bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Purification of human Neuronal PAS domain protein 2 (hNPAS2)
Authors: Moriel Karla , Xi Dongman , Sarabia Alejandro , Chauhan Neha , Ray Supriyo , Xiao Chuan
Published: June 2021

The circadian rhythm is an internal clock responsible for a variety of physiological processes occurring within a 24‐hour cycle. Synchronized by a master clock in the brain, the circadian rhythm consists of a transcriptional‐translational feedback loop (TTFL) capable of inducing oscillating gene expression in all the cells throughout the organism. The TTFL begins with the heterodimerization of two transcription factors, BMAL1 and CLOCK, resulting in the transcription and translation of many circadian‐related genes. The target protein of interest, human neuronal PAS domain protein 2 (hNPAS2) is a paralog of CLOCK that can serve as a positive regulator of the circadian rhythm by heterodimerizing with BMAL1. Previous research suggests that disruption in the expression of hNPAS2 may result in improper circadian functioning and development of sleeping and mental disorders. In this study, the full‐length eukaryotic form of hNPAS2 was cloned into a bacterial expression vector. However, bacterial expression has led to misfolding and aggregation of the recombinant hNPAS2, becoming the central issue of the study. In an effort to obtain soluble and homogeneous hNPAS2, multiple expression and purification methods have been developed. The resultant purified soluble protein will enable future structural and functional studies, which can bring new insights to the regulatory mechanisms of the circadian rhythm and understanding of circadian‐related disorders. Additionally, a portion of this research was integrated into a teaching module delivered by the leading student researcher to a senior undergraduate advanced biochemistry course. Multiple novel pedagogic techniques were employed in the teaching module with the objective of raising the students' interests to research as well as their abilities to apply the principles and concepts of biochemistry learned in the course in a real scientific study.

Support or Funding Information

Research reported in this poster was supported by the National Institute of General medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, AND UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health Research reported in this poster was supported by the National Research of General Medical Sciences of NIH under award number SC3GM109870 Research reported in this poster was supported has used core facility at BBRC supported by National Institute of Minority Health and Health Disparities under award number 5G12MD007592

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The IAPP Toxicity on Rats, Raccoons and Degus in HeLa Cells
Authors: Munoz Crystabel
Published: June 2021

Type 2 diabetes is the most common form of diabetes, where the body suffers from insulin resistance. The islet amyloid polypeptide (IAPP) is a secretory product of beta cells in pancreatic islets of Langerhans that also inhibits glucagon and insulin secretion. The aggregation of IAPP is present in pancreatic islet amyloid deposits seen especially in Type 2 diabetes, in humans and other mammalian species. The objective of this study was to determine the effects of the animal toxicity on mammalian cells. In this work, the human IAPP sequence and the effect on mammalian cells was compared to the IAPP from degu, rat, and raccoon. Molecular techniques such as MTT and LDH Cytotoxicity assay were used to test the effect of human and animal IAPP on HeLa cells. The MTT results show a consistent drop in cell viability as the concentration of IAPP from a human, degu, rat and raccoon increases. LDH cytotoxicity shows the highest toxicity in human IAPP compared to the rest of the studied animals. Although when mixing both the human and animal IAPPs, the toxicity of the human IAPP decreased whereas the viability had no significant effect. Further molecular analysis is necessary to determine the correlation between animal IAPP toxicity and the incidence of Type 2 diabetes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Catalytic Null Splice Variant of Human Leucyl‐tRNA Synthetase with Enhanced Non‐Canonical Function
Authors: Forsyth Connor Mackenzie , Baymiller Maxwell , Martinis Susan A.
Published: June 2021

At the center of translation are the ancient and essential aminoacyl‐tRNA synthetases (AARS). These proteins set the genetic code by attaching amino acids to their cognate tRNAs with very high accuracy. Over a lengthy evolutionary time, AARS have developed functions beyond aminoacylation through the addition of new domains and repurposing of existing structural motifs. Non‐canonical functions of AARS are especially widespread in eukaryotes and range from cell signaling to transcription and translation regulation. For example, human cytoplasmic leucyl‐tRNA synthetase (LeuRS) has been adapted as an intracellular leucine sensor for the mammalian target of the rapamycin complex 1 (mTORC1) master regulatory pathway (Han, 2012). Ten alternatively‐spliced variants of cytoplasmic LeuRS were identified in humans using RNA Seq (Lo, 2014). It is possible that each of these splice variants contributes to novel and idiosyncratic functions. We have found that one of these variants, LeuRS AS04, is overexpressed in both Jurkat T cells and peripheral blood mononuclear cells (PBMC), suggesting a potential immune‐cell specific role. Despite the absence of key catalytic residues, LeuRS AS04 is a potent and specific activator of mTORC1. In particular, downstream phosphorylation of mTORC1 target ribosomal protein S6 kinase beta‐1 (p70S6K1) was significantly enhanced in cells overexpressing AS04 compared to those overexpressing full‐length LeuRS. We hypothesize that this alternately spliced LeuRS variant might have evolved to enhance and regulate the non‐canonical function of LeuRS‐dependent mTORC1 signaling.

Support or Funding Information

W.M. Keck Foundation Biomedical Research Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of pathogenic mutations in human mitochondrial alanyl‐tRNA synthetase
Authors: Chihade Joseph , Kennicott Hannah , Jessica Makori , de Leon Samuel Diaz , Donnell Isaac , Heath Jacob
Published: June 2021

Translation in mitochondria is dependent on a set of mitochondrial specific aminoacyl‐tRNA synthetases. Defects in these enzymes are related to a range of human diseases. For example, mutations in the human mitochondrial alanyl‐tRNA synthetase (AARS2) have been associated with infantile cardiomyopathy and with adult‐onset leukoencephalopathy. Structural analysis of homology models and haplotype analysis suggests that all pathogenic mutations result to some degree in loss of AARS2 function, and that the severity of the mutation correlates with the observed disease phenotype.(1) We are testing this hypothesis in vitro by examining protein stability, RNA binding, and aminoacylation activity of these mutants.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of peroxide exposure on the transfer RNA of radiotrophic C. neoformans
Authors: Kelley Melissa , Myers Ryan , Jora Manasses , Addepalli Balasubramanyam , Limbach Patrick
Published: June 2021

Cryptococcus neoformans is a radiotrophic fungi capable of surviving in extreme environments. Understanding post‐transcriptional RNA modifications may lead to insight on translational regulation and protein synthesis. Little is known about the role of transfer RNA (tRNA) modifications in C. neoformans. Modification profiles change during oxidative stress and may play biological roles in cell survival. Here, the impact of peroxide exposure on C. neoformans tRNA is investigated through liquid chromatography coupled with mass spectrometry.

Support or Funding Information

Department of Defense: Defense Threat Reduction Agency (DTRA)University of Cincinnati

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A nucleotide‐dependent switch in proteasome assembly mediated by the Nas6 chaperone
Authors: Park Soyeon , Li Frances , Sokolova Vladyslava
Published: June 2021

The proteasome is a molecular machine essential for protein degradation. The proteasome is assembled via the 9‐subunit lid, 9‐subunit base, and 28‐subunit core particle (CP). Previous work has shown that the chaperones Rpn14, Nas6, Hsm3, and Nas2 each bind a specific ATPase subunit of the base, and antagonize base‐CP interaction. Here we show that the Nas6 chaperone also obstructs base‐lid association. Nas6 alternates between these two inhibitory modes according to the nucleotide state of the base. When ATP cannot be hydrolyzed, Nas6 interferes with base‐lid, but not base‐CP association. In contrast, under conditions of ATP hydrolysis, Nas6 obstructs base‐CP, but not base‐lid association. Modeling of Nas6 into cryo‐EM structures of the proteasome suggests that Nas6 controls both base‐lid affinity and base‐CP affinity through steric hindrance; Nas6 clashes with the lid in the ATP‐hydrolysis‐blocked proteasome, but clashes instead with the CP in the ATP‐hydrolysis‐competent proteasome. Thus, Nas6 provides a dual mechanism to control assembly at both major interfaces of the proteasome.

Support or Funding Information

Boettcher Webb‐Waring Biomedical Research Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relevance of the Non‐canonical Complex Formed by Proteasome Subunit α4
Authors: Panfair Dilrajkaur , Hammack Lindsay , Hopf Brett , Kusmierczyk Andrew R.
Published: June 2021

The 20S proteasome plays an essential role in maintaining protein homeostasis as part of the larger 26S proteasome complex. It is composed of seven distinct α subunits and seven distinct β subunits arranged as a stack of four heptameric rings in an α7β7β7α7 manner. Assembly of these 28 subunits is very efficient and aided by assembly chaperones that function, in part, to prevent off‐pathway interactions. Certain α‐subunits of the proteasome form non‐canonical ring complexes in vitro. The significance of such complexes is not known but they could simply represent dead‐end, or off‐pathway, assemblies. We recently provided the first in vivo evidence of such a non‐canonical ring complex formed by the yeast α4 subunit. Levels of this complex increase when proteasome function is compromised. Here, we aim to further investigate the relevance of this α4 high molecular weight complex (α4HMWC). Using a salt bridge disruption strategy, we achieved destabilization of the α4HMWC without affecting proteasome assembly. If the α4 HMWCs are important physiologically, we may observe growth phenotypes of the resulting yeast mutant lacking this complex. Our results will help determine whether this complex is an off‐pathway, or dead‐end, product or whether it may have an independent function within the cell.

Support or Funding Information

Research Support Funds Grant from Indiana University‐Purdue University Indianapolis to A.R.K

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the Role of the L/I Zipper in the Stability of Paramyxo‐ and Pneumovirus F Proteins
Authors: Branttie Jean Mawuena , Webb Stacy R. , Dutch Rebecca E.
Published: June 2021

Paramyxoviruses such as Hendra virus (HeV) and Measles virus (MeV) are important pathogens of worldwide significance. Others such as Parainfluenza virus (PIV5) are widely used as paramyxovirus model systems. Despite their prevalence, currently, no FDA‐approved vaccines or antiviral therapeutics are available against them. Paramyxoviruses consist of a single‐stranded negative sense RNA genome enveloped in a host‐derive membrane. Paramyxovirus glycoproteins play an important role in infectivity: the attachment protein (G, HN, or H) docks the viral particle to the host cell, and the fusion protein, (F), drives fusion with a host cell membrane, which allows the release of viral contents into the host cell. To promote successful fusion, the F protein undergoes dramatic, essentially irreversible conformational changes which must be carefully timed. To avoid unproductive triggering events, F is held in a metastable prefusion conformation until the right signal for fusion occurs. Previous studies in HeV F have shown that the transmembrane (TM) domain is important for prefusion stability with a leucine/isoleucine (L/I) zipper playing a key role. Furthermore, similar L/I zipper motifs are present across the paramyxovirus and the closely related pneumovirus families. To define the significance of the L/I zipper motifs in other paramyxo‐ and pneumovirus F proteins, we created a PIV5 F mutant protein which has alanine substitutions to the L/I zipper in the TM domain. Our preliminary data suggest that the mutant expresses; however, compared to wild type, which demonstrates a strong localization to the plasma membrane, the mutant shows a ubiquitous distribution in cells. This suggests that the mutant undergoes different trafficking events compared to the wild type. Further studies will be performed to quantify surface and total expression of the mutant, and also examine its stability and fusogenic activity. The effect of the L/I zipper will also be investigated in the context of Human metapneumovirus, a pneumovirus in order to determine whether the TM domain of paramyxo‐ and pneumoviruses present a broad therapeutic target.

Support or Funding Information

We gratefully acknowledge the NIH/NIAID for supporting our research grant R01 AI051517

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allosteric Inhibitor of ERAP1 Acts by Stabilizing a Closed Conformation
Authors: Maben Zachary , Arya Richa , Stern Lawrence
Published: June 2021

Endoplasmic‐reticulum associated aminopeptidase 1 (ERAP1) is a M1 family zinc aminopeptidase that generates peptides for class I major histocompatibility complex (MHC‐I) presentation and T cell surveillance. ERAP1 has been crystalized in open and closed states, and is postulated to convert between them during its catalytic cycle. Small‐angle X‐ray scattering (SAXS) studies show that in solution ERAP1 predominately adopts the open conformation. A highly specific small‐molecule inhibitor of ERAP1 causes it to adopt a closed conformation, suggesting an allosteric inhibition mechanism. The compound inhibits ERAP1 peptide processing activity but activates hydrolysis activity of a dipeptide analog, leucine‐7‐amido‐4‐methylcoumarin, supporting an allosteric mechanism. The inhibitor also differentially alters enzymatic behavior of naturally occurring ERAP1 variants with lysine or arginine at position 528, which differ in their solution conformational equilbrium. Arg/Lys 528 forms an electrostatic interaction in the closed conformation that is broken when ERAP1 adopts the open conformation; computational studies predict that substitution of arginine for lysine decreases the electrostatic potential by 1.67 kT/e. We propose that this polymorphism alters the stability of the closed conformation, and that the differential behavior of the inhibitor for the ERAP1 variants is directly related to the stability of the closed conformation. The role of conformation in the catalytic cycle may be a feature of the entire M1 aminopeptidase family.

Support or Funding Information

This research was supported by NIH National Institute of Allergy and Infectious Diseases (NIAID) under the National Institutes of Health award number R01AI038996.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Glucosome: A Metabolic Compartment for Glucose Metabolism in Living Cells
Authors: Jeon Miji , Kohnhorst Casey , Kyoung Minjoung , An Songon
Published: June 2021

Glycolytic enzymes have been suggested to form multienzyme complexes in a variety of organisms. Recently, we have shown that liver‐type phosphofructokinase 1 (PFKL) tagged with a fluorescent protein forms various sizes of cytoplasmic enzyme clusters in human cells. We also found that these PFKL clusters colocalize with other cytoplasmic rate‐limiting enzymes in glucose metabolism, supporting the spatiotemporal formation of a multienzyme complex, namely the “glucosome.” Importantly, quantitative high‐content imaging data unveil that the various sized cytoplasmic enzyme clusters play a functionally different role by shunting glucose flux into the pentose phosphate pathway or serine biosynthesis. We propose that the differing sizes of glucosome clusters represent various metabolic functions to guide the direction of glucose‐mediated carbon flux in the cell. Therefore, we envision that our study will advance the understanding of how the multienzyme metabolic assemblies are spatially compartmentalized in the cytoplasm to functionally contribute to regulate glucose flux in cells.

Support or Funding Information

National Institutes of Health Grant T32GM066706

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Organophosphate (OP) Conjugation on Structure and Dynamics of Human Acetylcholinesterase
Authors: Fajer Mikolai , Cheng Xiaolin , Kovalevsky Andrey , Gerlits Oksana , Blumenthal Donald , Taylor Palmer , Radić Zoran
Published: June 2021

Organophosphate (OP) conjugation of the human acetylcholinesterase (hAChE) active site leads to a deadly increase of the neurotransmitter acetylcholine in the nervous system. The slow reactivation rate of OP‐hAChE by current antidotes is a major problem in effective treatment. This is a possible consequence of sterically restricted antidote access to the conjugated phosphorus in already narrow active center gorge of hAChE emphasized by available static X‐ray structural snapshots. We have conducted inelastic neutron scattering (INS) experiments on both hAChE and paraoxon (POX) conjugated hAChE (POX‐hAChE) to better understand the influence of OP conjugation on hAChE dynamics and reactivation rate limitation. The measured dynamic structure factor S(q,ω) showed a statistically significant difference at high Q between the two samples. The corresponding vibrational density of states (DOS) indicated that the difference was a result of low frequency vibrations in the 30 to 50 cm−1 range. In order to identify the particular mode of motion that is affected by OP conjugation, we compared the three X‐ray structures of the POX‐conjugated hAChE with the apo‐hAChE, and found tertiary conformation deviations in three regions: the acyl pocket, C‐terminal helix bundle and the Ω loop. We then constructed computational models of the apo‐hAChE and POX‐hAChE powder samples each with eight randomly oriented monomers. After equilibration, multiple 10 ps simulations were performed to compute the vibrational DOS via the velocity autocorrelation functions of the protein heavy atoms. An additional set of 10 ps simulations were given an impulse to each of the three specific regions. These simulations confirmed that a motion closing the acyl loop gorge was in line with experimental INS data. This motion was further shown to be coupled with the helix bundle dynamics, providing an explanation for the change in hAChE quaternary structure after OP‐conjugation observed in the SAXS data. Taken together, our results reveal detailed molecular insights into how OP‐conjugation changes the dynamic behavior of hAChEs.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering yeast endosymbionts as a first step towards laboratory evolution of mitochondria
Authors: Mehta Angad , Supekova Lubica , Supek Frantisek , Schultz Peter
Published: June 2021

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in a protoeukaryotic cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between E. coli and the budding yeast S. cerevisiae. Fusion of yeast with E. coli ectopically expressing several genes from unrelated, intracellular bacteria was key for establishing endosymbiosis. ADP/ATP translocase‐expressing E. coli provided an energy source for a respiration‐deficient cox2 yeast mutant, enabling growth of yeast‐E. coli chimera on a non‐fermentable carbon source. Similarly, yeast provided a source of thiamin or NAD to an E. coli thiamin or NAD auxotroph respectively. Expression of several SNARE‐like protein on the surface of E. coli was also required to prevent lysosomal degradation. The engineered yeast‐E. coli chimeras sustained growth on selection medium in containing the antibiotic carbenicillin indicating the presence of intracellular E. coli which supports the growth by ATP synthesis on selection medium. Further, sf‐gfp expressing E. coli endosymbionts could be observed in the yeast by super resolution fluorescence microscopy after more than 40 doublings. This readily manipulated system should allow us to experimentally delineate host‐endosymbiont adaptations that occurred during evolution of the current much reduced mitochondrial genome

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Light Triggered Isoelectric Point and Solubility Shifts Applied to Photoactivated Insulin Depots
Authors: Nadendla Karthik , Friedman Simon H.
Published: June 2021

We previously described photoactivated insulin depot, a minimally invasive technique of insulin delivery (1). In this approach, an inert photoactivated depot is injected under the skin. Exposure of the depot to light triggers the release of native insulin into the blood. First‐generation materials were designed to have insulin chemically attached to an insoluble polymer (for insolubility) via photocleavable linker. Though this material is effective in studying the approach in vitro and in vivo (2), it has multiple problems associated with the insoluble polymer. About 95% of the depot consisted of the non‐therapeutic polymer, which is non‐biodegradable and difficult to inject into an animal.

In this work, we solved these problems by eliminating the polymer and reacting insulin with a photocleavable group containing positively charged groups. The result is an insulin with an altered pI and thus, with a different solubility (3). We achieved the insulin's insolubility by shifting its pI to physiological pH. We observed an increase 0.65 units of pI of insulin with the addition of a single positive charge. We raised the pI of insulin from 5.4 to 7.2, specifically by the addition of two positively charged groups and quenching one negative charge (carboxylate) on insulin. This material has high solubility in acidic pH, and hence can be a soluble formulation. But upon injection at skin pH, it is observed to precipitate as a depot. Upon exposure to light, the positively charged groups are removed to generate the native insulin, which can then be absorbed into the blood due to its high solubility. These materials have high insulin density (91% w/w), are soluble in acidic formulations and upon photolysis, will result in charged, soluble and excretable by‐products without leaving any traces of depot at the site of injection.

Support or Funding Information

NIH‐1DP3DK106921‐01, University of Missouri Fast Track Award and UMKC School of Pharmacy Dean's Bridge Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing an Efficient Method for the Incorporation of a Series of Fluorotyrosines in Peptides via Solid Phase Peptide Synthesis
Authors: Chau Noel , Colla Alexander , Schwans Jason
Published: June 2021

The incorporation of unnatural amino acids in peptides and proteins has furthered the investigation of various protein structures and enzyme function. Enzyme residues suggested to be important for catalysis are readily mutated and measured for energetic effects. However, the limited repertoire of naturally occurring amino acids constrains the available substitutions. To obtain a deeper understanding of how enzymes work, unnatural amino acids are used to systematically perturb enzymatic residues. Previous studies in the enzyme ketosteroid isomerase (KSI) used a series of fluorotyrosine analogs to perturb the tyrosine hydrogen bond donor. However, challenges in the synthesis of fluorotyrosine analogs limited their use. To overcome these challenges and extend the series of fluorotyrosines used in enzymatic studies, we are developing an approach to selectively incorporate fluorotyrosines in peptides using silyl‐based and fluorenylmethyloxycarbonyl (Fmoc) based chemistry. Introduction of Fmoc had moderate yields of up to 70%, while silylation of Fmoc‐tyrosine led to low yields of ~20%. Low yields may be attributed to presence of additional undesired silylation products. To overcome this limitation, a series of silylation conditions and additional protecting groups on the amino acid are being evaluated. After optimizing reaction conditions, the dually protected fluorotyrosines will then be used to generate synthetic peptides via solid phase peptide synthesis.

Support or Funding Information

This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 5UL1GM118979‐03; 5TL4GM118980‐03; 5RL5GM118978‐03. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Inhibitors of P‐Glycoprotein from In Silico Optimizations of Inhibitor SMU‐29 using the ChemGen Program
Authors: Ammerman Lauren E. , Nanayakkara Amila K. , Aljowni Maha , Lippert Alexander R. , Vogel Pia D. , Wise John G.
Published: June 2021

P‐glycoprotein (P‐gp) is an integral membrane efflux pump that is capable of lowering the intracellular concentration of many drugs to sub‐therapeutic levels. Consequently, P‐gp has been implicated in multidrug resistances (MDR) to cancer chemotherapies. One promising P‐gp inhibitor, SMU‐29, was previously identified by us using in silico methods (Brewer et al., Molecular Pharmacology 86, 716–726, 2014) and was later confirmed to reverse MDR in cancer cell culture experiments (Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). We created millions of SMU‐29 variants in silico using our novel medicinal chemistry program, ChemGen, in efforts to pharmacologically optimize SMU‐29. In one set of chemical variants, only part of the structure of SMU‐29 was varied; in a second set of variants, only the central structural scaffold of SMU‐29 was conserved. We used in silico docking to identify chemical variants of SMU‐29 that exhibited high estimated affinities for P‐gp's nucleotide binding domains (NBDs), and low estimated affinity for P‐gp's transmembrane drug‐binding domain (DBD). Using this approach, we identified over 2000 ChemGen‐created variants with equivalent or better estimated affinities at the P‐gp NBDs relative to SMU‐29. We selected a small set of these SMU‐29 variants for chemical synthesis and further screening in vitro against multidrug resistant cancer cell lines. All of the tested and synthesized SMU‐29 variants were observed to act as P‐gp inhibitors with improved PC50 values. Some of these variants were also observed to inhibit the breast cancer resistance protein (BCRP), a related ABC‐transporter that is also implicated in MDR in cancer. Our previous screens that identified SMU‐29 as a hit compound had a 33% P‐gp inhibitor identification success rate (hit rate); the present in silico optimization of SMU‐29 using ChemGen has demonstrated a very high success rate of identifying P‐gp inhibitors with improved affinities for the target protein.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In‐vitro compositional investigations of antioxidant, phytochemicals, nutritional and minerals in the fruit of Kigelia africana (Lam.) Benth
Authors: OSENI OLATUNDE ABASS
Published: June 2021

The health problems in the third world and the attendant complications resulting into death of many people mostly caused by cancer, hypertension and other heart disorders primarily because of the inability to afford adequate health conditions and maintain balanced nutrition status has called for alternative means of attaining the required global Millennium Developmental Goals (MDGs) status on health and also to prevent mortality among all ages. This study therefore aimed at investigating the natural antioxidants, phytochemicals, proximate and mineral elements composition in fruit of Kigelia africana popularly known as the sausage tree as an indication for utilization in pharmaceutical for human and animals' feed benefits. The results obtained for 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), total phenolic content, total flavonoid, Ferric reducing antioxidant power (FRAP), phytochemical constituents, proximate and mineral elements compositions of the fruits revealed high percentage of antioxidant potentials, presence of medicinal important phytochemicals, good quantity of nutritional and medicinally important mineral elements. However, from the results of the study, the fruit of Kigelia africana can be exploited for medicine to heal many diseases and food additives in animals' feed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GRAPES: A Versatile Tool for Analyzing Structural Variation From Whole‐Genome and Targeted DNA Sequencing Data
Authors: del Olmo Bernat , Matés Jesús , Mademont‐Soler Irene , Pérez‐Serra Alexandra , Coll Monica , Allegue Catarina , Puigmulé Marta , del Olmo Garcia‐Bassets Ivan , Pascali Vincenzo , Oliva Antonio , Brugada Ramon
Published: June 2021

The annotation of human genomic diversity is nowadays at an unprecedented rapid pace, but arguably still limited by the lack of computational approaches that integrate the analysis of multiple forms of genetic variation and are suitable for the analysis of both, whole‐genome sequencing and targeted sequencing strategies. For the analysis of structural variations (SVs), for example, which include >50 bp deletions, duplications, copy‐number variations (CNVs), inversions, mobile‐element insertions, and translocations, only a few computational tools integrate breakpoint signatures and read‐depth information in a single approach. And, none of them makes use, in the analysis of targeted sequencing, of breakpoint information to filter false‐positive CNVs when small adjacent introns are completely captured despite that targeted sequencing is still more popular than whole‐genome sequencing (e.g. exome‐sequencing and many common approaches of genetic‐testing are forms of targeted sequencing). Here, we present the development of GRAPES (Genomic Rearrangement Analyzer for Paired‐End Sequencing), which is as a versatile tool that allows the analysis of SVs on whole‐genome and targeted DNA sequencing data by integrating discordant‐pairs, split‐reads, read‐depth, and local assembly. In the case of targeted sequencing, GRAPES uses on‐ and off‐ target read depth coupled with breakpoint analysis to provide accurate predictions. We have validated GRAPES using the accurately annotated NA12878 genome, the ICR96 CNV exon validation series, and a panel of simulated datasets, and it achieves higher rates of sensitivity and specificity than previously developed approaches. We have tested GRAPES to annotate SVs from targeted sequencing data of patients affected with inherited cardiac disorders, uncovering complex SVs that were unresolved by using other algorithms. In sum, GRAPES not only is a comprehensive tool for the analysis of SVs from whole‐genome sequencing and targeted sequencing data, but also allows the annotation of SVs overlooked by current single‐approach approaches.

Support or Funding Information

International Doctorate Grant in Biomedical Research and Public health of the Università Cattolica del Sacro Cuore

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Population Genetics of Scaevola on Culebra, Puerto Rico.
Authors: Hodkinson Lauren , Witherup Susan
Published: June 2021

On the islands of Puerto Rico, a native species of coastal shrub, Scaevola plumieri, co‐occurs with an invasive species, Scaevola taccada, introduced from the indo‐pacific islands. We are interested exploring the genetic diversity of both S. plumieri and S. taccada on the island of Culebra, PR in order to characterize the differences in genetic variation of an endemic species versus an invasive species. We hypothesize that the S. plumieri will have a greater amount of genetic diversity than the invading species due to S. taccada's more recent establishment on the island. Previous studies in our lab have shown that the alleles for two microsatellite regions were distributed across all populations on Culebra and had no correlation between distance and genetic diversity. To improve our characterization of diversity, we developed novel species‐specific microsatellite primers for S. plumieri using whole genome sequencing data. Six pooled samples from different beaches across Culebra were sent for Illumina MiSeq paired‐end sequencing and a Python computer code was written to identify potential microsatellites from the raw sequencing data. Leaf samples from both S. plumieri and S. taccada populations were collected from Culebra during January of 2015 and March of 2017, dried with silica gel, and the genomic DNA from each leaf was extracted. Microsatellites in the S. plumieri samples were amplified using the newly developed primers whereas microsatellites in S. taccada were amplified using previously developed primers. Fragment peaks from analysis by capillary gel electrophoresis were investigated using the program Geneious and peaks representing the fragment size of the microsatellite alleles were identified and recorded for each individual. These data were then evaluated through the program Arlequin which implements a series of tests to determine molecular variance (AMOVA), identify deviations from Hardy Weinberg Equilibrium and correlate geographic distance and genetic variation. In the future we plan to further expand our analysis using these novel S. plumieri primers to incorporate plant samples form Vieques, PR and mainland PR for a complete characterization of Scaevola diversity across all islands of Puerto Rico.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Temporal Regulation and Functional Impact of ERK Activity Near the Plasma Membrane
Authors: Keyes Jeremiah , Ganesan Ambhighainath , Zhang Jin
Published: June 2021

The ERK1/2 pathway plays critical roles in eukaryotic biology by transducing extracellular signals into cell‐fate decisions. One conundrum is in understanding how disparate signals induce specific responses through a common, ERK‐dependent kinase cascade. One major mechanism to control ERK‐signaling to induce proper cellular responses is through precise regulation over the temporal dynamics of ERK activity. For example, in the pheochromocytoma PC‐12 cell line, ERK exhibits transient activity in response to Epidermal Growth Factor (EGF), ultimately resulting in proliferation. On the other hand, PC‐12 cells respond to Nerve growth factor (NGF) with sustained ERK phosphorylation and eventual differentiation into neuronal‐like cells. An additional mechanism of control over ERK is through spatial regulation of activity; however, it has been difficult to accurately examine differences in activity among specific subcellular locations. To address this issue, we have expanded the toolbox of FRET‐based ERK biosensors by creating a series of improved biosensors that are specifically targeted to various subcellular regions to measure spatiotemporal changes in ERK enzymatic activity in real‐time in living cells. Using these sensors, we have tested the hypothesis that a single extracellular signal can induce different temporal dynamics between subcellular compartments in PC‐12 cells. We report that while EGF induces transient ERK activity in the cytosol and nucleus, EGF simultaneously induces sustained ERK activity near the plasma membrane of PC‐12 cells. Furthermore, we report that PKA and cAMP play an integral role in the regulation of ERK at the plasma membrane. Specifically, we observe that increasing basal cAMP prior to EGF induces a more transient activity at the plasma membrane in a PKA‐dependent manner. However, our results also indicate that PKA activity post‐EGF is necessary for sustained ERK activity near the plasma membrane, revealing the importance of understanding the temporal dynamics of pathway crosstalk. Furthermore, we report that selective inhibition of ERK activity near the plasma membrane drastically alters PC‐12 cell morphology and adhesion. These data further our fundamental understanding of how the spatiotemporal dynamics and native biochemistry of ERK in living cells modulates cell behavior.

Support or Funding Information

IRACDA fellowship through NIGMS/NIH award K12GM068524, R01 DK073368, R01 MH111516, R35 CA7622 from NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the Spc105 Complex in Organization and Microtubule‐Binding Activity of the Budding Yeast Kinetochore
Authors: Kim Jae ook , Zelter Alex , Johnson Richard , MacCoss Michael J. , Asbury Charles L. , Davis Trisha N.
Published: June 2021

Equally partitioning duplicated chromosomes during mitosis is of paramount importance; failure can cause aneuploidy, which predisposes individuals to cancer and birth defects. Chromosome segregation is orchestrated by kinetochores, a super‐complex of proteins that assemble onto centromeric DNA. Kinetochores must strongly couple chromosomes to spindle microtubules and they must sense and correct aberrant microtubule attachments. The Spc105 complex is an essential part of the budding yeast kinetochore, as are its homologues found widely across eukaryotes; however, it has yet to be completely defined. Spc105 complex plays an important role in the mitotic checkpoint, which produces a soluble ‘wait’ signal to delay anaphase in the absence of proper kinetochore‐microtubule attachments. The Spc105 complex is also proposed to contribute directly to kinetochore‐microtubule coupling. However, the extent to which the Spc105 complex and its homologues in other organisms contribute to microtubule coupling is unclear because it has been extremely difficult to study the purified complex in vitro owing to its biochemical instability. Molecular understanding of the binding of Spc105 complex to other complexes within the kinetochore is also lacking. We purified the recombinant Spc105 complex for the first time through co‐expression of its two protein components, Spc105p and Kre28p. Chemical cross‐linking and mass spectrometry (XL/MS) reveal that Spc105p and Kre28p interact through a region of about 200 amino acids. Additional XL/MS and pulldown experiments suggest regions through which the Spc105 complex interacts with the microtubule‐binding Ndc80 complex. Total internal reflection fluorescence (TIRF) microscopy shows that the Spc105‐GFP complex binds directly on microtubules. The ability to recombinantly purify the Spc105 complex will allow molecular details of its organization at the kinetochore and its contribution to the kinetochore function to be studied directly.

Support or Funding Information

National Institutes of Health (R01 GM040506) ‐ Trisha N Davis

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Temporal control of growth factor‐mediated signaling pathways during cell differentiation and Xenopus embryonic development
Authors: Mondal Payel , Krishnamurthy Vishnu Vardhan , Khamo John , Sharum Savanna , Zhang Kai
Published: June 2021

Growth factor‐mediated signaling pathways regulate a wide‐spectrum of cellular functions such as survival, proliferation, differentiation, and apoptosis. Different growth factors typically trigger the activation of the same set of downstream signaling pathways which includes the mitogen activated protein kinase (MAPK), phosphoinositide 3‐kinase (PI3K)‐AKT, and phospholipase C (PLC) pathways Evidence suggests that precise spatiotemporal regulation of these signaling pathways is involved in generating diverse cellular outcomes. Although conventional genetic and pharmacological approaches have helped us understand different aspects of signaling pathways, a quantitative delineation of their signaling kinetics is pending due to a lack of tools that allow for precise temporal control of these pathways. Non‐neuronal optogenetics, an emerging technique that utilizes light to control intracellular signaling pathways, is a remarkable tool to help achieve this goal. Here, we introduce a bicistronic optogenetic system which we recently developed in our laboratory to achieve temporal control of the MAPK pathway. This tool uses the photoactivatable protein cryptochrome 2 (CRY2) and the N‐terminal domain of cryptochrome‐interacting basic‐helix‐loop‐helix (CIBN) from Arabidopsis thaliana. This novel system allows reversible, light‐mediated activation of the MAPK signaling pathway in intact mammalian cells as well as in developing Xenopus laevis embryos. In PC12 neuronal cells, light‐controlled, intermittent MAPK activity reveals a memory effect in light‐induced neurite outgrowth. In Xenopus embryos, developmental stage‐specific MAPK activation further reveals that this pathway can reprogram cell fate after a crucial time window when germ layers are specified. Our strategy can be generalized to control other kinase pathways with a similar activation mechanism. Results from our research will help resolve intracellular mechanisms of growth factor‐mediated signal transduction during cell differentiation and embryonic development.

Support or Funding Information

Kai Zhang, Department of Biochemistry, University of Illinois Urbana‐Champaign

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wnt16 regulates chondrocyte differentiation through Wnt/ planar cell polarity (PCP) pathway
Authors: ZENG Yelin , YUENG Wai , MAK King Lun , Zhao Hui
Published: June 2021

Wnt signaling, a highly conserved signaling pathway, plays important roles in endochondral ossification which is a key process for skeletal development and bone repair. Wnt16, as one of the nineteen Wnt ligands, is reported to repress osteoclastogenesis, prevent cortical bone fragility fractures and to be upregulated in osteoarthritis. But how Wnt16 mediates chondrocyte differentiation during endochondral ossification is still unclear. Here, we investigate the roles of Wnt16 specifically in chondrocytes during endochondral ossification. First, we generated Col2a1‐Wnt16 transgenic mice in which Wnt16 was overexpressed in chondrocytes under the control of Col2a1 promoter and enhancer. The transgenic mice showed a great reduction of tissue mineralization during embryonic development. We also genetically knocked out Wnt16 by generating Wnt16Loxp/Loxp;Col2a1‐Cre mutant mice to examine whether Wnt16 is required for skeletal development. The mutant mice showed no severe phenotype in early skeletal development. However, after 2‐month‐old, the mutant mice displayed a smaller body size and lower bone mass as compared to that of control littermates. In vitro, our studies showed that Wnt16 delays chondrocyte hypertrophy and subsequent maturation. Mechanistically, we found that Wnt16 mainly activates the planar cell polarity (PCP) pathway through activation of JNK in primary chondrocyte. After treated chondroprogenitor cell line ATDC5 with SP600125, a JNK specific inhibitor, Wnt16‐induced delay of chondrocyte hypertrophy is eliminated. In addition, our data suggest that Wnt16 mainly interacts with Ror2 or CD146, co‐receptors of PCP pathway, but not Vangl2 or Ryk. Collectively, our current study provides evidence that Wnt16 delays chondrocyte hypertrophy through PCP pathway partially by binding to Ror2 and CD146. Our findings deepen the understanding of chondrocyte differentiation during endochondral ossification.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DDX3 induces neural crest through activation of an Akt‐Wnt signaling axis
Authors: Perfetto Mark , Li Jiejing , Wei Shuo
Published: June 2021

DDX3 is a DEAD‐box RNA helicase critical for normal development. Recently, mutations in DDX3 were found to be associated with multiple abnormalities including cleft lip/palate, phenotypes that are often caused by aberrant neural crest development. Here we show that knockdown of DDX3 inhibits neural crest induction in Xenopus embryos. This is accompanied by reduction in Akt activity and canonical Wnt signaling. Blocking Akt activity phenocopies DDX3 loss of function in inhibiting Wnt signaling and neural crest induction, and a constitutively active mutant of Akt rescues the neural crest induction phenotypes caused by DDX3 knockdown. These results show for the first time that a DDX3‐Akt‐Wnt axis is required for neural crest induction.

Support or Funding Information

NIH Grant GM114105

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.