bluuebunny/crossref_metadata_2025 huggingface dataset:
Individuals with Acute Spinal Cord Injury Display Impaired Biomarkers of Cardiometabolic Health
Authors: Ely Matthew R. , Middelbeek Roeland J. , Goodyear Laurie J. , Taylor J. Andrew
Published: May 2022

Individuals with spinal cord injuries (SCI) have a greater incidence of cardiovascular disease (CVD) than the non‐injured. Contributing to the increased CVD is decreased cardiometabolic health including increased insulin resistance, central adiposity, and dyslipidemia. In fact, cross‐sectional analyses of individuals with chronic SCI (up to 50 years post injury) indicates that up to 70% are classified as overweight/obese and 25‐60% have either insulin resistance or dyslipidemia. However, current understanding of cardiometabolic health in SCI derives from those with chronic injury, hence it is unknown if biomarkers are adversely affected in those with more acute injuries. Therefore, we assessed cardiometabolic health in individuals with SCI <3 yrs. Cardiometabolic health was assessed through fasting blood glucose, insulin, triglycerides, low density lipoprotein (LDL), high density lipoprotein (HDL), total cholesterol, C‐reactive protein (CRP), and hemoglobin A1C (HbA1C). In addition, oral glucose tolerance testing (OGTT) was performed, and body composition was estimated via dual energy x‐ray absorptiometry (DXA). Five individuals (1F, 4M) aged 20 to 34 yo with SCI at neurologic level of injury ranging from spinal cord segments C6 to T4 (ASIA A and B) at 9 to 27 months post‐injury participated. All subjects displayed at ≥1 marker of impaired cardiometabolic health, while one displayed five markers. Body fat ranged from 27 to 42%, and one subject was classified as obese, two as overweight, and two as normal weigh based on BMI. Dyslipidemia was prominent in four of five subjects: one had elevated total cholesterol (>200 mg/dL), one had elevated triglycerides (>150 mg/dL), three had low HDL (<40 mg/dL), and two had elevated LDL (>100 mg/dL). Glucose tolerance test data and fasting insulin measurements showed normal fasting glucose (<100 mg/dl), HOMA‐IR (<2.0), and HbA1C (<5.7%) in all individuals, but one subject displayed impaired glucose tolerance during the OGTT (1 hr post >200 mg/dL, 2hr post >180 mg/dL). Three of five subjects had CRP levels >3.0, suggesting an increased inflammatory state. These preliminary results strongly suggest that cardiometabolic health may be impaired within 3 years after SCI. These biomarkers of altered cardiometabolic health, specifically related to lipid dysregulation, may reflect adipose tissue dysfunction, possibly in response to the accumulation of adipose tissue and/or the decreased physical activity, increased sedentary activity, and skeletal muscle atrophy that typically occurs following a spinal injury. More research is needed to determine the mediators of impaired cardiometabolic health in subjects with SCI and determine effective strategies to mitigate the detrimental effects of SCI on cardiometabolic health.
Synthesis of the transmembrane domain of the spike protein from SARS‐CoV‐2 using solid phase peptide synthesis and determination of its oligomerization state
Authors: Grimes Loyal T. , Reichart Timothy M.
Published: May 2022

As of today, nearly six hundred thousand lives have been lost to the disease COVID‐19 in the United States alone. This highly contagious disease has a proposed R0 value of 5.7, meaning that for every person infected, they transmit the virus to about 5 other people. This has caused researchers to expeditiously study Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Due to the state of the current pandemic, the distribution of effective vaccination and antiviral medication is paramount to global health. The Spike (S) protein in SARS‐CoV‐2 is largely responsible for the reproduction of this virus. The S protein consists of a receptor binding domain (RBD), which the virus uses to bind to angiotensin‐converting enzyme 2 (ACE2) receptors of host cells (REF) as well as a stem like structure binding to the membrane. The fusion of the viral and cell membranes allow for the release of the viral genome. It is known that the Spike protein has a trimerized oligomerization state, which is believed to be necessary to its function. The goal of this project is then to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the S protein in SARS‐CoV‐2. The native sequence of this peptide is the following:

WYIWLGFIAGLIAIVMVTIML

In order to determine the oligomerization state of the TM Domain of the S protein, that being how many times the peptide may repeat or come together with identical units of itself. By conducting a fluorescence‐based oligomerization assay this may be determined. This method relies upon having the native sequence of the domain separate from the entire protein to determine the specific effect of oligomerization state on the TM domain. The most practical method to synthesize the TM domain of the S protein is through Solid Phase Peptide Synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will additionally need to be purified and quantified through high‐performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography‐mass spectrometry (LCMS) to determine the prevalence and mass of the synthesized peptides. If the masses correlate to the same masses found in the S protein of SARS‐CoV‐2, then the correct peptides will have been synthesized correctly. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS‐CoV‐211‐13. The function of the S protein is believed to only present when the peptides from trimers. Therefore, the analysis of their oligomerization states, that is how well trimerization can occur, will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and TAMRA in the fluorescence assay.
Membrane phosphoinositides and renal epithelial cell polarity determination in the Xenopus pronephros in vivo
Authors: Schena Giorgia , Rajendran Vanathy , Khokha Mustafa , Caplan Michael
Published: May 2022

Background

Though they are only minor components of cell membranes, phosphoinositide lipids (PIs) participate in numerous signaling processes and in membrane identity determination. Many studies of cultured MDCK renal epithelial cells found that PIs are distributed with polarity among plasma membrane (PM) domains, and that their polarized distributions are required for the delivery of distinct populations of apical and basolateral membrane proteins. The extent to which PIs drive these processes in actual renal epithelial cells in vivo has never been examined. We investigate the distribution of PIs in vivo in the pronephros of Xenopus laevis tadpoles using MCherry‐tagged Pleckstrin Homology (PH) domains that selectively bind different PIs (PH‐AKT, PH‐PLCD1, which bind to (PI(3,4,5,)P3 and PI(4,5)P2 respectively) with the goal of assessing whether and how PI localizations affect cell polarity determination and the trafficking of proteins to their sites of ultimate functional residence in renal epithelial cells in situ.

Methods

mRNA encoding MCherry‐PH‐AKT or MCherry‐PH‐PLCD1 was injected into fertilized oocytes and their distributions were assessed in the developed pronephros at stage NF45 via fluorescence microscopy. Knockdown (KD) of PTEN, a lipid 3’ phosphatase that regulates membrane PI composition, was achieved via injection of targeted morpholinos and confirmed by western blotting. The effects of PTEN KD on MCherry‐PH‐AKT distribution were assessed by fluorescence microscopy.

Results

In MDCK cells PH‐AKT and PH‐PLCD1 localize to the basolateral and apical PMs, respectively. Their distributions are quite different in the pronephros, with both sensors localizing apically in the proximal portion of the tubule and basolaterally in the distal part. Interestingly, PTEN localization, at least in proximal tubules, is apical and not different than previously reported in cell models. Finally, PH‐AKT staining dramatically re‐distributes to the lateral domain of renal cells upon PTEN KD but this treatment does not alter the localization of protein markers of epithelial PM polarity.

Conclusions

These studies constitute the first effort to assess the role of PIs in establishing PM polarity in renal epithelial cells in vivo. Our data reveal important differences from observations previously made with in vitro systems. These findings highlight the need to explore the processes that produce renal epithelial cell polarity in vivo and in the context of intact renal tubules.
Postnatal age influences how Intermittent Hypoxia impacts hippocampal synaptic plasticity and NMDA receptor subunit expression
Authors: Arias‐Cavieres Alejandra , Garcia Alfredo
Published: May 2022

The ability to scale synaptic strength supports the robustness and flexibility of learning and memory throughout a lifetime. Intermittent Hypoxia (IH) is a hallmark of several clinical conditions such as sleep apnea and apneas of prematurity. These conditions are associated with neurocognitive deficits. We recently demonstrated that IH phenocopies aging by causing oxidative stress and suppressing NMDAr‐dependent synaptic plasticity. However, the basis of these IH‐dependent phenomena appears to be driven through a mechanism independent of aging where IH causes downregulation of GluN1, the obligatory subunit of NMDAr. Based on these findings, we sought do determine how IH exposure during early postnatal life impacts hippocampal neurophysiology. Electrophysiological studies and protein analyses were performed using hippocampal brain slices taken from mice (P14‐P15) exposed to ten days of neonatal IH (nIH, starting at P4‐P5). In contrast to the IH‐mediated effects in the adult hippocampus, nIH neither suppressed GluN1 expression nor eliminated NMDAr‐dependent synaptic plasticity, but rather caused NMDAr subunit remodeling by causing GluN2A downregulation and GluN2B upregulation. Moreover, the preserved synaptic plasticity after nIH was maintained was dependent on a larger contribution of GluN2B. These findings indicate that postnatal age is a significant factor influencing the mechanistic consequences of IH on hippocampal neurophysiology.
Neucode Tags for Highly Multiplexed Metabolomics
Authors: Grady Scott , Armbruster Michael , Edwards James , Arnatt Christopher
Published: May 2022

We describe the design and synthesis for neutron encoded (NeuCode) tags for highly multiplexed targeted sample analysis for use in high resolution mass spectrometry (HRMS) based metabolomics. A set of aldehyde and carboxylic acid reactive tags were synthesized with a quaternary ammonium polar head group which limits off‐target ionization and promotes coelution of differentially tagged metabolites. A four carbon linker allowed us to synthesize highly multiplexed NeuCode tags by providing many areas for isotope substitution and the obtained amine tags can be easily converted to other functional groups to target a wide array of metabolites. Tags were used to analyze levels of targeted metabolites in differentially treated cell lysates producing tagged samples which were mixed 1:1 and run in a single LC‐MS injection.
Size Characterization of Graphene‐coated Silver Nanoparticles for Drug Delivery System Design
Authors: Martinez Guadalupe Anna C. , Weiner Brad , Morell Gerardo , Medina Nataniel , Pantoja Wenndy , Lavin Alexis
Published: May 2022

Nanoparticles prove to be a promising tool for drug delivery due to their modifiable properties, such as size, which may offer more targeted delivery. Silver nanoparticles (AgNPs) have been shown to exert anticancer effects such as antiangiogenesis. Size control is crucial for the use of AgNPs as drug delivery systems because smaller AgNPs amplify cytotoxicity in both cancerous and healthy cells. Another way that AgNPs are cytotoxic is through the release of silver (Ag+) ions. It has been shown that AgNPs below 10 nm in size have a comparable toxicity to Ag+ ions. In this study, AgNPs were coated with graphene quantum dots. Graphene quantum dots (GQDs) are nanosheets of carbon. These nanosheets of carbon may control the release of silver ions and provide additional benefits such as increased surface area for drug loading and increased drug solubility. The objective of this study is to synthesize graphene quantum dot‐coated silver nanoparticles (AgGQDs) to serve as drug delivery systems. We hypothesize that our methods will produce AgGQDs larger than 10 nm to avoid excessive cytotoxicity. The GQDs were synthesized by pyrolysis of citric acid and used as reducing agents for the synthesis of AgGQDs. The GQDs were characterized using X‐ray Photoelectron Spectroscopy (XPS). The AgGQDs were characterized via Transmission Electron Microscopy (TEM) and in situ Energy‐dispersive X‐ray Spectroscopy (EDS). EDS confirmed that the images observed were silver nanoparticles. TEM images showed that the AgGQDs were spherical and had a size of approximately 20‐25 nm in diameter. Based on these results, we can conclude that our AgGQDs will not be drastically cytotoxic due to their intermediate size.
KSHV Manipulation of Antigen Presentation via the Ubiquitin Proteasome System
Authors: Causey Amerria M. , Ehrlich Elana S.
Published: May 2022

Kaposi Sarcoma‐associated herpesvirus (KSHV, HHV‐8) is a human oncovirus with a two‐stage life cycle consisting of latent and lytic replication. Lytic replication is characterized by the expression of late genes, the assembly of viral particles, and is initiated by the expression of RTA. In addition to being a transcription factor, RTA is an E3 ubiquitin ligase with the ability to target proteins for degradation via the ubiquitin‐proteasome system (UPS). Previously in our lab, through a comparative proteomics study, we identified proteins that displayed increased ubiquitination in the presence of RTA. Three proteins identified are known to be involved in the process of antigen presentation. We hypothesize KSHV RTA is targeting these proteins to maintain a persistent infection. Here we provide preliminary evidence that suggests that RTA targets the process of antigen processing and presentation. suggesting a novel mechanism for evasion of the immune response by KSHV RTA. We demonstrate degradation of HLA, PSMB3, and TAP2 cells expressing RTA. Cells expressing RTA exhibited decreased peptide loading on HLA. Our data suggests a novel mechanism for evasion of the host immune response by KSHV RTA.
B‐Raf: How One Mutation Leads to Melanoma
Authors: Zhao Hongru , Choe Jana , Leng Yunzhao , Alex Katherine
Published: May 2022

The MAPK/ERK signaling pathway is a kinase cascade that promotes cell proliferation. The signaling cascade starts with the binding of epidermal growth factor receptors and progresses to the cell nucleus, resulting in cell growth and division. B‐Raf is a signal transduction protein that is crucial in the MAPK/ERK signaling pathway. After interacting with GTP‐bound RAS proteins, B‐Raf phosphorylates the proteins MEK1 and MEK2, activating molecules that lead to cell proliferation. A mutation in B‐Raf contributes significantly to many types of cancers, especially melanoma. Most notably, a single nucleotide mutation resulting in a codon switch, V600E, prompts constitutive activity of B‐Raf, which leads to uncontrolled cell division. V600E is the major cause of B‐Raf mutation‐induced melanoma. Medications like vemurafenib and sorafenib can inhibit the hyperactivity of mutated B‐Raf and treat metastatic B‐Raf‐mutated melanomas. The Governor’s Academy MSOE Center for BioMolecular Modeling MAPS team used 3‐D modeling and printing technology to understand how the structures of B‐Raf contribute to the phosphorylation of MEK1 and MEK2 and to study the mechanisms of B‐Raf inhibitors. Our model highlights features including: the N terminus, the binding site of 14‐3‐3 protein; the catalytic C terminal region, featuring the kinase domain; and residue 600.
Exercise Preconditioning Alters Markers of Cellular Senescence and Prevents DOX‐Induced Cardiorespiratory Dysfunction
Authors: Doerr Vivian , Montalvo Ryan N. , Nguyen Branden L. , Boeno Franccesco P. , Ozdemir Mustafa , Bomkamp Matthew P. , Smuder Ashley J.
Published: May 2022

Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however its clinical use is limited due to cardiorespiratory complications, including the development of dyspnea, exercise intolerance, and cardiomyopathy. While the mechanisms responsible for DOX‐induced cardiorespiratory dysfunction remain unclear, evidence indicates that DOX administration causes DNA damage and promotes oxidative stress‐induced cellular senescence. Factors shown to contribute to DOX‐induced cellular senescence include cell cycle arrest, driven by signaling through the p16INK4a/Rb and p21/p53 pathways, impaired mitochondrial function, upregulation of pro‐survival pathways, increased lysosomal mass, and expression of the proinflammatory senescence‐associated secretory phenotype (SASP). In contrast, exercise has been shown to prevent cellular senescence in several tissues, leading to the hypothesis that endurance exercise preconditioning is sufficient to prevent senescence induced in the heart and diaphragm muscle following DOX chemotherapy treatment. To determine the relationship between DOX‐induced cardiorespiratory impairment, exercise preconditioning and the impact of accelerated cellular senescence, female Sprague‐Dawley rats underwent two weeks of endurance exercise training (treadmill running, 5 days/week, 60 min/day, 30m/min, 0% grade) before receiving a bolus dose of DOX (20mg/kg IP) or saline treatment. Functional measures were evaluated 48 hours after DOX or saline administration, followed by tissue collection for subsequent analysis of cellular senescence markers. Our results revealed that exercise preconditioning prior to DOX administration prevented diastolic and systolic dysfunction in the heart, with marked improvements in fractional shortening, myocardial performance index, and posterior wall shortening velocity compared to sedentary DOX‐treated animals. DOX‐induced weight loss, reduction in grip strength, and diaphragm weakness were partially rescued with exercise as well, with diaphragm specific force production preserved at frequencies of 60‐160Hz. Finally, both exercise and DOX appear to alter expression of proteins associated with the senescent phenotype in the heart and diaphragm. Therefore, the protective effects of exercise against DOX cardiorespiratory dysfunction may be related to changes in muscle cellular senescence.
Cervical Spinal Cord Injury Induces Distinct Changes in Gut Microbiome Composition Over Time in Rats
Authors: Newton Jessica , Britsch Daimen , Strattan Lydia , Taylor Chase , Calulot Chris , Alilain Warren
Published: May 2022

Nearly 60% of all spinal cord injuries (SCI) occur at the cervical level. These high‐level injuries can lead to quadriplegia, autonomic dysregulation, and interruption of the descending respiratory pathways required for breathing. As respiratory motor function is required for life, it is critical to restore breathing as soon as possible after cervical SCI. Indeed, therapeutic techniques in animal studies have been successful at restoring breathing function after SCI; however, these interventions appear to be more effective at chronic than acute timepoints post‐injury. One potential cause for this observation is the impact the injury has on the gastrointestinal (GI) tract and gut microbiome, which have previously been shown to have detrimental effects on recovery outcomes, including lower limb motor function and emotional affect as indicated by increased anxiety‐like behavior. However, the impact the gut microbiome has on the recovering spinal cord at different timepoints, levels, and injury severities still needs to be fully realized. We aimed to build upon these previous findings and investigate the impact of cervical SCI on the gut microbiome over time and up to chronic timepoints. We hypothesized that cervical SCI leads to transient changes in the gut microbiome, which are most severe acutely after injury impeding functional recovery of breathing, but resolve over time and thus eventually allow for more profound recovery. To test our hypotheses, we performed left C2 hemisections on adult female rats, collected fecal samples from injured, sham, and naïve animals, and assessed microbiome composition at various timepoints pre‐ and post‐injury. Preliminary results suggest that following cervical SCI (up to 12 weeks post injury), robust differences in gut microbiome are apparent compared to non‐injured animals. Future studies will classify bacterial identities and assess the impact of the post‐injury gut microbiome on respiratory motor function and plasticity, as well as inter‐institutional differences.
Carbon Fiber Multielectrode Arrays for Multiplexing Neurotransmitter Detection in Heterogeneous Brain Regions
Authors: Zestos Alexander G. , Rafi Harmain , Ardabili Negar G.
Published: May 2022

FSCV, or fast scan cyclic voltammetry, is an electrochemical technique that measures the oxidation and reduction of various molecules and proteins. Analytes are oxidized/reduced at specific voltages which serve as a “molecular fingerprint” for detection. Carbon fiber microelectrodes (CFMEs) have been extensively used to measure neurotransmitters with FSCV due to their ability to adsorb cationic monoamine neurotransmitters such as dopamine and serotonin, which are increased extracellularly be cocaine and amphetamine. Although this method provides high temporal and spatial resolution, only single channel potentiostats and electrodes have been primarily utilized. More recently, the need and use of carbon fiber multielectrode arrays has risen to target multiple brain regions simultaneously. Previously, we characterized a novel carbon fiber multielectrode array (MEA), a 4‐channel electrode, and found it comparable to the single channel CFME in sensitivity and selectivity. The MEA, along with a commercial potentiostat, had the additional capability of multiplexing neurotransmitter measurements ­in‐vitro by multi‐waveform application. We utilized the multielectrode array and four‐channel potentiostat to measure potassium‐stimulated release in the caudate putamen in coronal mouse brain slices ex‐vivo­­. This method yielded a sensitivity of 3.33 nA/µM with concentrations detected as low as 100 nM. In the present study we used the MEA to detect neurotransmitters in the brain using electrical stimulation and potassium‐chloride induced neurotransmitter release. We mapped out endogenous levels of neurochemicals such as dopamine, serotonin, and adenosine in regions including the ventral tegmental area, caudate putamen, raphe nuclei, and substantia nigra. This was accomplished by targeting the areas using mouse brain atlas coordinates and inserting the electrode into different brain slices. Successful sampling of neurotransmitter concentrations will aide in understanding complex brain heterogeneity, the dynamic neurochemical environment, complex behaviors, and how disease states or drugs affect separate brain areas concurrently. Specifically, we will use this method to measure the effects of psychostimulants such as cocaine and amphetamine on extracellular levels dopamine and other monoamine neurotransmitters.
Aquaporin 1 mediates microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary hypertension
Authors: Yun Xin , Philip Nicolas M. , Jiang Haiyang , Smith Zion , Huetsch John , Mahendra Damarla , Suresh Karthik , Shimoda Larissa
Published: May 2022

Pulmonary arterial hypertension (PAH) is a devastating disease, with increased pulmonary arterial pressure leading to right heart failure. The mechanisms underlying PAH are unclear, but increased pulmonary arterial smooth muscle (PASMC) and microvascular endothelial cell (MVEC) migration and proliferation leads to vaso‐occlusive lesions and vascular remodeling observed in PAH. Aquaporins regulate water transport and are expressed in many cells. We previously identified that hypoxia upregulates the expression of aquaporin 1 (AQP1) in PASMC and elevated AQP1 was required for hypoxia‐induced migration and proliferation. In this study, we wanted to elucidate whether the same is true in MVECs, and used the SU5416 plus hypoxia (SuHx) rat model, a robust model of PAH, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in MVECs and contributed to a hyperproliferative/hypermigratory phenotype.

Rats were injected with the vascular endothelial growth factor receptor inhibitor, SU5416, combined with hypoxia (3 wk at 10% O2) followed by 2 wk return to normoxia. Using MVECs isolated from the normoxia (N‐MVEC) or SuHx (SuHx‐MVEC) rats, we found that AQP1 protein levels were increased in SuHx‐MVECs and associated with upregulation of AQP1 mRNA levels, even in the absence of sustained hypoxia exposure. Silencing of AQP1 reduced migration and proliferation observed in SuHx‐MVEC, whereas augmenting AQP1 levels in N‐MVECs using forced expression with an adenoviral construct containing wild‐type AQP1 (AdAQP1) was sufficient to increase cell migration (transwell assay) and proliferation (BrdU incorporation) compared to control cells infected with green fluorescent protein (AdGFP). Since we formerly reported the C‐terminal tail of AQP1 contains putative calcium and protein biding sites and is required for enhanced PASMC migration and proliferation, we tested the role of the AQP1 C‐terminal tail in N‐MVEC. Cells were treated with AdGFP, AdAQP1 or adenovirus containing AQP1 lacking the entire cytoplasmic C‐terminal tail (AdAQP1CT). In contrast to the increase in migration and proliferation induced by AdAQP1, infection with AdAQP1CT did not alter N‐MVEC migration and proliferation.

Our results suggest a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in microvascular endothelial cells from a rodent model that reflects many of the features of human PAH. Interestingly, the AQP1 C‐terminal tail is required for regulating MVEC function, similar to our previous findings in PASMC.
Pathways of prevention and treatment of ischemic‐reperfusion injury as consequence of myocardial infarction
Authors: Abzalilov Timer A. , Samorodov Aleksandr V. , Nurlanova Sabina N.
Published: May 2022

Myocardial infarction and the phenomenon of ischemic‐reperfusion injury (IRI) which happens as adverse unwanted effect of the restoration of blood flow in the affected area in the post infarction period is one of the main causes of patient’s death all over the world, what makes the prevention and treatment of this condition a frequent issue for clinicians. For treatment patient undergoes mechanical reperfusion of the affected area of the heart and often uses potent antithrombotic agents. Ischemic‐reperfusion injury can happen consequently because of cellular mechanisms (like calcium overload in cardiomyocytes, excessive generation of reactive oxygen species, mitochondrial damage and others). It was discovered that cardiomyocytes can resist ischemic‐reperfusion injury because of specific mechanisms inherent to these cells; ability was named ‘cardio protection’. For future use for prevention and treatment of this health issue, research has been carried out on experimental heart conditioning and pre‐conditioning (it has low possibility of application in clinical practice), new pharmacological preparations are being searched for and their effectiveness is being verified in terms of their ability to induce a cardio protective effect.
The Impact of Aging on the Association between Aortic Stiffness and Cerebral Pulsatility Index
Authors: Fico Brandon G. , Miller Kathleen B. , Rivera‐Rivera Leonardo A. , Corkery Adam T. , Pearson Andrew G. , Eisenmann Nicole A. , Howery Anna J. , Rowley Howard A. , Johnson Kevin M. , Johnson Sterling C. , Wieben Oliver , Barnes Jill N.
Published: May 2022

The central arteries dampen the pulsatile forces from myocardial contraction, limiting the pulsatility that reaches the cerebral vasculature, although there are limited data on this relationship with aging in humans. The purpose of this study was to determine the association between aortic stiffness and cerebral artery pulsatility index in young and older adults. We hypothesized that cerebral pulsatility index would be associated with aortic stiffness in older adults, but not in young adults. We also hypothesized that both age and aortic stiffness would be significant predictors for cerebral pulsatility index. This study included 23 healthy older adults (aged 62 ± 6 years) and 33 healthy young adults (aged 25 ± 4 years). Aortic stiffness was measured using carotid‐femoral pulse wave velocity (cfPWV), while cerebral artery pulsatility index in the internal carotid arteries (ICAs), middle cerebral arteries (MCAs), and basilar artery were assessed using 4D Flow MRI. Cerebral pulsatility index was calculated as (maximum flow – minimum flow) / mean flow. In the combined age group, there was a positive association between cfPWV and cerebral pulsatility index in the ICAs (r = .487; p < 0.001), MCAs (r = .393; p = 0.003), and basilar artery (r = .576; p < 0.001). In young adults, there were no associations between cfPWV and cerebral pulsatility index in any of the arteries of interest (ICAs: r = .253; p = 0.156, MCAs: r = ‐.059; p = 0.743, basilar artery r = .171; p = 0.344). In contrast, in older adults there was a positive association between cfPWV and cerebral pulsatility index in the MCAs (r = .437; p = 0.037) and basilar artery (r = .500; p = 0.015). However, the relationship between cfPWV and cerebral pulsatility index in the ICAs of the older adults did not reach the threshold for significance (r = .375; p = 0.078). In conclusion, age and aortic stiffness are significant predictors of cerebral artery pulsatility index in healthy adults. This study highlights the importance of targeting aortic stiffness in our increasingly aging population to reduce the burden of age‐related changes in cerebral hemodynamics.
Single‐Cell Sequencing Reveals Distinct Expression Patterns of the Integrated Stress Response (ISR) Pathway Regulating Cell Survival in Secretory Cells in Colon of Hnrnp I Knockout Mice
Authors: Xu Guanying B. , Pan Yuan‐Xiang , Mei Wenyan , Chen Hong
Published: May 2022

Objectives

Heterogeneous nuclear ribonucleoprotein I (Hnrnp I) is an RNA‐binding protein functions during pre‐mRNA splicing and regulation of alternative splicing events. Previously we found that ablation of Hnrnp I activates NF‐κB signaling in intestinal epithelial cells (IECs), resulting in increased nuclear translocation of P65 (NF‐κB subunit) and upregulation of proinflammatory cytokines and chemokines. Integrated stress response (ISR) signaling was linked to activation of canonical NF‐κB pathway. Therefore, in this study, we aimed to investigate ISR in the colon of Hnrnp I knockout mice. We hypothesize that cellular stress from Hnrnp I knockout triggers ISR, consequently affects the cellular dynamics among secretory cell types on colon epithelium (enteroendocrine, Paneth and goblet cells).

Methods

Total RNA was isolated from total colon cell preparations from WT and KO mice. Single‐cell RNAseq libraries (biological replicates, n=3) were prepared using Chromium Single‐Cell Gene Expression NextGem (v3.1, 10X Genomics). Analyses from raw counts were performed using the Seurat (v3.2.0) R package using default parameters, a computational cell calling algorithm was used to identify cell type using two published annotated mouse cell datasets. Pathway analysis was performed for secretary cell clusters including enteroendocrine cells (EECs), Paneth cells, and goblet cells

Results

Comparing to WT, number of EECs in colon was significantly increased in KO mice. Total mRNA level of Atf4, the principal regulator of ISR, was significantly increased in colon secretory cells in KO mice. We also examined mRNA expression of ATF4 target genes in secretory cells. Specifically, gene encoding the regulator of apoptosis and autophagy, members of Bcl2 family, Bnip3, was significantly increased in secretory cells in KO mice, while Mlx and Gabarapl2 (Atg8), both are involved in apoptosis and autophagy signaling pathways, have a trend of increase in secretory cells in KO mice, all comparing to WT.

Conclusions

Cellular stress from Hnrnp I knockout activated ISR in IECs, specially in those secretory cell types – EECs, Paneth, and goblet cells. The activation of ISR led to both autophagy and apoptosis signaling in these cells through the upregulation of Bnip3, Mlxand Gabarapl2. Taken together, our results suggest that in response to the cellular stresses from the depletion of Hnrnp I in IECs, ISR activation in colonic secretory cells, stimulating apoptosis and autophagy pathway signaling, particularly in EECs, resulting in the proliferation of EECs in KO. The overall manipulation by ISR signaling in the colon secretary cells may present a critical early response to modulations in colon, potentially leading to perturbations to homeostasis among host colon epithelia, colon residential immunity, and gut microbiota.
A Cell Permeant Phosphopeptide Mimetic Of Niban Restores Endothelial Function And Inhibits p38 MAPK Activation After IL‐1β Induced Vascular Injury
Authors: Katta Anish , Sisung Amanda , Morelli Madeleine , Brophy Colleen , Cheung‐Flynn Joyce
Published: May 2022

Activation and dysfunction of the vascular endothelium play a significant role in the pathogenesis of many viral infections such as COVID‐19. The cytokine storm (systemic inflammation) present in COVID‐19 causes organ failure by inflaming the vascular endothelium, leading to endothelial dysfunction and eventually death. Previous studies in the laboratory show that vascular injury leads to endothelial dysfunction that is associated with activation of p38 MAPK signaling and altered phosphorylation of Niban. Based on these findings, a cell permeant phosphomimetic peptide of Niban (NiPp) was developed and shows inhibitory activities on p38 MAPK and restores endothelial dysfunction after vascular injury. The effects of NiPp on endothelial dysfunction induced by interleukin‐1β (IL‐1β), a cytokine that is increased during infections, were examined ex‐vivo using intact rat aortic tissue (RA) and primary human endothelial cells (HCAEC). IL‐1β treatment led to a reduction in endothelial‐dependent relaxation (EDR) in RA, and inhibition of p38 MAPK by NiPp or BIRB796, a potent p38 MAPK inhibitor, improved EDR in IL‐1β treated RA (Figure 1). Treatment of HCAEC with NiPp or BIRB reduced IL‐1β induced activation of p38 MAPK and its downstream substrate, CREB (Figure 2). These data show that p38 MAPK and Niban signaling play a role in endothelial function, especially in response to inflammatory injury. Niban may be an endogenous regulator of p38 MAPK activation. NiPp may serve as a potential therapeutic to ameliorate inflammation of vascular endothelium.
Changes in PVN Neurons after Low‐Frequency Acute Optogenetic Stimulation
Authors: Paundralingga Obed , Jia Shuping , Cunningham J. T.
Published: May 2022

The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center. It receives afferent inputs from many brain regions, including the median preoptic nucleus (MnPO). The connection between the MnPO and the PVN is particularly important in generating chronic intermittent hypoxia phenotypes such as increased sympathetic activity leading to the development of hypertension. PVN‐projecting MnPO neurons discharge frequency is <5Hz. To gain more insight into the acute change in PVN neuron properties after low‐frequency stimulation, whole‐cell patch‐clamp recordings were performed on PVN neurons. Adult 250‐350g male rats were injected bilaterally in the PVN with 100 nL retrogradely transported adeno‐associated virus encoding Channelrhodopsin (AAVrg‐CaMKIIa‐hChR2(H134R)‐mCherry) or with AAVrg‐CAG‐tdTomato as control. Three weeks after the injections, the rats were anesthetized with isoflurane (2‐3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. Postsynaptic currents were recorded from PVN neurons in whole‐cell voltage clamp configuration (Vhold=‐60 mV) under constant bath perfusion of normal aCSF (2‐3 ml/min). Axon terminals in the PVN were stimulated with 5Hz LED‐generated blue light (470nM) pulses of 50‐ms duration, 5 seconds off/on for a total of 1 min, delivered using optical fiber directed to the MnPO. Photo‐evoked currents were measured during 5 min before and after the photo‐stimulation train. Amplitude and frequency of spontaneous currents were compared before and after stimulation as well. At the end of each recording, cells were characterized as type I (magnocellular) or type II (parvocellular) PVN neurons based on the presence of transient outward rectification. Data were analyzed offline using Easy Electrophysiology v2.3.3 software. For both cell types, 5 Hz optogenetic stimulation caused a significant reduction in the optogenetic‐evoked current amplitude during the first 15 minutes after stimulation (before stimulation 94.88±2.85 %, n=9 cells vs. 15 minutes after stimulation 67.52±6.15 %, n=9. P=0.0027) before returning to baseline values. Post‐stimulation spontaneous postsynaptic current amplitude and frequency decreased in type II PVN neurons from retrograde ChR2 animals (pre‐stimulation amplitude 100±0.62 % vs. post‐stimulation amplitude 86.50±0.54 %, P=0.0371; pre‐stimulation frequency 3.82±1.7 Hz, post‐stimulation frequency 2.396±1.2 Hz, P= 0.0385) but not in type I PVN neurons from retrograde ChR2 animals or cells from control animals. Kinetic profiles of spontaneous current in type I and II PVN neurons did not change after 5 Hz optogenetic stimulation. The results suggest that low‐frequency stimulation discriminately alters spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.
Assessing Contractility of 3D iPSC‐derived Muscle Models for Safety and Discovery Using a Novel, High‐throughput, and Label‐free Instrumentation Platform
Authors: Geisse Nicholas , Berry Bonnie J. , Gray Kevin , Moerk Travis , Person Jason , Luttrell Shawn M. , Macadangdang Jesse
Published: May 2022

Stem cell models hold great promise for improving the predictive power of preclinical in vitro assays for new therapies, drug discovery, basic scientific research, and disease modeling. Complex 3D platforms, such as Engineered Muscle Tissues (EMTs) fabricated from primary or iPSC‐derived cells, can directly measure tissue contractility, which is challenging in conventional 2D platforms where cells are rigidly attached to a surface. However, traditional methods to fabricate EMTs demand extensive bioengineering expertise, and measuring contractility often involves laborious, serial, and low‐throughput optical measurements.

Here, we report on the design, fabrication, and validation of a novel 3D EMT platform that uses 1) facile and scalable bioengineering approaches to generate tissues from a variety of cell sources, and 2) a label‐free parallel measurement technique. Our tissue casting approach has a success rate of >96% (n > 100) and produces consistently‐sized constructs with a standard deviation of +/‐ 9% across 6 experiments. Tissue casting, media changes, and drug dosing are also highly amenable to automation.

The substrate features an embedded magnet; as tissues contract, the magnet’s displacement is quantitatively detected in a highly‐parallel manner using specialized sensors. We detected 24 contractions simultaneously with a measurement rate of 100Hz, which is suitable for measuring various aspects of contractility such as upstroke velocity, decay time, and fatigability. We demonstrated that the signal voltage changes are linear with respect to EMT contraction.

We will present data showing acute effects of drugs measured minutes after EMT dosing and chronic effects of structural cardiotoxicants like doxorubicin, sunitinib, and BMS‐986094 on EMTs. All chronically‐dosed tissues showed statistically significant dose‐dependent reduction in twitch frequency over a multi‐day time course (p <0.05). We will also show that our platform can be used to generate physiologically‐relevant skeletal muscle constructs and achieve tetanic responses upon stimulation.

In addition to modeling healthy tissues, our platform can also be used to study disease models. We are currently developing patient‐specific Duchenne Muscular Dystrophy models for the development of personalized gene therapies. EMTs can be made from cells sourced from patients and used to test whether a new therapy will improve or recover functional contraction.

We have designed a novel system that can leverage the complexity of 3D cellular models in a scalable format and can be tailored for specific applications, including personalized medicine or disease modeling. The Mantarray platform will provide a stand‐alone tool capable of screening significant numbers of compounds for the rapid safety evaluation of drug candidates thereby accelerating drug discovery and development.
Renal‐Tubular SGK1 is Involved in Blood Pressure Circadian Rhythm
Authors: Al‐Qusairi Lama , Debonneville Anne , Stifanelli Matteo , Basquin Denis , Staub Olivier
Published: May 2022

As many physiological functions, blood pressure (BP) follows a circadian pattern that rises during the active and decreases during the inactive phase of the day. BP rise at the beginning of active phase (known in human as the “morning surge”) coincides with the increase in circulating glucocorticoids and aldosterone. Serum‐ and glucocorticoid‐induced kinase (SGK1), a clock‐controlled and aldosterone‐induced gene, plays a role in BP regulation in human and animal models. However, a role of SGK1 in the BP circadian regulation has not been demonstrated.

Using telemetry, we analyzed BP in the inducible renal‐tubule specific Sgk1Pax8/LC1 model under basal and high‐potassium diet (HKD; 5%K+). Our data show that, under basal conditions, renal SGK1 plays no role in BP regulation. After one week of HKD and despite largely unaffected mean arterial BP (MAP), Sgk1Pax8/LC1 mice exhibit altered day/night variations. More specifically, the increase in BP during the active phase (BP surge) was blunted in both systolic and diastolic BP. After 7 weeks of HKD, more profound defect in BP was observed in the mutants as they exhibit decreased MAP, together with a dysregulated circadian rhythmicity. The BP defect was associated with abnormal NCC and ENaC expression and processing. Altogether, our data suggest that i) The kidney plays a major role in setting the BP circadian rhythm. ii) BP surge requires the renal‐tubule SGK1.
Renal chemosensitive and mechanosensitive afferent responses are preserved in TRPV1 knockout rats
Authors: DeLalio Leon , Stocker Sean
Published: May 2022

The transient receptor potential vanilloid type‐1 (TRPV1) channel is expressed in renal sensory nerves, and intrarenal administration of the TRPV1 agonist capsaicin increases renal afferent nerve activity (ARNA). Nonselective denervation of renal sensory nerves using high concentration capsaicin reduces arterial blood pressure (ABP) in experimental models of hypertension. Pharmacological blockade of TRPV1 using renal pelvic infusion of capsazepine attenuates ARNA responses to capsaicin, CGRP, and elevated pelvic pressure. Thus, TRPV1 channels mediate activation of renal sensory nerves.

To test this hypothesis, we generated a novel TRPV1 knockout rat (TRPV1‐/‐) using CRISPR/Cas9 to produce a 26bp deletion in exon 3. Multifiber ARNA responses to chemical (bradykinin and capsaicin) and mechanical (elevated renal pelvic pressure) stimuli were compared in Inactin‐anesthetized male and female wild‐type and TRPV1‐/‐ littermates (250‐400g). There were no significant differences in baseline mean ABP between wild‐type and TRPV1‐/‐ rats (122±2 mmHg vs 122±3 mmHg, respectively; p=0.48, n=8‐10), or baseline ARNA between wild‐type and TRPV1‐/‐ rats (14±3 Hz vs 18±5 Hz, respectively; p=0.24, n=8‐10).

Intrarenal artery infusion of capsaicin (0.1–10μM, 50μL per 15s) significantly increased ipsilateral ARNA in wild‐type but not TRPV1‐/‐ rats (Δ discharge 65±14 Hz vs 9±3 Hz at 10μM, respectively; p<0.05, n=7). As a second chemosensitive stimulus, intrarenal artery infusion of bradykinin (0.1–10μM, 50μL per 15s) produced significant increases in ARNA in ­both wild‐type and TRPV1‐/‐ rats that were not significantly different between groups (Δ discharge of 10μM: 52±11 Hz vs 76±21 Hz, respectively; p=0.33, n=7). Elevated renal pelvic pressure (0–20mmHg; 30s) significantly increased ARNA in both wild‐type and TRPV1‐/‐ rats; however, ARNA responses were significantly smaller in wild‐type versus TRPV1‐/‐ rats (Δ discharge with 20mmHg: 17±3 Hz versus 29±6 Hz, respectively; p=0.03, n=8‐9).

The preservation of ARNA responses in TRPV1‐/‐ rats contradicts prior studies using pharmacological TRPV1 antagonists. A final set of experiments tested whether intrapelvic capsazepine infusion (200μM, 200μl per 3 min) attenuates ARNA responses to elevated pelvic pressure (20mmHg) in both wild‐type and Trpv1‐/‐ rats. Interestingly, pelvic administration of capsazepine attenuated mechanosensitive ARNA responses in both wildtype (saline: 31±3 Hz vs capsazepine: 8±1 Hz; n=3) and TRPV1‐/‐ (saline: 20±7 Hz vs capsazepine: 6±1 Hz; n=3) rats thereby suggesting a TRPV1‐independent action of capsazepine.

In conclusion, TRPV1 channels are dispensable for the activation of chemo‐ and mechanosensitive renal sensory nerves.