bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing the emphasis of radiology in a first‐year medical gross anatomy course (536.7)
Authors: Darling Ryan , Lu Yuefeng , Conway Marianne , Maklad Adel , Sinning Allan
Published: June 2021

Radiology is a rapidly expanding field of medicine with continual advances in noninvasive imaging techniques such as CT and MRI. It is increasingly important to integrate radiological education into medical school curriculum due to the growth in use of these images by physicians, and because the USMLE is incorporating more radiology questions. The University of Mississippi Medical Center has been implementing this, starting as early as the students’ first semester in Gross Anatomy. Beginning in 2011, the course included physician guest lectures to introduce the students to the basic physics behind the techniques, as well as to provide examples of reading radiological images within their area of specialty. The students were also offered a more interactive learning environment via small discussion groups led by radiologists. Added to the curriculum in 2013, each student was given two sets of DICOM files that included a full body CT of a male and female cadaver in all three standard planes of section to aid in their learning of radiological anatomy in conjunction with atlas illustrations and their cadaveric specimens. In addition, students were given premium access to IMAIOS’s E‐Anatomy and periodically given short radiology quizzes for them to assess their progress. The students’ performance on four region‐based multiple choice exams, specifically on radiology questions, increased progressively ~7.5% over those 5 years. Individual question and region‐based analyses also indicated differences across years. After completion of the course, the final exam will be added and statistics updated.
Cytoskeletal organization during terminal differentiation and enucleation of mammalian erythroblasts (538.3)
Authors: Nowak Roberta , Casu Carla , Rivella Stefano , Fowler Velia
Published: June 2021

Terminal differentiation of red blood cells is a complex morphogenetic process involving chromatin condensation, cell polarization and nuclear expulsion. Microtubules are important for cell polarization, while F‐actin assembly and myosin activity are important for nuclear expulsion. In this study, we used confocal microscopy to examine erythroblasts from mouse fetal liver, adult bone marrow and phlebotomized mouse spleen. In non‐polarized erythroblasts, the F‐actin cytoskeleton is organized into a cage‐like structure surrounding the nucleus. In polarized erythroblasts from fetal liver or spleen, the F‐actin reorganizes to a bright spot at the side of the nucleus. Mitochondria are relocated from the periphery of the cell to this actin spot. This bright spot of F‐actin and the mitochondria travels with the rear of the nucleus as it is ejected. Additional foci of F‐actin are located at the neck separating the expelled nucleus from the reticulocyte. Linear cables of F‐actin are also observed in the nuclear ‘bud’ which are sometimes associated with nuclear pores. Notably, adult mouse bone marrow lacks the bright spot of F‐actin near the nucleus during nuclear expulsion. Arp2/3 and mDia2 are not associated with the bright F‐actin while myosinIIB is associated with these foci. We conclude that the actin cytoskeletal organization of enucleating erythroblasts is not conserved among different erythropoietic tissues.
Examining O‐xylosyltransferase shedding in Drosophila (539.1)
Authors: Palus Brooke
Published: June 2021

O‐xylosyltransferase (Oxt) is a Golgi transmembrane glycosyltransferase that initiates the first step in the heperan and chrondroitin sulfate biosynthesis on core proteoglycans (HSPG and CSPG). HSPGs and CSPGs are abundant extracellular matrix proteins present in virtually all tissues and have critical functions in articular cartilage in vertebrates. In this tissue, HSPGs and CSPGs are essential in regulating signaling and homeostasis. Disrupted homeostasis of articular cartilage leads to its degradation and osteoarthritis (OA). The onset of OA has been shown to result in increased serum levels of Oxt in mouse and humans. While the biosynthetic activity of Oxt has been studied, little is known about its function extracellularly. Understanding how and why Oxt is shed and its role extracellularly may provide further insight into how cell signaling and tissue homeostasis can be disrupted in OA patients. The goal of this study is to determine if Oxt is shed from Drosophila wing imaginal discs and what function extracellular Oxt has in organismal development. As expected, Oxt localizes to the Golgi and the ER the site of HSPG and CSPG biosynthesis. Oxt was also found extrcellularly suggesting it is shed in Drosophila. In loss‐of‐function and over expression studies the composition of the extracellular matrix and Oxt diffusion from it cell of origin were also examined.
Using a yeast‐2‐hybrid system to map chemotactic protein‐protein interactions in Epulopiscium sp. type b (539.5)
Authors: Conrad Arielle , Piefer Andrew
Published: June 2021

Chemotaxis is the process by which certain single and multicellular organisms direct their movement based on chemical signals found in their environment. Epulopiscium sp type b (Epulo) is an extremely large gram positive bacterium, whose chemotactic pathway has not yet been fully explored. The purpose of this study is to expand the knowledge base of the Epulo chemotactic pathway by examining the interactions that take place between the methyltransferase, a putative methyltransferase/methylesterase fusion protein, and the methyl accepting chemotaxis proteins. PCR was used to isolate the genes that encode these proteins from purified Epulo DNA. Subsequently, these genes were subcloned into yeast expression vectors for 2‐hybrid protein interaction studies.
Stimulation of inflammasome‐dependent innate response by group A streptococcus ADP‐ribosyltransferase protects host from bacterial infections (539.9)
Authors: Lin Ann , Beasley Federico , Keller Nadia , Hoffman Harold , Nizet Victor
Published: June 2021

Streptococcus pyogenes (GAS) is a causative agent of severe invasive infections including necrotizing fasciitis, bacteremia and toxic shock syndrome. SpyA (S. pyogenes ADP‐ribosyltransferase) is one of a subset of virulence factors significantly up‐regulated in the hyper‐invasive strain of a globally disseminated M1T1 GAS. Surprisingly, mice systematically challenged with GAS ΔspyA suffered significantly higher mortality and displayed reduced bacterial clearance. We recovered higher levels of ΔspyA GAS compared to the wild‐type (WT) strain from bone marrow‐derived macrophage (BMDM) killing assays. Complementation of the GAS ΔspyA mutant restored the WT BMDM survival phenotype, heterologous expression of SpyA in Staphylococcus aureus or the macrophage itself led to a significant increase in bacterial killing. Increased bacterial survival of the ΔspyA mutant was associated with severe attenuation of caspase‐1 dependent inflammasome activation and impaired IL‐1β production. BMDMs deficient in caspase‐1, NLRP3, ASC or pro‐ IL‐1β reversed the effect of SpyA expression on macrophage survival and abolished protection seen in WT infected mice. In conclusion, SpyA triggers caspase‐1 dependent inflammatory responses, or “pyroptosis” in macrophages, highlighting the importance of this macrophage innate immune response pathway in defense against invasive bacterial pathogens.

Grant Funding Source: Canadian Institutes of Health Research (CIHR)
Architectural changes of the extracellular matrix in compensatory lung growth (540.2)
Authors: Wagner Willi , Bennett Robert , Ysasi Alexandra , Belle Janeil , Ackermann Maximilian , Tsuda Akira , Mentzer Steven , Konerding Moritz
Published: June 2021

Compensatory growth of the remaining lung after murine pneumonectomy (PNX) involves the structural remodeling of the peripheral acini. Although the role of stem cells and local cellular proliferation is an area of active investigation, very little is known about the corresponding changes in the lung extracellular matrix (ECM). Post‐PNX adaptation of the elastin network, in particular, is essential for maintaining the mechanical and functional properties of the lung. Elastic fiber bundles concentrate particularly in the end bulbs of free septal alveolar edges shown in trans‐electron‐microscopic imaging and Verhoeff‐van‐Gieson staining. The elastic fibers form a continuous helical cable element, interconnecting alveolar entrance rings, spanning several levels of alveolar ducts. High‐resolution synchrotron radiation microscopy revealed this cord element running on the inside of alveolar ducts of infant murine lungs in the alveolar phase of lung development. The continuity of the elastic helix in adult c57bl/6 mice was visualized by scanning electron microscopy and by confocal microscopy of decellularized murine lung slices. Stereological estimates show an increased number of elastin‐containing septal edges during compensatory growth. The architectural changes in the murine ECM suggest active remodeling of the elastin network after PNX. The orientation of elastin in the septal tips of remodeling alveoli suggest the active participation of the elastin network in neoalveolarization. Consistent with these observations, the elastin cord element may be involved in the long‐range signaling essential for functional acinar remodeling.

Grant Funding Source: NIH
BRG1 deletion de‐represses p53 pathways inducing cell cycle inhibition and apoptosis during peri‐gastrulation (541.10)
Authors: Singh Ajeet , Boyle Michael C , Rubino Mark , Tandon Arpit , Shah Ruchir , Archer Trevor
Published: June 2021

BRG1, a catalytic subunit of the SWI/SNF ATPase chromatin‐remodeling complex, plays a vital role in gene regulation and tumorigenesis. Germline SWI/SNF function loss results in early embryonic lethality at approximately E3.5 in the mouse. BRG1 necessity during development beyond the pre‐implantation stage, however, has not been defined. The myriad temporal genomic changes during development require chromatin remodeling to facilitate differential gene expression. We hypothesized that BRG1 deficiency during post‐implantation development will cause gastrulation failure. Using a tamoxifen inducible conditional mouse model, we show that BRG1 ablation beginning at gastrulation results in arrested growth and early embryonic death. Mutant embryos failed to grow; histologic analysis revealed several developmental defects such as lack of normal cardiac structure and disrupted neural fold architecture. Immunohistochemistry analysis revealed aberrant expression of cell cycle and apoptosis marker proteins in BRG1 mutants. Microarray analysis revealed BRG1 ablation increased expression of cell cycle regulators, particularly within the p53 pathway. Co‐IP analyses reveal BRG1 interacts with CHD4 to suppress p53 pathways in P19 cells. These results suggest BRG1 plays a critical role in genomic surveillance essential for cell survival after the pre‐implantation period for normal cellular survival, proliferation and differentiation.

Grant Funding Source: Z01 ES071006‐13
Caffeine induces both short‐term and long‐term effects on gene expression and DNA methylation in the mouse heart (542.3)
Authors: Wendler Christopher , Poulsen Ryan , Fang Xiefan
Published: June 2021

Introduction: Evidence indicates that disruption of normal prenatal development influences an individual’s risk of developing cardiovascular disease as adults. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. One substance that fetuses are commonly exposed is caffeine, an adenosine receptor antagonist, with consumption during the first month of pregnancy being reported by 60% of women. Methods: Pregnant CD‐1 dams were injected with 20 mg/kg of caffeine once a day from E6.5‐E9.5. Hypothesis: Altered adenosine action in utero leads to adverse effects on adult heart function and morphology, and these effects are mediated by DNA methylation‐induced changes in cardiac gene expression. Results: Using an outbred strain of mouse, CD‐1, we demonstrated that in utero caffeine exposure induces increased wall thickness and increased left ventricular mass in adult hearts. When we examined gene expression in embryonic hearts following the same caffeine treatment, we demonstrated that enzymes responsible for DNA methylation and demethylation were affected. The DNA methylation genes DNA methyltransferase 1 and 3a were both down regulated and the Tet 1 enzyme which initiates DNA demethylation was up regulated following in utero caffeine exposure. These results are consistent with our observations that in utero caffeine exposure results in global hypomethylation in the adult heart. In addition, the cardiac structural genes Myh7, titan, and troponin I1 had altered gene expression. Conclusion: Caffeine affects the expression level of genes that control DNA methylation in utero, and caffeine leads to altered gene expression and cardiac morphology in adulthood.

Grant Funding Source: R01 HD058086
Fibulin‐1 suppresses EMT and apoptosis during semilunar valve morphogenesis (542.6)
Authors: Harikrishnan Keerthi , Cooley Marion , Sugi Yukiko , Kern Christine , Argraves W
Published: June 2021

Epithelial to mesenchymal transition (EMT) occurring in the cardiac outflow tract (OFT) is a process that is critical to semilunar valve development. Fibulin‐1 (Fbln1), an extracellular matrix protein, is a major component of the OFT that is expressed during OFT EMT (i.e., E9.5‐10.5). The aim of this study was to determine the role of Fbln1 during the earliest events in OFT EMT. At E9.5‐10.5, the proximal OFT cushions of Fbln1 null embryos were hyper cellular (i.e., 82% increase at E9.5, 43% increase at E10.5, n=5 per genotype; p<0.05) as compared to wildtype (WT). Further evidence that Fbln1 normally functions to suppress endocardial cushion EMT came from OFT cushion explant studies. The results showed that OFT explants from Fbln1 null embryos displayed a 137% increase in the number of cells that migrated from the explants as compared to WT (WT n=8, null n=4; p<0.001). Despite the finding that Fbln1 deficiency led to hypercellularity of OFT cushions at E10.5, we observed decreased cell numbers of OFT cushions at E12.5. Further analysis revealed a 172% increase in apoptosis in regions of EMT derived mesenchyme of Fbln1 nulls that accompanied a 35% reduction in cell numbers in the same regions (n=4 per genotype, p<0.05) at E12.5. Decreased cellularity and increased apoptosis persisted in the OFT prevalvular mesenchyme of Fbln1 nulls at E14.5. Morphometric analysis of 3D reconstructions of Fbln1 null OFT valves at E17.5 revealed that the anterior leaflet of the pulmonary valve, and the left and non‐coronary leaflets of the aortic were significantly reduced as compared to WT (30‐40% reduction; p<0.05. The reduced valve sizes at E17.5 correlated with decreased cell numbers (45%, 30% and 30%, respectively; WT n=6, null n=8; p<0.05). Taken together, Fbln1 regulates cellularization of the proximal endocardial cushion and prevalvular mesenchyme. Mechanistically, Fbln1 acts as a suppressor of OFT cushion EMT and apoptosis.

Grant Funding Source: Supported by HL095067
Non‐invasive vascular function measurement tools: the correlation between arterial distensibility and venous compliance in young males (546.8)
Authors: Bentley Danielle , Nguyen Cindy , Thomas Scott
Published: June 2021

Reliable non‐invasive tools for the determination of arterial and venous health permit repeated assessments in intervention studies. While arterial health is commonly estimated using tonometry and venous function estimated using venous plethysmography, no studies have reported on the relation of these measures of vascular function. We assessed the reproducibility (ICC) and correlation (Pearson r) of Augmentation Index (AIx) and Venous Compliance (dV/dP) in a sample (n=8) of healthy (SBP/DBP: 107.1±7.4 / 70.0±5.5mmHg), young (Age: 25.5±3.7 years), physically active (Rapid Assessment of Physical Activity: 6.6±0.5) men. All participants completed an initial familiarization session, followed by two experimental sessions where both arterial Augmentation Index and Venous Compliance were measured. AIx values were accepted only if the operator index > 90%. Participants had good but varied arterial health (AIx = ‐1.9±11.0; range ‐18 to 17) and venous health (.03±.00; range .02 to .05). Reliability between days for AIx was high (Cronbach’s α= .97) while dV/dP scores at 20mmHg were less reliable (Cronbach’s α =.46). There was no correlation between intra‐individual same day AIx and dV/dP variables (r = ‐.31, p >0.05). Based on these results, studies of vascular function should consider the arterial health measurement of Augmentation Index and the venous function measurement of Venous Compliance as independent variables.

Grant Funding Source: Supported by CIHR CGS Doctoral Research Award
p38α MAPK mediates saturated fatty acid‐induced cardiomyocyte apoptosis (547.19)
Authors: Oh Charles , Nguy Michael , Migrino Raymond , Thornburg Kent , Schwenke Dawn , Reaven Peter
Published: June 2021

BACKGROUND: Cardiomyocyte apoptosis mediated by a p38α MAPK‐dependent pathway may be a key step leading to lipotoxic/diabetic cardiomyopathy. Saturated fatty acid (SFA), elevated in type 2 diabetes, induce apoptosis in many cell types including cardiomyocytes. We tested the hypothesis that SFA‐induced cardiomyocyte apoptosis is dependent on p38α activation.METHODS AND RESULTS: Immortalized human adult ventricular cardiomyocytes (AC16 cells) were exposed to high physiological levels of a SFA, palmitate (PA). The apoptotic response was determined by flow cytometry, and the p38α‐dependent pathway evaluated using a p38 inhibitor, and by p38α siRNA knockdown.PA increased apoptosis in AC16 cardiomyocytes dose‐dependently; after 16 h exposure to PA (control‐2.6±0.7%, 150 μM PA‐3.5±1.1%, 300 μM PA‐11.5±3.4%, n=4, p<0.001). Apoptosis rose higher after 24 h treatment (control‐2.3±0.7%, 150 μM PA‐5.9±2.7%, 300 μM PA‐37.6±10.7%, n=3, p<0.02).PA did not change total p38α protein levels, but increased p38α phosphorylation. Specific p38α siRNA reduced the expression of p38α but not p38β, and markedly and dose‐dependently attenuated PA‐induced apoptosis (control siRNA‐7.7±1.0%, 300 μM PA‐34.4±5.0%, 300 μM PA+30 pmol siRNA‐23.7±4.4%, 300 μM PA+60 pmol siRNA‐19.7±2.6%, 300 μM PA+120 pmol siRNA‐17.3±2.8%, n=4, p<0.04). PD169316, which inhibits both p38α and p38β isoforms, tended to reduce PA‐induced apoptosis (n=4, p=0.05).Downstream, AP‐1 transcription factors, c‐fos and c‐jun (n=3, p蠄0.05), and caspase 8 (n=3, p=0.03) mRNA levels, and protein expression were increased by PA. CONCLUSIONS: This study in cardiomyocytes demonstrated that 1) PA induces p38α activation, 2) PA increases AP‐1 and caspase 8 expression, and 3) reduced p38α expression attenuates PA‐induced apoptosis. Weaker results with PD169316 may be due to concurrent inhibition of p38β. Our results suggest a potential mechanism by which high plasma SFA levels may lead to the development of diabetic cardiomyopathy.

Grant Funding Source: VA
Damage to mitotic telomeres results in DNA damage response activation, cell division delay, and micronucleation (548.1)
Authors: Alcaraz Silva Barbara , Stambaugh Jessica , Shah Jagesh , Berns Michael
Published: June 2021

Telomeres are nucleoprotein structures required to protect chromosome ends. Uncapping of telomeres during interphase elicits a DNA damage response causing cell cycle arrest, aberrant DNA damage repair resulting in, chromosomal end fusion, chromosome instability and eventually lead to tumorigenesis. In the present study we investigate the consequences of DNA damage occurring at telomeres during the mitotic phase of the cell cycle using laser microirradiation. Microirradiation was used in combination with GFP live‐cell imaging and dual‐labeling to study double‐strand break (DSB) repair by monitoring the co‐localization of γH2AX with DNA damage response (DDR) factors. We found DDR proteins such as ATM and WRN localize to damaged telomeres but not to other chromosomal sites of focal laser damage. Interestingly, damage at mitotic telomeres results in a delay in anaphase onset, which is dependent on the activity of ATM, Chk1 and the spindle assembly checkpoint kinase, Mps1. Telomere‐damaged cells exited mitosis with unresolved telomere repair and increased occurrence of micronuclei. Telomeric damage is therefore distinct from damage at other chromosomal loci during mitosis and elicits an intra‐mitotic delay that may permit timely repair.
Generation and characterization of topoisomerase IIα mutants (549.5)
Authors: Gibson Elizabeth , Straughn Lisa , Deweese Joseph
Published: June 2021

Topoisomerases are nuclear enzymes that regulate DNA topology and remove knots and tangles in the genetic material. While the anticancer agent etoposide is highly effective at inducing topoisomerase II‐mediated DNA damage, etoposide is also metabolized into a catechol and a highly active quinone. To further study the mechanism of the metabolites, we are developing human topoisomerase IIα mutants with altered sensitivity to etoposide. Based upon published studies, we identified amino acid positions that may influence enzyme response to etoposide. Using a PCR‐based mutagenesis process, we mutated Gly462 to Ala, Asp463 to Glu, Arg487 to Lys, Ala429 deletion, Gly534 to Arg, Gly449 to Asp, and Pro716 to Leu. We used biochemical assays to analyze the DNA cleavage and relaxation activities of the mutant enzymes in the presence and absence of etoposide. Our results demonstrate that five mutants are functional (R487K, D463E, ΔA429, P716L, and G534R), one mutant has activity too low to quantify (G462A), and one is being purified for analysis (G449D). In the presence of etoposide, D463E and ΔA429 cleave near wild‐type (WT) levels, while R487K, P716L, G534R respond less. When the ratio of enhancement in the presence of drug (versus no drug) is compared R487K, P716L, and G534R have the lowest level of enhancement. When looking at DNA relaxation with each mutant in the presence of drug, ΔA429 and P716L are stimulated to relax DNA, while R487K, D463E, and G534R are inhibited. Based upon cleavage enhancement in the presence of etoposide, R487K, P716L, and G534R are less sensitive to etoposide. These mutants will be used to analyze the activity and mechanism of the catechol and quinone metabolites of etoposide against topoisomerase II.
Characterization of HU‐331 as a cannabinoid quinone inhibitor of topoisomerase II (549.6)
Authors: Regal Kellie , Mercer Susan , Deweese Joseph
Published: June 2021

Topoisomerases are essential enzymes that are involved in DNA metabolism. Due to the double‐stranded DNA cleavage mechanism, topoisomerase II is an effective target for anticancer drugs, such as doxorubicin. However, doxorubicin causes cardiac toxicity and can induce DNA damage resulting in secondary cancers. A cannabinoid‐based quinone HU‐331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off‐target toxicity than doxorubicin. Reports indicate that HU‐331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We proposed that HU‐331 inhibits human topoisomerase IIα function by inhibiting the ATPase domain of the enzyme. We employed biochemical assays to study the mechanism of action of HU‐331 against purified topoisomerase IIα. These assays examined DNA cleavage, ligation, relaxation, and ATPase function of topoisomerase IIα. Our results demonstrate that HU‐331 inhibits topoisomerase IIα‐mediated DNA relaxation at micromolar levels. We find that HU‐331 does not induce DNA strand breaks, decreasing the risk of secondary cancers. When added prior to the DNA substrate, HU‐331 blocks DNA cleavage and relaxation activities of topoisomerase IIα. Our results also show that HU‐331 inhibits the ATPase function of topoisomerase IIα. Preliminary binding studies also indicate that HU‐331 decreases the ability of topoisomerase IIα to bind DNA. We have thus far concluded that HU‐331 impacts the catalytic activity of topoisomerase IIα by inhibiting the ATPase function. These results provide a promising foundation for the potential of HU‐331 as an anticancer drug. Future studies will explore the ability of HU‐331 to abrogate cleavage and relaxation activities. We will also further clarify the impact of HU‐331 on the enzyme:DNA interaction.
Probing the biological role a poor Km plays in the processivity and temporal control of CcrM (549.9)
Authors: Woodcock Clayton , Reich Norbert
Published: June 2021

Caulobacter crescentus is a model prokaryote involving asymmetric division which requires stringent temporal and spatial control of gene regulation attained through epigenetic silencing. The Cell cycle regulated Methyltransferase, CcrM plays as an essential role in this process, and is tightly regulated during cell division. The essential methylation of the entire X bp Caulobacter genome requires a highly efficient enzyme, and two prior reports are conflicting on CcrM’s methylation mechanism. We provide a rigorous test of the enzyme’s ability to methylate multiple recognition sites (processive catalysis) and show that indeed, the enzyme acts processively. Surprisingly, we also show that CcrM has an unusually poor affinity for its DNA substrates in comparison to other bacterial and eukaryotic DNA methyltransferases. We suggest that this poor DNA affinity may serve a biological role since CcrM is rapidly proteolyzed by Lon protease in vivo. We are currently determining if this efficient proteolysis occurs because CcrM is poorly associated with DNA.
HIPK2 regulates double‐strand break signaling by controlling WIP1 phosphatase at the protein level (550.1)
Authors: Choi Dong Wook , Na Wooju , Choi Cheol Yong
Published: June 2021

Double‐strand break (DSB) signaling, induced by ionizing radiation and other damage reagents, prevents genomic instability and tumoriogenesis. Features of DSB signaling are tight regulation of phospho‐dynamics of key players including ATM, H2AX, CHK2. In this study, we identified that WIP1, serine/threonine phosphatase involving termination of DSB signaling, is responsible for initiation of DSB signaling in a HIPK2‐dependent manner. Under normal condition, WIP1 maintained at a low level by proteasomal degradation through HIPK2‐mediated phosphorylation, which is important for proper activation of DSB repair signaling in response to ionizing radiation. After completion of DNA repair, HIPK2 is phosphorylated by AMPK, which induces dissociation of WIP1 from phosphorylated HIPK2. We found that HIPK2 is indispensible for DSB‐induced foci formation and cell cycle check point activation. In addition, using HIPK2 heterozygote mice, we demonstrated that HIPK2 has an essential role in survival of mice in response to ionizing radiation. Taken together, we suggest that AMPK‐HIPK2‐WIP1 axis plays a key role in the initiation and termination of DSB repair signaling cascade through fine‐tuning of WIP1 protein level.
The Mus81‐Mms4 structure‐selective endonuclease undergoes nicked junction‐induced conformational changes to bend its DNA substrates for cleavage (553.1)
Authors: Mukherjee Sucheta , Wright William , Ehmsen Kirk , Heyer Wolf‐Dietrich
Published: June 2021

Mus81‐Mms4 is a structure‐selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81‐Mms4 requires inherent flexibility in DNA substrates for optimal catalysis. Förster Resonance Energy Transfer (FRET) experiments further reveal that recognition of 3’‐flap and nicked Holliday junction substrates by Mus81‐Mms4 involves induction of a sharp bend with a 100° angle between the two duplex DNA arms. In addition, thiol crosslinking of Mus81‐Mms4 bound to DNA substrates demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches support a model for catalysis by Mus81‐Mms4 in which initial DNA binding is based on minimal structural requirements and is followed by a rate‐limiting conformational transition of the substrate and protein, which leads to a sharply kinked substrate that may fray the DNA four bp away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotides. These data suggest that mutually compatible conformational changes of Mus81‐Mms4 and its substrates tailor its incision activity to nicked junction molecules, resulting in products that cannot be directly ligated.

Grant Funding Source: Supported by NIH and TRDRP
Signaling control of proteasome, immunoproteasome, and PA28 regulator gene expression during adaptation to oxidative stress (555.11)
Authors: Davies Kelvin
Published: June 2021

Levels of oxidative stress change continuously, depending on environment, metabolism, foods, drugs and medications, and radiation. Young, healthy, mammalian cells can readily adapt to increases in oxidative stress such that they become (temporarily) much more resistant to oxidative damage. Intracellular proteins are major targets of oxidative stress and it is vitally important that cells rapidly and selectively degrade mildly oxidized proteins, before they undergo more severe oxidation, aggregation, and cross‐linking. Our studies show that such adaptive responses include the immediate disassembly of the 26S proteasome (catalyzed by HSP70 and Ecm‐29) to form free 20S proteasome and 19S regulator complexes, at which point ATP/Ubiquitin‐dependent proteolysis is temporarily lost. The additional free 20S Proteasomes (+/‐ 11S or PA28 regulators) are of immediate help in degrading oxidized proteins. The original 26S Proteasomes are re‐assembled, and ATP/Ubiquitin‐dependent proteolysis is restored over a three‐hour period. During this three‐hour period, and during the subsequent 17 hours, new 20S Proteasomes, Immunoproteasomes, and 11S (PA28) proteasome regulators are synthesized, partially under the control of the Nrf2 signal transduction pathway. We have now obtained similar findings in C. elegans worms, and D. melanogaster fruit flies. Proteasome and Immunoproteasome subunit genes, and the 11S (PA28) regulator appear to be true shock/stress genes that provide significant oxidative stress protection. In older cells, Proteasome activities decline, and adaptational responses mediated by Nrf2 become sluggish or even ineffectual. The antagonistic effects of Bach1 and/or Nrf1 may actually blunt adaptive proteolytic responses to stress in older cells and organisms. Studies in animals and humans suggest that declining Proteasome activities and, perhaps, declining responsiveness to stress, may contribute to the ageing process, and to various age‐associated diseases.

Grant Funding Source: NIH/NIEHS ES003598
Visualization of actin mono‐ADP‐ribosylation in iota toxin and actin complex (555.4)
Authors: Tsuge Hideaki , Tsurumura Toshiharu , Tsumori Yayoi
Published: June 2021

Clostridium perfringens iota‐toxin (Ia) mono‐ADP‐ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. We revealed the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole‐4‐carboxamide adenine dinucleotide) (ref1); however, the structures of the NAD+ bound form (NAD+‐Ia‐actin) and the ADP ribosylated form [Ia‐ADP ribosylated (ADPR)‐actin] remain unclear. Here we obtained the good diffraction apo crystals and found that ethylene glycol as cryo‐protectant inhibits ADP ribosylation. We used the apo crystal as the ADP‐ribosylation reaction chamber and applied NAD+‐soaking to this complex in different conditions. Finally, we obtained high‐resolution structures of NAD+‐Ia‐actin and Ia‐ADPR‐actin (ref2). The structures of NAD+‐Ia‐actin and Ia‐ADPR‐actin represent the pre‐ and postreaction states, respectively. The strain‐alleviation model of ADP ribosylation, which we proposed previously, became more plausible with additional snapshots of actin ADP‐ribosylation.

(1) Tsuge H, Nagahama M, Oda M, Iwamoto S, Utsunomiya H, Marquez VE, Katunuma N, Nishizawa M, Sakurai J (2008) PNAS 105 (21):7399‐7404.

(2) Tsurumura T, Tsumori Y, Qiu H, Oda M, Sakurai J, Nagahama M, Tsuge H (2013) PNAS 110 (11):4267‐4272.
A role for mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (557.1)
Authors: Abeliovich Hagai , Zarei Mostafa , Rigbolt Kristoffer , Youle Richard
Published: June 2021

Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells. Defects in mitophagy occur in degenerative disorders such as Parkinson’s disease, and decreases in the levels of mitophagy correlate with aging phenomena. An important goal is to achieve an understanding of the quality control mechanism(s) that specifically cull defective mitochondrial components or compartments, during mitophagy. Specifically, it is not known whether a small cohort of defective molecules will cause the ablation of an entire compartment, or whether an active process segregates defective molecules from functional ones and allows a more efficient culling mechanism. In this study we have combined a proteomic study with a molecular genetic and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We report that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism that couples a weak physical segregation with mitochondrial fission and fusion to achieve a distillation‐like effect. In agreement with this interpretation, the differential rates of mitophagy that we observed for different mitochondrial matrix proteins strongly correlate with the degree of physical segregation observed for these proteins, within the matrix.

Grant Funding Source: Supported by the Israel Science Foundation
Dysregulated ubiquitin proteasome system leads to neurodegeneration in a spontaneous obese rat model (559.1)
Authors: Reddy G , Reddy S , Reddy V , Raghu G , Giridharan N
Published: June 2021

Objective: A spontaneously developed obese rat (WNIN/Ob) was established from the existing WNIN rats by selective breeding. The WNIN/Ob rats show accelerated aging and develop age related diseases including neurodegeneration. The main aim is to investigate the role of ubiquitin proteasome system (UPS) in obesity‐associated neurodegeneration in WNIN/Ob rat model.

Methods: Various components of UPS like ubiquitin, UCHs (ubiquitin C‐terminal hydrolases), proteasomal sub‐units and endoplasmic reticulum (ER) stress markers were analysed by qRT‐PCR and immunoblotting. Chymotrypsin‐like activity of proteasome was assayed by fluorometric method. Apoptotis and morphology of neurons in the cerebral cortex was assessed by immunohistochemistry and transmission electron microscopy, respectively.

Results: The results indicated dysregulated UPS, increased ER stress and up‐regulation of apoptotic regulators in obese rats. Neurons in the cerebral cortex of obese rats showed swollen mitochondria, disrupted ER and degenerating axons, nucleus and finally neurons. It appears UCHL‐1 mediated apoptosis through stabilizing p53 as a key factor of neurodegeneration.

Conclusion: Alterations in the UPS may be causally related to neurodegeneration in WNIN‐obese rat. Further this rat model could be a suitable animal model for investigating the role of UPS in age related neurodegenerative diseases.
An RNA polymerase from the ocean improves in vitro RNA synthesis (560.1)
Authors: Zhu Bin , Tabor Stanley , Richardson Charles
Published: June 2021

Phages in the ocean harbor the greatest diversity of protein on the earth. Since a large portion of these proteins must play roles in nucleic acids metabolism, one would expect numerous novel mechanisms underlying the fundamental processes including transcription, DNA replication, and recombination and thus abundant molecular reagents therein. We recently characterized the single subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyzes RNA synthesis over a wide range of temperature and salinity. Its processivity is greater than 30,000 nt without any significant intermediate products. Most important, Syn5 RNA polymerase produces precise run‐off transcripts with homogeneous 3’ termini. The enzyme predominantly used for in vitro run‐off RNA synthesis‐T7 RNA polymerase synthesizes, in addition to run‐off products of precise length, transcripts with an additional non‐base‐paired nucleotide at the 3’‐terminus (N+1 product). Therefore, the enzyme is advantageous for the production of RNAs that require precise 3’‐termini, such as tRNAs and RNA fragments that are used for subsequent assembly.
Nucleolin‐enhanced deadenylation and bulk translation (566.1)
Authors: Woo Ho Hyung , Lee Sang , Chambers Setsuko
Published: June 2021

Rate of translation is one of the critical factors, determining protein abundance in a cell. After a pioneer round of translation in which 80S ribosome scans the linear mRNA, a cap‐dependent mRNP closed‐loop is formed for bulk translation.

Nucleolin interacts with the PABPC and works together for deadenylation and mRNP closed‐loop formation. Nucleolin interacts to the Pan2/Pan3 and CCR4‐NOT deadenylation complex, indicating nucleolin’s involvement in the mRNA deadenylation. Nucleolin also interacts with the eIF4F translation initiation complex, suggesting another role of nucleolin in translation initiation. We found that an increased level of deadenylated mRNAs is associated with heavy polyribosomes in nucleolin overexpressed cell lines. Together these results indicate that nucleolin‐mediated deadenylation is not followed by mRNA decay, but can lead to the bulk translation by forming poly(A)n‐less mRNP closed‐loop.

We propose that a non‐canonical mRNP closed‐loop lacking poly(A)n tail can be formed by nucleolin for bulk translation. In this model, nucleolin enhances deadenylation of the mRNA and induces the formation of poly(A)n‐less mRNP closed‐loop by binding dsRNA formed between the mRNA 5’‐ and 3’‐UTR complementary sequences. Nucleolin is proposed as a mediator for the transition from the canonical to non‐canonical poly(A)n‐less mRNP closed‐loop to enhance the bulk translation.
New structural insights into cap‐independent translation initiation (566.6)
Authors: Brilot Axel , Koh Cha San , Grigorieff Nikolaus , Korostelev Andrei
Published: June 2021

Conventional initiation of translation of cellular mRNAs depends on the m7G cap located at the 5' untranslated region (UTR) and on extraribosomal proteins called initiation factors. Translation of many viral mRNAs and some cellular mRNAs can be initiated in a cap‐independent manner, with fewer initiation factors, to promote expression under stress conditions when overall cellular translation is downregulated. In these mechanisms, several types of structured RNA at the 5' UTR are employed, called the internal ribosome entry sites (IRES). The structural basis for how an IRES binds to the ribosome and positions the open reading frame of mRNA is not fully understood. Here, we present molecular structures of ribosome complexes, which provide new structural insights into the mechanism of IRES‐dependent translation.

Grant Funding Source: Supported by HHMI and National Institutes of Health
Mechanistic studies of ribosome‐dependent toxins active during the stringent response (566.8)
Authors: Dunham Christine , Schureck Marc , Maehigashi Tatsuya , Ruangprasert Ajchareeya , Dunkle Jack , Miles Stacey
Published: June 2021

Regulation of protein synthesis is one mechanism by which bacteria alter their metabolic rates during times of stress. This process is known as the ‘stringent response’ where the presence of deacylated tRNAs bound to the ribosome is one signal that initiates a cascade of events ultimately resulting in the activation of toxin proteins. Type II toxin proteins halt translation by targeting mRNA bound to the ribosome for degradation in a codon‐dependent manner. Here, we present structural studies of different toxin proteins bound to target mRNAs on the 70S ribosome. Our structures reveal novel interactions between conserved toxin residues with the mRNA backbone in the ribosomal A site that helps to rationalize codon specificity by different toxins. Biochemical and in vivo growth assays complement our structural studies to provide a unified mechanism of toxin‐mediated mRNA degradation.

Grant Funding Source: Supported by NSF MCB 0953714 and Pew Scholars Program in the Biomedical Sciences
Functional characterization of natural occurring variants of the 70‐kDa heat‐shock protein, HSPA1A (567.10)
Authors: Siracusa Matthew , Nikolaidis Nikolas
Published: June 2021

Seventy‐kDa‐heat‐shock proteins (Hsp70s) are molecular chaperones essential for protein homeostasis and cell survival under both physiological and pathological conditions. Given this vital role of Hsp70s, it is important to investigate whether naturally occurring variants of these proteins have altered functional properties. In this study, the functional outcome of four non‐synonymous single nucleotide polymorphisms present in different human populations on HSPA1A was tested. Site directed mutagenesis was used to generate the HSPA1A mutations into both both bacterial and mammalian expression vectors. Through the use of a malachite green colorimetric ATPase assay, the mutant proteins’ ability to hydrolyze ATP at the protein’s ATP‐binding domain was tested. All four proteins were found to be functionally differentiated from each other and the WT. Based on the Km values obtained, the WT had the highest affinity for binding to ATP, but based on the Vmax values, all the mutants were able to hydrolyze ATP at a much faster rate, with the S16P mutant showing nearly a 4‐fold increase from the WT. The mutant proteins’ ability to properly localize intracellularly within HEK‐293 cells was also tested by tagging HSPA1A to GFP, using fluorescent dyes to stain the mitochondria, lysosomes, and plasma membrane, and viewing where the proteins colocalized via confocal microscopy. Under normal growth conditions all four mutants were found to localize to these regions in a similar manner to the WT indicating that these mutations did not affect the protein’s ability to localize within the cell.

Grant Funding Source: This project was supported by funds from CSUPERB and CSUF to NN, and HHMI to MS and NN
Phosducin‐like protein and CCT interact with G protein β‐like, β propeller proteins (567.4)
Authors: Tsang Tsz Ming , Mak Wai Shun , Chan Tsz Yin , Lukov Georgi
Published: June 2021

Previous studies have established that the cytosolic chaperonin complex, CCT and phosducin‐like protein (PhLP) are required for the proper folding of the G protein β subunit, and the assembly of the Gβγ subunit complex. The structure of the Gβ subunit is a seven bladed, β propeller. Previous studies have also shown that CCT has multiple binding partners, some of which have β propeller structure, similar to that of Gβ. Our objective is to investigate if proteins with seven bladed β propeller structure, similar to that of Gβ would associate with CCT and PhLP. We investigated the binding interaction of three Gβ‐like proteins, CDC20, TBL2 and MLST8, to CCT and PhLP. Using co‐immunoprecipitation approaches, we demonstrated that all three Gβ‐like proteins interact with CCT and PhLP. These findings suggest that CCT and PhLP may work together not only during the folding of the Gβ proteins, but they may be the folding machinery of all proteins with Gβ‐like, seven bladed β propeller structure.

Grant Funding Source: BYUH Mentored Research and Student Research Associateship Funds
Mapping the binding interactions between a specialized Hsp70 chaperone and its Hsp40 co‐chaperone: a direct role for the Hsp40 HPD motif (567.5)
Authors: Alderson T. , Kim Jin , Markley John
Published: June 2021

Proteins from the ISC operon of E. coli constitute the machinery used for the synthesis of iron‐sulfur clusters and their delivery to recipient apo‐proteins. [2Fe‐2S] cluster transfer from the holo‐scaffold protein IscU depends on ATP hydrolysis in the nucleotide‐binding domain (NBD) of HscA, a specialized Hsp70 chaperone. HscB, an Hsp40‐type co‐chaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer. However, the interactions between HscA and HscB have remained uncharacterized, and, moreover, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. We have created three variants of the isolated HscA NBD truncated at different residues within the linker, and have shown that the linker binds to the NBD and autoactivates ATP hydrolysis. Using solution‐state NMR spectroscopy and chemical shift perturbations, we mapped the apo‐HscA, HscA‐ADP and HscA‐ATP binding sites onto HscB. Our NMR data provide the first evidence of a structural role for the ubiquitously conserved J‐domain residues HPD of HscB, which we found selectively bind to the ATP‐bound form of HscA. Additionally, we have identified and characterized a previously unreported interaction between HscA and IscA, an iron‐binding protein putatively involved in [4Fe‐4S] cluster transfer, which competes with HscB for binding to HscA.

Grant Funding Source: Supported by NIH grants U01 GM094622 and P41 GM103399
The structural basis for recognition and modification of the 30S ribosomal subunit by an antibiotic resistance methyltransferase (569.3)
Authors: Dunkle Jack , Vinal Kellie , Zelinskaya Natalia , Conn Graeme , Dunham Christine
Published: June 2021

Ribosomal RNA contains modifications at specific nucleotides in all organisms, some of which control resistance to antibiotics. However, the mechanisms are not well understood controlling how modification enzymes recognize their specific target nucleotide and position it within the active site. We report the crystal structure of the pathogen‐derived aminoglycoside‐resistance rRNA methyltransferase NpmA bound to the 30S ribosomal subunit in a pre‐catalytic state. The structure reveals that NpmA recognizes conserved rRNA tertiary structure rather than RNA sequence. Additionally, the structure shows NpmA flips the target nucleotide from its position within the helical base stack to position it within the active site. The structure provides a general framework for investigating the mechanisms of other rRNA modification enzymes.

Grant Funding Source: Supported by National Institutes of Health