bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Hepatic Progenitor Cells in Hepatic Fibrosis of Elderly Cadavers: Lobular Distribution of Cytokeratin 7 and 19 Expressing Hepatic Progenitor Cells
Authors: Png Chien Yi M , Mark Ki M
Published: November 2023

Human hepatic progenitor cells (HPCs) are small oval‐shaped cells with an ovoid nucleus and scant cytoplasm. Cytokeratin (CK) 7 and CK19 are strongly expressed by HPCs and have been widely used as phenotypic markers for HPCs. In normal liver, HPCs reside in a quiescent state in the canals of Hering that are located in the periportal parenchyma. When the liver is damaged, as in alcoholic liver disease, nonalcoholic fatty liver disease, viral hepatitis, and chronic cholestasis, the HPCs become activated. Since elderly cadavers with diverse causes of death demonstrated progressive stages of liver fibrosis (Mak et al., 2012, Anat Rec 295:40–50), we aimed to evaluate whether the cadaveric livers served as a model for fibrosis‐associated HPC activation in the aged liver. To that effect, consecutive paraffin liver sections from embalmed cadavers were immunoperoxidase stained using monoclonal CK7 and CK19 antibodies, in conjunction with EDTA‐based antigen unmasking. Sections were post‐stained with Sirius red stain for assessment of fibrosis. Positive CK7 and CK19 staining of intraportal bile ducts and ductules validated the specificity of the immunoreaction. HPCs were identified by the CK7 or CK19 immunostaining of the cytoplasm. In livers with minimal fibrotic changes, CK7 and CK19 positive HPCs were generally distributed in proximity to the portal tracts, as well as in the lobular parenchyma at variable distance from the portal tracts. These CK7 or CK19 immunoreactive cells appeared either as individual cells, small clusters of cells or strings of cuboidal cells. In septal fibrosis, CK7 positive HPCs, along with occasional CK19 stained HPCs, could be seen in proximity to the developing septa, bridging septa and parenchymal fibrotic foci. In cirrhosis, the matrix of fibrotic bands often revealed CK7 positive HPCs, while the parenchyma of the nodules contained variable numbers of CK7 stained HPCs. Strikingly, CK19 immunoreactive HPCs were either rare or undetectable in cirrhotic livers.

Conclusions

The deep intralobular distribution of HPCs likely resulted from their migration from the periportal area into the lobular parenchyma, possibly relating to hepatic regeneration consequent to liver damage caused by fibrosis. CK7 expressing HPCs were found in all stages of fibrosis, while CK19 HPCs were absent from cirrhosis, suggesting that CK7 serves as a more sensitive phenotypic marker for HPCs than CK19. Hence, embalmed livers from elderly cadavers provide a potentially useful model for assessing HPC activation in association with liver fibrosis progression in the aged liver.
A rare case of bilateral supra‐piriformis pelvic exit of the common fibular nerve and its clinical relevance
Authors: Vasan Cheryl , Goldman Evan , Vasan Nagaswami
Published: November 2023

Bilateral supra‐piriformis pelvic exit of the common fibular nerve (CFN) was observed during dissection of the pelvis and gluteal region. The donor was a 70‐year old Caucasian female who died of coronary vascular disease. The sciatic nerve (SN) is an integrated lumbosacral plexus of nerves that exits the pelvis through the greater sciatic foramen inferior to the piriformis muscle and later divides into the tibial nerve (TN) and CFN. In the present case, the right and left CPN exited the pelvis superior to the piriformis muscle while the TN exited inferior to the muscle. Further pelvic dissection showed that TN and CFN followed an independent course and did not unite to form the SN prior to pelvic exit. The TN exited inferior to piriformis and descended in the middle of the thigh whereas the CFN pursued a lateral course through the gluteal region, posterior thigh and popliteal fossa. Variations in the pelvic exit of the TN, CFN and the SN have been observed and classified. Knowledge of the potential alterations in the course of the sciatic, TN and/or the CFN are of clinical importance when performing such procedures as hip arthroplasty, piriformis tendon release for treatment of piriformis syndrome or, sciatic anesthesia in the popliteal fossa for foot and ankle surgery. This poster will depict a number of actual images of the variation and its’ classification based on previously established classification systems.

Support or Funding Information

This study was supported by the Department of Biomedical Sciences.
A genome wide screen to identify genes that prevent the accumulation of mutations
Authors: Hombauer Hans Wolfgang , Schmidt Tobias , Cava Gloria Ximena Reyes , Ceylan Umran , Gries Kerstin , Meurer Matthias , Knop Michael
Published: November 2023

Synthesis of eukaryotic genomes requires the function of DNA replicative polymerases Polα (Pol1), Polɛ (Pol2) and Polδ (Pol3). Biochemical evidence supports the idea that Polα initiates DNA synthesis at replication origins as well as at every Okazaki fragment. This initial DNA synthesis is further extended by the proofreading proficient DNA polymerases Polɛ and Polδ.

The DNA lagging strand is synthesized by Polδ in a discontinuous manner, as 200–300 bases‐long Okazaki fragments. On the other hand, the leading strand is synthesized in a continuous manner; however, it is less clear if Polɛ, Polδ or a combination of both, are mainly required during this process.

In order to identify unrecognized genes that might affect DNA replication fidelity, we performed a genome‐wide screen in S. cerevisiae searching for synergistic mutator interactions with DNA polymerase mutants. We modified the Synthetic Genetic Array protocol to cross low‐fidelity DNA polymerase mutants pol1‐L868M, pol2‐M644G and pol3‐L612M (or wild‐type) against the non‐essential yeast gene deletion collection. Single and double mutants were tested for an increased mutator phenotype with in vivo mutator assays (lys2‐10A and CAN1 inactivation). Analysis of the single mutants identified ~45 genes, with well‐known functions in different DNA repair pathways, which prevented the accumulation of mutations. Moreover, we identified one additional gene deletion that has not been previously linked to a mutator phenotype. This mutant not only resulted in elevated mutation rates, but also increased gross chromosomal rearrangements.

Analysis of the double mutants identified a small number of gene deletions (including exo1Δ) that resulted in a strong synergistic mutator interaction with pol1‐L868M and pol3‐L612M, but not with pol2‐M644G. This unpredicted bias supports the hypothesis that leading and lagging strand are mainly synthesized by Polδ. Furthermore, we have identified a small number of genes whose roles become increasingly important under conditions of impaired DNA replication fidelity.

Support or Funding Information

Deutsches Krebsforschungszentrum (DKFZ). Marie Curie CIG grant “iMMR”.
Implications for 4‐Oxo‐2‐nonenal‐Derived Histone Adducts in Mediating Gene Expression
Authors: Camarillo Jeannie Marie , Galligan James J , Marnett Lawrence J
Published: November 2023

Histone modifications play a critical role in the maintenance of chromatin structure and the regulation of gene expression. For example, Lys acetylation to histone tails results in chromatin remodeling to allow transcriptional activation of targeted genes. Recent work has identified core histones as targets for adduction by the oxidative stress‐derived lipid electrophile, 4‐oxo‐2‐nonenal (4‐ONE). Here, we investigate the potential implications of these modifications on gene expression. Using an alkyne analog of 4‐ONE, a4‐ONE, we employed click chemistry to selectively tag modified proteins from RKO cells treated with electrophile. Our data reveal that a4‐ONE is a long‐lived histone modification, present up to 24 hours after treatment. Using pharmacological inhibitors of histone acetylation and deacetylation, we observe alterations in the acetylation of core histones when cells are co‐treated with a4‐ONE. These data suggest that a4‐ONE modifications occur at physiologically relevant sites that regulate chromatin structure and gene expression. Further, utilizing a combination of chromatin immunoprecipitation and click chemistry, we can selectively pull out DNA associated with adducted histones, further suggesting a putative role for these modifications in the regulation of gene expression. Together, our data suggest that histone adducts may play a role in chromatin structure and the mediation of gene expression.

Support or Funding Information

NCI F31 CA192861 (JMC) and NCI R37 CA087819 (LJM)
Reduced Expression of MicroRNA‐200 Family Members Lead to Epithelial‐Mesenchymal Transition of a Chemoresistant Colorectal Cancer Cell Line
Authors: San KayKay , Martino Alice , Ezekiel Uthayashanker
Published: November 2023

After a patient's tumor is removed, he/she undergoes adjuvant chemotherapy and/or radiotherapy. Both of these treatments lead to formation of resistant cells that become metastatic cancer cells. The cellular program responsible for the change of epithelial tumors (such as breast, colorectal, pancreatic, and ovarian) into metastatic mesenchymal cell types is Epithelial‐Mesenchymal Transition (EMT). EMT is a normal process during embryogenesis, where epithelial cells lose their characteristics and become motile mesenchymal cells. Invasive and metastatic characteristics of carcinoma cells in primary tumors are mediated by EMT. During EMT, the primary tumor cells lose cell‐cell adhesion, normally mediated by E‐cadherin, and attain mesenchymal markers. The cells exhibit increased intercellular separation and elongated shape with pseudopodia. We developed a chemoresistant colorectal cancer cell line (DLD‐1 OxR) by exposing DLD‐1 colorectal cancer cells to increasing concentrations of oxaliplatin (a chemotherapy drug used for colorectal cancer). DLD‐1 OxR cells exhibit increased intercellular separation, increased motility, and elongated spindle shape with pseudopodia. MicroRNAs (miRNAs) are small non‐coding RNAs that regulate gene expression postranscriptionally. MiRNA, with silencing complexes, bind 3′ untranslated regions which lead to degradation of RNA or translation repression. Recent studies show that miRNA‐200 family members regulate the EMT process and metastasis. Decreased expression of miRNA‐200 family members leads to metastatic transformation of cancer cells. One of the miRNA‐200 family members is miRNA‐200c. Our hypothesis is that miRNA‐200c expression is decreased in DLD‐1 OxR cells. Loss of miRNA‐200c leads to expression of several regulatory genes involved in EMT, such as, Suz12 (one of the polycomb repressive complex proteins) and claudin‐1 (an integral membrane protein). We have found that miRNA‐200c expression is lower in DLD‐1 OxR cells compared to the parental DLD‐1 cells. Also, the miRNA‐200c targets are upregulated in DLD‐1 OxR. In conclusion, our study suggests that miRNA‐200c is involved in the EMT process in colorectal cancer cells.

Support or Funding Information

KS and AM are supported by the DeNardo Education and Research Foundation.
Untethering glucocorticoid receptor‐mediated transcriptional repression
Authors: Weikum Emily Rye , Hudson William , Ortlund Eric
Published: November 2023

The glucocorticoid receptor (GR), a ligand regulated transcription factor that controls the expression of thousands of genes driving both up‐ and down‐regulation at equal frequencies. The mechanism of gene activation is quite well understood and involves GR cooperatively dimerizing onto DNA at glucocorticoid response elements (GREs). However, the mechanism of repression is much more debated and is thought to occur though two possible mechanisms, DNA‐dependent and DNA‐independent. The DNA‐dependent mechanism involves direct binding of GR to the newly discovered negative glucocorticoid response elements (nGREs). The DNA‐independent mechanism involves GRs interaction with activator protein‐1 (AP‐1), which is a central transcription factor in immune systems that drives expression of inflammatory response genes. This mechanism, known as “tethering”, is the prevailing model for GR‐mediated repression at inflammatory genes. However, recent discovery of GR‐nGRE interactions lead us to wonder if DNA‐dependent repression was possible at AP‐1 response elements (TREs). We report that GR is able to repress inflammatory genes in the absence of AP‐1, GR binds to TRE sequences in vitro, and crystal structures of GR bound to novel recognition sites located within canonical TRE‐sequences. This data represents a paradigm shift in our understanding of GR‐mediated repression and represents an alternative mechanism to the tethering hypothesis.

Support or Funding Information

F31 NRSA (1F31GM11397‐01A1)
Characterization of Mn(II) homeostasis within Bacillus subtilis
Authors: Baertlein Matt , Vos Cullen , Jeffries Will , Isaacson Erin , Gabriel Scott
Published: November 2023

Metal ion homeostasis is a critical component of all cellular life. Metals act as cofactors for many essential enzymes; yet, in excess, metals can cause oxidative stress and cell death. In order to further understand metal ion homeostasis, investigation of pathways that regulate the corresponding intake and efflux of metal ions is needed. While many of the pathways which control metal ion intake and efflux within B. subtilis have been characterized, Mn(II) efflux still remains unknown. Zone of inhibition (ZOI) data from our lab shows a ydfM knockout decreases the survivability of B. subtilis in the presence of toxic Mn(II) levels, while there was no significant difference in toxic concentrations of Cu(II), Cd(II), and Zn(II). Additionally, Beta‐galactosidase assays have shown that ydfM transcription increases with increasing Mn(II) concentration but not increasing concentrations of Cd(II), Cu(II), Fe(III), Ni(II), or Zn(II). These results have led us to hypothesize that that YdfM acts as an Mn(II) efflux protein within B. subtilis.

Concurrently, we also have aimed to identify the transcriptional regulator of YdfM. Interestingly, the gene immediately downstream of YdfM is predicted to be a metal efflux protein regulator. Initial bioinformatics work, shows that YdfL possesses a helix turn helix DNA binding motif as well as several conserved amino acid sequences which suggest it is similar to other metal ion influx and efflux regulators. ZOI data also shows there is a significant increase in survivability in the ydfL knockout when compared to wild type B. subtilis, further suggesting YdfL acts as a regulatory repressor of ydfM. Additional preliminary beta‐galactosidase, growth curve, and gel shift data currently ongoing will also be presented which aims to confirm YdfM's role in the efflux of Mn(II) as well as YdfL's role as a regulator of ydfM transcription.
Structure of the Bacterial Transcription Termination Factor Rho in Complex with the Regulatory Factor NusG
Authors: Lawson Michael , Berger James
Published: November 2023

All eukaryotic and prokaryotic organisms utilize Spt5‐like proteins to regulate transcription. The bacterial Spt5 homolog NusG binds to RNA polymerase with its N‐terminal domain, and to transcription factors such as Rho with its C‐terminal domain. Rho is a hexameric helicase that utilizes energy from ATP hydrolysis to directionally translocate specific nascent transcripts through its central pore, which generates a force that disrupts transcription by RNA polymerase at distinct sites in bacterial genomes. For reasons that remain unclear, NusG is strictly required for a subset of transcripts at Rho‐dependent termination loci.

In order to further elucidate the mechanism by which NusG aides Rho‐dependent transcription termination, we solved a 3.7 Å crystal structure of a complex of Rho, RNA, and the C‐terminal domain of NusG. The structure reveals that NusG binds to Rho in a fashion that orients Rho to translocate directly towards RNA polymerase, and positions the central pore of Rho near the RNA exit channel of the polymerase. We established both pulldown‐ and anisotropy‐based assays to track binding of NusG to Rho in vitro, and used the Rho/NusG complex structure to guide the engineering of point mutants that abrogate binding. We are using these mutants as tools to further probe the role of NusG in Rho‐dependent termination both in vitro and in vivo.

Support or Funding Information

This work was supported through funding from the G. Harold and Leila Y. Mathers Foundation and the NSF Graduate Research Fellowship Program.
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 Information

This 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).
Tumor Suppressor Phosphatase PHLPP Regulates Gene Expression Through Suppression of Transcription Factor Activation
Authors: Katsenelson Ksenya Cohen , Stender Joshua D. , Glass Chris , Newton Alexandra C.
Published: November 2023

Epigenetic mechanisms play a key role in cancer and are a major contributor in driving the aberrantly high levels of oncogenic receptor tyrosine kinases (RTK) in cancer. Understanding mechanisms leading to altered transcription of oncogenes and tumor suppressors has the potential to provide new therapeutic targets. Here we show that the PH domain leucine‐rich repeat protein phosphatase 1 (PHLPP1), a tumor suppressor that directly dephosphorylates and inactivates Akt, has an additional function of regulating approximately 10% of the transcriptome. Specifically, we used non‐biased high‐throughput RNA‐sequencing and chromatin immunoprecipitation‐sequencing techniques to analyze genome‐wide mRNA levels and acetylation and phosphorylation patterns of genes in cells from wild‐type and PHLPP1 knockout mice. De novo motif analysis of the promoter for the genes regulated by PHLPP1 identified enrichment in the recognition motifs for a number of transcription factors, including ones involved in inflammatory signaling. Biochemical analysis revealed that PHLPP1 regulates the phosphorylation and activation of these transcription factors, such that loss of PHLPP1 leads to enhanced inflammatory signaling. Our data support a model in which PHLPP1 dephosphorylates specific transcription factors to act as the brakes to inflammatory signaling, a hallmark of cancer.
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 Information

Biomedical 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.

Methods

We 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.

Results

The genome of a clinical isolate infected with TVV4 has been nearly completed. Virions were isolated by ultracentrifugation.

Conclusion

New 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 Information

This 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 Information

Neena 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 Information

This 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 Information

MOST 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 Information

This 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 Information

American 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 patients

Support or Funding Information

FONDECYT 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 Information

AHA 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 Information

Supported by Burroughs Wellcome Foundation Collaborative Travel Research Grant #1014031
The degradation requirements for topologically distinct quality control substrates in the yeast endoplasmic reticulum
Authors: Guerriero Christopher James , Reutter Karl Richard , Augustine Andrew , Brodsky Jeffrey L.
Published: November 2023

In eukaryotic cells, nearly one third of all newly synthesized proteins are translocated into the endoplasmic reticulum (ER). Inside the ER, proteins are post‐translationally modified and folded by molecular chaperones into their native states. Mutations, transcriptional/translational errors, aberrant post‐translational modifications, or misassembly can result in ER‐associated degradation (ERAD), a quality control pathway which recognizes and degrades misfolded proteins to prevent toxic gain‐of‐function phenotypes. Given the vast array of conformers that proteins adopt, it is not surprising that many diseases result from protein degradation by the ERAD pathway.

The rate‐limiting step during ERAD is substrate retrotranslocation from the ER into the cytoplasm, which occurs prior to proteasome‐mediated degradation. Therefore, the retrotranslocation step can tune degradation efficiency and potentially be targeted to mitigate ERAD‐related diseases. Retrotranslocation is driven by the AAA‐ATPase Cdc48p in yeast (p97 in mammals), which extracts integral membrane proteins either directly from the ER membrane or through an as‐yet‐elusive retrotranslocation channel. Although integral membrane proteins have diverse membrane spanning domains and orientation, little is known about how the placement of a misfolded domain in the ER lumen versus the cytoplasm influences ERAD efficiency. We predict that extraction of a large ER lumenal domain will slow retrotranslocation kinetics.

To test this hypothesis, a pair of model ERAD substrates were designed to contain the same truncated nucleotide‐binding domain from a well‐characterized ERAD substrate, Ste6p*, but on opposite sides of the ER membrane. We previously demonstrated that a soluble version of this domain, NBD2*, when expressed in the cytoplasm is degraded by the proteasome, and therefore can be used as a transposable degron (Guerriero et al., JBC, 2013). Our first model substrate, Chimera A*, is a dual pass membrane protein fused to NBD2* positioned in the cytoplasm. By contrast, Chimera N*, is a single pass membrane protein which deposits NBD2* in the ER lumen. Initial characterization confirms the proper topology of these substrates and proteasome‐dependent degradation. Moreover, the substrates rely on ER resident E3 ligases, Hrd1p and Doa10p, for efficient degradation. We are currently determining the relative dependence on Cdc48p for both degradation and retrotranslocation in order to better understand the energetic barriers to ERAD (Supported by NIH grants DK101584 to C.J.G. and GM075061 to J.L.B.).
A High‐Content RNAi Screen to Elucidate Novel Effectors of Cardiac Proteotoxicity
Authors: McLendon Patrick M , Davis Gregory , Gulick James , Shay‐Winkler Kritton , Molkentin Jeffery D , Robbins Jeffrey
Published: November 2023

Rationale

Proteotoxicity has emerged as an important underlying pathologic mechanism in cardiac disease. Many heart failure phenotypes, including ischemia/reperfusion, pressure overload‐induced hypertrophy and desmin‐related cardiomyopathy accumulate misfolded protein within the cytoplasm of cardiomyocytes, potentially leading to cell death and dysfunction. While recent success in treating these phenotypes has been achieved by inducing the protein degradation mechanisms that mediate protein quality control in cardiomyocytes, more study is needed to understand the players and processes involved in pathologic aggregation.

Objective

To uncover, in a non‐biased way, novel gene targets that reduce protein accumulation in a cellular model of proteotoxic heart disease.

Methods and Results

To address this, we developed a cellular model of cardiac protein aggregation in primary mouse cardiomyocytes by expressing a fluorescent aggregation‐prone protein, a mutated αλπηα−B‐crystallin (CryABR120G). The cells developed large intracellular aggregates, consistent with data from mice and humans. We subjected this cell model to a high‐content, genome‐wide RNAi knockdown screen using a lentiviral shRNA library (~ 16,000 genes). The effect of gene knockdown was calculated by measuring the quantity of punctate aggregate structures within cardiomyocytes. Using shRNA knockdown, we identified 236 genes in the mouse genome that could substantially ( > 50%) reduce aggregate content in cardiomyocytes. The hits span diverse and interesting cellular processes including mitochondrial and apoptotic cell death, calcium signaling, post‐translational modifications and metabolism and suggest overlapping therapeutic mechanisms between proteotoxicity and other cardiovascular pathologies.

Conclusions

While rigorous validation of our candidate list is ongoing, we have confirmed several candidates which can lead to reduction of proteotoxic inclusions in cardiomyocytes. We are confident that further interrogation of our candidate genes will contribute vital details on the molecular mechanisms of pathologic protein aggregation, as well as yield new and interesting therapeutic targets.

Support or Funding Information

This work is supported by funding from the National Institutes of Health (to PMM and JR) and by Le Fondation Leducq (to JR)
An inherited developmental disorder reveals functions for WHAMM and the actin nucleation machinery in early stages of autophagy
Authors: Campellone Kenneth , Mathiowetz Alyssa , Russo Ashley , Baple Emma , Crosby Andrew
Published: November 2023

Human cells rely on the actin cytoskeleton to properly organize, shape, and move their membrane‐bound organelles. However, the nucleation factors responsible for assembling actin filaments have not been well characterized in relation to human disease. Nephrocerebellar syndrome (NCS) is a recently‐described developmental disorder in which patients encode truncated mutants of the WD40‐repeat protein WDR73 and the actin nucleation factor WHAMM. Here we show that cells from NCS patients exhibit cytoskeletal irregularities, possess enlarged ER‐Golgi intermediate compartments, and have severe defects in the cytoplasmic degradation process of autophagy. Re‐introduction of wild type WHAMM into patient cells restored autophagosome biogenesis, whereas depletion or genetic inactivation of WHAMM in healthy human cell lines impaired both the lipidation of the autophagosomal protein LC3 and the clearance of ubiquitinated protein aggregates. Wild type WHAMM localized to subdomains of nascent autophagosomes, and proper WHAMM function involved binding to the phospholipid PI(3)P, the small G‐protein Rab1, and actin. These results reveal a cytoskeletal mechanism for controlling autophagosome remodeling that is perturbed in cells from NCS patients.