bluuebunny/crossref_metadata_2025 huggingface dataset:
Uranyl Ion Biosensors for Water Quality and Safety
Authors: Jett Susan J , Bonham Andrew J
Published: November 2023

We have developed a novel electrochemical biosensor for the purpose of detecting uranium, in the uranyl ion form, in untreated water samples at low levels for use in academic, research, and professional settings. This could lead to applications in ongoing water quality monitoring, as well as improve targeting and efficiency of remediation of contaminated water sources. Uranium contamination of a water source has far reaching implications as a connected water system allows for contaminates to pollute all connected water systems over many miles, in turn effecting the organisms that rely on that water supply. This system uses a modified uranyl‐binding aptamer that has been inserted into an oligonucleotide scaffold to create a conformation‐switching element. This oligonucleotide is then integrated into an electrochemical biosensor platform. Ongoing work is determining the detection limits and sensitivity to the target ion of our scaffold biosensor, with the goal of parts per billion detection of uranyl, allowing compliance measurement against EPA standards of uranium limits.
Antioxidant activity of the hexanic extract from Eryngium carlinae “in vitro” and in Saccharomyces cerevisiae
Authors: Peña‐Montes Donovan , Mejía‐Barajas Jorge , Clemente‐Guerrero Mónica , Salgado‐Garciglia Rafael , Cortés‐Rojo Christian , Montoya‐Pérez Rocío , Manzo‐Avalos Salvador , Saavedra‐Molina Alfredo
Published: November 2023

Oxidative stress has been associated with the onset of chronic degenerative diseases such as cancer, cardiovascular and neurodegenerative disorders. Epidemiological studies have shown the use of plants containing antioxidant is beneficial to health. Eryngium carlinae commonly known in México as “Hierba Del Sapo” in traditional herbal medicine, attributed with diuretic and healing properties. Currently, ingested as daily infusion is use to regulate blood pressure and as a hypolipidemic agent; however. Different assays (“in vitro” and “in vivo”) have been developed to evaluate the antioxidant activity of plant extracts. The identification and quantification of constituents, through gas chromatography–mass spectrometry (GC/MS) analysis. The antioxidant activity was evaluated using “in vitro” assays (total antioxidant capacity, DPPH radical scavenging activity, scavenging capacity towards hydrogen peroxide (H2O2)), and, analyzing the antioxidant effect on biomarkers of oxidative stress and its influence on the viability in cells of Saccharomyces cerevisiae treated with the hexanic extract, and exposed to H2O2. In chromatographic analysis, the majority constituents found in the hexanic extract of E. carlinae were terpenes and sesquiterpenes, (Z)β‐Farnesene (38.79 %) β‐Pinene (17.53 %), Calamene (13.30 %), α‐Farnesene (10.38 %). “In vitro” tests, showed a similar reducing capacity of H2O2, and an approximate effect of anti‐lipid peroxidation obtained with ascorbic acid was observed in our results. In S. cerevisiae, the results of viability are correlated in the reduction of levels of cellular lipid peroxidation, protein carbonylation and glutathione ratio in accordance with control. In conclusion, the hexanic extract of E. carlinae has antioxidant activity “in vitro” and in S. cerevisiae, reducing the levels of lipid peroxidation, protein carbonylation, and increasing viability, attributable to farnesene and pinene, and the synergism between the constituents of the extract.

Support or Funding Information

CONACYT 169093 to ASM; CIC‐UMSNH 2.16 to ASM.
Antioxidant activity of the seed oil extract from Eryngium carlinae “in vitro” and in Saccharomyces cerevisiae
Authors: Mejía‐Barajas Jorge A. , Rodríguez‐Lino Adriana Lizbeth , Torres‐Martínez Rafael , Salgado‐Garciglia Rafael , Cortés‐Rojo Christian , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo
Published: November 2023

Free radicals are unstable and reactive species, that modified molecules as carbohidrates, lipids, proteins or genetic material producing oxidative stress. A number of pathologies has been associated to this uncontrolled reactions. The importance to study new possibilities against the imbalance in organisms concern with antioxidants which are molecules that prevent or slow molecules oxidation. Eryngium carlinae is a plant with medicinal properties. A number of techniques has been developed to study antioxidant properties, as in vitro or using Saccharomyces cerevisiae yeast as biological model. We obtained the seed oil extract from Eryngium carlinae by Soxhlet method and evaluated its antioxidant capacity. Results obtained showed a higher antioxidant activity of seed oil from Eryngium carlinae than ascorbic acid as compared.

Support or Funding Information

CONACYT 169093 to ASM; CIC‐UMSNH 2.16 to ASM
Biological, Electrochemical and Spectroscopic Study of Cu (II) Complexes: Applications to the Development of Anticancer Metallodrugs
Authors: Lewis Adam , Fox Kristin , Tanski Joseph , Tyler Laurie
Published: November 2023

The activity of several Cu (II) complexes with three different biologically relevant ligand systems – benzimidazole, benzothiazole, or derivatized pyridine‐moieties – were assessed to determine structure‐function relationships important to the application of these complexes as anticancer agents. Topoisomerase I (Topo I) inhibition activity was evaluated by monitoring the complexes’ ability to prevent relaxation of supercoiled (SC) DNA via gel electrophoresis. Human Serum Albumin (HSA) binding ability was measured using a fluorescence quenching‐based assay. The Stern‐Volmer equation was then used to determine the binding constants for the complexes with HSA. The reduction potential for the Cu II/I redox couple for each complex was determined using cyclic voltammetry. The electrochemical and biological data will be discussed and related to the coordination sphere around the Cu(II).
Engineered Caspases with Altered Specificities Enable Identification of Exosites
Authors: Hardy Jeanne A , Hill Maureen , MacPherson Derek J , Wu Peng , Julien Olivier , Wells James A
Published: November 2023

Exosites for substrate binding are promising for development of inhibitors and activators with novel mechanisms of actions. Despite their potential, to date there is no systematic way to identify exosites on enzymes. We have developed a method for identifying exosites, and applied it to an important class of proteases, the caspases. To identify exosites, we built a version of caspase‐7 that maintained all exosites, but that has the active‐site specificity of caspase‐6. This new caspase allows us to categorize substrates as being active‐site or exosite driven. Simple substitution of the critical active‐site residues from caspase‐6 dramatically diminished caspase‐7 activity, but did not alter specificity, indicating that a more holistic evolutionary procedure would be needed to achieved the desired hybrid caspase. Using our GFP‐based reporter of caspase activity (CA‐GFP), we implemented a flow‐cytometry based directed evolution screen for caspase‐7 variants with caspase‐6 cleavage patterns. The caspase‐7 variants with the most strongly altered specificity displayed kinetic values similar to wild‐type caspase‐6 and mimicked caspase‐6 cleavage patterns with protein substrates. Our seven crystal structures of evolved caspases bound to either DEVD or VEID substrates allowed us to observe how the new residues in the active site contributed to the change in specificity by altering the conformation of a critical active site loop. Using N‐terminomics on the entire human proteome, we observed that the substrate specificity of the evolved caspase was superimposible with caspase‐6 and unlike the parent scaffold, caspase‐7, suggesting that our directed evolution was successful. This allowed us to identify Lamin A/C as an exosite dependent substrate. Ultimately we hope that this approach will identify exosites for recognition of just a subset of key regulatory or disease‐causing substrates, which will lead to more specific small‐molecule modulators of caspase function.

Support or Funding Information

This work was supported by the National Institutes of Health (GM80532) to JAH and (GM081051) to JAW.
Late endosomes and its unique anionic lipid bis(monoacylglycero)phosphate act as doorways for the endosomal escape and cytosolic entry of the fluorescently‐labeled dimeric cell‐penetrating peptide dfTAT and bioactive cargos
Authors: Erazo‐Oliveras Alfredo , Najjar Kristina , Truong Dat , Wang Ting‐Yi , Pellois Jean‐Philippe
Published: November 2023

One of the key challenges and a multibillion dollar issue in the field of delivery of biologics/therapeutics inside live cells is endosomal entrapment. Currently, there is an urgent need to define the mechanisms of cell penetration and endosomal escape at a cellular and molecular level. A better understanding of how cell penetration and endosomal escape occurs will allow the improvement of existing delivery strategies. Our laboratory has shown that dfTAT, a recently identified delivery tool, can circumvent this giant hurdle. In this work we describe the use of dfTAT, a dimeric fluorescently‐labeled peptide found to promote endosomal leakage extremely efficiently and without deleterious effects to cells1, as a model cell‐penetrating peptide that can reveal the cellular organelles and factors involved in endosomal escape. By controlling/fine‐tuning the trafficking of dfTAT within the endocytic pathway, we have identified late endosomes as the unique organelles rendered leaky by dfTAT and as the exit point for dfTAT endosomal escape. Following endocytic uptake and trafficking, dfTAT binds bis(monoacylglycero)phosphate (BMP), a negatively‐charged phospholipid enriched in late endosomes and, consequently, causes endosomal leakage in cellulo. Moreover, dfTAT binds and causes leakage of bilayers containing BMP but not bilayers containing two other negatively‐charged lipids, phosphatidic acid (PA) and phosphatydilglycerol (PG), in vitro (PG being a structural isomer of BMP). Finally, we establish that an anti‐BMP antibody is capable of blocking leakage in vitro and endosomal escape in cellulo. Together, these data not only identify late endosomes as the cellular gateways utilizable for efficient delivery protocols, but also sheds light on a mechanism that may provide a fundamental basis for the rational design of future cell‐permeable molecules that are highly active yet display low cytotoxicity.

Support or Funding Information

RO1GM087227, R01GM087981 and RO1GM110137
Mutagenesis Study to Reveal Important Determinants of XXT2 Catalytic Activity
Authors: Smith Adrienne Leah , Culbertson Alan T. , Zabotina Olga A.
Published: November 2023

Xyloglucan (XyG) is the main hemicellulosic component of the primary cell wall in eudicots and nongraminaceous monocots. Three xyloglucan xylosyltransferases, including XXT2, are known to encode XyG xylosyltransferases, key enzymes in the process of XyG biosynthesis.

A computational model of XXT2 was used to identify amino acids, F204, K207, D228, S229, D230, H378, putatively localized in the active site of XXT2 and mutagenesis study was performed to confirm predictions. All created mutants were expressed in E. coli SoluBL21 in pET20b plasmids containing an N‐terminal GB1 fusion tag. To increase yield and purity for future biophysical experiments, such as isothermal titration calorimetry and circular dichroism, the mutated genes were amplified from the pET20b plasmids, ligated into pGen2 plasmids and transfected into HEK293 cells. Results of the enzyme assay demonstrated that K207, D228, D230, and H378 are critical for enzyme activity due to their proposed coordination of the divalent cation. Reduced activity seen for the mutation made at F204 suggests that while this site is important for enzyme activity, it is not as crucial as the other sites tested. In addition, cysteine residues were selected for mutagenesis to investigate their effect on disulfide bond formation shown to be involved in hetero‐ and homo‐ dimer formations. Expression and analysis of these mutants is currently in process.

Truncations of XXT2 from both the N‐ and C‐ends were used to examine the contributions of these termini to protein solubility and activity. In N‐terminal truncations longer than 31 residues, both protein expression and activity of XXT2 were negatively affected, indicating a possible effect on protein folding. Increasing sizes of truncations from the C‐terminus showed an increase in protein expression but a decrease in its catalytic activity. We conclude that the C‐terminus is not involved in protein folding but rather substrate binding. This truncation identified a series of six positively charged residues between 24 and 44 amino acids from the C‐terminus that may play a role in substrate binding. Mutagenesis of these residues revealed that four of the mutants have reduced catalytic activity.

We conclude that the N‐terminal of XXT2 is involved in protein folding while the C‐terminal is involved in substrate binding. Furthermore, active site residues K207, D228, D230 and H378 are proved to be critical for enzymatic activity of XXT2.

Support or Funding Information

This research is supported by NSF‐MCB Grant #1121163
Structure and biosynthesis of complex N‐glycan cores and antennae in nematodes
Authors: Wilson Iain B. H. , Yan Shi , Paschinger Katharina
Published: November 2023

There are thousands of nematode species, some of which are parasites such as Ascaris suum, Trichuris suis or Haemonchus contortus; others such as the popular model organism, Caenorhabditis elegans, are free‐living. Although all are animals, they lack some of the characteristics of glycosylation found in vertebrates, but their ‘foreign’ nature is relevant to the interactions of nematode parasites with their hosts. The presence of complex modifications of the chitobiose core region of nematode N‐glycans and of phosphorylcholine on the glycan antennae are typical, but the details are species‐specific. Whereas Caenorhabditis N‐glycans can contain up to four fucose residues, this number is reduced in parasitic species. Also galactosylation, at unusual positions, is a recurrent theme. Of the three core fucosyltransferases and one fucose‐modifying galactosyltransferase known, unusual and unexpected enzyme specificities have been revealed by studying both enzyme specificities and the effect on the glycome of deleting the relevant genes; even the processing hexosaminidases of nematodes are only distantly related to their ‘cousins’ in other invertebrates which are responsible for formation of paucimannosidic glycans. Thereby, glycomics, genetics and biochemistry are not only important tools for understanding nematode glycan biosynthesis, but open the way to identifying carbohydrate motifs which play roles in the interactions of these organisms with their hosts.

Support or Funding Information

Austrian Science Fund (FWF) grants P21946 to K.P. and P23922 to I.W.
Bacterial glycosidases acting on red blood cells
Authors: Bourne Yves , Ponchel Guillaume , Roig‐Zamboni Véronique , Bennett Eric P , Henrissat Bernard , Clausen Henrik , Sulzenbacher Gerlind
Published: November 2023

Glycosidases represent the major catalytic machinery for the breakage of glycosidic bonds. A classification system for glycosyl hydrolases, based on sequence similarity, is available on the dedicated carbohydrate‐active enzymes (CAZy) database and has led to the definition of more than 130 different families (1). Among them, two families of bacterial glycosyl hydrolases, identified by a combination of screening and bioinformatics approaches, perform efficiently and specifically in conversion of red blood cells for the production of universal red blood cells (2,3). Most of the enzymes are found in symbiotic or soil‐borne bacteria. Because members of one family have essentially identical kinetic properties with both branched and linear substrates, they can also act in the enzymatic removal of immunodominant alpha3Gal xenotransplantation epitope (3). We will describe the molecular determinants responsible for the fine substrate specificity of these enzymes and emphasize the potential biomedical applications in remodeling glycan structures.

Support or Funding Information

Supported by the CNRS and DGA
Silencing Mannosylphospho Dolichol Synthase with shRNA Impacts Differentiation of Capillary Endothelial Cells
Authors: Baksi Krishna , Zhang Zhenbo , Banerjee Aditi , Serrano Jesus E , Perez Linyer E , Linares Lorraine , Seijo Arelis , Sanchez Neysharie , Katiyar Usha , Banerjee Dipak K
Published: November 2023

Mannosylphospho dolichol synthase (DPMS) is down stream of N‐acetylglucosaminyl 1‐phosphate transferase (GPT) in the dolichol pathway of asparagine‐linked (N‐linked) protein glycosylation. When capillary endothelial cells are treated with a GPT inhibitor tunicamycin, angiogenesis is inhibited quantitatively reducing the breast tumor growth in nude mice by inducing unfolded protein response (upr)‐mediated apoptosis. There was also a quantitative loss of DPMS catalytic activity. We, therefore, hypothesized that there is a cross‐talk between the GPT and DPMS, and DPMS independently plays a significant role in angiogenesis. To evaluate, we have isolated a stable capillary endothelial cell line in which DPMS has been knocked down after transfecting the cells with DPMS shRNA plasmid in pSilencer 4.1‐CMV neo vector. The control used a scrambled shRNA sequence. DPMS expression in each cell clone was monitored by measuring catalytic activity, qRT‐PCR for DPMS gene expression, western blotting and immunofluorescence microscopy for DPMS protein expression. They all have down regulated in the knockdown clone. Western blotting with Concanavalin A also indicated the presence of reduced level of N‐linked glycoproteins with terminally linked mannose. Interestingly, when the cellular morphology was followed as a function of time, the DPMS knockdown clone exhibited lumen‐like structure formation more frequently than the control. Clonogenic assay indicated 0% survival of DPMS knockdown cells after 48 hours of tunicamycin (1 μg/mL) treatment. We, therefore, conclude that DPMS silencing makes cells less angiogenic, more susceptible to the anti‐angiogenic glycotherapy tunicamycin and importantly impacts the differentiation characteristics of the cell.

Support or Funding Information

).Supported in part by grants from NIH/NIMHD 2G12MD007583 (KB) and NSF‐EPSCoR RII Track 1 Grant # EPS‐1002410 (DKB)
Elevated O‐GlcNAcylation levels improve mitochondrial function
Authors: Tan Ee Phie , Swerdlow Russell , Slawson Chad
Published: November 2023

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a highly dynamic post‐translational modification that involves an addition of a single N‐acetylglucosamine to serine and threonine residue in mitochondria, nuclear and cytoplasmic proteins. O‐GlcNAc cycling is the addition and removal of O‐GlcNAc by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) respectively. Disruptions in O‐GlcNAc cycling contribute to diseases such as diabetes, cancer, and neurodegeneration. Accumulative dysfunctional mitochondria can also lead to the development of neurodegenerative diseases, and importantly, O‐GlcNAcylation regulates mitochondrial function. Previously, using a proteomics based screen we found that altering the expression of OGT and OGA disrupted mitochondrial protein expression, including proteins involved in the respiratory chain and TCA cycle. Furthermore, mitochondrial morphology in the over‐expressing cells had reduced and disorganized cristae and altered shape and size. Both cellular respiration and glycolysis was impaired in the OGT/OGA gain of function cells. These data suggested that O‐GlcNAc cycling was essential for the proper regulation of mitochondrial function. We hypothesized that prolonged elevations in cellular O‐GlcNAc levels would alter the metabolic profile of the cell. We disrupted O‐GlcNAc cycling by either treating SH‐SY5Y neuroblastoma cells with low levels of Glucosamine (GlcN) or OGA inhibitor Thiamet‐G (TMG). Importantly, the metabolic substrate for O‐GlcNAcylation is UDP‐GlcNAc. UDP‐GlcNAc is synthesized via the hexosamine biosynthetic pathway, which is highly sensitive towards the availability of nutrients in the cellular environment. GlcN feeds into the hexosamine biosynthetic pathway after the first rate‐limiting enzyme GFAT1, glutamine fructose‐6‐phosphate aminotransferase 1, and additional supplementation of GlcN increases O‐GlcNAcylation via elevating the synthesis of UDP‐GlcNAc. We found that prolonged TMG/GlcN treatment improved mitochondrial function. In the TMG/GlcN treated cells, reactive oxygen species (ROS) production was significantly reduced. Interestingly, the protein expression of the mitochondrial antioxidant, manganese superoxide dismustase (MnSOD) was lower. Additionally, ATP production was lower in these TMG/GlcN treated cells. Furthermore, we found that mitochondrial were longer in shape and mitochondrial fusion/fission protein expressions were decreased. Altogether, these data demonstrated that prolonged alterations to the cellular homeostasis of O‐GlcNAc levels improve mitochondrial function likely through reprogramming of metabolic transcriptional programs. Further understanding of how O‐GlcNAc cycling regulates metabolism will provide new insights into metabolic diseases such as cancer and Alzheimer's.

Support or Funding Information

RO1DK100595
O‐GlcNAcylation of the Human Kinome
Authors: Liu Xin , Han Guanghui , Wells Lance , Graves Lee , Zhu Heng , Neiswinger Johnathan , Hart Gerald W.
Published: November 2023

O‐GlcNAc, first characterized more than 30 years ago, is an O‐linked β‐N‐acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residues. O‐GlcNAc has been found on over 4,000 cytoplasmic and nuclear proteins. The addition of O‐GlcNAc to proteins is catalyzed by O‐GlcNAc transferase (OGT), while its removal is catalyzed by O‐linked N‐acetyl‐β‐D‐glucosaminidase (OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, such as signal transduction, transcription, cell cycle progression and protein‐protein interactions. O‐GlcNAc plays important roles in human diseases, such as cancer, diabetes and neurological disorders.

Protein phosphorylation is a post‐translational modification that serves as a rapid and reversible means to modulate protein activity and transduce signals. The regulation of phosphorylation is a central mechanism in cell health and disease. O‐GlcNAcylation has extensive interplay with phosphorylation at the site level on proteins and by modifying kinases. However, the roles of O‐GlcNAcylation have only been studied on a few kinases.

In vitro O‐GlcNAcylation of large kinase microarrays indicates that well over one‐half of all human protein kinases are substrates for OGT. Multiplexed kinase inhibitor beads (MIBs) are being used to confirm the O‐GlcNAcylation of kinases in vivo. These affinity beads have enriched at‐least 117 kinases as determined by mass spectrometry and HCD and ETD fragmentation are being used to map their O‐GlcNAcylation sites.

Support or Funding Information

Supported by NIH P01HL107153. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU
Heterologous Expression of Multidrug Resistance Proteins
Authors: Marchesseault Collette A. , Vogel Pia D. , Wise John G.
Published: November 2023

One key to developing effective cancer treatments for many patients today involves the inhibition of multidrug resistance pumps overexpressed in cancer cells. These membrane proteins in the ABC transporter family act to remove the chemotherapeutics from the cell, rendering normal chemotherapies ineffective. Two ABC transporters that are especially problematic and that cause multidrug resistances are P‐glycoprotein (P‐gp) and the Breast Cancer Resistance Protein (BCRP). High‐throughput computer modeling was used to narrow the search for possible inhibitors of P‐gp and many of these inhibitors have been shown to be effective in biochemical assays (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular pharmacology, 86(6), 716–726) as well as in reversing multidrug resistances in human cell culture assays (Follit, C. A., Brewer, F. K., Wise, J. G., & Vogel, P. D. (2015) Pharmacology Research & Perspectives, 3(5) e00170, doi:10.1002/prp2.170). In order to expedite the production of P‐gp protein for inhibitor testing, structural and mechanistic studies, new bacterial expression systems are being developed. It is hypothesized that such systems would allow for an increase in protein yield and the overall efficiency of the isolation of the transporter protein. It should be noted that glycosylation of P‐gp is not essential for its folding or activity. To accomplish these aims, P‐gp was cloned into E. coli behind the glpF glycerol transporter as a fusion gene. GlpF is a native E. coli membrane protein known to be expressed at high levels and was included to aid with the expression, folding and insertion of P‐glycoprotein into the bacterial membrane. Clones were verified by DNA sequencing and protein expression was examined using Western blot analysis using anti‐His6 and anti‐P‐gp antibodies to screen for the presence of P‐gp. Purified sub‐bacterial particles and isolated transporter preparations were assayed for ATP hydrolysis by a coupled enzyme assay to assess catalytic activity. Positive preliminary results for the expression of protein in E. coli were obtained when the bacteria were grown in the presence of a known inhibitor of P‐gp. A similar bacterial expression has also been used for BCRP expression. Gene block assembly was used to synthesize a BCRP gene which was then verified by DNA sequencing. Preliminary results suggest that expression of BCRP in E. coli membranes has also been accomplished. Optimization of the growth and purification of P‐gp and BCRP from E. coli cells will be discussed.

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, Hamilton Undergraduate Research Scholarship, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, New England Biolabs / Lynn McBee, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Potent lipidated antagonists to protease‐activated receptor 2 (PAR2)
Authors: Boitano Scott , Hoffman Justin , Flynn Andrea , Sherwood Cara L. , Zhang Zhenyu , Patek Renata , Price Theodore J. , Vagner Josef
Published: November 2023

Protease‐activated receptor‐2 (PAR2) belongs to a four‐member family of G‐Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR2 signaling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased‐ligand signaling. We recently demonstrated that lipid tethering of known PAR2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL) increased their potency > 200 fold. In this study, we used lipid tethering (hexadecyl (Hdc) group with polyethylene glycol (PEG) spacers) and heterocycle (2‐aminothiazoyl; 2‐at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS) to elucidate novel PAR2 antagonists. Compound 562 (C562), 2‐aminothiazol‐4yl‐SKGRS‐PEG3‐Hdc blocks PAR2 Ca2+ signaling elicited via peptidomimetics (2‐at‐LIGRL‐NH2) or via asthma associated protease activation (Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o‐). This compound was a biased‐signaling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signaling, the other major signaling pathway activated via PAR2. A shortened version of C562, 2‐at‐SKGR‐PEG3‐Hdc (C595), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signaling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR2 agonist, 2‐at‐LIGR‐PEG3‐Hdc. Thus, we screened a series of potential PAR2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG3‐Hdc lipid tether. We describe several potent agonists, and one partial agonist (C608, 2‐at‐TIGR‐PEG3‐Hdc) that also acts as a potent, specific and biased signaling antagonist of PAR2. When used in nanomolar concentrations, C608 blocked PAR2‐dependent Ca2+ signaling via protease or peptidomimetics without effects on MAPK signaling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR2 full and biased ligand signaling.

Support or Funding Information

This work was primarily funded by a multi‐PI grant (NS 073664 to SB, JV and TJP) from the National Institutes of Health. Additional support for this work was from the following grants: National Institute of Health training grants (T32 HL 007249 for ANF; T32 ES 007091 for CLS), National Institutes of Health R01NS 065926 (TJP) and R01AI083403 (SB; Michael O. Daines, PI), State of Arizona Technology and Research Initiative Fund Awarded through Bio5 (JV).
Chronic Caloric Restriction Alters Nutrient Sensing in Skeletal Muscle in High‐Fat Diet Fed Rats
Authors: Hernandez‐Saavedra Diego , Chen Hong , Pan Yuan‐Xiang
Published: November 2023

Caloric restriction (CR) is regarded as one of the most effective strategies for weight loss, decreasing chronic disease risk markers. However, the reduction on body weight induces both lean and fat mass loss, and the molecular mechanism underlying excessive loss of skeletal muscle mass remains largely unknown. We hypothesize that CR alters nutrient sensing pathways in muscle, causing phenotypical loss of muscle mass. To that end, the aim of our study was to analyze the changes in body composition after prolonged CR and the effect on nutrient sensing pathways. In our study, 5 to 6 week old male Sprague‐Dawley rats were fed a high fat diet (45% Kcal from fat) Ad libitum for 3 months. Rats were then divided into HF group Ad libitum (HF), maintenance group fed a control diet (ML, 100% energy requirement at baseline adjusted with body weight), and the caloric restriction group (CR, 25% energy reduction of baseline requirement with age adjustment). Food intake, body weight, and body composition were regularly monitored and recorded. Six months after the dietary intervention rats were sacrificed, nutrient sensing intermediates in serum were measured, and skeletal muscle samples were analyzed for gene expression. The HF group showed an increase of 30.5% in body weight, of which 83.0% is fat and 7.0% is lean mass, respectively. At the same time, ML maintained body weight gain (8.4%). A negative balance was observed in the CR group, with a marked 16.9% decrease in body weight, and a significant fat and lean mass reduction of 61.0% and 12.4%, respectively at the end of the experiment. Gene expression analysis of skeletal muscle revealed a 2.2‐fold increase in the expression of Activating Transcription Factor 4 (Atf4) in CR group compared to HF, as well as a 3.1‐fold upregulation of proteosomal degradation factor Muscle‐Specific Ring Finger Protein 1 (Murf1) and a 2.3‐fold increase in master myogenic regulator Follistatin (Fst). Nutrient sensing during the caloric restriction is further characterized by investigation of regulatory mechanisms of these pathways. Overall, chronic caloric restriction resulted in the activation of nutrient sensing and protein degradation pathways, which correlates to the reduced fat mass and concomitant lean mass loss in response to the reduced energy.
Life Long High Fat Diet Causes Hepatic Lipid Accumulation by Programming Lipid Synthesis Genes in Liver of Male Rat
Authors: Jung Paul Mark , Zhou Dan , Moody Laura , Kriska Adam , Chen Hong , Pan Yuan‐Xiang
Published: November 2023

Life long high fat (HF) diet has been shown to cause more hepatic lipid accumulation in offspring as compared to a post‐weaning HF diet. However, it is still unclear what the epigenetic mechanisms are. We hypothesize that a life long HF diet will cause greater hepatic lipid accumulation as compared to a post‐weaning HF diet. The objective of this study is to understand the epigenetic mechanisms of hepatic lipid accumulation as an outcome of life long HF diet. Timed‐pregnant Sprague‐Dawley rats were given either a control (C, 16% fat) or HF (HF, 45% fat) diet during gestation and lactation. Pups from both C and HF mothers were weaned onto the same HF diet, creating two offspring groups: C/HF and HF/HF. Rats were sacrificed at 12 weeks of age and the left lobe of the liver was used for further analysis. Liver histology showed greater hepatic fat accumulation in the HF/HF group. Gene expression data was measured for multiple genes in the following pathways: glycolysis, gluconeogenesis, fatty acid synthesis, and triacylglycerol synthesis. Only glycerol‐3‐phosphate acyltransferase (GPAM) and fatty acid synthase (FASN) showed higher levels of mRNA. DNA methylation of specific genes was analyzed based on data obtained from methyl‐DNA immunoprecipitation with high‐throughput sequencing. Differential methylation was observed for both GPAM and FASN. Our data suggests that life long HF diet increases lipid storage by increased hepatic GPAM and FASN gene expression in male rats, potentially through epigenetic regulation.
Effect of frequent intake of sweeteners on the JAK2/STAT3 signaling pathway in the central nervous system of mice
Authors: Barrios Alberto Andrés , Contreras Irazú , Estrada José Antonio
Published: November 2023

Sweeteners are food additives that are commonly used worldwide. There are discrepancies in epidemiological studies evaluating their effect on body weight and appetite; this generates the need to further study the mechanism by which sweeteners could affect satiety and energy balance.

This study's main objective is to evaluate whether frequent intake of the commercial sweeteners; sucralose, steviol glycosides and sucrose could cause alterations in the leptin‐mediated JAK2/STAT3 signaling pathway, in the central nervous system of mice.

For this purpose, three study groups comprised of 14‐week‐old female BALB/c mice were established: sucrose, steviol glycosides and sucralose groups were supplemented with those sweeteners in their daily water for 6 weeks (from week 8 to week 14), additionally, a control group was established without sweetener supplementation. Mice were fed ad libitum and weight was measured weekly. The amount of food and water intake was measured daily. After 6 weeks, body composition of mice was determined using bioelectrical impedance and total brain proteins were obtained to determine the expression of total and phosphorylated JAK2 and STAT3 proteins by western blot.

Our preliminary results show that there is a higher consumption of water, but lower food intake in the sucrose group compared with the control and other experimental groups; there are no significant differences in body weight and adiposity among groups. Regarding JAK2/STAT3, expression of pJAK2 and JAK2 was found to be higher in the group supplemented with sucralose, whereas we did not observe changes in the STAT3 proteins. We also found that mice supplemented with steviol glycosides and sucrose had a lower expression of pJAK2 and JAK2 compared to control mice.

Our results suggest that frequent intake of commercial sweeteners may cause alterations in energy balance and appetite by altering the phosphorylation of JAK2, however, we need further study the effect of these sweeteners in the leptin‐receptor expression and serum leptin concentrations.
Mitochondrial Translocator Protein (TSPO) Is a Regulator of Fatty Acid Metabolism
Authors: Tu Lan N , Zhao Amy H , Hussein Mahmoud , Selvaraj Vimal
Published: November 2023

Translocator protein (TSPO), first identified as a secondary target of benzodiazepines and termed the ‘peripheral’ benzodiazepine receptor (PBR), is a highly conserved protein in the outer mitochondrial membrane. Despite intensive research for the past 25 years, the exact function of TSPO remains elusive. High TSPO expression seen in steroidogenic mitochondria, and induction of steroid hormone production by TSPO binding drugs, led to a proposed function for TSPO in mitochondrial cholesterol import, the rate‐limiting step in steroidogenesis. However, this prevalent model was recently refuted based on Tspo gene deletion studies in vivo and in vitro that indicated no deficits in steroidogenesis. Seeking a new functional definition for TSPO, we found that high TSPO expression is not exclusive to steroidogenic cells, but also to other cell types active in lipid metabolism such as liver and adipose tissues. We therefore hypothesized that TSPO plays an important role in mitochondrial fatty acid metabolism. Using steroidogenic MA‐10 Leydig cells that express very high levels of TSPO as the model, we generated CRISPR/Cas9‐mediated TSPO knockout MA‐10 clones (MA‐10:TspoΔ/Δ) and examined changes to core mitochondrial functions after TSPO deletion. MA‐10:TspoΔ/Δ cells showed a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species (ROS) production. Overexpression of TSPO using a lentiviral system reversely reduced expression of FAO genes in MA‐10 cells. Extending this in vitro observation to in vivo tissues in global TSPO knockout (Tspo−/−) mice, we observed that loss of TSPO in adrenal glands (a major steroidogenic tissue), and liver (the chief metabolic compartment), both significantly upregulated FAO genes compared to the control floxed (Tspofl/fl) tissues. These results demonstrate the first experimental evidence that TSPO affects mitochondrial energy homeostasis through controlling FAO at the level of outer mitochondrial membrane, possibly through regulating activity of carnitine palmitoyltransferase I (CPT1). Therapeutic modulation of TSPO function using available TSPO binding drugs could be beneficial for increasing energy expenditure through FAO in lipid metabolic disorders.

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Startup funds from Cornell University
Activation of Adiponectin Receptors Induces Proprotein Convertase Subtilisin Kexin Type 9 Expression by Activating PPARgamma Pathway
Authors: Sun Lei , Yang Xiaoxiao , Liu Ying , Chen Yuanli , Han Jihong , Duan Yajun
Published: November 2023

Adiponectin is a hormone derived from adipose tissues. Reduced adiponectin levels have been determined to be associated with the occurrence of obesity‐linked diseases including insulin resistance/type 2 diabetes and atherosclerosis. Adiponectin exerts its functions by activating the adiponectin receptors (AdipoRs). Proprotein convertase subtilisin kexin type 9 (PCSK9) facilitates the development of atherosclerosis by enhancing the degradation of low‐density lipoprotein receptor (LDLR) protein to increase the circulating LDL‐cholesterol levels. PCSK9 expression is regulated by peroxisome proliferator‐activated receptor gamma (PPARgamma) and sterol regulatory element‐binding protein 2 (SREBP2). However, it remains unknown if activation of AdipoRs can influence PCSK9 expression and its function. Herein, we report that activation of AdipoRs induced PCSK9 expression through activation of the PPARgamma pathway. At molecular and cellular levels, we determined that activation of AdipoRs induced PCSK9 promoter activity, mRNA and protein expression in HepG2 cells. Although PPARgamma expression and SREBP2 translocation can be induced by activation of AdipoRs, the promoter analysis demonstrated that the mutation of PPAR‐responsive elements (PPRE), but not the sterol regulatory element (SRE) in PCSK9 promoter, attenuated the AdipoRs activation induced PCSK9 promoter activity which indicates that activation of PPARgamma dominates AdipoRs induced PCSK9 expression. Meanwhile, we determined that LDLR expression was increased which was due to that activation of AdipoRs induced SREBP2 translocation and enhanced PPARgamma mediated SREBP2 processing. In addition, we found that activation of AMPKalpha was also involved in AdipoRs regulated PCSK9 and LDLR expression in HepG2 cells. In vivo, administration of AdipoRs activators increased PCSK9 and LDLR expression in mouse liver and enhanced PCSK9 secretion. Our results indicate that although AdipoRs activation increased PCSK9 expression, it simultaneously induced LDLR expression and the induction of LDLR expression can still partially make contribution to the anti‐atherosclerotic properties of adiponectin.
Robust Glucose and 1,5‐Anhydroglucitol Uptake Assays Using Novel Fluorescent Conjugates
Authors: Li Shunan , Khatwani Santoshkumar , Lin Yuan , Tchaga Grigority , Yan Gordon
Published: November 2023

Glucose is the primary carbon source for ATP production and biosynthesis in proliferating mammalian cells by either glycolytic or tricarboxylic acid (TCA) cycle pathways. These pathways are tightly regulated and their disruption leads to many metabolic diseases such as diabetes. Cancer cells exhibit increased glucose uptake to support their high rate of proliferation. The glucose uptake has been widely studied by a non‐radioactive assay using a fluorescently‐labeled glucose derivative, 2‐NBD‐Glucose. Despite its applicability, this conjugate exhibits photo bleaching and reduced fluorescence when used at low concentrations (i.e. <400 μM). BioVision has developed a novel bright green fluorescent 2‐deoxy‐glucose conjugate (GluTracker™) which, just like glucose and 2‐NBD‐Glucose, can be taken up by cells via glucose transporters. However, this glucose analog cannot be fully metabolized by cells, and thus accumulates inside cells. Fluorescence generated by this fluorescent glucose analog is proportional to the cellular glucose uptake and can be used to measure glucose uptake using fluorescence microscopy or flow cytometry. In addition, we have also developed a novel green fluorescent 1,5‐Anhydroglucitol conjugate (AGTrackerTM) to study the uptake of 1,5‐Anhdroglucitol (1,5‐AG). The blood plasma level of 1,5‐AG has been used in clinical studies to monitor short‐term glycemic control in diabetic patients. The AGTrackerTM conjugate can be used to study 1,5‐AG uptake by fluorescence microscopy or flow cytometry. In conclusion, BioVision's GluTracker™ (for Glucose) and AGTrackerTM (for 1,5‐AG) Uptake Assays provide valuable tools for drug discovery and identifying potential modulators in glucose‐dependent pathways.

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NA
Effects of aqueous cinnamon extract on 3T3‐L1 cell size, lipid volume and lipase activity
Authors: Stockert Amy , Newstead Sabrina , Freeh Ellen , Stephan Megan , Aulthouse Amy
Published: November 2023

Extracts of cinnamon have long been used medicinally and recent research has solidified its use for controlling blood glucose levels. The correlation between type 2 diabetes and obesity make research in carbohydrate and lipid metabolism essential. We utilized the 3T3‐L1 cells as a model to examine degrees of differentiation to mature adipocytes by examining lipid droplet accumulation at 0, 2, 12, 24, and 48 hours post treatment. Lipids from microdroplets were exchanged with oil red O as a marker of adipogenesis, and where appropriate the lipid volume was estimated and assayed. Culture periods were extended up to two weeks to reach mature adipocytes. A reduction is cell size was observed in both monolayer and three‐dimensional culture, but the size changes appear more significant were grown in three‐dimensional culture. Cinnamon treated cells appear to have more lipid droplet accumulation, yet the lipid volume did not correlate directly with the reduction in cell size, suggesting a true change in metabolic signaling rather than an osmotic contraction. In three‐dimensional culture, there was an obvious cluster of lipid droplets, yet in monolayer this was not as obvious due to the single layer of cells. Monolayer cells were counted in each quadrant of the culture to determine the percent of cells that had rounded morphology compared to those that remained flat and fibroblast‐like. Of the rounded cells, the portion containing at least 50% space fill with lipid droplets were considered adipogenic. In monolayer, estimate of lipid droplet volume was not reliable and a quantitation of extracted dye was necessary. Culture treatments were repeated in duplicate leaving one culture unfixed to allow assay of lipase activity.
Comparison of a Novel Commercial Immunoassay, AGILE Research, and the Meso Scale Discovery Array System for the Quantification of Insulin and Leptin in Human Plasma
Authors: Afsahi Savannah , Walker Amy , Pan Deng , Lerner Mitch , Sears Dorothy D , Goldsmith Brett , Barron Francie Elizabeth
Published: November 2023

Protein quantification for the purpose of diagnosis or monitoring of disease and disease states are integral to patient care. However, modern immunoassays tend to rely on indirect assays to provide quantitative measurements of target molecules, which increase the complexity of the assay and can introduce error into the results. For example, the Meso Scale Discovery (MSD) Insulin and Leptin Single Array kits use a sandwich immunoassay and an electrochemiluminescent response to measure analyte concentrations in serum or plasma samples. We present measurements determined using a novel direct detection approach that leverages advances in nanoelectronic sensors to obtaining quantification of protein concentrations in complex human samples. AGILE Research is a new, commercially available tool using graphene biological field effect transistors that eliminates the need for secondary labeled antibodies to obtain results. We present data from use of these tools in comparison to data collected using the MSD Insulin and Leptin Single Array assay.
Antidiabetic Effect of the Ethanolic Extracts of Azadirachta indica Leaves on Streptozotocin Induced Wistar Rats
Authors: Aniefuna Ejike Louis , Omorevbarhia Omorovbiye
Published: November 2023

The antidiabetic efficacy of aqueous extract of Azadirachta indica leaf in normal and streptozotocin induced diabetic rats were compared and evaluated. Phytochemical analyses as well as 12 hrs acute toxicity test of the orally administered ethanolic extract of Azardirachta inidica were carried out using standard methods. Diabetic condition was induced in rats by a single intraperitoneal dose of 200mg/kg b.w. of streptozotocin. The ethanolic extract was administered orally in 2 doses of 50mg/kg b.w. and 100mg/kg for 12 days. The 12 hrs acute toxicity test of the extract of A. indica leaves yield a LD50 of 4.50g/k.g b.w. Both doses of the extract were significantly (p<0.05) effective in reducing blood sugar levels of streptozotocin induced hyperglycaemic rats on the twelveth day of treatment when compared to the control diabetic rats that were treated with gubenclamide (a standard antidiabetic drug) at a dose of 0.2mg/kg b.w on the same day. Nevertheless, 100mg/kg b.w had the highest reduction in blood glucose level. These results suggests that the ethanolic leaf extract of A. indica has antidiabetic effects and could be of benefit in the management of diabetes mellitus as well as in preventing the onset of diabetes mellitus.

Support or Funding Information

Omorevbarhia Omorovbiye, graduate student member ASBMB, omonao.omo@gmail.com

Aniefuna Ejike, graduate student member, ASBMB, aniefunaejike@yahoo.com