Physiological Significance Research Articles

Verification of the analytical performance of the serum magnesium assay on Abbott Architect ci8200

Magnesium is recognized as the fourth most important cation in the body and the second most abundant intracellular cation, following potassium. Its physiological significance lies in its capacity to form complexes with crucial intracellular anionic ligands, notably ATP, and its ability to compete with calcium for specific binding sites on proteins and cell membranes. Over 300 enzyme reactions rely on magnesium. The concentration of free magnesium within cells determines the magnesium content of these enzymes. Magnesium influences myocardial contraction and the electrical activity of myocardial cells, as well as the specialized conduction systems of the fetus, by modulating the transmembrane movements of ions like sodium, potassium, and calcium. Furthermore, magnesium can affect the contractility of vascular smooth muscle, and alterations in intracellular magnesium levels may impact cell proliferation or contraction. Magnesium is involved in nucleic acid and protein metabolism, intermediary metabolism, energy production, and consumption reactions, and it plays specific roles in various physiological systems such as the neuromuscular and cardiovascular systems. Our research aims to assess the analytical accuracy of the serum magnesium assay method utilizing an Abbott kit on the Architect ci8200 automated system in the biochemistry laboratory of Mohammed VI University Hospital of Oujda. The evaluation of the kit's analytical accuracy was conducted within the flexible scope A by conducting a performance analysis on the Architect ci8200. We examined repeatability, reproducibility, measurement imprecision, and compared results between two Architect ci8200 instruments. All findings align with the acceptability criteria recommended by the supplier and the Valtec protocol of the Société Française de Biologie Clinique (SFBC), indicating overall satisfactory outcomes for the study. The Architect ci8200 automated system demonstrated satisfactory analytical performance for reliably determining serum magnesium levels. Method verification in medical laboratories is essential to guarantee the accuracy, precision, and reliability of test results. Verification entails confirming that the test method used is suitable for its intended purpose, yields result consistent with the claimed performance characteristics, and meets the laboratory's quality control and quality assurance standards.

Unresolved questions in the regulation of skeletal muscle insulin action by reactive oxygen species.

Reactive oxygen species (ROS) are well-established signaling molecules implicated in a wide range of cellular processes, including both oxidative stress and intracellular redox signaling. In the context of insulin action within its target tissues, ROS have been reported to exert both positive and negative regulatory effects. However, the precise molecular mechanisms underlying this duality remain unclear. This Review examines the complex role of ROS in insulin action, with a particular focus on skeletal muscle. We aim to address three critical aspects: (a) the proposed intracellular pro-oxidative redox shift elicited by insulin, (b) the evidence supporting that redox-sensitive cysteine modifications impact insulin signaling and action, and (c) cellular mechanisms underlying how ROS can paradoxically act as both enhancers and inhibitors of insulin action. This Review underscores the urgent need for more systematic research to identify specific reactive species, redox targets, and the physiological significance of redox signaling in maintaining insulin action and metabolic health, with a particular emphasis on human skeletal muscle.

Second-generation antipsychotic quetiapine blocks voltage-dependent potassium channels in coronary arterial smooth muscle cells.

Voltage-dependent K+ (Kv) channels play an important role in restoring the membrane potential to its resting state, thereby maintaining vascular tone. In this study, native smooth muscle cells from rabbit coronary arteries were used to investigate the inhibitory effect of quetiapine, an atypical antipsychotic agent, on Kv channels. Quetiapine showed a concentration-dependent inhibition of Kv channels, with an IC50 of 47.98 ± 9.46 μM. Although quetiapine (50 μM) did not alter the steady-state activation curve, it caused a negative shift in the steady-state inactivation curve. The application of 1 and 2Hz train steps in the presence of quetiapine significantly increased the inhibition of Kv current. Moreover, the recovery time constants from inactivation were prolonged in the presence of quetiapine, suggesting that its inhibitory action on Kv channels is use (state)-dependent. The inhibitory effects of quetiapine were not significantly affected by pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors. Based on these findings, we conclude that quetiapine inhibits Kv channels in both a concentration- and use (state)-dependent manner. Given the physiological significance of Kv channels, caution is advised in the use of quetiapine as an antipsychotic due to its potential side effects on cardiovascular Kv channels.

Differential and synergistic effects of left and right atrial ablation of paraseptal ganglionated plexuses in a cohort of consecutive patients undergoing cardioneuroablation

Abstract Background Cardioneuroablation (CNA) treats reflex syncope by ablating the main epicardial ganglionated plexuses (GPs) conveying vagal outputs to the sino-atrial (SA) and atrio-ventricular (AV) nodes. The two main GPs, superior paraseptal (SPSGP) and inferior paraseptal (IPSGP), regulate vagal tone to SA and AV nodes, respectively. Both GPs can be targeted with radiofrequency energy (RF) from the right (RA) and left atrium (LA). It is unclear if bi-atrial ablation yields more effective GP ablation or if the ablation sequence affects GP response. Purpose We aimed to assess the added efficacy of ablating the SPSGP and IPSGP in the LA after initial RA ablation, and vice versa, in a separate group of patients. Methods In a consecutive patient population undergoing CNA we evaluated the acute efficacy of RF delivery on the SPSGP and the IPSGP. The population was divided into two consecutive groups: one receiving RF initially in the RA and then in the LA (group 1), and the other in the reverse order (group 2). The acute effect of RF applications was measured as percentage increase in heart rate (HR) for the SPSGP and as reduction of PR interval for the IPSGP. Results Sixty-one patients (50±15 years; 61% males) underwent CNA. Of these, 17 underwent ablation of the SPSGP and IPSGP starting from the RA and completing the procedure in the LA (group 1), while in 44 patients, the procedure was performed in the opposite order (group 2). In group 1, RF delivery on the SPSGP from the RA prompted an increase in HR (54.1±11.1 vs 60.6±14.7 bpm; p=0.03; median HR increment 3.8%, IQR 0-38.3) that was further enhanced after LA ablation (from 57.5±11.9 to 64.5±13.5 bpm; p<0.001; median HR increment: 8.0%, IQR: 2.0-13.5). In group 1, RA ablation of the IPSGP reduced PR interval in a single patient (from 224 to 184 ms), without further shortening after LA ablation. Final CNA success was 82% in group 1. In group 2, RF delivery on the SPSGP from the LA induced a marked increase in HR (58.4±13.1 vs 76.8±16.1 bpm; p<0.001; median HR increase 30.0%, IQR 14.0-43.0). In the same patients, additional ablation from the RA prompted further but lower incremental effect (from 70.0±17.3 to 76.9±17.9 bpm; p<0.001; median HR increment 8.3%, IQR: 0-16.4). In group 2, 4 (9%) patients had PR shortening after LA ablation of the IPSGP (241±93 vs 172±14 ms; p value). Of these, a single patient had further PR reduction after IPSGP RA ablation (from 189 to 161 bpm). Final CNA success was 93% in group 2. Symptoms-free survival was 88% at 16±5 months in group 1 and 89% at 19±7 months in group 2 (p= ns). Conclusions Bi-atrial ablation of the SPSGP and IPSGP provides incremental acute efficacy compared to ablation from either the RA or the LA alone. LA ablation of the main GPs seems an essential component of CNA. The physiological significance of these findings requires further investigationGanglionated Plexuses Segmentation

Prediction of Protein‒DNA Interface Hot Spots Based on Empirical Mode Decomposition and Machine Learning

Protein–DNA complex interactivity plays a crucial role in biological activities such as gene expression, modification, replication and transcription. Understanding the physiological significance of protein–DNA binding interfacial hot spots, as well as the development of computational biology, depends on the precise identification of these regions. In this paper, a hot spot prediction method called EC-PDH is proposed. First, we extracted features of these hot spots’ solid solvent-accessible surface area (ASA) and secondary structure, and then the mean, variance, energy and autocorrelation function values of the first three intrinsic modal components (IMFs) of these conventional features were extracted as new features via the empirical modal decomposition algorithm (EMD). A total of 218 dimensional features were obtained. For feature selection, we used the maximum correlation minimum redundancy sequence forward selection method (mRMR-SFS) to obtain an optimal 11-dimensional-feature subset. To address the issue of data imbalance, we used the SMOTE-Tomek algorithm to balance positive and negative samples and finally used cat gradient boosting (CatBoost) to construct our hot spot prediction model for protein‒DNA binding interfaces. Our method performs well on the test set, with AUC, MCC and F1 score values of 0.847, 0.543 and 0.772, respectively. After a comparative evaluation, EC-PDH outperforms the existing state-of-the-art methods in identifying hot spots.

Motor network dynamic resting state fMRI connectivity of neurotypical children in regions affected by cerebral palsy

BackgroundNormative childhood motor network resting-state fMRI effective connectivity is undefined, yet necessary for translatable dynamic resting-state-network-informed evaluation in pediatric cerebral palsy.MethodsCross-spectral dynamic causal modeling of resting-state-fMRI was investigated in 50 neurotypically developing 5- to 13-year-old children. Fully connected six-node network models per hemisphere included primary motor cortex, striatum, subthalamic nucleus, globus pallidus internus, thalamus, and contralateral cerebellum. Parametric Empirical Bayes with exhaustive Bayesian model reduction and Bayesian modeling averaging informed the model; Purdue Pegboard Test scores of hand motor behavior were the covariate at the group level to determine the effective-connectivity-functional behavior relationship.ResultsAlthough both hemispheres exhibited similar effective connectivity of motor cortico-basal ganglia-cerebellar networks, magnitudes were slightly greater on the right, except for left-sided connections of the striatum which were more numerous and of opposite polarity. Inter-nodal motor network effective connectivity remained consistent and robust across subjects. Age had a greater impact on connections to the contralateral cerebellum, bilaterally. Motor behavior, however, affected different connections in each hemisphere, exerting a more prominent effect on the left modulatory connections to the subthalamic nucleus, contralateral cerebellum, primary motor cortex, and thalamus.DiscussionThis study revealed a consistent pattern of directed resting-state effective connectivity in healthy children aged 5–13 years within the motor network, encompassing cortical, subcortical, and cerebellar regions, correlated with motor skill proficiency. Both hemispheres exhibited similar effective connectivity within motor cortico-basal ganglia-cerebellar networks reflecting inter-nodal signal direction predicted by other modalities, mainly differing from task-dependent studies due to network differences at rest. Notably, age-related changes were more pronounced in connections to the contralateral cerebellum. Conversely, motor behavior distinctly impacted connections in each hemisphere, emphasizing its role in modulating left sided connections to the subthalamic nucleus, contralateral cerebellum, primary motor cortex, and thalamus. Motor network effective connectivity was correlated with motor behavior, validating its physiological significance. This study is the first to evaluate a normative effective connectivity model for the pediatric motor network using resting-state functional MRI correlating with behavior and serves as a foundation for identifying abnormal findings and optimizing targeted interventions like deep brain stimulation, potentially influencing future therapeutic approaches for children with movement disorders.

Differential Effect of Aortic Valve Replacement for Severe Aortic Stenosis on Hyperemic and Resting Epicardial Coronary Pressure Indices.

Coronary pressure indices to assess coronary artery disease are currently underused in patients with aortic stenosis due to many potential physiological effects that might hinder their interpretation. Studies with varying sample sizes have provided us with conflicting results on the effect of transcatheter aortic valve replacement (TAVR) on these indices. The aim of this meta-analysis was to study immediate and long-term effects of TAVR on fractional flow reserve (FFR) and nonhyperemic pressure ratios (NHPRs). Lesion-specific coronary pressure data were extracted from 6 studies, resulting in 147 lesions for immediate change in FFR analysis and 105 for NHPR analysis. To investigate the long-term changes, 93 lesions for FFR analysis and 68 for NHPR analysis were found. Lesion data were pooled and compared with paired t tests. Immediately after TAVR, FFR decreased significantly (-0.0130±0.0406 SD, P: 0.0002) while NHPR remained stable (0.0003±0.0675, P: 0.9675). Long-term after TAVR, FFR decreased significantly (-0.0230±0.0747, P: 0.0038) while NHPR increased nonsignificantly (0.0166±0.0699, P: 0.0543). When only borderline NHPR lesions were considered, this increase became significant (0.0249±0.0441, P: 0.0015). Sensitivity analysis confirmed our results in borderline lesions. TAVR resulted in small significant, but opposite, changes in FFR and NHPR. Using the standard cut-offs in patients with severe aortic stenosis, FFR might underestimate the physiological significance of a coronary lesion while NHPRs might overestimate its significance. The described changes only play a clinically relevant role in borderline lesions. Therefore, even in patients with aortic stenosis, an overtly positive or negative physiological assessment can be trusted.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes.

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurBand MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, result in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation, and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of PCOS induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

Low-density EEG-based Functional Connectivity Discriminates Minimally Conscious State plus from minus

Objective: Within the continuum of consciousness, patients in a Minimally Conscious State (MCS) may exhibit high-level behavioral responses (MCS+) or may not (MCS−). The evaluation of residual consciousness and related classification is crucial to propose tailored rehabilitation and pharmacological treatments, considering the inherent differences among groups in diagnosis and prognosis. Currently, differential diagnosis relies on behavioral assessments posing a relevant risk of misdiagnosis. In this context, EEG offers a non-invasive approach to model the brain as a complex network. The search for discriminating features could reveal whether behavioral responses in post-comatose patients have a defined physiological background. Additionally, it is essential to determine whether the standard behavioral assessment for quantifying responsiveness holds physiological significance. Methods: In this prospective observational study, we investigated whether low-density EEG-based graph metrics could discriminate MCS+/− patients by enrolling 57 MCS patients (MCS−: 30; males: 28). At admission to intensive rehabilitation, 30 min resting-state closed-eyes EEG recordings were performed together with consciousness diagnosis following international guidelines. After EEG preprocessing, graphs’ metrics were estimated using different connectivity measures, at multiple connection densities and frequency bands (α,θ,δ). Metrics were also provided to cross-validated Machine Learning (ML) models with outcome MCS+/−. Results: A lower level of brain activity integration was found in the MCS− group in the α band. Instead, in the δ band MCS− group presented an higher level of clustering (weighted clustering coefficient) respect to MCS+. The best-performing solution in discriminating MCS+/− through the use of ML was an Elastic-Net regularized logistic regression with a cross-validation accuracy of 79% (sensitivity and specificity of 74% and 85% respectively). Conclusion: Despite tackling the MCS+/− differential diagnosis is highly challenging, a daily-routine low-density EEG might allow to differentiate across these differently responsive brain networks. Significance: Graph-theoretical features are shown to discriminate between these two neurophysiologically similar conditions, and may thus support the clinical diagnosis.

PSI-17 Effect of timing of high dietary zinc supplementation on the microbiome of gestating sows and their piglets

Abstract The objective of this study was to assess if high dietary zinc (Zn) fed to gestating sows modulates the microbiome of the sow and that of their offspring. Sows were fed 1 of 3 dietary treatments: 1) Control: a corn-soybean meal-based diet containing 125 ppm total supplemental zinc, 2) Breed-to-Farrow: as Control + 141 ppm supplemental Zn as ZnSO4 fed from 5 d post-breeding to farrowing; and 3) Day 110-to-Farrow: as Control + 2,715 ppm supplemental Zn as ZnSO4 starting on d 110 of gestation until farrowing. A subset of third parity sows (n = 30; 10 per treatment) were selected to assess the microbiome of colostrum, milk, and rectal and vaginal surfaces of sows. At farrowing, 4 pigs per litter (n = 120) were selected based on birthweight (BiW; 2 average BiW pigs and 2 pigs with BiW below the litter average) were selected to assess the piglet gut microbiome on the day of birth (d 0) and d5 of age. 16S rRNA sequencing was implemented for colostrum and milk samples while all other sample types were sequenced using shotgun metagenomics to determine taxonomic and functional profiles. Measures of alpha and beta diversity were conducted along with determination of differentially abundant taxa and pathways in tandem with generalized linear mixed models. Greater dietary concentrations of Zn fed to gestating sows minimally affected their colostrum, milk, and vaginal microbiome. However, fecal samples of Breed-to-Farrow sows showed decreased abundance of potentially pathogenic bacteria such as Salmonella enterica and Shigella flexneri and greater abundance of bacteria typically involved in fiber fermentation compared with Control sows (Figure 1a). Piglets born to Day 110-to-Farrow sows also showed greater abundance of some taxa typically involved in fiber breakdown and short chain fatty acid (SCFA) generation on d 5 compared with Control pigs (Figure 1b). At d 0, Breed-to-Farrow piglets showed less abundance of methanogenic archaea and differential abundance of taxa with potentially pathogenic roles, compared with Control pigs (Figure 1c). Gene families and pathways playing roles in central metabolic functions (starch, pyruvate, sucrose, amino acid metabolism) were more abundant in Breed-to-Farrow piglets compared with pigs born to Control sows on d 0 and 5 of age. In conclusion, greater Zn fed to gestating sows impacted the abundance of gut taxa with potential roles in SCFA-production and pathogenicity in both sow and piglet, despite overall minor effects. However, the physiological significance of these findings for the sow and piglet remains unclear.

Spatially resolved analysis of microenvironmental gradient impact on cancer cell phenotypes.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

Light physical activity improves lower limb perfusion asymmetries in older sedentary adults — an exploratory study

Perfusion asymmetries between lower limbs are common even in healthy young individuals, although their physiological significance is unknown. Even in the absence of vascular disease, ageing is among the determinants that seem to accentuate those differences. While it is known that physical activity affects vascular hemodynamics, its specific impact on perfusion asymmetries in healthy elderly individuals is still unclear. This study aimed to identify vascular asymmetries and their variations after a regular program of light exercise in older individuals with age-related comorbidities in the absence of peripheral vascular disease (PVD). Ten participants, mean age 62.4 ± 5.6 years old, were selected following specific criteria. Participants were mostly sedentary, combining one or more common morbidities for this age group — in particular hypertension, overweight and / or diabetes (T2D). Exclusion criteria were an abnormal Ankle-Brachial Index assessed in a standing position at rest and evidence of compromised mobility. All procedures respected the principles of good clinical practice, being previously submitted and approved by the institutional Ethical Commission. Lower limb perfusion was assessed in both feet (anteromedial region) at two time points — day zero (D0) and thirty days later (D30) — using two optical-based technologies — Laser Doppler Flowmetry (LDF) and Polarized Spectroscopy (PSp). These two technologies measure at different depths, with LDF providing deeper tissue readings. During this evaluation period, participants underwent a daily structured program of light exercises based on the FITT principles (F, frequency; I, intensity; T, time and T, type of activity/activity performance) centred in the lower limb perfusion. The group revealed significant asymmetries at D0, where the right foot consistently showed higher values with both technologies correlating positively. At D30, LDF values increased by 57.5% in both feet, meaning that asymmetries disappeared. No significant changes were observed with PSp. Basic cardiovascular functions such as blood pressure and mean arterial pressure were also evaluated, showing significant decreases in systolic pressure and mean arterial pressure. These encouraging results suggest that regular physical activity, even if light in intensity and duration, improves overall hemodynamics and specifically raises perfusion in the lower limbs. These findings should be further explored with the aimof prevention and treatment of PVD. This research was financed by national funds through FCT - Foundation for Science and Technology, I.P. (Portugal), under the [UIDB/04567/2020] and [UIDP/ 04567/2020] programs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular detection of lacrimal apparatus and ocular surface - related ABC transporter genes

The ocular system is in constant interaction with the environment and with numerous pathogens. The ATP-binding cassette (ABC) transporters represent one of the largest groups among the transmembrane proteins. Their relevance has been demonstrated for their defense function against biotic and abiotic stress factors, for metabolic processes in tumors and for their importance in the development of resistance to drugs. The aim of this study was to analyze which ABC transporters are expressed at the ocular surface and in the human lacrimal apparatus. Using RT-PCR, all ABC transporters known to date in humans were examined in tissue samples from human cornea, conjunctiva, meibomian glands and lacrimal glands. The RT-PCR analyses revealed the presence of all ABC transporters in the samples examined, although the results for some of the 48 transporters known in human and analyzed were different in the various tissues. The present results provide information on the expression of ABC transporters at the mRNA level on the ocular surface and in the lacrimal system. Their detection forms the basis for follow-up studies at the protein level, which will provide more information about their physiological significance at the ocular surface and in the lacrimal system and which may explain pathological effects such as drug resistance.

Predicting red blood cell traffcking and capillary hemodynamics in angiogenic and tumor microcirculation in silico

Objective: Angiogenic and tumor microvasculatures are known to have abnormal topology due to the presence of frequent vessel junctions, irregular and deflated blood vessels, multi-furcations, and tessellated vessel organization. Although recent advances in imaging techniques in vivo have enabled mapping such vasculatures at high spatial resolution, simultaneous measurements of hemodynamic parameters, such as the wall shear stress (WSS) with full 3D details, remain a challenge. Theoretical network flow models, often used for hemodynamic predictions in such experimentally acquired images, cannot provide the full 3D hemodynamic details either, as these models treat each blood vessel as 1D segment and do not explicitly model red blood cells (RBCs). To overcome this limitation, we have developed a high-fidelity, 3D Computational Fluid Dynamics modeling to predict the flow of a large number of deformable RBCs through physiologically realistic tumor/angiogenic microvascular networks in silico. Methods: We use in vivo images to create such vascular networks in silico and then predict RBC traffcking and capillary hemodynamics. Deformation of every flowing RBC is considered with high accuracy, and 3D geometry of each vessel is accurately modeled. Flow is driven by specifying physiological pressure boundary conditions. Model predictions have been validated against in vivo data. This in-house predictive tool is versatile, can be applied to any microvascular network image obtained in vivo in any organ, and can predict trajectories of diverse cell types including leukocytes, platelets and circulating tumor cells, drug and molecular transport in capillary blood, and cell-vessel adhesion. Results: We provide quantitative differences between healthy microvascular networks and tumor/angiogenic networks in terms of RBC distribution, perfusion, and wall shear stress. Our model shows increased heterogeneity in RBC and flow distribution in both tumor and angiogenic vasculatures than the healthy one. Also, we predict reduced flow and hematocrit in several vessels in both tumor and angiogenic vasculatures. Interestingly, several vessels in the angiogenic vasculature are predicted to have higher flow than the healthy one, while most vessels in the tumor vasculature show flow reduction. This in silico prediction is consistent with a recent in vivo study which showed higher flow in peri-tumor region and reduced flow in tumor. We further predict a significant heterogeneity in WSS and WSS gradient, blood velocity profiles, and near-wall RBC-depleted region. Conclusion: In conclusion, we have developed a versatile, in silico model that allows high-fidelity prediction of capillary hemodynamics in tumor microcirculation and provide information on hemodynamic variables that are not readily measurable in vivo but have physiological significance in tumor progression and treatment. NIH (R01EY033003) and NSF (CBET1804591). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Insertion sequence IS6770 modulates potassium symporter kup transcription in Enterococcus faecalis JH2-2 under low pH conditions

Enterococcus faecalis is a phylogenetically and industrially relevant microorganism associated with Lactic Acid Bacteria. Some strains of this bacterium are employed as probiotics in commercial applications, while others serve as the principal component in starter cultures for artisanal regional cheese production. However, over the last decade, this species has emerged as an opportunistic multiresistant pathogen, raising concerns about its impact on human health. Recently, we identified multiple potassium transporter systems in E. faecalis, including the Ktr systems (KtrAB and KtrAD), Kup, KimA and Kdp complex (KdpFABC). Nevertheless, the physiological significance of these proteins remains not fully understood. In this study, we observed that the kup gene promoter region in the JH2–2 strain was modified due to the insertion of a complete copy of the IS6770 insertion sequence. Consequently, we investigated the influence of IS6770 on the expression of the kup gene. To achieve this, we conducted a mapping of the promoter region of this gene in the E. faecalis JH2–2 strain, employing fluorescence gene reporters. In addition, a transcriptional analysis of the kup gene was executed in a strain derived from E. faecalis V583 that lacks the IS30-related insertion element, facilitating the identification of the transcriptional start site. Next, the expression of the kup gene was evaluated via RT-qPCR under different pH stressful conditions. A strong upregulation of the kup gene was observed at an initial pH of 5.0 in the strain derived from E. faecalis V583. However, the activation of transcription was not observed in the E. faecalis JH2–2 strain due to the hindrance caused by the presence of IS6770.Besides that, our computational analysis of E. faecalis genomes elucidates a plausible association between transposition and the regulation of the kup gene. Remarkably, the ubiquitous presence of IS6770 throughout the phylogenetic tree implies its ancient existence within E. faecalis. Moreover, the recurrent co-occurrence of IS6770 with the kup gene, observed in 30 % of IS6770-positive strains, alludes to the potential involvement of this genomic arrangement in the adaptive strategies of E. faecalis across diverse niches.

Pkd1RC/RC mice demonstrate remodeling of purinergic receptors in ADPKD cysts

Polycystic kidney diseases (PKD) are characterized by development of multiple cysts, dilations of nephron segments, which replace normal tissues and lead to kidney insuffciency. To identify new gene pathways affected by cyst development in collecting ducts, we used a bulk RNAseq approach comparing gene expression of normal microdissected cortical collecting ducts (n=3 mice) vs cysts microdissected from Pkd1 RC/RC mice (n=4). Bulk-RNA analysis identified 18,000 genes and allowed statistical comparison of over 15,000 genes. Our data reveals that although cysts originate from normal collecting ducts, cystic epithelium show 2692 down-regulated and 2278 up-regulated genes (p<0.05 pAdj. FDR). Ingenuity Pathways Analysis identifies the following intracellular mechanisms mostly affected by transition: Rac and Rho signaling, fibrosis signaling, epithelial-to-mesenchymal transition, cytoskeleton rearrangement and ERK/MAPK signaling. Several of the differentially expressed genes were identified in GWAS of PKD, chronic kidney diseases and hypertension. Our previous publication reported that development of cysts in an autosomal recessive model of PKD is associated with a shift of P2Y to P2X receptor abundance. In the current study we found that in the autosomal dominant Pkd1 RC/RC mice purinergic signaling undergoes similar remodeling. The most abundant ionotropic receptors with reduced expression were P2ry2 -2.25; P2ry4 -1.37, log2 fold change, whereas ionotropic receptors P2rx5 and P2rx7 increased expression (2.88 and 1.53, log2 fold change, respectively). Additionally, analysis detected elevated abundance of P2ry6 and P2ry12 RNA level. We used a model of cystogenesis to test how P2X signaling affects growth of cysts formed by mpkCCDcl4 cells in Matrigel. Stimulation of P2X receptors with α,βMe-ATP significantly increases cyst growth. We hypothesize that the physiological significance of the predominant P2X signaling in the cysts include their role in regulation of ATP release via pannexin-1 channels. Abnormal ATP accumulation in the cyst space was shown earlier to contribute in cystogenesis and we previously showed that pannexin-1 mediates ATP release to the cyst lumen. Co-immunoprecipitatation experiments in mpkCCDcl4 cells revealed that P2X5 and P2X7 receptors bind pannexin-1 whereas P2Y6 and P2Y12 do not interact with pannexin-1. Interestingly, stimulation of cystogenesis with α,βMe-ATP was accompanied by increased abundance of pannexin-1. Lack of functional P2X5 in humans prioritizes P2X7 receptors as a therapeutic target in ADPKD. We conclude that development of ADPKD cysts involves massive transcriptome remodeling of collecting ducts which include a shift in purinergic signaling that facilitates pathogenic pannexin-1 hyperactivity. DK123266 and DK131114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Physiological and Pathogenesis Significance of Chorein in Health and Disease

This comprehensive review explores the physiological and pathophysiological significance of VPS13A, a protein encoded by the VPS13A gene. The VPS13A gene is associated with Chorea-acanthocytosis (ChAc), a rare hereditary neurodegenerative disorder. The review covers essential aspects, beginning with the genetics of VPS13A, highlighting its role in the pathogenesis of ChAc, and addressing the spectrum of genetic variants involved. It delves into the structure and function of the VPS13A protein, emphasizing its presence in various tissues and its potential involvement in protein trafficking and lipid homeostasis. Molecular functions of VPS13A in the brain tissue and other cell types or tissues with respect to their role in cytoskeletal regulation and autophagy are explored. Finally, it explores the intriguing link between VPS13A mutations, lipid imbalances, and neurodegeneration, shedding light on future research directions. Overall, this review serves as a comprehensive resource for understanding the pivotal role of VPS13A in health and disease, particularly in the context of ChAc. Key words: Chorein • Tumor • Actin • Microfilament • Gene expression • Chorea-acanthocytosis

Mechanisms and physiological relevance of acid-base exchange in functional units of the kidney.

This review discusses the importance of homeostasis with a particular emphasis on the acid-base (AB) balance, a crucial aspect of pH regulation in living systems. Two primary organ systems correct deviations from the standard pH balance: the respiratory system via gas exchange and the kidneys via proton/bicarbonate secretion and reabsorption. Focusing on kidney functions, we describe the complexity of renal architecture and its challenges for experimental research. We address specific roles of different nephron segments (the proximal convoluted tubule, the loop of Henle and the distal convoluted tubule) in pH homeostasis, while explaining the physiological significance of ion exchange processes maintained by the kidneys, particularly the role of bicarbonate ions (HCO3-) as an essential buffer system of the body. The review will be of interest to researchers in the fields of physiology, biochemistry and molecular biology, which builds a strong foundation and critically evaluates existing studies. Our review helps identify the gaps of knowledge by thoroughly understanding the existing literature related to kidney acid-base homeostasis.