bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining the effect nutrient availability has on the activity of the 3′‐5′ exoribonuclease Rrp6
Authors: Ryan Tessa , Callahan Kevin P
Published: November 2023

Eukaryotic cells contain quality‐control systems that monitor RNA biogenesis. These systems feature ribonucleases that prevent the accumulation of nonfunctional RNAs as well as regulate normal mRNAs. Rrp6 is a 3′‐5′ exoribonuclease that plays a critical role in the 3′‐end formation and degradation pathways in the nucleus. In Sacchromyces cerevisiae Rrp6 physically and functionally interacts with the exosome, a highly conserved RNA‐processing complex. Rrp6 possesses activities that are both dependent and independent on its association with the exosome. It has been well documented that the deletion of RRP6 (rrp6‐Δ) leads to increased levels of both coding as well as non‐coding RNAs. However few experiments have addressed how changes in nutrient source affect the activity of Rrp6, specifically its ability to regulate coding and non‐coding RNAs. To address this question we evaluated the level of several Rrp6 regulated RNAs in wild‐type and rrp6‐Δ cells grown in media containing either galactose, glycerol or glucose. We calculated the growth rate of the yeast under these conditions and used quantitative real‐time polymerase chain reaction (qRT‐PCR) and ΔΔ Ct analysis to determine changes in RNA expression. The goal of these experiments is to better understand the activity of Rrp6p, which is critical regulator of RNA levels in all eukaryotes.
A Trypsin‐like Protease from Alternaria alternata Allergens Promotes Airway Inflammation through Activation of Protease‐activated Receptor‐2/β‐arrestin Signaling
Authors: Yee Michael C. , Nichols Heddie L. , Pal Kasturi , Polley Danny , Lee Kyu J. , Ming Monica , Seigler Michael D. , Hollenberg Morley D. , Daines Michael O. , Boitano Scott , DeFea Kathryn A.
Published: November 2023

Fungal Alternaria alternata exposure is correlated with increased morbidity and a higher risk of fatal asthma attacks in asthmatic patients. Alternaria allergens contain serine proteases capable of activating proteinase‐activated receptor‐2 (PAR2) and promote airway inflammation that is dependent upon protease activity. We have previously shown that PAR2‐dependent airway inflammation is mediated through the β‐arrestin‐2 activated pathway. Thus, we hypothesized that proteases from Alternaria filtrates will promote activation of the β‐arrestin‐2 (βarr2) dependent cellular signaling pathways that mediate airway inflammation in murine models of Alternaria‐induced asthma. We identify a single alkaline serine protease, with trypsin‐like characteristics, AASP (Alternaria Alkaline Serine Protease) that can activate PAR2, as demonstrated by recruitment of β‐arrestin‐2 to PAR2 and mobilization of intracellular Ca2+ in cultured cells. Using histological and flow cytometric analyses, we demonstrate that Alternaria‐induced airway inflammation requires both PAR2 and βarr2, as demonstrated by the reduced recruitment of leukocytes (particularly eosinophils and CD4+ T‐cells) into the lung, goblet cell hyperplasia, and thickening of the lung epithelium in PAR2−/−or βarr2−/−, compared to wild‐type mice. These inflammation parameters are also reduced by treatment of mice with Soybean Trypsin Inhibitor (SBTI) during the administration of Alternaria allergens, indicating that AASP is likely necessary for the inflammation induced by Alternaria. These are the first experiments demonstrating that the PAR2‐β‐arrestin‐dependent signaling axis is important for asthma induced by household allergens. Thus, targeting this pathway via biased antagonism of PAR2 could be a new avenue of therapeutic intervention for asthma.
What pathological mutants tell us about synuclein aggregation pathways
Authors: Bhumkar Akshay , Gambin Yann , Leitao Andre , Bowden Quill , Giles Nichole , Francois Bill , Hunter Dominic , Boecking Till , Sierecki Emma
Published: November 2023

Misfolding and pathological aggregation of proteins is a hallmark of many neurodegenerative diseases. α‐synuclein (α‐Syn) is one of the major components of the Lewy bodies associated with Parkinson's disease and other neurodegenerative disorders called synucleinopathies. Mutations in the SNCA gene were the first reported links between familial sporadic Parkinson's disease and perturbations at the molecular level.

We compared the behavior of α‐synuclein and five pathological mutants (A30P, E46K, H50Q, G51D and A53T). To gain insights into the aggregagtion behavior of these proteins, we developed a method coupling single molecule detection and cell‐free expression to measure precisely the oligomerisation of proteins, without purification and denaturation steps, in completely undisturbed samples. In these conditions, α‐Syn oligomerisation and aggregation is a rapid process that occurred co‐translationally, at nM concentrations.

Surprisingly, the pathogenic mutants segregated into two classes: one group forms large aggregates and fibrils while the other tends to form smaller oligomers and fewer fibrils. Strikingly, co‐expression experiments reveal that members from the different groups tend to not interact with each other, both at the fibril and monomer levels. Further biochemical analyses revealed differences of structure between the aggregates. Therefore, the different mutants could provide access to different species formed along the fibrillation path of α‐Syn.

We then examined the effects of a variety of chaperones on the aggregation propensity of the different mutants. This uncovers the specificty of the different chaperones for specific species in the aggregation pathway and identifies new therapeutic targets in Parkinson's disease and multiple sceloris atrophy (MSA).
Dynamic Organellar Maps, a Tool to Study Proteome‐Wide Subcellular Localization Changes
Authors: Itzhak Daniel , Tyanova Stefka , Cox Jürgen , Mann Matthias , Borner Georg
Published: November 2023

Most biological process involve changes in protein subcellular localization that are critical for cellular function. However, there is currently no method for monitoring such translocation events on a systems‐wide level. We have combined subcellular fractionation with mass‐spectrometry based proteomics in a robust workflow for the reliable determination of protein subcellular localization. Using this method, we have localized ~8,700 proteins in cultured cells to 14 different subcellular compartments, making it the largest repository of human protein subcellular localization of a single system. The method is robust enough to apply the maps comparatively, which we assessed using EGF signaling as a model system. We captured ligand‐induced movement of EGFR from the plasma membrane to the endosome, as well as accompanying known and novel translocation events, validating the method for use in dynamic studies. We then investigated localization changes induced during treatment with several small molecules, leading us to a rapid understanding of the molecular mechanism of these compounds. In conclusion, we have generated an extensive resource for protein subcellular localization, and developed a method for determining protein subcellular localization changes at the proteome‐wide level. This method adds a novel dimension to the systems biologist handbook. It provides a powerful tool in drug discovery, and will also grant unprecedented insights into processes such as cell signaling, trafficking, and cellular differentiation.

Support or Funding Information

Max Planck Society
Targeted drug delivery system for ribonuclease A
Authors: Barceló‐Bovea Vanessa Celeste , Griebenow Kai , Cruz‐Montañez Alejandra
Published: November 2023

Ribonuclease A is a very accessible protein that has been tested as a therapeutic for cancer in vitro and in vivo due to its ability to hinder protein production and induce cell death. However, the results in vivo were only positive when high amounts of enzyme were injected directly in the tumors. To overcome this limitation we are testing the use of a drug delivery system consisting of nanoparticles made out of the protein itself coated with poly(lactic‐co‐glycolic acid)‐poly(ethylene glycol)‐folate conjugates (PLGA‐PEG‐FA), in which the folate moiety confers selectivity towards cells overexpressing folic acid receptors on their membrane. Using a solvent displacement nanoprecipitation method we obtained nanoparticles constituted of ribonuclease A. Nanoparticles obtained under different conditions were analyzed by scanning electron microscope and dynamic light scattering, confirming spherical shape and an average size of 600 nm. The FA‐PEG‐PLGA copolymer to coat the nanoparticles was synthesized by conjugating the free alcohol group in PLGA with the carboxyl group in the folate‐conjugated‐PEG. The structure of the product was confirmed by 1H‐NMR. Experiments for coating optimization are in process and the efficacy of the system will be tested in HeLa cells as well as normal cells to confirm the selectivity. This drug delivery system does not have the limitation of a maximum loading capacity, and has been proven by us to efficiently deliver other proteins to cancer cells. Therefore, it is expected to selectively deliver higher amounts of ribonuclease A to cancer cells than previously reported systems.

Support or Funding Information

Supported by the MSRC (Molecular Science Research Center)
Omics approaches for discovery of natural anti‐aging and anti‐cancer compounds and defining mechanisms of their action
Authors: Titorenko Vladimir , Leonov Anna , Arlia‐Ciommo Anthony , Medkour Younes , Svistkova Veronika
Published: November 2023

Caloric restriction (CR) and dietary restriction (DR) extend lifespan across species and improve health by delaying the onset of age‐related diseases. All currently known anti‐aging chemical compounds (1) mimic lifespan‐extending and health‐improving effects of CR and DR without restricting caloric and nutrient intake; and (2) target signaling pathways that are under the stringent control of calorie and/or nutrient availability. It was believed therefore that all longevity pathways are “adaptable” by nature because they modulate longevity only in response to certain changes in the extracellular and intracellular nutrient and energy status of an organism. However, it is possible that that some longevity pathways could be “constitutive” or “housekeeping” because they control longevity irrespective of calorie and/or nutrient availability. We designed a high‐throughput chemical genetic screen for compounds that increase the lifespan of yeast under CR by modulating such housekeeping pathways. Our screen identified lithocholic acid (LCA) as one of them. Using proteomics, lipidomics, metabolomics and cell biological approaches, we found that LCA delays aging by: (1) remodeling lipid metabolism in the endoplasmic reticulum, lipid droplets and peroxisomes ‐ thereby preventing liponecrotic programmed cell death caused by the age‐related accumulation of fatty acids; (2) remodeling the repertoire of mitochondrial membrane lipids ‐ thereby reducing the number of mitochondria, increasing their size, expanding their cristae, elevating the abundance of respiratory supercomplexes in the inner mitochondrial membrane, and altering the age‐related dynamics of changes in mitochondrial respiration, membrane potential and reactive oxygen species; (3) attenuating age‐related mitochondrial fragmentation ‐ thereby suppressing mitochondria‐controlled apoptosis; and (4) promoting “mitohormesis” through the activation of several stress response‐related processes in mitochondria. Our findings also imply that, in addition to its robust anti‐aging effect, LCA exhibits a potent and selective anti‐tumor effect in cultured human neuroblastoma, glioma, breast cancer and prostate cancer cells by activating both the intrinsic and extrinsic pathways of apoptotic death.

Support or Funding Information

Supported by NSERC of Canada.
A new role of cysteine residues in human H‐ferritin activity
Authors: Yeom Jae Yoon
Published: November 2023

Ferritin is an intracellular enzyme that catalyzes iron oxidation and hydration to store intracellular iron in ferritin cavity. It is a 24‐heteropolymer composed of various combination with two types of subunit; ferritin heavy chain (FTH) and ferritin light chain (FTL). Although ferritin enzymatic mechanism has been studied well, it is still unclear how and where electrons produced by ferroxidase reaction are accepted. Human ferritin H‐chain has three cysteine residues: Two, Cys103 and Cys131, are highly conserved among mammalian proteins and the other one, Cys91, present only in human protein. On non‐reducing SDS‐PAGE analysis of recombinant FTH, we observed the mobility shift of FTH band, implying the existence of an intramolecular disulfide linkage. LC‐MS/MS data showed that the intramolecular disulfide linkage was formed between Cys91 and Cys103. Using stopped‐flow spectrometer, two major reactions, DFP formation and iron mineralization, of FTH WT and cysteine mutants are analyzed and the results showed the lower enzymatic activity of cysteine mutants compared to WT. Together, our data suggest that catalytic efficiency of FTH is regulated by redox states of cysteine residues.
Structural Characterization of SOD5 Mutants from Candida albicans
Authors: Villarreal Johanna , Peterson Ryan L. , Taylor Alexander B. , Holloway Stephen P. , Galaleldeen Ahmad , Culotta Valeria Cizewski , Hart P. John
Published: November 2023

Candida albicans is the most prevalent human fungal pathogen and there is currently no vaccine to fight these infections. Colonization by this microbe can lead to acute infections of the mucosal surfaces in the oral and genital cavities, which for immunocompromised individuals can lead to life‐threatening systemic infections. Superoxide Dismutase (SOD) is an enzyme that catalyzes the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen. SOD5, a cell surface protein, is used by Candida albicans to defend itself against reactive oxygen species (ROS) released during the host immune response. SOD5 is related to Cu/Zn SODs but the structure shows a copper‐only active site and the protein is lacking the electrostatic loop common to Cu/Zn SODs. To better understand the biophysical characteristics of SOD5, mutants of SOD5 with single amino acid substitutions at positions 110 and 113 were generated to disrupt the hydrogen bonding network that stabilizes the active site. These mutants were over‐expressed in E. coli and, because of their insolubility, were denatured, refolded, purified, and set up for crystallization trials. Data were collected for crystals of the mutants and the structures were determined using the molecular replacement method. The structures reveal that the mutations did not alter binding of copper at the copper site.
The Energetic Cost of Improved Acid Resistance
Authors: Keffeler Erica Catherine , Payne Sophie , Blum Paul
Published: November 2023

Thermoacidophiles require specific mechanisms in order to withstand extreme temperatures and low pH. Sulfolobus solfataricus is an aerobic acidophile that is native to acidic geothermal environments. The Blum lab adapted S. solfataricus to possess a unique, heritable super acid resistance trait with the capability to survive in a pH that is exceedingly lower than its optimum. These newly adapted lineages were named super acid resistant Crenarchaeota (SARC). Energy is required for acid resistance, but it is predicted that SARC strains require an excess amount of energy to resist an extremely low pH. S. solfataricus receives its energy through their central carbon metabolism by generating electron carriers that are transferred to the electron transport chain to produce ATP. In this work, the transcriptomic data derived from the SARC lineages was analyzed to produce potential explanations on how the SARC strain met their energy demands in a more acidic environment. In addition to this work, chromatin proteins are also an interest due to their potential contribution to the SARC phenotype. A novel 8kDa chromatin protein is suspected to play a role in acid resistance gene expression, therefore it will be expressed and purified for downstream x‐ray crystallization studies.
Skin Preparations from Catfish (Arius bilineatus, Val.) Contain a Lipid Which Inhibits Cancer Cell Survival In Vitro
Authors: Al‐Hassan Jassim M. , Afzal Mohammad , Paul Bincy Maniyalil , Oommen Sosamma , Liu Yuan Fang , Pace‐Asciak Cecil
Published: November 2023

The Catfish (Arius bilineatus, Val.) secretes a gelatinous substance composed of biochemically active lipids and proteins from its skin upon stress or injury. Preparations from the skin have previously been shown to affect blood clotting, accelerates healing of non‐healing diabetic foot ulcers and relief from neuropathic pain in man1. Chromatographic separation of the crude lipid preparation on silica gel column gave a fraction eluted with chloroform that apoptosed human cancer cell lines (K562 and DU‐145) in vitro, observed IC50 was between 5 – 10 ug/0.1ml/1e4 cells. Identification of the active substance(s) is currently under way.

Support or Funding Information

(Supported by a grant from Kuwait Foundation for the Advancement of Science No. KFAS 201320701A‐D, and Kuwait University Research Grant No. SL03/14).
Antiproliferative effect of curcumin and kaempferol on colon cancer cells
Authors: Kaverimanian Arjun , Khatwani Natasha , Ezekiel Uthayashanker
Published: November 2023

Results of epidemiological studies show that people who consume diets rich in phytochemicals (such as individuals living in India) have lower rates of colorectal cancer. Occasional exposure of colonic cells to a single phytochemical may be insignificant in inhibiting growth of cancer cells, whereas regular and repeated exposure of colonic cells to phytochemicals alone or in combination with other phytochemicals may be efficacious in preventing colon cancer. Combinations of phytochemicals may provide additive or synergistic effects and could be a potent way to prevent or eliminate colon cancer initiation and/or progression. The focus of this project was to determine the effect of phytochemicals alone or in combination on cancer cell proliferation. Of interest were curcumin, a phytochemical in turmeric, and kaempferol, a phytochemical in many vegetables, fruits and beans, since they both have been reported to have anti‐cancer potential. Antiproliferative effects of curcumin and kaempferol were assessed using the DLD‐1 colon cancer cell line of epithelial origin. Initial findings indicated that both phytochemicals inhibited proliferation of DLD‐1 cells to varying extents. In particular, curcumin inhibited DLD‐1 proliferation at relatively low concentrations while kaempferol showed a significant antiproliferative effect only at high concentrations. When the concentration of curcumin was kept constant at 12.5 μM (IC50) and concentration of kaempferol was varied, a synergistic anti‐proliferative effect was observed. In fact, even the lowest concentrations of kaempferol combined with curcumin significantly inhibited colon cancer cell proliferation. Results of this study suggest that a combination of curcumin and kaempferol has significant inhibitory effects on proliferation of colon cancer cells.

Support or Funding Information

Financial support for AK, NK and TA was received from the DeNardo Education and Research Foundation.
ENGINEERED FGF‐1 DERIVATIVES LACKING CYSTEINE FOR TREATING CORNEAL ENDOTHELIAL DYSTROPHY
Authors: Eveleth David , Bradshaw Ralph , Blaber Michael
Published: November 2023

Fibroblast growth factor‐1(FGF‐1) binds and stimulates all members of the FGF class of tyrosine kinase receptors, thus making it a potent agent in tissue regeneration, but its value as a therapeutic is greatly compromised by poor stability and a dependency on heparin as a co‐factor. As the mature form is not exported from cells via the endoplasmic reticulum/Golgi system, its three cysteine residues are all present in reduced form and the presence of these free cysteines limit the biological half‐life of the protein considerably. Substitution of these and other residues has yielded a variety of engineered FGF‐1 derivatives with greatly improved stability and pharmaceutical properties (1), We have tested two of these derivatives, in which the Cys residues have been eliminated, for their ability to stimulate proliferation of corneal endothelial cells as a potential treatment of corneal endothelial dystrophies, such as Fuch's Dystrophy, a condition that leads to the deterioration of the endothelial layer on the back side of the cornea and can only be presently treated effectively by corneal transplants. THX 1011 is a triply substituted derivative in which the Cys at 16, 83 and 117 have been substituted by Val and THX 1114 has the Cys at 16 and 117 changed to Ser and Val, respectively, with the Cys at 83 converted to an internal disulfide bond by mutating the Ala at 66 to Cys. As compared to native FGF‐1 both derivatives were more active stimulators of proliferation of primary cultures of corneal endothelial cells from rabbits or humans with THX 1114 being an order of magnitude more potent than THX 1011. TTHX1011 potently accelerated the regeneration of the corneal endothelium in vivo following cryogenic injury in the rabbit. These data suggest that eFGF‐1s may be a useful therapeutic for corneal endothelial dystrophies.

Support or Funding Information

Trefoil Therapeutics LLC
L‐ to D‐ amino acid substitution impacts the cellular penetration activity and the intracellular behavior of the endosomolytic delivery agent dfTAT
Authors: Najjar Kristina , Erazo‐Oliveras Alfredo , Pellois Jean‐Philippe
Published: November 2023

The plasma membrane acts as a selectively permeable membrane hindering the entrance of large hydrophilic molecules into the cytosolic space of cells. Cell‐penetrating peptides (CPPs) have since been used to internalize macromolecules by using the endocytic pathway as a route of cellular entry. However, only about 1% of endosomal content is released making endosomal entrapment a severe bottleneck in the field of macromolecular delivery. Achieving efficient cytosolic release of macromolecules from endosomes could increase the potential use of CPPs for therapeutic and cell biology applications. In our laboratory, we have generated a peptide L‐dfTAT, derived from the prototypical CPP TAT, which penetrates live cells by escaping from endosomes with a particularly high efficiency. L‐dfTAT can deliver proteins into live cells through a simple co‐incubation protocol and shows no negative impact on cell physiology. L‐dfTAT delivery is efficient however; this peptide is synthesized with L‐amino acids making it susceptible to protease degradation, which could diminish its true cell permeation activity. In this study, we establish the effect of circumventing this issue by substituting L‐ with D‐amino acids, which are protease resistant. We show, D‐dftat is protease resistant and enters cells through a two‐step process similar to L‐dfTAT. D‐dftat impacts endocytic uptake negatively, however endosomal escape is enhanced. Interestingly, D‐dftat severely impacts cellular proliferation rates and has a dramatic effect on the transcriptome when compared to L‐dfTAT. With this, understanding the molecular basis that are required for the endosomolytic activity of L‐dfTAT is of great significance and have not been identified yet. Therefore, this project also focuses on synthesizing, a focused library of peptide analogues based on the L‐dfTAT prototype to establish the structure activity relationships that will facilitate in identifying the molecular features that are required for this efficient endosomolytic and cell permeation activity. This project also lays a foundation for the future design of delivery agents with improved therapeutic significance.

Support or Funding Information

This article was supported by Award Number R01GM110137, R01GM087981 and R01GM087227
Defects in protein O‐mannosylation result in abnormal muscle contractions and aberrant neural sensory feedback in Drosophila
Authors: Panin Vlad , Baker Ryan , Nakamura Nausuke , Lyalin Dmitry
Published: November 2023

Protein O‐mannosylation pathway is evolutionarily conserved in metazoan organisms. In a wide range of species, from Drosophila to humans, defects in protein O‐mannosylation was found to be associated with abnormal muscle and neural development, as well as postdevelopmental pathologies, including muscle degeneration. In humans, mutations in genes involved in the biosynthesis of O‐mannosyl glycans result in severe muscular dystrophies that are called dystroglycanopathies because of prominent defects in glycosylation and function of Dystroglycan, a cell membrane protein mediating interactions of cells with the extracellular matrix. While recent studies shed more light on important structural features of O‐mannosyl glycans and revealed numerous new targets of O‐mannosylation in mammals, the mechanisms that underlie functions of O‐mannosyl glycans in vivo remain poorly understood. In our study, we focused on elucidating these functions using Drosophila as a model system. Our experiments concentrated on analyses of phenotypes associated with defects in the protein O‐mannosyltransferase (POMT) genes, rotated abdomen (rt) and twisted (tw). We characterized temporal and cell‐specific requirements of POMT genes for normal neuromuscular physiology and proper body posture. Using a combination of genetic, immunofluorescent and live imaging approaches, we unveiled new phenotypes, including defects of embryonic body posture caused by uncoordinated muscle contractions. Our experiments indicated that POMT activity is simultaneously required in muscles and neurons, suggesting that POMT genes affect communication between these cells. We found that coordinated waves of muscle contractions induce rolling behavior, which also suggested an innate chirality of embryo ‐ shell interactions. Our data provided evidence that abnormal protein O‐mannosylation result in a defect of the sensory feedback that controls the pattern of muscle contractions. Our experiments also suggested that peripheral multidendritic neurons play an important role in the pathogenic mechanism of abnormal muscle contractions and the body rotation phenotype. Taken together, our results elucidated in vivo functions mediated by the POMT pathway in Drosophila, which could shed light on potential analogous mechanisms in mammals.

Support or Funding Information

This project was supported in part by NIH/NS075534
Lectin Nucleotide Phosphohydrolases may function as co‐receptors or modulators of several chitin‐based oligosaccharide signaling events in plants
Authors: Etzler Marilynn E. , Roberts Nicholas , Kalsi Gurpreet
Published: November 2023

Chitin oligosaccharidic signals play important roles in plant defense and the initiation of symbiotic interactions of plants with microorganisms. Two of these symbioses which have great agricultural importance are the symbiosis of arbuscular mycorrhizal fungi with about 80% of land plants that leads to the facilitation of nutrient absorption and the host/strain specific nitrogen‐fixing symbiosis between rhizobial strains of bacteria and leguminous plants. Similarities in structures of the Myc factor and Nod factor signals that initiate these two respective symbioses as well as some common features of the early stages of these symbioses have suggested that the rhizobium‐legume symbiosis may have evolved from the more ancient mycorrhizal symbiosis by gene duplication and divergence. This possibility is supported by our recent transgenic studies in which inhibition of expression of a Nod factor‐binding protein in legume roots inhibited both of these symbioses. The above Nod factor‐binding protein represents a new category of lectins (LNPs) that have both Lectin and Nucleotide Phosphohydrolase activity. These LNPs are members of the apyrase family of enzymes which are found in a wide variety of organisms. Phylogenetic comparisons have shown that the above LNP, (LNP1), represents a unique subcategory of apyrases found in leguminous plants. LNP1 is a peripheral membrane protein localized on the surface of root hairs in the nodulation zone of the root.

Using a combination of biochemical, molecular and cell biological techniques we identified a number of other apyrases in the tissues of the legume, Dolichos biflorus, several of which bind to the hog blood group A + H gastric mucin affinity resin used in the isolation of LNP1. We now report the comparison of these LNPs with respect to their structures, carbohydrate‐ and nucleotide‐binding activities and differential expression in the plant. Phylogenetic analysis shows that these other LNPs are not confined to leguminous plants. The data suggest that these LNPs may function as co‐receptors or modulators of other chitin‐related oligosaccharide signaling events found in plants.

Support or Funding Information

This research was supported by NIH Grant GM21882 and by Ceres, Inc.
Correlating the Reversal of Multidrug Resistance in Cancers by Novel P‐glycoprotein Inhibitors to the Expression of ABC Transporters
Authors: Sunshine Alexis L. , Follit Courtney A. , Marchesseault Collette , Wise John G. , Vogel Pia D.
Published: November 2023

P‐glycoprotein (P‐gp) is a transmembrane protein that has evolved to eliminate toxic compounds from cells. It functions by using the hydrolysis of ATP to generate a conformational change that physically pushes xenobiotics that are bound in the drug‐binding domain of P‐gp from the cytosolic side of the membrane into the extracellular space. This broad functionality of the pump relies on the inherently non‐discriminating character of multiple binding sites within the drug binding domains and allows P‐gp to transport a variety of substances including most currently approved chemotherapeutics. Overexpression of P‐gp in a cell, therefore, results in a remarkable resistance to the presence of toxic chemicals, even enabling cancerous cells to survive chemotherapy treatments, which ultimately renders such therapy ineffective. It is possible, however, for an inhibitory agent to bind to P‐gp and to inactivate its function. In recent computational ultra‐high throughput searches followed by biochemical, biophysical and cell culture evaluations, we have identified several compounds that reverse multidrug resistance in cancer cells in culture (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular pharmacology, 86(6), 716–726; Follit, C. A., Brewer, F. K., Wise, J. G., & Vogel, P. D. (2015) Pharmacology Research & Perspectives, 3(5) doi:10.1002/prp2.170). In the present study, we aim to extend these findings by quantifying the expression levels of P‐gp and other multidrug resistance proteins in various multi‐drug resistant cancer cell lines. Correlating the level of reversal of multidrug resistances with the level of expression of the various ABC transporter proteins that have been associated with the multidrug resistance phenotype, will allow us to determine the ABC transporter specificity of the discovered compounds. Such an approach may in the future be used to develop personalized medical treatment for patients with multi‐drug resistant cancers conferred by specific ABC transporters.

Support or Funding Information

This work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, New England Biolabs / Lynn McBee, the Hamilton Undergraduate Research Scholarship Program, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Inhibition of ABC‐Transporters by in silico Identified Small Molecules
Authors: Follit Courtney A , Nanayakkara Amila K , Wise John G , Vogel Pia D
Published: November 2023

Multidrug resistance (MDR) is a complex phenotype characterized by simultaneous resistance to structurally and functionally dissimilar drugs. MDR is frequently associated with overexpression of transmembrane efflux proteins belonging to the ATP‐binding cassette (ABC) transporter superfamily including P‐glycoprotein (P‐gp), multidrug resistance associated protein (MRP1), and breast cancer resistance protein (BCRP). Normally expressed at tissue barriers, these proteins protect vulnerable cells from xenobiotic attack by pumping a wide range of toxins out of the cell. Unfortunately, this broad substrate range, characteristic of these transporters, poses a major obstacle to the successful chemotherapeutic treatment of cancers since the majority of clinically relevant chemotherapeutic drugs can be effluxed by P‐gp, MRP1, and BCRP. Treatment and prevention of MDR have been studied extensively for decades, and yet, there are still no effective FDA‐approved therapies available.

Using targeted molecular dynamics and massively parallel drug docking studies, we have screened millions of drug‐like molecules and identified hundreds of compounds that are predicted to inhibit P‐gp by specifically interacting with its ATP binding domain. We have previously shown that of four of these in silico identified compounds inhibited P‐gp catalyzed ATP hydrolysis (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular Pharmacology, 86(6), 716–726). When tested in cell culture, three of these compounds restored chemotherapeutic sensitivity in a multidrug resistant human cancer cell line which overexpresses P‐gp (Follit CA, Brewer FK, Wise JG, Vogel PV (2015) Pharmacology Research & Perspectives 3(5): e00170). In this study, we expand our cell culture screening to evaluate 46 potential inhibitors identified by our in silico screening methods and report that over 30% of these compounds restored chemosensitivity to MDR human cancer cell lines. The best of these compounds will be further investigated to determine toxicity and specificity. With future chemical optimization, we are working towards developing powerful co‐therapeutics to treat and prevent MDR in human cancers.

Support or Funding Information

This work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Plasma Concentrations Of FAM3D Protein Is Higher In Individuals With Type 2 Diabetes Mellitus Treated With Sulfonylureas
Authors: NEVES JOSE ANTONIO JANUARIO , FELICIANO REGIANE , GIANNELLA‐NETO DANIEL
Published: November 2023

Background

Among the new cytokine Family with Sequence Similarity 3 (FAM3), a subfamily of proteins similar to cytokines known as FAM3D was identified. FAM3D is predominantly expressed in the gastrointestinal tract of normal individuals. Plasma concentrations of FAM3D range according to the nutritional status presenting a postprandial increase and a reduction in the later post‐absorptive period. It might be speculated that FAM3D is an inhibitor of insulin secretion, as it has been observed a significant increase in patients with pancreatic adenocarcinoma associated with T2D.

Aims

Quantitatively determine the plasma concentration of FAM3D, in a group of patients with T2D and comparing with patients with pre‐Type 2 Diabetes Mellitus (pT2D) and Control (CTRL) individuals paired according to gender, age and body mass index in order to better elucidate the physiology of FAM3D as well as its pathophysiological role in T2D.

Methods

We selected 90 patients comprising 15 male and 14 females diagnosed with T2D; 12 male and 15 female diagnosed pT2D; and 7 male and 9 female CTRL. Seventeen patients were excluded according to the inclusion and exclusion criteria. In total, 73 patients were included.

All were submitted to measurement of anthropometric and biochemical tests, including HOMA‐IR calculation and the determination of plasma concentrations of FAM3D by ELISA. The Kruskal‐Wallis tests were used in the comparison of study groups, and the regression coefficient r of Spearman was calculated on correlation between plasma concentrations of FAM3D and other anthropometric and biochemical variables. The level of statistical significance was set at p <0.05.

Results

Among the groups there were no significant difference in the concentration of FAM3D, and the median±Q for the CTRL group (24.92ng/mL ±9.11); pT2D (14.65ng/mL ±6.02) and T2D (19.87ng/mL ±5.39).

The group of DM2 patients on sulfonylurea (23.42ng/mL ±6.1) had higher values compared to the group treated with other oral antidiabetic drugs (14.9 ng/mL ± 7.30), p <0.05.

Conclusions

There was no correlation of FAM3D between groups. Patients with T2D on sulfonylurea presented a higher concentration of FAM3D when compared with T2D patients treated with other oral antidiabetic drugs. Although further studies are necessary, the increased concentrations of FMA3D in patients with T2D on sulfonylurea could be due to the inhibitory effect of the secretagogue on FAM3D.

Support or Funding Information

Universidade Nove de Julho
Liraglutide Induces Brown Adipocyte Specific Genes in Mice Skeletal Muscle Tissue
Authors: Li Lixin , Zhou Joseph Yi , Bahaee Jamsheed
Published: November 2023

Obesity is a risk factor for type 2 diabetes, coronary artery disease, and stroke. Glucagon like peptide ‐1 (GLP‐1) is synthesized by the L cells of ileal mucosa and is released after nutrients ingestion to potentiate glucose stimulated insulin secretion. Liraglutide, a full agonist of the GLP‐1 receptor (GLP‐R), has long‐lasting effects due to its increased resistance to enzymatic degradation. Clinical data has demonstrated the protective effect of liraglutide on weight gain in type 2 diabetes subjects. However, the underlying mechanisms of this protective effect have not been identified. Brown adipose tissue plays a major role in control of energy balance in rodents, whether GLP‐1 activate brown fat differentiation has not been studied. C2C12 myoblasts are known to be able to differentiate into adipocytes after stimulation. By treating the undifferentiated C2C12 myoblast with 5nM liraglutide, we observed a significant induction of GLP‐1 receptor after 24 h. C2C12 cells were cultured to confluence, and then exposed to brown adipocyte differentiation medium in the presence or absence of 5nM liraglutide, the mRNA level of brown fat enriched genes including peroxisome proliferator‐activated receptor α (PPAR‐α) and cell death activator‐A (Cidea) were upregulated after 6 days of induction of differentiation compare to control cells. Induction of peroxisome proliferator‐activated receptor‐γ coactivator (PGC)‐1α mRNA was observed as early as 3 d post‐induction. In vivo study was performed by injecting C57 black/6 mice with liragludie daily for 5 weeks. Both mRNA and protein levels of uncoupling protein‐1(UCP‐1), a brown fat specific gene, are significantly induced in skeletal muscle tissue after liraglutide treatment. Lirglutide also upregulated other brown fat enriched gene including PGC‐1α, Cidea, and PPAR‐α. Our study indicates that liraglutide induces brown adipogenesis. GLP‐1 and its analogues are potential therapies for obesity and obesity related metabolic disorders.

Support or Funding Information

Central Michigan University Faculty Startup Fund.
Effects of Paternal High Fat Diet on Plasma Leptin, Adiponectin, and Insulin in Offspring of C57BL/6 Mice
Authors: Zontini Alexis , Slyvka Yuriy , Zhang Yizhu , Nowak Felicia V
Published: November 2023

Parental obesity is a predictor of childhood excess weight. Having a father, mother or sibling with a BMI of 40 increases the risk of adult obesity 5 fold compared to individuals who have normal weight first degree relatives. The rise in obesity in the U.S. has played a major role in the increasing number of instances of cardiovascular disease, diabetes, and even some types of cancer. This study aims to determine phenotypic effects on leptin, adiponectin, glucose tolerance (GTT), and HOMA in offspring of non‐obese (Group 1) and high‐fat diet‐induced obese (Group 2) fathers in C57BL/6 mice. Male mice were fed a low fat diet (LFD, 10% fat) or a high fat diet (HFD, 45% fat) for 12 weeks then mated to females fed LFD. Pups were fed regular chow and tested at 6 weeks, and 6 and 12 months of age. Glucose was measured using One Touch glucometer. Circulating levels of insulin, leptin and adiponectin were quantified by immunoassay. The results showed that leptin is higher at 6 months of age in Group 2, and is significant in females. It tends to be lower in Group 2 at 12 months, but not significant. Adiponectin on the other hand is higher at 6 weeks and 12 months in Group 2 of either sex but lower at 6 months, and concentrations at 12 months show parallel sex and group differences with the high molecular weight (HMW) form. Leptin is higher overall in males than females while adiponectin is higher in females. Results from HOMA and GTT insulin show a significant increase in both parameters at 6 weeks in males from Group 2 with a similar trend in both sexes at 6 and 12 months. We can conclude that there is an overall increase in insulin resistance in Group 2 and the group differences of leptin and adiponectin are overall inversely related, as would be expected. As part of a larger study looking at paternal transmission of epigenetic modifications to offspring, the ultimate goal is to find candidate genes for targeted intervention aimed at preventing obesity and diabetes in high‐risk offspring.

Support or Funding Information

Heritage College Research Award and an Ohio University Research Council Award
Determining the Role of Skeletal Muscle microRNA‐133a in Early‐Onset Insulin Resistance
Authors: da Silva Rosa Simone , Mughal Wajihah , Nguyen Lucas , Chapman Donald , Aliani Michel , Pereira Troy , Rampitsch Christof , Dolinsky Vernon , Gordon Joseph
Published: November 2023

Fetal exposure to diabetes during pregnancy increases the risk for early‐onset insulin resistance in the offspring; however, the key molecular regulators responsible for fetal metabolic programming have not been characterized in muscle tissue. We demonstrate that prenatal exposure to gestational diabetes decreased the soleus muscle expression of microRNA‐133a in offspring. Using tandem mass spectrometry techniques we identify a conserved phosphorylation motif within the MEF2 and SRF transcription factors that is targeted by PKCδ and regulates microRNA‐133a expression and mitochondrial function in response to a lipotoxic signal. Reconstitution of MEF2 function in cultured myotubes by expression of a neutralizing mutation in this identified phosphorylation motif restores microRNA‐133a expression and mitochondrial membrane potential during lipotoxicity. Furthermore, overnight exposure to palmitate reduced mitochondrial oxygen consumption and insulin‐stimulated glucose uptake, which were reversed when cells were transfected with microRNA‐133a mimicking oligonucleotides. Mechanistically, we demonstrate that microRNA‐133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. In addition, Nix‐and palmitate‐induced mitochondrial depolarization were attenuated by the beta2‐adrenergic agonist clenbuterol. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin‐resistance, PKCδ activation, and elevated Nix expression as young adult rats. Our data identifies a novel pathway whereby fetal exposure to diabetes reduces microRNA‐133a, alters skeletal muscle metabolism, and may predispose the offspring to early‐onset insulin resistance.

Support or Funding Information

Supported by Children's Hospital Foundation of Manitoba, and NSERC Canada to Joseph Gordon and CIHR to Vernon Dolinsky.
Genesis of Antibiotic Resistance (AR) XVII Population Dynamics That contravenes Hardy‐Weinberg‐Castle Principle (HWCP) augment AR Pandemic
Authors: Moody Nadine , Carrizales Diana , Carrizales Tania , Kannan Subburaj
Published: November 2023

The impending onset of an AR pandemic is a plausible scenario. Based on the elements of HWCP, we have identified the potential contributors for this debacle. Here, we present the potential contributors of a fiasco that has a multi factorial disposition. Foremost of all factors, the medical professionals are constantly exposed a variety of infectious disease with diverse in patient and also outpatient population on a daily basis. These members often do not take time off the job to go to a doctor because they work with many doctors who can prescribe antibiotics at the workplace. The healthcare workers may expose other workers, patients, or their own family to the illness while waiting for the antibiotic to work. In the event that the antibiotic prescribed does not work or the illness is of a viral nature, a week later, many people may have been exposed. Based on a large sample of U.S. acute care hospitals, the survey found that on any given day, about 1 in 25 hospital patients has at least one healthcare‐associated infections (HAI). There were an estimated 722,000 HAIs in U.S acute care hospitals in 2011. About 75,000 hospital patients with HAIs died during their hospitalizations. More than half of all HAI occurred outside of the intensive care unit (N Engl J Med 2014; 370:1198–208). http://www.cdc.gov/HAI/surveillance/(Verbatim). Another key factor in the impending AR pandemic is that the under utilization of diagnostic assays is to be ascertain of microbe sensitivity to antibiotics. Incessantly, contrived and brooked deplorable economic disparity in general population like that of third world countries such as India, appropriate use of antibiotics is impractical. Taken together, both medical professionals, along with socioeconomically deprived section of the global population, pose an imminent risk factor for an impending AR pandemic.

Support or Funding Information

Supported by professional development funds by SWTJC, to S.Kannan for 2014–205
Spatial distribution of the abundance and antibiotic sensitivity of Staphlyococcus aureus in the Hillsborough River
Authors: Seferian Rhianna , Williams Ann , Froeschke Bridgette
Published: November 2023

The Hillsborough River is Tampa's primary potable water supply and used frequently for recreational activities including boating, kayaking, fishing, and swimming. However, the Florida Department of Environmental Protection (FDEP) has identified regions of the poor water quality. The overall purpose of our study was to quantify the abundance and determine antibiotic susceptibility of Staphylococcus aureus from seven discharge locations in a one‐mile section of Hillsborough River (from The University of Tampa (UT) to Tampa General Hospital). The concentration of S. aureus, at each site, was quantified using water filtration methods following EPA standards. Overall, there was a significant higher abundance (cfu/mL) of S. aureus at UT than any other site. Antibiotic susceptibility was tested using the Kirby‐Bauer method on 10 random isolates from each site for each event. Out of the 470 isolates, 166 were classified as Methicillin Resistant S. aureus (MRSA). The MRSA isolates were further tested with tetracycline and vancomycin. Out of the 166 MRSA isolates, 88 were also resistant to vancomycin (VRSA) and one MRSA isolate was resistant to both vancomycin and tetracycline. DNA extraction and Next Generation sequencing was conducted on 10 random isolates of collected MRSA, 9 random isolated of VRSA and 1 isolate resistant to vancomycin and tetracycline to examine the MecA gene, a candidate known to have multiple variants that allows for resistance in S. aureus. This can help aid in the correct administration of antibiotics for those infected with S. aureus from the river.

Support or Funding Information

University of Tampa: BIology Department Undergraduate Summer Research Fellowship

University of Tampa: Honors College Research Fellowship