bluuebunny/crossref_metadata_2025 huggingface dataset:
The Outcomes Logic Model: An Approach for Program Planning and Evaluation: Taking successful pilot programs to scale: Evolution of TBL anatomy program
Authors: Vasan Nagaswami , DeFouw David
Published: June 2021

In curriculum planning, program development and evaluation, most medical educators recognize that outcomes drive the planning, development and management of curriculum and outcomes are complex and intertwined with environmental factors over which they often have limited control. Medical education being dynamic, curriculum changes are often necessary to benefit both the learners and institutions. Furthermore, diminished resources for medical education and accountability necessitate planning programs with a ‘blueprint’ in place. The logic model makes explicit the logic of how a program achieves its objectives and goals. The model focuses on programs particular population or target groups (students, faculty or patients). The outcomes logic model explores the effect of the current program in terms of resources required to develop it, program activities and outputs‐on the target group, and enables production of a blueprint for future development, expressed as short‐term, medium‐term, and long‐term goals, with corresponding outcome measures. This blueprint is the core of the logic model. Team Based Learning in anatomy is used as an example to illustrate the outcomes logic model. It shows a step‐wise process in planning and implementing a new curriculum. Serve as a format for clarifying what the program hopes to achieve. Help programs stay on tracks. Document what the program is actually doing and its impact.
Student‐centered collaborative learning in a medical histology course through Virtual Microscopy and Team‐Based Learning
Authors: Enfield Grace , DeFouw David , Vasan Nagaswami
Published: June 2021

The New Jersey Medical School (NJMS) has transformed histology into an active, collaborative learning experience through use of Virtual Microscopy (VM) and Team‐Based Learning (TBL). VM and TBL are effective tools that promote interactive learning. At NJMS, the Integrated Structure and Function (ISF ‐ histology and physiology) course mainly consists of lectures on structural and functional features and VM lab. The VM system provides access to thousands of digitized images. VM is an invaluable tool because it allows students to conveniently study histological images online and work as a team, and allows faculty to teach in an interactive group format. VM images can be captured, labeled, saved for reference, allowing for interpretation, reinforcement, review, feedback from fellow classmates and instructors. In 2009, we piloted TBL strategy in histology because it requires consistent preparation and class attendance to ensure student participation. TBL encouraged peer teaching and interactive discussion that promotes better retention of knowledge. We found that student performance on the NBME Cell Biology and Histology subject exam showed improvement compared to previous years. Students and faculty have responded favorably to VM and TBL because it's effectiveness in collaborative learning. We plan to expand TBL more in the future. (Supported by the Department of Cell Biology and Molecular Medicine.)
A functional role for CHD8 in transcriptional regulation through the modulation of chromatin structure
Authors: Bochar Daniel A , Menon Tushar , Yates Joel A
Published: June 2021

ATP‐dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. The CHD family can be subdivided into three subfamilies: CHD1‐2, CHD3‐5, and CHD6‐9. Despite being linked to several human diseases, very little functional data exists for the CHD6‐9 family as compared to the other two subfamilies.

In order to understand the function of CHD8, we have isolated a CHD8 containing protein complex which includes WDR5, Ash2L, and RbBP5. These proteins are required components of the MLL/Set1 histone methyltransferase complex. We demonstrate that CHD8 localizes to the promoters of MLL regulated genes upon gene activation. During activation of these target genes, depletion of CHD8 enhances gene expression and causes a decrease in both histone H3 lysine 4 trimethylation and histone H3 lysine 27 trimethylation. Furthermore, depletion of CHD8 results in a loss of WDR5/Ash2L/RbBP5 subcomplex localization at target promoters. These studies suggest that CHD8 alters eukaryotic gene expression by modulating the methylation status of histones at target promoters through the recruitment of chromatin modifying enzymes.

This work was supported in part by an American Cancer Society Research Scholar Grant (RSG‐07‐189‐01) to D.A.B
Utilizing elongation factor Tu mutagenesis to incorporate large unnatural amino acids in vivo
Authors: Peeler Jennifer Carol , Mehl Ryan A.
Published: June 2021

Site‐specific in vivo incorporation of unnatural amino acids (UAAs) via aminoacyl tRNA synthetase (RS) mutagenesis has been widely used in protein structure and function studies. While a large, diverse family of UAAs has been incorporated into proteins, aminoacyl tRNA synthetases for certain UAAs seem problematic to evolve or generate. We developed a reporter system to explore substrate specificity of current UAA RSs. Using these permissive RSs, structural limitations have been explored. The photocrosslinking UAAs anthraquinonylalanine and 3‐(3’‐fluorenyl‐9’‐oxo)‐alanine should be incorporated by permissive RSs. However, incorporation of these UAAs into proteins has not yet been accomplished. It is the current understanding that elongation factor Tu (EF‐Tu) size limitation is preventing the incorporation of these UAAs. It is believed that UAAs that were previously too large to see incorporation with mutant RSs alone will hopefully be incorporated utilizing mutant RS and mutant EF‐Tu combinations in vivo. Here we report progress with mutant EF‐Tu and orthogonal aminoacyl tRNA synthetase pairs. Supported by F&M funds, NSF‐MCB‐0448297; Research Corporation (CC6364).
Career Envy: The Road to a Successful PUI Position
Authors: Cotten Myriam L. , Decatur Sean , Mehl Ryan , Parson Kathleen , Provost Joseph
Published: June 2021

Careers at Primarily Undergraduate Institutions (PUI) are exciting and present a rewarding opportunity for faculty to combine diverse interests in education with research. Faculty who chose this path as a career face a challenge in balancing teaching, research and service. This panel discussion brings together mid‐career and experienced faculty from private/independent and public institutions to discuss critical factors in choosing a PUI career and well‐established approaches for being successful as an undergraduate faculty. The discussion will include common themes of: how to prepare and be competitive for a PUI position, transitioning from a post doc into a teaching‐rich environment, aligning teaching with research to be more efficient and productive, approaching research with undergraduates, and working with administrators and find mentors to support your career. Students, postdoctoral fellows and faculty are welcomed to participate in the discussion. Supported by the Council for Undergraduate Research and Research Corporation for Science Advancement.
Molecular Mechanism of the Ubiquitin Conjugating Enzyme E2‐25K to Synthesize the Polyubiquitin Chains
Authors: Shin Dong Yeon , Park Eun Sil , Yoo Yung Joon
Published: June 2021

Ubiquitin(Ub) is involved in various cellular functions through the ubiquitination of many proteins. The assembly of polyUb chains with different topology determines the consequence of ubiquitinated proteins but the detailed mechanism for the synthesis of polyUb chains is largely unknown. In this study, we tried to elucidate the molecular mechanism of the K48‐polyUb synthesis. E2‐25K contains a C‐terminal Ub‐associated (UBA) domain and has a catalytic activity of E3‐independent ubiquitination. The most plausible mechanism of Ub chain synthesis is the aminolysis‐based transfer between a donor Ub on the active site cysteine(C92) and an acceptor Ub(s) interacting with UBA domain. However, UBA‐deleted E2‐25K mutant was able to synthesize polyUb although the catalytic activity was reduced. This results indicated that E2‐25K core domain alone could recruit the acceptor Ub(s) to synthesize the polyUb chains, facilitated by the UBA domain. In addition, we found that the polyUb chains were linked onto C92 of E2‐25K through the thioester bond using the nonreduced/reduced two‐dimensional electrophoresis, suggesting that protein substrates could be ubiquitinated en bloc by preassembled polyUb chains from E2‐25K in addition to conventional one‐by‐one ubiquitination. (Supported by a grant for the Study of Ubiquitome Functions from the KMEST and the Systems Biology Infrastructure Establishment Grant by GIST)
The Role of Arv1 in Sterol Metabolism and its Contribution to the Unfolded Protein Response
Authors: Shechtman Caryn Faith , Henneberry Annette , Seimon Tracie , Tabas Ira , Sturley Stephen L
Published: June 2021

Proper sterol distribution within the cell is a critical component of membrane homeostasis. The endoplasmic reticulum (ER)‐localized protein Arvl mediates sterol transport from the ER to the plasma membrane. Alterations in ER sterol distribution are a stimulus for unfolded protein response (UPR) activation. We hypothesize that the loss of Arv1p in yeast will increase the free sterol (FS) content of the ER, resulting in UPR induction. The UPR will also be induced in primary murine macrophages where ARV1 expression has been knocked down. Loss of yeast Arv1p function results activation of the UPR, mediated by Ire1p. The yeast arv1Δ ire1Δ homozygous haploid is inviable. Mutant ire1 core luminal domains, defective in sensing unfolded proteins, rescue the lethality of the arv1Δ ire1Δ haploid; these strains exhibited increased UPR induction that was synergistic with protein misfolding. We also demonstrate that decreased ARV1 expression in mammalian cells results in UPR induction, particularly an upregulation of CHOP. As a result of ARV1 knockdown, macrophages undergo apoptosis due to prolonged UPR induction and FS‐induced cytotoxicity. We demonstrate that defects in ER homeostasis, as a result of changes in ARV1 expression, exhibit UPR induction in both yeast and mammalian systems. The UPR appears to be a generalized response, resulting from perturbations in membrane structure, lipid metabolism and protein folding.
Endoplasmic Reticulum Stress Enhances Steroidogenic Acute Regulatory (StAR)‐Related Lipid Transfer Domain Protein 5 (STARD5) Expression and Cholesterol Efflux in HKC‐8 Human Renal Proximal Tubule Cells
Authors: Clark Barbara J , Chen Yuchyu , Brown Ryan , Carroll Julie A , Blaker Kristen M , Meier Renate K , Khundmiri Syed J , Lederer Eleanor D
Published: June 2021

STARD5 belongs to the START protein superfamily of lipid transport proteins and functions in intracellular non‐vesicular cholesterol transport. The kidney is a major site of expression and we reported STARD5 expression to be localized to the renal proximal tubules and increased in diabetic mouse kidney. The goal for this study was to characterize the HKC‐8 human proximal tubule kidney cells for STARD5 regulation by high glucose, endoplasmic reticulum (ER) stress, and oxysterols. Cells were treated with low (5 mM) or high (25 mM) glucose for 24 hr – 7 days and mRNA levels were measured by RT‐qPCR. STARD5 expression was decreased 50% under high glucose conditions, consistent with a decrease in ER stress as measured by decreased GRP78 mRNA levels. Oxysterols, ligands for the LXR nuclear receptor, had no effect on STARD5 expression but induced ABCA1 promoter activity and mRNA levels. Thus, HKC‐8 cells have an intact LXR response and express ABCA1. Tunicamycin‐induced ER stress increased STARD5 expression and enhanced cholesterol efflux from the cells but had no effect on ABCA1 mRNA levels. Our results suggest that high glucose conditions do not directly stimulate STARD5 expression or ER stress in HKC‐8 cells. Induction of ER stress, on the other hand, enhances cholesterol efflux, as well as STARD5 expression. A direct role for STARD5 in cholesterol efflux is currently under investigation.
A liver mitochondrial outer membrane (MOM) fatty acid transfer complex
Authors: Lee Kwangwon , Kerner Janos , Hoppel Charles L.
Published: June 2021

Carnitine palmitoyltransferase‐I (CPT‐I) catalyzes the regulated step in overall mitochondrial fatty acid oxidation. Here we present both a Blue‐Native PAGE (BNE) and an immunological approach to isolate and characterize the MOM CPT‐I and its interacting partners. When solubilized MOM was subjected to BNE, a series of high molecular weight bands greater than the monomeric CPT‐I (88 kDa) were identified. The proteins in these bands were characterized by in‐gel digestion and sequence analysis by LC‐MS/MS (Center for Proteomics and Bioinformatics). An immunocapture system was designed using an antibody mixture of five CPT‐I specific peptide‐derived IgGs. Immunocaptured proteins were subjected to LC‐MS/MS. Using this method, not only the expected CPT‐I, but also LCAS (long‐chain acyl‐CoA synthase), and VDAC (voltage‐dependent anion channel) isoforms were captured and identified. These accompanying proteins also were identified with BNE. Therefore, we conclude that the MOM contains a complex for fatty acid transfer that consists of LCAS, VDAC and CPT‐I.

Supported by the NIH grant DK‐066107.
Adipose‐specific deletion of SCD1 does not protect mice from obesity
Authors: Flowers Matthew , Strable Maggie , Ade Lacmbouh , Zhao Minghui , Ntambi James
Published: June 2021

A critical regulator of lipogenesis is stearoyl‐CoA desaturase 1 (SCD1), which catalyzes the synthesis of monounsaturated fatty acids (MUFA) from saturated fatty acids. To explore the role of SCD1 in the adipose tissue, we used the Cre‐lox system to delete SCD1 from white and brown adipose tissue (AKO) using the aP2‐Cre recombinase transgene. We also generated mice with simultaneous deletion of SCD1 from both adipose and liver (LAKO) or from liver alone (LKO) using the albumin‐Cre transgene. Mice were subjected to high‐fat diet‐induced obesity (DIO; Research Diets 12492) or genetically‐induced obesity using the leptin‐resistant yellow agouti (Ay/a) allele and compared to control mice not expressing Cre recombinase (LOX). Food intakes and changes in body weights were similar amongst the LOX, AKO, LKO and LAKO mice in both models of obesity. Additionally, fat pad mass (epididymal and subcutaneous) was unaffected by adipose and/or liver deletion of SCD1. Immunoblot analysis confirmed deletion of SCD1 and fatty acid composition analysis by gas chromatography revealed reduced MUFA content in the targeted tissues. In light of our previous observations that mice with a whole‐body or skin‐specific deletion of SCD1 are resistant to obesity, the current study suggest that SCD1 deletion from adipose and/or liver is insufficient to elicit protection from obesity.

Research supported by funding from NIH‐NIDDK and AHA.
Ribosomal protein sequence analysis reveals the order of evolution of bacterial classes, direct lineage of Cyanobacteria and Chloroplasts and that LUCA was an Actinobacteria
Authors: Duax William , Huether Robert , Hogan Dana , Teysir Jimmitti , Dziak David
Published: June 2021

Search vectors composed primary of Gly, Ala, Arg, and Pro residues (GARP) distributed across the entire protein sequence retrieve 98% of each of the ribosomal proteins in bacterial species with no false “hits”. Different combinations of G, A, R and P and insertions or deletions differentiate each ribosomal protein from all others. We are able to identify site mutations that subdivide each ribosomal protein ensemble into the individual classes of bacteria. For example, four fold variation in a single sequence position in S4 separates 490 gram‐positive bacteria that have Y and F in this position and 717 gram‐negative bacteria that have W in this position from 68 cyanobacteria and 601 chloroplasts that have an H in this position. Detection of co‐evolution of amino acids in four sequence positions makes it possible to divide the 1876 ribosomal proteins into 25 classes. Further subdivision into orders, families, genus, and species is trivial. While there are significant differences between the sequences of the ribosomal proteins in different classes of bacteria, within each class the amino acid sequences have remained highly conserved for millions of years. We have found that the total GARP content of the ribosomal proteins of each class is a marker of the order of evolution and that the last universal common ancestor (LUCA) appears to have been an Actinobacteria.
Investigating the molecular basis of temperature‐dependent sex determination in the painted turtle
Authors: O'Shaughnessy Katherine Lynn , Valenzuela Nicole , Neuwald Jennifer , Literman Robert , Harris Amanda , Manternach Tanya
Published: June 2021

Temperature dependent sex determination (TSD) is a commonly found mechanism in many reptilian species; however the molecular and evolutionary basis for this form of environmental sex determination remains unclear. This study used the painted turtle (Chrysemys picta) as a TSD model to analyze expression of two genes involved in vertebrate gonadogenesis, the Wilms’ tumor‐suppressor gene (Wt1) and steroidogenic factor 1 (Sf1) respectively. Previous study observed differential expression of Sf1 and Wt1 in C. picta at constant temperature incubation. In order to mimic more ecologically relevant conditions this study compared expression of Sf1 and Wt1 under fluctuating incubation temperatures. The results obtained demonstrate significant differences in Wt1 and Sf1 expression; however, the timing of the differential expression is shifted to an earlier developmental period than under constant incubation conditions. These findings suggest that fluctuating incubation temperatures might affect the timing of the thermal sensitive period (TSP) and consequent activation of the genetic network responsible for sex determination in TSD species. This study was funded in part by the National Science Foundation (NSF IOS 0743284) and REU supplement IOS‐0925486 to N. Valenzuela, and REU grant DBI‐0552371 to M. Rothschild.
Model for Protein Kinase A Type RIα Tetrameric Holoenzyme
Authors: Boettcher Angela J. , Wu Jian , Yang Jie , Cheung Nikki , Kornev Alexandr P. , Kim Choel , Taylor Susan S.
Published: June 2021

Protein kinase A holoenzyme contains two Catalytic (C) and two Regulatory (R) subunits, which dimerize through their N‐terminus Dimerization/Docking (D/D) domains. R‐subunit isoforms are highly conserved; the most divergent region lies at the linker connecting the D/D domain and the two tandem cAMP binding domains. Binding of four cAMP molecules to the R‐subunit dimer leads to significant structural changes that lead to the activation of the kinase. This process is highly cooperative in the tetrameric holoenzyme but the cooperativity is significantly reduced in the heterodimer which lacks the D/D domain and the linker. To understand the role of the linker region, we solved the structure of a PKA RIα(73–244)/C heterodimer, which contains the first cAMP binding domain and most of the linker. The overall structure is very similar to the previously solved RIα(91–244)/C heterodimer complex, however, the new structure allowed observation of 7 more residues (84–90) in the linker. Interestingly, this linker region docks to another symmetry related heterodimer, suggesting that one R‐subunit not only binds to one C‐subunit at multiple sites within the heterodimer but also cross‐talks with another heterodimer through the linker region when the tetrameric complex is formed. The β4‐β5 loop of the R‐subunit and the Y235PPPFF239 of the C‐subunit, which links αF and αG and is where PKI binds are also involved in this cross‐talk. By integrating small angle X‐ray/neutron scattering, molecular interface free‐energy calculations and other data, a model of RIα/C tetrameric complex is proposed.
An Inhibitor Against CKII Phosphorylation Downregulates VPAC1 Expression in HuT‐78 Cells
Authors: Nelson Ashley Marie , Benton Keith , Hermann Rebecca , Van der Steen Travis , Dorsam Glenn
Published: June 2021

Presently, Leukemia affects about 250,000 people in America and it is expected that 45,000 new cases will be diagnosed in 2009. We have previously published that Ikaros (IK), a tumor suppressor, downregulates vasoactive intestinal receptor 1 (VPAC1), an anti‐proliferative G protein coupled receptor, mediated by IK engagement to VPAC1. Currently, it is not known how post‐translational modifications, such as phosphorylation, regulate IK DNA binding activity. Therefore, we hypothesized that inhibition of casein kinase II, CKII, known to phosphorylate IK, would shift the equilibrium to favor its dephosphorylated IK state, causing greater DNA binding and repression of VPAC1. To test this, HuT‐78 cells were cultured in the presence and absence of 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazol (DRB) for 0, 12, and 24 h. The levels of VPAC1 mRNA expression were measured by TaqMan qPCR. After treatment with DRB, the levels of VPAC1 in the cells decreased from 0 to 12 h by about 70%, but were restored after 24 hours. These results support that the hypophosphorylated IK pool is most likely responsible for transcriptional regulation of VPAC1. Overall, DRB downregulates VPAC1 expression by inhibiting the phosphorylation of IK through the inhibition of CKII. Funding: NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.
GIT1 Act As GAP For Arf6 Downstream Integrin Outside‐In Signaling During Platelet Aggregation
Authors: Huang Yunjie , Sidney Whiteheart
Published: June 2021

Our previous work showed that ADP‐ribosylation factor (Arf) 6 is critical for platelet function. Arf6‐GTP is relatively high in resting platelet but quickly converts to Arf6‐GDP during platelet activation in response to primary agonists (thrombin, collagen, etc.). An additional control of Arf6 nucleotide state is hypothesized since blocking integrin‐mediated signaling with RGDS peptides leads to a partial recovery of Arf6‐GTP; this phenotype is also observed in β3 integrin knock‐out mice. This recovery of Arf6‐GTP could be through contact‐mediated signaling via an Eph kinase/ephrin mechanism or via an integrin‐based.

The G protein‐coupled receptor kinase interacting protein (GIT) 1 is present in platelet and upon platelet activation, GIT1 is tyrosine‐phosphorylated, a process that is blocked by RGDS peptide treatment. Since GIT1 has an N‐terminal Arf GTPase‐activating domain, we hypothesize that GIT1 acts as a GTPase‐activating protein (GAP) for Arf6 and is downstream of cell‐cell contact‐mediated signaling. Western blotting analysis demonstrates that GIT1 is present in the insoluble cytoskeletal pellet after thrombin activation of platelets. Inhibiting Arf6‐GTP loss affects this redistribution. Further study is required to determine if GIT1 Arf6‐GAP activity is regulated by phosphorylation or whether Arf6‐GTP targets GIT1 to the cytoskeleton.

Supported by NIH grants HL56652 and HL091893.
Monitoring GTPCH‐1 Interaction with GFRP Using Time‐Resolved Fluorescence Resonance Energy Transfer
Authors: Li Li , Du Yuhong , Fu Haian , Salerno John C , Chen Wei , Harrison David G
Published: June 2021

GTP Cyclohydrolase I (GTPCH‐1) is the rate‐limiting enzyme for tetrahydrobiopterin (BH4) synthesis and we have recently shown that it is inhibited by GTP Cyclohydrolase Feedback Regulatory Protein (GFRP) in the endothelium. The complex formation of GTPCH‐1 and GFRP is modulated by several factors including phosphorylation of the former and mechanical forces acting upon the endothelial cell. We have designed and optimized a time‐resolved fluorescence resonance energy transfer (TR‐FRET) assay to monitor the interaction of GTPCH‐1 with GFRP in the presence of GTP and BH4 as cofactors. This assay uses GTPCH‐1 labeled with a europium chelate as FRET donor and GFRP labeled with APC as acceptor and requires low concentrations of proteins to achieve an optimal FRET signal. This assay is highly sensitive and stable and has achieved a robust performance in 384‐well and 1536‐well formats with a signal‐to‐background ratio greater than 12 and a Z’ factor greater than 0.75. In the absence of GTP and BH4 this assay only generates a background signal, confirming that these are needed for protein interaction and demonstrating specificity of the assay. Because it features a one‐step “mix‐and‐read” format, this assay could be particularly suited for use in high throughput screening for GTPCH‐1 modulators. Supported by HL39006 and a VA Merit Grant.
DNA breaks at fragile sites generate oncogenic RET/PTC rearrangements in human thyroid cells
Authors: Dillon Laura , Gandhi Manoj , Pramanik Sreemanta , Pierce Levi , Nikiforov Yuri , Wang Yuh‐Hwa
Published: June 2021

Common fragile sites are regions of the genome that are prone to DNA breakage and frequently coincide with the location of genes involved in carcinogenic chromosomal translocations. In this study, we examined the direct involvement of fragile sites in the formation of RET/PTC rearrangements, which are commonly found in papillary thyroid carcinomas, and are all a result of a translocation event involving the RET gene. We demonstrated that fragile site‐inducing chemicals, including aphidicolin, can induce the formation of RET/PTC rearrangements in thyroid cells. We further studied fragile site‐induced DNA breakage within the genes (RET, CCDC6, and NCOA4) participating in the two major types of RET/PTC rearrangements, RET/PTC1 and RET/PTC3, through cytogenetic, molecular biology, and sequence analysis methods. Using FISH, we found the rate of DNA breakage at these genes to be similar to known fragile site‐induction levels of the common fragile sites containing these genes, FRA10C and FRA10G. We mapped the location of induced breakpoints using LM‐PCR and found them to be located near breakpoints observed in patients. Factors contributing to the formation of DNA breaks have been investigated using sequence analysis. These results provide the first direct evidence for the role of chromosomal fragile sites in the generation of cancer‐specific rearrangements in human cells. (Supported by the NCI, CA113863).
Phosphorylation of RPA2 Is Required For TopBP1 Recruitment and ATR Activation
Authors: Liu Shengqin , Glanzer Jason , Oakley Greg
Published: June 2021

Replication protein A (RPA) is a heterotrimer, single strand DNA binding protein involved in multiple aspects of DNA metabolism. The N‐terminus of the RPA2 subunit is phosphorylated at multiple sites during cell cycle progression and in response to DNA damage. To understand the role of RPA2 phosphorylation, we generated stable cell lines expressing wild‐type and mutant forms of RPA with down regulation of endogenous RPA2 by shRNA. The inability to phosphorylate Ser4 and Ser8 resulted in a loss of phosphorylation of Ser12 and Thr21, but not Ser33 in response to etoposide treatment. Cells expressing mutant RPA2 showed a defective G2/M checkpoint and failed to halt mitotic entry after etoposide treatment. Decreased phosphorylation of Chk1 and Mre11, suggested ATR activation in response to etoposide was compromised in cells expressing mutant RPA2. TopBP1, a key activator of ATR, is recruited to DNA lesions by Rad9. Rad9 also interacts with RPA through binding with RPA1. Defective phosphorylation of the mutant RPA increased interaction with Rad9 and inhibited phosphorylation of Rad9. The decrease in the phosphorylation of Rad9 correlated with a loss of TopBP1 recruitment to chromatin in response to DNA damage. Taken together, these results suggest that phosphorylation of RPA may regulate RPA function by altering its interaction with other key proteins in the DNA damage response pathway.
Computational prediction and experimental validation of 5 DNA‐binding residues of human flap endonuclease 1 (hFEN1)
Authors: Kanjanapangka Julie , Finger Lawrence David , Shen Binghui
Published: June 2021

Human FEN1 is a structure‐specific DNA nuclease involved in removing 5′ RNA primers of Okazaki fragments and in long‐patch base excision repair. Defects in FEN1 lead to increased genomic instability. Identification of DNA‐binding residues will further elucidate the molecular mechanisms behind genome instability initiation and facilitate cancer therapy linked to hFEN1 mutations. No crystal structure data is available of hFEN1 actively bound to DNA; however, a structure of an hFEN1 homolog, T4 phage RNase H in its active form (pdb 2ihn), is available as well as hFEN1 complexed with PCNA in the inactive form (pdb 1ul1). The 2ihn pdb structure shows 31 DNA‐interacting residues. Computational structural alignment of the two X‐ray crystal structures has revealed 5 residues within the 31 subset of RNase H that have a seemingly structurally conserved equivalent in hFEN1; these are K74, L29, V30, F53, and H174. These conserved equivalents in hFEN1 may therefore interact with DNA. To validate our predictions, we use HPLC to measure specific activity levels of hFEN1 mutant proteins versus the wildtype using fluorescein‐labeled DNA substrate. We found that the specific activities of the mutants are impaired relative to the wildtype. We will measure DNA binding activity of hFEN1 mutants using gel shift with biotin‐labeled DNA substrate to validate if the results we observe are due to defects in DNA substrate binding.

This work is funded by NIH R01CA076734: Fen1 in genome stability and cancer.
Analyses of Alternative Splicing in C. elegans Longevity Genes
Authors: Seipelt Rebecca , Saites Louis , Mohan Varuna , Winfrey Alexander
Published: June 2021

The purpose of this study was to investigate development and age‐specific novel alternative splicing patterns in the longevity genes age‐1 and clk‐1. The nematode Caenorhabditis elegans was used as the model organism, from which mRNA was harvested at different points in their developmental cycle (eggs, larval 3 (L3), dauer, adults, geriatrics). Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis was performed to detect the presence of mRNA of different sizes at different genetic locations throughout the gene, which would suggest alternative mRNA isoforms. The fragments were sequenced (eggs L3, geriatrics) to verify actual splicing reactions. The results indicate novel instances of intron inclusion and exon exclusion with the latter being associated specifically with developmental stage L3.
Insulin signaling via PKCδ isoforms in the diabetic brain promotes cognitive function
Authors: Patel Niketa A. , Cooper Denise R.
Published: June 2021

People with type II diabetes have significantly lower cognitive function. Strong links between insulin and cognitive function supported by epidemiological data from humans and animal studies and in vitro research. The consequences of protein kinase C (PKC) activation by insulin in the CNS influence memory, cognition, synaptogenesis, and neuronal repair. In addition, PKCδ, a novel PKC is implicated in memory, neuronal survival and proliferation. Insulin regulates the alternative splicing of mouse PKCδ splice variants: PKCδI promotes apoptosis while PKCδII is a pro‐survival protein. In vitro and in vivo studies demonstrated that apoptosis accounted for the neuronal loss and cognitive decline in the diabetic mouse models. Our data in mouse neural cells derived from the hippocampus demonstrates that insulin increases the expression of PKCδII and is not influenced by high glucose or thiazolidinediones. We used inhibitors of several pathways targeted by insulin to determine which pathways are involved in PKCδ alternative splicing in neuronal cells and show that insulin increases PKCδII expression via PI3K‐Akt pathway. Constitutively active Akt2 kinase mimicked insulin effects in increasing PKCδII levels while WT‐Akt2 kinase had no effect. Further, using over‐expression and siRNA we show that PKCδII increases expression of pro‐survival proteins Bcl‐2 and Bcl‐xL thereby increasing neurogenesis.
The effects of diet cycling on longevity
Authors: List Edward Owen , Wright‐Piekarski Jacob , Berryman Darlene E , Kopchick John J
Published: June 2021

Introduction

It has been suggested that diet cycling is unhealthy to the point that remaining obese is preferable to a fluctuating body weight.

Objective

To investigate the effects of diet cycling on longevity.

Methods

Male C57BL/6J mice were placed in one of three dietary regimes: a high fat (HF) diet, a low fat (LF) diet or a continuous diet cycle (4 weeks HF, 4 weeks LF). The diets were maintained for the entire lifespan of the mice. Body weight, food consumption, body composition, glucose tolerance and longevity were determined.

Results

As expected, body weight, body fat, food consumption and glucose tolerance were significantly elevated in the HF mice compared to LF controls. Diet cycling resulted in increases and decreases in the same parameters and the changes were concomitant with the HF and LF cycles. The HF and diet cycled mice consumed 61% and 20% more kcal/mouse/day than LF controls, respectively. At the time this abstract was written, 10% of the HF fed mice, 50% of the LF fed mice and 40% of the diet cycled mice remained alive, and the average lifespan of the HF, LF and diet cycled mice was 1.46, 2.38 and 2.15 years, respectively. Thus, the average lifespan of the HF fed mice was decreased by 38.7% compared to LF controls, while the diet cycled mice only exhibited a 9.7% decrease.

Conclusions

Since diet cycled mice lived longer than non‐diet cycled mice on a HF diet, diet cycling was healthier with respect to lifespan than not “dieting” at all.

This work was supported in part by the State of Ohio's Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, by the Diabetes Research Initiative at Ohio University, and by NIH grants DK075436, AG019899, AG031736.
Metabolic signaling in aging and disease
Authors: Pilgeram Laurence
Published: June 2021

The rate of biosynthesis and turnover of plasma protein fibrinogen is a marker of metabolic signaling in aging and disease. The rate in the young normal human subject of 0.260 mg/ml/24 hours increases to 0.378 in older normal subjects and to 0.466 in age matched coronary thrombosis patients measured by endogenous labeling of fibrinogen with L‐glutamic acid‐C14. This increased rate of fibrinogen turnover was traced to generation of occlusive fibrin polymers labeled by glycine‐C14 ethyl esters in the presence of active fibrin stabilizing factor. Circulating fibrin increased 520% (P<0.001) above normal in ischemic thrombotic cerebrovascular disease. Long chain saturated free fatty acids (FFA) exert not only primary control over incorporation of C14 amino acids into the fibrinogen structure but also activate the cascade sequence of reactions which convert fibrinogen into occlusive fibrin. FFA are normally transported by albumin to sites of energy metabolism. Decline in albumin synthesis in aging causes an increase in unbound FFA which induce not only increased fibrinogen synthesis but also activation of Hageman Factor generation of thrombin conversion of fibrinogen into occlusive fibrin. It has now been discovered that induction of a normal FFA/albumin ratio in plasma of coronary thrombosis and thrombotic stroke patients regenerates a normal level of fibrinogen synthesis by human hepatocytes.
Biochemical‐structural‐phenotypic correlation of adenylosuccinate lyase deficiency based on analysis of mutant enzyme complexes
Authors: Zikanova Marie , Skopova Vaclava , Hnizda Ales , Krijt Jakub , Kmoch Stanislav
Published: June 2021

Adenylosuccinate lyase (ADSL) deficiency is disorder of purine metabolism affecting mainly central nervous system. Three forms differing in severity of symptoms and correlating negatively with ratios of accumulating SAdo/SAICAr in body fluids were established. This diversity is not clear and may result from a genotype specific and thus structural related nonparallel loss of activity towards one of the substrates.

To test this we selected 16 patients with individual disease causing genotypes and cloned, expressed, purified and characterized individual mutant proteins and co‐expressed genotype specific mutant proteins. We also located mutations in the structural model of ADSL.

We found that phenotypic severity (eg. SAdo/SAICAr ratio) generally does not result from disproportional enzyme activities of mutant protein complexes but it correlates rather with their residual enzymatic activities and structural stability. We conclude that SAdo/SAICAr ratio value is probably not predictive of phenotype severity but rather it corresponds to degree of patient's development and brain maturation.

This work was supported by CR grants CSF 301070600 and MSM 20620806.
DNA methyltransferases in male offspring of sleep‐deprived mice
Authors: Calegare Bruno Frederico Aguilar , Fernandes Leandro , Tufik Sergio , D'Almeida Vânia
Published: June 2021

Growing interest has been driven by the discovery of a tight relationship between prenatal manipulations and development of short‐ and long‐term health disorders. Our objective was to analyze developmental programming effects of maternal sleep deprivation on offspring expression of DNA methyltransferases (DNMTs) in the hypothalamus. Pregnant female Swiss mice aged 3 months were distributed into 5 groups: One group of dams was continuously sleep‐deprived using the platform technique from gestational days 0 to 3 (PSD 72). Three additional groups were sleep‐deprived by gentle handling for six hours on gestational days 1 (GH 1), 2 (GH 2) or 3 (GH 3); and a last group which could sleep normally (CT). At the 3rd month of age, male offspring were euthanized and the hypothalamus dissected for RNA extraction. Real time PCR assays were used to measure levels of mRNAs encoding the enzymes in the tissue. We found no alteration in the expression of the DNMTs 1 and 3a. Although we found no differences in the DNMTs expression in the hypothalamus, epigenetic alterations could have important implications for development of health disorders. Support: FAPESP, CNPq and AFIP.
Phosphorylation of Thr522 activates SIRT1 and is required for DYRK‐mediated cell survival in response to DNA damage
Authors: Li Xiaoling , Guo Xiumei , Williams Jason , Bienstock Rachelle
Published: June 2021

SIRT1, a NAD+‐dependent protein deacetylase, plays an important role in stress response and energy metabolism. While the list of SIRT1 targets continues to grow, how SIRT1 activity is regulated remains unclear. Here we show that two pro‐survival DYRK kinases, DYRK1A and DYRK3, directly phosphorylate SIRT1 at Thr522, promoting p53 deacetylation and cell survival. A SIRT1 phosphorylation mimetic (T522D) displays elevated deacetylase activity, thus inhibiting cell apoptosis. Conversely, a SIRT1 dephosphorylation mimetic (T522V) fails to mediate DYRK‐induced deacetylation of p53 and cell survival. Furthermore, knockdown of DYRK1A and DYRK3 leads to hypo‐phosphorylation of SIRT1, sensitizing cells to DNA damage‐induced cell death. Finally, phosphorylation of Thr522 activates SIRT1 by decreasing its affinity to protein products but increasing its enzymatic turnover. Taken together, our findings demonstrate that anti‐apoptotic DYRKs promote cell survival through phosphorylation of SIRT1, which may have important implications in understanding the molecular mechanism of tumorigenesis and aging. This research was supported by the Intramural Research Program of the NIH, NIEHS to X.L.
Understanding molecular mechanisms in mitochondria underlying LRRK2‐related Parkinson's disease
Authors: LIU Huifang , Ho Wing Lok , Ho Wing Man , Zhou Zhong jun , Ho Shu Leong
Published: June 2021

Parkinson's disease (PD) is a common neurodegenerative movement disorder. Mutation of LRRK2 is pathogenic in PD. So far, more than 20 mutations of LRRK2 have been reported in Parkinson patients of different races. Some of the mutations were shown to be able to increase. We aim to elucidate the importance of LRRK2 in the pathogenesis of PD by studying the effects of this protein on vulnerability of neuronal cell death under oxidative stress.

Materials and Methods

We have developed stable HEK293 cells overexpressing wild‐type and mutated (R1441G, G2019S) LRRK2. The effects of LRRK2 over‐expression on various mitochondrial parameters were determined, e.g. cell viability, mitochondrial complexes (I–IV) activity and membrane potential, ATP/ADP ratio, and mitochondrial fragmentation after challenged with H2O2 to induce oxidative stress. We generated R1441G LRRK2 knockin (KI) mice. We are characterizing the LRRK2 R1441G KI mice and wildtype mice by immunohistochemistry of tyrosine hydroxylase (TH) to identify TH+ neurons after challenged with MPTP injection (IP) for 2 weeks to induce nigrostriatal degeneration.

Results

Our preliminary data showed that the intracellular expression pattern of mutant LRRK2 (G2019S) in HEK293 was different from the wild‐type under oxidative stress by H2O2. Mutant LRRK2 was translocated towards mitochondria after treatment with H2O2. LDH release and ATP/ADP ratio proved more serious trend of apoptosis in mutant LRRK2 cell lines. Mitochondrial complexes I activity and mitochondrial fragmentation level were also changed between wild type and single mutation proteins (R1441G and G2019S) in HEK293.

The results of this study have important implication on understanding the mitochondrial dysfunction in the pathogenesis of PD.
Oltipraz inhibits liver X receptor‐alpha‐dependent lipogenic gene induction and hepatic steatosis via AMPK‐S6K1 pathway
Authors: Kim Sang Geon , Hwang Seong Hwan , Ki Sung Hwan , Bae Eun Ju , Kim Hyun Eun
Published: June 2021

Nonalcoholic fatty liver disease is closely associated with metabolic syndrome and insulin resistance. This study investigated the role of S6K1 in LXRalpha activation and the effects of oltipraz on LXRalpha‐dependent lipogenesis in hepatocytes and high‐fat diet animal model. Oltipraz prevented the ability of T0901317 to activate SREBP‐1c, inhibiting its own mRNA and protein induction. The inhibition of S6K1 by oltipraz contributes to the repression of SREBP‐1c induction, whereas S6K1 activation antagonized this inhibitory effect. AMPK, whose activation leads to S6K1 inhibition, contributes to inhibit the induction of SREBP‐1c by T0901317. Oltipraz impaired T0901317‐induced CYP7A1 transactivation. Moreover, S6K1 and AMPK oppositely regulate LXRalpha activity through their phosphorylations at different residues. S6K1 inhibition antagonized CYP7A1 induction promoted by AMPK inhibition, whereas AMPK activation abrogated S6K1‐dependent CYP7A1 induction, supporting the opposing role of S6K1 and AMPK in LXR activity. Consistently, oltipraz inhibited hepatic triglyceride accumulation and lipogenic gene induction in mice. Our findings showing the role of AMPK‐S6K1 pathway in LXRalpha activity and S6K1‐dependent inhibition of LXRalpha‐induced lipogenic gene transactivation by oltipraz led to the identification of S6K1 as an attractive target for intervention in hepatic steatosis.
Akt1 and Akt2 exhibit isoform‐specific functions in HL‐1 cardiomyocytes
Authors: Raupach Annika , Möllendorf Sarah Lan , Gödecke Axel
Published: June 2021

To investigate isoform‐specific functions of the protein kinases Akt1 and Akt2 in cardiomyocytes, we generated cell lines derived from HL‐1 cardiomyocytes which stably express shRNA directed against Akt1 (ΔAkt1) and Akt2 (ΔAkt2). Cells expressing no target shRNA (shC) were used as controls. On the protein level, the successful knockdown led to a reduction in the range of 85–90% for either isoform and no compensatory upregulation of other Akt isoforms. Although Akt1 accounts for 75% of total Akt in HL‐1 cells, we found preferentially Akt2 to be phosphorylated on Ser473 after stimulation with insulin or IGF‐1 (insulin‐like growth factor 1). Phosphorylation of the downstream target GSK3β (Ser9) was reduced in both knockdown cell lines but this effect was not isoform specific. In contrast, the protein level of the RAB GTPase AS160, involved in insulin stimulated glucose uptake, was specifically reduced in ΔAkt2 cells. Moreover, a reduction of the insulin regulated GLUT4 glucose transporter was found in both knockdown cell lines. The insulin independent GLUT1 was not affected by either knockdown. Our data show that Akt2 represents the preferred target activated in HL‐1 cardiomyocytes by insulin/IGF‐1 stimulation. Akt2 regulates the protein levels of AS160 and therefore appears to play a major role in the context of glucose metabolism in HL‐1 cardiomyocytes. Supported by the DFG (SFB 612).
POSTNATAL ROSIGLITAZONE ADMINISTRATION TO NEONATAL RAT PUPS DOES NOT ALTER THE ADULT METABOLIC PHENOTYPE
Authors: Rehan Virender K. , Truong Nghia , Sakurai Reiko , Lee Wai‐Nang Paul , Torday John S.
Published: June 2021

We have previously shown that systemically administered Rosiglitazone (RGZ), a PPARγ agonist, up to 7 days of life significantly enhances lung maturation without affecting serum electrolytes, blood glucose, blood gases and serum lipid profile in the newborn period. However, whether this intervention in early neonatal life alters the adult metabolic phenotype is not known. Newborn Sprague Dawley rats were given either saline or RGZ (0.3 – 3 mg/kg BW) i.p. from postnatal day 1 to 7. Pups were allowed to breast feed ad lib and then weaned to rat chow on postnatal day 21. Following standard methods, glucose and insulin tolerance tests were performed at 14 wks of age. To assess the effects of early RGZ administration on glucose and fatty acid synthesis, deuterium labeling studies were performed. Animals were sacrificed, following which serum and tissues (skeletal muscle, liver, lung, brown and white fat) were collected for the expression of adipogenic differentiaton markers by Western blotting. There was no effect of early postnatal RGZ administration on BW, glucose or insulin tolerance, serum cholesterol and triglyceride levels, or triglyceride and fatty acid synthesis in both males and females. Treatment with the PPARγ agonist RGZ in early neonatal life does not alter developmental metabolic programming, and does not lead to an altered metabolic phenotype in the adult. Grant Support: NIH (HL75405, HD51857, HD058948).
Distinct phosphatase requirements and GATA factor responses to nitrogen catabolite repression and rapamycin treatment in S. cerevisiae
Authors: Cooper Terrance G. , Tate Jennifer J. , Georis Isabelle , Dubois Evelyne
Published: June 2021

The rapamycin (Rap) ‐sensitive TorC1 kinase complex and downstream phosphatases (Sit4, PP2A) are central components through which S. cerevisiae responds to its nutritional environment. TorC1 and the cell's internal nitrogen supply regulate the localization of Gln3 and Gat1, the transcriptional activators of Nitrogen Catabolite Repression (NCR) sensitive genes. In nitrogen excess, Gln3 and Gat1 are cytoplasmic and NCR‐sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear and transcription is derepressed. We have discovered distinct Sit4 and PP2A requirements for NCR‐sensitive and Rap‐induced nuclear Gat1 localization that differ markedly from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two separable but interconnected regulatory pathways receiving distinct input signals. Gln3 localization responds most strongly to intracellular nitrogen availability (NCR‐sensitive & methionine sulfoximine, Msx, induced) and more weakly to Rap inhibition of TorC1, whereas Gat1 localization responds most strongly to Rap‐inhibited TorC1 regulation and more weakly to NCR (insensitive to Msx). Supported by NIH grant GM‐35642‐22.