bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Prebiotic Fructooligosaccharides on Susceptibility to Dextran Sodium Sulfate‐Induced Colitis
Authors: Kaderly Brooke , Poku Caleb , Hart Jessica , Oswald Trenton , Walton Kristen L. W.
Published: May 2022

Inflammatory bowel disease (IBD) is a chronic intestinal disease that includes Crohn’s disease and ulcerative colitis. Previous studies have found alterations in the microbiome in IBD and in animal models of IBD, underscoring the role of the intestinal microbiota in IBD pathogenesis. Prebiotics are molecules in foods, such as fructooligosaccharides and other soluble fiber molecules, that support the growth or activity of beneficial probiotic microbes. Previous studies suggest that prebiotics can reduce symptoms in models of colitis. This study aimed to determine whether pretreatment with 1% short‐chain fructooligosacchardies (FOS) for 1 week, followed by 8 days of 3% DSS administration with continued FOS treatment, altered the severity of disease in C57Bl/6 mice. Clinical signs of colitis, including weight loss, fecal occult blood, and fecal consistency, were monitored daily during the DSS treatment period and the daily disease activity index was calculated. At the end of the experiment, samples were collected for histological scoring, and for RNA isolation and analysis of colonic pro‐inflammatory cytokine expression by RT‐PCR. Through the 8 days of DSS treatment, the disease activity index increased in both DSS‐treated and FOS/DSS‐treated mice compared to control non‐colitic mice, but was not significantly different between the two colitic groups. In addition, colon wet weight, colon length, and histological colitis scores were similar in colitic mice treated with FOS compared to colitic mice without FOS treatment. Colonic claudin mRNA expression was lower in DSS‐treated mice than control mice, but was not significantly altered by FOS treatment. These data suggest that treatment with fructooligosaccharides did not significantly alter susceptibility to DSS‐induced colitis.
Famotidine exerts anti‐inflammatory effects via a vagus nerve‐dependent mechanism
Authors: Yang Huan , George Sam J. , Thompson Dane A. , Brines Michael , Pavlav Valentin A. , Andersson Ulf , Chavan Sangeeta S. , Tracey Kevin J.
Published: May 2022

Background

The COVID‐19 pandemic in the U.S. caused > 48 million infections and >500,000 deaths. Famotidine, a histamine H2 receptor antagonist widely used to treat acid reflux and gastritis, is associated with improved survival and attenuated COVID‐19 disease severity (Mather JF et al. Am J Gastroenterol. 115:1617,2020; Freedberg DE, et al. Gastroenterology 159: 1129, 2020; Janowitz T et al. Gut, 69:1592, 2020). However, the mechanism of action for these beneficial effects is unknown. As hyperactive inflammatory responses from SARS‐CoV‐2 plays a critical role in disease progression in COVID‐19, here we studied the effects of famotidine in murine models of cytokine storm.

Methods

Male C57BL/6 mice were subjected to bilateral sub‐diaphragmatic vagotomy and were recovered for 7 days before experiments. Male C57BL/6 mice were given an injection of famotidine either intraperitoneally (4 mg/kg) or intracerebroventricular (0.4 mg/kg) prior to administration of LPS (7 mg/kg) given intraperitoneally. Mice were euthanized 2.5 hours after LPS administration and cytokine levels in sera were measured using ELISAs. For survival studies, male C57BL/6 mice were subjected to LPS (6 mg/kg) injected intraperitoneally followed by treatment with famotidine (intraperitoneal injection at 4 mg/kg) or vehicle (PBS) twice daily for 3 days, and survival was monitored for two weeks.

Results

Famotidine (4mg/kg, injected intraperitoneally at 30 min before LPS) significantly reduced LPS‐induced elevated serum TNF levels at 2.5 hours post‐LPS injection (serum TNF levels in LPS + vehicle = 1,491+ 129 vs. LPS + famotidine = 1,060 + 72* pg/ml, N=10 mice per group. *: P=0.001). Famotidine (4mg/kg, injected intraperitoneally, twice a day for 3 days) improved survival in endotoxemic mice (two‐week survival in famotidine group = 100% vs. 70% in the PBS‐control group, n=30/group, p=0.001). Famotidine administered intracerebroventricularly (0.4 mg/kg) also significantly reduced LPS‐induced serum TNF levels and was >10 times more potent via the intracerebroventricularly route as compared to the intraperitoneal route. Bilateral sub‐diaphragmatic vagotomy attenuated the cytokine inhibiting effects of famotidine (administered intracerebroventricularly) on LPS‐induced TNF release (serum TNF levels in LPS + vehicle = 592+ 55 vs. LPS + famotidine = 616 + 98 pg/ml, N=5 per group. P=N.S.).

Conclusions

Famotidine stimulates vagus nerve’s inhibition of LPS‐induced TNF release and attenuates lethality in endotoxemic mice. These anti‐inflammatory effects require the intact vagus nerve and via a brainstem‐dependent mechanism.
Determining the Time Management Techniques and Study Strategies of Master’s Students Enrolled in a Graduate Gross Anatomy Course: a Pilot Study
Authors: Boothe Casey , Schaefer Audra
Published: May 2022

Upon entering graduate or professional school, students are often overwhelmed by the robust amount of information being delivered and the short time frame in which they are expected to learn the content. Students who lack sufficient study skills and time management techniques typically struggle to adapt to the heightened demands of graduate school. However, students who are self‐regulated learners possess the skills to adequately manage their time and monitor their own learning. Self‐regulated learning is the ability to set learning goals, plan study time, and assess learning. At the University of Mississippi Medical Center, there is a one‐year Master’s program—the Master of Science in Biomedical Sciences (MS‐BMS)—intended for students who are pursuing a future career in healthcare. This program introduces students to the coursework often encountered within the first year of medical or dental school, and the academic demands of the program prepare them for the heightened rigor of professional school. This study (IRB Protocol 2020V0105) sought to describe the study strategies and time management techniques of students enrolled in the MS‐BMS Gross Anatomy course. Students who consented to participate in the study (n=84) were asked to complete study logs documenting when, how long, where, and how they studied. In addition, students were asked to complete reflection questions both prior to and following each exam. These questions prompted students to reflect on how they were preparing for upcoming exams, what strategies worked well in their exam preparation, and what strategies needed to be adapted or changed. The information provided in the study logs and student responses to the pre‐ and post‐exam reflection questions underwent thematic analysis to establish codes that capture the time management techniques and study strategies of the Master’s students enrolled in the graduate gross anatomy course. The codes were utilized to establish common themes to describe the predominating student approaches in studying gross anatomy content. Some students reported utilizing less useful methods such as rereading their notes, reading through the lecture presentations multiple times, and watching recordings of the lectures again. Other students implemented more effective methods like using flashcards, self‐quizzing, and creating their own resources such as diagrams and charts. Student perceptions of the assignments were also analyzed, and many students acknowledged the assignments were helpful, held them accountable in their studying, and encouraged them to reflect on the effectiveness of their time management techniques and study strategies. The results of this study will be used to direct the implementation of future assignments geared towards bettering the time management and study strategies of this population.
Expression and Characterization of Helminthic Aminoacyl‐tRNA Synthetases
Authors: Wilson David , Kammann Molly , Qi Leo , Williams Augustus , Chihade Joseph
Published: May 2022

Parasitic worms, broadly described as helminths, currently infect approximately 2 billion people worldwide. Existing treatment options are limited and many cause harmful side effects. We are working to characterize aminoacyl‐tRNA synthetases, enzymes that carry out an essential step in protein synthesis, as potential drug targets in these parasites. Using genomic data to predict the protein sequences, we have identified synthetases likely to differ most from human homologues. We have designed gene sequences to insert into bacterial expression vectors in order to of express, purify, and characterize the helminthic enzymes.

In order to facilitate kinetic assays with these enzymes, we have made progress in adapting and improving a non‐radioactive aminoacylation assay, which couples the formation of AMP during the aminoacylation reaction to the production of NADH, which can be measured spectrophotometrically at 340 nm.
Deficiency of Ataxia Telangiectasia‐mutated Kinase (ATM) Preserves Heart Function by Affecting Cardiac Remodeling in Response to Western‐type Diet in Female Mice
Authors: Ramirez Paulina , Wingard Mary , Shook Paige , Dalal Suman , Singh Mahipal , Singh Krishna
Published: May 2022

BACKGROUND

Western‐type Diet (WD) and deficiency of ATM protein independently associate with heart disease. Previous work demonstrated that WD in male ATM deficient mice induces accelerated body weight gain and heart dysfunction (Am J Physiol Heart Circ Physiol. 2021;320:H2324‐H2338). Conversely, WD in female ATM deficient mice attenuates weight gain and preserves heart function (unpublished data). Here, we investigated the mechanism by which ATM deficiency preserves heart function in female mice. It is hypothesized that ATM deficiency attenuates adverse myocardial remodeling in female mice, thereby preserving heart function.

METHODS

Female wild‐type (WT) and ATM heterozygous knockout (hKO) mice, aged 6 weeks, were fed with normal chow (NC) or WD for 14 weeks. Heart sections were stained with Masson’s trichrome to quantify fibrosis, TUNEL‐stained to quantify apoptosis, and WGA (wheat germ agglutinin)‐stained to quantify myocyte hypertrophy. Data were analyzed using ANOVA followed by Student‐Newman‐Keuls test.

RESULTS

ATM deficiency associated with increased fibrosis, apoptosis (myocytes and non‐myocytes) and hypertrophy in hKO‐NC vs WT‐NC (p<0.05; n=4). WD significantly increased fibrosis in WT‐WD mice, while no increase in fibrosis was observed in hKO‐WD. WD significantly increased apoptosis in both genotypes. However, the increase in apoptosis was significantly lower in hKO‐WD vs WT‐WD (p<0.05; n=4). WD increased hypertrophy in WT group. However, the hypertrophy remained significantly higher in hKO‐WD vs WT‐WD (p<0.05; n=4).

CONCLUSION

Decrease in myocardial fibrosis and apoptosis, and increase in myocyte hypertrophy may play a role in preservation of heart function in ATM deficient female mice in response to WD.
Cardioprotective Effects of Preconditioning Exercise in the Female Tumor‐Bearing Mouse
Authors: Tichy Louisa , Brantley Jason T. , Parry Traci L.
Published: May 2022

Background

Cancer cachexia, a metabolic wasting syndrome, affects up to 80% of cancer patients and leads to the death in up to 20% of cancer patients. While research is growing in the field, there are still no clear diagnostic criteria and cancer cachexia remains an untreated condition. Aerobic exercise has been shown to positively impact cachexia by slowing its development and attenuating muscle loss. The most effective timing, duration, and intensity of exercise as a preventative and protective measure against cancer cachexia remains questionable. Therefore, the purpose of this study was to examine the effects of preconditioning exercise as a protective measure for tumor‐mediated muscle wasting.

Methods

Female LC3 Tg+ and WT mice were randomly separated into four groups, sedentary non‐tumor bearing (SED+NT), sedentary tumor bearing (SED+T), treadmill exercise non‐tumor bearing (TM+NT), and treadmill exercise tumor bearing (TM+T). Mice underwent an 8‐week treadmill exercise training protocol (TM) or remained sedentary (SED). Next, mice were implanted with tumor cells (T group; 1x105 LLC cells in flank) or remained non‐tumor (NT) for 4 weeks. To examine the protective effects of exercise on tumor growth and muscle wasting during tumor‐mediated muscle wasting, grip strength and tumor growth evaluations were taken at baseline, 8‐week, and 12‐week time points. At the end of the 12‐week study, muscle and tumor tissue was collected and weighed.

Results

Tumor bearing resulted in a significant decline in cardiac function. SED+T showed a significant decrease in fractional shortening (P < 0.05) when compared to the other groups. This coincided with an increase in beclin‐1 expression in SED+T mice. Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to preserve cardiac function (TM+T not significantly different than SED+NT). Exercise‐mediated cardioprotection also coincided with abolished beclin‐1 signaling since this was not significantly elevated in TM+T mice. Additionally, TM resulted in a 20‐fold decrease in estimated tumor volume (P < 0.05) and a 60% decrease in tumor mass (P < 0.05) compared to SED tumors.

Conclusion

The data indicate potential cardioprotective effects of preconditioning exercise on preserving cardiac structure and function, as well as regulating autophagic pathways (beclin‐1) during tumor bearing. Preconditioning exercise may be an effective and accessible treatment intervention for early‐stage cancer survivors. This data is crucial in identifying the significance of exercise and the timing of exercise as a protective measure against the detrimental effects of cancer cachexia.
Identification and characterization of the domain that mediates actin binding and directs basal and TNF‐induced myosin light chain kinase 1 (MLCK1) recruitment to the perijunctional actomyosin ring
Authors: Chanez‐Paredes Sandra D. , He Weiqi , Zha Juanmin , Zuo Li , Graham W V. , Rosenberg Harry J. , Turner Jerrold R.
Published: May 2022

Two myosin light chain kinase (MLCK) splice variants are expressed within intestinal epithelial cells, MLCK1 and MLCK2. These differ by a single exon that completes an IgCAM domain (IgCAM3) that is only present in MLCK1. We have previously shown that a small molecule targeting IgCAM3 prevents TNF‐induced MLCK1 recruitment to the perijunctional actomyosin ring thereby blocking myosin regulatory light chain (MLC) phosphorylation and tight junction barrier loss. Here, we sought to determine whether IgCAM3 alone was sufficient or if a larger portion of MLCK1 is required to direct recruitment and to define the interactions of these domains with F‐actin. MLCK1‐EGFP or MLCK2‐EGFP were stably expressed in Caco‐2 monolayers. In the absence of exogenous stimuli, MLCK1‐EGFP preferentially localized to the perijunctional actomyosin ring relative to MLCK2‐EGFP, which was most concentrated within basal stress fibers. Truncated EGFP‐tagged constructs consisting only of IgCAM1‐4 of MLCK1 or MLCK2 without the catalytic domain localized similarly to full‐length proteins. Further analyses demonstrated that the minimal MLCK1 trafficking domain consisted of IgCAM2 and IgCAM3 as well as the unstructured sequences preceding each of these domains; IgCAM3‐EGFP was distributed throughout the cytoplasm and nucleus of Caco‐2 cells. Consistent with this intracellular localization, studies using recombinant MLCK constructs showed that MLCK1(IgCAM1‐4) and MLCK1(IgCAM1‐3) bound F‐actin with greater affinity than MLCK1(IgCAM3) or MLCK2(IgCAM1‐4). TNF induced recruitment of MLCK1‐EGFP and MLCK1(IgCAM1‐4)‐EGFP, but not MLCK2‐EGFP or MLCK2(IgCAM1‐4)‐EGFP, to the perijunctional actomyosin ring within Caco‐2 monolayers. Although IgCAM3 was not concentrated at the perijunctional actomyosin ring, it was able to act as a dominant negative inhibitor of TNF‐induced full‐length MLCK1 recruitment. Moreover, IgCAM3 prevented TNF‐induced increases in tight junction permeability. Thus, although IgCAM3 alone is insufficient for perijunctional localization, it can disrupt TNF‐induced MLCK1 recruitment to the perijunctional actomyosin ring. These data confirm our previous results showing that IgCAM3‐mediated interactions are required for TNF‐induced MLCK1 recruitment and increased tight junction permeability but also demonstrate that interactions mediated or stabilized by other MLCK1 domains are also critically involved in this process. The data also suggest that, although direct binding to F‐actin may contribute to MLCK1 localization, it is insufficient for TNF‐induced MLCK1 recruitment and barrier regulation.
Intestinal P‐glycoprotein (P‐gp) Contribution to Talinolol Pharmacokinetics in Human
Authors: Wan Christine , Ishida Kazuya , Lai Yurong
Published: May 2022

Objective

Understanding the mechanisms of intestinal secretion and recirculation is important to the realm of drug metabolism and pharmacokinetics (DMPK). P‐glycoprotein (P‐gp), one of the major efflux transporters in the intestine, has broad substrate specificity and could contribute to the intestinal secretion/recirculation of drugs. The pharmacokinetic (PK) model incorporating intestinal P‐gp contribution will be able to accurately capture the interactions for the compounds of P‐gp substrate, allowing the model to aid in determining first in‐human (FIH) dose predictions as well as related potential drug‐drug interactions (DDIs). Talinolol, a beta‐adrenergic receptor antagonist utilized for cardioprotective and antihypertensive treatments, is a good substrate of P‐gp, and a double peak of plasma concentration has been observed. We hypothesize that intestinal secretion/recirculation by P‐gp could contribute to this unique plasma concentration‐time profile of talinolol. The aim of this study was to assess whether the double peak of plasma concentration of orally administered talinolol can be captured using physiological‐based pharmacokinetic (PBPK) modeling.

Methods

The plasma concentration‐time profiles of talinolol were obtained by digitizing literature data. The P‐gp‐mediated transport kinetics of talinolol were also obtained from Caco‐2 assays present in literature. Using the plasma concentration of talinolol data after intravenous (IV) administration, the intestinal secretion clearance (CLGI) was estimated using SimCYP (v19). After estimating CLGI, the regional absorption of talinolol affected by intestinal P‐gp was assessed using plasma talinolol concentration data after oral administration and the advanced dissolution, absorption, and metabolism (ADAM) model was subsequently generated utilizing SimCYP.

Results

CLGI of talinolol was estimated through using plasma concentration data of the drug after IV infusion reported by Schwarz et al., and CLGI was estimated to be 12.6 L/hr. This CLGI value was validated by capturing another plasma concentration‐time profile of talinolol after IV infusion reported by Westphal et al. The dual‐peak of the plasma talinolol concentration after oral administration was captured using ADAM model, but this model will be further refined to better capture this dual‐peak after oral administration of talinolol in human with consideration of regional differences of absorption and/or recirculation (i.e., more P‐gp in ileum compared to duodenum and jejunum).

Conclusion

A model was successfully generated to accurately capture the PK profiles of talinolol after IV administration. Further refinement of the model will be required for oral administration. The outcomes from the investigation essentially broaden our overall understanding of the processes underlying intestinal secretion and recirculation.
Retroaortic Left Renal Vein: An Incidental Finding During Cadaver Dissection
Authors: Syed Mohsin , Akhter Noor , Haynie Cade , Hazeslip Lindsey , Held Michael , Hendrixson James , Phelan Kevin D. , Davies David L.
Published: May 2022

Developmental variations are frequently observed during the course of dissection in the gross anatomy laboratory and serve as valuable learning experiences for students. In part, because of the insights and motivation derived from such discovery opportunities, the classical method of teaching anatomy by cadaver dissection is still the preferred way of teaching in most medical schools in U.S. and globally. However, the shortage of qualified educators, and the resource intensive nature of the operating and maintaining gross anatomy laboratories have prompted consideration of alternative resources. Some U.S. medical schools have already adopted virtual dissection tools. Use of high quality plastinated specimens has been explored by some universities. Still, replacing cadaver dissection with virtual dissectors and computer‐generated images deprive students of beneficial learning experiences where they encounter structural variation. For instance, it has been reported that about 10% of clinical malpractice is attributed to the ignorance of anatomical variations. Here we report an example of an instructive congenital variation. The left renal vein normally passes through the angle between the aorta and superior mesenteric artery to drain into the inferior vena cava. Yet congenital variations in the position of left renal vein have previously been reported. During the cadaver dissection by first‐year medical students, we discovered a case where the left renal vein passed between the aorta and the lumbar vertebrae in an elderly female cadaver donor. This unusual position of left renal vein is termed retroaortic left renal vein (RLRV), which has a reported incidence from 0.5% to 3.6%. Careful dissection revealed the course of left renal vein and its relation with the surrounding structures. Noticeable compression of the LRV between the aorta and vertebral column was observed. The left kidney was smaller in size compared to the right kidney and darker in appearance. No significant change was observed in the renal parenchyma. The donor had significant plaque accumulation at the bifurcation of the common iliac arteries no other vascular congenital anomalies that can accompany a retroaortic left renal vein were noted in this donor. This study highlights the educational importance of learning about morphologic variations and their potential impact on adjacent organs.
Supernumerary Branches of the Dorsomedial Cutaneous Nerve of the Hallux Crossing the Extensor Hallucis Longus Tendon
Authors: Pocwierz Alexander J. , Dumford Emily R. , Gumble Zachary , Hess Allison J. , Quinn Makaela , Zdilla Matthew J. , Lambert H. Wayne
Published: May 2022

The dorsomedial cutaneous nerve of the hallux (DMCN) provides sensation to the skin of the dorsomedial hallux (big toe) and the first metatarsophalangeal joint. This cutaneous nerve, a terminal branch of the superficial fibular (peroneal) nerve, is located in the dorsum of the foot and crosses the extensor hallucis longus (EHL) tendon during its course. At this intersection point, the DMCN is at risk for injury during operations of the hallux and metatarsophalangeal joint including EHL tendon transfer, hallux valgus or hallux rigidus corrections, bunionectomy, cheilectomy, or injection procedures. Iatrogenic injuries often result in sensory loss, debilitating causalgia, or neuroma formation. The aim of this study was to identify anatomical variations at the intersection of the DMCN and the EHL tendon. Twenty‐three feet (12 left‐sided, 11 right‐sided) from 12 cadavers were dissected to follow the course of the DMCN and look at its intersection with the EHL tendon. Dissection resulted in damage to three DMCNs; however, 20 feet (11 left‐sided and 9 right‐sided feet) dissections resulted in excellent visualization of both the DMCN and the EHL tendon. Supernumerary branches of the DMCN were identified crossing the EHL tendon in multiple locations in fourteen of twenty feet (70.0%), including nine left feet (9/11, 81.8%) and five right feet (5:9, 55.6%). Of the fourteen feet with supernumerary branches, seven feet (50.0%) had two branches, four feet (28.6%) had three branches, and three feet (21.4%) had four‐to‐six branches of the DMCN crossing over the EHL tendon in multiple locations. The results of this study identify the complex branching patterns of the DMCN. Surgeons should be aware of that supernumerary branches of the DMCN exist, crossing the EHL tendon in multiple locations, and use ultrasonography to lower the risk of iatrogenic injury to the DMCN during surgical procedures.
Characterization and Cross‐Linking of a Novel Insect Cuticle‐Like Protein with Chitin and Graphene Binding Domains
Authors: Clay Ryan W. , Mitchell Jon C.
Published: May 2022

Chitin and carbon nanotubes can both confer extraordinary physical properties upon polymers that contain them. However, these materials are also highly insoluble in most common organic solvents, making them difficult to utilize without first modifying or functionalizing them, which can alter their properties. Natural organisms produce peptides that utilize or demonstrate affinity for these materials, which can be exploited by designing a protein capable of binding to both chitin and carbon nanotubes and of cross‐linking to form a biopolymer.

The conserved 65 amino acid motif called the extended Rebers and Riddiford (R&R) Consensus found in arthropod cuticle proteins has been experimentally confirmed to confer chitin‐binding ability to recombinant proteins that include it. Similarly, a phage‐derived 12 amino acid sequence (P1) has been shown to allow including recombinant proteins to coat and solubilize unfunctionalized carbon nanotubes. This work combines these segments in a single peptide along with flexible linker domains and cross‐linking domains derived from insect cuticle proteins to create a novel double‐binding cuticle protein (DBCP). Cross‐linking can be achieved through [Ru(bpy)3]2+‐mediated photo irradiation, a method showing increased promise for the creation of dityrosine cross‐links between organic molecules. This will allow ligand‐bound proteins to form a stable matrix, capturing chitin and carbon nanotubes to form a resilient biopolymer.

DBCP gene was designed and synthesized for use in heterologous expression in BL21 E. coli. Western blotting will confirm the expression and purification of the target protein. Ligand binding ability will be confirmed by solubilization assays introducing ligands to concentrated protein in common solvents. FTIR will further confirm ligand binding ability and the nature of the bonds or attractions involved. Cross‐linking will be induced using [Ru(bpy)3]2+‐mediated photo irradiation.

It is anticipated that use of the R&R consensus and P1 phage‐derived peptide in conjunction with resilin cross‐linking domains will create a recombinant protein capable of binding chitin and carbon nanotubes in addition to forming dityrosine cross‐links through [Ru(bpy)3]2+‐mediated photo irradiation, resulting in a more rigid biopolymer with than commonly produced protein hydrogels, expanding the range of possible use‐cases for laboratory‐derived biopolymers.
The rate‐limiting enzyme in the CDP‐choline pathway is regulated by phosphorylation‐domain charge density
Authors: Foster Jason R. , Dellaire Graham , Ridgway Neale
Published: May 2022

Eukaryotic cell membranes are primarily composed of phospholipids, the most abundant of which is phosphatidylcholine (PC). De novo synthesis of PC by the CDP‐choline pathway is under the control of the rate‐limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). The nuclear CCTa isoform is regulated by translocation to the inner nuclear membrane (INM) and nuclear lipid droplets (nLD) in response to lipid activators, such as oleate, or deficiency in PC content. CCTa association with the INM is mediated by a positively‐charged membrane‐binding M‐domain and a phosphorylated P‐domain containing 16 serine phospho‐sites. Mutation of all P‐domain serine residues to alanine (dephosphorylated mimic) enhanced association with nLDs in oleate‐treated U2OS cells, while mutation to glutamate (phosphorylated mimic) or mutation of M‐domain lysine residues ablated nLD association. However, it is unknown if phosphorylation regulation of CCTa association with the INM and nLDs involves specific serine residues or the net charge of the P‐domain. To address this question, we mutated 4 sets of 2‐3 serine residues in the P‐domain to alanine or aspartate. CCTa cDNAs expressing different combinations of these mutations were expressed in oleate‐treated U2OS cells and the frequency of nLD association was measured. The mutants were also expressed in CHO cells with a temperature‐sensitive CCTα isoform and INM translocation in response to oleate was determined. The greatest inhibitory effect on INM and nLD association was observed when all 4 sets of serine residues were mutated to aspartate and not by any one site, suggesting that net charge density of the P‐domain regulates affinity for the NE and nLD. Additionally, CCTa serine‐to‐alanine mutants expressed in U2OS cells were unstable compared to the corresponding aspartate mutants. We conclude that CCTa association with the INM and nLD is inhibited as overall P‐domain phosphorylation increases. In its active membrane‐associated, dephosphorylated state CCTa protein has decreased stability, a potently negative feedback mechanism to control PC synthesis. These results provide novel insight into how PC synthesis is regulated that will be essential for identifying kinases and phosphatases that act on CCTα.
Intracellular Delivery of Functional Proteins
Authors: Giancola JoLynn , Okon Aniekan , Raines Ronald
Published: May 2022

Leveraging proteins as therapeutics has revolutionized the way we treat diseases. Recent advances in protein biologics and antibody engineering represent promising new treatment strategies for addressing complex etiologies. Thus far, these advances have been restricted to only accessible extracellular proteins, leaving a significant portion of the proteome undruggable. Efficient intracellular delivery of proteins would reveal uncharted therapeutic avenues and cell surface engineering opportunities. This presentation describes combinatorial strategies towards delivering proteins to the cytosol. By modifying a protein of interest through surface esterification or functionalization with a cell penetrating peptide we have observed robust intracellular delivery. In this presentation, I will first describe the development of a functional assay using a model protein to assess the efficacy and localization of delivery agents. Applying these findings to HaloTag has demonstrated the generality of our approach. I will show that our delivery strategy has enabled stable cell surface engineering. This study highlights that multiple intracellular delivery strategies can be used synergistically to enable cytosolic access with functionally active protein/biologic cargo.
Untangling Frizzled functions
Authors: Hollis Katie , Williams Bart
Published: May 2022

Wnt signaling is a complex network of cellular signals with multiple downstream pathways regulated by signaling complexes comprised of combinations of the 19 Wnt ligands, 10 Frizzled (FZD) receptors, various co‐receptors and Dishevelled (DVL) second messengers. Up until now, Wnt receptors have either been functionally defined as whole families or individually by how they affect specific disease states when they are dysregulated, but the specific roles each receptor plays in Wnt signaling and activating the various pathways have yet to be fully understood.

CRISPR is a powerful tool that has allowed whole families of protein isomers to be knocked out. By using cellular models that have been modified by CRISPR/Cas9 and are missing key proteins in the Wnt signaling complex, we can determine their individual functions and redundancies.

We have determined that a FZD is necessary but any of the individual receptors is sufficient for DVL phosphorylation. Additionally, we have found that some Wnt signals can be rescued by single receptors, or just part of a receptor, while others seem to require multiple FZDs. The expression of these receptors is crucial for not only regulation within the Wnt family of pathways, but we also discovered a new arm of crosstalk with mTOR signaling.

Wnt signaling is known to play key roles in many aspects of development, but erroneous signaling can cause diseases such as cancer and osteoporosis. Current therapeutics act as a sledgehammer and target all Wnt signaling, which leads to many off‐target effects and unintended side‐effects. Since cell surface receptors are typically the easiest to therapeutically target, FZD receptors have considerable therapeutic potential. Understanding the roles of each FZD receptor will allow us to better target individual disease mechanisms.
Defining the Mutational Landscape That Affects the Histone Demethylase KDM6A/UTX in Human Cancer
Authors: Chi Young‐In , Stodola Timothy J. , de Assuncao Thiago Milech , Volkman Brian , Smith Brian , Mathison Angela J. , Lomberk Gwen , Zimmermann Michael T. , Urrutia Raul
Published: May 2022

We are developing, standardizing, and implementing advanced methods for genomics interpretation of disease‐associated mutations, as a fundamental step toward the discovery of mechanisms of diseases. A key example is the epigenetic regulator, KDM6A/UTX, whose gene and encoded product have elicited significant investigation. When mutated in the germline, KDM6A/UTX gives rise to a developmental disease (Kabuki syndrome), while in somatic cells it mediates cancer development, as a tumor suppressor. However, the full extent of the mutational landscape that affects this gene and the underlying mechanisms of dysfunction remain to be fully explored. Here, we report the results from a multi‐tiered approach evaluating the impact of 197 KDM6A/UTX somatic mutations, using information derived from combining conventional genomics bioinformatics with computational biophysics. Our approach incorporates multiple scores derived from alterations in protein sequence, structure, and molecular dynamics to classify the KDM6A/UTX mutations. We identified 136 (69%) as damaging variants, consisting of 15 Structural Variants (SV), 88 Dynamic Variants (DV), and 33 Structural and Dynamic Variants (SDV). Another 32 variants appear to be tolerated by KDM6A/UTX, while 29 still remain as genomic Variants of Uncertain Significance (VUS). Comparative analyses of these results with those obtained by classic genetic diagnostic pipelines demonstrated that our approach provides a more comprehensive evaluation of damaging potential and, more importantly, reveals possible mechanisms underlying dysfunctions of distinct genomic variants. Thus, these results should be taken into consideration for clinical classifications of genomic variation, as well as provide critical insights for experimental validation and the development of mutant‐specific drugs to fight diseases caused by KDM6A dysfunctions.
Elucidating the Antibiotic Resistance of Acinetobacter calcoaceticus Strains
Authors: Glover Janiece S. , Ticer Taylor , Engevik Melinda
Published: May 2022

Background

According to the Center for Disease Control (CDC), antibiotic resistance is one of the largest public health challenges facing modern society. It has been estimated that 2.8 million people each year in the US are infected with an antibiotic‐resistant organism, resulting in >35,000 deaths. One of the multi‐drug resistant microbes listed on the CDC’s urgent threats list is Acinetobacter. The Acinetobacter calcoaceticus–Acinetobacter baumannii complex (ACB complex) is a group of clinically relevant Acinetobacter species that can cause pneumonia, urinary tract, skin and soft tissue infections. Although a lot of work has focused on antibiotic resistance in A. baumannii, the resistance of A. calcoaceticus is underexplored. To address this gap in knowledge, we obtained three commercially available A. calcoaceticus strains and four clinical isolates. We hypothesized that clinical isolates of A. calcoaceticus would be well adapted to withstand traditional antibiotics.

Methods & Results

To test this hypothesis, we grew commercially available and clinical isolates of A. calcoaceticus in a fully defined bacterial media, termed ZMB1, which was then seeded into Biolog plates (12B) containing beta‐lactams (penicillin G, carbenicillin, oxacillin), tetracyclines (tetracycline, penimepicycline), aminoglycosides (polymyxin B, paromomycin, sisomicin, tobramycin, spectinomycin), glycopeptides (vancomycin), sulfonamides (sulfamethazine, sulfadiazine, sulfathiazole, sulfa‐methoxazole), aminocoumarins (novobiocin), pteridines (2,4‐diamino‐6,7‐ diisopropyl‐ pteridine), biocides (benzethonium chloride), macrolides (spiramycin), antimycobacterials (rifampicin), amphiphiles (dodecyltrimethyl ammonium bromide), and acids (D,L‐Serine hydroxamate, 5‐fluoroorotic acid, L‐aspartic‐β‐ hydroxamate). All A. calcoaceticusstrains were resistant to penicillin G, tetracycline, carbenicillin, and sisomicin. Interestingly, although our commercially available strains were sensitive to sulfamethazine, we observed that all of our clinical isolates were resistant to this antibiotic. We also identified that 2 of our isolates were resistant to polymyxin B and one isolate was highly resistant to dodecyltrimethyl ammonium bromide. These data suggest significant differences in antibiotic resistance between strains.

Conclusions

Collectively, these data demonstrate reveal a profile of resistance for A. calcoaceticus. This data also indicates that clinical isolates of A. calcoaceticus are much more resistant to antibiotics than lab adapted strains and should be incorporated into experimental study design.
A Tale of Two Scavenger Receptors: Structure‐Function Relationships of Purified SR‐B1 and CD36
Authors: Powers Hayley R. , Jenjak Shawn E. , Volkman Brian F. , Sahoo Daisy
Published: May 2022

Both CD36 and SR‐B1 are glycosylated integral membrane proteins belonging to the class B scavenger receptor family and are known to play roles in cardiovascular and atherosclerotic disease. CD36 serves as the receptor for oxidized low density lipoprotein (oxLDL) and facilitates cholesterol accumulation in macrophages and development of atherosclerotic plaques. SR‐B1 (scavenger receptor class B type 1) is the primary receptor for high density lipoprotein (HDL), which removes cholesterol from the peripheral atherosclerotic plaques and delivers it to the liver for excretion. Current structural information about class B scavenger receptors is limited to only purified extracellular regions, transmembrane segments, or transient transfection studies in cultured cells, and remains a roadblock to studying the isolated functions of these lipid and cholesterol transporters. Recently, we have successfully expressed and purified full‐length human SR‐B1 and CD36 using an Sf9 insect cell/baculoviral infection system, which allows for the expression of glycosylated proteins. Purified full‐length receptors are a valuable addition to our scientific toolbox and allow us to ask novel questions about the structure‐function relationships of scavenger receptors. To verify function within Sf9 cell membranes, we expressed human full‐length constructs in plated Sf9 cells and observed significantly increased binding of DiI‐HDL and DiI‐oxLDL, as well as increased DiI‐lipid uptake when comparing SR‐B1 and CD36 to empty vector infected cells, respectively. When purified and solubilized in detergent micelles, both receptors remain stable over time as shown by Prometheus thermal shift assays. We have also demonstrated that purified proteins are able to bind to their native ligands (HDL to SR‐B1 and oxLDL to CD36) with high affinity by microscale thermophoresis (MST). However, as SR‐B1 and CD36 are both scavenger receptors, they are able to bind to a variety of lipoprotein ligands and we have demonstrated these interactions with varying binding affinities by MST. Glycosylation of both CD36 and SR‐B1 has been shown to be important for cell surface expression, but the role of glycosylation in ligand binding has remained uncharacterized. We are the first to demonstrate that the glycosylation status of CD36 and SR‐B1 does not alter binding affinities or protein stability. These purified receptors and functional assays provide promise for understanding the ways in which class B scavenger receptors perform their critical functions and lay the groundwork for future structure‐function studies.
Individuals with a Previous Symptomatic COVID‐19 Infection Have Altered Blood Pressure and Heart Rate Variability During Acute Exercise
Authors: Chan Jillian , Homitz Jessica , Cashin Niamh , Guers John
Published: May 2022

As the number of COVID‐19 cases begin to diminish it is important to turn our attention to any long‐term issues that may be associated with a prior infection. Cardiovascular (CV) defects have been noted following infection. However, less is known how a previous COVID‐19 infection alters CV response during exercise (EX). Furthermore, differences may exist between COVID‐19 infected individuals who had symptoms (symptomatic) when compared with those that did not have symptoms (asymptomatic) during EX.

Hypothesis

Previously symptomatic (S) COVID‐19 recoveries have an altered CV response to acute EX relative to both control (CON; never infected), and asymptomatic (AS) individuals who were previously infected with COVID‐19.

Methods

Subjects (n=14; CON=4; S=5; AS=5; Age = 21 ± 2.3) filled out a questionnaire before performing EX to determine if they were AS or S and if symptomatic what symptoms they experienced. Subjects next performed a 5‐minute warm‐up followed by 25 minutes of treadmill EX (5‐6 METS). During EX, blood pressure was recorded on the brachial artery every 5 minutes and 3‐lead electrocardiography was measured continuously. Heart rate variability (HRV) was analyzed in the time‐domain using the standard deviation of the inter beat intervals of normal sinus beats (SDNN). Blood pressure variability (BPV) was computed as the average of the absolute differences between consecutive mean arterial pressure (MAP) measurements. A one‐way ANOVA with a Tukey’s post hoc was used to determine statistical differences between groups. Data are shown as mean ± SEM. Significance was set to p<0.05.

Results

Baseline MAP in a seated (CON=89 ± 6 mmHg; AS=92 ± 3 mmHg; S=87± 5 mmHg), standing (CON=86 ± 2 mmHg; AS=90 ± 7 mmHg; S=86 ± 3 mmHg) and supine (CON=86 ± 3 mmHg; AS=90 ± 6 mmHg; S=86 ± 4 mmHg) position were similar amongst all three groups. There were no differences (p>0.05) between HR (CON = 104 ± 4 BPM vs. AS=118 ±6 BPM vs. S=1112± 3 BPM) or MAP (Con = 108±4 mmHg vs. AS=105±13 mmHg vs. S=108±7 mmHg) between groups during EX. However, HRV (Con=139 vs. AS =156±6ms vs. S=77.7±11ms; S vs. CON; AS; p<0.05) and BPV (Con= 139 ± AS=13±1.1 mmHg vs. S=5±1.0mmHg; S vs. CON; AS; p<0.05) were different.

Conclusion

Previously symptomatic subjects may have altered HRV and BPV during EX and thus indicates that experiencing symptoms associated with COVID‐19 may impact the ANS.
Increased Endogenous Production of Pro‐inflammatory Adipokines by 3T3‐L1 Adipocytes Differentiated with Arachidonic Acid
Authors: Caldari‐Torres Cristina , Trinh Thy
Published: May 2022

Inflammatory factors derived from adipose tissue (AT) can set the stage for chronic, low‐grade inflammation. This meta‐inflammation can predispose the individual to further inflammatory‐based disorders and diseases. Polyunsaturated fatty acids (PUFAs) are known to have immune modulating effects, with omega‐6 (n‐6) PUFAs typically being associated with increased production of inflammatory molecules, and n‐3 PUFAs having been shown to be anti‐inflammatory, in both in vivo and in vitro studies. The 3T3‐L1 cell line is an embryonic mouse fibroblast line that can be differentiated into an adipocyte phenotype making it a suitable line for studies on the effects of PUFAs on inflammatory molecule production by AT. The aim of this research was to determine the effects of n‐6 and n‐3 PUFAs on the release of inflammatory adipokines in lipopolysaccharide (LPS)‐challenged and LPS‐unchallenged adipocytes. Fibroblasts were differentiated into adipocytes with the addition of 100 μM of linoleic acid (LA; n‐6), arachidonic acid (ARA; n‐6) or eicosapentaenoic acid (EPA; n‐3) throughout the differentiation period, followed by a 6 h, 1 μg/ml LPS challenge. Cell‐conditioned media was collected before and after the LPS challenge for interleukin (IL)‐6 and prostaglandin E2 (PGE2) quantification via enzyme‐linked immunosorbent assays. Differentiation of adipocytes with n‐6 ARA caused a significant increase in PGE2 levels compared to control cells, or cells differentiated with LA or EPA, both after LPS challenge (two‐way ANOVA; p<0.0001) and before LPS challenge (two‐way ANOVA; p<0.0001). We did not observe differences in PGE2 levels between LPS‐challenged and non‐challenged cells differentiated with ARA (matched pairs t‐test; p=0.42) and EPA (p=0.57), while control (p=0.0005) and LA‐treated cells (p=0.0003) produced higher amounts of PGE2 after LPS stimulation. Interestingly, quantified levels of IL‐6 across treatments did not differ before and after LPS stimulation (matched pairs t‐test; p=0.75), although, as observed with PGE2, there was a significant increase in IL‐6 production by ARA‐differentiated cells compared to control, LPS, LA, and EPA‐treated cells (two‐way ANOVA; p=0.004). In conclusion, use of ARA in the differentiation process of 3T3‐L1 fibroblasts into a final adipocyte phenotype increases their endogenous (non‐LPS stimulated) production of IL‐6 and PGE2.
Alterations in Genes Associated with Bioactive Lipid Pathways during Development of Pulmonary Hypertension in Rat SU5416‐Hypoxia Model of Severe Pulmonary Arterial Hypertension
Authors: El‐Rabahi Tony , Panchakshari Sheethal , Chaudhary Ketul R.
Published: May 2022

Background

Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vascular system characterized by sustained elevation in resting mean pulmonary arterial pressure (>25 mmHg) with presence of occlusive vascular remodeling and progressive loss of microvasculature. PAH patients have a poor five‐year survival rate of only ~50% and the primary cause of death for PAH patients is the right (‐sided) heart failure. Better understanding of pathophysiology of PAH and RHF is required to find novel therapies. In this project, using a rat model of severe PAH, we explored the alterations in expression of genes involved in bioactive lipid pathways in the lungs and the right ventricle (RV) during development of PAH.

Methods

Adult male Sprague Dawley rats were administered a single injection of SU5416 (SU; 20 mg/kg, subcutaneous) and subjected to 3 weeks of hypoxia (10% O2) followed by 4 weeks of normoxia (room air), SUHx model of sever PAH. Right ventricular systolic pressure (RVSP) was measured, and heart and lung samples were collected at 1, 3, 7, 21 and 49‐days post SU injection. Histological analysis was conducted to confirm vascular remodeling in the lungs and development of RV hypertrophy (RVH; RV/LV+S). RTqPCR was performed to evaluate gene expression changes in the genes related to bioactive lipids pathways.

Results

In response to SUHx, progressive increase in RVSP and RVH was observed. Histology of lungs and heart samples confirmed pulmonary vascular and RV remodeling at 7 weeks post SU in the SUHx model. We observed increase in expression of ALOX‐5 in the RV and lungs of SUHx rats, compared to controls. Furthermore, alterations in various cytochrome P450 (CYP) isozymes were observed in the RV and lungs of SUHx rats compared to controls.

Conclusions

Together, we have identified unique changes in bioactive lipid pathway genes in the lungs and the RV that were associated with development of PAH and RV remodeling in SUHx model. Further studies are ongoing to confirm the role of the candidate genes in pathophysiology of PAH and development of RHF.