bluuebunny/crossref_metadata_2025 huggingface dataset:
Dog Faces Are Faster than Wolf Faces
Authors: Burrows Anne M. , Omstead Kailey M.
Published: May 2022

Humans domesticated dogs with attention to the facial expressions that dogs produce, selecting for a suite of facial movement. Domestic dogs and humans are adept at accurately understanding one another’s facial expressions and movement around the eyes in dogs is highly valued by humans. This unique, mutual ability to accurately process facial expressions is part of the dog‐human bond. An accurate understanding of how dog and wolf faces differ from one another is fundamental to understanding the processes of animal domestication, evolution of the dog, and the origins of human behavior since the Upper Paleolithic. While we know that dogs have facial musculature that wolves do not regularly possess, our understanding of evolutionary muscle physiology between these two species is incomplete. The present study was designed to assess differential distribution of slow‐twitch (type I) and fast‐twitch (type II) myosin fibers in select facial muscles of dogs and wolves. We tested the hypothesis that dog facial muscle physiology would be more similar to humans than to wolves in terms of myosin fiber type distribution. We used samples from the orbicularis oris muscle (OOM) and the zygomaticus major muscle (ZM) from a range of domestic dog breeds (Canis familiaris) and gray wolves (Canis lupus) that were part of previous studies. Samples were processed for myosin immunohistochemistry (IHC) and we selected 3‐5 sections from each specimen stained for identification of each fiber type in both the OOM and ZM muscles. We created composite images of stained sections and assessed fiber type percentages using ImageJ. Percentage of fast‐twitch fibers in dog samples ranged from 66%‐95% and wolves averaged around 25%. Slow‐twitch fiber percentages in dogs averaged only 10% while wolves averaged 29%. The dog range of type II fibers mirrors the range found in human facial muscles, indicating that the domestication process in dogs also involved selection for rapid facial movement, similar to the movement seen in human faces.
Identification of protein interactors of BRAF via Chemical Induced Dimerization‐BRAF and LC‐MS/MS
Authors: Yu Alison , Pearce Margaret , Janetopoulos Christopher , Wang Zhihong
Published: May 2022

Rapidly Accelerated Fibrosarcoma (ARAF, BRAF, CRAF) Kinase is central to the MAPK Signaling Cascade (RAS‐RAF‐MEK‐ERK). Despite over three decades of scrutiny, the regulatory mechanisms of BRAF are not well understood. According to the accepted model, inactive RAF kinase is monomeric, autoinhibited, and cytosolic while activated RAF is membrane recruited via RAS‐GTP and dimerized; however—direct evidence and key details are still missing. Using a chemical biology approach to target this complex biological system, we seek to exert spatial control using the genetically encodable Chemically Induced Dimerization (CID)‐BRAF; CID‐BRAF exploits the FRB‐rapamycin‐FKBP system to membrane recruit BRAF. We hypothesize that membrane recruitment of BRAF enables diversification of PPI, which remain elusive due to the transient nature of these interactions. To capture transient protein‐protein interactions (PPI), cytosolic BRAF and membrane‐recruited BRAF cells were crosslinked, co‐immunoprecipitated, and identified via LC‐MS/MS. Current efforts are focused on the validation of putative MS identified PPI and identification of BRAF interaction sites. Identifying novel PPI will provide additional mechanistic insight to the regulation of BRAF and facilitate the development of next generation inhibitors of dysregulated BRAF.
NHE1 as a Target to Block Lung Fibrosis Progression
Authors: Provost Joseph , Desalegne Sara
Published: May 2022

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial pneumonia of unknown origin. Fibrosis, a wound healing process, occurs when fibroblasts increase proliferation, convert to myofibroblasts, and secrete extracellular matrix (ECM) proteins. As a result, tissue becomes scarred, reducing lung compliance and ultimately leads to organ dysfunction and death. IFP is characterized by profibrotic agonists and alpha‐smooth muscle actin (α‐SMA) expression. TGF‐β, IL‐1β, LPA, 5‐HT are agonists involved in cell migration regulation, proliferation, and cytokine production. Chronic agonists exposure causes α‐SMA expression which stiffens fibrotic tissue alongside ECM proteins. Prior studies have shown that the sodium hydrogen exchanger isoform 1 (NHE1) is a key regulator in cell migration and pathophysiological development of cancer. The characteristics of IFP are similar to that of cancer (i.e., response to growth signals, myofibroblast origin and behavior, altered cellular communications, and intercellular signaling pathways). Due to these similarities, we hypothesize that NHE1 plays a key role in IFP progression. Results show fibroblast to myofibroblast differentiation is induced in the presence of NHE1 and can also be blocked by the NHE1 inhibitor (EIPA) in mammalian hamster cells. Cells treated with agonists showed some to high α‐SMA expression. Cells treated with agonist and EIPA had no expression of α‐SMA (TGF‐β/EIPA: 35.67 ± 1.76; IL‐1β/EIPA: 11.67 ± 2.03, LPA/EIPA: 33.33 ± 2.19, 5HT/EIPA: 30.00 ± 1.15), and is comparable to the untreated control (20.83 ± 3.12). This improves our understanding of NHE1's role in IPF, and how NHE1 can be a target to impede or hopefully reverse myofibroblast differentiation in IPF patients.
Brain morphology and underlying genetic correlates in schizophrenia
Authors: Martínez Abadías Neus , Hostalet Noemí , Herrero Elena , Canales‐Rodríguez Erick J , Salvador Raymond , Pomarol‐Clotet Edith , Sevillano Xavier , Salgado‐Pineda Pilar , Fatjó‐Vilas Mar
Published: May 2022

Schizophrenia (SZ) is a psychiatric disorder associated with brain and neurodevelopmental alterations that affects nearly 1% of the worldwide population. SZ is a complex disorder, polygenic and multifactorial, and despite large research efforts, its etiopathophysiology is still largely unknown. In this study, we assessed whether polymorphic variation in genes of the Sonic Hedgehog signaling pathway were associated with brain shape variation and contributed to differences between individuals diagnosed with SZ and healthy controls (HC). We genotyped three SNPs in SUFU, SHH and GLI3 genes (SUFU‐rs10786679, SHH‐rs10949808, GLI3‐rs3735361) in a sample of 308 subjects (113 patients with SZ and 195 HC), and quantified brain shape differences in a subsample of 100 subjects using the cartesian coordinates of brain landmarks recorded on head magnetic resonance scans.

The genetic association analyses revealed that the T allele of the SHH‐rs10949808 was significantly associated with the risk for SZ, in both allelic (P=0.02586) and recessive (p=0.0312) models. Geometric Morphometric analyses showed that brain shape was significantly different between individuals diagnosed with SZ and HC. Discriminant functions correctly classified 72% of HC and 60% of patients based on brain morphology. Procrustes‐ANOVA analysis detected that diagnosis was a significant factor (P=0.001) and explained 5.11% of brain morphological variance; whereas any SNP presented significant effects in brain shape variance when analysed separately. However, when the three SNPs were combined into a single variable quantifying the number of risk variants (0 to 3) carried by each person, the analyses showed that patients with 3 risk variants presented a significantly different brain morphology as compared those carrying 2 (P=0.0297) or none risk variants (P=0.0487). These findings underscore the role of neurodevelopment‐related genes and Sonic Hedgehog pathway in SZ and the need to account for the polygenic nature of this disorder to further understand its etiology. Combining brain and genetic data, along with other morphological biomarkers, may open new lines of research in psychotic disorders.
Comprehensive Glycomic Profiling of Breast Cancer Patients
Authors: Funkhouser Avery , Chaballout Basil , Goodwin Jane , Martin Julie , Edenfield W Jeffery , Arthur Connie M. , Blenda Anna V.
Published: May 2022

Glycans are complex carbohydrates found on extracellular membrane lipids and proteins. They are known to be variably expressed by cancer cells, which can have partial or even novel glycan structures. Therefore, we hypothesize that the analysis of breast cancer patient glycomic profiles will result in elucidation of specific and generalizable signatures which will be useful for the diagnosis and treatment of breast cancer. Our study examines N‐glycan expression and its changes through breast cancer stages and compares them to healthy donor samples to create a catalogue of changes and profiles based on cancer stage and other tumor attributes. We are specifically investigating N‐glycans found in patient serum, on the tumor cells, and on adjacent normal tissue. Serum and tissue (tumor and normal) samples from breast cancer patients were analyzed using mass spectroscopy to determine the presence, type, and amount of over 300 unique glycan compositions. Bioinformatic analysis is being used to develop comprehensive glycomic profiles of patients and glycan signatures of cancer stages. We have found several sets of glycan compositions in the serum that appear to change over the progression of cancer. Most notably, glycans of low molecular mass (H4N2, H3N3, H5N2, H3N3F1, H4N3, H3N4, H3N5, H5N5) are rarely present in healthy samples, but are prevalent in all 4 stages of cancer patients. Alternatively, several glycans of varying molecular weight have low expression in healthy patients but higher expression in cancer patients (H4N4F1, H6N2, H5N4F1, H3N4F1, H5N4, H5N2, H4N5F1). Comparing serum and tumor glycan profiles to those of healthy sera and tissue controls will allow for a more comprehensive understanding of the aberrant expression of glycan structures in breast cancer. The collected information has the potential for more accurate diagnoses and personalized treatments. Prospective uses of differential glycomic profiles could allow oncologists and their patients a more nuanced understanding of the tumor and its characteristics.
Quantifying p53 Regulated Genes in a Zebrafish Melanoma Model
Authors: Lane Ethan D. , Freeman Edward , Callahan Kevin P.
Published: May 2022

Cancer is a prevalent disease and is one of the leading causes of death worldwide driven by mutations at the genetic level. An ideal model organism used for cancer research, specifically melanoma, is zebrafish as adult zebrafish display disappearance of their horizontal stripes and emergence of black masses. Zebrafish have highly conserved genes, comparable signaling pathways, and similar tumor development. This research project is designed to identify gene expression changes that are critical for the progression of melanoma in this model system. Wild‐type and heterozygous p53 deficient (M214K) will be exposed to ultraviolet (UV) radiation to promote tumorigenesis. The expression of multiple genes in the p53 pathway will be monitored via qPCR during progression of the melanoma. Genes that are identified as up‐regulated or down‐regulated will be targeted using CRISPR‐Cas9 to determine their role in the progression of the disease.
IL‐6 Signaling is Required for LPS‐induced Barrier Function Loss
Authors: Lu Shuhan , Martino Nina , Chuy Dareen , Ramos Ramon B. , Adam Alejandro P.
Published: May 2022

Interleukin‐6 (IL‐6) is a major mediator of the cytokine storm in response to severe systemic inflammation and sepsis. This cytokine promotes the activation of the transcription factor STAT3 via Janus kinases (JAK)‐dependent phosphorylation on tyrosine 705. Prior work in our lab demonstrated that overactivation of this pathway within the endothelium via an endothelial‐specific deletion of the negative regulator SOCS3 (SOCS3iEKO) leads to kidney failure and acute mortality after a single lipopolysaccharides (LPS) challenge that was non‐lethal in wild‐type mice. This mortality was associated with increased systemic IL‐6 levels and vascular leak in lungs and brain of SOCS3iEKO mice. Furthermore, translating ribosome affinity purification studies showed that in response to LPS, the kidney and brain endothelia expressed high levels of IL‐6. These findings suggested that IL‐6 signaling within the endothelium might be required to promote vascular leak. To sought to test this hypothesis in vitro. We found that LPS promoted a mild increase in monolayer permeability in HUVEC that was abrogated by pretreatment with the JAK inhibitor ruxolitinib. This loss of barrier function by LPS was associated with an increase in IL‐6 expression and a modest increase in STAT3 phosphorylation. Endothelial cells express very low levels of the gp80 subunit of the IL‐6 receptor. Thus, to test whether an IL‐6 autocrine loop induced barrier function loss, we treated cells with LPS in the presence or absence of a soluble form of gp80 (herein, LPS+R). LPS+R‐treated cells showed increased STAT3 phosphorylation and severe barrier function loss. A similar increase in STAT3 phosphorylation and monolayer permeability was observed when gp80 was added to TNF‐treated cells. In contrast, addition of gp80 to LPS‐ or TNF‐treated cells did not alter the levels of p65RelA or p38 phosphorylation. Pretreatment of LPS+R‐treated cells with the p38 inhibitor SB203580 showed that activation of p38 signaling was not required for STAT3 phosphorylation or barrier function loss. In summary, our data strongly suggest that the endothelium responds to strong inflammatory challenges with an increase in IL‐6 expression that may act in an autocrine fashion to promote strong barrier function loss, potentially explaining the systemic vascular leak observed in critically ill patients with high circulating IL‐6 levels.
Desmosome Dysregulation and MLC Phosphorylation Contribute to Loss of Barrier Integrity in DRA KO mice
Authors: Kumar Anoop , Anarwala Shawana , Abdul Qadeer Abdul , Kaur Jasleen , Jayawardena Dulari , Natarajan Anbazhagan Arivarasu , Priyamvada Shubha , Majumder Apurba , Saksena Seema , Alrefai Waddah A. , Gill Ravinder K. , Dudeja Pradeep K.
Published: May 2022

DRA or SLC26A3 is the key transporter for chloride absorption in the mammalian intestine. Our recent studies demonstrated that DRA deficiency increased paracellular permeability and altered tight and adherens junction protein expression in mouse colon. However, the mechanisms underlying increased paracellular permeability and the role of intercellular junctional proteins (desmosomes) that play a critical role in the maintenance of intestinal barrier integrity in DRA KO mice are not well understood. Aim The current studies were undertaken to examine the role of desmosomes and Rho‐ROCK‐MLC phosphorylation pathway in colonic barrier integrity in DRA KO mice.

Method

DRA KO and WT mice (M/F, 8‐10 weeks old) were used as an in‐vivo model. Protein expression was measured by immunoblotting and immunofluorescence, whereas mRNA expression was measured by QRT‐PCR and Rho A activity was examined utilizing the G‐LISA kit.

Results

Our results showed that the mRNA expression of desmosome proteins, desmoglein‐2 (DSG2) and desmocollin‐2 (DSC2) was significantly reduced (50‐70%, p<0.05) in DRA KO colons compared to wild‐type mice. Similarly, DSG2 protein expression was also significantly downregulated in DRA KO mice colon (~ 50%, p<0.05) as assessed by immunoblotting and immunostaining. We also examined the role of Rho‐ROCK‐MLC2 phosphorylation pathway in DRA KO mice. Interestingly, DRA KO mice colon showed a significant increase in Rho A protein expression (~5‐fold, p<0.01) and Rho A activity (~ 3.5‐fold, p<0.05) compared to WT. Furthermore, DRA KO mice also exhibited a significant decrease in ezrin phosphorylation (a downstream target of Rho A signaling) (~ 2‐fold, p<0.05) compared to WT mice. In addition, another downstream target of the Rho A pathway, MYPT‐1 (Myosin Phosphatase Target Subunit 1), also showed a significant decrease (~ 60%, p<0.05) in its protein expression in DRA KO mice. MYPT‐1 downregulation in DRA KO mice was associated with a significant increase in MLC‐2 phosphorylation (~ 2‐fold, p<0.05), which may have contributed to barrier dysfunction in colon of DRA KO mice. Since, our recent studies showed microbial dysbiosis in DRA KO mice, it was of interest to examine the role of gut microbiome by co‐housing WT and KO mice for 4 weeks for microbial exchange. Interestingly, co‐housed DRA KO mice also showed a significant but lower increase in Rho A expression (~ 3‐fold, p<0.05) and downregulation of MYPT‐1 expression (~ 60%, p<0.05) compared to co‐housed WT mice indicating that the regulation of Rho A and MYPT‐1 expression was only partly dependent on microbial dysbiosis observed in DRA KO mice.

Conclusion

Our studies highlight the novel role of desmosome dysregulation and increased MLC phosphorylation in compromised colonic barrier integrity in DRA KO mice.
Distinct Signaling of Native PTH‐related Peptide
Authors: Peña Karina A. , White Alex , Savransky Sofya , Vilardaga Jean‐Pierre
Published: May 2022

The parathyroid hormone (PTH) type 1 receptor (PTHR) regulates a variety of key biological processes, including mineral ion and bone homeostasis, through the action of its two natural peptide ligands: PTH and PTH‐related peptide (PTHrP). Particularly, PTHrP is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling. Although mature forms of PTHR ligands consist of 84 and 141/173 amino acids for PTH and PTHrP, respectively, our current understanding on how endogenous PTHR ligands transduce signals through PTHR is largely derived from studies done with the N‐terminal fragments of both peptides, PTH1‐34 and PTHrP1‐36. While both peptides induce acute intracellular Ca2+ (iCa2+) release, they differ in the location and duration of cAMP signaling. PTH1‐34 induces sustained cAMP production from PTHR signaling complexes in endosomes, whereas PTHrP1–36 induces transient cAMP production derived from ligand–PTHR interactions at the plasma membrane. Although the transient nature of PTHrP1‐36 signaling has been regarded as a main PTHrP feature, here we demonstrate that PTHrP1‐36 cannot be considered as an analogue of the native PTHrP1‐141. We show that PTHrP1‐141 triggers sustained cAMP signaling from the plasma membrane without inducing b‐arrestin recruitment. Additionally, PTHrP1‐141 fails to stimulate iCa2+ release. We also show that the molecular basis for signaling differences between PTHrP1‐36 and native PTHrP1‐141 are caused by the stabilization of distinct PTHR conformations. These results indicate that native PTHrP1‐141 displays distinct signaling features from PTHrP1‐36 and can be considered an endogenous biased agonist for PTHR.
GPR55‐Dependent Excitation of Dorsal Root Ganglion Neurons by Lysophosphatidylcholine
Authors: Bennett Aidan S. , Alward Taylor A. , Reed David E. , Lomax Alan E.
Published: May 2022

Irritable Bowel Syndrome is a functional gastrointestinal (GI) disorder that leads to chronic abdominal pain. The perception of this visceral pain involves peripheral mechanisms, such as nociceptors with cell bodies in dorsal root ganglia (DRG) and axons in the gut wall, and central mechanisms, such as neuroplasticity within the spinal cord and brain. Lysophosphatidylcholine (LPC), which has recently been shown to bind to GPR55 in human prostate carcinoma cells, is released from cell membranes and is elevated in patients with IBS. Dorsal root ganglion (DRG) neurons express GPR55, and activation of GPR55 has been implicated in inflammatory pain. Therefore, we hypothesised that elevated LPC during IBS contributes to pain due to its activation of GPR55 on dorsal root ganglion (DRG) neurons, leading to the excitation of visceral pain pathways. Current clamp recordings revealed that application of LPC (10 µM) to murine DRG neurons depolarised the resting membrane potential (p = 0.0001) by approximately 8 mV and decreased the rheobase (p < 0.05) by approximately 20%. Using ratiometric Ca2+ imaging using FURA‐2 AM, LPC (10 µM) doubled intracellular [Ca2+]i. This effect was significantly reduced by the selective GPR55 antagonist CID16020046 (10 µM) (p < 0.05), suggesting the response to LPC is at least partially mediated by GPR55. The source of the [Ca2+] elevation following LPC application was elucidated using cyclopiazonic acid (CPA; 10 µM), which depletes intracellular Ca2+ stores, and a 0‐Ca2+ external solution to remove the contribution of Ca2+ influx from extracellular sources. While both significantly decreased the Ca2+ influx elicited by LPC, the 0‐Ca2+ external solution almost abolished the effect (p < 0.0001) of LPC. Together, these data suggest that the increased [Ca2+] elicited by LPC activation of GPR55 is partially mediated through the release of Ca2+ from intracellular stores but is mostly due to influx of extracellular Ca2+.
Supplementing Gross Anatomy Pelvis and Perineum Content with Video Modules: Effects on Student Learning
Authors: Askew Emily B. , McLaughlin Scott C. , Harrell Kelly M.
Published: May 2022

Human gross anatomy and embryology (GAE) remains an integral piece in the first‐year medical student's education, even as medical schools across the United States condense their foundational phase curricula. In the setting of undergraduate medical programs that are making this transition, supplemental video modules are more frequently being utilized in gross anatomy learning. At Brody School of Medicine (BSOM) at East Carolina University (ECU), the GAE course is a 16‐week long course taught during the first block of the first‐year of medical school at BSOM. In addition to human gross anatomy (lecture and donor‐cadaver dissection), faculty also lecture on human embryology and radiology. In this project, we generated video modules focused on the genetic male pelvis and perineum and genetic female; these were then incorporated into the GAE course at BSOM. The videos included recordings of relevant anatomical features of donor‐cadaver prosections generated by BSOM anatomy faculty, as well as author generated illustrations highlighting key structural relationship, high‐yield embryology concepts, and voice‐over narration. The videos were uploaded to the BSOM learning management system, and learners instructed to watch the videos in preparation for the upcoming sessions focused on the pelvis and perineum (prosection demonstration and flipped‐classroom session). The aim of this study was to ascertain student self‐perception of comprehension of pelvis and perineum material with the incorporation of the video modules into the prosection laboratory and active‐learning session. The learners sampled were first‐year medical students enrolled in GAE at BSOM in Fall 2019 (n=87). In prior years, the pelvis and perineum unit included a three‐hour dissection followed by a flipped‐classroom session. Data analysis of survey results (n=86, 99% response rate) indicate that prior to reviewing the modules, 12% of respondents felt moderately or very comfortable with the content. After viewing the videos, 80% of respondents felt moderately or very comfortable with the content. Interestingly, all respondents (100%) agreed that it would be beneficial to their learning for similar videos to be included in future flipped‐classroom sessions. Collectively, these results provide insight into the positive effects of supplemental video modules in supporting student comprehension and retention of anatomical sciences knowledge when time constraints may require faculty to modify laboratory activities and active‐learning classroom sessions. Additional scholarship opportunities focus on evaluating the correlation between the use of video modules in GAE and learners' exam performance.
Normalization of Oxygen‐Induced Retinopathy in the Mouse Supplementation with Myo/Nog Cells
Authors: Sutera Christopher K. , Wilgucki Grace , Weyback Edgar , Tsai E‐Jine , Brahmbhatt Rushil , Martin Mark , Adams Sarah , Lecker Paul , Johal Karanveer , Spikes John , Gorski Grzegorz , Gerhart Jacquelyn , Philp Nancy , George‐Weinstein Mindy , Bravo‐Nuevo Arturo
Published: May 2022

Introduction

Retinopathy of Prematurity (ROP) is the leading cause of blindness in children, affecting 50% of infants born prior to gestational week 32. ROP is the result of postpartum supplemental oxygen administered to compensate for underdeveloped lungs. However, hyperoxia inhibits proper development supplying the retina. When the infants are returned to ambient air, an imbalance is present between the oxygen concentration and metabolic demands of retinal neurons. This leads to a rapid induction of angiogenesis, leaky blood vessels, neovascular tuft formation, and cell death. Myo/Nog cells that express MyoD, Noggin, and brain‐specific angiogenesis inhibitor 1 (BAI1) exhibit neuroprotective properties in the eye and brain and are proposed to play a role in regulating angiogenesis in ROP.

Methods

An Oxygen‐induced retinopathy (OIR) protocol was implemented in neonatal C57BL/6J mice by exposing them on postnatal days 7‐12 (P7‐P12) to hyperoxia (75% oxygen) and then returning them to normal air (21% oxygen) from P12 until P21. On P15, groups receiving treatments were intravitreally injected with 1 µL of phosphate‐buffered saline (PBS) containing 2000 Myo/Nog cells isolated from the brain with the BAI1 antibody (BAI1+ group), 2000 unsorted brain cells (BAI1‐ group), or PBS alone (PBS group). Electroretinography (ERG) was conducted on P21 to assess retinal function. Eyes were extracted and either dissected for flat‐mount observation of vasculature and tufts, or processed for histology. The thickness of the retina and number of TUNEL+ apoptotic cells were quantified in tissue sections of eyes processed for histology. Adobe Photoshop (Adobe, San Jose, CA) was used to splice together 9 images that made up each whole retina from flat‐mounted eyes. The blood vessels were selected using the “Lasso tool” on photoshop, quantified by number of pixels, and then compared to the total number of pixels in the whole retina.

Results

OIR induced neovascular tuft formation. Exogenously added Myo/Nog cells normalized the retinopathy induced by the OIR model, reducing the number of tufts and improving the quality of the vasculature. The vasculature was most developed in retinas injected with brain derived Myo/Nog cells. ERG data shows a trend towards improvement of visual function with Myo/Nog cell injection.

Conclusion

Myo/Nog cell regulation of retinal vascularization may be related to the production of BAI1. Improved visual function seen with injection of Myo/Nog cells may be related to both a reduction of neovascular tufts and direct effects on retinal neurons. Therefore Myo/Nog cells have therapeutic potential in the prevention of ROP by normalizing the vasculature and preventing neuronal cell death.
USP Potency Adjusted Bovine Mucosal Heparins are Comparable to Porcine Mucosal Heparin and May be Interchangeable for Anticoagulation
Authors: Olson Guy , Kouta Ahmed , Baig Nausheen , Jeske Walter , Hoppensteadt Debra , Iacobelli Massimo , Iqbal Omer , Bakhos Mamdouh , Cera Lee , Bansal Vinod , Fareed Jawed
Published: May 2022

Introduction

Most of the heparins used clinically are derived from porcine intestinal mucosa. In contrast to the porcine mucosal heparin (PMH), the active pharmaceutical ingredient (API) of bovine mucosal heparin (BMH) exhibit a somewhat weaker USP potency as cross‐referenced against PMH. We hypothesized that at equivalent potencies as adjusted by using the USP reference, the BMH may exhibit comparable effects.

Materials & Methods

Molecular weight profile and in vitro anticoagulant and antiprotease assays were used to compare the BMH with PMH. USP potency was measured in the amidolytic assays. Potency adjustments in reference to USP standard were made by weight basis. Potency adjusted BMH and PMH were injected at identical dosages (50 U/kg and 100 U/kg) to groups of human primates (n=4) via intravenous route and blood samples were collected for a 6 hour period of time.

Results

BMH exhibited higher molecular weight profiles compared to PMH as determined by size exclusion chromatography {BMH (Mw) 18.6 ± 0.5 kDa and PMH 15.4 ± 0.4 kDa}. BMH showed a potency of 130 U/mg whereas PMH showed a potency of 190 U/mg. When the BMH was compared at a potency adjusted concentration with PMH, it showed identical calibration curves in the aPTT and anti‐protease assays. Mean t1/2 ranged from 54 ± 11 min & to 71 ± 18 min for PMH & BMH respectively (p=>0.05). Mean AUC values based on anti‐Xa or anti‐IIa activities were comparable for both heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for both heparins.

Conclusion

Potency adjusted based dosing results in comparable anticoagulants and pharmacokinetic profiles for BMH and PMH. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations suggest that potency equated BMH and PMH produced comparable anticoagulant effects and may be interchangeable.
Potential Involvement of NHE1 in COVID‐19 Related Pulmonary Damage
Authors: Ta Lam , Vigilante Danielle , Iyer Meera , Provost Joseph
Published: May 2022

In some moderate and severe COVID‐19 infections lung scarring similar to fibrosis have been observed in survivors, many of which have persistent functional and radiographic abnormalities at the initial follow‐up suggesting a possible shared pathobiology between fibrotic lung disease and COVID‐19‐ lung disease. Fibrosis is an inflammatory response to pulmonary infections that activates fibroblasts and produces excessive extracellular matrix proteins that stiffen cells, leading to reduction in lung capacity and breathing deficiency. Several factors involved in the “cytokine storm” also are pre‐fibrotic leading to the advanced disease. This includes interleukin 6 and others. The focus of this study is on sodium hydrogen exchanger isoform 1 (NHE1) that plays a key role in intracellular pH regulation and cell movement and its effect on fibrosis development. Both cytokines IL‐1 and IL‐6 are used to show one measure of cell restructuring and stiffness by measuring stress fiber formation. To examine the role of NHE1 in fibroblast response, both IL‐1 and IL‐6 cells were treated with proinflammatory cytokines in the presence and absence of NHE1 inhibitor (EIPA). This study will provide an initial insight to the relationship between NHE1 and proinflammatory response for future experiments.
SPARC: Sex difference in colorectal afferent sensitization in zymosan‐induced visceral hypersensitivity as revealed by high‐throughput GCaMP recordings in thoracolumbar and lumbosacral DRG
Authors: Feng Bin , Guo Tiantian , Liu Jia , Chen Longtu , Bian Zichao , Zheng Guoan
Published: May 2022

Female patients with irritable bowel syndrome (IBS) report more intense visceral pain sensation than male patients. Female rats show more sensitive and cycle‐dependent visceromotor responses (VMR) to graded colorectal distension (CRD). Both clinical psychophysical evidence and preclinical behavioral data support an apparent sex difference in visceral nociception. However, very little is known regarding the potential differences in sensory neural encoding of distal colon and rectum (colorectum) between males and females. In this study, we systematically assessed the sex difference in colorectal neural encoding by conducting high‐throughput optical recordings in intact dorsal root ganglia (DRG) from control and visceral hypersensitive mice. We crossbred GCaMP6f mice with VGLUT2‐Cre mice to express GCamP6f gene in most colorectal sensory neurons. Mice receiving intracolonic treatment of zymosan (30mg/mL, 0.2mL) for three consecutive days developed behavioral visceral hypersensitivity as validated by enhanced VMR to CRD. We then harvested the colorectum with pelvic nerve, lumbar splanchnic nerve and T12 to S1 DRG in continuity in an ex vivo preparation for GCaMP6f recordings. A total of 2275 colorectal afferents were characterized from both sexes in control and zymosan‐treated groups. DRG neurons responding to either colorectal distension (15, 30, 45 and 60 mmHg) and/or mucosal shearing (20‐30 mL/min) were functionally categorized into four classes: mucosal, muscular‐mucosal and high‐ and low‐threshold muscular afferents. In control group, the number of colorectal afferents recorded per mouse were slightly lower in male mice than in female ones (38.7 vs. 45.2) with no significant difference in their distributions across thoracolumbar (TL) and lumbosacral (LS) DRG. In zymosan group, more afferents were recorded from each male mice than female ones (53.8 vs. 44.1) showing significantly higher TL innervation (T12, L1, and L2 DRG) in male group. Within the male groups, zymosan treatment resulted in a significant increase in the proportion of TL innervation as compared with saline treatment. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline‐ and zymosan‐treated groups. Our results have revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity. The current outcomes draw further focused research on the neurophysiological differences between sexes that could drive the differential gastrointestinal symptoms in male and female IBS patients.
Analysis of Academic Performance of Students Taking Physiology Reveals an Achievement Gap Relative to Student GPA
Authors: Wu Steven C. , Anderson Lisa C.
Published: May 2022

We previously reported that the use of small stakes assessments in an undergraduate/graduate physiology class (PHSL3061/5061, Principles of Physiology) contributed to improved average test scores for physiology and biomedical engineering majors and reduced the number of non‐passing physiology majors (EB 2021). Our overall hypothesis is that using small stakes assessments could improve outcomes for all students.

In this current study, we examined physiology majors from 2018‐2021 by race, gender, first generation status, class (sophomore, junior, senior), and indicated financial need. A typical physiology major taking PHSL3061/5061 tended to be white (60%), female (67%), non‐first generation student (75%), and in their senior year (60%).

When small stakes assessments were given, the number of students achieving below 70% was reduced as previously reported. However, those students that achieved a final score below 70% tended to be black, indigenous, person of color (BIPOC) (60%), female (80%), first generation student (75%), in their senior year (60%), and indicated some financial need (60%). Moreover, when term GPAs versus final scores were plotted, this group tended to have a term GPA between 3.0‐3.5/4.0, revealing an achievement gap in PHSL3061 relative to their predicted performance based on GPA.

We also compared academic performance (final score and term GPA at the end of the course) by race, gender, first generation, and class. The most significant differences in final score and term GPA were found between first generation and non‐first generation students.

Overall, our data revealed a group of students that are not achieving scores relative to their GPAs. We believe that these analyses can identify achievement gaps sooner. Taken together, the use of small stakes assessments can still lead to improved outcomes for physiology students. Further analyses of physiology students by category could reveal other potential areas where there are achievement gaps.
Mice with Brain‐Specific Deletion of Hsd11b2 have an impaired renal vascular and tubular response to high salt intake
Authors: Bailey Matthew A. , Krilis Georgios , Stewart Kevin , Grenier Celine
Published: May 2022

Loss‐of‐function mutations in Hsd11b2, the gene encoding the cortisol‐metabolising enzyme 11βHSD2, causes the hypertensive syndrome of Apparent Mineralocorticoid Excess. In affected individuals, overactivation of the mineralocorticoid receptor by cortisol is causative. Hypertension is salt‐sensitive, attributed to enhanced sodium reabsorption in the distal nephron and renal sodium retention. However, in the brain, 11βHSD2‐expressing neurons regulate salt appetite. We previously generated mice with conditional knockout of Hsd11b2 in the brain (Hsd11b2.BKO), which displayed enhanced salt preference and salt‐sensitive hypertension (DOI: 10.1161/CIRCULATIONAHA.115.019341). The mechanisms of salt‐sensitivity are unknown. In the current study we examined renal artery vasoreactivity and the in vivo acute pressure natriuresis response in male Hsd11b2.BKO mice and control littermates.

Mice (n=10 per genotype/diet) were fed either high salt (3% Na) or 0.3% Na diet for 7 days. Animals were humanely killed, the renal arteries were isolated and mounted on a wire‐myograph to generate cumulative concentration‐response curves to phenylephrine and the nitric oxide donor, sodium nitroprusside. High salt diet significantly increased the sensitivity of the renal artery to phenylephrine‐induced vasoconstriction in Hsd11b2.BKO mice (LogEC50 0.3% diet ‐6.41±0.07 vs high salt ‐6.91±0.11; p=0.0013) but not in control mice. Sodium nitroprusside induced concentration‐dependent relaxation of renal arteries: the maximum relaxation was significantly reduced from 88±3% to 65±4% in Hsd11b2.BKO after high salt feeding (p<0.01); this was not seen in control mice. In other experiments, mice (n=5‐7 per genotype/diet) were anesthetised (thiobutabarbital sodium, 120mg/kg IP) and fractional renal sodium excretion was measured at baseline and following acutely increased blood pressure, achieved by sequential arterial ligation. In all mice, sodium excretion increased with blood pressure. In the high salt group, the pressure natriuresis response was significantly attenuated in Hsd11b2.BKO mice, compared to control animals.

We find that genetic amplification of mineralocorticoid signalling in the brain attenuates the normal renal vascular and tubular adaptation to high salt intake. This regulation of renal salt excretion by the central nervous system may have implications for salt‐sensitive hypertension in humans.
Design and Evaluation of Cleavable CoQ‐triphenylphosphonium Analogs
Authors: Steenberge Laura H. , Pagliarini David J.
Published: May 2022

Coenzyme Q (CoQ, ubiquinone) is a ubiquitous redox‐active lipid that is required for oxidative phosphorylation, acts as a lipophilic antioxidant, and is a critical cofactor for numerous other processes, such as uridine biosynthesis and fatty acid oxidation. Primary defects in CoQ biosynthesis cause a variety of phenotypes, from nephropathy and myopathy to fatal multiorgan disease. Secondary defects in CoQ levels have been implicated in the pathophysiology of many common conditions, such as neurodegenerative diseases, cardiomyopathies, diabetes, and aging. Unfortunately, exogenous oral supplementation of CoQ aimed at alleviating or preventing disease is difficult due to its large molecular weight and hydrophobicity. Only 2% of orally administered CoQ reaches the bloodstream, and less than 15% of the CoQ in the blood becomes incorporated into mitochondria in tissues; therefore, is often impossible for patients reach an effective dose, even when taking grams of CoQ daily. More efficient means of delivering exogenous CoQ to mitochondria, its main site of action, need to be developed. Here, we describe the synthesis and evaluation of an initial set of compounds designed to deliver CoQ in larger quantities selectively to mitochondria. These compounds incorporate a mitochondria‐targeting triphenylphosphonium (TPP) moiety attached to CoQ through a reversible linker, and are designed to accumulate in mitochondria and be cleaved to release native CoQ. Our results indicate that select versions of these compounds can successfully be delivered to mitochondria in a cell model and be cleaved to produced CoQ, encouraging further development. Our ongoing efforts include assessing the efficacy of these compounds in rescuing CoQ‐deficient cells, and further optimizing design features to further promote effective cleavage and mitochondrial localization while minimizing toxicity.
An Evolutionarily Conserved Residue in ABCG2 Regulates the Transport Cycle and Inhibitor Activity
Authors: Gose Tomoka , Das Sourav , Wang Yao , Lynch John , Fukuda Yu , Wills Medb , Baril Stefanie , Shelat Anang , Schuetz John D.
Published: May 2022

ABCG2 is a medically significant ATP‐binding cassette (ABC) transporter restricting the cellular penetration of more than 200 toxins and drugs, as well as promoting physiological detoxification and multidrug resistance in cancers. Although ABCG2 cryo‐EM structural studies reveal an overlap between substrate and inhibitor binding sites in the ABCG2 binding pocket, it is unknown how much these substrate and inhibitor interactions will translate into functional knowledge that is exploitable in live cells.

We generated multiple single point‐mutations in the residues of the ABCG2 binding pocket to determine if there is a relationship between binding (defined by thermal shift stabilization) and transport. Unexpectedly, the relationship between substrate‐induced thermal stabilization and transport was poor and an alanine substitution in a highly conserved asparagine residue in ABCG2 markedly impaired substrate‐induced thermal stabilization, but paradoxically enhanced substrate transport. To determine if the substitution altered ABCG2 conformational response to substrate binding, we used live cells and performed copper phenanthroline induced disulfide cross‐linking analysis on cells expressing the appropriate cysteine substituted ABCG2 (1). Consistent with the enhanced transport, the substitution promoted a substrate‐induced conformational change from an inward‐ to an outward‐facing ATP‐bound state. Molecular dynamics (MD) simulations using an ABCG2 structure (PDB: 6VXH) suggested a mechanism for this enhanced transport by the alanine mutant: disruption of a water‐bridge interaction with the asparagine residue coupled with an alteration in contacts with certain residues in the binding pocket. We next investigated if this alanine substitution affected inhibitor binding and transport inhibition. The substitution strongly reduced inhibitor‐induced thermal stabilization and transport inhibition. The disulfide cross‐linking analysis revealed that the substitution abolished the ability of many inhibitors to block the transition to an outward‐facing state. For these inhibitors, MD simulations revealed that the substitution disrupted a hydrogen bond interaction with the asparagine residue and impacted inhibitor interactions with multiple residues. Overall these changes indicate that inhibitors have mostly lost their ability to act like “wedges” to lock ABCG2 into an inward‐facing conformation.

These findings reveal that an evolutionarily conserved asparagine residue, in the binding pocket, appears to have a key role in regulating the conformational dynamics of ABCG2.

Reference:

(1) Orlando, B. J. and Liao, M., ABCG2 transports anticancer drugs via a closed‐to‐open switch. Nat. Commun., 11, 2264 (2020)
Analytical Needs for Machine Learning‐based Image Analysis of Brainstem Astrocyte Cell Function
Authors: Blackburn Jessica , Czeisler Catherine M. , Otero José J.
Published: May 2022

Reactive astrogliosis represents a neuropathogical finding in most brain diseases across the lifespan. Despite its near ubiquitous presence in CNS pathologies, the consequences of reactive astrogliosis on cell function are poorly characterized. We address the capability gap within the glial biology community by applying machine learning image segmentation and multivariate analyses to understand the consequences of gliosis on astrocyte cell function. We utilized live cell imaging of astrocytic intracellular calcium transients as a measure of physiological responses to external stimuli (i.e., exposure to lipopolysaccharide and/or hypoxic (100% N2) gas challenges). After obtaining 300 GBytes of live cell imaging footage, we developed an efficient, unbiased image segmentation workflow to capture astrocytes during physiological challenges. In contrast to prior methodologies which extracted only mean fluorescence intensity of segmented objects, we utilized pixel textures and fluorescent propagation in our analysis. Following the recommendations set forth by the recent consensus statement (Escartin et al., 2021), we performed multivariate and clustering analyses to identify distinct groups based on intracellular calcium transients waveforms under physiological challenges. These analyses reveal a diversity of cellular processes amongst brainstem astrocytes.
Functional Characterization of Germline and Cancer‐specific Protein MAGEB2
Authors: Romney Carlan V. , Colemon Ashley , Ramanathan Saumya
Published: May 2022

Cancer is a leading cause of death in the United States of America and is a complex disease characterized by uncontrolled proliferation of cells. Current cancer therapies are promoting the use of personalized medicine to treat the disease, thus increasing survival rates. Melanoma antigen genes (MAGEs), a subgroup of cancer‐testis antigens (CTAs), are expressed in the male germline and aberrantly in many cancers (Type I) or ubiquitously expressed (Type II). This research focuses on MAGEB2, a virtually understudied Type I MAGE located on the X chromosome. Previous data from our lab indicate that expression of MAGEB2 in normal cells increases cell proliferation, while depletion of MAGEB2 from cancer cells results in lack of cell viability. Based on these data, we hypothesize that MAGEB2 expression provides a proliferative advantage to cancer cells. To determine the signaling pathways modulated by MAGEB2 expression, we performed RNA‐Sequencing on cells expressing MAGEB2 and then identified the most upregulated and downregulated genes with significance to cancer. We will validate our gene expression data and perform gene ontology analysis, followed by gain and loss of function studies to determine signaling pathways required for MAGEB2 to cause a proliferative phenotype. Our second approach will determine the proteins associated with MAGEB2. Using immunoprecipitation (IP)/Mass Spectrometry (MS), we have identified binding partners involved in DNA repair, apoptosis, cell cycle regulation, ribosome biogenesis, chromatin maintenance and remodeling, and protein synthesis. Understanding the mechanisms altering MAGEB2 expression can help scientists develop novel therapeutics to treat cancer.