bluuebunny/crossref_metadata_2025 huggingface dataset:
Aquaporin 1 Modulates Apoptosis Susceptibility in Pulmonary Hypertension
Authors: Niedermeyer Shannon , Yun Xin , Suresh Karthik , Damarla Mahendra , Shimoda Larissa
Published: May 2022

Pulmonary arterial hypertension (PAH) remains a fatal diagnosis despite available therapies. PAH is characterized by extensive remodeling of the pulmonary vasculature involving the formation of vaso‐occlusive lesions and a thickened medial layer of the vascular wall, both of which contain pulmonary arterial smooth muscle cells (PASMCs). We have demonstrated that PASMCs isolated from a well‐established rat model of PAH are resistant to apoptosis under both basal and stimulated conditions. The cell membrane protein aquaporin 1 (AQP1) was initially described as a water transport channel, but more recently has been implicated in other cellular functions including migration and proliferation, and in several distinct cancer types, has been associated with apoptosis resistance. AQP1 is upregulated in PASMCs isolated from rat models of PAH, although its role in apoptosis resistance is unclear. We hypothesized that increased expression of AQP1 contributes to apoptosis resistance in PASMCs.

Methods

Our animal model is the Sugen‐hypoxia (SuHx) rat model of PAH in which Sugen‐5416, a vascular endothelial growth factor receptor type 2 inhibitor is injected into rats before they are placed in chronic hypoxia (10% O2) for 3 weeks, then returned to normoxia (21% O2) for 2 weeks. Control animals receive vehicle injection and are kept in normoxic (21% O2) conditions. Cells from distal pulmonary arteries are obtained via removal of the endothelium and enzymatic digestion of the tissue to obtain a pure smooth muscle population. To deplete AQP1, we transfected control and SuHx PASMCs with small interfering RNA (siRNA) directed toward either the AQP1 protein (siAQP1) or a non‐targeted sequence (siNT). Apoptosis was measured under basal conditions by Hoechst staining and visualization of DNA condensation. In complementary experiments, control PASMCs were infected with adenoviral constructs to over‐express AQP1 (AdAQP1) or green fluorescent protein (AdGFP; infection control). Cells were treated with either PBS or the apoptotic stimulus hydrogen peroxide (250 µm for 24 h). Apoptosis was again measured by Hoechst staining and visualization of DNA condensation.

Results

In both control and SuHx PASMCs, AQP1 knock down increased apoptosis under basal conditions. In control PASMCs with increased AQP1 expression, there was no difference in apoptosis under basal conditions. To further provoke a response in these infected cells, the apoptotic stimulus hydrogen peroxide was applied; however, again there was no difference in apoptosis levels between AdAQP1 and AdGFP infected cells.

Conclusions

These data suggest that increased AQP1 may be necessary but not sufficient for apoptosis resistance. Future studies further characterizing the role of AQP1 in apoptosis resistance are necessary to understand molecular mechanisms governing resistance to apoptosis in PASMCs and provide novel pathways for new therapeutic agents targeting vascular remodeling with the potential to reverse disease.
Localized Heat Therapy Improves Mitochondrial Respiratory Capacity but Not Fat Oxidation in Human Skeletal Muscle
Authors: Marchant Erik D. , Hyldahl Robert D. , Gifford Jayson R. , Hancock Chad R.
Published: May 2022

Exercise training (EX) imposes stress on skeletal muscle, resulting in improved mitochondrial respiratory capacity and fat oxidation. Heat, which is one of the stresses of exercise, has been previously shown to increase mitochondrial respiration in human skeletal muscle. Still, it is unknown how the effects of localized heat therapy (HEAT) compare to the effects of EX, or if HEAT improves fat oxidation capacity. Therefore, the objective of this study was to determine the extent to which 6 weeks of HEAT improve mitochondrial function compared to EX. We hypothesized that HEAT would increase mitochondrial respiratory capacity, fatty acid oxidation and citrate synthase (CS) activity, but to a lesser extent than EX. Subjects were randomized into three groups: HEAT (n=11), EX (n=8), and a sham control group (CON, n=12). Subjects in the HEAT group were exposed to 2 hrs/day, 3 days/wk of short‐wave diathermy, which raised internal muscle temperature by 3.2 ± 0.33° C, while CON subjects experienced no change in muscle temperature during sham diathermy sessions. EX consisted of single‐leg extension exercise on a modified cycle ergometer for 45 min/day, 3 days/week, and resulted in a 1.8 ± 0.42° C increase in muscle temperature. Muscle biopsies were performed at baseline, and after 3 and 6 weeks of intervention. Small muscle fiber bundles (2‐4 mg wet wt.) were permeabilized, and mitochondrial respiration was assessed using high‐resolution respirometry (Oroboros O2k, Innsbruck, Austria). CS and 3‐hydroxyacyl‐CoA dehydrogenase (HAD) activity assays were performed in duplicate on supernatant from homogenized muscle samples. At 6 weeks, EX resulted in 27.9 ± 8.7% and 33.2 ± 11.7% increases in maximal coupled and uncoupled respiration, respectively, which were significantly different from baseline (p<0.05). At 6 weeks, HEAT resulted in 24.8 ± 6.2% and 24.5 ± 6.2% increases in maximal coupled and uncoupled respiration, respectively, which were significantly different from baseline (p<0.05), but not different from EX. Fatty acid oxidation was improved only in EX subjects after 6 weeks (29.5 ± 6.8%), compared to baseline (p<0.05), with no change in HEAT or CON subjects. CS activity increased in EX subjects (25.9 ± 10.5 µmol·min‐1·mg protein‐1 or 19.0 ± 7.4%) compared to all others (p<0.05), but no changes were observed in HEAT or CON subjects. Furthermore, no change in HAD activity occurred in any group. These results suggest that HEAT improves mitochondrial respiratory capacity to a similar extent as EX but does not result in other EX‐induced adaptations like increased fat oxidation and CS activity. Still, HEAT‐induced improvements in respiratory capacity may have therapeutic potential in the context of skeletal muscle disease or disuse, and further investigation is warranted.
Topographical Distribution and Morphology of CGRP‐IR Axons in the Flat‐Mounts of Whole Mouse and Rat Hearts
Authors: Chen Jin , Bendowski Kohlton , Bizanti Ariege , Zhang Yuanyuan , Madas Jazune , Ma Jichao , Cheng Zixi J.
Published: May 2022

To understand and treat cardiac pain, it is important to have an understanding of the topographical distribution of nociceptive axons in the heart. While previous studies have detected nociceptive axons in the sectioned heart, their full distribution has not yet been determined. In this study, we used a nociceptive marker calcitonin gene‐related peptide (CGRP) to determine the distribution of nociceptive afferent axons in the whole heart. We first removed the hearts of male Sprague‐Dawley rats (n=6), and C57BL/6 mice (n=6). The hearts were dissected into left and right atria and ventricles and interventricular septum, and prepared as flat‐mounts. These samples were then processed with immunohistochemical labeling using a primary antibody which binds to CGRP and an Alexa Fluro 488 secondary antibody for fluorescence. The flat‐mounts were imaged with a Zeiss M2 Imager (automatic fluorescence microscope) using a 488 nm excitation wavelength. In both rats and mice, we found that CGRP‐IR nerve bundles entered the heart and innervated the left and right atria and ventricles. For the right/left atria, CGRP‐IR axons entered as large bundles near the superior/inferior vena cava, left pre‐caval vein and the pulmonary veins before bifurcating into small branches, and finally formed single varicose axons and terminals which distributed throughout the tissue, including cardiac ganglia, the SA/AV nodes, auricles, and the blood vessels. In the right/left ventricles and interventricular septum, CGRP‐IR axons entered as large bundles through the base before bifurcating into small branches towards the apex Ultimately, single varicose axons and terminals innervated myocardium and blood vessels. Our data shows the distribution of CGRP‐IR axons in the whole heart at single cell/axon resolution. With this information, we will gain insight into the function of these axons and the pathways that they follow, paving the way for the development of better treatments for cardiac pain and disease. The first two authors contributed equally to this work.
Spexin Co‐localizes with Insulin in Pancreatic Islet Cells of Normal and Diabetic Rats
Authors: Adeghate Ernest , D'Souza Crystal , Ali Ifrah I. , Saeed Zulqarnain , Alharthi Saleh H. , Alhadhrami Zayed K. , Alhadhrami. Saif K. , Alhajri Helal S. , Tariq Saeed
Published: May 2022

Spexin is a newly discovered gastrointestinal tract (GIT) peptide. Preliminary biochemical analysis showed that spexin was localized to secretory granules in a transfected pancreatic cell line. Spexin was also expressed in the submucosal cells of the mouse stomach indicating that it is a resident peptide in the GIT and may thus influence pancreatic function. It has been suggested that spexin may play a role in the regulation of physiological homeostasis. Our aim was to determine whether spexin is present in the pancreas of normal and streptozotocin‐induced diabetic rats. We also wanted to know whether spexin co‐localizes with pancreatic hormones in the islet of Langerhans. Immunohistochemical, immunofluorescence and transmission electron microscopy (TEM) methods were used to determine the cellular localization of spexin in the endocrine pancreas of non‐diabetic and diabetic rats. Immunofluorescence study showed that a significant number of cells in the core of rat pancreatic islet contains spexin, where insulin‐positive cells are located. We used double labelling immunofluorescence method to determine whether spexin and insulin co‐localize together in the same cell. We observed that these spexin‐immunoreactive cells also contain insulin. There was no evidence of co‐localization of spexin with either somatostatin or pancreatic polypeptide. The number of spexin‐ and insulin‐immunopositive cells was significantly (p <0.05) reduced after the onset of diabetes mellitus. Moreover, TEM showed that the secretory granules of pancreatic beta cells contain spexin‐labelled immunogold particles. In conclusion, the presence of spexin in pancreatic beta cells suggests a role in the regulation of pancreatic endocrine function.
Dysbiosis in the Mouse Esophagus Caused by CaSR Deletion
Authors: Abdulnour‐Nakhoul Solange , Nakhoul Nazih
Published: May 2022

Background

We have previously reported a mouse model, where we knocked out the calcium sensing receptor (CaSR) in the esophageal stratified squamous epithelium (EsoCaSR‐/‐) using Cre recombination. The esophageal tissues showed morphological changes including elongation of the rete pegs, abnormal keratinization and stratification, and a remarkable bacterial build‐up on the luminal epithelial surface. In addition, the barrier properties of the epithelium were altered as indicated by a decrease in cell‐cell junction proteins and increased permeability.

Objective

We aimed to quantify and characterize the mucosal‐associated bacterial microbiome caused by deletion of CaSR in the esophagus.

Methods

To explore the bacterial buildup in EsoCaSR‐/‐ esophageal tissue, we analyzed the mucosal‐associated bacterial microbiome and compared it to the one in control EsoCaSR+/+ mice. Total bacterial load was estimated by quantitative polymerase chain reaction (qPCR) of 16S ribosomal DNA (rDNA) using SYBER green. We sequenced 16S rRNA targeting the hypervariable V3‐V4 region and performed gene profiling and metagenomic analysis.

Results

Quantification of 16S DNA by qPCR indicated ~1.5‐fold increase in bacterial load in EsoCaSR‐/‐as compared to control. Sequencing data indicated significant changes in the microbiome composition of EsoCaSR‐/‐. Phylum P‐Cyanobacteria was increased in EsoCaSR‐/‐, family F_Burkholderiacea and order o_Betaproteobacteria were decreased. The microbe species s_Rodentibacter, s_Lactobacillus_hilgardii were enriched in CaSR KO mice while s_Curvibacter_unclassified, s__Streptococcus_equinus, s_Lactobacillus_crispatus and s_Millionella_massiliensiswere decreased. Linear discriminant analysis (LDA) detected 3 bacterial species; g‐Rodentibacter, s_Rodentibacter_unclassified,and s_Lactobacillus_hilgardi showed statistically significant and biologically consistent differences between control and EsoCaSR‐/‐ tissues. Metagenomic analysis indicated a significant decrease in metabolic pathways and bacterial enzymes.

Conclusions

This is the first report on dysbiosis caused by CaSR deletion in the esophagus. Our data demonstrate a direct in‐vivo interaction between esophageal epithelia and bacteria and could shed light on cellular factors modulating the microbiome.
The Codependent Expression of the Essential Mammalian RNA Polymerase I PAF49/PAF53 Heterodimer
Authors: McNamar Rachel , Knutson Bruce , Rothblum Lawrence
Published: May 2022

Ribosome biogenesis (RB) is a complex process that plays a central role in maintaining cellular homeostasis and supporting cell growth. Therefore, dysregulation of RB contributes to pathologies such as cancer, ribosomopathies, neurodegenerative diseases, and aging. Our lab studies transcription of ribosomal RNA by RNA polymerase I (Pol I), the rate‐limiting step in ribosome biogenesis. Many studies have demonstrated that inhibiting Pol I transcription in cancer cells causes cell death while untransformed cells arrest, thereby making the Pol I apparatus a target for chemotherapeutic agents. This study focuses on two mammalian polymerase associated factors of the Pol I transcription apparatus, PAF49 and PAF53. The PAFs form a heterodimeric complex and are required for rDNA transcription and cell proliferation. The goal of this study is to determine the role(s) of PAF49 and PAF53 in rDNA transcription and understand how these two proteins are regulated.

To rapidly knock down (KD) either PAF49 or 53 in HEK293 cells, a novel system that utilizes CRISPR/Cas 9 and an auxin inducible degron was used. Currently, we are using this system to determine if KD of PAF49 effects the expression of PAF53 and vice versa. Our data demonstrates that KD of PAF49 causes the rapid depletion of PAF53 protein levels while KD of PAF53 has no effect on PAF49. Additionally, we have found that the dimerization domain of PAF49 is not sufficient to stabilize PAF53. Rescue of PAF53 protein levels requires PAF49 to dimerize and bind to the polymerase. Dimerization to PAF53 and binding to the polymerase is also necessary to support cell proliferation and rDNA transcription. Further, KD of PAF49 or PAF53 has no effect on the protein levels of the core subunits of Pol I. These findings are significant because they aid in further understanding the process of rDNA transcription by Pol I. Additionally, they will contribute to the discovery of novel drug targets that could be utilized in effective cancer treatments. For example, disrupting the interaction between PAF49 and Pol I should inhibit Pol I transcription and cause cell death in cancer cells.
Employing Spherical Harmonics in Assessing the 3D Shape Variation and Evolutionary Potential of Human Sphenoidal Sinuses
Authors: Ryan Katharine G. J. , Auerbach Benjamin , Sylvester Adam , Butaric Lauren
Published: May 2022

The evolutionary potential and shape variation of the human sphenoidal sinus are unknown. Moreover, a consensus about the function or evolutionary forces that influence variation of paranasal sinuses among humans remains unestablished. One initial approach to address this lies in determining the covariance of sinus shape with surrounding morphology. The sphenoidal sinus is centrally located within the basicranium at the intersection of the three cranial fossae, and is adjacent to the synchondroses where primary cranial base angulation occurs. The angulation of the cranial base and its relative orientation with the face is a distinctive, derived trait in Homo sapiens and is important to our understanding of the evolution of our species. Yet, the shape of the sphenoidal sinus is highly variable and cannot be quantified with traditional landmarking methods. Originally developed in gravitational physics, spherical harmonics has been successfully leveraged in biological morphological studies to quantify and assess 3D shape variation of complex structures. This research will use spherical harmonics to for the first time quantify the 3D shape of the sphenoidal sinuses and assess their covariation with the sphenoid body. By demonstrating the utility of spherical harmonics for human morphometric research, we are opening up new ways for investigating biological and evolutionary questions of human anatomical features that to date have been difficult to evaluate.

Here we preliminarily analyze the covariance of sphenoid sinus and basicranial shape. The sphenoidal sinuses of 120 human crania were segmented (Avizo) from CT scans. We quantified the three‐dimensional shape variation of sphenoidal sinuses using spherical harmonic analyses (SHARM, Matlab), producing coefficients comparable to traditional landmarks placed virtually on the basicranial surface. Landmarks were chosen that capture the general shape of the sphenoid body. Two‐Block Partial Least Squares (2B‐PLS) analyses revealed no significant covariation (p=0.59) between the shape of the sphenoidal sinus and its surrounding bony architecture. This indicates the establishment of sinus shape occurring independently of the surrounding bone. This is contrary to previous research, which has suggested that paranasal sinuses opportunistically pneumatize within the available space of their respective cranial bones. Future research will need to investigate this relationship further with additional sets of landmarks representing different developmental regions and functional matrices that interface with the sphenoid body. This research is also foundational to investigate to what extent mutual constraint among these traits may exist in the ability to respond to natural selection, i.e. evolvability.
Evaluation of Loaded Breathing on Inspiratory Intercostal Muscle Activation During High Frequency Spinal Cord Stimulation to Restore Breathing in Spinal Cord Injury – Animal Model
Authors: Kowalski Krzysztof E. , DiMarco Anthony F.
Published: May 2022

Background

High Frequency Spinal Cord Stimulation (HF‐SCS) is a unique method of inspiratory muscle activation and has the potential to restore breathing in ventilator dependent tetraplegic subjects. However, these subjects are 3‐4 times more likely to have sleep disordered breathing (SDB) and have a higher incidence of obstructive airways disease than individuals in the general population, resulting in an increased inspiratory load.

Objective

To examine the effects of increased inspiratory load (airway occlusion) on inspiratory intercostal muscle activation during HF‐SCS.

Methods

In 3 spinalized (C2 level) anesthetized dogs, recording electrodes were positioned in the parasternal (PA) and external (EI) intercostal muscles (2nd and 3rd interspaces respectively) to record multiunit (integrated) and single motor units (SMU) EMG activities. During HF‐SCS at functional residual capacity (FRC), respiratory muscle activity was assessed before and after single and multi‐breath airway occlusion.

Results

During HF‐SCS, tracheal occlusion resulted in augmentation of peak multi‐unit integrated EI, but little change in PA EMG activity. During the occluded breath, EI activity increased to 148 ± 3% of that observed during control breaths (p<0.01) whereas PA activity was little changed (105 ± 5%; NS). EI instantaneous SMU peak firing frequency (PFF) was also greater during the occluded breath (15.7 ± 1.6Hz vs. 11.3 ± 0.3Hz; p<0.05). whereas there was only a small increase in PA PFF (10.8 ± 0.6Hz vs. 10.0 ± 0.6Hz; NS).

HF‐SCS during airway occlusion results in significant increases in activation of the EI but not PA muscles.

Conclusion

HF‐SCS applied to support ventilation in high tetraplegics with airway obstruction may elicit compensatory increases in EI muscle activation. These results support the concept that spinal reflex effects remain functional during HF‐SCS. The EI have a much higher density of muscle spindles compared to the PA muscles, which may mediate the differential activation of these muscles.
Electrical Stimulation of the Dorsal Motor Nucleus of the Vagus Attenuates Serum Pro‐Inflammatory Cytokine Levels in Murine Models of Endotoxemia and Sepsis
Authors: Falvey Aidan , Palandira Santhoshi P. , Tracey Kevin J. , Pavlov Valentin A.
Published: May 2022

The inflammatory reflex is a vagus nerve‐mediated and brainstem‐integrated physiological mechanism that regulates immunity and inflammation (Annu Rev Immunol, 2018, 36:783). The efferent vagus nerve originates in brainstem nuclei, including the dorsal motor nucleus of the vagus (DMN). Recently, it was shown that optogenetic stimulation of cholinergic DMN neurons inhibits serum TNF levels during endotoxemia (Proc Natl Acad Sci USA, 2020, 117(47):29803). However, the effects of electrical DMN stimulation on cytokine levels in inflammatory conditions have not been previously evaluated. To provide insight, we examined the effect of electrical DMN stimulation in mice with endotoxemia and cecal ligation and puncture (CLP)‐induced polymicrobial sepsis. In the endotoxemia model, 10–12‐week‐old male C57BL/6 mice (n=14‐20 per group) were anesthetized using isoflurane and the head was positioned on a stereotaxic frame. A surgical intervention and a stereotaxic‐guided approach were used to insert a concentric bipolar electrode into the left DMN. Electrical stimulation (50 μA, 30 Hz, 260 μs) or sham stimulation was performed for five mins, followed by lipopolysaccharide (LPS, 0.5 mg/kg) i.p. administration. The mice were euthanized 90 mins after LPS and blood (via cardiac puncture) was collected and processed for cytokine analysis. In the CLP model, DMN stimulation or sham stimulation was performed as described above immediately after subjecting mice (n=14‐20 per group) to CLP, i.e., following a midline laparotomy in anesthetised mice, the cecum was isolated and the distal end was ligated and punctured. 24h later, the mice were euthanised and blood was collected and processed for analysis. In mice with endotoxemia, electrical DMN stimulation significantly decreased serum TNF (p = 0.0069) and IL‐6 (p = 0.0427) levels compared with sham stimulation. In addition, electrical DMN stimulation increased the serum levels of the anti‐inflammatory cytokine IL‐10 (p = 0.0284). In mice with CLP, electrical DMN stimulation resulted in significantly lower serum TNF levels compared with sham stimulation (p = 0.0291). These results indicate for the first time that electrical DMN stimulation alters circulating pro‐inflammatory and anti‐inflammatory levels in murine endotoxemia and sepsis. These findings are of interest for further development of bioelectronic approaches targeting DMN for therapeutic benefit in conditions, characterised by immune dysregulation and inflammation.
Structural analysis of G protein β5 subunit folding by the cytosolic chaperonin CCT/TRiC and its co‐chaperone phosducin‐like protein 1 reveal molecular mechanisms of chaperone‐mediated β‐propeller protein folding
Authors: Sass Mikaila , Ludlam William G. , Wang Shuxin , Kwon Yujin , Carter Ethan , Bohman Joseph , Shen Peter S. , Willardson Barry M.
Published: May 2022

The cytosolic chaperonin CCT and its co‐chaperone phosducin‐like protein 1 (PhLP1) play important roles in G protein complex assembly by folding G protein β subunits (Gβ) into β‐propeller structures. To understand this process at the molecular level, we have isolated the CCT‐Gβ5‐PhLP1 folding intermediate in both the open and closed CCT conformations and determined its structure by high resolution cryo‐electron microscopy (cryo‐EM). In the open structures, Gβ5 interact with the N‐ and C‐termini of the CCT subunits deep inside the folding chamber between the CCT rings in a closed β‐propeller conformation. PhLP1 allosterically enhances Gβ5 binding to CCT by interacting with the CCT apical domains at the rim of the folding chamber without contacting Gβ5 directly. In the closed CCT structures, Gβ5 move from between the CCT rings into one of the folding chambers, suggesting a path for release of Gβ5 from CCT during its ATPase cycle. These findings provide a molecular explanation for CCT‐dependent folding and release of Gβ5 to interact with RGS proteins and perform their essential functions in G protein signaling.
Small molecule inhibitors of human RGS2 developed by structure based drug design approach
Authors: Printy Natalie , Bair Adam , Choi So‐Hee , O'Brien Joseph , Wilkinson Joshua , Roman David , Kinder David , Mahfouz Tarek M.
Published: May 2022

Regulators of G‐protein signaling (RGS) is a family of approximately 30 proteins that bind to the alpha subunits of G‐proteins (Galpha) and accelerate their GTP hydrolysis rates. This deactivates the G‐protein and terminates G‐Protein Coupled Receptors (GPCRs) signaling. RGS proteins, therefore, play important roles in regulating GPCR signaling and most members are implicated in human diseases such as hypertension, depression, and others. Regulator of G‐protein Signaling 2 (RGS2) is a regulator of angiotensin‐II receptor signaling and a modulator of oxytocin receptor signaling as well. More importantly, RGS2 was reported to be over expressed in the majority of solid breast cancers and in metastatic prostate cancer. For this reason, we sought to develop RGS2 inhibitors as potential chemotherapeutic agents. We utilized structure‐based drug design approaches to develop inhibitors of RGS2‐Galpha‐q interactions. Available structures of the RGS2‐Galpha complex were used to extract a pharmacophore model for searching of commercially available chemical databases. The identified hits were docked to different RGS structures to screen for compounds with the highest binding potential and most selectivity towards RGS2. We report the first group of inhibitors of RGS2‐Galpha‐q interactions developed through rational drug design that interfered with RGS2 signaling in cell‐based assays. In addition, inhibitor AJ‐3 inhibited the growth of MCF‐7 breast cancer cells in cell culture assays, which suggests that RGS2 inhibitors may have a potential to be a new class of chemotherapeutic agents.
Biochemical Investigation of a Tryptophan Biosynthetic Enzyme in Plants
Authors: Li Miriam , Holland Cynthia
Published: May 2022

While humans require the essential amino acid Trp from their diet, plants, fungi, and bacteria have retained the enzymes necessary to synthesize Trp de novo. The five‐step pathway starts with the product of the shikimate pathway, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step of the pathway by anthranilate phosphoribosyltransferase (PAT1). Aside from the characterization of blue‐fluorescent trp1 mutants, PAT1 remains to be biochemically investigated. Using steady‐state kinetics, the regulation and substrate‐specificities of PAT1 from Arabidopsis thaliana and from Citrus sinensis were investigated. Active site mutants were generated to trace the evolution of specificity and regulation in these two enzymes. Because Citrus spp. siphon anthranilate from the Trp pathway and use it for synthesizing defense metabolites, like acridone alkaloids, our findings may explain how plants evolved a pool of anthranilate to use in specialized metabolism.
Effects of Poly‐Substituted Pyrroles on Pro‐Inflammatory Signaling in RAW264.7 Macrophages
Authors: Offenback Gabriel H. , Fishberg Cole L. , Wang Annie L. , Brown Madeline M. , Gupton John T. , Fischer‐Stenger Krista
Published: May 2022

Inflammation is the body’s natural means of protection against infection and the restoration to homeostasis after cellular and tissue damage from injury or metabolic stress. A multitude of complex signaling pathways are necessary for the activation of pro‐inflammatory signaling in macrophages, and the dysregulation of these pathways can contribute to disease states linked to chronic inflammation making them attractive targets for therapeutics. Our laboratory explores the potential of a class of poly‐substituted pyrrole compounds that are known microtubule depolymerizers to regulate the inflammatory response in macrophages. Previous work from our laboratory demonstrated that JG‐03‐14 (3,5‐dibromo‐4‐(3,4‐dimethoxyphenyl)‐1H‐pyrrole‐2‐carboxylic_acid ethyl ester) and its refined analog, NT‐07‐16, which possesses an additional methoxy group at the 2‐position of the phenyl ring decrease the production of pro‐inflammatory cytokines in lipopolysaccharide (LPS)‐activated RAW264.7 murine macrophages. Our results demonstrated that the reduction in pro‐inflammatory mediators is due, in part, to a decrease in the nuclear translocation of NFκB, a critical transcription factor for pro‐inflammatory signaling. These results are supported since microtubule dynamics is thought to be involved in NFκB nuclear translocation. Here we expand the scope of our previous work by exploring the effects of NT‐07‐16, and a potentially more potent stereoisomer mix of the NT analog, NT‐07‐45 on other pro‐inflammatory signaling pathways in the RAW264.7 macrophages. Recent studies have shown that exposure to NT‐07‐45 decreases the production of the antiviral and pro‐inflammatory cytokine interferon‐beta (IFN‐β) in macrophages. In addition, the NT analog alters the phosphorylation of the interferon regulatory factor (IRF3) which decreases its activation and could affect its ability to enter the nucleus and induce IFN‐β production. Our recent work has also found that exposure of RAW264.7 macrophages to the NT analogs alters the phosphorylation, and possibly the activation, of c‐Jun N‐terminal kinase (JNK), an important MAP‐Kinase (MAPK) involved in macrophage inflammatory signaling. These findings indicate that the decrease in the production of pro‐inflammatory mediators observed in RAW264.7 macrophages exposed to polysubstituted pyrroles, may be due to the inability of several transcription factors involved in the inflammatory response to efficiently translocate into the nucleus. While much is still to be learned about the anti‐inflammatory mechanisms of these pyrrole compounds it appears that the effects may go beyond their microtubule‐depolymerizing activity.
IRBIT as a Regulator of Bicarbonate Transport in the Small Intestine
Authors: Sarthi Jessica B. , van Unen Vincent , Chan Joshua E. , Abazari Shayda , Trumbull Annie , Guh‐Siesel Alexander , Kuo Calvin J. , Sellers Zachary M.
Published: May 2022

Background

Small intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5‐trisphosphate (IP3) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown.

Aim

To examine duodenal expression and function of IRBIT in regulating bicarbonate transport.

Methods

Duodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical‐in or flipped apical‐out enteroids. mRNA expression was evaluated using qPCR and re‐analysis of a duodenal sc‐RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back‐titration.

Results

IRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5‐fold higher expression than Calu‐3 airway submucosal gland cells or HEK293 kidney‐derived cells (n=1 each). Analysis of duodenal sc‐RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt‐villus axis, primarily localizing in E‐cadherin positive epithelial cells, but also present in the lamina propria. In patient‐derived duodenal enteroids, IRBIT was expressed in both crypt‐like (undifferentiated) and villus‐like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc‐RNAseq analysis, AHCYL1 and SLC26A3 were co‐expressed in 38% of all villus cells, compared to only 5% co‐expressing AHCYL1 and SLC26A6. CFTR‐independent duodenal bicarbonate secretion stimulated by linaclotide (10‐7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10‐6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh‐A250, 10‐5 M; 68±8%, n=10). Linaclotide stimulation of apical‐out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking.

Summary and Conclusions

IRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition.
Exploring Protein Structures of SARS‐CoV‐2 Variants of Concern within an Undergraduate Research Course
Authors: Van Stry Melanie , Billings Ryan , Cantero Alondra , McCullough Mariah
Published: May 2022

Lane College is a Historically Black College with a legacy of educating underserved minority students. As a primarily undergraduate teaching college, faculty and students have limited opportunities to engage in undergraduate research. To address this concern, the biology department created the undergraduate research course sequence (Undergraduate Research I and II) to provide course‐based undergraduate research experiences for students. During the Spring 2021 semester, to limit the spread of COVID‐19, the College began the first few weeks with complete online instruction before switching to hybrid or face‐to‐face modality. For the biology undergraduate research students, the projects were redesigned based on input from the students to explore the viral protein structural changes in newly emerging SARS‐CoV‐2 variants of concern (VOC). First, students explored the virology of SARS‐CoV‐2 and structures of key viral proteins using curriculum available from Rutgers University on the Protein Data Bank during the online instruction period. Then the students selected a variant of concern, B.1.1.7 (Alpha), B.1.351 (Beta), or P.1 (Gamma), and a specific viral protein containing at least one mutation for further study. During the hybrid instruction period, using open‐source bioinformatics tools, including NCBI SARS‐CoV‐2 genome sequences, PANGO Lineage VOC data, UCSC Genome Browser and SNAP Gene, the students mapped the location of the VOC mutation on their chosen protein’s known structure. They then modeled the effect of the mutation on the protein structure using Chimera or iCN3D, since Chimera is not compatible with Chromebooks. Students determined that the mutations found in nsp3 in Alpha variant and ORF8 in Gamma variant did not have a large impact on the overall structure of the proteins and predicted that there would not be significant effects on protein function. In contrast, several mutations found in Beta variant spike protein likely impact receptor binding domain interactions with angiotensin converting enzyme 2 receptor. The methods developed here will be useful for further investigations of protein structures from emerging variants of concern. This experience demonstrates the utility of bioinformatics research projects in online and hybrid courses and the importance of consideration of compatibility of various open‐source tools with student devices.
Neuroscience Core Concepts: Implementation in Varied Courses at Varied Institution Types
Authors: Schaefer Jennifer E. , Chen Audrey , Phillips Kimberley A. , Sonner Patrick M.
Published: May 2022

Consensus core concepts are overarching principles that provide a framework for organizing facts and understanding in higher education curricula. Physiology educators have long benefitted from published core concepts in physiology (Michael and McFarland, 2011; Michael et al., 2017), but neuroscience core concepts were only recently developed (Chen et al., in press). The need for core concepts to structure neuroscience education is particularly pressing given the rapid expansion of neuroscience knowledge, programs, and curricula in recent years. The eight empirically‐developed, consensus neuroscience core concepts are labeled as communication modalities, emergence, evolution, gene‐environment interactions, information processing, nervous system functions, plasticity, and structure‐function. This presentation will review these neuroscience core concepts and demonstrate how they can be implemented in different courses (Human Anatomy and Physiology, Introductory Neuroscience, Advanced Neuroscience, Neuroethics, and Sensation and Perception), for different levels (non‐majors, lower‐division majors, upper‐division majors), and at different institution types (small liberal arts, large state institution).
Do We Have a New Psoriasis Drug?
Authors: Šola Marija , Skroza Nevena , Mangino Giorgio , Škrtić Anita , Seiwerth Sven , Sikirić Predrag
Published: May 2022

Pentadekapeptide BPC 157 was used in numerous laboratory rat animal models with proven cytoprotective and organ protective properties. In different wound therapy (incisional/excisional wound, deep burns, diabetic ulcer, alkali burns) fast and complete tissue restitution was achieved. BPC 157 was found to rapidly stimulate the expression of various genes involved in the healing process of the skin and mucosal lesions in these models. Psoriasis is a chronic, relapsing, immune‐mediated skin disease pathogenically driven by proinflammatory cytokines of which: TNF‐alpha, IL‐12, IL‐17, IL‐23 play a key role. Since BPC 157 has shown a significant anti‐inflammatory effect in several studies, the aim of this study is to show that it has the same role in the regression of inflammatory skin lesions such as those in psoriasis. An experimental model of psoriasis in laboratory animals is easily feasible, using 5% imiquimod topically.

MATERIALS AND METHODS The experiment is done on male Wistar Albino rats. Animals are divided into groups of 6. „Psoriasis‐like” lesions are induced in all animals applying 5% imiquimod cream topically on shaved skin on their backs for 7 consecutive days. On the 7th day, 3 hours after applying imiquimod cream treatment starts usage BPC 157 in cream (conc. 1μg/g neutral cream) or only neutral cream (Belobaza) in control groups. Animals are treated and observed daily, video documentation was made. Animals are treated until the sacrifice day according to schedule. The severity of skin lesions is measured clinically using a modified version of Psoriasis Area Severity Index‐PASI score. Modified PASI includes scoring the following lesion features: erythema, infiltration, desquamation on scale 1‐4; maximum modified PASI value is 12. Two way ANOVA and Bonferroni post hoc test were used for statistical analysis. Data represented as mean SD for N=6/group.

RESULTS Animals treated using BPC 157 conc.1μg/g in a cream had significantly lower values of PASI score when compared to control group. Faster resolution of imiquimod induced „psoriasis‐like” lesions was achieved using BPC 157 when compared to control group.

CONCLUSION BPC 157 is well‐known peptide used in various experimental models without any side effects of it's use, with proven effects on the NO‐system, and excellent results in wound healing. Psoriasis is a rather common immune‐mediated skin disease driven by proinflammatory cytokines. TNF‐alpha, IL‐12, IL‐17, IL‐23 play a key role in the pathogenesis of psoriasis. It is undisputably that the use of BPC 157 topically successfully neutralizes the effects of imiquimod and induces regression of „psoriasis‐like“ lesions, the question that needs to be answered yet is whether BPC 157 has an impact on one of the above‐mentioned cytokines. It is yet to be explored which mechanism is the key to it´s beneficial effects in treating „psoriasis‐like“ lesions in experimental models.
Heart Rate Variability Parameters Indicate Altered Autonomic Tone in Patients with COVID‐19
Authors: Gruionu Gabriel , Gupta Anita , Rattin Megan , Nowak Thomas V. , Ward Matthew , Everett Thomas H.
Published: May 2022

The COVID‐19 disease induces long term heart health complications and may induce autonomic nervous system dysfunction. Heart Rate Variability (HRV) is a measure of sympathetic (SNS) and parasympathetic (PNS) control of heart function. Recently, studies have shown that HRV analysis may be used as a predictor of COVID‐19 symptoms and correlates with progression of the disease. We aimed to uncover the interplay between SNS and PNS in hospitalized COVID‐19 patients at the time of admission and compare it with similar measurements in healthy patients (no comorbidities) and patients with cardiovascular disease. We hypothesized that COVID‐19 would induce autonomic dysfunction similar to patients with cardiovascular disease (CVD). ECG telemetry recordings of 30‐60 minutes in duration were acquired from patients that were admitted to Indiana University Health system hospitals for either COVID‐19 complications or for complications associated with cardiovascular disease (CVD) states (arrhythmia, heart failure, coronary artery disease). In addition, 20‐minute ECG Lead I recordings were obtained from healthy volunteers with no associated comorbidities. HRV parameters were calculated during sinus rhythm in the time, frequency, and nonlinear domains from the ECG telemetry recordings. The patient population was composed of 50 COVID‐19 patients (average age 63, range 26‐94), 32 healthy (average age 32.7, range 17‐69) and 49 patients with cardiovascular disease (average age 65.4, range 30‐88) as control groups. The COVID‐19 group had a higher percentage of patients with BMI>30 (obese) than the control groups (55% vs 36%). Also, the COVID‐19 and CVD patients had significantly higher heart rate and time‐domain HRV parameters (including SDRR, RMSSD, SDSD) and SD1 in the non‐linear domain when compared to healthy patients (88.8±53.0 and 87.9±55.2 vs 49.5±31.3, p<0.01). In the frequency domain, the LF/HF ratio was significantly lower in the COVID and CVD groups compared to healthy controls (0.5±0.76 and 0.55±0.50 vs 1.05±0.96, p<0.01). COVID‐19 patients have significant HRV alterations which suggest increased vagal tone than in healthy volunteers but similar to patients with severe cardiovascular disease comorbidities. Even though the COVID patients had an increased heart rate, the results of the HRV analysis indicate increased vagal tone which would support autonomic nervous system dysfunction in these patients.
Investigating the Anticancer Activity of Novel Norethindrone Derivatives in PC3 Prostate Cancer Cells
Authors: Hollenbacher Courtney A. , Harris Katelyn A. , Reid Nicholas A. , Schneider Ryan A. , Dudley Richard W.
Published: May 2022

It is estimated that over 248,000 new cases of prostate cancer will be diagnosed this calendar year (2021) representing upwards of 20% of newly diagnosed cancer cases in men.1,2 Despite these numbers, prostate cancer‐related deaths have significantly declined over the period of time from 1993 to 2016, yet prostate cancer was expected to be responsible for almost 10% of cancer‐related deaths in 2018.1 Metastasis and cancer cell resistance results in treatment failure and progressive disease, and this remains a significant concern in the care of men with prostate cancer.

To address the significant need for novel agents in the treatment of resistant prostate cancer, we designed and synthesized a small library of norethindrone (NE) derivatives with varying substituents attached to the 17‐position (Compounds A‐D) with the hopes of identifying androgen receptor antagonists. Click chemistry was utilized to facilitate the formation of a triazole linking NE to alkyl and aryl substituents. The corresponding azides were synthesized in situ from alkyl bromides or aryl bromides in a one‐pot microwave‐assisted reaction in an Anton Parr Monowave‐50 reactor at 150℃ for 20 minutes.

Here we present initial data of our small library of NE derivatives which were screened for anticancer activity using the XTT cell viability assay in PC3 prostate cancer cells. Two of the compounds (A and D) failed to induce an anticancer response. Compound B demonstrated weak anticancer activity with an IC50 of greater than 100 µM, whereas Compound C demonstrated low micromolar activity with an IC50 of 19 µM. Further studies are ongoing to determine the mechanism(s) by which Compound C elicits its anticancer activity in the metastatic‐derived PC3 prostate cancer cells. Additionally, these results will assist with further rational design using NE as a scaffold in an attempt to increase the potency against PC3 and other prostate cancer cell lines.

1. American Cancer Society. Cancer Facts & Figures 2021. Atlanta: American Cancer Society; 2021

2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019 Jan;69(1):7‐34. doi: 10.3322/caac.21551. Epub 2019 Jan 8. PMID: 30620402.
A Novel Role for Glucocerebrosidase 1 (GBA1) in Parkinson’s Disease
Authors: Jayabalan Nanthini , Roper Katherine (Kerry) , Woodhouse Helen M. , Adam Robert J. , O’Sullivan John D. , Gordon Richard
Published: May 2022

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, yet the pathological mechanisms driving disease onset and progression are not well understood. Glucocerebrosidase 1 (GBA1), the gene encoding the lysosomal enzyme glucoslyceramidase (GCase), has been identified as the most important genetic risk factor contributing to the development of PD. In this study, we aimed to investigate the relationship between inflammasome activation and GCase dysfunction in immune cells in PD. Primary microglial was generated from brains of P0‐ P2 mouse pups. After overnight serum cells was primed with 200 ng/mL of ultrapure LPS for 3 hr followed by treatment with either 40 µM of LTI‐291 or 200 µM of Condruitol β‐epoxide (CBE) for an hour. The cells was activated with 5 mM of adenosine triphosphate (ATP). The supernatant and cells were collected for IL‐1β and GBA assay, respectively. For in vivostudies, 8‐ 12 weeks old male C57BL/6J mice were injected with 20 μg of 6‐hydroxydopamine (6‐OHDA). Mice were treated with 12 mg/kg daily doses of LTI‐291 via oral gavage commencing 24 hours prior to surgery. On day 3 post‐surgery, brains were collected for analysis of protein expression. Our findings demonstrate that NLRP3 inflammasome activation increases IL‐1β production which significantly decreased in the presence of GCase activator, LTI‐291. This indicates the decreased inflammasome activation in conjunction with increased GCase activity. Similarly, GCase activity in LTI‐291 treated cells was significantly increased when compared to control and LPS primed samples. In vivo,6‐ OHDA mice had significant loss of tyrosine hydroxylase (TH), indicative of the substantial dopaminergic neuronal death which was rescued in LTI‐291 treated mice. Overall, these findings identify the potential role of NLRP3 inflammasome activation in driving GCase dysfunction, which could contribute to PD pathogenesis. Furthermore, we demonstrated that pharmacological augmentation of GCase activity was protective, reducing NLRP3 inflammasome activation in vitro and dopaminergic neuronal loss in vivo.