bluuebunny/crossref_metadata_2025 huggingface dataset:
The Kratom‐Derived Alkaloid Mitragynine Enhances Cortisol Secretion from a Human Adrenocortical Cell Line
Authors: Kopf Phillip G. , Prozialeck Walter C.
Published: May 2022

Kratom is a tropical evergreen tree indigenous to Southeast Asia whose leaves and decoctions have long been used in traditional folk medicine for the relief of pain and enhancement of vitality. In recent years kratom use has increased in the United States, where an estimated 10‐15 million people use the herb for the self‐management of pain and opioid withdrawal. Kratom leaves contain over 40 active alkaloids, with the most important being mitragynine, 7‐hydroxymitragynine and paynantheine. To further explore the possible anti‐inflammatory actions of these kratom alkaloids, we examined their effects on cortisol secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or mitragynine, 7‐hydroxymitragynine, and paynantheine (10 μM). After 24, 48, 72, and 96 h, cortisol secretion was examined. Basal cortisol secretion was significantly increased by mitragynine at all timepoints, with maximal effect at 96 h (in ng/mL: vehicle, 8.9±1.1; 10 μM mitragynine, 47.3±0.7; n=4). Alternatively, 7‐hydroxymitragynine only significantly increased basal cortisol levels at the 96 h timepoint (in ng/mL: vehicle, 8.9±1.1; 10 μM 7‐hydroxymitragynine, 17.4±1.2; n=4), while paynantheine only significantly increased basal cortisol levels at the 24 h timepoint (in ng/mL: vehicle, 23.4±1.0; 10 μM paynantheine, 32.8±2.2; n=4). Additional studies are currently being done to characterize effects on cellular viability and concentration responses for each of these kratom alkaloids on cortisol secretion. These data indicate that mitragynine can enhance cortisol secretion in vitro and suggest that such a mechanism may contribute to the possible analgesic actions of kratom in vivo.
eDNA Analysis of Goat‐Grazed Rhamnus cathartica Soil Microbial Communities
Authors: Soneral Paula , Khomcyk Alesia , Enke Raymond , Nash Bruce , Seto Jeremy
Published: May 2022

Biological invasions of exotic species pose a significant threat to local biodiversity and ecosystem services. Redistribution of plant species in particular disrupts key structure‐function relationships in the soil by altering the composition of microbial taxa. This study combines environmental DNA analysis with a goat‐grazing intervention to investigate the taxonomic structure of soil microbiomes interacting with the invasive plant, Rhamnus cathartica (common Buckthorn). 16S (515F/806R) metabarcodes were amplified from soil DNA harvested before and after a goat‐grazing regimen on buckthorn‐invaded and control plots. Samples were sequenced on an Illumina iSeq, then analyzed using the DNA Subway Purple Line (QIIME 2.0) to process sequencing reads and calculate diversity indices. PCoA visualizations of Jaccard Beta diversity patterns revealed differences in community composition between buckthorn and control soils, but no effects from goat grazing. Five dominant taxa were identified in no‐buckthorn control soils, accounting for 35 +/‐ 9.6 SD% of the average relative abundance of total sequences. Genus DA101 notably demonstrated the largest decrease in average relative abundance in the buckthorn and goat‐grazed soils (% decrease = 6.4 +/‐ 2 SD), whereas the evenness of other taxa increased. Overall, buckthorn soil showed significant increases in Pielou’s taxonomical evenness (Kruskal‐Wallis H(2, N=29) = 10.9; p=0.004), but goat grazing had no impact on this score (p>0.01). Faith’s phylogenetic diversity scores showed no significant differences between buckthorn, goat‐grazed, and control soils (Kruskal‐Wallis H(2, N=31) = 2.2; p=0.32). Taken together, these preliminary results illustrate that buckthorn increases taxonomical evenness but has no effect on overall phylogenetic diversity of soil communities. Moreover, buckthorn goat‐grazing had little impact on soil community structure. Future studies will investigate the role of DA101 on buckthorn suppression as a possible mitigation strategy for this invasive species.
VCAM1 regulates Fra1/JunB mediated IL‐8 expression and pathological retinal neovascularization in a murine model of oxygen‐induced retinopathy
Authors: Kaur Geetika , Bisen Shivantika , Sharma Deepti , Singh Nikhlesh K.
Published: May 2022

Purpose

Pathological retinal neovascularization is a clinical manifestation of various proliferative retinopathies, including retinopathy of prematurity (ROP), diabetic retinopathy, and the wet form of macular degeneration. Over the last decade, numerous studies have reported an increased infiltration of hematopoietic stem cells (HSCs), macrophage/microglia in patients with intraocular angiogenic diseases.

Methods

Using a murine model of oxygen‐induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we are trying to understand the significance of IL‐33/Hypoxic induced VCAM1 expression in retinal neovascularization.

Results

Here, using a murine model of oxygen‐induced retinopathy, we found that NFkB and VCAM1 levels were highly up regulated in retina when new vessels were developing in the retina. Genetic deletion of IL‐33 resulted in reduced NFkB activation and VCAM1 levels in hypoxic retina, resulting in reduced neovascularization. These findings suggest a role for IL‐33‐NFkB‐VCAM1 signaling in pathological neovascularization. Our results demonstrate that IL‐33 induce tube formation in human retinal microvascular endothelial cells and depletion of VCAM1 levels significantly blocked it. Protein profiler analysis showed that IL‐33 regulates VCAM1 dependent IL‐8 expression in HRMVECs. In order, to understand how IL‐33 induced VCAM1 regulates the expression of IL‐8, we did sequence analysis of IL‐8 promoter and observed that IL‐8 promoter has AP1 binding site. We observed that IL‐33 induced the expression of Fra1 and JunB significantly in HRMVECs. Cloning and site directed mutagenesis of IL‐8 promoter revealed that the AP1 binds to IL‐8 promoter in response to IL‐33 and regulate the expression of IL‐8 in HRMVECs. Consequently, down regulation of VCAM1 levels significantly reduced IL‐33 induced Fra1 and JunB expression as well as IL‐8 expression.

Conclusions

These results strongly suggest that IL‐33/ST2L‐NFkB‐VCAM1‐AP1 regulates IL‐8 expression, playing a key role in retinal neovascularization and thus represents a new pharmacological target for the treatment of diseases where excessive neovascularization is the underlying pathology.
An Exercise‐Driven, Insulin‐Independent Glucose Uptake Pathway in Contracting Skeletal Muscle
Authors: Norman Natalie J. , Ghali Joyce , Radzyukevich Tatiana , Landero Julio , Heiny Judith
Published: May 2022

Glucose uptake by skeletal muscles, which import and store up to 80% of circulating glucose, plays a major role in glucose homeostasis. Prior research has focused largely on the insulin regulated GLUT4 transporter, which mediates glucose uptake in quiescent (non‐contracting) muscles using a canonical insulin‐dependent translocation mechanism. An ongoing paradox in the field is that actively contracting muscles take up glucose at 50‐100x greater rates than quiescent muscles, despite declining insulin levels. For this reason, exercise is a potent treatment for disorders of insulin insufficiency and insulin resistance. However, despite more than 30 years of study, the mechanism by which contracting muscles take up glucose without a requirement for insulin remains incompletely understood. It is proposed that GLUT4‐translocation occurs using signaling pathways that bypass the insulin receptor. However, no GLUT4 models have accounted for all the glucose taken up by contracting muscles or the great discrepancy between measured GLUT4 translocation (2‐fold increase in membrane density) and glucose uptake rate (50‐100‐fold increase), suggesting the existence of additional mechanisms. The goals of this study were to:i) develop a technology based on Inductively Coupled Plasma‐Mass Spectroscopy (ICP‐MS) that allows simultaneous detection of multiple transported species, including 13C‐labeled hexoses; and ii) test the hypothesis that contraction‐stimulated glucose uptake may be linked to Na,K‐ATPase (NKA) transport, which is also dramatically stimulated during muscle contraction. We measured uptake of Rb (a congener for K+), and 13C‐[6C]‐D‐glucose or 13C‐[6C]‐2‐deoxy glucose in isolated mouse EDL muscles stimulated to contract ex vivo. Muscles were obtained from WT mice. Glucose uptake was measured for 5 min at 32 C in a physiological solution containing 200 mM tracer RbCl and 11mM 13C glucose. Following uptake, the Rb and 13C content of the muscle were measured by ICP‐MS. We achieved a Limit of Detection of 54uM for 13C glucose in mouse muscle. This detection power for 13C by ICP‐MS is entirely new and was achieved by combining technical approaches: i) introduction of 13C‐based hexose substrates; ii) modifications to sample preparation that reduce background carbon; iii) ratiometric monitoring of the 13C/12C signal; and iv) 13C‐optimized instrument conditions. Contracting skeletal muscle took up 13C‐[6C]‐labeled‐D‐glucose at a rate of 10.8 mM/g‐min, and Rb at a rate of 1.2 mM/g‐min. The ability to detect glucose and other carbon‐based organic compounds by ICP‐MS is expected to advance studies of coupled transport and other biological processes that require multi‐species detection.
Studies on the function of myeloid‐derived Neuropilin‐1 in glioma: a focus on tumor hypoxia
Authors: Radin Daniel P. , Caponegro Michael , Smith Gregory , Moushiaveshi Victoria , Selwood David , Tsirka Stella
Published: May 2022

Glioblastoma (GB), is the most aggressive and most commonly diagnosed primary adult neoplasm in the CNS. As GB grows rapidly, it outstrips its oxygen supply, resulting in pockets of hypoxia throughout the developing tumor. Hypoxic glioma cells recruit microglia and macrophages to support angiogenesis to resolve hypoxia and promote an anti‐inflammatory tumor milieu. It was previously shown in peripheral cancers that Neuropilin‐1 (Nrp1) on macrophages governed entry into hypoxic niches, wherein these cells supported angiogenesis and bolstered immune evasion. We have previously shown myeloid specific Nrp1 deletion or pharmacological antagonism reduces angiogenesis and restores antitumor immunity. Herein, we demonstrate that Nrp1 deletion from myeloid cells reduces infiltration into hypoxic areas of developing gliomas. This results in an increase in tumor hypoxia and myeloid cell abundance. Additionally, we reveal that myeloid cells adopt an M2‐like phenotype in hypoxic areas, but that this phenotype is abolished in areas of hypoxia when Nrp1 is deleted, supporting multiple functions of this protein in myeloid cells. Finally, we verified that pharmacological antagonism of the Nrp1 b1 domain is sufficient to increase tumor hypoxia and GAM accumulation in developing gliomas. These data further support efforts co‐targeting Nrp1 and hypoxia‐associated survival pathways independent of glioma driver mutations.
The Effects of Exercise on the Sport Concussion Assessment Tool 3 (SCAT3)
Authors: Iring Stephanie , Favre Michelle , Tosto‐Mancuso Jenna , Ratnakumar Neethan , Lozhkin Aaron , Falvo Michael , Serrador Jorge
Published: May 2022

There are 3.8 million sports‐related concussions reported annually. Although there is a growing number of sports‐related concussions, there is a lack of evidence‐based methods to evaluate, treat, predict, and monitor recovery in athletes. Today, concussions are clinically diagnosed using self‐reported symptoms and by examining posture, neurocognition, ocular/vestibular control, and various imaging techniques. The Sport Concussion Assessment Tool (SCAT) is widely used on the sideline along with other methods to diagnose a concussion. However, current studies use control groups that are rested (i.e baseline) without considering the effect of intense exercise and contact (not related to head) that a player may have experienced during a game. To obtain this data we developed a mobile tent‐based laboratory that permits us to collect data on the field during a rugby match. SCAT3 questionnaires were performed within 3 hours following a rugby match in players who experienced a head injury or completed the match without a head injury. Additional measures included beat‐by‐beat blood pressure (Finometer), heartrate, common and internal carotid blood flow, and end‐tidal CO2 (nasal cannula) during rest, in the seated and supine position. A principal component analysis was performed on the 26 listed symptoms on the SCAT3 and Mann‐Whitney U‐test on the resulting 4 components. There were a total 209 players (67 female, 142 males; 33 ± 13 years) who just played a match, with 80 who experienced a head injury and 129 controls. Self‐reported symptoms were significantly greater in players who experienced a head injury 26.16 ± 17.6 vs control 8.93 ± 11.5, P<.001. A principal component analysis on our entire dataset produced 4 symptom components: (1) Cognitive‐Sensory (2) Emotional‐Affective (3) Hypersensitive (4) Headache. A Mann‐Whitney U‐tests showed significant differences between the concussed and control groups for the following symptom components: Cognitive‐Sensory (p<0.001), Headache (p<0.001), Emotional‐Affective (p<0.004), and Hypersensitive (p<0.001). This data suggests that the SCAT3 symptoms were greater in the players with a head injury, but that some of the control players who just played an intense match also show an increased number of symptoms and severity. Further analysis and violin plots on each of the 4 components showed that the symptom component named “Headache” composed of; Pressure in the Head, Headache, Feeling Slowed down, Dizziness, Don’t Feel Right may provide a better indicator of concussion in players that just played an intense match. Future work is needed to examine how these components can be used along with current physiological measures to better assess a concussion in players that just came off the field.
The 5‐HT1A Agonist 8‐OH‐DPAT Enhances Submental Laryngeal Elevator EMG Amplitude During Swallow
Authors: Musselwhite M. N. , Young Kellyanna C. , Frazure Michael L. , Greene Clinton L. , Iceman Kimberly E. , Pitts Teresa
Published: May 2022

Swallow is a critical behavior for sustaining life, important for both for the ingestion of nutrients and for protecting the airways. To produce an effective swallow, neural circuits must coordinate over 20 muscles across multiple cranial/spinal nerves to produce a rostral‐caudal pressure gradient to move the bolus from the mouth to the stomach. The medullary raphe nuclei are the main source of serotonin in the brainstem, and this region has been implicated in modulation of swallow and other airway protective behaviors. In order to explore the role of serotonin in swallow, we conducted experiments in adult cats (n = 6). Animals were anesthetized with intravenous sodium pentobarbital and tracheostomized. Fine wire intramuscular electromyograms (EMGs) were recorded from swallow and respiratory related muscles. The 5‐HT1A receptor agonist 8‐OH‐DPAT was delivered either intravenously through the femoral artery (n = 3) or intra‐arterially through the vertebral artery (n = 3) as a series of six cumulative doses (0.1‐30 ug/kg i.v.; 1‐300 ug/kg i.a.). Swallow was elicited by oral infusion water (3 ml) via syringe and was confirmed by the presence coordinated activity on swallow‐related EMGs. EMG amplitude was normalized prior to analysis. Administration of 8‐OH‐DPAT demonstrated a significant effect on the two primary submental muscles responsible for hyolaryngeal elevation necessary for full occlusion of the airway by the epiglottis. Mylohyoid amplitude increased by 45% (p = 0.006) and geniohyoid amplitude increased by 64% (p = 0.04) at the highest dose when compared to control. This is evidence of the raphe and its serotonergic network coordinating distributed aspects of the swallow control network as these two muscles are innervated via separate cranial nerves: the trigeminal for the mylohyoid and hypoglossal for the geniohyoid. Disordered hyolaryngeal function is a leading cause of aspiration across a multitude of neuro‐ degenerative and ‐traumatic diseases, and pharmacologic intervention with serotonergic agonists represents a promising agent for therapeutic interventions.
R‐(‐)2,5‐dimethoxy‐4‐iodoamphetamine (DOI) Blunts the Discriminative Stimulus Properties of Oxycodone
Authors: Salinsky Leah M. , Fox Robert , Anastasio Noelle , Cunningham Kathryn A.
Published: May 2022

Aims

A record ~100,000 fatalities due to drug overdoses was reported in the U.S. for the twelve months preceding April 2021. Opioids account for most deaths, and recurrent opioid use may lead to the development of opioid use disorder (OUD), a debilitating chronic disorder. Current medication‐assisted OUD treatment (e.g., buprenorphine) mitigates withdrawal, reduces mortality, and reduces intake of abused opioids. Still, the opioid epidemic crystalized the need to identify novel non‐opioid therapeutics to improve the odds of successful OUD recovery. In the last decade, there has been a resurgence of interest in serotonergic psychedelics for the treatment of psychiatric disorders, including substance use disorders. Psychedelic compounds act through agonist actions at the serotonin (5‐HT) 5‐HT2A receptor (5‐HT2AR) and there is some literature to suggest functional interactions between 5‐HT2AR and opioids. We tested the hypothesis that the psychedelic 5‐HT2AR agonist R‐(‐)2,5‐dimethoxy‐4‐iodoamphetamine (DOI) will disrupt the discriminative stimulus properties of the commonly misused prescription opioid oxycodone. The drug discrimination paradigm is a rigorous laboratory procedure for modeling the interoceptive effects of drugs and is widely recognized as useful in establishing ligand potency and effectiveness in vivo, as well as interrogating abuse liability.

Methods

Male, Sprague‐Dawley rats (n = 13) were trained to discriminate an injection (s.c.) of oxycodone (1 mg/kg) from saline (1 ml/kg) in a two‐lever, water‐reinforced paradigm. Pharmacological testing began upon establishment of the stable oxycodone vs. saline discrimination. Substitution tests with DOI (0‐0.2 mg/kg; s.c.) and combination tests with DOI plus oxycodone were conducted interspersed with maintenance sessions. Percent oxycodone‐lever responding, response rates and latency to the first response were analyzed with an experimentwise error rate of α=0.05.

Results

All rats acquired the oxycodone vs. saline discrimination to stability. Substitution tests indicated that all DOI doses resulted in percent oxycodone‐lever responding significantly different from the training dose of oxycodone (p< 0.05). Pretreatment with DOI (0.1, 0.2 mg/kg) significantly reduced the percent oxycodone‐lever responding (0.5 mg/kg), relative to pretreatment with vehicle (p < 0.05). The selective 5‐HT2AR antagonist M100907 prevented DOI‐induced suppression of oxycodone‐lever responding (p < 0.05).

Conclusions

The current studies reveal that the psychedelic DOI does not substitute for oxycodone, but does suppress the stimulus effects of oxycodone, an effect which is reversed by the 5‐HT2AR antagonist M100907. Taken together, these studies provide support for expanded interrogation of the efficacy of 5‐HT2AR agonists in counteracting the behavioral effects of opioids.
A NIRS‐derived Skeletal Muscle Oxygen Consumption Method Accurately Reflects the Gold Standard Direct Fick Method during Single‐leg Knee Extension Exercise
Authors: Craig Jesse C. , Broxterman Ryan M. , Barret‐O'Keefe Zachary , Wray D. Walter , Barstow Thomas J. , Richardson Russell S. , Trinity Joel D.
Published: May 2022

Background

Quantifying the oxygen consumption (V̇O2) of skeletal muscle, which represents the largest metabolically‐active tissue mass, is an important assessment in exercise physiology. The gold standard for quantifying muscle V̇O2 in the exercising human is the direct Fick method, however, technical challenges (e.g. arterial catheter insertion) preclude the widespread use of this technique, justifying the need for alternative approaches for the quantification of muscle V̇O2. Recently, we developed a noninvasive method of quantifying muscle V̇O2 using Doppler ultrasound measurements of blood flow (Q̇) and near‐infrared spectroscopy (NIRS)‐derived deoxygenated‐[Heme] in a modified Fick method calculation, but, to date, this has not been validated against the gold standard method.

Purpose and Hypotheses

Therefore, the purpose of this investigation was to compare NIRS‐derived and direct Fick quantification of muscle V̇O2 at multiple levels of single‐leg knee‐extensor exercise. We tested the hypotheses that: 1) the muscle V̇O2 assessed by direct Fick and the NIRS‐derived muscle V̇O2 would not be statistically different from each other across work rates, and 2) the V̇O2 assessed by the two techniques would be strongly related.

Methods

Seven healthy young volunteers (3 women, 4 men; 24 ± 5 years) performed an incremental work rate test on a dynamic single‐leg knee‐extensor ergometer. Measurements of femoral artery Q̇ (Doppler ultrasound), vastus lateralis deoxygenated‐[Heme] (NIRS), and femoral artery and vein oxygen content were taken at rest, 0, 5, 10, 15, and 20 Watts (W) and used to estimate direct Fick and NIRS‐derived muscle V̇O­2.

Results

NIRS‐derived V̇O2 was not different from direct Fick V̇O2 at any point (rest: 45 ± 21 vs 25 ± 17 ml/min; 0 W: 153 ± 70 vs 133 ± 68 ml/min; 5 W: 188 ± 81 vs 160 ± 65 ml/min; 10 W: 242 ± 123 vs 214 ± 91 ml/min; 15 W: 279 ± 131 vs 247 ± 89 ml/min; 20 W: 343 ± 163 vs 319 ± 122 ml/min; all P = 0.26 – 0.44). Muscle V̇O2determined by the two techniques was strongly related (r2: 0.97 ± 0.03) with a slope of 1.1 ± 0.3 and Y‐intercept of ‐21 ± 22 ml/min.

Discussion

Taken together, these findings indicate that the NIRS‐derived, noninvasive assessment of skeletal muscle V̇O2 accurately reflects the gold standard quantification of muscle V̇O2over a range of work rates (with a constant ≍20 ml/min offset). This investigation provides the first evidence to support the use of the NIRS‐derived muscle V̇O2 estimate during free‐flow conditions (i.e. not using venous or arterial occlusions like previous work), introducing a novel, noninvasive technique to quantify exercising skeletal muscle V̇O2.
Intermittent Hypoxia Promotes the Development of Abdominal Aortic Aneurysm in Male Mice
Authors: Sharma Neekun , Power Gavin , Soares Rogerio N. , Ramirez‐Perez Francisco I. , Khalyfa Abdelnaby , Gozal David , Martinez‐Lemus Luis A. , Manrique‐Acevedo Camila , Padilla Jaume
Published: May 2022

Introduction

Intermittent hypoxia (IH) is one of the hallmarks of obstructive sleep apnea (OSA). OSA is highly prevalent among patients with abdominal aortic aneurysm (AAA). However, it remains unknown whether IH is a risk factor for the development and faster expansion of AAA. The goal of this study was to determine whether exposure to chronic IH mimicking OSA increases the susceptibility to angiotensin (Ang) II‐induced AAA in mice.

Methods

Four‐ to six‐week‐old male C57BL/6 mice (n = 20/group) were subjected to IH (6.5%‐21% O2, 90 s/cycle, 12 h/day during daylight) or room air (21% O2, 24 h/day) for 6 months. During the final 14 days of IH or room air (RA) exposures, 10 mice from each group were infused with Ang II (1000 ng/kg/minute) using a subcutaneously implanted osmotic minipump to induce AAA. Sham surgery was performed in control mice (n = 10/group). Outcome was assessed by transabdominal ultrasound imaging of the abdominal aorta using a Vevo 2100 imaging system followed by histopathological examination.

Results

Ultrasound imaging revealed increased abdominal aortic diameter in RA mice following Ang II infusion compared to sham mice. Exposure to IH significantly augmented the Ang II‐induced increase in aortic diameter and incidence of AAA compared to RA Ang II mice. Histologically, IH along with Ang II infusion augmented medial elastin depletion and accumulation of collagen in the aorta. Additionally, IH was also associated with the pathological process of extensive matrix degradation as evidenced by increased matrix metalloproteinase (MMP9) and a disintegrin and metalloproteinase (Adam)17 expression in the aorta following Ang II infusion.

Conclusions

IH exposures simulating OSA promote the development of AAA. Clarification of the underlying molecular mechanisms may reveal novel therapeutic targets for the management of AAA.
Reliability of Heart Rate Variability during Polysomnographic Sleep
Authors: Kerkering Emma M. , Greenlund Ian M. , Bigalke Jeremy A. , Migliaccio Gianna C. L. , Tikkanen Anne L. , Nicevski Jennifer R. , Carter Jason R.
Published: May 2022

Spectral analysis of heart rate variability (HRV) is often used as a noninvasive index of cardiac sympathetic and/or parasympathetic activity during sleep. During controlled wake recordings, the low frequency (LF) component of HRV can be unreliable within experimental sessions and against other direct sympathetic measurements. In contrast, the high frequency (HF) component of HRV has higher reported reproducibility within controlled wake sessions. It remains unknown if the frequency component of HRV is reliable across sleep cycles within individuals during stable sleep. The purpose of the present study was to determine HRV reliability during stable stage II (N2) sleep, slow wave sleep (SWS) and rapid eye movement (REM) sleep. We hypothesized that both LF and HF components of HRV would be less reliable during REM sleep than during SWS and N2 sleep. Twenty‐seven participants (11 male, 16 female, 26±1 years, 27±1 kg/m2) were enrolled in the present study. Overnight polysomnography (PSG; NATUS, Middleton, WI) and continuous two‐lead electrocardiography (ECG) were recorded during an 8‐hour sleep opportunity. The nocturnal ECG recordings were imported into a custom software (WinCPRS, Absolute Aliens, Finland) for analysis of HRV via fast‐Fourier transformation function. Autonomic cardiac activity was quantified as LF (0.04‐0.15 Hz) and HF (0.15‐0.4 Hz) HRV. Two separate stable periods of sleep (range, 5‐10 min) absent of cortical arousal were selected from each sleep stage for comparison. Raw LF and HF HRV underwent logarithmic transformation due to non‐normal distribution. Statistical analysis included bivariate correlation of HRV across differing sleep stage cycles (α = 0.05). LF HRV was significantly correlated across two independent sleep cycles in N2 (r=0.694, p<0.001, n=26), SWS (r=0.765, p<0.001, n=25), and REM sleep (r=0.699, p<0.001, n=20). Similarly, HF HRV was reliable across two independent sleep cycles in N2 sleep (r=0.839, p<0.001, n=26), SWS (r=0.765, p<0.001, n=25), and REM sleep (r=0.881 p<0.001, n=21). Taken together, both LF and HF HRV measures were reliable across stable N2, SWS, and REM sleep. Our results support the reliability of frequency domain analysis of HRV during key PSG sleep stages within a single night, but further research across nights is warranted.
Schwann Cell Derived Signals Enhance AChR Clustering in C2C12 Myotubes
Authors: Guzman Steve D. , Abu‐Mahfouz Ahmad , Macpherson Peter C. , Brooks Susan V.
Published: May 2022

The neuromuscular junction (NMJ) is a tripartite structure made up of the pre‐synaptic motor neuron terminal, the post‐synaptic muscle end plate, and the perisynaptic Schwann cells (SC). Degeneration of the NMJ is strongly associated with age‐associated muscle mass and strength declines as well as other neurodegenerative diseases; however, the cellular interactions and signals that initiate the degeneration are poorly understood. In particular, understanding of the role of perisynaptic SCs in NMJ maintenance is scant, whereas the signaling between motor neurons and muscle fibers has been extensively studied. For example, agrin is a motor neuron derived secreted factor that is the primary signal for the induction of acetylcholine receptor (AChR) clustering in myofibers, and treatment of myotubes in culture with agrin has been shown to similarly promote AChR clustering in the absence of neurons. The presence of SCs at motor nerve terminals implicates signaling from these cells in both NMJ formation and maintenance. Thus, the goal was to determine if SC derived signals impact AChR clustering. We hypothesized that SC derived factors would potentiate agrin‐induced aneural AChR clustering in C2C12 myotubes. Differentiated C2C12 myotubes were treated with agrin for either 10 minutes or 4 hours to minimally or maximally induce AChR clustering, respectively. Additional myotubes were treated with agrin for 10 minutes followed by treatment with conditioned media (CM) derived from SW10 Schwann cell line cultures for the remaining 4 hours. Finally, additional myotube cultures were treated for 4 hours with CM alone or normal complete media as controls. The number of clusters and mean cluster area were increased in myotube cultures treated with the combination of a 10‐minute agrin pulse followed by and CM from SW10 cells compared to cultures treated for either 10 minutes or 4 hours with agrin. Neither cluster number nor area were elevated compared with controls in cultures treated with CM alone. These data suggest that SC‐derived signals can potentiate agrin‐induced post‐synaptic AChR clustering in vitro. We are currently investigating the critical SC‐derived signals that potentiate AChR clustering to determine factors that may provide therapeutic benefit in neurodegenerative phenotypes including aging.
Characterizing the Capacity of Acinetobacter calcoaceticusto Grow in Intestinal Conditions and Modulate the Gut Epithelium
Authors: Glover Janiece S. , Browning Brittney , Ticer Taylor , Engevik Amy , Engevik Melinda
Published: May 2022

Background

Acinetobacter species can occupy several ecological niches, including the mammalian intestine. Acinetobacter has been identified in the human fecal microbiota and it is speculated that the gut can serve as a reservoir for this pathobiont. The Acinetobacter calcoaceticus–Acinetobacter baumannii complex is a common cause of drug‐resistant pneumonia, urinary tract, skin and soft tissue infections. A. calcoaceticus has been considered to have relatively low virulence compared to its counterpart A. baumannii since colonization is more frequently noted more than infection clinically. However, few studies have dissected the interaction between A. calcoaceticus and the host, particularly in the gut. In this study, we sought to examine the ability of A. calcoaceticus strains to survive environmental stressors found in the gut. We also examined the reciprocal interactions of A. calcoaceticus strains on the host using colonic organoids.

Methods & Results

Three commercially available strains of A. calcoaceticus and four clinical isolates were grown aerobically in brain heart infusion (BHI) media, subcultured in a fully defined bacterial media termed ZMB1 and exposed to various concentrations of NaCl, ethanol, bile salts and pHs that were designed to mimic the gut. All A. calcoaceticusstrains grew robustly at NaCl concentrations that mirrored the small intestine, but differences were observed between strains at higher NaCl concentrations. Additionally, all strains grew well at pH 7 and pH 6, and exhibited growth reduction at pHs 5 and 4. All strains tolerated several concentrations of bile salts and ethanol; suggesting tolerance for intestinal stressors. Incubation of live A. calcoaceticus strains with inside‐out jejunum and colonic organoids significantly increased pro‐inflammatory cytokines (IL‐1α, KC/IL‐8, MCP‐1 and TNF) and decreased MUC2 and MUC13 transcripts, without altering tight junctions.

Conclusions

Collectively, these data demonstrate that A. calcoaceticus is well adapted to the gastrointestinal tract and points to the potential for Acinetobacter to influence the gut epithelium. Of note, we observed differences between lab adapted strains and clinical isolates, highlighting strain variation and indicating that clinical isolate strains should be incorporated into future work.
Role of SOX18 in Promoting Tumorigenesis in Pediatric Cancer Cell Lines
Authors: Rubannelsonkumar Cherubina , Ojile Jack , Mendez Jacqueline , Valle Durlin , Shackleford Terry
Published: May 2022

Sarcomas constitute a high percentage (∼13%) of cancer‐related deaths among pediatric patients between 0‐19 years of age, with Rhabdomyosarcoma (RMS) being the most common pediatric soft tissue sarcoma and Ewing Sarcoma being the second most common malignant bone tumor in children. Yet, survival for those who develop such metastatic sarcomas remains below 20‐30%. Interestingly, SOX family proteins are known to be up regulated in various cancer types and play a role in cancer progression (tumorigenesis, metastasis, etc.). More specifically, targeted knockdown of SOX18 has been shown to suppress various tumorigenic properties in cancer cell lines including osteosarcoma cells, hepatocellular carcinoma cells and breast cancer cells. Additionally, prior studies showed inhibition of IGFR leads to compensatory pathway activation via other RTK receptors resulting in continued cell survival. Upregulation of SOX18 was observed in such cases. Thus, it is important to clarify the role of SOX18 in RMS and ES. Ewing Sarcoma (ES8) and Rhabdomyosarcoma (RH41) cell lines were infected with shSOX18 to produce knockdown and selected via puromycin resistance. Western blotting and RT‐qPCR was used to analyze baseline and resultant protein and mRNA levels, respectively after knockdown. Results showed that the shRNA was effective in SOX18 knockdown and lead to reduced cell proliferation. Cell migration, cell proliferation and colony formation assays were used to characterize effects of knockdown. Further evaluation of knockdown effects on angiogenesis was analyzed using chick chorioallantoic membrane (CAM) assays. For ES8 and RH41 cell lines, SOX18 knockdown led to reduced cell migration, cell proliferation and colony formation. We anticipate characterization of the role of SOX18 in RMS and ES may inform future development of novel targeted treatments and increased understanding of role of SOX18 in activation of RTK compensatory pathways during IGFR inhibition in pediatric sarcomas.
β‐Adrenergic Receptor Signaling Reduces Pro‐Inflammatory Signaling and Promotes the M2 Phenotype in the RAW264.7 Macrophage Cell Line
Authors: Wu Albert , Cabrera‐Paz Francy , Fischer‐Stenger Krista
Published: May 2022

Catecholamines alter macrophage function by binding to adrenergic receptors on the cell membrane. Stimulation through β2‐adrenergic receptors is known to down regulate the activity of classically activated M1 macrophages while stimulation through α‐adrenergic receptors typically enhances their function. M1 macrophages initiate the pro‐inflammatory response to eliminate infection while alternatively activated M2 macrophages down regulate the immune response and promote tissue repair. Our studies with the RAW264.7 murine macrophage cell line as a model for macrophage function support these findings. Previously, we exposed macrophages to the selective β2‐adrenergic receptor agonist formoterol or the non‐selective α‐adrenergic receptor agonist clonidine prior to lipopolysaccharide (LPS) activation and analyzed them for production of the proinflammatory cytokines tumor necrosis factorα (TNFα) and interleukin‐6 (IL‐6) by ELISA. The results showed that formoterol decreased and clonidine increased macrophage cytokine production. The MAP‐Kinase (MAPK) signaling pathway plays an important role in the proinflammatory response, and our recent results obtained by western blotting show that norepinephrine (NE) reduces the activation of extracellular signal‐regulated kinase (ERK 1/2) by decreasing the time that the MAPK is phosphorylated. Further studies using quantitative PCR (qPCR) show that expression of inducible nitric oxide synthase (iNOS) mRNA was reduced while expression of arginase‐1 (Arg‐1) mRNA increased in macrophages pre‐treated with NE prior to LPS activation. The iNOS enzyme catalyzes the production of nitric oxide, which has antimicrobial properties, and is a marker for M1 macrophages. The Arg‐1 enzyme catalyzes the reaction of arginine to ornithine, a precursor to molecules participating in wound healing, and is indicative of M2 macrophages. Therefore, these findings suggest that stimulation of the adrenergic receptors by NE may also alter the phenotype of the macrophages. Taken together these findings suggest that exposure to NE may not only reduce the proinflammatory activity of macrophages but may also induce development of the alternatively activated M2 phenotype in RAW264.7 macrophages.
Characterization of the flavin dependent dimethylsulfone monooxygenase SfnG from Pseudomonas fluorescens
Authors: Gonzalez Reyaz , Soule Jess , Phan Ngan , Wicht Denyce K. , Dowling Daniel P.
Published: May 2022

Sulfur is one of the essential elements of life, being involved in pathways to generate sulfide, which is used downstream for amino acid, metabolite, and signaling molecule synthesis. Inorganic sulfate and sulfite are the typical sources of sulfur for these bacteria; however, limiting sulfate/sulfite conditions require bacteria to use alternative sulfur sources. Within Pseudomonas fluorescens a set of genes are expressed that allow them to utilize alkanesulfonates to generate sulfite in this limiting condition; the proteins expressed are two‐component flavin‐dependent monooxygenases (TC‐FMOs) involved in the desulfonation of the organosulfur compound and a flavin mononucleotide (FMN) reductase essential for the generation of reduced FMN. The first step in this pathway is SfnG, a TC‐FMO that acts upon dimethylsulfone to produce methanesulfinate, which is further acted on by enzymes in this pathway to generate sulfite. Current work has structurally characterized SfnG through X‐ray crystallography with the ligands FMN and DMSO2 to 1.75‐Å resolution and without ligands to 2.6‐Å resolution. The characterization of SfnG elucidated residues predicted to be important for binding of DMSO2 and FMN within the active site. Further structural analysis showed the active site of SfnG is enclosed by a lid region which is disordered without substrates bound, and the structural arrangement of ligands and the predicted oxygen binding site is in support of SfnG utilizing the recently proposed N5‐peroxyflavin intermediate. Further analysis of SfnG has revealed an interesting quaternary state reliant on substrate binding. The quaternary structure organization is different from other TC‐FMO’s involved in C–S bond cleavage, which was further explored through FMN binding studies. These results will contribute towards our knowledge of TC‐FMOs involved in C–S bond cleavage.
N‐cadherin signaling stabilizes occludin tight junction via PI3K p110β signaling
Authors: Lee Quinn , Kruse Kevin , Zhao Shuangping , Tai Leon , Komarova Yulia
Published: May 2022

The blood‐brain barrier (BBB) is a specialized microvasculature integral for brain tissue‐fluid homeostasis that is comprised of brain endothelial cells (BECs) and pericytes. Disruption of the BBB and subsequent vascular leakage of protein‐rich fluids is toxic to surrounding neurons and is an underlying risk factor in neurodegenerative disorders. BECs and pericytes form adhesions through the transmembrane protein Neural (N)‐cadherin. Although BEC‐pericyte interactions are critical for maintaining the BBB, the role of N‐cadherin adhesions in regulating the BBB remain unclear. Our previous work demonstrated that mutant mice lacking Cdh2 (N‐cadherin) in ECs or pericytes exhibited a size‐dependent increase in BBB permeability without affecting vessel or pericyte coverage. These findings raise the possibility that N‐cadherin junctions activate outside‐in signaling to strengthen the BBB. Analysis of tight junction (TJ) proteins occludin and claudins 1 and 5 in microvascular BECs of the cortex demonstrated a significant reduction in the accumulation of occludin, but not claudins, at TJs of KO mice. To delineate the signaling mechanism by which N‐cadherin adhesion‐mediated signaling regulates occludin TJs, we utilize biomimetic surfaces (Ncdh‐BioS) bearing covalently linked N‐cadherin extracellular domain to induce N‐cadherin adhesion in BEC monolayers in vitro. Consistent with our observations in mice, assembly of N‐cadherin junctions induced the accumulation of occludin and ZO1 at TJs in BEC monolayers. Depletion of N‐cadherin reversed these events, suggesting that N‐cadherin adhesion‐induced signaling assembles or stabilizes occludin TJs. Analysis of occludin‐Dendra 2 kinetics revealed decreased internalization rates of occludin from TJs in BECs grown on Ncdh‐BioS as compared to collagen, suggesting that N‐cadherin signaling stabilized occludin TJs. Furthermore, formation of N‐cadherin adhesion complexes led to activation of phosphoinositide 3‐kinase (PI3K) signaling as evidenced by the spatial redistribution of Akt to the plasma membrane as well as Akt phosphorylation. Pharmacological inhibition of class I PI3K with Copanlisib or depletion of PI3K p110β, but not of other PI3K catalytic isoforms, abolished Akt activation and increased the rate of occludin internalization from TJs in BECs grown on Ncdh‐BioS. Cumulatively, these data demonstrates that N‐cadherin outside‐in signaling strengthens the BBB by stabilizing occludin TJs through PI3K p110β signaling.
Elucidating the antibiotic sensing mechanism of VanB vancomycin‐resistant Enterococci
Authors: Rotsides Photis , Maciunas Lina J. , Lee Paula , Webber Nakoa , Beld Joris , Loll Patrick J.
Published: May 2022

The antibiotic vancomycin is used as a last resort to treat persistent infections caused by Gram‐positive pathogens. Vancomycin kills bacteria by binding a peptidoglycan precursor, thereby inhibiting cell‐wall biosynthesis. An alarming type of resistance to this antibiotic comes in the form of vancomycin‐resistant Enterococci(VRE). VRE have acquired genes that allow them to remodel the cell‐wall precursor and prevent vancomycin binding. Expression of these remodeling genes is under control of the VanSR two‐component system. VanS is a membrane‐bound sensor kinase that recognizes the vancomycin signal, and in response activates the transcription factor VanR, which activates expression of the remodeling genes. However, very little is known about how VanS senses the antibiotic.

To date, nine different types of VRE have been discovered, with VanA and VanB types responsible for the vast majority of human infections. Since vancomycin can induce the expression of both VanA and VanB resistance genes, we hypothesize that the VanS proteins from these types are activated by directly binding to vancomycin. We used in vitro autokinase assays to show that vancomycin directly activates VanS from VanB VRE (VanSB), while having no direct effect on VanSA­. We isolated the VanSB periplasmic sensor domain and used fluorescence anisotropy to show that it directly binds to a fluorescent vancomycin analog. Computational modeling predicts that the VanSB sensor domain adopts a PAS‐like fold, and HDX‐MS experiments supported this prediction and identified a potential vancomycin‐binding site. We also developed vancomycin photoprobes to confirm this binding site and to elucidate vancomycin’s orientation in the interaction. These results demonstrate how VanSBcan directly sense vancomycin in the environment to activate the resistance mechanism in VanB VRE, providing a promising therapeutic target to combat these dangerous pathogens.
Cumulative effects of H+ and Pi on peak power of skeletal muscle fibers from young and older adults
Authors: Sundberg Christopher W. , Teigen Laura E. , Hunter Sandra K. , Fitts Robert H.
Published: May 2022

The cellular mechanisms for the age‐related loss in muscle power output and increased fatigability during dynamic contractions remain unresolved. We recently observed that the depressive effects of fatiguing levels of acidosis (H+: pH 6.2) and inorganic phosphate (Pi: 30 mM) did not differ in muscle fibers from young compared with old men. However, the effects of these metabolites may have been saturated in the severe fatigue‐mimicking condition, potentially masking any age differences in the sensitivity of the cross‐bridge to these metabolites. Therefore, in this study we compared the contractile mechanics of myofibers (n = 327) from the vastus lateralis of 13 young (20‐32 years, 7 women) and 12 older adults (70‐90 years, 6 women) in conditions mimicking quiescent muscle and a range of fatiguing levels of acidosis (pH 6.8, 6.6, 6.2) and Pi(12, 20, 30 mM). The knee extensors of the older adults demonstrated hallmark signs of aging muscle including 19% lower thigh lean mass, 60% lower absolute power output, and ~1.8‐fold greater fatigability compared with young. Single fiber force, shortening velocity, and power decreased progressively with increasing concentrations of H+ and Pi; however, there were no age or sex differences in the depressive effects of these metabolites in any of the fatigue‐mimicking conditions. The cross‐sectional area of the fast fibers was 41% smaller in older compared with young adults, which corresponded with lower absolute fiber power. In contrast, there were no differences in fast fiber size‐specific power, indicating the age‐related decline in absolute fiber power was explained entirely by the differences in fiber size. Estimates of the amount of fast muscle in the thigh were associated with age‐related differences in whole‐muscle power output (r=0.78). These data suggest that the age‐related loss in whole‐muscle power output is primarily determined by the atrophy of fast fibers for both men and women, but that the age‐related increase in fatigability cannot be explained by an increased sensitivity of the cross‐bridge to H+ and Pi.