Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

Systematic Down-Selection of Repurposed Drug Candidates for COVID-19.

SARS-CoV-2 is the cause of the COVID-19 pandemic which has claimed more than 6.5 million lives worldwide, devastating the economy and overwhelming healthcare systems globally. The development of new drug molecules and vaccines has played a critical role in managing the pandemic; however, new variants of concern still pose a significant threat as the current vaccines cannot prevent all infections. This situation calls for the collaboration of biomedical scientists and healthcare workers across the world. Repurposing approved drugs is an effective way of fast-tracking new treatments for recently emerged diseases. To this end, we have assembled and curated a database consisting of 7817 compounds from the Compounds Australia Open Drug collection. We developed a set of eight filters based on indicators of efficacy and safety that were applied sequentially to down-select drugs that showed promise for drug repurposing efforts against SARS-CoV-2. Considerable effort was made to evaluate approximately 14,000 assay data points for SARS-CoV-2 FDA/TGA-approved drugs and provide an average activity score for 3539 compounds. The filtering process identified 12 FDA-approved molecules with established safety profiles that have plausible mechanisms for treating COVID-19 disease. The methodology developed in our study provides a template for prioritising drug candidates that can be repurposed for the safe, efficacious, and cost-effective treatment of COVID-19, long COVID, or any other future disease. We present our database in an easy-to-use interactive interface (CoviRx that was also developed to enable the scientific community to access to the data of over 7000 potential drugs and to implement alternative prioritisation and down-selection strategies.

LINC to Life: Evaluation of a Safety Planning Training Program with Clinicians and Mental Health Staff.

Suicide rates continue to rise, and clinicians and mental health staff play a critical role in keeping suicidal clients safe. Safety planning, including means safety, may help to decrease suicide risk. Unfortunately, availability and evaluation of safety planning training for these providers are scarce. The goal of the present study was to evaluate a safety planning training, LINC to LIFE Safety Planning (L2L SP). L2L SP is a 150-minute, face-to-face training program that teaches providers to engage clients in collaborative safety planning and means safety efforts, facilitate diverse client coping strategies, problem-solve, and involve close others, among other skills. These objectives are achieved through interactive content delivery, role-play, and corrective feedback. L2L SP was administered to 95 participants. Key determinants of behavioral change (e.g., knowledge, attitudes, perceived behavioral control [PBC]) were measured at pre, post, and six-month follow-up. Additionally, participants' behaviors and emotions in working with suicidal clients were measured at pretest and six-month follow-up. Paired sample t-tests, repeated measures MANOVA, and univariate ANOVAs with post-hoc testing using Bonferroni correction were conducted. Results supported significant improvements in knowledge, PBC, and intentions at post-test, and attitudes, PBC, and effective emotional responses at follow-up. Exploratory analyses suggested significant improvements in behaviors among clinicians and mental health staff who saw clients reporting suicidal ideation. The present study provides promising results regarding brief safety planning training. Declines in knowledge and PBC following the training highlight the potential need for booster sessions or more intensive initial training in these areas.HighlightsThe present study evaluated a comprehensive, interactive safety planning training.Knowledge, PBC, and intentions were significantly improved at post-test.Attitudes, PBC, and emotions were significantly improved at follow-up.

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

ß-Adrenoceptor regulation of metabolism in U937 derived macrophages.

Macrophages have important roles in the immune system including clearing pathogens and wound healing. Metabolic phenotypes in macrophages have been associated with functional phenotypes, where pro-inflammatory macrophages have an increased rate of glycolysis and anti-inflammatory macrophages primarily use oxidative phosphorylation. ß-adrenoceptor (ßAR) signalling in macrophages has been implicated in disease states such as cancer, atherosclerosis and rheumatoid arthritis. The impact of ßAR signalling on macrophage metabolism has not been defined. Using metabolomics and proteomics, we describe the impact of ßAR signalling on macrophages treated with isoprenaline. We found that ßAR signalling alters proteins involved in cytoskeletal rearrangement and redox homeostasis of the cell. We showed that ßAR signalling in macrophages shifts glucose metabolism from glycolysis towards the tricarboxylic acid cycle and pentose phosphate pathways. We also show that ßAR signalling perturbs purine metabolism by accumulating adenylate and guanylate pools. Taken together, these results indicate that ßAR signalling shifts metabolism to support redox processes and upregulates proteins involved in cytoskeletal changes, which may contribute to ßAR effects on macrophage function.

Ambient Discrimination, Victimization, and Suicidality in a Non-Probability U.S. Sample of LGBTQ Adults.

Individuals who identify as lesbian, gay, bisexual, transgender, or queer (LGBTQ) are at a higher risk for suicidality compared to the general population. A growing body of research has investigated this risk, particularly with attention to systemic factors such as discrimination and harassment. Unfortunately, research has only examined the impact of direct discrimination on suicidality and has neglected to examine how ambient discrimination (i.e., witnessing or being made aware of discriminatory behaviors directed at someone other than yourself in your group) relates to suicidality. Additionally, although some links exist between discrimination and suicidality, the mechanisms by which these are related are understudied. This study aimed to address these gaps by exploring the effect of ambient discrimination on suicidal ideation and examining psychological pain as a mediator in this relationship. Data were collected from a sample of 200 LGBTQ-identified individuals (M age = 35 years; 53.5% female; 86% White). Results of independent t tests and a one-way multivariate ANOVA revealed greater vulnerability for ambient/direct discrimination and psychache among individuals identifying as transgender, queer, and other. Regression and mediation analyses revealed that while both ambient and direct discrimination predicted suicidal ideation, only direct discrimination accounted for unique variance in the outcome; however, both ambient and direct discrimination contributed unique variance to psychological pain, which fully mediated their relationships to suicidal ideation. Results of this study may begin to provide insight into the pathways of risk and points of intervention for suicidality in the LGBTQ community.

Don't Invite Everyone! Training Variables Impacting the Effectiveness of QPR Trainings.

Gatekeeper trainings have been increasingly utilized in response to rising suicide rates in youths. However, the extent to which common factors inherent to gatekeeper trainings impact training outcomes is largely understudied. As such, the present study explored how factors such as training size and trainer engagement abilities influenced trainee participation and outcomes (i.e. changes in attitudes, perceived behavioral control (PBC), and knowledge regarding suicide prevention). All trainees participated in a gatekeeper training; trainers were not randomly assigned. Mediation and moderation analyses were completed using the PROCESS macro for SPSS (Hayes in Introduction to mediation, moderation, and conditional process analysis: a regression-based approach, Guilford Press, New York, 2013). Trainee participation in a training was examined as a mediator of the relationship between the training size and training outcomes, while trainer engagement was examined as a moderator of the relationship between size of training and trainee participation. Size of training was significantly related to lower changes in participant knowledge, along with lower trainee participation in gatekeeper trainings. Trainee participation significantly mediated the relationship between size of training, attitudes, and PBC. Additionally, trainer engagement significantly moderated the association between size of training and trainee participation. The results of this study suggest that general gatekeeper training-related variables may influence participant outcomes, specifically through trainee participation.

Frustration with feeling: Latent classes of non-suicidal self-injury and emotion regulation difficulties.

Although emotion regulation has been identified as a key function of non-suicidal self-injury (NSSI), it is unclear how specific indices of emotion regulation are associated with particular NSSI methods as markers of risk. This study used latent class analysis (LCA) to identify subgroups of individuals who engage in NSSI and their patterns of emotional regulation difficulties. Undergraduate students in the southeastern United States (N = 326) completed an online survey. LCA was used to identify subgroups of individuals engaging in NSSI and their associated emotion regulation difficulties. These subgroups were then compared across a variety of behavioral health outcomes (e.g. impulsive behavior, disordered eating, problematic alcohol use, suicide attempt history) to characterize specific risk profiles. The LCA revealed four subgroups who engage in NSSI and have specific emotion regulation difficulties. These subgroups were differentially associated with behavioral health outcomes, including suicide risk, disordered eating, and impulsive behavior. Results of this research could aid in clinical identification of at-risk individuals.

Optimized Method for Untargeted Metabolomics Analysis of MDA-MB-231 Breast Cancer Cells.

Cancer cells often have dysregulated metabolism, which is largely characterized by the Warburg effect-an increase in glycolytic activity at the expense of oxidative phosphorylation-and increased glutamine utilization. Modern metabolomics tools offer an efficient means to investigate metabolism in cancer cells. Currently, a number of protocols have been described for harvesting adherent cells for metabolomics analysis, but the techniques vary greatly and they lack specificity to particular cancer cell lines with diverse metabolic and structural features. Here we present an optimized method for untargeted metabolomics characterization of MDA-MB-231 triple negative breast cancer cells, which are commonly used to study metastatic breast cancer. We found that an approach that extracted all metabolites in a single step within the culture dish optimally detected both polar and non-polar metabolite classes with higher relative abundance than methods that involved removal of cells from the dish. We show that this method is highly suited to diverse applications, including the characterization of central metabolic flux by stable isotope labelling and differential analysis of cells subjected to specific pharmacological interventions.

An oncogenic role for sphingosine kinase 2.

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an anti-proliferative/pro-apoptotic function for SK2, while others indicate it has a pro-survival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.