bluuebunny/crossref_metadata_2025_split huggingface dataset:
Upregulation of CYP17A1 by Sp1‐mediated DNA Demethylation Confers Temozolomide Resistance through DHEA‐Mediated Protection in Glioma
Authors: Hsu Tsung‐I 
Published: November 2023

Steroidogenesis‐mediated production of neurosteroids is important for brain homeostasis. Cytochrome P450 17A1 (CYP17A1), which converts pregnenolone to dehydroepiandrosterone (DHEA) in endocrine organs and the brain, is required for prostate cancer progression and acquired chemotherapeutic resistance. However, whether CYP17A1‐mediated DHEA synthesis is involved in brain tumor malignancy, especially in glioma, the most prevalent brain tumor, is unknown. To investigate the role of CYP17A1 in glioma, we determined that CYP17A1 expression is significantly increased in gliomas, which secrete more DHEA than normal astrocytes. We found that as gliomas became more malignant, both CYP17A1 and DHEA were significantly upregulated in temozolomide (TMZ)‐resistant cells and highly invasive cells. In particular, the increase of CYP17A1 was caused by Sp1‐mediated DNA demethylation, whereby Sp1 competed with DNMT3a for binding to the CYP17A1 promoter in TMZ‐resistant glioma cells. CYP17A1 was required for the development of glioma‐cell invasiveness and resistance to TMZ‐induced cytotoxicity. Additionally, DHEA markedly attenuated TMZ‐induced DNA damage and apoptosis. Together, our results suggest that components of the Sp1‐CYP17A1‐DHEA axis, which promotes the development of TMZ resistance, may serve as potential biomarkers and therapeutic targets in recurrent glioma.Support or Funding InformationThis project was supported by grants from MOST (MOST 103‐2320‐B‐038‐046‐MY3, 105‐2911‐I‐038‐503 and 105‐2320‐B‐038‐062) provided by Ministry of Science and Technology in Taiwan.
Transcriptional Regulation by Mediator Kinases during Starvation or Proliferation
Authors: Rubin Jonathan D. , Dowell Robin D. , Taatjes Dylan J. 
Published: November 2023

The objective of this research is to better understand the role of Mediator kinase activity in transcriptional regulation. Mediator is a multi‐subunit protein complex necessary for regulated transcription of all RNA polymerase II (RNAPII) transcripts. Mediator kinases (CDK8 and CDK19) form 4 subunit modules which reversibly associate with Mediator affecting transcription in poorly understood ways. Overexpression of CDK8 has been shown to correlate with tumor aggressiveness and mortality in colorectal cancer patients and the kinase activity of CDK8 and CDK19 promotes proliferation of acute myeloid leukemia in cells and in vivo. The kinase activity associated with these enzymes and how this relates to transcriptional regulation is not well understood because specific inhibitors of Mediator kinase activity have only recently been discovered.The study of genome‐wide phenomena has been aided greatly by the development of next generation sequencing. A recent sequencing technique, global run‐on with sequencing (GRO‐Seq), measures genome‐wide nascent transcription and allows for the unprecedented characterization of general transcription machinery. Serum response facilitates the study of transcriptional regulation by coordinating cells and increasing transcription at known genes involved in serum response. We performed GRO‐Seq on cells during serum response with and without the specific Mediator kinase inhibitor Cortistatin A (CA).Preliminary results show that qualitatively, genes involved in serum response have increased GRO‐Seq read counts at initiation regions following Mediator kinase inhibition. RNAPII pausing as determined by a pausing index is significantly increased following CA treatment for a large number of genes. Gene ontology (GO) enrichment analysis revealed that these paused genes are associated with metabolic processes. Additionally, this dataset was analyzed using a newly developed technique which detects eRNAs from GRO‐Seq datasets and measures transcription factor (TF) motif enrichment at eRNA epicenters. We found that MNT was significantly more enriched at eRNAs following Mediator kinase inhibition with CA. MNT is a known antagonist of MYC, a major downstream effector of MAPK signaling, and is a key regulator of the MYC transcriptional network in vivo. These results suggest that Mediator kinase activity is involved in activating metabolic pathways during serum response potentially through the regulation of transcriptional pausing or elongation and TF activation.Support or Funding InformationNIH SCR Training Grant ‐ T32 GM08759
Haematological and miRNAs (let‐7g, miR‐21, miR‐141) expression modulation profile in serum samples of Human prostate cancer
Authors: JAMES AYORINDE BABATUNDE , FADAKA ADEWALE OLUWASEUN , MAGBAGBEOLA OLUBUNMI ABIOLA , OTURU ABAYOMI , KOLAWOLE OLUFEMI OMOWUNMI , OGUNJIMI ADEBISI , OSHODI TEMITOPE , HABEEBU MUHAMMED , ONAWOGA FATIMA OMODEINDE , AJOGBEJE EBENEZER OLALEYE 
Published: November 2023

MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. This study investigated the haematological and miRNA expression modulation profile in serum samples of human prostate cancer.Twenty‐two study participants with age range between 59 and 82yeaars were included in the study after informed consent. Blood samples of the participant were obtained and both haematological parameters and miRNA analysis (miR‐21, miR‐141 and let‐7g) were performed using Bc‐3200 auto‐haemalogical analyzer and RT‐PCR.The result shows that there was no correlation between the haematological profile and the modulation of miR‐21, miR‐141 and let‐7g. There was no significant difference in the haematological profile between the healthy control and prostate cancer (PCa) patients. However, miR‐21, miR‐141 and let‐7g (p<0.0001) expressions were significantly up regulated although, miR‐21 expression was more pronounced in the serum of the study participants relative to healthy control group.Analysis of miR‐21, miR‐141 and let‐7g might have clinical utility as a supplement to prostate specific antigen screening.
Cyclosporin A Alters Expression of Renal MicroRNAs: New Insights Into Calcineurin Inhibitor Nephrotoxicity
Authors: Bai Yun , King Clayton , Francis Cynthia , Gooch Jennifer 
Published: November 2023

BackgroundCalcineurin inhibitors are powerful immunosuppressants that revolutionized organ transplantation. However, non‐immune effects of the calcineurin inhibitor, cyclosporin A (CsA), have significantly hindered their use. Specifically, nephrotoxicity, which is associated with tubulointerstitial fibrosis, inflammation, and podocyte damage, affects up to half of all transplant patients. Calcineurin is involved in many aspects of kidney development and function. Therefore, mechanisms of CsA‐induced nephrotoxicity are complex and not yet fully understood. MicroRNAs (miRs) are short non‐coding RNAs that regulate protein‐coding RNA expression through post‐translational repression of target messenger RNAs (mRNAs). MicroRNA dysregulation is known to be involved in kidney diseases including fibrosis.ObjectiveThis study investigates the microRNA expression changes induced by cyclosporin A.Methods and ResultsWe compared the renal microRNA expression profiles between mice that received CsA (20mg/kg) or vehicle daily for 6 weeks. The results demonstrated that CsA induced significant changes in renal microRNA expression. We used combined criteria of False Discovery Rate (≤0.1), fold change (≥2) and median signal strength (≥50) and identified 76 expression level significantly changed microRNAs. This approach identified microRNAs previously linked to renal fibrosis including let‐7d, miR21, miR29, miR30, miR130, miR192, and miR200 as well as microRNAs that have not been reported to be related to nephrotoxicity or immunosuppression. Pathway analysis of microRNA/mRNA changes highlighted the Wnt, TGF‐β, mTOR, and VEGF pathways. The mRNA expression profiles were compared in the same samples. The change of mRNA and microRNA profiles showed close correlations. To validate that the observed microRNA and mRNA expression level changes in mice kidney tissue were directly related to CsA treatment, the expression of three microRNAs (miR21, miR186, and miR709) and three mRNAs (bmpr1a, smurf1 and smad 7) were compared in the HEK293 cell line. A similar trend of expression level change was induced by CsA treatment in all selected microRNAs and mRNAs in the in vitro cell model. These data provide a roadmap for future work to study the role of the known and novel candidate microRNAs in the mechanism of nephrotoxicity and future therapeutic potential.ConclusionsOur study profiled expression of microRNA and mRNA genes in mouse kidney after kidney fibrosis has been induced (the major sign of kidney toxicity) and compared the pairwise expression profiles. The results indicate that CsA induces an extensive change in both microRNA and mRNA gene expressions compared to control mice. In addition, the microRNA and mRNA profiling results show a close correlation.Support or Funding InformationNIH1R15M113120‐01
Non‐coding RNA Editing Involved in Adipose Dysfunction during Aging
Authors: Seidler Abbagael , Marcelo Aileen , Page Jamika , Santanam Nalini 
Published: November 2023

Adipose tissue is one of the key endocrine organs that maintain lipid and glucose homeostasis. Adipose dysfunction increases upon aging, and results in dramatic alterations in fat mass, distribution and function. This leads to age associated metabolic dysfunction and increased risk to cardiometabolic diseases. Continuous replenishment of mature adipocytes by differentiation of pre‐adipocytes is a must for normal adipose function. We recently showed decrease in adipogenesis during aging was due to dysfunction in microRNA's (miRNA) involved in adipogenesis (miR‐143). This dysfunction may be attributed to changes in enzymes involved in biogenesis of miRNA (DROSHA, DICER) or enzymes involved in modifying its function (ADAR). We determined the differences in the expression levels of these enzymes in Fisher 344 x Brown Norway hybrid rats (FBN) at ages 8 mo and 25 mo old (n=3). There were sex specific differences in the levels of both biogenesis and editing genes in peri‐gonadal adipose tissue from FBN rats with aging. The DROSHA and DICER levels were higher but editing enzymes (ADARs) lower in female rats and the reverse in male rats with aging. Sanger sequencing of miR‐143 (a key miRNA in adipogenesis) showed edited sequence in old rats compared to younger rats. Sex‐specific post‐transcriptional regulation of miRNA(s) involved in adipogenesis during aging in whole adipose tissue will also be explored in pre‐adipocytes and mature adipocytes from aging rats and correlated to metabolic changes leading to insulin resistance.Support or Funding InformationSupported by NIH‐NIA‐R15 AG051062 (NS); Marshall University Biomedical Sciences Graduate Program, the Marshall University Foundation Summer Research Internship for Minority Students Fund, and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence
“Listening in” ‐ The cross‐talk between mother and infant through exosomal microRNAs in breast milk
Authors: Kraft Jamie Dale , Altosaar Illimar 
Published: November 2023

Breast milk is an intricate source of key nutritional complexes required to nourish an infant as well as facilitate the development of the infant's immune system. During the postnatal period, the gastrointestinal tract (GIT) matures following enteral feeds and colonization with commensal bacteria. The development of the GIT could be delayed or altered in a premature infant, thus predisposing the infant to inflammatory diseases such as necrotizing enterocolitis (NEC). NEC is the leading cause of death among premature infants. The pathophysiology is currently unknown, however, prematurity, formula feeding and an abnormal intestinal microbiota are thought to cause an inflammatory response in the intestine which can lead to NEC and sepsis. Breast milk is the gold standard of infant nutrition and is largely thought to help prevent NEC.The components of breast milk responsible for the development of the GIT have yet to be identified, although, novel research has discovered the presence of microRNA (miRNA) in breast milk exosomes. Due to stable structure, exosomes are protected against degradation, suggesting breast milk exosomal miRNAs continue to be biologically active following ingestion by the infant. miRNAs have previously been described as functioning in cellular communication, implying a method of “listening in” to the cross‐talk between mother and infant via breastfeeding.To characterize miRNAs and identify the role they play in the development of the GIT, exosomes from breast milk must be isolated in a reproducible method to extract miRNAs. Isolating and storing procedures of unprocessed breast milk are underexposed. In recent studies, it has been revealed that storing unprocessed breast milk at −80°C or −4°C lead to cell death within the milk, causing contamination of the native breast milk exosomes. The profiling of miRNA fluctuates depending on the milk fraction analyzed, preparation of milk and exosome fraction as well as the miRNA extraction protocol. Extra care should be taken when preparing samples and following extraction procedures in this area of research. During this early stage of exploring the ways in which the mother communicates with her newborn, it is necessary to collaborate with other research groups to standardize procedures and to synchronize approaches.
Positive Charge in the n‐region of the Signal Peptide Contributes to Efficient Post‐translational Translocation of Small Secretory Proteins
Authors: Liu Ming , Sun Jinhong , Xiong Yi , Cui Jingqiu , Guo Huan 
Published: November 2023

A majority of secretory proteins in mammalian cells undergo signal recognition particle (SRP) dependent co‐translationally translocation across the endoplasmic reticulum (ER). However, due to minimal time for SRP recognition, many small secretory proteins can be post‐translationally tanslocated into the ER. Several chaperones and cellular machinery have been identified to be involved in post‐translational translocation of these small secretory proteins. However, the intrinsic signals of small secretory proteins that contribute to efficient post‐translational translocation remain unknown. Here, we analyzed signal sequences of eukaryotic secretory proteins and found that the percentage of the proteins with positive charge in the n‐region of their signal peptide (SP) increases substantially as the polypeptides become shorter. Eliminating the n‐region positive charge selectively impaired translocation of small secretory proteins. This translocation impairment appeared to be mainly caused by the defect of post‐translational translocation. Slowing down protein translational rate of small secretory proteins could alleviate their translocation defects caused by loss of SP N‐terminal positive charge. Together, these data revealed a previously unrecognized role of n‐region positive charge in ensuring efficiency of post‐translational translocation of small secretory proteins.Support or Funding InformationThis work was supported by NIH RO1‐DK088856, and by the research grants from The National Natural Science Foundation of China 81070629, 81370895, 81570699.
In vivo and in vitro studies of RRF (ribosome recycling factor) revealed that its major function is to release mRNA from the post‐termination complex and not splitting of the ribosomal subunits
Authors: Quaglia Fabio , Kaji Hideko , Kaji Akira , Inokuchi Yoshio 
Published: November 2023

In prokaryotes two adjacent ORF are often linked with an overlapping combination of termination and initiation codons with a total of 4 or 5 nucleotides, for example UAAUG or AUGA. In these junctions, ribosomes at the stop codon are released by RRF and some of them re‐bind to the nearby AUG and start translating the downstream ORF. In the absence of RRF, the ribosome at the stop codon remains on the mRNA and would read it in frame with the termination triplet. We studied the role of RRF in these junctions in vitro and in vivo. For in vivo studies we used an E. coli strain with a temperature sensitive mutation of RRF so that we were able to inactivate the function of RRF at the non‐permissive temperature (39°C). We show that for correct reading of the downstream ORF, AUG is essential. The shorter the upstream ORF the lower the downstream reading. Introduction of a complementary sequence to the 3′‐terminal region of 16S rRNA into the mRNA increased downstream reading from AUGA. Shortening of the upstream ORF to 4 codons completely abolished the downstream reading of UAAUG. This suggests that if Shine‐Dalgarno (SD) sequence is near the termination codon, the RRF‐released ribosome is attracted by the SD sequence to the extent that it loses the downstream movement after it is released.For in vitro studies we used the PURE system so that we were able to omit RRF in the reaction mixture. We have confirmed, in vitro, the essence of the in vivo observation described above using a mRNA having the following sequence: “GGGAAUUCAAAAAUUUAAACAGGUAUACAUACU AUG UUU ACG AUU ACU ACG AUC UUC UUU ACG AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG UAAUG CGU CUG CAG GCA UGC AAG CUA A24A” (Bold character is the junction sequence broken underline is the Shine‐Dalgarno sequence). In the presence of RRF, the downstream reading starts from AUG causing the incorporation of [14C]‐Leucine (CUG and CUA). On the other hand in the absence of RRF, the first triplet read was UGC of UAAUGCGUC and [14C]‐Valine was incorporated due to the codon GUC. Upstream reading was detected by [3H]‐phenylalanine incorporation (due to UUUs and UUCs). With this assay we also showed that Fusidic Acid was able to inhibit RRF at lower concentration than that necessary to inhibit translocation (monitored by the incorporation of [3H]‐phenylalanine), causing the inhibition of the incorporation of [14C]‐Leucine. Moreover, using ribosome with tethered subunits (1), we were able to show that the splitting of the ribosomal subunits was not necessary in the recycling reaction, demonstrating that recycling of the ribosome take place also in the absence of the splitting of the ribosomal subunits.
Deconstructing the Peptide Specificity of TCR Recognition
Authors: Riley Timothy P , Mendoza Juan , Hellman Lance , Garcia KC , Baker Brian 
Published: November 2023

T cell receptors (TCRs) have emerged as a new class of therapeutics, most prominently for the treatment of cancer where they are key components of new cellular therapies as well as soluble biologics. Although in many ways similar to antibodies, TCRs differ in the complexity of the receptor‐ligand interface: whereas antibodies can be elicited to almost any antigens, TCRs are restricted to linear peptides presented by class I or class II MHC proteins. Additionally, TCRs do not undergo affinity maturation, and bind with weak‐to‐moderate affinities and reduced specificity.The off target and organ‐specific toxicities observed in cancer immunotherapy present an obstacle to T cell based therapeutics. A recent clinical trial underscored the need for improved methods to define TCR specificity after melanoma patients treated with TCR engineered T cells suffered from fatal cardiovascular toxicity arising from the unpredicted recognition of a muscle specific peptide. To address this drawback to T cell based immunotherapies, we developed a novel protein engineering approach to define the peptide specificity of a given TCR. Here, a large scale peptide library underwent directed evolution in a yeast display system, where interactions with the melanoma reactive DMF5 TCR acted as the guiding selection pressure. After this technique identified a panel of putative cross reactive peptides, sequence analysis and computational modeling followed by kinetic binding experiments and X‐ray crystallography determined the DMF5 TCR recognizes two distinct classes of peptides through chemically distinct mechanisms. This information led to the rational, structure based design of additional cross reactive peptides and introduces a unique approach to screen the human proteome for TCR targets to avoid unanticipated autoimmune reactions in response to therapeutic TCRs.Support or Funding InformationThis project was supported, in part, with support from the Indiana Clinical and Translational Sciences Institute funded, in part by Grant Number UL1TR001108 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Determining histone deacetylase 8 substrates using non‐natural amino acids
Authors: Lopez Jeffrey , Haynes Sarah , Majmudar Jaimeen , Martin Brent , Fierke Carol 
Published: November 2023

The histone deacetylase family is comprised of 18 enzymes that catalyze the removal of an acetyl group from the ɛ‐position of a lysine residue. Lysine deacetylation is a post translational modification that affects over 6000 different macromolecules and has been implicated in various types of cancers and neurodegenerative diseases. To elucidate and identify HDAC substrates, we have incorporated the photo reactive, non‐natural amino acid – p‐benzonyl‐4‐phenylalanine (pBpa) into HDAC8, a structurally well characterized isozyme of this family with a largely unknown substrate pool. Using both photo crosslinking and proteomics will allow us to identify both substrates and binding partners of HDAC8 and enhance the understanding of how this enzyme is regulated in vivo. In addition, this will validate the approach as a feasible system to probe other HDAC isozymes and their specificities.Support or Funding Information5‐R01‐GM‐040602 C.A.F.; F31‐GM‐116619 J.E.L.
Characterization of ppGpp‐Binding Proteins in Escherichia coli
Authors: Nguyen Duc Tien , Jung Che Hun 
Published: November 2023

Guanosine‐5′‐diphosphate 3′‐diphosphate (ppGpp) is a bacterial signal molecule that generates stringent responses under conditions of deprivation and amino acid deficiency. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of ppGpp metabolism and ppGpp‐mediated regulation. Although many studies focus on studying direct and indirect targets of ppGpp regulation, the overall mechanism of ppGpp is still not clear. In this study, ppGpp was immobilized on NHS‐activated Sepharose. ppGpp binding proteins were isolated by affinity chromatography, and further separated by 2‐D gel electrophoresis. The resolved proteins were identified by MALDI‐TOF MS. The tryptic hydrolysates were also analysed by LC‐MS/MS. Among the identified proteins, some proteins of interest were cloned and over‐expressed. The cloned proteins (pepB, iadA, map, dld, ldh, gldA, lon, ycbZ, pyrD, lpdA) were tested for their binding to ppGpp by fluorescence spectroscopy and were indentified as ppGpp‐binding proteins with various Kds. Interestingly, we found ppGpp inhibit membrane D‐lactate dehydrogenase non‐competitively with Ki 48 mM and fermentative D‐lactate dehydrogenase competitively with Ki 120 mM. These results provide that this method will be very effective for screening proteins which new plausible ppGpp regulated proteins. And further studies on these proteins will expand the scope for the functions of ppGpp.Support or Funding InformationBK21 PLUS (Brain Korea 21 Program for Leading Universities & Students), National Research Foundation (NRF) of Korea