bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroanatomical variants of clinical importance in the population of Ethiopia
Authors: Wirtu Amenu T , Geneti Soressa A , Georgis Solomon T Haile , Gemechu Jickssa M
Published: November 2023

The causes of neuroanatomical variations are yet unknown despite understanding their presence will have significant impact in improving medicolegal issues, diagnostics, imaging and therapeutics in the future. Neuroanatomical variations in the branching patterns of the brachial plexus and sciatic nerve have been reported in several populations but rarely in Ethiopia. After an ethical approval by the institutions involved, twenty‐four bodies were examined in both sexes in accordance with the guidelines of Grant's dissection principle and Cunningham's manual of practical anatomy in sampled governmental and private Schools of Medicine in Ethiopia. Critical observation, careful dissection and imaging were done accordingly. We found anteriorly communicating fibers of musculocutaneous and median nerve in (6.3%), posteriorly communicating fibers of ulnar with radial nerve in (2.1%) and higher pelvic division of anterior and posterior fibers of sciatic nerve in (6.3%) of Ethiopians. Identification of these variants will help the neurologists to make a proper diagnosis of sensorimotor symptoms and of considerable significance to surgeons and orthopaedicians when dealing with patients of neural entrapment syndromes, posttraumatic evaluations and exploratory interventions. Altogether, the findings show the existence of neuroanatomical variants of clinical importance that needs special consideration in the treatment and diagnosis of this diversified population of diverse genetic and geographical origins, which also was assumed as the cradle of mankind.
Decreases in relative cerebellum volume are correlated with digit reduction and limb loss in squamates
Authors: Heesy Christopher Paul , Hall Margaret I , Ruiz Saul A , Wells Nora
Published: November 2023

Squamates (lizards and snakes) are a broadly diverse radiation ideal for the study of evolutionary questions, especially the underlying biological mechanism that relates brain to body size in vertebrates. Relatively large brain size has evolved multiple times among distantly related lizard groups. Importantly, smaller brain size has been suggested in elongate, and/or limbless vertebrates, including snakes and some clades of lizards. In this study, we examine the relationship between digit and limb loss on brain: body size scaling in lepidosaurs. We combine novel data on brain and body mass from squamates collected from the American Southwest as well as from the literature and also collate data on digit and limb number. Employing modern phylogenetic comparative methods, our analyses are consistent with the hypothesis that relative brain size decreases with digit and limb loss, and the relationship between brain: body size ratio and limb loss has evolved multiple times within lizards as well as in snakes. We find that relative cerebellum volume is the major brain component that is reduced in these taxa. Our interpretation is that relative brain size reductions in squamates with limb loss as reflecting lower innervation demands by body wall structures.
Using collaborative problem sets to facilitate learning in the online thermodynamics classroom
Authors: Dean Diane
Published: November 2023

Teaching thermodynamics online requires creativity and careful consideration of the psychology of learning. We have a required thermodynamics course in our online biochemistry and chemistry master's program. For many years we experienced poor performance from students on the midterm exam. The average was around 74%, a failing grade in a graduate level course. For two semesters, I implemented a collaborative problem set in the entropy lesson material. This resulted in a very small improvement to 76% as an average on the midterm exam. Although I did not interpret this to mean the change had significantly improved the instruction, students commented that the collaboration was very helpful. Hence, during the most recent semester I implemented collaborative problem sets throughout the course. The material has been designed into 4 separate 2.5 week lessons and each lesson has collaborative problem set. This has resulted in an improved performance on the midterm to a 90% average. The students are required to answer the problem set individually and share that attempt with a group of two to three additional students. The initial attempt is a starting point for the group's work. Many students can be motivated by a sense of community. The students feel empowered to support other students learning and attempt to produce high quality solutions thereby insuring the group's success. The students work together on private group discussion board to compare and analyze each student's attempt of the problems. The critical analysis encourages students to recognize their own mistakes and explain the mistakes of others. The group then produces a group solution which is graded for correctness. Finally, each individual student does a second individual problem set on the same concepts completing the unit's assessments. All of our online courses included collaborative discussion; however, these discussions were difficult in thermodynamics because of the problem based nature of the course. Using a large whole class discussion did not foster a sense of community. The isolation of the students resulted in a lack of in‐depth analysis of the solutions to the problems. Requiring students to determine a consensus answer has changed this dynamic creating a group for which each student felt responsible and increased the complexity with which the students consider different solutions thus improving the overall performance in the course.
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 metabolism

Support or Funding Information

This work was supported by a grant from the Andrew Mellon Foundation
Measurement of BCAA in Milks and Supplements With an Enzyme Assay: Confirmation of Results with HPLC
Authors: Keenan Kelly , Do Daniel , Ngo Kathleen
Published: November 2023

Branched chain amino acids (BCAA) are comprised of three types of amino acids (valine, leucine, and isoleucine) and are a common type of supplement or a key ingredient in various types of protein supplements. Given their popularity, a laboratory experiment was devised to measure the level of BCAA in various types of milks or supplements. To make this experiment accessible to many, an enzyme assay was adapted to measure the level of BCAAs. It is expected that this project can be done in advanced high school Biology laboratories or college Biochemistry laboratories. The samples are hydrolyzed to break all bonds releasing the free amino acids and the BCAAs are measured using an enzyme assay that detects only these amino acids. In order to validate this method, the samples were also measured for BCAA using reverse phase‐high performance liquid chromatography (RP‐HPLC). It was demonstrated that all three BCAA survived the hydrolysis process with high recoveries. When these amino acids were added to samples, the recoveries were also very high. The results from RP‐HPLC confirm the results from the enzyme assay and demonstrate that this method makes for a viable, accurate and easily accessible laboratory experiment.
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 Information

Welch 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 Information

Cornell 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 Information

Supported 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 Information

R01 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 Information

This 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 Information

ORSP 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

Objective

In this work we will find out whether Notch receptors serve as direct downstream targets of TGF‐β/Smad4 signaling in endothelial cells.

Methods

Realtime‐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.

Results

TGF‐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.

Conclusion

For 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 Information

NCGR and INBRE Funding ‐ 8P20GM103451‐12

NIH 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 Information

NIH 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 Information

NIH, NHLBI, NIDDK
A Non‐Canonical Function of Leucyl tRNA Synthetase Negatively Regulates Skeletal Myogenesis
Authors: Son Kook , Khanna Nidhi , Banerjee Aditi , Martinis Susan , Chen Jie
Published: November 2023

Aside from its house‐keeping function in protein synthesis, leucyl tRNA synthetase (LeuRS) has a non‐canonical function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also governs skeletal myogenesis, but its signaling network is distinct from that in cell growth regulation. We set out to investigate a potential role of LeuRS in skeletal myogenesis. Knockdown of LeuRS enhanced myoblast differentiation in vitro, whereas overexpression of LeuRS inhibited myogenesis. In a mouse model of injury‐induced adult muscle regeneration, local knockdown of LeuRS enhanced regeneration. Hence, LeuRS acts as a negative regulator of myogenesis both in vitro and in vivo. However, inhibition of the translational function of LeuRS by leucinol severely impaired myogenesis, consistent with the notion that protein synthesis is required for the myogenic process. These observations suggest a non‐translation role for LeuRS in suppressing myogenesis. Indeed, we found leucine‐binding but not tRNA charging activity of LeuRS to be necessary for its overexpression effect on myogenesis, suggesting that LeuRS may act as a leucine sensor in its function to ensure homeostasis of myogenesis. To probe the mechanism underlying this non‐canonical function of LeuRS, we examined the involvement of Rag GTPases, mediators of LeuRS activation of mTORC1 in cell growth. Constitutively active Rag reversed the effect of LeuRS knockdown on myogenesis and, conversely, Rag knockdown overcame the inhibitory effect of LeuRS overexpression. Rag knockdown also enhanced muscle regeneration in vivo, mirroring the effect of LeuRS knockdown. Taken together, our findings reveal for the first time a function for LeuRS in modulating the homeostasis of myogenesis. This new LeuRS function is mediated by a Rag‐mTORC1 pathway that negatively regulates the myogenic IRS1‐PI3K‐Akt pathway.

Support or Funding Information

Keck Foundation, NIH/NIAMS R01AR048914
Examining how allosteric mutations affect ligand binding and specificity on Dihydrofolate reductase
Authors: Alfonso Melany , Okondo Marian , Goodey Nina M
Published: November 2023

The purpose of my research is to study the enzyme Dihydrofolate reductase (DHFR) from the organism Bacillus stearothermophillus (Bs). DHFR is an important enzyme because it catalyzes the reduction of 7,8‐dihydrofolate (DHF) to 5,6,7,8‐tetrahydrofolate (THF) and THF is essential for DNA synthesis. For this reason, DHFR is the subject of many studies and is a pharmaceutical drug target to treat diseases like cancer and third world bacterial and protozoal infections. Drugs like methotrexate (MTX), pyrimathemine (PYR), and trimethoprim (TMP) are used to inhibit DHFR homologs from different pathogens and treat diseases caused by these pathogens. In many cases, however, it has been found that DHFR builds resistance to these antifolate drug compounds. Previously, it was found by Dr. Goodey and her students that drug binding and selectivity in DHFR has to do with the amino acid residue mutations in both the active site and in allosteric regions on the DHFR. For this project, we want to understand how these allosteric mutations affect ligand binding and specificity. The research was started by making two mutants (each have one allosteric mutation) and comparing them to the DHFR wild type. The first mutation distal from the active site was isoleucine in position 86 to alanine (I86A) and tyrosine in position 127 to alanine (Y127A). These DHFR mutants have been purified and a fluorescent dye molecule that act as a reporter tag was added to them. From these fluorescently labeled DHFR proteins I have worked on understanding the effect of these mutations on conformational motions associated with drug binding and selectivity by measuring the turnover number (kcat) and dissociation constant (Kd) of these proteins.
Cellular protein p32/gC1qR recruits PKC to Viral Protein ICP34.5 and Facilitates HSV Nuclear Egress
Authors: Zhang Cuizhu , Wu Songfang , Wang Yu , Zhang Liming , Pan Shuang , Liu Yaohui , Yang Mengmeng , Chen Da , Wang Jiyan , He Bin , Cao Youjia
Published: November 2023

The nuclear envelop is considered anatural barrier of virus budding and efficient replication. Several lines of evidence suggest that the disruption of the integrity of nuclear lamina correlate with the nucleocapsid budding of herpesviruses. Mammalian cellular protein p32 is identified as a new target for Herpes simplex virus 1 (HSV‐1)virulent protein, ICP34.5. We found that the efficient nuclear egress of HSV‐1 particles was impaired in the absence of cellular p32 protein. Lacking ICP34.5 encoding gene in HSV‐1 or reduced expression of host p32 severely affects viral replication. Here we report that the interaction betweenp32 and ICP34.5 of HSV‐1 facilitates nucleocapsid release to the cytoplasm. P32binds to the NH2‐terminus of ICP34.5 and recruits PKC to the nuclear lamina, where LaminA/C is phosphorylated and nuclear integrity disturbed. Unlike wildtype virus, an HSV mutant devoid of ICP34.5 or its amino‐terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In HSV infected cells, p32 is recruited to the nuclear membrane and triggers PKC activation whereas in cells infected by HSV with ICP34.5 mutants fail to exert any effect. These results suggest that disintegration of the nuclear lamina mediated by the association of p32 and ICP34.5 promotes HSV replication via PKC.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (31370862; 81672010 to YC)
Generation of TYRO3 Receptor Tyrosine Kinase Clones to Study Interactions with SH2 Domain Proteins in the Retinal Pigment Epithelium
Authors: Harris Lauren , Shelby Shameka
Published: November 2023

The retina is comprised of cone and rod photoreceptors that must continually be maintained in order to preserve visual acuity. Daily light exposure to the outer portions of the photoreceptors, termed outer segments (OS), leads to photo‐oxidative stress. To combat potential retinal damage caused by light exposure, the Retinal Pigment Epithelium (RPE) phagocytizes spent outer segments. Disruption of OS phagocytosis leads to the accumulation of debris that blocks the flow of oxygen and nutrients to the retina. This will eventually lead to atrophy of the retina and, ultimately, blindness. Previous studies have demonstrated the requirement of Mer Receptor Tyrosine Kinase (MERTK) in the process of OS phagocytosis. Protein interactions between MERTK and SH2 domain proteins Grb2, P85α, Src, and Vav3 have also been shown to be necessary for OS phagoctytosis. Recent studies suggest that TYRO3, a familial receptor tyrosine kinase to MERTK, can compensate in the absence of MERTK. As such, we hypothesized that TYRO3 may bind to SH2 domain proteins known to bind to MERTK. To test the similarities of interactions between MERTK, TYRO3, and associated SH2 domain proteins, various clones of TYRO3 were generated, and GST‐tagged SH2 domain proteins were overexpressed and purified. GST‐SH2 domain proteins Grb2, P85α, Src, Vav1, and Vav3 were overexpressed and purified via affinity chromatography, followed by gel‐filtration chromatography. Two truncated TYRO3 proteins that included the kinase domain and cytoplasmic tail (residues 470 – 809 and 498 – 809) were successfully cloned into a pRSET vector and recombinantly overexpressed. Additionally, full length TYRO3 was successfully cloned into pcDNA3.1 His vector for overexpression in mammalian cultured cells. Further experiments will adequately purify the truncated TYRO3 protein and identify interactions between TYRO3 and SH2 domain proteins recombinantly via Ni‐NTA pull‐down assays. Confirmation of interactions identified in‐vitro will be conducted in mammalian cells transfected with TYRO3. This study has generated the tools necessary to further identify components of the RPE phagocytic mechanism. Elucidation of this mechanism will be instrumental in identifying future retinal disease genes and understanding the impact on proteins that may be involved in Age‐related Macular Degeneration.

Support or Funding Information

Florida Southern College Faculty‐Student Summer Research Collaboration Grant
Toxic PR Poly‐Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers
Authors: Lin Yi
Published: November 2023

The most prevalent form of familial amyotrophic lateral sclerosis (ALS) involves an expansion of the GGGGCC (G4C2) hexanucleotide repeat located within the first intron of a gene designated C9orf72. While normal individuals typically carry 20–30 G4C2 repeats, affected patients exhibit up to 1,000 or more of the hexanucleotide repeats. Surprisingly, the extended G4C2 repeats can be both transcribed and translated. Translation of either the sense or antisense transcripts of the repeats generates five poly‐dipeptides, GAn, GPn, GRn, PAn and PRn. Two of these polydipeptides, GRn and PRn, show significant cellular toxicity.

Missing to date are unbiased studies of the direct intracellular targets of GRn and PRn that may explain the toxicity of these poly‐dipeptides. To address this question, we employed two independent methods, photo‐crosslinking and immunoprecipitation, to identify protein targets of PRn. Both approaches showed that the PRn targets are significantly enriched for proteins bearing low complexity (LC) sequences. By in vitro reconstitution of the interaction between PRn and hnRNP A1, we found that the LC domain of hnRNP A1 is both necessary and sufficient for the interaction with PR20 peptide.

When incubated at neutral pH and physiologic ionic strength, the LC domains of numerous RNA binding proteins polymerize into labile, cross‐β fibers. These LC domain polymers then proceed to form hydrogels that mimic the formation of RNA granules. Several independent assays give evidence that PRn binding requires that the LC domains of target proteins exist in a cross‐β polymeric state. First, a series of aliphatic alcohols (1,6‐hexanediol, 2,5‐hexanediol, etc) showed the same hierarchical order of specificity in melting cross‐β fibers, hydrogels as they did in disrupting the interaction between the hnRNPA2 low complexity domain and the PRn poly‐dipeptide. Second, 1,6‐hexanediol, the aliphatic alcohol that has the strongest effect in dissolving cross‐β fibers, was found to impede binding of the PRn poly‐dipeptide to its native, intracellular targets. Finally, a single amino acid substitution (D290V) in hnRNP A2, which strengthens cross‐β polymer formation, also strengthens its binding to the PRn poly‐dipeptide. By constrast, another single amino acid substitution (F291S), which significantly compromises polymer formation, also impedes hnRNPA2 binding to the PRn poly‐dipeptide.

In summary, our observations reveal that the intracellular targets of PRn are proteins with low complexity domains and offer mechanistic insight into the toxicity of poly‐dipeptides.
Using Biophysical Characterization to Explore Suppressor of IKKepsilon Structure
Authors: Machek Megan L. , Shikwana Flowreen , Graham Sasha‐Kaye I. , Minzer Ian D. , Wey Ryan , Cruz Richard , Bell Ellis , Bell Jessica K.
Published: November 2023

The innate immune system is the first line of defense against pathogens. Suppressor of IKKepsilon (SIKE) is a downstream component of the antiviral TLR3 innate immune pathway, although its function in this pathway has not yet been determined. In order to derive information about SIKE function, we undertook characterization of SIKE structure to determine a structure‐function relationship. Using PHYRE2, a SIKE model of the human isoform was predicted that consisted of a V‐shaped coiled coil fold. The secondary structure of SIKE was assessed via circular dichroism. Spectra were consistent with the predicted alpha helical model. Thermal melts (15–80°C) showed a linear, rather than sigmoidal, unfolding transition consistent with computational predictions (GlobPlot, DisEMBL, PONDR), which suggested SIKE contained 35% disordered regions. Addition of up to 25% trifluoroethanol to stabilize helical structure increased the helical content of SIKE by 35%. Fluorescence polarization and size exclusion chromatography assessed the solution size of SIKE (23 kDa) with respect to globular standards. SEC revealed a concentration dependent (0.9 – 3.6 mg/mL) association of SIKE into three distinct species of 79, 185 and 1,325 kDa. Fluorescence polarization of each species compared to standards of lysozyme (14 kDa), albumin (66 kDa) and glutamate dehydrogenase (355 kDa) were consistent with the SEC results. Limited proteolysis coupled to tandem mass spectrometry as well as crosslinking assessed by SDS‐PAGE mapped accessible SIKE sequences and association between SIKE monomers, respectively. Limited proteolysis at 1, 5, and 10 min identified trypsin cleavage sites consistent with a dimeric model of SIKE. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) identified predominantly a dimer species as well as a smaller proportion of a tetrameric species. In the cleft of the V‐shaped coiled coil, 3DLigandSite proposed a zinc binding site. To confirm an interaction between SIKE and divalent cations, SIKE‐ANS titration and fluorescence‐based thermal shift assays were completed +/− Mg, Mn, Ca, and Zn. In each case, a subset of ions perturbed SIKE structure, demonstrating a divalent cation interaction. In conclusion, using various biophysical techniques, we have found that SIKE's structure has regions of disorder bookended by alpha helices; SIKE monomers associate primarily into a dimeric species; divalent metals contribute to SIKE stabilization, and the homology model presented is consistent with these biophysical characterizations.

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Work supported by NIH grant R21AI107447 and USD SURE program.
Destabilization of the Non‐Polar Subdomain of Hemolysin A Inhibits Hemolysis
Authors: Brunner Madeline Rose , Grilley Daniel , Weaver Todd Michael
Published: November 2023

Hemolysin A (HpmA), found within Proteus mirabilis, was the focus of this research. HpmA is a member of the two‐partner secretion pathway (TPS), which is the most common mechanism of protein secretion implemented by gram‐negative bacteria. TPS pathways feature both an A and B component, where TpsB secretes and activates its associated TpsA partner across the gram‐negative bacterial outer membrane.

Functionally, the 1,557 residue full‐length HpmA protein (fHpmA) acts as a cytolysin and more specifically as a hemolysin. To investigate the hemolytic activity of HpmA, an amino‐terminal truncated version containing only the first 265 residues of the protein was engineered. The truncated form, HpmA265, is secreted by HpmB, its partner, but is incapable of hemolysis. Therefore, HpmA265 harbors a functional TPS domain, but lacks a functional hemolytic domain. Recent equilibrium unfolding studies have dissected HpmA265 into three subdomains: 1) polar core, 2) non‐polar core, and 3) carboxy‐terminal. Each subdomain has an independent ΔGH2Oo′. The objective of this project is to better understand the relationship between subdomain stability, secretion capability, and hemolytic function.

This research targeted a series of residues located within the non‐polar core subdomain. The interior of this subdomain, like many globular proteins, contains primarily non‐polar residues. Three residues, Leu188, Leu194, and Ile197 were targeted for mutagenesis based on their localization within the non‐polar core and their proximity to one another. To disrupt the architecture within the non‐polar subdomain, Leu188, Leu197 and Ile197 were replaced with polar residues. Circular dichroism denaturation studies of L194N illustrated an increase in structure loss during the first transition state indicating that the nonpolar core denatured in conjunction with the carboxy‐terminal subdomain. These results are consistent with variant I197H, which also showed an increased m‐value and ΔCD signal showing destabilization of the non‐polar subdomain. A template‐assisted hemolytic assay was used to monitor the impact of the histidine replacement at I197. Past functional results have noted a biphasic process comprised of an initial lag phase followed by a rapid lytic phase. The I197H variant displayed a 8‐fold increase in lag time and a 8‐fold decrease in the maximum rate of hemolysis. Therefore, the template‐assisted function of HpmA265 appears to reside within the non‐polar core subdomain. Results of this research will help us to better understand the two‐partner secretion pathway as related to bacterial virulence.

Support or Funding Information

This work was supported, in whole or in part, by National Science Foundation Grant MCB1050435 (TMW) and a University of Wisconsin‐La Crosse Faculty Research Grant (TMW).