bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Altered Non‐Canonical Wnt Signaling and Increased Tight Junction Remodeling in Cftr Knockout (KO) Mouse Small Intestine
Authors: Clarke Lane L. , Woode Rowena A. , Liu Jinghua L. , Walker Nancy M. , Strubberg Ashlee M.
Published: June 2021

Recent investigations have shown Cftr expression and its functional activity in the Lgr5+ intestinal stem cell (ISC) population of mice where it acts as a negative regulator of proliferation (Strubberg et al., CMGH 2017). In freshly‐isolated small intestinal crypts and crypts of cultured enteroids from Cftr KO mice, loss of Cftr activity results in increased Wnt/β‐catenin signaling and ISC hyperproliferation. A persistent alkaline intracellular pHi in Cftr KO ISC increases plasma membrane association of the Wnt transducer Disheveled (Dvl) thereby facilitating binding to the Wnt receptor Frizzled 7. Dvl also acts a transducer of non‐canonical Wnt signaling involved in directed cell migration and cell polarity, a necessary accompaniment to crypt proliferation. It is known that both cystic fibrosis (CF) patients and Cftr KO mice exhibit increased intestinal permeability. We hypothesized that increased pHi in progenitor cells of the crypt transit‐amplifying (TA) zone in Cftr KO mice increases plasma membrane Dvl association, potentially driving non‐canonical Wnt signaling for active tight junction (TJ) remodeling and increased permeability. Dvl2 association at the apical membrane was measured in TA cells of live WT and Cftr KO enteroids from mice expressing Dvl2‐EGFP (Dvl2 KO/Dvl2‐EGFP rescue) and membrane Tomato (RosamT/mG) using fluorescence confocal microscopy. Activity of the noncanonical Wnt receptor Frizzled 6 (Fz6) and the Rho GTPase Cdc42, an important regulator of intestinal polarity and migration, assessed TJ remodeling. Enteroid permeability was measured by FITC‐dextran (3 kD) diffusion into the lumen. Dvl2‐EGFP intensity at the apical membrane in TA zone crypt cells was increased ~2‐fold in Cftr KO crypts as compared to WT. Increased Dvl2‐EGFP apical membrane localization could be normalized in Cftr KO crypts by two days exposure to reduced medium pH (pH 6.6). Immunolocalization indicated the Fz6 was well expressed on the basolateral membranes near TJ of both WT and Cftr KO TA zone cells. Immunolocalization revealed increased Cdc42 activity at the TJ and immunoblots showed increased Cdc42 protein amounts in both fresh and enteroid crypts in Cftr KO intestine as compared to WT. Intestinal permeability was significantly increased in Cftr KO vs. WT enteroids. We conclude that cell alkalinity in the Cftr KO crypt epithelium affects Dvl transduction of both canonical and non‐canonical Wnt signaling thereby abnormally increasing proliferation and TJ remodeling. Increased TJ remodeling may contribute to increased intestinal permeability in CF patients.

Support or Funding Information

Supported by NIDDK 5R01DK048816 and the Cystic Fibrosis Foundation (CLARKE11G0 and LIU13Q0).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prorenin receptor regulates lipid metabolism via distinct actions on PPARγ and SREBP‐2 in liver of male mice
Authors: Gatineau Eva o. , Yiannikouris Frederique
Published: June 2021

Introduction and objectives

The epidemic of obesity is currently affecting more than 35% of the population in the United States. Non‐alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and metabolic syndrome. Our laboratory previously demonstrated that adipose prorenin receptor (PRR) KO mice developed lipodystrophy and liver steatosis associated with elevated hepatic triglycerides (TG) levels. Interestingly, hepatic PRR and PPARγ expression were up‐regulated in HF‐fed adipose PRR KO mice. Since body of evidence indicated that PPARγ becomes elevated in fatty livers and since we showed that PRR regulated PPARγ in adipose, we sought to investigate whether PRR regulated TG contents in liver through a PPARγ‐dependent mechanism.

Methods and results

PRR floxed male mice fed a standard diet were injected with a single dose of an adeno‐associated virus thyroxine‐binding globulin Cre (Liver PRR KO, n=5) or saline (Veh, n=6). Body weights were measured weekly for 3 weeks. The deletion of PRR in liver did not change body weight curves. Hepatic levels of TG were significantly decreased in liver PRR KO mice compared with control mice injected with saline (Veh, 53.22 ± 15.4 mg/g liver; AAV, 10.79 ± 1.61 mg/g liver, P<0.05). The deletion of PRR induced a significant decrease of PPARγ gene expression whereas SREBP‐1c expression was unchanged suggesting that PRR regulated TG contents through a PPARγ‐dependent mechanism. Surprisingly, liver weights were significantly higher in liver PRR KO mice compared to control mice injected with saline (Veh, 1.82 ± 0.12 g; Liver PRR KO, 2.37 ± 0.08g, P<0.05). Plasma cholesterol levels (Veh, 143 ± 10 mg/dL; Liver PRR KO, 383 ± 14g mg/dL, P<0.05) and hepatic total cholesterol levels (Veh, 3.64 ± 0.09 mg/g liver; Liver PRR KO, 10.47 ± 0.55 mg/g liver) were elevated in liver PRR KO mice. Interestingly, the increase in total cholesterol content in liver was associated with an increase in SREBP‐2 and HMGCoA‐reductase gene expression suggesting that the deletion of PRR stimulated cholesterol synthesis. Additionally, ABCG5 gene expression was increased, reflecting an increased cholesterol efflux.

Conclusion

Taken together our results demonstrated for the first time that PRR is an important modulator of lipid metabolism in liver. In agreement with our hypothesis, hepatic PRR positively regulated TG contents through a PPARγ‐dependent mechanism. Interestingly, the deletion of hepatic PRR stimulates cholesterol synthesis via SREBP‐2 up‐regulation indicating that PRR acts at the crossroad between triglycerides and cholesterol metabolism.

Support or Funding Information

COBRE ‐ P20 GM103527

American Heart Association ‐ 13SDG17230008

University of Kentucky, Center for Clinical and Translational Sciences, CCTS pilot grant, CCTS small grant, NIH T32HL091812

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative study of the intestinal microbiota in children from Mexican mothers with or without gestational diabetes
Authors: de Leon‐Solis Andrea Garcia , Torres‐Trueba Mariana , Palacios‐Gonzalez Berenice , Aguilar‐Reyes Jaqueline , de Leon‐Mendez Maria del Carmen Garcia , Lara‐Anaya Monica , Vadillo‐Ortega Felipe , Meraz‐Cruz Noemí
Published: June 2021

Pregnancy includes the development of an individual, from fertilization to labor. This development is influenced by genetic and environmental modifications during the intrauterine period, which continues until the first 1000 days of life. Important lapse in health and disease programming. The presence of gestational diabetes (GDM) favors the development of metabolic diseases in adult life and has an 18% prevalence in Mexico. The diversity of the intestinal microbiota is affected by maternal diabetes status, and this could contribute to the abundance of different phylas that contribute to the generation of new associated diseases. The aim of this study was to compare the fecal microbiota of children between 3 months and 3 years of age born to mothers with and without GDM. Anthropometric measures and fecal sample were collected from 29 patients (without GDM=24; GDM=5). The V4 region of bacterial 16S rRNA gene was sequenced by Illumina MiSeq. There were no significant differences between the birth weight of the infant and the weeks of gestation. Our data showed differences in composition of the microbiota in the study groups. We observed a decrease in Firmicutes, in addition with an increase in Bacteroidetes and Proteobacterias in GDM group. Also, LefSe analysis indicated a significant increment in Desulfovibrionaceae in this group compared with the control group. These results suggest the presence of dysbiosis in child from gestational diabetic mothers.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beta‐hydroxy‐beta‐methylbutyrate (HMB) augments muscle contraction‐induced protein synthesis via mTORC1 signaling in cultured L6 myotubes
Authors: Sato Satoko , Takamura Yusuke , Yamana Ikko , Nomura Mitsuru , Uchiyama Akira , Furuichi Yasuro , Manabe Yasuko , Fujii Nobuharu L.
Published: June 2021

Introduction

Beta‐hydroxy‐beta‐methylbutyrate (HMB), a metabolite of L‐leucine, has been shown to enhance resistance exercise‐induced protein synthesis in human and animal studies. Since there is no suitable contraction model using skeletal muscle cell line for analyzing protein synthesis level, the combined effects of HMB and muscle contraction on the protein synthesis is still unknown.

Objective

The present study aimed to establish a new muscle contraction model in L6 myotubes for analyzing protein synthesis. Using this model, we investigated the effects of HMB on the contraction‐induced protein synthesis via the mammalian target of rapamycin complex 1 (mTORC1) pathway.

Methods

L6 myotubes seeded into 4‐well rectangular plates were placed in contraction system equipped with carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 100 Hz for 10 min followed by the HMB treatment. Cells were collected at 3 hours poststimulation and evaluated mTORC1 signaling pathway activation by Western blot analysis.

Results

The phosphorylation level of Akt which is upstream components of mTORC1 pathway and the phosphorylation of 70‐kDa ribosomal protein S6 kinase (p70S6K) which is downstream components of this pathway were significantly increased 3 hours after electric stimulation (p<0.05). Furthermore, the phosphorylation level of p70S6K in contraction cells combined with HMB was significantly increased compared with that in contraction‐only cells (p<0.05).

Conclusion

We established a new muscle contraction model for analyzing protein synthesis in cultured L6 myotubes. Additionally, we found that HMB enhances muscle contraction‐activated mTORC1 signaling pathway using this model.

Support or Funding Information

This work was funded by Lion Corporation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiorespiratory Fitness Discriminates Sarcopenic (Abdominal)Obese from Sarcopenic Aging Subjects
Authors: Burini Roberto Carlos , Manda Rodrigo Minoru , Teixeira Okesley
Published: June 2021

The present study aimed to characterize sarcopenic obesity (SpOb) and its related morbidities in free‐living adults clinically selected for a lifestyle modification program(LiSM).

The study analyzed retrospectively baseline data from 523 individuals, of both genders that spontaneously joint the LiSM (Move for Health_Mexa‐se pro‐saude) program. The selected sample had data of medical anamneses, anthropometry, physical (and cardiorespiratory) fitness and blood biochemistry. Abdominal obese (Ob) definition, was defined by altered waist circumference (NCEP‐ATP III). Sarcopenia (Sp) was defined by the lower quartile of muscle‐mass index (kg muscle mass/m2 height=MMI). Statistical analyzes were performed by SAS software 9.2 (p <0.05).

The sample was 54.74 ± 10.18 years old, 72.7% females, 28.5% sarcopenic, 69.6% (abdominal) obese and 20.6% SpOb. Ob subjects presented higher BMI, % BF and MMI, along with higher values of triglycerides, blood glucose, HOMA‐IR, CRP, uric acid and reduced values of plasma HDL‐c, trunkal flexibility and VO2max. Sp was associated with lower values of handgrip strength, HDL‐c and higher HOMA‐IR. SpOb Individuals were older and presented lower muscle strength and aerobic fitness as well lower plasma HDL‐c and higher HOMA‐IR. After adjustments cardiorespiratory fitness was the only age‐dependent discriminant of (abdominal) obese sarcopenia.

Thus, The anthopometric diversion of SpOb keeps the pro‐inflammatory status (higher HOMA‐IR and lower HDL‐c) of its components (Ob and Sp) and has cardiorrespiratory fitness, as the sole aging‐dependent discriminant.

Support or Funding Information

Support CNPq and CAPES

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Shrinking the Mystery of Tsantsa Identification: Using Qualitative and Quantitative Measurements to Distinguish Natural and Imitation Shrunken Heads
Authors: Vinovskis Carissa , Oya Mayu , Royer Danielle
Published: June 2021

INTRODUCTION

During the 17th through mid‐20th centuries, the Jivaro tribes of South America participated in ritual head shrinking as part of a religious ceremony following battle. In the late 19th and early 20th centuries, travelers to South America took interest in these shrunken heads, known as tsantsas, and brought them back home as curios, inspiring the production of fake shrunken heads for the tourist trade. Numerous tsantsas, both real and fake, have ended up in museums or private collections worldwide. As many museums have very specific rules about repatriation of human remains, understanding differences between authentic and imitation tsantsas is imperative.

GOALS

The purpose of this study was to qualitatively and quantitatively describe the five tsantsa specimens held in the Anthropology Collection curated at the Denver Museum of Nature and Science (DMNS).

METHODS

First, each specimen was scored independently by two observers for 19 qualitative features typically associated with authentic tsantsas. Each observer also collected 29 standard facial measurements from four key locations (eye = 6, nose = 9, mouth = 4, ear = 10). Each facial measurement was size‐corrected using the geometric mean of all measurements for the specimen. The size‐corrected facial measurements were analyzed using principle component analysis (PCA).

RESULTS

Inter‐observer agreement on scoring of the qualitative features was high, and analysis of the facial measurements revealed no significant differences between raters. The qualitative features suggested the presence of two distinct groups within the DMNS sample: A Probably‐Authentic Group (three specimens) exhibiting 15 or more out of the 19 authentic characteristics, and a Probably‐Imitation Group (two specimens) exhibiting 8 or fewer of the 19 characteristics. PCA of all size‐corrected variables identified two factors accounting for 78% of the variance in the sample (factor 1 = 47%; factor 2 = 31%). The PCA revealed that the five specimens clustered within two distinct groups, distinguished primarily along factor 1. The group composition determined by PCA was concurrent with the two groups indicated by the qualitative traits.

CONCLUSIONS

Agreement between analysis of qualitative tsantsa characteristics and facial morphometric measurements suggests the presence of three authentic and two imitation tsantsas in the DMNS collection. To affirm these findings, microscopic hair analysis is also performed to establish the presence of human hair. However, DNA analysis is needed to determine with confidence whether the DMNS tsantsas are human or fabricated.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allometry of Felid Knee and Elbow Effective Mechanical Advantage
Authors: Harper Christine M. , Sylvester Adam D.
Published: June 2021

Body size has a profound impact on animal locomotion because all terrestrial animals must support and move their mass against gravity. If large animals were isometrically scaled versions of smaller animals, then larger animals would have relatively weaker muscles and bones, as both are a function of area. The loads associated with locomotion however, are largely equivalent to body mass, a function of volume. To contend with this problem, larger animals have been observed to use more extended limb postures; increasing the effective mechanical advantage (EMA) of their muscles. Felids however, maintain the same limb posture across body sizes, suggesting they are not increasing EMA through limb extension. To investigate how felids maintain the same posture across body sizes we analyzed the relationship between the EMA of the triceps brachii and quadriceps and body mass in nine species of felids with kinematic data from the literature.

Ulnae (n=37) and femora (n=30) of nine species of felids were surface scanned using a 3D NextEngine laser scanner. Linear measurements were taken on the tibia, patella, talus, calcaneus, navicular, third cuneiform and third metatarsal. Ulna and femur surface models were aligned in virtual space using anatomical and biomechanical axes. Triceps brachii EMA was calculated as the ratio of the muscle lever arm to the GRF lever arm, which were both collected on the ulna surface model. The quadriceps muscle and GRF lever arms were calculated using the femur surface models, linear measurements of limb and foot bones and kinematic data. Joint EMAs, muscle lever arms and GRF lever arms were regressed against body mass using PGLS regressions incorporating a phylogeny from 10kTrees. Pagel's λ was zero for all analyses, so data were reanalyzed using reduced major axis (RMA) regressions without phylogenetic information.

The triceps brachii and quadriceps EMA scale to body mass with slopes of 0.15 and 0.13, respectively, indicating positive allometry, meaning that larger felids have a relatively greater EMA and suggesting that some aspect of felid joint morphology is altered. For both joints neither the muscle lever arms nor the GRF lever arms are statistically different from isometry, however the muscle lever arms generally trend toward positive allometry, while the GRF lever arms lean toward negative allometry. This indicates that either there are subtle changes to both lever arms or that different felid species accomplish an increased EMA in distinct ways.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Variations in Nuclear Localization Strategies Among Pol X Family Enzymes
Authors: KIRBY THOMAS , PEDERSEN LARS C. , GABEL SCOTT A. , GASSMAN NATALIE R. , LONDON ROBERT E.
Published: June 2021

The pol X family enzymes DNA polymerase β (pol β), DNA polymerase λ (pol λ), DNA polymerase μ (pol μ), and terminal deoxynucleotidyl transferase (TdT) have critical roles in DNA repair. Nevertheless, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional NLS in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak in and out of the nucleus. Here we further explore the active nuclear transport of the three remaining human pol X enzymes by identifying and structurally characterizing their functional NLS sequences. We used X‐ray crystallography and fluorescence polarization binding assays to characterized the three pol X NLS sequences and their interactions with Importin α that lacks the Importin β binding domain (ImpαΔIBB). All three polymerases use NLS sequences located near their N‐terminus; the pol μ and TdT NLS sequences are monopartite, while pol λ utilizes a bipartite sequence, unique among the pol X family members. We confirmed the results by fluorescence‐based cellular localization studies, which also verified the bipartite nature of the pol λ NLS. This bipartite sequence, was somewhat unexpected, since NLS prediction programs either identified very low probability possibilities or completely failed to identify a potential NLS. Further validation of the identified NLS sequences was conducted by fluorescence polarization peptide‐binding to ImpαΔIBB, where TdT and pol λ demonstrated submicromolar dissociation constants, while pol μ had a low μM Kd value. Nuclear targeting is unique to each pol X family enzyme with variations in sequence context and affinity likely regulating the import and retention of these enzymes in the nuclear compartment.

Support or Funding Information

This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, project number ZIA ES050111 to REL.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and mechanism of DNMT3A‐mediated DNA methylation
Authors: Song Jikui , Zhang Zhi‐Min , Lu Rui , Wang Gang Greg
Published: June 2021

Mammalian DNA methylation is an important epigenetic mechanism crucial for gene silencing and imprinting, X‐inactivation, genome stability, and cell fate determination. It is established mainly at CpG dinucleotides by de novo methyltransferases DNMT3A and DNMT3B, and subsequently maintained by DNA methyltransferase 1 (DNMT1) in a replication‐dependent manner. Deregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. We determined the crystal structure of the DNMT3A – DNMT3L tetramer in complex with a DNA substrate containing two target sites separated by fourteen base pairs. Structural analysis of the DNMT3A – DNMT3L – DNA complex reveals that the two DNMT3A monomers simultaneously attack the two target sites, supporting the notion that DNMT3A homodimer can co‐methylate two adjacent CpG dinucleotides in one DNA‐binding event. Importantly, we identified that a residue from the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Mutational, enzymatic and cellular analyses further demonstrate that the hematological cancer‐associated somatic mutations of the substrate‐binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A‐mediated DNA methylation.

Support or Funding Information

Sidney Kimmel Foundation for Cancer Research;March of Dimes Foundation (1‐FY15‐345);National Institute of Health (1R35GM119721)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening and identification of four serum miRNAs as novel potential biomarkers for cured pulmonary tuberculosis
Authors: Wang Chong , Yang Su , Liu Chang‐Ming , Jiang Ting‐Ting , Chen Zhong‐Liang , Tu Hui‐Hui , Li Ji‐Cheng
Published: June 2021

Rapid and efficient methods for the determination of cured pulmonary tuberculosis (TB) are lacking. We screened serum miRNAs using the Solexa sequencing method among untreated TB patients, two‐month treated TB patients, cured TB patients, and healthy controls. A total of 100 differentially expressed miRNAs were identified in cured TB patients, including 37 up‐regulated (fold change >1.50, P<0.05) and 63 down‐regulated (fold change <0.60, P<0.05) miRNAs. Gene ontology (GO) enrichment analysis revealed that most of the predicted genes were present in the nucleus with a strong protein binding function. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis strongly suggested alterations in the metabolic pathways. Following quantitative real time chain reaction (qRT‐PCR), significantly reduced expression levels of miR‐21‐5p (0.59, P=0.039), miR‐92a‐3p (0.46, P=0.038), and miR‐148b‐3p (0.96, P=0.049) were found in the cured TB patients compared with the untreated TB patients. Also, significantly increased expression levels of miR‐92a‐3p (2.82, P<0.001), miR‐125a‐5p (5.09, P<0.001), and miR‐148b‐3p (4.25, P<0.001) were found in the untreated TB patients compared with the healthy controls. We established a cured TB model with 73.08% accuracy by four miRNAs (miR‐21‐5p, miR‐92a‐3p, miR‐148b‐3p, and miR‐125a‐5p), and also established a diagnostic model with 71.43% accuracy. Our study provides experimental data for establishing objective indicators of cured TB, and also provides a new experimental basis to understand the pathogenesis and prognosis of TB.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (No. 81573709), the Zhejiang Provincial Natural Science Foundation of China (No. LQ18H040009), and Zhejiang Provincial Medical Science and Technology Foundation of China (2017KY550).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenium Regulation of Selenoprotein Enzyme Activity and Transcripts in a Pilot Study with Founder Mouse Strains from the Collaborative Cross
Authors: Sunde Roger A.
Published: June 2021

Rodents and humans have 24–25 selenoproteins, and these proteins contain the 21st amino acid, selenocysteine, incorporated co‐translationally into the peptide backbone in a series of reactions dependent on at least 6 unique gene products. In selenium (Se) deficiency, there is differential regulation of selenoprotein expression, whereby levels of some selenoproteins and their transcripts decrease dramatically in Se deficiency, but other selenoprotein transcripts are spared this decrease; the underlying mechanism, however, is not fully understood. To begin explore the genetic basis for this variation in regulation by Se status in a pilot study, we fed Se‐deficient or Se‐adequate diets (0.005 or 0.2 μg Se/g, respectively) for eight weeks to the eight Founder strains of the Collaborative Cross. We found rather uniform expression of selenoenzyme activity for glutathione peroxidase (Gpx) 3 in plasma, Gpx1 in red blood cells, and Gpx1, Gpx4, and thioredoxin reductase in liver. In Founder mice, Se deficiency decreased each of these activities to a similar extent. Regulation of selenoprotein transcript expression by Se status was also globally retained intact, with dramatic down‐regulation of Gpx1, Selenow, and Selenoh transcripts in all 8 strains of Founder mice. These results indicate that differential regulation of selenoprotein expression by Se status is an essential aspect of Se metabolism and selenoprotein function. A few lone differences in Se regulation were observed for individual selenoproteins in this pilot study, but these differences did not single‐out one strain or one selenoprotein that consistently had unique Se regulation of selenoprotein expression. These differences should be affirmed in larger studies; use of the Diversity Outbred and Collaborative Cross strains may help to better define the functions of these selenoproteins.

Support or Funding Information

Supported by the Wisconsin Alumni Foundation Selenium Nutrition Research Fund (No. 12046295)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Cell Stress and 3′ End Alterations Control the Metabolism of a Cellular Non‐Coding RNA
Authors: Rivas Thomas , Kugel Jennifer F. , Goodrich James
Published: June 2021

RNA Polymerase II (Pol II) canonically acts as a DNA‐dependent RNA polymerase (DdRP), using double stranded DNA to synthesize protein‐encoding mRNAs and some non‐coding RNAs. Pol II also has RNA‐dependent RNA polymerase activity (RdRP), which uses an RNA as a template to synthesize RNA. An example of Pol II RdRP activity is the 3′ end extension of the non‐coding B2 RNA to generate extended B2 (eB2) RNA. B2 RNA binds to Pol II and is a substrate for a Pol II‐dependent 18 nucleotide RdRP‐catalyzed extension to form eB2 RNA. eB2 RNA can be detected in cells and has a reduced half‐life as compared to B2 RNA.

B2 RNA is transcribed by Pol III from Short Interspersed Elements (SINEs), which exist in over 350,000 copies in the mouse genome due to retrotransposition. Upon cellular stress, transcription of non‐coding B2 RNAs from B2 SINEs is greatly increased, thereby increasing the likelihood of retrotransposition of B2 SINEs. The RdRP activity of Pol II could control the levels of B2 RNA post‐transcriptionally by generating eB2 RNA to promote its degradation, thereby impacting the frequency of retrotransposition.

To study the effects of 3′ end extension and cellular stress on the lifecycle and metabolism of B2 RNA we are developing a heterologous expression system. Because the B2 SINE sequence is embedded in many mouse mRNA transcripts (e.g. UTRs and introns), it is impossible to perform hybridization and PCR‐based experiments to distinguish between a Pol III‐derived B2 RNA and those sequences within mRNA transcripts. To overcome this, we express B2 and eB2 RNA in human cells, which do not contain B2 SINEs, using a human Pol III promoter. The expressed RNAs are the correct size, have the correct 5′ ends, have the correct 3′ secondary structure, and exogenous eB2 has decreased stability compared to B2 RNA. Therefore, the heterologous system mimics characteristics of B2 RNA and eB2 RNA in mouse cells.

Using this system, ongoing experiments are investigating how extension alters the cellular localization of B2 RNA and retrotransposition efficiency in unstressed and stressed cells. Altogether, these experiments will determine the interplay between B2 RNA expression, Pol II RdRP extension, cellular localization, and cellular stress on the life cycle of this ncRNA.

Support or Funding Information

R01 GM68414 T32

GM008759

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enzymatic Site‐Specific Labeling of RNA for Affinity Isolation of RNA‐Protein Complexes
Authors: Busby Kayla N. , Devaraj Neal K.
Published: June 2021

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Building on our recently developed enzymatic technique, RNA‐TAG (Transglycosylation At Guanosine), we demonstrate the site‐specific labeling and affinity purification of RNA transcripts. This technology takes advantage of a bacterial tRNA guanine transglycosylase (TGT) for the incorporation of nucleobase derivatives bearing functional probes, such as biotin, into an encodable RNA stem loop recognition motif. Affinity purification enables high levels of enrichment of target RNA complexes and analysis of bound proteins. Using this methodology, we have demonstrated that an RNA of interest can be selectively biotinylated in live mammalian cells or cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient methodology for the elucidation of RNA function via affinity purification of cellular RNA‐protein complexes.

Support or Funding Information

This work is supported by the NIH under grant R01 GM123285. K.N.B. is supported by the Chemical Biology Interfaces Training Grant, NIH Grant T32 GM112584.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermal Stability of Type II Polyketide Acyl Carrier Proteins
Authors: Rivas Marco , Courouble Valentine ,   2016 Haverford College Biochemistry Superlab, Sisto Ashley , Beld Joris , Kokona Bashkim , Charkoudian Louise K.
Published: June 2021

Engineering biosynthetic pathways in microorganisms is a compelling route to gain access to novel molecules with drug‐like properties. Polyketide synthases (PKSs) utilize acyl carrier proteins (ACPs) to convert simple acetate building blocks into structurally complex molecules, many of which are antibiotics and anticancer agents. In a process known as combinatorial synthesis, biochemists have attempted to mix‐and‐match enzymes from different PKSs to create new hybrid synthases capable of synthesizing novel polyketide natural products. However, the ACP has proven to be a roadblock in these studies, as little is known about how the ACP functions and interacts with other enzymes due to its small and dynamic nature. To further our knowledge about the structure and function of ACPs, we cloned, expressed, and characterized a subset of 14 type II PKS ACPs. Temperature‐dependent circular dichroism (CD) experiments were conducted to determine the thermostability of the ACPs in the presence or absence of divalent cations. Our results indicate that despite strong sequence similarity, ACPs display a range in thermal stability. Further, the presence of metal ions confers additional stability to the ACP, presumably by decreasing electrostatic repulsive forces. Analytical ultracentrifugation‐sedimentation velocity experiments revealed that all ACPs sediment as monomers, and that despite sharing a common ancestor with fatty acid synthases, only one of the PKS ACPs studied interacted with the E. coli fatty acid ketosynthase, FabF.

Support or Funding Information

We would like to acknowledge an NSF CAREER Award #R15GM120704 and an NIH Award #CHE‐1652424 to Louise K. Charkoudian, as well as the Beckman Scholars Program, as sources of funding for this project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Site‐selective alterations within the hemolysin A non‐polar core
Authors: Grosskopf Julian David , Weaver Todd Michael , Grilley Daniel Patrick
Published: June 2021

The secretion of virulence factors often aids bacterial pathogenesis. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system. In fact, whooping cough, meningitis, and certain food‐borne illnesses have been attributed to TPS pathway containing gram‐negative bacterial species. Systematically, TpsA members are activated concomitant with Tps‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration. Collectively, these TpsA functions provide advantageous invasion and proliferation strategies within host cells. Structurally, TpsA members can be divided into a two‐partner secretion (TPS) domain and a functional domain. All TPS domains are constructed from a 300‐residue right‐handed, parallel β‐helix structure, and recognize their cognate TpsB partner. In order to further understand the relationship between TPS domains and TpsA structure and function, we have implemented a truncated form of hemolysin A (HpmA265), a TpsA member from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Specifically, these research investigations targeted valine 158 (V158) and phenylalanine 215 (F215) located within the first and last parallel β‐strands of the non‐polar core subdomain. A series of site‐selective variations were established at both V158 and F215. These variant forms of HpmA265 were characterized structurally via protease sensitivity and protein folding techniques, while functionality was ascertained within a template‐assisted hemolysis assay. Structurally, the V158 variants have destabilized the unfolding transitions associated with both the polar and non‐polar core subdomains, while leaving functionality unaffected. Site‐selective variants at F215 have selectively destabilized the non‐polar core subdomain, while leaving the unfolding transition attributed to the polar core subdomain unaffected. Additionally, the F215 variants do not affect template‐assisted hemolysis. Therefore, our results have been able to dissect the structural stability within the non‐polar core subdomain from template‐assisted function. These results have expanded the understanding for the implementation of TPS domains within gram‐negative bacteria.

Support or Funding Information

Funding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (JDG).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How and why internal cavities destabilize proteins
Authors: Mulder Frans AA. , Xue Mengjun , Yoshimura Yuichi , Kitahara Ryo
Published: June 2021

Proteins are marginally stable as a consequence of the large and opposing contributions from enthalpy and entropy to the folded state. The microscopic contributions to the total system ΔS and ΔH are still highly enigmatic. Here, we use hydrostatic pressure ‐– coupled to NMR spectroscopy detection – to explore the protein energy landscape in residue‐specific detail. As the conjugate of pressure is volume, this approach offers a mild and clearly defined perturbation of the system, as opposed to mutagenesis, temperature or denaturants. We could thus directly examine the Van der Waals contribution to stability. Pressure‐variation also allowed us to detect several partially unfolded states that consist of cooperative modules, a result that argues for a rather discrete route that connects the folded and unfolded forms of a protein.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LDLR Processing Dysregulation by Statin Treatment of Class II Transport Mutant Cells
Authors: Omer Linda , Boyd Nolan L.
Published: June 2021

The familial hypercholesterolemia (FH) Class II transport mutation is believed to be the most common defect in the low‐density lipoprotein receptor (LDLR). Class II mutations cause mis‐folding of the LDLR precursor preventing its transport from the ER for maturation in the Golgi and transport to the plasma membrane. The homozygous 3‐nucleotide deletion, c.654_656delTGG (FH‐Piscataway), in LDLR exon 4, caused a Class II mutation resulting in serum LDL over 800mg/dl in a 9yo female (GM03040, Coriell). Induced pluripotent stem cells (iPSC) derived from this patient's fibroblasts showed restored LDL‐receptor mediated endocytosis in homozygous CRISPR corrected clones. When statin treated, LDLR in non‐corrected FH‐iPSC (FH‐iPSC‐NC) persisted in the precursor (120kDa) form, while corrected cells (FH‐iPSC‐C) processed the LDLR to the mature form (160kDa). Quantification of total LDLR (immature + mature) showed 2.2‐fold more protein in NC cells. LDLR transcript quantification of statin treated NC, C and LDLR normal cells (H1 human embryonic stem cells, hESC) showed no difference, indicating LDLR accumulation is a dysregulation of post‐translational protein processing. This data and evidence in the literature led to our hypothesis that statin‐treatment of Class II LDLR mutant cells causes ER accumulation of mis‐folded LDLR and ER stress, leading to the unfolded protein response activation, which is reversed by LDLR correction. Since LDL cholesterol is primarily regulated by hepatocytes, we differentiated FH‐NC, C and H1 to hepatocyte‐like cells (HLC) using a modified version of Hay's protocol (PMID: 25081564) where stage 3 HLC were formed into spheroids of ~50/aggregate using AggreWell 400 plates. The HLC spheroids were treated with 5 mM Lovastatin or ethanol control in 5% lipoprotein‐deficient serum media overnight after which protein or RNA lysate was collected for Western blotting or qPCR, respectively. LDLR transcript levels were unchanged between groups, indicating that dysregulation is occurring at the post‐translational level in HLC. This work will provide new information as to whether statin treatment is implicated in inducing ER stress in Class II LDLR mutations and contribute to understanding the downstream effects and significance of statin treatment in patients with Class II LDLR FH.

Support or Funding Information

Jewish Heritage Fund for Excellence (NB)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Molecular Mechanism of Intermolecular Signal Transduction in Cystathionine‐β‐Synthase (CBS)
Authors: Chandrasekaran Sita S. , Ruiz Donovan George , Kennedy Dana Renae , Esquerra Raymond M.
Published: June 2021

Human cystathionine‐β‐synthase (hCBS) is a pyridoxal phosphate‐dependent (PLP) enzyme that contains heme and catalyzes the β‐replacement reactions of serine (L‐Ser) by homocysteine (L‐Hcy) to produce cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which leads to aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role that heme plays in regulating the catalytic process. My project seeks to develop a continuous, kinetic assay to measure CBS activity. Two approaches are being explored: 1) A coupled kinetic assay, in which the cystathionine produced by CBS is used by Cystathionine‐β‐lyase (hCBL) to generate pyruvate, and pyruvate production is measured phototrically; 2) A novel nuclear magnetic resonance assay, in which we will monitor the direct production of cystathionine over time. These methods will be compared to the standard, but discontinuous, radiometric assay. These assays will be used to understand the effect of small ligand binding to the heme and point mutations at the heme cofactor in relation to the enzyme activity. The long‐term goal of this work is to use time‐resolved absorption and magnetic circular dichroism spectroscopy to understand how small ligand binding to the heme induces conformational changes at the PLP active site in CBS and to measure activity of CBS with various mutated active site residues.

Support or Funding Information

Arnold and Mabel Beckman Foundation; NIH MARC: T34‐GM008574

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Menkes Syndrome on the Immune System
Authors: Engelhart Morgan , Zhang Jessica , Gotschall Jennifer , Huffman David , Miller Keith Russell
Published: June 2021

Menkes syndrome is an inherited disorder that affects copper levels in the body because of a mutation in the enzyme ATP7A. This protein is found in all cells throughout the body, except liver cells, and is essential for shuttling copper between two cellular locations. ATP7A can be found within the Golgi apparatus to supply copper to certain enzymes, but if copper levels are elevated in the cells environment, it can move to the cell membrane and shuttle copper out of the cell. When this protein is mutated, copper becomes poorly distributed throughout the cells of the body and can lead to reduced activity of many copper containing enzymes. This leads to brain abnormalities, bone weakness, degeneration of retina, and slowed development and growth. However, effects on the immune system and specifically macrophages are still unclear. These cells can become stimulated by foreign pathogens and secrete a cytokine, called Interferon‐gamma (IFN‐γ), which activates and recruits surrounding immune cells for an immune response. IFN‐γ stimulates production of reactive oxygen species (ROS) from metals within the cell to kill pathogens after phagocytosis. Due to its capability to aid in an immune response, it is hypothesized that IFN‐γ coupled with copper—a producer of reactive oxygen species—would activate macrophages and improve the cell's bactericidal effects. However, if ATP7A is mutated, and copper cannot be transported into the cell, its capacity to kill will decrease. Before experimentally determining the effects of IFN‐γ and copper on the immune system, the cytokine itself was synthesized. IFN‐γ encoding cDNA was amplified using PCR, and then annealed to the pET‐32‐Xa/LIC vector. The newly formed vector was successfully produced as determined by restriction endonuclease digestion, and then transformed into BL21‐DE3 E. coli. Induction of protein expression using IPTG was completed and visualized using SDS‐PAGE. IFN‐γ was isolated and purified using a HisTrap Nickel affinity column, and a SDS‐PAGE showed two bands, at 39 kDa (IFN‐γ) and 22 kDa (contaminated band). These results suggest a lack of purity due to an absence of protease inhibitors in solution, or low binding affinity to the nickel affinity column. Ongoing studies are observing macrophage phagocytosis and killing of bacteria in the presence and absence of IFN‐γ and copper to further understand the impact of Menkes syndrome on the immune system.

Support or Funding Information

Funded by The Giving Well Family Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beneficial Metabolic Consequences of Acyl‐CoA Synthetase ACSVL3 Knockout in Glioma Cells
Authors: Watkins Paul , Shi Xiaohai , Kolar Elizabeth , Clay Emily , Liu Yanqiu
Published: June 2021

We previously reported that knockdown or knockout (KO) of the fatty acid metabolic enzyme Very Long Chain Acyl‐CoA Synthetase 3 (ACSVL3; SLC27A3) in glioblastoma cells was beneficial. KO U87 glioma cells grew at a slower rate, became adherence‐dependent, and produced fewer, slower‐growing subcutaneous and intracranial tumors when implanted in NOD‐SCID mice. To understand the mechanisms underlying these changes, we investigated several possibilities. We initially demonstrated that lack of ACSVL3 did not slow the synthesis of membrane phospholipids necessary for the rapid proliferation of tumor cells in culture. We then investigated the possibility that ACSVL3 KO affected either apoptosis or autophagy. However, no differences in these processes between control and KO U87 cells were found. We next looked at cell cycle, and found that KO produced significant changes. In non‐synchronized cells, control U87 cells were mainly in G0/G1, while ACSVL3 KO cells were mainly in S‐phase. Both control and KO cells synchronized by serum starvation started out mainly in G0/G1, but by 12 hours KO cells shifted significantly toward S‐phase. Identification of the cyclins and cyclin‐dependent kinases that may be involved is in progress. Proteomic and metabolomic profiling indicated that in addition to effects on lipid metabolism, ACSVL3 KO produced changes in glucose and energy metabolism. Although glucose transporters GLUT1 and GLUT3 protein levels were reduced by KO, cellular uptake of labeled 2‐deoxyglucose was unaffected. However, oxidation of glucose to CO2 was reduced 40% by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Interestingly, lactate production and the levels of both LDHA and LDHB were elevated by ACSVL3 KO, and pentose phosphate pathway activity was lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria had a more punctate morphology in U87 cells, but were more elongated in KO cells. In conclusion, depletion of ACSVL3 affects many metabolic processes not directly related to lipid metabolism in human glioblastoma cells.

Support or Funding Information

Kennedy Krieger Research Administration funds

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