Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia.

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

Systemic elevation of PTEN induces a tumor-suppressive metabolic state.

Decremental loss of PTEN results in cancer susceptibility and tumor progression. PTEN elevation might therefore be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with PTEN expression elevated to varying levels by taking advantage of bacterial artificial chromosome (BAC)-mediated transgenesis. The "Super-PTEN" mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake and increased mitochondrial oxidative phosphorylation and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and -independent pathways and negatively impacting two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect.

PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation.

PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation.

Evaluation of objective tools and artificial intelligence in robotic surgery technical skills assessment: a systematic review.

BACKGROUND: There is a need to standardize training in robotic surgery, including objective assessment for accreditation. This systematic review aimed to identify objective tools for technical skills assessment, providing evaluation statuses to guide research and inform implementation into training curricula. METHODS: A systematic literature search was conducted in accordance with the PRISMA guidelines. Ovid Embase/Medline, PubMed and Web of Science were searched. Inclusion criterion: robotic surgery technical skills tools. Exclusion criteria: non-technical, laparoscopy or open skills only. Manual tools and automated performance metrics (APMs) were analysed using Messick's concept of validity and the Oxford Centre of Evidence-Based Medicine (OCEBM) Levels of Evidence and Recommendation (LoR). A bespoke tool analysed artificial intelligence (AI) studies. The Modified Downs-Black checklist was used to assess risk of bias. RESULTS: Two hundred and forty-seven studies were analysed, identifying: 8 global rating scales, 26 procedure-/task-specific tools, 3 main error-based methods, 10 simulators, 28 studies analysing APMs and 53 AI studies. Global Evaluative Assessment of Robotic Skills and the da Vinci Skills Simulator were the most evaluated tools at LoR 1 (OCEBM). Three procedure-specific tools, 3 error-based methods and 1 non-simulator APMs reached LoR 2. AI models estimated outcomes (skill or clinical), demonstrating superior accuracy rates in the laboratory with 60 per cent of methods reporting accuracies over 90 per cent, compared to real surgery ranging from 67 to 100 per cent. CONCLUSIONS: Manual and automated assessment tools for robotic surgery are not well validated and require further evaluation before use in accreditation processes.PROSPERO: registration ID CRD42022304901.

BACKGROUND: Robotic surgery is increasingly used worldwide to treat many different diseases. The robot is controlled by a surgeon, which may give them greater precision and better outcomes for patients. However, surgeons' robotic skills should be assessed properly, to make sure patients are safe, to improve feedback and for exam assessments for certification to indicate competency. This should be done by experts, using assessment tools that have been agreed upon and proven to work. AIM: This review's aim was to find and explain which training and examination tools are best for assessing surgeons' robotic skills and to find out what gaps remain requiring future research. METHOD: This review searched for all available studies looking at assessment tools in robotic surgery and summarized their findings using several different methods. FINDINGS AND CONCLUSION: Two hundred and forty-seven studies were looked at, finding many assessment tools. Further research is needed for operation-specific and automatic assessment tools before they should be used in the clinical setting.

The Lipid Kinase PI5P4Kß Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kß, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kß preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kß is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kß is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kß. The critical role of the GTP-sensing activity of PI5P4Kß in cancer signifies this lipid kinase as a cancer therapeutic target.

A Deep Learning Approach to Classify Surgical Skill in Microsurgery Using Force Data from a Novel Sensorised Surgical Glove.

Microsurgery serves as the foundation for numerous operative procedures. Given its highly technical nature, the assessment of surgical skill becomes an essential component of clinical practice and microsurgery education. The interaction forces between surgical tools and tissues play a pivotal role in surgical success, making them a valuable indicator of surgical skill. In this study, we employ six distinct deep learning architectures (LSTM, GRU, Bi-LSTM, CLDNN, TCN, Transformer) specifically designed for the classification of surgical skill levels. We use force data obtained from a novel sensorized surgical glove utilized during a microsurgical task. To enhance the performance of our models, we propose six data augmentation techniques. The proposed frameworks are accompanied by a comprehensive analysis, both quantitative and qualitative, including experiments conducted with two cross-validation schemes and interpretable visualizations of the network's decision-making process. Our experimental results show that CLDNN and TCN are the top-performing models, achieving impressive accuracy rates of 96.16% and 97.45%, respectively. This not only underscores the effectiveness of our proposed architectures, but also serves as compelling evidence that the force data obtained through the sensorized surgical glove contains valuable information regarding surgical skill.

Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine resistance in breast cancer.

Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α-positive (ERα+) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)-dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2's cis-regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.

Author Correction: MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.

Tumour microenvironment factors shaping the cancer metabolism landscape.

Cancer cells exhibit metabolic alterations that distinguish them from healthy tissues and make their metabolic processes susceptible to pharmacological targeting. Although typical cell-autonomous features of cancer metabolism have been emerging, it is increasingly appreciated that extrinsic factors also influence the metabolic properties of tumours. This review highlights evidence from the recent literature to discuss how conditions within the tumour microenvironment shape the metabolic character of tumours.

Gas Chromatography Coupled to Mass Spectrometry (GC-MS) to Study Metabolism in Cultured Cells.

Cancer cells exhibit characteristic patterns of metabolic behaviour that can be exploited for therapeutic purposes. Conditions found within the tumour microenvironment, such as hypoxia and selective nutrient availability, are known to influence the metabolism of cancer and stromal cells. Understanding cancer metabolism requires the use of analytical methods that allow detection and quantification of many metabolites simultaneously. Gas chromatography-mass spectrometry (GC-MS) is a versatile method to quantify metabolite abundance and, in combination with stable isotope labelled compounds, can yield important insights into the activity of metabolic pathways in cancer cells. This chapter provides an overview of the use of GC-MS for metabolic analysis of adherent cancer cells with an emphasis on the technical background that should be taken into consideration when designing and executing GC-MS-based metabolomics experiments.

Degradation of activated K-Ras orthologue via K-Ras-specific lysine residues is required for cytokinesis.

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

Teaching Green Chemistry and Engineering through the Epoxidation of Poly-ß-myrcene.

This study explores the application of the epoxidation process of poly-ß-myrcene, a constituent of the natural resin from Chios Mastic trees (Pistacia Lentiscus L.), as an educational instrument for teaching Green Chemistry and Engineering to students at various academic levels. The study provides a comprehensive presentation of foundational knowledge essential for interpreting the subsequent experimental data. Consequently, the production process that leads to the production of Mastic Epoxide (MASTEP) stands as an invaluable pedagogical resource, enabling educators to impart crucial principles of Green Chemistry and Engineering to both pre-graduate and post-graduate students. By employing MASTEP as a case study, this educational approach actively involves students in a dynamic learning environment. Through this methodology, learners develop a profound comprehension of sustainability, innovation, and good practices. The integration of the MASTEP concept into the curriculum would foster a deeper understanding of responsible methodologies among aspiring chemical engineers and scientists, equipping them to make substantial contributions towards a more sustainable global landscape. This educational model aims to contribute to preparing future generations for a pivotal role in fostering a sustainable world through their professional endeavors.

Vesical fungal bezoars on post-TURBT scar tissue causing obstruction and mimicking urothelial carcinoma.

Fungal bezoars (fungal balls) are rarely reported in the upper or lower urinary tract. They can be the cause of severe morbidities such as urinary tract obstruction, renal failure and fungaemia. Hereby, we present a rare case of a male patient who underwent transurethral resection of bladder tumour (TURBT), and during his postoperative period, he was diagnosed with bladder fungal bezoars adherent to his resection area. The fungal bezoars were covering an extended area of the right lateral bladder wall, including the right ureteric orifice and causing right urinary tract obstruction. Those findings were manifested only after a relooked cystoscopy and histological evaluation.We aim to present a rare example of fungal bezoars mimicking other pathologies in the urinary tract and review the current literature for similar documentation. We underline the necessity of follow-up examinations for urologists performing TURBT surgeries, including urinalysis, imaging modalities and cystoscopy.

Metformin: a case of divide and conquer.

Metformin is a widely prescribed anti-diabetic drug and its use is associated with lower cancer incidence. The mechanisms by which metformin attenuates tumorigenesis are not clearly understood. In a paper published in Proceedings of the National Academy of Sciences of the United States of America, Hirsch and colleagues show that metformin interferes with a signaling pathway, mediated by the transcription factor NF-κB, which drives cell transformation and is required for the maintenance of cancer stem cells.

Systematic diet composition swap in a mouse genome-scale metabolic model reveals determinants of obesogenic diet metabolism in liver cancer.

Dietary nutrient availability and gene expression, together, influence tissue metabolic activity. Here, we explore whether altering dietary nutrient composition in the context of mouse liver cancer suffices to overcome chronic gene expression changes that arise from tumorigenesis and western-style diet (WD). We construct a mouse genome-scale metabolic model and estimate metabolic fluxes in liver tumors and non-tumoral tissue after computationally varying the composition of input diet. This approach, called Systematic Diet Composition Swap (SyDiCoS), revealed that, compared to a control diet, WD increases production of glycerol and succinate irrespective of specific tissue gene expression patterns. Conversely, differences in fatty acid utilization pathways between tumor and non-tumor liver are amplified with WD by both dietary carbohydrates and lipids together. Our data suggest that combined dietary component modifications may be required to normalize the distinctive metabolic patterns that underlie selective targeting of tumor metabolism.

Poorly differentiated muscle-invasive giant cell tumor of the bladder leads to unfavorable clinical outcome.

Urothelial carcinoma of the urinary bladder is a common clinical entity. Recently, researchers focused on the emerging clinical significance of histologic variants, because they may need special therapy and their prognosis differs. Hereby, we describe a case of a giant cell osteoclast-like urothelial carcinoma of the urinary bladder.

MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine.

α-ketoglutarate (αKG) is a central metabolic node with a broad influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving compound stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that, under our experimental conditions, glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.