bluuebunny/crossref_metadata_2025_split huggingface dataset:
Patient‐specific Autoantigen Sample Preparation and Analysis Using the Reversible Protein Tag ProMTag
Authors: Minden Jonathan S. , Ascherman Dana , Lucas Amber 
Published: May 2022

Autoimmune diseases affect >20 million people in the US today. Currently, disease‐specific autoantibodies are thought to be the best biomarkers for diagnosis. Conventional immunoprecipitation methods have been used to identify autoantigens from the most common autoimmune diseases. However, these diseases account for only 6.5 million of the 20 million patients suffering from autoimmune diseases, leaving many without diagnoses until irreversible damage occurs. The remaining 13.5 million patients have >70 autoimmune disorders without well characterized autoantibodies. The state‐of‐the‐art diagnostic test of these remaining diseases relies on gel electrophoresis of immunoprecipitated radiolabeled proteins, which cannot be identified by MS due to safety issues and the overwhelming presence of immunoglobulins.We have created an immunoprecipitation method that uses serum from patients with any autoimmune disorder to identify patient‐specific autoantigen proteins. This method uses a reversible click chemistry tag, called ProMTag. One end of the ProMTag forms a reversible, covalent bond with protein by coupling to lysines and amino termini. The other end of the ProMTag can form an irreversible, covalent bond with a solid bead support via a click chemistry, methyltetrazine‐TCO, pairing. In this study, the proteins of cell lysates that contain potential autoantigens were labeled with ProMTag. The ProMTagged‐proteins were exposed to patient antibodies bound to Protein A beads, thus capturing the ProMTagged autoantigens. All proteins were released from the Protein A beads, including ProMTagged‐autoantigens and untagged‐antibodies. The ProMTagged‐autoantigens were subsequently coupled to TCO beads, and the untagged‐antibodies were washed away. The linkage between the ProMTag and autoantigens was then reversed, yielding autoantigen proteins with greatly reduced antibody contamination ready for MS analysis. MS analysis successfully identified autoantigens from patient serums with rheumatoid arthri. This autoimmune biomarker discovery method can accelerate sample testing for known autoantigens and facilitating rapid discovery of novel autoantigens for both diagnostic and predictive biomarkers.
Exercise Training Attenuates Radiation‐Induced Impairments in Endothelium‐Dependent Vasodilation of Coronary Arterioles in Rats
Authors: Kunkel Olivia , Baumfalk Dryden , Horn Andrew , Behnke Brad 
Published: May 2022

BackgroundRadiation is an important and effective anti‐cancer therapy, but is associated with many side effects. Radiation exposure may cause damage to the heart and coronary vasculature. Exercise training is currently being investigated in cancer research as a treatment or adjuvant to anti‐cancer therapies, and has been shown to mitigate deleterious changes in coronary vascular function, such as with aging. We hypothesized that impairments in endothelium‐dependent vasodilation of coronary resistance arterioles that occur in response to radiation exposure would be mitigated with exercise training.Methods12 male Fischer 344 rats (3‐4 months old) were randomly assigned to one of four groups: control (CON; n=3), exercise (EX; n=3), control radiated (RAD; n=3), or exercise radiated (EX RAD; n=3). After increasing intensity over the first 3 weeks, EX and EX RAD animals ran on a motorized treadmill 5 days/week for 60 minutes/day, at an intensity of 15 m/min and 15° incline for the remainder of 12 weeks of training. After the conclusion of exercise training, all groups of animals were subjected 2 Gy whole body radiation by X‐Rad 350 x‐ray irradiator and euthanized within 8 hours. Hearts were harvested and left anterior descending (LAD) arteriole sections were isolated and fixed on glass pipets. Vessels were pressurized at 60 cm H2O. Once a minimum of 20% tone was developed, vessels were exposed to cumulative doses of bradykinin (10‐13‐10‐7 M), an endothelium‐dependent vasodilator, and vessel diameter was recorded at each dose.ResultsNo differences were detected for CON vs. EX or CON vs. EX RAD. Vasodilation for RAD was significantly reduced vs. CON (p = 0.007). EX RAD was significantly increased vs. RAD (p = 0.025).ConclusionsThere were no differences between CON and EX; however, vasodilation was impaired for radiated animals, suggesting even one dose of 2 Gy radiation can cause significant impairment in endothelium‐dependent vasodilation of coronary arterioles. Exercise training attenuated this impairment and restored endothelium‐dependent vasodilation to levels not different from control animals. These findings are significant, given that exercise training may be used as a simple and safe method to preserve coronary arteriole function in patients receiving radiation therapy.
Loss of Autotaxin inhibits Toll‐like receptor4‐mediated immune mechanisms and promote spontaneous colitis in Il10‐ko mice
Authors: Rhee Sang H. , Kim Su Jin , Howe Cody , Jeong Sihyun , Im Eunok 
Published: May 2022

Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl‐phosphorylcholine into lysophosphatidic acid and sphingosine 1‐phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll‐like receptor 4 (TLR4) and its co‐receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4‐mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe‐induced intestinal inflammation.To study this hypothesis, we generated myeloid cell lineage‐restricted Atx‐knockout (ko) mice (Atx‐dME/dME) to investigate TLR4‐mediated immune and inflammatory responses and examined LPS‐receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Phagocytic activity of macrophages was assessed. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using Atx‐dME/dME;Il10‐/‐ mice that have both Atx‐deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients.With peritoneal macrophages from Atx‐dME/dME mice, we identified that Atx‐ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4‐mediated responses were substantially reduced in Atx‐ko macrophages. TLR4‐induced innate immunity such as phagocytosis was attenuated in Atx‐ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of Atx‐dME/dME mice compared to that of Atx‐wt littermates. Consequently, Atx‐dME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10‐/‐ mice, combining Atx‐dME/dME with Il10‐/‐ mice (Atx‐dME/dME;Il10‐/‐) did accelerate spontaneous colitis development. Atx‐dME/dME;Il10‐/‐ mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log‐rank P=0.0046), while Atx‐wt;Il10‐/‐ littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P<0.001), indicating an association of reduced ATX levels with intestinal inflammation.Collectively, we found that Atx deficiency suppresses TLR4‐mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe‐induced gut inflammation.
Conformations of p90 Ribosomal S6 Kinase (RSK) Activation
Authors: Kobori Evan , Chen Katherine , Taylor Susan 
Published: May 2022

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie downstream of the Ras‐MAPK pathway and regulate cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N‐terminal kinase (NTK), and a C‐terminal kinase (CTK) in the same polypeptide chain. The NTK is a member of the AGC kinase superfamily, and the CTK is a member of the CAMK family. A flexible linker bridges the NTK and CTK, and embedded within the linker is the highly conserved tail of AGC kinases (AGC tail) which contains the conserved turn and hydrophobic motifs (HF). RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal‐regulated kinase (ERK2) and 3‐phosphoinositide‐dependent protein kinase 1 (PDK1). ERK2 phosphorylates the CTK and the turn motif. The phosphorylated CTK is then able to phosphorylate the linker at the HF, this enables PDK1 to bind the linker and then phosphorylate and activate the NTK2. The precise structural details of RSK kinase activation are not fully understood and a more rigorous biochemical characterization could provide valuable insight into how kinase complexes assemble and how the linker facilitates multiple binding and phosphorylation events.To gain a better understanding of the FL RSK2‐ERK2 signaling complex we have used cryoEM and hydrogen deuterium exchange mass spectrometry (HDX) to gain insight into the overall shape and solvent accessibility of the overall complex. We have determined a low‐resolution density map of the RSK2‐ERK2 complex, revealing a compact shape. Additionally, all three kinase domains are relatively well protected from solvent, with the exception of the activation loops of the CTK, and the aC helix of the NTK. While the tails of RSK2 and linker (AGC tail) are highly solvent exposed. We hypothesize that the linker region needs such solvent accessibility in order to enable the various interactions and phosphorylation events necessary for RSK activation. We have also utilized peptide array binding experiments to determine the residues within the linker that are necessary to enable ERK2 phosphorylation of the turn motif, CTK phosphorylation of the HF, and PDK1 binding and subsequent phosphorylation of the NTK activation loop. ERK2 appears to bind to the linker and NTK in a multivalent fashion via the C‐lobe (300‐320) and the N‐lobe. The CTK seems to primarily interact with HF in the linker. And PDK1 binds to both the HF and a conserved FDX1‐2Y/F motif in the linker.
Ephrin Ligands are Upregulated in the Saliva of SARS‐CoV‐2 Infected Patients
Authors: Egal Erika , Helms My N. , Liou Theodore G. , Mimche Patrice 
Published: May 2022

BackgroundPatients infected with SARS‐CoV‐2 may develop severe lung disease characterized by respiratory distress, systemic thrombosis, or death. Molecular mechanisms responsible for severe cases of COVID‐19 remain unclear. We investigated the role of the Eph family receptor‐interacting ligands (ephrin‐A1 and ephrin‐B2) in COVID‐19 patients. Ephrin binding to its tyrosine kinase Eph receptor plays important roles in injury and inflammation. Serum levels of ephrin‐A1 ligand correlates positively with COVID‐19 severity (Mendoza et al, CellPress 2021). Based on this recent report, and the emerging role of Ephrin‐Eph signaling in lung injury, we hypothesize that ephrin‐mediated cytokine signaling is upregulated in epithelial and immune cells assayed from saliva samples obtained from seriously ill COVID‐19 patients seeking emergency evaluation and treatment.MethodsSaliva samples were collected from patients (± SARS‐CoV‐2) in the University of Utah Emergency Department (ED) with IRB‐approved written consent. Saliva samples were incubated 1:1 in Streck Preservative (prior to EpCAM and CD45 labeling) or with 0.008% glutaraldehyde (following ELISA and multiplex immunoassays) in order to inactivate virus.ResultsFlow cytometric evaluation of saliva for EpCAM+ and CD45+ cells indicated that SARS‐CoV‐2 positive samples contain both epithelial and immune cell types (see Fig 1). Uninfected saliva samples (control) primarily contained EpCAM+ cells. COVID‐19 samples (n=67 patients) expressed higher levels of type 1 interferon [IFNγ1 and IFNα2 (p< 0.001)] vs control (n=64 patients). Interferon induced protein [IP‐10] was also upregulated in COVID‐19 patients (p<0.001), as well as several cytokines in the interleukin family [IL‐1β, IL‐6, IL‐8 (p<0.001) and IL‐12p70 (p<0.05)]. TNF‐α did not differ significantly between the ± SARS‐CoV‐2 samples evaluated. Using a subset of the +SARS‐CoV‐2 samples (n=20) and uninfected controls obtained from the ED (n=20), we show that both ephrin‐A1 and ephrin‐B2 ligands are significantly elevated in +SARS‐CoV‐2 samples vs. ED control samples (p<0.001). Ephrin‐B2 levels correlated negatively with IL‐12p70 (Pearson Correlation =‐0.53, p=.01).ConclusionSaliva is low risk to obtain and is a biologically relevant sample that can be used to reliably measure changes in epithelial and immune cell responses of COVID‐19 patients. Specifically, our study showed that ephrin‐A1 and ephrin‐B2 ligands are upregulated in the saliva of COVID‐19 patients in a TNF‐α independent manner and that ephrin‐B2 correlates negatively with IL‐12p70 (a potent pro‐inflammatory cytokine). Future studies are aimed at establishing ephrin ligands as a biomarker for severity of lung injury and improving understanding the patho‐physiology of SARS‐CoV‐2 infection.
Membrane Protein Comparison Between Cell Membranes and ExtracellularVvesicle Membranes of S. pneumoniaeProvide Insights into Extracellular Vesicle Formation and Shedding
Authors: Biedka Stephanie , Eutsey Rory , Hiller Luisa , Minden Jonathan S. 
Published: May 2022

Extracellular vesicles (EVs) are complex, cell‐derived nanoparticles generated by all cell types. EVs are composed of lipid bilayer membranes and their associated membrane proteins, nucleic acids, and luminal proteins. The mechanism by which Gram‐positive bacteria shed EVs is still unknown. EVs from the Gram‐positive human pathogen S. pneumoniae, which is a major cause of otitis medi and pneumonia, are of particular interest because of how they EVs modulate the host immune response. To uncover possible mechanisms for EV production and shedding in S. pneumoniae, we have performed a comparative proteomics analysis of EV membrane proteins versus whole‐cell membrane proteins.Membrane proteins were enriched from intact S. pneumoniae cells or their EVs using a ProMTag labeling and capture workflow. ProMTag is a bifunctional protein tag where one moiety of the tag is able to form a reversible, covalent link to primary amines on proteins. The other moiety is methyltetrazine, which can form an irreversible, covalent bond with trans‐Cyclooctene (TCO) on the surface of beads to capture ProMTagged proteins for cleanup and elution. Using this workflow plasma membrane proteins can be tagged, captured, washed to remove non‐plasma membrane proteins, and then eluted in their original, unmodified state.In this study, intact cells and EVs from S. pneumoniae cultures were separated and the extracellular domains of membrane proteins in these two fractions were labeled with ProMTag. The membrane proteins were then enriched, washed, and eluted using the ProMTag workflow. These membrane protein populations were then TMT labeled and analyzed using mass spectrometry. Comparative analysis revealed membrane proteins that are concentrated or absent in EV membranes relative to bulk plasma membrane from whole cells, indicating a selective process for EV formation in S. pneumoniae. With this information, we present a new model for EV formation and shedding in S. pneumoniae.
Sex Differences in Antioxidant Gene Expression in response to Chronic Arsenic Exposure
Authors: Berry Savannah R. , Dresler Sara R. , Propper Catherine R. , Kellar Robert S. , Traustadóttir Tinna 
Published: May 2022

Inorganic arsenic is highly toxic metalloid considered a water contaminant that is naturally occurring through geological formations where it leaches into ground water sources and is anthropogenically sourced through pollution and found in surface water as a contaminant. Acute and chronic arsenic exposure can stimulate the production of reactive oxygen species and increase systemic oxidative stress. The aim of the present study was to investigate sex differences in the redox response induced by chronic arsenic exposure in a murine model.Twenty‐four (12 female & 12 male) 10‐wk old mice were randomized to arsenic treatment or control. Arsenic groups were exposed to controlled arsenic levels (10µM = ppb = ug/L) via drinking water for 8‐wks. Following the treatment, mice were euthanized, and skeletal muscle was collected. The tissue was homogenized and processed for measures of gene expression for seven antioxidant genes (Sod2, Cas1, Gpx, Gst, Gsr, Gclc, Nrf2). One‐way ANOVA was performed with post‐hoc comparisons depending on normality of data. Interactions between sex and arsenic were determined using univariate testing.There was a significant sex‐by‐treatment interaction for Cas1, Gpx1, Gsr, Gclc, and Nrf2 where the female mice treated with arsenic responded by upregulating these genes in comparison to controls (p<0.05), whereas the males did not. Only Sod2 was lower in the arsenic treated female mice versus controls and there were no effects on Gst across the 4 groups.These data demonstrate that sex differences only emerged under stimulated conditions (arsenic exposure) and support the protective role of estrogen in antioxidant defenses.
Interplay between intestinal microbiota and PRDM16‐mediated metabolic regulation of intestinal stem and progenitor cells
Authors: Stine Rachel R. , Seale Patrick 
Published: May 2022

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. Previously, we have shown that the transcription factor PRDM16 is a critical regulator of intestinal metabolism and differentiation. Acute deletion of Prdm16 in adult mice causes intestinal wasting, apoptosis, and an accumulation of poorly differentiated cells in the small intestinal crypt, primarily in the duodenum and proximal jejunum. PRDM16 has emerged as a regulator of metabolic signaling in multiple tissues including thermogenic adipose tissue and the small intestine. RNA‐seq and tracer metabolomic studies show that PRDM16 regulates fatty acid oxidation (FAO) in the intestinal crypt, leading to impaired differentiation of stem and transit amplifying cells into mature cells.Our current studies have focused on the effects of the intestinal microbiota, which produces significant quantities of metabolites that could affect cells in the digestive tract. We postulated that altering the microbiome of mice in the context of Prdm16 deletion could modify the phenotype. Strikingly, pre‐treatment of animals with an antibiotic cocktail including ampicillin, neomycin, vancomycin and metronidazole almost completely rescues the Prdm16 deletion phenotype. While deletion of Prdm16 typically leads to death within 10 days, antibiotic treated mice survive at least 12 weeks after deletion. An early hallmark of the Prdm16‐deletion phenotype is the induction of the p53 pathway in the crypt. Apoptotic cells are present in high numbers, particularly in transit amplifying progenitors. Gene expression profiling of isolated small intestinal crypts indicates that the early p53 signature that is typical of Prdm16 deletion does not develop in antibiotic treated mice. Furthermore, pretreatment of mice with antibiotics prevents the severe defects normally observed when Prdm16 is deleted ex vivo in organoid cultures. However, treatment of organoids with the same antibiotics only in culture does not lead to a phenotypic rescue. We are currently characterizing how deletion of Prdm16 alters the intestinal microbiota and the effects that those changes could have on metabolite levels in the tissue. Overall, these studies aim to understand how changes in metabolic signaling can affect stem and progenitor cell behaviors and how the microbiome and, potentially, antibiotics themselves can affect the function of the intestine.
Functional Morphology of Sensory Organs of the House Cricket, Acheta domesticus (L.): A Morphological and Physiological Investigation
Authors: Shields Vonnie D. , Weaver Cameron E. , Farmer Shannon 
Published: May 2022

Insects have paired antennae that bear many sensory organs or sensilla. These sensilla allow them to gather information about olfactory, gustatory, and mechanosensory environmental cues. Both antennae of male and female house crickets, Acheta domesticus, bear approximately 200 segments. The first aim of this study was to morphologically characterize the sensillum types on the antennae. Scanning and transmission electron microscopy studies revealed that each antennal segment bears seven sensillum types. Four types have external morphological features suggestive of olfactory sensilla, namely an abundance of cuticular pores. Two of these types resemble short to medium‐sized pegs, a third resembles a short peg with longitudinal ridges or “fingers,” while a fourth resembles a short peg recessed in a deep pit. Each antenna also bears two sensillum types devoid of external cuticular pores, suggestive of a mechanosensory function. One of these types is a long hair‐like sensillum with conspicuous ridges, while the other is a small circular depression on the antennal surface. Another medium‐sized sensillum bears diagonal circular ridges and a single apical pore, indicative of a gustatory function.The second aim of this study was to determine which odorants elicited positive or negative anemotaxis (orientation toward or away from a food source, respectively), suggestive of possible food lures or repellents. Odorants permeate through the environment and bind to receptor sites in olfactory neurons. Once these molecules bind, chemical signals are converted to electrical ones. Olfactory signals are then transmitted to higher‐order olfactory processing centers in the insect’s brain. Depending on the odorant, this may trigger a behavioral response(s). Odorants may be used to detect and identify food, mating partners, and avoidance of predators. Previous studies in the lab have focused on odorants associated with some fruits, vegetables, and proteins. In this study, we tested some odorants associated with amines derived from ammonia. We hypothesized that such odorants would elicit negative anemotaxis. We carried out each experiment over a 24‐hour period in plastic bins. We found that the crickets elicited positive anemotaxis to the odorants tested, leading us to reject our hypothesis. These findings are important as house crickets are pests and known to contaminate food sources. This finding raises concerns about foodborne illnesses associated with microbial pathogens and can create health concerns especially in developing countries. The results of this study may prove to be helpful in the suggestion of lures as effective biocontrol measures against pests.