bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Heteropolysaccharide Adhesion to the Corneal Glycocalyx
Authors: Liu Betty S. , Liao Matthew , Wagner Willi , Khalil Hassan , Chen Zi , Ackermann Maximilian , Mentzer Steven J.
Published: May 2022

The cornea presents unique challenges to wound care. The cornea is constantly exposed to bacterial colonization, has ongoing risk of wound‐related astigmatism, and is cumbersome for traditional wound dressings. A potential solution to these challenges is provided by the corneal glycocalyx.

Recent high pressure rapid freezing fixation techniques suggest that the glycocalyx is much thicker than previously believed. In the case of the cornea, the glycocalyx may provide an opportunity for wound care anchored to the transmembrane carbohydrate surface layer.

To probe the surface glycocalyx we used a plant‐derived structural heteropolysaccharide called pectin. Pectin has been shown to mirror the structure of the glycocalyceal surface layer of visceral organs. To test the adhesivity of pectin to the corneal glycocalyx, high methoxyl citrus pectin was molded into tape form. Bovine globes were obtained from a local abattoir and used same day. Experiments were performed with custom fixtures using a materials analyzer (TA‐XT plus, Stable Micro Systems). To assess pectin adhesion to cornea, pectin was compressed onto the corneal surface then withdrawn, recording force and distance. A corneal incision was sealed with pectin and subject to increased pressure, until leak. At least three biologic replicates were analyzed, expressed as mean ± one standard deviation. Significance was assessed by Student’s T‐test. Pectin films on cornea were also fixed with formalin and imaged using a scanning electron microscope (Philips XL30 ESEM).

Pectin films were flexible, translucent, thin (80um), and did not distort an Amsler grid. Pectin was significantly more adherent (3.2±0.54 N) to the bovine cornea than control polymers nanocellulose fibers (1.2±0.4 N), sodium hyaluronate (1.7±0.7 N), and carboxymethyl cellulose (1.3±0.1)(p<0.05). The work of adhesion, reflecting the area under the adhesion curve, was also significantly greater (p<0.05, asterisk, Figure 1). The cornea incisions sealed with pectin were resistant to pressure fluctuations ranging from ‐51.3±8.9 mmHg to +214.4±68.0 mmHg, both extremes beyond physiologic human intraocular pressures. Sudden or complete sealant failure was not observed. Consistent with these findings, scanning electron microscopy demonstrated a low‐profile tape densely adherent to the bovine cornea (Figure 2).

The tensile and peel adhesive strength of the pectin biopolymer suggests a mechanism of polymer‐polymer entanglement with the corneal glycocalyx. As a therapeutic option for corneal wounds, the entangled pectin provides not only a protective barrier, but also an opportunity for pectin‐embedded drug delivery.
Investigation of the Role of Wnt/Delta‐Catenin Signaling in Regulating Opioid Pain Relief After Heat Shock Protein 90 Inhibition
Authors: Mahrer Callie A. , Streicher John M.
Published: May 2022

Opioid receptors are important regulators of pain, and opioid drugs like morphine are used in a wide variety of clinical settings throughout the world. In spite of their clear utility and efficacy concerning the treatment of pain, the addictive nature of opioids limits their use as an option for the treatment of chronic pain. This illustrates the great medical and social need to improve opioid therapy, both enhancing analgesia and reducing side effects like addiction. More recently, we showed that Heat shock protein 90 (HSP90), a molecular chaperone that participates in a vast array of critical molecular processes in the cell, has been implicated as an important modulator of opioid‐induced pain relief within the spinal cord. We found that inhibition of HSP90 improved the therapeutic index of opioids, boosting pain relief and reducing side effects. In an effort to elucidate the molecular pathways responsible for these benefits, we performed proteomic analysis of spinal cord tissue from male and female CD‐1 mice treated with intrathecal Vehicle or 17‐AAG (0.5 nmol, 24 hrs), an HSP90 inhibitor. We found that the signaling molecule delta‐catenin2 was reduced by 27.6% by 17‐AAG treatment in spinal tissue. Since delta‐catenin2 stabilizes and enables Wnt signaling, this suggests that Wnt signaling is also reduced by HSP90 inhibition. As Wnt has been shown to promote pain, this fits with our model that suppressed Wnt/Catenin signaling could enhance opioid pain relief after HSP90 inhibition. We thus report here our efforts to test this hypothesis by using a Wnt activator (AMBMP) and Wnt inhibitor (JW74) combined with 17‐AAG and morphine in a mouse tail flick pain model; we also report our efforts to confirm molecular Wnt signaling changes in spinal cord by Western Blot and qPCR for Wnt itself along with established Wnt signaling targets VEGF‐A and LEF1. Together these studies will establish a new mechanistic node linking HSP90 to the regulation of opioid pain relief in the spinal cord. This will enhance our basic science understanding of this system and could lead to novel therapeutic targets.
Investigating Cell Fate Specification upon Nkx2‐1Loss
Authors: O'Neill Marisol A. , Bush Jeffrey O.
Published: May 2022

Nkx2‐1 has long been known to be essential for proper respiratory development, as its loss in mice results in an unseparated trachea with underdeveloped cystic lungs. However, how NKX2‐1 regulates distinct cell fates in the proximal airways is unknown. While Nkx2‐1 has been extensively studied for its role in lung adenocarcinomas, few studies have described the effects of Nkx2‐1 loss on the trachea beyond its role in early tracheal separation morphogenesis. In previously published work we have shown that the majority of genes differentially expressed in the trachea and esophagus, during early foregut development are not regulated by NKX2‐1 and maintain their expected dorsoventral expression patterns in the unseparated foregut upon NKX2‐1 loss. These results suggested that Nkx2‐1 may not be a master regulator of early respiratory fate, but may instead regulate specific cell fate programs during tracheal development. To address this question, we examined E16.5 and E18.5 Nkx2‐1Δ/Δand control mouse tracheas by immunofluorescent staining for tracheal cell types. Secretory cells (MUC5AC), multiciliated cells (acetylated α‐TUBULIN and FOXJ1), and Club cells (SCGB1A1) were all found in Nkx2‐1Δ/Δventral foreguts by immunofluorescence. While all expected proximal airway cell types were present in Nkx2‐1Δ/Δ tracheas, a disproportionate number of multiciliated cells were present in the Nkx2‐1Δ/Δforegut compared to control. This was not a consequence of effects on early respiratory induction, because tamoxifen‐induced loss of NKX2‐1 in Nkx2‐1CreER/loxembryos at E12.5 also resulted in overabundance of FOXJ1 positive cells at E16.5. Based on these results, while we conclude that NKX2‐1 is not required to define the early proximal respiratory epithelial lineage, it may instead be required for specification of proper proportions and distribution of respiratory cell types.
Implementation of a method for analysis of Gi/o‐coupled orphan GPCR signaling
Authors: Franchini Luca , Watkins Lyndsay , Orlandi Cesare
Published: May 2022

G‐protein coupled receptors (GPCRs) are currently the target of almost 30% of the marketed drugs. However, only a small percentage of the whole GPCR repertoire is well characterized for both endogenous ligands and signaling pathways, with ~100 GPCRs still considered orphans (oGPCRs). Nonetheless, oGPCRs are increasingly recognized for their essential pathophysiological roles. This pool of uncharacterized GPCRs has therefore a considerable untapped potential as new pharmacological targets. Scientists exploited several indirect approaches to discover new ligand‐receptor pairs and characterize receptor‐signaling couplings, however in the last few years the number of deorphanized GPCRs has not grown as expected. According to the “GPCR database” almost two thirds of GPCRs are coupled to Gi/o/z proteins, for which currently available assays display low sensitivity. To address part of this problem, we recently used different reporter systems in combination with Gα‐protein chimeras to uncover Gα‐protein coupling by measuring the constitutive activity of a library of oGPCRs expressed in a heterologous system. Using this assay we identified 8 out of 19 oGPCRs tested as coupled to Gi/o/z proteins. In the present project, we aim at setting up a new in vitro high‐throughput screening assay for Gi/o/z‐coupled oGPCRs, combining Gi/o/z‐protein chimeras and cAMP measurements using GloSensor. Our preliminary data obtained studying traditional Gi/o/z‐coupled receptors suggest that expression of the GsGz chimera in combination with Pertussis Toxin (PTX) results in an assay displaying the highest sensitivity and signal amplitude. Considering the high expression of many oGPCRs in the brain, we hypothesized that their endogenous ligands would be also localized there. We therefore tested the activation of transfected oGPCRs using our optimized system by mouse brain extract, as a mixture of candidate ligands. The success of this preliminary analysis validates our approach for future screening purposes. Overall, implementation of this method for screening medium to large libraries of molecules would allow to deorphanize oGPCRs, providing new insights in GPCR pharmacology and the discovery of new ligand‐receptor pairs.
HIF1α and NFκB Regulate Mineralocorticoid Receptor Gene Expression in Aging Human Vascular Smooth Muscle Cells
Authors: Ibarrola Jaime , Lu Qing , Zennaro Maria‐Christina , Jaffe Iris Z.
Published: May 2022

Background

Mineralocorticoid receptor (MR) activation drives aging‐associated cardiovascular diseases including hypertension, vascular stiffness and heart failure. Vascular MR expression increases with age and drives the aging phenotype but the mechanism of MR gene regulation is unknown. The MR has two mRNA isoforms (1α and 1β) that differ in only the 5’‐untranslated region (exon 1α or 1β) and in the promoter sequence (P1 or P2, respectively). Thus, we investigated the transcriptional mechanisms regulating MR gene expression in aging human smooth muscle cells (SMC).

Methods

MR mRNA and protein expression were quantified in primary human aortic SMC (HASMC) and human aortic tissue comparing adult (average age 40s) and aged (average age 70s) male and female donors and comparing adult HASMC or Pac1 (rat SMC line) treated with H2O2 to induce aging. MR promoter activity was tested by luciferase reporter assays. In silico analysis of the MR promoters identified putative transcription factor (TF) binding sites and inhibitor studies and chromatin immunoprecipitations (ChIP) were performed to validate the predicted regulatory mechanisms.

Results

Increased SMC donor age or H2O2 treatment were associated with significantly increased expression of both of the MR mRNA isoforms, MR protein level, and activity of both MR promoters in HASMCs. The hypoxia‐inducible TF, HIF1α, and the inflammatory TF, NFκB, were predicted in silico to bind to the MR promoters and found to increase significantly with age in HASMCs. HIF1α was induced by H2O2 in HASMCs and this was associated with significant enrichment of HIF1α binding specifically at a site in the 1a exon, near the MR P1 promoter. HIF1α inhibition attenuated expression specifically of the MR‐1α mRNA isoform resulting in decreased MR protein in HASMC. NFκB also increased with age in HASMCs and contributes to MR expression. In response to H2O2,NFκB binding is enriched at to two different binding sites, one each in the P1 and P2 promoters and NFκB inhibition decreased expression of both MR mRNA isoforms (1α and 1β) and MR protein in SMC. Finally, HIF1α and NFκB expression each positively correlated with donor age and with MR expression (p<0.0001) in human aortic tissue from males and females.

Conclusion

The MR gene is regulated by the inflammatory transcription factor NFkB and by the hypoxia/oxidative stress‐induced TF HIF1α, to increase MR expression in SMC of the aging human vasculature. This molecular insight may explain enhanced MR activity in cardiovascular aging and could potentially contribute to other cardiovascular conditions driven by oxidative stress and inflammation.
A Novel Non‐Linear Approach to Characterizing Astrocytic Neurovascular Coupling with Optogenetics and Computational Modeling
Authors: Suarez Alejandro , Fernandez Lazaro , Balachandar Lakshmini , Riera Jorge
Published: May 2022

The role of astrocytes in neurovascular coupling remains hotly contested with various proposed mechanisms of hemodynamic response. Past findings have proven calcium activity to be the essential component in all astrocytic pathways of vascular control. However, these findings have proven contradictory over the last two decades, suggesting that an additive approach may not be appropriate for the quantification and characterization of astrocytic neurovascular coupling. We hypothesize that these astrocyte‐governed biochemical pathways have an unequal contribution and that they enact hemodynamic change in a dynamic, non‐linear fashion. Using a tetracycline‐based transgenic mice model, expressing ChR2‐EYFP solely in astrocytes, we optogenetically stimulated the production of a calcium wave in mice cortical astrocytes and evaluated hemodynamic change with a Laser Doppler Flowmetry (LDF) modality. Under these conditions, a sustained increase in localized perfusion of around 20% was found within stimulated areas. Subsequent pharmacological inhibition of phospholipase (PLA2) governed pathways facilitated the characterization of PLA2isoforms within cortical astrocytes. Furthermore, we created a computational biophysical model of hemodynamic response aiming to reproduce the experimental data generated from this study and previous ones. This data better elucidates the role of astrocytes in regional resource delivery, suggesting a new perspective in the diagnosis, treatment, and prevention of neurodegenerative diseases with vascular presentations such as Alzheimer’s Disease. We believe our findings provide a new approach by which future studies may revisit the use of pharmacology to characterize the neurovascular coupling phenomenon.
Genetic Variant (rs2304672) in 5’ UTR region in PER2 gene and its association to Parasomnias and other sleep disorders in Puerto Rico
Authors: Pagan‐Gonzalez Gabriel O. , Ramirez‐Aponte Edwin G. , Martinez‐Cruzado Juan C.
Published: May 2022

Sleeping is one of the main physiological processes of cellular and physiological restoration. Inadequate sleep patterns can impede adequate systemic restoration and can lead to the development of chronic health conditions. In 2014, both World Health Organization (WHO) and the Central Disease Control (CDC) in the United States categorized inadequate sleep patterns as a public health crisis. Both entities majorly agree that, approximately 40% of the general population suffers from sleep disorders, with 60% of these opt for medication to induce sleep. Researchers from Epidemiologist suggest that a series of external factors, have been theorized to contribute to lack in sleep quality. Including high levels of stress, sounds, and light quality. Alternatively, there has been a limitation regarding studying internal factors affecting sleep quality and sleep behavior. Parasomnia is an abnormal sleep behavior in any of the five sleep stages characterized by sleep walking, sleep talking, bedwetting, nightmare disorders, and sleep paralysis principally. To date, little is known about this sleep abnormality and its origins. In this study, we referenced data from the 1000 Genome Project to study the genetic variant (rs2304672) of the PER2 gene in Puerto Rico, whose general population houses the highest percentage of the variant in all the America’s. Provided data suggests that this genetic variant is linked to poor sleep quality and parasomnias. In Puerto Rico, modern demographics evidence that the population is comprised of three main ethnic groups. We theorize that a bottleneck effect is an influential factor in the distribution and prevalence of said genetic variant in the island. Our results found that the genetic variant is common in the southwestern and southeastern coastal regions of Puerto Rico. Additionally, we found that the genetic variant frequency in the entire island is of 3.83% in the general population of Puerto Rico.
The LP07 and LLC pre‐clinical models of lung cancer present divergent anabolic deficits and expression of pro‐inflammatory effectors of muscle wasting
Authors: Belcher Daniel J. , de la Rosa Guitart Maria , Hain Brian , Kim Hyo‐Gun , Waning David , Barreiro Esther , Nader Gustavo A.
Published: May 2022

Pre‐clinical models of lung cancer have been instrumental in uncovering the mechanisms of muscle wasting. However, differences in genetic background, immune status, tumor origin, and administration route can introduce confounding variables, limiting the understanding of muscle wasting in lung cancer. In this study, we examined anabolic deficits and the expression of pro‐inflammatory cytokines in the skeletal muscle of LP07 and Lewis lung‐cell carcinoma (LLC) pre‐clinical models of lung cancer. Female mice were injected with either LP07 or LLC tumor cells in their right flank. Tumor growth occurred over a 28–30‐day period and resulted in gastrocnemius muscle depletion with more pronounced wasting in the LLC model (LP07: ‐20.3%, P=0.004; LLC: ‐31.9%, P<0.001). The LP07 mice experienced a loss in ribosomal (r) RNA (‐34.9%, P=0.015), which was likely due to decreased rDNA transcription elongation, evidenced by a lower internal transcribed spacer (ITS) signal (‐47.9%, P=0.017). Reductions in RPS6 (LP07: ‐60.8%, P=0.020; LLC: ‐83.0%, P=0.003) and 4E‐BP1 (LP07: ‐31.4%, P<0.001; LLC: ‐36.5%, P<0.001) phosphorylation was similar in both models but was independent of changes in mTOR phosphorylation which was less pronounced in the LP07 (‐2.8%, P=0.997) compared with the LLC model (‐44.2%, P=0.042). Additionally, IL‐6 and TNF‐α mRNA levels were similarly elevated in the LLC (IL‐6: 750.7%. P<0.001; TNF‐α: 156.5%, P<0.001) and LP07 models (IL‐6: 719.4%, P<0.001; TNF‐α: +100.0%, P=0.28). In summary, while both models underwent muscle depletion, only the LP07 model displayed a reduction in rRNA and rDNA transcription. RPS6 and 4E‐BP1 phosphorylation’s were similar in both models, however, the increase in pro‐inflammatory factors was more pronounced in the LLC model. Our findings reveal a significant divergence in the anabolic deficits and expression of pro‐inflammatory effectors of muscle wasting in the LP07 and LLC pre‐clinical models of lung cancer.
Cytokine and Chemokine Release from Immortalised Human Myotubes in Culture: Effect of Contractions and Comparison with Isolated Intact Mature Adult Murine Muscle Fibres
Authors: Neiland Harvey J. , Staunton Caroline A. , Owen Euan , Kumiscia Jekaterina , Shigdar Shahjahan , Jones Samantha W. , Jackson Malcolm J. , McArdle Anne
Published: May 2022

Skeletal muscle releases a number of signalling molecules, including immunomodulatory cytokines and chemokines which can act in an autocrine, paracrine and endocrine fashion. For example, neuromuscular integrity is dependent on efficient and appropriate signalling from muscle to nerve and vice versa and when such signalling fails then tissue deterioration occurs. The aim of this study was to characterise the pattern and time course of release of pro‐ and anti‐ inflammatory cytokines and chemokines from cultured immortalised human myotubes at rest and following contractions compared with release from isolated viable intact mouse muscle fibres. Immortalised human myoblasts were fused to mature into myotubes for 7 days (Donna et al, 2003). Alternatively, single muscle fibres were isolated from the flexor digitorum brevis (FDB) muscle of adult C57Bl/6 mice (Pye et al, 2007). Cells and isolated fibres were subjected to electrical field stimulation with trains of biphasic square wave pulses of 2 ms duration for 0.5 s repeated every 5 s at 50 Hz and 30 V per well of fibres for a total stimulation time of 15 min. Media from all samples was analysed for cytokine content at 15 minutes and 24 hours following contractions using a multiplexed cytokine assay (Luminex) and data were standardised to protein content of the well. At 15 minutes following contractions of isolated muscle fibres, a significant increase in a number of cytokines and chemokines were seen in the media, including IL‐5, IL‐6, IL‐10 and G‐CSF although this was not fully replicated in 2D cultured myotubes. At 24 hours following contractions, IL‐6 content was significantly increased in the media of both isolated fibres and 2D cultured myotubes. Thus, data demonstrate some commonality of cytokines/chemokines released but future studies will examine release from more physiological 3D muscle constructs as well as the effects of such release on neuronal cell structure and function.

Pye et al, J Physiol. 2007 May 15; 581(Pt 1): 309–318.

Donna et al, Mol Cancer Res. 2003 (1) (9) 643‐653.
Autophagy Impairment and Sarcopenia in Type‐Identified Muscle Fibers of Aging Extensor Digitorum Longus Muscle
Authors: Pareja‐Cajiao Miguel , Barrera Maria A. , Ducaji Rachel A. , Gransee Heather M. , Hellyer Nathan J. , Sieck Gary C. , Mantilla Carlos B.
Published: May 2022

Sarcopenia is the aging‐related loss of force‐generating capacity and atrophy of skeletal muscles and is a major contributor to morbidity and mortality in the elderly. The molecular mechanisms underlying sarcopenia remain to be elucidated. Skeletal muscles exhibit different rates of sarcopenia, generally reflecting their specific muscle fiber type composition. Muscles primarily composed of type IIx and/or IIb muscle fibers (i.e., extensor digitorum longus; EDLm) develop sarcopenia at a faster rate than other muscles. In the diaphragm, muscle fiber cross‐sectional area (CSA) is reduced in old age, predominantly in type IIx and/or IIb fibers. Autophagy is a multistep, catabolic process that targets dysfunctional cytoplasmatic structures including proteins and organelles for degradation and recycling. Autophagy is impaired in old age across several tissues, including motor neurons and skeletal muscle. Expression of core autophagy proteins can indicate changes in autophagy, e.g., LC3 reflects initiation/elongation steps and p62 clearance reflects degradation. We hypothesize that impaired autophagy is associated with the development of sarcopenia in type‐identified muscle fibers of the EDLm. Indeed, at 24‐months of age, the CSA of type I and IIa fibers was not reduced, whereas the CSA of type IIx and/or IIb fibers was ~25% smaller (p<0.01 in both cases), compared to 6‐month‐old mice. These findings are consistent with fiber type selective sarcopenia in the EDLm. Expression of LC3 and p62 in the EDLm of mice at 6‐ and 24‐ months of age was evaluated via immunofluorescence by quantification of the number of LC3 and p62 puncta per muscle fiber type. In type I and IIa fibers, the number of LC3 puncta increased by ~30% and of p62 increased three‐fold in 24‐ compared to 6‐month‐old mice (p<0.01 in both cases). In type IIx and/or IIb fibers, the number of LC3 puncta decreased by ~60% and p62 puncta increased by ~50% in 24‐ compared to 6‐month‐old mice (p<0.01 in both cases). Accordingly, compared to 6 month‐old mice, autophagic degradation is impaired across muscle fiber types in 24‐month old mice, but likely to a greater extent in type I and IIa fibers of old mice given the increase in both LC3 and p62. Autophagic initiation is primarily reduced in type IIx and/or IIb fibers in the EDL as indicated by the reduced LC3 expression. Taken together these findings indicate that fiber type selective sarcopenia may reflect differences in autophagy across muscle fiber types.
North American River Otters Exhibit Direction Asymmetry in Limb Bone Shape
Authors: Valdez Dominik R. , Lynch Leigha M.
Published: May 2022

The occurrence of handedness has been widely observed and studied among humans. There is evidence that suggests the asymmetric pressures of using one limb more prominently can have a developmental effect on bone size and shape. The correlation of handedness with directional asymmetry of limb bones has propagated the question of whether this instance also occurs across the animal kingdom, especially within species that use their limbs to move and manipulate objects. Using the North American River Otter (Lontra canadensis), we compared differences in bone size and shape of the left and right limbs. The assessments were made by taking a variety of length and width measurements, as well as integrating 3D geometric morphometrics to compare the bone profiles. Using a t‐test, we found non‐significant (p>.05) differences in the length and width measures of the left and right bones. However, results of an ANOVA on Procrustes aligned shape coordinates indicate significant (p<.05) differences in bone shape. Our results suggest that the shape of left and right limb elements in both the forelimb and hindlimb illustrate directional asymmetry. Future research will provide insight as to whether the shape asymmetry is a product of handedness or other environmental factors.
Sex Differences in the Relationship between Exercise and the Brain
Authors: White Zach , Hall Stephanie
Published: May 2022

Introduction

Exercise is an effective, non‐pharmacological therapy to promote brain health. Sex differences in cognitive changes through the lifespan and in response to exercise training are documented, yet more information is needed to characterize these disparities. We hypothesized that aerobic exercise training would improve memory, coordination, and muscular strength in female rats and have no significant effect in male rats.

Methods

Thirty‐two Fisher 344 animals (16 males and 16 females) were housed 2 per cage, on a 12:12 light:dark cycle with food and water ad libitum. At two months old, following a 1‐week acclimatization period where the non‐compliant runners were identified, they were randomly assigned to sedentary or exercise training groups. Those in the exercise training group completed a 10 week treadmill training protocol. The protocol was administered during the animal’s dark cycle and began at 15 cm/s for 15 minutes and increased up to 35 cm/s for 60 minutes. Animals in the sedentary group were restricted to cage activity. Memory (Morris water maze), coordination (Rotarod) and muscular strength (grip strength) were assessed following the exercise intervention.

Results

A significant exercise training effect was seen in females but absent in males. Female runners showed greater performance in the Morris water maze (9.2 sec. vs 32.7, p <.05) and in the Rotarod (69 sec. vs 47.5, p <.05). No significant training effect was documented in the male groups. Also, no training effect was documented in grip strength in either males or females.

Conclusion

In the present study, memory and coordination improvements as a result of aerobic exercise training were seen in female but not male rats. This work adds to existing literature which investigates sex differences in cognitive health and adaptations to exercise training.
Riding a VeSPha in the classroom: A Versatile Simulation tool for effective didactic illustration of Pharmacodynamic concepts
Authors: Iniguez‐Lluhi Jorge
Published: May 2022

A solid mastery of Pharmacodynamics concepts is a cornerstone of effective pharmacology education. Understanding the meaning and appropriate use of pharmacodynamics terms such as potency, affinity, intrinsic efficacy, or receptor reserve is extremely valuable but challenging for most trainees. The onus, therefore, is on the educators to devise effective strategies and tools to facilitate the acquisition of this critical competency.

When approaching pharmacodynamics, the ability of learners to explore the influence of driving parameters on the magnitude and position of concentration response curves is a powerful way to visualize and make concrete the basic concepts of pharmacodynamics. To this end, I have developed VeSPha, a Versatile Simulation tool for Pharmacodynamics based on a rapid equilibrium generalization of Furchgott’s views on agonism. Through multiple sliders, the user can vary the parameters of the system, such as the concentrations of orthosteric and allosteric ligands, their respective affinities, the allosteric coupling constant, the intrinsic efficacies of each of the receptor species, the total number of receptors and the sensitivity of the system to the stimulus. A graph panel allows the display of the resulting response curves as a function of the concentration of ligand as well as orthosteric and allosteric ligand occupancy curves. Dropdown lines with corresponding EC50 as well as apparent Kd values for each ligand are also displayed. VeSPha also includes bar graphs as well as an intuitive graphic representation of the model displaying the values of the parameters, the distribution of the receptor species at the chosen state, the total stimulus as well as the resulting responses.

The tool allows easy exploration of a wide gamut of possibilities that illustrate basic concepts of affinity, intrinsic efficacy, partial agonism, constitutive activity, inverse agonism, competitive antagonism, as well as allosteric modulation of both affinity and efficacy in any combination. The tool is implemented as an easy‐to‐use Excel file. This allows for flexible standalone use and for straightforward embedding into PowerPoint presentations for real‐time illustrations during lectures. The tool also readily generates simulated data including sampling errors for composing realistic, internally consistent assessment items such as quiz, problem set and exam questions.

Implementation of this didactic tool is straightforward and effective in the author’s experience across a wide range of learners spanning from undergraduates, Master’s, Ph.D. M.D. Pharm D and DDS students. The tool, including a tutorial video can be made available to the Pharmacology teaching community upon request and an online version is under development.
1,3‐Butanediol Mitigates High‐Salt Diet Induced Cognitive Impairment in Aging Mice
Authors: Behera Jyotirmaya , Petit Rubins H. , Zheng Yu‐Ting , Tyagi Neetu
Published: May 2022

Vascular dementia (VaD) is the second most common form of dementia after Alzheimer’s disease (AD). Hypertension is the second leading risk factor for developing neurodegenerative disease. A recent study reported that Dietary salt promotes cognitive impairment through tau phosphorylation. However, the effect of ketone bodies or the precursor molecule (1,3‐butanediol) supplementation on cognitive impairment in aged mice or the absence of high salt has not been investigated. The study tests the hypothesis that 1,3‐butanediol attenuates the progression of vascular dementia mediated through histone acetylation mechanism in hypertensive aged mice. To test this hypothesis, we employed old male and female wild‐type (WT) mice. For three months, the mice were fed with a high salt diet (HSD) (8%). HSD mice have supplemented with ketone bodies precursor, 1,3‐Butanediol (2%), and ketone bodies compound β‐hydroxybutyrate (BHB, 0.2%) in drinking water for 12 weeks. Blood pressure was measured after 12 weeks of treatment by tail‐cuff plethysmography. After 12 weeks, urine and tissues were collected. The results show that 1,3‐Butanediol or BHB treatment reduced blood pressure and miRNA‐128‐3p in aged hypertensive mice. Further, the data analysis revealed the loss of short‐term and long‐term memory in HSD‐hypertensive mice compared to HSD+BHB or HSD+ 1,3‐Butanediol mice. This mechanistic study shows that HSD+ 1,3‐Butanediol mice display an increased mitochondrial TFAM dependent upregulation of brain‐derived neurotrophic factor (BDNF) and nuclear receptor subfamily 4A (Nr4a) in the mouse hippocampus via activation of the H3K27ac acetylation at the promoter. In addition, the anti‐miR‐128 treatment restored neuroprotective function and reduced the dementia pathology in old‐hypertensive mice. The present data demonstrate that 1,3‐Butanediol or BHB increases ketone bodies production and improves cognitive functions in aged hypertensive mice and could be a novel nutritional intervention for vascular dementia treatment.
Systematic Conformation‐to‐Phenotype Mapping via Limited Protein Sequencing
Authors: Serebryany Eugene , Trauger Sunia A. , Budnik Bogdan , Shakhnovich Eugene I.
Published: May 2022

Non‐native conformations drive protein misfolding diseases, frustrate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well‐suited for elucidating them, and computational techniques lack training data. Deep mutational scanning can comprehensively map complex protein fitness landscapes but not their conformational landscapes. We describe an approach to systematically discover, stabilize, and resolve native and non‐native conformations of a protein, in vitroor ex vivo, and directly link them to their molecular, organismal, or evolutionary phenotypes, using high‐throughput disulfide scanning (HTDS) of the entire protein. To reveal characteristic disulfide bonding patterns of chromatographically resolved conformers, we devised a limited protein deep‐sequencing method that identifies the exact sequence positions of two Cys residues simultaneously within the same polypeptide chain. HTDS of the E. coli periplasmic chaperone HdeA revealed phenotypically distinct classes of disordered hydrophobic conformers. HTDS can determine phenotypic effects of non‐native conformations for many proteins that function in disulfide‐permissive environments.
Rac and Cdc42 Inhibitors in Rheumatoid Arthritis Therapy
Authors: Borrero‐Garcia Luis D. , Torres Anamaris , Dorta‐Estremera Stephanie , Dharmawardhane Suranganie
Published: May 2022

Rheumatoid Arthritis (RA) is the most common autoimmune disorder characterized by chronic inflammation due to synovial fibroblast proliferation, neovascularization, leukocyte extravasation, and progressive joint destruction. Most RA medications focus on relieving symptoms of chronic inflammation, rather than prevent the inflammatory cells from attacking the joints and the underlying cartilage and bone. These initial processes have been shown to be under the regulation of key signaling proteins Rac and Cdc42. Therefore, the objective of this study was to demonstrate the effect of our patented Rac/Cdc42 inhibitors MBQ‐167 and EHop‐016 in inflammatory cells in vitro. Our hypothesis is that inflammatory cells in the synovium can be inhibited by Rac/Cdc42 inhibitors that have the potential as a RA therapeutics. To test this hypothesis, macrophage‐like cell lines WBC264‐9C human leukocytes or THP1 human macrophage were incubated with 0‐500 nM of MBQ‐167, lysed and subjected to Rac.GTP pulldown assays. Results showed that Rac activation was inhibited by 46‐56% at 250 nM and by 60‐77% at 500 nM MBQ‐167 for both cell lines. Total Rac levels were not significantly changed by MBQ‐167 in concentrations up to 500 nM. Moreover, preliminary results on synovial fibroblasts SW‐982 demonstrated inhibition of Rac activation in response to MBQ‐167 at 750 nM or higher. Next, macrophage‐like cells were treated with the Rac/Cdc42 inhibitors and tested for cell viability using a MTT assay. Results showed no effects on cell proliferation at high µM concentrations of Rac/Cdc42 inhibitors. Since Rac activation regulates cell migration, which is central to the inflammatory response in synovial joints, we tested the effect of MBQ‐167 in a wound healing assay. Incubation with MBQ‐167 at 250 or 500 nM inhibited macrophage migration. At 24h, 500 nM MBQ‐167 reduced macrophage cell migration by 80%. In conclusion, this data demonstrates that our Rac/Cdc42 inhibitors may be used to inhibit inflammatory cell action and their migration at sites of inflammation in RA, without affecting macrophage numbers, indicating no toxic effects in the immune system.
Pyruvate Kinase M2: a novel regulator of adipogenesis
Authors: Dowker Presley D. , Demeter Jenna B. , Bettaieb Ahmed
Published: May 2022

Obesity is the cornerstone of many other conditions, with far‐reaching impacts on numerous body systems and quality of life. Obesity is closely and likely causally associated with metabolic syndrome and consequent atherosclerosis, heart disease, stroke, and type 2 diabetes as well as diseases such as cancer and osteoarthritis. Recent studies suggest exploring the possibility of activating brown adipose tissue as a potential anti‐obesity strategy. However, understanding the mechanisms underlying the processes of brown adipogenesis and browning is foundational for effectively promoting this strategy as a primary, secondary, and/ or tertiary preventive measure against obesity. Additionally, recent studies have identified the glycolytic enzyme pyruvate kinase M2 (PKM2), as a novel regulator of white and brown adipogenesis. Yet, the molecular mechanisms remain to be elucidated. In the current study, we investigated the effects of PKM2 deficiency on white and brown fat adipogenesis using shRNA‐mediated lentiviral approach. We also investigated the underlying molecular mechanisms. Preliminary findings demonstrate that deletion of PKM2 enhances brown fat adipogenesis via modulation of the β‐catenin signaling pathway. Additionally, disruption of β‐catenin activity altered adipocytes differentiation and mimicked the effects of PKM2 deficiency on adipogenesis. Taken together, our study suggests that targeting PKM2/β‐catenin axis may serve as a strategy to promote brown fat adipogenesis, enhance whole body energy expenditure, and prevent obesity and its associated complications.
Vaccine Immunogenicity assay in bovines against fasciola hepatica
Authors: Zambrana Ana I. , Checa Jackeline , Goyeche Antonella , Alonzo Pablo , Silveira Fernando , Cancela Martín , Maggioli Gabriela
Published: May 2022

Fasciolosis affects cattle health and production around the world, resulting in economic loss for farmers. Current treatment against this parasite in cattle is based in drugs, which do not prevent infection and/or liver damage. In this work, we present the results of immunogenicity assays by ELISA which will lead to vaccine trials against F. hepatica with a Uruguayan and native plant derived adjuvant. Using four different vaccine formulations and a total of 27 cows, my PhD project currently aims to select the best formulation through ELISA studies from blood samples collected in 2021. The novel natural adjuvant production system (ISCOM) shows a promising replacement for current synthetic adjuvants due to its protective properties. Also, the vaccine formulations are produced with recombinant proteins that are present in the parasite (FhLAP1, FhLAP2, FhStf1 and FhStf2).

With these results that I will present in my poster, I will further analyze samples through RNA seq. so that I can select the best formulation for a first vaccine trial and fasciola hepatica infection in 2023.
Augmented Urinary Excretion of Angiotensinogen Precedes Albuminuria in Human Young Adults with Overweight and Mice with High Fat Diet‐Induced Type 2 Diabetes
Authors: Gonzalez Alexis A. , Rivera Patricia , Miranda Catalina , Reverte Virginia , Prieto Minolfa C.
Published: May 2022

Obesity is linked to the development of hypertension and type 2 diabetes (T2D) and both causing chronic kidney disease (CKD). Increased body mass index (BMI) and high fat diet (HFD) contribute to activation systemic renin angiotensin system (RAS). In addition, activation of intrarenal and intratubular RAS has been associated with increased pressure (BP), CKD, and augmentation of urinary excretion of angiotensinogen (uAGT) during T2D. However, whether augmentation of uAGT reflect intrarenal/intratububular RAS activation in subjects with overweight, remains unclear. Here, we tested the hypothesis that increases in uAGT precedes renal dysfunction in mice and human subjects with overweight,

Methods

C57BL/6 mice (N = 10) were fed either a normal fat diet (NFD) or HFD (45% Kcal from fat) for 28 weeks. During this period, metabolic phenotype along with urinary albumin, creatinine, reactive oxygen species (ROS) and AGT, were measured every 4 weeks. In addition, for evidence in humans (age 19‐22 years‐old), 11 individuals with overweight and 17 individuals with normal weight, were studied. In these subjects, anthropometric parameters, systolic and diastolic BP, fasting blood glucose (FBG), and urinary mRNA levels of AGT and kidney injury markers IL‐18 and connecting tissue growth factor, were determined. Patients with history of cardiac or cerebrovascular events and endocrine diseases, anti‐hypertensive treatment (ACE inhibitors, ARBs) and/or anti‐diabetic drugs, were excluded

Results

In mice with metabolic alterations overtime, systolic BP significantly increased after 18 weeks in HFD but not NFD mice. Intrarenal ROS increased in HFD mice. These changes paralleled the augmentation uAGT excretion, which occurred in the absence of overt albuminuria. In human subjects with overweight, systolic and diastolic BP, urinary transcripts levels of AGT, renin, and injury markers IL‐18 and CTGF were increased as compared to normal weight individuals. However, no significant changes were observed in proteinuria or albuminuria.

Conclusions

Our results indicate that augmentation of uAGT precedes albuminuria in young human adults with overweight and mice during the development of HFD‐induced T2D. These findings emphasize the relevance of uAGT and indicator of intrarenal/intratubular RAS activations and its potential as early biomarker of kidney injury during metabolic diseases as obesity and diabetes.
FoxO1 Promotes Liver Fibrosis through TGF‐β1 mediated Hepatic Stellate Cell Activation
Authors: Pan Quan , Gao Mingming , Ai Weiqi , Shen Zheng , Yang Wanbao , Sun Yuxiang , Qi Yajuan , Guo Shaodong
Published: May 2022

The O‐class of the forkhead transcription factor FoxO1 is a crucial factor mediating the action of insulin→PI3K→Akt and governs diverse cellular processes. To investigate the role of FoxO1 in control of liver fibrosis, we generated liver‐specific deletion of FoxO1 gene in mice (L‐F1KO). Using the mouse model, we injected intraperitoneally (i.p.) corn oil or 20% solution of carbon tetrachloride (CCL4) twice per week for 4 weeks (with 2.5 ul/g body weight) in WT and L‐F1KO mice. H&E staining and Sirius Red staining revealed that CCl4‐induced liver injury and fibrosis were significantly appeared, but the effects were largely attenuated in L‐F1KO mice. Moreover, we found that both mRNA and protein levels of TGF‐β1, a cytokine that activates hepatic stellate cell (HSC) for fibrogenesis, significantly decreased in the liver L‐F1KO mice compared to WT mice in response to CCL4 treatment. In primary hepatocytes, with FoxO1‐loss of function and gain‐of function, we confirmed the regulation of TGF‐β1 expression by FoxO1. Moreover, conditional medium (CM) collected from WT hepatocytes treated with CCL4 activated human HSC cell line (LX‐2), such effect was attenuated by FoxO1 deletion in primary hepatocytes or neutralization of TGF‐β1 in the CM using TGF‐β1 antibody. These results indicate a crosstalk between hepatocytes and HSC via FoxO1‐regualted TGF‐β1. To further confirm this concept in vivo, we overexpressed FoxO1 in the liver of WT and liver‐specific TGF‐β1 deficient (L‐TGF‐β1KO) mice with adenovirus expressing FoxO1 (adFoxO1), and injected (i.p.) these mice with CCL4. We found that FoxO1 overexpression in WT mice promoted CCL4‐induced liver fibrosis and HSC activation, and such effect was blocked in L‐TGF‐β1KO mice. Taken together, our data indicate that hepatic FoxO1 promotes CCL4‐inducled liver fibrosis via upregulating TGF‐β1 expression in hepatocytes and stimulating TGF‐β1‐mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.
Effects of physiological and supraphysiological concentrations of cannabidiol on pre‐adipocyte proliferation
Authors: Huang Mingyang , Zhu Ziyan , Caldari‐Torres Cristina
Published: May 2022

Cannabidiol (CBD) is one of the non‐psychotropic endocannabinoids from the Cannabis sativa plant. Studies support that CBD can modulate inflammation and lipid production in various cell lines, but its anti‐obesity effects are not well defined. Specifically, the effects of CBD on differentiation and proliferation of adipocytes are still unknown. Excessive fat accumulation can result in obesity, a worldwide public health problem with an increasing trend. Obesity increases the risk of developing chronic diseases. The purpose of this study was to determine the effects of different concentrations of CBD (0.2 [physiological] and 20 μM [supraphysiological]) on proliferation of pre‐adipocytes (3T3‐L1 cell line). Cells were cultured at 37°C in a humidified atmosphere (5% CO2) until they became pre‐confluent. Three groups of cells were set up: control (cells in complete growth media [CGM]), CGM supplemented with 0.2 μM CBD, and CGM supplemented with 20 μM CBD. Cells were plated in 96‐well plates in octuplicates in media containing the corresponding treatment (C, 0.2 μM CBD, or 20 μM CBD). Cells were incubated for 6, 12, 24 and 48 h, at which time points cell proliferation was measured using an MTS assay. At the 24 and 48 hour time points, cells treated with 20 μM CBD had higher cell proliferation (24 hours: 0.299 ± 0.006; 48 hours: 0.449±0.016) compared to the 0.2 μM group (24 hours: 0.262± 0.006; 48 hours: 0.217± 0.016) and the control group (24 hours: 0.249± 0.006; 48 hours: 0.312± 0.016) (p<0.0001, full‐factorial ANOVA). Interestingly, cells treated with 0.2 μM CBD had decreased proliferation at 48 hours compared to control cells, while there was no difference in proliferation between these two groups at 24 hours. Our results suggest that physiological and supraphysiological concentrations of CBD affect pre‐adipocyte proliferation differently and that further research is needed to clarify these effects.
Structural basis for Rab6 GTPase activation by the Ric1‐Rgp1 complex
Authors: Feathers J. R.
Published: May 2022

Membrane trafficking is a highly coordinated process in eukaryotes essential for maintaining lipid homeostasis and sorting proteins to the proper location inside cells. At the hub of this dynamic set of pathways, the Golgi complex directs much of this sorting through vesicle mediated transport. Vesicles of varying lipid composition loaded with different protein cargo are in constant flux through the Golgi and precise coordination of these processes requires communication between compartments. Rab GTPases are small signaling proteins that act as molecular switches to regulate vesicular trafficking. Rab6 functions at the Golgi and is important for endosome to Golgi retrograde transport in both yeast and metazoans and for anterograde exit from the TGN in metazoans. The Ric1‐Rgp1 protein complex is a highly conserved guanine nucleotide exchange factor (GEF) required for activation of Rab6. In mammalian cells Ric1‐Rgp1 is known to be regulated by the Rab33 GTPase, but it remains unresolved how the activity of the complex is regulated and how it interacts with Rab6. Furthermore, very little is known about the structure and domain organization of Ric1‐Rgp1. We have used cryo‐EM to determine the high‐resolution structure of the yeast Ric1‐Rgp1‐Rab6(Ypt6) complex, representing the key intermediate of the nucleotide exchange reaction. This structure reveals the overall architecture of the complex and has enabled us to identify the specific interactions that govern activation of Rab6. Ongoing studies aim to test the physiological significance of these interactions and determine the orientation of this complex on the Golgi membrane surface.