Programming a Ferroptosis-to-Apoptosis Transition Landscape Revealed Ferroptosis Biomarkers and Repressors for Cancer Therapy.

Ferroptosis and apoptosis are key cell-death pathways implicated in several human diseases including cancer. Ferroptosis is driven by iron-dependent lipid peroxidation and currently has no characteristic biomarkers or gene signatures. Here a continuous phenotypic gradient between ferroptosis and apoptosis coupled to transcriptomic and metabolomic landscapes is established. The gradual ferroptosis-to-apoptosis transcriptomic landscape is used to generate a unique, unbiased transcriptomic predictor, the Gradient Gene Set (GGS), which classified ferroptosis and apoptosis with high accuracy. Further GGS optimization using multiple ferroptotic and apoptotic datasets revealed highly specific ferroptosis biomarkers, which are robustly validated in vitro and in vivo. A subset of the GGS is associated with poor prognosis in breast cancer patients and PDXs and contains different ferroptosis repressors. Depletion of one representative, PDGFA-assaociated protein 1(PDAP1), is found to suppress basal-like breast tumor growth in a mouse model. Omics and mechanistic studies revealed that ferroptosis is associated with enhanced lysosomal function, glutaminolysis, and the tricarboxylic acid (TCA) cycle, while its transition into apoptosis is attributed to enhanced endoplasmic reticulum(ER)-stress and phosphatidylethanolamine (PE)-to-phosphatidylcholine (PC) metabolic shift. Collectively, this study highlights molecular mechanisms underlying ferroptosis execution, identified a highly predictive ferroptosis gene signature with prognostic value, ferroptosis versus apoptosis biomarkers, and ferroptosis repressors for breast cancer therapy.

Reduction in metabolic noise reveals rejuvenation following transient severe caloric restriction.

Among land vertebrates, the laying hen stands out due to its great reproductive efficiency: producing an egg daily all year long. This production rate makes the laying hen a special model animal to study the general process of reproduction and aging. One unique aspect of hens is their ability to undergo reproductive plasticity and to rejuvenate their reproductive tract during molting, a standard industrial feed restriction protocol for transiently pausing reproduction, followed by improved laying efficiency almost to peak production. Here we use longitudinal metabolomics, immunology, and physiological assays to show that molting promotes reproduction, compresses morbidity, and restores youthfulness when applied to old hens. We identified circulating metabolic biomarkers that quantitatively predict the reproduction and age of individuals. Lastly, we introduce metabolic noise, a robust, unitless, and quantifiable measure for heterogeneity of the complete metabolome as a general marker that can indicate the rate of aging of a population. Indeed, metabolic noise increased with age in control hens, whereas molted hens exhibited reduced noise following molting, indicating systemic rejuvenation. Our results suggest that metabolic noise can be used as a quick and universal proxy for assessing successful aging treatments, accelerating the timeline for drug development.

Dynamic Contrast-enhanced CT Lymphangiography to Quantify Thoracic Duct Lymphatic Flow.

Background CT lymphangiography has been used to image the lymphatic anatomy and assess lymphatic abnormalities. There is, however, a need to develop a method for quantification of lymphatic flow rate in the thoracic duct (TD). Purpose To develop and validate a TD lymphatic flow measurement technique using dynamic contrast-enhanced CT lymphangiography. Materials and Methods Lymphatic flow rate was measured with two techniques: a first-pass analysis technique based on a single compartment model and a thresholding technique distinguishing between opacified and nonopacified voxels within the TD. The measurements were validated in a swine animal model between November 2021 and September 2022. CT images were acquired at 100 kV and 200 mA using a fast-pitched helical scan mode covering the entire TD following contrast material injection into the bilateral inguinal lymph nodes. Two helical CT scans, acquired at the base and peak contrast enhancement of the TD, were used to measure lymphatic flow rate. A US flow probe surgically placed around the TD provided the reference standard measurement. CT lymphatic flow measurements were compared with the reference US flow probe measurements using regression and Bland-Altman analysis. Repeatability was determined using repeated flow measurements within approximately 10 minutes of each other. Results Eleven swine (10 male; mean weight, 43.6 kg ± 2.6 [SD]) were evaluated with 71 dynamic CT acquisitions. The lymphatic flow rates measured using the first-pass analysis and thresholding techniques were highly correlated with the reference US flow probe measurements (r = 0.99 and 0.91, respectively) and showed good agreement with the reference standard, with Bland-Altman analysis showing small mean differences of 0.04 and 0.05 mL/min, respectively. The first-pass analysis and thresholding techniques also showed good agreement for repeated flow measurements (r = 0.94 and 0.90, respectively), with small mean differences of 0.09 and 0.03 mL/min, respectively. Conclusion The first-pass analysis and thresholding techniques could be used to accurately and noninvasively quantify TD lymphatic flow using dynamic contrast-enhanced CT lymphangiography. © RSNA, 2023 See also the editorial by Choyke in this issue.

Fluorescence-guided surgery with indocyanine green to identify an idiopathic chyle leak-case report.

Background: Chylothorax is the leakage of chyle into the pleural space and is associated with up to 50% morbidity. Although, the identification of traumatic chylothoraces is well described, non-traumatic chylothoraxes, mostly idiopathic, present therapeutic challenges as they are difficult to localize. We describe an attempt at localizing and treating an idiopathic chylothorax refractory to conservative and minimally invasive techniques. This was done using indocyanine green (ICG) and was a joint case between a thoracic surgeon and an interventional radiologist. Case Description: A 50-year-old female with a recent history of coronavirus disease (COVID)-19 presented with shortness of breath. She was found to have a right pleural effusion and was admitted to the hospital, where a chest tube was inserted and pleural fluid analysis confirmed the diagnosis of a chylothorax. Conservative management was attempted but with little success. Initial magnetic resonance lymphangiogram (MRL) revealed abnormal enhancing lymphatic masses in the right paraspinal thoracic space as well as lympho-venous junction obstruction with large neck collaterals. She then underwent percutaneous lympho-venous junction angioplasty followed by multiple rounds of glue embolization without clinical improvement. The decision was then made to proceed with a thoracotomy, identification of the site of thoracic duct (TD) leakage, and a mechanical pleurodesis assisted by intraoperative imaging. Ten mg of ICG was injected into the inguinal lymph nodes. Using a camera capable of detection of near-infrared (NIR) light, we were able to visualize the site from which the ICG was extravasating in the chest. Glue was then injected in that area to further help in reducing the leak. After keeping her nil per os (NPO) and requiring one more ligation, a repeat MRL showed a markedly decreased leak into the right pleural space. Two weeks later, she was seen in clinic and reported significant improvement in her symptoms. Conclusions: This is the case of a 50-year-old female who was found to have a significant right chylothorax. She underwent conservative management, followed by tube thoracostomy, and TD ligation but was refractory to treatment. Fluorescence-guided surgery was pivotal to localize the leakage site and help seal it intraoperatively.

2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation.

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules' outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques.

Endocannabinoid basis of personality-Insights from animal model of social behavior.

Rationale: The endocannabinoid system is known to be involved in learning, memory, emotional processing and regulation of personality patterns. Here we assessed the endocannabinoid profile in the brains of mice with strong characteristics of social dominance and submissiveness. Methods: A lipidomics approach was employed to assess the endocannabinoidome in the brains of Dominant (Dom) and Submissive (Sub) mice. The endocannabinoid showing the greatest difference in concentration in the brain between the groups, docosatetraenoyl ethanolamine (DEA), was synthesized, and its effects on the physiological and behavioral responses of Dom and Sub mice were evaluated. mRNA expression of the endocannabinoid receptors and enzymes involved in PUFA biosynthesis was assessed using qRT-PCR. Results: Targeted LC/MS analysis revealed that long-chain polyunsaturated ethanolamides including arachidonoyl ethanolamide (AEA), DEA, docosatrienoyl ethanolamide (DTEA), eicosatrienoyl ethanolamide (ETEA), eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) were higher in the Sub compared with the Dom mice. Untargeted LC/MS analysis showed that the parent fatty acids, docosatetraenoic (DA) and eicosapentaenoic (EPA), were higher in Sub vs. Dom. Gene expression analysis revealed increased mRNA expression of genes encoding the desaturase FADS2 and the elongase ELOVL5 in Sub mice compared with Dom mice. Acute DEA administration at the dose of 15 mg/kg produced antinociceptive and locomotion-inducing effects in Sub mice, but not in Dom mice. Subchronic treatment with DEA at the dose of 5 mg/kg augmented dominant behavior in wild-type ICR and Dom mice but not in Sub mice. Conclusion: This study suggests that the endocannabinoid system may play a role in the regulation of dominance and submissiveness, functional elements of social behavior and personality. While currently we have only scratched the surface, understanding the role of the endocannabinoid system in personality may help in revealing the mechanisms underlying the etiopathology of psychiatric disorders.

A conserved family of immune effectors cleaves cellular ATP upon viral infection.

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. Here, we report a family of immune effectors in bacteria that, upon phage infection, degrade cellular adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems, including cyclic oligonucleotide-based antiviral signaling systems (CBASS), prokaryotic argonautes, and nucleotide-binding leucine-rich repeat (NLR)-like proteins, and we show that ATP and dATP degradation during infection halts phage propagation. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a family of bacterial defense systems with a two-component phosphotransfer-signaling architecture. The immune ATP nucleosidase domain is also encoded within diverse eukaryotic proteins with immune-like architectures, and we show biochemically that eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

Kynurenic acid, a key L-tryptophan-derived metabolite, protects the heart from an ischemic damage.

BACKGROUND: Renal injury induces major changes in plasma and cardiac metabolites. Using a small- animal in vivo model, we sought to identify a key metabolite whose levels are significantly modified following an acute kidney injury (AKI) and to analyze whether this agent could offer cardiac protection once an ischemic event has occurred. METHODS AND RESULTS: Metabolomics profiling of cardiac lysates and plasma samples derived from rats that underwent AKI 1 or 7 days earlier by 5/6 nephrectomy versus sham-operated controls was performed. We detected 26 differential metabolites in both heart and plasma samples at the two selected time points, relative to sham. Out of which, kynurenic acid (kynurenate, KYNA) seemed most relevant. Interestingly, KYNA given at 10 mM concentration significantly rescued the viability of H9C2 cardiac myoblast cells grown under anoxic conditions and largely increased their mitochondrial content and activity as determined by flow cytometry and cell staining with MitoTracker dyes. Moreover, KYNA diluted in the drinking water of animals induced with an acute myocardial infarction, highly enhanced their cardiac recovery according to echocardiography and histopathology. CONCLUSION: KYNA may represent a key metabolite absorbed by the heart following AKI as part of a compensatory mechanism aiming at preserving the cardiac function. KYNA preserves the in vitro myocyte viability following exposure to anoxia in a mechanism that is mediated, at least in part, by protection of the cardiac mitochondria. A short-term administration of KYNA may be highly beneficial in the treatment of the acute phase of kidney disease in order to attenuate progression to reno-cardiac syndrom and to reduce the ischemic myocardial damage following an ischemic event.

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways.

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa.

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.

N-acetylneuraminic acid links immune exhaustion and accelerated memory deficit in diet-induced obese Alzheimer's disease mouse model.

Systemic immunity supports lifelong brain function. Obesity posits a chronic burden on systemic immunity. Independently, obesity was shown as a risk factor for Alzheimer's disease (AD). Here we show that high-fat obesogenic diet accelerated recognition-memory impairment in an AD mouse model (5xFAD). In obese 5xFAD mice, hippocampal cells displayed only minor diet-related transcriptional changes, whereas the splenic immune landscape exhibited aging-like CD4+ T-cell deregulation. Following plasma metabolite profiling, we identified free N-acetylneuraminic acid (NANA), the predominant sialic acid, as the metabolite linking recognition-memory impairment to increased splenic immune-suppressive cells in mice. Single-nucleus RNA-sequencing revealed mouse visceral adipose macrophages as a potential source of NANA. In vitro, NANA reduced CD4+ T-cell proliferation, tested in both mouse and human. In vivo, NANA administration to standard diet-fed mice recapitulated high-fat diet effects on CD4+ T cells and accelerated recognition-memory impairment in 5xFAD mice. We suggest that obesity accelerates disease manifestation in a mouse model of AD via systemic immune exhaustion.

Imaging and Interventions for Lymphatic and Lymphatic-related Disorders.

The lymphatic system is critical in fluid balance homeostasis. Yet, until recently, lymphatic imaging has been outside of mainstream medicine due to a lack of robust imaging and interventional options. However, during the last 20 years, both clinical lymphatic imaging and interventions have shown dramatic advancement. The key to imaging advancement has been the interstitial delivery of contrast agents through lymphatic-rich tissues. These techniques include intranodal lymphangiography and dynamic contrast-enhanced MR lymphangiography. These methods provide the ability to image and recognize lymphatic anatomy and pathologic conditions. Percutaneous thoracic duct catheterization and embolization became the first widely accepted interventional technique for the management of chyle leaks. Advances in interstitial lymphatic embolization, as well as liver and mesenteric lymphatic interventions, have broadened the scope of possible lymphatic interventions. Also, recent techniques of lymphatic decompression allow for the treatment of a variety of lymphatic disorders. Finally, immunologic studies of central lymphatic fluid reveal the potential of lymphatic interventions on immunity. These advances herald an exciting new chapter for lymphatic imaging and interventions in the coming years.

On the Origins of Enzymes: Phosphate-Binding Polypeptides Mediate Phosphoryl Transfer to Synthesize Adenosine Triphosphate.

Reactions involving the transfer of a phosphoryl (-PO32-) group are fundamental to cellular metabolism. These reactions are catalyzed by enzymes, often large and complex, belonging to the phosphate-binding loop (P-loop) nucleoside triphosphatase (NTPase) superfamily. Due to their critical importance in life, it is reasonable to assume that phosphoryl-transfer reactions were also crucial in the pre-LUCA (last universal common ancestor) world and mediated by precursors that were simpler, in terms of their sequence and structure, relative to their modern-day enzyme counterparts. Here, we demonstrate that short phosphate-binding polypeptides (∼50 residues) comprising a single, ancestrally inferred, P-loop or Walker A motif mediate the reversible transfer of a phosphoryl group between two adenosine diphosphate molecules to synthesize adenosine triphosphate and adenosine monophosphate. This activity, although rudimentary, bears resemblance to that of adenylate kinase (a P-loop NTPase enzyme). The polypeptides, dubbed as "P-loop prototypes", thus relate to contemporary P-loop NTPases in terms of their sequence and function, and yet, given their simplicity, serve as plausible representatives of the early "founder enzymes" involved in proto-metabolic pathways.

Microsurgical Peritoneovenous Bypass for the Treatment of Recalcitrant Chylous Ascites.

BACKGROUND: New treatments for recalcitrant chylous ascites are needed to avoid sequelae associated with increased intraabdominal pressures, chyle loss, and diminished quality of life. An autologous microsurgical technique was developed to treat recalcitrant chylous ascites and restore normal physiology. METHODS: A retrospective case series was performed for patients with recalcitrant chylous ascites surgically treated from 2018 to 2020. The authors included all patients with recalcitrant chylous ascites refractory to current standard-of-care interventions such as diet modifications, pharmacologic therapies, and peritoneovenous mechanical shunts. All were treated with microsurgical peritoneovenous bypass with a minimum follow-up of 12 months. RESULTS: Six patients were included over a 2-year period. Surgery was aborted for two patients (33%) with intraoperative venous reflux of the deep inferior epigastric vein, negative on preoperative ultrasound. One patient had a successful reoperation using the contralateral greater saphenous vein; the other elected for a chronic indwelling drain for chyle drainage. Among the five successful procedures (83%), ascites drainage decreased from a median preoperative volume of 1 L/day to postoperative volume of 0.06 L/day. Median hospital length of stay was 7 days (range, 212 to 194 days). Three patients had one complication each, including vancomycin-resistant Enterococcus , spontaneous bacterial peritonitis, and pulmonary embolism. All complications resolved with additional interventions. Median follow-up was 13.5 months (range, 12 to 27 months). CONCLUSION: Microsurgical peritoneovenous bypass was a reliable and reproducible autologous surgery for the treatment of recalcitrant chylous ascites at a minimum follow-up of 12 months. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Lymphatic Intervention, the Frontline of Modern Lymphatic Medicine: Part I. History, Anatomy, Physiology, and Diagnostic Imaging of the Lymphatic System.

Recent advances in lymphatic imaging have provided novel insights into the lymphatic system. Interventional radiology has played a significant role in the development of lymphatic imaging techniques and modalities. Radiologists should be familiar with the basic physiology and anatomy of the lymphatic system to understand the imaging features of lymphatic disorders, which reflect their pathophysiology. This study comprehensively reviews the physiological and anatomical aspects of the human lymphatic system as well as the latest lymphatic imaging techniques.

Lymphatic Intervention, the Frontline of Modern Lymphatic Medicine: Part II. Classification and Treatment of the Lymphatic Disorders.

Lymphatic disorders encompass a broad spectrum of diseases involving the lymphatic system, ranging from traumatic lymphatic leaks to lymphatic malformations. Lymphatic disorders can be categorized into traumatic and non-traumatic disorders according to their etiology. These two categories may be further divided into subgroups depending on the anatomical location of the lymphatic pathology and their association with clinical syndromes. Thoracic duct embolization was a milestone in the field of lymphatic intervention that encouraged the application of percutaneous embolization techniques to treat leaks and reflux disorders in the lymphatic system. Additional access routes for embolization, including retrograde thoracic duct and transhepatic lymphatic access, have also been developed. This article comprehensively reviews a variety of options for the treatment of lymphatic disorders, from conservative management to the most recent embolization techniques.

Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry.

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.