bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Binding to Artificial Lipid Droplets Demonstrates the Importance of Phospholipid Composition
Authors: Paal Matthew , Gillingham Jacob , Furcich Justin , Brown Margaret , Listenberger Laura
Published: April 2020

All cells and tissues store excess lipid in intracellular structures known as lipid droplets. Each lipid droplet consists of a neutral lipid core (primarily made of triacylglyceride and cholesteryl ester) surrounded by a phospholipid monolayer. Several proteins interact specifically with the surface of the lipid droplet and control their growth and degradation. Here, we share the development of an in vitro binding assay, that utilizes synthetic lipid droplets and partially purified lipid droplet proteins, to study protein binding to lipid droplets. We separate bound from unbound proteins with sucrose gradient ultracentrifugation and detect lipid droplet proteins following SDS‐PAGE and western blotting. We have used this assay to show that changing the phospholipid composition of the lipid droplet surface alters interactions with perilipin 2, a ubiquitously expressed lipid droplet protein that is predicted to interact with lipid droplets through an amphipathic alpha helix. Specifically, perilipin 2 association with synthetic lipid droplets was increased when the amount of phosphatidylethanolamine relative to phosphatidylcholine was increased. We hypothesize that the smaller headgroups of phosphatidylethanolamine allows additional interaction with the underlying neutral lipid. These results may be relevant to the formation of lipid droplets in alcoholic fatty liver disease, where lipid droplets form with altered levels of phosphatidylethanolamine and phosphatidylcholine.

Support or Funding Information

Funding for this research was provided by an ASBMB Undergraduate Research Award and the Lowell and Doris Kispert Endowment at St. Olaf College.
Metformin Treatment Attenuates Non‐Small Cell Lung Carcinoma Cell Viability In Vitro.
Authors: Stott Nicole L. , Beach Cassandra , Marino Joseph S.
Published: April 2020

Lung cancer, the second most common cancer, is one of the largest causes of cancer‐related mortalities worldwide. Non‐Small Cell Lung Carcinoma (NSCLC) comprises 80–85% of all new lung cancer diagnoses, suggesting a great need to continue finding measures to combat this mounting disease. Metformin induces an anti‐proliferative effect in many other cancers, including breast, prostate and obesity‐activated thyroid cancer. The purpose of this study was to determine how Metformin treatment influences NSCLC cell viability in vitro. NSCLC cells were grown for 24 hours and serum‐starved overnight with serum‐free media to induce quiescence. Cells were treated with equal volumes of fresh growth media followed by Metformin treatment with a range of concentrations from 5mm to 15mM. NSCLC cell viability was assessed every 24 hours for 3 days via an MTT assay using 3‐(4,5‐Dimethyltiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide. Proliferating NSCLC treated with Metformin showed significantly reduced cell viability (p=0.0109), after 48 hours of treatment, irrespective of Metformin concentration. The results of this study indicate that Metformin is effective at decreasing NSCLC viability. Experiments are ongoing to investigate alterations in cell cycle regulators and metabolic signaling pathways.

Support or Funding Information

Targeted Research Internal Seed Program
Peripheral Inflammation Drives TLR2 and IL‐1R1 Mediated Neurodegeneration in Parkinson’s Disease
Authors: Bearoff Frank , Johnson Marguerite , Dhavale Dhruva , Nonnemacher Michael , Kotzbauer Paul , Kortagere Sandhya
Published: April 2020

Current evidence suggests a causal role of inflammation in the pathogenesis of Parkinson’s Disease (PD). PD is a progressive neurodegenerative disorder that is associated with loss of dopaminergic neurons and the aberrant accumulation of alpha‐synuclein fibrils in Lewy bodies present in surviving dopaminergic neurons in the substantia nigra. Importantly, alpha‐synuclein fibrils can interact with toll‐like receptor 2 (TLR2) on both neurons and microglia to induce a neuroinflammatory response. Previous work has investigated the effects of pre‐existing inflammation on neurodegeneration. However, the effect of pre‐existing peripheral inflammation on alpha‐synuclein fibril induced neuroinflammation in PD has not been well understood. We hypothesize that peripheral inflammation promotes crosstalk between TLR2 and IL‐1R1 receptors when activated by alpha‐synuclein fibrils. Our preliminary results in SH‐SY5Y cells demonstrate that activation of either TLR2 or IL‐1R1 promotes synergistic interaction between the receptors leading to an increase in p‐ERK1/2 levels in a MYD88 dependent manner. In the in vivo model, prior exposure to a low dose of lipopolysaccharide (LPS) systemically increased the expression of proinflammatory cytokines such as IL‐1b and TNFα, and nuclear factor of kappa light polypeptide gene enhancer in B‐cells, alpha (IkBa) compared to animals that received saline. Treatment with alpha‐synuclein fibrils post LPS treatment significantly enhanced IL‐1b, TNFα, p‐ERK1/2 expression suggesting a synergistic effect from TLR2 and IL‐1R1 activation by alpha‐synuclein and IL‐1b respectively. In addition, these animals showed a significant increase in GM‐CSF expression compared to LPS only or saline treated animals suggesting alpha‐synuclein fibril treatment lead to an increase in infiltration of T‐cells, neutrophils and macrophages that may be causal for aberrant neuroinflammatory response in PD

Support or Funding Information

Drexel Coulter Foundation
Insight into Parkinson’s Disease from Yeasts: Combined Impact of Covalent Modifications and Familial Mutations on α‐Synuclein
Authors: Borland Carris Temera , Ganev Yoan , Thomas Rosemary , Roman Alexandra , Grauer Thea , Mwale Chisomo , Jones Paul , Balaram Ariane , Tcaturian Estella , Debburman Shubhik
Published: April 2020

Parkinson’s Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α‐synuclein protein. α‐Synuclein is highly post‐translationally modified in healthy and diseased states. While phosphorylation and nitration of α‐synuclein are well‐studied as contributors to PD pathology, less is known about SUMOylation, acetylation, and glycation. Also, the combined effects of these modifications remain largely unclear, on both wildtype α‐synuclein (linked with sporadic PD) and the six mutant forms (A30P, E46K, H50Q, G51D, A53T, A53E) linked with early‐onset familial PD. We first evaluated the effects of blocking SUMOylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more toxic and aggregated, losing its membrane localization. Second, we found that SUMOylation and phosphorylation counteract each other in toxicity and localization. Third, we expanded our investigation to two newly reported modifications – acetylation and glycation. We found that acetylation is protective and glycation is harmful. When we combined acetylation or glycation manipulations with SUMOylation and phosphorylation alterations on the α‐synuclein level, we found that the effects of these modifications are not additive – the impacts of acetylation and glycation depends on phosphorylation status. Furthermore, we found that decreased nitration protects against toxicity and reduces α‐syn aggregation in the mutants modifying SUMOylation and phosphorylation. Finally, we investigated the effects of the familial mutants in tandem with altered acetylation, glycation, or nitration. We report two familial mutant‐specific effects: H50Q is surprisingly sensitive and non‐toxic in a hyper‐acetylation environment, while A53E is highly toxic with hypo‐glycation. We also found that too much nitration exacerbates toxicity of the familial mutants relative to low nitration. Specifically, mutant A53T is highly sensitive to excess nitration while H50Q is non‐toxic in such environment. These studies show the relevance of covalent modifications in sporadic and familial PD, and their in‐tandem effects that underlie toxicity mechanisms.

Support or Funding Information

YG= supported by Parkinson's foundation‐American Parkinson's disease association, A.B,C.M, A.R and P.J: Research grants from Neurosci
Disease‐Causing Mutations Shift the pK of the pH‐Sensitive H+‐Conductor Slc4a11
Authors: Quade Bianca Natalia , Marshall Aniko , Parker Mark Daniel
Published: April 2020

SLC4A11 is a H+‐conducting membrane protein that is sensitive to changes in both intracellular and extracellular pH. It is expressed in corneal endothelial cells (CECs) where it functions alongside other acid/base transporters such as MCT1 and NBCe1. These proteins function to maintain the osmotic balance and clarity of the cornea. Mutations in SLC4A11 cause congenital hereditary endothelial dystrophy (CHED) and Fuchs’ endothelial corneal dystrophy (FECD). Signs of these corneal dystrophies include loss of normal CEC morphology and corneal edema. Previously—using the Xenopus oocyte expression system, two‐electrode voltage clamp circuitry (to monitor conductance), and H+‐selective microelectrodes (to monitor intracellular pH)—we determined that the intracellular pK of wild‐type (WT) mouse Slc4a11 activity is 7.15 ± 0.04 (n=13, SD, pHe=8.5). In this study we use the same experimental setup to explore whether disease‐causing mutations can affect the intracellular‐pH dependence (pK) of Slc4a11.

We chose FECD mutation E399K and CHED mutations T401K and R804H for comparison to WT due to the loss or gain of a titratable residue. We find that Slc4a11‐‘E399K’ exhibits a pK value of 7.02 ± 0.03 (n = 6, SD) and that Slc4a11‐‘T401K’ exhibits a pK value of 7.09 ± 0.03 (n = 6, SD); both significantly acid‐shifted compared to WT. On the other hand, ‘R804H’‐Slc4a11 exhibits a significantly alkali‐shifted pK value of 7.29 ± 0.04 (n = 6, SD) [Statistics are the result of two‐tailed unpaired t‐tests with Bonferroni correction].

Thus we describe three disease‐causing mutations that cause a shift in the intracellular pK value of Slc4a11. If the pH‐dependence of the H+ conductance mediated by Slc4a11 is critical to the physiological role of Slc4a11, these shifts in pK could contribute towards the etiology of corneal dystrophy.

Support or Funding Information

This work was supported by NIH R01‐EY028580 to MDP.
Anticancer Properties of Secondary Metabolites Isolated from Lichen Parmelia vagans
Authors: Bondarenko Vladimir , Raymond Ky
Published: April 2020

This study aims to analyze the antiproliferative properties of the secondary metabolites extracted from lichen Parmelia vagans collected from the semi‐deserts of Russia. Identification of novel natural compounds that can selectively slow down or stop proliferation of cancer cells can enhance development of anticancer therapeutics.

Various lichen extracts were prepared using either water or organic solvents. We assessed the antiproliferative activity of the extracts against several human cancer cell lines; human malignant melanoma (A375), human mammary gland adenocarcinoma (MCF7). Human dermal fibroblasts (HDFa) were used as control. The same number of cells of different cell lines were plated into a 96‐well plate and allowed to adhere overnight in CO2 incubator. Different dilutions of the lichen extracts were added to the cell cultures next day, and the plate and incubated at 37°C for 6–72 hours. By the end of the incubation time a picture of each well was taken to assess cell confluency and morphology. To asses the effect of the lichen extracts on cell growth and proliferation we used a Resazurin assay. After Resazurin assay, the cells in each well were treated with trypsin, suspended in growth medium and counted using Bio‐Rad cell counter to evaluate a percent of live cells.

Our experimental data from cell viability assay and live cells count indicate that ethyl acetate extracts of P.vagans significantly suppress (30% – 60%) the growth of human malignant melanoma but have a limited effect on human mammary gland adenocarcinoma. We also found that cancer cells subjected to lichen extract change their morphology, and these changes resemble an activation of apoptosis.

Our findings indicate that Parmelia vagans extracts contain secondary metabolites that slow cancer cells growth and proliferation. Furthermore, the extracts affect cancer various cells differently, suggesting that the active compound(s) utilizes a distinct mechanism of action on different types of cancer. Future experiments will focus on the isolation and characterization of the compounds from P.vagans extracts that are responsible for the inhibition of cancer cell growth. Another important direction of future research is to identify the cellular target(s) of the antiproliferative compound(s) and its molecular mechanism of action. In summary, the lichen’s secondary metabolites may hold vast medicinal potential and could be a viable source of a novel of anticancer drugs.

Support or Funding Information

The funding for this project was provided by Touro University Nevada research grant.
Impact of heat therapy on exercise performance, collateral artery growth and skeletal muscle capillarization in a peripheral artery disease model
Authors: Kim Kyoungrae , Ro Bohyun , Damen Frederic , Gramling Daniel P. , Lehr Trevor D. , Song Qifan , Goergen Craig J. , Roseguini Bruno T.
Published: April 2020

Introduction

Repeated exposure to heat therapy (HT) has been shown to improve exercise tolerance in patients with symptomatic peripheral artery disease (PAD), but the mechanisms underlying this effect remain unknown. We investigated the effects of repeated exposure to HT on endurance running capacity, collateral growth, and skeletal muscle capillarization in a preclinical model of PAD.

Methods

Male C57Bl/6 mice (32‐week‐old) were fed a high‐fat diet for 10 weeks. At 42 weeks of age, the animals underwent bilateral ligation of the femoral artery to induce hindlimb ischemia. After two weeks of recovery, the animals were randomly allocated to receive HT (n=12) or a control intervention (n=13). Heat therapy was applied by placing the animal in a flat bottom restrainer and immersing the lower half of the body in a glass container filled with water at 39°C for 30 min daily over 3 consecutive weeks. Animals assigned to the control group were also restrained, but were placed in an empty container. At baseline and one day after the last treatment session, the animals completed a treadmill exercise test (10 m/min for 5 min at 10% grade followed by an increase in speed by 1 m/min every minute until exhaustion) for the assessment of endurance exercise performance. One day after the final treadmill test, the animals were anesthetized, the infrarenal aorta was cannulated with polyethylene‐50 tubing, and a vascular casting compound (Microfil) was injected into the cannula after perfusion‐fixation. Microcomputed tomography (micro‐CT) was used for the measurement of the average diameter of the anterior and posterior gracilis arteries. Skeletal muscle capillarization was assessed in the soleus and extensor digitorum longus (EDL) muscles in a subset of animals (Control: n=8 vs. HT: n=7).

Results

The change from baseline to 3 weeks in maximum running speed (Control: 1.0±1.1 m/min vs. HT: 3.5±0.7 m/min, p=0.072), running distance (Control: 25.7±23.5 m vs. HT: 82.2±15.8 m, p=0.065) and total running time (Control: 62.7±63.5 s vs. HT: 217.3±39.0 s, p=0.056) tended to be greater in the animals exposed to HT as compared to the control group. However, there were no differences between groups in the average diameter of the upper and lower gracilis arteries (Control: 0.14±0.00 mm vs. HT: 0.13±0.00 mm, p=0.119) or the number of capillaries around a fiber in both the EDL (Control: 4.61±0.24 vs. HT: 4.64±0.16, p=0.923) and the soleus (Control: 5.17±0.33 vs. HT: 5.28±0.39, p=0.834) muscles.

Conclusion

These findings indicate that the improvement in exercise tolerance following repeated exposure to HT does not appear to stem from collateral artery enlargment or enhanced muscle capillarization in a mouse model of PAD.

Support or Funding Information

Funding: Ralph W. and Grace M. Showalter Research Trust Award
Isolation and Single Cell Chemical Biological Analysis of Natural Product Producing Cave Microorganisms
Authors: Algoo Jonathan David , Penton Kathryn , Bachmann Brian
Published: April 2020

Microbial secondary metabolites (natural products) constitute a large fraction of pharmaceuticals, with applications as antibiotics and anti‐cancer therapeutics. However, the discovery of new natural product drug leads is increasingly difficult as isolation studies using traditional methods, and from traditional sources, have resulted in progressively fewer novel compounds discovered. Historically, searching for new sources of biodiversity from unique ecosystems has resulted in increased discovery rates. The cave environment, a previously uninvestigated ecosystem, is a promising terrain to discover bacteria with novel natural products because of its unique conditions, which include low light, relatively constant temperature year‐round, abundant oxygen content, and low nutrient content (oligotrophy). We hypothesize that the oligotrophic environment present in caves forces microorganisms to compete or cooperate with each other, which require the development of natural products as communication, offensive, or defensive chemicals. Non‐Streptomyces actinomycetes are a group of bacteria found in caves that represent good candidates for the isolation of new natural products. We acquire bacterial samples by swabbing rock formations and collecting sediment inside a cave. These samples are diluted and incubated on a number of different agar plating conditions. Individual colonies are then isolated and identified by their 16S rRNA sequences. Isolated actinomycetes are stimulated using a battery of chemical and biological conditions known to elicit secondary metabolism (coculture strains, heavy metals, and antibiotics), and then culture extracts are analyzed for possible cellular activity using multiplexed biological assays against mammalian cells. Specifically, extracts are tested using single cell analysis via a flow cytometry platform termed Multiplexed Activity Metabolomics (MAM), which identifies compounds for isolation by analyzing extracts and metabolomic arrays against multiple cell status markers.

Support or Funding Information

National Cancer Institutes R01 CA226833
Renal Olfr1393 may contribute to glucose metabolism
Authors: Hotait Zahraa S. , Shepard Blythe D.
Published: April 2020

Olfactory receptor 1393 (Olfr1393), a G‐protein coupled sensory receptor most commonly associated with olfaction, has been located in the kidney, liver, and other vital organs. We have recently discovered that renal Olfr1393 has a regulatory role in glucose handling via the proximal tubules’ essential sodium glucose cotransporters 1 and 2 (SGLT1, SGLT2). These studies demonstrated that Olfr1393 knockout (KO) mice exhibited improved glucose tolerance as compared to their wild type (WT) counterparts, presenting with euglycemia and glycosuria. Furthermore, we recently determined that the KO mice also exhibit an improved diabetic phenotype when challenged with a high fat diet (type II diabetes) or streptozotocin (type I diabetes). Because of its involvement in glucose reabsorption, we postulated that Olfr1393 may also alter glucose metabolism in the kidney and liver, the two organs capable of gluconeogenesis. To begin, RNA was isolated from kidneys obtained from euglycemic and type I diabetic Olfr1393 WT and KO mice. The RNA samples were reverse transcribed into cDNA, and quantitative polymerase chain reactions (qPCR) was performed to determine the expression levels of glycolytic and gluconeogenic enzymes in each sample. The enzymes selected for this study are those involved in the rate limiting steps of these metabolic processes, and include gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PEPCK), glucose‐6‐phosphatase (G6Pase), fructose 1,6 bisphosphatase (FBP), and glycolytic enzymes phosphofructokinase, liver type (PFK‐L), and pyruvate kinase, liver type (PK‐L). Thus far, the qPCR data ‐ normalized to 18s rRNA for each sample ‐ shows an overall trend towards higher cycling threshold (Ct) values for the KO samples in normal chow mice, indicating lower expression of these enzymes in the murine kidney. Additionally, we find that gluconeogenesis is differentially regulated in the kidneys from diabetic WT and KO mice with an overall trend toward decreased enzyme expression in the Olfr1393 KO samples. Emphasis is currently focused on confirming these findings at the protein level. Further efforts to understand the influence of Olfr1393 on wholebody glucose metabolism will also examine hepatic glucose metabolism. Collectively, these studies suggest that Olfr1393 influences crucial glucose handling processes reaching beyond proximal tubule reabsorption and may even act as a global regulator of glucose handling.

Support or Funding Information

NIDDK
Characterization of the Putative Cytosine RNA Methyltransferase TbCRMT5 from Trypanosoma brucei
Authors: Schultz William C. , Militello Kevin T.
Published: April 2020

RNA methylation is a type of posttranscriptional modification that plays an important role in controlling gene expression. The organism Trypanosoma brucei, the protozoan parasite responsible for Human African Trypanosomiasis, does not seem to have abundant promoter regions or transcriptional regulation machinery. Thus, RNA methylation may play an especially important role in regulating gene expression in this organism. Recently, we have identified seven putative cytosine RNA methyltransferase (CRMT) genes in T. brucei. One of the putative CRMTs, termed CRMT5, is required for maximum parasite
growth. The purpose of this research is to provide evidence for genuine RNA methyltransferase activity. CRMT5 was expressed in E. coli with an N‐terminal 6x‐histidine tag and purified using a His‐affinity column. Purified CRMT5 was used in a series of methyltransferase assays using luciferase activity as a readout. CRMT5 addition resulted in luciferase activity in the presence of cytosine‐containing RNA (T. brucei total RNA and Poly‐IC RNA). There was little to no luciferase activity observed in the presence of RNA that lacks cytosine or when a mock purification from E. coli without the CRMT5 gene was used. Furthermore, a CRMT5 mutant was created changing a putative active site cysteine to alanine (C439A). We expect the mutant to show reduced luciferase activity in the methyltransferase assay. Future experiments will include performing a methyltransferase reaction with CRMT5 and subsequently isolating the RNA for bisulfite sequencing to confirm the methylation of cytosine bases, performing methyltransferase assays with different substrates to determine if there is a binding preference, and confirming our methyltransferase assay results with a radioactivity assay. Evidence for the presence of 5‐methylcytosine and RNA methyltransferases indicates the presence of a process to create an epitranscriptome in T. brucei.

Support or Funding Information

NIH grant 1R15AI133428‐01 to KTM
Effects of a combination of arginine silicate complex and magnesium biotinate on the corneal phototoxic effect of UV exposure in rats
Authors: Sahin Nurhan , Orhan Cemal , Tuzcu Mehmet , Komorowski James R. , Ojalvo Sara Perez , Sylla Sarah , Sahin Kazim
Published: April 2020

The purpose of this study was to investigate the effects of a combination of inositol‐stabilized arginine silicate complex (ASI) and magnesium biotinate (MgB) on the prevention of corneal tissue damage after UVB exposure in rats. Twenty‐eight Sprague‐Dawley rats were randomized into one of the following groups; 1: Control, 2: UV (exposed to UV radiation with no other treatment), 3: UV+MgB Low Dose [L, 140 mg human equivalent dose (HED) of ASI and 1.5 mg HED of Biotin (B)], 4: UV+ASI+ MgB High Dose (H, 140 mg HED of ASI and 10 mg HED of B. Post UV exposure, ASI+MgB administration enhanced the reduced levels of corneal brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in rats. In addition, UV exposure increased the apoptotic protein Bax and glial fibrillary acid protein (GFAP), while decreasing the expression of Bcl‐2, neuronal remodeling marker protein growth‐associated protein‐43 (GAP43), and matrix metallopeptidase 9 (MMP‐9). However, ASI+MgB reduced the corneal Bax and GFAP levels and reversed the reduction of Bcl‐2, GAP‐43 and MMP‐3 levels after UV exposure. These results suggest ASI+MgB combination might be a potential complex that can prevent corneal damage from chronic UV exposure.

Support or Funding Information

Nutrition 21 Turkish Academy of Science (K.Sahin)
Temporal Changes in Pathologic Fibrosis Following Volumetric Muscle Loss Injury
Authors: Hoffman Daniel B. , Sorensen Jacob R. , Call Jarrod A. , Corona Benjamin T. , Greising Sarah M.
Published: April 2020

Volumetric muscle loss (VML) is a devastating orthopaedic injury that results in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. Clinically, two major problems exist in the care of VML injuries: 1) no standard of care to address the loss of function and skeletal muscle mass, and 2) the pathologic fibrotic response impedes muscle healing and regeneration, causing destruction of muscle architecture and altering of the microenvironment of the muscle. However, to date there is only limited mechanistic understanding of the VML‐induced fibrosis. Our recent work indicates that through 21 days post‐VML there is a 22% loss in TA mass, 40% loss in total protein and mitochondrial content, and 45% loss in maximal isometric torque; however, how total collagen content changes over this time is unknown. We examined the temporal changes in the fibrotic deposition early after VML injury to identify a potential window of effective intervention. Adult male Lewis rats (n=42) underwent a full thickness ~20% (~85mg) VML injury to the tibialis anterior (TA) muscle or sham injury and were harvested for genetic and histologic evaluation. First, at 14 and 56 days post‐VML, fibrotic genes probed had biologically significant upregulation compared to control. Collagen I (Col1a1) and collagen III (Col3a1) had a 42‐ and 26‐fold upregulation in expression after 14 days, respectively. The upregulation was dampened at 56 days, yet still significant with Col1a1 and Col3a1 up 10‐ and 5‐fold from baseline. Interestingly, IL‐6 had the strongest upregulation between the 14 and 56‐day time points, from 4‐ to 59‐fold compared to controls. Second, in order to temporally track fibrotic changes more specifically, we evaluated extracellular proteins at 0, 3, 7, 14, 28, and 48 days post VML‐injury both biochemically and histologically. Histologic results indicate that following VML injury that significant fibrotic deposition begins to develop as early as 7 days post‐VML and there is a substantial cellular infiltration into the VML defect area. This suggests there is an initial attempt to follow a normal healing process; however, it is ultimately failed by a vast overcompensation by the immune system, which is supported by the escalating increase in IL‐6 expression. Additional work is needed to precisely probe the crosslinking of collagen fibers and overall makeup of the extracellular matrix across the early time frame after VML injury. Future work will help to identify potential therapeutic targets and assess the efficacy and timing of early rehabilitation, increased physical activity, and pharmacologic approaches to mitigate the fibrotic development after VML injury.

Support or Funding Information

None.
Ablation of PDE4B Protects from Lung Injury in a Mouse Model of Acute P. aeruginosa Infection
Authors: Saleh Lina Abou , Aragon Ileana V. , Boyd Abigail , McDonough Will , Koloteva Anna , Spadafora Domenico , Mneimneh Wadad , Barrington Robert A. , Richter Wito
Published: April 2020

Rationale

Non‐selective inhibitors of Type 4 cAMP‐phosphodiesterases (PDE4s) exert well established anti‐inflammatory effects in non‐infectious models of lung inflammation, such as upon exposure to lipopolysaccharide or ovalbumin, and have demonstrated clinical efficacy in the treatment of COPD. However, they also produce side effects, including emesis, nausea, diarrhea, and weight loss, that limit their clinical utility. The PDE4 family comprises 4 subtypes (PDE4A‐D). As each plays unique and non‐overlapping physiological roles, targeting individual subtypes may serve to separate the therapeutically beneficial from side effects resulting from non‐selective PDE4 inhibition. PDE4B has been proposed as a primary target by which PAN‐PDE4 inhibitors exert anti‐inflammatory effects. Here, we assessed whether genetic ablation of PDE4B is per se sufficient to protect mice infected with live P.aeruginosa from acute lung injury and whether PDE4B ablation can dampen immune responses without worsening the bacterial infection.

Methods

PDE4B knockout (PDE4BKO) mice and their wildtype (WT) littermates were infected intranasally with P.aeruginosa lab strain PA01 and bronchoalveolar lavage fluid (BALF) and lungs were harvested 16 h later to assess lung infection/inflammation.

Results

At 16 h post‐infection, the levels of pro‐inflammatory cytokines in BALF and lung tissue (TNFα, IL1β, IL‐6, and KC) as well as lung histopathological scores were reduced in PDE4BKO mice compared to their WT littermates indicating anti‐inflammatory benefits of PDE4B ablation, whereas the total number of leukocytes or neutrophils in BALF was unchanged. Unexpectedly, despite dampening the immune response, bacterial load in BALF and lungs was significantly decreased in PDE4BKO compared to WT controls. The ability of PDE4BKO mice to clear the bacteria more efficiently correlates with their ability to alleviate the significant PA‐induced hypothermia observed in WT controls. Indeed, when differences in hypothermia between WT and PDE4BKO mice are equalized by externally warming the animals, the bacterial load in WT and KO mice are comparable whereas anti‐inflammatory benefits, such as reduced cytokines levels in PDE4BKO mice, are retained.

Conclusions

Our results suggest that ablation of PDE4B per se is sufficient to alleviate lung inflammation in a model of acute P.aeruginosa infection without worsening the bacterial infection. Thus, PDE4B may represent a therapeutic target for inflammatory lung diseases associated with P.aeruginosa infections, such as ventilator associated pneumonia or cystic fibrosis.

Support or Funding Information

Supported by grants from the Cystic Fibrosis Foundation (SALEH18H0, RICHTE16GO) and the NIH (HL76125, HL141473, HL066299).
Elevated Plasma Activin A Levels during Pregnancy Impairs Global Longitudinal Strain in the Heart
Authors: Bakrania Bhavisha A. , Shahul Sajid , Hall Michael E. , Granger Joey P.
Published: April 2020

Preeclampsia (PE) is a disorder prevalent in 3–8% of pregnancies, characterized by maternal hypertension, endothelial dysfunction and impaired cardiac function. PE is thought to develop in response to impaired spiral artery remodeling, leading to poor perfusion of the placenta. As a result, the ischemic placenta releases several anti‐angiogenic and pro‐inflammatory factors into the maternal circulation leading to widespread endothelial activation. Studies have also shown that preeclamptic women exhibit decreased cardiac global longitudinal strain (GLS), a sensitive measure of systolic function that indicates fibrosis and tissue injury. Importantly, these findings were true independent of high blood pressure, suggesting that placental factors could play a role in the development of cardiac dysfunction in PE. A recent study showed Activin A, a pro‐fibrotic factor that is released by the placenta, is shown to be increased during PE pregnancy. Furthermore, increased circulating Activin A has been shown to be predictive of impaired GLS one year postpartum. PE is a multifaceted disease, and whether increased circulating Activin A alone induces cardiac dysfunction is not known. We hypothesized that chronic excess levels of Activin A during pregnancy induces cardiac dysfunction. To address this, rats were administered vehicle (n=6) or Activin A (n=6/group; A1, 1.25 μg/day, A2, 1.9μg/day, A3, 3 μg/day, A4, 6μg/day) via osmotic pump on gestational day (GD) 14. All animals had an indwelling carotid catheter placed on GD 18, followed by a comprehensive echocardiography assessment and blood pressure measurement on GD19. Activin A infusion resulted in significant increases in circulating Activin A in all treated groups (Vehicle, 585±47 pg/mL, A1, 2028±210 pg/mL, A2, 1895±382 pg/mL, A3, 2416±168 pg/mL, A4, 2864±181 pg/mL, P<0.01). At higher doses, Activin A infusion was associated with significantly impaired GLS (Vehicle, −22.1±0.8 %, A3, ‐ 16.9±0.9 %, P<0.01, A4, −14.9±0.5 %, P<0.01). Interestingly, mean arterial pressure was not significantly different between groups. These findings suggest that Activin A causes cardiac dysfunction in a dose dependent manner and may induce impaired GLS during pregnancy, independent of increases in blood pressure. This model could be useful in understanding the mechanisms by which Activin A induces these cardiac abnormalities in PE and whether blockade of this factor could improve outcomes for these women.

Support or Funding Information

This research was supported by the American Heart Association (18POST33990293) and the National Institutes of Health (P01HL051971, P20GM104357).
BDNF Augments NMDA‐signaling and Reduces GABAA‐signaling in the Paraventricular Nucleus of the Hypothalamus (PVN) to Elevate Blood Pressure
Authors: Thorsdottir Daniella , Einwag Zachary , Givant Madeleine , Erdos Benedek
Published: April 2020

PVN presympathetic neurons, projecting to the rostral ventrolateral medulla (RVLM) or the spinal cord, play a critical role in the regulation of blood pressure. BDNF is a neurotrophic factor known to be upregulated in the PVN in response to hypertensive stimuli such as stress. However, the mechanisms by which BDNF modulates cardiovascular regulation in the PVN remains unclear. BDNF alters glutamatergic and GABAergic signaling, as well as neuronal morphology broadly in the central nervous system. Within the PVN, glutamatergic synaptic input and NMDA‐signaling are elevated, while GABAergic inhibition is reduced in hypertensive models. We tested the hypothesis that increased BDNF expression in the PVN elevates blood pressure by enhancing NMDA‐signaling, diminishing GABAA‐signaling and altering morphology of RVLM‐projecting PVN neurons. To simulate stress‐related upregulation of BDNF in the PVN, an AAV2 viral vector expressing BDNF was bilaterally injected into the PVN of Sprague Dawley rats, while control rats received AAV2‐GFP injections. Three weeks later, blood pressure and heart rate responses to PVN injections of NMDA and GABAA agonists and antagonists were recorded under anesthesia. Our results showed that inhibition of NMDA receptors with AP5 induced significantly larger decreases in blood pressure and heart rate in the BDNF group (−23±4 mmHg and −56±17 BPM) compared with the GFP group [5±1 mmHg (p<0.01) and −5±1 BPM (p<0.05)]. In contrast, activation of NMDA receptors with NMDA induced similar cardiovascular responses in GFP and BDNF animals. Inhibition of GABAA receptors with gabazine induced significantly smaller blood pressure increases in the BDNF group (32±6 mmHg) compared to GFP (61±11 mmHg, p<0.05), but heart rate responses were similar in the two groups. Activation of GABAA receptors with muscimol caused significantly reduced hypotensive responses in the BDNF group (−14±2 mmHg) compared to GFP (−23±4 mmHg, p<0.05), while changes in heart rate were similar. Quantitative RT‐PCR from PVN brain punches indicated that expression of NMDAR1 or GABAA‐Δ was unaffected by BDNF overexpression. However, immunofluorescence imaging in PVN brain sections demonstrated that BDNF significantly increased the soma size of RVLM‐projecting PVN neurons. In summary, our findings indicate that BDNF augments glutamate signaling in the PVN, primarily by increasing presynaptic glutamate release, and reduces GABA inhibition possibly through postsynaptic mechanisms. Finally, BDNF may also affect the balance of excitatory/inhibitory synaptic balance and regulation of blood pressure by increasing the soma size of PVN‐RVLM neurons.

Support or Funding Information

R01 HL133211‐01A1.
Estrus Cycle Stages Differentially Alter Ventilatory Pattern Variability (VPV) in Septic Female Rats
Authors: Clifford Caitlyn , Campanaro Cara , Tackett Shiloh , Horton Kofi-Kermit , Nethery Dave , Hsieh Yee-Hsee , Jacono Frank , Dick Thomas
Published: April 2020

The sex hormones, estrogen and progesterone, affect central respiratory control. Progesterone, increases respiratory frequency (fR). Sepsis, a disease process caused by a dysregulated systemic inflammatory response, also affects respiratory control that can be quantified by Ventilatory Pattern Variability (VPV). We hypothesized that natural hormone fluctuations during the female estrus cycle affect VPV and its differential control during sepsis. The estrus cycle of female Sprague Dawley rats (n = 19) was assessed by collecting, hematoxylin/eosin staining, and identifying vaginal epithelial cells. Proestrus, in which progesterone peaks, was determined by the absence of both leukocytes and cornified multinucleated epithelial cells. Rats were inoculated with fibrin pellets containing 0 (D0) or 100x 10^ 6 (D100) E. coli cells during proestrus. Before and 12 h after inoculation, we recorded the ventilatory pattern in conscious unrestrained rats using whole‐body plethysmography. VPV [coefficient of variation (CV) for the duration of the respiratory cycle (CV‐Ttot), autocorrelation function (AC), mutual information (MI), sample entropy (SampEn), and nonlinear complexity index (NLCI)] was compared across four experimental groups: 1) D0 proestrus rats (n = 3), 2) D100 proestrus rats (n = 7), 3) D0 non‐proestrus rats (n = 4), and 4) D100 non‐proestrus rats (n = 5) using one‐way analysis of variance (ANOVA). In the non‐proestrus groups, SampEn was greater in D0 than D100 (1.31±0.011 vs 1.14±0.002 Nats, p = 0.006), but in proestrus rats, SampEn did not vary with inoculation (D0, 1.26±0.003 vs D100, 1.26±0.004 Nats, NS). However, CV‐Ttot, AC and MI trended toward but did not reach significance given the low n. These preliminary data suggest that a decrease in VPV due to septicemia occurs in nonproestrus stages, whereas did not occur in proestrus.

Support or Funding Information

Funded By: VA Research Service (I01BX004197); NIH (U01 EB021960)
The Influence of Behavioral Selection on Dental Variation in Domesticated Silver Foxes
Authors: Wood Emma K. , Maddux Scott D. , Southard Thomas E. , Kharlamova Anastasiya , Trut Lyudmila , Franciscus Robert G.
Published: April 2020

The relationship between canine tooth size and social signaling has long been observed in wild carnivore and primate populations. In several of these species, it has been well‐established that larger canine size is directly correlated to social structures where agonistic interactions are commonplace. However, canine teeth are difficult to study in the wild because they serve multiple roles in addition to social signaling, such as food acquisition and guiding functional occlusion of the jaws. The Russian silver fox experiment represents a long‐term, controlled study where foxes have been selected for a behavioral phenotype toward either non‐aggression or hyper‐aggression. Results from the experiment have demonstrated that an increase in the range of variation of several soft tissue features accompanies selection for non‐aggression. In the absence of food‐related selective pressures, the influence of behavioral selection on dentition can be observed. The objective of this study is to compare the variation present in the gross dental sizes of canine teeth, which serve both social and food‐related roles, and carnassial teeth (maxillary P4 and mandibular M1), which play critical roles in mastication, as a result of selection solely on behavioral phenotype. We employed univariate and multivariate comparisons of maxillary canine, maxillary fourth premolar, mandibular first molar mesiodistal length and buccolingual breadth, as well as maximum height and crown height of the canine. These data were collected in sex‐balanced samples of unselected (n=51), tame (n=48), and hyper‐aggressive (n=50) experimental strains. Results indicate that the canine tooth is the most variable tooth across all three strains. This is in line with observations recorded in wild fox populations. However, foxes in the aggressive‐selected strain exhibit significantly reduced variation in canine crown height compared to the tame and control groups, further supporting hypotheses that the canine is strongly influenced by behavioral selective pressures favoring aggression. The patterns of variability in the tame‐selected strain tend to mirror those in the unselected strain, albeit with greater degrees of sexual dimorphism present in the variation. This provides further evidence that canine teeth are under strong selection relative to behavioral pressures and supports hypotheses that selection on behavior can affect the rate of variability of structures.

Support or Funding Information

University of Iowa College of Dentistry; University of Missouri; University of North Texas Health Science Center
Stent Graft Expulsion Outside the Skin: A Rare Case
Authors: Kalkan Asim , Bas Behlul , Kocer Taylan , Atis Seref Emre , Unver Sevilay Sema , Koca Yavuz Selim
Published: April 2020

Patients who undergo stent grafting may present to the ED some time after the procedure with various related symptoms. The most common of these are stent graft occlusions or hematoma, although infection or abscess also may develop. In this case report, a 58-year-old man presented to the ED with a purulent wound on the stump of an amputated leg and a foreign body protruding from the wound site. The patient had a history of stent insertion with femoropopliteal bypass 11 years before this incident and an above-the-knee amputation because of stent occlusion 8 years prior. This wound had appeared with reddening of the skin 1 month before presentation, followed by the emergence and protrusion of a foreign body.
Pandemics and Their Impact on Medical Training: Lessons From Singapore
Authors: Liang Zhen Chang , Ooi Shirley Beng Suat , Wang Wilson
Published: April 2020

The ongoing coronavirus disease 2019 (COVID-19) crisis has hit Singapore hard. As of February 25, 2020, Singapore had the fourth highest number of confirmed COVID-19 infections outside of China, only trailing behind South Korea, Italy, and Japan. This has had reverberating effects on Singapore’s health care system, and has, consequently, also affected medical education all the way from the undergraduate to the postgraduate level. While efforts are underway to contain disease spread and transmission, the authors believe that this is an opportune time to examine and reflect on the impact that medical crises like COVID-19 can have on medical training and education and to evaluate “business continuity plans” to ensure quality medical education even in the face of constant disruptions from pandemic outbreaks. Medical training is as important a mandate as patient care and service. The authors believe that even in trying times like this, rich and precious lessons can be sought and taught, which will immensely benefit medical students and residents—the health care leaders of tomorrow. In this Perspective, the authors discuss the various ways in which the COVID-19 crisis has affected medical instruction in Singapore and explore pertinent practical and creative solutions for the continuity of medical training in these trying times, drawing on their previous experience with the Severe Acute Respiratory Syndrome outbreak in 2003 as well as the current ongoing COVID-19 crisis.
Minimally Invasive Chevron Akin (MICA) for Correction of Hallux Valgus
Authors: Redfern David
Published: April 2020

The Minimally Invasive Chevron Akin (MICA) technique continues to gain in popularity among surgeons worldwide as a reliable technique for the surgical treatment of hallux valgus. This article provides an update of surgical techniques as described by the originators and review of current literature on the topic.
Dexmedetomidine suppresses the progression of esophageal cancer via miR-143-3p/epidermal growth factor receptor pathway substrate 8 axis
Authors: Zhang Peisen , He Hefan , Bai Yuyan , Liu Weifeng , Huang Lirong
Published: April 2020

Esophageal cancer is one of the fatal cancers around the world. Dexmedetomidine (DEX) is widely used during anesthesia of esophageal cancer surgery. Nevertheless, the role of DEX in the progression of esophageal cancer remains barely known. The proliferation, apoptosis and metastasis of esophageal cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, transwell migration and invasion assays and Western blot assay. The expression of miR-143-3p was measured by quantitative real-time PCR in esophageal cancer tissues and cells. The binding sites between miR-143-3p and epidermal growth factor receptor pathway substrate 8 (EPS8) were predicted by Starbase online software, and the combination was verified by dual-luciferase reporter assay. The murine xenograft model was established using KYSE150 cells to verify the function of DEX in vivo. DEX inhibited the proliferation and metastasis while accelerated the apoptosis of esophageal cancer cells. The abundance of miR-143-3p was lower in esophageal cancer tissues and cells than that in paring normal tissues and normal esophageal mucosal cells Het-1A. MiR-143-3p could be induced by DEX treatment in esophageal cancer cells, and miR-143-3p also suppressed the development of esophageal cancer. EPS8 was a functional target of miR-143-3p, and it played an oncogenic role in esophageal cancer. DEX inhibited the growth of tumor via miR-143-3p/EPS8 in vivo. DEX suppressed the growth and metastasis while facilitated the apoptosis of esophageal cancer cells through upregulating the abundance of miR-143-3p and reducing the level of EPS8 in vivo and in vitro, providing promising target for the treatment of esophageal cancer.
Growth inhibition and apoptosis of human multiple myeloma cells induced by 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid derivatives
Authors: Rogers Lanerica Jamese , John Teena , Park Jonathan , Tucker Michael , Ma Handong , Wu Yan , Rayalam Srujana , Wang Xinyu
Published: April 2020

Multiple myeloma is a blood cell cancer and can cause symptoms such as bone loss and fatigue. Recent studies have shown that the bone marrow microenvironment may mediate tumor proliferation, drug resistance, and migration of the multiple myeloma cells. Synthetic triterpenoids have been used for the treatment of cancer due to their antiproliferative and anti-inflammatory effects. The objective of this study is to examine the effect of 2-cyano-3, 12 dioxoolean-1,9-dien-28-oic acid (CDDO) derivatives on human multiple myeloma cells. Three CDDO derivatives, such as CDDO-methyl ester, CDDO-trifluroethyl amide, and CDDO-imidazolide (Im), were tested on the growth of human multiple myeloma cells. Our results show that all CDDO derivatives decrease the viability of multiple myeloma cells in a dose- and time-dependent manner, with CDDO-Im being the most potent. CDDO-Im was selected to investigate whether its inhibitory effect on multiple myeloma cell growth is due to cell cycle arrest and induction of apoptosis. The results suggest that CDDO-Im may inhibit cell cycle progression in the G0/G1 phase and induce the intrinsic apoptotic pathway. The effect of CDDO-Im on multiple myeloma cells was evaluated in a Transwell model using myeloma cells co-culturing with human HS-5 stromal cells to simulate the bone marrow microenvironment in vitro. The results showed that CDDO-Im induced multiple myeloma cell apoptosis in the presence of HS-5 cells, albeit to a lower extent than in multiple myeloma cells cultured alone. In conclusion, our data suggest that CDDO-Im inhibits the growth of multiple myeloma cells, either cultured alone or co-cultured with bone marrow stromal cells, through the induction of apoptosis.