bluuebunny/crossref_metadata_2025_split huggingface dataset:
A study of putative genetically determined defects of de novo purine synthesis using CRISPR‐Cas9 genome‐edited HeLa cells
Authors: Skopova Vaclava , Baresova Veronika , Souckova Olga , Krijt Matyas , Kmoch Stanislav , Zikanova Marie 
Published: November 2023

ObjectivesThe diagnosis of patients with neurological impairment can be set down only in 2% of studied cases. The reason might be the inability to capture new, to date unknown, diseases. One of the metabolic pathways whose malfunctions could lead to neurological impairment is de novo purine synthesis (DNPS). Currently, two genetically determined defects of DNPS enzymes, ADSL deficiency and AICA‐ribosiduria, are known. Therefore, the existence of other defects manifested by neurological symptoms and by accumulating DNPS intermediates in body fluids, is highly presumable.MethodsWe knocked out the expression of DNPS enzyme‐coding genes in HeLa cells using GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit. We tested the cells for presence of mutations by DNA sequencing, for the presence of knocked‐out proteins by Western blot, and we analyzed DNPS substrates accumulation in cell lysates and growth media by LC‐MS/MS. We enzymatically prepared the substrates of DNPS enzymes for use as standards. We also analyzed the ability of the knockout cells to form purinosome by immunofluorescence.ResultsWe prepared CRISPR‐Cas9 genome edited HeLa cells deficient for particular steps of DNPS to model possible genetically determined defects of the DNPS enzymes. We developed an analytical method for screening of these defects based on LC‐MS/MS analysis of the accumulated enzyme substrates. The precise detection of the metabolites was accomplished using the prepared DNPS intermediates. In all prepared model cells except of one we detected an accumulation of the substrate(s) for knocked‐out enzyme. The purinosome formation was disrupted in cell lines where the DNPS enzymes GART, ADSL and ATIC were knocked‐out. In cell lines with knock‐out of the other proteins of DNPS was the purinosome formation reduced.ConclusionWe conclude that, similar to ADSL deficiency and AICA‐ribosiduria, the deficiency of other DNPS enzymes will manifest with accumulation of substrates for these enzymes in the body fluids of patients. Our data allow the detection of PAICS and PFAS deficiency in a population of patients with severe neurological impairment that have not yet been diagnosed.Support or Funding InformationThis work was supported by grants AZV 15‐28979A from the Ministry of Health, CR, PRVOUK‐P24/LF1/3 programs of Charles University in Prague, CR, and NPU from the Ministry of Education, Youth and Sports, CR.
Role of Wnt Signaling in Colon Cancer Cell Metabolism
Authors: Tanio Stephanie Sprowl , Pate Kira , Waterman Marian L. 
Published: November 2023

Over eighty percent of colon cancers derive from mutations that overactivate the Wnt pathway and cause cell transformation. These mutations stabilize β‐catenin protein, leading to its accumulation and nuclear translocation. Nuclear localization enables interaction with LEF/TCF transcription factors and the transcriptional overactivation of Wnt target genes, or gene programs. Identifying these gene programs and defining how they drive both the cancer cell phenotype and the tumor response to drug treatment is critical for developing therapies that block tumor progression. We and others have discovered that metabolism is one of the gene programs regulated by Wnt. Constitutively active Wnt signaling drives cells toward a glycolytic phenotype even in the presence of oxygen, a phenomenon known as Warburg metabolism. Using Gene Expression and ChIP‐seq analysis of colon cancer cells expressing dominant negative LEF‐1 and TCF‐1 isoforms (dnLEF/TCFs) we identified direct Wnt target genes that regulate this metabolic phenotype. Gene ontology analysis identified a subset of metabolism‐linked genes, including PDK1 (Pyruvate Dehydrogenase Kinase 1) and SLC16A1/ MCT1 (monocarboxylic acid transporter 1). PDK1 is a mitochondrial kinase that suppresses respiration and increases rates of glycolysis. MCT1 is a membrane‐bound transporter critical for maintaining the homeostasis of glycolytic cancer cells through import and/or efflux of lactate. PDK1 and MCT1 RNA and protein levels are significantly and specifically down regulated by dnLEF/TCFs in multiple colon cancer cell lines (SW480, SW620, HCT116 and DLD1). ChIP‐seq analysis with dnTCF1 identified regulatory regions with Wnt Response Elements (WREs) in distal and promoter proximal locations for each locus. Each peak of occupancy could confer Wnt regulation in luciferase reporter assays. c‐Myc, a Wnt target gene and known regulator of SLC16A1 transcription, regulates the locus additively but not synergistically with LEF/TCFs. Work by others has shown MCT1 to be necessary for the import of 3‐bromopyruvate, a potential cancer therapeutic that inhibits glycolysis through a mechanism not well understood. Our data suggests that colon cancer cells are sensitive to 3‐bromopyruvate in a Wnt dependent manner, and that co‐treatment of cells with Wnt inhibitors (e.g. XAV939, a tankyrase inhibitor) promotes resistance and recovery from 3‐bromopyruvate treatment. Overall this data suggests that Wnt signaling is important for maintaining a Warbug phenotype through PDK1 and MCT1, and that MCT1 may serve as an important indicator for drug sensitivity in colon cancer therapy.Support or Funding InformationNIH R01 CA108697 (PI: Waterman), President's Dissertation Year Fellowship, UC Irvine 2015–2016
Allyl‐isothiocynate (AITC) ameliorates LPS induced NF‐κB mediated neuroinflammation
Authors: Subedi Lalita , Lee Taek Hwan , Chae Jae Kyoung , Venkatesan Ramu , Kim Sun Yeou 
Published: November 2023

Toxins‐induced microglial activation can initiate the inflammatory cascade in most of the neurodegenerative as well as neuroinflammatory conditions that leads to the neuronal cell death. Chronic activation of microglia is dangerous and the strategy to control the microglial over activation might be protective in neurodegenerative diseases. Natural compounds especially nutraceuticals are good examples for the treatment of such condition without cellular toxicity. In this study, we investigated the promising antineuroinflammatory activity as well as neuroprotective efficacy of allyl‐isothiocynate (AITC) from W. japonica against LPS activated BV‐2 cells and its toxicity to neuroblastoma cells called N2a cells. We evaluated the neurotrophic effect of AITC in neuronal astrocytes as well. AITC inhibited the LPS activated BV2 induced inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), nitric oxide (NO), prostaglandin E2 (PGE2), Tumor necrosis factor‐a (TNF‐a), interleukin‐6 (IL‐6), MAPK signaling, and NF‐κB mediated transcription. Additionally, AITC reduced the activated microglia induced neuronal toxicity via downregulating the neuronal apoptosis together with enhanced neurite outgrowth in neuronal cells. Furthermore AITC also increased the neurotrophins production through neuronal astrocytes. In conclusion, AITC might be a potential neuroprotective agent.Support or Funding InformationThis research was supported by High Value‐added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (114006041HD020)
Metabolome Dynamics Platform Reveals Endocrine‐metabolic Crosstalk in PCOS and Its Reprogramming of Lipid Metabolic Pathways with 3‐Iodothyronamine
Authors: Assadi‐Porter Fariba M , Selen Ebru , Bolandnazar Zeinab , Sabatin Martina , Rogowski Michael , Chiellini Grazia 
Published: November 2023

ObjectivesPolycystic ovary syndrome (PCOS) is a complex metabolic and endocrine disorder, and a leading cause of infertility in women. The PCOS metabolic phenotype in women and mice is associated with defective lipid and glucose metabolism that is linked to dysregulation of steroidogenesis through unknown mechanism(s). Here, we investigated tissue specific treatment effects of 3‐Iodothyronamine (T1AM), a natural analog of thyroid hormone, action on metabolic pathways and associated transcriptional and gene signaling in vivo in order to understand the crosstalk between endocrine and lipid metabolism as drug targets in PCOS pathophysiology.MethodsWe analyzed plasma and tissue extracts using multidisciplinary omics and biochemical approaches. We used 1H‐NMR spectroscopy using metabolome dynamics platform (MDP) to analyze metabolomics profiles and complemented with monitoring transcription levels of major targeted metabolic regulatory genes and proteins.ResultsAdministering with the low sub‐chronic application of T1AM (10mg/Kg/Day) in vivo induces a profound anti‐lipogenic effect by lowering gene expression of key regulators of lipid metabolism, Ptpb1 and Plin2, and significant increased levels of metabolites (glucogenic amino acids, carnitine and acetate) in a tissue specific manner, while enhancing protection against oxidative stress in liver and muscle. In contrast, T1AM has an opposing effect on the regulation of estrogenic metabolic and gene pathways in the ovary by upregulating Star, Cyp11a1 and Cyp17a1s. Biochemical measurements provide further evidence of a significant reduction of plasma total cholesterol and triglycerides after T1AM treatment.ConclusionsOur MDP analysis indicate a novel mechanism for T1AM in regulating lipid metabolism, which untangles endocrine‐metabolic pathways, thus elucidating the intricacies within the pathophysiology of PCOS at the tissue specific level in contrast to overall systemic effect. This study opens new avenues to design new approaches for essential therapeutic targets in order to improve quality of life in complex diseases such as PCOS with endocrine‐metabolic dysfunction.Support or Funding InformationThis research was supported by RC4 EY021357 and a Wisconsin Institute of Discovery Grant (WID‐135A039) to F.A‐P. This study made use of the National Magnetic Resonance Facility at Madison for data collection, which is supported by NIH grant P41RR002301 (NIGMS).
A structural imbalance of the brain and the whole body spinal column associated with the temporomandibular disorders
Authors: Lee Somin , Jung Jeeyoun , Jerng Ui Min , Kwon O sang , Choi Kwangho 
Published: November 2023

BackgroundThe most commonly reported that the temporomandibular disorders (TMD) refer to the causes symtoms are numerous include systemic, iatrogenic disease, occlusal trauma and mental health disorders. These TMD symptoms are related to muscle sensitivity through palpation, restricted mouth opening, asymmetric mandibular movements, joint sounds, pain and otologic signs and symptoms. However, it is not fully reports about correlation of TMD and the systemic spinal column.MethodsA survey was performed using the electronic databases (PubMed, SCOPUS, Oasis, Korean TK, RISS, KISS) between 2006 and 2016. The following search keywords were selected using TMD pain, TMD, TMJ, TMJ disorders, systemic spinal column, occlusal splint, TMD physiotherapy, TMJ rheumatoid.ResultsResults of the number of reports, TMD correlation between systemic spinal column. These data were TMD with cervical spine disorders has been reported by several researches. Furthermore, TMD relevant reported that functional cerebrospinal therapy (FCST) (Lee young Jun clinics who developed) is markedly improvement of systemic imbalances in the spine.ConclusionIn the conclusion, improvement of the temporomandibular joint disorder can be utilized for fundamental treatment that can improve the imbalance of the whole body spinal column.Support or Funding InformationThis work was supported by a Korea Institute of Oriental Medicine (C16100).
Markers of Muscle Protein Synthesis and Breakdown in Slow‐twitch Skeletal Muscle of Rodents Aged 3 to 24 months
Authors: Haun Cody T. , Mumford Petey W. , Mobley Brooks C. , Kephart Wesley C. , Holland Maleah , Osburn Shelby C. , Martin Jeffrey S. , Young Kaelin C. , Kavasis Andreas N. , Lowery Ryan P. , Wilson Jacob M. , Roberts Michael D. 
Published: November 2023

PURPOSEMuscle protein synthesis (MPS) and muscle protein degradation (MPD) are well known modulators for muscle mass maintenance. Given that slow‐twitch skeletal muscle atrophies with aging, we sought to examine the effects of aging on MPS and MPD markers in the soleus muscle of rodents ranging from 3 to 24 months.MEHTODSMale Fischer 344 rats (300–600g) were successfully aged to 3, 6, 12, 18 and 24 months, were injected with puromycin (0.02 mg/g body mass), euthanized, soleus muscle was extracted and wet skeletal muscle weights were obtained. Muscle tissue was then processed for biochemical processing. MPS was assessed via the SUNsET method, and MPD was assessed using whole lane densitometry via ubiquitin immunoblotting.RESULTSRelative soleus masses revealed significant between‐group differences throughout the lifespan (p=0.004); specifically, masses were 20% greater in 3 versus 18 month old rats (p=0.010) and 20% greater in 3 versus 24 month old rats (p=0.013). Interestingly, MPS levels were not different between any of the age groups. Poly‐ubiquinated protein levels were trending (p=0.051), but not different between any of the age groups.CONCLUSIONIt appears that soleus muscle atrophy occurs with aging. However, this does not appear to be reflected via decrements in MPS and increases in protein poly‐ubiquination. Mechanisms related to slow‐twitch skeletal muscle atrophy remain to be determined and should be further investigated.Support or Funding InformationACKNOWLEDGEMENTS: This study was supported in part by a contract from The University of Tampa (to M.D.R. from J.M.W.) as well as laboratory start‐up funds from M.D.R.
“Translation Speed is Genetically Encoded and Influences Protein Folding”
Authors: Spencer Paige , Siller Efrain , Anderson John , Barral Jose 
Published: November 2023

The degeneracy of the genetic code allows most amino acids to be encoded by multiple codons. The distribution of these so‐called synonymous codons among protein coding sequences is not random and multiple theories have arisen to explain the biological significance of such non‐uniform codon selection. Many ideas revolve around the notion that certain codons allow for faster or more efficient translation, whereas the presence of others result in slower translation rates. The presence of these different types of codons along a message is postulated in turn to confer variable rates of emergence of the nascent polypeptide from the ribosome, which may influence its capacity to fold towards the native state, among other properties. Previous studies have reported conflicting results with regards to whether certain kinds of codons correlate or not with particular structural or folding properties of the encoded protein. We believe this has arisen, in part, because different criteria have been traditionally used for predicting whether a codon will be translated quickly or slowly in a given organism, including its frequency of occurrence among highly expressed genes and the concentration of tRNA species capable of decoding it, which do not always correlate. We have developed a metric to predict organism‐specific polypeptide elongation rates of any mRNA based on whether each codon is decoded by tRNAs capable of Watson‐Crick, non‐Watson‐Crick or both types of interactions. We demonstrate by pulse‐chase analyses in living E. coli cells that sequence engineering based on these concepts predictably modulates translation rates due to changes in polypeptide elongation and show that such alterations significantly impact the folding of proteins of eukaryotic origin. We also demonstrate that sequence harmonization based on expression‐host tRNA content designed to mimic ribosome movement of the original organism can significantly increase the folding of the encoded polypeptide. This body of work could provide insight into how synonymous nucleotide substitutions result in altered protein function and disease.Support or Funding InformationThe Welch Foundation and The Sealy Center for Structural Biology and Molecular Biophysics
Profiling stage‐related tissue proteomes to discover potential diagnostic and prognostic biomarkers for lung adenocarcinoma
Authors: Hsu Chiung‐Hung , Wu Chih‐Ching , Wang Chih‐Liang , Hsueh Chuen , Wu Yi‐Cheng , Hsu Chia‐Wei , Liu Chin‐Ching , Lai Ping‐Chang , Yu Jau‐Song , Chang Yu‐Sun , Yu Chia‐Jung 
Published: November 2023

Lung cancer is the leading cause of cancer‐related death worldwide. Both diagnostic and prognostic biomarkers are urgently needed to increase patient survival. In this study, we identified/quantified 1763 proteins from paired adenocarcinoma (ADC) tissues with different extents of lymph node (LN) involvement using an iTRAQ‐based quantitative proteomic analysis. Based on a bioinformatics analysis and literature search, we selected six candidates (ERO1Lα, PABPC4, RPS25, TARS, NARS, and RCC1) from 151 proteins with a 1.5‐fold increase in ADC tumors without LN metastasis compared with adjacent normal tissue. These six proteins underwent further verification using immunohistochemical staining and western blotting. The protein levels of these six candidates were overexpressed in tumor tissues compared with the adjacent normal tissue. The area under the receiver operating characteristic curve was 0.864 for the combination of the two potential biomarkers (ERO1Lα and NARS) in discriminating tumor tissues without LN metastasis from adjacent normal tissue. Importantly, ERO1Lα and NARS levels were positively associated with LN metastasis, and ERO1Lα overexpression was correlated with poor patient survival. Moreover, we found that the knockdown of either ERO1Lα or NARS reduced the viability and migration ability of ADC cells. Our results collectively provide a potential biomarker dataset for ADC diagnosis/prognosis and reveal novel roles of ERO1Lα and NARS in ADC progression.Support or Funding InformationThis work was supported by grants from the Ministry of Science and Technology, Taiwan, R.O.C. (101‐2320‐B‐182‐035‐MY3 and 104‐2320‐B‐182‐027), the Chang Gung Medical Research Fund (CMRPD3E0081‐2 and CMRPD1C0092) and the Ministry of Education, Taiwan, R.O.C. (EMRPD1E1401).
Site‐Specific Covalent Labeling of RNA with RNA‐TAG: A Platform for the Elucidation of RNA Function
Authors: Busby Kayla N , Alexander Seth C , Devaraj Neal K 
Published: November 2023

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Here, we demonstrate the site‐specific covalent labeling of RNA transcripts using the RNA‐TAG (transglycosylation at guanosine) methodology. This technology takes advantage of a bacterial tRNA transglycosylase (TGT) capable of incorporating nucleobase derivatives bearing functional probes into an encodable RNA stem loop recognition motif. Using this methodology, we have successfully labeled mRNA transcripts with a wide variety of probes including biotin, BODIPY, and Cy7 in a single enzymatic step. Endogenously transcribed and processed RNAs containing the RNA‐TAG recognition hairpin have been successfully visualized in cell imaging studies. Furthermore, we have demonstrated that an RNA of interest can be selectively biotinylated and affinity purified from a complex mixture of RNAs and cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient platform for the elucidation of RNA function via cellular localization studies and affinity purification of RNA‐protein complexes.Support or Funding InformationThis work was supported by the Department of Defense Army Research Office Contract W911NF‐13‐1‐0383. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1144086 (fellowship to K.N.B.).
Production and Characterization of Polyclonal Generic Phosphotyrosine‐specific Antibodies
Authors: Pelech Steven , Yue Lambert , Lai Shenshen , Winkler Dirk , Shi Jane , Zhang Hong 
Published: November 2023

Reversible protein‐tyrosine phosphorylation plays critical roles in cell regulation under normal and pathological conditions, which has made generic phosphotyrosine (pY) antibodies invaluable tools for biomedical research. A recent study that compared the specificities of three widely‐used monoclonal pY antibodies has raised concerns about strong sequence motif selectivity and low overall coverage rates. These issues can potentially introduce significant bias in pY site identification and quantification. In this study we describe a novel strategy of generating a pool of polyclonal antibodies from a large set of physiological pY‐site sequences. Over 400 peptides spanning 7 to 20 amino acids in length, each featuring 1 to 7 phosphorylation sites and representing over 1000 phosphosites, were used to immunize over 100 rabbits. The sera from these rabbits were subjected to ammonium sulphate fractionation, pooled, and then affinity‐purified on pY‐agarose columns to produce the PYK antibody preparations. The specificity of the purified PYK antibody was assessed with phosphopeptide microarrays, including the Jerini Peptide Technologies Phosphatase Peptide Microarray, which features 6,099 peptides representing diverse human pY‐sites. The rabbit polyclonal PYK phosphotyrosine antibody proved to perform better than the well known 4G10, PY20 and PY100 mouse monoclonal antibodies. PYK was extremely stable to repeated freeze thaw, and found to be 4‐fold or more sensitive for detection of phosphopeptides on arrays and for proteins following Western blotting of EGF‐treated A431 cell lysates than the monoclonal antibodies. Like the other monoclonal pY antibodies, PYK reactivities with phosphothreonine and phosphoserine were over 200‐ and 300‐fold less, respectively, when compared to phosphotyrosine on phosphopeptide microarrays. PYK also detected a larger number of diverse pY‐containing peptides than 4G10, PY20 and PY100 on phosphopeptide microarrays, and unlike these other antibodies, acidic amino acid residues surrounding the pY‐sites were not negative determinants for antibody recognition. Since acidic amino acids are important positive determinants for substrate recognition for most protein‐tyrosine kinases and flank most protein‐tyrosine phosphosites, our findings indicate that the PYK antibody may be more useful for enrichment and analysis of physiological pY‐sites than the commonly used mouse monoclonal antibodies for such purposes. Further purification of the PYK antibody over an IgG‐agarose column also allowed development of preparation of a reporter antibody that was suitable for use in sandwich antibody microarrays to assess changes in the tyrosine phosphorylation status of over 500 signaling proteins simultaneously with as little as 50 μg of crude lysate protein from cells and tissues.Support or Funding InformationSupported by Kinexus Bioinformatics Corporation.