bluuebunny/crossref_metadata_2025_split huggingface dataset:
The relationship between the parotid fascia and the deep fascia of the infraorbital region: a new concept in descriptive and applied anatomy
Authors: Amodeo Chiara Andretto , Delmas Vincent , Casasco Andrea , Keller Gregory S 
Published: November 2023

This study aims to demonstrate that a relationship exists between the parotid fascia and the deep fascia of the infra orbital region, recently described by the first author, that continues from the temporal region and covers the mimetic muscles that lift the upper lip.Methods24 hemifaces were dissected to show the relationship between these two deep fascial layers, a preauricular incision was performed, the Superficial musculo‐aponevrotic layer was dissected and reflected. The dissection was then performed deep to the parotid fascia to reach the anterior border of the parotid gland.Another incision was placed in the temporal area, the temporoparietal fascia was elevated, the superficial layer of the deep temporal fascia was incised and dissected toward the zygomatic arch and the infraorbital region.Resultsin all specimens a relationship was present between the parotid layer and the superficial layer of the deep temporal fascia, that continued from the temporal region to the infraorbital one, after crossing the zygomatic arch.The two layers were in continuity in the superior third of the parotid gland, at the anterior border of the gland where the branches for the zygomaticus major, minor and the other muscles that lift the upper lip leave the gland to reach the muscles.Conclusionsa relationship exists between the deep fascia of the infraorbital region, this relationship was observed in the superior third of the anterior border of the parotid gland at the level of the emergency of the branches of the facial nerve for the muscles that lift the upper lip and it was connected to the ligaments present in this area.
Olive Leaf Metabolites as Covalent Human Topoisomerase II Poisons
Authors: Vann Kendra R , Sedgeman Carl A , Gopas Jacob , Golan‐Goldhirsh Avi , Osheroff Neil 
Published: November 2023

Topoisomerase II plays many essential roles in genome maintenance. To carry out its physiological functions, the enzyme generates transient double‐stranded breaks in the DNA to help resolve topological problems that naturally occur. Thus, while essential to cell survival, topoisomerase II has the potential to fragment the genome. Beyond its critical cellular functions, human topoisomerase II is the target for a number of widely prescribed anticancer drugs that act by increasing levels of enzyme‐mediated DNA strand breaks. Many of these “topoisomerase II poisons” are derived from natural sources. Identification and characterization of topoisomerase II poisons from natural sources can provide novel drug scaffolds. Therefore, a series of water‐soluble extracts from Mediterranean plants were screened for activity against human topoisomerase IIα. An extract from Phillyrhea latifolia L., a member of the olive tree family, significantly enhanced DNA cleavage mediated by human topoisomerase IIα. P. latifolia L. contains a number of phenolic metabolites, including antioxidants associated with several human health benefits. Oleuropein, hydroxytyrosol, and verbascoside enhanced topoisomerase IIα‐mediated DNA cleavage ~4‐fold. Analysis of topoisomerase II‐mediated DNA cleavage carried out in the presence of oleuropein, hydroxytyrosol, and verbascoside indicated that these compounds displayed hallmark characteristics of “covalent topoisomerase II poisons.” First, the ability of the metabolites to poison topoisomerase II was enhanced substantially in the presence of an oxidant, but was abrogated in the presence of a reducing agent. Second, the metabolites required the N‐terminal region of topoisomerase IIα to enhance DNA cleavage. Third, the metabolites inhibited topoisomerase II activity when incubated with the enzyme prior to the addition of DNA. Phenolic olive plant metabolites retained the ability to act as topoisomerase IIα poisons in complex dietary herbal formulations, such as commercial olive leaf extracts and extra virgin olive oils. These results strongly suggest that olive plant metabolites can act as dietary topoisomerase II poisons. These studies may provide a platform for developing new anticancer or chemopreventative therapies. Current studies are focused on further elucidating the interaction of covalent poisons with topoisomerase II. Results from ongoing mutagenesis studies may provide additional insight into the mechanism by which covalent poisons interact with human topoisomerase II.Support or Funding InformationGM033944, GM062459, GM08320, ICA (Israel), Deutsche Forschungsgemeinschaft, and R. H. Holzer Foundation.
A nucleolar role for the Fanconi anemia pathway protein, FANCI, in rDNA transcription
Authors: Sondalle Samuel B. , Longerich Simonne , Sung Patrick , Baserga Susan J. 
Published: November 2023

Fanconi anemia (FA) is a disease of DNA repair because proteins mutated in patients with FA are impaired in removing interstrand DNA crosslinks. We propose a new nucleolar role for the FA protein, FANCI, in ribosome biogenesis based on extensive preliminary data. Ribosome biogenesis is the process of making ribosomes that initiates in the cell nucleolus with the transcription and processing of the pre‐ribosomal RNA (rRNA) followed by the assembly of mature ribosomes. Immunoaffinity purification of FANCI from HeLa cell nuclear extract followed by mass spectrometry revealed co‐purification with nucleolar proteins required for ribosome biogenesis, including PES1. Using subcellular fractionation, we have found that FANCI is indeed enriched in the nucleolus. In particular, the deubiquitinated form of FANCI is by far the most predominant form of nucleolar FANCI, even under conditions of DNA damage by mitomycin C, which stimulate monoubiquitination of FANCI. Immunoprecipitation revealed that deubiquitinated FANCI, but not its binding partner in DNA repair, FANCD2, is co‐immunoprecipitated by the nucleolar protein, PES1. To probe a possible nucleolar function for FANCI, we depleted FANCI in human cells and observed reduced transcription by RNA polymerase I (RNAPI). In addition, FANCI is co‐immunoprecipitated by the large subunit of RNAPI, indicating a physical and functional role for FANCI in efficient pre‐rRNA transcription. Finally, our results demonstrate novel regulatory mechanisms by deubiquitinases on the ubiquitination state of FANCI in the nucleolus. While our model does not contest the well‐established underlying DNA repair defect observed in cells from FA patients, it does suggest a dual pathophysiology for FA that includes defects both in DNA repair and in ribosome biogenesis.Support or Funding InformationThis work is funded by the NIGMS and NCI.
Participation of Distal RRRY Motif in Strand Displacement DNA synthesis by A‐Family DNA Polymerases
Authors: Modak Mukund J , Ketkar Amit , Singh Kamal 
Published: November 2023

Strand displacement synthesis of DNA (SDSD) is an essential activity during Okazaki fragment maturation process. We have previously shown that residues in fingers subdomain and RRRY motif of E.coli DNA polymerase I participates in SDSD and 3′ – 5′ exonuclease activity, respectively (J. Biol. Chem. 2008, 283: 17979–17990). We further investigated the function of the conserved RRRY motif in the function of DNA polymerase I enzymes with or without associated 3′‐5′ exonuclease activity. Using DNA polymerase I from M. tuberculosis, which lacks 3′ – 5′ exonuclease activity and E.coli DNA polymerase I, which contains the 3′ – 5′ exonuclease activity, we now show that the RRRY motif is essential for SDSD. A detailed biochemical properties of individual arginine to alanine mutant enzymes showed that the first and the third arginine (R796 and R798) in M. tuberculosis polymerase I are essential for the SDSD, while, the 2nd arginine (R797) and 4th tyrosine (Y799) are not required. Similar properties were displayed by the mutant derivatives of equivalent residues in E.coli polymerase I. The loss of strand displacement DNA synthesis activity resulted from the significant reduction in the DNA binding affinity by mutant enzymes. Two most affected mutant enzyme species were further found to be defective in the formation of the enzyme‐DNA‐dNTP ternary complexes. We further find that displaced strand cleavage is mediated by 3′ – 5′ exonuclease activity of E.coli DNA polymerase I, in RRRY‐dependent manner. Our data, for the first time, demonstrate dual function of a conserved motif despite its distal location from the active sites.Support or Funding InformationNone
Human Y‐family DNA polymerase kappa is more tolerant of amino acid substitutions in the active site loop region than its E. coli ortholog DinB
Authors: Antczak Nicole M , Ippoliti Paul , Walsh Jason , Packer Morgan , Beuning Penny 
Published: November 2023

More than 100,000 lesions are generated on DNA in each cell per day due to such agents as reactive oxygen species and ultraviolet radiation. Y‐family DNA polymerases are specialized to carry out translesion synthesis by replicating damaged DNA and are conserved in all domains of life. There are two Y‐family polymerases in E. coli, DinB (Pol IV) and UmuD′2C (Pol V), one in the archaeon Sulfolobus solfataricus, Dpo4, as well as several in humans including Pol κ (hPol κ), which is a DinB ortholog. Both DinB and hPol κ are known to bypass minor groove adducts of guanine whereas Dpo4 can bypass both minor and major groove adducts. Using primer extension assays on DNA substrates containing the minor groove adduct N2‐furfuryl‐dG as well as undamaged templates combined with site‐directed mutagenesis to construct polymerase variants, we found that loop 1 residues of DinB near the catalytic site are important for activity as well as the selection of the correct nucleotide. There is a similar trend in the loop 1 region of hPol κ. In single nucleotide incorporation assays, wild‐type and variants of both polymerases show a tendency to insert incorrect dT as well as the correct dC opposite the N2‐furfuryl‐dG adduct. This is also seen for wild‐type and variants of hPol κ on an undamaged DNA template. These observations suggest that the loop 1 region of both polymerases is important in correct nucleotide incorporation during translesion synthesis. Furthermore, although both DinB and hPol κ are inhibited by major groove modifications, hPol κ is more active than DinB on DNA with major groove adducts. In order to probe this further, we used primer extension assays with DNA substrates containing the minor groove adduct N2‐furfuryl‐dG, major groove adduct N6‐furfuryl‐dA, as well as undamaged templates, with loop swap constructs of DinB and hPol κ, we found the loop regions of DinB and hPol κ near the catalytic site are important for bypass of different types of DNA damage and insertion of the correct nucleotide. Specifically, hPol κ is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of minor groove adducts by DinB and hPol κ can be accomplished.Support or Funding InformationSupport from American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.
Age‐related epigenetic modifications contribute to dysfunction of male germline progenitor cells
Authors: Lee Tin‐Lap , Liao Jinyue , Chan Wai‐Yee 
Published: November 2023

Ageing is a complex time‐dependent biological process that takes place in every cell and organ, eventually leading to degenerative changes that affect normal biological functions. In the past decades, the number of couples to conceive children at advanced ages has increased significantly. It is widely recognized that advanced maternal age poses higher birth defect risk due to oocyte ageing. However, the effect of advanced paternal age on male germ cell quality is largely overlooked, and the impact has been implicated in a variety of disease conditions affecting the offspring. Mammalian spermatogenesis is a tissue‐specific, stem cell‐dependent process that relies on self‐renewal and differentiation of male germline stem cells to support continual production of spermatozoa. Currently, the mechanisms regulating these processes remain largely unknown, and emerging evidence has suggested that epigenetic profiles shift dramatically during aging. Epigenetic modifications on DNA methylation and demethylation pathways through formation of 5‐methylcytosine (5mC) and 5‐Hydroxymethylcytosine (5hmC) have profound influences on developmental programs, but its role remains elusive in male germ cell development. In this study, we examined the dynamics of DNA methylation and gene expression in undifferentiated and differentiating cell populations in early male germ cell development by performing RNA‐seq and whole genome 5mC/5hmC profiling at single‐‐base resolution. We defined signaling and transcriptional difference involving key self‐‐renewal pathways in undifferentiated and differentiated cell populations and found that differentially methylated genes were enriched in various age‐related cellular developmental processes. We also observed methylation changes associated with OCT4 and GDNF, which are both important for undifferentiated spermatogonia maintenance. Finally, 5hmC was enriched specifically in proximal upstream and downstream regions relative to TSS and its presence correlates positively with gene expression, we found genes enriched in undifferentiated populations display higher promoter 5hmC levels, indicating 5hmC is associated with stemness‐‐related genes and stem cell capacity.Support or Funding InformationHong Kong Research Grant Council (RGC) ‐General Research Fund project no. 469713, 468312
Regulation of kcnd2 by microRNA‐30e in rat trigeminal ganglions following chronic constriction injury of infraorbital nerve
Authors: Dong Yingchun 
Published: November 2023

BackgroundTrigeminal neuralgia is a severely debilitating orofacial neuropathic pain and is difficult to treat since the pathophysiology is not well clear.ObjectiveThe present study aimed to detect the expression of voltage‐gated potassium channel gene Kv4.2 (kcnd2) and microRNA‐30e in rat trigeminal ganglions (TGs) after chronic constriction injury of infraorbital nerve (ION‐CCI), and explore the mechanism of the decreased expression of kcnd2 under microRNA‐30e regulation.MethodThe rats are divided randomly into normal control and ION‐CCI group. The expressions of kcnd2 and microRNA‐30e were detected in rat TG. Luciferase assay was performed to detect whether the expression of kcnd2 was regulated directly by microRNA‐30e.ResultThe expression of kcnd2 was decreased while microRNA‐30e expression was increased significantly in ipilateral TG after ION‐CCI compared to the control. The decreased expression of kcnd2 is negatively correlated with the increased microRNA‐30e expression and positively correlated with the head withdrawal threshold reflecting pain. Luciferase assay showed that binding of microRNA‐30e to the 3′UTR of kcnd2 gene suppressed the transcriptional activity, and downregulation of microRNA‐30e significantly increased the kcnd2 mRNA level in F11 cells.ConclusionOur results show that microRNA‐30e is negatively correlated with the development and maintenance of orofacial neuropathic pain after ION‐CCI via regulating the activity of kcnd2. MicroRNA‐30e may be a target for the therapy of trigeminal neuralgia.Support or Funding InformationNational Natural Science Foundation of China (No. 81100768) and Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (No. YKK11040; QRX11123).
ACA11, a Small Nucleolar RNA Activated in Multiple Myeloma, Stimulates Proliferation by Inactivating NRF2 and Increasing Redox Signaling
Authors: Mahajan Nitin , Weber Jason D. , Maggi Leonard B. , Tomasson Michael H 
Published: November 2023

Multiple myeloma (MM) is a malignancy of antibody secreting plasma B cells. The t(4;14) translocation is detected in 20% of MM and is associated with shortened patient survival. Previously, we identified a novel orphan box H/ACA class small nucleolar RNA (snoRNA), that is located within intron 18–19 of the MMSET gene, and is expressed co‐transcriptionally with MMSET in MM cells harboring the t(4;14) translocation. H/ACA small RNAs are an evolutionarily conserved class of noncoding RNAs (ncRNA) involved in a diverse range of processes including posttranslational modifications, pre‐ribosomal RNA processing, and telomere maintenance. RNA‐Seq analysis performed on CD138+ tumor cells isolated from t(4;14) positive and negative MM patients revealed a unique signature of upregulated genes implicated in mitochondrial function and oxidative phosphorylation. We hypothesized that ACA11 helps to drive MM by increasing oxidative stress. We found that acute overexpression of ACA11 led to the increased levels of reactive oxygen species (ROS) in human myeloma cell lines, human foreskin BJ fibroblasts, murine fibroblasts and primary splenic B‐cells. Cells overexpressing ACA11 showed enhanced cell proliferation, colony formation, and elevated ERK1/2 phosphorylation, a downstream consequence of oxidative stress. In addition, nuclear factor erythroid‐derived 2‐related factor 2 (NRF2), which regulates anti‐oxidant signaling, was inactivated in ACA11 overexpressing cells. Together our results suggest that overexpression of ACA11 suppresses the ability of NRF2 to induce target anti‐oxidant genes, resulting in increased oxidative stress and ultimately cell proliferation. These ACA11‐dependent changes likely contribute to the malignant phenotype in cancers that overpass the MMSET gene.Support or Funding InformationThe work is supported by NIH R01‐CA174743 to MHT and LBM
Characterization and Expression Analysis of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle
Authors: Orsech Hannah , Waddell David 
Published: November 2023

Skeletal muscle atrophy is a serious medical condition resulting from an array of physiological conditions such as aging, corticosteroid use, cancer and loss of innervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel genes showing differential expression in response to neurogenic atrophy. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C2C12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, several fragments of the proximal regulatory region were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with a myogenin expression plasmid, resulting in myogenin dependent induction of the Zfp593 reporter genes. Bioinformatic analysis of the promoter region of Zfp593 revealed two conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Myogenin is a member of the myogenic regulatory factor family of transcription factors, which are important in muscle cell differentiation and are also significantly induced in response to neurogenic atrophy. Interestingly, Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Therefore, in order to determine if Zfp593 is localized to the nucleus of muscle cells, the Zfp593 cDNA was amplified from C2C12 cells and sub‐cloned into an expression plasmid that produces a GFP‐Zfp593 fusion protein. The GFP‐Zfp593 expression plasmid was then transfected into proliferating C2C12 cells followed by visualization using confocal microscopy. Cells transfected with the GFP‐Zfp593 expression plasmid showed fluorescence that is exclusively localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.Support or Funding InformationUniversity of North Florida Transformational Learning Opportunity Grant
GSK3β Modulates Excess Nutrient‐Induced Autophagy Impairment in Human Aortic Endothelial Cells via FOXO1
Authors: Weikel Karen , Ruderman Neil , Ido Yasuo 
Published: November 2023

Dysregulated autophagy, the process by which damaged proteins and organelles are turned over, and decreased AMP‐activated protein kinase (AMPK) activity are each associated with type 2 diabetes and atherogenesis. In a human aortic endothelial cell (HAEC) model of poorly‐controlled type 2 diabetes, we observed that the same concentrations of excess nutrients that increased inflammation and apoptosis also decreased AMPK activity and autophagy. Our current objective was to determine whether glycogen synthase kinase 3β (GSK3β), an enzyme whose hyperactivity has been associated with diabetes, atherogenesis and inhibition of AMPK activity, attenuates autophagy and AMPK activity in nutrient‐laden HAECs.AMPK activity was measured by protein levels of p‐AMPKThr172 and the SAMS peptide assay. GSK3β activity was measured by protein levels of p‐GSK3βSer9. GSK3β activity was increased by treating cells with Akt inhibitor VIII (Akti) and GSK3β activity was suppressed by infecting HAECs with lentivirus knocking down GSK3β (shGSK3β) or expressing the kinase‐dead (K85A) mutant. Changes in Lysotracker staining reflected changes in lysosome acidity. Autophagosome formation was evaluated by measuring bafilomycin‐induced changes in protein levels of LC3‐II, by both Western blot and immunofluorescence. P‐FOXO1Thr24 and mRNA levels of PI3KC3 and SOD2, FOXO1 target genes, were used to evaluate FOXO1 signaling.A combination of 25 mM glucose and 0.4 mM palmitate (excess nutrient conditions) inhibited basal autophagy and AMPK activity and also increased GSK3β activity. Treatment of HAECs with Akti decreased Lysotracker staining under both control and excess nutrient conditions. Suppression of GSK3β activity attenuated excess nutrient‐induced losses in Lysotracker staining. Under both control and excess nutrient conditions, suppression of GSK3β activity increased LC3‐II protein levels and knockdown of GSK3β increased FOXO1 signaling.These data suggest that increases in GSK3β activity may impair autophagy in our HAEC model of poorly‐controlled type 2 diabetes by decreasing lysosome acidity and autophagosome formation. The mechanism by which GSK3β affects each of these aspects of autophagy remains to be clarified, but may involve modulation of FOXO1 signaling.Support or Funding InformationThis research was supported by NIH T32 Multidisciplinary Training in Cardiovascular Research HL007224.