bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial dynamics in hepatitis B and C virus persistent viral infections (575.9)
Authors: Kim Seong‐Jun , Syed Gulam , Khan Mohsin , Sohail Muhammad , Siddiqui Aleem
Published: June 2021

Human hepatitis B and C virus (HBV and HCV) infections are the leading cause of chronic liver disease. Mitochondrial liver injury is associated with HBV and HCV infections. The objective of our studies is to demonstrate viral involvement in mitochondrial dynamics. Results: We show here that both HBV and HCV triggered mitochondrial fission and mitophagy. Both viruses induced perinuclear clustering of mitochondria, stimulated the gene expression of dynamin‐related protein 1 (Drp1) and its mitochondrial receptor, mitochondrial fission factor (Mff). Both viruses promoted Drp1 phosphorylation at Ser616 and its mitochondrial translocation followed by mitochondrial fission. Both viruses also stimulated the expression of Parkin, PINK1, and LC3B and induced Parkin recruitment to the mitochondria followed by mitophagy. Upon translocation to mitochondria, E3 ubiquitin ligase Parkin was self‐ubiquitinated and facilitated the ubiquitination and degradation of its substrate Mitofusin 2, a mediator of mitochondrial fusion. Interference of virus‐induced aberrant mitochondrial fission and mitophagy by silencing Drp1 and Parkin suppressed viral assembly and secretion with a concomitant decrease in cellular glycolysis and ATP levels. More importantly, it caused significant increase in apoptotic signaling evidenced by a notable increase in release of cytochrome C from the mitochondria, caspase‐3/7 activity, cleavage of poly(ADP‐ribose) polymerase (PARP) and caspase‐3, and apoptotic cell death. Conclusions: Aberrant mitochondrial dynamics induced by HBV and HCV infections attenuates apoptosis of infected hepatocytes. These events may also contribute to the persistent viral infection.

Grant Funding Source: NIH DK 077704
Inhibition of cholesterol and fatty acid synthesis by inhibiting SREBPs prevent diabetic nephropathy in db/db mice with type 2 diabetes (579.2)
Authors: Luo Yuhuan , Wang Xiaoxin , Levi Moshe
Published: June 2021

Sterol Regulatory Element Binding Proteins (SREBPs) are master transcriptional regulators of fatty acid and cholesterol synthesis. In this study we examined the effects of SREBP inhibition on diabetic nephropathy. To this end we treated db/db mice of Betulin for 12 weeks.

We found increased renal lipid accumulation in the kidneys of db/db mice mediated by increased expression of nuclear SREBP‐1 and SREBP‐2 protein. Treatment with Betulin prevented the upregulation of SREBPs in the kidney resulting in a correspondent reduction of kidney triglyceride and cholesterol accumulation. SREBP inhibition prevented the increases in urine albumin excretion (db/db: 59±13 µg/24hr; db/db‐betulin: 21±3 µg/24hr; p<0.05) and mesangial matrix expansion as shown by PAS staining of the kidneys of db/db mice (mesangial matrix index: db/db, 2.25±0.08; db/db‐betulin, 1.60±0.11; p<0.05). The treatment also prevented podocyte loss determined by staining with p57 and WT‐1. Furthermore, improvement in oxidative stress and inflammation was also observed in the treatment groups (urinary TBARS: db/db, 181±27 mmol/day vs. db/db‐betulin, 103±24 mmol/day, p<0.05; urinary MCP‐1: db/db, 146±22 mmol/day vs. db/db‐betulin, 83±15 mmol/day, p<0.05). To determine if Betulin has direct effects in kidney podocytes, we cultured human podocytes in high glucose conditions. Betulin prevented the high glucose induced increased the expression of SREBP‐1 and SREBP‐2 and lipid accumulation in human podocytes.

Our study therefore indicates a renoprotective role for SREBP inhibition in diabetic nephropathy by preventing lipid accumulation and modulating oxidative stress and inflammation.

Grant Funding Source: NIH, VA
Upregulation of endothelial cell notch ligand jagged 1 promotes tumorigenesis and metastasis in breast cancer (58.2)
Authors: Ararso Yonathan , Jiang Jie , Rafii Shahin
Published: June 2021

Angiogenesis and the establishment of a vascular network by tumor‐associated endothelial cells (ECs) are well‐described in cancer. Although ECs play an essential role in tumorigenesis by constructing vasculature, other mechanisms through which ECs support tumor survival and metastasis is largely unknown. Since Notch signaling is critical for breast carcinogenesis, we investigated the potential role of ECs in mediating Notch activation. We propose that by upregulating their expression of Notch ligand Jagged 1(Jag 1), ECs induce aberrant paracrine Notch signaling in breast cancer cells, thereby providing an instructive signal for tumor growth and progression. To determine the role of EC Jag 1 expression on breast carcinogenesis, we established 3D co‐cultures of human breast cancer cells (MDA‐MB‐231) with either wild‐type or Jag 1 knockdown (KD) human umbilical vein ECs (HUVECs). We found that Jag1 loss in ECs induced decreased tumor growth, mammo‐angioshpere formation, and CD40 and CD133 expression in MDA‐231 cells. Co‐injection of Jag1 KD co‐cultures into immunocompromised mice also resulted in decreased tumor weight and volume compared to wild type co‐injections. Conditional Jag1 deletion in ECs of PyMT‐MMTV transgenic mice also led to decreased primary tumor growth and lung metastasis. These results suggest that EC Jag1 upregulation supports Notch‐dependent breast cancer cell growth and metastasis

Grant Funding Source: Supported by the Howard Hughes Medical Institute
Triple negative human breast cancers accumulate significantly less Arg‐pyrimidine moieties, than other subtype lesions (58.3)
Authors: Castronovo Vincent , Chiavarina Barbara , Nokin Marie‐Julie , Durieux Florence , Bianchi Elettra , Irigaray Philippe , Uchida Koji , Belpomme Dominique , Bellahcène Akeila
Published: June 2021

Metabolic disorders influence breast cancer development and progression.

Methylglyoxal (MGX), is a highly reactive glycolytic byproduct able to inflict carbonyl stress to proteins, lipids and DNA. The possible role of MGX in breast cancer development has not been yet extensively explored. We analyzed the accumulation of Arg‐pyrimidine adducts, in a series of more than 100 human breast adenocarcinoma and we observed a consistent increased of MGX adducts in cancer cells compared to the non‐tumoral counterpart. Most triple negative lesions examined exhibited low accumulation of Arg‐pyrimidine residues compared other subtypes. Intriguingly, the level of expression of glyoxalase 1 (Glo‐1), an enzyme that detoxifies MGX, was similar in both triple negative and other subtype lesions, suggesting a potential difference in the Glo‐1 activity. This hypothesis is supported by our preliminary results on breast cancer cell lines, showing that triple negative cells are more efficient to respond to treatment with MGX by increasing the activity of Glo‐1. Our study represent the first observation that Arg‐pyrimidine adducts accumulation is a consistent event in human breast cancer with a differential detection between triple negative and other breast cancer subtypes. Our preliminary data suggest that methylglyoxal production and detoxification could be a molecular link between metabolic diseases and breast cancer.

Grant Funding Source: This study is supported by Founds for National Research (FNRS), Belgium
Myoferlin is a key regulator of HER receptor family function in breast cancer (58.5)
Authors: Turtoi Andrei , Blomme Arnaud , Bellahcène Akeila , Hennequière Vincent , Lifrange Eric , Delvenne Philippe , Castronovo Vincent
Published: June 2021

Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair and endocytosis events. Although few reports associate myoferlin with cancer, its actual role in tumor biology remains to date unveiled. Owing to a proteomic approach, we discovered myoferlin as an overexpressed cell membrane associated protein in human breast adenocarcinoma. We validated this observation in 150 breast cancer cases. We further investigated the biological role of myoferlin in breast cancer and found that it is a key functional regulator of HER receptor family. Myoferlin depletion blocked EGF‐induced breast cancer cell migration and epithelial‐to‐mesenchymal transition. Mechanistically, lack of myoferlin led to impaired degradation of phosphorylated EGFR via dysfunctional caveolae. Inability to degrade pEGFR caused sustained activation and malfunction of downstream targets like AKT. NRG1 stimulation of myoferlin silenced, HER2 positive, breast cancer cells demonstrated similar dysfunctions in HER2 and HER3 activity. Using two different animal models of breast cancer, we show that myoferlin depletion significantly suppressed tumor and metastasis development. Although the effects observed with different HER family members appear to involve distinct mechanisms, the data strongly evidence a paramount role of myoferlin in HER receptor function in cancer.

Grant Funding Source: This work is supported by the National Fund for Scientific Research (FNRS, Belgium) and ULG.
Asporin is a key stromal regulator of TGF‐β‐1 function in breast cancer (58.7)
Authors: Maris Pamela , Blomme Arnaud , Bianchi Elettra , Castronovo Vincent , Turtoi Andrei
Published: June 2021

Asporin is extracellular matrix protein that has been associated with bone and joint diseases. Using innovative proteomic analysis we have previously found asporin as overexpressed in pancreas and breast cancer. In the current work we explore for the first time the functional significance of asporin expression in cancer. We show in 150 cases of breast carcinoma that asporin is a consistent feature of the tumor, whose high levels positively correlate with the good patient outcome. On the molecular level we demonstrate that only tumor‐associated fibroblasts are able to secrete asporin after being reprogrammed by the cancer cells. We found that TGF‐β‐1 alone is able to induce asporin expression and in turn asporin can bind TGF‐β‐1 to inhibit its action on TGF‐β receptors. The main interaction between the two proteins occurs via asporin 159‐205 amino acid region. Synthetic peptide corresponding to this key asporin segment is able to inhibit TGF‐β‐1 induction of SMAD2 phosphorylation in breast cancer cells. Using a TGF‐β‐1‐based model of epithelial to mesenchymal transition (EMT) in A549 cells, we further show that asporin peptide is able to inhibit TGF‐β‐1‐induced EMT.

Our work identifies asporin as a natural inhibitor of TGF‐β‐1 activity in cancer, offering a novel possibility of targeting TGF‐β‐1‐driven tumor progression.

Grant Funding Source: University of Liege
New arylamidas and arylimidas as potential cholinesterase inhibitors for treatment of Alzheimer’s disease (582.2)
Authors: Cipres‐Flores Fabiola , Trujillo‐Ferrara Jose , Cuevas‐Hernandez Roberto , Correa‐Basurto Jose , Soriano‐Ursua Marvin
Published: June 2021

Alzheimer’s disease (AD) is a pathology for which major features are by progressive memory lacking as well as gradual and irreversible disruption of cognitive functions that eventually leads to dementia. Furthermore, Alzheimer’s disease presents a loss of forebrain cholinergic neurons and a progressive decreasing of acetylcholine (ACh). ACh activity in the brain is regulated by the hydrolytic action of cholinesterase (AChE). Therefore, the palliative treatment of Alzheimer's disease is given by the AChE inhibitors.

The aim of this work was to evaluate 4 arylamides and 4 arylimides synthesized as potential inhibitors of the AChE according to previous evaluations carried out by means of in silico assays. The selected compounds were synthesized from 4‐chloroaniline or N, N‐dimethyl‐P‐phenyl‐endiamine with maleic anhydride and/or succinic anhydride. The synthesis procedure and chemical characterization is in agreement with proposed structures. The evaluation of these compounds as AChE inhibitors allow us to select two of them which showed notably greater inhibition on the enzyme. Thus, these two compounds could have potential therapeutic in AD.

Grant Funding Source: Supported by Consejo Nacional de Ciencia y Tecnología (62488), COFAA and SIP of IPN‐Mexico
Heritable transformation of adaptive landscapes elicited by transient expression of intrinsically disordered proteins (586.2)
Authors: Jarosz Daniel
Published: June 2021

Many environmental stresses elicit specific responses that promote survival. Whether such transient changes in gene expression have additional long‐term consequences is unknown. Here we examine the overexpression phenotypes of each open reading frame in the yeast genome under a diverse set of growth conditions. Over fifty proteins created an enduring change in phenotype when overexpression was stopped. The vast majority of these proteins harbor intrinsically disordered domains, and many are also regulated by environmental stress. Most of the traits produced by transient overexpression were beneficial and obeyed the non‐Mendelian patterns of inheritance typical of yeast prions. Like well‐known yeast prions, these traits critically depended on cellular protein homeostasis networks. However, unlike most other yeast prions, inheritance of these phenotypes often did not involve the formation of amyloid by the causal protein or require the protein disaggregase Hsp104. Rather, these traits depended on the protein folding activity of the Hsp70 chaperone to be transmitted from mother cells to their daughters, thus defining a new type of protein‐only inheritance. Taken together, our findings suggest that transient bursts in gene expression can heritably reshape adaptive landscapes by driving the acquisition of self‐perpetuating protein conformations.
Hepatic metabolism of 3‐hydroxypropionate, a toxic byproduct in propionic acidemia (590.2)
Authors: Wilson Kirkland , Han Yong , Koshy John , Brunengraber Henri , Tochtrop Gregory , Zhang Guofang
Published: June 2021

Propionic acidemia (PA) is a rare autosomal recessive disorder leading to inhibition of propionate entering an anaplerotic pathway, via propionyl‐CoA, of the citric acid cycle (CAC). PA is characterized by a defect in the conversion of propionyl‐CoA to methylmalonyl‐CoA through propionyl‐CoA carboxylase (PCC). Decreased activity of PCC shunts propionyl‐CoA into alternative metabolic pathways forming 3‐hydroxypropionate (3‐HPA), which increases in concentration to toxic levels. We investigated the metabolic fates of 3‐HPA in rat livers using the combination of metabolomics studies and mass isotopomer analysis. Isolated rat livers were perfused with recirculating buffer containing 2 mM (un)labeled 3‐HPA ([13C3] and [1‐13C]3‐HPA). Perfusate and liver tissue were analyzed via GC‐MS and LC‐MS/MS. 3‐HPA was shown to be converted to acetyl‐CoA, with loss of carbon‐1 from 3‐HPA rather than enter into the CAC anaplerotically via propionyl‐CoA. The proposed pathway of 3‐HPA conversion to malonyl‐CoA was disproven using [13C3] 3‐HPA labeling studies. Instead β‐alanine was labeled with further conversion to acetyl‐CoA proposed. The newly identified metabolic pathway of 3‐HPA in the rat liver may play important role in the detoxification of this compound.

Grant Funding Source: Supported by CRTP T32 Training Grant
Quantitative shotgun proteomics reveals early markers of colorectal carcinogenesis (591.1)
Authors: Uzozie Anuli , Nanni Paolo , Staiano Teresa , Grossmann Jonas , Barkow‐Oesterreicher Simon , Shay Jerry , Tiwari Amit , Buffoli Federico , Laczko Endre , Marra Giancarlo
Published: June 2021

Colorectal adenomas are cancer precursor lesions of the large bowel. Using iTRAQ 8‐plex labelling, OFFGEL electrophoresis, and LC‐coupled tandem MS, we investigated the proteome of 30 colorectal adenomas and paired normal mucosal samples (prospectively collected during colonoscopy), and that of normal (HCEC) and cancerous (SW480, SW620, CACO2, HT29, CX1) colon epithelial cell lines. A total of 4325 and 2017 non‐redundant protein families were identified and quantified in tissue and cell lines, respectively. Principal component analysis clearly distinguished adenomas from normal mucosa, and cancer cell lines from HCEC cells. Two hundred twelve proteins displayed significant adenoma‐related expression changes (q‐value<0.02, mean fold change +/‐1.4), which correlated (r=0.74) with similar changes previously identified by our group at the transcriptome level. Fifty one proteins displayed directionally similar expression changes in colorectal cancer cells (vs HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited protein expression changes previously documented only in advanced colorectal cancers. They also disclosed several novel changes with potentially important roles in early‐stage large bowel tumorigenesis, including the marked upregulation of a key enzyme in the polyol pathway.

Grant Funding Source: Supported by the Swiss National Science Foundation
Characterization of the binding of antiproliferative factor to cytoskeleton associated protein 4 by surface plasmon resonance (592.1)
Authors: Planey Sonia , Ling Jun
Published: June 2021

Interstitial cystitis (IC) is a chronic, debilitating bladder disease that affects mostly women and is frequently misdiagnosed due to lack of a non‐invasive test to detect the disease. While the cause remains unknown, biomarkers for IC have been described, including antiproliferative factor (APF), a glycopeptide that is detectable in the urine of 95‐97% of IC patients vs. normal controls. Validation of APF as a biomarker and etiologic agent for IC has been hindered by the absence of robust assays to detect and measure its concentration in patient urine. In this study, we evaluated the utility of surface plasmon resonance (SPR) to detect and quantitate APF in solution through binding to its cellular receptor, cytoskeleton associated protein 4 (CKAP4). We purified histidine‐tagged CKAP4 (HT‐CKAP4) and immobilized it on a NTA Sensor Chip surface via ionic interaction. Robust binding to CKAP4 antibody indicated that the purified HT‐CKAP4 was active and in native conformation; moreover, we demonstrated direct binding to APF analyte at a level detectable above the background control. Specificity of the HT‐CKAP4/APF interaction was confirmed by a competition assay demonstrating titration of CKAP4 antibody binding to HT‐CKAP4 by increasing concentrations of APF analyte. Future studies will determine the ability this SPR‐based assay to detect APF in urine from patients with IC with the goal of developing a non‐invasive, point‐of‐care diagnostic test for IC.

Grant Funding Source: USA MED Research ACQ Activity W81XWH‐13‐1‐0454
Using fluorescence recovery after photobleaching to calculate viscosity and velocity in the phloem sap of roots in Arabidopsis thaliana (594.2)
Authors: Beecher Sierra , Knoblauch Michael , Jensen Kaare
Published: June 2021

The sieve tube system (STS) of phloem is the conduit by which energetic molecules for plant growth and storage are transported from the leaves, where they are constructed (sources), to non‐photosynthetic regions of the plant (sinks). A clear understanding of the physics and physiology of this system is crucial, as seeds and other storage organs of plants are staple foods for most human diets, and plant growth is dependent on this phenomenon. The pressure flow hypothesis, presented in 1930 by Ernst Munch states that a pressure differential built from a concentration gradient of photosynthate (mainly sucrose, in the case of A. thaliana) causes sap to flow from sources to sinks in vascular plants. If this is the case, in vivo measured parameters of this system should conform to the Hagen‐Poiseuille equation for pressure flow. Using confocal laser scanning microscopy (CLSM) to image young root phloem in Micro‐ROCS© (Rhizosphere Observation Chambers) has allowed measurement of several geometric features of phloem relevant to the Hagen‐Poiseuille equation. By making films of the SES refilling with dye‐loaded sap after photobleaching in specific regions, translocation can be observed and measured. Analyzing intensity recovery at regions along this refilling pathway generates curves that reveal not only translocation, but also diffusive properties of the fluorophore. Small capillary tubes were used as “simulated phloem” to verify this method with known concentrations of the fluorophore dissolved in solutions of known viscosity. Similar FRAP methods were used to analyze parameters of conditions in the tubes, and compare them with the measurements obtained from plants.
Dietary glutamate: interactions with the enteric nervous system (596.1)
Authors: Wood Jackie , Wang Guo‐Du , Wang Xi‐Yu
Published: June 2021

Luminal glutamate concentration rises during protein digestion in the small intestine and passes the ENS during absorption. Glutamate, in the brain (CNS), is an excitatory neurotransmitter. We assessed a premise that glutamatergic signalling in the ENS recapitulates its transmitter role in the CNS. Pharmacological analysis with receptor agonists and antagonists together with immunohistochemical localization of glutamate transporters and receptors was used. Analysis focused on electrical and synaptic behavior of ENS neurons, stimulation of mucosal secretion by secretomotor neurons and muscle activity mediated by musculomotor neurons. Immunoreactivity for glutamate was expressed in ENS neurons. ENS neurons expressed immunoreactivity for the EAAC‐1 glutamate transporter. Neither L‐glutamate nor glutamatergic receptor agonists excited ENS neurons. Metabotropic glutamatergic agonists did not stimulate neurogenic mucosal chloride secretion. Neither L‐glutamate nor the metabotropic glutamatergic agonist, ACPD, changed the mean amplitude of spontaneously occurring contractions in circular or longitudinal strips of intestinal wall from guinea pig or human small intestine. Early discoveries, for glutamatergic neurotransmission in the CNS, inspired expectation that study in the ENS would yield discoveries recapitulating the CNS glutamatergic story. We found this not to be the case.

Grant Funding Source: The work was supported in part by National Institutes of Health grant: NIH 2 R01 DK 037238 and a gra
Mast cell activation by glia maturation factor, 1‐methyl‐4‐phenylpyridinium and α‐synuclein: implications for Parkinson’s disease (596.5)
Authors: Zaheer Asgar , Kempuraj Duraisamy , Khan Mohammad , Javed Hayate , Zaheer Smita
Published: June 2021

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra. We have previously reported elevation of glia maturation factor (GMF) in the brains of Alzheimer’s disease and Multiple Sclerosis. Mast cells are both sensors and effectors in communication between nervous, vascular and immune systems, and also mediate inflammation. We have analyzed the effect of GMF on mouse bone marrow‐derived mast cells (BMMCs), human umbilical cord blood‐derived mast cells (hCBMCs) and mouse embryonic brain‐derived astrocytes. We demonstrate that GMF activate BMMCs to release chemokine CCL2, and that BMMCs‐derived from GMF‐KO mice released less CCL2 when compared to BMMCs obtained from Wild type mice. We also report that GMF, and PD‐relevant toxins α‐synuclein and MPP+, a metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) activated hCBMCs to release IL‐8. We also found novel GMF expression in both hCBMCs as well as BMMCs by immunocytochemistry. Flow cytometric analyses have demonstrated that GMF and MPP+ increased the expression of IL‐33 in mouse astrocytes. GMF also induced the expression of costimulatory protein CD40 in astrocytes. Our findings that activation of mast cells by GMF, MPP+ and α‐synuclein to release proinflammatory molecules along with GMF expression by mast cells may provide a new drug target for neurodegenerative diseases.

Grant Funding Source: Supported by VA Merit Review award and NINDS grant NS073670
The amyloid precursor protein of Alzheimer’s disease localizes to a domain adjacent to synaptic vesicles of the presynaptic terminal (596.9)
Authors: Protsenko Ekaterina , DeGiorgis Joseph
Published: June 2021

Alzheimer’s disease (AD) afflicts an estimated 26 million individuals worldwide and its incidence is predicted to reach 100 million by 2050. While abnormal aggregation of the A‐beta component of the amyloid precursor protein (APP) has been causally linked to AD, APP’s endogenous function remains elusive. While a number of reports claim that APP associates with synaptic vesicles, our recent experiments by confocal microscopy suggest that APP localizes to a region near the synaptic vesicles but does not directly overlap with the vesicular domain. Synaptic vesicles were identified by antibodies against the synaptic vesicle protein SV2, while APP was identified by an antibody against the C‐terminus of human APP. Similar experiments using antibodies against APP and the post‐synaptic marks PSD‐93 and PSD‐95 show that APP also does not overlap with the post‐synaptic density (PSD). We are currently immune‐localizing APP at the EM level in goldfish spinal cord using nano‐gold conjugated secondary antibodies and silver enhancement. All microscopy work was performed using goldfish and lamprey tissues. Sequencing of the species’ respective APP genes shows that these sequences share high sequence homology with the human APP responsible for the disease.

Grant Funding Source: Supported by the Rhode Island INBRE Program NIMGS grant 5 P20 GM103430‐13
Desensitization of DMSO‐treated platelets to common agonists via membrane modulation (598.5)
Authors: Yamaguchi Jonathan , Tran Phat , Sen Nivedita , DeCook Tracy , Slepian Marvin
Published: June 2021

Thrombosis and thromboembolic complications continue to plague mechanical circulatory support devices (MCS). Biochemical pathways involving prostaglandin and ADP are known targets for aspirin and thienopyridines to modulate platelet activation and aggregation, respectively. However, a recent study in our lab has demonstrated the limited ability of these agents to alter shear‐mediated platelet activation. Therefore, this study examines the impact of altering the platelet membrane fluidity by treatment with dimethyl sulfoxide (DMSO) and its inherent effects on platelet activation by standard agonists. DMSO was chosen due to its previously reported antithrombotic properties. Although it was reported that DMSO inhibits platelet activation through cyclooxygenase‐1 inhibition, we believe that the alteration in membrane fluidity also plays a key role in desensitization.

Human venous blood was collected from healthy volunteers in citrated buffer with UA‐IRB consent. Platelet rich plasma (PRP) was isolated and treated with DMSO ranging from 0.95%‐60% (v/v). Light transmission aggregometry (LTA) was performed to detect platelet desensitization using arachidonic acid, ADP, collagen, epinephrine, and ristocetin. Platelets were fixed and visualized at the varying DMSO treatments under scanning electron microscopy (SEM).

Collagen and epinephrine had limited effect on PRP aggregation ranging from 0.95% to 10% of DMSO. ADP and arachidonic acid showed a clear inhibition at 10% and 5%, respectively. DMSO had no effect on ristocetin‐induced platelet aggregation. SEM of platelets at 5% and 10% showed significant reduction in filopodia.

DMSO altered platelet membrane fluidity and desensitized their aggregability from certain common agonists; thus reflecting the role of DMSO in some biochemical pathways that inhibit platelets. Next, we will measure the platelet membrane fluidity using TMA‐DPH and DPH fluorescence anisotropy in the presence of DMSO to further validate our results.
Intestinal epithelium specific knockout of nonmuscle myosin II A disrupts epithelial barrier and exaggerates experimental colitis (60.5)
Authors: Naydenov Nayden , Feygin Alex , Conti Mary , Adelstein Robert , Ivanov Andrei
Published: June 2021

The integrity of epithelial monolayers depends on the formation of tight junctions (TJ) and adherens junctions (AJ). TJ and AJ associate with underlying actomyosin bundles that are critical for their stability and function. Our previous in vitro studies have identified nonmuscle myosin II A (NM IIA) as a unique regulator of AJ/TJ structure and barrier properties in the intestinal epithelium. In this study, we investigated the roles of NM IIA in the maintenance of the intestinal epithelial barrier in vivo. Mice carrying a floxed allele of NM IIA were crossed with villin‐cre mice. The progeny demonstrated Mendelian inheritance, suggesting that this NM IIA conditional knockout (cKO) is not embryonically lethal. Immunoblotting indicated that NM IIA expression was specifically ablated solely in the intestinal epithelium, but not in the brain, lung, kidney or liver. NM IIA cKO showed an increased baseline mucosal permeability and exaggerated dextran sodium sulfate (DSS)‐induced breakdown of the intestinal barrier. Furthermore, NM IIA cKO animals demonstrated increased body weight loss, amplified disease activity and epithelial damage during DSS colitis. These findings highlight NM IIA as an important regulator of barrier properties of normal and inflamed intestinal mucosa in vivo. Supported by the Crohn’s and Colitis Foundation of America (NGN) and NIH grants DK083968 and DK084953 to AII.

Grant Funding Source: Supported by the Crohn’s and Colitis Foundation of America (NGN) and NIH grants DK083968 and DK08495
Necrotizing enterocolitis leads to renal abnormality mediated through tight junction proteins (60.8)
Authors: Garg Parvesh Mohan , Tatum Rodney , Ravisankar Srikant , Hewan‐Lowe Karlene , Shekhawat Prem , Chen Yan‐Hua
Published: June 2021

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Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Clinically, it is associated with hyponatremia & fluid imbalance. NEC is associated with changes in the intestinal tight junction (TJ) proteins, but its effect on kidney TJ proteins is unknown. We investigated the expression of TJ proteins in kidney using a mouse NEC model. Four day old C57BL/6 pups were divided into 2 groups: the control group was dam‐fed; the experimental group was fed 50 µl 33% Esbilac formula every 3 h and subjected to asphyxia ( 100% N2 x 60 s) and cold stress (4°C for 10 min) twice a day to develop NEC. After 4 days of treatment, kidneys and intestines were collected. NEC was confirmed in intestines by histological exam. We found that the kidneys from the NEC group displayed interstitial edema. On western blot analysis, claudin‐1, ‐2, ‐3, and ‐7 were increased in the NEC kidney group compared to dam‐fed controls while the claudin‐5 expression remained unchanged. These findings were also confirmed on immunofluorescence. The levels of NF‐κB and p‐ERK1/2 in kidneys were elevated in the NEC group. No obvious apoptotic bodies and necrosis was seen in NEC group compared to controls. In conclusion, expression of several TJ proteins in kidneys was increased in the NEC group which may explain the reason behind kidney abnormality.

Grant Funding Source: Supported by NIH grant number HL085752
Global lipocalin 2 deletion does not reverse high‐fat diet‐induced obesity resistance in mice lacking skin stearoyl‐CoA desaturase‐1 (605.10)
Authors: Friedlander Nicholas , Burhans Maggie , O'Neill Lucas , Ade Lacmbouh , Ntambi James
Published: June 2021

In the past forty years, obesity has grown into a pressing health issue with global implications. Obesity significantly increases the risk of developing secondary conditions ranging from insulin resistance and atherosclerosis to certain types of cancer. Our lab previously determined that mice lacking stearoyl‐CoA desaturase‐1 (SCD1) in the skin (SKO) were lean and protected from high‐fat diet (HFD) induced adiposity and hepatic steatosis. Moreover, expression of lipocalin 2 (Lcn2), a gene with a controversial role in mediating obesity, was significantly elevated in the skin and plasma of these mice. In the current study, we hypothesized that Lcn2 may mediate some of the effects of the protection against HFD‐induced obesity in SKO mice. To analyze this, we crossed Lcn2‐deficient mice with SKO mice to produce Lcn2‐/‐; SKO, or double‐knockout (DKO) mice. Surprisingly, DKO mice remained protected from HFD‐induced obesity and hepatic steatosis relative to SKO mice. In conclusion, our data strongly suggests that, although Lcn2 is significantly upregulated in SKO mice, it does not mediate the beneficial metabolic effects of reduced adiposity and liver triglyceride accumulation under HFD feeding conditions.

Grant Funding Source: Supported by NIH
Oxytocin attenuates beta oxidation and fatty acid synthesis through CPT1α and FABP4 (605.8)
Authors: Han Jen Lin , Mersmann Harry , Shin Torng Ding
Published: June 2021

Oxytocin has a role in regulating body energy homeostasis. It’s demonstrated that central and subcutaneous oxytocin infusions stimulated lipid metabolism both in standard and high fat diet mice and rats. To study the effects of oxytocin on lipid metabolism, we tested series concentrations of oxytocin (0, 10, 33, 100, 150, 333 nM) to 3T3L1 adipocytes for both long (216 hours) and short (24 hours) time periods. Our results showed that short term treatment of 150 nM oxytocin significantly increased triglyceride accumulation, but decreased mRNA expressions of forkhead box protein O1 (FoxO1), carnitine palmitoyltransferase Iα (CPT1α), fatty acid binding protein 4 (FABP4); however, only CPT1α decreased after long term treatment of 150 nM oxytocin. To expand the in vitro results, we further treated oxytocin on adipocytes isolated and induced from mesenchymal stem cells of tibia and femur in mice. After short term treatment of oxytocin, the mRNA expression of FABP4 had the same tendency as in 3T3L1 adipocytes. Taken together, oxytocin may modulate lipid metabolism through decreasing beta oxidation and affecting fatty acid synthesis.
The omega 3‐6 balance in foods predicts 3:6 proportions in tissue HUFA (605.9)
Authors: Lands Bill
Published: June 2021

Objective: Many chronic health disorders are unintended results of eating foods with imbalanced amounts of n‐3 and n‐6 essential fatty acids (EFA) that accumulate in tissues as highly unsaturated fatty acids (HUFA) and lead to excessive chronic actions by n‐6 eicosanoids. To help people prevent chronic tissue imbalances, we made a simple tool to predict the impact of a food item on tissue HUFA.

Methods: An empirical equation successfully predicts the quantitative impact of nutrient EFA on tissue HUFA proportions. Using it with data from the USDA Nutrient Database for eleven n‐3 and n‐6 nutrients in terms of 3‐6 differences led to Omega 3‐6 Balance Scores for over 5,000 foods (Lands B, Lamoreaux E. Nutrition & Metabolism 2012; 9:46‐54). The Score is a single number that gives the likely impact of a food on the proportions of n‐3 or n‐6 acids which will develop in the HUFA of a person’s finger‐tip blood sample.

Results: Foods with more positive 3‐6 Balance Scores raise the % omega‐3 in HUFA, whereas those with more negative Scores raise the % omega‐6. Calorie‐weighted average daily Scores for ethnic food styles worldwide range from +3 to ‐7. The 3‐6 Balance Scores at the public website http://www.fastlearner.org/Omega3‐6BalanceApp.htm can be downloaded to mobile devices to help guide personal food choices when shopping, preparing meals or discussing foods with friends. Examples are: broccoli, +3; mackerel, +52; flax oil,+46; potato, 0; rice, 0; salmon, +46; spinach,+5; avocado, ‐10; hummus, ‐ 16; peanut butter, ‐19; potato chips, ‐ 22; tofu, ‐26; vegetable oil (corn, ‐59; soybean, ‐50); walnuts, ‐44.

Conclusions: Explicit Scores replace vague, general advice to eat “healthy meals”. To lower the risk for diseases mediated by n‐6 eicosanoids, people can select more positive Scores as they “NIX the 6 and EAT the 3”. Effective health care works better when preventing the cause rather than just treating symptoms created by the cause.
Overcoming blood‐brain barrier by targeted drug delivery to brain tumor vasculature with carbohydrate mimetic peptide in mouse glioma model (607.13)
Authors: Suzuki‐Anekoji Misa , Yeh Jiunn‐Chern , Nonaka Motohiro , Shibata Toshiaki , Nakayama Jun , Akama Tomoya , Sugihara Kazuhiro , Fukuda Minoru , Fukuda Michiko
Published: June 2021

Although glioma cells cultured in vitro are responsive to several anti‐cancer drugs, it is very difficult to treat glioma in vivo due to blood‐brain barrier (BBB). In the previous study, we identified a carbohydrate mimetic peptide, designated as IF7, that homes tumor vasculature. IF7 binds to annexin A1, which is expressed specifically on the endothelial cell surface of malignant tumors. We showed that IF7 conjugated anti‐cancer drug SN38 (IF7‐SN38) suppressed growth of colon, melanoma, breast, prostate and lung tumor models in the mouse. Since IF7 was thought to be transported across endothelial cells through transcytotic pathway, we hypothesized that IF7 can deliver anti‐cancer drug to brain stroma overcoming BBB.

In this study, we produced glioma tumors in mouse brains by intracranial injection of C6 cells. To determine targeting of IF7 peptide, Alexa 488‐tagged IF7 (IF7‐A488) was injected intravenously. IF7‐A488 targeted tumor vasculature within minutes, penetrated through endothelial cell wall and spread to the glioma sphere, while control peptide conjugate did not. We injected luciferase‐expressing C6 (C6‐Luc) cells into mosue brains, and monitored growth of C6‐Luc tumor by Xenogen IVIS imager. Daily intravenous injection of IF7‐SN38 reduced size of C6‐Luc glioma with minimum effective dose at 2.7 µmoles/kg, which is 15 times lower than reported dose of SN38. To compare targeting efficacy of IF7‐SN38 between brain tumor and subcutaneous tumor, two B16‐Luc tumors, one in the brain and another under the skin, were generated in a mouse. Intravenous injection of IF7‐SN38 suppressed growth of these tumors with comparable efficiency regardless of their locations at concentration of 5 µmoles/kg, while unconjugated SN38 suppressed growth of only subcutaneous tumor at 50 µmoles/kg but not the brain tumor, demonstrating the efficacy of IF7‐SN38 in overcoming BBB.

These results strongly encourage clinical application of IF7‐SN38 to glioma tumors.

Grant Funding Source: Supported by NIH grant CA33859
SLC2A11 (GLUT11) as mannose preferential transporter (607.15)
Authors: Ichikawa Mie , Losfeld Marie‐Estelle , Freeze Hudson
Published: June 2021

Most studies of hexose transporters (GLUT family) focus on glucose and fructose import for energy generation. Few studies examine their role in delivering sugars for biosynthetic pathways. Exogenous mannose can directly contribute nearly 50% of the mannose found in glycoproteins, even though its extracellular concentration (50 µM) is 100‐fold lower than physiological glucose (5mM). To explain this preference, we hypothesized the existence of a high affinity mannose‐preferential hexose transporter. We identified a patient with damaging mutation in SLC2A11 (Glucose transporter, GLUT11) and applied our new GC‐MS method to determine the contributions of 13C‐glucose and 13C‐mannose to N‐glycans. Patient fibroblasts showed reduced direct contribution of mannose, prompting us to investigate GLUT 11 as potential mannose transporter. RNAi knockdown of GLUT11 confirmed that it directly contributes mannose to N‐glycans , especially at the physiological range (50 µM) suggesting that GLUT11 has low Kuptake for mannose. The reported Km for glucose is 160µM. Preliminary results show that knockdown of GLUT 1 has no effect on mannose flux into N‐glycans. We suggest that GLUT11 is the first mannose‐preferential transporter for N‐glycan synthesis. Other mannose preferential transporters may exist.

Grant Funding Source: R01DK55615 and The Rocket Fund
How gene knockouts perturb the N‐glycome of Caenorhabditis elegans (608.3)
Authors: Wilson Iain , Yan Shi , Paschinger Katharina
Published: June 2021

Although the model nematode Caenorhabditis elegans is considered to be rather simple, its N‐glycome proves to be one of the more complex amongst lower multicellular eukaryotes. Indeed, the number of types and the unusual structure of N‐glycans in this organism seemingly outmatches that of plants or insects. Underlying this is a wealth of glycosyltransferases; as judged by homology, there are well over twenty fucosyltransferases encoded by the worm genome, but the biochemical functionality of only a minority has been assigned. Glycomic analyses would suggest that at least five fucosyltransferases act on N‐glycans of the nematode: beyond the ‘typical’ core α1,3‐ and core α1,6‐fucosyltransferases modifying the distal core N‐acetylglucosamine residue, one fucosyltransferase transfers to the proximal residue of the core, at least one to the periphery of paucimannosidic glycans and at least one to the ‘Galβ1,4Fuc’ epitope. Furthermore, N‐acetylglucosaminyltransferases, processing hexosaminidases and Golgi mannosidase II play an important role in shaping the final N‐glycome. Using a mix of typical assays, glycan microarrays and glycomic analyses of knock‐out strains, we have determined the biochemical function of a number of relevant enzymes. It is also remarkable how novel structures become apparent when analysing the glycomes of ‘multiple‐knock‐out’ strains. Thus, at least under laboratory conditions, a high degree of plasticity of the worm glycome is possible.

Grant Funding Source: Austrian Science Fund (FWF) and European Commission (Euroglycanarrays ITN)
IR‐induced phosphorylation of rpS3 and TRAF2 regulates radioresistance in non‐small cell lung cancer cells (609.1)
Authors: Kim EunGi , Kwon TaeWoo , Son Beomseok , Lee Jongbong , Youn BuHyun , Lee Jung Sub
Published: June 2021

Radioresistance is a main impediment to effective radiotherapy. However, the exact molecular mechanism of radioresistance has not been understood yet. To elucidate the mechanism in lung cancer, we compared radiation responses in two types of non‐small cell lung cancer (NSCLC) cells with different radiosensitivity. In radioresistant NSCLC cells, ionizing radiation (IR) led to CK2α‐ and PKC‐mediated phosphorylation of rpS3 and TRAF2, respectively, which induced dissociation of rpS3‐TRAF2 complex and NF‐кB activation, resulting in up‐regulation of prosurvival genes. Also, dissociated phospho‐rpS3 translocated into nucleus and bound with NF‐кB complex, contributing to p65 DNA binding property and specificity. However, in radiosensitive NSCLC cells, IR‐mediated rpS3 phosphorylation was not detected due to the absence of CK2α overexpression. Taken together, our findings revealed a novel radioresistance mechanism through functional orchestration of rpS3, TRAF2, and NF‐кB in NSCLC cells. Moreover, we provided the first evidence for the function of rpS3 as a new TRAF2‐binding protein and demonstrated that phosphorylation of both rpS3 and TRAF2 is a key control point of radioresistance in NSCLC cells.

Grant Funding Source: Supported by the Radiation Technology R&D Program (2013M2A2A7042502)
Effect of pathway‐interconnectors in SEB‐induced apoptosis related events in human PBMCs (609.19)
Authors: Waldschmidt James , Kivel James , Wielnau Melissa , Mendis Chanaka
Published: June 2021

Effect of Pathway‐Interconnectors in SEB‐Induced Apoptosis Related Events in Human PBMCs Staphylococcus enterotoxin B (SEB) is produced by the bacterium Staphylococcus aureus. SEB is the most common cause of food poisoning often accompanied with nausea, diarrhea, and intestinal cramping. The purpose of this study is to further investigate human peripheral blood mononuclear cells (PBMCs) in order to better understand the apoptosis related events induced by SEB. C‐Jun N‐Terminal Kinase (JNK) has been previously identified to be induced by SEB, and has known to inter‐connect multiple signaling pathways. As a crucial pathway inter‐connector in SEB‐induced human PBMCs, we believe that JNK may possess inhibitory effects of SEB‐induced apoptosis. Here we will attempt to evaluate the type of pathway (intrinsic [caspase 3, MCLR1, BAX, etc.], extrinsic [caspase 8, caspase 10] utilized by SEB by studying the gene expression profile using RT‐PCR. We will also investigate the Protein expression patterns of some genes through ELISA. We believe that our work will allow us to better understand the complex interactions of multiple signal transduction pathways induced by SEB.
Modulation of Notch signaling by intracellular bacterial toxins (610.3)
Authors: Larabee Jason , Ballard Jimmy
Published: June 2021

The Notch signaling pathway has recently been recognized as a critical signaling pathway in macrophages. This study examines how Notch signaling functions in the context of infectious disease as well as how contrasting intracellular bacterial toxins modify this pathway. In this work, we investigate how the Notch signaling pathway is altered in macrophages by Bacillus anthracis edema toxin (ET) and Clostridium difficile toxin B (TcdB). In preliminary studies, ET was found to activate and augment Notch signaling in macrophages through a mechanism involving transducin‐like enhancer of split (TLE) and recombinant recognition sequence binding protein at the Jκ site (RBP‐J). Because ET, an immunosuppressive toxin, activates Notch signaling in macrophages, we hypothesized that TcdB, an inflammation stimulating toxin, may have an alternative effect on this signaling pathway. To address this possibility, the Notch signaling pathway was examined in primary macrophages exposed to TcdB, and results from these studies found that Notch signaling was suppressed by TcdB. Further analyses have suggested that the TcdB induced suppression of Notch signaling is through a mechanism involving the stimulation of interferon signaling. These results provide insight into mechanisms utilized by disparate bacterial toxins to modify Notch signaling in macrophages.
Regulation of actin dynamics during Legionella pneumophila infection: a novel function of Naip5 (611.2)
Authors: Caution Kyle , Akhter Anwari , Hassan Hoda , Tazi Mia , Voss Oliver , Doseff Andrea , Amer Amal
Published: June 2021

Clearance of Legionella pneumophila (Legionella), the causative agent of Legionnaire’s disease, a potentially fatal pneumonia, requires efficient phagolysosomal fusion. Regulation of the host actin cytoskeleton is crucial for proper vesicle trafficking, which in turn promotes efficient phagosome‐lysosome fusion and destruction of invading pathogens. Naip5 is an intracellular NOD‐like receptor (NLR) that detects and promotes the degradation of Legionella via its flagellin. The molecular mechanisms of Naip5‐mediated clearance are unknown. It is this study’s objective to decipher the effect of Naip5 on actin dynamics and its upstream regulators to promote phagosome‐lysosome fusion and restriction of the bacterium.

During Legionella infection, Naip5‐/‐ bone marrow‐derived macrophages (BMDMs) exhibited less phagosome‐lysosome fusion events, which was due to lack of caspase‐7 activation. The absence of active caspase‐7 in Naip5 cells inhibited the dephosphorylation (activation) of cofilin, blocking formation of new actin filaments. This lack of F‐actin formation hinders efficient phagolysosomal fusion and therefore allows intracellular replication of Legionella.

Taken together, these data establish for the first time that Naip5 regulates actin polymerization during infection with Legionella by activating caspase‐7 to modulate the activation of the upstream regulator of actin: cofilin. Understanding the novel molecular mechanisms of Naip5 regulation of the host cytoskeletal network will provide novel targets to develop therapeutic interventions for numerous infectious diseases.

Grant Funding Source: Supported by: R01HL094586