bluuebunny/crossref_metadata_2025 huggingface dataset:
Biophysical Analysis of Human Neuropeptide Y: Mutations in the Hairpin Core Reveal Unusual Thermal Stability Linked to Higher‐Order Self‐Association
Authors: Hopkins Mandi M. , Bain David L.
Published: June 2021

Neuropeptide Y (NPY) is a highly conserved 36‐residue peptide, abundantly present in the central and peripheral nervous systems. NPY interacts with G‐protein coupled receptors (GPCRs) to regulate processes including energy homeostasis, affective behavior, plasticity, and memory function. The N‐terminal polyproline sequence of NPY folds back onto a C‐terminal α‐helix to form a hairpin structure. The hairpin undergoes transient unfolding to allow the monomer to interact with its target GPCRs, and to form reversible dimers in solution. Previous computational studies predicted that increasing the hydrophobicity of the hairpin core should result in a more thermodynamically stable hairpin, and decreased functional activity. To experimentally test this hypothesis, we characterized the role of two conserved tyrosines (Y20 and Y27) located within the hydrophobic core of the hairpin fold by mutating them to more hydrophobic phenylalanines. Consistent with the computational prediction, these mutations increased the thermal stability of NPY as observed by circular dichroism studies. However, mutant stability relative to wild‐type was higher than expected—melting temperatures increased by approximately 20 °C for the single mutants (Y20F and Y27F), and by 30 °C for the double mutant (Y2027F). These findings suggested that the mutations were not simply enhancing hairpin stability, but might also be driving self‐association to dimer. Using sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, we determined that the mutations indeed increased self‐association, but shifted the equilibrium toward hexamer‐like species. Notably, these later species were not unique to the NPY mutants, but were found to pre‐exist in the wild‐type population, contrary to previous reports. Collectively, the findings indicate that NPY self‐association is more complex than previously recognized, and that Y20 and Y27 may play a modulatory role in hairpin stability. Specifically, the ensemble of NPY quaternary states is adjustable by modulating the hydrophobicity of the NPY hairpin core, which may have implications for the development of new NPY‐based therapeutics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of 19F NMR to probe conformational changes of arrestin
Authors: Stout Rebecca Mae , Zhan Xuanzhi , Carroll William
Published: June 2021

Signal specificity is achieved through specific and highly‐regulated molecular recognition. Arrestins, a small family of multi‐functional adaptor proteins, mediate surprisingly complex cellular signaling events. To elicit the appropriate cellular response, arrestins have to ensure that the correct components are linked together to prevent the formation of mismatched protein complexes. It is now clear that arrestins undergo significant conformational movements in many regulating processes; however, previous studies of arrestin conformational changes are almost completely focused on receptor‐induced conformational changes, and little is known about binding‐induced conformations that allow arrestins to accomplish their diverse signaling functions. We hypothesize that binding induced conformational changes play decisive roles in regulating the recruitment of binding partners. 19F NMR has proven to be a powerful technique in the study of protein structure and dynamics because the 19F nucleus is easily incorporated into protein through specific labeling. The chemical shift of 19F is extremely sensitive to the changes of local micro‐environment caused by the conformational movements of protein, making it possible to analyze the minor movements within a protein molecule. 19F NMR is performed by incorporating a fluorine label into the protein structure Fluorine labeling can be accomplished in a variety of ways. Here, we developed a 19F NMR‐based method to detect the conformational movements of arrestins by employing a thio‐based method to incorporate 19F to the specific residues on arrestin. We have observed the release of C‐tail in arrestin‐3 upon binding of IP‐6 (inositol hexaphosphate). The methods developed here can be employed to detect the conformational changes of arrestin with many other binding partners.

Support or Funding Information

This project is supported by NSF MRI project (1531870), TTU faculty research grant (XZ) and Student Research Grant (RS)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual function of the trigger factor chaperone in nascent protein folding
Authors: Kaiser Christian , Liu Kaixian , Maciuba Kevin
Published: June 2021

Multi‐domain proteins – constituting a large group in all proteomes – often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. We have used optical tweezers to study the folding of elongation factor G (EF‐G), a model multi‐domain protein, as it emerges from the ribosome. We find that the N‐terminal G‐domain in nascent EF‐G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non‐native state. This non‐native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and non‐productive interactions among unfolded domains are efficiently prevented by the ribosome‐binding chaperone trigger factor. Thus, our single‐molecule measurements of multi‐domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding early folding defects is crucial, since they can propagate and result in misfolding of the entire protein. Our experiments define the folding pathway for a complex multi‐domain protein and shed light on the molecular mechanism employed by molecular chaperones to ensure productive and efficient folding.

Support or Funding Information

CMK acknowledges support by the Pew Scholars Program in the Biomedical Sciences and the National Institute of General Medical Sciences (1R01GM121567)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effectiveness of in silico Engineering of the β‐glucosidase B Enzyme
Authors: Boulanger Kyle R. , Hall Bonnie L.
Published: June 2021

Enzymes are used in a wide range of commercial and medical applications, and it is desirable for these enzymes to have maximum efficiency. A common approach to engineering a more efficient enzyme is generating random mutations and selecting those that increase enzyme activity in vitro. In silico design utilizes molecular modeling software to more rapidly predict structure‐function relationships, thus identifying more efficient enzymes without massive in vitro screening efforts. The drawback with these predictions is a lack of accuracy, yielding correct results as low as ten percent of the time (Carlin, 2016). To improve the algorithms used for molecular modeling, a large pool of enzyme mutants would need to be analyzed both in silico and simultaneously characterized kinetically under consistent experimental conditions. We are characterizing the BglB protein, as it is easily overexpressed and its efficiency can be measured using a simple colorimetric assay. We have engineered a pool of BglB mutants in silico using FoldIt, and compared the computational predictions with the in vitro kinetic results. Based on kcat and Km values for the various BglB mutants, we found FoldIt was able to correctly predict structure‐function relationships for BglB approximately 40% of the time. The remaining 60% of the time, the software predictions did not match the in vitro results. These BglB data, as a part of the larger SEEK database, can help refine the algorithms used in the FoldIt modeling software. Hopefully, molecular modeling will eventually be able to accurately predict structure‐function results in silico, saving both time and money in the enzyme engineering process.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineer Alkane Hydroxylating Properties into Phenol Hydroxylase: A Look at the 2nd Coordination Sphere
Authors: Guillen Erik , Sazinsky Matthew
Published: June 2021

Bacterial Multicomponent Monooxygenases (BMMs) are diiron enzymes characterized by the ability to hydroxylate a variety of hydrocarbon substrates. BMMs are naturally found in bacteria whose natural source of energy is derived from small organic compounds requiring initial hydroxylation for bacterial aerobic respiration. BMMs are of interest because the substrate transformations they perform can be useful in removing atmospheric greenhouse gases (i.e. methane) from the environment and such biocatalysts have potential applications in industrial organic syntheses. Phenol hydroxylase (PH) from Pseudomonas sp OX1 is a BMM family member essential for carrying out the oxidation of phenol to catechol but unlike the highly homologous soluble methane monoxygenase, it is unable to carry out oxidation on alkanes. Various studies suggest that the diiron coordination sphere in PH and sMMO are nearly identical, however, it is not understood how the protein scaffolds tune their reactivity toward different substrates. Here we present our efforts to explore the effects of the 2nd coordination sphere on reactivity and report on the development a screen to that would allow us search for alkane‐hydroxylating properties among a library of PH 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.
Mini‐Ins: a Monomeric Human Insulin Inspired From Cone Snail Venom Peptides
Authors: Chou Danny
Published: June 2021

For people with diabetes, maintaining blood glucose levels within the nondiabetic range is effective in delaying or even preventing long‐term complications, but achieving near normoglycemia is still challenging for these patients. This is due to the fact that the current fast‐acting insulin, Humalog, still has relatively slow absorption after subcutaneous injection which leads to delayed insulin response and sub‐optimal control of glycemia. An even faster‐acting insulin requires a monomeric insulin molecule. However, the region of the insulin molecule responsible for self‐association is also responsible for insulin receptor binding. Therefore, despite millions of dollars invested, a truly monomeric, ultrafast‐acting insulin remains to be invented. In this project, we take a bio‐inspired approach to utilize a venom insulin as a potential candidate with ultrafast‐acting properties. Recently, we and collaborators reported that a cone snail venom insulin, Con‐Ins G1, is monomeric in nature due to its shortened B‐chain. Furthermore, Con‐Ins G1 is bioactive toward human insulin signaling, albeit with 20‐fold reduction in potency. Learning from the structural insights, we performed a series of structure‐activity relationship studies to identify the key mutations from Con‐Ins G1 that lead to increased bioactivity. We have identified A8H, A9R, B10E, B20Y Des‐octapeptide (B23–30) insulin “mini‐Ins” as the shortest bioactive human insulin analog discovered to date. In vitro and in vivo experiments demonstrated equal biological potencies between native insulin, Humalog and mini‐Ins. The discovery of mini‐Ins demonstrates a promising therapeutic potential as a monomeric insulin.

Support or Funding Information

This work was supported by Utah Science Technology and Research Agency and the Margolis Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of Polymer Based Protein Engineering and ATRP to Modulate Substrate Size Specificity of Avidin
Authors: Winegardner Nicole P. , Adams Haili , Lucas Amber , Kaupbayeva Bibifatima , Murata Hironobu , Russell Alan S. , Minden Jonathan S.
Published: June 2021

Avidin and biotin interact very strongly and are commonly used to tag and purify proteins or peptides for proteomic analysis. Using a pH dependent reversible biotin tag that targets primary amines, all the proteins can be tagged at a slightly basic pH. After labeling proteins with the biotin tag, any excess unreacted biotin left over in solution must be dialyzed away to prevent competition for binding spots on the Avidin beads. Tagged proteins can then be bound to a solid surface Avidin bead and contaminants washed away. Proteins can then be released from the beads in their original unmodified state by lowering the pH. While this workflow has been shown to be very useful for purifying proteomes, the requirement for dialysis after the initial tagging step makes this workflow laborious and difficult to automate. This step is time consuming, can result in protein loss or precipitation, and makes automation impossible. In this study we seek to utilize polymer based protein engineering to circumvent this issue.

Atom transfer radical polymerization (ATRP) has been utilized in recent years to specifically modify and fine tune the behavior of proteins. By adding polymers of a specific composition and length in targeted areas of a protein, the behavior of a protein can be modified. In this study, we utilized ATRP to grow a dense polymer network off the surface of Avidin to act as a molecular sieve; The large polymers allow small molecules of biotin to bind to the binding site, while larger biotinylated proteins are excluded. The resulting “Caged Avidin” can then be used in the proteome purification workflow in the place of dialysis by binding and sequestering any unreacted free biotin, allowing the entire labeling, binding, washing, and eluting workflow to take place in a single tube with no bias and no sample loss. This can revolutionize sample preparation in biomarker and drug discovery by allowing for automation, and thus improved yield and reliability, of sample preparation.

Support or Funding Information

This work was supported in part by the Center for Polymer Based Protein Engineering at Carnegie Mellon University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fast and Accurate Evaluation of Oxidation‐Induced Destabilization of mAbs
Authors: Piatti Patricia , Mohamadi Mariam , Tschammer Nuška , Breitsprecher Dennis , Fung Peter A.
Published: June 2021

The applicability of monoclonal antibodies (mAbs) in therapeutic research continues to rise — they now account for almost 50% of protein‐based drugs. Monitoring their quality as well as binding properties are critical as these parameters provide insight into mAb functionality and efficacy as potential drugs.

Here we study trastuzumab, a monoclonal antibody that has been used to successfully treat patients with certain forms of breast cancer. Trastuzumab acts by binding to and interfering with the HER2/neu receptor in cancer patients. Using two complementary technologies, we examine how targeted oxidation affects trastuzumab structure and therefore its binding capabilities to protein A. First, a rapid analysis of oxidation‐induced changes in mAb folding and stability was performed. The same samples were then analyzed to determine how oxidation compromises mAb interactions. Oxidational stress directly correlated with weaker binding affinities of the Fc region towards protein A, showcasing fast and accurate characterization of trastuzumab sample quality.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cooperative Treatment of Breast Cancer Using an Irinotecan/IR‐820 Co‐loaded Hollow Mesoporous Silica Nanoparticles Nanoplatform
Authors: Li Tingting , Geng Yue , Chen Zhongyuan , Yang Hong , Wu Chunhui , Liu Yiyao
Published: June 2021

Background

Nanomaterial‐based multimodal systems are promising candidates for exerting synergistic therapeutic effects in cancer therapy.1 Combination therapy through different mechanisms can cooperatively inhibit cancer progression and overcome the limitations of monotherapies.2 In this study, we used hollow mesoporous silica nanoparticles (HMSNs) as a biocompatible carrier to co‐load chemotherapy drugs Irinotecan and near‐infrared (NIR) IR‐820 dye, which enhanced anti‐tumor efficacy by combining chemotherapy and phototherapy.

Methods

Hollow mesoporous silica nanoparticles (HMSNs) were prepared by an established method 1, then IR 820 and Irinotecan were loaded into the HMSNs by electrostatic attracting method. The successful synthesis of HMSNs/Irinotecan/IR820 (HMII) nanocomplex were confirmed by Fourier Transform Infrared (FTIR) spectroscopy and Fluorescence spectra. Confocal laser scanning microscopy (CLSM) was used to detect the cellular uptake of HMII. The photothermal conversion efficiency and anti‐tumor efficiency in mouse mammary cells (EMT‐6) bearing mice were further evaluated.

Results

The mean diameter of HMII were around 80 nm, which possessed the advantages of homogenous particle size, high stability, and good safety. The results demonstrated that HMII enhanced the delivery of Irinotecan and IR‐820 into EMT‐6 cells. HMII generated a high temperature upon a NIR laser irradiation (808 nm), and showed higher therapeutic efficacy in EMT‐6‐bearing mice compared to either HMII without laser or free drug with a laser

Conclusion

By combining chemotherapy and phototherapy (PTT), HMII efficiently inhibited the growth of breast cancer in mice. This nanosystem provides promising applicability for combination therapy of breast cancer.

Support or Funding Information

We would like to thank the National Natural Science Foundation of China (81671821, 81471785, 31470959, 31470906, and 11502049)

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 Bioinformatics Approach to Discover the Evolutionary Origin of the PTBP Splicing Regulators
Authors: Pina Jeffrey , Ontiveros Robert Jordan , Keppetipola Niroshika , Nikolaidis Nikolas
Published: June 2021

Polypyrimidine binding tract proteins (PTBPs) are members of the hnRNP family and are involved in the modulation of alternative splicing, serving as both repressors and activators of cassette exon inclusion in mature mRNA (Kafasla et al, 2012). The human genome encodes for three different PTBP paralogs, PTBP1, PTBP2 and PTPB3. These homologous proteins are highly similar in primary structure and domain organization, which is characterized by a N‐terminal domain and four RNA recognition motifs (RRMs) connected via linker regions. PTBPs share >70% sequence identity and differ in their tissue expression with PTBP1 being expressed nearly ubiquitously while PTBP2 and PTBP3 show more tissue specific expression (Keppetipola et al, 2012; Tan et al, 2015). Changes in the expression of PTBP1 and PTBP2 have been found to be critical in the process of neuronal differentiation and maturation (Li et al, 2014). PTBP1 has been observed to be overexpressed in ovarian cancer cells (He et al, 2007) and PTBP1 and PTBP2 have been implicated in the splicing of transcripts that promote proliferation of glioma (Cheung et al, 2009). Inhibition of PTBP3 expression in gastric cancer cells induced apoptosis (Liang et al, 2017). Therefore, further understanding of PTBPs may provide insight to how related proteins exert differential splicing outcomes and may provide therapeutic targets for drug design. In this study, we sought to characterize PTBP by elucidating its evolutionary history via sequence and phylogenetic analyses. Homologous sequences, including vertebrate, invertebrate, plant and fungi sequences, were identified by using BLAST search tools across various species databases. Multiple sequencing alignments (MSAs) and phylogenetic analyses were performed using different methods in MEGA6. Our current results reveal that the three human PTBP paralogs have orthologs found ubiquitously amongst jawed vertebrate species. In contrast, jawless vertebrate and invertebrate species contain only one homologous protein. This finding suggests that the gene duplication events that produced the three human PTBPs occurred within the ancestor of all jawed vertebrates. Plant species also have two or three PTBP homologs; however, these do not cluster with their animal homologs. This suggests that in both animal and plant species the genes multiplied independently. Fungi, which are unicellular organisms, contain one or no PTBP homologs. MSAs have also revealed a variety of residues within the RRMs that are highly conserved across the various PTBP orthologs surveyed, which suggests their functional and structural importance. We are conducting analysis of intron and synteny conservation as well as 3D structure comparisons to further develop the tentative conclusions posited.

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 Genome‐Wide CRISPR‐Cas9 Screen Identifies Importin‐β11 as a Required Factor for β‐catenin Signaling in Colon Cancer
Authors: Mis Monika , Steinhart Zachary , Angers Stephane
Published: June 2021

Over 25000 Canadians were diagnosed with colorectal cancer last year. The majority of colorectal adenocarcinomas (CRC) have a mutation in APC (adenomatous polyposis coli), a key component of the Wnt signaling pathway. In the absence of Wnt, APC is part of a protein complex that marks the Wnt signaling effector, β‐catenin, for ubiquitin‐mediated proteolysis. In the presence of Wnt ligands, β‐catenin escapes degradation, accumulates in the nucleus and regulates context‐dependent transcriptional programs regulating stem cell renewal, proliferation and differentiation. Inactivating APC mutations lead to hyperactive Wnt‐ β‐catenin signaling, driving tumor initiation and growth. Our objective was to identify novel regulators of the β‐catenin signaling pathway downstream of APC mutation in the context of colon cancer.

We have developed a positive‐selection genetic screen to identify genes required for Wnt‐ β‐catenin signal transduction. This system leverages the DLD‐1 colon cancer cell line, which contains an inactivating APC mutation and therefore has high constitutive Wnt signaling, and an inducible synthetic reporter gene system that leads to apoptotic cell death when the Wnt‐ β‐catenin signaling pathway is activated. This reporter consists of β‐catenin‐ responsive LEF‐TCF elements driving the expression of an inducible version of Caspase9, a member of the intrinsic apoptotic cascade. To identify genes required for β‐catenin signaling in these cells, a CRISPR‐Cas9 functional screen was performed. This genome‐wide screen applies the TKO sgRNA lentiviral library that contains over 90000 unique sgRNAs that target over 17500 human genes. Upon induction of Caspase9 activity, only cells harbouring loss‐of‐function within genes essential for Wnt‐ β‐catenin signaling are able to grow. The sgRNA conferring growth‐rescue were amplified from genomic DNA and identified using next generation sequencing.

The top genes enriched in the resistant cell population included known members of the Wnt‐ β‐catenin signaling pathway as well as a gene, which has no previous link to the Wnt signaling pathway, IPO11. Knockout of IPO11 confirmed its requirement for Wnt‐ β‐catenin signal transduction by luciferase assay and qPCR of β‐catenin target genes. IPO11 encodes for a nuclear import protein, importin‐ β11, which transports cargo into the nucleus. Knockout of IPO11 reduces β‐catenin nuclear localization. Importin‐ β11 associates with β‐catenin in cells, and this interaction is competed by dominant‐negative Ran mutant (Q69L). Knock‐out of IPO11 reduces proliferation of patient‐ derived, colorectal cancer cells, three‐dimensional organoid model, which mimics the physiological conditions required for cancer stem cell maintenance.

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 Role of TDP‐43 in the Pathogenesis of Frontotemporal lobar degeneration
Authors: Tsai Kuen‐Jer
Published: June 2021

Frontotemporal lobar degeneration (FTLD) is one of the most common neurodegernation diseases, especially in the dementia. TDP‐43 is a multifunctional DNA/RNA‐binding factor, which has been identified as the major disease protein of frontotemporal lobar degeneration with ubiquitin‐positive inclusions (FTLD‐U) and amyotrophic lateral sclerosis (ALS). TDP‐43 has been known mainly as a nuclear protein, with two RNA‐recognition motifs (RRM), RRM1 and RRM2, and a glycine‐rich region in its C‐terminus. However, the role of TDP‐43 in the pathogenesis of FTLD‐U has remained unknown. Therefore, to know how TDP‐43 pathology results in neuronal death in FTLD‐U may provide a therapeutic strategy to FTLD‐U. In this study, to test whether aberrant cell cycle activity and DNA damage involve in the neuronal death results from TDP pathology, the expression level of cell cycle associated genes and DNA damage marker had been examined. Form the result, cell cycle re‐expression and DNA damage occur in the TDP‐43 Tg mice and human brain tissues.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CDK5: The Connection Between Alzheimer's Disease and Type 2 Diabetes?
Authors: Mora Kevin Edgar , Aguanno Ann
Published: June 2021

Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) are two of the fastest growing diseases in the United States. T2DM is a complex, onset disorder arising from pancreatic beta cell failure and often associated with altered sensitivity of target tissues to insulin. AD is a neurodegenerative disorder causing a decline in cognitive ability, that is associated with two neuropathological hallmarks: neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau; and plaques comprised of aggregated amyloid beta proteins. Recent evidence suggests that insulin deregulation in the brain may play a role in the pathology of AD. Cyclin dependent kinase 5 (CDK5) is an unusual member of the cyclin dependent kinase family, because instead of a role in the cell cycle, it is predominately active in terminally differentiated neurons. CDK5 has been shown to be required for proper central nervous system development and for neuronal patterning and migration. Aberrant activation of CDK5 has been implicated in several neurodegenerative diseases, including AD, where its deregulation contributes to the formation of NFTs, among other protein aggregation and misfolding events. In addition to its role in neuronal development, CDK5 has also recently been associated with insulin exocytosis in the pancreas. An emerging correlation between TIIDM and AD suggests a connection between insulin deregulation and neurodegeneration. Because CDK5 is active in both tissues, it represents a possible common feature in these two pathologies. To study this connection, our lab uses the neuronal cell line model, PC12 which we transdifferentiate using neurotrophic growth factors. Once differentiated, we assess the extent of differentiation via qualitative and quantitative methods in the presence or absence of CDK5 chemical inhibitors and quantify the morphological changes using digital photomicroscopy. Our results indicate that aberrant CDK5 activity contributes to alterations in neuronal architecture. These along with changes in CDK5's kinase activity, confirms the effectiveness of our chemical inhibitors in altering CDK5 activity. Using this model, we have now begun to examine the role of normal and aberrant CDK5 activity on the response of differentiated neuronal‐like PC12 cells to varying concentrations of glucose and insulin in culture. Since a number of reports suggest that dysfunction in cerebral glucose metabolism and deregulation of brain insulin signaling are early abnormalities in AD, this model system provides a means to examine the potential role of CDK5 as a link between AD and Type II diabetes.

Support or Funding Information

Marymount Manhattan College Faculty Research Award

Sister Collette Mahoney Science Research Grant

Badgeley Foundation

Joseph A. Alexander Foundation

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 Susceptibility Screen of Phytochemicals Against Staphylococcus aureus
Authors: Mak Victoria P. , Heuertz Rita M.
Published: June 2021

Given the current and emerging levels of bacterial resistance to existing antimicrobic agents, evaluative assessment of plant‐derived compounds for antibacterial potential was deemed important and timely. The focus of this research was to determine the effect of phytochemicals on Staphylococcus aureus using the Kirby‐Bauer susceptibility assay, a method widely used in clinical laboratories as standard operating procedure for determination of bacterial susceptibility to antibiotics. Whatman filter disks were saturated with phytochemical and placed onto a Mueller Hinton agar plate swabbed with bacteria using the standard 3‐way streak method. Bacterial plates were incubated and then susceptibility was determined by measurement of resultant zones of inhibition. Antibiotic disks were assessed in parallel with phytochemical‐impregnated disks. The hypothesis was that specific phytochemicals inhibit bacterial growth. S. aureus (n=4 ATCC strains) displayed large zones of inhibition for cinnamaldehyde (31±12 mm: mean±SD), epigallocatechin gallate (14±1 mm) and plumbagin (16±3 mm). Inclusion of cinnamaldehyde, epigallocatechin gallate or plumbagin as anti‐Staphylococcus agents as direct therapeutic agents or as combination treatment strategies with current antimicrobial drugs may offer treatment options for patients with multi‐drug resistant bacterial infections.

Support or Funding Information

Financial support for VPM was received from the DeNardo Education and Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resensitizing Multidrug Resistant Bacteria to Antibiotics by Targeting Bacterial Response Regulators
Authors: Milton Morgan Eilise , Minrovic Bradley M. , Harris Danni L. , Draughn G Logan , Kang Brian , Jung David , Lewis Caleb P. , Thompson Richele J. , Melander Roberta J. , Zeng Daina , Melander Christian , Cavanagh John
Published: June 2021

Many of the currently available antibiotics target a narrow spectrum of metabolic processes required for cellular growth. The rate at which bacteria are developing resistance leads to traditional approaches no longer offering long‐term protection. Bacteria often avoid antibiotics through the use of biofilms. Biofilms act as a protective barrier against harsh environments and leave bacteria resistant to antibiotic treatments. As such, understanding how biofilms are formed and developing small molecule therapies are vital steps in combating antibiotic resistance. Formation of biofilms is controlled in part by transcription factors known as response regulators. Response regulators are part of two‐component systems which allow the cell to detect and respond to changes in its environment and are used to trigger quorum sensing and virulence factors. Response regulators have been highly sought after as drug targets. Knocking out function with drug therapies would likely inhibit pathogenic behavior, making response regulators a potent new approach that has not yet been exploited. The dynamic nature of response regulators makes structure based drug design challenging. Fortunately, a class of 2‐aminoimidazole (2‐AI) compounds can bind bacterial response regulators, inhibiting biofilm formation and resensitize multidrug resistant bacteria to antibiotics. This work highlights the interactions between 2‐AIs and the biofilm response regulators BfmR from Acinetobacter baumannii and QseB from Francisella tularensis. These studies have led to a model for response regulator inhibition by 2‐AIs. Understanding the impact of 2AIs on response regulators and their mechanism of interaction will lead to the development of more potent compounds that will serve as adjuvant therapies with broad‐range antibiotics.

Support or Funding Information

This project has been funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201500010C, grant R01‐GM055769 and by the V Foundation for Cancer Research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rv1495 toxin as a model for inhibitors of Mycobacterium tuberculosis DNA topoisomerase I
Authors: Moreno Pamela Katherine Garcia , Tse‐Dinh Yuk‐Ching
Published: June 2021

Topoisomerases overcome topological barriers encountered during DNA replication, transcription, recombination and repair. The genomes of mycobacteria encode only one type IA topoisomerase; Mycobacterium tuberculosis DNA topoisomerase I (Mtb Top I) is thus essential for cellular survival and proliferation, and has been validated as a novel TB drug target. Inhibitors of Mtb Top I may be useful for development of much needed new therapy for drug resistant TB.

Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the antitoxin), with endoribonuclease activity. Besides being an endoribonuclease, Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C‐terminal domain of Mtb Top I for mutual inhibition of catalytic activity. The C‐terminal domains of bacterial topoisomerase I have diverged in evolution with zinc ribbon domains found in most bacterial topoisomerase I as well as human type IA topoisomerases, while mycobacterial topoisomerase I have C‐terminal domains do not bind Zn(II). The mode of Mtb Top I inhibition by Rv1495 could be informative for the design and discovery of small molecule drugs that can target topoisomerase I in M. tuberculosis and pathogenic NTM (non‐tuberculosis mycobacteria).

Previous studies have revealed that inhibition of Mtb Top I is due to the direct physical interaction of the N‐terminal domain of Rv1495 toxin with the 33 kDa C‐terminal domain of Mtb Top I. However, no molecular details about this interaction have been reported. We have established an in vivo genetic selection scheme to identify specific amino acid residues in Mtb Top I required for this protein‐protein interaction and inhibition by Rv1495. E. coli AS17 cells have a temperature sensitive mutation in topA coding for topoisomerase I. Recombinant Mtb Top I expressed by plasmid pLICMTOPI can complement the topAts mutation for growth of AS17 at the non‐permissive temperature of 42°C. This complementation is abolished by co‐transformation of AS17 with a second plasmid pBADRv1495 expressing Rv1495. We have constructed a library of pLICMTOPI plasmid with random mutations in Mtb Top I coding sequence. Following transformation of AS17/pBADRv1495, mutated Mtb Top I clones that can complement growth of AS17 at 42°C in the presence of Rv1495 expression have been isolated successfully. These mutants retain the catalytic function of Mtb Top I to support viability at 42°C, but are resistant to inhibition by Rv1495 toxin. The results from analysis of the mutations involved should allow identification of a potential binding site on Mtb Top I for small molecules that can target mycobacteria topoisomerase I specifically.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Native American Adolescent Obesity and Nutrient Intake
Authors: Dai Hanchu , Zheng Shasha
Published: June 2021

Improving nutrition and declining the rate of obesity have been demonstrated through many reports. Identifying obesity among adolescent minority populations have been long recognized, but little research has been done on Native Americans adolescents. Using anthropometric measurements and dietary assessments, the findings within each study have shown to obtain baseline measures to determine the prevalence of obesity within the Sherman Native American adolescent population. Data of each assessment appears to be of use for predicting obesity and creating effective future interventions. Compiling data using the Harvard School of Public Health Youth/Adolescent Questionnaire (HSPH YAQ), a semi‐quantitative food frequency questionnaire allowed significant data to be found between normal and obese weight students. Utilizing each finding allows more effective ways to target and reverse the inclining rate of obesity among Native American adolescents. Results show that antioxidants being examined on such as vitamin E and lycopene, are beneficial in lowering the obesity rate among Native American adolescents. Level of fiber, thiamin and folate consumption was significantly lower among obesity population in Sherman Indian High School's Native American adolescents. Dietary mineral intake was shown to be lower among obese Native American adolescents. As result, dietary consumption of these nutrients can be used to correlate and predict obesity and developing effective interventions for Native Americans in general.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HMG‐CoA Reductase Inhibitors Do Not Ameliorate Progressive Atherogenic Changes In Human Macrophages Treated With Systemic Lupus Erythematosus Patient Plasma: Implications For Statin Use and Novel Insight Into Systemic Lupus Erythematosus
Authors: Arain Hirra A. , Petri Michelle , Kasselman Lora J. , Renna Heather A. , Zhen Juan , De Leon Joshua , Carsons Steven E. , Reiss Allison B. , Dores Michael R.
Published: June 2021

Systematic lupus erythematosus (SLE) is an inflammatory autoimmune disease that predisposes patients to develop atherosclerotic cardiovascular disease (CVD). Like the disease itself, the etiology by which SLE promotes CVD remains largely unknown. The most prevalent treatment for CVD is HMG‐CoA reductase inhibitors, used due to its lipid lowering and inflammation attenuating properties. Due to the need for CVD treatment in SLE, the Lupus Atherosclerosis Prevention Study (LAPS) investigated the efficacy of the HMG‐CoA reductase inhibitor atorvastatin in lowering plaque progression in SLE patients and revealed that statins had no effect on atherosclerotic progression. From these findings, we investigated the impact of the HMG‐CoA reductase inhibitor atorvastatin on the reverse cholesterol transport profile in a subset of human macrophages using plasma from SLE subjects in the LAPS trial. These experiments probe how statins may modulate conversion to the foam cell phenotype induced by the autoimmune SLE environment. Previously, we demonstrated that SLE promotes pro‐atherogenic changes in macrophages by decreasing the amount ATP‐binding transporters (ABCA1 and ABCG1) available. ABCA1/ABCG1 are important for transporting cholesterol out of the cell by packaging into nascent high‐density lipoproteins. Here, we demonstrate that statin treatment was not able to attenuate the pro‐atherogenic effects by not reducing the foam cell phenotype produced from the SLE plasma using differentiated macrophages from a human leukemia cell line. Further, our data reveal that the SLE autoimmune environment promotes a progressive atherogenic change in macrophages over time by increasingly suppressing the amount of ABCA1 and ABCG1 transporters while increasing the amount of low‐density lipoprotein receptor (LDLR), the receptor responsible for low‐density lipoprotein uptake. Liver X receptor‐α (LXR‐ α) and peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), two nuclear receptors responsible for up regulating expression of reverse cholesterol transporters, revealed no change in expression in the presence of statins, indicating a possible post‐transcriptional regulation of the ABC transporters by the lupus environment over time. These findings demonstrate that SLE induces progressive atherogenic changes that could not be rescued by HMG‐CoA reductase inhibitors.

Support or Funding Information

This research was supported by the Elizabeth Daniell Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Fenton and photo‐Fenton pretreatments on solubilization of lignocellulosic biomass
Authors: De la Rosa Shadid Fernanda
Published: June 2021

Non‐edible lignocellulosic biomass materials are attracting increasing attention as renewable, economical, and abundant resources to reduce dependency on petroleum resources and minimize energy and material feedstock costs. Despite their potential, the complex and rigid structures of these materials limit their use in such applications. Biomass materials must first be broken down into components with smaller molecular weights (e.g., oligo‐ and monosaccharides) in order to be efficiently converted into a wide range of value‐added products. Hydrolysates from biomass can be used for producing various value‐added products, including biofuels (ethanol, hydrogen, etc.), industrially important chemicals (e.g., solvents), and food products (sugar and sugar alcohols, etc.).

Success of using lignocellulosic biomass for biofuels and other useful chemical productions depend largely upon pretreatment and break down methods applied as well as physical and chemical properties of the biomass. The pretreatment is an essential prerequisite to make biomass accessible and reactive by altering its structural features such as, removal of lignin and reduction of cellulose crystallinity, which thereby increases in porosity. The goal of this project was to evaluate Fenton and photo‐Fenton processes as pretreatments methods for biomass (corn stover and corn husk) solubilization and reduce extensive use of toxic and hazardous chemicals or energy inputs in the hydrolysis process. Fenton and Fenton‐UV experiments were carried out in a glass reactor using 100 mL pH=3 solution (HCl). The biomass (2.5g) and 1mmol Fe2+ were added as well as freshly prepared H2O2 solution (3 × 0.7 mmol H2O2; 15 min intervals). In the Fenton‐UV reaction, light source (Pen Ray 254 nm, 40W) was placed in the center of the reactor. Further hydrolysis of Fenton and Fenton‐UV treated samples occurred using mild acid hydrolysis (4% H2SO4) to calculate hydrolysis percentage of each filtered mixture and biomass residue. IR and SEM measurements were performed on solid biomass residue and hydrolysate was analyzed for total organic carbon (TOC) content. The hydrolysis percentage between different pretreatments showed that Fenton in corn stover and corn husk resulted in 3.8% and 1.6% hydrolysis respectively while the Fenton‐UV‐Acid hydrolysis treatment in corn stover and corn husk resulted in 32.4% and 51.8% hydrolysis. From this we concluded that Fenton‐UV‐Acid hydrolysis was the best pretreatment for the solubilization of corn biomass.

Support or Funding Information

University of Nebraska‐Lincoln: Undergraduate Creative Activities and Research Experience.

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 a Short Form of Perilipin 5
Authors: Dalton Rebekah C. , Tansey John T.
Published: June 2021

Many modern health issues arise from aberrant lipid metabolism. Among these are type II diabetes and non‐alcoholic fatty liver disease. One commonality these diseases share is neutral lipid metabolism and storage. Perilipins are a family of five conserved proteins that are found on the surface of lipid storage droplets and play a central role in the regulation of cellular neutral lipid metabolism. Perilipin 5 is expressed in tissues with a high capacity for fatty acid oxidation such as heart, oxidative muscle, and fasting liver. Using western blotting and reverse transcriptase PCR we have identified a shortened form of perilipin 5 we have termed perilipin 5B. This short form of the protein retains the amino terminal 35 kDa of the protein but lacks the 4 helix bundle found in the C‐terminus. Based on reports in the literature, this protein would lack the carboxy terminal domains reported to be necessary for interactions with lipases and other proteins but would retain serine 155 that is requisite for PKA phosphorylation and translocation to the nucleus. We have constructed a carboxy terminal truncation of perilipin 5 and are expressing it in CHO cells to further define the function of this protein. Like the full length protein, this truncation targets to lipid storage droplets. Collectively these studies indicate that perilipin 5B plays a role in lipid storage, but its function is still unknown.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidation of the N‐terminal Structure and Characteristics of Perilipin 5
Authors: Wei Derek T. , Tansey John T.
Published: June 2021

The United States and other nations are experiencing an epidemic of lipid‐related diseases. Understanding the etiologies of these diseases is an increasingly imperative objective. Finding the function and mechanisms of the molecules involved in these lipid‐related diseases could show new and enhanced treatments for those suffering from these conditions. The perilipins, a family of five conserved lipid droplet proteins, are a chief subject of study in this field. However, an abundance of structural data is absent and would facilitate major strides in clarifying perilipin function. There is three‐dimensional structural data on the carboxyl terminus of one member in the perilipin family, perilipin 3, but not for other family members. The current study is aimed to characterize and determine the structure of the N‐terminal domain of perilipin 5. Perilipin 5 is of interest because of its implications in numerous of the metabolic conditions observed in the population. The N‐terminus is the concentration of the project because the C‐terminus of the protein has a high sequence homology with that of perilipin 3. Therefore, the C‐terminus of perilipin 5 is predicted to take on a similar structure to the C‐terminus of perilipin 3. A cDNA fragment coding for the N‐terminal region of perilipin 5 was PCR amplified from the full‐length Mus musculus perilipin 5 gene. It was then transformed into Escherichia Coli to produce the protein fragment of interest. Isolation of the protein fragment is undergoing via nickel affinity chromatography. Theoretical secondary and tertiary structures of the N‐terminal fragment of perilipin 5 have been obtained using the programs Phyre2, SCRATCH, GOR4, CFSSP, and Jpred. Computer models predict the sequence's secondary structure to contain numerous alpha helices with increasing frequency near the back half of the fragment. In conclusion, determination of the N‐terminal structure of perilipin 5 will further enable research into the functions of the perilipin family.

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