bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Topic‐based Approach for Teaching Metabolism in a Flipped Classroom
Authors: Johanson Kelly E 
Published: November 2023

The biochemistry curriculum at Xavier University of Louisiana is split into three junior/senior level courses with one course focusing entirely on the study of biochemical energetics and the many biochemical pathways of carbohydrate, lipid, protein, and nucleic acid metabolism. The emphasis in this course is the integration and regulation of pathways based on the fuel state and energy demands of the organism. In previous years the course was taught as a traditional lecture with pathways covered in the order they appeared in the textbook. Despite different efforts to strengthen critical‐thinking skills, students continually exhibited difficulty with understanding the connections between pathways and struggled with answering discussion questions that required knowledge of basic content and conceptual understanding of pathway integration and regulation. In order to better emphasize the integration of the metabolic pathways the course material was split into four modules, each focusing on a specific fuel state and energy demand. Several pathways were represented in more than one module, allowing for reinforcement of critical reactions and regulation steps. To increase the effectiveness of content delivery, the module material was presented as a series of daily questions that were answered through small group discussion rather than traditional lecture. This flipped style was designed to allow for greater time in class to discuss difficult concepts and to allow students to make connections with support from the instructor. Student responses to to pre‐class quizzes and follow‐up questions after each class allowed for assessment of the effectiveness of the flipped classroom and of the topic‐based approach. Presenting the new format in a flipped fashion has been very successful in increasing student comprehension and retention of complex material. Student performance on discussion questions that require critical thinking and analysis of the pathways increased significantly in the two semesters of this new format as compared to previous years. Students also reported a greater understanding of the connections between pathways and of pathway regulation. Overall, the flipped classroom style is preferred by students and, combined with the content reorganization, has proved to be an effective way of teaching metabolismSupport or Funding InformationThis work was supported by a grant from the Andrew Mellon Foundation
Design of a robust undergraduate biochemistry laboratory course based on a modified and expanded bovine serum albumin purification scheme
Authors: Odunuga Tayo , Cheatwood Nicholas , Mullins John , Harris Michele 
Published: November 2023

The ASBMB curriculum for an undergraduate degree recommends a set of skills that can be acquired only through laboratory courses and research experience. Based on a modified and expanded purification scheme for BSA, we designed a robust, reproducible, budget‐friendly, safe and inquiry‐based undergraduate biochemistry laboratory course that encompasses a lot of the skill‐sets recommended in the ASBMB curriculum. The purification scheme employed in this work has been reported in a research paper. This work not only modifies certain steps in the scheme, it includes additional steps to enhance student learning and skill acquisition. Salt precipitation, ion exchange and size exclusion chromatography were employed by students to purify BSA from cow plasma. Presence of major contaminants of BSA purification, IgGs and nucleases, were tested in the purified sample by western blotting and DNase I assay respectively. The DNase assay step provides an opportunity for students to learn basic molecular biology techniques such as plasmid isolation and restriction digestion. One major addition to the purification process is the bromocresol green‐BSA complex formation assay to precisely quantitate BSA at each step and generate a purification table. Comparison of sequences and other parameters of albumin proteins from common animals provides a bioinformatics twist to student experience. Abundance of albumin from the plasmas of common animals, cow, pig and chicken, allows for variation in the design of the course, and students can work in groups or individually. The course could be designed as a half‐semester or full‐semester biochemistry laboratory module, and a sample schedule is provided.Support or Funding InformationWelch Foundation Grant # AN‐0008
A Novel Reductase‐Independent Role of Nuclear Ribonucleotide Reductase
Authors: Aye Yimon 
Published: November 2023

Since the origins of DNA‐based life, the enzyme ribonucleotide reductase (RNR) has spurred proliferation because of its rate‐limiting role in de novo deoxynucleoside‐triphosphate (dNTP) biosynthesis. Unsurprisingly RNR is overexpressed in cancer cells, rendering RNR a major chemotherapeutic target. Paradoxically, the large subunit, RNR‐a, of this obligatory two‐component complex plays a stage‐/context‐specific tumor‐suppressive role. Little explanation has been offered to resolve this dichotomy. Here, we show that nuclear RNR‐a has an unappreciated DNA‐replication‐inhibition function, leading to growth retardation and ultimately suppression of focus formation. A large‐scale protein‐interaction study uncovered that nuclear RNR‐α is an inhibitor of a novel nuclear protein which we identify as a promoter of DNA‐synthesis. This non‐reductase‐role of RNR‐α is regulated by exclusion from the nucleus, and promoted by nucleotide‐induced RNR‐a‐oligomerization that elicits rapid nuclear import. The newly‐discovered nuclear signaling axis acts as a primary defense against elevated/imbalanced dNTP‐pools that can exert mutagenic effects irrespective of the cell cycle. (Fu and Long et al ‐ Under Peer Review‐2016 Nat Commun).Support or Funding InformationCornell University and Weill Medicine seed grants and Cornell University start‐up funds (for Nucleotide Signaling research program in the Aye lab), and NSF CAREER, NIH Director's New Innovator, Beckman Young Investigator, and Sloan Fellowship programs (for Redox Signaling research program in the Aye lab).
Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD
Authors: Murison David Andrew , Timson Rebecca , Beuning Penny 
Published: November 2023

The SOS response, an induced DNA damage response pathway in Escherichia coli, is temporally separated into two stages. The initial stage affords an opportunity for the cell to utilize accurate repair mechanisms such as homologous recombination and nucleotide excision repair. During the later stage, cells employ a potentially mutagenic pathway termed translesion synthesis (TLS) to tolerate damage and promote survival. The umuD gene products are upregulated during the SOS response and play a role in both early and late stages. Full‐length UmuD is expressed as a homodimer of 139‐amino‐acid subunits which eventually cleave their N‐terminal 24 amino acids to form UmuD′. The cleavage product, UmuD′, together with UmuC, form the Y‐family polymerase DNA Pol V (UmuD′2C) capable of performing TLS. UmuD and UmuD′ exist as homodimers, but their subunits can readily exchange to form UmuDD′ heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway for the pro‐mutagenic UmuD′. Full‐length UmuD is targeted for degradation by the ClpXP protease due to a recognition sequence located on the N‐terminal arm of UmuD. Formation of UmuD′ via cleavage removes this recognition sequence, making UmuD′ invisible to ClpX. Thus, UmuD in the context of heterodimer acts as a delivery factor for UmuD′. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange for a number of single‐cysteine UmuD variants. By conjugating fluorescent molecules at different positions on the protein we were able to observe their effect on exchange kinetics. Interestingly, the most dramatic differential effects on exchange were observed at two adjacent positions in the peptide backbone. These findings provide insight into the molecular surfaces responsible for mediating exchange.Support or Funding InformationSupported by NSF CAREER MCB‐0845033 and American Cancer Society RSG‐12‐161‐01‐DMC.
Chemical Tools to Study the Molecular Mechanisms of the CoREST Complex‐Chromatin Interactions
Authors: Wu Mingxuan , Hayward Dawn , Kalin Jay H , Song Yun , Schwabe John W.R. , Cole Philip A 
Published: November 2023

Chromatin remodeling and histone modifications are important for regulating gene activation or silencing in physiologic and disease processes. Several cancers exhibit a strong dependency on histone deacetylases (HDACs) and lysine specific demethylase 1 (LSD1) activities and this has prompted aggressive efforts in academic and industrial research labs to deepen and broaden our understanding of these enzymes. The CoREST complex which can deacetylate and demethylate chromatin but the interplay between subunits of this complex and their interactions with nucleosomes is largely unknown. We generated histone H3 containing post‐translational modifications (PTMs) using engineered sortase that ligates H3 N‐tail depsipeptides and globular histone H3 with excellent conversions. Following octamer refolding and nucleosome reconstitution, we made a groups of nucleosome substrates containing H3K4‐dimethyl and assorted H3 acetyl‐Lys modifications. Using a purified core CoREST complex, we explored its enzymatic features in processing modified H3 N‐tail peptides and reconstituted nucleosomes. Our experimental findings and their significance will be discussed.Support or Funding InformationR01 GM062437
Understanding the role of the histone demethylase LID in the SIN3 histone modifying complex in Drosophila melanogaster
Authors: Chaubal Ashlesha , Pile Lori 
Published: November 2023

SIN3 is a master transcriptional regulator, conserved from yeast to mammals, that acts as a scaffold protein for a histone modifying complex. Though the SIN3 complex is canonically known to be a histone deacetylase complex, research from multiple groups using distinct model systems demonstrates that SIN3 can associate with both a histone deacetylase (HDAC) and a histone demethylase (HDM). In Drosophila, a single Sin3A gene is alternatively spliced to produce distinct SIN3 isoforms; SIN3 220, SIN3 190 and SIN3 187, that differ only at their C‐terminus. These isoforms are differentially expressed during development and form distinct SIN3 complexes. Interestingly, previous work in our lab has shown that SIN3 187 interacts with a single catalytic enzyme, the HDAC RPD3, while SIN3 220 interacts with two enzymes, RPD3 and the HDM dKDM5/LID. This differential interaction of SIN3 isoforms with distinct histone modifying activities gives us a unique opportunity to interrogate the role played by LID in the SIN3 complex. Using previously published transcriptome data, we have identified common and unique gene targets of SIN3 and LID. In Drosophila S2 cells, knockdown of LID results in an increase in the level of H3K9ac, H3K14ac and H3K27ac at genes commonly regulated by SIN3 and LID. Additionally, a reduction in SIN3 binding is observed at these genes upon loss of LID. We are currently investigating whether LID knockdown also affects the recruitment of RPD3 at common and unique gene targets. Furthermore, we are interested in determining whether presence of LID has an effect on the HDAC activity of the complex. We have established Drosophila cultured cells which express either the SIN3 187 or the SIN3 220 complex. Since LID preferentially interacts with the SIN3 220 complex we want to determine how loss of LID affects the histone modification patterns established by the two SIN3 complexes. These studies will help us understand the interplay between two distinct histone modifying activities and provide further insight into the molecular mechanisms of epigenetic control of gene expression by histone modifying complexes.Support or Funding InformationThis work is supported by a National Institutes of Health grant (1R01GM088886‐01A2) to Dr. Lori A. Pile
Biochemical Analysis of the Zinc Uptake Regulator (Zur) from Klebsiella oxytoca
Authors: Khacheryan Levon , Xie Yujie , Gallas Genna , Hernandez Jose 
Published: November 2023

Zinc metabolism is critical to maintain bacterial pathogenesis in their respective hosts. Research has shown that altering the zinc homeostasis of bacteria via mutation of genes critical for regulation, export or uptake functions, such as Zur, ZntR, ZntA or ZnuABC, induces metal toxicity (excessive increase in a metal concentration) or insufficient intracellular zinc concentration. Prolonged disruption of intracellular zinc ion concentration homeostasis leads to attenuated bacteria and ultimately, cell death.The Zinc uptake regulator (Zur) protein has been shown to act as a master regulator of gene transcription of proteins associated with zinc uptake transporters such as ZnuABC. A particular intracellular zinc concentration is necessary in bacteria for proper structure and function of essential biological enzymes. When zinc levels within the cell are sufficiently high, Zn2+ binds with Zur inducing a conformational change, which allows the Zn2+‐Zur complex to bind to the promoter region of its target DNA. This promoter region is upstream of the gene for the uptake transporter (ZnuABC); thus, causing repression of transcription of genes necessary for zinc acquisition. When zinc levels drop, the Zn2+ is released from Zur; thus reducing Zur's affinity for the DNA and normal transcription activity of ZnuABC ensues.In our research, we aimed to identify conserved amino acids that are essential for Zur functioning in Klebsiella oxytoca. We performed site‐directed mutagenesis and used an in silico analysis to identify a target mutation in the protein, which resulted in the H159A mutant. The effects of the mutation were studied in vivo with β‐galactosidase assays, which showed no effect of H159A mutation on the Zur protein function.Metal and oxidative stress viability effects were tested on a strain overproducing the Zur protein. Zur overexpression resulted in increased sensitivity to oxidative stress with H2O2 and Zn2+ compared to wild type due to increased cell inhibition with increasing concentrations of each condition. On the other hand, there was no increase in sensitivity with Zur overexpression for varying concentrations of Fe2+, Mn2+, and methyl viologen exposure.Finally, the effects of zinc availability on Zur and Superoxide Dismutase expression were also evaluated. Three isoforms of superoxide dismutase (MnSOD, Cu/ZnSOD and FeSOD) were expressed in K. oxytoca, which were grown in rich‐medium. In‐gel activity assays indicate cells overexpressing Zur lower Cu/ZnSOD and increase MnSOD activity compared to wild type. These results suggest Zur plays a crucial role in altering SOD activity in various isoforms to ultimately, inhibit oxidative stress and prolong cell survival.Overall, our results identify the presence of active Zur and three isoforms of SOD in K. oxytoca, and prove the relevance of Zur in the sensitivity to oxidative stress.Support or Funding InformationORSP Summer fellowship. Arizona College of Osteopathic Medicine. Midwestern University.
Smad4‐Dependent TGF‐β Signaling Directly Up‐Regulates Notch Receptor in Cerebrovascular Endothelial Cells
Authors: Lan Yu , Cheng Xuan 
Published: November 2023

ObjectiveIn this work we will find out whether Notch receptors serve as direct downstream targets of TGF‐β/Smad4 signaling in endothelial cells.MethodsRealtime‐PCR and Western Blot were performed to verify whether the expression of Notch1 and Notch4 were regulated by TGF‐β pathway. Luciferase reporter assay was employed to investigate how promoter of Notch1 and Notch4 been regulated by TGF‐β. Then ChIP assay will confirm whether promoter of Notch1 and Notch4 physically interact with Smad protein.ResultsTGF‐β1 and bone morphogenetic protein 4 (BMP4) treatments increased the expression of both Notch1 and Notch4 at transcriptional level. In addition, Smad4 physically associates with the Smad binding sites on the Notch4 promoter, which is largely enhanced under the treatment of TGF‐β1 and BMP4. Importantly, TGF‐β1 and BMP4 fail to transactivate Notch4 in the absence of endogenous Smad4 or the Smad binding regions on the Notch4 promoter.ConclusionFor the first time, the expression of Notch receptor could be directly up‐regulated by Smad4‐mediated TGF‐β/BMP signaling in cerebrovascular endothelial cells.
Zinc‐dependent Transcriptional Regulation in Paracoccus denitrificans
Authors: Neupane Durga P 
Published: November 2023

Zinc is an essential trace element required for every organism but it is toxic when present in high concentrations. Therefore, maintaining zinc homeostasis within a narrow range is a critical process for the growth and survival of bacteria that is also essential for bacterial virulence. Zinc homeostasis in bacteria is maintained in response to diverse environmental Zn concentration by a set of different importers and exporters. Importers include high‐affinity Zn binding ABC transporters of cluster A‐I family, which are under the control of the zinc uptake regulator (Zur). Zur is a transcription factor that binds promoter elements upstream of these genes only in the zinc‐bound form, thereby mediating zinc‐dependent repression. Here we have employed RNA‐Seq to identify genes differentially expressed under conditions of zinc limitation and study if these genes are regulated by Zur in Paracoccus denitrificans. The results showed the differential expression of 147 genes, of which 133 genes were up‐regulated under conditions of zinc limitation while only 14 were significantly down‐regulated. Up‐regulated genes included several transcription factors including zur, transition metal importers including Zn importers, metabolic genes and other hypothetical proteins. Intriguingly, we have also observed very high expression of components of denitrification such as nitric oxide reductase (norCB) and nitrite reductase (nirS). These were found to be directly regulated by Zur thereby showing the link between denitrification and zinc homeostasis in P. denitrificans.Support or Funding InformationNCGR and INBRE Funding ‐ 8P20GM103451‐12NIH SC2 GM111170‐01
The Neuroprotective role of miR‐1017, a 3′ Tailed Mirtron
Authors: de Cruz Matthew , Flynt Alex 
Published: November 2023

microRNAs (miRNAs) regulate the expression of most animal mRNAs. Due to the pervasive activity of these genes many roles have been identified in development and physiology, however many remain uncharacterized, especially non‐canonical family members. Here we identify a function of a 3′ tailed mirtron, miR‐1017. miR‐1017 is encoded within an intron of an Acetylcholine receptor, nAChRα2. Visualizing GFP driven by nAChRα2 transcriptional enhancer sequences showed the miR‐1017 host transcript is present in adult gustatory organs and the associated region of adult brains, specifically the suboesophageal ganglion. Consistent with a role in neurobiology, many miR‐1017 predicted targets have roles in neurons. Using mir‐1017 knock out (KO) flies, we found derepression of two targets: nAChRα5 and its host transcript nAChRα2. GFP driven by nAChRα2 in the mir‐1017 KO showed a broader expression pattern, suggesting a role in a negative feedback loop that modulates sensitivity of neurons to Acetylcholine (Ach). Numerous neurodegenerative diseases including Alzheimer's disease (AD) have shown that increased receptor activity causes reactive oxygen species to develop, which leads to neurodegeneration. Indeed, miR‐1017 KO presents reduced lifespan, poor climbing ability, and an increase in Cleaved Caspase staining in the CNS. miR‐1017 may function to limit Ach activity and protect from excitotoxic‐induced neurodegeneration. Interestingly, ectopic expression of miR‐1017 can rescue lifespan and neurological function in a fly AD model. This research demonstrates the importance of miRNA‐mediated gene regulation, even when enacted by non‐conventional species.Support or Funding InformationNIH R15
Functional Characterization of Diabetes‐Induced long non‐coding RNA Dnm3os in macrophages
Authors: Das Sadhan C , Reddy Marpadga A , Senapati Parijat , Wang Mei , Lanting Linda , Oh Hyung , Devaraj Sridevi , Natarajan Rama 
Published: November 2023

Macrophages play key roles in pathophysiological processes such as inflammation, pathogen response, and tissue repair. Diabetic complications are associated with enhanced macrophage activation and inflammation. Long noncoding RNAs (lncRNAs) regulate gene expression in diverse biological contexts via multiple mechanisms ranging from modulation of chromatin structure and function to post‐transcriptional mechanisms such as mRNA stability. Dysregulation of lncRNA function has been implicated in several diseases, but their role in diabetes complications is unclear. Using high throughput RNA‐sequencing, we recently profiled the transcriptome alterations including lncRNAs in bone marrow derived macrophages from type 2 diabetic (T2D) db/db mice compared to control db/+ mice. In these studies we demonstrated function of a lncRNA in promoting macrophage proinflammatory phenotype. Since the role of other lncRNAs in diabetes complications is largely unknown, we further characterized the function of lncRNA Dynamin 3 opposite strand (Dnm3os), which was upregulated in macrophages from db/db mice. Interestingly, Dnm3os was also increased in high fat diet‐induced insulin‐resistant T2D mice, as well as in monocytes from human T2D patients. Dnm3os was also induced in mouse macrophages treated with diabetogenic agent palmitic acid and proinflammatory agent LPS. RNA‐seq analysis in mouse macrophages stably overexpressing Dnm3os revealed that 167 genes were upregulated and 84 were downregulated compared with control macrophages. DAVID analysis revealed that Dnm3os regulated coding genes were associated with phagocytosis, inflammation and immune response. In contrast, siRNA mediated knockdown of Dnm3os attenuated expression of genes involved in inflammation, immune response and cell activation. RNA‐FISH and RT‐qPCR analysis of subcellular fractions showed that Dnm3os is predominantly localized in the nucleus. Overexpression of Dnm3os altered global histone modification marks in macrophages indicating its role in transcription. In order to determine the molecular mechanisms involved in Dnm3os functions, we performed in vitro RNA pull down assays using biotinylated Dnm3os transcripts combined with mass spectrometry in macrophage nuclear extracts. Results identified that Dnm3os interacts with Nucleolin, ILF2, ARP3 and other RNA binding proteins. RNA‐FISH coupled with Immunofluorescence analyses further confirmed the co‐localization of Dnm3os partially colocalized with nucleolin and ILF2 in the nuclei of macrophages. Collectively, these results demonstrate that Dnm3os has novel functions in diabetic macrophages to promote cellular dysfunction and activation via nuclear mechanisms involving interactions with nucleolin and ILF2. These findings could lead to lncRNA‐based therapies for inflammatory diabetes complications.Support or Funding InformationNIH, NHLBI, NIDDK