bluuebunny/crossref_metadata_2025 huggingface dataset:
Heart rate variability during different phases of menstrual cycle
Authors: Saperova Elena V. , Filippova Irina
Published: May 2022

The purpose of the present study was to determine whether there is a menstrual cycle effect on heart rate variability. Ten healthy regularly cycling females (age 19‐23 years) were studied during the follicular phase (FP) and luteal phase (LP) of menstrual cycle over two month. We found significant changes in heart rate (65,35±0,68 beats/min in FP vs 73,18±0,92 beats/min in LP, p<0,0005), AMo and stress index during the menstrual cycle with a minimum in the follicular phase (36,81±1,14 % and 76,86±5,74 p.d.u. respectively) and maximum in the luteal phase (42,05±1,27 % and 107,70±7,77 p.d.u. respectively, p<0,003). The HF and LF components decreased more during the luteal phase (655,56±46,64 ms2 and 981,85±66,64 ms2 respectively) than during the follicular phase (997,42±83,36 ms2 and 1524,66±145,17 ms2 respectively, p<0,05). In the follicular phase SDNN, pNN50, MxDMn were significantly higher (0,057±0,002 s, 41,49±1,79 % and 0,333±0,012 s respectively) than in the luteal phase (0,051±0,002 s, 29,73±1,88 % and 0,303±0,012 s respectively, p<0,05). Furthermore, the VIK was higher in the luteal phase compared to the follicular phase (p=0,003). These findings indicate that sympathetic nervous activity in the luteal phase is greater than in the follicular phase, whereas parasympathetic nervous activity is predominant in the follicular phase. A difference of the balance of ovarian hormones may be responsible for these changes of autonomic functions during the menstrual cycle.
Adipose tissue expression of HuR modulates cardiac pathology via adipose tissue‐derived extracellular vesicles
Authors: Anthony Sarah R. , Guarnieri Adrienne R. , Green Lisa C. , Slone Sam , Nieman Michelle L. , Owens A. P. , Kanisicak Onur , Tranter Michael
Published: May 2022

Obesity is widely associated with increased cardiovascular disease (CVD) extending beyond common co‐morbidities such as dyslipidemia and diabetes, as evidenced by increased CVD risk in ‘metabolically healthy obese’ individuals. Adipose tissue serves a broad role as an endocrine organ and has been shown to have a multitude of effects on cardiac physiology depending on metabolic state, adipose depot location, and primary cell type. We have previously shown that adipose tissue‐specific deletion of HuR (Adipo‐HuR‐/‐) reduces BAT‐mediated adaptive thermogenesis, accompanied by the spontaneous development of cardiac hypertrophy and fibrosis in mice. We and others have also shown that expression of HuR in subcutaneous white adipose tissue (scWAT) is decreased in obesity, suggesting a potential mechanistic link between obesity, HuR expression in scWAT, and CVD.

To identify underlying mechanisms, we applied both RNA‐seq and weighted gene co‐expression network analysis (WGCNA) across both cardiac and adipose tissue (BAT, scWAT, and perigonadal WAT) to correlate adipose tissue gene expression with the phenotype of cardiac fibrosis. Our results suggest HuR‐dependent adipose tissue‐derived extracellular vesicles (Ad‐EVs) as the mediator of cardiac pathology. To provide functional evidence for the role of Ad‐EVs in driving cardiac pathology in Adipo‐HuR‐/‐ mice, we demonstrate that plasma‐isolated vesicles from Adipo‐HuR‐/‐ mice, but not wild‐type littermate controls, induced a significant increase in hypertrophic signaling in primary isolated mouse cardiomyocytes.

In conclusion, our results demonstrate that loss of HuR expression in adipose tissue, as observed in the setting of obesity, is sufficient to induce cardiac hypertrophy and fibrosis, in part through endocrine‐mediated Ad‐EV signaling.
Studying Quantitative Relationships Between the Indicators of Physical Development at Birth and the Functional State of the External Respiratory System
Authors: Filippova Irina , Saperova Elena V.
Published: May 2022

The external respiration system is studied in various areas of human physiology and ecology in order to both identify the patterns of functioning of this system and to assess the nature and extent of the influence of environmental factors on respiratory function.

Indicators of the external respiration system are considered as individual indicators of the body's adaptation to the environment. The result of the influence of unfavorable environmental factors is most pronounced in the period of intrauterine development and largely determines the functional capabilities of the organism in the later periods of ontogenesis.

The aim of our work was to study the relationship between birth weight and expiratory flow rates. 500 children aged 4 to 7 years were examined. With age, there is an increase in the volumetric expiratory flow rate, which is associated with the processes of general development and growth of the body. The results of the analysis of variance also indicate that this relationship is statistically significant. The value of the Fisher coefficient (F) is 6.73 at a significance level of 0.00021. The greatest increase in expiratory volume flow rates is observed between four and five years (0.54 l/s or 54%), between six and seven years (0.36 l/s or 36%), and the increase between five and six years is insignificant (p>0.05).

In the course of our research, the dependence of the indicators of the volumetric expiratory velocity on the floor was also established. In boys, this figure was 1.34 l/s, in girls ‐ 1.19 l/s. This difference was also significant after taking into account age (p<0.05).

To identify the nature of the influence of anthropometric parameters at birth on the value of the volumetric expiratory flow rate, a multiple regression analysis was carried out, taking into account birth weight, birth length and age. To clarify the nature of the relationship under study, partial correlation coefficients were calculated, which showed that age and birth weight have the greatest influence on expiratory PTM. Body length at birth does not have a significant effect on external respiration. The partial correlation coefficients are as follows: body weight at birth – 0.244451; age – 0.569063; body length at birth – ‐0.156432.

Since we found that there are certain differences in the expiratory flow rate between boys and girls, we performed a regression analysis for each group. Thus, we can conclude that the indicators of external respiration increase with age due to the processes of growth and general development of the body. The results of the regression analysis of the influence of anthropometric parameters on the volumetric expiratory flow rate indicate the greatest influence on PTM of age and body weight at birth, both in boys and girls.
A New Method for Undruggable Targets: Amino Acids as Fragments of Inhibitors
Authors: Castillo Abdul , Fuglestad Brian
Published: May 2022

Over the last decade, protein‐protein interactions (PPIs) have emerged as promising targets in the precise treatment of serious diseases. Many essential cellular pathways that are implicated in human diseases are controlled by intracellular PPIs. Such PPIs could be potential drug targets, and thus the ability of molecules to inhibit specific PPIs has remarkable therapeutic value. Unfortunately, PPI interfaces are generally large and present great difficulty for small molecule inhibitor design. Alternate strategies are being developed towards drug design and discovery for this type of target. For example, the synthesis of unnatural amino acids allows production of extremely large libraries of highly modified peptides, which may be screened against a PPI target. Peptide hits are a potential source of lead compounds in the search for new pharmaceutical agents and are promising for PPI inhibition. It is in this context that selection of the right unnatural amino acid becomes crucial.

For this reason, developing new strategies to solve this problem is a key factor to find more efficient and specific inhibitors to PPI’s.

In this study, we introduce a simple, Nuclear Magnetic Resonance (NMR) based methodology that screens natural and unnatural amino acids as “fragments” for development of new peptide‐based inhibitors for specific PPI’s. We validate this new method against MDM2, a well‐characterized and important negative regulator of the p53 tumor suppressor with known small peptides that inhibit its interaction with p53. A central tryptophan is the largest contributor to binding in this interface, and the corresponding MDM2 binding pocket is where we focused our efforts. Using this methodology, we screened non‐natural tryptophan derivatives, and several displayed a better binding affinity compared with the natural counterpart. Our simplified method allows priority ranking of amino acids according to their affinity to the protein. The highest‐ranking amino acids will be incorporated into peptides synthetically to confirm that improved free amino acid affinity translates to enhanced peptide binding and inhibition. In addition, this serves as a convenient and robust method to ‘pre‐screen’ amino acids for incorporation into in vitro peptide selection via mRNA display.
Effect of Bacopa Monnieri and Memantine in LPS‐induced neuronal toxicity
Authors: Thakor Archi , Benzeroual Kenza
Published: May 2022

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the extracellular accumulation of Amyloid‐β (Aβ) plaques causing neurotoxicity and leading to neurodegeneration. The present study investigated the neuroprotective effects of Memantine (MN), an FDA approved drug, and the Ayurvedic nootropic drug Bacopa Monnieri (BM) on the amyloid precursor protein (APP) which is the precursor of the Aβ isoform, and the apoptotic pathway in LPS‐induced SHSY5Y cells toxicity. Our results showed that the pretreatment with MN of LPS‐induced toxicity in SHSY5Y cells significantly increased cell viability and decreased APP expression, thus limiting further neurotoxicity. In parallel, the apoptotic pathway triggered by LPS was reduced as represented by a dose‐dependent increase in the expression of Bcl2, an anti‐apoptotic marker, and a decrease in the expression of NFKB, cytochrome C and caspase‐3. On the other hand, pretreatment with BM also protected the cells from LPS‐ induced neurotoxicity by significantly increasing cell viability and decreasing APP expression. In addition, BM also prevented apoptosis by decreasing the expression of cytochrome C and caspase 3. Overall, both MN and BM show anti‐amyloidogenic and antiapoptotic effect in neuronal cells. The present study suggests that BM can be considered as a potential adjuvant therapy for preventing or delaying the progression of neurodegeneration in AD
Chronic Unpredictable Stress impairs Relaxation Responses to Acetylcholine in Resistance Arteries from Mice
Authors: Arishe Olufunke O. , Wilczynski Stephanie , Crockett Alexia , Priviero Fernanda , Hollis Fiona , Webb Clinton
Published: May 2022

Background

Exposure to chronic stress is considered a major environmental factor that causes vascular aging, giving rise to the development of cardiovascular diseases. The chronic unpredictable stress (CUS) protocol has emerged as a translationally relevant model for studying the pathophysiology of stress exposure in rodents. The CUS protocol relies on heterotypic, unpredictable, and uncontrollable stressors that prevent adaptation and lead to disrupted stress response systems, mimicking the real‐life situation. CUS induces changes in cardiovascular function that are accompanied by the release of inflammatory cytokines. Indeed, previous studies have reported endothelial dysfunction in humans exposed to chronic stress and experimental animal models of chronic unpredictable stress. However, the mechanisms through which CUS induces cardiovascular injury are still unknown and require investigation.

Hypothesis

This study tests the hypothesis that mice exposed to CUS will have impaired endothelial function.

Methods

Vascular function using wire myographs was performed on isolated mesenteric resistance arteries (MRAs) from male and female C57BL/6 mice subjected to CUS for 28 days. The arterial rings were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration‐response curves to phenylephrine (PE; 10‐9‐3x10‐5M) were performed in some arterial rings, whereas concentration‐response curves to acetylcholine (ACh; 10‐9 – 3x10‐5M) were performed in arterial rings precontracted with phenylephrine (PE 3x10‐6M). Concentration‐response curves were analyzed using non‐linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data were analyzed with GraphPad Prism 9.2.0.

Results

In the MRA of both males and females, relaxation responses to ACh were impaired in the animals exposed to CUS when compared to their controls. There were no differences in the contractile responses to PE between controls and mice exposed to CUS.

Conclusion

Our results show that relaxation responses to acetylcholine are impaired in mesenteric resistance arteries from mice exposed to chronic unpredictable stress. This suggests that exposure to chronic unpredictable stress causes endothelial dysfunction and may have important implications for the role of stress on the development of cardiovascular diseases, particularly, hypertension.
Characterizing the Luminal Microenvironment of Human Organoids for Studies of Gastric pH Regulation
Authors: Lyon Katrina , Sidar Barkan , Burger Clarice , Sebrell Thomas A. , Wilking James , Bansil Rama , Bimczok Diane
Published: May 2022

The stomach is notorious for having the steepest proton gradient in the body, which pathogenic Helicobacter pylori must traverse to infect the epithelium. This gradient across the 200‐300 μm‐thick gastric mucus layer enables the stomach to maintain a near‐neutral pH at the epithelial surface and a strongly acidic environment in the stomach lumen. In this study, we use human gastric organoids to study the maintenance and regulation of this pH gradient. Human gastric organoids are 3D cellular constructs derived from patient biopsies that mimic the microanatomy and physiology of the stomach, as they contain both mucus and gastric acid. Organoids have been used to investigate H. pylori‐induced gastritis, peptic ulcers, and stomach cancer, which claims 700,000 lives each year. Gastric mucus is critical for protection against H. pylori infection, which is the leading cause of gastric cancer. It is presently unclear how the gastric mucosal pH gradient is maintained, and how closely organoid models can mimic this microenvironment. First, we detected mucus within human gastric organoids using histological staining and bead microrheology. We also used a FITC‐conjugated lectin to visualize the mucosal matrix inside of the organoids. These data were supported by our observation that a clear viscous material was produced on the apical side of two‐dimensional, organoid‐derived cell monolayers. Additionally, we developed a novel method for the measurement of intraluminal pH of the gastric organoids. Using a micromanipulator and stereomicroscope to maneuver a glass microelectrode into the organoid lumen, we measured and manipulated luminal pH using histamine stimulation. We determined that pH remains consistent across organoid lines, and that the intraluminal space is more acidic than the surrounding environment. In ongoing studies, we will develop the spatial resolution of our organoid microprofiling technique to fully characterize the proton gradient within the mucus‐filled lumens gastric organoids. Our studies are the first to utilize pH microsensors in these models, and will lead to improved molecular and biophysical understanding of gastric physiology and potential new treatment approaches for disease conditions (such as peptic ulcers and gastric cancer) that involve dysregulated gastric acid secretion.
Structure Insights into Biased Signaling of kappa Opioid Receptor
Authors: El Daibani Amal , Paggi Joe , Kim Kuglae , Laloudakis Yianni , Popov Petr , Bernhard Sarah , Olsen Reid H , Diberto Jeffrey , Katritch Vsevolod , Wunsch Bernhard , Dror Ron , Che Tao
Published: May 2022

κ‐opioid receptor (KOR) has been known as an attractive drug target for pain management because it offers anti‐nociception without causing constipation, euphoria, or respiratory depression that accompany traditional analgesics targeting the μ‐opioid receptor (MOR). Although negative side‐effects of KOR activation prevent the adoption of most KOR‐based analgesics such as hallucinations, dysphoria, and sedation, nalfurafine is the only clinically available KOR agonist that shows atypical properties relative to other KOR agonists such as no hallucination and no dysphoria. To better understand the molecular determinants that drive KOR’s therapeutic property, here we reported the crystal structure of KOR bound to nalfurafine and identified the structural features that contribute to nalfurafine’s high potency and unique signaling profile. Using atomic‐level molecular dynamics (MD) simulations of KOR bound to G protein‐biased nalfurafine and other differently biased ligands, we identified receptor conformations that cause nalfurafine’s favorable therapeutic profile and provide a hypothesis for the structural basis for signaling bias at KOR in general.
Benztropine Reduces Reacquisition of Alcohol Self‐Administration in Rats with Stress History: Role of FKBP5
Authors: Sakoulas Eleanna M. , Bertotto Luisa B. , Logrip Marian L. , Lin Katrina , Pimentel Anastasia E. , Cruz Bryan , Vozella Valentina , Roberto Marisa , Zorrilla Eric P.
Published: May 2022

Post‐traumatic stress disorder (PTSD), which can affect 1 in 11 adults during their lifetime, is often comorbid with chronic alcohol use problems and increases alcohol relapse risk. PTSD and alcohol‐use phenotypes are associated with variants in the FKBP propyl isomerase 5 (FKBP5) gene, which encodes FK506‐binding protein 5 (FKBP5), a chaperone modulator of glucocorticoid receptors (GR) that is functionally implicated in stress‐related psychiatric disorders and alcohol withdrawal severity. Data suggests that FKBP5 inhibition may decrease alcohol intake, but its role in the post‐traumatic recurrence of alcohol drinking is unknown. Here, we tested the hypotheses that amygdala Fkbp5 expression is increased in rats with a history of traumatic stress and is associated with greater reacquisition of ethanol self‐administration. Then, we tested the hypothesis that benztropine mesylate, an FDA‐approved drug that disrupts FKBP5‐GR binding, reduces reacquisition of ethanol self‐administration in rats with a history of traumatic stress. Separate cohorts (for mRNA vs. benztropine studies) of male (n = 43) and female (n = 20) Wistar rats received 3 sessions of light‐cued footshock stress (30 min session, 60 footshocks, 1‐s 0.4 mA) before acquiring operant ethanol (10% v/v) self‐administration (1‐h sessions, fixed ratio1‐3), followed by extinction (16 sessions), and then renewed alcohol access. Amygdala Fkbp5 expression correlated significantly with increased ethanol self‐administration (rs(23) = 0.463, p = 0.023) during renewal of ethanol access. Subchronic (3 days) benztropine pretreatment (‐2 h, i.p., 0, 5, and 10 mg/kg [n = 10, 14, and 14]) significantly reduced the reacquisition of ethanol self‐administration. On days 2 and 3 of reacquisition, self‐administration was significantly reduced in drug treated animals (Dose x Day: F(4,64)=2.83, p < 0.04), culminating in a 68% reduction in males (0.78±0.16 vs. 0.25±0.13 g/kg) and a 41% reduction in females (0.88±0.14 vs. 0.52±0.13 g/kg) at the 10 mg/kg dose. Overall, the results warrant further study of benztropine and its potential inhibition of FKBP5 to reduce post‐traumatic alcohol relapse risk.
Effects of the Rho Kinase Inhibitor Fasudil on Adrenergic Contractions of Rat Aorta and Mouse Spleen
Authors: Docherty James R. , Alsufyani Hadeel A.
Published: May 2022

G‐protein coupled receptors such as α1‐adrenergic receptors (AR) are linked via the G‐protein Gq/G11 to both Ca2+ entry and release of Ca2+ from stores, but can also activate the small GTP binding protein RhoA and Rho kinase to produce contractions involving Ca2+ sensitization. In rat portal vein, tonic but not phasic components of contractions to the α1‐AR agonist phenylephrine involve predominantly the α1B‐AR subtype, and are at least partly mediated by mechanisms involving Rho kinase sensitive to fasudil (Alsufyani & Docherty, 2021). In this study we examined the involvement of Rho Kinase in contractions involving α1‐AR in two further tissues to investigate the predominant α1‐AR subtype involved. We have examined the Rho kinase inhibitor fasudil in rat aorta and mouse spleen, tissues containing multiple α1‐AR subtypes. Male Wistar rats (250‐300g) and C57 mice were killed by CO2 overdose and cervical dislocation, and rat aortic rings and mouse spleens were mounted in organ baths under 0.5 g tension, and bathed with Krebs‐Henseleit solution at 37oC. Tissues were contracted with cumulative additions of norepinephrine (NE) in 0.5 log unit increments, over the concentration range 10‐8‐10‐3M. Both α2‐ and α1‐ARs mediate contractions of mouse spleen and the major α1‐AR involved is the α1B‐adrenoceptor, and this receptor interacts with an α1D‐AR at low, and an α1A‐AR at high, NE concentrations (Alsufyani et al., 2021). In mouse spleen, fasudil (3 μM) reduced contractions to both low and high concentrations of NE. In rat aorta, contractions to NE are reported to be mediated by two subtypes of α1‐AR: α1B‐ and α1D‐ARs, with responses to low but not high concentrations of NE being sensitive to the antagonist BMY7378. Responses to high concentrations of NE in rat aorta are sensitive to the non‐selective α1‐AR antagonist prazosin and the putative α1B‐AR antagonist cyclazosin. Fasudil (3 and 10 μM) significantly reduced contractions to both low and high concentrations of NE in rat aorta in a dose dependent manner in terms of maximum response. It is concluded that α1‐AR mediated contractions to NE in both rat aorta and mouse spleen are sensitive to non‐competitive block by the Rho kinase inhibitor fasudil in concentrations of 3‐10 μM. Although multiple subtypes of α1‐adrenoceptor are involved in contractions of both rat aorta and mouse spleen, the evidence suggests that, as previously reported in rat portal vein, α1B‐adrenoceptors are most likely linked to the Rho kinase pathway

Alsufyani HA & Docherty, J.R. Can J Physiol Pharmacol. 2021 Jun;99(6):654‐659. Alsufyani HA, et al. Basic Clin Pharmacol Toxicol. 2021 Dec;129(6):416‐426.
DNSP‐11 reduces 6‐OHDA‐induced caspase 3/7 activation in an SH‐SY5Y cell model: Potential Role of ERK signaling pathway?
Authors: Parmar Mayur S. , Allen Erika , Narain Shreya , Khanna Deepesh , Cavanaugh Jane
Published: May 2022

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to be a promising therapeutic molecule for treating Parkinson’s disease (PD). However, because of the failure of GDNF infusion in clinical trials, due to its poor bio‐distribution, there was a need to develop other molecules with similar properties to GDNF that have improved bioavailability and administration. Dopamine neuron stimulating peptide‐11 (DNSP‐11) is a synthetic, amidated 11‐amino acid neuroactive peptide derived from the human proGDNF domain. DNSP‐11 has been shown to protect dopaminergic cells in vitro and restore dopaminergic activity in vivo. Therefore, the objective of our study was to test if DNSP‐11 protects human dopaminergic neuroblastoma SH‐SY5Y cells against 6‐OHDA induced toxicity and to elucidate the protective mechanism of action. DNSP‐11 reduced the 6‐OHDA–induced increase in caspase‐3/7 activity in SH‐SY5Y cells at early time points, indicating possible protection against apoptosis. DNSP‐11 also activated the cell survival signaling pathways, ERK1, 2, and 5 in SH‐SY5Y cells. In addition, a single injection of DNSP‐11 in adult rat striatum leads to modulation of these ERK proteins in the substantia nigra. Overall, the results indicate that DNSP‐11 protects human dopaminergic neuroblastoma cells against apoptotic cell death and activates neuroprotective ERK signaling pathways, suggesting its potential therapeutic role in Parkinson’s disease (PD).
Generation and characterization of Elovl4bknockout zebrafish as a model for Juvenile‐Onset Macular Degeneration
Authors: Nwagbo Uzoamaka , Bernstein Paul S.
Published: May 2022

Purpose

Very long‐chain polyunsaturated fatty acids (VLC‐PUFAs) are a distinct class of lipids with chain lengths greater than 24 carbons. ELOVL4 is a protein responsible for VLC‐PUFA biosynthesis. However, in patients with autosomal dominant Stargardt 3 disease, the gene encoding ELOVL4 is mutated, and as a result, a dysfunctional, truncated protein is produced instead. It is unclear whether the disease phenotype is caused by truncated protein aggregates, the absence of VLC‐PUFAs, or a combination of these factors. Moreover, homozygous elovl4 ablation is neonatal lethal in mice. This study aimed to delineate the effect of VLC‐PUFA loss on photoreceptor function.

Methods

Elovl4b knockout zebrafish were generated by creating a deletion mutation in exon 2 of the elovl4b gene using CRISPR‐Cas9. Mutant zebrafish were screened for non‐specific mutations by outcrossing mutant adults with wild‐type fish. Their eyes were isolated, total fatty acids extracted, and the quantity of VLC‐PUFAs determined through gas chromatography and mass spectrometry.

Results

We found that homozygous Elovl4b mutant zebrafish eyes had altered lipid profiles and a significantly lower abundance of C30 to C34 VLC‐PUFAs compared to age‐matched wild‐type controls. We also predict diminished a‐ and b‐wave amplitudes from ERG recordings – indicative of a loss of photoreceptor function and a thinner outer nuclear layer than controls.

Conclusion

Our data indicate that the loss of Elovl4b in zebrafish can cause an ocular phenotype comparable to juvenile‐onset macular degeneration. The availability of a homozygous animal model of ELOVL4 deficiency will also allow for further studies on other ELOVL4‐related diseases.
Characterizing the structure and function of phosphatidylinositol‐3‐phosphate‐binding L. pneumophilaeffectors
Authors: Grossi Marina , Neunuebel Maria R.
Published: May 2022

Phosphoinositides (PIPs) are distinctly‐phosphorylated lipids that govern the fate of endocytosed cargo in eukaryotic vesicular trafficking. A growing number of pathogens manipulate these lipids to infect and survive within human cells. The intracellular bacterial pathogen L. pneumophila translocates a large number of effectors, some of which target PIPs to interfere with their metabolism or to anchor on to specific host membranes. This allows the phagocytosed bacterium to evade degradation and to proliferate inside host macrophages. Therefore, understanding how and why L. pneumophila effectors bind PIPs is crucial for uncovering their roles during infection. Most of the PIP‐binding effectors do not share homology with conserved eukaryotic PIP‐binding domains, making them difficult to identify and characterize. This work aims to identify the structures of the PIP binding regions for two effectors, Lpg2409 and Lpg0160, as well as to uncover the role of Lpg2409 during infection. We demonstrate that both effectors can be purified on a suitable scale for crystallography, and show that Lpg0160 has a modest binding affinity for a fluorescently‐labeled PI3P analog in vitro. Additionally, immunoprecipitation experiments with overexpressed mCherry‐Lpg2409 in HEK293T cells revealed binding to scaffolding proteins involved in E3 ubiquitin ligase complexes, as well as mitochondrial proteins, suggesting a role in regulating the turnover of mitochondrial proteins. These findings suggest that Lpg2409 may have a previously unidentified role during L. pneumophilainfection.
High‐intensity, low‐volume inspiratory muscle strength training blunts the cardiovascular response to constant load respiratory testing
Authors: DeLucia Claire M. , Tavoian Dallin , Snell E. W. , DeBonis Dean R. , Schwyhart Sarah M. , Bailey E. F.
Published: May 2022

Previous studies indicate inspiratory muscle strength training (IMST) may attenuate blood pressure (BP) and heart rate (HR) responsiveness to sustained respiratory muscle work. However, these previous studies used a less vigorous IMST intervention and did not have subjects gauge perception of respiratory effort. Our objective in this study was to evaluate the effects of 6 weeks high intensity (75% maximum inspiratory pressure), low volume (5 sets of 6 breaths/day) IMST on BP and HR responses to a resistive breathing trial (RBT) to exhaustion (target pressure = 65% of maximal inspiratory pressure or PImax; duty cycle = 0.7; breathing frequency matched to eupnea). We recruited 10 (7M; 3F) healthy young adults to complete two RBT, pre‐ and then again post‐, 6 weeks IMST. Throughout the RBT we assessed beat‐to‐beat changes in BP and HR, inspiratory pressure, respiratory muscle work (mmHg.s) and recorded participant ratings of perceived exertion (RPE) via the Borg scale. We report significant IMST‐related improvements in endurance time (PRE: 362.0 ±46.6s vs. POST: 663.8 ±110.3s, p=0.003) and respiratory muscle work (PRE: ‐9445 ±1562mmHg.s vs. POST: ‐16648 ±3761mmHg.s, p=0.069). Remarkably, despite significant gains in work and endurance, the latter stages of the post RBT were characterized by a reduced cardiovascular responsiveness (i.e., SBP, DBP and HR) and lower RPE, consistent with a blunting of the metaboreflex.
Transmembrane Protein 184A and Syndecan‐4 function in the same pathway to promote angiogenesis
Authors: Altenburg Leanna M. , O'Toole Rachel A. , Ciabattoni Grace O. , Papov Isabella V. , Pasch Leah M. , Maslonka Brianna E. , Lowe‐Krentz Linda J.
Published: May 2022

Morpholino (MO) knockdown (KD) of the novel vascular heparin receptor, Transmembrane protein 184A (TMEM184A), increases endothelial cell (EC) proliferation rates, leading to disorganized and stunted vessel outgrowth in the regenerating zebrafish caudal fin. A similar vascular phenotype is observed in the developing zebrafish embryo, where TMEM184A KD reduces the number and length of intersegmental vessels (ISVs), as well as total Vascular Endothelial Cadherin (VEC) protein in ISV ECs. The Heparan Sulfate Proteoglycan Syndecan‐4 (Sdcn‐4) is required for VEC internalization in angiogenesis and interacts with membrane proteins to promote cellular signaling and molecular processes through interactions with its attached heparan sulfate (HS) chains. Since HS is nearly identical to heparin in structure and both TMEM184A and Sdcn‐4 appear to be required for stable angiogenesis, we asked whether TMEM184A and Sdcn‐4 interact and function in the same pathway to promote vascular sprouting and regeneration. Using MO knockdown of TMEM184A and Sdcn‐4 in the regenerating zebrafish caudal fin, we demonstrate that co‐injected subthreshold concentrations of both MOs synergize to produce the same vascular defect. In cells, we find that immunostaining of TMEM184A, Sdcn‐4, and VEC show trimeric colocalization at the cell surface, that in vesicle puncta they colocalize with the recycling marker Rab5a, and that TMEM184A precipitates Sdcn‐4 in whole cell lysate. In TMEM184A overexpression (OE) backgrounds and in ECs treated with heparin, immunostaining of VEC show increased expression levels in adherens junctions. Collectively, these results suggest that TMEM184A and Sdcn‐4 function in the same pathway to promote stable angiogenic repair, that TMEM184A interacts with both Syndecan‐4 and VEC in ECs, that they form a complex that is potentially recycled at the membrane surface, and that TMEM184A overexpression or activation stabilizes VEC in adherens junctions. Further study is required to determine whether TMEM184A and Syndecan‐4 function together to regulate VEC trafficking and promote angiogenesis or whether TMEM184A expression impacts on VEC membrane stability are distinct from the TMEM184A–Sdcn‐4 molecular mechanism that regulates angiogenic repair.
Exploring the role of redundant RFamide neuropeptide pathways during sleep in Caenorhabditis elegans
Authors: Conrad Paige , Olenginski Jordan , Nelson Matthew D.
Published: May 2022

RFamide neuropeptide signaling is required for sleep in vertebrates and invertebrates, suggesting an ancient evolutionary role during sleep regulation. In fruit flies and zebrafish, a single RFamide gene encodes neuropeptides that signal through one or possibly two receptors to regulate sleep. In contrast, the genetically tractable nematode, Caenorhabditis elegans, possesses >30 FMRFamide‐related peptide genes (flp), multiple of which have been shown to be either required for sleep or capable of inducing sleep when overexpressed. Moreover, their genome codes for >15 FMRFamide peptide receptors (frpr). We sought to explore the function of these pathways during sleep by measuring total sleep in single, double and triple mutants of flp and frpr genes. During these analyses, we found that two neuropeptide genes, flp‐3 and flp‐33, are required for sleep and likely function redundantly. Also, when either the flp‐3 or flp‐33 gene is overexpressed, a strong inhibition of movement, reminiscent of sleep, occurs. Additionally, we identified a redundant sleep role for the related GPCR‐encoding genes frpr‐4, frpr‐6, and frpr‐16 and a unique role for the highly similar frpr‐11 and frpr‐12 genes. We are currently assessing peptide‐receptor interactions using in vitro cell culture systems and testing for genetic interactions of the FLP‐33 pathways in vivo. Our work demonstrates an expansive network of RFamide signaling during sleep in C. elegans. Our work identifies novel mechanisms for sleep regulation in C. elegans. It remains unclear why such a diverse collection of pathways evolved in this simple organism but not in more complex animals like vertebrates.
Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC
Authors: Katti Sachin S. , Krieger Inna V. , Sacchettini James C. , Igumenova Tatyana I.
Published: May 2022

Diacylglycerol (DAG)‐sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High‐resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane‐mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high‐resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH‐836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor‐promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC‐mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG‐sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can “cage” chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.
Anatomy‐Focused Exercise Videos to Combine Studying with Wellness
Authors: Knox Michael , Brackmann Megan , Mehta Sejal , Schaefer Maureen , Tubbs Ryan
Published: May 2022

Study Objective and Hypothesis

The link between exercise and mental wellness is well appreciated. Yet, many medical students struggle to find the time to incorporate physical activity into their daily lives. A new and innovative teaching style that incorporates movement with Anatomy was developed at Michigan State University. Students are provided a series of supplemental videos that utilize exercise and choreography to reinforce difficult anatomical concepts. Previously published research by Guzzardo et al. demonstrated a link between movement, anatomy learning, and enjoyment. This current study deepens that understanding by gathering student feedback that qualifies the effects of the videos on their wellness.

Methods

First and second year medical students at Michigan State University were provided links to videos housed on the Knockout AnatomyYouTube channel during their Anatomy or Neuromuscular courses. All students within those two cohorts were invited to participate in our IRB‐approved qualitative study. Nineteen students completed virtual interviews that asked questions pertaining to their current level of activity, the importance of movement in their life, their motivation behind watching the videos, how the videos made them feel afterward, and if the videos helped them learn the material.

Results

Student responses were broken into four major themes. 1) All interviewees stated they view exercising as important for mental health and academic performance, and nearly all reported their time spent exercising has decreased since starting medical school. 2) After participating in the movement‐based videos, most interviewees reported a positive increase in mood and energy levels. 3) Several interviewees reported that participating in the videos reminded them of the positive benefit of routine exercise, and they were more motivated to achieve their weekly exercise goals on a more consistent basis. 4) Academically, most interviewees reported that the videos helped them retain anatomy information, particularly with challenging anatomy topics such as brachial plexus, dermatomes, and myotomes.

Conclusion

Many medical students feel they are unable to reach their exercise goals due to time constraints. Professors in all disciplines can better understand the positive effects of exercise on student wellness and challenge themselves to explore new and innovative ways to bring movement into the learning environment.

References

Guzzardo, G., Gilbert, M., Schaefer, M. and Nazaroff, C. (2021). Exercise Anatomy: A Flexible Approach. The FASEB Journal, 35:S1.
Improving the Percutaneous Approach for the Management of Trigeminal Neuralgia
Authors: Sowinski Halee N. , Bandak M. C. , Nestor Nicholas S. , Zdilla Matthew J.
Published: May 2022

Trigeminal neuralgia, a debilitating pain disorder affecting the trigeminal nerve distribution, is often treated through percutaneous procedures using the route of Härtel. Miscannulation and excessive attempts at traversing the foramen ovale can negatively affect surgical outcomes. Complications attributed to miscannulation include blindness, brainstem hematoma, temporal lobe hematoma, and carotid artery hemorrhage. Currently, there is disagreement regarding the best skin entry point for the percutaneous approach. However, identifying an optimum entry point for cannulation can decrease the number of attempts necessary for a successful cannulation and also decrease the amount of fluoroscopic radiation exposure for both the patient and the surgeon. Therefore, this cadaveric study assessed a total of 56 cadavers to identify the optimum skin entry point. Calvariae, brains, and trigeminal nerves were removed, and the foramina ovalae were exposed. A lumbar needle was inserted through each porous trigeminus and foramen ovale at varied angles. The cannulation procedure was “reversed” by inserting the needle from the interior of the cranium through the porous trigeminus, foramen ovale, and skin. The exit location of the needle relative to facial structures was assessed. The exit points from the facial skin, which would otherwise represent the optimal entry points for the percutaneous procedure, were recorded. The results of this study represent an improvement in the percutaneous approach for the management of trigeminal neuralgia.
The Antiarrhythmic Effect of Inhibition of the Mitochondrial Calcium Uniporter under Catecholamine Overload is Partly Dependent on Mitochondrial Function Preservation
Authors: de Jesús Salazar Ramírez Felipe , Bernal‐Ramírez Judith , Luévano‐Martínez Luis , García‐Rivas Gerardo
Published: May 2022

Introduction

Ventricular arrhythmias are a major cause of mortality in cardiovascular patients. Catecholamines have been associated with the development of ventricular arrhythmias and no new intervention has demonstrated efficacy in reducing mortality since the use of β‐blockers. Mitochondrial calcium transport has been deemed necessary for an adequate adrenergic response and constant adrenergic stimulation leads to mitochondrial calcium overload with subsequent mitochondrial dysfunction. Inhibition of the mitochondrial calcium uniporter (MCU) have been described to reduce asynchronized contraction in cellular and animal models. In this study, we assessed the effects that MCU inhibition by administration of Ru360, a MCU inhibitor, has on ventricular arrhythmia incidence in a model of catecholamine toxicity and characterize cellular and mitochondrial function to describe the subcellular mechanisms involved.

Methods

The study followed the national guidelines for laboratory animal use and care and was approved by the Animal Care and Use Committee. 12‐15‐week‐old C57bl/6 male mice received Ru360 or normal saline via IV, and a baseline ECG was recorded. Afterwards, Isoproterenol (400mg/kg) was administrated subcutaneously, and ECG recording was kept for another 20 minutes. Finally, hearts were excised, and cardiomyocytes and mitochondria isolated for further characterization. Data is presented as mean ± SEM. Fisher exact test was performed to evaluate arrhythmia incidence and one‐way ANOVA followed by Holm‐Sidak method for mitochondrial experiments and heart rate measurements.

Results

Ru360 completely prevented the development of ventricular arrhythmias at a dose of 50nmol/kg. Mitochondrial Ca2+ transport inhibition preserved mitochondrial function and membrane integrity demonstrated by a preserved respiratory control and calcium retention capacity and a partially preserved mitochondrial membrane potential. MCU inhibition also prevented mitochondrial membrane potential loss upon the addition of a Ca2+ insult. The effects shown appear to be caused by a reduced oxidative stress, shown by a higher cytochrome c oxidase activity, that helps preserve cellular Ca2+ homeostasis as seen in confocal microscopy.

Conclusion

Ru360 prevents arrhythmias possibly by reducing mitochondrial Ca2+ overload, preserving mitochondrial function, preventing mitochondrial ROS formation and oxidative damage, and conserving cellular Ca2+ homeostasis.
Mesenteric Artery Reactivity in Young APOE3 and APOE4 Mice
Authors: Wagner Katherine L. , Dunlap Jazmin N. , Gusek Brikena , Jones Carleton (. , Eckman Delrae M.
Published: May 2022

Introduction

Variant isoforms of apolipoprotein E (APOE) are risk factors for both Alzheimer’s disease (AD) and cardiovascular disease (CVD), with the APOE4 isoform increasing risk compared to the most common isoform, APOE3. Allelotype alters cerebral artery reactivity and cardiac indices in aged animals (ongoing studies in our lab), yet little is known about how allelotype affects systemic circulation. Thus, we hypothesized that APOE allelotype alters mesenteric arteries (MA) in mice. We determined the mechanical characteristics and vascular reactivity of 3rd‐order MA isolated from young (Y, 3 to 4 mo), male and female mice expressing human‐ApoE targeted replacement of APOE3 (B6.129P2‐Apoetm2(APOE*3)Mae N8) and APOE4 (B6.129P2‐Apoetm3(APOE*4)Mae N8) (Taconic Labs).

Methods

MAs were isolated, cleaned of connective tissue, cut into 8mm segments, and cannulated in an arteriograph chamber to assess vascular reactivity via myogenic tone (MT) and phenylephrine (PE)‐induced constriction (1µM). Passive mechanical properties assessed at intraluminal pressures of 10mm Hg to 140mm Hg included lumen diameter, wall thickness, wall tension, stress vs pressure, distensibility and arterial stress/strain. Data were analyzed using two‐way ANOVA and Students t‐test.

Results

MT was greater in E3 mice compared to E4 mice (P<0.001). K+‐ and PE‐induced constriction were similar between E3 mice and E4 mice. Subtle, yet significant changes were observed in passive mechanical properties. Lumen diameter and wall tension was greater in E4 mice compared E3 mice (P<0.01). Wall thickness was greater in E3 mice vs E4 mice (P<0.05). Stress vs pressure was greater in E4 mice vs E3 mice (P<0.05). E4 mice tended to have greater stress vs pressure and distensibility values than E3 mice, but they did not reach statistical significance. Finally, stress vs strain values in MA from E3 mice and E4 mice were similar.

Summary/Conclusions

These data indicate allelotype alters MA vascular reactivity and structural properties. Additional studies are warranted to determine whether these changes in systemic circulation persist with ageing and contribute to altered cerebral and cardiac structure/function observed in ongoing studies in our lab.
Modified Biocultural Stress Model
Authors: Gremba Allison
Published: May 2022

The biocultural stress model, first defined by Goodman and colleagues (1984) and then revised by Goodman and Armelagos (1989), is expanded and modified to include a more complete depiction of the relationship of environment and culture to disease. The modified biocultural stress model (figure 1) incorporates more contemporary theoretical concepts, such as the osteological paradox (Wood et al. 1992) and new models of biocultural interactions (e.g., Schell 1997), into the previous models. This represents a more accurate pathway of the embodiment of stress into the body as skeletal stress markers. The modified biocultural stress model was used as a framework for understanding structural violence in the Hamann‐Todd Human Osteological Collection and the Robert J. Terry Anatomical Skeletal Collection.
Molecular Basis for Regulation of Mammalian Fatty Acid Synthase
Authors: Schultz Kollin , Marmorstein Ronen
Published: May 2022

De novo lipogenesis (DNL) is an essential metabolic process that converts excess cellular glucose to fatty acids. Fatty Acid Synthase (FASN) is a key enzyme in the DNL pathway and has shown promising pre‐clinical results as a therapeutic target in a variety of metabolic diseases, including cancer, non‐alcoholic fatty liver disease (NAFLD), and most recently COVID‐19. Mammalian FASN is a large multifunctional enzyme with 6 catalytic domains and a flexibly tethered acyl carrier protein (ACP). The ACP shuttles covalently linked intermediates between catalytic domains to facilitate palmitate synthesis. Previous negative stain electron microscopy data has shown that mammalian FASN is a dynamic enzyme that adopts multiple conformations in solution, but the role of these conformational dynamics in the mechanism of FASN is yet to be determined. To understand how the conformational dynamics of mammalian FASN contribute to the overall mechanism and its’ regulation, we have generated multiple constructs and characterized their enzymatic activity using diverse biochemical assays. Additionally, we have made progress towards understanding the enzyme dynamics using Hydrogen‐Deuterium Exchange Mass Spectrometry (HDX‐MS). We are actively pursuing structural studies to implement an integrative approach towards advancing our understanding of the complex mechanism of mammalian FASN.