bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining Big Data Using Information Flow Analysis – Application to the Functional Characterization of Plant Derived Therapeutics
Authors: Hensley Preston , Logging William , Fliri Anton
Published: June 2021

Biological complexity results from redundancy among systems and processes which allow growth, reproduction and responses to the environment. Understanding their connectivity requires integrative, multi‐scale analytical approaches. However, this integration has been frustrated by heterogeneity in the sorts of data sets available, i.e., types of molecules, scales of measurement, types of model systems. Here, we describe an approach called Information Flow Analysis which works through alignment of cause‐effect spectra. These spectra link a defined vocabulary causes (for instance drugs) with a defined vocabulary of effects (for instance physiological responses). An individual spectrum might be an array row with one drug element and a list of effect elements. Each effect element would record the number of co‐citations for a drug and a particular physiological effect, occurring over the sum of all biological abstracts, for instance. Elements could be full or empty. As these elements are dimensionless, they eliminate data heterogeneity problems and the associated spectra are thus directly comparable. These strings of numbers (spectra) can be hierarchically clustered, with effects clustered by similarity in causes and causes clustered by similarity in effects. This type of analysis is scalable, recapitulates known biology, and elicits new relationships. Here, we apply this approach for the functional characterization active the components from the medicinal plant, Eugenia jambolana.
Influence of serum and hypoxia on endogenously synthesized lipids and lactate from radiolabeled glucose or glutamine in murine glioblastoma cells
Authors: Ta Nathan , Seyfried Thomas
Published: June 2021

Glucose and glutamine are essential energy metabolites for brain tumor growth and survival under both normoxic and hypoxic conditions. These metabolic substrates contribute their carbons to lipid biosynthesis. We used uniformly labeled [14C]‐U‐D‐glucose and [14C]‐U‐L‐glutamine to examine the de novo biosynthesis of all major phospholipids, cholesterol, and gangliosides in the VM‐M3 murine glioblastoma cells. Endogenous lipid synthesis, using either glucose or glutamine, was greater in media without fetal bovine serum (FBS) than in media containing 10% FBS under normoxia. De novo lipid synthesis was greater using glucose carbons than glutamine carbons under normoxia. The reverse was observed for most lipids under hypoxia suggesting an attenuation of glucose entering the TCA cycle. Lactate was produced largely from glucose carbons with minimal lactate derived from glutamine under either normoxia or hypoxia. Accumulation of triacylglycerols, containing mostly saturated and mono‐unsaturated fatty acids, was observed under hypoxia using carbons from either glucose or glutamine. The incorporation of labeled glucose and glutamine into most synthesized lipids was dependent on the type of growth environment, and that the VM‐M3 cells could acquire lipids, especially cholesterol, from the external environment for growth and proliferation. These results can help clarify the origin of lactate synthesis and the role of exogenous lipids, acquired from the microenvironment, in driving tumor cell growth.
Phosphorylation/dephosphorylation of Yeast Pah1p Phosphatidate Phosphatase Regulate Its Ubiquitin‐independent Proteasomal Degradation
Authors: Hsieh Lu‐Sheng , Su Wen‐Min , Carman George
Published: June 2021

PAH1‐encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol in the yeast Saccharomyces cerevisiae. The accumulation of triacylglycerol in the stationary phase of growth is dependent on Pah1p phosphatidate phosphatase activity. Yet, Pah1p is barely detectable in late log phase. The proteasome inhibitor MG132 prevents the growth phase‐dependent loss of Pah1p abundance. Unphosphorylated Pah1p was degraded by the 100,000 x g pellet fraction from stationary phase cells, and this degradation was prevented by MG132. Pah1p abundance in vivo was stabilized in a pre1 pre2 mutant with impaired proteasome function (i.e., chymotrypsin‐like activity). The 20S and 26S proteasomes were purified from yeast strains expressing tandem affinity purification‐tagged versions of Pre1p and Rpn11p, respectively. The 20S proteasome, but not the 26S proteasome, degraded unphosphorylated Pah1p in a time‐ and dose‐dependent manner with an optimum pH of 7.0. Phosphorylated Pah1p isolated from yeast was also subject to 20S proteasome‐mediated degradation. The Nem1p‐Spo7p‐mediated dephosphorylation of Pah1p stimulated the proteasomal degradation. Phosphorylation by Pho85p‐Pho80p and protein kinase A decreased the proteasomal degradation of Pah1p, whereas phosphorylation with protein kinase C stimulated the degradation. Human Pah1p orthologs lipin 1 and lipin 2 were also degraded by the 20S proteasome, but not the 26S proteasome. (Supported by NIH grant GM028140)
The Perinuclear Endoplasmic Reticulum as the Site of Phosphatidylethanolamine Synthesis in a Model Eukaryote
Authors: Buetikofer Peter , Farine Luce
Published: June 2021

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP‐ethanolamine and CDP‐choline branches of the Kennedy pathway represent major – and often essential –routes for the production of PE and PC, respectively. While ethanolamine and choline phosphotransferases (EPT and CPT, respectively) catalyze the last reactions in the respective pathways, choline/ethanolamine phosphotransferase shows dual specificity for both CDP‐ethanolamine and CDP‐choline as substrates

Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular model eukaryote, Trypanosoma brucei, both branches of the Kennedy pathway are essential for parasite growth in culture, making T. brucei an ideal organism to study eukaryotic phospholipid synthesis. We have previously reported that EPT catalyzes the production of alk‐1‐enyl‐acyl PE molecular species while CEPT synthesizes diacyl‐type PE and PC molecular species. We now show that the two enzymes localize to different sub‐compartments of the ER. By expressing a series of tagged EPT and CEPT enzymes in T. brucei parasites, EPT was found exclusively in a distinct area located close to but different from the nuclear membrane, which we term perinuclear ER. In contrast, CEPT was detected in the bulk ER. The different localization pattern of EPT and CEPT was confirmed by subcellular fractionation experiments in combination with enzyme activity measurements and identification of the tagged proteins by immmunoblotting.
FAM129B, a Novel Protein, Suppresses the TNFα Apoptotic pathway and Promotes the NF‐kB Survival Pathway in Cancer Cells by Interacting with KEAP1
Authors: Hachem Fatme , Chen Song , Evans Hedeel , Evans David
Published: June 2021

FAM129B, is a member of a family of novel proteins that include FAM129A and FAM129C.Little is known about the structure and functions of these proteins. Previously; we have shown that FAM129B suppresses the TNFα induced apoptotic pathway in HeLa cells (Song, Evans, & Evans (2010), JBC 286, 10201‐09). To elucidate the mechanism by which FAM129B suppresses this apoptotic pathway, we investigated the effect of FAM129B on several components of the TNFα apoptotic pathway: IkB kinase (IKKβ), the transcription factor NF‐kB, and the anti‐apoptotic proteins c‐FLIP, and cIAP. Silencing FAM129B expression using siRNA in HeLa cells promoted IKKβ degradation and decreased NF‐kB translocation to the nucleus and the expression of c‐FLIP and cIAP following induction of apoptosis. Also, over‐expression of recombinant FAM129B in HEK293 cells decreased IKKβ degradation, thus promoting NF‐kb activation. We also show that KEAP1, a protein that promotes IKKβ degradation, forms a complex with FAM129B in vivo and in vitro. The ETGE motif in FAM129B and the KELCH repeats in KEAP1 are the sites of interaction between the two proteins. Using site directed mutagenesis, we changed the ETGE motif into ETAA. This mutation disrupted the interaction with KEAP1 in vivo and in vitro .Over expression of recombinant FAM129B with the mutated ETGE motif in HEK293 cells, in contrast to the wild type protein, did not suppress apoptosis, inhibit IKKβ degradation or activate NF‐κB.We propose that FAM‐129B sequesters KEAP‐1, and blocks apoptosis by inhibiting the NF‐κB signaling pathway and the expression of anti‐apoptotic proteins.
Expression and Purification of the Metacaspase Protein Scp3 from the Fungus S. commune
Authors: Barker Travis , Gagliardi Connor , Fox Kristin
Published: June 2021

The objective of the present study was to obtain pure, active Scp3 protein for enzymatic and structural studies. Previous work with Scp3 yielded insoluble aggregates, so the protein Small Ubiquitin Modifying protein (SUMO) was fused to the N‐terminus of Scp3. SUMO has previously been shown to drastically increase the solubility of foreign proteins in E. Coli (our model organism). When Scp3 was ligated into the SUMO vector, a stop codon was introduced just before the gene code for Scp3, preventing the fusion of SUMO to Scp3. Site directed mutagenesis was used to remove the stop codon, allowing for continuous translation in the region between SUMO and Scp3. The vector containing SUMO and Scp3 was transformed into chemically competent E. coli cells, which in turn produced the SUMO/Scp3 fusion protein. The cells were grown at 15°C for about fifteen hours in order to produce the SUMO/Scp3 complex. This was confirmed by SDS‐polyacrylamide gels and western blots. A His‐tag was attached to the N‐terminus of the SUMO protein, so a His‐tag‐antibody was used in the western blots. The protein was then purified using a nickel affinity column to 95 percent. Thus far we have produced ample amounts of Scp3, however only a small fraction appears to remain soluble. This fraction becomes even more dilute during the purification process, making it difficult to work with. Currently, we are working on scaling up the procedure such that soluble protein is produced and can be harvested.
Cytochrome C Binds Complement 3b
Authors: Sibulo Lemuel , Rhodes Diana
Published: June 2021

The purpose of this study was to investigate the ability of cytochrome c (CytC) to bind to complement 3b (C3b). Using an enzyme‐linked immunosorbent assay (ELISA), bovine and equine CytC bound C3b with high affinity (KD ~ 10‐10 M) in low ionic strength buffers. This affinity decreased with increasing ionic strength; however, CytC still bound C3b with a KD ~ 10‐7 M in physiologic strength buffers. Western blot analysis indicated that CytC preferentially bound the α' chain (compared to the β chain) of C3b. SDS‐PAGE gels were used to monitor the effect of CytC on C3b degradation. These studies suggested that soluble CytC neither altered the degradative capacity of Factor H and Factor I towards C3b, nor did CytC behave as a cofactor itself in C3b degradation. A competition ELISA demonstrated that soluble CytC was unable to effectively compete for C3b's binding to immobilized CytC. In conclusion, immobilized CytC binds C3b with higher affinity in low ionic‐strength buffers than in physiologic buffers. CytC binds preferentially to the α' chain of C3b. Soluble CytC does not appear to significantly affect the cofactor‐mediated degradation of C3b nor the binding of C3b to immobilized CytC. It may be of functional significance that soluble CytC interacts with C3b differently than immobilized and thus possibly partially denatured CytC, since CytC switches from its native form to a partially unfolded conformation during its role in apoptosis. Thus, the interaction of complement with apoptotic cells may be regulated partly by the binding of C3b to the partially unfolded CytC present during apoptosis, but not to the native CytC normally found in mitochondria. Funded by the PNWU Research Seed Program.
Probing Conformational Change During Radical Propagation in the E.coli Class 1a RNR Using 3‐aminotyrosine as a Radical “Sink”
Authors: Lee Wankyu , Minnihan Ellen , Stubbe JoAnne
Published: June 2021

The E. coli class Ia ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxynucleoside 5ʹ‐diphosphates (dNDPs) using nucleoside 5ʹ‐diphosphates (NDPs) as substrates. The catalytic mechanism of RNR requires an active site thiyl radical, which is generated via reversible oxidation by a tyrosyl radical cofactor located over 35 Å away. The model for the long range oxidation involves multiple proton‐coupled electron transfer (PCET) steps through aromatic amino acid residues. Efforts to study the radical hopping mechanism have been challenging due to kinetic masking of the chemistry by a conformational gate. Lowering the midpoint potentials of pathway residues by using the amber stop codon suppression method to site‐specifically incorporate the unnatural amino acid (UAA) 3‐aminotyrosine (NH2Y) acts as a radical “sink” on pathway and changes the rate limiting step of the radical propagation mechanism from a conformational change to a PCET step. NH2Y‐incorporated RNR (NH2Y‐RNRs) have allowed the detection of radical intermediates on this PCET pathway and have established the role of three tyrosines, Y356, Y730 and Y731, in the PCET pathway. Despite the large perturbation in midpoint potential, NH2Y‐RNRs can also do multiple turnovers and produce dNDPs. The study of the rapid kinetics of NH2Y● and dNDP formation demonstrates the kinetic and chemical competence of the NH2Y● intermediates. Additionally, insight into the kinetic model of the NH2Y‐RNRs give rise to further details of multiple, more subtle conformational changes within the PCET pathway mechanism. Specifically, the NH2Y● populations exist in two distinct conformations, with one less competent in nucleotide reduction.
Inhibition and Kinetic Isotope Effect Studies to Further Characterize the Mechanism of Maleamate Amidohydrolase
Authors: Malani Karan , Snider Mark
Published: June 2021

Maleamate amidohydrolase (NicF) is involved in the degradation of nicotinic acid, catalyzing the deamination of maleamate to form maleate. To provide further insight concerning the mechanism of this enzymatic reaction, kinetic and UV spectroscopy assays were conducted to characterize the binding of (Z)‐4‐oxobut‐2‐enoic acid by NicF. The results indicate that this molecule behaves as a reversible competitive inhibitor with a Ki of 1.3 (± 0.13) x 10‐8 M. Overall, (Z)‐4‐oxobut‐2‐enoic acid was shown to be bound 10 x more tightly than the previously studied inhibitor maleic anhydride, and 400,000 times more tightly than the product, maleate. The observed tight binding of this aldehyde inhibitor is consistent with the proposed addition‐elimination mechanism for catalysis. Additionally, D2O kinetic isotope effects were measured to determine the number of protons essential for the rate limiting step. The Dkcat was observed to be insensitive to increasing mole fraction of D2O, whereas Dkcat/KM was inverse, increasing exponentially with increasing D2O. Thus, substrate binding appears to be accompanied by at least 3 proton transfers leading up to the first irreversible step.
Characterization of Human Polyamine Oxidase
Authors: Zamora Diana , Tormos Melendez Jose , Galaleldeen Ahmad
Published: June 2021

Polyamine Oxidase (PAO) is a flavoprotein that is necessary for the catabolism of polyamines. PAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine, respectively. Normal levels of polyamines are needed for cell growth, yet their actual function is not well understood. The crystal structures of maize and yeast PAO (Fms1) are available but no mammalian PAO structure has been determined to date. The identity between human PAO (hPAO), Fms1, and maize PAO is only 20%. Sequence homology and mutational analysis showed that a conserved histidine residue in mammalian PAOs that corresponds to position 67 of Fms1 helps to properly position the amine substrate for oxidation. Determining the crystal structure of hPAO will help identify the other residues that play a role in substrate binding and catalysis.

hPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. hPAO was expressed and purified using affinity and ion exchange columns. The his‐tag was removed and the protein is being assayed for activity. Analytical ultracentrifugation sedimentation velocity experiments are also underway to determine the oligomerization state of the protein in solution in addition to crystallization trials to determine the structure of hPAO.
Analyzing the Mechanistic Role of an Active Site Lysine Residue in Monofunctional and Bifunctional Proline Dehydrogenases
Authors: Floth Joshua , Becker Donald
Published: June 2021

The catabolism of proline to glutamate is a critical pathway in all organisms and holds roles in mental illness and cancer. The two enzymes utilized in the oxidation of proline are proline dehydrogenase (PRODH) and Δ1‐pyrroline‐5‐carboxylate dehydrogenase (P5CDH). In certain organisms, such as gram‐negative bacteria, these enzymes are fused into a single polypeptide named Proline Utilization A (PutA). In gram‐positive bacteria and eukaryotes, these enzymes exist as two separate polypeptides. A highly conserved active site lysine residue in PRODH enzymes has been shown by structural studies to be in proximity to the N1 hydrogen of proline. Previously, this lysine residue was proposed to deprotonate the N1 hydrogen of proline in Mycobacterium tuberculosis PRODH (MtPRODH), which is the first step in the mechanism of converting proline into glutamate (Serrano, H., and Blanchard, J. S., 2013. Biochemistry 2013, 52, 5009−5015). Substitution of this lysine residue with alanine eliminated catalytic activity in MtPRODH (Serrano, H., and Blanchard, J. S., 2013. Biochemistry 2013, 52, 5009−5015). To see if the role of this lysine is conserved in bifunctional PRODH/PutAs, we made the analogous mutation in PutA from Escherichia coli (EcPutA) and monofunctional PRODH from Thermus thermophilus (TtPRODH). Unexpectedly, this mutation only minimally affected the activity of EcPutA, whereas activity was completely lost by the mutation in TtPRODH, as observed previously for MtPRODH. These results indicate that there might be differences in the active site environments of monofunctional and bifunctional PRODH/PutAs
High‐Resolution 31P Field Cycling NMR as a Probe of Substrates Dynamics on GMP Reductase
Authors: Rosenberg Masha , Redfield Alfred , Roberts Mary , Hedstrom Lizbeth
Published: June 2021

GMP reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. The reaction proceeds in two steps: first GMP reacts with cysteine to create a covalent thioimidate intermediate E‐XMP* and ammonia, followed by hydride transfer from NADPH to E‐XMP* creating IMP. The crystal structure of GMPR and IMP/NADPH revealed the presence of two different NADPH conformations ‐ “in” and “out”. In the “in” conformation, the NADPH and IMP poised for the hydride transfer, while the “out” conformation is proposed to be important in the deamination step. Here we measured the 31P relaxation rates of substrates and cofactors over a large magnetic field range using high‐resolution field cycling NMR. Dipolar relaxation rates (R1) of 31P provide dynamic information about bound ligands. We show that the 31P relaxation rates of both bound substrates and cofactor increased dramatically at very low fields. These enhanced rates arise from dipolar interactions of the ligand 31P bound to the large protein. There are differences in the low field R1 profiles for the 31P of GMP compared to IMP in their respective NADP+ complexes that reflect changes in ligand binding dynamics. Different 31P relaxation profiles consistent with different conformations in E•IMP•NADP+ and E•GMP•NADP+ complexes that are considered to represent different steps of the GMPR reaction. These results demonstrate the feasibility of using field cycling to investigate the dynamics of protein‐bound substrates.
Investigating the Role of 3‐Phosphoinositide‐Dependent Protein Kinase 1 in the Spatiotemporal Regulation of the Purinosome
Authors: Sundaram Anand , An Songon
Published: June 2021

The enzymes involved in human de novo purine biosynthesis form a transient multienzyme complex, the “purinosome,” under purine starvation. To determine cellular regulatory mechanisms of purinosome assembly/disassembly, fluorescence live‐cell imaging has been used to monitor purinosome dynamics following small molecular inhibition/activation of protein kinases involved in cell signaling. To date, we observe that pharmacological inhibition of 3‐phosphoinositide‐dependent protein kinase 1 (PDK1) resulted in purinosome formation, but not purinosome dissociation, in HeLa and Hs578T cells. Subsequent inhibition of protein kinase C, one of downstream kinases of PDK1, resulted in purinosome formation as well. However,small‐molecule inhibition of other downstream targets of PDK1, including Akt and mammalian Target of Rapamycin (mTOR), exhibited no effect on purinosome assembly. We hypothesize that PDK1‐associated signaling pathways negatively regulate purinosome formation in an Akt‐independent manner. Collectively, our work has the potential to demonstrate the functional role of PDK1 on de novo purine biosynthesis, thus providing a novel target for anti‐cancer drug discovery.
Active Role of the Substrate During Catalysis by the Therapeutic Enzyme L‐Asparaginase II
Authors: Rempe Susan , Vanegas Juan , Anishkin Andriy , Rogers David , Sukharev Sergei
Published: June 2021

Bacterial type II L‐Asparaginases (ASPII) have been used for over four decades to treat acute lymphoblastic leukemia, yet a full reaction mechanism remains unknown. ASPII enzymes catalyze the deamidation of both asparagine (Asn) and glutamine (Gln), which results in the formation of aspartate (Asp) and glutamate (Glu) respectively, and the by‐product ammonia. Proposed ASPII mechanisms to date have yet to explain the absolute requirement of a substrate α‐carboxyl group, and clearly identify the role of the catalytic threonines T12 and T89. Here, we study the reaction mechanism of asparagine degradation by ASPII through ab initio molecular dynamics (MD) simulations. We select a reduced system from the substrate‐bound enzyme obtained by classical MD, and explore different initial reaction pathways by driving the system in steered simulations. Our results show that direct nucleophilic attack by T12 produces a highly unstable substrate‐enzyme intermediate, as the stabilization provided by the nearby protons (i.e., the “oxyanion hole”) is insufficient to sustain the high energy state. We find that the substrate‐enzyme intermediate can be stabilized by first protonating the substrate's amide oxygen through the K162‐T89 proton bridge. Furthermore, the α‐carboxyl of the substrate acts as a proton acceptor for the hydroxyl sidechain of T12 during nucleophilic attack. We conclude by showing that a complete deamidation mechanism may require a sequence of several nucleophilic attacks by both T12 and T89, with K162 playing a critical role as a proton buffer during the course of the reaction.
FQQI motif in the C‐terminus of Guanylyl‐cyclase/Natriuretic Peptide Receptor‐A mediates intracellular trafficking in mouse mesangial cells
Authors: Mani Indra , Pandey Kailash
Published: June 2021

Binding of cardiac hormone, atrial natriuretic peptide (ANP) to the guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), induces the rapid internalization and trafficking via the endo‐lysosomal compartments with concurrent generation of cGMP. To delineate the critical role of endocytic signal in intracellular sorting of receptor into these compartments, we have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AAAA) in enhanced green fluorescence protein (eGFP)‐tagged NPRA (eGFP‐NPRA) cDNA sequence. Mouse mesangial cells (MMCs) were transiently transfected with both mutant and wild‐type constructs. The internalization studies revealed the immunofluorescence colocalization of mutated motif (FQQI/AAAA) receptor with pan‐Cadherin, a plasma membrane marker, and showed a decreased internalization of eGFP‐NPRA by 49.23% compared with the wild‐type receptor. However, subcellular trafficking studies indicated that colocalization of mutated receptor with early endosome antigen‐1 (EEA‐1), lysosome‐associated membrane protein‐1 (LAMP‐1), and Rab 11 marker, was decreased into early endosome by 57.49%, lysosome by 31.98%, and recycling endosome by 25.43% respectively, compared with wild‐type receptor. The results suggest that Phe790 and Ile793 constitute important element in FQQI motif, which are essential for the internalization and trafficking of NPRA. The findings may provide the basis for new molecular association of single nucleotide polymorphisms in Npr1 gene with susceptibility to diseases and /or resistance to drugs in translational studies.
Modulation of Neuronal GIRK Channels by Cholesterol
Authors: Rosenhouse‐Dantsker Avia , Bukiya Anna
Published: June 2021

In recent years, cholesterol emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels is a decrease in channel activity. We have recently shown that unexpectedly cholesterol enrichment up‐regulates G‐protein gated inwardly rectifying potassium (GIRK or Kir3) currents in atrial myocytes. Here we focus on neuronal GIRK channels, and determine the effect of cholesterol on their function. Several GIRK subunits are expressed in the hippocampus: GIRK1, GIRK2a, GIRK2c and GIRK3. Among these, GIRK2 may be expressed as a homomer or as a heteromer with GIRK1 and/or GIRK3, both of which do not express as homomers in the plasma membrane. We first studied the effect of cholesterol on a pore mutant of GIRK2, GIRK2^ (GIRK2_E152D). We show that when GIRK2^ is expressed in Xenopus oocytes, its currents are up‐regulated by cholesterol. Next, using planar lipid bilayers we demonstrate that cholesterol significantly increases the open probability of the GIRK2^ channels. No apparent change was observed in the unitary current amplitude. Finally, we show that in‐vitro cholesterol enrichment of freshly isolated hippocampal neurons results in a strong increase in physiological GIRK currents. These findings point at cholesterol as a critical lipid regulator of GIRK channel activity in the brain.
Monoubiquitination Regulates Golgi Membrane Dynamics In The Cell Cycle
Authors: Wang Yanzhuang , Zhang Xiaoyan , Huang Shijiao
Published: June 2021

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of post‐mitotic Golgi membrane fusion. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in membrane fusion during post‐mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97‐mediated Golgi membrane fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi‐localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly; reduced HACE1 activity is human cancer such as Wilms' tumor results in fragmented Golgi. We have also discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required for post‐mitotic Golgi reassembly. VCIP135 activity is regulated by phosphorylation in the cell cycle; it is inactivated by phosphorylation in metaphase and activated upon dephosphorylation in telophase. Recently, we have identified the substrates on the Golgi membranes whose ubiquitination and deubiquitination are regulated by HACE1 and VCIP135 in the cell cycle. These data suggest that cycles of addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell division.
A New approach to SNAP‐tag technology as a possible imaging tool for translational research
Authors: Sumzin Nikita , Moroz Maxim , Ponomarev Vladimir
Published: June 2021

The SNAP‐tag is a modified protein designed from the DNA repair protein O6‐alkylguanine‐DNA‐alkyltransferase that recognizes benzylguanine derivatives and covalently attaches to the tag (Johnsson, 2003). To advance this method for in vivo and translational research we need better ways to modify target cells as well as improvements in probe design. We developed a retroviral vector bearing SNAP‐ADRB2 protein and Green Fluorescent Protein (GFP) for the generation of permanently transduced cancer cell lines. The SNAP‐ADRB2 sequence from a commercial plasmid was cloned into p28z‐IRES‐hsvTKGFP. The chimeric antigen receptor p28z of this plasmid was replaced with SNAP‐ADRB2 to construct the final vector SNAP‐ADRB2iTG. Supernatants from H29 virus producer cells were collected at 48 and 72 hours following transfection and used to permanently transduce C6 (rat glioma) and U87 (human glioma) cells in culture. Vector presence in the C6 and U87 cells was confirmed by GFP imaging and expression analysis using flow cytometry. Functionality of the SNAP‐tag system was assessed by in vitro labeling with fluorescent Surface‐549 substrate. The externalized SNAP protein was confirmed by fluorescent microscopy after in vitro labeling, which demonstrated a localized signal on the membrane of target cells. GFP and SNAP fluorescence in target cells persisted for at least two weeks. We have successfully created a new retroviral vector that allowed the generation of human and rat cancer cell lines with constitutive expression of the SNAP‐tag that could be used for in vitro and in vivo imaging. Further development of probes for this system could open the possibility of translational applications.
Cancer Cell‐induced Suppression of Cytotoxic Alu RNA Biosynthesis in the Co‐cultured Macrophages
Authors: Singhal Ashutosh , Mishra Smita , Chaudhuri Gautum
Published: June 2021

Macrophages are among the immune cells that are recruited at the site of solid tumor to phagocytose tissue debris and to destroy the tumor cells through phagocytosis‐induced inflammatory immune response. Thus, unless manipulated by the cancer cells, the inflammatory response induced in the macrophages should kill the macrophages bringing collateral damage to the tumor cells. One of the emerging mechanisms of induction of inflammatory response in the macrophages is the increase in the level of cytotoxic scAlu RNA in these cells. Through the enhancement of reactive oxygen species levels, scAlu RNA activates NLRP3 inflammasome in these cells to cause cell death via the caspase pathway. We found that macrophages co‐cultured with aggressive, but not with the non‐aggressive, breast cancer cells are manipulated by the suppression of their scAlu RNA biosynthesis. Alu RNA, the precursor of scAlu RNA, is transcribed by RNA polymerase III and requires TFIIIC110 for their expression. We found that the metalloproteinase MMP1, secreted by the aggressive breast cancer cells, proteolytically activates the G‐protein‐coupled receptor PAR1 on the surface of the macrophages. Activation of PAR1 elevated the cytosolic Ca2+ levels through the Galpha‐q/PIPLC/PIP3 pathway thereby activating the Ca2+‐dependent protease mu‐calpain. The TFIIIC110 protein, which has a mu‐calpain target PES sequence, was degraded by mu‐calpain resulting in the suppression of Alu RNA biosynthesis. Suppression of Alu RNA levels consequently decreased the levels of its processed derivative, the scAlu RNA, preventing the induction of inflammatory response in the macrophages. This research is supported in part by NIH/NCI grant 1R21CA181920‐01 to GC.
Determination of 12 Minerals in Human Milk by Inductively Coupled Plasma ‐ Mass Spectrometry: a GEHM Study of Three Global Cohorts through the First Year of Lactation
Authors: Gray Michael , Maria Sarah , Labhart Beau , Phillips Shay , McMahon Robert , Davidson Barbara , Morrow Ardythe
Published: June 2021

Elemental composition of human milk may be influenced by several factors, including dietary intake and genetic background, though variation in milk content over lactation and by geographic region has not been well characterized. A newly validated simultaneous method allows rapid and accurate determination of concentrations for 12 elements, P, Na, K, Ca, Mg, Mn, Cu, Zn, Fe, Mo, Cr, and Se, in human milk via inductively coupled plasma‐mass spectrometry (ICP‐MS). As part of the Global Exploration of Human Milk (GEHM) collaboration, human milk collected during the first year of lactation from three global cohorts (Mexico City, Mexico, Shanghai, China, and Cincinnati, United States) was analyzed at 4, 26 and 52 weeks lactation. Samples underwent acidic microwave digestion followed by ICP‐MS quantification, and analysis included suppression of known polyatomic interferences using collision cell technology. Milk from distinct geographic regions exhibited similar mineral concentrations as well as temporal patterns between 4 and 52 weeks of lactation. Among several observations, some elements showed consistent decreases in concentration between 4 and 52 weeks postpartum, including Ca (295 to 220 µg/mL, ↓25%), K (545 to 431 µg/mL, ↓21%), P (163 to 130 µg/mL, ↓20%), Cu (416 to 140 ng/mL, ↓66%) and Zn (2520 to 617 ng/mL, ↓76%), while Fe and Mn remained fairly stable with 蠄10% change in concentration over the course of lactation. This research was supported by Mead Johnson Pediatric Nutrition Institute.
Replacement of Sugar‐Sweetened Beverages with Water and its Impact on Insulin Sensitivity Among Overweight Adolescents and Young Adults
Authors: Jin Ran , Collin Lindsay , Vos Miriam , Welsh Jean
Published: June 2021

Background

Intake of sugar‐sweetened beverages (SSBs) has dramatically increased over recent decades. Habitually high consumption of SSB has been

linked to multiple adverse health consequences including insulin resistance. High consumers are encouraged to reduce their SSB consumption and drink more water in their place but little is known about the impact of this replacement.

Objective

We aimed to evaluate whether 1‐month replacement of SSBs with plain water would improve insulin sensitivity.

Methods

A total of 22 overweight males and females age 16‐21 y, who consumed 蠅 3 servings of SSBs daily at baseline, were recruited in a 1‐month cross‐over beverage trial. All subjects were instructed to replace all sweet beverages (SSBs, diet drinks, juices) with study provided

bottled water (3 daily) for 1 month and otherwise maintain their diet and activity level as usual. Their plasma levels of glucose and insulin sensitivity were compared at baseline and after one month of water replacement, using Wilcoxon test (SPSS, version 17.0, SPSS Inc., Chicago, IL). Insulin sensitivity was assessed by calculating HOMA‐IR (fasting glucose * insulin / 405) and adipose IR (fasting insulin * free fatty acid).

Results

After the replacement of their sugar‐sweetened beverages with plain water for 1 month, subjects had reduced concentration of plasma glucose (p = 0.048), and improved insulin sensitivity as indicated by both HOMA‐IR (p = 0.042) and adipose IR (p = 0.050).

Conclusion

The results of this study suggest that insulin sensitivity and plasma glucose level regulation among overweight high consumers improves after one month of replacing SSBs with water and in the absence of other changes in diet or physical activity.
HYDRAGUT study: Influence of HYDRAtion status on the GUT microbiota and their impact on the immune system.
Authors: Redondo Useros Noemi , Gheorghe Alina , Serrano Labajos Ruth , Nova Rebato Esther , Marcos Sanchez Ascension
Published: June 2021

While a good hydration state is essential for health maintenance, the dehydration state, produced by the imbalance between fluid intake and fluid losses, could be related to an inflammatory situation. A good mucosal hydration is also necessary for a normal protective intestinal barrier. We performed this study in one hundred twenty‐three healthy young adults from Madrid to address the influence of hydration status on the gut microbiota balance and their impact on the immune system. Blood and stool samples were collected from all volunteers. Beverage patterns, sodium and osmolality levels, intestinal microbiota, leukocyte subpopulations and cytokine levels were analysed.

Men showed statistically higher levels of osmolality and liquids intake than women (P<0.05), and osmolality had a significant positive correlation with beer intake in men and with soft drink intake in women (P<0.05). Men showed a trend to increased values of hydration parameters with TNF‐α rises. Sodium levels were influenced by Bacteroidetes (P=0.029), whereas Clostridium affected osmolality levels (P=0.043), suggesting an influence on cellular transport of solutes and thus contributing to the hydration state.Bifidobacterium composition influenced T lymphocytes levels (P<0.001), reflecting an interaction with the immune response of the host. It might be interesting to evaluate this effect in euhydration and dehydration states, and thus relating possible changes in dehydration biomarkers with changes in microbiota community and the immune response.
Leukocyte proliferation in iron‐deficient conditions
Authors: Contreras Irazú , Alvarado‐Guadarrama Michelle , Erazo‐Muñoz María , Estrada José
Published: June 2021

Iron is an element with crucial functions in the metabolic processes of the human body; therefore, the maintenance of its optimal concentration is essential for maintaining homeostasis and correct function. Iron deficiency is considered one of the major health problems worldwide, primarily affecting children and pregnant women. This condition´s harmful effects have been observed in the central nervous system, affecting cognitive processes, as well as in the immune system, affecting its functions, diminishing its ability to respond to harmful stimuli. Due to its effects on leukocytes, the aim of this study was to analyze the effect of iron deficiency on the proliferative capacity of immune cells by assessing cell cycle progression. Iron deficiency was induced from gestation in a group of BALB/c mice. Prior to mating, females were separated in two groups: one group was fed an iron‐deficient diet (10 ppm iron) and another an iron‐sufficient diet (100 ppm iron). After birth, both groups followed this diet for a period of 60 days. After treatment, mice were sacrificed and the spleen and thymus were extracted. Leukocytes from these organs were purified and analyzed by flow cytometry, in order to determine the amount of cells undergoing cell cycle using propidium iodide. Our results show that iron deficiency decreases the amount of leukocytes going through cell cycle progression, particularly B and T lymphocytes in spleen and T lymphocytes in thymus. It can be suggested that iron deficiency negatively affects the immune system, decreasing the proliferative capacity of leukocytes.
Grit is Negatively Associated with Body Weight, Body Mass Index and Disinhibition and Positively Related to Cognitive Eating Restraint
Authors: Metzgar Catherine , Nickols‐Richardson Sharon
Published: June 2021

Grit, the perseverance and passion for pursing long‐term goals, may be a personality trait of interest in weight management as grit may be related to body weight (BW) modification through the control of food intake. This study explored the association between grit and cognitive eating restraint (CER), disinhibition and hunger, three factors known to affect BW regulation. Eighty women [mean±SD age: 31.4±8.2 y, BW: 76.2±19.1 kg, body mass index (BMI): 27.9±6.9 kg/m2] completed the Short Grit Scale (Grit‐S) and Eating Inventory (EI) upon enrolling in a 1‐year weight gain prevention trial. Grit‐S scores ranged from 2.0 to 4.6 (1‐5 scale); the average Grit‐S score was 3.4±0.7. EI scores for CER (0‐21 items), disinhibition (0‐16 items) and hunger (0‐14 items) ranged from 2‐17, 1‐14 and 0‐13, respectively, with average scores of 9.1±3.5, 7.6±3.2 and 5.6±2.8, respectively. Pearson's correlation was used to examine the relationship between Grit‐S score and BW, BMI, CER, disinhibition and hunger. Grit was negatively associated with BW (r=‐0.24, p<0.05), BMI (r=‐0.23, p<0.05) and disinhibition (r=‐0.47, p<0.01) and positively associated with CER (r=0.23, p<0.05). There was no significant relationship between grit and hunger. These findings suggest that women who have a higher Grit‐S score may have better control of BW management as reflected in lower BW and BMI, higher level of CER and lower level of disinhibition. Further research is warranted to understand whether grit may be a useful predictor of BW status, prevention of BW gain, attainment of BW loss and/or success with BW‐loss maintenance.

Funded by an Illinois Agricultural Experiment Station/National Institute of Food and Agriculture research grant (#100125).
Plasma Acylcarnitines and Development of Metabolic Syndrome in a Middle‐Aged and Older Chinese Population
Authors: Lin Xu , Wu Jia‐Rui , Zeng Rong , Ye Xingwang , Gao Xianfu , Sun Liang , Li Rongxia
Published: June 2021

Incomplete fatty acid oxidation leads to accumulation of intermediates such as acylcarnitines. The aim of this study was to investigate associations of carnitine and acylcarnitines with development of metabolic syndrome (MetS) in a middle‐aged and older Chinese population. In 2005, 3289 participants, aged 50‐70 years, were recruited from Beijing and Shanghai, China, and were resurveyed in 2011. Concentrations of baseline plasma free carnitine and acylcarnitines were quantified using liquid chromatography‐tandem mass spectrometry. After 6‐year follow up, 421 out of 1208 participants without MetS at baseline developed MetS. Free carnitine, propionylcarnitine, valerylcarnitine, octanenoylcarnitine, palmitoylcarnitine and 3‐Hydroxy‐octadecenoyl carnitine were each statistically significantly associated with incident MetS after correction for multiple testing. In a stepwise logistic regression including all of these (acyl)carnitines simultaneously, only free carnitine and octanenoylcarnitine remained significantly associated with incident MetS. Compared with those in the first quartile of free carnitine, those in the fourth quartile had 2 fold increased risk [odds ratio: 2.00, 95% confidence interval (CI): 1.40, 2.86] for incident MetS. Similar trend was observed for octanenoylcarnitine [OR, 95% CI: 2.21 (1.56, 3.14) comparing extreme quartiles]. In conclusion, accumulation of free carnitine and octanenoylcarnitine may increase the risk of MetS in middle‐aged and older Chinese adults.
Quenching of Cytotoxic Reactive Carbonyl Species by Black Rice with Giant Embryo
Authors: Colzani Mara , Lee Yoon‐Mi , Han Sang‐Ik , Aldini Giancarlo , Yeum Kyung‐Jin
Published: June 2021

Reactive carbonyl species (RCS) are cytotoxic molecules originating from lipid peroxidation and sugar oxidation. A robust and efficient method based on high resolution mass spectrometry was used to test the ability of water‐soluble rice extracts to inhibit protein carbonylation induced by RCS. The model protein ubiquitin was incubated in vitro with different RCS such as 4‐hydroxy‐trans‐2‐nonenal (HNE) in presence of increasing concentration of various rice extracts. After 24 hours of incubation, the reaction was stopped and ubiquitin was analyzed by HRMS to determine the extent of protein modification due to the RCS, and to observe its modulation in presence of the extracts. The different rice extracts inhibited the formation of RCS‐induced protein modificationsto different extents. A black sticky rice with giant embryo (IC50=~29.1) strongly inhibited protein modification by the HNE than white rice (IC50=~151.0). These findings suggest that certain bioactive components in water‐soluble fractions of specific rice varieties effectivelysequester RCS, and that such rice variety can be valuable for dietary strategy for optimal health.

This work was supported by Rural Development Administration (PJ010059), Republic of Korea.
Rutin Reduces Body Weight with an Increase of Muscle Mitochondria Biogenesis and Activation of AMPK in Diet‐induced Obese Rats
Authors: Kim Seunghae , Seo Sangjin , Lee Mak‐Soon , Jang Eugene , Shin Yoonjin , Oh Soojung , Kim Yangha
Published: June 2021

Rutin is a flavonoid which exerts several beneficial properties in the health. The specific aim of this study was to investigate anti‐obesity effects of rutin in high fat diet‐induced obese rats. Male Sprague‐Dawley rats were randomly divided into 3 groups(n=8) and fed with normal diet(NOR), 45% high fat diet(HF) or 45% high fat diet containing 0.1% rutin (HF+Rutin) for 12 weeks. Compared to HF control, rutin supplementation significantly reduced body weight. Muscle mitochondrial number and size were decreased by HF, which were fully reversed by rutin. The mRNA levels related with mitochondrial biogenesis and β‐oxidation such as NRF1, Tfam, PGC‐1α, SIRT1 and CPT1 in muscle were significantly up‐regulated in HF+Rutin group. The AMPK activity was stimulated by rutin in not only muscle but also adipose tissue. In addition, rutin significantly decreased the mRNA levels of adipogenic genes such as PPAR‐γ, SREBP‐1c, and aP2 in white adipose tissue with the reduction of adipose tissue weight. Our results indicate that rutin decreased high fat diet‐induced body weight gain through PGC‐1α and AMPK activation in accompany with the decrease of adipogenesis in adipose tissue and the improvement of mitochondria function in muscle.
Association between ApoE (rs429358 and rs7412), SLC16A11 (rs13342692) and ABCA1 (rs9282541) polymorphisms and biochemical parameters in subjects with metabolic syndrome
Authors: Orozco‐Ruiz Ximena , Guevara‐Cruz Martha , Ayala‐Alanis Brenda , Medina‐Vera Isabel , Jimenez‐Rolland Edith , Aguilar‐Lopez Miriam , Tovar Armando , Torres Nimbe
Published: June 2021

Approximately one of two Mexican adults have metabolic syndrome (MetS). Some single nucleotide polymorphisms (SNPs) highly prevalent in the Mexican population, particularly ApoE, SLC16A11 and ABCA1 SNPs have been associated with dyslipidemia, diabetes and obesity. Thus, the aim of this work was to study the association of these SNPs with the presence of several factors of MetS.

We studied 880 Mexican overweight and obese subjects (164 men, 716 women). The frequency of ApoE, SLC16A11 and ABCA1 were 25.5%, 64.4% and 20.6% respectively. Subjects with the ApoE or SCL16A11 or the presence of both showed a higher waist circumference, serum triglycerides (TG), glucose, total cholesterol, blood pressure (BP) and lower HDL cholesterol (HDL‐C). Patients with MetS and ABCA1 SNP showed higher TG and BP and lower HDL‐C than those without MetS. Subjects with MetS and three SNPs showed higher TG (249 mg/dl) and lower HDL‐C (32 mg/dl). Interestingly subjects with ApoE and SCL16A11 or ApoE and ABCA1 had 1.5 fold more risk to develop MetS. Thus, the presence of specific gene variants could increase the risk to have MetS and possibly the risk of cardiovascular disease.

Supported by: MEDIX to NT and CONACYT 181685 to MGC