bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Utero Electroporation(IUE) and mouse genetic model approach to assess PITP‐dependent inositol lipid signaling in NSC pool maintenance in embryonic brain
Authors: Hur Seong Kwon , Xie Zhigang , Bankaitis Vytas 
Published: November 2023

Inositol (Ins) and phosphoinositide (PIP) signaling pathways are major intracellular regulatory systems of eukaryotic cells. Phosphatidylinositol transfer proteins (PITPs) execute novel modes of inositol lipid signal control by tightly channeling PIP production to specific biological outcomes. PITPα and PITPβ are highly expressed in mouse embryonic neocortex. We exploit an in utero electroporation approach to investigate the role of PITP‐dependent inositol lipid signaling in the embryonic neural stem cell (NSC) pool. We found that the combination of a PITPα null mouse line and PITPβ silencing evokes a dramatic depletion of NSC pools in embryonic brain. And, we finally have generated PITPαflox/flox and PITPβflox/flox single mutants, and PITPαflox/flox, PITPβflox/flox double mutant mouse line. In an experiment using Emx1‐Cre driver, which is forebrain specific driver, eviction of both PITPα and PITPβ in forebrain leads to a mouse that is born but has an amazing microcephaly that is due to virtual loss of the forebrain. Neither PITPα nor PITPβ eviction alone has any such effect. This experiment result is consistent with In utero electroporation data. Thus, we demonstrate that PITPα and PITPβ redundantly control NSC homeostasis in mouse brain development. Based on these data, we will observe carefully daughter cell fate during NSC division to investigate whether PITPs deficiency defeats NSC division program, asymmetric self‐renewing/differentiating cell divisions. Moreover, we will exploit our unique library of mutant PITPs with defined biochemical defects, to assess whether PtdCho‐ and PtdIns‐binding are required for PITP function in embryonic NSCs. Our research will generate completely new information regarding how lipid signaling contributes to NSC homeostasis in the developing mammalian brain.Support or Funding InformationNIH 1R01GM112591‐01A1
Acute exercise rescues high fat diet induced reductions in PGC‐1alpha protein content in mouse cortex
Authors: MacPherson Rebecca EK , Wright David C 
Published: November 2023

High fat diet induced obesity and insulin resistance have been directly implicated in the neuropathology of Alzheimer's disease. Specifically, previous work has demonstrated that a HFD suppresses PGC‐1alpha in male mice, leading to a down‐regulation of estrogen receptor alpha and increased inflammation. Exercise can improve neuroinflammation but the underlying mechanisms remain unknown. The purpose of this study was to determine the effects of a single bout of exercise on PGC‐1alpha, ER‐alpha, and markers of inflammation in brains from mice fed a high fat diet. Male C57BL/6 mice were fed a low (LFD, 10% kcals from lard) or a high fat diet (HFD, 60% kcals from lard) for 7wks. HFD mice underwent an acute bout of exercise (treadmill running: 15m/min, 5% incline, 120min) followed by a recovery period of 2h. The HFD increased body mass and glucose intolerance (both p<0.05). The HFD resulted in a decline in both PGC‐1alpha and ER‐alpha protein content (p<0.05). There were no diet‐induced alterations in either PGC‐1alpha or ER‐alpha mRNA expression. Similarly, the HFD did not alter the mRNA expression of inflammatory markers (IL‐6, TNF‐alpha, or IL‐1beta). Acute exercise rescued PGC‐1alpha protein content (p>0.05) and increased ER‐alpha protein content. Exercise did not affect gene expression of PGC‐1alpha, ER‐alpha, or inflammatory markers, 2 hours post‐exercise. This indicates post‐translational modification of PCG‐alpha and ER‐alpha proteins post‐exercise. Our findings demonstrate for the first time that an acute bout of exercise can increase PCG‐1alpha and ER‐alpha protein content in obese male mice.Support or Funding InformationRebecca EK MacPherson is supported by a Postdoctoral Fellowship from the Alzheimer's Society of Canada
Using Optical Structural Maps to Improve the Canine Genome
Authors: Silver Kristopher , Coleman Michelle , Biswell Rebecca , Lillich James , Brown Susan J 
Published: November 2023

Genome models have almost unlimited potential for understanding the biology and origins of disease. Efforts on the human genome have resulted in significant advances in human medicine and understanding of human biology. Similarly, recent efforts have resulted in the completion of a high‐quality, sequence‐based assembly of the genome of the domestic dog (Canis lupus familiaris). Though comprehensive, the canine genome assembly, based solely on sequence data, may have misassembled contigs and scaffolds in addition to having multiple gaps in it (more than 3000 sequences are still unanchored to the assembly). We are validating the current genome assembly and incorporating orphaned sequences into the dog genome assembly by using next generation mapping technology to create a structural map based on detection of fluorescent tags in ultra‐long molecules of DNA. The original sequence of the canine genome was based on a purebred boxer named Tasha. Accordingly, we selected three closely related boxers (a dame and two sibling offspring) to produce optical maps as similar to each other and Tasha as possible for maximum resolution. Ultra‐high molecular weight DNA from these three individuals was labeled at BspQ1 sites and imaged in nanochannels designed to contain single DNA molecules. Whole genome restriction maps were produced by overlap layout consensus for comparison to the CanFam3.1 assembly. Our results lay the groundwork for improving the canine genome assembly and for developing this technology as a potential tool for identifying significant chromosomal alterations that are associated with disease in dogs.Support or Funding InformationThis work was supported by a grant from Morris Animal Foundation.
Acylation of SOD1 Provides Tool to determine if Mitochondrial Aggregation of SOD1 is Main Driver of ALS
Authors: Banks Courtney , Rodriguez Nathan , Gashler Kyle , Bikman Benjamin , Andersen Joshua 
Published: November 2023

Most cases of ALS are sporadic but about 10% are familial (referred to as fALS), caused by mutations in a few known genes, including the ROS‐scavenging enzyme superoxide dismutase Cu/Zn (SOD1). These fALS‐linked mutations in SOD1 have no consistent effect on SOD1 ROS‐scavenging but rather are thought to cause a poorly understood toxic gain‐of‐function, believed by some groups to be an aggregation of mutant SOD1 in mitochondria that leads to neuronal death. However, the link between SOD1 mitochondrial aggregation and ALS is based on correlative data and has yet to be proven experimentally. Our preliminary experiments identified a novel mode of post‐translational SOD1 regulation by acylation (both acetylation and succinylation) at lysine 123 (K123). We observed that while acyl‐mimicking SOD1 mutants, K123E and K123Q, do not alter SOD1's ability to dimerize or scavenge ROS, they do abolish SOD1 mitochondrial localization even when combined with the fALS‐causing G93A mutation that is known to aggregate in the mitochondria. We also found that the acyl‐mimicking mutants of SOD1 abolish SOD1's ability to suppress respiration. In yeast, SOD1 is thought to suppress respiration in the cytosol through stabilization of casein kinase‐γ1 and the WNT pathway. However, the fact that the acyl‐mimicking mutants of SOD1 do not import into the mitochondria and also cannot suppress respiration suggests that SOD1's anti‐respiratory effect may be linked to SOD1's accumulation in mitochondria. We are now using the acyl‐mimicking SOD1 mutants as tools to determine if mitochondrial aggregation of mutant SOD1 is the main driver of ALS and if SOD1 mitochondrial localization is necessary for its anti‐respiratory activity.Support or Funding InformationSimmons Center for Cancer Research, BYU Internal Funding
Metal Specificity of Protein Kinases ‐ Implications in Pseudokinase Function
Authors: Herberg Friedrich W. , Knape Matthias J. 
Published: November 2023

cAMP‐dependent protein kinase (PKA) is regulated primarily in response to extracellular physiological signals, while nucleotides and metal ions may provide fine‐tuning inside the cell.PKA can use different metal ions for phosphoryl transfer, yet some, like Ca2+, do not support steady‐ state catalysis. Surface Plasmon Resonance (SPR) was used to study how metals can act differentially as a result of their inherent coordination properties. Ca2+ works in a single turnover mode to allow for phosphoryl‐ transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with substrate in the presence of Mg2+ATP. This allows us to depict the entire kinase reaction including complex formation, transition state as well as release of the phosphorylated substrate. In contrast to Mg2+, Ca2+ apparently slows down the enzymatic reaction. Ca2+ is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg2+. We therefore hypothesize that the physiological metal ions Mg2+ and Ca2+ both play a role in kinase activity and regulation under different circumstances. In a more detailed approach, the role of the metal binding sites 1 and 2 were also investigated in the context of pseudokinase function. The invariant DFG‐Asp184, coordinating both the lynchpin and the low affinity metal site, and the secondary metal binding site Asn171, were modified using site directed mutagenesis to mimic the situation in pseudokinases.
Engineering Protein Matrices for Production of β‐like Cells for Treatment of Type 1 Diabetes
Authors: Chigumba Desnor Nicole , Tirrell David A , Kozlowski Mark T 
Published: November 2023

Type 1 diabetes is a chronic condition in which patients cannot produce their own insulin. Previous studies have shown that it is possible to manipulate post‐natal progenitor cells to differentiate directly into β‐cells for transplantation by using laminin and methylcellulose to provide the appropriate biochemical and mechanical cues respectively. By using engineered protein hydrogels with an encoded laminin sequence and leucine zippers, we can make a material that provides both cues. As the cell culture is 3‐dimensional, this would enable us to suspend the cells in solution. Our primary objective was to produce a hydrogel which would be soluble at 37°C. Previous protein constructs were not soluble and we suspect this was because they had low LCSTs. Therefore, we altered the sequence to increase solubility. We cloned a PC10‐lam‐C10P protein where the C domain is hydrophilic and does not have an LCST. PE6‐lam‐E6P, PE6‐trunc lam‐E6P and PE6‐lam‐E6P C3A were also constructed as modifications of a previous elastin PEP construct. All constructs were soluble and MALDI‐TOF MS suggests we obtained our protein of interest. All constructs are sent to City of Hope Hospital for cell culture studies.Support or Funding InformationCalifornia Institute of Technology SURF program
Mutation of the Omega‐Loop Impacts Stability and Function of Human Cytochrome C
Authors: Saxena Manoj , Ramirez‐Paz Josell , Ponce De León Guzmán Solimar L. , Sharma Rohit kumar , Griebenow Kai 
Published: November 2023

Cytochrome c (cyt c) is an evolutionarily conserved globularprotein often used as a model for studying the protein stability and folding. Although its role as an electron carrier in the electron transport chain is well documented, findings about its role as a protein that could trigger apoptosis upon its release from mitochondria have reignited interest in cyt c structure and function. We have studied the role of two alanine‐to‐cys mutants that were designed in the omega loop of the cytochrome c. The importance of this loop is evident from the finding that the only reported natural variants associated with disease in human cyt c are found in this loop. Also, in vitro studies have shown that mutations in this loop impact the apoptotic function of cyt c. Our DSC data suggests that the A44C and A51C mutants are less stable than the wild type protein. A44C being least stable while the A51C's stability being intermediate between A44C and the wild type, as judged by the decline in their Tm. Additionally, the preliminary results from the in vitro cell free caspase 3 activity assay indicate altered apoptotic activities of these mutants. Results from the secondary structure comparison using CD suggests that the secondary structure of these mutants is not changed much while a change in the tertiary structure is clearly evident. We propose that on mutating Ala to Cys at these locations a possible conformational change occurs which could facilitate the change in caspase activity while decreasing the overall stability of the protein.Support or Funding InformationThis work was supported in part by the Institute for Functional Nanomaterials (NSF Cooperative Agreement 1002410)
Regulation of Protein Kinase CK2 Catalytic Activity by Protein Kinase C and Phospholipase D2
Authors: Bae Young‐Seuk , Lee Young‐Hoon , Park Jeong‐Woo 
Published: November 2023

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase that catalyzes the phosphorylation of a large number of cytoplasmic and nuclear proteins. The holoenzyme of CK2 is a heterotetramer, composed of two catalytic (α and/or α’) and two non‐catalytic (β) subunits. The β subunit enhances the catalytic activity of the α or α’ subunit and mediates tetramer formation and substrate recognition. We have previously demonstrated that phospholipase D2 (PLD2) overexpression antagonizes protein kinase CK2 (CK2) inhibition‐mediated cellular senescence. In the current paper, we show the molecular mechanism of CK2 activation by PLD2 and protein kinase C (PKC). Elevated expression and chemical activation of PLD2 increased the catalytic activity of CK2 and PKC in human colon cancer HCT116 and embryonic kidney HEK293 cells, whereas utilization of PLD2 chemical inhibitors and siRNA suppressed this activity. Inhibition of PKC in these cells suppressed PLD2‐induced CK2 activation, suggesting that PLD2 enhances CK2 activity through PKC. Overexpression of the PKC isoforms: PKCα, PKCβ, and PKCζ, but not PKCγ, stimulated CK2 activity in the cells. Importantly, purified conventional PKC (cPKC) and atypical PKC (aPKC) enzymes enhanced CK2 activity and phosphorylated serines 194 and 277 within CK2α, as well as serine 148 within CK2β. Furthermore, PKC‐mediated phosphorylation of these serines on CK2a was crucial for the stimulation of CK2 activity. Taken together, the present results suggest that PLD2 can stimulate CK2 activity through PKC‐mediated CK2α phosphorylation.Support or Funding InformationThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010‐0024102) and the Ministry of Science, ICT and future Planning (NRF‐2015R1A2A2A01004593).
Zinc finger protein ZPR9 interacts with MPK38, an AMPK‐related serine/threonine kinase, and stimulates MPK38‐dependent ASK1‐, TGF‐β‐, and p53 function
Authors: Ha Hyunjung , Seong Hyun‐A 
Published: November 2023

Murine protein serine‐threonine kinase 38 (MPK38) is a member of the AMP‐activated protein kinase (AMPK)‐related serine/threonine kinase family and plays a role in inducing ASK1‐, TGF‐β‐, and p53‐mediated activity. Here, zinc finger protein ZPR9 was found to be a positive regulator of MPK38. The redox‐dependent association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252, indicating ZPR9 as an endogenous substrate for MPK38. Expression of wild‐type ZPR9, but not the ZPR9 mutants C305S/C308S and T252A, enhanced MPK38‐induced ASK1, TGF‐β, and p53 function by stabilizing MPK38. Consistently, knockdown of endogenous ZPR9 using an inducible ZPR9 shRNA system showed an opposite trend and inhibited the MPK38‐induced ASK1, TGF‐β, and p53 function. These results suggest that ZPR9 functions as an activator in MPK38‐dependent ASK1‐, TGF‐β‐, and p53 signaling pathways.Support or Funding InformationThis work was supported by National Research Foundation of Korea Grant 2015R1A2A2A01006098
Effect of Targeting pathway inter‐connectors on gene expression pattern in Lipopolysaccharide (LPS) Induced Human Peripheral Blood Mononuclear Cells (PBMCs)
Authors: Mendis Chanaka 
Published: November 2023

Lipopolysaccharide (LPS) is an endotoxin that induces the secretion of pro‐inflammatory cytokines in many cells and has been known to induce septic shock in humans. This project is aimed at developing a rapid diagnostic method to combat the effects of LPS and eventually other toxins by utilizing signal transduction pathways induced by of LPS in Peripheral Blood Mononuclear Cells (PBMC) as a prototype module. We have identified a set of LPS specific genes in human PBMCs through micro array analysis as well as a central signaling pathways consisting some of the LPS specific genes. Previously identified pathway inter‐connectors such as JNK and p38 were then blocked by specific inhibitors and the gene expression pattern of the same set of LPS specific markers were evaluated. The main objective was to investigate the effect of two inhibitors on the time dependent gene expression pattern and then, the impact on the components that are indirectly associated with the inhibited signaling pathways. In addition, the specific inhibitors were evaluated by correlating the observed gene expression pattern though reverse transcription polymerase chain reactions (RT‐PCR) to the protein expression pattern though enzyme‐linked immunosorbent assay (ELISA)Support or Funding InformationN/A
BioCyc: Online Resource for Genome and Metabolic Pathway Analysis
Authors: Caspi Ron , Billington Richard , Foerster Hartmut , Fulcher Carol A. , Keseler Ingrid , Kothari Anamika , Krummenacker Markus , Latendresse Mario , Mueller Lukas A. , Ong Quang , Paley Suzanne , Subhraveti Pallavi , Weaver Dan S. , Karp Peter D. 
Published: November 2023

BioCyc.org is a genomic resource that contains more than 7600 Pathway/Genome Databases (PGDBs) for organisms whose genomes have been completely sequenced. Although most PGDBs are bacterial, PGDBs exist for key experimental organisms, including humans (HumanCyc), yeast (YeastCyc), and Arabidopsis (AraCyc).Each BioCyc database integrates the genome sequence of one organism with its predicted metabolic network, including metabolic pathways, reactions, and chemical compounds, all presented via a user‐friendly graphical interface. The BioCyc databases contain additional computed information such as predicted operons and candidate enzymes for catalyzing predicted enzymatic steps that have no assigned enzymes in the genome annotation.The BioCyc.org website enables users to perform many types of searches and data analyses online. Analysis tools include comparisons of pathways and genomes among multiple organisms, alignment of orthologous genes in a multi‐genome browser, and searching for minimal‐cost metabolic network routes that connect two substrates. Tools are also provided for visualizing omics data – such as metabolomics and gene expression data – on individual pathway diagrams, on personalized groups of multiple pathway diagrams called pathway collages, and on diagrams depicting the full metabolic network of an organism.A powerful tool in BioCyc is Smart Tables, which are user‐defined groups of metabolites or genes that can be transformed easily into other types of objects, analyzed, saved in a BioCyc account, and shared with other users.All BioCyc databases save one were created computationally using MetaCyc as a reference. MetaCyc (MetaCyc.org) is a comprehensive manually‐curated metabolic database whose contents are derived from experimentally studies. It currently contains more than 2,400 experimentally determined metabolic pathways from more than 2,700 organisms, curated from more than 47,800 scientific publications. It also contains extensive information on metabolic enzymes, reactions, and substrates.The BioCyc databases can be accessed through the BioCyc.org web site and can be downloaded. In addition, the Pathway Tools software can be downloaded and installed locally to create BioCyc‐like databases for more genomes, curate existing databases, or perform additional analyses that are not available online.Support or Funding InformationNational Institute of General Medical Sciences of the National Institutes of Health (NIH) [GM080746, GM077678, GM75742].
Enhancing the educational experience of summer interns through a laboratory techniques bootcamp
Authors: Silveyra Patricia , Crowder Roslyn , Matters Gail 
Published: November 2023

Underrepresented college students’ interest in STEM fields poses challenges, the most critical of which is having students stay the science course beyond their sophomore year until graduation. The Penn State College of Medicine provides annual summer internship opportunities for students interested in medicine and science careers through pipeline programs. One of the most challenging aspects for incoming summer students, particularly those coming from minority‐serving institutions, is their lack of hands‐on experience and knowledge of common laboratory methods and basic research techniques. To overcome these difficulties, we dedicated the summer programs’ first full week to introducing students to contemporary technologies, which would both improve their knowledge base and simultaneously impart instruction in techniques they may be required to perform in their laboratories. The goal of the course was to smooth the transition of summer students into their summer research labs and enhance student preparation and research productivity. The technologies were broad in scope and were explored five hours a day over the first four days of the summer program. Protocols were disseminated electronically, and instruction was provided by junior faculty, postdoctoral fellows, and graduate students, some of which also served as junior mentors for the students. The nature of the Bootcamp's capstone experience provided available time to step outside of the laboratory and learn valuable concepts and common laboratory practices like buffer preparation, calculations, and general good laboratory practices. The summer bootcamp experience terminated with a student lunchtime get‐together in which techniques were revisited, technical problems were dissected, data were analyzed, and students presented and discussed the results of their mini‐experiments. Overall, the student's feedback was positive and the course was well‐received by both students and research faculty. The success of the program resulted in its extension to all incoming students in its third year.Support or Funding InformationNIH NHLBI R25 HL103166 (M. Chorney, PI)NIH NIDDK R25 DK078381 (W. Reeves, PI)