bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Hypoxia on Gene Expression in the Chick Embryonic Heart
Authors: Nanka Ondrej , Krejci Eliska , Barth Jeremy , Sedmera David 
Published: June 2021

Our previous results showed that normal embryogenesis involves physiological tissue hypoxia. Hypoxic avian hearts show abnormal myoarchitecture, differences in the extent and distribution of hypoxic regions and increased HIF1α. Here we have used microarray analysis to identify genes differentially expressed between normoxic and hypoxic conditionsChicken eggs were incubated at 38°C under normoxic (21% O2) conditions for the first 48 h and subsequently under hypoxic (16% O2) conditions for 24 h (acute hypoxia) or 144 h (chronic hypoxia). Total RNA was extracted from control and hypoxic chick embryonic hearts, converted into biotin‐labeled material, and then hybridized to Affymetrix Chicken Genome Arrays according to manufacturer's protocols. Transcripts differentially expressed in the short term and long term hypoxic states were identified by the criteria fold change >1.5 and p <0.05 (Student's t‐test). 79 transcripts were significantly up‐regulated and 75 down‐regulated in the hypoxic samples. Functional categorization of these genes detected significant enrichment of biological processes such as cell growth, differentiation, muscle contraction and signal transduction. Additionally, we performed a focused examination of genes previously linked to hypoxia adaption, identifying 10 transcripts that were significantly affected in our hypoxic samples (p<0.05). Given that important hypoxia‐regulated genes (e.g., HIF1α) are known to be regulated at the protein, rather than the mRNA level, our results add significant insight into the transcriptional response that occurs in response to hypoxia.Supported by PRVOUK P35/LF1/5 and P302/11/1308.
Cyclophilin A, a Novel Component of Listeria monocytogenes Comet Tails
Authors: Dhanda Arandeep , Guttman Julian 
Published: June 2021

The intracellular bacterium Listeria monocytogenes hijacks the actin polymerization machinery within human cells for its entry, intracellular movement and subsequent spreading from cell‐to‐cell. Intracellular motility of Listeria involves Arp2/3‐mediated actin polymerization at one pole of the bacteria, resulting in the generation of structures known as comet tails. During cell‐to‐cell spreading, comet tails protrude from the host cell membrane from structures referred to as listeriopods. Although the minimal protein constituents required for these actin‐based structures were identified years ago, a complete understanding of the molecular constituents and their specific functions at these sites remains to be investigated. Cyclophilin A (CypA) is a cytosolic protein involved in protein folding and potentially actin nucleation. Thus, because Listeria depends on actin‐based structures for crucial events in its disease process, we tested the hypothesis that CypA is recruited to Listeria comet tails and sites of cell‐to‐cell bacterial spreading. Through the use of fluorescently‐tagged CypA DNA constructs and time‐lapse imaging, we demonstrate that CypA is present at 1) the surface of Listeria prior to comet tail formation, 2) comet tails themselves and 3) listeriopods. Our data reveals a new component in the actin‐based motility of Listeria and suggests that CypA may play a crucial role in the cell‐to‐cell spreading of Listeria.Grant Funding Source: NSERC and departmental funds
Probing the Inflammatory Response Behind Diabetes and Obesity via the Biochemical Characterization of NOD1, an Innate Immune Receptor
Authors: Rivas Thomas , Lauro Mackenzie , Grimes Catherine , Bahnson Brian 
Published: June 2021

Innate immunity is an intricate system employed by humans to regulate the trillions of bacteria that live in our body. One protein integral to the innate immune response is called nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1). NOD1 is a cytosolic, membrane‐associated protein that is linked to irritable bowel syndrome, asthma, diabetes, and obesity. These diseases are thought to arise from improper mRNA splicing of NOD1 transcripts leading to a poorly functioning NOD1 protein receptor that can no longer regulate an overstimulated inflammatory response. Activation occurs when NOD1 binds to a bacterial cell wall component called iE‐DAP, which then signals the the NF‐κB cascade to produce pro‐inflammatory cytokines and chemokines.Current research is trying to describe the binding of iE‐DAP to NOD1, however, these studies almost exclusively use expensive and low‐yielding insect cells to obtain their active protein. This project aims to develop a bacterial expression system to express wildtype NOD1 and the LRR domain, which is the region thought to be involved in ligand binding. Currently I have produced both constructs in high yield from an E. coli system to allow their biochemical characterization through peptidoglycan pull‐down experiments and circular dichroism. Future work aims to obtain crystal structures of NOD1 bound to its ligand and perform NMR studies to observe ligand binding events.
Broader Impact Careers for Scientists in Science Education and Communication
Authors: Jacque Berri , Tammen Stephanie , Meiri Karina 
Published: June 2021

Employment data from the NIH and NSF show a dramatic overproduction of scientists relative to the number of job openings for permanent positions. This has created a bottleneck where many scientists get stuck in a training whirlpool. At the Center for Translational Science Education (CTSE) at Tufts Medical School we work to bring cutting edge biomedical science education to high school students and teachers around the country, while providing scientists opportunities to explore a range of diverse careers in science education. Many graduate students and postdoctoral fellows come to the CTSE hoping to gain teaching experience or to impact the community through outreach in local high schools. Other scientist work as full time postdoctoral scholars, studying curriculum design and evaluation, as they transition to careers with broader scientific impacts, such as teaching, museum curator, K‐12 outreach project manager, or primary investigator performing science education research. In this way scientists apply their skills to solve urgent public health and science education challenges, while gaining skills and experience to better compete in a diverse range of careers in science education and communication.Funded through: NIH Grant R25OD010953‐04, a gift from Boston Scientific and a grant from Cubist Pharmaceuticals
Insulin and Angiotensin II Regulate Titin Alternative Splicing via Rbm20
Authors: Zhu Chaoqun , Yin zhiyong , guo wei 
Published: June 2021

ObjectiveThis study is to determine whether titin alternative splicing is regulated by insulin and angiotensin II via a muscle‐specific splicing factor, RNA binding motif 20 (Rbm20).MethodsIn primary cultures, neonatal cardiomyocytes (CMs) were isolated from day 1 rats. CMs from wild type (WT), heterozygous (HT), and homozygous (HM) Rbm20 deficient rats were cultured in serum‐free medium and treated with insulin, insulin plus LY294002 (LY) and angiotensin II for 5 days. In in vivo assay, HT rats were treated with streptozotocin (STZ) for 8 weeks. Titin isoforms were detected by 1% SDS‐agarose gel. Band intensity was quantified with imageJ. Western blot and QRCR were performed for protein and gene expression.ResultsInsulin and angiotensin II increased the expression of smaller titin isoform (N2B) in both WT and HT CMs, but no N2B was rescued by insulin and angiotensin II in HM CMs. The phosphorylation of Akt was increased in WT, HT and HM CMs. LY can inhibit Akt phosphorylation and titin isoform transition in both WT and HT insulin treated CMs. STZ treatment decreased the expression of smaller titin N2B in HT rats. The mRNA levels of titin isoforms were consistent with protein level. Rbm20 expression was increased in insulin treated CMs.ConclusionTitin alternative splicing has been shown to be largely responsible for the diastolic properties of the heart. Rbm20 is the major regulator of the titin alternative splicing. These results indicated that insulin and angiotensin II regulated titin alternative splicing is Rbm20 dependent, and Akt phosphorylation can enhance Rbm20‐regulated titin alternative splicing.Supported by NIH P20 GM103432, University of Wyoming Agricultural Experimental Station and USDA‐NIFA
Dissecting Musashi1 role in chromatin remodeling and its consequences to DNA repair and radio‐resistance
Authors: Rosa de Araujo Patricia , Silva Acarizia , Penalva Luiz 
Published: June 2021

Musashi1 (Msi1) is an evolutionarily conserved RBP with well establish roles in neuronal stem cell self‐renewal and differentiation. In the context of tumorigenesis, Msi1 has been described to be highly expressed in gliomas, medulloblastoma, lung, colon and breast cancers and linked to a poor prognosis. Its expression is required for tumor maintenance and possibly initiation. We recently established that a decrease in Msi1 expression makes glioblastoma cells more radio‐sensitive: Msi1 KD cells subjected to ionizing radiation showed reduced proliferation as evident from colony formation assays; moreover, an increase in DNA damage was observed via comet assay, γ‐H2AX expression levels and 53BP1 ionizing radiation induced foci.Msi1 has been always defined as a translation regulator. However, our recent data shows that Msi1 is often present in the nucleus of tumor samples and cell lines. Moreover, CLIP assays identified a large number of Msi1 interacting sites in intronic regions. We suggest then that Msi1 displays additional regulatory functions in the nucleus that could equally contribute to its role in tumorigenesis. Supporting this idea, among Msi1′s protein partners identified by yeast two‐hybrid and mass spectrometry are several chromatin remodeling proteins including Ino80C. The Ino80 complex has been associated with transcription factors involved in cell proliferation, differentiation and embryonic development. Moreover, Ino80 interacts directly with γH2AX during DNA double‐strand break repair. We are currently conducting experiments to explore the hypothesis that Msi1 influences radio‐resistance and DSB repair via its association with the chromatin remodeling machinery.
The Complex Role of Sequence on the Stability and Function of a Thermophilic TIM Barrel Protein
Authors: Chan Yvonne , Zeldovich Konstantin , Matthews C 
Published: June 2021

TIM barrel proteins are encoded by highly divergent sequences, but fold to highly conserved (βα)8 structures with varying stabilities and functions. The indole‐3‐glycerol phosphate synthase (IGPS) TIM barrel protein, required for tryptophan biosynthesis in yeast, was selected as a target for investigating how sequence influences structure, stability, and function. IGPS from the S. solfataricus (SsIGPS) thermophile was subjected to the EMPIRIC assay to assess the change in fitness accompanying saturating mutagenesis on the 8 β‐stands and the preceding loops, known to be important for protein stability. The strategy was designed to test the presumption that mutations in the β‐barrel would provide an in vivo estimate of protein stability by decreasing fitness. The majority of the mutations in the β‐strands and preceding loops decreased the fitness, as expected. Surprisingly, mutations that remove any of the four‐fold symmetric TIM barrel βα hairpin clamps provided a fitness advantage. In vitro studies on one such mutant protein, I107K, was found to be destabilized to urea denaturation. A functional assay revealed that the I107K mutation increases the Vmax as well as the substrate binding affinity, indicated by lower Km. We speculate that the decrease in stability from these mutations is accompanied by an increase in the dynamics of the platform and, therefore, an increase in the fitness of a thermophilic protein at a suboptimal temperature in a mesophilic host.Funding source: NIH GM23303
Interactome Analysis of BetaM Protein Encoded by ATP1B4 Gene Using Two‐hybrid Systems
Authors: Pestov Nikolay , Korneenko Tatyana , Modyanov Nikolai 
Published: June 2021

The BetaM, a type II integral membrane protein, became a muscle‐specific protein of inner nuclear membrane in eutherians through co‐option of the ATP1B4 gene that encodes Na,K‐ATPase β‐subunit in lower vertebrates. Earlier we determined that BetaM directly interacts with transcriptional co‐regulator SKIP and functions as a regulator of gene expression. Here we present the expanded search for BetaM interactors by screening of cDNA libraries using conventional yeast two‐hybrid and split‐ubiquitin systems. The list of identified BetaM interactors includes BetaM itself, SKIP, Syne‐1, sarcoglycan β, LZIP, RCN 3, LAP1, HO 1, HO2, PHD finger 3, cadherin16, frizzled 1, SC65, tetraspanins 3 and CD63, TGN29, ERGIC 3, BCRAP‐31, C4orf3 and SftpC. Only sarcoglycan β was identified by both assays. Analysis of BetaM mutants revealed that deletion of 56 N‐terminal residues including the Arg‐rich peptide and a half of Glu‐rich stretch does not disrupt the BetaM dimerization and other identified interactions. Transmembrane and/or C‐terminal domains are required for interactions with LZIP, Syne, reticulocalbin 3 and sarcoglycan β. N‐terminal domain seems to be sufficient for interaction with SKIP, LAP‐1, PHF‐3 and heme oxygenases. Further analysis indicates that the second half of the Glu‐rich intrinsically disordered N‐terminal domain is required for the interaction with SKIP. We also show that the relatively conserved fragments of N‐terminal domain adjacent to the membrane are sufficient for BetaM dimerization.Supported by RFBR (Grant 13‐04‐01413) and by Dept of Physiology & Pharmacology, University of Toledo College of Medicine.
The Effect of Divalent Cations on Complex Phosphoinositide/Lipid Monolayers at the Air/Water Interface
Authors: King Katrice , Qifti Androniqi , Gericke Arne 
Published: June 2021

ObjectiveInvestigate the effect of common inner leaflet plasma membrane lipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), on phosphoinositide domain behavior in the presence of cholesterol and/or divalent cations.BackgroundPI and phosphoinositides (PIPs) may only comprise a small percentage of the inner leaflet of the plasma membrane, but they have major implications in a variety of signaling events. Previously, we have observed the absence of macroscopically discernible domains in mixtures of PI/PE and PI(4,5)P2/PE. Upon the addition of cholesterol to these mixtures, we observe condensation of the monolayer and domain formation.MethodsWe use epifluorescence microscopy to image domain formation in lipid monolayers at the air/water interfaceResultsWe find that the addition of varying concentrations of Ca2+ to PE/PI(4,5)P2/Cholesterol monolayers result in a decrease in the size of the domains. Our hypothesis is that this is due to a penetration of the Ca2+ ions into the PI(4,5)P2 headgroup region, leading to a disruption of the hydrogen bond network formed by the PI(4,5)P2 headgroup and cholesterol. For both PE/PI(4,5)P2 and PI/PI(4,5)P2, we find homogeneous mixing of the monolayer. Alternatively, when cholesterol is added to PE/PI(4,5)P2 we observe the formation of small domains at low pressures. In addition to cholesterol, we also investigate the effect of divalent cations on these lipid mixtures.
Signaling Regulates Activity of DHCR24, the Final Enzyme in Cholesterol Synthesis
Authors: Luu Winnie , Zerenturk Eser , Kristiana Ika , Sharpe Laura , Brown Andrew 
Published: June 2021

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis (3β‐hydroxysterol Δ24‐reductase; DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its post‐translational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell‐line (Chinese hamster ovary‐7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling. Moreover, we propose that post‐translational modifications such as phosphorylation are a valuable resource for mapping the topology of membrane‐associated proteins such as DHCR24.The Brown lab is funded by grants from the National Health and Medical Research Council.
Inhibition of Apoptosis by Angiotensin 1‐7/mas Pathway through MKP‐2 Activation in Alveolar Epithelial Cells
Authors: Gopallawa Indiwari , Uhal Bruce 
Published: June 2021

Study Objective:The autocrine generation of angiotensin II (ANGII) and c‐Jun‐N‐ terminal kinase phosphorylation (pJNK) are both required in alveolar epithelial cell (AEC) apoptosis which contributes to lung diseases. Angiotensin 1‐7 (ANG 1‐7) protects against AEC apoptosis which is induced by ANG II. However the exact inhibitory mechanisms of ANG 1‐7 and the mas receptor are currently unclear in AECs. The objective of the current research is to identify the role of map kinase phosphatase (MKP‐2) in AEC survival. We hypothesized that ANG 1‐7 activates MKP‐2 and maintains low pJNK levels thereby preventing apoptosis.MethodsTo determine the functional role(s) of MKP‐2, AECs were transfected with MKP‐2 siRNAs in the presence of ANG II (0.1µM) and/or ANG I‐7 (0.1µM). Then the cells were subjected to determine pJNK and apoptosis by Western blotting and nuclear fragmentation assay respectively. The loss of mitochondrial membrane potential (MMP) was also measured using a lipophilic probe. To test the involvement of mas receptor the cells were transfected with anti‐sense oligonucleotides in the presence of ANG 1‐7 followed by Western blotting to detect MKP‐2ResultsThe current data show the induction of pJNK (p<0.01), nuclear fragmentation (p<0.01) and MMP (p<0.001) when MKP‐2 is knocked down. The data also show that silencing MKP‐2 prevented the blockade of apoptosis by ANG 1‐7. Moreover the blockade of the mas receptor by the antagonist (A779) or anti‐sense oligonucleotides attenuated the induction of MKP‐2 by ANG 1‐7ConclusionsThe results from these studies suggest that ANG 1‐7/mas activation prevents JNK phosphorylation by activating the JNK selective phosphatase MKP‐2 and further it shows the involvement of mas receptor in MKP‐2 activation.
Structural Properties in the FMN Reductase of the Alkanesulfonate Monooxygenase System that Dictate Oligomeric States and Catalysis
Authors: Musila Jonathan , Ellis Holly 
Published: June 2021

The alkanesulfonate monooxygenase system is expressed in many bacteria when sulfur is limiting to enable the organism to utilize a diverse range of alkanesulfonates. This system is comprised of a NADPH‐dependent FMN reductase (SsuE) that reduces flavin and transfers it to the monooxygenase enzyme (SsuD) for the desulfonation of alkanesulfonates 1. The SsuE enzyme exists in different oligomeric states, but it is unclear how these oligomeric states relate to catalysis.A conserved π‐helix is located in the tetramer interface of SsuE which results from the insertion of Tyr118 in the α4‐helix. This Tyr118 residue forms a hydrogen bond with the carboxyl group of Ala78 across the tetramer interface to enhance the protein architecture 2. Variants of Y118 SsuE were generated to evaluate the role of the π‐helix in SsuE. Interestingly, the Y118A and Y118S SsuE variants were FMN‐bound as purified, while the Y118F and ΔY118 SsuE variants were flavin‐free. The Y118 SsuE variants were able to effectively bind FMN giving similar Kd values as wild‐type SsuE. This flavin affinity correlated with the ability of Y118 SsuE to reduce flavin with kcat/Km values similar to that of wild‐type SsuE in flavin reductase assays. While desulfonation activity was observed with the Y118F SsuE variant, there was no activity in the coupled assays monitoring SsuD desulfonation with the Y118A, Y118S and the ΔY118 variants of SsuE. This would indicate that the Tyr118 is vital for the transfer of FMNH2 to SsuD. The results from these studies suggest that multiple non‐covalent interactions are important for the structural stability of SsuE. These findings provide insights on the role of Tyr118 in SsuE. 1. Eichhorn, E. et al. (1999) J. Biol. Chem. 274, 26639‐46. 2. Driggers, C. M. et al (2014) Biochem. 53, 3509‐19.
Electron Microscopic Analysis of Lens Gap Junction Connexin50 Hemichannels and Channels
Authors: Sosinsky Gina , Ren Cynthia , Ambrosi Cinzia 
Published: June 2021

The mammalian lens is an avascular organelle that depends on gap junction (GJ) mediated intercellular communication for homeostasis, growth and development. Connexin50 (Cx50) is one of three lens GJ proteins and one of the largest members of the connexin protein family. Cx50 is present in all cells of the lens, first synthesized in the epithelium and persisting at high levels in differentiating fibers. Cases of human hereditary cataracts caused by >20 Cx50 distinct mutations demonstrate its crucial role in lens function and Cx50 knockout mice develop cataracts. Little is known about Cx50 structure except through homology with Cx26, a small connexin solved to 3.5 Å resolution by X‐ray crystallography and NMR of Cx50 C‐terminal peptides. Cx26 and Cx50 have strong sequence conservation and folding in their transmembrane and extracellular domains, but Cx26 lacks the extensive, non‐conserved C‐terminus present in Cx50. We have examined the full‐length Cx50 hemichannels and channels by single particle EM methods, the only structural biology technique that directly addresses how the large Cx50 C‐terminus is organized within the full‐length channel. Using baculovirus expression in Sf9 cells, we have purified both full‐length mouse Cx50 (440 amino acids) and a truncated form (Cx50del250‐440, 249 amino acids long) missing most of the C‐terminus such that it is slightly longer than Cx26 (226 amino acids). Electron micrographs of purified Cx50 hemichannels show a range of morphologies due to the way the oligomers are oriented on the grid. In contrast, Cx26 and Cx50del250‐440 channels are more homogeneous, displaying the canonical “doughnut‐like” appearance in projection because their structure.Funding: NIH grants GM065937 (GS) & GM103412 (NCMIR)