Increased peritoneal B1-like cells during acute phase of human septic peritonitis.

Sepsis is a life-threatening condition caused by dysregulated host responses to infection. Myeloid cell accumulation and lymphocyte decline are widely recognized phenomena in septic patients. However, the fate of specific immune cells remains unclear. Here, we report the results of a human explorative study of patients with septic peritonitis and patients undergoing abdominal surgery without sepsis. We analyzed pairwise peritoneal fluid and peripheral blood taken 24 h after surgery to characterize immediate immune cell changes. Our results show that myeloid cell expansion and lymphocyte loss occur in all patients undergoing open abdominal surgery, indicating that these changes are not specific to sepsis. However, B1-like lymphocytes were specifically increased in the peritoneal fluid of septic patients, correlating positively with sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation II (APACHE-II) clinical severity scores. In support of this notion, we identified an accumulation of peritoneal B1b lymphocytes in septic mice.

Type-I interferon shapes peritoneal immunity in cirrhosis and drives caspase-5-mediated progranulin release upon infection.

BACKGROUND & AIMS: Gut bacterial translocation contributes to immune dysfunction and spontaneous bacterial peritonitis (SBP) in cirrhosis. We hypothesized that exposure of peritoneal macrophages (PMs) to bacterial DNA results in type-I interferon (IFN) production, shaping subsequent immune responses, inflammasome activation, and the release of damage-associated molecular patterns (DAMPs). METHODS: PMs from patients with cirrhosis were stimulated with E. coli single-stranded DNA (ssDNA), lipopolysaccharide LPS, and IFN or infected with E. coli, S. aureus, and Group B streptococcus in vitro. Cytokine release, inflammasome activation, and DAMP release were quantified by quantitative-PCR, ELISA, western blots, and reporter cells employing primary PMs, monocytes, and caspase-deficient THP-1 macrophages. Serum progranulin concentration was correlated with transplant-free survival in 77 patients with SBP. RESULTS: E. coli ssDNA induced strong type-I IFN activity in PMs and monocytes, priming them for enhanced LPS-mediated tumor necrosis factor production without toll-like receptor 4 tolerance induction. During in vitro macrophage bacterial infection, type-I IFN release aligned with upregulated expression of IFN-regulatory factors (IRF)1/2 and guanylate binding proteins (GBP)2/5. PMs upregulated inflammasome-associated proteins and type-I IFN upon E. coli ssDNA exposure and released interleukin-1ß upon bacterial infection. Proteomic screen in mouse macrophages revealed progranulin as being caspase-11-dependent during E. coli infection. PMs and THP-1 macrophages released significant amounts of progranulin when infected with S. aureus or E. coli via gasdermin-D in a type-I IFN and caspase-5-dependent manner. During SBP, PMs upregulated IRF1, GBP2/5 and caspase-5 and higher serum progranulin concentrations were indicative of lower 90-day transplant-free survival after SBP. CONCLUSIONS: Type-I IFN shapes peritoneal immune responses and regulates caspase-5-mediated progranulin release during SBP. IMPACT AND IMPLICATIONS: Patients with cirrhosis exhibit impaired immune responses and increased susceptibility to bacterial infections. This study reveals that type-I interferon responses, triggered by pathogen-associated molecular patterns, are crucial in regulating macrophage activation and priming them for inflammatory responses. Additionally, we elucidate the mechanisms by which type-I interferons promote the release of progranulin from macrophages during spontaneous bacterial peritonitis. Our findings enhance understanding of how bacterial translocation affects immune responses, identify novel biomarkers for inflammasome activation during infections, and point to potential therapeutic targets.

Additive Manufacturing of Head Surrogates for Evaluation of Protection in Sports.

Head impacts are a major concern in contact sports and sports with high-speed mobility due to the prevalence of head trauma events and their dire consequences. Surrogates of human heads are required in laboratory testing to safely explore the efficacy of impact-mitigating mechanisms. This work proposes using polymer additive manufacturing technologies to obtain a substitute for the human skull to be filled with a silicone-based brain surrogate. This assembly was instrumentalized with an Inertial Measurement Unit. Its performance was compared to a standard Hybrid III head form in validation tests using commercial headgear. The tests involved impact velocities in a range centered around 5 m/s. The results show a reasonable homology between the head substitutes, with a disparity in the impact response within 20% between the proposed surrogate and the standard head form. The head surrogate herein developed can be easily adapted to other morphologies and will significantly decrease the cost of the laboratory testing of head protection equipment, all while ensuring the safety of the testing process.

Advances and Challenges in Sepsis Management: Modern Tools and Future Directions.

Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and metabolic changes. The complexity of sepsis stems not only from its pathophysiology but also from the heterogeneity of patient responses, posing significant challenges in developing universally effective therapies. This review emphasizes the importance of phenotyping in sepsis to enhance patient-specific diagnostic and therapeutic strategies. Phenotyping immune cells, which categorizes patients based on clinical and immunological characteristics, is pivotal for tailoring treatment approaches. Flow cytometry emerges as a crucial tool in this endeavor, offering rapid, low cost and detailed analysis of immune cell populations and their functional states. Indeed, this technology facilitates the understanding of immune dysfunctions in sepsis and contributes to the identification of novel biomarkers. Our review underscores the potential of integrating flow cytometry with omics data, machine learning and clinical observations to refine sepsis management, highlighting the shift towards personalized medicine in critical care. This approach could lead to more precise interventions, improving outcomes in this heterogeneously affected patient population.

Immunosenescence and vaccine efficacy revealed by immunometabolic analysis of SARS-CoV-2-specific cells in multiple sclerosis patients.

Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments.

Pediatric Plexiform Fibromyxoma: A Case Report.

The plexiform fibromyxoma is a rare mesenchymal tumor in adults that generally originates in the antrum of stomach, being its occurrence in pediatric patients exceptional. It was classified as a distinct entity by World Health Organization in 2010. No recurrences and metastases have been documented in many of the reported patients to date, being the surgical treatment curative. We report the case of a 3-month-old infant who presented to the emergency department with an episode of intestinal subocclusion requiring an emergent surgery. During the surgical intervention, a mass was identified in the jejunum, causing partial occlusion of its lumen. The surgical pathology report revealed an infiltrative tumor composed of spindle-shaped cells disposed in a stroma with a plexiform pattern alternating myxoid areas. These findings and the immunohistochemical characteristics of the neoplastic cells led to classify the tumor as a plexiform fibromyxoma. A description of the immunophenotype of this tumor is made and differential diagnosis with other gastrointestinal tumors is also discussed.

The effects of photoactivated ciprofloxacin and bile acids on biofilms on bile duct catheters.

OBJECTIVES: This study examined the potential of a novel photoactivatable ciprofloxacin to act against bacterial infections and microbiomes related to biliary diseases. It also evaluated treatment by combining the impact of bile acids and antibiotics on biofilms. Innovative strategies were evaluated to address the elusive bile duct microbiome resulting in biofilm-related infections linked to biliary catheters. The healthy biliary system is considered sterile, but bile microbiomes can occur in disease, and these correlate with hepatobiliary diseases. Causes include biofilms that form on internal-external biliary drainage catheters. These biliary catheters were used to noninvasively study the otherwise elusive bile microbiome for a pilot study. METHODS: A new photoactivatable antibiotic was tested for efficacy against human-derived pathogenic bacterial isolates - Salmonella enterica and Escherichia coli - and catheter-derived bile duct microbiomes. In addition, the effect of bile acids on the antibiotic treatment of biofilms was quantified using crystal violet staining, confocal laser scanning microscopy, and biofilm image analysis. Two novel approaches for targeting biliary biofilms were tested. RESULTS: A photoactivated antibiotic based on ciprofloxacin showed efficacy in preventing biofilm formation and reducing bacterial viability without harming eukaryotic cells. Furthermore, combination treatment of antibiotics with bile acids, such as ursodesoxycholic acid, mildly influenced biofilm biomass but reduced bacterial survival within biofilms. CONCLUSION: Bile acids, in addition to their endocrine and paracrine functions, may enhance antibiotic killing of bacterial biofilms compared with antibiotics alone. These approaches hold promise for treating biliary infections such as cholangitis.

Profiling the dysregulated immune response in sepsis: overcoming challenges to achieve the goal of precision medicine.

Sepsis is characterised by a dysregulated host immune response to infection. Despite recognition of its significance, immune status monitoring is not implemented in clinical practice due in part to the current absence of direct therapeutic implications. Technological advances in immunological profiling could enhance our understanding of immune dysregulation and facilitate integration into clinical practice. In this Review, we provide an overview of the current state of immune profiling in sepsis, including its use, current challenges, and opportunities for progress. We highlight the important role of immunological biomarkers in facilitating predictive enrichment in current and future treatment scenarios. We propose that multiple immune and non-immune-related parameters, including clinical and microbiological data, be integrated into diagnostic and predictive combitypes, with the aid of machine learning and artificial intelligence techniques. These combitypes could form the basis of workable algorithms to guide clinical decisions that make precision medicine in sepsis a reality and improve patient outcomes.

Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.

The non-canonical inflammasome activators Caspase-4 and Caspase-5 are differentially regulated during immunosuppression-associated organ damage.

The non-canonical inflammasome, which includes caspase-11 in mice and caspase-4 and caspase-5 in humans, is upregulated during inflammatory processes and activated in response to bacterial infections to carry out pyroptosis. Inadequate activity of the inflammasome has been associated with states of immunosuppression and immunopathological organ damage. However, the regulation of the receptors caspase-4 and caspase-5 during severe states of immunosuppression is largely not understood. We report that CASP4 and CASP5 are differentially regulated during acute-on-chronic liver failure and sepsis-associated immunosuppression, suggesting non-redundant functions in the inflammasome response to infection. While CASP5 remained upregulated and cleaved p20-GSDMD could be detected in sera from critically ill patients, CASP4 was downregulated in critically ill patients who exhibited features of immunosuppression and organ failure. Mechanistically, downregulation of CASP4 correlated with decreased gasdermin D levels and impaired interferon signaling, as reflected by decreased activity of the CASP4 transcriptional activators IRF1 and IRF2. Caspase-4 gene and protein expression inversely correlated with markers of organ dysfunction, including MELD and SOFA scores, and with GSDMD activity, illustrating the association of CASP4 levels with disease severity. Our results document the selective downregulation of the non-canonical inflammasome activator caspase-4 in the context of sepsis-associated immunosuppression and organ damage and provide new insights for the development of biomarkers or novel immunomodulatory therapies for the treatment of severe infections.

Host extracellular vesicles confer cytosolic access to systemic LPS licensing non-canonical inflammasome sensing and pyroptosis.

Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.

Drilling of Cross-Ply UHMWPE Laminates: A Study on the Effects of the Tool Geometry and Cutting Parameters on the Integrity of Components.

Ultrahigh-molecular-weight polyethylene (UHMWPE) is used in the defence industry mainly owing to its properties, such as excellent dimensional stability, excellent ballistic performance, and light weight. Although UHMWPE laminates are generally studied under impact loads, it is crucial to understand better the optimal machining conditions for assembling auxiliary structures in combat helmets or armour. This work analyses the machinability of UHMWPE laminates by drilling. The workpiece material has been manufactured through hot-pressing technology and subjected to drilling tests. High-speed steel (HSS) twist drills with two different point angles and a brad and spur drill that is 6 mm in diameter have been used for this study. Cutting forces, failure, and main damage modes are analysed, making it possible to extract relevant information for the industry. The main conclusion is that the drill with a smaller point angle has a better cutting force performance and less delamination at the exit zone (5.4 mm at a 60 m/min cutting speed and a 0.05 mm/rev feed) in the samples. This value represents a 46% improvement over the best result obtained in terms of delamination at the exit when using the tool with the larger point angle. However, the brad and spur drill revealed a post-drilling appearance with high fuzzing and delamination.

Immunosuppressive effects of circulating bile acids in human endotoxemia and septic shock: patients with liver failure are at risk.

BACKGROUND: Sepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk. METHODS: To induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within < 24 h) and severe septic shock (norepinephrine dose > 0.4 µg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula. RESULTS: Although experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p = 0.0139). CONCLUSIONS: Circulating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients.

Raman Spectroscopy Profiling of Splenic T-Cells in Sepsis and Endotoxemia in Mice.

Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the immune cells. However, the host response toward sepsis is heterogeneous, and the roles of the individual immune cell types have not been fully conceptualized. During sepsis, the spleen plays a vital role in pathogen clearance, such as bacteria by an antibody response, macrophage bactericidal capacity, and bacterial endotoxin detoxification. This study uses Raman spectroscopy to understand the splenic T-lymphocyte compartment profile changes during bona fide bacterial sepsis versus hyperinflammatory endotoxemia. The Raman spectral analysis showed marked changes in splenocytes of mice subjected to septic peritonitis principally in the DNA region, with minor changes in the amino acids and lipoprotein areas, indicating significant transcriptomic activity during sepsis. Furthermore, splenocytes from mice exposed to endotoxic shock by injection of a high dose of lipopolysaccharide showed significant changes in the protein and lipid profiles, albeit with interindividual variations in inflammation severity. In summary, this study provided experimental evidence for the applicability and informative value of Raman spectroscopy for profiling the immune response in a complex, systemic infection scenario. Importantly, changes within the acute phase of inflammation onset (24 h) were reliably detected, lending support to the concept of early treatment and severity control by extracorporeal Raman profiling of immunocyte signatures.

Starvation Protects Hepatocytes from Inflammatory Damage through Paradoxical mTORC1 Signaling.

Background and aims: Sepsis-related liver failure is associated with a particularly unfavorable clinical outcome. Calorie restriction is a well-established factor that can increase tissue resilience, protect against liver failure and improve outcome in preclinical models of bacterial sepsis. However, the underlying molecular basis is difficult to investigate in animal studies and remains largely unknown. METHODS: We have used an immortalized hepatocyte line as a model of the liver parenchyma to uncover the role of caloric restriction in the resilience of hepatocytes to inflammatory cell damage. In addition, we applied genetic and pharmacological approaches to investigate the contribution of the three major intracellular nutrient/energy sensor systems, AMPK, mTORC1 and mTORC2, in this context. RESULTS: We demonstrate that starvation reliably protects hepatocytes from cellular damage caused by pro-inflammatory cytokines. While the major nutrient- and energy-related signaling pathways AMPK, mTORC2/Akt and mTORC1 responded to caloric restriction as expected, mTORC1 was paradoxically activated by inflammatory stress in starved, energy-deprived hepatocytes. Pharmacological inhibition of mTORC1 or genetic silencing of the mTORC1 scaffold Raptor, but not its mTORC2 counterpart Rictor, abrogated the protective effect of starvation and exacerbated inflammation-induced cell death. Remarkably, mTORC1 activation in starved hepatocytes was uncoupled from the regulation of autophagy, but crucial for sustained protein synthesis in starved resistant cells. CONCLUSIONS: AMPK engagement and paradoxical mTORC1 activation and signaling mediate protection against pro-inflammatory stress exerted by caloric restriction in hepatocytes.

Control of TurboID-dependent biotinylation intensity in proximity ligation screens.

Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system's inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilized avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions. SIGNIFICANCE: Proximity biotinylation screens using the last generation biotin ligase TurboID represent a powerful approach for the characterisation of transient protein-protein interaction and signaling networks. However, a constant and high basal biotinylation rate and the associated cytotoxicity often preclude the use of this method in proteomic studies. We report a protocol based on modulation of free biotin levels that prevents the deleterious effects of TurboID while allowing inducible biotinylation, even in subcellular compartments such as the endoplasmic reticulum. This optimised protocol greatly expands the applications of TurboID in proteomic screens.

Label-Free Characterization of Macrophage Polarization Using Raman Spectroscopy.

Macrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes.

The TLR-chaperone CNPY3 is a critical regulator of NLRP3-inflammasome activation.

TLRs mediate the recognition of microbial and endogenous insults to orchestrate the inflammatory response. TLRs localize to the plasma membrane or endomembranes, depending on the member, and rely critically on ER-resident chaperones to mature and reach their subcellular destinations. The chaperone canopy FGF signaling regulator 3 (CNPY3) is necessary for the proper trafficking of multiple TLRs including TLR1/2/4/5/9 but not TLR3. However, the exact role of CNPY3 in inflammatory signalling downstream of TLRs has not been studied in detail. Consistent with the reported client specificity, we report here that functional loss of CNPY3 in engineered macrophages impairs downstream signalling by TLR2 but not TLR3. Unexpectedly, CNPY3-deficient macrophages show reduced IL-1ß and IL-18 processing and production independent of the challenged upstream TLR species, demonstrating a separate, specific role for CNPY3 in inflammasome activation. Mechanistically, we document that CNPY3 regulates caspase-1 localization to the apoptosis speck and autoactivation of caspase-1. Importantly, we were able to recapitulate these findings in macrophages from an early infantile epileptic encephalopathy (EIEE) patient with a novel CNPY3 loss-of-function variant. Summarizing, our findings reveal a hitherto unknown, TLR-independent role of CNPY3 in inflammasome activation, highlighting a more complex and dedicated role of CNPY3 to the inflammatory response than anticipated.

Regulation of the Small GTPase Ras and Its Relevance to Human Disease.

Ras research has experienced a considerable boost in recent years, not least prompted by the Ras initiative launched by the NCI in 2013 ( https://www.cancer.gov/research/key-initiatives/ras ), accompanied and conditioned by a strongly reinvigorated determination within the Ras community to develop therapeutics attacking directly the Ras oncoproteins. As a member of the small G-protein superfamily, function and transforming activity of Ras all revolve about its GDP/GTP loading status. For one thing, the extent of GTP loading will determine the proportion of active Ras in the cell, with implications for intensity and quality of downstream signaling. But also the rate of nucleotide exchange, i.e., the Ras-GDP/GTP cycling rate, can have a major impact on Ras function, as illustrated perhaps most impressively by newly discovered fast-cycling oncogenic mutants of the Ras-related GTPase Rac1. Thus, while the last years have witnessed memorable new findings and technical developments in the Ras field, leading to an improved insight into many aspects of Ras biology, they have not jolted at the basics, but rather deepened our view of the fundamental regulatory principles of Ras activity control. In this brief review, we revisit the role and mechanisms of Ras nucleotide loading and its implications for cancer in the light of recent findings.