bluuebunny/crossref_metadata_2025_split huggingface dataset:
CTEN Is a Novel ΔNp63α Target for the Regulation of Prostate Cell Adhesion and Is Associated with Prostate Cancer Progression
Authors: Liao Yi‐Chun , Wu Wei‐Ming , Yang Kuan , Chen Ya‐Chi , Lo Su Hao 
Published: November 2023

p63 is a member of the p53 transcription factor family and a linchpin of epithelial development and homeostasis. p63 drives the expression of many target genes involved in cell survival, adhesion, migration and cancer. In this study, we identify CTEN as a downstream target of ΔNp63α, the predominant p63 isoform expressed in epithelium. ΔNp63 and CTEN are both highly expressed in normal prostate epithelial cells and are down‐regulated in prostate cancer. In addition, reduced expression of CTEN and ΔNp63 is correlated with prostate cancer progression from primary tumors to metastatic lesions. Silencing of ΔNp63 leads to decreased mRNA and protein levels of CTEN. ΔNp63α induces transcriptional activity of the CTEN promoter and a 140‐bp fragment upstream of the transcription initiation site is the minimal promoter region required for activation. A putative binding site for p63 is located between −61 and −36 within the CTEN promoter and mutations of the critical nucleotides in this region abolish ΔNp63α‐induced promoter activity. The direct interaction of ΔNp63α with the CTEN promoter was demonstrated using a chromatin immunoprecipitation (ChIP) assay. Moreover, impaired cell adhesion caused by ΔNp63α depletion is rescued by over‐expression of CTEN, suggesting that CTEN is a downstream effector of ΔNp63α‐mediated cell adhesion. In summary, our findings demonstrate that ΔNp63α functions as a trans‐activation factor of CTEN promoter and regulates cell adhesion through modulating CTEN. Our study further contributes to the potential regulatory mechanisms of CTEN in prostate cancer progression.Support or Funding InformationThis study was supported by grants from the Ministry of Science and Technology, Taiwan (NSC‐102‐2628‐B‐002‐028‐MY3) and National Taiwan University (NTU‐CESRP‐104R76262).
Biochemical Characterization of Zinc‐Responsive Transcription Factors Zur and ZntR in Klebsiella oxytoca
Authors: Crawford Kyler 
Published: November 2023

Zinc is an essential micronutrient that is sequestered by human innate immune defenses during infection. Bacterial acquisition via outer membrane transporters is necessary, but accumulation of zinc is toxic. The opportunistic human pathogen Klebsiella oxytoca is a member of the Enterobacteriaceae family that expresses the ZnuABC transporter complex to import zinc, and expresses the ZntABC transporter complex to export zinc. Though K. oxytoca infections have become an emergent problem, the regulation of this critical balance and the mechanism for zinc sensing have never been characterized. We hypothesize two transcription factors Zinc Uptake Regulator (Zur) and Znt Regulator (ZntR) are responsible for repressing import and activating export, respectively. In the current study, we applied in silico methods to identify a Zur (Zinc Uptake Regulator) and a ZntR (Znt Regulator) homologue in K. oxytoca. Both K. oxytoca Zur and ZntR were recombinantly overexpressed and purified using affinity chromatography. The target proteins aggregate in inclusion bodies found with the insoluble component of cellular fractions, so a protocol involving in vitro urea denaturation and refolding was developed. The DNA‐binding activity of the Zur and ZntR purified fractions was modeled with electrophoretic mobility shift assays (EMSA). A thorough biochemical protein characterization was conducted on Zur and ZntR, involving mass spectrometry, metal content quantification and chemical cross‐linking analyses. Additionally, we used the bacterial luxCDABE operon to create a bioluminescent reporter system that measured the expression of the znuA and zntA promoters. Overall, our results identify the presence of active Zur and ZntR regulators in K. oxytoca, and prove the metal‐dependent control of the znuA and zntA promoters.Support or Funding InformationBiomedical Sciences Department ‐ Midwestern University ‐ Glendale, AZ
Ribosomal Frameshifting of Trichomonas vaginalis virus 2, 3, and 4 Isolates
Authors: Ai Lizhuo 
Published: November 2023

Trichomoniasis, the most common non‐viral sexually transmitted disease with an estimated 2 million new infections every year in the world, caused by a flagellated protozoan parasite Trichomonas vaginalis. T. vaginalis itself harbors up to four strains of non‐segmented double stranded RNA Trichomonas vaginalis virus (TVV1–4). T. vaginalis virus encodes two genes, an upstream capsid open reading frame (CP ORF) and a downstream fusion RNA‐dependent RNA polymerase (RdRp ORF). Literature studies suggest TVV2 and TVV3 might utilize −1 bp ribosomal frameshifting, while there is strong evidence that TVV1 undergoes −2 bp frameshifting during translation of RdRp in fusion with its capsid. However, there are not many available known genome sequences or many studies done on TVV4 ribosomal frameshifting. Here, we begin to investigate TVVs 2–4.MethodsWe used the only three known consensus sequence of TVV4, and designed sequencing primers. We also used cell cultivation, ultracentrifugation, mass spectrometry, RNA isolation, SDS‐PAGE analysis, RT PCR, qRT PCR, gel electrophoresis, and DNA isolation and purification.ResultsThe genome of a clinical isolate infected with TVV4 has been nearly completed. Virions were isolated by ultracentrifugation.ConclusionNew samples of TVV4 genome have been sequenced in this research for future studies, including LC‐MS analysis of tryptic digests that will allow elucidation of the frameshifting mechanism in TVVs 2–4.Support or Funding InformationThis work was supported by the Mississippi INBRE, funded by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103476
Ethylene Glycol Linkers in Nucleic Acids
Authors: Ortega Alyssa Marie 
Published: November 2023

Single stranded RNA folds upon itself to create structural motifs, such as, hairpin loops, internal loops and bulge loops. Additional hydrogen bonding and ion interactions occur in the loops regions forming unique structural features that are recognized in RNA‐protein interactions. A helical structure interrupted by bulges is typically lower in stability than a continuous helix. In this study we designed RNA and DNA constructs containing varying length of ethylene glycol linkers to measure the stability of constructs that have no additional hydrogen bonding interactions in the bulge regions. The role of metal ions in RNA and DNA is complex as the negatively charged phosphate backbone of RNA and DNA molecules interacts with positively charged metal ions. In addition, loop regions often bind to metal ions to stabilize particular structural features. Thus, examining the stability of linker constructs in various ionic conditions provides further insights into factors that contribute to RNA and DNA stability. Thermodynamic denaturation experiments were performed in 10 mM MgCl2 and 1M KCl buffer solutions at a pH of 7. DNA internal loops and bulge loops showed a decrease in stability with an increase in linker size. Most constructs showed an increase in stability in the presence of magnesium ions as compared to 1 M KCl. A comparative analysis of RNA and DNA bulge loops and internal loops constructs with linkers will be presented.Support or Funding InformationNeena Grover and Colorado College Biochemistry Department
MicroRNA miR‐1207‐5p‐repression of ATRA receptors determine retinoid‐resistance in breast cancer
Authors: Chaudhuri Gautam , Misra Smita 
Published: November 2023

Major objective of this research is to understand the mechanism of retinoid‐resistance of the basal‐type breast cancer (BLBC) cells. BLBC are high‐grade, invasive cancers that are characterized by the “triple‐negative” phenotype (ER−/PR−/HER2−), lacking expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2. BLBC account for approximately 15% of human breast cancers, tend to occur in younger women, and account for a disproportionate amount of breast cancer deaths. As BLBC do not respond to either hormonal treatment or HER2‐targeted treatment, there are at present no targeted therapies for this aggressive cancer. Our long term goal is to develop chemical management strategies against BLBC which are resistant to retinoid therapy. The development of new therapeutic approaches has been invigorated by the discovery of miRNA molecules that can regulate gene expression and many important biological processes including breast cancer. While studying possible role of miRNAs in retinoid‐resistance of the BLBC cells, we found that these cells are low in the levels of three all‐trans‐retinoic acid (ATRA) receptor proteins: RARα, RXRα and RARγ. ATRA triggers antiproliferative effects in tumor cells and therefore ATRA and its synthetic analogs have great potential as anticarcinogenic agents. We report here that retinoid‐resistance in the BLBC cells is mediated by a microRNA, miR‐1207‐5p which binds to the RARα, RXRα and RARγ mRNAs and prevent the biosynthesis of these receptors. Retinoid‐resistant BLBC cells are unusually high in the level of the oncogenic long non‐coding RNA (lncRNA) PVT1. The microRNA miR‐1207‐5p is mitron, as it is encoded by an intron in the PVT1 pre‐RNA. Our research indicates that miR‐1207 gene itself may also have its own promoter which is regulated by the redox‐regulator transcription factor NRF2. We found that miR‐1207‐5p miRISC tightly binds at the 3′‐UTR of the RARα, RXRα and RARγ mRNAs as was revealed by iCLIP analysis. The levels of RARα, RXRα and RARγ mRNAs and proteins are inversely related to miR‐1207‐5p level in the BLBC cells. This research implicates miR‐1207‐5p in the regulation of retinoid‐resistance in the BLBC cells and reflects on potential use of a combination of miR‐1207‐5p antagonists (antagomir and sponges) and ATRA as potent chemotherapy against hard‐to‐cure BLBC.Support or Funding InformationThis research is supported in part by DOD‐CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920‐01 to GC
Pro‐inflammatory cytokines induce mesenchymal stem cell secretory phenotype by increasing autophagosomes and lysosomes
Authors: Hsueh YingChang , Chen HuaLing , Chang TzuChing , WU Kenneth Kun‐Yu 
Published: November 2023

Mesenchymal stem cells (MSCs) undergo premature senescence when challenged with oxidative and metabolic stresses. Pro‐inflammatory cytokines were reported to activate or damage MSCs. It is unclear whether cytokines provoke the cellular changes via premature senescence. To determine whether cytokines induce premature senescence, we treated bone marrow‐derived MSCs (BM‐MSC) with IL‐1β, TNFα or vehicle for up to 48h. Cell growth, p16 and p21 expressions, senescence‐associated β‐galactosidase (SA‐βGal) as well as cell morphology were analyzed. Neither IL‐1β nor TNFα induced growth arrest or expression of p16 or p21. Cell morphology was not overtly altered when compared with control cells. By contrast, SA‐βgal was increased by IL‐1β and TNFα. Since SA‐βgal increase was reported to be due to expansion of lysosome mass, we analyzed lysosomes by Lysotracker staining. Lysotracker staining was enhanced in cells treated with TNFα or IL‐1β. TNFα and IL‐1β induced autophagy as evidenced by increased LC3‐II on western blotting and LC3 immunofluorescence staining. Co‐localization analysis revealed increased auto‐lysosomes. However, LC3‐positive autophagosomes exceeded lysosomes. TNFα and IL‐1β‐induced autophagosomes were correlated with increased expression of cyclooxygenase‐2, prostaglandin E synthase, IL‐6, IL‐8 and TSG‐6. Work is in progress to determine whether inhibition of lysosomes and/or autophagosomes influences cytokine expression. Our findings indicate that TNFα and IL‐1β induces BM‐MSC secretory phenotype without accompanied cell senescence through activating autophagy and increasing autolysosomes.Support or Funding InformationMOST 104‐2321‐B‐039‐003
Depletion of BMI1 Induces Autophagy Mediated Necroptosis
Authors: Dey Anindya , Banerjee Soumyajit , Saha Sounik , Dwivedi Shailendra , Mukherjee Priyabrata , Bhattacharya Resham 
Published: November 2023

The clonal self‐renewal property conferred by the polycomb complex protein BMI1 (BMI1 proto‐oncogene, polycomb ring finger) is instrumental in maintenance of not only normal stem‐cells but also cancer initiating cells from several different malignancies and represents a major challenge to chemotherapy. Realizing the immense pathologic significance, PTC‐209, a small molecule inhibitor of BMI1 transcription has recently been described. We report here that, genetic or pharmacologic inhibition of BMI1 significantly impacts clonal growth and induces autophagy in ovarian cancer (OvCa) cells through ATP depletion. While autophagy can promote survival or induce cell death, targeting BMI1 engages the PTEN induced putative kinase 1 (PINK1) ‐ parkin RBR E3 ubiquitin protein ligase (PARK2) dependent mitochondrial pathway and induces a novel mode of non‐apoptotic, necroptosis mediated cell death. In OvCa, necroptosis is potentiated by activation of the receptor (TNFRSF)‐interacting serine‐threonine kinase 1 (RIPK1) ‐ receptor‐interacting protein kinase 3 (RIPK3) complex that phosphorylates its downstream substrate, mixed lineage kinase domain‐like (MLKL). Importantly, genetic or pharmacologic inhibitors of autophagy or necroptosis rescue clonal growth in BMI1 depleted cells. Thus, we have established a novel molecular link between BMI1, clonal growth, autophagy and necroptosis. In chemo‐resistant OvCa where apoptotic pathways are frequently impaired, autophagy mediated necroptotic cell death modalities provide an important alternate strategy that leverage overexpression of BMI1.Support or Funding InformationThis study was supported by the National Institutes of Health (NIH) CA 157481 to RB.
PGC‐1alpha a Novel Regulator of Autophagy
Authors: Salazar Gloria , Huang Jingwen , Zhao Yitong 
Published: November 2023

Aging or senescence is associated with increased predisposition to cardiovascular diseases, a leading cause of death worldwide. Two unrelated processes, mitochondrial biogenesis and autophagy, decline with age. However, whether regulators of mitochondrial biogenesis also regulate autophagy is incompletely understood. Mitochondrial biogenesis is regulated in part by the peroxisome proliferator‐activated receptor gamma (PPARgamma) coactivator‐1 alpha (PGC‐1alpha) a central organizer of metabolic function and oxidative states. Deficiency of the PGC‐1alpha gene promotes vascular senescence in mice. Autophagy contributes to cellular homeostasis because it mediates the degradation of old and dysfunctional cellular components, such as proteins and organelles including mitochondria. Using vascular smooth muscle cells (VSMCs) isolated from PGC‐1alpha knock out mice, we show that PGC‐1alpha deficiency strongly down‐regulates the expression of the autophagy adaptor p62/sequestosome 1 and prevents autophagy, measured by reduced LC3‐I to LC3‐II conversion and reduced number of autophagosomes. Consistent with a causative role of p62 downregulation in PGC‐1alpha‐deficiency phenotype, p62 down‐regulation by siRNA induces senescence and inhibits autophagy in VSMCs. Further, p62 knock out mice show increased senescence in the aorta tissue. Our data suggest that PGC‐1alpha stimulates p62 expression to up‐regulate autophagy leading to the degradation of dysfunctional mitochondria that prevents vascular senescence. These studies will provide a foundation for therapeutic interventions to up‐regulate PGC‐1alpha/p62 pathway to promote healthier aging and prevent/delay age‐related diseases including atherosclerosis.Support or Funding InformationAmerican Heart Association (14GRNT20180028)
IP3 Receptor Determines Increased Basal Mitochondrial Calcium And Autophagy Regulation In Mdx Skeletal Muscle
Authors: Valladares Denisse , Utreras‐Mendoza Yildy , Morales Camilo , Campos Cristian , Westermeier Francisco , Lavandero Sergio , Jaimovich Enrique 
Published: November 2023

Several reports describe that IP3 receptor (IP3R) is essential for efficient mitochondrial respiration and maintenance of cellular bioenergetics. This is due to the participation of this receptor in calcium transfer from the ER to the mitochondria. Changes in IP3R function can compromise mitochondrial function, changing ATP production and finally altering autophagy. Nevertheless until now there is no detailed study of the IP3R/calcium/autophagy axis in DMD. Our aim was to investigate the participation of IP3R in the regulation of mitochondrial calcium and autophagy in fibers from a DMD animal model, mdx. We analyzed protein levels of LC3, p62, atg5, beclin and bcl‐2 in FDB muscle of mdx (5–7 weeks), electroporated with a short‐hairpin for IP3R. We also evaluated the calcium transfer with mitochondria‐targeted probes that can be electroporated and also mitochondrial potential with TMRE in skeletal muscle fibers. The basal levels of expression of LC3II are diminished in mdx fibers compared with controls. This result correlates with an increased expression of p62 in mdx fibers. We also found differences in other autophagy proteins like atg5, beclin1 and Bcl‐2. When we performed the knockdown for IP3R we observed differences in the expression of almost all of the proteins analyzed. The expression of LC3II was increased in the electroporated mdx fibers with a decrease in p62 expression. We also confirmed that basal mitochondrial calcium level was increased in mdx fibers. Moreover, when we decreased the expression of IP3R, the basal mitochondrial calcium levels returned to normal. Similar result was obtained when we measured the mitochondrial potential in mdx fibers. Collectively, these results suggest that IP3R regulates mitochondrial calcium flux in adult skeletal muscle and is responsible for the increased mitochondrial calcium in mdx fibers. Moreover, knockdown of IP3R can regulate the expression levels of proteins associated with autophagy. Finally, we propose that some inhibitors of the IP3 pathway can be used as a new treatment for the muscle weakness observed in DMD patientsSupport or Funding InformationFONDECYT 3140491(DV), 3140532(FW), 1151293(EJ), ACT‐111 (EJ, SL), FONDAP 15130011(SL)
ULK1‐Mediated Autophagy is Required for Skeletal Muscle Recovery From Acute Ischemia‐Reperfusion Injury
Authors: Wilson Rebecca June , Drake Joshua C , Zhang Mei , Yan Zhen 
Published: November 2023

Ischemia‐reperfusion (IR) injury may occur in skeletal muscle under acute and chronic disease conditions, in which skeletal muscle is temporarily deprived of oxygen and nutrients due to loss of circulation (ischemia) followed by reestablishment of the circulation (reperfusion). IR initiates a cascade of events that result in skeletal muscle dysfunction and death. As a consequence, skeletal muscle undergoes regeneration for recovery. Autophagy is the highly regulated, critical cellular process of degrading damaged proteins and organelles; however, little is known about the role of autophagy in muscle recovery following IR‐injury. Here, we induced acute IR injury in mouse skeletal muscle by application of a rubber band tourniquet above the femur of one limb for 1 hour and followed by reperfusion by removing the tourniquet. We harvested the gastrocnemius muscles at different times (0, 3, 12, 24, 48, 72 hours, 7 and 14 days) post reperfusion. IR resulted in increased phosphorylation of the upstream autophagy protein unc‐51 like activating kinase‐1 (ULK1) at serine555 (activating) between 12 hours and 7 days and increased content of the autophagy machinery proteins microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and p62 between 0 hours to 14 days. These findings suggest there is activation of autophagy and enhanced autophagy protein expression during recovery from IR injury. Additionally, IR resulted in significant loss of muscle function with nearly undetectable force production at 72 hours and 7 days following IR but only recovered to ~50% by day 14. We also found a significant loss of muscle mass at 72 hour (~15%), day 7 (~25%) day 14 (~40%) compared to Con. In order to investigate the importance of autophagy in recovery from IR injury we subjected skeletal muscle‐specific ULK1 inducible knockout (MKO) mice and their wild‐type littermates (WT) to IR or a sham procedure (Sham) followed by assessment of mass and maximal tetanic force at day 14. WT mice had ~50% recovery of force production (vs. Sham), while MKO had only 25% recovery (p<0.002 vs. WT). Both WT and MKO had significant loss of muscle mass with no difference between the genotypes. These data strongly suggest that ULK1‐mediated autophagy is required for functional recovery.Support or Funding InformationAHA predoctoral fellowship 14PRE20380254
Role of the CHIP/Hsp70 Protein Quality Control Complex as an Information Processor
Authors: Page Richard C 
Published: November 2023

Intracellular protein quality control fulfills the need for a rapid and robust response to protein misfolding that is ubiquitous in human physiology. Defects in protein quality control pathways can lead to a wide range of diseases including cystic fibrosis and recessive hereditary cerebellar ataxias. Within the field of protein quality control, the ubiquitin ligase CHIP and chaperone Hsp70 play crucial roles in directing misfolded client proteins to either the refolding, or degradative pathways. Together, CHIP and Hsp70 form a complex that exhibits functions characteristic of an information processor. Our results suggest that the complex takes inputs from both dynamic motions and posttranslational modifications in determining the fate of the misfolded protein. Guided by our 2.91 Å resolution structure of the TPR domain of CHIP in complex with the α‐helical “lid” subdomain and unstructured “tail” of Hsc70, we can now assemble full‐length models of the CHIP/Hsp70 complex. This structural data, combined with nuclear magnetic resonance, small angle X‐ray scattering, electron paramagnetic resonance, and biochemical data identify the mechanisms by which the function of the CHIP/Hsp70 complex is regulated. Surprisingly, the CHIP‐TPR interacts with determinants within both the Hsc70‐lid subdomain and the C‐terminal GPTIEEVD motif of the tail, exhibiting a novel mode of chaperone‐TPR domain interaction. This interaction mode provides sensitivity to posttranslational modifications of CHIP and Hsp70 and enables dynamic motions within CHIP to modulate activity of Hsp70.Support or Funding InformationSupported by Burroughs Wellcome Foundation Collaborative Travel Research Grant #1014031