bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Structure Initiative:Biology‐Materials Repository: Developing a Public Resource for Structural Biology Plasmids
Authors: Cormier Catherine , LaBaer Joshua
Published: June 2021

The PSI:Biology‐Materials Repository (MR; http://psimr.asu.edu) provides centralized archival and distribution of the annotation and samples for plasmids created by the Protein Structure Initiative (PSI). The MR developed an informatics and sample processing pipeline to fully track, sequence analyze, and annotate each plasmid deposited in the MR. Plasmid annotations, including the full length sequence, vector information, and associated publications, are stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each plasmid links to external resources, including the PSI Structural Biology Knowledgebase facilitating cross‐referencing of plasmids to additional protein annotations and experimental data. To expedite and simplify plasmid distribution, the MR developed an Expedited Material Transfer Agreement (MTA) where researchers from network institutions can order and receive PSI plasmids without delay. Currently over 35,000 protein expression plasmids and 75 empty vectors from the PSI are available upon request from DNASU. Overall, this repository of expression‐ready plasmids allows the research community to quickly and easily dissect the biological function of proteins whose structures have been identified by the PSI.

This work was supported by the National Institute of General Medical Sciences.
A novel physiological role for Na, K‐ATPase: Maintenance of nucleoplasmic calcium
Authors: Galva Charitha , Artigas Pablo , Gatto Craig
Published: June 2021

Na, K‐ATPase (NKA) is the integral membrane protein responsible for establishing and maintaining the Na+ electrochemical gradient across the plasma membrane of animal cells by actively exporting 3Na+ in exchange for 2K+. In this report, we present evidence suggesting that NKA builds a maintains a Na+ gradient across the nuclear envelope (NE) membrane, extending our earlier observation that NKA functions within the Endoplasmic Reticulum (Gatto et al., AJP‐cell phys., 281, C982‐C992, 2001). Immunocytochemistry of isolated nuclei from HEK‐293 cells with commercially available alpha and beta antibodies revealed that NKA was located on the nuclear envelope. In addition, membrane preparations from isolated nuclei had ouabain‐sensitive ATPase activity. Moreover, immunostaining of nuclei with antibodies against the Na/Ca‐exchanger (NCX) and NKA reveals an overlap between localization of nuclear NKA and NCX (Na/Ca‐exchanger). Given that NCX is driven by a Na+ gradient, we propose that the intracellular NKA function is utilized by NCX to extrude Ca2+ from the nucleus. We measured NKA function in isolated HEK cell nuclei via CoroNa green fluorescence. We observed an ATP‐dependent, strophanthidin‐sensitive Na accumulation in the nuclear envelope loaded with the Na‐sensitive dye. Analogous experiments are underway utilizing Ca‐sensitive probes. This work is supported by NIH grant DK083859.
Adipose tissue triglyceride lipase mRNA is present in the small intestine and increased in response to acute and chronic high fat feeding in mice
Authors: Uchida Aki , Whitsitt Mary C. , Lee Bonggi , Slipchenko Mikhail N. , Cheng Ji‐Xin , Buhman Kimberly K.
Published: June 2021

Adipose triglyceride lipase (ATGL or PNPLA2) specifically catalyzes the initial removal of a fatty acid from triacylglycerol (TG) to generate diacylglycerol and fatty acid. Recent studies show that ATGL is highly abundant in adipose and liver tissue and critical for TG turnover. However, expression and regulation of ATGL in the small intestine is unknown. We determined that ATGL mRNA is expressed along the length of the small intestine at similar levels. To determine whether ATGL mRNA levels are regulated by acute high fat feeding, we measured mRNA levels in the jejunum of mice fed 200 μl olive oil via oral gavage. We found that ATGL mRNA levels were higher two hours post gavage and returned to control levels by four hours post gavage. Similarly, enterocyte TG storage observed by coherent anti‐Stokes Raman scattering microscopy found higher TG storage at 2 hours post gavage and decreased TG storage at 4 hours post gavage. We also observed that mice fed a high fat diet ad libitum for 3‐weeks had a 4.3 fold higher level of ATGL mRNA in the jejunum. Our results indicate that ATGL mRNA levels are regulated acutely and chronically by dietary fat and correlates with TG storage in enterocytes.
Demonstration of interaction of full‐length PPARα and LXRα
Authors: Balanarasimha Madhumitha , Rider S. Dean , Oswal Dhawal , Hostetler Heather A.
Published: June 2021

The two ligand‐activated nuclear receptors: peroxisome proliferator activated receptor (PPAR) and liver‐X receptor (LXR) are known to play important roles in fatty acid (FA) metabolism. PPARα and LXRα are known to interact and function as heterodimeric partners; however, their specific activity is still unknown. Further, previous studies utilized either murine or truncated forms of these proteins, and whether full‐length PPARα and LXRα interact is unknown. Prior studies indicate that PPARα is activated by polyunsaturated fatty acids, saturated and/or unsaturated long chain fatty acyl‐CoA. However, not much is known about the effects of these ligands on the function or interaction of PPARα and LXRα. In the present study, the effects of FA and their CoA derivatives (FA‐CoA) on the interaction of full‐length human PPARα and LXRα were studied. Circular dichroic spectra of the two proteins in the presence or absence of FAs and their FA‐CoA were determined. Analyses showed that most FA do not significantly alter the structures, while FA‐CoA displayed structural changes indicating that the FA‐CoA affect PPARα and LXRα interaction. As high levels of FA are associated with certain metabolic disorders and also serve as natural ligands for PPARα, changes in structure and/or interaction between PPARα and LXRα may have significant effects on the normal functioning of a cell. This work was supported by USPHS NIH grant DK77573.
Investigation of the Mechanism of Hydrogen Sulfide Activation of Protein Kinase C
Authors: Stahelin Robert V , Gunasekaran Christopher
Published: June 2021

Hydrogen Sulfide (H2S) has been shown to be an endogenously produced second messenger, affecting a variety of physiological and cell signaling processes. It is present at micromolar levels within the body and is produced via two key enzymes in the cysteine biosynthesis pathway: cystathione‐β synthase, and cystathione‐γ lyase. Despite its prior reputation as a noxious gas, H2S has been shown to have significant involvement in protection of cardiac tissue following myocardial infarction. These protective properties may be due to a role of H2S in control of mitochondrial oxidation, in addition to its ties to the localization and signaling of Protein Kinase C. Recent studies have indicated an involvement of H2S in the localization of certain isoforms of Protein Kinase C to the cellular membrane (epsilon and eta). In particular, PKC epsilon knockout has been found to be deleterious to cardiac recovery and also has probable involvement in the process of mitochondrial respiration. We are investigating the molecular basis of H2S induced translocation of PKC isoforms to the cellular membrane. The molecular basis of PKC recruitment to cellular membranes by H2S signaling will be presented.
Increases in physical activity reduces BMI gain in Mexican obese girls attending elementary school
Authors: Jauregui Alejandra , Villalpando Salvador , Rangel‐Baltazar Eduardo , Lara‐Zamudio Yaveth , Castro‐Hernández Jessica
Published: June 2021

Background

In México the prevalence of unhealthy weight jumps from 5% at 5 y to 25% at 7 y, making this a window of opportunity to better understand the pathogenesis of obesity.

Objective

To explore the association between PA patterns and obesity over the transition from kindergarten (K) to elementary school (ES) in a cohort of Mexican children.

Methods

The PA (5‐d accelerometry) and BMI of 211 children (5–6 y at the beginning of the study) were measured while in K, 1st and 2nd grade of ES. Associations between PA changes and BMI changes were examined using multiple, longitudinal regression models stratified by sex, controlling for energy intake and other demographic variables.

Results

Overweight/obese girls who had increased their PA level from 1st to 2nd grade ES had a lower BMI gain compared to obese girls who decreased or maintained their PA (0.02 kg/m2, 95%CI: −1.8, 1.9 vs 2.8 kg/m2, 95%CI: 2.2, 3.3, respectively). This association was not observed comparing changes from K to 1st or 2nd grade ES. In boys, changes in PA were not associated with change in BMI.

Conclusion

In obese girls, an increase in PA in the early years of ES may result in decreased adiposity overtime.
Purification and characterization of the lipid A 1‐phosphatase (LpxE) from Francisella novicida
Authors: Gillespie Robert A , Raetz Christian RH
Published: June 2021

The outer monolayer of the gram‐negative outer membrane is composed of the diglucosamine saccharolipid lipid A, the hydrophobic anchor of lipopolysaccharide. The lipid A of E. coli is derivatized with phosphate moieties at the 1‐ and 4′‐positions. Removal of the 1‐phosphate of lipid A results in low‐level polymyxin resistance and reduced activation of the human TLR4/MD‐2 innate immune receptor. Some gram‐negative species, including Francisella novicida, enzymatically remove the 1‐phosphate by LpxE, a lipid A 1‐phosphatase. To understand LpxE of Francisella novicida on a mechanistic level, the enzyme has been purified to homogeneity. A highly sensitive, in vitro phosphatase activity assay was developed. LpxE shows optimum in vitro activity at a pH of 5, consistent with the proposed mechanism of integral membrane lipid phosphate phosphatases. Alanine point mutants of the eleven conserved lipid phosphate phosphatase residues have been constructed. Four point mutants, R142A, S169A, R204A, and D214A are inactive, whereas H210A and H171A are 500‐ and 100,000‐fold less active than wild‐type, respectively. The effect of divalent cations and detergent concentration on activity has also been determined. Francisella LpxE activity is highly dependent upon the presence of two 3‐deoxy‐d‐manno‐oct‐2‐ulsonic acid residues in the 6’‐position of lipid A. This research was supported by NIH grant GM51796 to C.R.H. Raetz.
Evaluation of the binding interaction between LL‐37 and DNA fragments containing CpG motifs
Authors: Lao April , Gupta Angela , Dodson CarolAnn , Deeble Paul , Craig Maria Lee
Published: June 2021

LL‐37 is an antimicrobial peptide that has been implicated in autoimmune diseases. LL‐37 binds to DNA and delivers it across the cell membrane, and LL‐37‐mediated uptake by immune cells may lead to an anti‐self‐DNA response. We have examined the interaction between LL‐37 and DNA with a focus on DNA sequence and fragment size. In particular, we have studied the binding of LL‐37 to DNA of varying lengths that contain unmethylated CpG sequences known to activate TLR9 pathways. We have used gel shift assays to measure the affinity of LL‐37 for DNA fragments, observing sigmoidal binding behavior and obtaining binding constants on the order of 104‐105M−1. We also observe similar binding strengths for the CpG sequence and a GpC control and find that the binding strength decreases as DNA fragment length decreases from 400 to 100 bp. Our results are consistent with a model in which the binding of LL‐37 to DNA is both cooperative and sequence‐independent. Finally, we will test the effects of complexes between LL‐37 and CpG DNA on the invasiveness of prostate cancer cells, given conflicting studies identifying LL‐37 or CpG as anti‐tumorigenic in ovarian cancer cell lines but pro‐invasive in prostate cancer cell lines. This work was supported by NIH EARDA grant 5G11HD037064‐08 and a Virginia Foundation of Independent Colleges Summer Undergraduate Research Fellowship.
TAK1 is activated by two independent mechanisms in the regulation of epithelial cell survival
Authors: Takaki Emily Omori , Inagaki Maiko , Mishina Yuji , Matsumoto Kunihiro , Ninomiya‐Tsuji Jun
Published: June 2021

TAK1 (TGF‐β‐activated kinase 1) is a key signaling intermediate in cytokines such as IL‐1 signaling leading to cell survival. We have previously reported that skin and intestinal epithelium‐specific TAK1 knockout mice are lethal in early neonatal stage due to accumulation of reactive oxygen species and subsequent induction of cell death. TAK1 has three high affinity binding proteins, TAB1, TAB2 and TAB3. TAB3 is structurally very similar to TAB2 and both TAB2 and TAB3 activate TAK1 through recruiting TAK1 to the polyubiquitin chains. TAB1 shares no homology with TAB2/3 and functions to oligomerize and activate TAK1. Although TAB2/3 have been reported to mediate TAK1 activation in cytokine signaling, we found that TAK1 activation was intact in TAB2 and TAB3 double‐deficient cells in response to IL‐1. Unexpectedly, we discovered that TAK1 activation was abolished in TAB1 and TAB2 double‐deficient cells. Furthermore, epithelium‐specific TAB1 and TAB2 double but not TAB1 or TAB2 single knockout mice phenocopied epithelium‐specific TAK1 knockout mice. TAK1 kinase activity was abolished in TAB1 and TAB2 knockout in vivo epithelial tissues, while it was maintained in TAB1 or TAB2 single knockout epithelium. These results demonstrate that TAB1‐ and TAB2‐mediated mechanisms independently activate TAK1 in the in vivo epithelial cells, and one mechanism is sufficient for maintenance of TAK1 activity.

This work was supported by National Institutes of Health Grant GM068812 and GM084406 (to J. N‐T.)
Sumoylation and localization of Nrf2 to promyelocytic leukemia nuclear bodies are involved in its degradation in the nucleus
Authors: Malloy Melanie Theodore , Arinze Ifeanyi J.
Published: June 2021

Nrf2 (nuclear factor erythroid 2‐related factor 2) induces gene transcription by binding to the antioxidant response element(s) in target gene promoters. It is degraded primarily in the cytoplasm but can also be degraded in the nucleus. The mechanism of this nuclear degradation has not been elucidated. In HepG2 cells, we found that in the presence of the proteasome inhibitor MG‐132, GFP‐Nrf2 localized to promyelocytic leukemia nuclear bodies (PML‐NBs) in a sumoylation‐dependent manner. In vitro and in vivo sumoylation assays demonstrate that Nrf2 is sumoylated by SUMO‐1 and SUMO‐2/3. Short‐term treatment with As2O3 enhances polysumoylation by SUMO‐2/3 and targets Nrf2 to PML‐NBs. Given that polysumoylation has been shown to precede ubiquitylation and subsequent degradation of at least two proteins in PML‐NBs, we explored the possibility that this mechanism may be involved in the nuclear degradation of Nrf2. Desumoylation conditions stabilize Nrf2, and a sumoylation‐deficient mutant has a longer half‐life than the wild‐type. These results implicate sumoylation and trafficking of Nrf2 to PML‐NBs in the degradation of Nrf2 in the nucleus. Supported by NIH grants # SC1CA143985 and T32HL007737
Vitamin D2 enriched mushrooms significantly reduced inflammatory cytokine and chemokine expression in the spleens of LPS challenged rats
Authors: Babu Uma S. , Garthoff Larry H. , Balan Kannan V. , Calvo Mona S.
Published: June 2021

Vitamin D2 (VitD) from UVB‐exposed mushrooms (MR) has been shown to stimulate natural killer cell activity and modulate plasma cytokine response to lipopolysaccharide (LPS) challenge in rats. In this study, female rats were fed 1 of 5 diets for 10 weeks followed by challenge with pyrogen‐free saline or a low dose of LPS (100 μg/kg BW). All 5 experimental diets were based on AIN‐93G formulation with varying levels of VitD: 1) control levels of VitD; 2) no VitD; 3) no VitD + 5% unexposed MR; 4) no VitD + 2.5% UVB‐exposed MR, and 5) no VitD + 5% UVB‐exposed MR. Spleens were evaluated by microarray (Qiagen, Frederick, MD) for changes in expression of inflammatory cytokines and their receptors 3 h following LPS challenge. Several inflammatory chemokines, including Ccl2, 3, 4, 7 and 9, as well as Cxcl1, 2, 6, 9, 10 and 11 and cytokines, including interferon‐γ, IL‐1α and IL‐10 were significantly up‐regulated in spleens of LPS treated rats from all the dietary groups compared to those treated with saline. However, this up‐regulation was significantly reduced in the VitD enriched mushroom groups (2.5% & 5% UVB‐exposed MR) compared to those fed the control diet, no VitD‐control diet or 5% unexposed MR diet.

Conclusions

Microarray analysis of spleens confirmed earlier findings that rats fed UVB‐exposed MR diets with higher VitD experienced significant immune modulation with anti‐inflammatory effects.
Lipid modulators of PP2A activity are efficacious in models of neurodegenerative disorders
Authors: Stock Jeffry B
Published: June 2021

Hyperphosphorylation of tau in Alzheimer's Disease (AD) and α‐synuclein in Parkinson's Disease (PD) play a central role in disease progression and pathology. Use of kinase inhibitors to reverse these processes has been ineffective. Phosphatase activation is an alternative approach. The major neuronal protein phosphatase, PP2A, is regulated by reversible carboxyl methylation. Deficiencies in PP2A methylation have previously been linked to AD, providing a potential target mechanism for therapeutic intervention through inhibition of the PP2A demethylase, PME‐1. Lipid mediators have been identified that enhance PP2A methylation by inhibiting PME‐1 activity. The presence of these lipids in certain botanical extracts is consistent with epidemiological evidence of neuroprotective efficacy. Preliminary results indicate that in mice, oral administration of a naturally occurring serotonin derivative found in coffee, eicosenoyl‐5‐hydroxytryptamide (EHT), blocks PP2A demethylation and reduces P‐tau and P‐α‐syn in animal models for AD and PD. These data support the notion that dietary lipids that modulate PP2A may be useful for the prevention and treatment of neurodegenerative disorders.
Subfunctionalization of the calcineurin gene family in paramecium tetraurelia
Authors: Welker Jordan , Herring Kelsie , Van Houten Jason , Barrington William , Fraga Dean
Published: June 2021

Calcineurin is a serine/threonine protein phosphatase known to play a role in the motor function of cilia. A typical eukaryotic organism has between two and four copies of the gene for calcineurin, however Paramecium tetraurelia has 14 copies of this gene grouped into 7 isoforms or ohnologs. This multiplicity of the gene for calcineurin is suspected to be the result of whole genome duplication events during the course of evolution of Paramecium. We hypothesized that each isoform plays a different role in Paramecium rather than simply being redundant copies, as previously proposed. To test this hypothesis we utilized RNAi to systematically silence the 7 isoforms of the calcineurin catalytic subunit. The silenced cells were tested for abnormalities in various cilia mediated swimming behavior. We found that CaNA‐2, CaNA‐4, CaNA‐5, and CaNA‐6 decreased time spent swimming backwards. Previous work has shown that silencing of CaNA‐3 and CANA‐7 causes Paramecium to swim backwards for extended amounts of time, and that silencing of CaNA‐1 shows no phenotype in wild type. Forward swimming speed was also measured and found to show variations from wild type. We present our hypotheses that these duplicated genes have been maintained due to subfunctionalization processes and are not being retained strictly for redundancy.
G‐protein regulator LGN inhibits the activity of nitric oxide receptor soluble guanylyl cyclase
Authors: Chauhan Swati , Jelen Filip , Sharina Iraida , Martin Emil
Published: June 2021

Soluble guanylyl cyclase (sGC) is a heme‐containing enzyme which converts GTP to cGMP. SGC is referred to as nitric oxide (NO) receptor, as it is strongly stimulated by NO. Although the mechanism of sGC activation by NO received a lot of attention, the processes of sGC deactivation are less understood. In this report we demonstrate inhibition of sGC by a LGN‐dependent mechanism. LGN is a known regulator of Gα proteins. We identified LGN as a protein interacting with both α1 and β1 sGC subunits by yeast two‐hybrid screening and by co‐immunoprecipitation of sGC and LGN from BE2 human neuroblastoma cell line. Transient expression of LGN in MDA468 breast cancer cells markedly decreased sGC activity in a gene‐dose dependent fashion. When the effect of purified LGN protein on the activity of purified sGC was tested in vitro, we found no evidence of inhibition, suggesting that LGN does not directly affect sGC function. However, when the same experiment was performed in the lysate of COS7 cell, we observed a marked inhibition of sGC. These data suggest that LGN may function as an adaptor protein for additional cellular factors inhibiting sGC. Since LGN is known to regulate G‐proteins by association with Gα subunit, it may function as an adaptor protein for a complex consisting of sGC, LGN and Gα, thus connecting the NO‐dependent and G‐protein signaling pathways.
The expression, purification, and crystallization of the HNOX regulatory domain of bovine soluble guanylate cyclase
Authors: Taylor Leida , Garcin Elsa D
Published: June 2021

Soluble guanylate cyclase (sGC) converts GTP to cGMP, which plays a crucial role in vasodilation. The β subunit contains a heme NO/oxygen‐binding (HNOX) domain. NO binding to HNOX likely induces a conformational change leading to sGC activation. We aim to elucidate the mechanism of sGC activation using structural studies.

Our early results revealed that the β193‐HNOX construct is poorly expressed in E. coli. To solve this problem, we made 12 truncations (from residues 187 to 198) by site‐directed mutagenesis. Cells were transformed with all 12 mutant plasmids and expression conditions were tested. We found that 3 constructs were expressed at high levels, and that only 2 proteins were soluble. Ion exchange and size exclusion chromatography yielded large amounts of partially pure samples. The UV/Vis spectra of both proteins revealed that the heme pocket is intact and that NO is bound to the heme. We are now using immunoaffinity purification to improve the sample purity and attempt crystallization.

In summary, we used a systematic mutagenesis approach to obtain novel bovine HNOX sGC constructs with high levels of expression and solubility in E. coli. These constructs will be key to solve the first x‐ray structure of a mammalian HNOX domain that will provide a structural basis to understand the mechanism for NO activation.

Work supported by American Heart Association Scientist Development Grant (E. Garcin)
TESTS OF INTEGRIN TRANSMEMBRANE DOMAIN HOMO‐OLIGOMERIZATION DURING INTEGRIN LIGAND BINDING AND SIGNALING
Authors: Wang Wei , Zhu Jieqing , Springer Timothy A. , Luo Binghao
Published: June 2021

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside‐out and outside‐in signaling, the role of TM homomeric association remains elusive. Formation of TM homo‐oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo‐oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding, or during inside‐out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide‐bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo‐oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside‐in signaling. Therefore, integrin TM homo‐oligomerization is not required for integrin activation, ligand binding or signaling.
Investigation of NO‐mediated synthesis of c‐di‐GMP and its role in bacterial biofilm formation
Authors: Boon Elizabeth M
Published: June 2021

Bacteria colonize most surfaces, forming multicellular communities called biofilms. Biofilms are estimated to be responsible for up to 80% of human disease. The signaling molecule nitric oxide (NO) has been implicated in biofilms through an unknown pathway. In the genomes of many bacteria, an H‐NOX (Heme Nitric oxide/OXygen binding domain) is found next to a di‐guanylate cyclase (DGC) protein. H‐NOX domains define a new family of putative NO sensing proteins. The product of DGC proteins, cyclic di‐GMP, is a bacterial second messenger that is implicated in regulating the motility/biofilm growth switch in bacteria. We propose that H‐NOX responds to NO through the DGC, resulting in biofilm formation. We have characterized the DGC and determined the effect of NO/H‐NOX on cyclase activity. Additionally, we are detailing the effects of NO/H‐NOX on biofilm development via biofilm growth studies and genetic manipulations.
Small molecules abolish detrimental effects of apolipoprotein (apo) E4 in vitro by inhibiting domain interaction and correcting structure
Authors: Chen Hung‐Kai , Liu Zhaoping , Brodbeck Jens , Meyer‐Franke Anke , Ji Zhong‐Sheng , Miranda R. Dennis , Pleiss Michael , Hess J. Fred , McComas Casey , Freedman Stephen , Weisgraber Karl , Huang Yadong , Hargreaves Richard , Mahley Robert W.
Published: June 2021

ApoE4, the major risk factor for Alzheimer's disease (AD), assumes a pathological conformation—intramolecular domain interaction. ApoE structure correctors that abolish domain interaction were identified by fluorescence resonance energy transfer (FRET) assay. Screening a ChemBridge library identified CB9032258 (a phthalazinone derivative, N‐(2‐hydroxy‐3,5‐dimethyl‐phenyl)‐2‐(3‐methyl‐4‐oxo‐3,4‐dihydro‐phthalazin‐1‐yl)‐acetamide) that reduces the FRET signal dose‐dependently (IC50: 4.2 μM). Chemical modification of CB9032258 yielded well‐defined structure‐activity relationships (e.g., N‐(4′‐cyano‐biphenyl‐3‐yl)‐2‐(3‐methyl‐4‐oxo‐3,4‐dihydro‐phthalazin‐1‐yl)‐acetamide and 4‐{4‐[2‐(3‐methyl‐4‐oxo‐3,4‐dihydro‐phthalazin‐1‐yl)‐acetylamino]‐benzyl}‐piperazine‐1‐carboxylic acid tert‐butyl ester) with enhanced FRET potencies (IC50: 23 and 116 nM, respectively). ApoE4 domain interaction decreases mitochondrial cytochrome c oxidase subunit 1 levels, reduces mitochondrial motility, and inhibits neurite outgrowth. The compounds restored function in apoE4‐expressing neurons by disrupting domain interaction and reversing apoE4 detrimental effects. ApoE4 structure correctors negate detrimental apoE4 effects in vitro and could be an AD therapeutic. Supported by Merck Research Laboratories.
Hi Tmtc4 Interacts with C3G, Wntless, and Zfhx4: A Yeast Two‐Hybrid Trap for Proteins Associated with Temtamy Syndrome
Authors: Popli Tanav , Lee Jiang , Sherr Elliott
Published: June 2021

Agenesis of the corpus callosum (ACC), a failure of the corpus callosum to develop, is linked to over 50 congenital syndromes. One syndrome which exhibits ACC as a symptom is Temtamy Syndrome, which also presents with craniofacial abnormalities, ocular colobomata, and mild mental retardation. Studies of some of these cases have provided possible candidate genes which may play a role in the etiology of the disorder. Transmembrane tetratricopeptide protein 4 (TMTC4) and Ankyrin repeat and MYND domain containing protein 1 (ANKMY1) were both identified as candidate genes through chromosomal analysis of individuals affected with Temtamy Syndrome. A yeast two‐hybrid protein trap was performed for both of these protein products, and interaction partners for each were identified. Fourteen protein‐coding sequences were identified as interaction partners for Tmtc4, and five protein‐coding sequences were identified as partners for Ankmy1. Only three of the proteins interacting with Tmtc4 were linked to embryonic brain development, and none of the five Ankmy1 interactors have known roles in brain development. The three interacting proteins found were Zfhx4, which regulates neural differentiation, Wntless, a member of the Wnt family and integral Wnt regulator, and C3G, a guanine exchange factor in the Ras pathway which regulates neural precursor population and neuron migration in the cerebral cortex. Future work should confirm these interactions in brain tissue, as well as investigate the effects of the aberrant forms of Tmtc4 on these interactions. These findings could provide much insight into the etiology of Temtamy Syndrome, and ultimately bring us closer to understanding the mechanisms of ACC.
Genome‐wide expression profiling in capillary endothelial cells following Tunicamycin‐induced unfolded protein response
Authors: Banerjee Dipak K , Banerjee Aditi , Baksi Krishna
Published: June 2021

Angiogenesis is a hallmark of breast tumor growth and progression. Our objective has been to understand the dynamic relationship between asparagine‐linked (N‐linked) protein glycosylation and angiogenesis. We have used a protein N‐glycosylation inhibitor Tunicamycin and observed that it arrests cells in G1 and induces apoptosis by unfolded protein response mediated by transcriptional (ATF6) and translational (PERK) attenuation. The effect is stable under tumor microenvironment. Differential gene expression identified that out of 20,908 genes 133 and 464 genes are involved at 3h and 32h of Tunicamycin treatment sharing only 10 genes. 72.2% and 77.6% of these are upregulated whereas 27.8% and 22.41% are down regulated, respectively. 45 genes encode for transcription, 75% and 20% of which are upregulated whereas 15 genes encode cell cycle and 40% and 20% of which are upregulated at 3h and 32h, respectively. Only 10 genes encode for translation, 80% of which are upregulated at 32h. Interestingly, only one gene is upregulated at 32h for protein folding, DNA inhibition and angiogenesis. Real‐time PCR validated and confirmed the expression pattern of a number of these genes. To our knowledge, this first detailed study assessed the significance of genome‐wide expression profiling for glycotherapeutics treating breast cancer. Supported in part by Susan G. Komen for the Cure BCTR056206 (DKB) and NIH/NCRR/RCMI G12‐RR03035 (KB) grants..
Like Glucocorticoids (GCs), Mineralocorticoids (MCs) Inhibit Activation‐induced Apoptosis and Cytokine Production in Human Peripheral Blood T Lymphocytes
Authors: DONG LIHUA , Wang Shuibang , Tropea Margaret , Ferreyra Gabriela A. , Danner Robert
Published: June 2021

Both lymphocyte apoptosis and cytokine production, somewhat paradoxically, have been linked to death from sepsis. Corticosteroids improve hypotension in septic shock, but have inconsistent effects on survival possibly due to adverse effects. Although better known for inducing thymocyte apoptosis, GC receptor (GR) agonists inhibit activation‐induced T‐cell death via repression of CD95 ligand. Closely related to GR, MC receptor (MR) is highly expressed in T‐cells and therefore may regulate T‐cell function. Dexamethasone (DEX), a GR agonist, and deoxycorticosterone (DOC), a MR agonist, were studied in human T‐cells. Both DEX and DOC dose‐dependently inhibited PHA‐induced T‐cell apoptosis as measured by caspase 3 and 9 activity and annexin V with propidium iodide flow cytometry. Preliminary experiments using staphylococcal enterotoxin B (a superantigen) to stimulate T cells showed similar results. DEX and DOC suppressed FasL and IL‐2, but not Fas mRNA and protein. Moreover, DEX and DOC inhibited NFκB‐ and AP1‐driven reporter assays in PHA/PMA‐stimulated Jurkat cells, consistent with a nuclear receptor trans‐repression mechanism. MR, like GR can inhibit apoptosis and inflammatory responses in activated human T‐cells. MR agonists and antagonists may be useful for modulating T‐cell apoptosis and function in sepsis and other states of immune hyperactivity. Research Support: intramural NIH
Characterization of next‐generation anti‐androgens as potential prostate cancer therapeutics
Authors: Jobbagy Soma , Sikes Robert Allen , Koh John Tze‐tzun
Published: June 2021

Anti‐androgens are the gold standard of care for treatment of advanced prostate cancer (PCa). The purpose of this study was to characterize novel anti‐androgens PLM1, PLM6, and PAN52 designed and synthesized in the Koh laboratory. PLM6 and PAN52 but not PLM1 are thought to have unique mechanisms of action based on their ability to evade progression to anti‐androgen resistance in vitro. Western blot analysis of nuclear fractions showed correlation between prevention of AR nuclear translocation and progression of anti‐androgen resistance in prostate cancer cell lines. Some differences in localization effects were also observed between LNCaP and CWR22Rv1 PCa models that may reflect AR mutants present in these cell lines. The compound PLM6 and PAN52 found to down‐regulate the androgen receptor (AR) in PCa cell lines. These compounds thus appear to hold promise as therapy for advanced prostate cancer.

This work funded by the Beckman Foundation and the National Institutes of Health 5R01DK054257
EFFECT OF VISCUM ALBUM ON METOCLOPRAMIDE (MAXOLON) INDUCED HYPERPROLACTINEMIA AND HEPATOTOXICITY
Authors: Nwoke Stella C , Magbagbeola Olubunmi A , Ebuehi Albert O , Afolayan A T
Published: June 2021

The use of herbal concoctions for the management of infertility by Nigerian women is becoming a common trend. One of these herbal plants used is Viscum album. This research work studied the reversal effect of Viscum album on metoclopramide (maxolon) induced hyperprolactinemia and hepatotoxicity.

The level of Prolactin, Aspartate Aminotransferse (AST) and Alanine Aminotransferase ALT) activities were assayed in 25 female albino rats, which were divided into 5 equal groups. Group 1 control, while group 2 (negative control) treated with maxolon alone, group 3, 4 and 5 animals treated with maxolon, for 7 days, then treated with 62.5g, 125g and 250g of Viscum album extract respectively.

The result showed a significant increase (P< 0.05) in prolactin level (32.36ng/ml) in group 2 animals compared to the control group 1 (12.36ng/ml), while its level decreased in group 3, 4, 5 respectively (23.24ng/ml, 16.06ng/ml, 13.40 ng/ml).

The value of serum ALT of group 2 (25.00U/I) significantly increased (P < 0.05) as compared to group 1 (17.20U/I) while in group 3, 4, and 5, no significant increase in ALT was observed (20.40U/I, 20.00U/I, 21.40U/I). Activity of AST in group 2 animals (35.80U/I) was high as compared to the control group (27.60U/I) while in group 3, 4, and 5, the AST activity reduced.

In conclusion, the results show the overall effect of Viscum album extract was dose dependent, hepatoprotective and has hypoprolactinemia.
Vitamin D receptor and megalin gene polymorphisms and their associations with longitudinal cognitive change among US adults
Authors: Beydoun May A , Ding Eric L , Beydoun Hind A , Tanaka Toshiko , Ferrucci Luigi , Zonderman Alan B
Published: June 2021

We examined associations of single nucleotide polymorphisms (SNP) in the vitamin D receptor (VDR; CdX‐2, BsmI, ApaI, TaqI) and Megalin (rs2075252, rs4668123, rs3755166) regions with longitudinal annual rates of cognitive change (LARCC) using data from 702 Non‐Hispanic White participants in the Baltimore Longitudinal Study of Aging. LARCC were predicted with linear mixed models using all time points (Prediction I) or time points before dementia onset (Prediction II). In regression models of three SNP latent classes (SNPLC) per region, there were significant changes in cognitive decline on a number of tests, including Trails A (Prediction I) and B (Predictions I and II) in men when contrasting VDR2:CdX‐2[‐‐]/BsmI[AA]/ApaI[‐‐]/TaqI[GG] with VDR1:CdX‐2[‐‐]/BsmI[AG]/ApaI[‐‐]/TaqI[GA] SNPLC; and California verbal learning Task‐Delayed Recall (CVLT‐DR) in men (Predictions I and II) when comparing VDR3:CdX‐2[‐‐]/BsmI[GG]/ApaI[‐‐]/TaqI[AA] to VDR1. Megalin2:rs3755166[‐‐]/rs2075252[TT]/rs4668123[T‐](vs.Megalin1:rs3755166[‐‐]/rs2075252[CC]/rs4668123[‐‐]) was associated with accelerated decline on CVLT‐DR in men and overall (Prediction II) and CVLT‐List A in men (Prediction II). Variations in VDR and Megalin genes may influence cognitive decline among US adults in a sex‐specific fashion.
Manual Activities of Meal Preparation and Consumption Play a Significant Role in Musculoskeletal Nutrient Intake among Homebound Older Adults
Authors: Sharkey Joseph R
Published: June 2021

Purpose

Musculoskeletal nutrients (MN) – vitamin D, calcium, magnesium, and phosphorus – are associated with lower extremity physical function (balance, mobility, and strength) and with performance of basic tasks of daily living. Considering that manual, daily activities that involve meal preparation and consumption (AMPC) are important to older adults, especially those who are homebound, this study examined the influence of difficulties in nutrition‐related AMPC on usual intake of MN.

Methods

Self‐reported measures at baseline (demographic, psychosocial and health‐related factors, dietary intake of MN from 3 24‐hour dietary recalls, and seven nutrition‐related manual AMPC tasks) from a randomly recruited sample of 345 homebound older adults were examined. Ordered logistic regression was used to evaluate the relationship between level of difficulty in AMPC and dietary intakes <2/3 age‐ and gender‐specific Recommended Dietary Allowance/Adequate Intake (RDA/AI) in vitamin D, calcium, magnesium, and phosphorus.

Results

Almost 30% (n = 102) found 1–2 AMPC were very difficult/impossible; 13.9% (n = 48) reported ≥3 of 7 AMPC. Dietary intake <2/3 RDA/AI was reported by 92% for vitamin D, 72% calcium, 59% magnesium, and 3% phosphorus. Forty‐three percent reported <2/3 RDA/AI for 1–2 of the 4 MN; 52% for 3–4 MN. Independent of other factors, increased number of MN <2/3 RDA/AI was associated with gender, education, and difficulty ≥3 AMPC [odds ratio (OR) = 2.1, 95% confidence interval (CI) = 1.1–4.1].

Conclusions

This study suggests that manual activities of meal preparation and consumption may play a significant role in MN intake. As such, future interventions should focus on improving AMPC and the type of food containers used by older adults, which may also improve nutritional health and quality of life.
Effect of different nutritional formulas on postprandial glucose and inflammatory responses in healthy subjects and impaired fasting glucose (IFG)
Authors: Kwon Hye Young , Lee Eun Ju , Ryu Na Hee , Kim Ji Yeon , Kwon Oran
Published: June 2021

Evaluation of postprandial glycemic and inflammatory response to four nutritional feeds with different macronutrient and fiber composition. This study was designed to a double‐blind, placebo‐controlled, crossover trial. We measured glucose, insulin and several inflammatory maker, such as Free Fatty Acids, TNF‐α, sICAM‐1, NF‐κB and IL‐6. Plasma and mononuclear cells were collected at 0, 30, 60, 120, 180 min after the consumption of each products and placebo(glucose) in 8 healthy and 8 impaired fasting glucose(IFG) subjects. The fasting plasma glucose, insulin and inflammatory maker in IFG patients were higher than healthy subjects. In the postprandial challenge, the formula having high fiber contents significantly was low in peak glucose concentration (p>0.0001), IAUC, excursion, peak height (p<0.05) compared to glucose, whereas there were no differences in the mean insulin response. We noted significant increases in plasma IL‐6 concentrations at 180min after after the consumption of each products and placebo(glucose) in 8 healthy and 8 IFG subjects(p<0.05). Postprandial plasma TNF‐α, FFA, sICAM‐1 concentrations was not affected by food intake. Special nutritional formulas having high fiber improve glycemic response in healthy and IGF subjects. Plasma cytokine levels in IGF patients were higher than healthy subjects.
Effects of whole and refined grains on cardiometabolic risk factors: designing a controlled‐feeding study
Authors: Harris Kristina Arline , West Sheila , Heuvel Jack Vanden , Kris‐Etherton Penny
Published: June 2021

Dietary recommendations to reduce risk of major chronic diseases consistently include the substitution of whole grains for refined grains. However, the strength of evidence to support this is “moderate” due to reliance on epidemiological data and mixed results from free‐living clinical trials. Therefore, a 12‐week randomized, parallel‐arm, controlled‐feeding, weight‐loss study was designed to determine the biological effects of chronic whole and refined grain intake on cardiometabolic risk factors in participants with “pre‐metabolic syndrome (MetSyn).” Overweight or obese (BMI 25–42 kg/m2) men (n=25) and women (n=26) aged 35–55 with increased waist circumference and at least 1 other MetSyn characteristic were randomized to either a whole (WG) or refined grain (RG) diet. Weight was maintained for 6 weeks then weight loss was induced for 6 weeks via reduced‐calorie diet (~500 kcal/d). The macronutrient profiles of WG and RG diets were matched (~54% CHO, ~17% PRO, ~29% FAT, ~6.7% SFA) with the exception of fiber due to the whole grains (WG: 7 serv WG/d, 39 g fiber/d; RG: 0 WG serv/d, 22 g fiber/d in 2100 kcal/d diet). We hypothesize that a diet containing whole versus refined grains will elicit improvements in abdominal adiposity, inflammation, and endothelial function, which are clinical targets of cardiometabolic disease that present in MetSyn. This study is supported by General Mills.

Grant Funding Source: General Mills
The influence that the fractions reaches to the antiobesity key enzyme when partitioning the green pepper water extracts according to a solvent
Authors: Baek Jongmi , Kim Taewoo , Kim Jeongmi , Kim Taehyuk , Choe Myeon
Published: June 2021

We measured an effect by using the enzyme activity of the antiobesity key enzyme. Lipoprotein lipase (LPL) catalyses the hydrolysis of the triacylglycerol component of circulating chylomicrons and very low density lipoproteins, thereby providing non‐esterified fatty acids and 2‐monoacylglycerol for tissue utilisation. Also activation of fatty acids by acyl‐CoA synthetase(ACS) and carnitine acetyl transferase(CAT) enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. It is known that the capsaicin of the red pepper controls a fat already and there is an effect. But we result of partitioning the water extracts of the green pepper according to a solvent and evaluating that, the antiobesity effect was exposed to be shown in the fractions using the water. Therefore, the antiobesity effect of the green pepper is considered to be besides a capsaicin.

Thanks to financially support the Economy(MKE) through the fostering project of the Human Resource Training Project for Regional Innovation.
The effect of an emulsified fish oil preparation on cardiovascular risk factors in an overweight young adult population
Authors: Root Martin , McGinn Megan , Collier Scott , Zwetsloot Kevin
Published: June 2021

Background

Long chain omega‐3 fatty acids (O3) are known to have beneficial effects on a number of hemodynamic and vascular risk factors in at‐risk populations. The effects of a highly bioavailable emulsified preparation on a young adult population were less well known.

Methods

Young adults, age 18–30, with body mass indices (BMIs) greater than 23 (average = 28.1) were administered 1.7 g of fish oil O3 per day in an emulsified preparation (N=30) (Coromega, Inc.) or safflower placebo (N=27) for 4 weeks in a double‐blind randomized design. Blood was drawn and anthropometric measurements taken before and after the dosing period. Central pressures, arterial distensibility, inflammatory cytokines, fasting serum lipids, glucose, and C‐reactive protein were measured.

Result

None of the 5 American Heart Association metabolic syndrome measures improved over the dosing period. None of the hemodynamic measures (pulse wave velocity, augmentation index, or aortic systolic blood pressure), inflammatory cytokines (IL‐6, IL‐8, IL‐10, or TNF‐α), C‐reactive protein, or lipids (LDL, HDL, or triglycerides) improved over the dosing period.

Conclusions

These data suggest that O3 supplementation has no effect in this relatively healthy overweight cohort. This may be due to the relatively short supplementation time.
Pinto beans as part of a normal diet do not improve glycemic control in adults with type 2 diabetes
Authors: Hutchins Andrea , Winham Donna
Published: June 2021

This study examined the impact of daily consumption of ½ c pinto beans, compared to a control (½ c green beans), on changes in fasting glucose, postprandial glucose and HbA1c concentrations in men and women with type 2 diabetes (controlled by diet or metformin). Although some health professionals are promoting the benefits of dry beans for glucose control, confusion exists regarding the glycemic properties of dry beans in the context of a normal diet. After a baseline wash‐in period, 16 subjects were randomly assigned to 2 treatment periods [control period and pinto beans], each 12‐weeks in duration with a 4‐week wash‐out period. Eight subjects completed both treatment periods. During the wash‐in period and interventions, on days a diet record was kept (28 total days), subjects measured a postprandial capillary glucose concentration using a glucometer (provided by study) 1 hour after the meal during which the pinto beans or control were consumed. Before and after each intervention, subjects provided a fasted venous blood sample for glucose and HbA1c analyses. There were no statistically significant changes (p<0.05) in fasting glucose, HbA1c or average postprandial glucose values between the 2 interventions. The results of this study did not demonstrate improved glycemic control when pinto beans were incorporated into the normal diet of men and women with type 2 diabetes.

Research was supported by the US Dry Bean Council.
Test‐retest reliability of peripheral arterial tonometry (PAT): Implications for trial design in cardiovascular nutrition research
Authors: McCrea Cindy E , Skulas‐Ray Ann C. , Sauder Katherine A. , West Sheila G. , Kris‐Etherton Penny M.
Published: June 2021

Vascular endothelial dysfunction (ED) is an important step in the development of atherosclerosis and many foods and nutrients have been shown to improve ED. However, early measurement techniques were either invasive (requiring catheterization), or highly variable and operator dependent (e.g. FMD via ultrasound). Peripheral arterial tonometry (Endo‐PAT, Itamar, Israel) has been proposed as an alternative for assessing ED in clinical trials, particularly those with multiple sites. Few studies have assessed repeatability of PAT measures, and little published information is available to guide sample size calculations. In a sample of 20 healthy adults, we measured reactive hyperemia index (RHI, a purported measure of ED) during identical tests conducted 1 week apart. Participants consumed a standardized, low antioxidant diet for 48 hours prior. As the patient reclined, a blood pressure cuff was placed on the forearm and finger‐tip pulse amplitude was measured before, during, and after 5 min cuff inflation. Paired measurements were significantly correlated (r = 0.68). The coefficient of variation was 12.2 and the interclass correlation was 0.74. We confirmed that RHI scores can be reproducibly measured under controlled conditions. Our presentation will examine the meaning of these findings for statistical power and clinical study design.

The study was supported by NIH Grant M01 RR 10732.

Grant Funding Source: NIH Grant M01 RR 10732