Metabolic Changes in Living Human Lymphoma Cells Intervening NAD + Metabolism as Revealed by NAD (P)H‐Fluorescence Lifetime Imaging and Para‐Hydrogen‐Induced Polarization NMR

ABSTRACT Proliferating cells have a sustained high demand for regeneration of electron acceptors as nicotinamide dinucleotide (phosphate) (NAD(P) + /NAD(P)H) is involved in a number of critical redox reactions within cells. However, their analysis in living cells is still challenging. We propose that combining label‐free NADH and NADPH fluorescence lifetime imaging (NAD(P)H‐FLIM) and signal‐enhanced nuclear magnetic resonance (NMR) spectroscopy allows new, deeper insights into changes in specific metabolic pathways in living cells. For proof of principle, NAD + ‐metabolism was perturbed by specific inhibition of the rate‐limiting enzyme of the NAD + “Salvage pathway” Nicotinamide phosphoribosyltransferase (NAMPT) by FK866 in RAMOS human lymphoma cells. FK866 treatment leads to NAD(H) reduction, followed by reduced RAMOS cell proliferation. The NAD(P)H‐FLIM analysis revealed increased general NAD(P)H‐dependent metabolic activity indicated by increased ratios of enzyme‐bound to total NAD(P)H concentration upon NAMPT inhibition. More importantly, a marked reduction in lactate dehydrogenase (LDH) activity accompanied by NADPH oxidase activity increase is observed. Using signal‐enhanced NMR spectroscopy a reduced flux of pyruvate to lactate catalyzed by LDH is detectable in real time in living cells. This strongly supports NAD(P)H‐FLIM analysis and demonstrates that intervening in the NAD + “Salvage pathway” can have specific and global consequences for cells. Our principle study shows how spatially‐resolved metabolic imaging techniques, that is, NAD(P)H‐FLIM, are complemented by real‐time NMR, paving the way toward a comprehensive spatiotemporal understanding of metabolic pathways in living cells.Deutsche Forschungsgemeinschaft https://doi.org/10.13039/501100001659HORIZON EUROPE European Research Council https://doi.org/10.13039/10001918

Expected and Unexpected "Guests" at the Active Site of Human Orotidine 5'-Monophosphate Decarboxylase

With an extraordinary rate enhancement of 1017 compared to the uncatalyzed reaction and no need for a cofactor, orotidine 5'-monophosphate decarboxylase (OMPDC) is considered one of the most efficient enzymes. Its mechanism has fascinated researchers for over 50 years. In this study, we used high-resolution X-ray crystallography to examine the molecular interactions between the active site of human OMPDC and various natural and synthetic ligands, including transition-state and product analogues, at the atomic level. Additionally, we evaluated their binding affinities with isothermal titration calorimetry (ITC). During protein expression and subsequent structure analysis, we identified nucleotides xanthosine-5'-monophosphate (XMP) and thymidine-5'-monophosphate (dTMP) bound to the active sites of OMPDC and its Thr321Asn variant, respectively, and confirmed their high binding affinities through ITC. Chemically, we investigated the role of the ribose 2'-OH group using 2'-deoxy OMP and 2'-SH UMP, focusing on validating key binding interactions within the nucleoside moiety. To further explore these interactions, we modified the heterocycles (e.g., GMP and CMP) and synthesized a new transition-state analogue, cyanuryl-5'-monophosphate (YMP). YMP exhibited strong affinity for OMPDC and formed an additional hydrogen bond with a nearby water molecule. However, this enthalpically favorable interaction resulted in an entropic penalty compared to the best-known OMPDC inhibitor, BMP, leading to similar affinities. To address this, we synthesized 5-methyl OMP to further improve ligand-enzyme interactions. This modification enhanced stabilization within the hydrophobic pocket through van der Waals forces, paving the way for designing more effective OMPDC inhibitors with specific substitutions aimed at optimizing binding affinity and enzyme inhibition

Analyzing Gaze and Hand Movement Patterns in Leader-Follower Interactions During a Time-Continuous Cooperative Manipulation Task

In daily life, people often interact by taking on leader and follower roles. Unlike laboratory experiments, these interactions unfold naturally and continuously. Although it is well established that gaze typically precedes object manipulation, much less is known about how gaze–hand patterns evolve in interactive settings where one person must take the other’s actions into account. Here we examine predictive, planning-related behavior in a two-player tabletop game called “do-undo.” Participants alternated as Leader and Follower. The Leader performed simple pick-and-place actions to alter the arrangement of objects, while the Follower used other objects to restore the previous configuration. We recorded eye and hand movements, along with object trajectories, using a system that combined eye tracking with multi-camera motion capture. Touch sensors on the players’ hands provided precise timing of contacts, allowing us to segment cooperative action into well-defined temporal intervals. As expected, eye fixations consistently preceded manipulation, but clear role differences emerged. Leaders looked more often and earlier at target objects. In many trials, their gaze anticipated not only their own actions but also those required of the Follower. Leaders also more frequently checked the outcome of the do-undo sequence. Both roles showed gaze patterns consistent with memorization, but alternating gazes between objects and destinations were much more common in Leaders. Some patterns suggested longer-term planning beyond the immediate action. These findings reveal distinct decision-making and planning strategies in Leaders and Followers. Leaders consider not only their own next moves but also the potential actions of their partners, shedding light on the complex cognitive processes that underly everyday human interaction

The Prognostic Role of Frailty and Its Recognition With Simple FRAIL and Fried Frailty Questionnaires in Advanced Cancer Patients

ABSTRACT Background Patients with advanced cancer frequently suffer from frailty associated with vulnerability and adverse outcomes. Our aim was to assess the prevalence of frailty and elucidate the utility of two commonly used frailty questionnaires in an advanced cancer population. Methods The Fried Frailty Phenotype (FFP) and Simple FRAIL Questionnaire (SFQ) were assessed in hospitalized patients with mostly advanced cancer. Patients were classified by both questionnaires as frail (3–5 points), pre‐frail (1–2 points) and robust (0 points) and followed up for all‐cause mortality. Utility was evaluated with correlation and survival analysis. Results From 11/2017 to 02/2020, 251 mostly advanced cancer patients (61 ± 13 years, 53% men, BMI 25.3 ± 4.8 kg/m 2 , 78% cancer stage ≥ 3) were prospectively enrolled. In cancer patients, according to the FFP and SFQ, 17%/13% were frail, 52%/41% prefrail and 31%/47% robust. The correlation between both scores was strong ( r s  = 0.65, p  < 0.001). Both scores were predictors of mortality of cancer patients in univariable and multivariable Cox proportional hazards analyses (multivariable adjusted: per 1 point: FFP: HR 1.36, 95% CI, 1.15–1.61, p  < 0.001; SFQ: HR 1.29, 95% CI, 1.09–1.52, p  = 0.003—adjustment for age, cancer stage/type, anti‐cancer therapy naïve, sex, BMI, CKD and anaemia). The time‐dependent multivariable adjusted area under the receiver operating characteristic curve for 6‐/24‐month survival follow‐up for the FFP was 0.78 (95% CI, 0.70–0.86)/0.92 (95% CI, 0.87–0.98) and for the SFQ was 0.79 (95% CI, 0.69–0.88)/0.90 (95% CI, 0.83–0.97). Conclusion Frailty and pre‐frailty as assessed by FFP and SFQ are commonly found in advanced stage cancer patients. Both questionnaires have a strong correlation and are associated with all‐cause mortality in this population. Since the SFQ is easier and quicker to perform, it can be used remotely, and with untrained staff, it might facilitate earlier preventive measures and initiate further actions to mitigate its impact

Forebrain-specific loss of erythropoietin provokes compensatory upregulation of different EPO receptors

The procognitive growth factor erythropoietin (EPO) and its canonical receptor, EPOR, have long been recognized to be expressed by most cell types in the brain. Cognitive domains, improved by injections of exogenous EPO or by endogenous, hypoxia-stimulated EPO, include important forebrain functions, namely attention, working memory, drive, and executive performance. To gain mechanistic insight into the involvement of forebrain-expressed EPO, we deleted EPO in mice using as specific cre-driver Emx1. Here, we report that these mutant mice act comparably to their wildtype littermates in a comprehensive behavioral test battery. Importantly, we find that the transcripts of both EPOR and a novel, brain-expressed EPO receptor, EphB4, respond to EPO deletion with compensatory upregulation. EphB4 expression in brain and its increase upon forebrain erasure of EPOR are confirmed by in situ hybridization and immunohistochemistry. The augmented expression of both EPOR and EphB4 and their regulatory intercorrelation may explain why EmxEPO mutants show an even superior performance in the most challenging working memory task. Using the previously published single-nuclei-RNA-seq dataset, we further confirm the suggested compensatory mechanism, wherein EPO loss or reduction drives elevated EPOR expression, adding another layer to the intricate regulation of EPO signaling in hippocampal pyramidal neurons. Collectively, these data may explain the lack of behavioral and negative cognitive consequences upon forebrain-wide EPO elimination

Uncertain Short‐Run Restrictions and Statistically Identified Structural Vector Autoregressions

ABSTRACT This study proposes a combination of a statistical identification approach with potentially invalid short‐run zero restrictions. The estimator shrinks towards imposed restrictions and stops shrinkage when the data provide evidence against a restriction. We demonstrate that incorporating valid restrictions through the shrinkage approach enhances the efficiency of the statistically identified estimator, and the impact of invalid restrictions vanishes as the sample size increases. Applying the estimator to an oil market model indicates that incorporating stock market data into the analysis is crucial, as it enables the identification of information shocks, which are shown to be important drivers of the oil price

Deterioration rate of below-ground organic matter fractions depends on the degradation intensity of above-ground vegetation cover

http://dx.doi.org/10.13039/501100008257 Tarbiat Modares Universit