bluuebunny/crossref_metadata_2025_split huggingface dataset:
The effect of a 4th year anatomy teaching course on anatomical knowledge and teaching confidence (533.2)
Authors: Lufler Rebecca , Stefanik Joshua 
Published: June 2021

There has been a push by the Liaison Committee on Medical Education (LCME) and Accreditation Council for Graduate Medical Education (ACGME) to integrate the basic sciences into the 4th year medical curriculum and inculcate medical students with teaching skills. The objective of this study is to determine the effectiveness of a 4th year medical school elective course focused on teaching gross anatomy to address these concerns.4th year medical students enrolled in the course were administered a pre‐test to assess their gross anatomy knowledge and a survey to assess their confidence in gross anatomy knowledge and teaching skills. After a 4‐week course involving dissection and teaching 1st year medical students, they were given the same confidence survey and a post test assessing the same anatomical concepts using different questions.11 students completed the course, 81.8% (9) were male and 18.2% (2) were female. The mean age (standard deviation) of the students, anatomy knowledge pre‐test score and post‐test score was 27 (2.2), 53.9 (15.7) and 88.2 (7.2), respectively. The post‐test score was significantly higher than the pre‐test score (p <0.001). The median anatomy confidence before and after the course was 8.0 and 8.0, respectively. The median teaching confidence before and after the course was 8.0 and 9.0, respectively.Based on these preliminary results, a 4th year medical school elective course focused on teaching gross anatomy appears to increase anatomy knowledge but not measures of self‐reported confidence in teaching and anatomy knowledge.Grant Funding Source: Supported by Tufts University School of Medicine Innovations in Education Intramural Grant
Face and content validity of three assessment tools developed to evaluate cerebral angiography performance (536.4)
Authors: Nguyen Ngan , Eagleson Roy , Boulton Mel , de Ribaupierre Sandrine 
Published: June 2021

Cerebral angiography (CA) is considered the gold standard for diagnosing primary neurovascular diseases. Although guidelines have been established to ensure adequate CA experience before independent practice, current tools used to assess CA performance during training have questionable validity and reliability. The initial phases of validation often involve determining whether the tool appears to measure the construct of interest (face validity; FV) and whether the items in the tool cover a representative sample of the construct (content validity; CV). This study proposes to establish the FV and CV of 3 assessment tools (task‐specific checklist, error‐based scale, and global rating scale) developed to evaluate CA performance. FV and CV were established by asking expert catheter‐based physicians to judge whether each tool appears to be a practical and pertinent measure of CA performance and to evaluate the appropriateness of each tool item. Based on experts’ recommendations, each tool was modified to improve its ease‐of‐use, organization, and clarity. Confirmation of the FV and CV of the 3 tools will contribute to ensuring that future assessment of CA performance is both accurate and consistent.Grant Funding Source: Supported by MITACS
CCN2 induction by TGF‐β1 requires osteoblast specific promoter motifs (541.6)
Authors: Martinkovich Stephen , Silverstein Rachel , Geisinger Max , Planey Sonia , Arnott John 
Published: June 2021

Connective tissue growth factor (CCN2; formerly CTGF) is a 38kDa, cysteine rich, extracellular matrix protein that belongs to the CCN family of proteins. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF‐B1 and acts as a mediator of TGF‐B1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of CCN2 promoter motifs involved in CCN2 induction by TGF‐B1 in primary osteoblasts. We used a combination of luciferase promoter reporter assays and gel shift studies to demonstrate the novel requirement of both the TGF‐B1 response element (TRE) and Smad binding element (SBE) for CCN2 promoter trans‐activation in osteoblasts. We propose that the TRE and SBE function as cognate motifs for CCN2 induction by TGF‐B1 in an osteoblast specific manner.Grant Funding Source: Supported by NIAMS
The type III TGFβ receptor is required for hyaluronan‐mediated epicardial invasive cell motility (542.5)
Authors: Allison Patrick , Espiritu Daniella , Barnett Joey , Camenisch Todd 
Published: June 2021

The epicardium originates from the proepicardial organ (PEO). Both the PEO and epicardium require directed cell‐motility to cover the heart and instruct epicardially derived cells for coronary vasculature formation. The type III transforming growth factor‐beta receptor (Tgfbr3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(‐/‐) e13.5 mouse embryos , we show that Tgfbr3(‐/‐) epicardial cells are 34.4% deficient in 2D migration relative to wild type. Furthermore, we show that high‐molecular weight Hyaluronan (HMWHA) drives 3D cellular invasion in wild type epicardial cells, but not Tgfbr3(‐/‐) epicardial cells. We show Src kinase is required for HMWHA stimulated filamentous actin (f‐actin) polymerization in wild‐epicardial cells. Furthermore, HMWHA stimulates Y416Src phosphorylation in wild type, but Src is not activated in Tgfbr3(‐/‐) epicardial cells. RhoA and Rac1 GTPase are downstream of Src kinase, and directly upstream of f‐actin polymerization; HMWHA robustly induces RhoA and Rac1 GTP binding and activity in wild type, but not Tgfbr3(‐/‐) epicardial cells. This deficit in RhoA and Rac1 activation results in reduced formation of f‐actin stress fibers in HMWHA stimulated Tgfbr3(‐/‐) epicardial cells. Taken together, we demonstrate that the type III TGFβ receptor is required for activation of basic cell motility pathways stimulated by Hyaluronan that are required for proper coronary vasculature formation.Grant Funding Source: NIH ES 04940; ES06694; SWEHSC P30ES006694
To activate or destroy: regulation of the RNA Pol II elongation factor and ubiquitin ligase Elongin (550.3)
Authors: Weems Juston , Hall Shawn , Slaughter Brian , Unruh Jay , Gilmore Joshua , Washburn Michael , Florens Laurence , Conkright Julianna , Weems Lauren , Conaway Joan , Conaway Ronald 
Published: June 2021

Lesions induced by a variety of DNA damaging agents can block RNA polymerase II (Pol II) elongation. Mechanisms that reactivate or remove Pol II that has become stalled or arrested at such lesions are critical for resumption of gene expression after DNA damage repair and, in addition, may contribute to efficiency of the DNA repair process by enhancing access of the damage repair machinery to the lesion. Following exposure of cells to UV irradiation or other DNA damaging agents, arrested Pol II is ubiquitinated and degraded by the proteasome.Elongin was first identified and purified showing an activity that stimulates the rate at which Pol II elongates nascent transcripts in vitro and is composed of 3 subunits, Elongin A (EloA), B (EloB), and C (EloC). Although the majority of Elongin A isolated from cells contains the three subunit Elongin complex, it can also be linked to a heterodimeric module composed of the Cullin family protein Cul5 and the RING finger proteins Rbx1 or Rbx2 to form a larger complex that targets stalled Pol II for ubiquitination. A previous study used indirect immunofluorescence to provide evidence for increased nuclear colocalization of EloA and Cul5 after UV‐induced DNA damage, raising the possibility that DNA damage may promote assembly of the EloA ubiquitin ligase complex. Using a combination of MudPIT‐based mass spectrometry and FRET‐based approaches, we have obtained evidence that supports this model. In ongoing studies, we seek to define mechanisms by which stalled Pol II regulates assembly and activity of the Elo A ubiquitin ligase complex.
Comparative mutagenicity amongst c‐family bacterial DNA pol III’s (551.2)
Authors: Morris Justin , Mukherjee Purba , Pata Janice 
Published: June 2021

As evidence points to the use of multiple DNA pol III’s by gram‐positive bacteria, a question remains: which biochemical factors (domain composition, DNA oligonucleotide (oligo) length, etc.) serve as the strongest determinants of fidelity? We have characterized various patterns of relative mutagenicity amongst the essential C‐family bacterial DNA polymerases found in Staphylococcus aureus. These include two PolC constructs: PolC‐Exomut and PolC‐ΔNΔExo, as well as DnaE. In vitro assays were performed using various primer‐template (p/t) DNA substrates and nucleotides. Additional assays paired the polymerase with the β clamp. DnaE showed the highest frequency of nucleotide misincorporation, especially on longer p/t DNA substrates, as well as in bypassing 8‐oxoguanine lesions. PolC‐Exomut, the construct with both N‐terminal and Exonuclease (Exo) domains, consistently showed the most fidelity. A direct relationship between fidelity and DNA substrate length was also observed. The presence of secondary proteins consistently increased overall addition, but not fidelity. Overall, these assays indicate DnaE as a more error‐prone polymerase compared to its PolC counterparts. Additionally, oligo length was shown to influence the accuracy of nucleotide addition. Future assays will further examine the effects of oligo length as well as fidelity patterns specifically attributable to the N‐terminal domain.Grant Funding Source: Supported by NIH Grant R01‐GM080573
Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets R‐X‐R sites in lysine and arginine‐rich regions (555.1)
Authors: Feng You , Clarke Steven 
Published: June 2021

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remains controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω‐monomethylarginine residues and confirm its activity as the prototype type III PRMT. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post‐translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine‐ and arginine‐rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (R‐X‐R motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.Grant Funding Source: Supported by NIH R01GM026020
A new family of lysine methyltransferases preferentially targets molecular chaperones to orchestrate the functional organization of the human proteome (555.12)
Authors: Cloutier Philippe , Coulombe Benoit 
Published: June 2021

In efforts aimed at identifying and characterizing protein substrats and regulators of all known and putative human methyltransferases using cell compartment affinity purification coupled with mass spectrometry (MCC‐AP‐MS), our laboratory has discovered a novel family of lysine methyltransferases that preferentially target and regulate molecular chaperones. Our results indicate that methylation of molecular chaperones VCP by METTL21D and Hsp70 by METTL21A regulates the activity of both chaperones. In the case of VCP, trimethylation of lysine 315 by METTL21D is stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G and R191Q) known to cause Inclusion Body Myopathy with Paget's disease of bone and Fronto‐temporal Dementia (IBMPFD) and/or some familial forms of Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCP's first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts the ATPase activity of this molecular chaperone. In the case of Hsp70, methylation of lysine 561 by METTL21A was shown to decrease formation of stress granules in response to arsenite treatment, a model that is widely used to study improper protein folding in degenerative disorders. In addition to having implications for the development of therapeutics for degenerative neuromuscular disorders such as ALS and IBMPFD, the discovery of a family of chaperone‐targeting methyltransferases led us to propose the existence of a chaperone posttranslational modification code, that we termed the “chaperone code”, which is at play to orchestrate the proper folding and assembly of protein complexes that make up the human proteome. We will report on our most recent results relating to decryption of the chaperone code and its role in disease.Grant Funding Source: Muscular Dystrophy Association
Arkadia mediates sumoylation‐dependent degradation of Nrf2 in promyelocytic leukemia‐nuclear bodies (555.5)
Authors: McIntosh Deneshia , Arinze Ifeanyi 
Published: June 2021

The cellular abundance of the transcription factor Nrf2 is largely controlled by Keap1, an inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf2 by the Cullin3/RING box1 (Cul3‐Rbx1) E3 ubiquitin ligase complex, thereby targeting Nrf2 for proteasome‐dependent degradation in the cytoplasm. It is becoming increasingly apparent that degradation of Nrf2 can also occur by Keap1‐independent mechanisms. Recently, we showed that Nrf2 is a SUMO‐modifiable protein and that polysumoylated Nrf2 can be polyubiquitylated by the poly‐SUMO‐specific E3 ubiquitin ligase known as RING finger protein 4 (RNF4) and subsequently degraded in PML‐NBs. The role of sumoylation in the turnover of Nrf2, independent of Keap1, is a novel aspect of the biology of Nrf2. Here, we explored whether sumoylated Nrf2 is a substrate for Arkadia, another polySUMO‐specific E3 ubiquitin ligase (also known as RING finger protein 111 [RNF111]). Our results indicate that Arkadia can ubiquitylate polysumoylated Nrf2 in PML‐NBs, suggesting that Arkadia can target polysumoylated Nrf2 for degradation in these nuclear domains. This work provides support to the idea that post‐translational modification of Nrf2 by sumoylation impacts the turnover of Nrf2 in the nucleus.Grant Funding Source: Supported by NIH grants # SC1CA143985, 5T32HL007737, and 2 SD1MD000104
Sgtα: a novel independent regulator of steroid hormone receptors (567.8)
Authors: Gutierrez Omar , Garcia Yenni , Cox Marc 
Published: June 2021

Steroid hormone receptors (SHRs) are essential for the regulation of developmental, reproductive, and physiological homeostasis in eukaryotes. SHRs belong to the nuclear receptor superfamily, and comprise the androgen (AR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and estrogen receptors (ER). Understanding the regulation of SHRs is critical for the development of novel therapeutic strategies for SHR‐mediated diseases. Heat shock protein (Hsp) 90/70 kDa assembly is essential for the dynamic chaperone complex that directs SHRs folding. A recently identified cochaperone, human small glutamine‐rich TPR‐containing protein alpha (SGTα), has been shown to negatively regulate AR by retaining AR in the cytoplasm. Using functional yeast and mammalian reporter assays, we have demonstrated that SGTα can also regulate GR and PR. Interestingly, when co‐expressed with immunophilin FKBP52, a positive regulator of the same receptors, SGTα continues to abrogate receptor activity. Another TPR‐containing protein present in yeast, CPR7, can positively regulate AR. Thus, we assessed the effects SGTα has in a yeast model system using a CPR7 deletion strain. We observed the same pattern seen with FKBP52, suggesting that the regulation by SGTα is not solely based on its TPR domain. Future studies aim to determine the role for SGTα at the transcriptional level.Grant Funding Source: Supported by the NIH/SCORE grant from the NIGMS (1SC1GM084863) and COURI
Characterizing an engineered release factor capable of reading all three stop codons (569.2)
Authors: Korkmaz Gürkan , Lind Christoffer , Åqvist Johan , Sanyal Suparna 
Published: June 2021

Translation termination is the last step of protein synthesis where the newly synthesized protein is released from the ribosome. For this, a class I release factor (RF1 and RF2) in bacteria binds to one of the three stop codons (UAA, UAG and UGA) and hydrolyze the ester bond between nascent protein and the peptidyl‐tRNA. RF1 and RF2, although similar to each other, read the stop codons in a semi specific manner i.e. RF1 reads UAA and UAG, and RF2 reads UAA and UGA codons. According to current literature [1] these factors discriminate highly against sense codons. Although molecular dynamics simulation experiments [2] have proposed a network of interactions through which RF1 and RF2 recognizes their respective stop codons, there exist very little biochemical experiments to explain how this high specificity is achieved.Using site directed mutagenesis we generated several variants of RF1 and RF2 and characterized those in competition and kinetic assays. One of our engineered release factors showed omnipotent property in stop codon recognition. We have characterized this engineered release factor with respect to other related codons especially in comparison with other release factors. Further we were able to validate our biochemical experiments using computer simulations. Our experiments pinpointed the crucial residues on the class‐I release factors for stop codon specificity and enabled us to engineer a class I release factor capable of reading all three stop codons.Grant Funding Source: Supported by; Swedish Research Council, Sven and Lilly Lawski Foundation
In vivo receptor mediated near‐infrared transferrin uptake using fluorescence lifetime fret: implications in the targeted drug delivery (58.9)
Authors: Rajoria Shilpi , Zhao Lingling , Intes Xavier , Barroso Margarida 
Published: June 2021

Traditional cancer therapy generally leads to harmful side effects, thus warranting development of targeted therapies which are better tolerated by cancer patients. The main objective of current study is to develop unique in vivo non‐invasive optical imaging assays for the optimization of targeted therapy and diagnostic tools. We propose to quantify the internalization of therapeutic drugs conjugated to transferrin (Tfn), the native ligand of transferrin receptor (TfnR) using a Förster Resonance Energy Transfer (FRET) based imaging technology in vivo. Quantification of in vivo FRET donor % (FD%) in tumor xenografts, indicates a higher proportion of FD% with increasing acceptor:donor ratio demonstrating tumor uptake of NIR‐Tfn. Immunohistochemistry analysis of tumors suggests that the injected NIR‐Tfn is translocated to the tumor site. In conclusion, our novel NIR FRET assay successfully demonstrates a quantitative receptor‐mediated uptake of Tfn in human breast tumors in vivo, allowing for the development of Tfn‐based targeted therapy by means of not only delivering the drug at target site but also potentially block the iron‐internalization function of TfnR, thus directly killing the cancer cell. More importantly, our data supports the quantitative accuracy and sensitivity of a novel NIR FLIM FRET imaging assay to be used non‐invasively through living tissues in a small animal model.Grant Funding Source: National Institutes of Health grant R21 CA161782‐01
Biochemical characterization of 2, 3‐butanediol dehydrogenase from Taiwanofungus camphorata (583.2)
Authors: Wen Lisa , Tsai Wei‐Wei , Huang Jenq‐Kuen , Lin Chi‐Tsai 
Published: June 2021

The enzyme 2,3‐Butanediol dehydrogenase (BDH) plays important roles in reduction of acetoin to 2,3‐butanediol which is an important platform chemical with many industrial applications. Here, a TcBDH cDNA (1348 bp) encoding a putative BDH was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is similar to the BDHs from other species. A 3‐D structural model of TcBDH has been constructed based on the known structure of Pseudomonas putida formaldehyde dehydrogenase (PpFdh, PDB code 1KOL). To characterize the TcBDH protein, the coding region was subcloned into an expression vector pYEX‐S1 and transformed into Saccharomyces cerevisiae. The recombinant His6‐tagged TcBDH was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. The purified enzyme showed a single band of ~50 kD on 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (KM) value of the recombinant enzyme for acetoin was 8.46 mM. The enzyme’s optimal pH was 6.Grant Funding Source: National Science Council of the Republic of China, Taiwan under grant NSC 100‐2313‐B‐019‐003‐MY3
Biochemical characterization of a functional recombinant aryl‐alcohol dehydrogenase from Taiwanofungus camphorata (583.4)
Authors: Huang Jenq‐Kuen , Chang Che‐Chi , Ken Chuian‐Fu , Wen Lisa , Lin Chi‐Tsai 
Published: June 2021

A TcAAD cDNA encoding a putative aryl‐alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata (Tc). The deduced amino acid sequence is conserved among the reported AADs. A 3‐D structural model of the TcAAD has been created based on the known structure of voltage‐dependent potassium channels subunit beta‐2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6‐tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12 % SDS‐PAGE. The molecular mass determined by MALDI‐TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied.Grant Funding Source: National Science Council of the Republic of China, Taiwan under grant 100‐2313‐B‐019‐003‐MY3
High throughput screening of natural products for LDH inhibition as an anti‐mitotic therapeutic target (585.3)
Authors: Deiab Shihab , Mazzio Elizabeth , Zarmouh Najla , Mack Nzinga , Soliman Karam 
Published: June 2021

A pathological feature hallmark of solid tumors is an over‐functional energy metabolism which results in high level production of lactic acid from glucose. The key enzyme in the production of lactate is LDH, which is highly expressed in tumor tissue and regulates substrate utilization of pyruvate to form lactate. In this study, LDH isolated from human muscle was screened using over 905 plant derived extracts to discover natural products [0.0005‐.77mg/ml] with capability to inhibit LDH. Extracts were tested using an enzyme micro‐array format and extracts containing inhibitory properties were then ranked according to their IC50's The data clearly showed that the most potent plant based LDH inhibitor was Rhus chinenesis. Through bioactivity guided chemical separations and identifications as well as kinetic and docking studies, we showed that pentagallolyl glucose, PGG, is the entity responsible for the non‐competitive inhibition of LDH‐A. We further examined several cancer cell lines viability and lactic acid production in the presence of PGG and found a decrease in viability consistent with the decrease in lactic acid production. However further studies will be required to assess if and what role inhibiting the LDH enzyme can provide in terms of adaptability and survival mechanisms involved with energy metabolism of tumor cells.Grant Funding Source: Supported by NIH grants from NIMHD G12 MD007582 and P20 MD006738