bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fascial Tissue Reconstruction Using Acellular Collagen Matrix
Authors: Eberli Daniel , Yoo James , Atala Anthony 
Published: June 2021

Statement of Purpose: Acellular collagen matrices have been used experimentally and clinically for several applications, including urethra and bladder reconstruction. We investigated the feasibility of using a porcine collagen‐based matrix as an off‐the‐shelf biomaterial for fascial tissue repair in 3 different tissue systems.Methods: Biocompatibility of the acellular matrix derived from porcine bladders was assessed using cell viability, MTT and apoptotic index. The acellular collagen matrices were used as a sling material for the treatment of incontinence, abdominal hernias and penile tunica reconstruction in 24 rabbits. The animals were evaluated up to 3 months after implantation.Results / Discussion: The acellular matrix did not induce significant changes in cell viability, proliferation and apoptotic activity when compared with the controls. All animals survived without any untoward effects. The matrices remained intact at their respective implantation sites and demonstrated the maintenance of fascial tissue function. Histologically, there was only a minimal inflammatory response, which gradually decreased over time.Conclusions: These results show that the acellular collagen matrix derived from porcine bladders is biocompatible, durable and safe for use in vivo.
Molecular Basis of dTTP Specificity by Gene 4 DNA Helicase of Bacteriophage T7
Authors: Lee Seung‐Joo , Richardson Charles C 
Published: June 2021

Translocation on ssDNA and the unwinding of dsDNA into ssDNA is mediated by DNA helicases at the expense of hydrolysis of nucleotide triphosphate (NTP). While most of helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To understand the molecular basis of this specificity we have examined the efficiency of unwinding of dsDNA by T7 helicase using a variety of NTPs and their analogs. The 5‐methyl group of thymidine is critical for the efficient unwinding of DNA although the presence of a 3′‐ribosyl hydroxyl group partially overcomes this requirement. In order to identify specific interaction between the NTP and residues within the NTP binding pocket of the protein, several amino acid residues, selected from structural data, were replaced with random amino acid residues. The resulting library of altered gene 4 proteins led to the identification of those residues (T320, R504, Y535, and L542) that are critical for NTP utilization by T7 helicase. Interestingly, some altered proteins containing a single amino acid replacement exhibit NTP utilization profiles distinct from the wild‐type helicase. One of these examples is gp4‐R504G that utilizes dATP more efficiently than dTTP. This altered protein can also utilize dCTP which is barely used by the wild‐type protein. We suggest that the NTP must be stabilized by specific interactions within the NTP binding site of the protein to achieve efficient hydrolysis. These interactions dictate the NTP specificity.This work was supported by Public Health Service Grant GM 54397‐4.
Analysis of the mechanism by which telomerase impacts chromosomal stability and cell viability in Ku mutants
Authors: Holland Cory Lee , Lewis Lysle Kevin 
Published: June 2021

The Ku complex is a heterodimer comprised of two subunits, Ku70 and Ku80, which binds double‐stranded DNA (dsDNA) ends. Ku has multiple roles in DNA metabolism. The presence of Ku at the ends of the chromosomes protects the telomeres from degradation by nucleases and may also aid in recruitment of telomerase to DNA. Interestingly, Ku has also been shown to play a critical role in the non‐homologous end‐joining (NHEJ) pathway of repair of induced double‐strand breaks in DNA.In budding yeast (Saccharomyces cerevisiae), inactivation of either of the Ku subunit genes, YKU70 or YKU80, abolishes NHEJ DNA repair and makes cells incapable of growing at elevated temperatures (37°C) due to telomere instability. The precise cause of telomere instability in yku70 mutants at high temperatures is unknown, but cell death can be alleviated by increasing levels of TLC1, the RNA subunit of telomerase, as well as the polymerase subunit Est2. We have shown that overexpression of the Exo1 exonuclease accelerates the death of Ku mutants at 37°C and that TLC1 RNA and Est2 are able to rescue the accelerated death of Ku mutants. A series of yku70 double mutants that were constructed demonstrated that other telomere associated proteins such as Mlp2, Rif2, and Rad50 are not required for rescue. Funding for this project was provided in part by the Department of Chemistry and Biochemistry, Texas State University – San Marcos and Research Corporation.
MAMMALIAN TETHERED CATALYSIS (MTeC): A NOVEL METHOD TO IDENTIFY MODIFICATION‐SPECIFIC BINDING PROTEINS IN VIVO
Authors: Spektor Tanya Magazinnik , Rice Judd Christopher 
Published: June 2021

Protein:protein interactions within the cell are critical for most biological processes. How proteins bind each other and how they regulate this interaction are fundamental questions in biology. One of the best examples of this phenomenon in the nucleus is the interaction of post‐translationally modified histone proteins with key transcriptional regulatory proteins that control gene expression.In order to identify modification‐specific binding proteins in vivo, we have created a novel method called Mammalian Tethered Catalysis, or MTeC. First, a tandem epitope‐tagged eukaryotic expression plasmid is created that contains the portion of the target protein that will be modified (“bait”) fused to the catalytic portion of an enzyme that will post‐translationally modify the target residue within the bait. A negative control is also created by mutating this target residue. Each construct is transfected into a cell line of choice and purified by chromatography and/or immunoprecipitation(s). The resultant proteins within the purified material are identified by mass spectrometry and proteins unique to the experimental sample are analyzed further. Using histone modifications as a proof‐of‐principle, we have successfully employed this method to identify a novel interaction between monomethyled lysine 9 of histone H3 and chromatin‐modifying protein, G9a.
Mapping the site of inositol 1,4,5‐trisphosphate receptor ubiquitination
Authors: Sliter Danielle , Kirkpatrick Donald , Alzayady Kamil , Gygi Stephen 
Published: June 2021

Inositol 1,4,5‐trisphosphate (IP3) receptors form tetrameric channels in endoplasmic reticulum (ER) membranes, and mediate IP3‐induced calcium mobilization in mammalian cells. In response to various extracellular stimuli that persistently increase phospholipase C activity, IP3 receptors are ubiquitinated and then degraded by the 26S proteasome. We have previously shown that IP3 receptor ubiquitination is triggered by the combined action of IP3 and calcium, is mediated by the ubiquitin‐conjugating enzyme, mamUbc7, and is processed by the ER‐associated degradation pathway. Here, for endogenous type 1 IP3 receptor processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin chains. Using mass spectrometry we found that a total 43 of the 167 lysines in the receptor are ubiquitinated, with three lysines, K1771, K1884, and K1901, being most frequently identified. Additionally, using ubiquitin‐AQUA analysis we found that most of these sites (~85%) are occupied by monoubiquitin and that both K48 and K63 linkages are abundant in attached chains. Overall, these data show that an endogenous substrate ubiquitinated in situ by endogenous ligases is tagged widely with an array of ubiquitin species.This research was funded by a grant to RJH Wojcikiewicz by the National Institues of Health.
Increase of Glucose‐6‐Phosphate Dehydrogenase in Macrophage Is Associated with Enhanced Inflammatory Responses in Obesity
Authors: Ham Mira , Lee Joo‐Won , Choi A Hyun , Masuzaki Hiroaki , Kim Jae Bum 
Published: June 2021

The role of Glucose‐6‐phosphate dehydrogenase(G6PD), NADPH producing enzyme, has been received a great attention in metabolic disorder such as obesity, diabetes and atherosclerosis. Previously, we have demonstrated that increased expression of G6PD in adipose tissue of obese animals provokes insulin resistance and stimulates inflammatory response and ROS production in adipocytes. To further understand the role of G6PD in metabolic disorders, we investigated G6PD in macrophages. Interestingly, the level of G6PD expression in stromal vascular fraction of adipose tissue was increased in obese animals, which exhibit enhanced infiltrating monocyte and macrophages. Additionally, the expression of G6PD in macrophage was stimulated by inflammatory signals such as LPS. Moreover, overexpression of G6PD in macrophage stimulated the expression of inflammatory genes including MCP‐1, IL‐6 and iNOS, implying that enhanced G6PD activity is associated with augmentation of inflammatory response in adipose tissue of obese subjects. Together, it is plausible to speculate that abnormal increase of G6PD in macrophage would modulate expression profile of pro‐inflammatory cytokines, which could deteriorate whole body inflammatory signaling in obese subjects.This work was supported in part by grants from the Molecular and Cellular BioDiscovery Research Program (M10748000258‐07N4800‐25810) and the National Research Laboratory Program (R0A‐2004‐000‐10359‐0) of Korea Science and Engineering Foundation (KOSEF). J.B.K. and M.H. was supported by a BK21 Research Fellowship from the Ministry of Education and Human Resources Development.
New pyranine receptor for use in endovesiculation assay
Authors: Wood Ryan Matthew , Sidorov Vladimir 
Published: June 2021

The endovesiculation assay is a method for determining the amount of external surroundings that have been internalized into large unilamellar vesicles (LUVs). In our assay, internalization of the fluorescent 8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS, pyranine) is used to quantify the extent of endovesiculation. After endovesiculation is induced, a receptor for HPTS is applied. The fluorescence of the external dye is quenched by the receptor, and the residual fluorescence corresponds to the amount of dye internalized by endovesiculation.The amount of HPTS dye internalized in the assay is low, and a receptor that is highly sensitive is needed. The dynamic quencher p‐xylene‐bis‐pyridinium bromide (DPX) provides the necessary sensitivity because of its 99.7% quench of HPTS fluorescence.1 However, DPX requires 40 mM concentrations that do not mimic biological conditions. A class of cyclen‐based receptors offers a better alternative because they operate at significantly lower concentrations.2 Of the cyclen‐based derivatives that we have synthesized, nitrophenyl‐urea‐cyclen has the highest fluorescence quench (97.6%) and can be applied to the endovesiculation assay.
Large scale purification of recombinant bovine deoxyhypusine hydroxylase
Authors: Smith Joshua L , Wang Yinglu , Huang Jenq‐Kuen , Wen Lisa 
Published: June 2021

The mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine. Hypusine synthesis is a posttranslational modification catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) which is essential for the function of eIF5A in cell proliferation and survival. It has been suggested that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy. A detailed knowledge of structure‐function relationship of either enzyme will help in designing better inhibitors thus better control of proliferative diseases.Recently, we have cloned and expressed functional bovine DOHH (bDOHH) (Huang et al., Protein Expr. Purif. 54, 126–133, 2007). Here we report large scale purification of bDOHH. The GST‐tagged DOHH fusion protein was overexpressed by IPTG induction and the fusion protein purified by affinity chromatography with a glutathione agarose column. The GST tag was removed by thrombin digestion. The bDOHH protein was then separated from the GST carrier by glutathione agarose. The monomic bDOHH protein was separated from aggregates by gel filtration on a Superdex‐200 column. The purified protein is being used for initial crystallization screening.Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.
Purification of MBP‐tagged proteins using new prepacked columns
Authors: Karlsson Anneli Birgit Marianne , Bergh Ann , Busson Karolina , Carlsson Marianne , Heijbel Anna , Lundqvist Jon , Maier Esther , Zimmerman Rhyenne , Zou Jinyu 
Published: June 2021

The success of protein production and subsequent analysis are dependent on the expression levels, solubility and purification of the protein. Maltose Binding Protein (MBP) is often used as an affinity fusion tag. Several studies have shown that MBP‐tagged proteins have high solubility compared to other tagged proteins.The new MBPTrap™ HP consists of an affinity medium, Dextrin Sepharose™ High Performance, packed in 1‐ml and 5‐ml HiTrap™ columns. The medium has high specificity and capacity. Additionally, it can withstand harsh Cleaning In Place (CIP) procedures, like sodium hydroxide, without losing binding capacity. The HiTrap format enables automated protocols to be used on ÄKTAdesign™ systems as well as with syringe or laboratory pump.In this work, MBP‐tagged proteins from E. coli were purified on prepacked 1‐ml or 5‐ml MBPTrap HP columns. The purity of the eluted target proteins was greater than 90 %. A stability study was performed, six purification runs each followed by a CIP run (0.5 M NaOH) were made on the same column. The results show no significant change in yield or purity. The results of an automated two step purification on ÄKTAxpress™ is shown. The affinity chromatography step on MBPTrap HP was followed by a gel filtration step.In summary, the results show that the prepacked MBPTrap HP column has high binding capacity and the eluted target protein is very pure. Furthermore, the medium is stable in 0.5 M NaOH.
Mapping and Characterizing aura, a Cytokinesis Mutant
Authors: Solanki Bharti Teresa , Pelegri Francisco 
Published: June 2021

A fundamental biological phenomenon during embryogenesis in animal cells is the process of cell division, an event that divides daughter cells in two. The completion of cytokinesis often results in the asymmetric segregation of cell determinants, thereby allowing the two daughter cells to proceed towards diverse cell fates. Many of these processes are driven and regulated by maternal products already present in the unfertilized egg. A recessive maternal‐effect mutation, aura, was identified in a zebrafish gynogenesis‐based screen. Preliminary evidence indicates that aura affects several events during early development that may be dependent on microtubule cytoskeletal rearrangements. aura mutants exhibit defects in cortical granule exocytosis, the recruitment of cell adhesion components to the furrow, germ plasm segregation and late cytokinesis. A positional cloning approach was used to determine the location and identity of aura. A lesion in a candidate gene has been found and rescue efforts are now underway to confirm the identity of the gene. Cell biology, genetic, and experimental approaches are being used to characterize aura gene function further. Together, these studies will contribute to understanding the function and regulation of aura and its role in the cytokinesis pathway.Sources of funding: NIH Ruth L. Kirschstein NRSA MARC Fellowhship and NIH R01 Grant.
CDK5: A Contributor to the Insulin Phenotype of Transdifferentiated β Cells
Authors: Stevens Jessica Lee , Aguanno Ann 
Published: June 2021

We investigate the role of CDK5 in mammalian development. Although CDK5 belongs to the cyclin dependent kinase family of proteins involved in regulating the cell cycle, CDK5 itself has been shown to influence differentiation of non‐dividing nervous tissues. Previously our lab has used PC12 cells to study CDK5's role in neuronal development. Experiments involving growth factor (NGF, FGF) induced differentiation of PC12 cells in the presence of a CDK5 inhibitor resulted in drastic morphological changes. Recent reports suggest CDK5 may also play a extra neuronal role. Therefore, we have explored the impact of CDK5 inhibition in pancreatic tissue using the acinar cell line AR42J as our model. We have investigated insulin production in the presence or absence of CDK5. To achieve this goal, the cells were differentiated into insulin‐producing cells by the administration of the chemical exendin‐4. CDK5 activity was inhibited by exposure to olomoucine, and the two flavonoids, kampherol and quercetin. Our results suggest that CDK5 plays a role in both the secretion and production of insulin.This research was supported by the Rose M. Badgley Charitable Trust, The Murray Foundation and the Division of Sciences, Marymount Manhattan College.
Nde1/Ndel1 are required to load dynein onto microtubules in Xenopus egg extracts
Authors: Kijanska Monika Anna , Zylkiewicz Eliza , Choi Won‐Chan , Derewenda Zygmunt S. , Stukenberg P. Todd 
Published: June 2021

Dynein is the minus‐end directed microtubule motor, required for membrane transport, nuclear and neuronal migration and regulation of the mitotic spindle. To perform so many functions, the loading of dynein onto microtubules must be tightly regulated. Nde1 and Ndel1 are closely related proteins that have been implicated in a number of dynein related processes. Nde1 and Ndel1 are coiled‐coil proteins with the central Lis1 interacting domain and the highly phosphorylated C‐terminus that interacts with regulatory proteins.Addition of constitutively active Ran to Xenopus laevis egg extract nucleates microtubules that become substrate for motors and form asters with the minus ends of microtubules bundled in a central focus. We find extracts depleted of Nde1 and Ndel1 are unable to form asters. The addition of Ndel1 lacking the C‐terminal domain can rescue these extracts, suggesting that the C‐terminal domain is not essential for aster formation. Surprisingly, point mutants in the Lis1 interaction domain also partially rescue, indicating that Ndel1 may have Lis1 independent functions. Dynein and dynactin were not loaded onto microtubules in the absence of Nde1/Ndel1. This is surprising because in a purified system dynein/dynactin load onto microtubules independently of cofactors. However our data show that in a complex system dynein loading requires Nde1/Ndel1 function.Supported by NIH grant NS 036267.
Conserved C‐terminal arginines are crucial for targeting PNPLA5/GS2‐like triglyceride lipase to lipid droplets
Authors: Murugesan Sricharan , Goldberg Elysa B. , Brown William J. 
Published: June 2021

Lipid droplets (LDs) are highly conserved storage depots for triglycerides (TGs) and cholesterol esters. The Patatin‐like Phospholipase Domain containing protein A (PNPLA1‐9) family of neutral lipases play key roles in the hydrolysis of stored TGs and in the regulation of LD metabolism. Previous work in our lab has uncovered a conserved charge‐dependent LD targeting motif (LTM) in the C‐terminal third of human PNPLA5 and Brummer Lipase (BL), the Drosophila homolog of human Adipose Triglyceride Lipase (PNPLA2/ATGL). The LTM of PNPLA5 is a conserved basic patch containing three arginine residues, two of which, if mutated to alanines abolish LD association. Here we report that each of these arginine residues is crucial for targeting PNPLA5 to LDs. Transient transfection of full‐length PNPLA5 containing all possible arginine to alanine changes revealed that altering any one of the arginines abolished LD association in vivo. However, mutations of similar putative basic patches in human PNPLA2/ATGL revealed only mild mislocalization. These results show that although conserved arginines in basic patches of PNPLA5 and BL identify a conserved LTM, not all family members are recruited to LDs in an identical manner. This correlates with evidence that PNPLA family members serve overlapping, but not identical, roles in TG homeostasis.This work supported by Hatch Project NYC‐165415 and NIH grant DK51596 to WJB.
Comparison of caspase‐8 fluorescent substrates for development of microtiter plate homogeneous cell‐based assay
Authors: Rakhmanova Vera , Han Xing , Tong Xiaohe , Hong Anita L. , Meyer Rich 
Published: June 2021

Caspase‐8 is an initiator enzyme accepting signals from death receptors and activating downstream effectors caspases (caspase‐3, ‐7) through proteolytic cleavage. Caspase‐8 has substrate specificity for the amino acid sequence, Ile‐Glu‐Thr‐Asp (IETD). Although there are a number of assays using IETD based caspase‐8 substrates, those protocols are not optimized for 96‐well plate format and include additional steps of cell harvesting, lysate preparation and subsequent aliquoting of prepared lysates into 96‐well plates.To develop homogeneous 96‐well plate caspase‐8 fluorimetric assays, we compared a series of fluorophores (AMC, AFC and Rh110) linked to IETD peptide. AMC and AFC‐linked substrates tested with purified enzyme gave at least a 2‐fold higher signal/background ratio and better kinetic parameters than the Rh110 labeled substrate. When applied to cells grown and lysed in 96‐well plates, the Ac‐IETD‐AMC substrate showed a high background. The longer‐wavelength spectra of the AFC (Ex/Em=380 nm/500 nm) provided greater sensitivity and less interference from cell media. The use of Ac‐IETD‐AFC substrate for detection of caspase‐8 activity in cells enabled the development of a homogeneous assay with a wide linear range and high signal/background ratio. This assay is ideal for measuring enzyme activity in cells grown in 96‐well plates and for screening of apoptosis inducers or inhibitors.
The Immune System: Defenders of Our Health
Authors: Withum Elizabeth Anne , Mcloughlin Rachel 
Published: June 2021

The Immune system is one of many specialized systems of the body. This system has developed over millions of years to identify, label and protect us from foreign pathogens such as bacteria, virus, parasites and protozoans. These lessons are designed for middle school as an introduction to the human bodies Immune System and how we detect that the immune system is there and working properly. A specific focus will be on the neutrophil as the defender against bacterial attack.A modified ELISA has been developed using a commercially available ELISA kit purchased from an educational biological supplier. The ELISA will be used to detect chemokine levels in a mock patient infection sample. Students will interpret the ELISA results to establish chemokine levels in their patient samples. Finally, students will make the connection between chemokine levels, neutrophil recruitment and bacterial clearance that is necessary for a ealthy immune response.