bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of learning progressions through the chemistry curriculum to biochemistry
Authors: Wolfson Adele J. , Mercer Allison M. , Offerdahl Erika G. , Lewis Jennifer E.
Published: November 2023

Learning progressions describe the stages– not necessarily linear ‐ that students take as they develop more sophisticated ways of knowing a particular concept. Many concepts in biochemistry are introduced in earlier courses, including general chemistry and organic chemistry. We are developing learning progressions for several key concepts in biochemistry informed by interviews with faculty and students and by survey instruments. Faculty interviews and focus groups yielded topics for the progressions, and follow‐up interviews with students after they had completed the surveys allowed us to refine the survey. Students at two different institutions have participated in the survey as pre‐ and/or post‐course assessment. The results will allow us to propose learning progressions related to acid/base equilibria, energy diagrams and kinetics, and thermodynamics of ATP hydrolysis.. We hope to use the progressions to develop teaching materials and to foster discussion among faculty so that articulation from one course to the next is productive.

Support or Funding Information

NSF‐DUE‐1503980 (IUSE)
CRISPR in the Undergraduate Classroom: a CURE
Authors: Evans Anderson Heather J.
Published: November 2023

The CRISPR/Cas9 system has revolutionized modern science by simplifying access to targeted genetic editing. Use of this cutting‐edge technology is possible in an undergraduate course and has the potential to be a powerful teaching tool to promote student engagement and encourage further participation in undergraduate research. A CRISPR‐focused semester long project using mammalian cell culture was implemented in a standard 3hour per week lab across 15 weeks for a mid‐level undergraduate Cell Biology course. The methods used in this project included: basic mammalian cell culture method, primer design, PCR, genomic DNA isolation, cell transfection and selection, cell viability assay as well as a T7 endonuclease assay and DNA sequencing to determine if genetic editing occurred. At the end of the project, students wrote a lab report to summarize their work and present accumulated data. Remarkably, students were able to successfully edit their targeted gene; however, the ultimate goal of this implementation was to expose an entire class to an exciting research project in order to promote engagement with undergraduate research. A classroom undergraduate research experience (CURE) post‐course survey was administered to measure student experiences. The benefits of undergraduate research are widely cited but most undergraduate students do not get the opportunity to do independent research. This approach fulfills the 5 tenets that define a CURE (Auchincloss et al. CBE – Life Sciences Education, 2014), and expands the opportunity to engage a wider population of students in an undergraduate research experience than a traditional apprenticeship model. Furthermore, this approach also provides faculty a means for building independent undergraduate research programs. This project was supported within a typical lab course budget and can easily be modified to fit a wide variety of undergraduate students as well as easily be scaled up to large populations. Overall, the implementation of a semester long CRISPR project was an exciting way to engage an undergraduate class in a novel and relevant research project that was mutually beneficial to both students and the instructor.
Th1 Effector T cells induce TGFβ mediated Cardiac Fibrosis in Pressure Overload induced Heart Failure by adhering to Alpha 4 Integrin and mediating Fibroblast Transformation
Authors: Nevers Tania
Published: November 2023

Cardiac fibrosis (CF) is a key factor contributing to the pathogenesis of the deadly syndrome of heart failure (HF). Recent emerging data demonstrate a role for T cells in pro‐fibrotic cardiac repair and healing post‐ischemia. However, very little is known about how T cells directly impact cardiac fibroblasts (CFB) to promote CF and cardiac dysfunction in non‐ischemic HF. Recently, we reported that T cells infiltrated the left ventricle (LV) of the heart in patients with end‐stage non‐ischemic HF, and also in the mouse model of non‐ischemic HF induced by thoracic aortic constriction (TAC). Interestingly, mice deficient in T cells (TCRα−/− mice) are protected from cardiac fibrosis (CF) and cardiac dysfunction. Thus, we hypothesize that T cells activated in such context orchestrate the activation and differentiation of fibroblasts to myofibroblasts. To test this hypothesis, we first co‐cultured adult CFB with T cells isolated from mice 4 weeks post TAC or Sham surgery. TAC but not Sham T cells firmly adhered to CFB and mediated myofibroblasts transformation as demonstrated by increased α‐SMA fluorescent expression. Co‐culture of various CD4 T cell subsets with CFB demonstrated that Th1 effector T cell subset, in contrast to naïve T cells, adhered directly to CFB through alpha 4 integrin and mediated myofibroblasts transformation in a TGFβ dependent manner. Moreover, adoptive transfer (AT) of effector Th1 cells, but not naïve T cells into TCRα−/− mice at 48 hours and 2 weeks post TAC surgery exclusively induced perivascular fibrosis that co‐localized with T cell and leukocyte infiltration and TGFβ expression. AT of Th1 cells also induced LV contractile and systolic dysfunction and a shift in the fetal gene expression of the myosin heavy chain α toβ which occurs in pathological cardiac remodeling. Taken together, these findings identify a novel role for Th1 cells in cardiac fibrosis and function and may provide innovative insights in the treatment of chronic non‐ischemic HF.

Support or Funding Information

NIH HL094706
Atrogin‐1 Transgenic (AT1 Tg+) Mice Have Age‐Dependent Cardiac Dysfunction With Atrogin‐1 Mediated Glucocorticoid Receptor (GR) Expression Down Regulation
Authors: Mota Roberto , Parry Traci , Willis Monte
Published: November 2023

Previous studies have identified the muscle‐specific ubiquitin ligase Atrogin‐1 (MAFbx) as a critical regulator of pathologic and physiologic cardiac hypertrophy. Mechanistically, Atrogin‐1 regulates these processes by ubiquitinating transcription factors (NFAT and FOXO1/3, respectively) to inhibit these processes. However, their role in regulating transcription factors in aging has not previously been described.

Methods

Transgenic mice with increased cardiomyocyte‐specific (alpha‐MHC promoter driven) Atrogin‐1 were phenotypically characterized at baseline and up to one year by conscious echocardiography. In parallel, HL‐1 cells were cultured with increasing adenovirus expressing myc‐Atrogin‐1 (Ad.Atrogin1) or GFP control (Ad.GFP) at a multiplicity of infection (MOI) of 0, 10, 20, 50, 100 and 200. Data were subject to statistical analysis with two‐tailed t‐test and/or one‐way ANOVA using GraphPad 7.0 for Mac.

Results

Post‐transduction, HL‐1 cells were lysed and protein was collected. Immunoblots were then performed to identify potential Atrogin‐1 substrates. Conscious echocardiographic analysis of Atrogin‐1 Tg hearts revealed not differences in phenotype up to four months of age. However, by 12 months, Atrogin‐1 hearts exhibited significantly decreased ejection fraction, fractional shortening and left ventricular (LV) mass (p>0.05). This aging phenotype was reminiscent of recent studies where the cardiac GR was deleted, so we next investigated if Atrogin‐1 potentially targeted GR for degradation in the HL‐1 cardiomyocyte‐derived cell line. In HL‐1 cells with increasing Ad.Atrogin‐1 MOI, GR protein expression was significantly decreased in a dose‐dependent manner compared to controls (Ad.GFP) (p<0.05).

Conclusion

These results suggest that chronically increasing cardiomyocyte Atrogin‐1 can lead to an age‐dependent decrease in systolic function, mimicking the alterations in GR signaling reported in humans. In the heart, the muscle‐specific ubiquitin ligase Atrogin‐1 may down‐regulate cardiomyocyte GR by mediating its polyubiquitination and degradation by the 26S proteasome. Future studies will delineate the molecular details in which Atrogin‐1 regulated GR levels post‐translationally so that Atrogin‐1's role as a therapeutic target may be evaluated further in aging.

Support or Funding Information

Supported by the Leducq Foundation.
Splenectomy‐Induced Diastolic Dysfunction With Normal Ejection Fraction
Authors: Huber Jason S , Ruggiero Sara A , Romanova Nadya , Blois Shauna L , Brunt Keith R , Simpson Jeremy A
Published: November 2023

Background

Understudied and often disregarded, the spleen is perceived as redundant, non‐vital or even a vestigial organ. Conversely, epidemiological studies indicate splenectomy (or splenic injury) increase the prevalence of cardiovascular diseases and associated risk factors, suggesting the physiological function of the spleen is not yet realized. Recent investigations have examined the role of the spleen in experimental heart failure, however, the effect of long‐term splenectomy on cardiac function in healthy models remains to be determined. Thus, the aim of this study was to reconcile the observations made in the epidemiological studies by investigating cardiac function and morphology following splenectomy in healthy rodents.

Methods

Splenectomized and sham male Wistar rats were assessed at 5, 9, and 18 weeks post‐surgery by echocardiography, invasive hemodynamics, cardiac histology, and thromboelastography. Serum IL‐10 was measured by ELISA and left ventricular free wall was used for the analysis of select molecular markers.

Results

Splenectomy caused progressive left ventricular diastolic dysfunction (i.e., increases in dP/dt min, Tauw, and LVEDP), with no evidence of cardiac decompensation or systolic dysfunction. Left ventricular remodeling (fibrosis, hypertrophy and decreases in capillary to myocyte cross sectional area) were associated with decreases in serum IL‐10 and ventricular BNP expression. Increases in blood coagulability following splenectomy are associated with increased risk of developing thromboembolic pulmonary hypertension. In our model however, we found no evidence of pulmonary arterial hypertension or lung remodeling despite a ~2‐fold increase in coagulability.

Conclusions

Splenectomy in healthy rodents caused progressive diastolic dysfunction and significant cardiac remodeling (i.e., fibrosis, hypertrophy and decreased capillary density), demonstrating an underappreciated role of the spleen in cardiac homeostasis. Future studies need to characterize the mechanism(s) by which the spleen contributes to a healthy cardiovascular phenotype.

Support or Funding Information

This work was supported by the J.P. Bickell Foundation (to JA Simpson), the Natural Sciences and Engineering Research Council (to JA Simpson and KR Brunt), and the Ontario Veterinary College Pet Fund (to SL Blois). JA Simpson is a new investigator with the H&SF of Ontario.
Live cell microscopy of intestinal organoid oxygenation
Authors: Dmitriev Ruslan I. , Okkelman Irina A. , Foley Tara , Papkovsky Dmitri B.
Published: November 2023

Oxygenation of stem cell niche is important metabolic activity indicator for the intestine in normal and diseased states. In vivo, it can be affected by such factors as diet, drugs, metabolites produced by microbiota, and inflammation. We hypothesized that O2 must be also very important for adequate development of organoids and organs‐on‐chip, growing as 3D cultures. We took advantage of cell‐penetrating O2‐sensitive probes and phosphorescence lifetime imaging microscopy (PLIM), which, in contrast to the end‐point hypoxia stains and electrode‐based measurements, provides high‐resolution real‐time visualization of O2. Due to the specialised cultivation conditions for different types of organoids and varied ability for staining with live cell dyes and probes, we optimized the staining parameters with O2‐sensitive probes. Using staining with small molecule Pt‐Glc probe [1] and PLIM technique we analyzed O2 distribution in organoids from small intestine of mouse. We were able to directly measure oxygenation of organoids without their recovery from Matrigel matrix and multiplexed it with number of live fluorescent probes (Hoechst 33342, Cholera Toxin‐Alexa 488, TMRM, CellTox Green, Calcein Green) and immunofluorescent staining of fixed samples. We next analysed the oxygenation of live organoids treated either with mitochondrial uncoupler FCCP or anti‐diabetic drug metformin, inhibiting activity of mitochondrial complex I [2]. We found that even when produced from the same animal, resting organoids displayed highly heterogeneous oxygenation, from 27 to 88 μM (approx. 2.7~8.8% of air saturation). This can be explained by the growth at different depths in Matrigel matrix and limited diffusion of O2 and/or highly variable respiration between organoids. Treatment with FCCP, increasing oxygen consumption, resulted in decrease of organoid oxygenation, confirming that the intestinal epithelia tissue has active mitochondria. In contrast, metformin treatment significantly increased the overall oxygenation of organoids in comparison to non‐treated culture. However, the magnitude of response to metformin varied for samples taken from different animals. This data confirms that when accumulating metformin, intestinal epithelia undergoes inhibition of mitochondrial respiration, which can change its oxygenation and lead to glycolytic switch of metabolism. Altogether, our results point at the importance of analysis of organoid culture oxygenation in tissue engineering, and demonstrate how this information can be translated in studies of molecular mechanisms of diabetes.

Support or Funding Information

Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.
DDI1 and DDI2‐dependent removal of Replication Termination Factor Domain Containing 1 (RTFDC1) from replication forks is necessary for proper response to replication stress
Authors: Kottemann Molly C , Conti Brooke , Lach Francis P , Smogorzewska Agata
Published: November 2023

Accurate replication of the genome is a fundamental responsibility for the cycling cell, but DNA replication is itself a source of damage. Replication forks can stall at DNA lesions and physical barriers, in difficult to replicate regions, or due to nucleotide depletion. We have identified DDI1 and DDI2, mammalian homologs of the yeast UBL‐UBA shuttle Ddi1, as participants in DNA damage response due to replication stress. Pulldown of GFP‐DDI2 followed by mass spectrometry identified members of the proteasome and the replisome. Depletion of DDI1/2 caused significant hypersensitivity to replication stress drugs hydroxyurea (HU) and aphidicolin (Aph), and overexpression of either DDI1 or DDI2 rescued the sensitivity. During replication stress, DDI1/2 depletion caused fork restart defects, hyperactivated damage signaling, and increased accumulation of RPA‐coated single stranded DNA. Ultimately, these defects led to severe chromosomal instability and cell death by apoptosis. Using isolation of proteins on nascent DNA (iPOND), we identified proteins that are enriched at the replication fork in DDI1/2 depleted cells. The most enriched protein was Replication Termination Factor Domain Containing 1 (RTFDC1), the mammalian homolog of S. pombe Rtf2. Reducing excess RTFDC1 at the fork by siRNA treatment in the DDI1/2 depleted cells rescued the aberrant phenotypes seen during replication stress response, placing RTFDC1 as the key DDI1/2 target at the replisome. This work establishes a novel fork protection pathway that requires DDI1/2 to remove RTFDC1 from the replisome during replication stress response and we show that this pathway is necessary for maintenance of genome stability.

Support or Funding Information

MCK was supported by the Women and Science Fellowship and by the American Cancer Society‐J.T.Tai Postdoctoral Fellowship. This work was supported by the Pershing Square Sohn Prize for Young Investigators in Cancer Research and The Gabrielle H. Reem and Herbert J. Kayden Early‐Career Innovation Award. AS is an HHMI Faculty Scholar.
The Effects of Replication Fork Restart on CAG Repeat Instability in S. pombe
Authors: Gold Michaela , Freon Karine , Su Allen , Lambert Sarah , Freudenreich Catherine
Published: November 2023

Trinucleotide repeats are a category of repetitive DNA elements that can form secondary structures and cause many diseases. Expansions of CAG repeats in particular can lead to numerous neurodegenerative polyglutamine diseases, including Huntington's disease and some spinocerebellar ataxias. These expansions could occur because the secondary structures the repeats form can stall DNA replicative polymerases, and fork restart may be prone to repeat instability. In order to test this idea, we have used the Schizosaccharomyces pombe fission yeast Replication Fork Barrier (RFB) system to induce fork restart through a CAG repeat tract and study the effects on CAG repeat instability (expansions and contractions). We created constructs with 70 CAG repeats inserted at different locations relative to the RFB. With the repeats either 1.9 kb or 6.7 kb after the RFB, increased CAG repeat instability was detected after RFB induction, indicating that fork restart can be a source of repeat instability.

In order to test the mechanism of repeat instability during fork restart, key proteins potentially involved in the process were deleted. In contrast to wild‐type, in cells missing the Rad52 protein, which is involved with homologous recombination (HR) and required for restart in this system, induction of the RFB did not further increase repeat instability. This suggests that HR could be a leading cause of RFB‐induced repeat instability. Msh2 has been shown to bind CAG repeats and cause repeat expansions in mouse models. In S. pombe strains without Msh2, expansions decreased irrespective of RFB status. These results suggest that Msh2 is involved in the CAG expansion mechanism as in vertebrates, but likely not during fork restart, and S. pombe can be used to study this phenomenon. In summary, we have identified two mechanisms of CAG instability in S. pombe: expansions caused by presence of the Msh2 protein unrelated to fork restart, and a second novel mechanism of HR‐dependent fork restart that causes both expansions and contractions of this unstable repeat.

Support or Funding Information

This research was funded by National Institutes of Health grant P01‐GM105473/402373 and the Curie Institute Rothschild‐Yvette Mayent Award to C.H.F., ANR 14‐CE10‐0010‐01 and FRM label funds to S.L., and Tufts University Sandler and Undergraduate Research Funds to C.H.F. and M.A.G..
Mutagenesis of HEK293 Cells Reveals Phenotypic and Genotypic Variants Involved in Hsp90‐Mediated Glucocorticoid Signaling
Authors: Forst Thomas M. , Ryan Sean M. , Pelle Neiproi , Murphy Patrick J. M.
Published: November 2023

Hsp90 is a highly conserved eukaryotic chaperone protein responsible for mediating a multitude of intracellular signaling pathways, including those associated with the glucocorticoid receptor (GR), hsp70, and stress response. The goal of this study was to investigate the effects of mutagenesis on human embryonic kidney (HEK293) cells in the presence of a GR agonist (dexamethasone) as well as an hsp90 antagonist and clinically relevant benzoquinone ansamycin (17‐AAG, a geldanamycin derivative). HEK293 cells were incubated with increasing concentrations of dexamethasone and 17‐AAG in order to determine cell growth inhibition and cytotoxicity. Cell lysate was prepared when growth approached 80% confluency and analyzed for hsp90 and hsp70 via western blot. Relative concentrations for the proteins were qualitatively measured via ImageJ analysis software. The concentration of each drug necessary for producing an effect in hsp90 concentration while not exhibiting a significant decrease in cell viability was determined. HEK293 cell growth was assayed over 7 days in the presence of one or both drugs and then cultured in increasing concentrations of 17‐AAG in order to select for a drug resistant phenotype. The growth characteristics, phenotype, and genotype of drug resistant cells were then examined. Western blot analysis determined a significant increase in hsp90 concentration at 10 μM dexamethasone and an increase in hsp70 concentration in cell populations at all drug conditions tested relative to control. The growth curve performed at 10 μM dexamethasone reflected a disruption in normal cell cycle progression. A concentration of 1 μM 17‐AAG was shown to significantly decrease the concentration of hsp90 and 10 μM of the drug was determined to be cytotoxic. Growth characteristics of cells exposed to increasing concentrations of 17‐AAG over an extended time period were quantified, and exons of HSP90AA1 and HSP90AB1 were assayed via Sanger sequencing. Increases in hsp90 concentrations in the presence of dexamethasone and possible genetic alterations following prolonged 17‐AAG incubations presumptively indicate cells with directional hsp90 regulation can better survive exposure to high concentrations of clinically relevant glucocorticoids as well as hsp90 inhibitors.
Role of Exo1 nuclease in telomere DNA degradation in yku70/yku80 mutants of S. cerevisiae
Authors: Ream Jennifer Anne , Lewis L. Kevin
Published: November 2023

DNA is constantly damaged within the cell by endogenous and environmental mutagens such as oxidative chemicals and radiation. Double‐strand breaks (DSBs) resulting from this damage are repaired by two pathways, homologous recombination (HR) and nonhomologous end‐joining (NHEJ). The molecular machinery involved in HR and NHEJ are conserved in vertebrates and yeast. The Ku protein complex is fundamental to initiation of the NHEJ pathway. The yeast Yku70/Yku80 protein complex is integral to the stabilization and replication of telomeres, in addition to its involvement in DSB repair. Budding yeast (Saccharomyces cerevisiae) yku70 mutants have been shown to be defective in NHEJ repair and have short telomeres. These mutants also display temperature sensitive growth at 37 °C, caused by telomere cap instability and telomere DNA degradation by Exo1 exonuclease at high temperature. Our genetic experiments demonstrate that pathways other than Exo1 telomere digestion are crucial for cell survival at 37 °C. Results of this work involving gene knockout experiments and cell survival assays will be presented.
Toward the Molecular Identification of Afr2, A Gene Implicated in Liver Cancer
Authors: Grimes Zach , Seipelt‐Thiemann Rebecca , Paterson Martha , Spear Brett
Published: November 2023

In humans, liver cancer is the 5th leading cause of cancer death in men, and the 9th in women. Alpha‐fetoprotein (AFP) is a fetal protein that is active during liver development and hepatocellular differentiation. After the development stage, AFP is transcriptionally silenced due to the action of alpha‐fetoprotein regulator 1 (Afr1). However, during liver regeneration and tumorigenesis, AFP expression is reactivated by the action of α‐fetoprotein regulator 2 (Afr2). It is this observation that has led to the use of AFP levels as a diagnostic marker for liver cancers. Two strains of mice differ in their ability to develop liver tumors and reactivate expression of AFP. C3H/HeJ mice develop liver tumors and express high AFP levels in the regenerating liver while C57/BL6 do neither. Heterozygote mice express an intermediate level of AFP and show reduced tumorigenesis. Recombination mapping showed that Afr2 is located on chromosome 2. The purpose of this study was to use a refined genetic map and existing gene variation and expression data to identify Afr2 candidate genes. First, genetic variation for the region of interest was compared using genomic data from both the Mouse Genome International and the Sanger Institute. Gene ontology and domain identification databases were used to gain information about the genes that showed variation between strains. Several genes with the potential to regulate gene expression were identified. Second, microarray studies of control and reactivated liver in both strains were compared for genes that were upregulated in C3H/HeJ reactivated liver and found in the select region of chromosome 2. Through the combinatorial analysis of these lists, four genes were identified as candidates. Finally, studies are underway to investigate whether any of these candidate genes is able to activate transcription of a reporter gene cloned near the reported AFR2‐binding region. Identification of Afr2 may lead to better therapeutics for treatment of liver cancer due to its pivotal role in directing liver‐specific gene expression.
Retention of CENP‐A nucleosomes during DNA replication
Authors: Zasadzinska Ewelina , Huang Jiehuan , Bailey Aaron O. , Lee Nancy S. , Wong Kelvin A. , Jakubaszek Marta R. , Guo Lucie , Black Ben E. , Foltz Daniel R.
Published: November 2023

Centromeres are chromosomal loci that define the site of kinetochore formation and ensure equal chromosome segregation during mitosis and genomic stability. Centromeric identity is epigenetically regulated by the incorporation of the nucleosome containing the centromere specific CENP‐A histone H3 variant. In contrast to the canonical H3.1, CENP‐A deposition is independent of DNA replication, and occurs in early G1. Therefore new CENP‐A deposition and retention of CENP‐A during S‐phase are key events in epigenetic inheritance. DNA replication presents a challenge for CENP‐A inheritance because nucleosomes are removed to allow passage of the replication fork. However, CENP‐A nucleosomes are stably retained across S‐phase. We hypothesize that CENP‐A retention at centromeres during S phase is facilitated by a unique mechanism that distinguishes CENP‐A containing nucleosomes from bulk histones. We fused CENP‐A to the promiscuous biotin ligase (BirA*) to chemically label and affinity purify proteins transiently associated with CENP‐A during S‐phase. SILAC labeling and Mass Spec were used to identify S‐phase specific interactions. This analysis identified known G1 coupled CENP‐A deposition proteins such as HJURP and Mis18BP1 as well as novel factors, as associating with CENP‐A during S‐phase. Therefore the CENP‐A deposition proteins appear to play dual roles in S‐phase and G1. We demonstrate that HJURP is required for the inheritance of existing CENP‐A nucleosomes during DNA replication. MCM2 subunit of the replicative helicase binds HJURP and directly interacts with CENP‐A. Our data suggests that mechanism of transmission of CENP‐A nucleosomes requires CENP‐A specific deposition machinery including HJURP together with the activity of MCM2.

Support or Funding Information

AHA Predoctoral Fellowship
Sequence Analysis of Mutant Chick Embryo DNA
Authors: Vo Jessica , Byk Jennifer , Smith Sheila R. , Benore Marilee Ann
Published: November 2023

Riboflavin Binding Protein is a transport glyco‐phospho‐protein found in all oviparous species, required for transport of the vitamin riboflavin needed for proper embryonic development. A mutant strain of chickens cannot synthesize the protein; the embryos lacking riboflavin die after the thirteenth day of incubation. DNA was extracted from the embryos. Several kits were utilized and compared to determine the best method to isolate pure DNA in large yield. Sequence comparisons to wild‐type white leghorn chickens are used to confirm the DNA sequence causing the mutation.

Support or Funding Information

University of Michigan Dearborn Campus Grants
DNMTi Decitabine Induces a Type I Interferon Response in Leukemia Cell Lines
Authors: Nicholas David J , Srivastava Pragya , AlShangity AlMujtaba , Nemeth Michael J
Published: November 2023

Decitabine (DAC) is an FDA approved DNA methyltransferase inhibitor (DNMTi) currently used for treatment of Myelodysplastic Syndrome and Acute Myeloid Leukemia. Only 40% of patients respond to DAC treatment. Therefore, discovering and enhancing DAC's mechanism of action is of paramount importance. Recent literature suggests that DNMTis demethylate and induce expression of latent dsRNA retroviral species in ovarian cancer and colorectal cell lines. This viral mimicry triggers an interferon response that results in both apoptosis and targeting of treated cancer cells. To test whether a similar mechanism of action occurs in myeloid cancer, the leukemia cell lines U937, HL60, and KG1a were evaluated for gene expression changes in the interferon response pathway after two treatments of 0.5um DAC using RTqPCR. Up regulation of Interferon response genes DDX58, MDA5, OASL, MAVS, ISG15, IFN B, and IRF7 were observed in KG1a cells with reported fold changes of 3.45, 1.88, 54.40, 2.04, 3.25, 5.88, and 80.00 respectively. In addition, the ERV MLT1C49 was upregulated by a fold change of 2.46. In the U937 cell line, DDX58, MDA5, OASL, MAVS, and ISG15, were upregulated by 1.59, 2.02, 3.62, 1.468, and 6.3646, respectively. For the HL60 cell line, DDX58, OASL, and ISG15 were upregulated by 1.79, 5.16, and 1.87, respectively. In summary, our data suggests Decitabine induces a type I interferon response in leukemia cells, giving promise to the combinatorial treatment of DAC with emerging immunotherapy treatments.

Support or Funding Information

NIH R25CA181003 and SUNY Geneseo Biology Department
Epigenetic Regulation through UHRF Proteins
Authors: Rothbart Scott B , Vaughan Robert M , Cornett Evan M , Dickson Bradley M
Published: November 2023

The epigenetic inheritance of DNA methylation requires UHRF1, a histone‐ and DNA‐binding RING E3 ubiquitin ligase whose misregulation may contribute to the hallmark aberrance of DNA methylation patterning seen in cancer. UHRF1 facilitates recruitment of DNMT1 to replicating chromatin through orchestrated recognition of histones and DNA, which converge on ubiquitination of histone H3, a binding site for DNMT1. UHRF2 shares a high degree of structural homology with UHRF1, but surprisingly lacks functional redundancy as a DNA methylation regulator. Through comprehensive and comparative biochemical analysis of UHRF1 and UHRF2, we uncover mechanisms of intra‐ and inter‐molecular crosstalk regulating the binding and enzymatic activities of these chromatin regulators and reveal a molecular mechanism that uncouples UHRF2 from the DNA methylation maintenance program.

Support or Funding Information

This work was supported in part by the Van Andel Research Institute and a research grant from the National Institutes of Health to S.B.R. (Grant No. CA181343).
Unraveling The Role of Helicases in Nonfunctional rRNA Decay
Authors: Yu Victor , LaRiviere Frederick
Published: November 2023

rRNAs are essential, long‐lived, and highly abundant, comprising up to 90% of total cellular RNA. However, when structurally intact rRNAs in Saccharomyces cerevisiae are functionally compromised, they are instead rapidly degraded through a quality control pathway termed nonfunctional ribosomal RNA decay (NRD). Currently, two mechanistically distinct types of NRD are known: 18S NRD and 25S NRD. 18S NRD is translation dependent and removes rRNAs with loss of function mutations in the small subunit decoding site. The 18S NRD mechanism involves the translation factor‐like protein Dom34 and its binding partner Hbs1. On the other hand, 25S NRD, which occurs prior to subunit joining, is translation independent and removes nonfunctional rRNAs containing mutations in the large subunit peptidyl transferase center. The 25S NRD mechanism involves polyubiquination of proteins associated with a nonfunctional 25S rRNA through the E3 ligase components Rtt101 and Mms1. While some of the proteins that are involved in either 18S or 25S NRD are known, we have not yet elucidated the complete molecular mechanism of NRD. Since RNA helicases have been implicated in other RNA degradation pathways, we hypothesize that one or more helicases may be important for either or both NRD mechanisms. S. cerevisiae contains 44 different helicases, 14 of which are non‐essential. Thus, we are initially investigating whether any of these 14 helicases are involved in NRD using single gene deletion yeast strains. We transformed BY4741 and the 14 non‐essential helicase knock‐out strains with various rRNA expression plasmids that encode for functional and/or nonfunctional versions of the 18S and 25S rRNAs. These plasmid‐derived rRNAs also contain short, unique sequence tags that allows for specific detection of these rRNAs in the presence of total cellular rRNA. We measured the growth phenotypes of these transformed strains at various temperatures in liquid cultures. At standard temperatures, we see a slow growth phenotype in some of the deletion strains transformed with either nonfunctional 18S or 25S rRNA expression plasmids. Since some helicase deletion strains exhibit either cold‐ or temperature‐sensitivities, we are currently measuring growth phenotypes at these normally restrictive temperatures. In addition, we are measuring the rRNA expression levels in these deletion strains using an RT‐qPCR assay to quantify the level of NRD activity. We hope this work will provide insight into the role of helicases in RNA degradation mechanisms and contribute to our understanding of NRD.

Support or Funding Information

This work is supported by the Howard Hughes Medical Institute.
Alternative Splicing of hnRNPA2/B1 by SRSF2 and Its Effects on Stress Granule Formation in Myelodysplasia
Authors: Ardasheva Anastasia , Vasic Radovan , Halene Stephanie
Published: November 2023

Myelodysplastic syndrome (MDS) is a type of hematopoietic malignancy commonly diagnosed in patients >65 years old. Although the exact cause for MDS is not known, mutations in the cell splicing machinery are a common feature among patients diagnosed with this disease. One of the most common mutations occurs in the serine/arginine‐rich splicing factor 2 (SRSF2), converting Proline 95 to Histidine. We previously identified recurrent mis‐splicing of the heterogeneous ribonuclear protein A2/B1 (hnRNPA2/B1) in patients bearing the SRSF2 P95H mutation. In the actively translating cell, hnRNPA2/B1 is capable of forming non‐membrane droplets called stress granules that preserve stalled translational complexes under conditions stress. The four isoforms of hnRNPA2B1 are characterized by the presence or absence of exons 2 & 9. Studies on neuronal cells have shown that these isoforms localize differently within the cell under homeostatic conditions. We therefore hypothesized that the alternative splicing of hnRNPA2/B1 in patients bearing the SRSF2 mutation results in the expression of isoforms with altered ability to participate in stress granule formation, resulting in consequent defects in RNA metabolism. To assess the ability of the four isoforms to participate in stress granules, we transfected 293FT mammalian cells with each isoform and assessed cellular localization following induction of stress granule formation with sodium arsenite treatment. These experiments may allow for a greater understanding of the mechanisms that contribute to cellular dysfunction in MDS.
Biochemical Characterization of Evolutionary Divergent Vertebrate LARP6 Proteins
Authors: Castro Jose Miguel , Horn Daniel A , Lewis Karen A
Published: November 2023

The La‐related proteins (LARPs) contain a conserved bipartite RNA binding domain, called the La module, which consists of the La motif and an RNA recognition motif (RRM). Across the different LARP subfamilies, the La module interacts with a variety of RNAs, allowing this protein superfamily to perform roles as chaperone proteins, post‐transcriptional regulatory factors, regulators of RNA metabolism, and perhaps even tumor suppressors. The ligand specificity of the LARP6 subfamily is still unknown. The only endogenous ligand for LARP6 is found in the 5′ untranslated region of collagen type I mRNA. Animal studies confirmed that the binding of a particular RNA structure by LARP6 was critical for collagen production and muscle development. In this study, we are using the LARP6 proteins from zebrafish (Danio rerio), platyfish (Xiphophorus maculatus) and human to carry out detailed biochemical analyses of vertebrate LARP6 RNA binding activity. The purpose is to determine if there is a species‐specific protein‐RNA interaction due to co‐evolution of the ligand‐binding surface within the LARP6 RRM and the RNA ligand sequence and/or structure. Recombinant fish and human LARP6 proteins were expressed in E. coli with an N‐terminal SUMO tag. A filtration screening assay was used to identify additives that increase solubility and stability to the fish proteins. In order to robustly compare the binding affinity of the fish proteins to the human LARP6, we then optimized conditions for SUMO cleavage by Ulp1. Limited proteolysis studies were used to compare the domain topology of the fish LARP6 proteins to the human homolog. The RNA binding activity of all LARP6 proteins for the COL1A1 stem loop RNA from the appropriate species was measured by electrophoretic mobility shift assays (EMSAs). In order to determine if the La motif and RRM co‐evolved within species, interspecies chimeras were produced. These studies will explore whether the RRM determines the ligand specificity of the LARP6 protein.

Support or Funding Information

Funding provided by the Texas State University Department of Chemistry and Biochemistry; the National Institutes of Health through the South Texas Doctoral Bridge Program (GM102783 to J.M.C.) and an AREA grant (GM119096 to K.A.L.).
Understanding the role of post‐translational modifications on the splicing activity of two related RNA binding proteins
Authors: Reynaga Janice Mireya , Pina Jeffrey , Keppetipola Niroshika
Published: November 2023

The process of alternative splicing is regulated in part by RNA Binding Proteins, many of which occur as gene families with members sharing high primary structure identity and similar domain organization but have tissue specific expression patterns and different splicing activities. The Polypyrimidine Tract Binding Protein 1 (PTBP1) and Polypyrimidine Tract Binding Protein 2 (PTBP2) are paralogous RNA binding proteins. The two proteins share 74% primary structure identity and similar domain organization with 4 RNA binding domains connected via 3 linker regions. Albeit the structural similarity, PTBP1 and PTBP2 show differences in their splicing repression activity, where some exons are repressed by both and others repressed more strongly by PTBP1. The two proteins also have non‐overlapping expression patterns where PTBP1 is expressed in neuronal progenitor cells but is replaced by PTBP2 in differentiating neurons. This change in protein expression levels is accompanied by a transition in the neuronal splicing program that is critical for neuronal development and maturation. Previous studies investigating regions of primary structure that dictate higher PTBP1 repression activity on certain exons such as the c‐Src N1 exon highlighted that multiple regions confer splicing repression on PTBP2. Recent reports indicate that the two proteins are post‐translationally modified. Thus, we hypothesize that post‐translational modifications dictate the different splicing activity of PTBP1 and PTBP2. To test this, we have overexpressed PTBP1 and PTBP2 in mouse neuroblastoma (neuro 2A) cells and probed for protein phosphorylation, via Phos‐Tag Gel Electrophoresis and for ubiquitination and acetylation via Western Blot. In parallel, we purified Flag‐tagged PTBP1 and PTBP2 via Flag‐Immunoprecipitation and analyzed the proteins for PTM's via Mass Spectrometry. Preliminary results indicate that the two proteins are modified in neuro 2A cells. We are currently conducting site‐directed mutagenesis of modified residues to understand the role of these modifications on PTBP1 and PTBP2 splicing activity.

Support or Funding Information

This work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612‐20]
Thermodynamic analysis of a 4×4 internal loop in magnesium riboswitch
Authors: Gilbertson Ellen
Published: November 2023

A riboswitch is a segment of noncoding RNA that regulates the transcription of a gene in response to external cellular events. The magnesium riboswitch specifically regulates the expression of a transmembrane magnesium transport protein, mgtA, in response to intracellular magnesium ion concentration. The magnesium riboswitch in Salmonella enterica is a 161‐nucleotide sequence that exhibits conformational change upon magnesium binding. A 2.6 Å resolution crystal structure shows 6 magnesium‐RNA binding sites. This study used thermal denaturation and isothermal titration calorimetry to characterize the thermodynamic properties of Core 2, where two of the six magnesium ions are expected to bind. The Core 2 wildtype RNA construct conserves the adenine‐rich 4×4 internal loop sequence, including the magnesium‐binding sites A63 and A80. Modifications to positions 63 and 80 were also designed. All constructs were thermally denatured in 1 M KCl and 10 mM MgCl2 buffers at pH 5.5 and 7.0. In 1 M KCl, the RNA wildtype construct is more stable at pH 5.5 than at pH 7.0 by 1.5 kcal/mol, indicating a potential protonation‐dependent hydrogen bond. The wildtype gained ~3 kcal/mol of stability in 10mM magnesium ions as compared to 1M KCl in both pH buffers, as might be expected for a magnesium riboswitch. The magnitude of these interactions provides a benchmark for understanding the contributions of magnesium ions to RNA stability. Modifications of adenine at positions 63 and 80 also showed greater stability at the lower pH. These modified constructs showed a significant loss of the additional stabilization in magnesium over 1 M KCl at both pH conditions. Isothermal analyses of magnesium binding to RNA are also being performed. Thermodynamic examination of wild type and modified RNA constructs and their stability in magnesium ions will be presented.

Support or Funding Information

Grover Laboratory at Colorado College, Colorado College Department of Biochemistry, Colorado College Venture Grant, Crecelius Family Award for Outstanding Research in Biochemistry
Enhancement of Transgene Production in a Vaccinia Virus‐based Expression System
Authors: Richard Brent , Flores Julia , Jacobs Bertram , Chaput John , Wellensiek Brian
Published: November 2023

Poxvirus‐based vaccinations, i.e., vaccinia viruses (VACV), have been used more extensively for human immunization than any other vaccine in human history. VACV‐based vaccines are of particular interest due to the relative ease with which the VACV genome can be modified to express genes from a variety of sources. In recent years, there have been great advances in VACV‐based vaccines, including the generation of replication‐competent (non‐attenuated) and replication‐incompetent (attenuated) strains of VACV. While non‐attenuated VACV are highly immunogenic due to their ability to replicate in human cells, vaccines generated using these strains have higher than normal complication rates and are restricted in the population that can receive them. Attenuated strains lack the ability to replicate in human cells, resulting in a dramatic decrease in side effects, however this increase in safety comes at the cost of a reduced immune response. To address this issue, our current research is focused on increasing viral protein production in an attenuated VACV. In previous studies, we have discovered a human translation enhancing element (hTEE‐658) capable of serving as a VACV promoter. Characterization of this sequence has demonstrated that when this element is used in a VACV‐based expression system, it leads to increased protein expression levels over those driven by standard VACV promoters. Further optimization of expression levels driven by hTEE‐658, through varying the length of the transgene leader sequence, has resulted in the generation of a sequence element capable of driving high levels of protein production in an attenuated VACV system. These results provide a promising option for the production of safe, highly immunogenic VACVs that can be used to develop vaccines against a wide range of infectious diseases.

Support or Funding Information

Funding for this research was provided by Midwestern University intramural funds awarded to BPW and an NIH grant awarded to JCC
Phosphorylation of eIF2 directs multiple mechanisms of preferential translation for cell adaptation to environmental stress
Authors: Wek Ronald C , Young Sara K , Willy Jeffrey A , Fusakio Michael E
Published: November 2023

Phosphorylation of eIF2 (eIF2‐P) lowers protein synthesis to conserve resources and facilitate preferential translation of specific transcripts that promote adaptation to environmental stresses. Central to the mechanisms of preferential translation are upstream ORFs (uORFs) embedded within the 5′‐leaders of mRNAs, which serve as “bar codes” that help ribosomes determine the levels of translation of mRNA coding sequences (CDS) during eIF2‐P. We have been studying the functions of uORFs that demarcate translational control and the roles that these preferentially translated genes play in cell adaptation to stress. Preferentially translated GADD34 (PPP1R15A) and constitutively expressed CReP (PPP1R15B) each function to dephosphorylate eIF2‐P and restore protein synthesis. Both GADD34 and CReP mRNAs contain two upstream uORFs, and using biochemical and genetic approaches we show that distinct features of these uORFs are critical for differential CDS translation. In the absence of stress and low eIF2‐P, translation of an inhibitory uORF in GADD34 acts as a barrier by inefficient translation termination that prevents subsequent reinitiation at the GADD34 CDS. Induced eIF2‐P during stress directs ribosome bypass of the inhibitory uORF, leading to enhanced translation of the GADD34 CDS. By comparison, CReP expression occurs independent of eIF2‐P via an uORF that allows for translation reinitiation at the CReP CDS independent of stress conditions. Alterations in the GADD34 uORF not only affect the status of eIF2‐P and translational control, but also cell adaptation to stress. Translation of other preferentially translated genes also feature bypass of uORFs, although by mechanisms featuring different uORF properties. For example, bypass of a single uORF directs preferential translation of CHOP (DDIT3/GADD153), a transcription factor that triggers cell death during periods of extended stress. We discovered that the CHOP uORF serves to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP CDS. Deletion of the CHOP uORF increases the levels and alters the pattern of induced CHOP expression during eIF2‐P. Elevated CHOP expression leads to increased cell death in response to stress. Finally, we determined that bypass of uORFs in IBTKalpha enhances translation of the CDS in response to eIF2‐P. Elevated levels of IBTKalpha localize to endoplasmic reticulum exit sites and facilitate initiation of autophagy. These examples and others indicate that the proper mixing and matching of uORFs with distinct properties are central for directing gene‐specific translation during eIF2‐P. Preferentially translated genes are critical for the timing and extent of translation and transcription, and alter metabolism and reclamation processes such as autophagy, which together can determine cell survival during environmental stresses.

Support or Funding Information

NIH GM049164