bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Kynurenine Signaling Decreases Glioblastoma Multiforme Genomic Instability and Sensitizes Cells to Chemotherapeutic Treatment
Authors: Reed Megan R , Maddukuri Leena , Helm Emily , Bostian April C.L. , Zafar Maroof K , Eoff Robert L 
Published: November 2023

Glioblastoma multiforme (GBM) is an aggressive grade IV astrocytoma that is commonly treated with genotoxic compounds. Aberrant activation of the aryl hydrocarbon receptor (AhR) through mis‐regulation of the kynurenine pathway (KP) in GBM cells contributes to the malignant properties of these tumors. Our group determined that AhR activation by the KP led to an increase in chromosomal damage and that this was due, in part, to increased expression of the translesion DNA polymerase hpol κ. Based on these results, we hypothesized that blocking the rate‐limiting step in kynurenine (KYN) production through inhibition of the enzyme tryptophan 2,3‐dioxygenase (TDO) would sensitize GBM cells to genotoxic treatment by reducing the basal levels of proteins and enzymes that facilitate tolerance/repair of DNA damage. GBM cells pre‐treated with the TDO inhibitor 680C91 were found to be more sensitive to the DNA damaging agents cis‐diamminedichloroplatinum (CDDP) and bis‐chloroethylnitrosourea (BCNU). Flow cytometry analysis of GBM cell lines treated with 680C91 in combination with CDDP or BCNU revealed an enrichment in cells with 4N (G2/M) and 1N (sub‐G1) DNA content. These results are consistent with the promotion of cell cycle arrest and death following DNA damage when KYN/AhR signaling is blocked. To study global changes in replication fork dynamics, replication‐associated proteins were enriched using affinity‐mediated isolation of proteins on nascent DNA (AMIPOND). Analysis of the resulting mass spectrometry data indicate that when GBM cells are treated with 680C91 there is a broad depletion of DNA damage response and repair proteins near sites of active DNA synthesis. Immunoblotting was used to support trends observed in the proteomics results. In conclusion, our study provides support for a model in which inhibition of TDO sensitizes GBM‐derived cells to genotoxic drugs through depletion of DNA repair/damage tolerance factors.Support or Funding InformationSupport or Funding Information: Supported by NIH grants CA183895 to RLE and CA140409 to PAC. This research was also supported by a grant from the Arkansas Breast Cancer Research Program, the UAMS CTSA and a Seeds of Science Award from the Winthrop P. Rockefeller Cancer Institute.
The Role of Noncoding Genetic Elements in the Transcriptional Regulation of the Voltage‐Gated Sodium Channels SCN1A and SCN8A
Authors: Inglis George Andrew Stanowski , Escayg Andrew 
Published: November 2023

Epilepsy is a common neurological disorder that affects over 50 million people worldwide, and is characterized by recurrent, unprovoked seizures. Altered brain expression of the voltage‐gated sodium channel (VGSC) α subunit gene SCN1A has been shown to directly cause some forms of epilepsy. Heterozygous loss‐of‐function mutations in the VGSC gene SCN1A (encoding the protein Nav1.1) are responsible for 70–80% of cases of Dravet Syndrome (DS), a severe, treatment‐resistant form of childhood epilepsy. A significant decrease in SCN1A mRNA levels is also associated with temporal lobe epilepsy (TLE), the most common form of treatment‐resistant epilepsy in adults. Restoring SCN1A expression through increased gene expression is predicted to be therapeutic in these cases. Conversely, loss‐of‐function or reduced expression in the VGSC gene SCN8A (encoding the protein Nav1.6) has been demonstrated to increase seizure resistance and quality of life in mouse models of DS and TLE. However, despite the important role of the VGSC genes in epilepsy, and the clinical potential of modulating SCN1A and SCN8A expression as a therapy for patients with epilepsy, we have limited knowledge about the genetic elements or factors responsible for the transcriptional regulation of these two genes.By analyzing several genomic markers of open or actively transcribed chromatin across neuronal cell types, we identified several genetic elements in both SCN1A and SCN8A with putative transcriptional regulatory ability. We have cloned these elements into a vector upstream of a human SCN1A promoter driving luciferase reporter expression. A number of these constructs resulted in altered luciferase activity relative to empty vectors or constructs with just the SCN1A promoter, suggesting the cloned elements have functional significance. Additional analyses, including identification of proteins responsible for transcription, are being conducted on a subset of these elements.Support or Funding InformationThis work was supported through funding from the NIH National Institute of Neurological Disorders and Stroke (NINDS) and the Emory University Genetics & Molecular Biology T32 Training Grant.
Rdl Expression Impacts Circadian Rhythm and Locomotion in Drosophila melanogaster
Authors: MacDonald Stephanie , Rogers Ryan P 
Published: November 2023

The genes that modulate circadian rhythm in Drosophila melanogaster have been at the forefront of sleep research in an effort to characterize the impact of circadian rhythm on other biological processes and identify potential overlap in human sleep disorders. In flies, Resistant to Dieldrin (Rdl) encodes a subunit of the GABA receptor, which is upregulated by the circadian oscillator, WAKE. This interaction promotes RDL localization to the plasma membrane to modulate sleep; however, the specific function of RDL and its downstream interactions to facilitate sleep/wake switching have yet to be characterized. Prior research has shown that Rdl can suppress kazachoc (kcc), which in turn suppresses paralytic (para). para is known to control voltage‐gated sodium channels (VGSC's) in the same way that the human gene SCN1A does. This is noteworthy because mutations in SCN1A are linked to epilepsy in humans, and mutations in para result in seizure‐like behavior in Drosophila. The Drosophila Activity Monitor TriKinetics System and a custom‐built light controlling apparatus was employed to characterize locomotion in control and RdlMD‐RR mutant flies. Since circadian rhythm is largely influenced by light and dark patterns, the flies were monitored during 12‐hour light and dark cycles over a period of 5 days to determine any variation between genotypes. Analysis of the data show control flies exhibit consistent sleep and wake patterns with peaks of activity in daylight hours, and activity in darkness is significantly reduced. Conversely, RdlMD‐RR flies show less activity during daylight hours, and near paralysis in darkness. These data suggest that Rdl expression is related to restricting locomotion during sleep and that levels may be modulated by circadian oscillation. Future research will focus on characterizing the effect of Rdl on para expression using RT‐qPCR to elucidate the role of RDL in circadian regulation.Support or Funding InformationThis work was supported by funds from the Department of Sciences at Wentworth Institute of Technology.
Transcriptome‐wide mapping of the miR‐122 targetome revealed its mechanistic role in the maintenance of liver homeostasis
Authors: Barajas Juan Martin , Luna Joseph , Teng Kun‐Yu , Darnell Robert , Ghoshal Kalpana 
Published: November 2023

Liver cancer has the second highest mortality rate among all malignancies, which implies a lack of molecular understanding of disease development and progression necessary to combat this deadly disease. miR‐122 is a conserved liver‐specific miRNA, that maintains metabolic homeostasis, suppresses tumor development, and promotes HCV replication. Loss of miR‐122 is associated with loss of hepatic phenotype, gain of malignancy‐associated features, and poor patient prognosis in hepatocellular carcinoma (HCC) patients. Recent in vivo work in our miR‐122 knockout (KO) mice have established miR‐122 as a bona fide tumor suppressor. These data suggest it is critical to identify the miR‐122 targetome in the liver to better understand its function. Previously, in silico prediction was the go‐to‐method for identification of miRNA targets. However, these methods are dependent on known or evolutionarily conserved functionality of the miRNA‐Ago complex and without reference to cellular context, can result in a high number of false positives and false negatives. To overcome this, the laboratory of Dr. Robert Darnell introduced a novel unbiased high‐throughput method known as Ago‐dHiTS‐CLIP (Ago‐CLIP) which has been applied in wild‐type (WT) and miRNA KO platforms to systematically and biochemically identify transcriptome‐wide miRNA‐mediated Ago maps. To this end, in collaboration with Dr. Darnell's laboratory, we performed Ago‐CLIP analysis in livers of 6 week‐old WT and KO mice as well as in benign human liver expressing relatively high levels of miR‐122 and matching HCC tumor exhibiting down‐regulation of miR‐122. Ago‐CLIP identified ~800 miR‐122 targets in mice and ~1400 in humans with only 309 targets conserved in both. Surprisingly, a majority of miR‐122 sites are in coding exons (30%) and 3′‐UTRs (30%) along with sites in the 5′UTR, introns, etc. RNA‐seq analysis revealed specific upregulation of these 3′UTR and CDS targets in miR‐122 KO livers, suggesting these are functional miR‐122 targets. Furthermore, Liver and Hepatocellular Carcinoma (LIHC) data from The Cancer Genome Atlas (TCGA) revealed alterations in 26 conserved miR‐122 target genes are predictive of overall patient prognosis. BCL9, an exclusive CDS target and a critical component of Wnt/β‐catenin signaling, was found to be significantly predictive of patient survival and may play a key role in liver tumor progression. In summary, our analysis identified key targets involved in liver homeostasis and revealed a miR‐122 target‐signature predictive in human HCC.Support or Funding InformationThis work was supported by NIH grants RO1CA193244 and R01CA19324406S1 (K. Ghoshal).
miRNA‐mediated crosstalk between Wnt3a and TGFβ3 in osteoblast differentiation
Authors: Fushimi Shigeko , Nohno Tsutomu , Nishimatsu Shin‐ichiro , Katase Naoki , Terada Kumiko , Katsuyama Midori , Demura Masashi , Saijoh Kiyofumi , Nagatsuka Hitoshi , Katsuyama Hironobu 
Published: November 2023

miRNAs play roles in regulating gene expression at the post‐transcriptional level through fine‐tuning of the protein‐encoding gene expression, and are involved mainly in regulating important biological processes in different cell and tissue types, including osteoblast differentiation. Canonical Wnt signaling is also known to play pleotropic roles in regulating cell differentiation during mammalian osteogenesis, although involvement of miRNAs is not established for Wnt signaling in osteoblast differentiation. We examined here the role of candidate miRNAs expressed differentially after Wnt3a expression during osteoblast differentiation.Overexpression of Wnt3a resulted in increased transcription of the alkaline phosphatase gene, but in decreased transcription of the Runx2, collagen 1a and osteocalcin genes in osteoblastic MC3T3‐E1 cells. Analysis of miRNA expression profile showed that 14 miRNAs were up‐regulated and 21 miRNAs were down‐regulated after Wnt3a overexpression in MC3T3‐E1 cells. TGFβ3 is considered to be a candidate target gene that was down‐regulated by one of miRNAs with database search. Transfection of the miRNA mimic into MC3T3‐E1 cells significantly inhibited TGFβ3 mRNA level and then resultant protein expression, whereas overexpression of Wnt3a increased TGFβ3 mRNA level and protein expression.These results suggest that the miRNA is involved in osteoblast differentiation as a critical regulatory factor, mediating Wnt3a signaling pathway and TGFβ3 signaling pathway.Support or Funding InformationAuthors declare that we have no conflicts of interests.
Role of Long Non‐Coding RNA Alive in Response to Angiotensin II in Vascular Smooth Muscle Cells
Authors: Amaram Vishnu , Das Sadhan , Leung Amy , Reddy Amar , Lanting Linda , Natarajan Rama 
Published: November 2023

Angiotensin II (Ang II) is a key pro‐inflammatory and growth‐promoting factor involved in the development of cardiovascular diseases (CVDs) like hypertension and atherosclerosis by promoting vascular smooth muscle cell (VSMC) dysfunction. Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are associated with disease processes. Using high throughput RNA‐sequencing, we recently showed that several novel lncRNAs are differentially regulated by Ang II in VSMC. Here we investigated the role of one of these novel lncRNAs in Ang II mediated VSMC dysfunction. We found that an Ang II regulated lncRNAs in VSMC is transcribed from H3K27ac marked active enhancer regions and perhaps functions as an enhancer RNAs (eRNAs). This lncRNA, which we named “Ang II induced long noncoding RNA in vascular cells with enhancer function” (Alive), was upregulated by Ang II in rat and human VSMCs in a time dependent manner. Alive expression was also induced by platelet derived growth factor (PDGF) in rat VSMC. The expression of the mouse ortholog of Alive was increased in vivo in aortas of Ang II infused mice. Reporter luciferase assays showed that the putative Alive enhancer region indeed functions as a transcriptional enhancer. RNA‐FISH and subcellular fractionation by RT‐qPCR experiments showed that Alive is predominantly localized in the nucleus. Alive transcript is located 117 kb upstream of an Ang II responsive protein coding neighboring gene Aggrecan (Acan) that codes for an extracellular matrix protein and is associated with CVDs. Functional analysis revealed that knockdown of Alive with siRNAs or GapmeRs in rat VSMC decreased the expression of not only Alive, but also its neighboring gene Acan. Furthermore, knockdown of Alive resulted in the upregulation of inflammatory genes such as IL6 and Ptgs2. Currently, CRISPR‐dCas9 mediated activation of Alive expression is being tested to determine the effects of gain‐of‐function of Alive in VSMCs. These results suggest that Alive functions as a novel anti‐inflammatory lncRNA and its expression is increased in response to Ang II to protect against the Ang II associated VSMC dysfunction involved in CVDs.Support or Funding InformationNIH, NHLBI.
Regena/NOT2 is Essential for Gene Silencing by MicroRNAs
Authors: Zbornik Tika , Andersen Katherine , Bowden Sean , Reinke Catherine 
Published: November 2023

A forward genetic screen in Drosophila melanogaster has yielded genetic evidence demonstrating a requirement for the CCR4/NOT complex subunit Regena/NOT2 in gene silencing. A single mutation in Regena/NOT2 generated by ethyl methanesulfonate mutagenesis leads to increased expression of a GFP reporter that is fused to the 3′UTR of the Drosophila Bearded gene and sensitive to microRNA‐mediated gene silencing. MicroRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). MicroRNA‐mediated silencing is accomplished by miRISC through translation block, target mRNA degradation, or a combination of these mechanisms. To determine whether the observed requirement of Regena/NOT2 for gene silencing is dependent upon the activity of microRNAs, the effect of a mutation in Regena/NOT2 on a GFP‐based reporter with disrupted microRNA‐binding sites and insensitive to microRNA‐mediated gene silencing was assayed. No change in reporter expression was observed in the presence of a Regena/NOT2 mutation when the reporter lacked microRNA‐binding sites, supporting a microRNA‐dependent role for Regena/NOT2 in gene silencing. We are further exploring the specific role of Regena/NOT2 in gene silencing by determining whether this CCR4‐NOT complex component is required for translational block, transcript degradation, or both, and characterizing a putative role for Regena/NOT2 in microRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the forward genetic screen aim to identify additional essential miRISC components, to elucidate the complex molecular mechanisms of microRNA‐mediated silencing.Support or Funding InformationThis work was supported by a grant from the M.J. Murdock Charitable Trust
A Bifunctional Fusion Enzyme with DNA polymerase and dUTPase Activities
Authors: Dash Asim Kanti , Bhamidipati Sravya , Rashid Mohammad Bazlur 
Published: November 2023

Construction of a fusion enzyme by recombinant DNA technology could be useful in obtaining more efficient bifunctional enzymes. However, two open reading frames in tandem often interfere with each other's functions. We have previously demonstrated that when a relatively smaller dUTPase enzyme is tagged on either side of a thermostable DNA polymerase it did not affect polymerization function. Here, we will further reveal that both the enzymatic domains of these fusion enzymes also do not muddle each other's functions indeed. Furthermore, both of these affinity purified fusion proteins appeared to be ‘undisruptable dimers’ in denaturing protein gels and as confirmed by MALDI‐MS/MS analyses.Support or Funding InformationFaculty Research Support Fund from the Office of Sponsored Programs at the University of Houston‐Clear Lake, Texas.
Extra‐ribosomal function of bacterial ribosomes: Modulation of enzyme activities
Authors: Shekhtman Alexander , DeMott Chris , Majumder Subhabrata , Reverdatto Sergey 
Published: November 2023

The intracellular concentration of bacterial ribosome is linearly proportional to the growth rate and can occupy up to 20% of the cell volume or about 50 mg/mL. This high concentration facilitates weak binding between cytosolic proteins and ribosomes and can affect protein biochemistry. By using a combination of in‐cell and in vitro biophysical techniques, we show that adenylate kinase (ADK) and dihydrofolate reductase (DHFR) bind to ribosomes via specific interaction surfaces, and the respective co‐factors ATP and NADPH also bind to ribosomes both specifically and non‐specifically. As a consequence, ribosomes modulate the enzymatic activity of ADK and DHFR by interfering with substrate availability. Ribosome binding also impedes protein diffusion, an important determinant for reaction rates. Using fluorescence recovery after bleaching (FRAP), we show that green fluorescent protein (GFP) diffusion is significantly slower in the presence of ribosomes in vitro, comparable to that observed intracellularly. These results strongly suggest that ribosomes play a previously under‐appreciated, non‐translational role in regulating the cellular activity of enzymes.Support or Funding InformationThis work was supported by National Institutes of Health Grant 5R01GM085006 to A.S.
Assembly and disassembly of the hibernating bacterial 100S ribosome
Authors: Basu Arnab 
Published: November 2023

Translation is an important biological process critical for all life. Suppression of ribosome biogenesis and translation have been linked to bacterial persistence and slow growth inside the animal hosts. In Gram‐positive human pathogen Staphylococcus aureus, a ribosome‐binding protein called hibernation‐promoting factor (HPF) dimerizes the 70S ribosome to form the translationally inactive 100S complex. The formation of 100S ribosome in S. aureus is distinct than that of the majority of the gammaproteobacteria. In S. aureus the formation of 100S ribosome dimer begins at log phase and continues throughout the stationary phase, whereas the 100S dimer formation is specifically induced during the stationary phase in gammaproteobacteria (such as E. coli). Importantly, E coli requires a helper protein called ribosome modulation factor (RMF) in addition to HPF to promote 70S dimerization. The biological significance of the bacterial 100S ribosome has been elusive for six decades. We found that the loss of hpf reduces S. aureus survival and causes rapid degradation of ribosomes during stationary phase. Disrupting the 100S formation only derepresses the translation of a subset of genes. The reason for the selectivity of HPF‐dependent translation attenuation is unclear. Our recent work suggests a model by which the 100S ribosome serves as a reservoir for long‐term S. aureus viability because the 100S ribosomes may be more resistant to ribonuclease degradation. We propose that during stationary phase, the 100S ribosomes in the wild‐type are dissembled by an unidentified dissociation factor into subunits and may be recycled for a new round of translation. In contrast, the hpf mutant exhausts all ribosome pools upon entry to the late stationary phase, leading to cell death. Using biochemical and genetic approaches, we identified a bona fide dissociation factor responsible for the disassembly of 100S ribosome. Because the dissociation factor antagonizes the stability of 100S ribosome, we examined the temporal expression of HPF and the dissociation factor to elucidate how they impact the homeostasis of 100S ribosomes. We also performed chemical crosslinking to map the position of HPF on the ribosome that may account for its translational inhibition function. These results provide a plausible explanation for the constitutive production of 100S ribosome that is intimately related to S. aureus pathophysiology.Support or Funding InformationEdward Mallinckrodt Jr Foundation, and The Pew Charitable Trusts.
Probing the Pal‐Peptidoglycan Interaction
Authors: Phadke Shivani , Stanton Sarah , Pierce Jeanetta , LaClair Ciara , Hall Cianna , Michel Lea 
Published: November 2023

Peptidoglycan‐associated lipoprotein (Pal) is a dual‐oriented lipoprotein in Escherichia coli (E. coli) that interacts non‐covalently with peptidoglycan (cell wall) and other outer membrane and periplasmic proteins. By comparing Pal to its homologue in another Gram‐negative bacterium, our team was able to design point mutations at possible interaction sites between Pal and peptidoglycan. Recombinant Pal proteins (wild‐type and mutants) were expressed in E. coli and purified using His‐tag column chromatography. Multiple protocols were employed to purify peptidoglycan from Escherichia coli cells, with varying results. Using an enzyme‐linked immunosorbent assay (ELISA), we were able to assess the binding affinity of each Pal mutant to peptidoglycan, compared to wild‐type protein. Preliminary data suggest that several key residues help to modulate the Pal‐peptidoglycan interaction, and site‐directed mutants of Pal bind differentially to peptidoglycan compared to wild‐type Pal.Support or Funding InformationThe Camille and Henry Dreyfus Foundation: Special Grant Program in the Chemical Sciences (L. Michel).