bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural Lignan Justicidin A‐Enhanced Mitophagy and Mitogenesis in Cytotoxicity of Grade III Human Bladder Cancer Cells
Authors: Su Chun‐Li , Chang Kai‐Hsun
Published: June 2021

Mitochondria are crucial organelles for energy metabolism, cell signaling, and apoptosis in mammalian cells. To maintain cellular homeostasis, the cell has evolved complex systems for the quality control of mitochondria. Justicidin A (JA) is a novel and structure‐defined arylnaphthalide lignan isolated from Justicia procumbens, which has been used as an herbal remedy in Taiwan and China for fever, cancer, and pain. Our previous reports show that JA suppressed the growth of human hepatocellular carcinoma Hep 3B and human colorectal cancer HT‐29 cells in NOD‐SCID mice, and the JA‐induced cytotoxicity was observed via induction of autophagy and mitochondrion‐related apoptosis. JA also preferentially inhibited the population growth of many other cancer cell types including grade III human bladder cancer T24 cells, compared to that of normal human peripheral blood mononuclear cells (PBMC). The 50% population growth inhibition of JA for PBMC was at least 1000‐fold higher than that for T24 cells. In the present study, we reveal that JA‐induced autophagy in T24 cells, characterized by increases in LC3‐II expression and acidic vesicular organelles formation, was paralleled by inhibition of mTOR, production of Atg5‐Atg12 complex, and activation of MEK/ERK. The JA‐triggered alternation of mitochondrial membrane potential, determined by staining with rhodamin123 followed by flow cytometry, in combined with the occurrence of autophagy implies the degradation of damaged mitochondria by a form of selective autophagy (mitophagy). Elevation of phospho‐Parkin and BNIP3 expressions by Western and co‐localization of BNIP3 and LC3 puncta by confocal microscopy indicate that the JA‐induced mitophagy proceeded via the Parkin‐ and BNIP3‐related pathways. Since mitophagy is balanced by mitochondrial biogenesis for generation of new mitochondria (mitogenesis), the increase in the intensity of Mito‐Tracker deep red and nonyl acridine orange as well as upregulation of mitochondrial UQCRC1 and COXIV protein expressions reveal the induction of mitogenesis in JA‐treated cells. Nevertheless, co‐administration of JA enhanced cytotoxicity of T24 cells to a combination of cisplatin and gemcitabine, the standard first‐line treatment for patients with locally advanced and metastatic bladder cancer, and produced a synergistic effect. Overall, our data indicate a possible role of mitophagy and mitogenesis in JA‐induced cytotoxicity of bladder cancer cells.

Support or Funding Information

Supported in part by the Ministry of Science and Technology, Taiwan, No. MOST 105‐2320‐B‐003‐003 and 106‐2320‐B‐003‐006‐MY3

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Secondary structure analysis of intramolecular DNA four‐way junction composed of phosphorothioate bonds
Authors: Troisi Maria , Parker Elliot , Klein Mitchell , Moorhead Gaston , Alvarado Alice , Herrada Hector , Ghebreigziabher Daniel , Shur Courtney , Bell Anthony Jerome
Published: June 2021

Research shows that cruciform and bent DNA can target the DNA‐binding cytokine, High Mobility Group B1 (HMGB1). HMGB1 was initially classified as a highly abundant architectural nuclear protein. It is now clear that HMGB1 also functions as a damage associated molecular pattern molecule (DAMP). And several studies link deleterious HMGB1 proinflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 garners a great deal of attention as a disease biomarker and therapeutic target. We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of 4WJs for ex vivo studies, the helical termini are modified to generate intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the intramolecular junction lattice to enhance nuclease resistance. In this study, the secondary structure of five intramolecular 4WJs are investigated vs. the DNA control, J1. J1 is an immobilized 4WJ that is composed of four asymmetric strands of DNA. Circular dichroism (CD) studies show that the secondary structure of each intramolecular 4WJ (DNA and PS constructs) is composed of B‐ and A‐form helices. Whereas, the DNA control J1 is devoid of A‐form helices. The melting temperature (Tm) of each intramolecular 4WJ is significantly higher than J1. The Tm values for the intramolecular 4WJs are 25° – 30°C higher than J1. Electrophoretic mobility shift assays show that HMG proteins bind intramolecular 4WJs with similar affinity to J1. The data clearly shows that the lattice of an immobilized DNA 4WJ can be redesigned to enhance conformational stability and maintain: i) native secondary structure and ii) HMG binding affinity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Yiqi‐Huoxue Granule ameliorates the apoptosis induced by hydrogen peroxide through modulating UCP2 in H9c2 cells
Authors: WU HONG
Published: June 2021

The conservation of Yiqi‐Huoxue Granule (YQHX) on patients with cardiovascular diseases is associated with the alleviation of oxidative stress‐mediated injury; however, the underlying mechanism(s) is unclear. Our purpose was to explore the protective effects of YQHX on apoptosis induced by hydrogen peroxide (H2O2) in H9c2 cells. H9c2 cells were pretreated with YQHX for 16 h before being exposed to 200 μM H2O2 for 6 h. The apoptosis induced by H2O2 was determined using hoechst 33342 staining and Annexin‐V FITC/PI method. The expression level of UCP2, Bcl‐2, Bax, and caspase‐3 were measured using Western blotting. The effects of UCP2 knockdown with Lenti‐shUCP2 vector on cell apoptosis and production of intracellular ROS were also explored. H2O2 stimulation resulted in prominent activation of oxidative stress followed by enhanced apoptosis and ROS production, as well as decreased UCP2 expression. YQHX at the concentration of 0.75 or 1.5 mg/ml significantly decreased the expression of Bax and caspase‐3, whereas increased the expression of Bcl‐2 and UCP2. The above effects were weakened by transgenic UCP2 knockdown. In short, the present study suggested that YQHX ameliorates the apoptosis induced by H2O2 through modulating the expression of UCP in H9c2 Cells, indicating that YQHX is a potential therapeutic medication for the treatment of I/R injury‐mediated apoptosis.

Support or Funding Information

The present work was supported by grants from the National Natural Science Foundation of China (No. 81473453, 81673800) and the Science and Technology Innovation Talents in Universities of Henan Province (No. 14HASTIT028).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wenyang‐Yiqi Granule inhibits OGD‐induced autophagy by activating mTOR pathway
Authors: Yu Xiaofang , Wu Hong , Han Lihua , Wang Zhentao
Published: June 2021

Our aim is to explore the effects of Wenyang‐Yiqi Granule (WYYQ) on autophagy induced by oxygen glucose deprivation (OGD) in H9c2 cells. The cultured H9c2 cells were divided into control group, OGD group, Rapamycin group, WYYQ group, and WYYQ plus Rapamycin group. The cells in each treatment group were exposed to OGD for 6 h. The mRNA expression of Beclin‐1 and microtubule‐associated protein light chain 3 (LC3) was assessed using real‐time PCR. The expression of Beclin‐1, p62, LC3‐II/LC3‐I ratio, and the phosphorylation of mTOR pathway were detected using Western bolt. The apoptosis was measured using flow cytometry. The results showed that the expression of Beclin‐1 and LC3 increased whereas the expression of P62 decreased in OGD‐treated H9c2 cells. WYYQ significantly inhibited the expression of Beclin‐1 and LC3 and increased the expression of P62 at mRNA and protein level. The phosphorylation of mTOR pathway was suppressed in OGD condition, and WYYQ enhanced the phosphorylation of mTOR pathway. Rapamycin, an mTOR inhibitor, abrogated the upregulation of mTOR phosphorylation by WYYQ, however, it did not abolish the downregulation of autophagy‐related genes and proteins by WYYQ under OGD condition. Taken together, WYYQ ameliorates OGD‐induced autophagy, relating to its activation of mTOR signaling pathway.

Support or Funding Information

This work was financially supported by grants from the National Natural Science Foundation of China (No. 81373610).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of 31P NMR and ITC to Study the Binding of ATP, ADP, and AMP with Metal Ions
Authors: Zea Corbin , Donels Jordan , Greenwood Brandon , Akers Isabell , Thomsen Alli , Limani Arijan , Kanne Dillon
Published: June 2021

Magnesium is known to be an important cofactor in many enzymatic processes involving adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). The binding of magnesium to the phosphate end of the nucleotide phosphate is well known and crucial to the catalytic activity of many enzymatic reactions. There is high selectivity for magnesium and other metals are not found to participate in the binding and enzymatic catalysis of nucleotide phosphates. We aim to study the binding of various metals in addition to magnesium to see if it can be determined why other metals are not as crucial to enzymatic binding. To answer this question we assessed metal ion binding to ATP, ADP, and AMP in pH 6.9 and 8.0 HEPES buffer using 31P Nuclear Magnetic Resonance (NMR) as well as Isothermal Titration Calorimetry (ITC). These measurements will correlate the conformational changes seen in the 31P NMR to the binding energy measured in ITC. With magnesium we see increasing conformational changes in the 31P NMR that correlate with a large binding energy seen in ITC. With other metal ions we do see a conformational change in the NMR but the binding energy is not as large as that found for magnesium.

Support or Funding Information

Instrumentation support was provided by the Carver Foundation and the R.W. and Mary Nelson Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kangxian‐Yixin shortens the duration of paroxysmal atrial fibrillation in rats
Authors: Dong Lisha , Wang Zhentao , Chang Hongbo , Wu Hong
Published: June 2021

Our aim is to observe the effects of Kangxian‐Yixin concentrated pill (KXYX) on the duration of atrial fibrillation in rats with paroxysmal atrial fibrillation. Rat models of paroxysmal atrial fibrillation were induced by tail vein injection of acetylcholine as well as calcium chloride. Fifty male Wistar rats were randomly divided into model group, propranolol group and KXYX groups in high, medium and low dose. The model group was administered of saline in equal volume, while the other groups were given the corresponding dose of medicines one time a day for 1 week. After 7 days, the rat model of paroxysmal atrial fibrillation was generated and used to record the duration of atrial fibrillation. Compared with the model group, the duration of atrial fibrillation in treatment groups were significantly shortened (P<0.01 or P<0.05). And compared with the propranolol group, the duration in the medium‐dose and low‐dose KXYX groups were markedly longer (P<0.01 or P<0.05). The duration in the high and medium‐dose groups were shorter than that in the low‐dose KXYX group (P<0.01 or P<0.05). Taken together, KXYX shortens the duration of paroxysmal atrial fibrillation, suggesting that it may be a promising therapeutic medication for intervention of paroxysmal atrial fibrillation.

Support or Funding Information

This work was supported by grant from the National Natural Science Foundation of China (No. 81573920).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Specific Photocleavage Activity of N‐Methylmesoporphyrin IX on G‐Quadruplex DNA Structures
Authors: Escobedo Ruby A , Long Kimberly J , McBrayer Dominic N , Schoonover Michelle , Kerwin Sean M
Published: June 2021

G‐rich regions, at which G‐quadruplexes (G4) are proposed to form, are associated with promoter regions within proto‐oncogenes and may play a role in cell proliferation and carcinogenesis. The ability to identify the formation of G4 structures in these regions, including the temporal aspects of G‐quadruplex formation, is essential to establish the role of these structures in transcriptional regulation. We have focused on identifying highly selective G4 photocleavage agents as molecular probes for establishing the location and timing of G4 formation. N‐methylmesoporphyrin IX (NMM) has long been known as a G4 ligand; however, there has been little investigation into its DNA photocleavage activity. Here we report the ability of NMM to act as a highly selective photocleavage agent for specific G4 structures. We first studied the interaction of NMM with the G4 structures formed by DNA oligonucleotides containing the c‐MYC promoter or human telomeric (hTel) sequences formed in the presence of either Na+ or K+‐containing buffers using a FRET‐based Tm assay and CD spectroscopy. We employed a fluorescence‐based solution assay for G4 cleavage to establish that NMM effects photocleavage (420 nm) of c‐MYC more efficiently than the hTel G4 under both Na+ and K+ conditions, and effects photocleavage of c‐MYC G4 even under white light irradiation. Using denaturing polyacrylamide gel electrophoresis (PAGE), we confirmed this selective photocleavage and demonstrate that cleavage occurs at specific guanine residues of the c‐MYC G4. In order to determine if the photocleavage proceeds through the generation of reactive oxygen species like singlet oxygen, we carried out photocleavage reactions in the presence of H2O and D2O, and employed a turn‐on fluorescent singlet oxygen sensor to detect 1O2 generation. We find that NMM acts to destabilize specific G4 topologies, but oligonucleotides annealed in the presence of NMM form alternative topologies that are thermally stabilized. NMM photocleavage is highly selective for G4 DNA versus duplex DNA, and the K+‐stabilized c‐MYC G4 is preferentially cleaved compared to the Na+‐stabilized structure and the hTel G4 in either Na+ or K+. PAGE analysis indicates that NMM photocleavage of c‐MYC G4 occurs preferentially at the 3′ G‐tetrad residues. This photocleavage does not appear to be due to 1O2. These results indicate that NMM may be a useful tool for elucidating the role of promoter G‐quadruplex structures.

Support or Funding Information

Support for this work by the Cancer Prevention and Research Institute of Texas (RP160852) and Texas State University is gratefully acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer Effect of Curcumin‐Silibinin B Combination in Colorectal Cancer Cells is Mediated by Apoptosis and Autophagy
Authors: Horita Megan Ann , Reed Megan , Ganapathy Aravinda , Ezekiel Uthayashanker
Published: June 2021

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in men and women in the US. Epidemiological studies have shown that a diet high in fruits, vegetables, and spices is strongly associated with a reduced risk of developing cancer. Dietary cancer protection may be attributable to synergy between low dose phytochemicals present in the diet. We have found that curcumin, the major component of turmeric, and silibinin B, the major component of milk thistle, exert synergistic anticancer activity in CRC. When both compounds are used to treat the CRC cell line, more cells undergo cell death than when the compounds are used independently. We found that the combination of curcumin plus silibinin B mediated cell death by both apoptosis and autophagy.

Support or Funding Information

MH, MR, and AG are supported by DeNardo Education and Research Foundation Fellowship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design of antibody‐blocking monobodies: A novel therapeutic approach for autoimmune kidney disease
Authors: DIPAK GALANI Aryan , Stoddard Shana Victoria
Published: June 2021

Autoimmune diseases arise when antibodies erroneously attack healthy cells. Primary membranous nephropathy (PMN) is a kidney specific autoimmune disease affecting 10–12 million individuals worldwide. Currently non‐specific immunosuppressants which weaken the body's entire immune system are used to treat many autoimmune disorders. These non‐specific treatments decrease the patient's ability to fend off basic infections. Thus, there is a need for more targeted approaches to autoimmune diseases therapies. Therefore the goal of this project is the development of an antigen specific approach for autoimmune disease, through the design of protein monobodies capable of preventing autoantibody binding to the antigen. The PMN antigen, thrombospondin type‐1 domain‐containing 7a (THSD7A), was used as a target for cap development. Epitope sites were identified using an in silico homology model for the THSD7A antigen. The work here focuses on targeting two of the 17 extracellular domains which were identified to contain epitope sites, (specifically domains 9 and 10). In silico mutagenesis to the 3QHT monobody template was performed using Chimera and Phyre2 programs. Interface scores measured in Rosetta energy units (REU) were improved from −3.129 (3QHT template) to −3.927 in the 3QHT‐015‐AG cap. Data shows that introduction of hydrophobic residues enhanced binding. Introduction of a Phe residue, creating the 3QHT‐023‐AG mutant, permitted a cation‐pi interaction to domain 9 of THSD7A resulting in an increased binding score of −6.617 REU. Currently protein expression and purification is underway to allow for ELISA assays and further evaluation of these epitope caps binding to domain 9 of the THSD7A antigen. Through the development of antigen specific blocking monobodies, successful caps could provide a much safer and accurate treatment for autoimmune diseases.

Support or Funding Information

Faculty Development Endowment Grant, Rhodes College

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of the Human SULT1A1 Polymorphisms on the Sulfation of Acetaminophen, O‐Desmethylnaproxen, and Tapentadol
Authors: Rasool Mohammed , Liu Ming‐Cheh
Published: June 2021

Background

Non‐opioid and opioid analgesics, as over‐the‐counter or prescribed medications, are widely used for the management of a diverse array of pathophysiological conditions. Previous studies have demonstrated the involvement of human cytosolic sulfotransferase (SULT) SULT1A1 in the sulfation of acetaminophen, O‐desmethylnaproxen (O‐DMN), and tapentadol. The current study was designed to investigate the impact of single nucleotide polymorphisms (SNPs) of the human SULT1A1 gene on the sulfation of these analgesic compounds by SULT1A1 allozymes.

Methods

Human SULT1A1 genotypes were identified by database search. cDNAs corresponding to nine SULT1A1 nonsynonymous missense coding SNPs (cSNPs) were generated by site‐directed mutagenesis. Recombinant wild‐type and SULT1A1 allozymes were bacterially expressed and affinity‐purified. Purified SULT1A1 allozymes were analyzed for sulfating activity using an established assay procedure.

Results

Compared with the wild‐type enzyme, SULT1A1 allozymes were shown to display differential sulfating activities toward three analgesic compounds, acetaminophen, O‐desmethylnaproxen (O‐DMN), and tapentadol, as well as the prototype substrate 4NP.

Conclusion

Results obtained indicated clearly the impact of genetic polymorphisms on the drug‐sulfating activity of SULT1A1 allozymes. Such information may contribute to a better understanding of the differential metabolism of acetaminophen, O‐DMN, and tapentadol in individuals with different SULT1A1 genotypes.

Support or Funding Information

This work was supported in part by a grant from National Institutes of Health (Grant #R03HD071146).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacterial Community Composition and Environmental Factors in a Hypereutrophic Watershed
Authors: Cooper Carolyn , Belica Christopher , Pearch Abby , Schumann Eric , Brokus Sarah , Moen Francesco , Wade Randall , Best Aaron , Krueger Brent P , Pikaart Michael
Published: June 2021

Lake Macatawa is a drowned river mouth of Lake Michigan impacted by agricultural activity and urbanization. These impacts include high sediment, nutrient and fecal coliform loading. We are monitoring this watershed in order to aid decisions about remediations and public access to these recreational waters. Weekly samples over the course of a one‐year period from twelve representative lake and stream sites were analyzed for microbiological content by community 16S rRNA sequencing, fecal indicator bacteria presence, and Escherichia coli whole‐genome sequencing. Along with biological characterization, we have also profiled chemical and physical parameters including dissolved oxygen, nutrients, temperature, and pH. Results indicate continued high phosphorus, total suspended solids, and E. coli levels, exceeding total maximum daily load targets, or other benchmarks, for the watershed. Patterns in microbial communities show variation influenced by season and geographic location; for example, diversity of microbial communities increases in late fall and winter. Identification of microbial taxa acting as signatures of environmental conditions could lead to new methods of monitoring water quality. These data provide a baseline for monitoring remediation efforts in the Macatawa Watershed and serve as a comparison for hypereutrophic watersheds around the nation.

Support or Funding Information

NSF RUI Award 1616737

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Search for the Genetic Correlation between Coat Pigmentation, Blue Eyes and Cochleosaccular Deafness in a Pedigree of Spanish Mustangs
Authors: Erkey Celine , Lipsett Koren A
Published: June 2021

The correlation between loss of pigmentation, blue eyes and congenital deafness has been studied in various mammalian species. One pedigree of Spanish Mustangs has displayed this correlation, having two offspring from the same sire exhibit loss of pigmentation and congenital deafness. Previously, candidate gene studies were conducted to determine the link between white coat pigmentation, blue eyes and cochleosaccular deafness solely on exonic space. We examined candidate genes previously studied in horses, cats and dogs. KIT, a receptor tyrosine kinase in the pigmentation pathway, has shown to be the principle gene found to link deafness and loss of pigmentation in multiple mammalian species. A recessive mutation in 5′ splice junction in exon 17 has suggested a correlation to the loss of pigmentation but has not been linked to deafness. Similarly, a feline endogenous retrovirus 1 interruption in Exon 1 of KIT has been linked to affect melanocyte cells in cats with the same phenotype as our pedigree.

To expand into the intronic regions, whole genome sequencing was conducted on an offspring with the loss of pigmentation, blue eyes and congenital deafness. Due to the size of the horse genome, megadata analytics were required for quantitative bioinformatic analysis. In partnering with Amazon Web Services (AWS) and Illumina, the sequence data was processed and analyzed to identify specific single nucleotide polymorphisms (SNP), insertions and deletions within the entire genome. Specific parameters were designed using Linux systems in order to output the correct data for this particular research. We report here novel sequence variations of the original target genes within our pedigree genome.

Support or Funding Information

Provost's Senior Research Fund, X‐SIG Fund, & Albaugh Fund, Gettysburg College, Gettysburg, PA 17325

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrative Genomic Analysis of Ribosomal DNA in the Mouse Oocyte and Early Embryo
Authors: Garner Tyler B , Diaz Francisco J
Published: June 2021

Ribosomal RNAs compose the majority of the total transcripts present in cells and their expression is tightly controlled through changes in epigenetic modifications, chromatin structure, and DNA accessibility. These mechanisms conserve translational capacity for homeostatic maintenance and survival or stimulate ribosome biogenesis for growth and proliferation, such as in the maturing oocyte where gene expression is virtually arrested until after fertilization when transcription is upregulated to promote embryonic development. Next‐generation sequencing technologies have vastly improved our understanding of gene regulation during the egg to embryo transition, however most studies thus far have neglected rDNA. Here, we have integrated genomic analyses of histone modifications (ChIP‐seq), DNA methylation (RBBS‐seq), chromatin accessibility (ATAC‐seq and DNase‐seq), and transcriptome profiling (RNA‐seq) in oocytes and pre‐implantation embryos to the mouse rDNA repeat. We observed both low chromatin accessibility and enrichment of the active transcription markers H3K4me3 and H3K27ac in oocytes which increase significantly at the 2‐cell and 8‐cell stages, particularly along specific loci within the intergenic spacer (IGS). Conversely, repressive histone patterns associated with heterochromatin and Polycomb silencing (H3K9me3 and H3K27me3 respectively) were reduced from the oocyte to zygote and nearly abolished by the 8‐cell stage. Consistent with these findings, RNA‐seq coverage along the 18S, 5.8S, and 28S coding regions increased throughout development in addition to the appearance of signals in the external and internal transcribed spacers at the 2‐cell and 8‐cell stages, indicative of active rRNA transcription. Interestingly, uniquely‐aligned transcripts were detected at distinct loci along the IGS and promoter regions in 2‐cell and 8‐cell embryos that were absent in oocytes and zygotes. The significance of these stage‐specific, non‐coding transcripts in embryogenesis will be a focus of further investigations. Together, these analyses detail the rDNA genomic landscape from the egg to early embryo and present novel avenues for exploration of ribosomal regulation during this critical period of development.

Support or Funding Information

NIHT32GM108563‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cloning of Cia7 Gene and Generation of Transgenic Mutants of Chlamydomonas Reinhardtii
Authors: Vazquez Cano Erick U. , Ynalvez Ruby A.
Published: June 2021

Chlamydomonas reinhardtii is a green haploid microalga that stands as an exemplary model organism in the study of heavy metal tolerance and homeostasis of photosynthetic organisms. Within C. reinhardtii's genome resides Cia7, a novel gene that encodes a protein (CIA7) of 104 amino acids with a conserved sequence of four cysteine residues. Given that thiol moieties are essential to metal‐binding proteins, it has been hypothesized that CIA7 might play a role in heavy metal homeostasis of C. reinhardtii. In the path of elucidating the function of the gene, previous studies have focused on exposing C. reinhardtii cells to heavy metal stress and metal bioaccumulation studies. This research study, on the other hand, aimed to isolate and overexpress the CIA7 protein in Escherichia coliand in C. reinhardtii. Polymerase chain reaction (PCR) was used to amplify Cia7 from C. reinhardtii's cDNA library. The PCR product was purified and ligated to the maltose‐binding protein expression vector pMAL‐c5x. The recombinant DNA with the Cia7 insert was used to transform E. coli. The presence of the insert was verified by restriction enzyme digests. In this study, we report the protein overexpression of CIA7. The overexpressed protein will be used for the in vitro studies of the novel protein, CIA7. In addition, a recombinant DNA generated by using the psL72 vector and the Cia7 insert was transformed into C. reinhardtii cells (wild type, cc4425 and mutant of Cia7, cc5013). The obtained transgenic algae will be used in the future for metal bioaccumulation analysis. Overall, this study will provide the clones and transgenic algae necessary to characterize the function of the gene and to determine whether Cia7 confers heavy metal resistance to C. reinhardtii.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Role of NHE1 Palmitoylation in the Regulation of Cell Proliferation and Migration
Authors: Hanowski Stephanie A. , Provost Joseph J. , Wallert Mark A.
Published: June 2021

The Na+‐ H+ Exchanger Isoform I (NHE1) is a key regulator of cell proliferation and migration in a range of cell types. Activation of NHE1 is an initial step in the development of the transformed phenotypes of cancer cells and therefore a key driver for tumor development and cancer progression. NHE1 also contributes to two of the hallmarks of cancer, the activation of invasion and metastasis and sustained proliferative growth. We have identified for the first time, that NHE1 undergoes reversible palmitoylation, the addition of a palmitic acid residue to the cytoplasmic regulatory domain of NHE1. This suggests that targeting the potential palmitoylation sites on NHE1 may provide a therapeutic target for cancer treatment. This research evaluates the role of NHE1 palmitoylation in two cell lines: 1) Chinese hamster lung fibroblasts expressing human NHE1 (PSN) and 2) Human embryonic kidney (HEK293). The HEK293 were initiated by the transformation and culturing of normal HEK cells with sheared adenovirus 5 DNA in order to employ CRISPR‐CAS 9 gene‐editing techniques. The HEK293 cells were analyzed in the same ways the PSNs were to show a correlation within the different conditions being experimented with. We hypothesize the process of inhibiting palmitoylation will alter regulation of NHE1 and will play a role in the control of cell proliferation and migration. Using the palmitoylation inhibitor, 2‐bromopalmitate (2BP), we evaluated cell growth and migration in the presence and absence of palmitoylation. We also evaluated cell growth and migration in the presence and absence of the chemical inhibitor of NHE1, Cariporide (C). In proliferation assays, the rate of cell proliferation increased in both cell lines by approximately 5‐fold when the serum present in the growth media was increased from 0.5% to 10%. Under both conditions, the addition of 15 μM 2BP or 10 μM cariporide dramatically reduced or eliminated the increased proliferation. We also evaluated the rate of cell migration and once again, the rate was reduced by 50% or more in the presence of 15 μM 2BP or 10 μM cariporide. These data demonstrated that inhibiting the palmitoylation of NHE1 decreases both cell proliferation and the rate of cell migration.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid bilayer stress‐activated IRE‐1 modulates autophagy during endoplasmic reticulum stress
Authors: Thibault Guillaume , Koh Jhee Hong , Wang Lei , Beaudoin‐Chabot Caroline
Published: June 2021

Metabolic disorders, such as non‐alcoholic fatty liver disease (NAFLD), are emerging as epidemics that affect the global population. One facet of these disorders is attributed to the disturbance of membrane lipid composition. Perturbation of endoplasmic reticulum (ER) homeostasis through alteration in membrane phospholipids activates the unfolded protein response (UPR) and causes dramatic transcriptional and translational changes in the cell. To restore cellular homeostasis, the three highly conserved UPR transducers ATF6, IRE1 (also known as ERN1 in mammals) and PERK (also known as EIF2AK3 in mammals) mediate adaptive responses upon ER stress. The homeostatic UPR cascade is well characterised under conditions of proteotoxic stress, but much less so under lipid bilayer stress‐induced UPR. Here, we show that disrupted phosphatidylcholine (PC) synthesis in Caenorhabditis elegans causes lipid bilayer stress, lipid droplet accumulation and ER stress induction. Transcriptional profiling of PC‐deficient worms revealed a unique subset of genes regulated in a UPR‐dependent manner that is independent from proteotoxic stress. Among these, we show that autophagy is modulated through the conserved IRE‐1–XBP‐1 axis, strongly suggesting of the importance of autophagy in maintaining cellular homeostasis during the lipid bilayer stress‐induced UPR.

Support or Funding Information

This work was supported by the Nanyang Assistant Professorship programme from the Nanyang Technological University, the Ministry of Education ‐ Singapore Academic Research Fund Tier 1 (2016‐T1‐001‐078), and the Nanyang Technological University Research Scholarship to J.H.K. (predoctoral fellowship).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Life and Death: The Role of ADOR‐1 in Mediating Activation of the CEP‐1‐Apoptotic Pathway and IGF‐1 Signaling Pathway in Caenorhabditis elegans Exposed to Caffeine
Authors: Zarrinnegar Hanna , Adams Clay , Avida Natalie , Fong Ethan , Leeman Jacob , Madejski Sophia , Sweeney Robert , De Luke
Published: June 2021

Caffeine is the most widely used psychoactive drug in the world and forms a mild dependency in users who overuse the drug for an extended period of time. Other psychoactive drugs, including methamphetamine, have been shown to induce apoptosis in the brain's reward pathways, leading to lasting neuronal damage. Similarly, caffeine has been shown to activate the p53‐mediated apoptotic pathway in JB6 C141 cells derived from mice. However, other studies suggest that caffeine produces contradictory effects. Caffeine exposure has been reported to extend the lifespan of Caenorhabditis elegans through activation of the IGF‐1 signaling pathway. Additionally, caffeine has been shown to bind to ador‐1 receptors, the homolog of ADORA2A in humans, in C. elegans. We suspect that the seemingly contradictory effects of caffeine exposure are mediated by ador‐1, signifying that p53 and IGF‐1 activation occur downstream to adenosine receptors. Here we examine the role of ador‐1 in activation of the CEP‐1 apoptotic pathway, the C. elegans homolog of the p53‐apoptotic pathway in humans, and the IGF‐1 signaling pathway in C. elegans. Our current data clarifies the relationship between caffeine exposure and CEP‐1. We plan to further investigate the relationship of the CEP‐1 and IGF‐1 pathways in order to determine whether their activation is mediated by ador‐1.

Support or Funding Information

This work was supported by The Nueva School.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Microbial Population in the Nasal Cavity of Pigs during Post‐Weaning in Response to Probiotic Supplementation
Authors: Alcorn Paulyna , Keener Robert , Kobayashi Yass
Published: June 2021

Probiotics are commercially available for human consumption. Although it is a common dietary supplement, it has not been approved by the FDA. Probiotics are advertised to affect microbial population in the gastrointestinal system and improve immunity. Additionally, probiotic supplements have been used as an alternate growth and health promoter in domestic animals, such as pigs. However, the definitive effects of probiotic supplementation on these parameters are unknown. The objective of this study was to characterize the microbial population profile of the nasal cavity in post‐weaning pigs in response to probiotic supplementation. Nasal swipes were collected from 5 pigs that were fed with commercial feed containing mannan‐oligosaccharides plus β‐glucans and essential oils for 43 days. Additional samples were collected from 5 pigs that were fed with the identical commercial feed with essential oils alone for the same duration. DNA was isolated from each nasal swipe and V3/V4 region of 16s rDNA was amplified using PCR. Each PCR product was indexed. The indexed PCR products were sequenced to characterize the microbial population profile of each pig. DNA was successfully isolated and amplified for each sample. An analysis of the microbial population profile of each sample is being completed as well as an examination of whether the probiotic supplementation affects the microbial population profile in the nasal cavities of the pigs. In the future, effects of maternal probiotic supplementation on microbial population of piglets during weaning period and their effects on post‐weaning immune and stress responses will be examined.

Support or Funding Information

Kansas IDeA Network of Biomedical Research Excellence (NIH/NIGMS Grant #P20GM103418)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Growth of Cultures of Alternaria alternata from leaves of Actinidia arguta are inhibited by 1,4‐dimethylnaphthalene
Authors: Till Jessica , Campbell Michael
Published: June 2021

Alternaria alternata is a pathogenic fungus known to cause leaf spot in many plant species such as apples, purple coneflower, onions and many others. Actinidia arguta (hardy kiwifruit) is an alternative crop which has commercial potential in the United States. Field trials of A. arguta, located at Lake Erie Regional Grape Research and Extension Center, presented leaf lesions which suggested the presence of a pathogen. Leaf samples were taken from multiple symptomatic vines and allowed to incubate in empty sterile petri dishes. Symptomatic sections, approximately 1 cm2, were cut from the leaves and placed onto fresh potato dextrose agar (PDA) plates and incubated at 25°C for three days. Several fungal species were present on the plates with one fungal species common among all leaf samples plated. All visibly different species were isolated onto separate PDA plates, incubated, and re‐plated until purified cultures were obtained. PCR amplification of the 18 ITS region was accomplished using degenerate primers and DNA products were identified using Sanger sequencing. Resultant sequences were analyzed with BLASTn against the NCBI database. Cultures of A. alternata were identified as present on multiple leaf lesions. Established cultures were treated with 1,4‐dimethylnaphthalene (DMN) in a 9.5 Liter BBL GasPak chambers for two days. Mycelial growth was inhibited by DMN relative to control cultures. This is the first report of A. alternata isolated from the leaves of A. arguta and a demonstration that DMN can inhibit growth of this fungal species.

Support or Funding Information

Funded in part from a grant from the 1,4‐Group of Meridian, ID and the Behrend College Undergraduate Research Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of bacterial communities associated with laboratory cultures of Amphidinium carterae by metabarcoding
Authors: Gillespie Kathleen , Bachvaroff Tsvetan , Jagus Rosemary
Published: June 2021

In the ocean, dinoflagellates are associated with a microbial community that provides some of their essential nutrients. In the laboratory, most dinoflagellates are cultured in seawater supplemented with nutrients, although little effort has been made to maintain them axenically. This can complicate the study of their physiological processes such as protein synthesis and its modulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80 % of protein synthetic activity appears to be bacterial based on their responses to inhibitors of protein synthesis. The bacterial community associated with a nonaxenic culture of A. carterae was examined by combining mass‐amplification of short DNA sequences encoding 16S rRNA (barcodes) with high‐throughput sequencing. The Illumina MiSeq high‐throughput sequencing platform was used to sequence 600–1500 bp16S amplicons of the microbial community using universal primers for 16 rRNA genes. The dominant 16S sequences at the phylum level were assigned to Alphaproteobacteria, Rhodobacteraceae, Hyphomonadaceae and Gammaproteobacteria. Axenic cultures, maintained in antibiotic mixtures of kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS) grew as well as the nonaxenic and to slightly higher cell densities. They showed little evidence of bacterial protein synthesis based on responses to inhibitors of protein synthesis and a dramatically lower bacterial presence. However, over time in culture Proteobacteria, Cyanobacteria, and Actinobacteria gradually emerged, probably reflecting less than adequate sterile technique and less than optimal antibiotics.

Support or Funding Information

NOAA‐EPPLMRCSCNASF research award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Environmental Microbiomes During the Recycling of Produced Water using MALDI‐TOF‐MS
Authors: Reyes Michelle , Martin Misty , Santos Ines , Hildenbrand Zacariah , Schug Kevin
Published: June 2021

Hydraulic fracturing is the process of injecting large amounts of water under high pressure into a bedrock formation to increase oil and/or gas flow. To reduce use of freshwater resources, new techniques for the treatment of produced oilfield wastewater have been explored. One important concern is removing bacteria particularly sulfate‐reducing and iron‐oxidizing bacteria as these can contribute to costly degradation of product quality and biocorrosion of metal infrastructure. Therefore, identification and quantification of bacteria present after remediation is important to determine efficiency of the treatment and quality of the water. Matrix‐assisted laser desorption time‐of‐flight mass spectrometry (MALDI‐TOF MS) is a more efficient method of identifying microbes as opposed to other conventional identification methods, which have proven to be more time consuming and costly. In this work, MALDI‐TOF MS was used to identify microorganisms and assess the changes in microbiome diversity present in produced water (PW) before, during, and after treatment through various disinfection techniques. The raw PWs displayed unique microbial profiles that fluctuated throughout treatment with the five different modalities. Most of the microorganisms identified are commonly found in the environment such as Pseudomonas, Aeromonas, and Bacillus. UV treatment was the most effective disinfection modality (28.5% decrease in total CFUs/mL).

Support or Funding Information

Challenger Water Solutions

Collaborative Laboratories for Environmental Analysis and Remediation

Inform Environmental

Shimadzu

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Finding in Oxidative Phosphorylation: Isothermal Utilization of Latent Heat Energy by Electrostatically Localized Protons at Liquid‐Membrane Interface
Authors: Lee James Weifu
Published: June 2021

Upon application of a newly formulated equation for the proton motive force (pmf) across a biological membrane which takes electrostatically localized protons into account in elucidating the bioenergetics of oxidative phosphorylation in alkalophilic bacteria, it was shown that the localized protons at the liquid‐membrane interface dominate the total pmf. The decreasing trend of the pmf with higher pH values is due to the increasing exchange of cations with the localized protons and this trend matches excellently with the observed rapid increase in the bacteria population growth cell doubling times. It was revealed that the pmf from the isothermal latent heat utilization through the electrostatically localized protons is not constrained by the overall energetics of the redox‐driven proton pump system because (i) the localized protons are not free to move away from the membrane surface, (ii) the proton pumps embedded in the cell membrane extend beyond the localized proton layer, and (iii) the protonic inlet mouth of the ATP synthase that accepts protons is located within this layer. Remarkably, this new understanding agrees excellently with the true structures of mitochondrial respiratory protein complexes. It therefore appears that the oxidative phosphorylation system is able to isothermally utilize the latent heat energy effectively through electrostatically localized protons to help driving the synthesis of ATP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Energetic and Gluconeogenic Response in Intermittent Fasting (IF)
Authors: Cervantes Silvia Cristina , Gasca Deysi , Muñoz Mauricio Díaz
Published: June 2021

Physiology evolved during fluctuations in energy supply and demand. Coping with these changes has entrained the organisms to manage energy metabolism for optimal substrate storage and use during feeding or fasting, and periods of either rest or energy demand. During fasting, the liver is responsible for maintaining blood glucose levels by regulating metabolic pathways, such gluconeogenesis (GNG). GNG aims to form glucose de novo, its regulation occurs due to the activity of two limiting enzymes: phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (G6Pase), and it is highly regulated (Petersen et al., 2017).

The ability to efficiently adapt metabolism depending on demand or supply is known as metabolic flexibility (Smith, Soeters, Wüst, & Houtkooper, 2018). Intermittent fasting (IF) is a dietary protocol that consists of periods without access to food, alternating with periods with free access to food (ad libitum) (Chausse et al., 2015). The chronic regimen of the IF carries different responses to those observed in an acute fasting or continuous restriction (Secor and Carey, 2016).

We evaluate the adaptation and metabolic flexibility that happened in IF to maintain homeostasis. Chronicity in IF elicit physiological and metabolic changes, such as the increase in the size of the liver after feeding to cope with total energy restriction, and increase in gluconeogenic enzymatic activity to keep normal values of glucose. Experiments of indirect calorimetry show that the values of RQ in IF on feeding conditions are restored to the first AL of the treatment until the 4th cycle of IF, which are equivalents to their pairs in the AL group.

Support or Funding Information

Funded by Project Conacyt 284–557

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.