bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic Regulation of Macrophage Linage During Acute Lung Injury and Resolution
Authors: Rochford Ian , Joshi Jagdish C , Mehta Dolly
Published: November 2023

Macrophages coordinate both the initiation and resolution of acute lung injury (ALI), a chronic lung disease which causes significant mortality and morbidity. Macrophages are divided into multiple functionally plastic and phenotypically distinct subpopulations based on their differences in anatomical location and cytokine signaling. Here, we used a mouse model which expresses GFP under the control of the myeloid‐specific Lys‐M promoter and exposed them to pneumonia‐induced ALI to identify shifts in macrophages lineage during inflammatory and resolution phases of lung injury. Flow cytometric analysis showed a dynamic shift in macrophages subpopulations on the onset versus resolution of injury. We found that in naïve lungs, CD11c+/Cd11b+ macrophages, which represent both recruited and lung resident macrophage populations, represent 24.45% of total lung hematopoietic cells. At 4 hours post LPS inhalation, which marks the peak of injury phase, this population decreased to 6.7%. C11b+/CD11c+ then rebound back and their number increased to 59.34% and 49.74% respectfully, during resolution of injury at 24 and 48 hours. On the other hand, CD11c+/CD11b‐macrophages, which constitute alveolar macrophages population, continue to drop from a value of 34.6% at basal state to 29.5% during injury at 4 hours and to 5.9% at 24 hours post LPS. Interestingly, a CD11c+/CD11b (Low) population emerged at 48 hours amounting 17.9% of total macrophages. QPCR analysis of lungs indicated an increase in the expression of HDAC1, an epigenetic modifying enzyme, also at 48 hours. LyzM‐GFP bone marrow derived macrophages also showed distinct morphological phenotypic changes after stimulation with LPS from 0, 4, 18 and 24 hours, indicating differential lineage. We propose that characterization of the genetic and phenotypic signatures of CD11b/Cd11c macrophages of different lineages which disappear and emerge during the onset of injury and resolution phase of ALI may provide insight to their therapeutic potential as a cell‐based therapy for resolving ALI.

Support or Funding Information

Supported by PPG2 2POHL077806‐11 and T32‐HL007829
Adenosine 2A (A2A) receptor expression in rats with motor neuron death from intrapleural CTB‐saporin injections
Authors: Craig Taylor A. , Brown Scott M. , Nichols Nicole L.
Published: November 2023

When injected intrapleurally, cholera toxin B fragment conjugated to saporin (CTB‐SAP) mimics aspects of ALS including the amount of phrenic motor neuron death observed in ALS rats (Nichols et al., 2015). This selective phrenic motor neuron death is sufficient to enhance phrenic long‐term facilitation (pLTF, a form of phrenic motor facilitation; Nichols and Mitchell, ibid, 2014) after 7, not 28, days of CTB‐SAP. pLTF in normal rats predominately requires 5HT2 receptors and the new synthesis of BDNF, although there is an alternative BDNF independent mechanism for pLTF that requires A2A receptors and the new synthesis of TrkB. Preliminary data suggest that the mechanism responsible for pLTF following 7 days of 25mg CTB‐SAP is TrkB dependent (Nichols et al. ibid). Here, we tested the hypothesis that A2A receptor expression is upregulated on phrenic motor neurons following 7, not 28 days, of 25mg CTB‐SAP using immunohistochemistry. A2A receptor expression was studied in Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). Our preliminary data suggest that following CTB‐SAP injection, total A2A receptor positive pixels appear to be increased in the phrenic motor nucleus and non‐phrenic ventral horn at 7 (82±45 and 93±55 pixels, respectively; n=9), not 28 (8 and 8 pixels, respectively; n=2), days later vs. controls (51±24 and 27±15 pixels, respectively; n=6). This suggests that A2A receptors are upregulated to enhance pLTF at 7, not 28, days post‐CTB‐SAP. Future studies will be focused on completing this histology analysis and testing whether pLTF in 7, not 28, day CTB‐SAP treated rats is abolished following C4 intrathecal, A2A receptor antagonist treatment. This project increases our understanding of the expression of receptors associated with respiratory plasticity and its implications for breathing following motor neuron death.

Support or Funding Information

Supported by NIH K99/R00 HL119606
Endo‐lysosomal Escape of Cholera Toxin B‐Modified Protocells in Motoneurons
Authors: Giambini Sebastian , Porras Maria Alejandra Gonzalez , Brinker Jeffrey , Sieck Gary , Mantilla Carlos Bernardo
Published: November 2023

Mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a stable, versatile and biocompatible delivery platform that allows surface engineering to target specific cells and intracellular cargo delivery. The endo‐lysosomal escape of this type of drug carriers is important to enhance the therapeutic efficacy of their cargo. Motoneurons can be specifically targeted by surface‐coating protocells with the subunit B of cholera toxin (CTB‐protocells). The central hypothesis was that CTB‐protocells escape endo‐lysosomal compartments after internalization by motoneurons. In a first study, fixed NSC‐34 motoneuron‐like cells were treated with fluorescently labeled CTB‐protocells (for 1, 3, 6 and 24 hours), immunostained for lysosomes (with anti‐LAMP1) or early endosomes (with anti‐EEA1) and imaged using multicolor confocal microscopy. In a second study, NSC‐34 cells treated with CTB‐protocells and labeled with the acidic vesicle tracker LysoTracker were visualized using time lapse confocal imaging. In these two studies, colocalization of CTB‐protocells with lysosomes or endosomes was analyzed using ImageJ and found to remain less than 20% up to 24 hours. The fraction of CTB‐protocells in endo‐lysosomes increased only slightly between 1 and 24 hours. In a third study, transmission electron microscopy was used to confirm the intracellular localization of CTB‐protocells in NSC‐34 motoneuron‐like cells. CTB‐protocells were visualized in the cytosol and were not detected within larger vacuole compartments after 24‐hour incubation. In conclusion, CTB‐protocells escape lysosomal degradation in motoneurons making them a suitable vehicle to target drug delivery to motoneurons.
Hyperbaric oxygen treatment following mid‐cervical spinal contusion injury ‐ diaphragm outcomes
Authors: Turner Sara M , Schuster Cassandra M , Morton Aaron B , Hinkley J. Matthew , Fuller David D , Smuder Ashley J
Published: November 2023

Oxidative damage to the diaphragm as a result of cervical spinal cord (cSCI) injury promotes muscle atrophy. However, prevention of cSCI‐induced oxidative stress in the diaphragm via antioxidant therapy can reduce diaphragm atrophy. Hyperbaric oxygen treatment (HBOT) can modulate expression of reactive oxygen species (ROS) and antioxidant enzymes in skeletal muscle. Although mounting evidence suggests HBOT exposure acutely following spinal cord injury (SCI) is associated with a small but functionally beneficial impact on spinal cord pathology, the impact of HBOT on skeletal muscles after SCI is unknown. We tested the hypothesis that HBOT exposure initiated immediately following cervical SCI would alter the expression of diaphragm genes and proteins related to oxidative stress and skeletal muscle atrophy. Rats were randomly divided into the following groups: 1) naïve (no prior treatment), 2) lateral contusion at C3/4 (cSCI) and, 3) cSCI + daily HBOT. The HBOT consisted of a 1‐hr exposure to 2.0 atmospheres of 100% O2, beginning on the day of SCI and continuing for 10‐days. After the 10th treatment day, diaphragm muscle was harvested for analyses of mRNA and protein expression related to muscle proteolysis, oxidative stress, and mitochondrial function. Compared to naïve rats, the cSCI cohort showed increased expression (p<0.05) of markers related to atrophy (Atrogin‐1, MuRF‐1, IL‐1, TWEAK), oxidative stress (ROS, 4‐HNE), and mitochondrial dysfunction (decreased respiratory chain ratio; RCR). The cohort of cSCI rats treated with daily HBOT had significantly lower expression of the markers of proteolysis and oxidative stress, and a significantly greater RCR compared to untreated cSCI rats (p<0.05). In addition, daily HBOT increased antioxidant enzyme expression (SOD2, GPX1). Our data show that daily HBOT has a substantial impact on the diaphragmatic response to cervical SCI. In particular, the reduction in atrophy‐related gene expression raises the possibility that HBOT may have value as an adjunctive therapy to promote health in the phrenic neuromuscular system following cervical SCI.

Support or Funding Information

Craig H. Neilsen Foundation 313369 (SMFT); 1 R01 NS080180‐01A1 (DDF), State of Florida Brain and Spinal Cord Injury Research Program (DDF)
Toll‐like receptor 9‐dependent phosphorylation of AMPKα occurs via TAK1 and inflammatory signaling in vascular smooth muscle cells
Authors: McCarthy Cameron G. , Wenceslau Camilla F. , Ogbi Safia , Szasz Theodora , Webb R. Clinton
Published: November 2023

Toll‐like receptor (TLR)9 is a pattern recognition receptor of the innate immune system. Recently, a non‐canonical stress tolerance pathway was proposed for TLR9 in non‐immune cells, independent of (canonical) inflammatory signaling. It was reported that TLR9 inhibited sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA)2, increasing cytosolic Ca2+, and resulting in 5′ AMP‐activated protein kinase (AMPK)α activation. In our laboratory, we have reported that TLR9 treatment causes arterial dysfunction in vivo, as well as AMPKα phosphorylation (Thr172) ex vivo. However, this dysfunction and phosphorylation does not appear to be initiated by SERCA inhibition. Therefore, the mechanism by which AMPKα is phosphorylated in the vasculature is still unknown, as well as whether it contributes to TLR9‐mediated arterial dysfunction. We hypothesized that transforming growth factor β‐activated kinase 1 (TAK1), a kinase involved in TLR9 inflammatory signaling, was mediating AMPKα phosphorylation. Vascular smooth muscle cells (VSMCs) cultured from aortae of male Sprague‐Dawley rats were treated with TLR9 agonist (ODN2395; 2 μM) for 20 min. Some cells were pre‐treated with inhibitors of TLR9 (ODN2088; 10 μM), TAK1 [(5Z)‐7‐Oxozeaenol; 100 nM], or AMPKα (compound C; 10 μM) for 30 min. Western blot analysis revealed that ODN2395 increased phospho‐TAK1Thr184/187 (1.4 fold vs. Veh, p<0.05), and this increase was prevented in the presence of ODN2088. As previously reported in isolated arteries, ODN2395 increased phospho‐AMPKThr172 (1.6 fold vs. Veh, p<0.05), and this increase was prevented in the presence of (5Z)‐7‐Oxozeaenol. Finally, ODN2395 increased phospho‐myosin phosphatase target subunit 1 (MYPT1)Thr696 (1.5 fold vs. Veh, p<0.05), and this increase was prevented in the presence of compound C. These results reveal that TLR9‐dependent phosphorylation of AMPKα in VSMCs occurs via TAK1, and that AMPKα activation by TLR9 can mediate arterial dysfunction via inhibition of myosin light chain phosphatase.

Support or Funding Information

American Heart Association (AHA) and National Institutes of Health (NIH)
Effects of 1‐Kestose Feeding on Cecal Microbiota which Produce Lactate and Short‐chain Fatty Acids and on Blood Components in Rats
Authors: Shimomura Yoshiharu , Tochio Takumi , Kitaura Yasuyuki , Takahashi Motoki , Endo Akihito
Published: November 2023

Fructooligosaccarides (FOS) are known as prebiotics to improve the intestinal environment. The FOS are composed of 1‐kestose, nystose, and fructofranosylnystose, which have 1–3 fructose monomers linked with sucrose via beta‐2,1 glycosidic bonds. In the present study, we examined feeding of 0.5–5% 1‐kestose diets on cecal microbiota which produce lactate and short‐chain fatty acids by fermentation and on blood components in rats. The results showed that dietary 1‐kestose induced cecal hypertrophy and alterations in the cecal microbiota composition, including a marked increase in the cell number of Bifidobacterium spp, which induced significant increases in acetate and lactate, and a marked increase in butyrate in cecal contents. The feeding of 2.5–5% 1‐kestose diet induced a significant decrease in serum insulin concentration. These findings suggest that 1‐kestose is a primary component of the FOS to improve the intestinal environment.
Clodronate Does Not Alter Bone Remodeling in Young Horses
Authors: Richbourg Heather A , Mitchell Colin F , McNulty Margaret A
Published: November 2023

The bisphosphonate drug clodronate has been shown to treat diseases of abnormal bone remodeling (e.g., navicular disease) in horses, with the goal of systemically reducing osteoclast‐mediated bone loss. Recently FDA‐approved for use in horses, clinical trials have determined its effectiveness in reducing lameness in adult horses by 60 days post treatment. However, despite prolific clinical use, studies have not identified a systemic effect on normal bone remodeling in the horse, either in adults or young horses that are still undergoing tremendous bone growth and remodeling. Therefore, this study aimed to evaluate effects of clodronate on normal bone remodeling in young horses. In addition, the effect on bone healing was assessed using a bone defect model. Two‐ and three‐dimensional quantitative morphological evaluations of bone biopsies of the tuber coxae were utilized. We anticipated increases in bone volume, and decreases in bone remodeling and osteoclast number and function due to treatment compared to saline. Baseline biopsies were collected on Day 0 from nine horses aged 2–4 years old. Following the initial biopsy, horses were randomly assigned to a treatment group (control n=5, treated n=4) and treatment was administered intravenously. Oxytetracycline was administered at Day 47 and 57 as a fluorescent bone label. On Day 60, an additional bone biopsy was collected from the contralateral side, as well as a re‐biopsy of the initial side as a model of bone healing. Biopsies were evaluated via both micro‐computed tomography (microCT) and histomorphometry. Statistical analyses included paired and unpaired student's t‐tests. Horses tolerated surgery well with no post‐surgical complications. All biopsies were adequate for both microCT and histological evaluations. There was no significant effect of clodronate treatment on bone volume or trabecular structure parameters as evaluated by microCT. Clodronate did not significantly alter bone remodeling parameters after 60 days as evaluated by dynamic histomorphometry. Overall, clodronate does not appear to negatively affect normal bone structure and remodeling after 60 days in young horses. In addition, it appears that clodronate has little impact on bone remodeling short term, potentially due to its' relatively‐low potency compared to other bisphosphonates available. These data have significant clinical impact regarding the effectiveness of clodronate to treat diseases of bone remodeling in horses in general, and demonstrates that there is no negative effect of clodronate administration on normal remodeling processes in young horses.

Support or Funding Information

Louisiana State University Charles V. Cusimano Equine Health Studies Program Grant
Palisade Endings in Cat Extraocular Muscles Develop Postnatally Following Different Time Courses
Authors: Streicher Johannes , Davis López de Carrizosa María A. , de la Cruz Rosa R. , Loro Ángel Manuel Pastor , Blumer Roland
Published: November 2023

Proprioception from extraocular muscles (EOMs) provides the brain with eye position signals. Surprisingly, classical proprioceptors (muscle spindles and Golgi tendon organs) are absent in the EOMs of most mammals. Instead, palisade endings (PEs) are found as unique to EOMs of mammals. They are regularly present in frontal‐eyed but infrequent in lateral‐eyed species. PEs are formed by axons which extend onto the tendon where axons make a u‐turn and divide into axonal branches that establish nerve terminals around the tip of a single muscle fiber. For a century, PEs have been considered as sensory structures substituting classical proprioceptors in EOMs. The interest in PEs has newly aroused when molecular analysis and recent neuronal tracing experiments have shown that PEs are cholinergic and originate from the EOM motor nuclei.

Since the development of PEs is unknown, we have analyzed their postnatal maturation in a frontal‐eyed species (cat). Cats of different ages (P0, P8, P22, and adult) were used and whole mount preparation of the rectus EOMs were triple‐immunolabelled and analyzed in the CLMS. Triple labelling included antibody against neurofilament (to label axons), anti‐synaptopysin (to label nerve terminals), anti‐growth associated protein43 (GAP43, to label developing axons), and phalloidin (to label muscle fibers).

At P0, no PEs were found in the rectus muscles. However, in the medial rectus we found axons extending straight onto the tendon where they extensively sprouted to establish synaptophysin‐positive contacts. At P8, premature PEs were found in the medial rectus but not in the other rectus muscles. At P22, the complexity of PEs in the medial rectus had increased noticeably and formation of PEs had started in the inferior rectus. At this stage PEs were still absent in the lateral rectus and superior rectus muscle. In the adult animal, all rectus muscles contained PEs. GAP43, a marker for developing axons was highly expressed in premature PEs. GAP43 expression decreased with age and was nearly absent in PEs of adult animals.

Here we show that the development of PEs in the rectus muscles occurs in a staggered fashion and is more accelerated in the medial rectus. Recently we have shown that in frontal‐eyed species many more PEs are present in the medial rectus compared with the other rectus muscles. The preponderance and the quicker maturation of PEs in the medial rectus support the notion that PEs could play a role in convergence.
Genomic cell lineage analysis uncovers novel endothelial gene expression programs for metabolic and immune regulation
Authors: Klomp Jeff , Malik Asrar
Published: November 2023

Endothelial cells (ECs) take on a variety of cellular phenotypes that depend on their niches, making it difficult to discern lineage‐wide traits. Therefore, we sought to identify novel aspects of EC biology by defining a core EC transcriptional signature shared amongst ECs. We accomplished this by condensing multiple sources of EC transcriptomic and genomic data sets using multivariate statistics and phylogenetic analyses. Our comparative multi‐tissue analyses confirmed expected gene expression patterns such as Flt1, Pecam1, Cdh5, Tek, and Ptprb as core elements of ECs. We also observed strong statistical associations for a variety of genes without prominent roles in ECs, such as the oncostatin M receptor (Osmr), cysteine and tyrosine rich 1 (Cyyr1) and an uncharacterized cDNA sequence (BC028528) that were always positively associated with ECs compared to other cell types. This implied that associations with tightly regulated genes can be obscured by EC variability but may be important for fundamental EC biology. Strikingly, hallmark interferon alpha and gamma gene sets were associated with all EC subtypes and ECs showed highest enrichment in an interferon alpha activation gene signature (p<1e‐15). More broadly, glycolytic and mitochondrial metabolic gene sets were strongly down‐regulated in resting ECs while regulatory transcriptional and translational program gene sets were up‐regulated in these cells. Together, these findings support the existence of key metabolic and immune regulators that are necessary for constitutive EC function in all lineages that can be carefully scrutinized by gene modulation in experimental systems.

Support or Funding Information

NIH T32
Deciphering the dynamics of alternative pre‐mRNA processing of glutaminase in ovarian cancer
Authors: LaFontaine Patrick R , Gibbs Bettine , Masamha Chioniso Patience
Published: November 2023

Ovarian carcinoma is the fifth leading cause of cancer death among women in the United States and has the highest mortality rate of all gynecologic malignancies. Patients with ovarian cancer are often diagnosed with advanced stage, metastatic disease due to lack of specific symptoms and adequate reliable tumor specific biomarkers for early disease detection. An emerging hallmark of cancer is the metabolic reprogramming of transformed cells to support the energy and biosynthetic needs of the rapidly dividing and metastatic tumor phenotype. These metabolic changes facilitate increased glucose and glutamine uptake and metabolism. Recent studies suggest that highly invasive ovarian cancer cells show a remarkable dependence on glutamine, making glutamine metabolism a viable therapeutic target in ovarian cancer. There are several clinical trials focused on inhibiting glutaminase, the enzyme that catalyzes the first step of glutaminolysis and converts glutamine to glutamate. There are two genes that code for glutaminase in the human genome, glutaminase 1 (GLS1) located on chromosome 2 and glutaminase 2 (GLS2) encoded by chromosome 12. GLS1 is the enzyme most associated with tumor metabolism. There are two annotated alternative isoforms of GLS1, GAC and KGA, which result from alternative splicing. Different tumors express either or both isoforms. Under normal conditions the production of the KGA isoform is repressed by miR‐23 which binds to the long 3′UTR of KGA and silences its expression. We recently found that depletion of a 3′end processing factor, CFIm25, results in alternative polyadenylation of the KGA isoform resulting in a novel KGA transcript that has a truncated 3′UTR and is able to evade miR‐23 regulation. In addition, knockdown of CFIm25 also switched splicing to preferentially facilitate splicing to KGA instead of the GAC isoform. Our goal is to determine the different glutaminase isoforms in metastatic ovarian cancer cells and determine how they are regulated for therapeutic targeting using isoform specific siRNAs. We have developed isoform specific amplicons to measure levels of GAC, long KGA (with long 3′UTR) and short KGA (with short 3′UTR). Using qRT‐PCR we show that the highly metastatic ovarian cancer cell lines express high levels of the GAC isoform. The cisplatin resistant cell line SKOV3 also expresses KGA in addition to GAC and we detected this at both the mRNA and protein level. Inhibition of GLS1 using inhibitors resulted in decreased cell viability of highly metastatic ovarian cancer cell lines. Based on our findings, we hypothesize that expressing both the KGA and GAC isoforms facilitates the chemotherapeutic drug resistance of highly metastatic ovarian tumors.

Support or Funding Information

Funding: Butler University Holcomb research award, Senior Research Grant from the Indiana Academy of Sciences
The Role of the C1q Domain of Otolin1 in Otolith Morphogenesis
Authors: Das Olipriya
Published: November 2023

Otolin1 is an extracellular matrix protein of otoliths (“ear stones” of fishes), and otoconia (“ear dust” of higher vertebrates). These acellular biominerals are essential for the sense of balance; dislodging results in the most common human balance disorder, benign paroxysmal positional vertigo. Otolin1 comprises of a collagen and a C1q domain, similar to atypical collagens VIII and X. We propose that Otolin1 forms a scaffold to which other otolith proteins, Ca2+ and CO2− ions bind during otolith morphogenesis, with C1q trimers as hubs and collagen triple‐helices as spokes. To test this model, we investigated whether the Otolin1 C1q domain is necessary and sufficient to form trimers and higher complexes in vitro. The zebrafish Otolin1a C1q domain was expressed in bacteria either alone or fused to thioredoxin. Multimerization of affinity purified recombinant proteins was assessed by size‐exclusion chromatography and gel electrophoresis. Under denaturing conditions, recombinant C1q proteins formed monomers and trimers, but not dimers or higher‐order complexes. Under native conditions, they formed only higher‐order complexes. In contrast, thioredoxin alone only appeared as a monomer. Our results are consistent with the proposed ability of the Otolin C1q domain to form trimers that, in turn, assemble into higher‐order complexes. Experiments are underway to characterize the various complexes in more detail and to test whether the C1q domain is necessary to trimerize of full‐length Otolin1a.

Support or Funding Information

Supported by NIH R01DC006962
Quantitative Sequence Specific Protein Footprinting Reveals Structural Details of Amyloid‐β (1‐42) Peptide Oligomerization
Authors: Klinger Alexandra L , Kiselar Janna , Nix Andrew J , Paravastu Anant , Rosenberry Terrone L
Published: November 2023

Synchrotron based hydroxyl radical footprinting (HRF) was employed to study the formation of Aβ42 oligomers, believed to be the neurotoxic species in Alzheimer's disease. Rates of oxidative modification for seventeen individual residues (F4, Y10, H13, H14, L17, F19, F20, V24, K28, I31, I32, M35, V36, V39, V40, I41) were determined for monomer, 2‐4mer, and oligomer Aβ42 relative to intrinsic rates of modification determined for the same residues in fragment peptide. The ratio of measured and intrinsic rates reveals protection from hydroxyl radical attack due to reduced side chain solvent accessibility for structured Aβ42. Protection observed in the intrinsically disordered Aβ42 monomer is consistent with transient C‐terminal β‐strand formation and partial folding in the hydrophobic core region. Our results also reveal dimerization (and further assembly) contacts that are consistent both with oligomer C‐terminal antiparallel β‐sheet formation previously indicated from published solid state NMR work studies and newly detected N‐terminal structure.
Transport of the Alzheimer's Disease Associated Amyloid‐Beta Peptide by P‐Glycoprotein
Authors: McCormick James Warren , Nanayakkara Amila K , Vogel Pia D , Wise John G
Published: November 2023

The broad range of substrates that can be transported by the transmembrane protein, P‐glycoprotein (P‐gp), has been well established. While best known for the ability to confer general multidrug resistance to a population of cancer cells and for its pharmacokinetic implications in being a critical transporter in the blood brain barrier (BBB), P‐gp action has also been implicated in Alzheimer's disease (AD). One clearance pathway across the BBB for amyloid‐β, which is thought to be a causative agent in neurodegeneration and symptom onset in AD, was hypothesized to be catalyzed by P‐gp. A dynamic P‐gp model similar to that used in McCormick et al. (Biochemistry 2015) was used here to allow docking of amyloid‐β and an unrelated peptide of the same size to P‐gp. Simulations of the catalytic cycles through four different crystallographic conformations of P‐gp‐related proteins using targeted molecular dynamics (TMD) were performed. Even though movement of amyloid‐β was not targeted by the method, transport of amyloid‐β through the membrane was observed during the putative catalytic cycles. These simulations started with amyloid‐β bound to P‐gp in conformations that were open to the cytoplasm and ended with amyloid‐β bound to P‐gp in conformations that would allow amyloid‐β access to the extracellular space. In simulations using a control peptide of the same length, no transport was observed. In experiments to physically evaluate the computational results, synthetic amyloid‐β 42 was fluorescently labeled with carboxyfluorescein and incubated with cells having P‐gp overexpressed. The experiments showed that cell lines with high levels of P‐gp had measurably less accumulation of the fluorescent amyloid‐β 42 than cells with low expression levels of P‐gp. Amyloid‐β accumulation was inhibited in the presence of P‐gp specific inhibitors, supporting the hypothesis that P‐gp plays a role in the clearance of amyloid‐β 42 from the brain.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM09477102] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Genetic Requirements for the Degradation of Translocon‐Associated Proteins
Authors: Indovina Christopher Joseph , Engle Sarah M , Watts Sheldon G , Rubenstein Eric M
Published: November 2023

The endoplasmic reticulum (ER) translocon is a molecular channel responsible for moving secreted and endomembrane system proteins across the ER membrane. Proteins that stall within or otherwise aberrantly engage the translocon channel may impair transport of other proteins across the membrane. Proteins that persistently or aberrantly engage the translocon are targeted for degradation via the conserved ER‐resident ubiquitin ligase Hrd1. A better understanding of this system may prove useful in treating elevated cholesterol in humans as, under certain circumstances, the major protein component of low‐density lipoproteins (LDL; bad cholesterol) stalls within the translocon prior to LDL synthesis. Protein co‐factors that assist Hrd1 in targeting other substrates for degradation are dispensable for Hrd1‐dependent degradation of translocon‐obstructing proteins. We hypothesize that Hrd1 requires the function of a distinct cohort of protein co‐factors to recognize and ubiquitylate proteins that aberrantly engage the translocon. We have developed a growth‐based screen to identify genes in Saccharomyces cerevisiae that mediate the degradation of proteins that aberrantly engage the translocon. We fused the His3 enzyme, which is required for histidine biosynthesis, to a model protein that aberrantly engages with the translocon. In this way, histidine prototrophy is an indicator of impaired protein degradation. We introduced this fusion protein into the non‐essential yeast gene knockout collection using Synthetic Genetic Array (SGA) technology. Genes found to be required for degradation of the model translocon‐associated protein may represent therapeutic targets for patients with cholesterol‐related pathology.

Support or Funding Information

National Institutes of Health Grant R15 GM111713 (to EMR). Funds from the Ball State University Provost's Office and Department of Biology (to EMR). Ball State University Chapter of Sigma Xi Research Award (to CJI).
The Effect of A Y39A Substitution in Trypsin And Its Interaction with Macromolecules ‐ Implications for The Development of Protease Therapeutics
Authors: Adame Andrea , Huang Peishan , Baird Teaster
Published: November 2023

The majority of serine proteases that are used as therapeutic agents, such as tissue and urinary plasminogen activators, share the structural scaffold of trypsin. Trypsin is one of the most well‐studied proteases and is quintessential as a model for examining serine protease interactions with substrates. Previous work has shown that positions 39 and 60 play important roles in inhibitor‐binding to trypsin; an amino acid change results in a decreased affinity due to disrupted prime‐side, backbone interactions. A single amino acid substitution of alanine for tyrosine at position 39 in trypsin (Y39A‐Tn) was found to be less sensitive to inhibitors and has the potential to improve the protease half‐life. In Y39A‐Tn, hydrogen bonding with lysine 60 (K60) on trypsin and with the inhibitor residue at the P4′ position is prevented. Position Y39 of trypsin is well‐conserved among trypsin‐fold serine proteases and its resistance to naturally occurring inhibitors could suggest the same effect for interactions with a protein substrate. To examine the overall catalytic activity toward such a substrate, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and gel imaging were used to analyze digestion product band intensities. Preliminary data suggests that the Y39 variant has similar proteolytic activity towards protein substrates as wild‐type trypsin. Due to increased inhibitor resistance, Y39A variant can serve as a good model for other proteases in the therapeutics field.
Deciphering the Selectivity and Reactivity of Ebselen Derivatives toward Mycobacterium tuberculosis Antigen 85C
Authors: Goins Christopher M , Dajnowicz Steven , Thanna Sandeep , Sucheck Steven J , Parks Jerry M , Ronning Donald R
Published: November 2023

Ebselen was previously found to potently inhibit the essential Mycobacterium tuberculosis (Mtb) Antigen 85s through the allosteric, covalent oxidation of a solvent exposed, conserved cysteine residue. In this study, we sought to better understand the in vitro inhibitory properties of a recently synthesized 1st generation library of ebselen derivatives with respect to Ag85C. To better understand protein‐inhibitor interactions, Ag85C was successfully co‐crystalized with two bulky, chemically dissimilar derivatives, resulting in two X‐ray crystal structures of 2.01 Å and 1.30 Å. Both inhibitors displayed an aromatic stacking interaction with Phe253 and the aromatic ring of the ebselen scaffold in addition to a cationic interaction between the side chain of Arg239 and the respective chemical derivative. Aside from these interactions with Ag85C, the ebselen derivative was oriented towards solvent. To better understand how these two interactions potentially facilitate initial drug binding, resulting in the unique in vitro properties of the various derivatives, interaction energetics were computed for both interactions with respect to the two derivatives using Density Functional Theory. When comparing the energetic values to inhibitory activities, we found derivatives capable of forming favorable interactions with Arg239 have higher levels of inhibition. Furthermore, we looked into the electronic effects the derivatives have on the reactivity of selenium towards the oxidation of Cys209 using Density Functional Theory. The various derivatives were found to have little too no effect on reactivity, strengthening the importance of interactions with Arg239. Both novel structures displayed a greater shift in movement to helix α9 than what was observed in the previous Ag85C Ebselen structure. This movement caused the elongation and restructuring of a loop connecting helix α9 to the preceding β‐strand. We questioned whether this movement affected protein stability and found using differential scanning fluorimetry that Ag85C modified by ebselen or its derivatives decreased the melting temperature by 14 °C. This shift results in the unfolding of protein at physiological temperatures. Upon closer inspection of both structures, a solvent channel leading from the active site to the modified Cys209 is observable. This channel is of great intrigue to us as a future path for drug development towards Mtb Ag85s.

Support or Funding Information

NIH grant AI105084
AF4‐AF9 Protein‐Protein Interaction Inhibitor: Synthesis and Biological Evaluation
Authors: Roseman Shelby , Bao Wei , Koh John
Published: November 2023

In normal human cells, the Mixed Lineage Leukemia (MLL) gene is believed to encode proteins involved in the transcriptional regulation of developmental processes. However, MLL can undergo chromosomal translocations to join with over sixty partner genes and produce fusion proteins that play key roles in the transformation of hematopoietic cells into leukemia cells. These genetic fusions are observed in eighty percent of infant Acute Lymphoblastic Leukemia patients and sixty percent of infant Acute Myeloid Leukemia patients, and the most common fusion partner is AF4. MLL‐AF4 interacts with transcription factor AF9, and it has been shown that inhibiting this interaction leads to leukemia cell death. As such, the AF4‐AF9 interaction is a target for disrupting the MLL fusion pathway and a potential point of intervention for pediatric leukemia.

This study aims to evaluate and improve upon a small molecule inhibitor of the AF4‐AF9 interaction identified from high‐throughput screening. Though the compound disrupts AF4‐AF9 association, its mechanism of action is unclear and difficult to study. AF4 and AF9 are intrinsically unstable proteins and the initial hit compound is prone to oxidation. In this study, more stable analogs of the hit compound were synthesized. In addition, AF9 was expressed in high yield as a fusion with AF4 containing a proteolytic site in the linker. The fusion protein can be cleaved, but separation of AF4 and AF9 has proved challenging due to strong intermolecular association. Interactions of the complex with the target molecule were evaluated by circular dichroism and fluorescence anisotropy. Surface plasmon resonance will also be employed to study inhibitor binding to AF4 and AF9.

Support or Funding Information

Support for this project is provided by Alex's Lemonade Stand Foundation. Additionally, summer research funding was provided by the Plastino Scholars program and University of Delaware Summer Scholars.
Purification and Quantification of the Chemical Markers of Melanin to Enhance Early Diagnosis of Melanoma
Authors: Weddle Carly , Sienerth Karl , Glass Keely
Published: November 2023

Melanoma is characterized by overproduction of the pigment melanin, which is present in two forms in human skin – eumelanin and pheomelanin. High discordance rates among experienced pathologists in the diagnosis of questionable malignant epidermal samples demonstrate a need for more robust and objective diagnostic tools. Although optical imaging qualitatively suggests that a high ratio of eumelanin to pheomelanin corresponds to the incidence of malignant melanoma, no chemical analysis to date has successfully quantified this ratio. Reproducible techniques for quantifying the natural forms of melanin may provide insight into the molecular mechanisms underlying melanoma and may be used to determine whether a high eumelanin: pheomelanin ratio is indicative of malignant melanoma. Current protocols for analyzing melanin entail chemical degradation of the pigment into markers specific to each of the two natural forms. These chemical markers are separated and quantified by high performance liquid chromatography with ultraviolet detection (HPLC‐UV). While this characterization has proved useful, identification of the chemical markers in complex biological matrices is limited by interfering signals. This study introduces two new methods for identifying and quantifying the chemical markers of melanin: (1) sample purification with solid‐phase extraction (SPE) and (2) selective identification of degradation markers with mass spectrometry (HPLC‐MS). Application of these techniques to melanoma cells derived from a metastatic lung site and normal skin fibroblast cells demonstrate enhanced selectivity and reproducibility in the quantification of chemical markers of melanin in complex biological matrices. Sample preparation with SPE improves clarity in HPLC chromatograms and MS detection allows unambiguous assignment of individual peaks to specific degradation markers of melanin. Enhanced selectivity and accuracy in the quantification of melanin may ultimately contribute to standardization of techniques in pigment cell research and be used as a robust diagnostic tool for detecting early stage, thin lesion melanomas.

Support or Funding Information

Elon University Honors Program

Elon University Undergraduate Research Program

Elon University Lumen Prize
Glucocorticoid‐induced inhibition of AKT leads to CREB phosphorylation and increased myostatin expression via a PDE/cAMP/PKA pathway in skeletal muscle
Authors: Xie Yang , Zhang Peng , Espinoza Daniel , Perry Ben , Rahnert Jill , Zheng Bin , Price Russ
Published: November 2023

Muscle atrophy in diseases like chronic kidney disease and diabetes is associated with dysfunctional regulation of insulin/Akt/FoxO3 and myostatin‐SMAD3 signaling pathways. We recently found CREB, a transcription factor that regulates maintenance and regeneration of skeletal muscle, is phosphorylated in wasting conditions. In silico analysis identifies a CREB binding site in the myostatin promoter. This study investigated the mechanism by which atrophy‐inducing conditions regulate CREB phosphorylation and its impact on myostatin expression. Differentiated C2C12 myotubes were incubated with combinations of dexamethasone (Dex), LY294002 (PI‐3 kinase inhibitor), H89 (protein kinase A inhibitor), isobutylmethyl xanthine (IBMX, a general phosphodiesterase (PDE) inhibitor), milrinone (PDE3 inhibitor), or rolipram (PDE4 inhibitor) for up to 24 h. Cells were harvested for analysis of proteins or mRNAs. Treatment of myotubes with Dex for 6–24h increased CREB phosphorylation (pCREB) within 3 h and sustained the increase over 24h. Dex also inhibited Akt phosphorylation (pAkt) over a similar time frame. Inhibition of pAkt by LY294002 mimicked Dex by increasing pCREB; the actions of LY294002 and Dex on pCREB were not additive. Since Akt can activate PDE3/PDE4 and reduce cAMP/PKA signaling in non‐muscle cells, we tested whether Dex‐induced inhibition of PDE3/4 increases pCREB. Without Dex, IBMX, milrinone, and rolipram increased pCREB. The Dex‐induced increase in pCREB was attenuated by the PKA inhibitor H89. When added individually, Dex, milrinone, rolipram and IBMX increase myostatin mRNA and protein significantly. Our study identifies a new mechanism by which atrophy‐inducing conditions activate CREB and contribute to myostatin expression. Signals that attenuate insulin/Akt signaling reduce the PDE3/4 activity. This leads to an increase in cAMP, PKA activity, CREB phosphorylation and myostatin expression.

Support or Funding Information

Funded by NIH RO1 DK95610 and VA Merit X01BX001456
Puringeric Signaling in Bone as a Potential Mechanism in Prostate Cancer Proliferation and Cancer‐Induced Bone Pain
Authors: Wilson Michael , Duncan Randall , Boggs Mary
Published: November 2023

Prostate cancer (PCa) is the second leading cause of cancer‐related deaths in men in the US. PCa bone metastasis occurs in approximately 2 out of 3 patients whose cancer has spread to other parts of the body. In particular the majority of PCa that metastasizes to bone results in osteosclerotic tumors. Osteosclerotic tumors arise from interactions between cancer cells and osteoblasts that results in increased bone formation, but reduced bone strength and increased mechanical strains. The increased load in bone resulting from tumor formation correlates with increased pain sensation. Cancer‐induced bone pain (CIBP) is one of the most detrimental symptoms to patients with bone‐localized cancer, greatly reducing patient's quality of life. Current treatment for CIBP is ineffective. A better understanding of the molecular mechanisms of CIBP is necessary for proper treatment. We propose that mechanical load of PCa cells results in ATP release which can activate local neurons through P2X3 receptors resulting in CIBP. Additionally, we propose that nerve‐growth factor (NGF), a molecule implicated in PCa proliferation, enhances the potential for CIBP by through PCa cell proliferation and ATP signaling. To determine the effect of mechanical load on ATP release from PCa cells and activation of neurons, we mechanically loaded human bone‐derived PCa cells, C4‐2B4, via hypotonic swelling. Resultant C4‐2B4 load‐response conditioned media (L‐CM) +/− apyrase or various concentration of ATP were applied to primary mouse dorsal root ganglia (DRG) and changes in intracellular calcium were measured. To determine the role of P2X3 in response to L‐CM, TNP‐ATP, a P2X3 specific antagonist was utilized. Data indicated that mechanical load increased ATP release from C4‐2B4 cells and that L‐CM increased intracellular calcium in DRG which was reduced with apyrase. TNP‐ATP did not significantly block response to L‐CM suggesting another purinergic receptor may be responsible for this response.

To address the role of NGF in PCa proliferation and purinergic signaling, we applied NGF to C4‐2B4 and counted cells daily for 4 days. To determine the effect of NGF on C4‐2B4 response to ATP, we applied various concentrations of ATP to C4‐2B4 cell pretreated with NGF for 48 hrs and measured intracellular calcium. NGF treatment demonstrated a 32% increase in cell number at day 4 when compared to control. Calcium imaging studies indicated that response to ATP varied with addition of NGF indicating a potential pathway for altered sensitivity to ATP. These studies indicate that NGF may play an important role in the growth of metastatic prostate cancer cells in bone. Taken together these data indicate purinergic receptors as a potential therapeutic target for CIBP.

Support or Funding Information

Delaware INBRE program

NIH‐NIGMS (8 P20 GM103446‐15)

Charles Peter White Fellowship
Elucidating the Reproductome: System‐wide Regulation of Reproductive Neuropeptides
Authors: Glucksman Marc , Philibert Keith , Woitowich Nicole , Urban Janice , DeJoseph Gina
Published: November 2023

Kisspeptin (Kiss), neurokinin B (NKB) and dynorphin A (DynA), collectively known as the KNDy neurons in the hypothalamus, drive the activity of the reproductive axis through the regulation of gonadotropin releasing hormone (GnRH) release. These neurons project to the rostral hypothalamus and median eminence to relay peripheral signals such as estrogen levels and energy homeostasis to the GnRH neuron. As neuropeptides, the actions of Kiss and DynA are required to be modified enzymatically in the extracellular space to modulate downstream signaling, though the regulator of this process is unknown. Furthermore, Phoenixin, a recently identified neuropeptide produced in the hypothalamus, increases GnRH, GnRH receptor and Kisspeptin mRNAs, increases GnRH secretion and mediates ovarian cyclicity. EP24.15 (EC 3.4.24.15, thimet oligopeptidase) is a metallo‐endopeptidase, expressed throughout the hypothalamus and other brain regions, that cleaves GnRH and inactivates binding to its cognate receptor.

To authenticate a biochemical and physiological regulatory mechanism for the functioning of Kiss and DynA, we utilized a high throughput workflow schema by first identifying potential active site binding and cleavage sites both in silico and by mass spectrometry; measured enzymatic parameters; performed structural studies. Finally, we determined if EP24.15 and both Kiss and DynA are co‐expressed in the brain, leading to the elucidation of a functional neuroendocrine circuit whereby EP24.15 could regulate Kiss‐ or DynA ‐induced reproductive hormone secretion. EP24.15 and Kiss were co‐incubated and subjected to MALDI‐TOF mass spectrometry to confirm that EP24.15 cleaves full length Kiss as well as the fully functional Kiss10 decapeptide between S5–F6 in vitro (Vmax = 2.37±0.09 pmol/min/ng, Km = 19.68±2.53 μM). Dynorphin A (1–8) is cleaved between F4–L5 and L5–R6 (Vmax = 30.9±0.48 pmol/min/ng, Km = 14.62±0.89 μM). Phoenixin is cleaved between K8–V9 in vitro (Vmax = 2.90±0.20 pmol/min/ng, Km = 25.81±5.40 μM). These novel substrates are cleaved at rates comparable to other known neuropeptides. We have also demonstrated that both Kiss and DynA immunoreactivities are co‐expressed and localized with EP24.15 in the hypothalamus. These data demonstrate that EP24.15 cleaves Kiss, DynA, and Phoenixin in vivo to unveil an additional layer on the exquisite control mechanism of the mammalian reproductome. Further understanding the role of EP24.15 in neuropeptide metabolism will provide insight into the circuitry within the hypothalamus and provide targets for modulating various neuroendocrine axes, including reproductive function.

Support or Funding Information

Supported by NIH OD010662 (MJG).