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Abstract INTRODUCTION Neurofilament light chain (NfL) quantification aids in diagnosing and predicting neurological disorders, but clinical and laboratory practices vary across centers. Differences in result interpretation and reporting further challenge test commutability. This study aimed to review the global analytical and post‐analytical methods used for NfL measurement in routine clinical practice across different contexts. METHODS We established an international working group (WG) and distributed a survey to its members to gather information on context of use (COU), (pre) analytical methods, cutoff usage, as well as the interpretation and reporting of NfL measurements. RESULTS Among the centers, 63% measured NfL in cerebrospinal fluid (CSF), 87% in blood, and 53% in both. COU was widespread, with 50% defining pathological cutoffs based on publications and 42% considering age. Reporting was primarily done through numeric results (95%). DISCUSSION Harmonizing cutoffs, reporting, and interpretation across various clinical contexts will facilitate the incorporation of this biomarker into routine clinical practice. Highlights Unique international overview of current analytical and post‐analytical methods for neurofilament light chain (NfL) measurement in routine clinical practice. Tailored sheets for each neurological application. Strategies to harmonize cutoffs, reporting, and interpretation of NfL's measurement.

Somali Americans, originating from East Africa, are a fast-growing minority in the United States. Emerging data suggest Somali individuals exhibit increased cardiovascular (CV) risk compared to non-Hispanic White adults. Previous data from our laboratory demonstrated elevated ambulatory arterial stiffness in Somali Americans, as derived from 24-hour blood pressure monitoring. We sought to confirm these vascular stiffness findings via gold-standard applanation tonometry and examine potential sex differences, given that females of African ancestry are at elevated risk of CV disease (CVD). We therefore examined aortic hemodynamics and carotid-femoral pulse wave velocity in male and female Somali Americans. We hypothesized that female Somali Americans would present with greater aortic wave reflection and arterial stiffness compared to males. We further examined the accuracy of prediction of aortic wave reflection from a predominantly White population, when applied to the Somali American population. We utilized applanation tonometry to quantify arterial stiffness in 24 female (age: 27±10 years, BMI: 27±6 kg/m 2 ) and 19 male (age: 22±6 years, BMI: 27±5 kg/m 2 ) Somali Americans without CVD. Each participant arrived in the laboratory at Mayo Clinic, fasted for at least three hours, and did not consume caffeine or exercise for at least 12 hours prior to testing. After 15 minutes of supine rest, duplicate recordings of aortic blood pressure and augmentation index (AIx) were obtained via pulse wave analysis. Similarly, aortic stiffness was quantified via duplicate carotid-femoral pulse wave velocity recordings. Statistical analysis included independent samples t-tests to test for sex differences in aortic hemodynamics, and paired samples t-tests to determine differences between estimated and obtained AIx (α≤0.05). Aortic systolic arterial pressure (111±11 vs. 108±7 mmHg, p=0.295) was similar between female and male Somali Americans. Aortic diastolic arterial pressure (75±7 vs. 67±7 mmHg, p<0.001) and heart rate (67±9 vs. 57±7 beats/min, p<0.001) were higher in female Somali adults. Similarly, AIx (28±14 vs. 19±12%, p=0.045) and AIx normalized to 75 heart beats/minute (24±13 vs. 10±13%, p=0.001) were higher in female Somali Americans. Carotid-femoral pulse wave velocity (5.4±0.9 vs. 5.5±0.8 m/s, p=0.661) was similar between sexes. Estimated AIx (corrected for sex, age, height, and resting heart rate) was markedly lower than observed AIx in both female (13±7 vs. 28±14%, p<0.001) and male (6±5 vs. 19±12%, p<0.001) Somali Americans. These findings, obtained in young, healthy Somali adults are suggestive of elevated CVD risk, evident at an early age. These data are particularly concerning regarding vascular dysfunction in Somali females and suggest that prediction models for arterial stiffness should factor in race to optimize performance. Future work into mechanisms underlying vascular dysfunction in Somali Americans is warranted. Sleep Number Grant to Mayo Clinic and by NIH R01-HL160619, R01-HL169320, T32-DK007013, K12 AR084222, and AHA 24POST1241616. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Mitochondrial volume and quality are tightly regulated by two key mechanisms: removal of dysfunctional/damaged mitochondria by selective autophagy (mitophagy) and mitochondrial biogenesis. Disruption of either one or both processes results in the accumulation of dysfunctional mitochondria and increased oxidative stress. Emerging evidence suggest that mitophagy and mitochondrial biogenesis are tightly coordinated, working in concert to maintain mitochondrial homeostasis and quality control. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway disease, promotes mitochondrial biogenesis by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling, the nodal regulator of mitochondrial biogenesis, thereby increasing mitochondrial volume density in human airway smooth muscle (hASM) cells. Additional evidence indicates that TNFα induces mitochondrial depolarization, which is known to trigger a mitophagy pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, an E3 ubiquitin ligase. Mitochondrial depolarization facilitates the mitochondrial accumulation of PINK1 and its activation by autophosphorylation at ser228 (pPINK1 s228 ). pPINK1 s228 promotes the recruitment and phosphorylation of Parkin at ser65 (pParkin s65 ), which ubiquitylates several mitochondrial associated proteins promoting the removal of damaged mitochondria. The decrease in the number of functional mitochondria by mitophagy can be offset by increasing mitochondrial biogenesis through the upregulation of PGC-1α signaling. Therefore, we hypothesize that PARIS (ZNF746), a transcriptional repressor of PGC-1α, is a target of PINK1/Parkin-mediated ubiquitination and degradation in hASM cells. In the present study, primary hASM cells were isolated from bronchiolar tissue of 6 subjects with no history of chronic pulmonary disease or smoking and separated into the following treatment groups: 1) FCCP (1 µM, 6 h; positive control), a proton ionophore that induces mitochondrial depolarization; 2) TNFα (20 ng/mL, 6 h); and 3) untreated control. We observed that FCCP and TNFα-induced mitochondrial depolarization triggered mitochondrial translocation of PINK1 and increased phosphorylation of pPINK1 s228 . Additionally, phosphorylation of pParkin s65 and ubiquitination of several mitochondrial associated proteins was increased. Immunoprecipitation of ubiquitinated proteins revealed increased ubiquitination of PARIS, while total expression of PARIS was decreased. Mitochondria and lysosomes were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively, and imaged in 3D (Z optical slice of 0.5 μm) to demonstrate the coefficient for colocalization of mitochondria with lysosomes. We observed increased formation of mitophagosomes, characteristic of mitophagy. Our findings demonstrate that TNFα-induces both mitophagy and mitochondrial biogenesis via the activation of PINK1/Parkin-mediated mitophagy pathway. Such coordination of mitophagy and mitochondrial biogenesis has significant ramifications for the maintenance of mitochondrial homeostasis, quality control and cellular energy metabolism. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Airway inflammation occurs as a defense mechanism in response to pathogens, allergens, toxins, or pollutants. The physiological responses of airway inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), that causes airway remodeling, characterized by airway smooth muscle (ASM) hyperreactivity and proliferation. The molecular mechanism underlying TNFα-induced ASM cell proliferation is not fully understood. Previous studies from our lab demonstrated that TNFα exposure induces accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggering an ER stress response that involves autophosphorylation of inositol requiring enzyme 1α at Serine 724 (pIRE1α S724 ). pIRE1α S724 promotes alternative splicing of X-box binding protein 1 (XBP1s) mRNA. XBP1s acts as a ubiquitous transcription factor in ASM cells, targets a number of genes associated with different cellular responses. In the present study, we hypothesized that TNFα promotes XBP1s mediated transcriptional activation of the Cyclin B1, a protein essential for the control of the cell cycle, thus promoting ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples collected during lung surgery from female and male patients (49 to 61 years of age) with no history of chronic respiratory diseases or current smoking. Isolated hASM cells from the same patient were serum-deprived for 48 h and separated into two groups: 1) untreated controls, and 2) treated with TNFα (20 ng/ ml) for 6 h. To confirm the effect of TNFα on hASM cell proliferation, loss of function experiments were employed by: 1) transfecting hASM cells with a non-spliceable XBP1 mutant, and 2) treating the cells with 4μ8C, a pharmacological inhibitor for IRE1α mediated XBP1 splicing. hASM cell proliferation was measured using CyQuant cell proliferation assay. The binding site sequence of XBP1s to the Cyclin B1 promoter was identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. mRNA and protein expressions of Cyclin B1 were determined by qRT-PCR and Western blot respectively. Nuclear localization of Cyclin B1 was measured by nuclear fractionation, followed by western blot of nuclear and cytosolic fraction. Results were analyzed by paired t-tests. TNFα treatment for 6 h induced pIRE1α S724 phosphorylation and XBP1s splicing. Bioinformatic analysis revealed the presence of an XBP1s binding site on the Cyclin B1 promoter. The ChIP assay confirmed that XBP1s binds to the promoter region of the Cyclin B1 genes, and the binding affinity was increased in TNFα-treated hASM cells. Consistent with these results, we found that TNFα treatment increased both mRNA and protein expressions of Cyclin B1 in hASM cells. Nuclear localization of Cyclin B1 increased significantly in TNFα treated cells consistent with an increase in hASM cell proliferation. Inhibition of XBP1 splicing reduced the expression of Cyclin B1 in hASM cells and hASM cell proliferation. In summary, TNFα induces an ER stress response involving pIRE1α S724 phosphorylation and XBP1s splicing. In turn, XBP1s transcriptionally targets expression of Cyclin B1 that mediates hASM cell proliferation. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Introduction: Throughout the lifetime of an asthmatic, the lung undergoes changes in the composition of its extracellular matrix (ECM) and becomes more fibrotic, a characteristic feature of airway remodeling. Due to this, lung functionality decreases as the lung becomes less elastic and airway resistance increases. The ECM proteins constantly network with their respective mechanosensory receptors and regulatory proteins on the cell membrane. These signaling interactions allow for continuous interactions and feedback between the ECM and airway smooth muscle cells (ASM). ASM cells are one of the major structural cells that regular airway remodeling and are the primary target for treating asthma. In this study, we investigated the specific alterations that occur in the ECM of ASM cells treated with TGF-β1. Methods: Human primary airway smooth muscle (ASM) cells were grown to confluency in DMEM/F-12 with 10% FBS and 1% AbAm in standard conditions. Cells were serum deprived for 48 hours prior to experiments. Cells were treated with TGF-β1 (2 ng/mL) in serum free media. Cells were harvested after 48 hours and 200 µg of protein lysate was prepared. Data-independent acquisition (DIA) mass spectrometry was performed at IDeA National Resource for Quantitative Proteomics. Fold changes for ECM class proteins were collected from DIA results. Pathway analysis was then done using Qiagen ingenuity pathway analysis. Furthermore, expression patterns of select proteins were confirmed via western blotting. Results: The effects of TGF-β1 on specific types of collagens, integrins, laminins, and additional ECM proteins were analyzed. Western blotting confirmed similar changes in expression as measured by DIA. TGF-β1 exposure upregulated LAMA4, LAMB2, and LAMC1 which is likely to increase the prevalence of Laminins 221 and 421. TGF-β1 augmented integrin signaling by upregulating ITGA7, ITGA11, ITGA1, ITGB1 and downregulating ITGA2 and ITGB8. TGF-β1 increased expression of TSP1, TSP2, TSP3, TNS-C, FB, Elastin and Versican while downregulating TNS-X. Several interactions between ECM proteins and their sensors and receptors were highlighted for their enrichment. Conclusion: The DIA analysis shows an upregulation of proteins involved in airway remodeling by TGF-β1 in ASM cells. These findings substantiate our previous results as well as provide insights into a more targeted approach in studying ECM-Cell interactions. NIH R01HL171245 (Venkatachalem), R01HL146705 (Venkatachalem), R01HL142061 (Pabelick, Prakash). and IDeA National Resource for Quantitative Proteomics (R24GM137786). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Allergens from the fungus Alternaria alternata promote secretion of alarmins from the airway epithelium including ATP, IL-33 and both genomic and mitochondrial DNA fragments, which produce type 2 immune responses in airways. Alternaria -induced oxidative stress activates ATP release by opening apical voltage-dependent anion channels (VDAC-1) and stimulating exocytosis of ATP. This investigation demonstrated that treatment of human bronchial epithelial (hBE) cells with a selective peptide antagonist or small molecule inhibitor (FPS-ZM 1 ) of the receptor for advanced glycation end products (RAGE), reduced Alternaria -evoked ATP secretion, decreasing the initial rate by ~40%. Furthermore, the Alternaria -induced increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) was inhibited by ~75% following pretreatment with RAGE antagonist peptide. Intranasal administration of Alternaria extract (50 μg) produced a significant increase in IL-33 secretion into bronchoalveolar lavage (BAL) fluid, which was inhibited in RAGE-deficient ( Ager -/- ) mice. Moreover, Alternaria -induced increases in IL-5 and IL-13 secretion into BAL fluid were also significantly reduced in Ager -/- mice. These findings support the conclusion that Alternaria -dependent ATP release and Ca 2+ mobilization was dependent in part on RAGE activation and that RAGE antagonists have the potential of reducing type 2 immune responses in airways following Alternaria allergen exposure. This study was supported by grants from the NIH (R01A1128729 & 1R01AI169530) to HK and SMO and by the Minnesota Agricultural Experiment station grant (AES0016134) to SMO. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Rationale: Asthma is a chronic inflammatory disease of conducting airways characterized by airway hyperresponsiveness and remodeling. Airway smooth muscle (ASM) is a major structural cell in the airway that contributes to airway hyperresponsiveness and remodeling. Mitochondrial dynamics is a fission/fusion process crucial for optimal function and signal transduction. Inflammation alters the mitochondrial dynamics in ASM, thereby disrupt mitochondrial function and contributes to asthma pathophysiology. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cellular homeostasis. Recently, we found upregulation of AhR in response to inflammation and asthma in ASM. Additionally, AhR activation inhibited inflammation-induced ASM proliferation and remodeling. However, the role of AhR in regulating mitochondrial dynamics in ASM during inflammation and asthma is obscure. Objective: The aim of this work is to unveil the role of AhR in regulating mitochondrial structure and function in human ASM during inflammation and asthma. Methods: Primary non-asthmatic and asthmatic human ASM cells isolated from surgical lung sections were cultured in DMEM-F12 medium supplemented with 10% FBS and 1% AbAm. Cells were serum deprived, treated with AhR agonist 6-Formylindolo[3,2-b]carbazole (FICZ: 10 nM) and antagonist CH223191 (10 μM), with/or without TNFα (20 ng/mL). Mitochondrial bioenergetics was determined by Seahorse XFp analyzer. Mitochondrial morphology was visualized using mitotracker dye. Expressions of fission and fusion mRNA and protein were determined by qPCR and immunoblotting. Additionally, AhR role in fusion and fission was confirmed in shRNA mediated AhR knockdown and AhR overexpressed ASM cells. Results: AhR activation by FICZ alleviated TNFα-induced disrupted mitochondrial bioenergetics in non-asthmatic ASM cells as well as in asthmatic ASM. Furthermore, TNFα-induced increased mitochondrial fission and reduced fusion was restored by AhR activation in both asthmatic and non-asthmatic ASM. AhR inhibition by CH223191 and knockdown promoted mitochondrial fission and blunted fusion in non-asthmatic ASM cells. Interestingly, AhR overexpression and activation by FICZ restored the mitochondrial dynamics altered by inflammation and asthma in ASM cells. Conclusion: Collectively, AhR activation promotes mitochondrial fusion and abolishes fission induced by inflammation and asthma in human ASM, thereby maintains mitochondrial structure and homeostasis. Therefore, AhR is a potential therapeutic target to mitigate altered mitochondrial dynamics in ASM during inflammation and asthma. Supported by NIH grants R01-HL171245 (Venkatachalem and Britt), R01-HL146705 (Venkatachalem), R01-HL155095 (Britt), R01-HL142061 (Pabelick, Prakash) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: During acute hypoxia, Thehuman brainemploys complex regulatory mechanisms to compensate for high O 2 demand and low O 2 blood saturation. High hemoglobin-oxygen (Hb-O 2 ) affinity improves arterial oxygen saturation (S a O 2 ) in low oxygen environments providing a potential physiologic benefit in humans. However, the impact of high Hb-O 2 affinity on cerebral blood flow (CBF) and cerebral tissue oxygenation (S t O 2 ) has yet to be examined. This work aimed to determine the influence of an acute increase in Hb-O 2 affinity from Etavopivat, a selective red blood cell pyruvate kinase activator, on CBF regulation and S t O 2 during handgrip exercise in normoxia and hypoxia. Methods: 16 healthy adults (31±8 yr, 5 females) completed two visits; before and after Etavopivat (400 mg daily, 7 days) administration,. Each visit included graded handgrip (~3 min stages each) exercise (rest, 10% maximal voluntary contraction (MVC), 20% MVC) while participants inspired normoxic (NORM, 21% O 2 ), moderate (MH, 15% O 2 ), and severe (SH, 10% O 2 ) hypoxic gas mixtures. Hb-O 2 binding affinity (P 50 ), was measured using dual-wavelength spectrophotometry (pH~7.4 and 37ºC). Blood sampling through catheterization of the non-exercising arm measured S a O 2 . CBF was approximated by the peak middle cerebral artery velocity, measured with a 2MHz transcranial doppler ultrasound through the transtemporal window. S t O 2 was measured using frequency-domain near infrared spectroscopy of the frontal cortex. CBF and S t O 2 between visits were compared using separate two-way RM ANOVA models to determine the effects of Etavopivat [before (baseline) or after 7d Etavopivat exposure (post)] and exercise intensity [rest, 10% MVC, 20% MVC] for each inspirate. Results: P 50 was reduced after Etavopivat exposure (Baseline: 28.6±1.6 mmHg, Etavo: 24.8±1.3, P <0.001) without impacting hematocrit ( P =0.97). S a O 2 was higher following Etavopivat exposure during each inspirate ( P <0.001 for each). There was no significant effect of handgrip exercise on CBF or S t O 2 within any inspirate. Cerebral S t O 2 was higher during each inspirate (all P <0.002)– NORM (Baseline: 65.3±12.3%, Post: 68.8±9.8%), MH (Baseline: 61.0±11.7 %, Post: 66.1±10.5%) and SH (Baseline: 47.7±13.5%, Post: 56.6±11.5%). In addition, CBF was increased during each inspirate (all P <0.001)– NORM (Baseline: 62.2±12.2 cm/s, Post: 69.2±12.5 cm/s), MH (Baseline: 60.8±13.1 cm/s, Post: 67.9±12.3 cm/s) and SH (Baseline: 69.8±16.3 cm/s, Post: 79.2±18.0 cm/s). Conclusion: The acute pharmacological induction of high Hb-O 2 affinity leads to increased CBF and cerebral S t O 2 during acute hypoxic exposure in conditions of mild muscular exertion. Whether the improvement of cerebral S t O 2 is associated increased S a O 2 or CBF (or both) is unclear. These findings are the first to investigate the complex determinants of cerebral S t O 2 as a function of an acute increase in Hb-O 2 affinity. These improvements in S t O 2 during hypoxic exposure may lead to a host of theoretical benefits such as improved hypoxia tolerance, and mitigated decrements in cognitive function during hypoxic exposure. This study was funded by Forma Therapeutics Inc, which was acquired by Novo Nordisk on October 14, 2022, Novo Nordisk A/S. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Dynamic cerebral autoregulation (CA) refers to the brain's ability to maintain stable cerebral blood flow despite fluctuations in blood pressure (BP). Impaired CA has been associated with cardiovascular disease severity and cognitive decline. On the other hand, enhanced cardiorespiratory fitness appears to be protective against both cerebrovascular dysregulation and cognitive decline, with an unclear link between the two. However, among individuals with cardiovascular disease, the impact of exercise-based cardiac rehabilitation (CR) on CA remains unclear. Therefore, the aim of this study was to test the hypothesis that a 12-week CR program would improve dynamic CA in adults with CVD. To address this question, data were collected from 14 patients enrolled in the Mayo Clinic CR program following a cardiac-related hospital admission (age: 43-70 yrs; n=3 females), which included moderate-high intensity aerobic exercise 3 times/week for 12-weeks. Continuous measures of middle cerebral artery velocity (MCAv; transcranial doppler) and finger BP (photoplethysmography) were obtained during 3-minutes of rest and a 3-minute sit-to-stand protocol (0.025Hz frequency) pre- and post the CR program. Transfer function analyses were used to provide an index of dynamic CA. BP-MCAv coherence in the low frequency (LF) and very low frequency (vLF) domains at rest were not different pre- and post- CR (LF: 0.48±0.19 vs 0.43±0.16; P=0.54; vLF: 0.51±0.12 vs 0.47±0.16; P=0.14). Similarly, gain and phase values at rest were not different pre- and post- CR (gain LF: 0.66±0.37 vs 0.58±0.33; P=0.16; vLF: 0.87±0.53 vs 0.74±0.43; P=0.21; phase LF: 0.41±0.30 vs 0.43±0.26; P=0.64; vLF: 0.72±0.53 vs 0.66±0.16; P=0.18). In contrast, during the sit-to-stand protocol, coherence was lower following CR (pre vs post LF: 0.81±0.13 vs 0.79±0.16; p<0.01; vLF: 0.80±0.09 vs 0.79±0.14; p<0.01). In addition, gain was reduced post CR (pre vs post LF: 0.59±0.35 vs 0.55±0.31; P=0.02; vLF: 0.76±0.33 vs 0.61±0.37; p<0.01), suggesting CR-induced improvements in CA efficiency. However, phase remained unchanged pre- and post- CR during the sit-to-stand protocol (LF: 1.01±0.71 vs 0.84±0.58; P=0.17; vLF: 1.16±0.69 vs 1.08±0.60; P=0.19). Taken together, these findings demonstrate that a 12-week exercise-based CR program improves dynamic CA efficiency after a cardiac event. These novel results highlight the potential role of exercise training in improving cerebrovascular regulation. Funding: NIH AG073726 and Mayo Clinic This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.