bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationAuburn University, Auburn, AL King Faisal University, Hofuf, Saudi ArabiaBiofilm 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 1Determination 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

PurposeThis study characterized components of carbon dioxide expiration (VCO2) and their relationship with performance fatigability (PF) in adaptation to chronic exercise perturbation.Methods20 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).ResultsSignificant 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*).ConclusionThe 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 InformationThis 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 InformationAmerican 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.Figure 1Multiplex monitoring of apoptosis in real‐time: combining readouts from cellular impedance and live cell imaging.Figure 2
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 cancerSupport or Funding InformationPharmacyclics 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 InformationPresident's Disseration Year FellowshipFigure 1
Blocking Store‐Operated Ca2+ Entry to Protect Cardiomyocytes from Epirubicin‐Induced Toxicity
Authors: Liu Xian , Chang Yan , Choi Sangyong , Pan Zui 
Published: April 2020

Epirubicin (EPI) is one of widely used anthracycline chemotherapy drugs, yet its cardiotoxicity severely limits its clinical application. Altered intracellular Ca2+ homeostasis has been linked to epirubicin‐induced cell death and hypertrophy. While store‐operated Ca2+ entry (SOCE) recently has been linked with cardiac hypertrophy and heart failure, its role in epirubicin‐induced cardiotoxicity remains unknown.METHODHL‐1, a cardiomyocyte cell line derived from adult mouse atria, was used in this study. Intracellular ROS was measured by
dihydroethidium(DHE) fluorescence and intracellular Ca2+ was monitored by ratiometric dye Fura‐2 on a spectrofluorometer (excitation λ = 350/385nm and emission λ = 510nm; Photon Technology International, NJ). Upon depletion of ER Ca2+ stores by 10μM thapsigargin (TG) in 0mM Ca2+ extracellular solution, SOCE was activated when extracellular solution was rapidly exchanged to 2mM Ca2+. Apoptosis assay was conducted by nuclei staining, F‐actin phalloidin staining and Western Blotting of caspase‐3. While cell size was compared in HL‐1 cells with different treatments using Image J, the mRNA of Orai1, Orai3, STIM1 and B‐type natriuretic peptide (BNP) were also compared using real time qPCR.RESULTSIncubation of 1uM EPI for 30min induced significant ROS production in cultured HL‐1 cells compared with vehicle control (10410±1492 vs. 2762±939, p<0.0001). EPI induced an enhanced SOCE (0.1214±0.0104 vs. 0.0682±0.0062, n=32, p=0.0008), likely through increased expression of SOCE components Orai1, Orai3, and STIM1 compared with control cells. Apoptosis was observed upon EPI treatment, indicated by the condensed and fragmented nuclei, degradation of F‐actin and increased cleavage of caspase‐3 protein. EPI treatment also resulted in larger cell size (3894±120 μm2) compared with non‐treated control cells (2452±57 μm2, n=581, p<0.0001). The hypertrophy marker BNP was up‐regulated upon EPI treatment as well. BTP‐2, a known SOCE blocker, could block this EPI‐induced SOCE and rescue HL‐1 cells from EPI‐induced apoptosis. BTP‐2 also prevented HL‐1 cells from EPI‐induced hypertrophy in terms of cell size (see Figure
, 2548±69 μm2) and also reduction of BNP expression.CONCLUSIONEpirubicin upregulated SOCE, induced apoptosis and hypertrophy in HL‐1 cardiomyocytes. Blocking SOCE by BTP‐2 could at least partially rescue HL‐1 cells from epirubicin‐induced apoptosis and hypertrophy.Support or Funding Information(1) NIH S10 OD025230 to Dr. Zui Pan(2) University of Texas at Arlington Center for Research and Scholarship Pilot Grant for Trainee to Xian LiuFigure 1