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Abstract Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

Objective: NF1 is a tumor suppressor gene and its protein product, neurofibromin, is a negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27 % of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) – a disorder caused by germline NF1 mutations – have an increased risk of dying from breast cancer [1–4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-deficient breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER + breast cancer. Methods: We employed two ER+ NF1-deficient breast cancer models: (1) an NF1-deficient MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF-related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U–13C]-glucose and [U–13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy. Results: Our results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis. Conclusions: NF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies.

The intensification of Northern Hemisphere glaciation (iNHG) at 2.73 Ma is associated with a reorganization of the subarctic Pacific Ocean and abrupt drop in opal mass accumulation rates. Uncertainty, however, remains around the extent to which these changes altered carbon dynamics and contributed to a reduction in atmospheric pCO2 and global temperatures. These issues are addressed here using the boron isotope (δ11B) proxy in diatom frustules to reconstruct past changes in the pH and pCO2 of ambient seawater. Diatom δ11B and [B] indicate a subarctic Pacific surface water increase of 0.3–0.5 pH units over the iNHG. This confirms that delivery of carbon and nutrients into surface waters was reduced at this time, explaining the drop in opal productivity and limiting CO2 outgassing from the ocean interior. We consider two hypotheses to explain this based on potential changes in circulation from the late Pliocene to early Pleistocene: "ventilation to stratification" or "stratification to ventilation." The ventilation to stratification hypothesis, which posits a switch from vigorous Pacific Meridional Overturning Circulation in the Pliocene to stratification over iNHG, has received more attention in the literature. The stratification to ventilation hypothesis, which posits a modest increase in ventilation, is more consistent with modern and late Pleistocene analogs, the majority of models and δ13C data. These late Pliocene changes in the subarctic Pacific, in conjunction with other external and internal processes including those in the Southern Ocean, would have contributed to a lowering of atmospheric pCO2 and the long‐term expansion of ice‐sheets across the Northern Hemisphere. Key Points: δ11B and [B] in diatom frustules reveal a surface water increase of 0.3–0.5 pH units in the subarctic Pacific Ocean over the iNHGThese changes would have lowered atmospheric pCO2 and contributed to the long‐term expansion of Northern Hemisphere ice‐sheets [ABSTRACT FROM AUTHOR]

Summary Measuring cellular energetics is essential to understanding a matrix's (e.g. cell, tissue, or biofluid) metabolic state. The Agilent Seahorse machine is a common method to measure real-time cellular energetics, but existing analysis tools are highly manual or lack functionality. The Cellular Energetics Analysis Software (ceas) R package fills this analytical gap by providing modular and automated Seahorse data analysis and visualization. Availability and implementation ceas is available on CRAN (https://cran.r-project.org/package=ceas). Source code and installable tarballs are freely available for download at https://github.com/jamespeapen/ceas/releases/ under the MIT license. Package documentation may be found at https://jamespeapen.github.io/ceas/. ceas is implemented in R and is supported on macOS, Windows and Linux. [ABSTRACT FROM AUTHOR]

Tobacco use is the leading cause of preventable death worldwide, and relapse during abstinence remains the critical barrier to successful treatment of tobacco addiction. During abstinence, environmental contexts associated with nicotine use can induce craving and contribute to relapse. The insular cortex (IC) is thought to be a critical substrate of nicotine addiction and relapse. However, its specific role in context-induced relapse of nicotine-seeking is not fully known. In this study, we report a novel rodent model of context-induced relapse to nicotine-seeking after punishment-imposed abstinence, which models self-imposed abstinence through increasing negative consequences of excessive drug use. Using the neuronal activity marker Fos we find that the anterior (aIC), but not the middle or posterior IC, shows increased activity during context-induced relapse. Combining Fos with retrograde labeling of aIC inputs, we show projections to aIC from contralateral aIC and basolateral amygdala exhibit increased activity during context-induced relapse. Next, we used fiber photometry in aIC and observed phasic increases in aIC activity around nicotine-seeking responses during self-administration, punishment, and the context-induced relapse tests. Next, we used chemogenetic inhibition in both male and female rats to determine whether activity in aIC is necessary for context-induced relapse. We found that chemogenetic inhibition of aIC decreased context-induced nicotine-seeking after either punishment- or extinction-imposed abstinence. These findings highlight the critical role nicotine-associated contexts play in promoting relapse, and they show that aIC activity is critical for this context-induced relapse following both punishment and extinction-imposed abstinence.

Abstract. Trauma management in Australia is predominantly that of blunt mechanism trauma spread across a geographically large and sparsely populated country. A complex network of patient care has evolved to manage major trauma. Over recent decades, focus has been given to improving and co-ordinating transfer of patients into major trauma centers and improved data collection with the corresponding improved patient outcomes. This article provides an overview of the nature and structure of the Australian trauma system and its regulation.

Reflection and reflective practice are much discussed aspects of professional education. This paper conveys our efforts to problematise teaching reflective practice in human resources (HR) education. The research, on which the paper is based, engages with stakeholders involved in the professional learning and education of reflective practice in three UK universities to provide a critical understanding of the complexities involved. Our research surfaces a level of conceptual ambiguity which creates an uneven landscape in terms of the teaching of reflective practice. Workplace cultures which do not support reflective practice, a focus on performance review and disparate stakeholder views, highlight competing discourses of performance-based reflection and critical management reflection and suggest a fundamental dissonance between a perspective that reflection in professional work warrants a critical character, and one which is based on a relatively simple "acquisition of knowledge" model of continuous professional development. The analysis helps assess the teaching challenge within HR professional learning. Similar intricacies may affect teaching in other professions and consequently this article offers a contribution of relevance and interest to others involved in teaching reflective practice.

Recent computational models of sign tracking (ST) and goal tracking (GT) have accounted for observations that dopamine (DA) is not necessary for all forms of learning and have provided a set of predictions to further their validity. Among these, a central prediction is that manipulating the intertrial interval (ITI) during autoshaping should change the relative ST-GT proportion as well as DA phasic responses. Here, we tested these predictions and found that lengthening the ITI increased ST, i.e., behavioral engagement with conditioned stimuli (CS) and cue-induced phasic DA release. Importantly, DA release was also present at the time of reward delivery, even after learning, and DA release was correlated with time spent in the food cup during the ITI. During conditioning with shorter ITIs, GT was prominent (i.e., engagement with food cup), and DA release responded to the CS while being absent at the time of reward delivery after learning. Hence, shorter ITIs restored the classical DA reward prediction error (RPE) pattern. These results validate the computational hypotheses, opening new perspectives on the understanding of individual differences in Pavlovian conditioning and DA signaling.

The boron isotope pH proxy in sediment-core planktic foraminifera is used as a tracer of oceanic CO.sub.2 outgassing to show that surface waters which derive partly from deep water upwelled in the Southern Ocean became a significant source of carbon to the atmosphere during the last deglaciation. Oceanic CO.sub.2 loss during the last deglaciation It is thought that ventilation of a deep ocean carbon reservoir in the Southern Ocean played an important role in the deglacial atmospheric CO.sub.2 rise, but there has been no direct documentation of changes in surface ocean carbon content during deglaciations. Boron isotope pH proxy data, a more direct tracer of oceanic CO.sub.2 outgassing, now show that surface waters -- partly derived from deep water upwelled in the Southern Ocean -- became a significant source of carbon to the atmosphere during the last deglaciation. Atmospheric CO.sub.2 fluctuations over glacial-interglacial cycles remain a major challenge to our understanding of the carbon cycle and the climate system. Leading hypotheses put forward to explain glacial-interglacial atmospheric CO.sub.2 variations invoke changes in deep-ocean carbon storage.sup.1,2, probably modulated by processes in the Southern Ocean, where much of the deep ocean is ventilated.sup.3. A central aspect of such models is that, during deglaciations, an isolated glacial deep-ocean carbon reservoir is reconnected with the atmosphere, driving the atmospheric CO.sub.2 rise observed in ice-core records.sup.4,5,6. However, direct documentation of changes in surface ocean carbon content and the associated transfer of carbon to the atmosphere during deglaciations has been hindered by the lack of proxy reconstructions that unambiguously reflect the oceanic carbonate system. Radiocarbon activity tracks changes in ocean ventilation.sup.6, but not in ocean carbon content, whereas proxies that record increased deglacial upwelling.sup.4,7 do not constrain the proportion of upwelled carbon that is degassed relative to that which is taken up by the biological pump. Here we apply the boron isotope pH proxy in planktic foraminifera to two sediment cores from the sub-Antarctic Atlantic and the eastern equatorial Pacific as a more direct tracer of oceanic CO.sub.2 outgassing. We show that surface waters at both locations, which partly derive from deep water upwelled in the Southern Ocean.sup.8,9, became a significant source of carbon to the atmosphere during the last deglaciation, when the concentration of atmospheric CO.sub.2 was increasing. This oceanic CO.sub.2 outgassing supports the view that the ventilation of a deep-ocean carbon reservoir in the Southern Ocean had a key role in the deglacial CO.sub.2 rise, although our results allow for the possibility that processes operating in other regions may also have been important for the glacial-interglacial ocean-atmosphere exchange of carbon., Author(s): M. A. Martínez-Botí [sup.1] , G. Marino [sup.2] [sup.3] , G. L. Foster [sup.1] , P. Ziveri [sup.2] [sup.4] [sup.5] , M. J. Henehan [sup.1] [sup.6] , J. W. [...]

Introduction: The move from senior student to novice/advanced beginner registered nurse has been noted to be a stressful period. This longitudinal study follows the experiences of five cohorts of graduate New Zealand registered nurses throughout their first year of practice (n = 530). Methods: Data were collected via self-report survey using the Casey-Fink Graduate Nurse Experience Survey© at three points throughout the first year of practice. Results: Exploratory factor analysis (EFA) resulted in a 5-factor solution for 24 items from section two of the survey: professional support, professional role, professional concerns, professional communication and personal stress. Most respondents (93%) were female, New Zealand Europeans (80%) working in either surgical or medical clinical areas. Professional support from preceptors and exposure to positive role models contributed to a sense of being supported for 81% of the respondents. Satisfaction with choosing the professional role of nursing remained constant with over 90% agreeing or strongly agreeing they had made the right career choice. Across time, respondents reported few professional concerns and few concerns about professional communication. Concerns about personal stress were consistent, with 50% reporting stress related to issues such as finances, childcare and job performance. Conclusion: Contrary to other research, a honeymoon period was not evident (other than the initial high satisfaction with salary), rather the nurses' satisfaction with nursing remained high throughout the year. [ABSTRACT FROM AUTHOR]