bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical Analysis of a Patient with Fumarate Hydratase Deficiency
Authors: Berkmen Melanie Barker , Grocott Olivia , Phanor Sabrina K. , Fung France , Thibert Ronald L. 
Published: April 2020

Fumarate hydratase deficiency (FHD) is a rare autosomal recessive metabolic disease with approximately 100 patients reported worldwide. FHD presents in early infancy and clinical features include developmental delays, brain malformations, hypotonia, poor growth, and seizures. Severely affected individuals often do not survive past early childhood. FHD is caused by two defective copies of the FH gene, which encodes the Krebs cycle enzyme fumarase found in mitochondria and the cytosol. We describe the symptoms, treatment, and biochemical analysis of a patient with FHD. While infantile spasms are common in FHD, the patient presented with epileptic spasms later in childhood. Unexpectedly, the patient responded excellently to lacosamide for her seizures. To biochemically analyze the patient’s two fumarase variants, we introduced each of the mutations (E432Kfs*17 and D65G) into a plasmid harboring the recombinant human FH gene. We purified and characterized the fumarase variants alongside wild‐type fumarase. E432Kfs*17 was extremely defective for both catalysis and oligomerization. The large decrease in catalytic efficiency of E423Kfs*17 is due to a large decrease in turnover (~1500‐fold lower kcat). The novel variant D65G formed homotetramers and had a turnover (kcat) similar to wild‐type, but required ~2‐fold higher substrate concentration to achieve one‐half maximum velocity (2‐fold higher Km). We propose that the relatively mild defect of the D65G variant may have played a role in the patient’s milder clinical severity.Support or Funding InformationThis research was funded by Suffolk University.
Time Course of the Effects of Tributyltin Exposures on eIF4B, eIF4E, and S6 in Human Immune Cells.
Authors: Ruff Amanda , Brown Shyretha , Hamza Nafisa , Whalen Margaret 
Published: April 2020

Tributyltin (TBT) is an environmental contaminant due to its use in a variety of applications. It has been used to prevent the growth of fouling organisms on the hulls of ships and due to this use, as well as others, it has entered the food chain. It is found in human blood (ranging as high as 261 nM). Inflammatory cytokines are important mediators of the response to injury or infection. However, if their levels are increased in the absence of a needed immune response, they can lead to chronic inflammation that is associated with a number of pathologies including, rheumatoid arthritis, Crohn’s disease, atherosclerosis, and cancer. TBT can increase the synthesis of pro‐inflammatory cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL‐1β), and interleukin 6 (IL‐6) in human immune cells. TBT appears to utilize the ERK1/2 and/or p38 MAPK pathways to stimulate pro‐inflammatory cytokine production by immune cells. MAPK pathways have the capacity to regulate translation including processes leading to the phosphorylation (activation) of eukaryotic initiation factor 4E (eIF4E), eIF4B, and the S6 ribosomal subunit. The current studies examine the levels and phosphorylation state of eIF4E, eIF4B and S6K after 10 min, 1 h, 6 h, and 24 h exposures to TBT in monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). Initial results indicate that TBT caused increased phosphorylation (activation) of eIF4B (S406) and S6 within 10 minutes of exposure. These results suggest that TBT is elevating the synthesis of key pro‐inflammatory cytokines in immune cells by its ability to activate translation.Support or Funding InformationThis work was supported in part by grant 5U54CA163066 from the National Institutes of Health/National Cancer Institute.
Comparison of Double‐Stranded RNA Binding Motifs between Different Organisms
Authors: Brown Courtney L. , Macbeth Mark R. 
Published: April 2020

Transmission of genetic information from DNA to protein passes through a messenger‐RNA intermediate. This mRNA is extensively processed after transcription, and one enzyme that does so is
adenosine
de
aminase that acts on
RNA (ADAR). ADARs catalyze the deamination of adenosines into inosines on double‐stranded RNA (dsRNA) substrates, ultimately leading to a mutation in their encoded protein. These mutations are essential not only for a properly functioning nervous system, but for survival. The ADAR enzyme is composed of two domains: a catalytic domain and an RNA binding domain that consists of one or more double‐stranded RNA binding motifs (dsRBMs). Determining the structures of these dsRBMs could lead to insight on the substrate specificity observed in ADARs, as it is known they are not sequence specific. To do so, dsRBMs from various organisms were subcloned into an expression plasmid, expressed, purified, re‐folded, and will be crystallized to determine their 3D structure by X‐ray diffraction. The structures of these dsRBMs from different organisms will be compared for similarities and differences to gain understanding into the mechanism by which they recognize their specific substrates.Support or Funding InformationButler Summer Institute (CLB, Summer 2019)Holcomb Awards Committee Grant (MRM, 2019)
A High Throughput Approach to Biochemically Characterizing Firefly Luciferase for Use in Multi‐Component Bioluminescent Imaging
Authors: Reid-McLaughlin Edison Esme , Flanagan Lyndsey , Leconte Aaron 
Published: April 2020

Bioluminescent imaging (BLI) is a foundational tool for in vivo imaging routinely used to accurately visualize cells within mouse bodies and brains. However, these probes are currently limited in their ability to image multiple targets at once as well as by their brightness. While directed evolution is commonly used to improve these enzymes, it is a slow approach and is often limited by a lack of biochemical knowledge to guide amino acid changes. Here, to rapidly generate high‐value biochemical information on the enzyme luciferase, we have developed a high‐throughput platform to categorize substrate recognition and brightness for many possible amino acid changes in a single experiment. Our approach couples screening small libraries followed by next‐generation sequencing to rapidly create sequence‐function maps. As an initial foray, we screened all mutations at three amino acid positions—A482, N229, and G246; as expected, each position showed unique patterns for their impact on function. Generally, mutations at the 482 position resulted in either inactivation of the protein or no change or slight decrease in function, while mutations at the 229 position resulted in decrease in function and a large shift towards red light emission, and mutations at the 246 position resulted in inactivation of the protein except for a handful of mutants that showed increased functionality. In addition, two mutants, N229G and G246S, were observed to have an increase in brightness with a modified luciferin substrate accompanied by a small decrease in brightness with the natural substrate, resulting in a specificity shifted towards the analogous substrate without compromised brightness. Collectively, these data illustrate that our novel, high‐throughput approach is reliable in creating diverse libraries of mutants and detecting true enrichment in function. In addition, we have identified a handful of mutants at two of the three residues investigate here that have improved brightness with non‐native substrates. This approach can be used to rapidly build a sequence‐function map at any amino acid position, facilitating future protein engineering and improving our basic biochemical understanding of this important enzyme.Support or Funding InformationFunding for this research has been provided by the Research Corporation for Science Advancement.
Exploring the effects of environmental changes on bacteriophage infection potential
Authors: Gocal Jakub 
Published: April 2020

Bacteriophages are viruses which are capable of infecting and killing bacteria. They have become a topic of interest over the last decade due to their possible role in phage therapy, the treatment of antibiotic resistant bacteria using bacteriophages.Mycobacterium Phage MichaelPhcott is a B1 cluster bacteriophage, which was isolated from a soil sample collected in Woodland Park, Philadelphia. Bacteriophages were extracted from the soil sample. After multiple rounds of purification, a pure population of phage MichaelPhcott was isolated. Phage MichaelPhcott was one of twelve phages annotated by the 2017–18 Drexel SEA‐PHAGES students. Bioinformatic programs: DNA Master, Glimmer, Starterator, and GeneMark were used to identify gene start sites within the genomic sequence of phage MichaelPhcott. PECAAN software (including databases NCBI Blast, Phamerator, and HHPred) was used to identify and assign gene functions to each gene. MichaelPhcott was found to be 68,494 base pairs long, with a G/C content of 66.4%, and exactly 100 protein‐coding genes.Phage MichaelPhcott may have a potential application within antibiotic resistance. It was found that exposure to whey protein generated a protective effect and the phage is able to survive and infect bacteria when grown over a range of different pH levels and temperatures.Antibiotics usage has resulted in antibiotic‐resistant bacteria that negatively impact the agricultural industry. Whey protein concentrate (WPC) is a filtrate from cow’s milk that reportedly has protective effects on lactic bacteriophages when exposed to changes in heat and pH levels. The goal of this experiment was to see the protective effects of WPC on bacteriophage MichaelPhcott when exposed to varying levels of pH and heating time.Support or Funding Information&[bull]Bacteriophages are viruses which infect bacterial host cells and use the bacterial cellular machinery to replicate the viral genome and generate viral protein. The bacteriophage viruses assemble and then exit the bacterial cell, killing it. &[bull]Mycobacterium phages infect and replicate within Mycobacterium smegmatis, a bacteria which is commonly found in soil. &[bull]In this project Mycobacterium phage MichaelPhcott was isolated from soil collected in Woodland Park, Philadelphia. &[bull]Genomic sequencing and characterization of the phage MichaelPhcott genome will allow us to understand more about the genes present within the genome, this may provide information about the lifecycle of this phage and other genetically similar phages.
DEVELOPMENT OF 19F NMR AS A NOVEL TOOL TO STUDY P450 REDOX PROTEIN INTERACTIONS
Authors: Campomizzi Christopher Sante , Estrada D. Fernando 
Published: April 2020

Cytochromes P450 are a ubiquitous family of hemoproteins which, in humans, play an indispensable role in the metabolism of vitamins, steroids, and pharmaceutical compounds. All P450s rely on an interaction with a redox partner for catalytic activity; these interactions have also been shown to regulate P450 function. In order to study the interaction between the vitamin D metabolizing enzyme, CYP24A1, and its redox partner, adrenodoxin (Adx), our group has previously relied on Nuclear Magnetic Resonance (NMR) titration of CYP24A1 against 15N labeled Adx. This approach allows for a 1:0.2 Adx:P450 complex to be studied before the NMR signals are broadened beyond detection. The application of NMR to study mammalian P450s is limited by the relatively large size of these proteins and their tendency to form multimers in solution. Nevertheless, this technique has revealed important details of this interaction that has led us to hypothesize that substrate may modulate the interaction between the two proteins.In order to overcome the limitations posed by traditional NMR nuclei, we have employed the NMR active nucleus, 19F. This nucleus is more sensitive than traditional NMR observable nuclei and is not naturally occurring in proteins, which drastically reduces the complexity of the spectra. Using the cysteine reactive label 3‐bromo‐1,1,1‐trifluoroacetone (BTFA), we have attached an 19F probe to helices 1 and 3 of Adx. Upon titrating ketoconazole‐bound CYP24A1 into 19F labeled Adx, our binding data show differential line broadening on helix 3, consistent with other studies implicating this helix as the P450 recognition site. Interestingly, we also observe changes in heterogeneity of the signal at the recognition site, suggesting that this complex undergoes conformational selection upon binding. Future studies include the monitoring the effect substrate has on the protein interaction, as well as labeling sites of interest on the P450 itself.Support or Funding InformationNIH R35GM133375
In Vitro Assay for the Characterization of RquA
Authors: Monlux Melina May , Jacobs Evan , Roger Andrew , Langelaan David , Cronk Jeff , Shepherd Jennifer 
Published: April 2020

To survive in low‐oxygenic environments, microbial organisms employ anaerobic respiration. In certain organisms, the terpenoid quinone known as rhodoquinone (RQ) plays a key role in this process, though few details are known about how RQ itself is produced or what impact it may have on the cellular respiration process from a biomedical standpoint. Investigations into the rquA gene product (RquA), an enzyme that is required for the production of RQ in Rhodospirillum rubrum and other microbial species, and its role in anaerobic energy metabolism may provide a new target for antibacterial drug treatments through inhibition. Previous findings have shown that the in vivo expression of RquA results in the production of RQ in two species that do not naturally produce it, E. coli and yeast. RquA is responsible for the conversion of ubiquinone (Q) to RQ but it is not clear what additional cofactors are required for the conversion. Using in vitro assays with synthetic Q, purified RquA or whole cell lysate, potential amino donors, and a variety of co‐factors such as S‐adenosyl methionine, we investigated the essential chemical components to perform this conversion. With these results, potential inhibition pathways may be identified and further investigated through assays and visualization methods such as X‐ray crystallography.Support or Funding InformationNew Frontiers in Research Fund, Gonzaga Science Research Program, CURCI
Chronic‐Low Dose Cadmiun Exposure Alters Sperm DNA Methylation in Mice
Authors: Saintilnord Wesley Nuack Gesner , Tenlep Sara , Pearson Kevin , Fondufe-Mittendorf Yvonne 
Published: April 2020

Millions of people are exposed to cadmium and chronic‐low dose exposure has been linked to increased risks of breast and endometrial cancer, osteoporosis, renal dysfunction, cardiovascular disease and reproductive defects. One possible mechanism is dysregulation of gene expression through oxidative stress. Since cadmium is not a mutagen, we hypothesize that it could also exert its effects through epigenetic reprogramming, modulating regulatory targets with consequences in gene expression. Studies have reported that these dysregulations in gene expression is transgenerational; however these studies have focused heavily on maternal exposure while the impact on sperm and fertility has received very little attention. This discrepancy is due to the long‐held belief that, DNA methylation, one of the most stable epigenetic marks, is erased during fertilization. Recent evidence now suggests that at least some methylation marks are retained and can be passed on to the next generation. Here, we investigate the impact of chronic‐low dose cadmium exposure on DNA methylation patterns in sperm using a mouse model and an environmentally relevant cadmium dose. We hypothesize that chronic‐low dose exposure to cadmium modifies DNA methylation patterns in sperm leading to developmental effects. Our preliminary data shows that exposure to cadmium did not cause any histological changes to the testes of cadmium‐treated compared to control animals. However, using the 5‐mC DNA ELISA method we demonstrated a global decrease in DNA methylation in our treatment group. Additionally, Reduced Representation Bisulfite Sequencing (RRBS) which measures DNA methylation changes at specific genomic loci, further validated the impact of cadmium exposure on DNA methylation. A stringent statistical cut‐off (q<0.01 with at least 25% difference between the groups and at least 10X reads), showed that chronic‐low dose cadmium exposure leads to 1,788 differentially methylated sites. Importantly, some of these sites, which we are now validating, were at promoters and are likely to have functional consequences in gene expression. Our results show for the first time that mice sperm methylome is altered in response to cadmium exposure while the testes integrity remain intact. Our data highlight a foundational framework to study gene expression patterns that may affect offspring in a paternal inheritance context.Support or Funding InformationUK‐CARES Grant P30 ES026529
Gene Expression Regulation of the Type I Interferon‐Induced Protein Schlafen 11
Authors: Borrego Andre Raymundo , Corona-Ayala Christian , Salvador Julienne Christa , Valdez Federico Christa , Llano Manuel 
Published: April 2020

We have recently discovered a major role of the type I interferon (IFN‐I)‐induced protein Schlafen (SLFN) 11 in the control of flavivirus infection and verified its effects on HIV‐1. This protein is also relevant in controlling chemosensitivity of cancer in patients, and animal and cellular models. Therefore, understanding the mechanisms regulating SLFN11 expression during infection and chemotherapy is relevant. We have found that infection of human cells by the flavivirus West Nile virus (WNV) or HIV‐1 upregulated the expression of SLFN11. Characterization of the pathways implicated that while WNV increases SLFN11 expression via IFN‐I signaling, HIV‐1 uses an IFN‐I‐independent pathway. To further map the latter signaling pathway, we evaluated the ability of a panel of inflammatory stimuli to induce SLFN11 in primary CD4+ T cells isolated from two healthy donors. Interestingly, phorbol myristate acetate (PMA), ionomycin, and phytohemagglutinin (PHA) potently induced this protein in both donors. These compounds also upregulated SLFN11 expression in cell lines of different histological origin. However, both IFN‐I‐dependent and ‐independent pathways failed to stimulate expression of a 1.3 kb promoter cloned in a gene reporter system, suggesting that this promoter lacks cis‐acting regulatory elements that are relevant in the regulation of SLFN11 expression in cells. In summary, our data indicate that SLFN11 expression is controlled via IFN‐I‐dependent and ‐ independent pathways.Support or Funding InformationResearch Initiative for Scientific Enhancement funded by a grant (#R25GM069621‐11) from the National Institute of General SciencesUniversity of Texas at El Paso
Synthesis, characterization and antibacterial activity of metal complexes of Ni(II) with 7‐iminobenzo[b]chromeno[4,3‐e][1,4]diazepin‐6(7H)‐one
Authors: Odai Sevdije Govori , Haziri Arben , Daci Majlinda , Ibrahimi Hamide 
Published: April 2020

Two novel metal complexes from 7‐iminobenzo[b]chromeno[4,3‐e][1,4]diazepin‐6(7H)‐ones with Ni (II) were synthesized. The ligands and their metal complexes have been characterized by IR, 1H‐NMR, 13C‐NMR, Mass spectra and Elemental analysis. The synthesized ligands and metal complexes were screened for microbiological activity against Escherichia coli ATTC 8739 and Staphylococcus aureus ATTC 25923. The results showed that the metal complexes were found to be more active than free ligands.Support or Funding InformationSupport for this study was provided by the Ministry for Education, Science and Technology of the Republic of Kosovo. Grant number:2‐5521‐2
SAXS‐enabled Macromolecular Engineering at SIBYLS
Authors: Hodge Curtis D. , Hura Greg L. 
Published: April 2020

Macromolecular engineering provides novel opportunities in many fields. However, progress can be slow when structural analysis relies on low‐throughput structural techniques. Employing unique and high‐throughput X‐ray scattering (SAXS) tools, we have engineered a Self‐Assembling Protein Nanoparticle to display Antibodies (SAPNA) with application in CAR T Cell therapy. Adoptive CAR T Cell therapy is a highly effective, but extremely expensive new cancer treatment with costs in the range of ~$1 million per patient (hospital fees included). The therapy requires >100 million CAR T cells per infusion per patient. A key step in the CAR T manufacturing process is polyclonal expansion of the patient‐ or donor‐derived T cells. This is currently achieved through CD3/CD28 T cell receptor stimulation by agonist antibodies (either soluble or chemically linked to magnetic beads). We have shown our engineered SAPNA solution is highly effective relative to existing technologies at expanding CAR T cells with potential application in fighting cancer. High‐Throughput SAXS (HT‐SAXS) was an integral technique that enabled structural insights into engineered constructs. A crucial step was monitoring macromolecular changes in response to mutations and solution conditions. We also utilized Size Exclusion Chromatography SAXS (SEC‐SAXS) to assess the antibody display, while providing important information on the structural dynamics of the antibodies themselves. Herein, we detail the SAXS platform (sibyls.als.lbl.gov) we used to rapidly converge on our functional SAPNA and SAPNA’s potential application in CAR T Cell therapy.Support or Funding InformationUS DOE‐BER, Integrated Diffraction Analysis Technologies (IDAT) program. Additional support from NIGMS project ALS‐ENABLE (P30 GM124169) and a High‐End Instrumentation Grant S10OD018483.
Binding, Release, and Detection of Ligands by Ultrabithorax Protein‐Based Materials
Authors: Booth Rebecca , Churion Kelly , Bystroff Chris , Bondos Sarah 
Published: April 2020

Proteins can bind, release, and detect molecules with unparalleled specificity. Incorporating proteins into materials can confer the ability to recognize specific ligands. We have leveraged the ability of materials composed of the Drosophila transcription factor Ultrabithorax (Ubx), a partially intrinsically disordered protein, to bind ligands, with the goals of delivering DNA to cells and creating biosensors. DNA binding and delivery is accomplished via the Ubx homeodomain, which retains its ability to recognize specific DNA sequences in Ubx materials. Ubx materials reversibly bind DNAs harboring the Ubx target DNA sequence and can protect these DNAs from degradation. DNA sequences with multiple binding sites have a slower rate of release from Ubx fibers. Ubx fibers can deliver DNA to transform E. coli, sustaining DNA release for up to 60 days.To build a biosensor, an additional protein (LOO8GFP) is incorporated into the materials via gene fusion. In this approach, the loo8gfp gene is placed adjacent to the ubx gene without intervening stop codons. When transformed into bacteria, this fused gene produces a LOO8GFP‐Ubx fusion protein. The intrinsically disordered regions of Ubx enable LOO8GFP incorporation without disrupting materials assembly or structure. The proof‐of‐concept sensor system is leave‐one‐out GFP (LOO8GFP), in which the 8th β‐strand has been ‘left out’ of circularly permuted GFP. Without this β‐strand the GFP is unable to fold properly and is not fluorescent. Fluorescence can be reconstituted with the addition of the left‐out peptide, making this construct a self‐reporting biosensor for the missing piece of its sequence. However, LOO8GFP can oligomerize, which (i) hampers ligand binding and (ii) leads to fluorescence in the unbound state. Ubx offers a transparent support system for LOO8GFP that maintains LOO8GFP stability and binding affinity while preventing LOO8GFP aggregation. To assess the utility of this composite material, we are examining assembly of LOO8GFP‐Ubx fibers, measuring binding affinities, and quantifying on‐ and off‐rates for the left‐out peptides. In the long term, the LOO8GFP‐Ubx system can be engineered to bind and detect other peptide sequences. Thus, Ubx materials can be engineered for variety of potential functions and uses based on incorporating bioactive DNAs and/or proteins.