Biomedical Research Laboratories Research Articles

Nurturing Undergraduate Researchers in Biomedical Sciences

Undergraduate researchers are the next-generation scientists. Here, we call for more attention from our community to the proper training of undergraduates in biomedical research laboratories. By dissecting common pitfalls, we suggest how to better mentor undergraduates and prepare them for flourishing careers.

Exogenous IL‐4 Promotes Myocardial Infarction Repair by Turning off Pro‐Inflammation in Neutrophils while Stimulating Anti‐Inflammation in Macrophages to Induce Neutrophil Phagocytosis

Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While IL‐4 is an in vitro stimulus for anti‐inflammation, the MI myocardium does not express IL‐4. We hypothesized that continuous exogenous IL‐4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. C57BL/6J male mice (3–6 months of age) were subcutaneously infused with either saline (n=17) or IL‐4 (20 ng/g/day; n=17) and evaluated at MI day 3. Macrophages and neutrophils were isolated ex vivo from the infarct region and examined for pro‐inflammatory genes (Ccl3, Ccl5, Il1b, Il12a, and Tnfa) and anti‐inflammatory genes (Arg1, Ym1, Mrc1, Il10, and Tgfb, Il6). Exogenous IL‐4 decreased pro‐inflammatory Ccl3, IL‐12a, Tnfa, and Tgfb1 in neutrophils while increasing anti‐inflammatory Arg1 and Ym1 in macrophages (all p<0.05); none of the other markers were different. Tissue clearance by IL‐4 treated neutrophils was not different, while phagocytosis of neutrophils, but not myocytes, doubled in IL‐4 treated macrophages (p<0.05). Because macrophage cell physiology was influenced by IL‐4, whole transcriptome analysis of macrophages isolated from the infarct regions of IL‐4 (n=10) or saline (n=11) treated mice was performed by RNA‐sequencing. Of the 24,341 genes sequenced, 2,042 genes were differentially expressed with IL‐4 stimulation (all p<0.05). Pdgfc gene expression was ranked first by p value, increasing from 1.8±0.3 in saline treated macrophages to 5.4±0.2 FPKM units in macrophages stimulated with IL‐4 (p=1×10−9). Importantly, changes in macrophage physiology and transcriptome occurred before differences in cardiac physiology evaluated by echocardiography, which allowed the isolation of effects in cell phenotype not induced by global myocardial effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF‐CC protein (10 μg/ml) or PDGF‐CC blocking antibody (10 μg/ml) revealed no effect on Arg1 or Ym1 expression, indicating the in vivo effect of IL‐4 to stimulate macrophage gene expression was independent of PDGF‐CC. Altogether, our results indicate that exogenous IL‐4 promotes inflammation resolution by turning off pro‐inflammation in neutrophils while stimulating anti‐inflammation in macrophages to induce neutrophil phagocytosis.Support or Funding InformationNational Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505

LOX‐1 is the prime target for mesenchymal stem cell exosome mediated cardioprotective effects

ObjectiveMyocardial infarction (MI) is the leading cause of mortality worldwide. Attempts have been made to exploit stem cells by implanting them in the damaged cardiac tissue in hopes of regenerating/repairing cardiac muscle, limiting infarct size and improving cardiac function. However, the results of studies with the use of stem cells have been inconsistent. Recent studies show that use of mesenchymal stem cell (MSC) exosomes may limit cardiac injury, and even reverse cardiac damage. We studied the effect of MSC exosome administration on the extent of cardiac injury and function in an ischemia model.MethodsIschemia was induced in 8–10 week old healthy male wild‐type mice by ligating the left coronary artery (LCA). Mice were pretreated with MSC exosomes or saline as control. At 7 days after LCA ligation, the animals were euthanized and hearts were isolated. Hearts were sectioned into infarct and remote non‐infarct areas and used for further studies.ResultsWe observed that the infarct size was smaller in the exosome‐treated mice than in the saline‐treated mice (9±1 % vs. 12±1 %, respectively, p<0.01) and cardiac contractile function (fractional shortening) was preserved (42±4 % vs. 28±2 %, p<0.01) at 1 week after LCA ligation. Left ventricular wall thickness was preserved in exosome treated mice hearts (0.65±0.06 mm vs 0.33±0.02 mm, p<0.02). White blood cell accumulation in and around the infarct area was also reduced in exosome‐treated mice hearts (346±15 cells/hpf vs. 750±85 cells/hpf in saline‐treated hearts, p<0.01). Further, we observed that LOX‐1 and cleaved caspase‐3 were overexpressed in infarct areas of saline‐treated mice hearts. MSC exosome treatment reduced expression of LOX‐1 and cleaved caspase‐3 in infarct areas of the LCA ligated mice hearts (p<0.01). Expression of NLRP3 and TLR4 was also reduced in MSC‐exosome treated hearts. Proteomic analysis of the infarct areas of saline‐ and exosome‐treated LCA ligated hearts revealed differentially expressed proteins. The infarct areas of exosome‐treated (vs. saline‐treated hearts) showed upregulation of 75 specific proteins and downregulation of 37 proteins. Comparison of biological processes in infarct areas of exosome‐treated (vs saline‐treated LCA ligated heart) showed enrichment of processes governing cell communication, anaerobic respiration, apoptotic cell death while showing depletion of processes governing cell death, cardiac muscle cell apoptotic processes, angiotensin activated signaling, and activation of certain kinase pathways. The results of proteomic studies were concordant with the limitation of infarct size and preservation of functional data in the exosome‐treated hearts.ConclusionsMSC exosomes exert their cardioprotective effects by downregulating expression of LOX‐1 and caspase‐3 in LCA‐ligated mice hearts. Presumably, LOX‐1 inhibition led to inhibition of inflammatory pathways leading to mitigation of ischemic tissue damage.Support or Funding InformationVA Merit Review (Grant‐in‐Aid from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, Washington, DC)MSC exosomes exhibit cardioprotective function by inhibiting LOX‐1Figure 1

Restoration of the Blood‐Spinal Cord Barrier after Spinal Cord Injury through 5‐HT1F Receptor Mediated Mitochondrial Biogenesis

Vascular and mitochondrial dysfunction are well‐established consequences of spinal cord injury (SCI). Evidence suggests mitigating these dysfunctions may be an effective approach in treating SCI. We propose to restore vascular and mitochondrial dysfunction following SCI through pharmacological induction of mitochondrial biogenesis (MB). We previously reported that 5‐hydroxytryptamine receptor 1F (5‐HT1F) agonism induces MB in the spinal cord and promotes locomotor recovery with drug initiation beginning at a clinically relevant time point of 8h post‐SCI. The goal of this study was to elucidate if MB induction with the newer selective 5‐HT1F receptor agonist lasmiditan can enhance restoration of the blood‐spinal cord barrier (BSCB) after SCI. C57BL/6J mice were subjected to moderate SCI using a force‐controlled impactor‐induced contusion model followed by daily administration of lasmiditan (0.1 mg/kg, i.p.) beginning 1h after injury. Integrity of the BSCB was assessed using Evans Blue dye extravasation. SCI increased dye extravasation at 1, 3, and 7d, and lasmiditan‐treated mice displayed reduced dye accumulation at 7d post‐SCI, suggesting accelerated BSCB recovery. Levels of tight junction proteins occludin and zonula occludens‐1 were decreased in vehicle‐treated mice and returned to sham levels with lasmiditan treatment by d7. These findings led us to investigate if lasmiditan induces MB in endothelial cells. Primary cultures of cerebral microvascular endothelial cells from C57BL6 mice were treated with lasmiditan. A Seahorse Biosciences flux analyzer was used to quantify FCCP‐uncoupled oxygen consumption rate (OCR), a marker of MB. Lasmiditan increased FCCP‐uncoupled OCR, nuclear‐ and mitochondrial‐encoded proteins (PGC‐1a, COX1, TFAM), and mitochondrial number, confirming MB. Lastly, treatment with lasmiditan enhanced endothelial branching morphogenesis and migration, indicating a role of the 5‐HT1F receptor activation in angiogenic pathways. Collectively, these data reveal that 5‐HT1F receptor agonist‐induced MB occurs in endothelial cells and may accelerate restoration of the BSCB after SCI.Support or Funding InformationThis study was supported by the National Institutes of Health National Institute of General Medical Sciences: GM084147 (R.G.S), and the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs: BX: 000851 (R.G.S.).

Myocardial Oxygen Saturation Measured by Photoacoustic EKV Imaging

Cardiac remodeling following myocardial infarction (MI) leads to structural and vascular changes in the myocardium. To better understand the mechanisms involved, we used photoacoustic ECG‐gated Kilohertz Visualization imaging (PA EKV) to measure oxygen saturation in the myocardium. Photoacoustic imaging is a noninvasive imaging technique that uses near‐infrared laser light to optically excite molecules in vivo. Oxygenated hemoglobin absorbs laser light at a different wavelength (850nm) from non‐oxygenated hemoglobin (750nm) which allows for the detection of the relative oxygen saturation (sO2) of a tissue and the amount of hemoglobin present (HbT). In this study, photoacoustic imaging was combined with EKV imaging for continuous measurement of sO2 and HbT throughout the contraction cycle.Imaging was done on C57Bl/6J mice (F, 4–5 months of age) to measure baseline sO2 levels in the anterior and posterior myocardium. An MI was induced by ligation of the left anterior descending coronary artery. Mice were imaged at days 1, 7, and 14 after MI to measure changes in myocardial sO2. All data presented here are from diastole of the long axis orientation and similar results were obtained from short axis measurements. Oxygen saturation on the anterior myocardial wall decreased from 74.7% pre‐MI to 19.9% 1 day after MI (p<0.0001). Oxygen saturation showed recovery at 7 (54.9%, p=0.023) and 14 (49.9%, p=0.007) days post infarct, yet remained significantly reduced from pre‐MI levels. Oxygen saturation was lower on the posterior myocardium (57.2%) compared to the anterior myocardium but did not change following MI.Our results demonstrate oxygen saturation begins to recover 7 days after MI as the heart adapts to ligation and neoangiogenesis is stimulated. This study validates the use of PA EKV for the measurement of oxygen saturation in the infarcted myocardium.Support or Funding InformationWe acknowledge funding from National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505. The UNMC Ultrasound Core is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institution of Health under grant P30 GM127200.

The Secretome of Female CD8+ T‐cells Increases Monocyte Phagocytosis

Cardiovascular diseases including myocardial infarction (MI) are the leading cause of death in the US. Elevated CD8+ T‐cell counts (>1065 cells/mm3) have been linked to increased mortality rate (2‐fold) in post‐MI patients. In a previous study conducted by our lab, we concluded that CD8+ T‐cells regulate macrophage recruitment and impair collagen deposition during the early phase post‐MI, leading to adverse cardiac remodeling and decreased survival. Interestingly, survival was improved in male but not female mice lacking functional CD8+ T‐cells (p<0.05) compared to WT animals from the respective sex. Based on these data, we hypothesized in the current study that CD8+ T‐cells have detrimental effects in males during the post‐MI remodeling process through cellular communications with macrophages. In order to address this hypothesis, we employed C57Bl/6J mice (WT; n=3/sex), and mice with non‐functional CD8+ T‐cells (CD8atm1mak; n=3/sex). The animals underwent permanent occlusion of the left anterior descending coronary artery to induce the MI, and macrophage activation and tissue necrosis was evaluated by immunoblotting and flow cytometry at post‐MI day 3. Male CD8atm1mak mice have more necrotic myocytes within the day 3 infarct compared to CD8atm1mak female mice and WT animals of either sex. Protein assessment of macrophage phagocytosis maker, Mertk, revealed that WT females had lower Mertk shedding compared to both WT males and CD8atm1mak female mice, indicating that Mertk‐mediated phagocytosis is higher in WT females than the other groups. To determine if the secretion profile of CD8+ T‐cells was regulating macrophage physiology, we isolated CD8+ T‐cells from the spleens of male and female WT mice and cultured them in RPMI supplemented with 1% antibiotics and 10% FBS (positive control) or 0.1% FBS (unstimulated, negative control). To test influence of effector and memory CD8 T‐cells, cells were stimulated in vitro for 24 hours towards a memory phenotype (1.5 ng/mL IL4) or effector phenotype (1 ng/mL IL12), and secretome was collected from all groups. The collected secretome was used to stimulate bone marrow monocytes isolated from female mice (2‐hour stimulations). Bone marrow monocytes stimulated with the secretome from female effector CD8+ T‐cells had increased phagocytosis mirroring what was observed in vivo. In conclusion, CD8 T‐cells in females but not males correlated with decreased shedding of Mertk and increased macrophage‐mediated removal of necrotic debris. Our data suggests that this may be due to sex differences in the effector phenotype post‐MI.Support or Funding InformationThis work was supported by the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award IK2BX003922 and the National Institutes of Health award number U54DA016511. This work was also financially supported, in part, by the 2019 S&R Foundation Ryuji Ueno Award that was bestowed to Dr. DeLeon‐Pennell by the American Physiological Society. This work was also supported by the Ralph H. Johnson VA Medical Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Veterans Administration, or the American Physiological Society.

Cadherin‐3 and Catenin‐α2 are novel MMP‐12 substrates

Adverse cardiac remodeling after myocardial infarction (MI) is a major cause of heart failure. Degradation of the existing extracellular matrix (ECM) and subsequent formation of a new ECM scar are prominent components of cardiac remodeling. Matrix metalloproteinases (MMPs) increase after MI to dictate the degradation component. MMP‐12 inhibition after MI worsens dilation of the left ventricle by increasing its wall thinning, suggesting a possible protective role for MMP‐12 in MI remodeling. We hypothesized that identifying MMP‐12 substrates could provide mechanistic insight into MI roles. We used the Mouse Heart Attack Research Tool 1.0 (mHART 1.0) database and performed regression analysis to identify ECM genes that correlated with MMP‐12 expression. Of the 83 ECM genes compared to MMP‐12, 41 showed significant regressions (all p<0.05). This indicates a strong relationship between ECM breakdown after MI and MMP‐12. Synaptotagmin‐1, Contactin‐1, MMP‐10, MMP‐13, and Catenin‐α2 were the top 5 genes with significant positive correlation (Pearson coefficient r>0.99 and p<0.0001 for all). By Enrichr analysis for transcription factor pathway exploration, Yap1 signaling had a top combined score of 9.37 and p‐value 0.0033, indicating the upregulation of HIPPO signaling as the most enriched pathway. Catenin‐α2(Ctnna2), Cadherin‐1 (Cdh1), and Cadherin‐3 (Cdh3) were the three genes linked to Yap1‐ Hippo signaling. By substrate cleavage assay using 0.1 μg MMP‐12 enzyme to 0.5 μg substrate, Cdh3 and Ctnna2 but not Cdh1 were identified as novel MMP‐12 substrates. Cdh3 and Ctnna2 showed robust cleavage at 15 min, and Ctnna2 was completely processed by 3h. Our combined results reveal that MMP‐12 actions in MI remodeling could be attributed in part to Cdh3 and Ctnna2 proteolytic processing.Support or Funding InformationWe acknowledge funding from the National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, and the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505.

Acetaminophen‐induced Liver Injury Induces DNA Damage and Activation of Ataxia Telangiectasia Mutated in Mice

Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. At therapeutic doses it is safe but at higher dosages APAP causes acute liver failure (ALF). Drug‐induced liver toxicity is the most common cause of liver damage, accounting for 50% of the cases of liver failure. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then aids in driving further progression of APAP‐induced liver injury.MethodsC57Bl/6 mice were injected with APAP (300mg/kg) do induce liver injury. Liver tissue was collected after 1, 2, 4, 6, 12, and 24 hours. Histology was performed on paraffin‐embedded sections using antibodies specific for phospho‐ATM (ser 1981) and γH2AX (ser 139). Replication protein A (RPA), Ataxia Telangiectasia mutated (ATM) and 53BP were assessed by qPCR. Phospho‐ATM and RPA were assessed by immunoblotting.ResultsAPAP injected mice showed a marked increase in the accumulation of DNA damage when compared to saline injected mice in the liver. Markers for DNA double strand breaks (DSB), γH2AX and 53BP, were shown to be elevated 2 hours after APAP injection followed by a gradual decrease. RPA, a single stranded DNA‐binding protein, was also shown to be elevated 4 hours after APAP injection indicating an accumulation of single strand breaks. ATM, a DDR maker, and the phosphorylated form of ATM were shown to also be elevated after APAP injection after onset of DSB, with peak expression at 4 hours.ConclusionDuring APAP‐induced liver injury, there is an elevated level of DNA damage found within the liver. This elevated amount of DNA damage results in the activation of ATM and the DDR. ATM‐mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM‐mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP‐induced liver damage.Support or Funding InformationThis study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This work was completed with support from the Veterans Health Administration and with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

CT derived radiomic score for predicting the added benefit of adjuvant chemotherapy following surgery in stage I, II resectable non-small cell lung cancer: a retrospective multicohort study for outcome prediction

Use of adjuvant chemotherapy in patients with early-stage lung cancer is controversial because no definite biomarker exists to identify patients who would receive added benefit from it. We aimed to develop and validate a quantitative radiomic risk score (QuRiS) and associated nomogram (QuRNom) for early-stage non-small cell lung cancer (NSCLC) that is prognostic of disease-free survival and predictive of the added benefit of adjuvant chemotherapy following surgery. We did a retrospective multicohort study of individuals with early-stage NSCLC (stage I and II) who either received surgery alone or surgery plus adjuvant chemotherapy. We selected patients for whom we had available pre-treatment diagnostic CT scans and corresponding survival information. We used radiomic texture features derived from within and outside the primary lung nodule on chest CT scans of patients from the Cleveland Clinic Foundation (Cleveland, OH, USA; cohort D1) to develop QuRiS. A least absolute shrinkage and selection operator-Cox regularisation model was used for data dimension reduction, feature selection, and QuRiS construction. QuRiS was independently validated on a cohort of patients from the University of Pennsylvania (Philadephia, PA, USA; cohort D2) and a cohort of patients whose CT scans were derived from The Cancer Imaging Archive (cohort D3). QuRNom was constructed by integrating QuRiS with tumour and node descriptors (according to the tumour, node, metastasis staging system) and lymphovascular invasion. The primary endpoint of the study was the assessment of the performance of QuRiS and QuRNom in predicting disease-free survival. The added benefit of adjuvant chemotherapy estimated using QuRiS and QuRNom was validated by comparing patients who received adjuvant chemotherapy versus patients who underwent surgery alone in cohorts D1-D3. We included: 329 patients in cohort D1 (73 [22%] had surgery plus adjuvant chemotherapy and 256 (78%) had surgery alone); 114 patients in cohort D2 (33 [29%] had surgery plus adjuvant chemotherapy and 81 (71%) had surgery alone); and 82 patients in cohort D3 (24 [29%] had surgery plus adjuvant chemotherapy and 58 (71%) had surgery alone). QuRiS comprised three intratumoral and 10 peritumoral CT-radiomic features and was found to be significantly associated with disease-free survival (ie, prognostic validation of QuRiS; hazard ratio for predicted high-risk vs predicted low-risk groups 1·56, 95% CI 1·08-2·23, p=0·016 for cohort D1; 2·66, 1·24-5·68, p=0·011 for cohort D2; and 2·67, 1·39-5·11, p=0·0029 for cohort D3). To validate the predictive performance of QuRiS, patients were partitioned into three risk groups (high, intermediate, and low risk) on the basis of their corresponding QuRiS. Patients in the high-risk group were observed to have significantly longer survival with adjuvant chemotherapy than patients who underwent surgery alone (0·27, 0·08-0·95, p=0·042, for cohort D1; 0·08, 0·01-0·42, p=0·0029, for cohorts D2 and D3 combined). As concerns QuRNom, the nomogram-estimated survival benefit was predictive of the actual efficacy of adjuvant chemotherapy (0·25, 0·12-0·55, p<0·0001, for cohort D1; 0·13, <0·01-0·99, p=0·0019 for cohort D3). QuRiS and QuRNom were validated as being prognostic of disease-free survival and predictive of the added benefit of adjuvant chemotherapy, especially in clinically defined low-risk groups. Since QuRiS is based on routine chest CT imaging, with additional multisite independent validation it could potentially be employed for decision management in non-invasive treatment of resectable lung cancer. National Cancer Institute of the US National Institutes of Health, National Center for Research Resources, US Department of Veterans Affairs Biomedical Laboratory Research and Development Service, Department of Defence, National Institute of Diabetes and Digestive and Kidney Diseases, Wallace H Coulter Foundation, Case Western Reserve University, and Dana Foundation.

Visualization and Quantification of 3D Tumor Cell Migration under Extracellular Stimulation.

To investigate tumor cell migration capability, the scratch/wound healing assay and the Transwell assay are the most commonly used assays in the current biomedical research laboratory. However, both assays have their limitations and may mislead the interpretation of the results. In the current study, visualization and quantification of tumor cell migration process was realized in a three-dimensional (3D) environment. The tumor cells horizontally migrated along a Matrigel-filled microchannel under extracellular stimulation. The cell migration process was visualized under a microscope, and the migration speed could be calculated based on the traveling distance of the cells and the time required. Here, three demonstrations were conducted, respectively, including cells attracted by nutrient gradient, stimulated by cytokine, and coculturing with fibroblasts. The results revealed that the cell migration capability could be visually and quantitatively correlated to the extracellular stimulation. The current protocol is compatible to the existing laboratory setup and provides a persuasive result for the study of the 3D cell migration process. Understanding of the molecular and intercellular mechanism of cancer metastasis can potentially develop effective therapeutic strategy.

Cardiac Tissue Engineering as Use Case to Connect Biomedical Research Laboratories to an Emerging Global Data Infrastructure.

Methods for cardiac tissue engineering and application in experiments are core technologies developed at the Institute of Pharmacology and Toxicology in Göttingen. As is the case in many academic research laboratories data capture and documentation may be improved to latest methods of digital research. A comprehensive information system infrastructure is the foundation of further advances toward automation of lab processes. A data management system concept is proposed and prototypically deployed that enables traceability of assets within the lab and reproducibility of published assays and results. The prototype integrates existing electronic lab notebook, experiment result database, and a newly introduced research data management system by means of a custom developed portal and integration component. The architecture concept and developed integration tools explore connection of routine experimental work in a biomedical research lab to a universal infrastructure of data.

Protein binding kinetics quantification via coupled plasmonic-photonic resonance nanosensors in generic microplate reader

Almost no analytical assays, either colorimetric or fluorescence assays, for generic microplate readers is capable of dynamic measurements of protein-protein binding or the quantification of kinetic association and dissociation constants of protein interactions. On the other hand, protein binding kinetics quantification can be uniquely done on special expensive surface plasmon resonance (SPR) sensing equipment. Here we report the integration of coupled plasmonic-photonic resonance nanosensors in standard 96-well plate format and by using which, for the very first time, the demonstration of label-free dynamic SPR-like protein binding measurement and kinetics quantification in a generic microplate reader. Our low-cost label-free nanosensor plate enables very sensitive detection of immobilized protein interactions based on the transmission optical density (OD) value changes at specific wavelengths measured in a generic microplate reader. The relative end-point OD value changes show a good linear response with protein concentrations (from 0.05 to 50 μg/ml). And the protein quantification in serum results are consistent with the concurrent hospital lab tests. Most importantly, the kinetic association and dissociation constants of protein interactions in our sensor plate wells are determined by time-lapse dynamic OD value measurement in the generic microplate reader. Enabled by our unique nanosensor plate, SPR-like measurement of protein binding kinetics is now available using generic microplate reader ubiquitous in many chemistry and biomedical research labs.

Synthetically Scalable Poly(ampholyte) Which Dramatically Enhances Cellular Cryopreservation.

The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether-alt-maleic anhydride). This poly(ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies.

Novel Inflammatory Signaling via Interleukin‐13 Decreases Potassium Channel Expression and Increases Vascular Smooth Muscle Excitability

The transcriptional repressor element‐1 (RE1) silencing transcription factor (REST), also known as neuron‐restrictive silencer factor (NRSF) is a gene silencing transcription factor, which has been characterized as a master regulator of the neuronal phenotype. REST plays a key role in the development of the nervous system and is a critical player in the etiology of neurodegenerative diseases such as Huntington's Disease, though little is known about the role of REST in non‐neuronal cells. We are exploring a potential role of REST as a mediator between inflammation and changes in smooth muscle phenotype. Kv7.5 voltage‐dependent potassium channels, encoded by the KCNQ5 gene, are expressed in smooth muscle cells where they play an important role in determining resting membrane potential. Interleukin‐13 (IL‐13) is an inflammatory cytokine, which is implicated as a primary mediator of the phenotypic changes induced by allergic inflammation in many tissues. In the present study, we hypothesized that IL‐13 can increase nuclear REST and decrease KCNQ5 mRNA expression and Kv7.5 protein in vascular smooth muscle cells, providing a potential link between inflammation, REST and potassium channel‐mediated vascular hyper‐responsiveness. In support of this hypothesis, using A7r5 rat aortic smooth muscle cells, we found that 1) IL‐13 treatment significantly increased nuclear REST, and 2) significantly suppressed the expression of KCNQ5 and Kv7.5 currents, resulting in increased sensitivity of A7r5 cells to arginine vasopressin (AVP). 3) REST overexpression also significantly decreased Kv7.5 currents, and 4) significantly increased the sensitivity of A7r5 cells to AVP. We conclude that inflammation that induces IL‐13 may increase nuclear REST levels and thereby decrease KCNQ5 expression and Kv7.5 currents, ultimately increasing vascular smooth muscle cell excitability and contributing to vascular hyper‐responsiveness.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research &amp; Development Service of the VA Office of Research and Development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD8 T‐cells regulate macrophage recruitment leading to exacerbated cardiac remodeling

Elevated CD8 T‐cell counts (&gt;1065 cells/mm3) have been linked with increased mortality rate (2‐fold) in patients after myocardial infarction (MI). However, the exact mechanisms by which CD8 T‐cells impair cardiac wound healing post‐MI are not totally understood. We hypothesized that CD8 T‐cells regulate the inflammatory response leading to decreased survival and cardiac function post‐MI. Permanent occlusion of the left anterior descending artery was performed on C57BL/6J (WT; 4.5±0.1 months of age; n=6 males and females/day) and CD8atm1Mak mice (CD8KO; 4.1±0.2 months of age; n=6 males and females/day). CD8KO mice had improved day 7 survival (73%) compared to WT mice (50%; p&lt;0.05). Interestingly, 100% of the CD8KO mice who died did so due to cardiac rupture of the left ventricle (LV; rupture/total deaths; 3/3) whereas 50% of WT animals (5/10; p&lt;0.05) died of cardiac rupture. Strain analysis showed that as early as post‐MI day 3, global longitudinal strain was improved in CD8KO animals (−5.3±0.1%) compared to WT (−2.8±0.1%; p&lt;0.05) suggesting WT mice have increased myocardial wall stress. Echocardiography measured at day 7 post‐MI showed improved cardiac physiology, as indicated by fractional shortening in CD8KO mice (11±2%) compared to WT mice (5±1%; p&lt;0.05) implicating CD8 T‐cells as a negative regulator of wound healing during the later phase post‐MI. Proteomic plasma analysis of 111 proteins indicated CD8KO had elevated immune response at both post‐MI day 3 and day 7. This was supported by increased macrophage numbers at day 3 post‐MI in CD8KO animals compared to WT mice (p&lt;0.05) as shown by Mac3 staining of the infarct. Collagen within the infarct was also elevated at day 3 in CD8KO compared to WT (p&lt;0.05) as shown by picosirius red staining implicating CD8 T‐cells in regulation of myocardial fibrosis. In conclusion, CD8 T‐cells regulate macrophage recruitment and impair collagen deposition during the early phase post‐MI leading to adverse cardiac remodeling and decreased survival.Support or Funding InformationThis work was supported by the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award IK2BX003922 to KYD‐P. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Veterans Administration.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thrombospondin‐1 worsens acetaminophen‐induced acute liver failure in mice through its activation of TGFβ1

IntroductionAcute liver failure results from a significant loss of liver function and has few effective treatments and poor clinical outcomes. Previously we have demonstrated that transforming growth factor beta 1 (TGFβ1) is upregulated during azoxymethane‐induced acute liver failure in mice and this signaling pathway promotes hepatic encephalopathy progression. Recently TGFβ1 has been shown to promote acetaminophen‐induced liver injury by promoting senescence of hepatocytes. TGFβ1 requires interactions with other proteins, such as thrombospondin‐1 (TSP‐1), to become activated and to be capable of binding its receptors. Therefore, we hypothesize that TSP‐1 activates TGFβ1 during acetaminophen‐induced acute liver failure worsening outcomes.MethodsMale C57Bl/6 mice or TSP‐1 knockout mice were injected with acetaminophen (APAP; 300 and 600 mg/kg) to generate acute liver injury. APAP‐treated mice had serum and liver collected at up to 24 hours post injection. Liver pathology was assessed by H&amp;E staining, TUNEL staining, malondialdehyde assay, glutathione assay, nitrotyrosine expression and transaminase assessments. TGFβ1, TSP‐1, IL‐1β, IL‐6, TNF‐α, SOD1, iNOS and phosphorylated/total JNK expression were assessed by immunoblotting, immunohistochemistry, immunofluorescence, ELISA and/or real‐time PCR assays.ResultsMice injected with APAP had elevated hepatic TGFβ1 and TSP‐1 levels, which co‐localized primarily to hepatocytes. APAP‐treated mice with genetic knockout of TSP‐1 had reduced hepatic injury and hepatocyte necrosis as evident by improved serum transaminases, restored glutathione levels, reduced phosphorylated JNK signaling and less TUNEL staining compared to wildtype APAP‐treated mice. Expression of oxidative and nitrosative stress markers, as well as proinflammatory cytokines, were reduced in TSP‐1 knockout mice compared to wildtype mice administered APAP.ConclusionsStrategies employed to reduce TSP‐1 signaling in APAP‐treated mice reduced liver damage and pathology. Therefore, targeting TSP‐1 signaling may be a novel therapeutic target for the management of APAP‐induced acute liver failure.Support or Funding InformationThis study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This study was also funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. McMillin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of miR‐92a Results in Glucose Intolerance via Impaired Pancreatic Beta Cell Function

MicroRNAs (miRs) are small non‐coding RNA that regulate numerous physiological and pathological processes by repressing protein translation. MiR‐92a, a member of the miR‐17–92 cluster, is altered during development and in a host of disease states, with pancreatic miR‐92a levels reduced in animal models of diabetes. Here, we tested the hypothesis that miR‐92a is a critical regulator of metabolism that modulates pancreatic beta cell function and glucose homeostasis. Glucose tolerance and insulin sensitivity were assessed by glucose (2 g/kg, ip) and insulin (1 U/kg, ip) tolerance tests, respectively in wildtype (WT: C57Bl/6) and whole body miR‐92a (miR‐92a−/−) knockout mice. Deletion of miR‐92a impaired glucose tolerance (p&lt;0.01), but not insulin sensitivity (p&gt;0.05), compared to WT mice. To explore the potential causes of this glucose intolerance, we assessed both fasted insulin and glucose‐stimulated (1 g/kg, ip, 5 min) insulin secretion (GSIS), and found both to be lower in miR‐92a−/− mice compared to WT mice (both p&lt;0.01). To examine if miR‐92a modulates insulin secretory capacity, we administered 10% L‐arginine (1 g/kg, ip) and a β‐adrenergic agonist (CL 316,243: 1 mg/kg, ip) following a 4–6 hr fast and plasma insulin was assessed at time 0, 5 and 15 min. Fasted plasma insulin was ~70% lower in miR‐92a−/− mice compared to WT mice (p&lt;0.01). In response to L‐arginine, plasma insulin was lower in miR‐92a−/− mice compared to WT mice (1.80 ± 0.52 vs 4.57 ± 0.62, 5 min, p&lt;0.01; 1.23 ± 0.42 vs 6.30 ± 2.66, 15 min, p&lt;0.05). Likewise, plasma insulin was ~3 and ~8‐fold lower at 5‐ and 15‐min after β‐adrenergic stimulation in miR‐92a−/− mice compared to WT mice (both p&lt;0.01). Taken together, these results indicate a reduced insulin secretory capacity in response to glucose, amino acid and lipid stimulation in miR‐92a−/− mice. Subsequent histological analysis demonstrated that both average islet size and extrapolated total islet mass were ~50% less in miR‐92a−/− mice compared to WT mice (both p&lt;0.05). In conclusion, our findings support a critical role for miR‐92a in pancreatic beta cell function and whole body metabolic homeostasis.Support or Funding InformationThis work was supported in part by awards from the National Institute on Aging, R01 AG048366, R01 AG050238, and K02 AG045339, and by Merit Review Award 1I01BX002151 from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory Research and Development Service.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuron‐specific knockout of FXR attenuates the neurological complications of chronic liver disease

Hepatic encephalopathy (HE) describes the neurological deficits that result from liver impairment. Liver disease is associated with an increase of circulating bile acids that can cross the blood brain barrier and activate FXR receptors in neurons. We have previously demonstrated that aberrant bile acid signaling via activation of neuronal FXR contributes to HE pathogenesis in rodent models of acute liver failure. However, a role for FXR‐mediated bile acid signaling in HE due to chronic liver cirrhosis is undefined.MethodsNeuron‐specific FXR knockout mice were generated by crossing Floxed FXR mice (FXRfl) with SNAP‐25 cre recombinase mice. The resulting mice were designated FXRΔneu. C57Bl/6 (WT), FXRfl, and FXRΔneu were treated with carbon tetrachloride (CCl4; 1 ml/kg) by oral gavage twice per week for 12 weeks. Neurobehavioral indices and neuromuscular deficits were assessed by open field test, rotarod, grip strength test and gait analysis. After 12 weeks, tissue was collected and liver damage was assessed by serum chemistry and H&amp;E staining. Total bile acid content was assessed in the cortex and cerebellum using colorimetric assays. The expression of ASBT, FXR, and its downstream effector SHP was assessed by qPCR and immunofluorescence. Microglia activation was assessed by Iba1 immunofluorescence. The expression of proinflammatory cytokines were assessed by qPCR and EIA. In parallel, total bile acids, ASBT and FXR expression were assessed in brain tissue from cirrhotic patients with HE, compared to cirrhotics without HE and age‐ and gender‐matched controls that had been collected and banked by the Australian Brain Bank Network.ResultsTotal bile acid content was elevated in the cortex and cerebellum in CCl4‐treated WT mice and in cirrhotic patients with HE, compared to cirrhotics without HE and non‐liver impaired controls. Furthermore, ASBT, FXR and SHP expression were increased in the frontal cortex of mice and humans with HE, but not in non‐HE control samples. WT and FXRfl mice treated with CCl4 had significant deficits observed in every neurobehavioral and neuromuscular test performed, as well as marked microglia activation and increased proinflammatory cytokine expression compared to vehicle‐treated mice. These neurological deficits and neuroinflammation were attenuated in FXRΔneu mice after CCl4 treatment, even though liver damage was comparable in all genotypes of mice used.ConclusionsTaken together, these data indicate that neuronal expression of FXR plays an important role in the development of HE. Specific targeting of FXR activation in the brain may prove fruitful in the development of novel therapeutics for the management of HE.Support or Funding InformationThis study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This study was also funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. McMillin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of dynamic social material conditions on cognition in the biomedical research laboratory

The modern biomedical research laboratory is increasingly defined by dynamic social material conditions requiring researchers to traverse multiple shifting cognitive ecologies within day-to-day practice. Although the complexity of biomedical research is well known, the mechanisms by which the social and material organisation of this space is negotiated has yet to be fully considered. Integrating insights from Material Engagement Theory and Enactive Cognition with observations undertaken within a biomedical research laboratory, this paper develops an understanding of how actors are able to respond to the dynamically changing world that is enacted with and around them.

The mutational landscape of recurrent versus nonrecurrent human papillomavirus-related oropharyngeal cancer.

Human papillomavirus-related (HPV-related) oropharyngeal squamous cell carcinomas (OPSCCs) have an excellent response rate to platinum-based chemoradiotherapy. Genomic differences between primary HPV-related OPSCCs that do or do not recur are unknown. Furthermore, it is unclear if HPV-related OPSCCs that recur share a genomic landscape with HPV-negative head and neck cancers (HNCs). We utilized whole exome sequencing to analyze somatic nucleotide (SNVs) and copy number variants (CNVs) among a unique set of 51 primary HPV-related OPSCCs, including 35 that did not recur and 16 that recurred. We evaluated 12 metachronous recurrent OPSCCs (7 with paired primary OPSCCs) and 33 primary HPV-unrelated oral cavity and OPSCCs. KMT2D was the most frequently mutated gene among primary HPV-related OPSCCs (n = 51; 14%) and among metachronous recurrent OPSCCs (n = 12; 42%). Primary HPV-related OPSCCs that recurred shared a genomic landscape with primary HPV-related OPSCCs that did not recur. However, TSC2, BRIP1, NBN, and NFE2L2 mutations occurred in primary OPSCCs that recurred but not in those that did not recur. Moreover, primary HPV-related OPSCCs that recur harbor features of HPV-unrelated HNCs, notably including MAPK, JAK/STAT, and differentiation signaling pathway aberrations. Metachronous recurrent OPSCCs shared a genomic landscape with HPV-unrelated HNCs, including a high frequency of TP53, CASP8, FAT1, HLA-A, AJUBA, and NSD1 genomic alterations. Overall, primary HPV-related OPSCCs that recur share a genomic landscape with nonrecurrent OPSCCs. Metachronous recurrent OPSCCs share genomic features with HPV-negative HNCs. These data aim to guide future deescalation endeavors and functional experiments. This study is supported by the American Cancer Society (RSG TBG-123653), funding support for RAH (T32DC00018, Research Training in Otolaryngology, University of Washington), funds to EM from Seattle Translational Tumor Research (Fred Hutchinson Cancer Research Center), and center funds from the Fred Hutchinson Cancer Research Center to EM. UD is supported by the Department of Veterans Affairs, Biomedical Laboratory Research and Development (BLR&D), grant IO1-oo23456, and funds from the Pittsburgh Foundation and PNC Foundation.