bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Hepatogenic Differentiation of Human Adipose Tissue‐derived MSCs Using a Silicone‐based Cell Culture System
Authors: Stock Peggy , Hildebrand Janis , Böttcher Gunter , Christ Bruno
Published: April 2020

Conventional two‐dimensional as well as established three‐dimensional cell culture systems exhibit a high modulus of elasticity, yet, without representing the stiffness of human organs. Previously, we established a protocol for the culture of primary rat hepatocytes on a silicone of low stiffness, which sustained their physiological functions. In terms of the development of human‐based tests to evaluate the potential risk of novel drugs, the method was adjusted for use of human adipose tissue‐derived mesenchymal stromal cells (hAT‐MSC). Aim of the study was to improve hepatogenic differentiation of hAT‐MSC on fibronectin‐coated 3D silicone scaffolds with low stiffness (E‐modulus approx. 250 kPA) mimicking the stiffness of human liver including surface modifications to increase hydrophilic properties.

hAT‐MSCs were cultured on 3D silicone scaffolds. Hepatogenic differentiation was initiated by the 2 step protocol. The capability of the hepatocyte‐like cells to store glycogen, to synthesize urea and albumin as well as the enzyme activities of CYP1A1 and Cyp2B1/2 were analysed and compared to the conventional 2D cell culture and hepatogenic differentiation on polystyrene (E‐modulus 103 MPa).

hAT‐MSC attached to the 3D silicone scaffolds and formed cell clusters. 10 days after starting the hepatogenic differentiation until the end of the culture on day 24, the cells on 3D silicone scaffolds displayed more intensive PAS staining for glycogen as compared to the cells on 2D cultures indicating improved glycogen storage. The urea synthesis rate [nmol/10.000 cells/24h] of these cells was significant higher as compared to cells on 2D culture throughout the culture period, though declining with ongoing culture; 10 d: 2D vs 3D 0.05±0.01 vs 1.68±0.05 and 24 d: 0.03±0.007 vs 0.4±0.04, respectively. The albumin synthesis rate [pg/10.000 cells/24h] was significant higher in cells differentiated for 24 d as compared to undifferentiated cells: 48.5±11 vs 7.4±1.9. Enzyme activity of CYP1A1 after 24 d was 3‐fold higher in cells differentiated on 3D silicone scaffolds as compared to cells cultivated on 2D and the enzyme activity of CYP2B1/2 was 12‐fold higher.

With these results, medically approved silicone treated with plasma was identified to improve hepatogenic differentiation of hAT‐MSC, likely by providing the appropriate three‐dimensional growth conditions. Thus, the silicone scaffolds represent an important tool for the development of cell cultures featuring organ‐like stiffness and improving physiological functions of hepatogenic differentiated MSC.

Support or Funding Information

The study was supported by the European Regional Development Fund and the Free State of Saxony (SAB) 100282466 to J.H. and P.S.
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 Information

This 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 Information

This 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 Information

Butler Summer Institute (CLB, Summer 2019)Holcomb Awards Committee Grant (MRM, 2019)
Neuronal Calcium Sensor 1 (NCS1) as a Potential Drug Target for Treatment of Wolfram Syndrome
Authors: Fischer Tom T. , Nguyen Lien D. , Ehrlich Barbara E.
Published: April 2020

Wolfram syndrome (WS) is an orphan, autosomal recessive neuroendocrinological disease that affects approximately 1 in 500,000 people worldwide. Patients develop diabetes mellitus, diabetes insipidus, optical atrophy, and hearing loss and usually die in their 30s. The majority of cases are attributed to mutations in a single gene, WFS1, which encodes for the protein wolframin. Despite the known genetic cause, there is currently no direct treatment for WS. This lack of therapy is because the regular functions of wolframin, and the pathophysiological consequences following the loss of intact WFS1, remain elusive. Here, we further examined the function of WFS1 in the context of glucose toxicity, to address the earliest diagnosed symptom of WS which is the onset of diabetes mellitus near age 6. Based on a recent study
[1], we aimed to show that WFS1 interaction with a calcium binding protein, neuronal calcium sensor 1 (NCS1), is important for its normal functions. NCS1 is known to regulate exocytosis, promote cell survival, and maintain calcium homeostasis. We showed that knocking out WFS1 in rat insulinoma (INS1) cells resulted in increased baseline calcium, reduced ATP‐evoked inositol‐trisphosphate receptor (InsP3R)‐dependent calcium response, reduced phospho‐Akt (Ser473), and increased vulnerability to high glucose treatment. Furthermore, both INS1 control (CTRL) and WFS1 knockout (KO) cells showed increased NCS1 mRNA following high glucose treatment. However, only the CTRL cells showed increased NCS1 protein expression, whereas WFS1 KO cells showed decreased NCS1 expression. These results suggest that NCS1‐WFS1 interaction protects NCS1 from degradation, potentially by the calcium‐dependent protease calpain. Lastly, we showed that overexpression of NCS1, or treatment with a putative NCS1‐binding drug, rescued the deficits observed in WFS1 KO cells. Overall, we demonstrated a physiological function of the WFS1‐NCS1 interaction and that protecting NCS1 levels can ameliorate the deficits caused by loss of WFS1. These findings will facilitate the discovery of drugs that can prevent or reduce the symptoms of WS.
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 Information

Funding 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 Information

NIH 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 Information

New Frontiers in Research Fund, Gonzaga Science Research Program, CURCI
Protein and membrane trafficking in a dextran sulfate sodium‐induced ER stress in absorptive intestinal Caco‐2 cells
Authors: Wanes Dalanda , Toutounji Mohamad , Ngezahayo Anaclet , El-Sabban Marwan , Rizk Sandra , Naim Hassan Y.
Published: April 2020

A key morphological feature during the pathogenesis of inflammatory bowel disease (IBD) is the increased permeability of intestinal epithelial cells. Here, we address the implication of ER stress in protein and membrane trafficking in a dextran sulfate sodium (DSS)‐induced IBD‐like phenotype of absorptive intestinal Caco‐2 cells. We show that DSS treatment significantly reduces the transepithelial electric resistance (TEER) of Caco‐2 cells, while keeping the cell viability intact. The altered cell polarity is associated with an increase in the expression levels of the ER stress protein markers, BiP, CHOP, ATF4 and XBP1 as well as an impaired intracellular trafficking and polarized sorting of sucrase‐isomaltase (SI). SI associates normally with lipid rafts and the impaired sorting in DSS‐treated cells is likely due to reduced cholesterol levels and subsequent distortion of the lipid rafts. We conclude that perturbation of ER homeostasis in DSS‐treated Caco‐2 cells impairs membrane and protein trafficking resulting in altered cellular integrity.
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 Information

UK‐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 Information

Research 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 Information

Support 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 Information

US 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.
A Locomotor Activity Study of the Effects of L‐Serine to Neurodegeneration Impacts of Beta‐Methylamino‐L‐alanine in Drosophila melanogaster
Authors: Nguyen Quang Thanh , Rivera Lemuel Vince , Goto Joy June
Published: April 2020

Amyotrophic Lateral Sclerosis‐Parkinsonism Dementia Complex (ALS‐PDC) is a neurodegenerative disease that described by loss of motor function, tremors, and dementia due to neurodegeneration. The disease is thought to be caused by Beta‐Methylamino‐L‐Alanine (BMAA), a neurotoxin from blue‐green algae. Since it has a structure that is similar to glutamate, BMAA act as an agonist to the glutamate receptor N‐methyl‐D‐aspartate which lead to excitotoxicity and degenerate neurons. L‐serine is an essential amino acid that has shown to interfere with the effect of BMAA in vivo studies. The purpose of this research is to further test the novel impact of L‐serine to BMAA‐induced fly in long‐term study. The interaction of BMAA and L‐serine was analyzed through circadian rhythm activity and viability of wild‐type Drosophila melanogaster in ALS‐PDC model. We used two Drosophila Activity Monitor (DAM) systems to measure activity of 64 fruit flies (Gender and Age‐Match). The experimental groups were fed with L‐serine, BMAA, BMAA+L‐serine and the control group was fed with standard fly agar. Fly activity was recorded by the number of fly pass the IR beam in the monitor for 10‐day (12L/12D at 22°C). After 10 days, circadian rhythm data showed BMAA flies increased activity over the first few days, and decreased gradually to the end of the period. BMAA‐induced flies (n = 12) does not show pattern of regular activity in L/D as compared to Control (n = 15) and L‐serine (n = 16). The co‐fed BMAA+L‐serine (n = 7) showed a similar pattern as the Control. For daytime vs nighttime activity (mean ± s.e.m), Control (n = 15) shown regular activity (day: 137 ± 13%, night: 21 ± 5%) as compared to BMAA (n = 12) that shown high activity in dark cycle (day: 162 ± 10%, night: 155 ± 21%). Moreover, the cofed L‐serine + BMAA (n = 7) exhibited normal daytime activity but not nighttime activity (day: 138 ± 14%, night: 167 ± 31%). The data from DAM shows the abnormal activity of flies in long term when inducing with BMAA such as abrupt sleep due to high activity during dark phase. L‐serine shows to reduce down the high activity when cofed with BMAA. This supports our hypothesis of L‐serine ameliorates the effects of BMAA in fly. This preliminary data is a starting point for further research in drug discovery for ALS‐PDC.

Support or Funding Information

Acknowledgements

This work was funded by the Fresno State Undergraduate Research Grant Award (2019–2020), and Howard P. Kubo, M.D. Award for Outstanding Achievement in Biomedical Science.
Detecting reactive oxygen species in yeast at early growth points
Authors: Steward-Blanke Lottie R. , Bestwick Megan L.
Published: April 2020

Reactive Oxygen Species (ROS) at low levels can act as important signaling molecules; however, when there is a surplus of ROS this can lead to harmful and or lethal results for the cell. ROS are primarily generated in the mitochondria of cells. This development occurs in the electron transport chain (ETC) which is embedded in the inner membrane of the mitochondria. Production of the mitochondrial superoxide anion (a type of ROS), occurs at redox active prosthetic groups, or electron carriers, bound to ETC proteins where kinetic factors favor O2 to becoming the superoxide anion. These kinetic factors can include the inhibition of ETC by small molecules. If an electron interacts “early” with O2 then superoxide will form, which could ultimately result in peroxides or more toxic hydroxyl radicals (HO•). Both proteins and small molecules are used within cells to mediate ROS levels and maintain redox states within these mechanisms to ensure ROS levels do not reach damaging levels. The accumulation of too much ROS can prove to be harmful to the organism. Mutations and dysfunctional molecules can result from ROS reacting with both nuclear and mitochondrial DNA, proteins, and lipids; however, more recent research shows that low levels of ROS provide important signaling mechanisms. Our interest is in how metal cofactors are incorporated into the ETC protein complexes of yeast and how misincorporation or modulation of available metals, such as copper and iron, in yeast mitochondria leads to the production of ROS and how under these conditions ROS changes during yeast lifespan. This project is particularly interested in how ROS changes “early” in yeast lifespan, meaning timepoints before yeast cells complete stationary phase and begin to enter the death phase. To detect the ROS superoxide, yeast cultures were grown in rich media for one to three days and then stained with dyhydroethidum (DHE). DHE is a fluorescent indicator for cytosolic superoxide, and cells were assayed utilizing both a fluorescence plate reader and FACS. To enhance superoxide production cells were also treated with the ETC inhibitor Antimycin A (a Complex II inhibitor), and the control inhibitor Oligomycin A (an ATPase inhibitor). Our results indicate that we are able to detect cellular superoxide using the fluorescent dye DHE and that cells cultured in the presence of the inhibitor Antimycin A have higher DHE fluorescence values compared to cells cultured in the presence of Oligomycin A. Since Antimycin A inhibits Complex III in the ETC, these results are consistent with ROS being generated from this complex. Our current work is to culture cells in supplemental copper ranging from no treatment to 0.5 mM CuSO4, or bathocuproine disulphonate (BCS) a copper chelator. Ultimately, we intend to use DHE fluorescence to assess superoxide levels in multiple yeast strains to provide insight into how ROS changes during yeast lifespan.
Identification Of An Actin Fold Containing Metabolic Enzyme That Undergoes Regulated Polymerization
Authors: Wilhelm Jim , Carver Michael , Kyriakakis Phillip , Monfort Elena , Barry Rachael , Leschziner Andres
Published: April 2020

Cytoskeletal actins are thought to have evolved from actin fold enzymes, such as hexokinase or hsp70, that acquired the ability to polymerize while losing all other enzymatic functions apart from ATP hydrolysis. However, the discovery of metabolic enzymes, such as CTP synthetase, whose enzyme activity is regulated by self‐assembly into filaments has suggested another route to evolving cytoskeletal polymers: a filament forming metabolic enzyme that lost its role in metabolism and acquired novel cellular functions. In order to investigate this possibility, we surveyed all of the actin fold sugar kinases in yeast for their ability to form filaments. This screen identified one metabolic actin, the yeast glucokinase, Glk1p, that was capable of forming filaments in vivo. We have reconstituted Glk1p polymerization in vitro and have found that either ATP/Glucose or glucose‐6‐phosphate is sufficient to trigger filament formation. Consistent with this, we have also found that increasing glucose‐6‐phosphate levels in vivo triggers Glk1p polymerization. Furthermore, the addition of glucose and nonhydrolyzable ATP analogs to Glk1p also causes filaments to form. Thus, ATP hydrolysis is not required for Glk1p polymerization. Interestingly, the addition of ADP causes rapid disassembly of glucose‐6‐phosphate/Glk1p filaments suggesting that the stability of the filament is sensitive to the hydrolysis of ATP. While this is reminiscent of the dynamic behavior of cytoskeletal actins, our studies of Glk1p argue that it forms filaments that are structurally distinct from cytoskeletal actin filaments. Since the early steps of glycolysis consume ATP before generating ATP, we propose that Glk1p polymerization acts as a brake on glycolysis to prevent catastrophic depletion of ATP under certain metabolic conditions. We are currently using our structural and biochemical insights into Glk1p polymerization to test this possibility. Because Glk1p is the first filament forming actin that has more enzymatic functions than simple ATP hydrolysis, our results provide novel insights into both the evolution of the actin cytoskeleton and how the plasticity of the actin fold can support both polymerization and essential enzymatic activities.

Support or Funding Information

This work was supported by a HHMI Collaborative Innovation Award
The Lipid Anticancer Drug Edelfosine Affects Gene Expression Regulation
Authors: Ponce Maria Laura Sosa , Cobb Jennifer A , Zaremberg Vanina
Published: April 2020

Sirtuins are highly conserved regulators of gene expression. A genetic screen performed by our group using budding yeast found that sensitivity to the anticancer lipid drug edelfosine is alleviated in cells lacking sirtuins (SIR complex in yeast). The mechanism by which sirtuins mediate sensitivity to edelfosine, which localizes to the nuclear envelope (NE), is unclear. Here, we investigated the effect of edelfosine on the nuclear membrane and regulation of the nucleus. Using fluorescence microscopy, we observed NE deformation and changes in clustering of the telomere‐binding proteins Rap1 and Sir4. Transcriptomic analysis of cells treated with edelfosine showed major downregulation of genes known to be bound by Rap1, with enrichment of ribosomal protein genes. The overall effect of edelfosine on gene expression was reduced in sir4Δ mutant cells. We also found that constitutive deacetylation of histone H4 Lys16, a target of the SIR complex, caused hypersensitivity to edelfosine. We speculate that edelfosine disrupts genomic regulation via a SIR‐mediated mechanism. We show here that lipid anticancer drugs can affect DNA regulation by disrupting the NE, which has interesting implications for potential combination therapies.

Support or Funding Information

This work has been financially supported by the Natural Sciences and Engineering Research Council of Canada to VZ and JAC. Also supported by a QEII scholarship to MLSP.
Evaluating the Metabolite Profile of HER2‐positive Breast Cancer Cells
Authors: Abuelreich Sarah , Leon Nicole , Medina Daisy , Sahni Malika , Krishnan Krish , Bush Jason
Published: April 2020

Human epidermal growth factor receptor 2 (HER2)‐positive breast cancer represents about 20% of breast cancer cases and although treatments are available, some patients develop resistance to multiple exposures of anti‐HER2 therapies. New strategies need to be developed for a more accurate selection of patients who can benefit from these treatments. While much is known about HER2 biology, the biochemical impact of HER2 on breast cancer cell metabolism is not well understood. The goal of this project is to better understand HER2 metabolomics and associated molecular mechanisms using Nuclear Magnetic Resonance (NMR) spectroscopy. A series of eight different breast cancer cell lines ranging from low to high HER2 expression were adapted to a common media with negligible morphological change or alteration in HER2 expression levels. Metabolite profiles for experimental replicates of each cell line were determined using high‐resolution 1H‐NMR, principal components, and pathway analyses. This study identified pathways associated with metabolic reprogramming of nonessential amino acids. Specifically, pathway and enrichment analyses identified glycine and serine as the most significant which are known pathways supporting one‐carbon metabolism. Furthermore, the data showed an increased consumption of glutamate, which is necessary for energy production and nucleotide biosynthesis. These pathways support the altered metabolism and inefficient production of energy via aerobic glycolysis in breast cancer cells promoting tumor growth and progression. Ultimately, we hope to exploit these discoveries for metabolic targeting as a therapeutic strategy against HER2‐positive breast cancer cells.

Support or Funding Information

NIH grant 5SC3GM122630 to JAB
Scaffolding Protein IQ Motif Containing GTPase Activating Protein 2 Regulates Liver Metabolic Homeostasis
Authors: Sen Anuhna , Youssef Sara , Wendt Karen , Schmidt Valentina , Anakk Sayeepriyadarshini
Published: April 2020

The liver plays a central role in maintaining metabolic flexibility by regulating signaling pathways. Disruption in this process can lead to metabolic disease, such as obesity and cardiovascular risk. Scaffold proteins are well known to coordinate many signaling cascades. Since IQ Motif Containing GTPase Activating Protein 2 (IQGAP2) is a highly expressed scaffold protein in the liver, we examined its role in regulating metabolic programs. Adult wild type and Iqgap2−/− mice were fed ad libitum or fasted for 24 hours (9.00 am, or ZT4) and both male and female mice were used in this study to determine for sex differences in metabolism. We examined their gross morphology, performed serum biochemical assays and quantified expression of key enzymes regulating metabolic pathways including fat and carbohydrates. We found deficiency of IQGAP2 did not alter gross morphological features, liver‐to‐body weight ratio or white adipose tissue (WAT) weight. Iqgap2−/− male mice did not show pronounced differences in metabolic gene expression or fuel accumulation. In contrast, Iqgap2−/− female mice showed decreased expression of gluconeogenic, ketogenic and fatty acid oxidation genes. This basal decrease was compensated upon fasting with higher induction of ketogenic and gluconeogenic programs in Iqgap2−/− female mice. Our preliminary data suggest female Iqgap2−/− mice have defective nutrient storage. The two main hepatic fuel stores are glycogen and triglycerides. While hepatic triglycerides were not altered in female Iqgap2−/− mice, glycogen accumulation was significantly reduced. Histological analysis of hepatic glycogen accumulation showed distinct pericentral zonation which was apparent in the fed state. Consistent with this result, female Iqgap2−/− livers displayed reduced expression of genes regulating glycogen synthesis and degradation. Taken together, these data suggest a novel role for IQGAP2 in regulating hepatic fuel storage, especially glycogen levels.

Support or Funding Information

Illinois Campus Research Board and American Cancer Society
The C‐terminal Domain of AAGAB Mediates Tetramerization and Stability
Authors: Yin Qian , Tian Yuan , Crisman Lauren Christine , Wang Bing , Li Suzhao , Shen Jingshi
Published: April 2020

In clathrin‐mediated endocytosis (CME), the coat protein clathrin relies on
cargo adaptors such as the heterotetrameric AP2 adaptor complex to recruit cargo proteins to endocytic sites. It was previously thought that multimeric cargo adaptors such as AP2 adaptor assemble spontaneously. However, we recently discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB). Without the assistance of AAGAB, AP2 adaptor fails to form, leading to CME defects. Heterozygous AAGAB mutations cause human diseases such as puncate palmoplantar keratoderma type 1 (PPKP1), a skin disorder characterized by lesions on palms and soles. However, the biochemical property and structure of AAGAB remain to be established. AAGAB is a soluble protein with two conserved regions – an N‐terminal G protein‐like domain (G domain) and a C‐terminal domain not similar to any known protein domain. We found that the full‐length AAGAB exists entirely as a homotetramer in solution and the tetramerization is mediated by its C‐terminal domain. Using X‐ray crystallography, we solved the atomic structure of the C‐terminal tetramerization domain (TD). The structure unveiled that the TD tetramer is formed by dimerization of two dimers. Furthermore, we observed that the expression level of the TD‐deficient AAGAB mutant (AAGABdeltaTD) in cells was drastically reduced compared to full‐length AAGAB. Together, our findings demonstrated that the TD mediates AAGAB tetramerization and is required for the stability of free AAGAB proteins prior to AP2 binding. These data also provide the first molecular explanation for
the disease‐causing nonsense mutations found on AAGAB lacking the C‐terminal tetramerization domain.

Support or Funding Information

Florida State University startup funds to Yin; NIH/NIDDK R56 DK095367 to Shen; NIH/NIAID R21 AI128443 to Shen and Li.
Sputtering of Nanometric Films As a Tool to Enhance Antimicrobial Properties
Authors: Beckford Daihlia , Ward Zack , Phillips Jacey , Repa Kristen Stojak , Miller Casey , Michel Lea Vacca
Published: April 2020

Sputtering is a high vacuum physical vapor deposition technique during which atoms are excited from the surface of a target by a plasma. The energized atoms create a thin layer (thin film) on the surface of a chosen substrate material. Film thickness is determined by the deposition time (longer time thicker films). Our project aims to enhance the antimicrobial properties of a variety of textiles, polymers, and other surfaces by sputtering a nanometric film of different metals such as copper (Cu), gold (Au), and silver (Ag). Here, we describe our efforts to develop and optimize our experimental methods to assess the antibacterial properties of our sputtered surfaces, using Escherichia coli (E. coli) as the bacterial source. Preliminary data from our experiments suggest that our sputtering technique, which employs a “seed layer” of metal, successfully deposits a metallized film onto some surfaces, and that some metals are more easily washed off those surfaces than others. We also describe our different approaches toward the assessment of the antibacterial properties of the metallized surfaces.

Support or Funding Information

School of Chemistry and Materials Science, Rochester Institute of Technology