bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased Cell Proliferation with 2, 3‐ Dichloro‐5, 8‐Dimethoxy‐1, 4‐Naphthoquinone and 4‐Hydroxy Tamoxifen in Triple Negative Breast Cancer Cell Lines
Authors: Robinson Anastasia G.J. , Copeland Robert L.
Published: April 2020

Triple negative breast cancer (TNBC) is a subtype of breast cancer (BC) that makes up 10–15% of BC diagnosis. This form of the disease disproportionately affects African American (AA) women as compared to women of other ethnicities. TNBC is classified by lack of expression of three receptors: Estrogen alpha (ERα), progesterone (PR) and human epidermal growth factor 2 (HER2). These receptors are used as therapeutic targets for BC that express them. As specific targets are not present, surgery, radiation and chemotherapy have been the mainstay of therapy for TNBC. Recently, newer drugs have become available based on specific genetic and epigenetic mutations like BRCA1, however more treatment options need to be produced to reduce the mortality rate in this disease. A novel compound, 2, 3‐dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone (Z285) belongs to the class of naphthoquinones that demonstrate activities as anticancer, antimalarial, and antibacterial compounds. These compounds have also been shown to cause an increase in reactive oxygen species (ROS) production as well as affect signaling pathways associated with epidermal growth factor receptor (EGFR). Moreover, it has been reported that treatment with tamoxifen can cause cell death. This is based on the accumulation of the drug and its active metabolites within cells that may cause an increase in the ROS production ultimately leading to cellular death, even in ERα negative cell lines.

In the current study, three triple negative cell lines, HCC1806, MDA MB 231 and HS578T were used. These cells were treated with Z285 and 4‐hydroxy tamoxifen (4OH‐Tam) for 24 or 72hr with 1, 2, 4, 8, 16μM and 3, 6, 12, 24, 48μM respectively. Synergestic activity was demonstrated using SynergyFinder as well as the Chou‐Talalay method (CompuSyn). In other experiments, cells were treated with the IC50 of Z285 which was established in prior experiments. mRNA and protein were isolated between 6 and 24hr. Results showed alterations of several mRNA in response to these treatments. In addition, there were modifications to Nrf2 protein concentration, a transcription factor associated with oxidative stress. Therefore, these results indicate that combination treatment of Z285 and 4OH‐Tam induce cell death at lower concentrations. Thus this novel compound which causes an increase in ROS in these TNBC cells, may render them more susceptible to tamoxifen therapy.

Support or Funding Information

Charles and Mary Latham Fund
Characterization of Desulfinase for the Removal of Recalcitrant Sulfur Compounds in Petroleum
Authors: Dodd Cameron
Published: April 2020

Biodesulfurization provides a method to remove the sulfur from organosulfur compounds found in petroleum. This multi‐step pathway is able to remove sulfur from dibenzothiophene (DBT), one of the most recalcitrant compounds found in petroleum and crude oil. A critical enzyme in this pathway is an aromatic desulfinase (DszB) that catalyzes the final rate‐limiting step of the desulfurization. The DszB enzyme from Nocardia asteroides A3H1 was overexpressed in E. coli, purified using Ni2+‐affinity chromatography and characterized kinetically. Kinetic assays revealed a sigmoidal response when the velocity was plotted against substrate concentration, calling into question the monomeric structure of the enzyme previously reported by crystallography studies. Size exclusion chromatography and cross‐linking experiments were conducted to further investigate the oligomerization of HPBS desulfinase. Attempts to further purify the enzyme using Co2+‐affinity chromatography provided further evidence that under certain conditions DszB may exhibit cooperativity.

Support or Funding Information

JMU Department of Chemistry and Biochemistry Dr. Linette M. Watkins NSF Grant No. CHE‐1757874
An Anti‐Mesothelin Recombinant Immunotoxin Based on Deoxyribonuclease‐I
Authors: Sprehe Ruby , Baker Jillian , Weldon John
Published: April 2020

Recombinant immunotoxins (RITs) are fusion proteins consisting of a toxin attached to an antibody fragment. Unlike traditional chemotherapy, RITs used in cancer therapy can be specifically targeted to tumor cells while sparing healthy cells. One shortcoming of RITs is their immunogenicity, which is the immune response of patients to treatment with RITs. Patients with intact immune systems often produce antibodies to the toxin that neutralize its function. For this experiment, we attempted to prepare a RIT using deoxyribonuclease‐ I (DNase‐I) instead of a typical bacterial or plant‐derived toxin. DNase‐I is an endogenous human protein that catalyzes the degradation of DNA into its component nucleotides. Previous research shows that DNase‐I can be toxic to cells if delivered through targeted endocytosis. Furthermore, we predict that DNase‐I will be less immunogenic than other toxins, because it is a human protein. We successfully cloned an expression vector joining DNase‐I to an anti‐mesothelin antibody fragment and expressed it in E. coli. The resulting protein, however, was unstable and we were unable to purify it after refolding from inclusion bodies. We concluded that the design of the fusion protein needs improvement to enhance its stability. It is also possible that the refolding conditions need further optimization. Future directions include exploring both of these possibilities.

Support or Funding Information

I would like to thank the Towson Bridges Program and CCBC for the opportunity. Thank you to Dr. Laing, Dr. Snyder, Dr. Berlyn and Trudymae for guidance throughout the program. Thank you to the NIH Bridges Grant 5 R25 GM058264 16 for funding. Special thanks to Dr. Weldon, Jillian Baker and all students that have helped throughout the project.
Antioxidant Activities of Green Synthesized Zinc Oxide Nanoparticles and its Curative Influence in Carbon tetrachloride‐Induced Hepatorenal Damage in Adult Rats
Authors: Otuechere Chiagoziem A. , Ekozin Adriel , Adewuyi Adewale
Published: April 2020

The objective of this study was to synthesize Zinc Oxide nanoparticles coated with Pterocarpus mildbraedii leaf extracts (PmZnONPs) by an eco‐friendly method, and investigate its antioxidant activity and curative potential against carbon tetrachloride (CCl4) ‐ induced hepatorenal toxicity in rats. PmZnONPs was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), fourier transform infra‐red spectroscopy (FTIR), alongside zeta potential measurement. In vitro, free radical activities using 1, 1‐diphenyl‐2‐ picrylhydrazyl (DPPH) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS) showed strong antioxidant activity compared to ascorbic acid. In rats, PmZnONPs (1 and 3 mg/kg p.o.) significantly prevented CCl4‐induced elevations of alkaline phosphatase, gamma‐glutamyl transferase, creatinine, urea, aspartate aminotransferase, alanine aminotransferase, and total bilirubin. Moreover, PmZnONPs restored the decreased levels of plasma uric acid, hepatorenal antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, glutathione transferase, and glutathione, that were significantly induced by CCl4 treatment. Histological studies indicated that CCl4‐induced lesions in the liver and kidney were alleviated by the treatment of PmZnONPs. Immunohistochemical studies showed that PmZnONPs significantly suppressed the high immunoreactivity of cyclooxygenase‐2, interleukin‐6 and NF‐κB arising from CCl4 intoxication. These results indicated that PmZnONPs protected the rats from CCl4‐induced hepatorenal damage via its antioxidant properties as well as its inhibition of pro‐inflammatory proteins.

Support or Funding Information

This research did not receive any support from any funding organization.
Assessment of Collagen Metabolism by Human Gingival Fibroblasts Reared in Serum‐free Conditions
Authors: Laird Patrick , Mattioli Patrisia , Maloney Trevor J. , Otto Ashley , Musiol Joseph , Nguyen Stephanie , Popper Hannah , Morris Gregory Philip , Beqo Belinda , Isaac Tamika , Green Kevan , Shamseddin Seyed , Borghaei Ruth , D’Angelo Marina
Published: April 2020

Periodontal disease is present in 48% of people over the age of 30 years and 70% over the age of 65 years. It is marked by an inflammatory state that affects the gum tissue, periodontal ligaments, and the alveolar bone itself. It has several different causes, but a significant risk factor is smoking because it impairs the healthy biological pathway of repair of the tissues. In a generally healthy patient, there is a balance of broken down collagen and repair with intact collagen. The enzyme responsible for this collagen type 1 degradation is matrix metalloproteinase‐8 (MMP‐8). In this study, the metabolic activity of primary human gingival fibroblasts (HGVF) isolated from inflamed non‐smoker (IFN) and inflamed smoker (IFC) were cultured in serum‐free media over a 48 hour period. We measured production of intact and degraded collagen type I in the secreted fraction (conditioned media) and in the incorporated extracellular matrix. In the secreted and incorporated fractions at both time points (24 and 48hr), HGVF isolated from a smoker (IFC) produced more intact collagen (100% more secreted and incorporated) and more degraded collagen (97% more secreted and 95% more incorporated) than HGVF isolated from the non‐smoker (IFN) (p<0.001). These data support the hypothesis that inflamed tissue isolated from a current smoker exhibits a profile of collagen metabolism in line with more acute diseased state (fibrotic collagen profile). In addition, the culture of primary human gingival fibroblasts in serum‐free medium for at least 48 hours is a viable method to study the disease mechanism of periodontitis and potential therapeutics to treat the disease.

Support or Funding Information

This work is supported by the Division of Research at PCOM
Non‐Tuberculous Mycobacteria Infections Increase NOS2 While Decreasing NO Levels
Authors: Lin Kuo-Chuan , Bedi Brahmchetna , Sadikot Ruxana T. , Sutliff Roy
Published: April 2020

Non‐tuberculous mycobacteria (NTM) are rod‐shaped bacilli that cause lung infections in patients with weakened immune system or underlying lung disease such as cystic fibrosis, COPD, or prior tuberculosis. While it is well‐established that NTM cause lung disease, the host response to infection with NTM has not been well‐characterized. The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by innate immune cells is considered a major defense mechanism against mycobacteria. Nitric oxide (NO), a product of inducible nitric oxide synthase (NOS2) in macrophages, plays an essential role in M. tuberculosis (MTb) infection through its bactericidal and anti‐inflammatory characteristics. Therefore, we hypothesized that NTM infection may upregulate NOS2 expression, which in turn may contribute to the host response to NTM infection. RAW 264.7 and MH‐S cells, virus transformed murine macrophages and alveolar macrophages, respectively, were exposed to NTM (M. avium‐intracellulare, or MAI) at a MOI of 25 and NO levels in cell media were monitored 4, 8, 12, 24, and 48 hours after NTM infection using a colorimetric GSNO assay. mRNA expression of NOS isoforms (NOS1, NOS2, and NOS3) in MH‐S and RAW cells were analyzed 24‐ and 48‐hours post‐infection. MH‐S cells were also transfected with siRNA to NOS2 and infected with MAI for 0.5, 4, and 24 hours, and their intracellular NTM were quantified to assess macrophage function. NO levels decreased following 4 hours of NTM exposure and remained decreased for 48 hours (P<0.05). NTM infection increased the expression of NOS2 without affecting levels of other NOS isoforms (P<0.05). siNOS2 transfected cells infected with MAI for more than four hours had significantly greater levels of intracellular MAI compared to scrambled negative control (P<0.05). Our preliminary data are the first to demonstrate that NTM increase NOS2 expression while decreasing NO levels. Furthermore, decreasing NOS2 expression exacerbates NTM infections. Future studies will examine mechanisms whereby NTM decrease NO levels. Therefore, we propose that approaches that restore NO can enhance macrophage host defense against NTMs and improve infection clearance.

Support or Funding Information

This work was supported in part by NIH Grant R01HL102167 (RLS), Merit Review funding from the Department of Veteran’s Affairs, Office of Research and Development (RTS). NTM isolates are Isolates were provided by Emory University Hospital
Chronic Ablation of Hindlimb Afferents Containing TRPV1 Receptors Attenuates Cardiovascular Responses to Muscle Metaboreflex Activation
Authors: Mannozzi Joseph Thomas , Senador Danielle , Al-Hassan Mohamed , Lessanework Beruk , Alvarez Alberto , O’Leary Donal S.
Published: April 2020

Increases in sympathetic nerve activity during exercise occur, in part, due to activation of the muscle metaboreflex – a pressor reflex triggered via stimulation of type III and IV skeletal muscle afferents. Some of these afferents express capsaicin sensitive transient receptor potential vanilloid 1 (TRPV1) channels. Epicardial and intrathecal administration of resiniferatoxin (RTX), an ultra‐potent analog of capsaicin, attenuates responses to cardiac sympathetic afferent activation and provides relief of limb pain due to bone cancer. We investigated whether administration of intrathecal RTX (2μg/kg, L4–L6) would attenuate the muscle metaboreflex (induced by partial reductions in hindlimb blood flow during steady‐state mild exercise; 3.2 km/h). Experiments were performed using conscious, chronically instrumented canines before and 10–42 days after RTX. RTX markedly attenuated metaboreflex‐induced increases in mean arterial pressure (+27.0 ± 5.0 mmHg vs. +5.9 ± 8.2 mmHg) and cardiac output (+1.40 ± 0.20 l/min vs. +0.28 ± 0.12 l/min). Terminal experiments under isoflurane anesthesia performed 78 ± 14 days after RTX showed attenuated pressor responses to hindlimb intra‐arterial infusion of 20μg/kg capsaicin (+45.7 ± 6.5 mmHg pre RTX vs. +19.7 ± 3.1 mmHg post RTX). We conclude that intrathecal RTX chronically attenuates muscle metaboreflex pressor responses. Thus, RTX could be an effective treatment to reduce excessive sympatho‐activation often seen during exercise in patients with heart failure or other diseases when the metaboreflex is overstimulated due to underperfusion of the active skeletal muscle.

Support or Funding Information

HL‐55473, HL‐126706, HL‐120822 and GM‐058905
Salt‐dependent protein splicing of an intein from the halophile Haloferax volcanii
Authors: Cawood Alexandra , Ruiz Mercedes , Mills Kenneth
Published: April 2020

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an intein, or internal protein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitant with the ligation of the exteins. The extremophile Haloferax volcanii grows in high salt environments. We are interested in the salt‐dependence of the protein splicing and nuclease activity of the intein that has invaded its DNA Polymerase beta gene. We have shown that we can induce protein splicing of the intein in vitro as a function of salt concentration and time. The intein has an intervening homing endonuclease domain and we are interested to learn if mutations within this domain would influence splicing. Characterization of a conditionally‐active nuclease may have utility in biotechnology applications.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138), National Institutes of Health (grant 1R15GM132817‐01), and by the Camille & Henry Dreyfus Foundation
Salmonella biases epithelial differentiation in murine enteroids through Wnt and Notch signaling, which may contribute to diarrheal pathogenesis
Authors: Quach Andrew , Jayaratne Rashini , Ibeawuchi Stella-Rita , Das Soumita , Barrett Kim
Published: April 2020

Salmonella enterica serovar Typhimurium is one of the most burdensome foodborne diarrheal pathogens worldwide. However, despite its prevalence, the mechanism by which Salmonella causes diarrhea is not entirely known. We have shown in mice that Salmonella infection decreases expression of SLC26A3 (Down‐Regulated in Adenoma; DRA), a chloride/bicarbonate exchanger. In order to understand the mechanism of this effect, we employed an enteroid model from murine colon. Enteroid‐derived monolayers (EDM) that recapitulate both the absorptive and secretory cell lineages of the native intestinal epithelium (enterocytes vs. enteroendocrine, goblet, and Paneth cells, respectively) were infected apically with Salmonella and relevant protein expression was studied using qRT‐PCR and western blot. Infection significantly reduced expression of DRA (40%, p = 0.0124) and Hes1 (45%, p = 0.0004) while upregulating expression of ATOH1 (3‐fold, p = 0.0292) and mucin 2 (Muc2) (2‐fold, p = 0.0134). Hes1 is a Notch pathway downstream signaling molecule and thus a precursor to the absorptive epithelial lineage that expresses DRA. ATOH1, on the other hand, is a Wnt pathway downstream signaling molecule and a precursor to secretory lineages, including goblet cells that express Muc2. Infection of EDMs also reduced levels of the Notch intracellular domain. The involvement of Notch was further investigated by inhibiting Notch signaling using a γ‐secretase inhibitor, which reproduced the downregulation in Hes1 and DRA and upregulation in ATOH1 and Muc2 as seen with infection. Because the balance of Wnt and Notch signaling determines relative intestinal epithelial differentiation, our findings suggest that the diarrheal pathogenesis of Salmonella may reflect Notch inhibition and an accompanying shift in epithelial differentiation from absorptive to secretory cell types, a decreased capacity for absorption, and thus the accumulation of diarrheal fluid.
Cardiac and Medullary Neuroinflammatory Pathways Trigger Androgenic Incitement of Cardiovascular Sequels of Endotoxemia in Rats
Authors: Sallam Marwa Y. , El-Gowilly Sahar M. , El-Mas Mahmoud M.
Published: April 2020

Inconsistent reports are available on the role of testosterone in end organ damage caused by endotoxemia. Here, pharmacologic, surgical, and molecular studies were employed to assess the testosterone modulation of cardiovascular and autonomic derangements caused by endotoxemia and the role of peripheral and central inflammatory pathways in this interaction. Studies were performed in conscious male rats pre‐instrumented with femoral indwelling catheters for hemodynamic monitoring and subjected to castration or pharmacologic interventions that interrupt the biosynthetic cascade of testosterone. Compared to the effects of lipopolysaccharide (LPS, 10 mg/kg i.v.) in sham operated rats, 2‐week castration significantly reduced the LPS‐evoked: (i) falls in blood pressure, (ii) decreases in time‐ and frequency domain indices of heart rate variability (HRV), (iii) shifts in cardiac sympathovagal balance towards parasympathetic dominance as evidenced by decreases in low‐frequency to high‐frequency ratio of spectral powers, and (iii) increases in protein expressions of toll‐like receptor 4 and monocyte chemoattractant protein‐1 in the heart as well as in medullary neurons of the nucleus tractus solitarius and rostral ventrolateral medulla. The favorable influences of castration on inflammatory and cardiovascular abnormalities of endotoxemia were replicated in intact rats pretreated with degarelix (gonadotropin‐releasing hormone receptor blocker) or finasteride (5α‐reductase inhibitor), but not formestane (aromatase inhibitor). The data signifies the importance of testosterone and its biosynthetic enzymes in cardiovascular and autonomic insults induced by the endotoxic inflammatory response. Clinically, the interruption of testosterone biosynthesis could offer a potential therapeutic strategy for endotoxemia.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grants No. 14895)
Signaling Pathways Involved in Enhanced 5‐HT‐Induced Contraction of Pulmonary Artery Segments From Guinea‐Pigs With Hyperthyroidism
Authors: Oriowo Mabayoje , Oommen Elsie
Published: April 2020

We have previously reported that hyperthyroidism in rats resulted in enhanced 5‐HT‐induced contractions of pulmonary artery segments by a mechanism involving activation of calcium‐activated chloride channels. We have investigated other possible pathways that might be involved in this response in this study. Pulmonary artery ring segments were set up in Krebs’ solution for isometric tension recording. 5‐HT‐induced contraction was significantly enhanced in main pulmonary artery segments from guinea‐pigs with hyperthyroidism compared with a similar preparation from control guinea pigs. Nifedipine (10−6M) and tempol (3×10−3M) significantly reduced 5‐HT‐induced contraction in artery segments from thyroxine‐treated while having no effect in artery segments from control guinea pigs. Y‐27632 (10−5M) and SB 202190 (10−5M) but not PD 98059 (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated guinea pigs. However, the reduction was greater in artery segments from thyroxine‐treated guinea pigs. Genistein (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control or thyroxine‐treated guinea pigs. Genistein was equipotent in both cases. These results would suggest that hyperthyroidism‐induced increased 5‐HT‐induced contraction of the guinea pig pulmonary artery is mediated by multiple signaling pathways including increased influx of extracellular calcium through L‐type dihydropyridine channels, tyrosine kinase pathway, Rho‐kinase pathway of calcium sensitization, increased generation of reactive oxygen species and p38 MAPK pathway.
Sympathomotor activation by midbrain locomotor neurons is mediated through glutamate release in the rostral ventrolateral medulla
Authors: Koba Satoshi , Kumada Nao , Watanabe Tatsuo
Published: April 2020

Our pilot data showed that a portion of neuronal cells in the mecencephalic locomotor region (MLR) sends axonal projections to the rostral ventrolateral medulla (RVLM) (MLR‐RVLM pathway), being capable of eliciting sympathomotor activation in rats. Here, we examined the effect of ionotropic glutamate receptor blockade in rat RVLM on sympathomotor activation by the MLR‐RVLM pathway. Male rats received microinjections bilaterally in the RVLM with a retrograde adeno‐associated virus that encoded a channelrhodopsin with green fluorescence protein. Under anesthesia, 473 nm wavelength laser illumination of the MLR after administration in the RVLM with a cocktail of AP5 and CNQX, NMDA and AMPA glutamate receptor blockers, respectively, elicited 29% less (P < 0.05) renal sympathoexcitation than that after saline administration. Likewise, under decerebration without anesthesia, ventral root fiber excitation in response to MLR photostimulation seen after saline administration in the RVLM was suppressed to a 35% extent (P < 0.05) by preliminarily administration of the glutamate receptor blockers. These observations suggest that glutamatergic neurons in the MLR‐RVLM pathway function in generating central command function.

Support or Funding Information

Supported by JSPS KAKENHI 18H03151 (S. Koba).
Inconsistent effects of surgical and chemical castration on arterial baroreceptor dysfunction and cardiac and brainstem inflammation in endotoxic rats
Authors: Sallam Marwa Y. , El-Gowilly Sahar M. , El-Mas Mahmoud M.
Published: April 2020

Impaired cardiac autonomic and arterial baroreflex activity has been implicated in cardiovascular derangements induced by endotoxemia. In this communication, we tested the hypothesis that androgenic hormones contribute to endotoxic manifestations of arterial baroreflex dysfunction and related cardiac and central neuroinflammatory pathways in male rats. Baroreflex curves relating changes in heart rate to increases or decreases in blood pressure evoked by phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious sham‐operated and castrated rats treated with or without lipopolysaccharide (LPS, 10 mg/kg i.v.). Slopes of baroreflex curves were used as measures of baroreflex sensitivity (BRS). In sham rats, LPS caused significant reductions in BRS and increases in the immunohistochemical expression of NFκB in cardiac tissues as well as in brainstem neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM). The baroreflex depressant effect of LPS was maintained in CAS rats despite significant attenuation of the inflammatory response in cardiac and brainstem areas. We also evaluated the effects of pharmacologic inhibition of androgenic biosynthetic pathways on LPS responses. Whereas none of the LPS effects were altered after prior exposure to formestane (aromatase inhibitor, 15 mg/kg s.c)‐pretreated rats, the LPS‐evoked BRSPE, but not BRSSNP, depression and cardiac and neuronal inflammation disappeared in intact rats pretreated with degarelix (gonadotropin‐releasing hormone receptor blocker, 1 mg/kg s.c.). On the other hand, the prior inhibition of 5α‐reductase by finasteride (20 mg/kg s.c) did not affect baroreflex dysfunction induced by LPS and reduced cardiac, but not neuronal, NFκB expression. The data highlight advantageous effects for the inhibition of hypothalamic‐pituitary‐gonadal axis in counteracting baroreflex dysfunction caused by endotoxemia and predisposing cardiac and neuroinflammatory signals.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)
Investigating the Effects of Gpa2 Phosphorylation on Yeast Sporulation
Authors: Cheranda Nina Kiran , Huang Shan , Wang Yuqi
Published: April 2020

Gpa2 is a G alpha protein in budding yeast, Saccharomyces cerevisiae, that functions in the nutrient signaling pathway. In diploid yeast cells, Gpa2 interacts with the cytoplasmic protein Ime2 and acts as an inhibitor to prevent Ime2 from inappropriate activation of sporulation. It is unclear how the inhibitory effect of Gpa2 on Ime2 is regulated. Recently, we found that Gpa2 undergoes phosphorylation and that its level of phosphorylation increases upon nitrogen starvation, a condition that triggers sporulation in yeast. Interestingly, phosphorylation enhances plasma membrane localization of Gpa2, raising the possibility that Gpa2 phosphorylation may play a role in the regulation of yeast sporulation. Accordingly, we investigated the sporulation efficiency of diploid cells that express either wild type Gpa2 or Gpa2‐10SA mutant, which lacks 10 serine residues required for phosphorylation. Using confocal microscopy to analyze the cells with clear indicators of the presence of spores, we found that more cells expressing wild type Gpa2 underwent sporulation than cells expressing Gpa2‐10SA mutant did. Our findings suggest that regulated phosphorylation of Gpa2 is a potential mechanism that relieves the inhibitory effect of Gpa2 on Ime2.

Support or Funding Information

Knoedler Undergraduate Fund from Saint Louis University
Exposure to Fullerene C60 Nanoparticles Impairs Lung Mechanics and Mitochondrial Function
Authors: Zin Walter A. , Caldeira Dayene F. , Pinheiro Felipe G. , Maciel Leonardo , Oliveira Dahienne F.
Published: April 2020

C60 fullerene (C60) carbon‐based nanoparticles are widely used because of their physicochemical properties, and are released into the atmosphere from anthropogenic or natural sources. Due to its small size (0.65–0.7 nm) and electronegative chemical behavior, C60 can overcome natural biological barriers and deposit onto the respiratory system. However, the literature does not fully describe its putative pulmonary burden. C60 interaction with double membrane organelles like mitochondria, housing enzyme systems that convert metabolism by‐products into usable energy (ATP) and are an important source of oxygen‐derived free radicals, has been recently described. We hypothesized that C60 impairs lung function and alters its inflammatory status via mechanisms possibly involving mitochondria. Hence, we addressed pulmonary mechanics, histology and mitochondrial function after a single exposure to C60. Our Institutional Ethics Committee approved the study (code: 155/18). Thirty‐five male BALB/c mice were randomly divided into 2 groups intratracheally (it) instilled with vehicle (0.9% NaCl + 1% Tween 80, CTRL) or C60 (1.0 mg/kg, FUL). Twenty‐four hours after exposure, 15 FUL and 8 CTRL mice were anesthetized (10 mg/kg xylazine and 120 mg/kg ketamine), paralyzed (pancuronium bromide, 0.1 mg/kg) and mechanically ventilated for the determination of lung mechanics. They were then euthanized by sectioning of abdominal aorta and vena cava; the lungs were extracted at FRC for histological processing. Results are expressed as median, 25th and 75th percentiles. FUL group showed increased lung tissue elastance (FUL=38.21, 32.43, 39.83 vs CTRL=31.63, 29.31, 33.41 cmH2O/mL, p=0.0049) and viscance (FUL=4.44, 3.72, 4.76 vs CTRL=3.73, 3.36, 3.87 cmH2O/mL, p=0.0225). In addition, lung histology disclosed increased inflammatory cell number (FUL=16.0, 14.6, 18.8 vs CTRL=9.2, 8.8, 9.6 10−3/μm2 cells, p=0.0022) and alveolar collapse (FUL=11.33, 10.34, 13.79 vs CTRL=5.78, 4.69, 7.71%; p=0.0286), septal thickening and pulmonary edema. The remaining 6 FUL and 6 CTRL mice were expertly euthanized by cervical dislocation. Mitochondrial function expressed reduction in state 1 respiration in the presence of pyruvate‐malate substrates [FUL=3.0±1.14 vs CTRL=4.46±0.9 (SEM) nmol O2/min/mg protein, p=0.0210], ATP production (FUL=122.6±18 vs CTRL=154.5±14 μmol/100 μg protein, p=0.0340), and higher generation of reactive oxygen species (ROS) (FUL 733.1±169.32 vs CTRL=486.39±73.1 nmol/100 μg protein, p=0.0313). In conclusion, exposure to fullerene C60 impaired lung mechanics and mitochondrial function, increased ROS concentration, and triggered pulmonary tissue inflammation.

Support or Funding Information

CNPq, FAPERJ, FINEP, CAPES
A Study of Nonstructural Protein 1 of SARS Coronavirus
Authors: Nag Anita , Valafar Homayoun , Pandala Niharika
Published: April 2020

Severe acute respiratory syndrome coronavirus (SARS‐CoV) encoded nonstructural protein 1 (nsp1) orchestrates a multi‐pronged mechanism to down regulate host gene expression, also known as host shutoff. Nsp1 is a 180 amino acid long protein with primarily flexible structure that allows nsp1 to interact with various cellular factors during host shutoff to inhibit translation and promote mRNA decay. Mutations of the surface residues of nsp1 either attenuates or augments its host shutoff function prompting the hypothesis that these residues bind cellular proteins that facilitate nsp1 during host shutoff. To identify cellular proteins that interact with nsp1 and assist its function, we conducted a quantitative proteomics experiment by comparing protein complexes associated with nsp1 and its inactive mutant (K164AH165A) and computer modeling to predict structural changes. By comparing protein complexes, we identified a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts nuclear‐cytoplasmic transport of biomolecules. Expression of nsp1 in 293 cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation of the protein while other nucleoporins and the nuclear lamina remain unperturbed, suggesting a significant alteration of the nuclear pore complex in the presence of nsp1. However, expression of mutant nsp1, which does not bind to Nup93, does not alter the nuclear pore complex. To better understand the structural basis of this protein’s function, we have utilized computational modeling tools, more specifically, I‐TASSER (Iterative Threading ASSEmbly Refinement). Structural models of various mutants of nsp1 were obtained (by using I‐TASSER) in order to explore the structural impact of different mutations. Our preliminary explorations show significant topological changes on nsp1’s surface for specific mutations. These perturbations of surface properties can provide a steric‐based mechanism of interfering with nsp1’s binding or recognition function therefore, aggravating its host shutoff function.
Effect of Continuous‐Flow Left Ventricular Assist Device Support on Coronary Artery Endothelial Function in Ischemic and Non‐Ischemic Cardiomyopathy
Authors: Symons J David , Deeter Lance , Deeter Nicholas , Bonn Trevor , Cho Jae Min , Ferrin Peter , McCreath Lauren , Diakos Nikolaos , Taleb Iosif , Alharethi Rami , McKellar Stephen , Wever-Pinzon Omar , Navankasattusas Sutip , Selzman Craig , Fang James , Drakos Stavros
Published: April 2020

The coronary vasculature encounters a reduction in pulsatility after implementing durable continuous‐flow left ventricular assist device (CF‐LVAD) circulatory support. Evidence exists that appropriate pulsatility is required to maintain endothelial cell homeostasis. We hypothesized that coronary artery endothelial function would be impaired after CF‐LVAD intervention. Coronary arteries from patients with end‐stage heart failure caused by ischemic (ICM, n=16) or non‐ischemic (NICM, n=22) cardiomyopathy were isolated from the LV apical core which was removed for the CF‐LVAD implantation. In 11 of these patients, paired coronary arteries were obtained from an adjacent region of myocardium after the CF‐LVAD intervention (n = 6 ICM, 5 NICM). Vascular function was assessed ex vivo using isometric tension procedures in these patients and in 7 non‐failing donor controls. Maximal endothelium‐dependent vasorelaxation to bradykinin (BK, 10−6 to 10−10 M) was blunted (p<0.05) in arteries from patients with ICM compared to NICM and donor controls, whereas responses to sodium nitroprusside (SNP, 10−4 to 10−9 M) were similar among the groups. Contrary to our hypothesis, vasorelaxation responses to BK and SNP were similar before and 219±37 days after CF‐LVAD support. Of these patients, an exploratory subgroup analysis revealed that BK‐induced coronary artery vasorelaxation was greater (p<0.05) after (87±6%) vs. before (54±14%) CF‐LVAD intervention in ICM patients, while SNP‐evoked responses were similar. Coronary artery endothelial function is not impaired by durable CF‐LVAD support, and in ICM patients appears to be improved. Investigating coronary endothelial function using in vivo approaches in a larger patient population is warranted.

Support or Funding Information

JDS (AHA16GRNT31050004, NIH RO3AGO52848, NHLBI RO1 HL141540); LD, ND, TB (University of Utah Undergraduate Research Opportunities Program); JMC (University of Utah Graduate Research Fellowship); SGD (AHA Heart Failure Strategically Focused Research Network 16SFRN29020000, NHLBI RO1 HL135121, NHLBI RO1 HL132067).
A Case Study on Glucose Phosphate Isomerase Deficiency: An End of the Semester Review
Authors: Nag Anita
Published: April 2020

Problem based learning has been extensively used in medical and business schools ensuing enormous benefit for the students. However, science majors (biology and chemistry), destined to attend professional schools, have very little exposure to problem based learning in their regular science curriculum. The first semester biochemistry course at USC Upstate covers several new materials while developing on concepts already taught in general chemistry and biology courses. During the course of a semester, students often study materials in isolation without connecting them with each other. To assist students retain these concepts, a student‐centered activity has been designed to connect multiple biochemistry concepts into one intertwined case study, which is used as the end of the semester student‐led review session allowing students integrate research based data and concepts in the context of a real‐life scenario. The case describes an infant suffering from an inherited genetic disorder in glucose phosphate isomerase (GPI), which is supported by laboratory test results of the defective enzyme and research data on GPI. This end of the semester case study on GPI incorporates chemistry of water, nucleic acid chemistry, carbohydrate chemistry, metabolism and protein chemistry including isolation, characterization, enzyme kinetics and mechanism. Relating multiple concepts to one mutant enzyme involved in glycolysis not only allows students to appreciate how these concepts apply to real life problems but also to retain the material in the context of a biomedical problem. Students’ gain in knowledge is evaluated using pre‐tests, post‐tests and other assessments.
p38 MAPK Isoforms as Modulators of Oncogenic Phenotypes in Inflammatory Breast Cancer
Authors: Rivera Jeisac Guzman , Peterson Esther A.
Published: April 2020

Inflammatory Breast Cancer (IBC) is a rare, aggressive, and highly metastatic subtype of breast cancer; accounting for approximately 1–6% of all breast cancer diagnoses in the United States and 7–10% of breast cancer‐associated deaths in Western countries. The aggressiveness of this disease is generally attributed to the formation of diffuse tumor cell clusters, known as tumor emboli, that block the dermal lymphatic vessels near the chest wall, a key hallmark of IBC. Clinically, it has been documented that most of IBC cases are either HER2 amplified or Triple Negative (TN‐IBC), the latter being the most difficult to treat. As a result, there is a dire need to understand the molecular profile of IBC to further develop more efficient targeted therapies. It has been reported that 17β‐Estradiol (E2) exerts non‐genomic signaling in ER‐negative IBC and non‐IBC cells using estrogen receptors GPR30 and estrogen receptor alpha variant 36 (ERα‐36), which activate the PI3K/AKT/ERK pathways involved in oncogenic phenotypes. p38 isoforms alpha, beta, gamma, and delta have been demonstrated to modulate proliferation, migration, apoptosis, and cell senescence via stress‐induced pathways or mitogen‐activated pathways. Also, it has been shown that these isoforms have context‐dependent functions depending on tissue type, expression and activation level, and the pathways involved. Nevertheless, the effects of these isoforms have yet to be elucidated in IBC. Preliminary data suggest that estrogen mediates p38 activation when IBC cells are treated with E2. For this reason, we hypothesize that estrogenmediated activation is involved in p38‐induced oncogenesis via non‐genomic signaling in an isoform‐dependent manner in IBC cell lines. A comparison of p38 isoforms expression was done on both IBC and non‐IBC cell lines via Western blot and qRT‐PCR, presenting differential expression profiles. This data was then used to design a knockdown model to study the effects of the most predominant isoforms on oncogenic phenotypes. Treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines when performing a dose response curve. Ongoing studies include assessing the effects of p38 inhibition and knockdown on proliferation, invasion, migration, and tumor emboli formation under basal and endocrine conditions. To recapitulate, understanding E2 responsiveness by ER‐negative cell lines may provide great insight into novel targeted therapies using the non‐genomic signaling pathway; in particular, p38 signaling appears to be a key effector of prooncogenesis in IBC.
Glypican‐1 Is a Trigger for Pulmonary Edema Development During Acute Heart Failure
Authors: Chignalia Andreia Z. , Isbatan Ayman , Black Stephen M. , Dull Randal O.
Published: April 2020

Heart Failure is a growing global epidemic with a prevalence of over 37.7 million cases. Accumulating evidence indicate that the lungs actively participate in the pathogenesis of Heart Failure and, in particular, in the development of pulmonary edema associated with acute heart failure (AHF). The molecular mechanisms by which pulmonary edema develops during AHF are not clarified but increases in pulmonary capillary pressure may play a role. Herein we sought to investigate if glypican‐1 (Gpc1), a heparan sulfate proteoglycan known to be activated by pressure, contributes to hyperpermeability and pulmonary edema during AHF. We hypothesize that Gpc1 induces pulmonary edema via eNOS uncoupling and RhoA nitration. The isolated perfused lung preparation was used to simulate AHF in male CD‐1 (wild type, WT) and glypican‐1 knockout (Gpc1KO) mice (6 weeks old). Control experiments were done setting left atria pressure (PLA) at 3cm H2O for 10 minutes; AHF was simulated by setting PLA at 10 cm H2O for 10 minutes. Lung edema was assessed by lung wet‐to‐dry ratio; heart function was assessed by echocardiography, reactive oxygen species (ROS) production was assessed by lucigenin enhanced chemiluminescence. PKCa activation, eNOS phosphorylation at T495 and Rho nitration were assessed by immunoblotting. An in vitro approach was taken to further investigate the molecular mechanisms by which increased pressure leads to endothelial hyperpermeability: human lung microvascular endothelial cells (HLMEC) were exposed or not to high hydrostatic pressure (30cm H2O) for 10 minutes and analysis of activation of PKC‐eNOS‐RhoA axis was performed by immunoblotting. WT and Gpc1KO mice showed similar ejection fraction and increase in pulmonary artery pressure. Deletion of Gpc1 protected mice from pulmonary edema associated with AHF and prevented increase on ROS production. Inhibition of eNOS with L‐NIO mitigated pulmonary edema during AHF in WT mice. NOC‐9, a NO donor, restored the ability of GPC1KO lungs to develop edema during AHF. Moreover, WT mice showed increased eNOS phosphorylation at T495, PKCa activation and RhoA nitration during AHF, effects not seen in Gpc1KO. In vitro, increased hydrostatic pressure induced activation of the same pathways as seen in the in situ model. Taken together, these results suggest that activation of Gpc1 leads to eNOS uncoupling and RhoA nitration that results in endothelial hyperpermeability and pulmonary edema during AHF. These data evidence the lung endothelium as an active player during AHF and glypican‐1 as a gatekeeper of barrier function.

Support or Funding Information

P01 HL134610 (SMB) and funds from the Department of Anesthesiology, College of Medicine, University of Arizona, Tucson and the Department of Anesthesiology, College of Medicine, University of Illinois at Chicago.
Dsg2 Upregulation as a Rescue Mechanism in Pemphigus
Authors: Sigmund Anna M. , Egu Desalegn T. , Waschke Jens , Vielmuth Franziska
Published: April 2020

In the bullous autoimmune disease pemphigus, autoantibodies directed against the desmosomal cadherins desmoglein (Dsg)1 and 3 cause loss of intercellular adhesion clinically manifested as flaccid blisters of the skin and mucous membranes. We showed that homophilic Dsg3 interactions are directly inhibited upon autoantibody binding and recently observed that Dsg3 also undergoes heterophilic interactions with Dsg2. Thus, we here investigated the impact of this heterophilic interaction in pemphigus. Dsg2, which is almost absent in healthy, adult epidermis, was upregulated in epidermis of pemphigus patients. Similarly, pemphigus autoantibodies induced upregulation of Dsg2 in a human ex‐vivo skin model. Further, a newly generated stable murine Dsg3‐deficient keratinocyte cell line showed severely disturbed intercellular adhesion in keratinocytes dissociation assay paralleled by upregulation of Dsg2 in the cytoskeletal‐bound membrane fraction. Heterophilic interactions were confirmed by Dsg2‐Dsg3 co‐immunoprecipitation in human keratinocytes. We next characterized heterophilic Dsg2‐Dsg3 interactions by cell‐free atomic force microscopy, which showed binding frequency, ‐strength, Ca2+‐dependency and a catch‐bond behavior comparable to the homophilic Dsg3 interaction. A pathogenic pemphigus aDsg3 antibody from a pemphigus model, AK23, and autoantibodies from a pemphigus patient directly interfere with homophilic Dsg3 binding. Interestingly, direct inhibition was significantly less pronounced for heterophilic Dsg3‐Dsg2 interactions. In contrast, an aDsg2 blocking antibody reduced heterophilic Dsg3‐Dsg2 and homophilic Dsg2‐Dsg2 binding to the same degree. Consequently, the aDsg2 antibody severely impaired already disturbed intercellular cohesion of Dsg3‐deficient keratinocytes, underlining the importance of Dsg2 for intercellular adhesion if Dsg3 is missing. Moreover, heterophilic Dsg2‐Dsg3 interactions showed a longer life‐time compared to homophilic Dsg2‐Dsg2 interactions which may contribute to their biological importance when Dsg3 homophilic interactions are compromised. Taken together, the data show that Dsg2 undergoes heterophilic interactions with Dsg3, which may serve as a compensatory mechanism to attenuate autoantibody‐induced loss of keratinocyte adhesion in pemphigus. This further reflects the biological significance of heterophilic Dsg3 interactions under pathological conditions.

Support or Funding Information

DFG FOR 2497 to JW, Else‐Kröner‐Fresenius‐Stiftung 2016_AW157 to FV
Exploring the Rap1A Active Site Through Accelerated Molecular Dynamic Simulations
Authors: Schwabe Michael , Holzapfel Genevieve , Mattos Carla
Published: April 2020

GTPases are hydrolytic proteins that convert GTP to GDP and serve as bivalent switches, where the GTP‐bound state is active, and the GDP‐bound state is inactive. Although Ras is the archetypal GTPase, Rap1A is closely related with 50% sequence homology and similar tertiary structure; a key difference occurs at position 61. In Ras Q61 participates in the hydrolysis mechanism and mutation leads to oncogenesis, while T61 in Rap1A is not thought to be involved in catalysis. The goal of this project is to explore the Rap1A active site and method of intrinsic hydrolysis. Two crystal structures were obtained with differing conformations of T61 in the Rap1A active site. Accelerated molecular dynamic simulations were run for 200 ns to sample the variety of motion present in Rap1A. Results indicate coordinated movements between the switch I, switch II, and helix 3 regions. Further, switch II appears less mobile than simulations with Ras isoforms.

Support or Funding Information

MCB‐1517295, MCB‐0818678