bluuebunny/crossref_metadata_2025 huggingface dataset:
A Thermodynamically Balanced Kinetic Model of the Mitochondrial Na+‐Ca2+ Antiporter
Authors: PRADHAN RANJAN KUMAR , BEARD DANIEL A. , DASH RANJAN K.
Published: June 2021

The Na+‐Ca2+ antiporter is the primary efflux pathway for Ca2+ in respiring mitochondria, and hence is a key regulator of mitochondrial Ca2+. Although experimental studies on the kinetics of Na+‐Ca2+ antiporter are available, the composition of its functional unit and kinetic mechanisms associated with the antiporter are still not clear. In order to have a quantitative biophysical understanding of its role in regulating mitochondrial Ca2+, we developed here a kinetic model of the Na+‐Ca2+ antiporter on the basis of its kinetic properties which is thermodynamically balanced and satisfactorily describes available data obtained under various experimental conditions. The model is based on a combination of multi‐state catalytic binding mechanism for carrier‐mediated facilitated transport and Eyring's free energy barrier theory‐based transformation mechanism for electrodiffusion. The model appropriately depicts the activating effects of membrane potential for a 3:1 electrogenic Na+‐Ca2+ exchange, and the inhibitory effects of protons at high and low pH, seen experimentally. The proposed model can be useful for further development of mechanistic integrated models of mitochondrial bioenergetics and Ca2+ handling, which can be helpful in understanding the mechanisms by which Ca2+ plays a role in mediating signaling pathways and modulating energy metabolism.
Claudin‐18 May Contribute to the Increase of Protein Permeability in Cultured Human Alveolar Epithelial Type II Cells Exposed to Proinflammatory Cytokines
Authors: Fang Xiaohui , Lee Jae‐Woo , Neyrink Arne , Gonzales Linda W , Ballard Philip L , Matthay Michael A
Published: June 2021

Our previous study demonstrated that proinflammatory cytokines increase protein permeability across human alveolar type II (AT II) cell monolayers (J Biol Chem 2007). However, the mechanisms are not fully understood. The family of claudin transmembrane proteins had been implicated in the regulation of epithelial paracellular permeability. In this study we determined the gene profile of cultured human AT II cells exposed to cytomix (TNF‐α, IL‐1β and IFN‐γ) to identify candidate genes that may contribute to the increase in protein permeability. Human AT II cells were isolated and cultured on collagen‐coated wells. After 48 hr, the cells were exposed to serum‐free medium (control) or cytomix for 72hr. cDNA was prepared and hybridized to Affymetrix U133A microarray chips. Western blot was used to test for protein expression of candidate genes. mRNA was detected at a significant level for 50% of the probes, representing approximately 7200 genes. Although mRNAs for claudin 1, 3, 4, 6, 7, 8, 9, 10, 15, 18 were detected, only claudin 18 mRNA was significantly decreased (95%) by cytomix exposure. Western blot confirmed that protein expression of claudin‐18 was significantly reduced by cytomix. The reduction of claudin 18 expression was restored by the NF‐kB inhibitor, BMS‐345541. We conclude that claudin 18 is regulated by cytokines and may play a role in protein permeability change across human ATII cell monolayers.
Systems Level Analysis of Cell‐Specific AQP2 Gene Expression in Collecting Duct
Authors: Yu Ming‐Jiun , Rinschen MM , Khositseth S , Braucht DWW , Uawithya P , Chou C‐L , Pisitkun T , Knepper MA
Published: June 2021

We investigated the basis of cell‐specific expression of aquaporin‐2 (AQP2) in the collecting duct. Bioinformatic analysis of the 5′‐flanking region of the AQP2 gene (GenomatixTM) revealed 11 conserved transcriptional regulator (TR) binding element (BE) motifs corresponding to SF1, NFAT, Forkhead, AP2, SRF, CREB, GATA, Ets, E‐Box, RXR and Homeobox TR families. To identify TRs that potentially bind to these sites, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TR from these families that are also expressed in native inner medullary collecting duct (IMCD). One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD clones expressing various levels of AQP2 protein (Ets1) and two showed a significant negative correlation (Elf1 and Nr1h2). Affymetrix profiling in native proximal tubules (PT), thick ascending limbs (TAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or TAL (candidate AQP2 enhancers), and 5 TRs (including HoxA5, HoxA9,and HoxA10) expressed in PT and TAL but not in IMCD (candidate AQP2 repressors). The results point to Ets and Homeobox as potentially important BEs in cell‐specific expression of AQP2 in the collecting duct. Full transcriptomic data are available online (Google "transcriptome database").
Functional role of multiple phosphorylation sites in the carboxyl‐terminal tail of the water channel Aquaporin‐2
Authors: Moeller Hanne B , MacAulay Nanna , Knepper Mark A , Fenton Robert A
Published: June 2021

Phosphorylation of Aquaporin‐2 (AQP2) at serine 256 (S256) is essential for its accumulation in the plasma membrane of collecting duct principal cells. We examined the role of additional phosphorylation sites in the C‐terminal tail on AQP2 function. Lack of AQP2 phosphorylation at S256 (S256A‐AQP2) reduced water permeability 3‐fold compared to wildtype (WT) AQP2‐injected Xenopus oocytes. Prevention of AQP2 phosphorylation at S261, S264, and S269 had no effect on water permeability. Oocytes expressing AQP2 mimicking phosphorylation at S264 and S269 had similar water permeabilites to WT‐AQP2‐expressing oocytes. Microscopy and biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A‐AQP2, had equal abundance in the oocyte plasma membrane. Water permeability relative to plasma membrane abundance demonstrated that lack of phosphorylation at S256, S261, S264 or S269 had no effect on AQP2 unit water transport. No effect on AQP2 unit water transort was observed for the 264D and 269D forms, indicating that phosphorylation of the C‐terminal tail of AQP2 is not involved in channel gating. Phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior S256 phosphorylation. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.
Effects of nitric oxide donors on hIK1 cell surface expression
Authors: O'Rourke Kellie Leatha , Jones Heather Marie
Published: June 2021

The human intermediate conductance ion channel, hIK1, is a calcium (Ca2+) activated potassium (K+) channel that has a variety of roles in physiological functions. These functions include cancer development, cell volume regulation, cell growth and cell proliferation. Nitric oxide (NO) is a signaling molecule increased by activation of nitric oxide synthase. Treatment of hIK1 transfected HEK cells with NO‐donors, SNAP and PAP‐NONOate, showed increased hIK1 protein expression levels. Additionally, cell surface immunoprecipitation experiments demonstrated increased levels of hIK1 at the cell surface with NO‐donor treatment. Cell surface expression of hIK1 in untreated HEK cells was evaluated using biotin ligase labeled cells which were treated with strepavidin‐Alexa 488 and detected using confocal microscopy. Channels in untreated HEK cells were rapidly endocytosed and, following a one hour incubation, a majority of the channel was located within endocytic vesicles. Treatment with the NO donors increased cell surface expression of hIK1 after one hour and a decrease in the amount of channel located in endocytic vesicles. These results suggest that NO may regulate hIK1 endocytosis and lead to an increase expression of active channel at the cell surface.
TRPC1 Mediates Capacitative Calcium Entry through Activation of STIM1 in Mouse Pulmonary Arterial Smooth Muscle Cells
Authors: Ng Lih Chyuan , McCormack Mary D. , Airey Judith A. , Singer Cherie A. , Shen Xiao‐Ming , Keller Phillip S. , Hume Joseph R.
Published: June 2021

The present study aimed to determine if TRPC1 and STIM1 mediate capacitative Ca2+ entry (CCE) in mouse pulmonary arterial smooth muscle cells (PASMCs). In primary cultured PASMCs loaded with fura‐2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). The transient but not sustained rise in [Ca2+]i was partially inhibited by nifedipine but they were both inhibited by SKF 96365, Ni2+, La3+ and Gd3+. In addition, CPA increased the rate of Mn2+ quench of fura‐2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. The nifedipine‐insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells pretreated with antibodies raised against extracellular epitopes of TRPC1 and STIM1. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca2+]i and Mn2+ quench rate caused by CPA, and they were reduced by TRPC1 antibody. RT‐PCR and Western blot analysis revealed TRPC1 and STIM1 mRNAs and proteins. Furthermore, TRPC1 was found to co‐immunoprecipitate with STIM1. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry and CCE. These data provide direct evidence that TRPC1 channel mediates CCE through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]
Arboreal quadrupedalism and its impact on the mouse body plan
Authors: Byron Craig Daniel , Kunz Hawley Elise , Matuszek Heather Lynn
Published: June 2021

Several models that explain the origin of primates involve the specialization of a micro‐mammal ancestor (possessing non‐grasping and clawed digits) to a fine‐branch arboreal setting. In this analysis laboratory mice are used as a model organism to demonstrate the adaptive response of skeletal connective tissues to a fine‐branch setting. Animals were weaned at 15 days postnatal and placed into one of three experimental groups: control, treadmill‐running, or climbing. Climbing mice trained on a matrix for 30 minutes per day for one month. This structure simulates a thin, branching arboreal substrate and required mice to grasp and ambulate using above‐branch quadrupedal postures. Observations suggest that climbing exercise successfully encouraged grasping as well as greater mobility in arm and leg joints. Gastrocnemius and anterior tibialis mass were enhanced in the climbing group as well as forelimb elongation. Histology of the hands and feet suggest that climbing treatment also affects the divergence of the metatarsals and metacarpals as well as altering their cross‐sectional geometry. These results suggest that mice can be used to probe growth plasticity in anatomical regions that are important to grasping thin branches. These data can enhance our understanding of function and phylogeny within the earliest fossil primates.
Short‐term hindlimb unloading induces translocation of nNOS from the sarcolemma to sarcoplasm in skeletal muscle
Authors: Macias Brandon Richard , Kim Jong‐hee , Courtney Sean , Canon Claire , Lawler John
Published: June 2021

Long duration spaceflight missions cause skeletal muscle atrophy in astronauts. We hypothesize that neuronal Nitric Oxide Synthase (nNOS) and dystrophin translocate from the sarcolemma to the sarcoplasm following 48‐hrs of simulated microgravity as compared to ground controls. Eight Fisher‐344 rats underwent 48‐hour simulated microgravity using the well established hindlimb unloading model, and eight rats were used as ground controls. Hindlimb muscles were carefully dissected, snapped frozen, and stored for analysis. Cryostat sections of soleus muscle (10 micrometer thick), were prepared for immunohistochemical analysis of nNOS and dystrophin localization. Hindlimb unloading induced translocalization of nNOS from the sarcolemma to the sarcoplasm. However, dystrophin was localized at the sarcolemma in the hindlimb unloaded group. Both nNOS and dystrophin were localized to the sarcolemma in the ground control group. These data are the first to demonstrate that nNOS delocalizes from the sarcolema during short‐term hindlimb unloading (48‐hrs). Future studies should investigate the localization of other dystroglycan proteins such as B1‐syntrophin and alpha‐dystrobrevin. Supported by the Texas A&M University Training Grant in Space Life Sciences with funding from NSBRI (NASA NCC 9‐58).
Reactive Species‐Mediated Covalent Modification of cGMP‐Protein Kinase I
Authors: Ramchandran Ramaswamy , Schilling Alexander , Sewite Negash , Kumar Harshavardhan , Raghavan Aarti , Turaka Prasanna , Sarkar Joy , Raj Usha
Published: June 2021

We have shown in the fetus and newborn, that in pulmonary vascular relaxation induced by cGMP‐elevating agents, cGMP‐dependent protein kinase I(Prkg1) plays a primary role. Hypoxia attenuates cGMP‐dependent protein kinase(Prkg1)‐mediated relaxation in pulmonary vessels. We have shown that peroxynitrite, an important effector of oxidative stress, produced in pulmonary vascular smooth muscle cells (PVSMC) during hypoxia, decreases Prkg1 protein levels as well as Prkg1 activity. In this study we test the hypothesis that hypoxia alters Prkg1 protein by ROS/RNS‐dependent covalent modification of critical amino acids which decreases its kinase activity. Ovine PVSMCs were exposed to hypoxia (3 % O2) or normoxia (20% O2) and the purified Prkg1 protein was analyzed by FTMS. Two peptides containing cysteine were among those identified for Prkg1 and were found to exhibit definitive transformation under hypoxic conditions. These results suggest that hypoxia‐induced reactive oxygen/nitrogen species can modulate the activity of Prkg1 by modifying the cysteine residues which may be relevant to the pathophysiological conditions associated with increased hypoxia‐induced oxidative stress. Support NIH R01 HL075187‐05A1
The effects of genistein on MCF‐7 and MDA‐MB‐231 breast cancer cell proliferation
Authors: Jordan Clayton , Munzlinger Megan , Potterfield April , Mayne Jeff
Published: June 2021

Genistein, an isoflavonoid present in soybeans and soy products, is said to exhibit anti‐carcinogenic effects. However, blood plasma phytoestrogen levels over 10 μM, which are generally considered inhibitory, are difficult to achieve physiologically. In the present study, MCF‐7 and MDA‐MB‐231 breast cancer cells were treated with genistein at 2, 7 and 12 μM after 24 hours, refreshed on day 3, and cell proliferation was assessed on day 6 using the trypan blue method for both cell types, and MTT assays for the MDA‐MB‐231 cells. It was found that genistein stimulated cell proliferation by 52.06% at 2 μM and 53.26% at 7 μM for MCF‐7 cells, while there was no stimulation at 12 μM. Genistein stimulated cell proliferation by 77.95% at 2 μM for MDA‐MB‐231 cells, while not at 7 or 12 μM. The results from this study would suggest that physiologically relevant levels of phytoestrogens might have a proliferative effect on both estrogen‐receptor positive and negative breast cancers.
Ribosomal protein genes form a barrier to horizontal gene transfer
Authors: Sorek Rotem
Published: June 2021

Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to that of another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted transfer of 246,045 genes from 79 prokaryotic genomes into Escherichia coli and identified genes that consistently fail to transfer. A dominant group among these genes included ribosomal protein genes. Our data suggest that toxicity to the host inhibited transfer of these genes regardless of the species of origin and that increased gene dosage may be the predominant cause for the observed toxicity. Therefore, increased dosage of specific ribosomal proteins is toxic to bacterial cells. Possible mechanisms of toxicity and evolutionary consequences will be discussed.
Small molecule screen reveals differences in the structural landscape of amyloid‐beta oligomers and fibrils
Authors: Reinke Ashley Anne , Seh Han Yiau , Gestwicki Jason E.
Published: June 2021

Aggregated amyloid‐β (Aβ) peptide is implicated in the pathology of Alzheimer's disease (AD). In vitro and in vivo, these aggregates are found in a variety of configurations, including globular oligomers and linear fibrils. Despite being composed of identical monomer units, these different quaternary configurations give rise to distinct biological effects; for example, recent evidence suggests that oligomers might play a key role in AD pathology. To identify unique molecular features that might differentiate these architectures, we employed a fluorescence‐based interaction assay to screen a collection of 68 known Aβ ligands. Using this approach, we found that most of the small molecules, including the well‐known compounds Bis‐ANS and Congo Red, interact equally well with both Aβ oligomers and fibrils. However, the fluorescence of five indole‐based compounds was selectively quenched in the presence of oligomers, but remained unchanged after addition of fibrils. These results suggest that amino acids capable of quenching indole fluorescence, such as Lys16, Phe19, Phe20, or Lys28, are relatively exposed in oligomers and buried in fibrils. These differences in accessibility may encompass one mechanism by which different amyloid structures give rise to distinct biological outcomes.
Baicalein protects hippocampal neuronal HT22 cells from ER stress‐induced apoptosis by modulating unfolded protein responses‐associated proteins
Authors: Choi Ji Hyun , Kang Insug
Published: June 2021

The endoplasmic reticulum (ER) stress has been suggested to be involved in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Here we examined the effect of baicaleine (BE), a flavonoid, on ER stress‐induced apoptosis in mouse hippocampal neuronal cell line HT22. BE inhibited apoptosis induced by the ER stress inducers thapsigargin (TG) and brefeldin A (BFA). We showed that BE blocked ER stress‐induced unfolded protein response (UPR), including expression of CHOP and spliced form of XBP‐1 and phosphorylation of eIF2α, JNK, p38MAPK and the cleavage of ATF‐6. Moreover, we showed that BE blocked the accumulation of ROS and maintained mitochondrial membrane potential in ER stress‐induced HT 22 cells. Similar to the effect of BE, N‐acetyl‐cysteine (NAC), an antioxidant inhibited TG‐ or BFA‐induced apoptosis and changes in UPR‐associated proteins. Taken together, these results suggest that BE protects HT22 neuronal cells against ER stress‐induced apoptosis by inhibition of ROS accumulation and regulation of ER stress associated proteins.

This work was supported by grants from KOSEF of Korea Government.
Shotgun Proteomics Identifies Protein Biomarkers Specific for Acute Renal Transplant Rejection
Authors: Sigdel Tara , Kaushal Amit , Gritsenko Marina , Norbeck Angela , Xiao Wenzhong , Qian Wei‐Jun , Camp David G. , Sarwal Minnie M
Published: June 2021

Background

Proteomics can help in biomarker discovery for different diseases. Since, acute rejection (AR) remains the primary risk factor for the renal transplant outcome; development of non‐invasive biomarkers for AR diagnosis is important.

Materials and Method: We analyzed 82 urine samples that included patients with biopsy proven AR and stable grafts (STA), nonspecific proteinuria (NS), and healthy controls (HC). The samples were fractionated and processed by LC‐MS. Protein‐level spectral counts were obtained by which were used to get quantitative information. ELISA assays on UMOD, pigment epithelium‐derived factor (PEDF) and CD44 were performed to verify the trueness of the discovery.

Results

A total of 1446 unique urinary proteins were identified including 22 AR specific proteins a number of which were involved in antigen presentation and complement system pathways. We performed ELISA assay on UMOD, PEDF and CD44 on an independent set of urine samples and demonstrated that the observation from LCMS experiment was true and these proteins can serve as potential biomarkers to discriminate AR urine from STA, NS and HC.

Conclusion

A number of potentially AR specific biomarkers were identified. The validation of these potential biomarkers will offer a clinically applicable, non‐invasive urinary diagnostic assay for AR.
Development of a novel fluorescent nitric oxide synthase assay
Authors: Stahler Shannon Lynn , Huang Xinyi , Suhadnolik Lorraine , Bray Jennifer , Wilhelm James , Aulabaugh Ann , Harrison Richard
Published: June 2021

Many therapeutic indications exist for nitric oxide synthase (NOS) making it a target of interest for drug discovery. The NOS family of enzymes, which include inducible NOS, endothelial NOS, and neuronal NOS, regulate functions such as cell communication, immune defense, and vasodilation. NOS enzymes convert L‐arginine and NADPH to L‐citrulline and nitric oxide (NO). NO is the agent responsible for the downstream physiological effects of NOS. In the absence of any downstream targets, NO is rapidly converted to nitrite in in vitro assays.

Several NOS assays exist, but lack the sensitivity required for HTS‐compatible formats. This poster describes a new NOS assay using a fluorescent detection system. The detection system utilizes 4,5 Diaminofluorescein (DAF2) in which the probe reacts with nitrite under acidic conditions. Initial concentrations of various assay components were selected based on each component's individual effect on the detection system. The assay was further optimized based on the overall effect of all components on both the enzyme reaction and the detection system. Several known NOS inhibitors were evaluated under the final assay conditions resulting in a validated, robust HTS‐compatible assay.
Does immortalization of primary endothelial cells by overexpression of human telomere‐reverse transcriptase impair typical endothelial characteristics?
Authors: Baumer Yvonne , Schlosshauer Burkhard
Published: June 2021

To study endothelial remodelling or regeneration in vitro mostly primary endothelial cells (EC) are used. However, primary EC are short living and often high priced. It would be desirable to use immortalized EC that are not transformed by oncogenes and display most EC‐characteristics of naïve EC. Therefore, we have used human telomere‐reverse transcriptase (hTERT)‐transduced primary human umbilical vein EC (HUVEC). Thus, we studied both HUVEC cell types. Comparative analysis included: 1) cell adhesion to 14 different extracellular matrix proteins analysed on a multisubstrate array (MSA), 2) protein‐expression of 9 endothelial and non‐endothelial markers by fluorescence‐microscopy (IF) and western blot‐analysis 3) uptake of Dil‐Ac‐LDL as revealed by IF and fluorescence activated cell sorting (FACS)‐analysis, 4) upregulation of ICAM‐1 after TNFa / interferon y‐treatment, 5) tube formation in hydrogel cultures, and 6) permeability properties. We conclude that immortalization of primary EC by hTERT retains the typical endothelial characteristics. Thus, hTERT‐EC represent a valuable tool for in vitro research.

Supported by the German Ministry of Education and Research (BMBF) #01GU0727. Cells kindly provided by Dr. C. Fricke.
Glutaredoxin Family in Breast, Colon, and Lung Cancer With Emphasis on Glutaredoxin2 and Glutaredoxin3
Authors: Cha Mee‐Kyung , Kim Il‐Han
Published: June 2021

Glutaredoxin (Grx) maintains and regulates the cellular redox state and redox‐dependent signaling pathways. Grx has been implied in various disease‐related conditions. The human Grx family has four distinct members (Grx1‐3 and Grx5) in various subcellular locations. We investigated levels of expression of all Grx genes in various cancer tissues by real‐time polymerase chain reaction. Levels of Grx2 and Grx5 messenger RNA (mRNA) in breast cancer tissue exceeded that in other solid cancers. Moreover, the level of Grx2 mRNA in breast cancer was the highest among those of other mRNAs including Grx, thioredoxin (Trx1, Trx2), and peroxirdoxin (Prx I‐VI) families. Among members of the Grx family, Grx3 gene was preferentially induced in lung and colon, whereas not in thyroid, lymphoma, liver, and kidney. Western blot analysis of different and paired breast tissues revealed consistent and preferential expression of Grx3 in lung and colon cancers. The expression of Grx3 gene was correlated with that of survivin gene, suggesting its reverse‐association with apoptosis. Taken together, the striking and preferential inductions of Grx2 in breast cancer and Grx3 in lung and colon may enable their use as corresponding cancer markers. *This work was financially supported by Regional Research and Development Cluster Project (B0009735) funded by the Ministry of Knowledge Economy (MKE) of Korea.
Metalloproteinases as key molecules to target invasiveness in solid tumors
Authors: Karamanos Nikos K , Staxtea Xanthi , Gialeli Chrisostomi
Published: June 2021

Metastasis of cancer cells requires degradation of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are the principal ECM‐degrading enzymes. Paracrine interactions between stroma fibroblasts and cancer cells are also involved in the expression and activity of MMPs. The aim of this study was to examine whether gene expression of MMPs is related with the invasive potential of colon cancer. In vitro studies on a panel of cell lines in the presence specific tyrosine kinase inhibitors and monoclonal antibodies were performed.

The results showed that the invasive properties of cancer cells are associated with significant changes in gene expression of MT1‐MMP and MMP‐2. The expression of MT1 from HT‐29 colon cancer cells is increased by the conditioned media (CM) obtained by fibroblasts. MT1 contributes to the activation of proMMP‐2. STI571 suppressed the expression of MT1 in HT‐29 colon cancer cells as well as pro‐ and active MMP‐2. Panitumumab that target EGF‐R in colon cancer affects also the expression of MMPs.

It is concluded that paracrine interactions is a determining factor for tumor cell behavior and suppression of certain MMPs may be of great value in molecular targeted therapy.
Phosphorylation of Ser205 by Casein kinase II persists on Pax3‐FOXO1a, but not Pax3, throughout myogenic differentiation
Authors: Dietz Kevin N. , Miller Patrick J. , Hollenbach Andrew D.
Published: June 2021

The myogenic transcription factor Pax3 plays an essential role in early skeletal muscle development and is a key factor in Alveolar rhabdomyosarcoma (ARMS), a childhood solid muscle tumor. ARMS is associated with an unfavorable prognosis due to the propensity for wide dissemination and a poor response to chemotherapy. The characteristic translocation in ARMS results in the fusion of the 5' Pax3 sequences to the 3' FOXO1a sequences to encode a fusion protein, Pax3‐FOXO1a. Post translational modifications such as phosphorylation are common mechanisms of regulating transcription factors, including Pax3 and FOXO1a. The Pax3 domain of Pax3‐FOXO1a is phosphorylated at multiple sites which positively influences the DNA‐binding activity and subsequent transcriptional activity of Pax3‐FOXO1a. Here, we show that both Pax3 and Pax3‐FOXO1a are phosphorylated at Ser205 in proliferating mouse primary myoblasts and identify Casein kinase II as the kinase that phosphorylates Pax3 and Pax3‐FOXO1a at Ser205. Furthermore, we demonstrate that Pax3‐FOXO1a, but not Pax3, is constitutively phosphorylated at Ser205 throughout differentiation of mouse primary myoblasts. Finally, we show that Pax3‐FOXO1a is phosphorylated at Ser205 in a variety of translocation containing ARMS cell lines. These results suggest a possible mechanism by which the disregulation of Pax3‐FOXo1a may contribute to tumorigenesis.
Modulation of PHLPP activity: screening for specific inhibitors
Authors: Sierecki Emma , Newton Alexandra C
Published: June 2021

PHLPP (PH domain Leucin repeats Protein Phosphatase) has been shown to directly dephosphorylate Akt and PKC, leading to the desactivation of the Akt pathway and degradation of PKC. PHLPP also inhibits the Erk pathway and regulates the levels of tyrosine kinase receptors, in an indirect manner. In order to decipher the role of PHLPP in these different pathways, we need chemical tools to modulate PHLPP activity. However, the phosphatase domain of PHLPP belongs to the PP2C family of phosphatases that are characterized by their requirement of manganese/magnesium for their activity and by their resistance to commonly used phosphatase inhibitors such as okadaic acid or calyculin. To find specific inhibitors and activators, we screened the Diversity Set of the NCI for inhibitors of PHLPP 2. We designed a medium throughput colorimetric assay, using pNPP as a substrate. Compounds were tested at 100 µM and cutoff was set at 30% activity. We then evaluated the specificity of the compounds against the other member of the PHLPP family, PHLPP 1 and against another member of the PP2C family, PP2Cα. We determined the IC50 for the most promising compounds. Molecules with a IC50 of ≈1µM were also tested in cells and their effect on PHLPP was accessed by monitoring the phosphorylation state of Akt.

This work was supported by the JDFR 3‐2008‐478 and NIH 067946.
Synthetic Melanins with Antiviral Activity
Authors: Holmes Earle W. , Thompson Kenneth D.
Published: June 2021

Enveloped viruses can cause severe, and occasionally life‐threatening, infections in humans and animals. We have prepared a family of water‐soluble synthetic melanin polymers that have differing physicochemical properties and distinctive selectivities for preventing the infection of animal cells by enveloped viruses in vitro. For example, our series of 20 compounds with anti‐HIV activity have been shown to have ED50's in the range of 4 to 216 ng/mL (http://www.bioactivepolymers.com ). Recently we submitted 10 synthetic melanins to the NIAID's Antimicrobial Acquisition and Coordinating Facility for in vitro activity testing against 7 herpesviruses, 7 respiratory viruses, 2 orthopoxviruses, and 10 bioterrorism viruses. The compounds were tested for inhibitory activity using the NIAID's standard screening screening assays as described on their website (http://niaid‐aacf.org/screeningassays.htm ). The results showed that the most potent of our synthetic compounds had the following ED50's (μg/mL) for the 20 viruses tested thus far: HSV1, 1.4; HSV2, 0.06; CMV, 3.4; VZV, < 0.03; EBV, 98; Dengue C., 26; Rift Valley Fever, 26; Tacribe, 41; RSV A., 32; Cowpox, 27; Vaccinia, 10; SARS, 13; H1N1, 2.5; H3N2, 3.2; H5N1, 0 .21; Flu B, 2.0. ED50's of > 100 μg/mL were observed for Yellow Fever, Adenovirus, PIV, and Rhinovirus. The inhibitory activities of screen‐positive compounds with moderate to high activities were confirmed by supplementary testing. The compounds typically had low toxicities toward animal cells, with CC50's of >100 μg/mL. The results suggest that synthetic melanins are promising candidates for the development of topical microbicides for use in the prevention of STD's and for use in the prevention and treatment of other viral infections in humans and animals.
Cardiac and pulmonary arterial changes by fat embolization in the rat
Authors: Haileselassie Bereketeab , Adler Federico , McIff Terence , Poisner Alan , Herndon Betty , Molteni Agostino
Published: June 2021

Fat embolization syndrome (FES) is a respiratory syndrome that may follow bone fractures with fat embolism. A model uses intravenous (iv) triolein. We found pulmonary arterial vasoconstriction in rats after triolein, improved by an ACE‐ inhibitor (captopril) or A2‐type 1 receptor blocker (losartan). We report cardiac morphology induced by triolein and drug treatment (5‐6 / group). Rats received 0.2 ml triolein with necropsy at 24, 48, 96, or 264 hrs. Others received triolein + 50 mg/kg captopril or 10 mg/kg losartan or both (5/group; 48 hr necropsy). Saline controls were necropsied at 24 hrs and 264 hrs (4‐5/group). Measurements were made on cardiac cross sections. Right ventricle (RV)/ left ventricle(LV) areas were analyzed. Triolein alone produced a time‐dependent pulmonary vasoconstriction. RV/LV ratio increased vs saline at 96 hrs (p<0.05) but returned to control values at 264 hr. At 48 hr, captopril, losartan or both groups showed RV/LV ratios comparable to controls, improved from ratios of groups receiving only triolein. The FES model produces RV dilation secondary to pulmonary vessel constriction, clearing without treatment by 264 hrs. Captopril and losartan preserve pulmonary arterial lumen patency and decrease RV dilation. This suggest both the reversibility of FES‐related cardiovascular changes and the role of the renin‐angiotensin system in its pathogenesis.

Funding: MA & EJ Asher Ortho. Res. Endow.
Fenretinide is active in osteosarcoma in vitro by inhibition of cholesterol esterification
Authors: Gasperi‐Campani Anna , Batetta Barbara , Baiocchi Daniela , Putzolu Marirosa , Baldini Nicola , Giunti Armando , Roncuzzi Laura , Dessì Sandra
Published: June 2021

Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolescents. Although the combination of systemic chemotherapy and surgery enables long‐term survival in most cases, the poor prognosis of patients with metastatic or recurrent disease and the lack of establishment of second‐line chemotherapy suggest that novel therapeutic strategies for this tumor are needed. Here we show that fenretinide, a synthetic derivative of all‐trans‐retinoic acid, is active against osteosarcoma in vitro, at concentrations identical or lower to those detectable in breast cancer patients plasma during chemopreventive clinical trials. Cell lines were HOS and MG63, known to be resistant to methotrexate and cyclophosphamide, drugs in use in osteosarcoma treatment. We demonstrate for the first time that the molecular basis of fenretinide activity is the inhibition of cholesterol esterification, with down‐regulation of ACAT and MDR1 mRNA, followed by downregulation of caveolin‐1 protein expression. This result is in line with our recent results obtained in VSMC and in leukemia cells. The confirm and extention of these data to different human tumors will provide evidence of a novel mechanism of either cancer or human vascular proliferation disease control by the inhibition of cholesterol esterification.
Bifunctional magnetic nanoparticles for early detection and magnetic hyperthermia cancer therapy
Authors: Balivada Sivasai , Rachakatla Raja Shekar , wang Hongwang , Samarakoon Thilani N. , Dani Raj Kumar , Chikan Viktor , H.Bossmann Stephen , Troyer Deryl L.
Published: June 2021

Magnetic hyperthermia offers promise as cancer therapy. We have tested bimetallic core/shell Fe/Fe3O4 magnetic nanoparticles (MNP) for in vivo diagnosis and targeting of MNP to tumor tissue. We have received the MNP from NanoScale Corporation. These particles have a Dopamine‐PEG layer, to which a porphyrin 'A' is attached; another porhyrin 'B' is also attached to the MNP via a peptide that represents the urokinase cleavage site. MNP were tested in B16F10 melanoma cells and neural stem cells for loading using Ferrozine assay.. For in vivo tumor detection, the MNP were injected intravenously into tumor bearing mice and fluorescence was detected after one and two days. Heat distribution studies were conducted in live mice by injecting MNP into mouse leg; temperatures were measured using a fiber optic probe. Short exposure time hyperthermia studies were conducted by exposing tumor bearing mice to alternating magnetic field (AMF) for 10 min (336KHz frequency and 5KA/m amplitude) after injecting theMNP intratumorally. MNP loaded efficiently into mouse cells with little toxicity. We detected fluorescence in tumors during laser imaging of live mice. Mouse subcutaneous melanomas were markedly attenuated; we observed 76% tumor inhibition after short exposure to AMF. These data indicate that bi‐ functional core shell MNP are effective for hyperthermia therapy and detection of subcutaneous melanoma.
CIB1 Is Important For Tumor‐Induced Pathological Angiogenesis
Authors: Zayed Mohamed Adel , Yuan Weiping , Chalothorn Dan , Barry William , Faber James , Parise Leslie
Published: June 2021

Pathological angiogenesis contributes to various malignant, ocular, and inflammatory disorders, emphasizing the need to further understand it on a molecular level. We previously demonstrated that CIB1 is essential for endothelial cell (EC) function and ischemia‐induced pathological angiogenesis. We therefore hypothesized that CIB1‐KO mice may also have a defect in tumor‐induced angiogenesis. To test this hypothesis, either B16 melanoma or Lewis lung carcinoma cells were xenografted in age‐matched wildtype and CIB1‐KO mice. For 14 days, growth and intra‐tumoral perfusion were monitored non‐invasively with a scanning laser‐Doppler perfusion imager. Tumors that developed in CIB1‐KO mice were grossly necrotic in appearance, demonstrated less perfusion, and were smaller in weight and volume (p < 0.05). Biopsies obtained from melanoma tumors in CIB1‐KO mice demonstrated significantly reduced intra‐tumoral microvessel density (p = 0.002). Biopsies also showed a trend towards more tumor necrosis and hemorrhage, but this was not statistically significant (p = 0.06, and p = 0.13, respectively). Preliminary microarray data suggests that CIB1‐KO ECs have altered expression of various pro‐ and anti‐angiogenic genes. These data demonstrate that CIB1 plays an important role in tumor‐induced pathological angiogenesis. This work was supported by NIH2‐P01‐HL45100 (LVP) and NIH HL‐62584 (JEF).
Translational aspects of the common 14q13 amplicon in human lung cancer
Authors: Mu David , Hsu David S. , Acharya Chaitanya R. , Riedel Richard F. , Potti Anil
Published: June 2021

Previously we discovered and characterized the second most common amplicon in lung cancer, located at chromosomal region 14q13 and harboring three transcription factor genes ‐ TTF1, NKX2‐8, and PAX9. We showed that these three genes work in various combinations to cause malignant transformation (Kendall et al. 2007 PNAS 104: 16663). To further explore the clinical relevance of the three coamplified oncogenes, using stable transfectants of human bronchial epithelial cells we determined the RNA expression signatures that represent activation of the biological pathway defined by each of the three genes. By risk stratifying a non‐small cell lung cancer clinical dataset with the expression signatures, we discovered that co‐activation of TTF1 and NKX2‐8 pathways identified a cluster of patients (20%) with poor survival. We demonstrated that poor patient survival may be a reflection of resistance to platinum compounds, the standard care of lung cancer. Finally, we provide evidence that lung cancer cells with activated TTF1/NKX2‐8 pathways are sensitive to a RAS pathway inhibitor, providing a blueprint for future research regarding the treatment strategy of such type of lung cancer.
NF‐¿B inhibition protects against tumor‐induced cardiac atrophy in vivo
Authors: Willis Monte S , Wysong Ashley , Rodriguez Jessica E , Baldwin Al , Couch Marion
Published: June 2021

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation, which occurs in ~80% of patients with advanced malignancy, resulting in 20‐30% of all cancer‐related deaths. The pro‐inflammatory cytokines released during cancer‐cachexia induce muscle loss by increasing ubiquitin ligases that degrade the sarcomere, in a NF‐¿B dependent manner. Using a recently described mouse model of tumor‐induced cancer cachexia, we tested the hypothesis that inhibiting cardiac NF‐¿B activity in this model would protect against cardiac atrophy. An adenocarcinoma cell line (C26) was implanted in the hind limb of mice and allowed to form a tumor for 6 days. On day 7, mice were treated daily with either Compound A or Nemo‐binding domain (NBD) peptides to inhibit NF‐¿B. At day 17 after implantation, we identified that NF‐¿B inhibition prevented the development of tumor‐induced cardiac atrophy in a dose‐dependent manner by echocardiographic and histological analyses. We also identified that tumor‐induced cardiac systolic dysfunction returned to baseline levels with NF‐¿B inhibition. This study identifies for the first time that NF‐¿B inhibition is cardioprotective against cancer cachexia‐induced cardiac atrophy and systolic dysfunction, suggesting that NF‐¿B inhibition may help reduce cardiac‐associated morbidities found in advanced malignancies.
Flash‐heating breastmilk is feasible in Dar es Salaam, Tanzania
Authors: Young Sera Lewise , Chantry Caroline , Ngonyani Monica , Israel‐Ballard Kiersten , Ash Deborah , Nyambo Margaret
Published: June 2021

WHO recommends HIV+ mothers exclusively breastfeed (EBF) for 6 mos unless replacement feeding is acceptable, feasible, affordable, sustainable and safe. After 6 mos, adequate replacement foods are often unavailable and are immunologically inferior. Heat‐treating breastmilk is currently recommended by WHO as an infant feeding option for PMTCT of HIV but its feasibility has not been evaluated. Flash‐heating (FH) is a simple method for home pasteurizing breastmilk that inactivates HIV while preserving milk's nutritional and anti‐infective properties. Our objective is to determine the feasibility of FH once complementary foods are introduced. In this ongoing, longitudinal study, community health workers (CHWs) in Dar es Salaam, Tanzania, visit 100 HIV(+) mothers weekly from 2‐9 mos postpartum and counsel on FH if infant is HIV(‐) at introduction of complementary foods. Mothers are surveyed weekly about infant health and feeding practices and unheated and heated milk samples are collected biweekly. 24/61 (39%) mothers with HIV(‐) infants have chosen FH. Mean frequency of manual expression is 4.8 times daily (range 1‐7). Mean daily milk volume is 435 mL (range 60‐1080mL). 23/73 unheated milk samples contained pathogens; all FH samples are bacteriologically negative. 50% of FH mothers have disclosed their HIV status to their spouse; CHWs observed that stigma may be an obstacle to FH for some women. These data suggest that FH is a simple method for reducing MTCT of HIV that women can successfully use during times of high risk to potentially improve their infant's health and diet. FH may be most successful among women who have disclosed their status. Based on this feasibility data an efficacy study of FH on infant health outcomes is warranted.
Effect of Green Tea Flavonoid Supplementation on Features of Metabolic Syndrome (MeS)
Authors: Penugonda Kavitha , Sanchez Karah , Leyva Misti , Aston Christopher , Lyons Timothy , Basu Arpita
Published: June 2021

Green tea, rich in flavonoids, has been shown to possess cardiovascular health benefits. This is a preliminary report on a randomized controlled trial investigating whether green tea beverage or extract supplementation improved the cardiovascular risk profile associated with MeS. Age and sex‐matched trios of participants with MeS were randomly assigned to control (4 cups water/day), green tea beverage (4 cups/day), or green tea supplement (2 capsules & 4 cups water/day) group for 8 weeks. Fasting blood samples, physical measurements were taken at screening, 4 & 8 weeks. Blood samples were analyzed for lipid, glucose, catechin and nitric oxide (NOx) levels. Body weight decreased in green tea (‐1.9 Kg) vs control (+0.1 Kg). There is an increasing trend in HDL cholesterol in green tea (+1.0 mg/dl) vs control. A decrease in ox‐LDL was found in green tea group vs baseline. Interestingly, no consistent effects were seen on glucose levels. No significant difference in the plasma catechin and serum NOx concentrations among the three groups. However, green tea beverage group showed a decreasing trend in serum NOx levels (p<0.1) compared to baseline, indicating anti‐ inflammatory effect. Green tea beverage or supplements may aid weight loss, raise HDL levels and may help in reducing ox‐LDL and NOx levels in MeS subjects. Thus, chronic green tea consumption may promote cardiovascular health by reducing oxidative stress and inflammation in at risk subjects.

Funded by CHES, OSU
Nutritional Contribution of White Potatoes in the Diets of U. S. Children and Adults: NHANES, 2003‐2006
Authors: Fulgoni Victor L , Keast Debra R , Slavin Joanne L
Published: June 2021

Using 24‐hr recall dietary data from the National Health and Nutrition Examination Survey (NHANES) 2003‐2006 we determined the contribution white potatoes (excluding potato chips) make to the diets of children 2‐18 years (n=7,332) and adults 19+ years (n=9,490). In children, the average consumption of white potatoes was 31.7 g/d per capita and 89.8 g/d for consumers only. In consumers, white potatoes made significant contributions (percentage of intake in parentheses) of the following nutrients: fiber (18.7%), potassium (16.9%), vitamin B6 (15.5%), copper (13.0%), and magnesium (10.7%). White potatoes provided 8.4, 9.4, 8.9, and 6.1% of calories, carbohydrates, total fat, and saturated fat, respectively in consumers; additionally white potatoes provided meaningful amounts (=5% of intake) of other nutrients, e.g., vitamin K (9.5%), vitamin C (8.5%), thiamin (8.4%), niacin (7.9%), phosphorus (6.4%), vitamin E (5.5%), iron (5.5%), and folate (5.0%). In adults the average consumption of white potatoes was 39.3 g/d per capita and 110 g/d for consumers only. In consumers white potatoes provided 7.6, 10.6, 6.1, and 4.4% of calories, carbohydrates, total fat, and saturated fat, respectively, while making a significant contribution to the daily intake of the following nutrients: fiber (17.4%), potassium (16.8%), vitamin B6 (16.3%), copper (13.5%), vitamin C (12.4%), and magnesium (10.0%). Additionally, white potatoes provided meaningful amounts (=5% of intake) of many other nutrients, e.g., thiamin (8.3%), niacin (7.4%), phosphorus (6.0%), vitamin K (5.4%), iron (5.3%), and folate (5.0%). White potatoes benefit both children and adults by providing significant quantities of nutrients to the American diet. Supported by McCain Foods Limited.
Efficacy of a plant sterol‐fortified low fat soy beverage on cholesterol metabolism in normal to mildly‐hypercholesterolemic subjects
Authors: Chan Yen‐Ming , Rideout Todd C. , Jones Peter J.H.
Published: June 2021

The objective of this study was to evaluate the efficacy of plant sterol‐enriched low‐fat soy milk (PS‐LS) on lipid levels in individuals with normal to elevated low density lipoprotein‐cholesterol (LDL‐C) levels, compared to 1% dairy milk (DM). Thirty‐three subjects (LDL‐C >2.1 mmol/L) were fed a controlled low fat diet supplemented with DM or PS‐LS providing 1.95 g of PS/d, using a cross‐over study. Each dietary phase was 4 wk in duration, separated by a 4 wk wash‐out period. Blood samples were taken at the beginning and the end of each phase. Consumption of PS‐LS lowered (P<0.0001) plasma total cholesterol (T‐C) levels by 10%, compared to DM feeding (5.26±0.16 vs 5.80±0.17 mmol/L). A 13% reduction (P<0.0001) in plasma LDL‐C levels was observed following PS‐LS consumption, relative to DM intake (3.20±0.15 vs 3.66±0.15 mmol/L). Compared to the DM control, PS‐LS supplementation lowered T‐C/HDL‐C (P=0.0011) and LDL‐C/HDL‐C (P=0.0008) ratios (T‐C/HDL‐C=4.71±0.27 and 5.05±0.26 for PS‐LS and DM, respectively; LDL‐C/HDL‐C=2.74±0.20 and 3.21±0.20 for PS‐LS and DM, respectively). Results promote the use of PS‐LS as an effective functional food, which may exert more profound cardio‐protective effects, compared to the choice of DM, in normal and hypercholesterolemic populations. Sponsored by WhiteWave Foods Company.