bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Kinase C Fusions Reveal Another Mechanism for Loss of Protein Kinase C Function in Cancer
Authors: Van An-Angela N. , Kunkel Maya T. , Baffi Timothy R. , Antal Corina E. , Newton Alexandra C.
Published: April 2020

Over 60 protein kinase C (PKC) fusion genes have been identified in a wide variety of cancers, making PKC the most frequently fused AGC kinase. Our laboratory previously revealed that cancer‐associated point mutations annotated for PKC family members are generally loss‐of‐function, with functional studies revealing a tumor suppressive role for PKC. Here, we sought to determine whether PKC fusions may also be loss‐of‐function. In our analysis, we characterized TANC2‐PRKCA, a fusion identified in lung squamous cell carcinoma that retains the catalytic domain of PKCα, and PRKCA‐CDH8, a fusion identified in breast cancer that retains the regulatory domain of PKCα. Overexpression of TANC2‐PKCα in cells showed that the fusion protein is constitutively active; however, it lacks phosphorylation at key sites required for the stability of the enzyme. Most importantly, without its amino‐terminal pseudosubstrate, the fusion protein is unable to adopt the autoinhibited, stable conformation of wild‐type PKC, rendering it markedly unstable. To determine whether the fusion protein could accumulate in cells given this instability, we utilized CRISPR/Cas9‐mediated gene editing to express TANC2‐PRKCA. Indeed, while the fusion mRNA was detected in the CRISPR‐edited clones, we were unable to detect the fusion protein. Thus, although the fusion is rendered constitutively active through loss of autoinhibition, its subsequent instability prevents its accumulation in cells, making it paradoxically loss‐of‐function. In contrast to fusions that retain the PKC catalytic domain, fusions that retain the PKC regulatory domain are intrinsically loss‐of‐function with respect to PKC activity. However, we were interested in exploring whether regulatory domain fusions could also act in a dominant‐negative manner. Overexpression of PKCα‐CDH8 in cells suppressed endogenous PKC activity, both basally and following agonist stimulation. This suggests that PKC regulatory fusions are not only loss‐of‐function by loss of the catalytic domain, but also dominant negative by competition of the ligand‐sensing modules for binding the allosteric activator diacylglycerol. Taken together, our data demonstrate that both catalytic and regulatory domain PKC fusions are loss‐of‐function, revealing another mechanism by which PKC activity is lost in cancer and supporting a tumor suppressive role for PKC.

Support or Funding Information

This work was supported by NIH R35 GM122523 (ACN). ANV, TRB, and CEA were supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), and CEA was supported by an NSF Graduate Research Fellowship (DGE1144086).
Stability‐function trade‐offs during the evolution of antimicrobial resistance
Authors: Matange Nishad
Published: April 2020

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic‐target proteins. In the present study, I use trimethoprim (TMP)‐resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) to exemplify how drug‐resistance can often be associated with protein misfolding and aggregation. I show that misfolding of TMP‐resistant DHFR is associated with its reduced steady state levels, resulting in a fitness cost. Importantly, the effect of destabilizing mutations on drug‐resistance is contingent on the folding environment of the cell. In particular, the presence or absence of the lon‐protease, a quality control protease in E. coli, determines the genotype‐phenotype relationship between DHFR mutations and their level of trimethoprim‐resistance. The status of lon‐protease also determines the mutational repertoire accessible to EcDHFR to evolve drug resistance. This study demonstrates that trade‐offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.

Support or Funding Information

DST‐INSPIRE Faculty Fellowship, Department of Science and Technology, Govt. Of India

Figure 1Figure 2
Molecular and structural characterization of a TEAD mutation at the origin of Sveinsson’s chorioretinal atrophy
Authors: Bokhovchuk Fedir , Mesrouze Yannick , Izaac Aude , Meyerhofer Marco , Zimmermann Catherine , Fontana Patrizia , Schmelzle Tobias , Erdmann Dirk , Furet Pascal , Kallen Joerg , Chène Patrick
Published: April 2020

Four TEAD transcription factors (TEAD1–4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson’s chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol–1. We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H‐bonds and the loss of van der Waals contacts, which explains why the Tyr421HisTEAD1 mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex.

Close view of the region surrounding key Tyr/His429 in the superimosed wtYAP:wtTEAD4 and wtYAP:Tyr429HisTEAD4 complexes

Figure 1
Vascular Expression of Toll‐Like Receptor 4 and Myeloid Differentiation Factor‐88 Mediate Endothelial Dysfunction in Response to Lipopolysaccharide Independent of Circulating Immune Cells
Authors: Coate Ian D. , Didion Sean P.
Published: April 2020

The goal of this study was to test the hypothesis that vascular expression of toll‐like receptor 4 (TLR4) and its primary downstream signaling pathway, myeloid‐differentiation factor‐88 (MyD88), mediate endothelial dysfunction in response to lipopolysaccharide (LPS). Carotid arteries from wild‐type (C57Bl/6), Tlr4‐Lps‐d (Tlr4 mutant mice), and Myd88‐deficient (Myd88−/−) mice were incubated with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS, but not vehicle, treatment produced concentration‐dependent impairment (P<0.05) of endothelium‐dependent relaxation to acetylcholine (ACh) in wild‐type mice. In contrast, in carotid arteries from Tlr4‐Lps‐d or Myd88−/− mice endothelial responses to ACh following LPS incubation were found to be similar (P>0.05) to responses in wild‐type mice incubated with vehicle. LPS has no effect (P>0.05) on vascular response to sodium nitroprusside in any of the six experimental groups. Both basal and NADPH‐stimulated superoxide levels were selectively increased (P<0.05) in vessels from wild‐type, but not Tlr4‐Lps‐d or Myd88−/−, mice treated with LPS. RNA expression revealed that both TLR4 and MyD88 are indeed expressed in the vascular wall under basal conditions and that LPS induces differential expression of a number of inflammatory gene products, including Tlr4, Myd88, and Nfkb1. Pathway analysis revealed a linkage between TLR4 and MyD88 in the vessels wall with IL‐6 and downstream targets such as Nox2 and Nox4. These findings serve to link activation of TLR4 and downstream signaling through MyD88 and NFkB1 with increases in vascular superoxide and functional impairment of endothelial function in response to LPS. Taken together, these findings demonstrate that both Tlr4 and Myd88 expression and activity within the vascular wall contributes to LPS‐induced impairment of endothelial function. The fact that experiments were performed ex vivo, demonstrate that the effects of LPS on endothelial function occur directly within the vascular wall independently of circulating immune cell activation.

Support or Funding Information

Supported in part by NIH/NIBIB R01‐EB2006.
Breathing Pattern Differs Between Male and Female Rats in Escherichia coli Induced Septicemia
Authors: Campanaro Cara , Clifford Caitlyn , Tackett Shiloh , Horton Kofi-Kermit , Nethery David , Hsieh Yee-Hsee , Dick Thomas , Jacono Frank
Published: April 2020

Septicemia is a potentially fatal infection marked by bacteria in the bloodstream coupled with a dysregulated inflammatory response. Escherichia coli (E. coli) is a common cause of septicemia. We hypothesized that in E. coli induced septicemia, respiratory frequency (fR) and the ventilatory pattern variability (VPV) would depend on sex. Male (n=6) and female (n=6) Sprague Dawley rats were inoculated with E. coli (100×106 colony forming units) contained within a clot formed of thrombin, fibrinogen, and phosphate buffered saline. We measured ventilation in unanesthetized rats with whole‐body plethysmography before (0h) and 12h after inoculation. Before inoculation, the fR male rats was greater than that of females (fR: 78.4 ± 11.9 vs. 58.7±6.98 breaths per minute (brthspm); p=0.007). In contrast, VPV did not differ between males and female (autocorrelation coefficient (r) at 1 cycle length 0.58±0.17 vs 0.56±0.13 and sample entropy 1.24±0.04 vs 1.34±0.11 Nats). However, 12h after inoculation, fR in male rats was much greater than female (142.73±22.03 vs. 64.59±6.61 brthspm; p= 0.0001) and analysis of VPV found differences in autocorrelation (0.814±0.084 vs. 0.578±0.160; p=0.01) and sample entropy (0.98±0.10 vs. 1.19±0.13 Nats; p=0.01). Further, in both male and female rats, fR increased 12h after inoculation rats (males, p=0.001; females p=0.02). These preliminary data indicate that male rats have a higher resting fR and greater ventilatory response to septicemia than female rats. Pending analysis of pro‐inflammatory cytokines will indicate whether the magnitude of these ventilatory response correlates to the magnitude of the inflammatory response.

Support or Funding Information

Funded by: VA Research Service (I01BX004197); NIH (U01 EB021960)

Figure 1
Connectivity Enhanced Structure Activity Relationship (ceSAR): A Novel Approach to Increase Accuracy and Speed of Virtual Drug Discovery
Authors: Thorman Alexander William , Reigle James , Chutipongtanate Somchai , Shamsaei Behrouz , Pilarczyk Marcin , Fazel-Najafabadi Mehdi , Adamczak Rafal , Kouril Michal , Medvedovic Mario , Meller Jarek
Published: April 2020

Experimental drug discovery and repurposing is a costly and time‐consuming process. Here, a novel approach, dubbed connectivity enhanced structure activity relationship (ceSAR) is introduced. ceSAR combines traditional virtual screening methodologies with cheminformatics and Library of Integrated Network‐Based Cellular Signatures (LINCS) gene expression signatures of genetic and chemical perturbations. This methodology combines structural similarity of small molecules with transcriptional connectivity and virtual screening‐predicted specific interacting molecules for the enhancement of structure activity relationship (SAR) and lead compound discovery. The transcriptional connectivity aspect of ceSAR is implemented as an R Shiny package, titled Sig2Lead, that employs connectivity between chemical and genetic signatures included in the LINCS library to identify small molecule inhibitors of user‐targeted pathways. Chemical libraries are reduced with ceSAR by first measuring concordance of transcriptomic signatures between genetic and chemical perturbation, identifying putative pathway inhibitors, followed by traditional docking approaches to re‐score the remaining library to identify compounds likely to interact with the target of interest. This combination identifies compounds that are both pathway inhibitors and have shape complementarity to the target of interest, making the most likely scenario that the molecules are directly interacting with the target protein of interest to modulate the pathway. Through integration of these orthogonal approaches, both an increased accuracy and speed can be observed, with the speed increase being several orders of magnitude.

Support or Funding Information

This work was supported in part by the National Institutes of Health grants U54 HL127624

ceSAR combines transcriptional connectivity and virtual screening to identify novel inhibitors from a screened chemical library. The library is first reduced using transcriptional connectivity to a knockdown of the target gene to restrict the search to putative pathway inhibitors. These pathway inhibitors are then reduced further using traditional virtual screening approaches to find compounds with shape complementarity to the protein of interest.

Figure 1

Comparison of results of an early drug screen using a combination of docking with AutoDock4.2.6 and Sig2Lead shows a dramatic reduction in compounds without losing the best‐performing compounds. If used in this case, the ceSAR method would have excluded 51 of the 147 tested compounds, of which 45 had no observable IC50 via a Fluorescence Polarization assay, thus driving library enrichment. This reduction would have saved time and reagents through preventing testing of as many spurious compounds.

Figure 2
Human Lung Microvascular Endothelial Cell Death in Response to Cell‐free Hemoglobin
Authors: Tomasek Toria , Meegan Jamie E. , Ware Lorraine B. , Bastarache Julie A.
Published: April 2020

During sepsis, circulating levels of cell‐free hemoglobin (CFH) are often elevated due to the lysis of red blood cells, and higher concentrations are associated with worse clinical outcomes. Sepsis is caused by a widespread inflammation response, which leads to the release of pro‐inflammatory molecules and cytokines into the circulation. These compounds can prime and activate the endothelium rendering it more susceptible to damage from other circulating molecules. Sepsis is a leading cause of acute respiratory distress syndrome (ARDS), which is characterized by vascular leak and fluid accumulation in the lungs. In our previous studies in murine models of sepsis, lung endothelial cell death was observed, which may contribute to the alveolar capillary barrier dysfunction that characterizes ARDS. Here we investigate whether CFH can initiate cell death in vitro and whether this is potentiated by exposure to pro‐inflammatory signals. Primary human lung microvascular endothelial cells (HLMVECs) were cultured to confluence and exposed to various clinically relevant concentrations of CFH diluted in complete media for up to 24 hours or pre‐treated for 4 hours with 5 ng/ml Cytomix (equal parts IL‐1β, TNF‐α, and INFγ), followed by treatment with 1.0 mg/ml CFH for 24 hours. Cell viability was determined using Draq7, a cell membrane impermeable nuclear stain. Cell death was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which detects DNA fragmentation from late apoptosis or necrosis. While there was a time‐ and concentration‐dependent trend, no significant differences between CFH treated and untreated cells were seen in the absence of cytomix, even at the highest concentration of 1.0 mg/ml CFH at 24 hours for the TUNEL assay (57 TUNEL+
cells per FOV in CFH‐treated vs. 17 in control, p=0.8245). However, there was a significant increase in Draq7 staining with 1.0 mg/ml CFH treatment after 24 hours (7.9% Draq7+ cells in CFH‐treated vs. 2.3% in control, p=0.0240). Exposure to cytomix augmented the decrease in viability from CFH as measured by Draq7 assay (32.4% Draq7+ cells in Cyto + CFH‐treated cells vs.
14.5% in CFH‐treated cells, p<0.0001). These findings indicate that CFH alone has modest effects on lung endothelial cell viability. However, during sepsis when circulating cytokine levels are increased, the effect of CFH on endothelial cell death is enhanced, which may contribute to the microvascular leak and alveolar fluid accumulation that are characteristic of acute lung injury.

Support or Funding Information

This work is funded by NIH HL135849, HL103836, and HL094296.

Figure 1
Effect of URAT1 inhibition with verinurad on proximal tubule intracellular lactate: A mathematical modeling analysis and hypothesis for antiproteinuric effect
Authors: Nieves Emily , Hallow Melissa , Greasley Peter , Helmlinger Gabriel , Parkinson Joanna
Published: April 2020

Verinurad (RDEA3170) lowers serum uric acid by inhibiting URAT1, a urate/carboxylate co‐transporter in the proximal tubule (PT) of the kidney. In a recent clinical trial, verinurad also reduced proteinuria, but the mechanism remains unknown. One proposed hypothesis is that the inhibition of URAT1 augments protein reabsorption by increasing intracellular lactate in proximal tubule cells. The increased availability of lactate may lead to a restoration of the reabsorption of protein by increasing ATP levels and/ or improving endocytosis of protein by lowering endosomal pH. The goal of this study was to mathematically evaluate the effect of URAT1 inhibition with verinurad on intracellular lactate concentration.

A mathematical model of urate and lactate transport in the rat proximal tubule was adapted to simulate transport in humans. The transporter model was further adjusted to represent states of hyperuricemia and altered expression of uric acid transporters that likely occur in hyperuricemia. The transporter model was linked to a systems description of plasma uric acid and the pharmacokinetics and pharmacodynamics of verinurad were incorporated into the model.

The model was able to reproduce observed verinurad pharmacokinetics, as well as the observed changes in plasma and urinary uric acid. The model predicted that URAT1 inhibition with verinurad increases intracellular lactate concentration in the human PT by 26% in normouricemic and 20% in hyperuricemic conditions.

Model simulations indicate that verinurad may substantially increase the availability of lactate inside PT epithelial cells. Since lactate is used as a fuel by the PT, increased availability of lactate may potentially help restore protein reabsorption capacity in fuel‐deficient PT cells by increasing ATP levels. In addition, increased intracellular lactate could help maintain the low endosomal pH necessary for protein reabsorption. This hypothesis remains to be experimentally proven; further non‐clinical/clinical data would be valuable to support model predictions.

Urate and lactate proximal tubule transport model.

Figure 1
Essential Anatomy for Primary Care vs. Non‐Primary Care Clerkships and Electives
Authors: Hankin Mark , Harmon Derek , Martindale Jim , Keim Sarah , Topping Daniel , Farias Anna , Harrell Kelley , Marzban Hassan , Cotter Meghan , Royer Danielle , Davies David , Lopez Lisa , Carpenetti Tiffany , Downs Mary Beth , McNary Thomas , Latacha Kimberly , Zumwalt Ann , Mork Amy , Wineski Lawrence , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S. , Brooks William , Kirera Francis , Lewis Steven , Granite Guinevere , McWhorter David , Pratt Rebecca , Notebaert Andrew , Miriyala Sumitra , Gest Thomas , Doroudi Majid , Giannaris Eustathia Lela , Kar Rekha , Sakaguchi Alan , Lackey-Cornelison Wendy , Hisley Kenneth
Published: April 2020

Introduction

To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim

To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in clerkships and electives to identify the anatomy they consider essential for their specialty.

Methods

An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the nominal relationship between groups of primary care (Family Medicine, General Internal Medicine, General Pediatrics) and non‐primary care specialties, and whether they considered a given anatomical region important to their clerkship/elective. Second, the study compared the rank assigned by each specialty to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results

At the time of abstract submission, data had been collected from 598 clinicians (clerkship/elective directors and attending physicians) in 19 clerkships/electives at 33 medical schools (allopathic n=28; osteopathic n=5). Relative to Non‐Primary care physicians, Primary Care physicians showed a statistically significant, higher percentage of “Yes” responses for all seven anatomical regions (Table
1). Further data analysis has identified the highest ranking anatomical topics within each region for Primary Care and Non‐Primary Care Specialties.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.

Responses to the question: Is the anatomy of this body region important to your clinical specialty?

Table 1
Using Breath Metabolic Profiling to Identify Genetic Variants in Alcohol Metabolism
Authors: Kang Sunwoo , White Joseph Robert , Gross Eric Richard
Published: April 2020

Aldehyde dehydrogenase 2 (ALDH2) metabolizes acetaldehyde to acetic acid. An ALDH2 genetic variant, ALDH2*2, limits acetaldehyde metabolism. As a result, after consuming alcohol, the ALDH2*2 genetic variant causes acetaldehyde accumulation, facial flushing, and tachycardia; commonly referred to as Asian glow. Here we describe how we developed a machine learning algorithm to distinguish between genetic variants of ALDH2 using data collected from the human breath mass spectrum.

To develop this machine learning algorithm, we recruited human volunteers to perform an alcohol challenge after obtaining IRB approval. For these volunteers, we performed genotyping in addition to subjecting volunteers to a 0.25g/kg alcohol challenge. During the alcohol challenge, we measured the human breath mass spectra for 16 people (8 ALDH2*1*1 and 8 ALDH2*1*2) using selective ion flow mass spectrometry. This created a metabolic profile for each person of the full mass spectrum which consisted of 1,155 ion measurements complemented with 14 metabolite panel measurements.

To develop a machine learning algorithm to predict ALDH2 genotype, standard scaler (SS) and principal component analysis (PCA) were applied to normalize the variance in ion counts and maximize high dimensional input. The visualization of metabolomics data showed two distinct clusters that are dependent on the ethanol intake (Figure
1A) and on ALDH2 genotype (Figure
1B). Based on the cluster distinction, we were able to verify that our data could characterize ethanol metabolism. Next, we applied ~20 classifier algorithms such as Support Vector Machine, Perceptron, Gaussian Naïve Bayes, Stochastic Gradient Descent, and Decision trees. The prediction through Random Forest yielded three novel features from the raw mass spectra that are not included in metabolite panel. These algorithms were able to achieve a training accuracy above 90% threshold for identifying between ALDH2 genotypes. These finding suggests a non‐invasive method to determine ALDH2 genotype that may be used to complement existing methods to determine the response a person has to alcohol.

Support or Funding Information

This research received grant funding from Stanford Chem‐H and NIH NIGMS GM119522.

Visualization of Temporal HEB Metabolomics Profiling. Figure 1A: 3D PCA on Ethanol Catabolism with SVM distinction kernel. Blue=baseline, red=after ethanol exposure / Figure 1B: 3D Visualization of Partial Gradient of Acetaldehyde and Ethanol. Blue=ALDH2 cohort, red=ALDH2*2 cohort

Figure 1
A Larval Zebrafish Model for Assessing Hypoxic‐Induced In Vivo Cardiomyocyte Damage: Time Course for Induction and Cardiac Output Recovery
Authors: Burggren Warren , Abramova Regina , Bautista Naim , Danielson Regina Fritsche , Gupta Avi , Hansson Kenny , Iyer Neha , Jagadeeswaran Pudur , Jennbacken Karin , Patel Vishal , Raman Revathi , Trivedi Hersh , Wang Qing-Dong
Published: April 2020

Larval zebrafish are used to study regenerative properties of cardiac muscle and treatments that may enhance this process. Cardiac injury models in zebrafish larva currently include cryoinjury, laser ablation, pharmacological treatment and cardiac dysfunction mutations. Effective in damaging cardiomyocytes, these models lack the critical element of tissue hypoxia, which induces molecular cascades within cardiac muscle. We have developed a potentially more realistic model for studying cardiac arrest and recovery in larval zebrafish (Danio rerio) by using acute, severe hypoxic exposure to briefly induce cardiac arrest. Exposure to PO2s of 6–9 mmHg induces loss of mobility followed by cardiac arrest within 120 min in 5 days post fertilization (dpf) and within 40 min at 10 dpf. Approximately 90% of 5dpf larvae survived acute, severe hypoxic exposure, but survival fell to 30% by 10 dpf. Heart beat resumed in surviving larvae and returned to air‐saturated water at 4 min in 5 dpf and 6–8 min at 8–10 dpf. Heart rate, stroke volume and cardiac output in control 6–7 dpf larvae before hypoxic exposure were 188±5 bpm, 0.20±0.001nl and 35.5.3±2.2 nl/min (n=35), respectively. In larvae with hypoxia‐induced cardiac arrest, heart rate returned to control values by 24 h of recovery, but stroke volume and cardiac output remained ~50% depressed from control values at 24 h of recovery. Full restoration of cardiac performance was evident at 72h, indicating that regenerative properties of the zebrafish heart extend to critical physiological measures. Molecular markers for apoptosis (Caspase‐3) were elevated >5‐fold immediately after resumption of heart beat, peaking after 4 hours into the recovery period. A cell regeneration marker (PCNA) increased >4‐fold beginning after a lag of 2 hours, and was still elevated ~6 hours after resumption of heart beat, presumably contributing to recovery of heart function. The highly reproducible cardiac effects of acute hypoxia‐induced cardiac arrest in the larval zebrafish presents an alternative, realistic model for studying cardiac tissue hypoxia injury and recovery of function.

Support or Funding Information

AstraZeneca

Time course of changes in heart rate, stroke volume, and cardiac output in 5–7 day larval zebrafish following induction of cardiac arrest by acute hypoxic exposure (PO2 ~ 7–9 mmHg).

Figure 1
Real‐time Monitoring of Staphylococcus aureus Biofilm Formation Under Flow Condition in Microfluidic Chambers
Authors: Al Mouslem Abdulaziz , Feliciano Eber , De La Fuente Leonardo , Panizzi Peter R.
Published: April 2020

Staphylococcus aureus (S. aureus) causes most of life‐threatening infections such as endocarditis, osteomyelitis and sepsis. Since biofilm development is often a key factor in the development of an infection of indwelling medical devices, we sought to study biofilm formation in real‐time using a lab‐made microfluidic device with the goal of assessing the efficacy of therapeutic agents. In this study, we monitored the bacterial growth of S. aureus in side‐by‐side microfluidic channels. The total volume of an individual microfluidic channel was 0.148 mm3. Each independent micro‐channel has three separated ports (two inlets and an outlet for waste) to allow for constant flow of both growth medium and the bacterial cells. Biofilm coverage of S. aureus was characterized under various flow conditions ranging from 0.1 to 1 μL/min. Optimal biofilm formation was evident at 0.5 μL/min, therefore that flow rate was used in all subsequent experiments. To more closely mimic the human plasma environment, the effect of fibrinogen (Fbg) supplementation was tested. We found that Fbg enhanced the process of biofilm formation (P < 0.05) (Figure
1). To determine if we were monitored true biofilm formation rather than simple bacterial grow, we infused calcofluor white (CFW) into the channels and imaged by a real‐time fluorescence microscope. We verified true biofilm formation by visualizing the production of expolysaccharides matrix by S. aureus indicated by positive CFW staining. These results may help to better understand the biofilm formation process and discover new drugs targets for the treatment of staphylococcal infections.

Support or Funding Information

Auburn University, Auburn, AL King Faisal University, Hofuf, Saudi Arabia

Biofilm formation of S. aureus in microfluidic chambers.

A segment of bright‐field image of microfluidic channels after 24 hours of growing S. aureus in the presence (upper) and the absence (lower) of Fbg.

Figure 1
SUMOylation Site Identification Via FRET Analysis
Authors: Way George , Xiong Zhehao , Liao Jiayu
Published: April 2020

This year’s influenza is estimated to affect more than 20 million people in the United States alone. Although influenza has been extensively researched, it still remains a major public health threat every year. Current methods to cull the spread of the influenza virus are vaccination and anti‐influenza virus drugs. Although these methods can be effective, the infuenza’s capacity to mutate and undergo antigenic shift make these methods fall short. SUMOylation is a post‐translational modification that is important for cellular processes as well as protein function and stability. The dysregulation of SUMOylation has been observed in neurodegenerative diseases, many different types of cancers, and viral infections. SUMOylation has been shown to be an important host factor for the influenza A virus but the mechanism still needs to be better understood. Currently, the methodology to identify SUMOylated lysine residues relies on immunodetection after site‐directed mutagenesis. Although this method has yielded great success, it is prone to false positives due to a lack of sensitivity or the breakdown of the isopeptide bond from endogenous SENP. We have developed a highly‐sensitive FRET‐based methodology to identify the exact lysine residue that is targeted by SUMOylation in the NS1 protein of the influenza virus (Figure
1). Using our method, we found lysine 131 was SUMOylated, contrary to previous findings (Figure
2). Our method can be applied to other proteins and ubiquitin‐like modifications to identify important residues and evaluate posttranslational modifications as druggable targets for cancer and antiviral therapies.

Development of in vitro FRET‐based SUMOylation assay of IAV NS1. A) FRET‐based SUMOylation assay of NS1. If a lysine residue is responsible for SUMOylation, the mutation of the lysine residue to alanine will result in a decrease of the FRET emission at 530 nm given a 414 nm excitation. B) The EmFRET RFU is specific to the SUMOylation of NS1, provided the requirements of SUMOylation (SAE1, E2, and ATP).

Figure 1

Determination of SUMOylated lysine residues of NS1. A) Table of NS1 lysine mutants for the FRET assay. B) Systematic mutation of NS1 lysine residues to alanine.

Figure 2
Characterization of CO2 Expiration and its Relationship with Performance Fatigability in Adaptation to Chronic Exercise Perturbation
Authors: Wooten Liana C. , Neville Brian T. , Chin Lisa M.K. , Guccione Andrew A. , Keyser Randall E.
Published: April 2020

Purpose

This study characterized components of carbon dioxide expiration (VCO2) and their relationship with performance fatigability (PF) in adaptation to chronic exercise perturbation.

Methods

20 healthy men (n=9, age=44.7±13.9 ) and women (n=11, age=50.3±11.1) completed peak cardiopulmonary exercise (CPET) and constant work rate tests (CWRT) on separate days before and after a vigorous, 4‐week aerobic exercise training (AET) regimen. Each test was followed by 10‐minutes of passive recovery and an endurance test at 70% of the peak watts attainted during the CPET. PF was indexed by the endurance test durations following both the peak CPET (End1) and CWRT (End2), peak CPET time (pk‐Time), and peak CPET watts (pk‐Watts). Metabolic indices were calculated as total VO2 (tVO2), total VCO2 (tVCO2), metabolic VCO2 (m‐VCO2), and non‐metabolic VCO2 (nm‐VCO2). Data were analyzed for significant (p<0.05) changes and determinant relationships using paired t‐tests, and Pearson coefficients of determination (r2).

Results

Significant reductions (Table
) in PF measures along with significant increases in tVO2, tVCO2, and nm‐VCO2 were observed after AET. A change in m‐VCO2 was not observed. End1 (s) End2 (s) pk‐Time (s) pk‐Watts tVO2 (ml) tVCO2 (ml) m‐VCO2 (ml) nm‐VCO2 (ml) 265±337 p<0.01* 321±392 p<0.01* 63±40 p<0.001* 24±19 p<0.001* 1127±1569 p<0.01* 1512±2225 p<0.01* 904±2255 p=0.089 608±666 p<0.001* nm‐VCO2 was strongly determinant of performance fatigability (pk‐Time r2=0.858, pk‐Watts r2=0.832, end1 r2=0.533, end2 r2=0.682, p<0.001*).

Conclusion

The current study demonstrated significant increases in the buffering component of total carbon dioxide expiration but not in the component associated with energy repletion. The buffering component was directly associated with measures of performance, providing plausibility that the buffering component may be a determinant of performance fatigability.

End1 (s)

End2 (s)

pk‐Time (s)

pk‐Watts

tVO2 (ml)

tVCO2 (ml)

m‐VCO2 (ml)

nm‐VCO2 (ml)

265±337

321±392

63±40

24±19

1127±1569

1512±2225

904±2255

608±666

p<0.01*

p<0.01*

p<0.001*

p<0.001*

p<0.01*

p<0.01*

p=0.089

p<0.001*
The Effect of a Humanized Anti‐Cocaine Monoclonal Antibody on the In Vitro Metabolism of Cocaine
Authors: Turner Mackenzie E. , Crutchfield Christopher A. , Bell-Horwath Tiffany R. , Wetzel Hanna N. , Norman Andrew B.
Published: April 2020

The humanized anti‐cocaine monoclonal antibody (mAb) h2E2 is a lead candidate for treating cocaine‐use disorders and relapse. When cocaine binds to h2E2, it is hypothesized to be unavailable for hydrolysis by endogenous esterases such as liver carboxylesterase I (CES1) and butyrylcholinesterase (BChE). Therefore, h2E2 is expected to slow the clearance of cocaine. However, pharmacokinetic studies in rats and mice demonstrate an apparently unchanged elimination half‐life (t1/2β) of cocaine in the presence of h2E2. To explore this paradoxical finding, we investigated the effect of h2E2 on the in vitro metabolism of cocaine by CES1, which hydrolyzes cocaine to benzoylecgonine (BE), and by BChE, which hydrolyzes cocaine to ecgonine methyl ester (EME). Cocaine hydrochloride (20 μM) was incubated at 37°C with a range of concentrations of either CES1 or BChE in the presence or absence of h2E2 (10 μM). Aliquots were collected at designated time points over 60 minutes. Cocaine and metabolite concentrations in each sample were measured using LC‐MS/MS. In CES1 reactions, there was a time‐dependent formation of BE in the absence of h2E2 (n=5). The mAb dramatically inhibited the production of BE by greater than 90% at all enzyme concentrations. Preliminary data (n=1) also suggest h2E2 prevents the hydrolysis of cocaine to EME by BChE. The dramatic decrease in both the production of BE and EME in the presence of the antibody is consistent with h2E2 reducing the concentration of free cocaine by acting as a chemical antagonist. In addition, it is possible that the substantial h2E2‐induced increase in plasma cocaine concentrations may alter the in vivo metabolism of cocaine in favor of butyrylcholinesterase, thereby increasing the relative proportion of EME. This and the paradoxical elimination of cocaine from plasma in vivo should be resolved through a more extensive investigation of the pharmacokinetics of cocaine in the presence of the antibody.

Support or Funding Information

This work was supported by the National Institutes of Health, National Institute on Drug Abuse grant number U01DA039550 (ABN).

displays benzoylecgonine production (ng/mL) over time (minutes). Samples containing CES1, h2E2, and cocaine (n=5) are shown in black. The non‐h2E2 group (n=5) is shown in blue. Data were normalized with baseline cocaine readings (t=0). The graph displays an initial timecourse of 60 minutes.

Figure 1
Elucidating the Role of the Skin Barrier Protein Filaggrin in the Adaptive Immune Response to Commensals
Authors: Gonzalez Jeanmarie R. , Scharschmidt Tiffany C.
Published: April 2020

Atopic dermatitis (AD), commonly known as eczema, is a chronic inflammatory skin disease affecting millions of Americans. Disruption of skin barrier physiology is central to its pathogenesis. Mutations in the gene encoding filaggrin are present in up to 40% of patients and predispose patients to severe and early‐onset AD as well as systemic atopy (asthma and allergic rhinitis). Filaggrin, expressed in the upper layers of the skin epithelium, is a protein critical to the structural integrity of the epidermis and its key physiological functions of maintaining proper skin moisturization and pH. The epidermal barrier also plays a central role in the skin’s response to bacteria. In AD, disease flares are accompanied by skin microbial dysbiosis, specifically increased abundance of Staphylococcus epidermidis (S. epi), a common skin commensal, and Staphylococcus aureus, a pathobiont. Whereas the role of S. aureus in AD is relatively well‐studied, comparatively little is understood about the impact of impaired skin epithelial barrier function (e.g. flg mutations) on the immune response to commensal bacteria and how this might contribute to skin and systemic inflammation in atopy. I hypothesize that early life deficiency of skin barrier function alters the quality of the commensal‐specific T cell response, which contributes to later pro‐inflammatory responses to these bacteria. Using a novel system allowing me to track CD4+ T cells specific for S. epi I have found that mice lacking filaggrin (flg−/−) differ from wildtype (wt) littermates in their immune response to this commensal. Our lab has previously shown that wt mice colonized early in life develop immune tolerance to S. epi via enrichment of antigen‐specific regulatory T cells (Tregs), an immune cell population that controls inflammation. In contrast, S. epi colonization of flg−/− mice elicits reduced antigen‐specific Tregs and increased numbers of antigen‐specific CD4+ effector T cells (Teffs). Ongoing studies are focused on elucidating the mechanistic basis of this key finding and determining its contribution towards propensity for skin and systemic type‐2 inflammation. In summary, our work suggests that skin barrier physiology significantly impacts the quality of the commensal‐specific CD4+ response early in life, which may have enduring consequences for skin immune homeostasis.

Support or Funding Information

American Physiological Society: Porter Physiology Development Fellowship
Combining Live Cell Imaging with Cellular Impedance to Monitor Apoptotic Cell Death in Real Time
Authors: Lamarche Brandon J. , Yang Grace , Zhang Jiaming , Ye Peifang , Li Nancy , Abassi Yama , Zhang Jing
Published: April 2020

Although apoptosis can be tracked using various biomarker‐based assays, these typically require multiple manual handling steps and only yield endpoint measurements. Requiring just a cell seeding step and a drug addition step, we used the Agilent xCELLigence RTCA eSight to continuously monitor drug‐mediated apoptosis over the course of multiple days. Providing a direct and objective assessment of cell number, cell size, cell‐substrate attachment strength, and cell barrier function, impedance biosensors embedded within the base of eSight microplates quantitatively track early (cell shrinkage) to late (fragmentation) apoptotic events with high analytical sensitivity. Concurrently, eSight captures live cell images in brightfield and three fluorescence channels (red, green, and blue), providing an orthogonal readout of the apoptosis process. By combining the strengths of real‐time impedance monitoring (simplicity, analytical sensitivity, and objectivity) with that of live cell imaging (specificity of the readout), eSight increases the information richness of the apoptosis assay without increasing the workload. Importantly, the drug EC50 values determined using these two approaches are nearly identical, suggesting that eSight can simultaneously provide both the primary and secondary (confirmatory) readouts for apoptosis studies.

Corroborating the impedance response with live cell images. Image panels demonstrate the progression of apoptosis 20 and 40 hours after treating A549‐Blue cells with 5.5 μm MG132. White arrows denote large membrane blebs that contain phosphatidylserine in their outer leaflet.

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Multiplex monitoring of apoptosis in real‐time: combining readouts from cellular impedance and live cell imaging.

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Ibrutinib treatment inhibits breast cancer progression and metastasis by inducing conversion of myeloid‐derived suppressor cells to dendritic cells
Authors: Varikuti Sanjay , Singh Bhawana , Volpedo Greta , Ahirwar Dinesh , Jha Bijay , Saljoughian Noushin , Viana Agostinho , Hamza Omar , Halsey Gregory , Holcomb Erin , Maryala Ritvik , Oghumu Steve , Ganju Ramesh K. , Satoskar Abhay R.
Published: April 2020

Ibrutinib is a known irreversible inhibitor of Bruton tyrosine kinase (BTK) and interleukin‐2‐inducible kinase (ITK). Ibrutinib has multiple immunomodulatory effects and is currently approved for treatment of chronic lymphocytic leukemia (CLL) and other cancers. Although previous studies have shown that ibrutinib inhibits growth of breast cancer cells, its impact on treatment of breast cancer and its metastasis is not determined. Myeloid‐derived Suppressor Cells (MDSCs) are immature myeloid cells present in tumors that can subdue the development of important tumoricidal cell populations, and their presence has been linked to poor prognosis in human and murine models. Here, using an orthotopic mouse breast cancer model, we show that ibrutinib effectively inhibits progression and metastasis of breast cancer. Mice treated with Ibrutinib display significantly lower tumor burdens and metastasis compared to controls. Furthermore, spleens and tumors from Ibrutinib‐treated mice showed higher numbers of mature DCs and lower numbers of myeloid‐derived suppressor Cells (MDSCs) which promote disease progression and are linked to poor prognosis in human and murine models. We also confirmed that ex vivo treatment of MDSCs with ibrutinib switched their phenotype to mature DCs and significantly enhanced MHCII expression. Further, ibrutinib treatment promoted T cell proliferation and effector functions leading to induction of anti‐tumor TH1 and CTL immune responses. Taken together, our results indicate that Ibrutinib inhibits tumor development and metastasis in breast cancer by promoting development of mature DCs from MDSCs and hence could be a novel therapeutic agent for treatment of breast cancer

Support or Funding Information

Pharmacyclics LLC, an AbbVie Company
CaMKIIδ differentially alters the passive mechanics of myocardium
Authors: Valdez William Ricardo , McCulloch Andrew
Published: April 2020

Abnormal diastolic function as seen in failing hearts with preserved ejection fraction (HFpEF) has been linked to alterations in the passive material property of myocardium. Specifically, the non‐crossbridge forming protein named titin contributes a stiffness that varies with fluctuating calcium concentration and has been thought to dominate the passive stiffness of myocardium throughout normal physiological range.

Titin is expressed in two isoforms in the heart: the N2B, which has a single spring‐like element, and a more compliant isoform, N2BA, which contains two spring‐like domains (the N2B and N2A). The N2B element exists in all isoforms and is cardiac specific and consists of a large 575 residue unique sequence (N2B‐Us), three flanking immunoglobulin domains, and two other smaller unique sequences. Titin‐based stiffness of myocardium has been shown to be modulated by altered ratio expression of titin isoforms as well as post‐translational modification with unbalanced phosphorylation of cardiac titin characterizing HFpEF. Several proteins can alter titin stiffness including protein kinase (PK) G, extracellular signal‐regulated kinase 2 (ERK‐2), PKC, PKA and calcium calmodulin‐dependent protein kinase II (CaMKII) with varied effects depending on the target site location on titin.

CaMKII is a dodecameric enzyme consisting of subunits encoded by four different genes known as CaMKII α,β,γ, and δ. CaMKIIδ is the predominant isoform in the heart and is alternatively spliced in the heart to generate δb and δc differing only by an 11‐amino acid nuclear localization signal. CaMKIIδ heteromultimerization is permissive, however, in that the enzyme may contain subunits from multiple CaMK genes and splice variants of those genes. Thus, it is hypothesized that the general ratio of CaMKIIδ subtypes determine its exact localization within the cardiomyocyte.

CaMKIIδ knock‐out has recently been shown to ameliorate heart failure development in response to pressure overload, Gαq expression, and isoproterenol infusion while CaMKIIδ subtypes show a differential regulation of calcium handling in‐vitro and infarct formation following ex‐vivo myocardial ischemia/reperfusion. The idea that the specific CaMKIIδ subtypes differentially regulate cardiac mechanics is interesting yet remains unresolved.

In our work, 5–6 weeks old mice models overexpressing CaMKIIδb and CaMKIIδc were bred against a CaMKIIδ knock‐out background, called KOB and KOC respectively. Results indicate that KOB mice show increased passive stiffness (12 mN/mm^2 +− 3 mN/mm^2) compared to KOC passive stiffness (6 mN/mm^2 +− 1 mN/mm^2) at 5–6 weeks prior to heart failure phenotype. Furthermore, as the mice progress into heart failure, the heart passive mechanics of KOB and KOC are consistent, thus, highlighting CaMKII as a precursor to heart failure development.

Support or Funding Information

President's Disseration Year Fellowship

Figure 1