

Immunometabolic adaptation and immune plasticity in pregnancy and the bi-directional effects of obesity.

Mandatory maternal metabolic and immunological changes are essential to pregnancy success. Parallel changes in metabolism and immune function make immunometabolism an attractive mechanism to enable dynamic immune adaptation during pregnancy. Immunometabolism is a burgeoning field with the underlying principle being that cellular metabolism underpins immune cell function. With whole body changes to the metabolism of carbohydrates, protein and lipids well recognised to occur in pregnancy and our growing understanding of immunometabolism as a determinant of immunoinflammatory effector responses, it would seem reasonable to expect immune plasticity during pregnancy to be linked to changes in the availability and handling of multiple nutrient energy sources by immune cells. While studies of immunometabolism in pregnancy are only just beginning, the recognised bi-directional interaction between metabolism and immune function in the metabolic disorder obesity might provide some of the earliest insights into the role of immunometabolism in immune plasticity in pregnancy. Characterised by chronic low-grade inflammation including in pregnant women, obesity is associated with numerous adverse outcomes during pregnancy and beyond for both mother and child. Concurrent changes in metabolism and immunoinflammation are consistently described but any causative link is not well established. Here we provide an overview of the metabolic and immunological changes that occur in pregnancy and how these might contribute to healthy versus adverse pregnancy outcomes with special consideration of possible interactions with obesity.

Quantification of hordenine in a complex plant matrix by direct analysis in real time-high-resolution mass spectrometry: Application to the "plant of concern" Sceletium tortuosum.

Recently, there has been an increase in the recreational abuse of several psychoactive plants, resulting in the United Nations Office on Drugs and Crime creating a list of "plants of concern." One such material is Sceletium tortuosum and products derived from it. Regulation of these materials is challenging because of their innocuous appearance, the cumbersome sample preparation steps required to render the material into a form amenable to analysis by conventional techniques, the requirement for nuanced sample analysis protocols, and lengthy analysis times. It is demonstrated here that direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) can be used to not only identify S. tortuosum material based on the detection of characteristic biomarkers including hordenine and several mesembrine alkaloids, but also quantify the amount of hordenine present. Using hordenine-d6 as an internal standard, a protocol, validated according to US Food and Drug Administration (FDA) Guidelines for the Development and Validation of Bioanalytical Methods, was devised for the quantification of the psychoactive component hordenine. The method was then applied to the quantification of hordenine in six commercially available products derived from the foliage and stems of S. tortuosum. By this method, the lower limit of quantification (LLOQ) was found to be 1 µg/ml. Observed hordenine concentrations ranged from 0.02738 to 1.071 mg of hordenine per gram of plant material. The developed technique provides an effective and quick means for the detection and quantification of hordenine in S. tortuosum, which can be extended to analysis of other hordenine-containing products.

Detection and Quantification of Psychoactive N,N-Dimethyltryptamine in Ayahuasca Brews by Ambient Ionization High-Resolution Mass Spectrometry.

The United Nations Office on Drugs and Crime designated twenty psychoactive botanical species as "plants of concern" because of their increased recreational abuse. Four of these are used to prepare ayahuasca brews. The complexity of the plant matrices, as well as the beverage itself, make the identification and quantification of the Schedule I component, N,N-dimethyltryptamine (DMT), a time-consuming and resource-intensive endeavor when performed using conventional approaches previously reported. Reported here is the development of a rapid validated method for the quantification of DMT in ayahuasca by direct analysis in real time-high-resolution mass spectrometry (DART-HRMS). This ambient ionization approach also enables identification of ayahuasca through detection of the secondary metabolites associated with its plant constituents. Analysis of six ayahuasca brews created using different combinations of DMT/harmala alkaloid-containing plants resulted in beverages with DMT levels of 45.7-230.5 mg/L. The detected amounts were consistent with previously reported values determined by conventional approaches.

Structural and hemodynamic properties of murine pulmonary arterial networks under hypoxia-induced pulmonary hypertension.

Detection and monitoring of patients with pulmonary hypertension, defined as a mean blood pressure in the main pulmonary artery above 25 mmHg, requires a combination of imaging and hemodynamic measurements. This study demonstrates how to combine imaging data from microcomputed tomography images with hemodynamic pressure and flow waveforms from control and hypertensive mice. Specific attention is devoted to developing a tool that processes computed tomography images, generating subject-specific arterial networks in which one-dimensional fluid dynamics modeling is used to predict blood pressure and flow. Each arterial network is modeled as a directed graph representing vessels along the principal pathway to ensure perfusion of all lobes. The one-dimensional model couples these networks with structured tree boundary conditions representing the small arteries and arterioles. Fluid dynamics equations are solved in this network and compared to measurements of pressure in the main pulmonary artery. Analysis of microcomputed tomography images reveals that the branching ratio is the same in the control and hypertensive animals, but that the vessel length-to-radius ratio is significantly lower in the hypertensive animals. Fluid dynamics predictions show that in addition to changed network geometry, vessel stiffness is higher in the hypertensive animal models than in the control models.

Recruiting Research Participants via Traditional Snowball vs Facebook Advertisements and a Website.

This article describes the use of Facebook, a website targeting nurses, and snowball sampling for recruitment of registered nurse participants in a qualitative study exploring measurement-driven clinical behavior and metric-driven harm. Previous studies suggest that social media can be a successful and cost-effective sampling strategy, increasing the numbers of participants, their diversity, and their representativeness of the population of interest. This study, however, found traditional snowball sampling to be far more effective than advertisements via Facebook and a professional website. Lessons learned are detailed, including cost and technical issues encountered. Suggestions for nurse researchers considering using Facebook for participant recruitment are described. Methodological research that could enhance the empirical-base supporting effective social media recruitment of research participants is offered for consideration by nurse researchers.

Macrophage Plasticity in Reproduction and Environmental Influences on Their Function.

Macrophages are key components of the innate immune system and exhibit extensive plasticity and heterogeneity. They play a significant role in the non-pregnant cycling uterus and throughout gestation they contribute to various processes underpinning reproductive success including implantation, placentation and parturition. Macrophages are also present in breast milk and impart immunomodulatory benefits to the infant. For a healthy pregnancy, the maternal immune system must adapt to prevent fetal rejection and support development of the semi-allogenic fetus without compromising host defense. These functions are dependent on macrophage polarization which is governed by the local tissue microenvironmental milieu. Disruption of this microenvironment, possibly by environmental factors of infectious and non-infectious origin, can affect macrophage phenotype and function and is linked to adverse obstetric outcomes, e.g. spontaneous miscarriage and preterm birth. Determining environmental influences on cellular and molecular mechanisms that control macrophage polarization at the maternal-fetal interface and the role of this in pregnancy complications could support approaches to alleviating adverse pregnancy outcomes.

Rapid detection and validated quantification of psychoactive compounds in complex plant matrices by direct analysis in real time-high resolution mass spectrometry - Application to "Kava" psychoactive pepper products.

RATIONALE: Classified by the UNODC as a top 20 plant of concern, Piper methysticum (also known as Kava) is being increasingly abused recreationally for its mind-altering effects. It is of significant forensic relevance to establish methods to rapidly identifyand quantify psychoactive compounds, especially those yet to be scheduled ascontrolled substances and which have exhibited various noteworthy health concerns. METHODS: Direct analysis in real time high-resolution mass spectrometry (DART-HRMS) demonstrated the ability to detect a range of kavalactones in Pipermethysticum derived products and plant material with no sample preparation. Inaddition, a validated method using calibration curves developed with a deuteratedinternal standard was used for the quantification of the psychoactive moleculeyangonin in various products. RESULTS: DART-HRMS detected the protonated masses of six major kavalactonesand three flavokavains in 18 commercial Kava products. A method consistent withFDA validation guidelines was established for the quantification of yangonin in thevarious complex matrices. Implementation of this method, with an LLOQ of 5 mg/mL, enabled successful quantification of yangonin in 16 Kava products.Concentrations for solid products ranged from 2.71 to 8.99 mg/g, while that forliquid products ranged from 1.03 to 4.59 mg/mL. CONCLUSIONS: Rapid identification and quantification of psychoactive smallmolecules in plant material can be accomplished using a validated DART-HRMSprotocol. This work illustrates an approach to qualitative and quantitative analysesof a wide variety of complex matrices derived from plants, and demonstrates thatthe commercially available products analyzed are P. methysticum derived and docontain psychoactive yangonin at quantifiable levels.

Akt and STAT5 mediate naïve human CD4+ T-cell early metabolic response to TCR stimulation.

Metabolic pathways that regulate T-cell function show promise as therapeutic targets in diverse diseases. Here, we show that at rest cultured human effector memory and central memory CD4+ T-cells have elevated levels of glycolysis and oxidative phosphorylation (OXPHOS), in comparison to naïve T-cells. Despite having low resting metabolic rates, naive T-cells respond to TCR stimulation with robust and rapid increases in glycolysis and OXPHOS. This early metabolic switch requires Akt activity to support increased rates of glycolysis and STAT5 activity for amino acid biosynthesis and TCA cycle anaplerosis. Importantly, both STAT5 inhibition and disruption of TCA cycle anaplerosis are associated with reduced IL-2 production, demonstrating the functional importance of this early metabolic program. Our results define STAT5 as a key node in modulating the early metabolic program following activation in naive CD4+ T-cells and in turn provide greater understanding of how cellular metabolism shapes T-cell responses.

Application of Direct Analysis in Real Time-High Resolution Mass Spectrometry to Investigations of Induced Plant Chemical Defense Mechanisms-Revelation of Negative Feedback Inhibition of an Alliinase.

Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 µM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.

Assessment of children coming into care: processes, pitfalls and partnerships.

Children in out-of-home care (OOHC) present with high levels of physical, developmental and emotional and behavioural difficulties, yet often fail to receive appropriate services. This article describes a joint health and welfare service specifically developed to provide comprehensive physical, developmental and mental health assessments to a cohort of children entering long-term care in one region of Sydney, New South Wales (NSW), Australia. Paediatric, allied health, dental and psychosocial assessments were co-ordinated from a single referral from the child's welfare case manager. Follow-up appointments were held 6-12 months later to assess the outcomes of recommendations. Physical, mental health and developmental difficulties in the children are reported, the implications for service requirements are presented and process blocks described. There is a need for a specific co-ordinating service to overcome the inherent fragmentation of this group (related both to transience and change in the welfare sector, and levels of comorbidity and chronicity in health presentations). Health and Welfare services must operate together, with an awareness of the processes and resource constraints in each sector, if they are to deliver sustainable and reliable health care to this vulnerable group.