Faculty Bibliography

Neurons undergo extensive metabolic reprogramming during differentiation; this reprogramming leads to specific changes in the kinetics and concentrations of metabolites. We developed an in-cell NMR-based method that monitors this metabolic transformation with subminute time resolution. Undifferentiated SH-SY5Y human neuronal precursor cells were encapsulated into alginate gel beads and differentiated inside the gel. Real time pulse chase (RTPC) in-cell NMR was used to measure relative steady state concentrations of glycolysis and TCA cycle metabolites and the kinetics of metabolite production and clearance in differentiated and undifferentiated cells. Neuronal differentiation slowed glycolysis, increased TCA cycle activity and glutamate production. Fructose 1,6 bisphosphate and glutathione were identified as major biomarkers of undifferentiated and differentiated cells, respectively. The results demonstrate that RTPC-NMR analysis of neuronal cells is an effective method for studying changes in metabolite profiles induced by stress and drug-induced stimuli.

Subarachnoid hemorrhage (SAH) due to the rupture of an intracranial aneurysm is a highly fatal type of stroke. Cerebral vasospasm (CVS) is a major post-SAH complication leading to delayed ischemic neurological deficits, thereby worsening patient outcomes. Previously, we found that lower plasma levels of the soluble receptor for advanced glycation end products (RAGE) predict symptomatic CVS in patients with SAH. However, the molecular mechanisms underlying CVS remain unclear. Here, using an SAH mouse model with endovascular perforation, we found that neurological deficits, CVS in the circle of Willis, and impaired cortical microarterial perfusion were markedly ameliorated in RAGE-deficient mice. Neutrophils accumulated in the cerebral perivascular space of WT mice after SAH but were markedly reduced in RAGE-deficient mice. Myeloid lineage-targeted deletion of RAGE, including neutrophils, improved CVS after SAH. Inhibition of the high mobility group box 1 (HMGB1)/RAGE axis or neutrophil elastase ameliorated CVS. In a transwell assay, the HMGB1/RAGE axis drives neutrophil migration and NETosis. These findings indicate that neutrophil RAGE signaling contributes to cerebrovascular dysfunction after SAH and suggest that RAGE-mediated neutrophil inflammation may be a therapeutic target in the hyperacute phase to mitigate early brain injury.

BACKGROUND:mice) translates into restrained CC and increased survival in tumour-bearing mice. RAGE, which is not expressed in adult healthy myofibres, is re-expressed in atrophying myofibres in cancer conditions. However, the specific contribution of muscular RAGE to CC was unknown. METHODS:mice were subcutaneously injected with Lewis lung carcinoma (LLC) cells, and body changes and survival were monitored until 25 dpi, when histological, molecular and proteomic analyses were performed in tumour-bearing and control mice. Muscle samples of pre-cachectic and cachectic pancreatic cancer patients were analysed to validate the results. RESULTS:muscles showed increased amounts of several enzymes involved in glycolysis and glucose catabolism, typical of Warburg metabolism. Noteworthy, muscles of pre-cachectic and cachectic cancer patients showed ~3-fold increase (p < 0.05) in RAGE amounts and reduced Akt-GSK-3β-PGC-1α pathway, compared with healthy control subjects. CONCLUSIONS:Our data provide evidence that RAGE engagement at myofibre level drives loss of body and muscle weights and inflammation in cancer conditions. RAGE ablation in muscles confers resistance to CC through myofibre remodeling and glycolytic reprogramming. On the clinical side, the overexpression of RAGE is an early event in muscles of cancer patients, suggesting a role for RAGE in the onset of the cachectic syndrome. Thus, the molecular targeting of RAGE might be useful to counteract cachexia and prolong survival in cancer patients.

Ageing is the primary non-modifiable risk factor for cardiovascular diseases (CVDs), with older women facing a greater risk of CVDs than age-matched men. Vascular smooth muscle cells (VSMCs) dysfunction and impaired calcium (Ca²⁺) handling are recognized as central contributors to arterial stiffening and vascular calcification. However, the molecular and functional determinants of Ca²⁺ clearance that drive vascular ageing remain poorly understood. We identify the (Na⁺)-sodium/Ca²⁺-calcium (K⁺)-potassium-dependent exchanger 4 (NCKX4) as a critical functional regulator of VSMC Ca²⁺ clearance and vascular integrity. Expression of NCKX4 (encoded by Slc24A4) was significantly reduced in aortae from aged (72-78 weeks) mice. Aged-related loss of NCKX4 impaired Ca²⁺ clearance function and increased Ca²⁺-phosphate mineralization. Notably, young (12-15 weeks) Nckx4^-/- mice exhibited elastic fibre fragmentation, collagen accumulation, arterial wall thickening and extracellular matrix (ECM) remodelling, which are hallmarks of vascular ageing that closely resembled those observed in aged wild-type mice. Transcriptomic profiling revealed that loss of NCKX4 alters pathways associated with Ca²⁺-integrin signalling, ECM turnover and mineralization, including dysregulation of anchorage integrins, microfibril-stabilizing components, osteogenic drivers and profibrotic integrins. Collectively, these findings demonstrate that impaired Ca²⁺ clearance promotes maladaptive inside-out integrin signalling, disrupting VSMC anchorage, ECM homeostasis and mineralization. Our results establish NCKX4 as a previously unrecognized determinant of vascular ageing, and its decline accelerates premature arterial remodelling and calcification. This study positions NCKX4 as a mechanistic link between age-dependent vascular vulnerability and arterial stiffening, with implications for novel therapeutic strategies targeting Ca²⁺ handling to prevent CVDs. KEY POINTS: Dysfunctional calcium (Ca²⁺) handling in vascular smooth muscle cells (VSMCs) increases susceptibility to cardiovascular diseases (CVDs), vascular calcification, stiffness and aortic remodelling. The K⁺-dependent Na⁺/Ca²⁺ exchanger NCKX4 mediates high-capacity Ca²⁺ clearance extrusion, thereby maintaining VSMC Ca^{2 +} homeostasis. NCKX4 (encoded by Slc24a4) expression markedly declines in the aorta and VSMCs of aged (72-78 weeks) mice. Genetic deletion or age-related loss of NCKX4 impairs Ca²⁺ clearance, leading to enhanced VSMCs calcification, premature arterial remodelling through disruption of Ca²⁺-mediated integrin-ECM signalling. NCKX4 is a newly identified mechanistic driver of vascular ageing and a potential therapeutic target for early detection, preservation of vascular integrity and mitigation of age-related CVDs risk.

MOTIVATION/BACKGROUND:Large-scale prospective cohort studies collect longitudinal biospecimens alongside time-to-event outcomes to investigate biomarker dynamics in relation to disease risk. The nested case-control (NCC) design provides a cost-effective alternative to full cohort biomarker studies while preserving statistical efficiency. Despite advances in joint modeling for longitudinal and time-to-event outcomes, few approaches address the unique challenges posed by NCC sampling, non-normally distributed biomarkers, and competing survival outcomes. RESULTS:Motivated by the TEDDY study, we propose "JM-NCC", a joint modeling framework designed for NCC studies with competing events. It integrates a generalized linear mixed-effects model for potentially non-normally distributed biomarkers with a cause-specific hazard model for competing risks. Two estimation methods are developed. fJM-NCC leverages NCC sub-cohort longitudinal biomarker data and full cohort survival and clinical metadata, while wJM-NCC uses only NCC sub-cohort data. Both simulation studies and an application to TEDDY microbiome dataset demonstrate the robustness and efficiency of the proposed methods. AVAILABILITY/BACKGROUND:Software is available at https://github.com/Zhaoyn-oss/JMNCC and archived on Zenodo at https://zenodo.org/records/18199759 (DOI: 10.5281/zenodo.18199759). SUPPLEMENTARY INFORMATION/BACKGROUND:Supplementary data are available at Bioinformatics online.

RAGE and its intracellular effector molecule, the actin polymerase DIAPH1, mediate inflammation and the complications of diabetes. Using NMR spectroscopy and mass spectrometry, we built a structural model of the RAGE-DIAPH1 complex, revealing how binding of the cytoplasmic tail of RAGE (ctRAGE) to DIAPH1 stimulates its actin polymerization activity, which is inhibited by a small molecule antagonist of RAGE-DIAPH1 interaction, RAGE406R. The solution structure of the RAGE406R - ctRAGE suggests that RAGE406R prevents the formation of the RAGE-DIAPH1. FRET, actin polymerization assays, smooth muscle cell migration, and THP1 cell inflammation experiments, together with the in vivo interrogation of the effects of RAGE406R in mouse models of inflammation and diabetic wound healing, support this mode of RAGE-DIAPH1 antagonism. Finally, the treatment of macrophages differentiated from peripheral blood-derived mononuclear cells from humans with type 1 diabetes with RAGE406R reduces the mRNA expression of the chemokine CCL2, diminishing the expression of a key node in the inflammatory response.

Ischemic heart disease is the leading cause of heart failure with reduced ejection fraction in the developed world. An evolution of background medical therapy over the past decade has spurred improvement in symptoms and a reduction in morbidity and mortality with ischemic cardiomyopathy. However, there is still ongoing debate about the role and impact of revascularization. Much of the societal guidance regarding revascularization with coronary artery bypass grafting in ischemic cardiomyopathy comes from the STICH trial (Surgical Treatment for Ischemic Heart Failure) which predates improvements in medical therapy. More recently, the REVIVED-BCIS2 trial (Revascularization for Ischemic Ventricular Dysfunction-British Cardiovascular Intervention Society) failed to show a benefit of percutaneous coronary intervention on heart failure hospitalization and mortality in ischemic cardiomyopathy over contemporary medical therapy alone. Yet, there are outstanding questions regarding the role and modality of revascularization required to improve outcomes. We review current data and future directions in the management of ischemic cardiomyopathy and the potential role of revascularization.

OBJECTIVE:This study examined racial and ethnic differences in percent total weight loss (%TWL) and glycemic improvement following sleeve gastrectomy (SG) and explored the role of socioeconomic and psychosocial factors in postsurgical outcomes. METHODS:This longitudinal study included patients who underwent SG between 2017 and 2020, with follow-up visits over 24 months. RESULTS:Non-Hispanic Black (NHB) participants had lower %TWL at 3, 12, and 24 months compared with Hispanic (H) and non-Hispanic White (NHW) participants. Fat mass index was initially lower in NHB, with smaller reductions over time and significant group differences persisting at 24 months. NHB participants had higher baseline fat-free mass index values; by 24 months, fat-free mass index values were lower in H participants. Hemoglobin A1c decreased across all groups but remained consistently higher in NHB and H compared with NHW at 24 months. NHB participants reported higher perceived discrimination, sleep disturbance, and perceived stress than H and NHW participants at all time points. Employment status predicted %TWL at 12 months. There was a significant interaction between race and ethnicity and employment status observed at 12 and 24 months, suggesting that employment-related disparities could impact surgical outcomes. CONCLUSIONS:NHB participants experienced less favorable outcomes following SG, emphasizing the need for tailored interventions addressing socioeconomic and psychosocial disparities.

DIAPH1 is a member of the family of Diaphanous Related Formins (DRFs) implicated in cell migration and cytokinesis. DRFs are maintained in an autoinhibited state by the intramolecular association between diaphanous inhibitory (DID) and diaphanous autoregulatory (DAD) domains. Actin polymerization requires the binding of activated RhoA to the GTPase binding domain (GBD) of DIAPH1 and the dissociation of DAD. In the presence of excess RhoA, actin polymerization is only partially activated. Using monomeric domain constructs of DIAPH1, the sequential binding affinities of RhoA and DAD to GBD-DID were characterized. Binding of RhoA and DAD were negatively cooperative requiring a 100-fold greater concentration of DAD to achieve saturation when RhoA binding site was occupied. The unimolecular architecture of full length DIAPH1 establishes an effective concentration of DAD in the micromolar range, which is 100-fold larger than the intrinsic affinity of DAD for DID. The effective concentration is large enough to maintain DIAPH1 autoinhibition, yet small enough to permit partial activation of DIAPH1 after RhoA binding. By exploiting negative cooperativity, DIAPH1 maintains a reserve of inactivated molecules enabling gradual responses to cellular processes that require prolonged and sustained regulation. The proposed mechanism is extended to other DIAPH1 activating ligands and broadly applicable to all DRFs.