bluuebunny/crossref_metadata_2025_split huggingface dataset:
Establishment of fetal brain atlases during the early second trimester
Authors: Liu Shu‐Wei 
Published: November 2023

ObjectiveThe use of spatiotemporal atlases can significantly improve the results and efficiency of automated analysis of fetal brain MRI data. The current study aims to establish age‐specific fetal brain atlases of the early second trimester (15–22 gestational weeks).Materials and MethodsThe thirty‐four postmortem fetal specimens of 15–22 weeks GW were collected and performed in a 7.0T Micro‐MRI. Both the data conversion and bias field correction were implemented as Pipeline workflows developed by Laboratory of Neuroimaging of University of Southern California. Manual removal of the non‐brain tissue was performed using BrainSuite software. For each cortex, 4 complementary global shape metrics were computed using LONI ShapeTools pipeline library – volume, surface area, shape index and curvedness. The script buildtemplateparallel.sh of Advanced Normalization Tools (ANTs) developed by University of Pennsylvania was run in each week to build the average template of each week. Three‐Dimension surface reconstruction of these templates were performed by BrainSuite. The general template was built from these weekly templates.ResultsFrom 15 to 22 gestational weeks, growth trajectories of brain area and volume is fitted well by linear regression model. The whole brain increased in volume by approximately 4‐fold and about 2.5‐fold in area. To reduce the bias effects of different demographic distributions of different numbers within each week, we built the templates each week first. After 3D surface reconstruction of these templates, we found that the 15th week still has some trails of neural folding in the early stages. The brain surface of 22nd week still looks smooth, but the whole brain's general shape looks more mature. The aggregated general template was then built based on these weekly templates. Four layers of lamination structures are displayed, from outer to inner, these layers are: cortical plate, subplate zone, intermediate zone, ventricular zone. The composed parts of basal ganglia could also be distinguished.ConclusionOur study first established the spatial‐temporal atlas of the early second trimester. And we also obtained the general developmental trajectories of the early fetal brain development. These atlases based on the high‐field MRI data provide good resolution and contrast, which will enable to characterize the dynamic anatomical changes of fetal brain development.Support or Funding InformationThe National Natural Science Foundation of China(No. 31071050, No. 31571237)
Formation of Dystrophic Neurites in Alzheimer's Disease
Authors: Yan Xiao‐Xin 
Published: November 2023

Dystrophic neurites (DNs) are abnormal neuronal processes characterized microscopically by aberrant sprouting, dystrophic expansion, and accumulation of various cellular organelles and cytoskeletal/signaling proteins. DNs occur around neuritic plaques in the brains of subjects with Alzheimer's disease (AD) and various other neurological conditions. Despite decades of research, the mechanism underlying the development of DNs remains unclear. This talk will discuss the understanding of DNs in historic perspectives, with a focus on the relationship between the formation of DNs and beta‐amyloid deposition according to recent studies using postmortem human brains and AD animal models.Support or Funding InformationAAA Symposium: Progress of Human Brain Banking in China: Construction and Research. Supported by AAA (American Association of Anatomists) and CSAS (Chinese Society for Anatomical Sciences).
Genetic Delivery of RNA Molecules to Alter Expression of EGFR in Brain Cancer Cells
Authors: Sivetz Nicole , Falotico Sarah C. , Nekrasov Peter , Hicks Martin J. 
Published: November 2023

Glioblastoma multiforme (GBM) is an incurable and aggressive type of brain tumor. It is the most common central nervous system malignancy with a median survival of only 14 months. GBM is characterized by increased activation of one or more types of cell membrane receptors that drive the proliferation of tumor cells. One receptor in particular, epidermal growth factor receptor (EGFR), is dysregulated in about 60% of GBM tumors. The amplification and overexpression of the EGFR gene makes it a primary target for therapy. Although a great deal is known about the biology of EGFR‐activated GBM tumors, therapies against its biologic processes are limited by the blood‐brain barrier. Therapeutic strategies targeting splicing of the pre‐mRNA transcript, before the mRNA is translated into an active peptide, show great promise. In our current approach, we have designed and cloned a therapeutic gene that encodes a pre‐trans‐splicing RNA (PTR). Using anti‐sense targeting, the therapeutic PTR delivers an early stop codon and polyadenylation signal into the EGFR pre‐mRNA transcript, leading to a short, non‐membrane bound soluble peptide. To optimize the PTR we are testing alternative antisense binding sequences, as well as distinct splicing elements along the EGFR transcript. We make use of multiple GBM cell lines and RT‐PCR to measure changes in expression of the EGFR transcript. In addition, we are developing a novel approach using a fluorescent PTR (fPTR) to quickly measure changes in expression. Results show that our vectors were expressed at high levels with subsequent changes in EGFR mRNA expression. Our approach to deliver the DNA encoding anti‐EGFR PTRs to alter pre‐mRNA splicing of the EGFR mRNA transcript in GBM cells has the potential to work from the inside‐out, bypassing systemic barriers.Support or Funding InformationMonmouth University School of Science Research Program, Bristol‐Myers Squibb, Independent College Fund of New Jersey, Johnson & Johnson
Understanding how two Similar RNA Binding Domains Mediate Different Protein‐Protein Interactions.
Authors: Marshall Collin 
Published: November 2023

The process of alternative splicing is partly regulated by RNA binding proteins that bind to cis‐elements on the pre‐mRNA transcript and influence the assembly of a functional spliceosome at adjacent splice sites. Many RNA binding proteins occur as gene families with members sharing high sequence identity and domain arrangement yet have non‐overlapping expression patterns and different splicing activity. Polypyrimidine Tract Binding Protein 1 (PTBP1) and Polypyrimidine Tract Binding Protein 2 (PTBP2) are paralogous RNA binding proteins that share 74% sequence identity and a similar domain arrangement, which consists of four RNA binding domains connected by 3 linker regions. The two proteins differ in their splicing activity, where both proteins repress certain target exons well while others are more strongly repressed by PTBP1. Recent studies conducted to identify regions of primary structure that contribute to the different repression activity highlighted that RNA binding domain 2 (RRM2) of PTBP1 confers higher splicing repression compared to PTBP2. The RRM2 region of PTBP1 and PTBP2 share 87% sequence identity and have identical RNA interacting residues. PTBP1 RRM2 binds strongly to co‐repressor Raver 1. PTBP2 RRM2 has counterparts of most of the residues in the Raver interacting motif, however interacts weakly with Raver 1. These results indicate that the two proteins engage in different protein‐protein interactions albeit the high sequence identity in the RRM2 region. Thus we hypothesize that the RRM2 region is post‐translationally modified and plays a role in protein‐protein interactions, which lead to differences in splicing repression activity. To test this, we have over‐expressed Flag‐tagged PTBP1 RRM2 and PTBP2‐RRM2 in mouse neuro 2A cells, purified via Flag‐immunoprecipitation and analyzed for modifications via Mass Spectrometry. Preliminary results indicate that the region is modified in the two proteins. We are currently conducting site‐directed mutagenesis to identify the role of modifications on protein‐protein interactions and splicing repression activity.Support or Funding InformationHoward Hughes Medical Institute (HHMI)
The Sbp1p Paralog Rnp1p Has A Role In P‐body Assembly Without Affecting Translational Repression
Authors: Segal Scott P , Chanhthee Alvin , Mackay Katherine 
Published: November 2023

Global translational repression occurs in response to many physiological stresses, including glucose deprivation, heat stress and genotoxic stress. Upon exit from the polysomes, mRNA can localize to either stress granules to be stored or P‐bodies to possibly be degraded. In yeast, a number of proteins are involved in translational repression in response to physiological stress. Among these proteins is Sbp1p, which contains two RRM domains that flank a central RGG box. Sbp1p, in part, acts to repress translation by binding eIF4G through its RGG box. Additionally, Sbp1p can also localize to both P‐bodies and stress granules. Since Sbp1p has the ability to interact with the translation initiation machinery, this suggests that this protein may have a greater role in mRNA storage in stress granules.Rnp1p is an Sbp1p paralog and structurally shares the two RRM domains, but does not contain the central RGG box. Interestingly, Rnp1p appears to have no effect on translational repression in response to genotoxic stress. However, overexpression of Rnp1p can drive assembly of P‐bodies, but not stress granules. Moreover, strains lacking Rnp1p are deficient in P‐body assembly in response to physiological stresses that result primarily in P‐body assembly, but not assembly of stress granules. Taken together, these data suggest that Rnp1p may have a role in the assembly of P‐bodies. Further work will ascertain whether Rnp1p can localize to P‐bodies and with which proteins Rnp1p interacts.
Investigating Contributions of the N and C Terminus to FOXO1 and DNA Interactions
Authors: Stenson Jazmin Marie 
Published: November 2023

FOXO1 is a mammalian transcription factor that belongs to the FOXO protein family. FOXO1 regulates glucose and fatty acid metabolism in the liver, and changes in FOXO1 expression have also been identified in a variety of cancer cells. All FOXO proteins bind to two consensus sequences known as the Daf‐16 binding element or DBE (5′‐TT (A/G)TTTAC‐3) and the insulin‐response element or IRE (5′‐TT(G/A)TTT(G/T)(G/T) ‐3′). FOXO1 binding can be enhanced by the presence of multiple copies of these consensus sequences although the mechanism behind this enhancement is not known. Molecular Dynamics Simulation and in vitro assays indicate that Wing 1 of the FOXO1 DNA‐binding domain makes contact with DNA bases outside of the consensus sequence. However, this data does not explain the enhancement in binding observed with multiple copies of the IRE or DBE that extend beyond the potential reach of Wing 1 indicating the need to explore additional mechanisms for this enhancement. We are therefore interested in determining how the N and C terminus of FOXO1 might contribute to the interaction with DNA. Bacterially expressed and purified versions of FOXO1 lacking specific domains were used to determine the effects on DNA binding through in vitro assays. Understanding the contribution of the entire protein structure to DNA binding may allow for a better understanding of how FOXO proteins bind and recognize specific target sequences.Support or Funding InformationNIH MARC 5T 34GM07716‐37; NIH BUILD RL5GM118966‐02, NIH RCMI 2G12MD007595‐06
Characterization of a novel apolipophorin‐III/apolipoprotein E C‐terminal domain chimera
Authors: Kakutani Leesa Miiko 
Published: November 2023

Human apolipoprotein E (apoE) is a two‐domain anti‐atherogenic lipid transport protein that mediates plasma cholesterol homeostasis by serving as a ligand for the low density lipoprotein (LDL) receptor. Of its three isoforms E3, E2, E4, the latter two have been associated with increased risk of cardiovascular and Alzheimer's disease respectively, yet all three contain an identical C‐terminal (CT) domain. The CT domain (residues 201–299) is responsible for tetramerization and has been found to initiate lipid binding that influences critical functional features of apoE. The N‐terminal (NT) domain of apoE has four α‐helices arranged in a bundle similar to apolipophorin III (apoLp‐III), a model insect apolipoprotein containing five α‐helices in one domain. To better understand the role apolipoprotein domains play in structure and function, a novel chimeric apolipoprotein was designed by attaching apoE‐CT to apoLp‐III. A disulfide bond was introduced to apoLp‐III to lock the helix bundle and eliminate its lipid binding. Recombinant apoLp‐III/apoE‐CT, apoE3, apoE‐CT, and apoLp‐III were expressed in bacterial cells, purified by affinity chromatography, and the purity verified by SDS‐PAGE. Western blot analysis using monoclonal apoE‐CT specific antibody confirmed the presence of apoE‐CT in the chimera. Crosslinking studies using dimethylsuberimidate revealed that the apoLp‐III/apoE‐CT chimera formed oligomers similar to apoE, while apoLp‐III was monomeric. Far UV circular dichroism showed increased α‐helical content, and incubation with 1‐anilinonaphthalene‐8‐sulfonic acid showed increased fluorescence intensity when apoE‐CT was added to apoLp‐III, consistent with the presence of water‐shielded dye binding exposed hydrophobic regions. Our results suggest that the chimeric protein has structural similarities to the parent proteins, and that self‐association properties of apoE can be transferred to apoLp‐III through the addition of the CT domain. Future studies will include measuring the ability of the chimera to solubilize phospholipid bilayers and binding to low density lipoproteins. The chimeric approach offers the potential to obtain insight into domain interactions and the structure‐function relationships in apolipoproteins.Support or Funding InformationThis research was supported by the National Institute of General Medical Sciences of the NIH (8UL1GM118979‐02, 8TL4GM118980‐02, 8RL5GM118978‐02, GM089564 and GM105561).
DNA Binding by a HEAT‐Repeat Subunit of Saccharomyces cerevisiae Condensin
Authors: Sarkar Rupa , Rybenkov Valentin V. 
Published: November 2023

Condensins play a crucial role in higher order chromosome organization in both prokaryotes and eukaryotes. In budding yeast Saccharomyces cerevisiae, condensin complex consists of five subunits: two evolutionary‐conserved structural maintenance of chromosome (SMC) subunits and three non‐SMC subunits. SMC subunits adopt a V‐shaped structure, in which two globular head domains are connected to a central hinge domain via two long coiled coils. Non‐SMC complex consists of a kleisin, which binds the head domains of SMC, and two HEAT‐repeat subunits Ycs4 and Ycg1. DNA binding and organization primarily resides in the SMC proteins. Non‐SMC subunits contribute to the activity of SMC and regulate the recruitment of holoenzyme to the chromosome. Unlike in bacteria, the non‐SMC complex of yeast S. cerevisiae binds DNA. In this study, we mapped the DNA binding site of non‐SMC complex using mass spectrometry based DNA footprinting. The DNA binding site is located on the C‐terminal domain of Ycs4 next to the HEAT‐repeat cluster. We verified the implicated amino acids using site‐directed mutagenesis and observed a reduction of DNA binding by Ycs4. Furthermore, we found that the mutations affect the cellular growth of S. cerevisiae and the activity of the holoenzyme condensin in vitro. These results show that DNA binding of Ycs4 is essential for the activity of condensin in vitro and in vivo.Support or Funding InformationOCAST HR 14‐042
Selecting Medically Relevant In Vitro Protein Characterization Projects with a High Potential For Success
Authors: Zajac Diana , Tucker Brady , Cotsoradis Alicia , Mallmann Clara , Cassidy Christine , Gurnon Daniel 
Published: November 2023

As of November 2016, well over one‐third of the more than 170,000 unique variation records in ClinVar are of unknown importance; these variants are either (a) characterized as variants of unknown significance (VUS), (b) have no clinical significance provided, or (c) list conflicting reports of clinical significance. The vast number of ambiguous entries in ClinVar offer an opportunity for researchers seeking new projects in the area of in vitro protein characterization; not only are there many variants to choose from, the size of the dataset provides potential to select targets that are a good fit for the capabilities of the lab. The opportunity to preselect medically relevant projects with a higher degree of success is especially intriguing for researchers at undergraduate institutions, who do not have the resources of larger labs.We describe the development of a selection algorithm for choosing an in vitro protein characterization project that is amenable to undergraduate research. Our approach combines information from ClinVar and the Protein Databank to select enzymes with published crystal structures of the human protein, that have been previously expressed and purified in e. coli, and that feature at least one missense VUS entry in ClinVar.We also describe progress on the characterization of four variants of unknown significance in Glucose‐6‐Phosphate Dehydrogenase, an enzyme associated with hemolytic anemia.Support or Funding InformationSupport provided by DePauw University and the Howard Burkett Scholar Fund.
Effects of Endoplasmic Reticulum Stress on Protein Quality Control
Authors: Buchanan Bryce William , Rubenstein Eric M 
Published: November 2023

Protein quality control is critical for cellular health. The Endoplasmic Reticulum (ER)‐Associated Degradation (ERAD) protein quality control system degrades aberrant proteins at the ER membrane. ERAD can be divided into multiple pathways, including ERAD‐L (lumen), ‐M (membrane), ‐T (translocon), ‐C (cytosol), and ‐RA (ribosome associated). In general, each ERAD pathway degrades proteins with aberrations in specific positions relative to the ER membrane. In ERAD‐L and ‐M, the ubiquitin ligase Hrd1 recognizes proteins with aberrations in ER luminal or transmembrane segments. Hrd1 also functions in ERAD‐T, in which proteins that persistently or aberrantly associate with the translocon are targeted for destruction. In ERAD‐C, the ubiquitin ligase Doa10 recognizes soluble or transmembrane proteins with cytosolic aberrations. In ERAD‐RA, the ubiquitin ligase Rkr1/Ltn1 recognizes translationally stalled ER‐targeted proteins. ER stress occurs when misfolded proteins accumulate in the ER. Multiple human diseases, including Parkinson's disease and diabetes, are characterized by elevated levels of ER stress. The extent to which ER stress contributes to pathology is not clear. In this study, we systematically evaluate the effect of ER stress on ERAD in the model organism Saccharomyces cerevisiae by comparing the abundance and rate of degradation of a panel of model substrate proteins. Our data suggest that the ERAD pathways are differentially affected by ER stress. A better understanding of the effects of ER stress on protein quality control pathways may contribute to an improved understanding of the consequences of ER stress on cellular physiology and facilitate the discovery of therapeutic targets for diseases characterized by increased levels of ER stress.Support or Funding InformationNational Institutes of Health Grant R15 GM111713 (to EMR).Funds from the Ball State University Provost's Office and Department of Biology (to EMR).Ball State University ASPiRE research awards (to BWB).
A neuronal‐specific surface‐exposed membrane proteasome complex modulates neuronal signaling through extracellular signaling peptides
Authors: Ramachandran Kapil V , Margolis Seth S 
Published: November 2023

Proteasome‐dependent proteolysis is a tightly regulated process that is essential for life in all archaeal and eukaryotic cells. In the nervous system, proteasome function regulates a wide variety of cellular processes that are critical for proper neuronal function. Largely, the roles of proteasomes in the nervous system have been attributed to modulating the levels of proteins critical for particular cellular processes. However, certain rapidly‐occurring processes such as neuronal transmission, vesicle recycling, and calcium signaling are also affected by acute inhibition of proteasome function. It remains unclear how proteasomes can acutely regulate a cellular process within seconds to minutes, as this remains inconsistent with their canonical role in proteostasis. Here, we made the discovery of a nervous system‐specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is tightly associated with the neuronal plasma membrane, exposed to the extracellular space, and is catalytically active. We have determined this complex largely to be made up of 20S proteasome subunits, with little to no 19S cap. Analysis of this membrane proteasome complex by mass spectrometry revealed nervous‐system specific auxiliary membrane glycoproteins which were sufficient for membrane proteasome expression. Selective inhibition of this membrane proteasome complex by a cell‐impermeable proteasome inhibitor blocked extracellular peptide production and attenuated neuronal activity‐induced calcium signaling within seconds. Moreover, membrane proteasome‐derived peptides are sufficient to robustly and rapidly induce neuronal calcium signaling, in part by acting through NMDA receptors. The nature of proteasomal membrane attachment, association with auxiliary transmembrane proteins, exposure to the extracellular space, and capability to signal across the membrane are characteristic of a transmembrane complex. Our discoveries challenge the prevailing notion that proteasomes primarily function to maintain protein homeostasis, and highlight the existence of a form of rapid neuronal communication through a novel transmembrane proteasome complex.Support or Funding InformationThis work was funded by institutional funding and the following grants to S.S.M. (R01 MH102364‐02). K.V.R. was supported by a training grant T32 GM007445 and NSF Graduate Research Fellowship DGE‐1232825.