bluuebunny/crossref_metadata_2025 huggingface dataset:
“Personalized mechanisms of Lafora disease using Differential Scanning Fluorimetry”
Authors: Donohue Kit , Brewer M. Katherine , Vander Kooi Craig W , Gentry Matthew S
Published: June 2021

Lafora disease is an autosomal recessive neurodegenerative disease. Patients grow up normally for their first decade of life, then develop severe epilepsy, and die in their 20s or 30s from massive neurodegeneration. The field has demonstrated that the pathogenic agent of the disease are aberrant glycogen molecules.

Glycogen phosphorylation impacts the branch point and chain length distribution within the glycogen molecule, determining both the granule shape and its accessibility to regulating enzymes. Laforin is the only known mammalian phosphatase capable of modifying the phosphorylation status of glycogen. When laforin is mutated, aberrant glycogen forms inclusion bodies and drives Lafora disease progression. Therefore, it is important to understand how different mutations in laforin affect its ability to bind and dephosphorylate glycogen. However, defining protein‐carbohydrate interactions can be challenging because many binding techniques cannot accurately measure low binding affinities.

We utilized differential scanning calorimetry (DSC) in combination with differential scanning fluorimetry (DSF) and a unique algorithm to consistently predict a true Kd using DSF data. Through the use of short polysaccharides that approximate glycogen micro‐structures, we show that the DSF method can be used in a high‐throughput assay to assess laforin‐carbohydrate interactions. With this data, we compare how different mutations impact the binding affinity of laforin to glycogen and how changes to glycogen structure alter the ability of laforin to bind and dephosphorylate glycogen. These results provide unique insights into personalized mechanisms of Lafora disease.

Support or Funding Information

This work was supported by the National Science Foundation through NSF MCB1817417 and NSF OIA 1355438

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microbial VOC Fingerprints: Rapid Detection of Antimicrobial Resistance in Pathogenic Bacteria
Authors: Dailey Allyson , Saha Jessica , Zaidi Fatima , Couch Robin
Published: June 2021

The US Centers for Disease Control and Prevention (CDC) estimates that at minimum, 2 million people in the United States are infected with antimicrobial resistant (AMR) bacteria, resulting in 23,000 deaths each year. With no projected decrease in the number of deaths and the evergrowing threat of multi‐drug resistant bacteria, the development of a rapid and accurate diagnostic test is crucial, not only to reduce the burden on the public healthcare system, but to save countless lives.

Microbial volatile organic compounds (mVOCs) are a structurally diverse group of microbial‐derived metabolites, generally related by their volatility at ambient temperature. There is great diagnostic potential for mVOCs, as numerous studies have identified unique mVOC‐fingerprints indicative of specific bacterial pathogens. In addition, specialized headspace sampling methods, such as solid‐phase microextraction (hSPME), coupled to gas chromatography, have enhanced the isolation, preconcentration, and analysis of mVOCs from biological specimens. Herein, we describe the development and assessment of our newly developed and patented metabolomics device and technique known as “simultaneous multifiber headspace solid‐phase microextraction (simulti‐hSPME),” for the rapid and minimally invasive preparation of mVOC fingerprints. This method is evaluated against antimicrobial susceptible and resistant strains of Yersinia pestis and Francisella tularensis.

Support or Funding Information

Funding for this project is provided by DTRA (Grant Number: HDTRA 1‐16‐1‐0040).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Longitudinal Analysis of DNA Methylation Status Linked to Post‐Traumatic Stress Disorder in Deployed Service Members
Authors: Gautam Aarti , Yang Ruoting , Miller Stacy , Chakraborty Nabarun , Abu‐Amara Duna , Blessing Esther , Hammamieh Rasha , Marmar Charles , Jett Marti
Published: June 2021

Pre‐deployment identification of risk and resilience factors is crucial in developing strategies to reduce or prevent post‐traumatic stress (PTSD) symptoms in military personnel. Combat and deployment itself can result in drastic changes in activity, diet, and stress, often with detectable molecular consequences. This longitudinal study was designed to assess the effects of deployment on DNA methylation in peripheral blood. Blood samples were collected pre‐deployment, immediately post‐deployment, and at a three month follow‐up. A battery of clinical tests and self‐reported psychological data were included in the study. DNA methylation assays, followed by repeated‐measures ANOVA, correlation analysis, and PTSD differential analysis were carried out to characterize methylation status alterations associated with significant PTSD Check List (PCL) changes (PCL difference >10). We identified 64 subjects with increases in PCL scores of 10 across the three phases of the sample collection. Repeated‐measures ANOVA testing, adjusted for cell composition and age, was conducted on 2415 methylation probes. 72 significantly differentially methylated probes were identified that were associated with Netrin, mTOR, hypoxia, and ubiquitination pathways. Correlation and differential analysis between PTSD and control samples identified 51 genes passing mean p‐value cutoffs across analyses. These genes are involved in the cAMP, mTOR, opioid, and CREB signaling pathways. Our findings will contribute to understanding the pathogenesis of PTSD co‐morbid conditions and provide mechanistic insights which may aid in averting the onset of chronic PTSD. Future work will integrate these epigenetic data with other molecular ‐omics and clinical findings to better understand PTSD co‐morbidities, as well as susceptibility and resiliency.

Disclaimers:

Research was conducted in compliance with all Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

Support or Funding Information

This study was supported by USAMRMC grant No: 09284002.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
WWOX drives UV/cold shock‐induced bubbling cell death whereas without WWOX cells pop out
Authors: Chang Nan‐Shan , Tsai Cheng‐Chang , Sheu Hamm‐Ming
Published: June 2021

Bubbling cell death (BCD) is known as release of an enlarging nuclear bubble from the surface of each cell at room temperature or lower post exposure of the cells to UV irradiation and cold shock. BCD concerns the severity of frostbite and UV. However, the molecular mechanism is largely unknown. Here, we determined that UV/cold shock induces tumor suppressor WWOX‐expressing L929S cells to undergo typical BCD. UV energy is rapidly taken up by the nuclear DNA to transmit signals and simultaneous rapid influx of calcium ion, followed by nuclear accumulation of numerous cytosolic and membrane proteins (e.g. p53, WWOX, TRAF2, NOS2, NF‐κB, C1q and Hyal‐2). Subsequently, release of nucleolar content leads to irreversible bubble formation and death without cell shrinkage. BCD is blocked at 37°C in L929S and other WWOX‐positive cells. Notably, transient overexpression of TIAF1 and SH3GLB2 restores BCD in TNF‐sensitive L929S cells at 37°C. In contrast, UV/cold shock‐treated WWOX‐deficient cells undergo shrinkage, then rapid nuclear bubble formation and final whole cell pop‐out, designated pop‐out death (POD). Antiapoptotic TRAF2 binds WWOX and the binding is abolished during BCD, suggesting that the dissociated WWOX is needed in inducing BCD.

Support or Funding Information

Research was supported by the Ministry of Science and Technology, Taiwan (MOST 105‐2320‐B‐006‐046, 105‐2320‐B‐006‐036,106‐2320‐B‐006‐017, and 106‐2320‐B‐006‐061), the Department of Defense, USA (W81XWH‐08–1‐0682), the National Health Research Institute, Taiwan (NHRI‐EX107‐10734NI) to NS Chang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPV4 regulates P. gingivalis lipopolysaccharide‐induced exacerbation of oxidized LDL‐mediated foam cell formation
Authors: Rahaman Shaik , Gupta Nabyendu , Goswami Rishov
Published: June 2021

P. gingivalis, a predominant causative agent of periodontitis, has been linked to lipid‐laden macrophage foam cell formation, a critical process in atherogenesis. Emerging data support a role for both a biochemical factor, e.g., lipopolysaccharides (LPS), and a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function and atherogenesis. We have obtained evidence that TRPV4, a mechanosensitive channel of the transient receptor potential vanilloid family, is the likely mediator of P. gingivalis lipopolysaccharide (PgLPS)‐induced exacerbation of oxidized low‐density lipoprotein (oxLDL)‐mediated macrophage foam cell formation. Specifically, we found that: i) genetic deletion of TRPV4 or antagonism of its function by a chemical inhibitor blocked PgLPS‐induced exacerbation of oxLDL‐induced macrophage foam cell formation; ii) macrophage TRPV4 activity was increased in response to both PgLPS and pathophysiological range matrix stiffness, and iii) TRPV4 deficiency blocked PgLPS‐induced exacerbation of matrix stiffness‐induced macrophage foam cell formation. Mechanistically, we show that TRPV4 regulates PgLPS‐induced oxLDL uptake but not its cell surface binding in macrophages. Altogether, these findings identify a critical role for TRPV4 in regulating PgLPS‐induced exacerbation of macrophage foam cell formation by modulating uptake of oxLDL.

Support or Funding Information

AHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik Rahaman

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional analysis of a biflavone, as a novel inhibitor of TRPV4‐dependent macrophages foam cell formation
Authors: Rahaman Shaik , Alharbi Mazen , Goswami Rishov
Published: June 2021

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory disease, is the most dominant underlying pathology. Macrophages, an essential cell type in atherogenesis, are known to directly respond to changes to their surrounding mechanical environment. As TRPV4 (transient receptor potential channel of the vanilloid subfamily) channels can be sensitized by changes in matrix stiffness, we hypothesize that, TRPV4 may upregulate inflammatory/atherogenic macrophage functions. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay; we identified ginkgetin, a biflavone, as a novel antagonist to TRPV4. Primary and secondary screening was performed with normal human dermal fibroblasts and mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐elicited Ca2+ influx after TRPV4 activation with its selective agonist GSK1016790. In our hand's, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐elicited Ca2+ influx. We show that antagonism of TRPV4 channel by ginkgetin blocks oxidized LDL (oxLDL)‐induced macrophage foam cell formation in a concentration dependent manner. Mechanistically, we show that ginkgetin attenuated oxLDL uptake but not its cell surface binding in BMDMs. Altogether, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated macrophage foam cell formation by modulating uptake of oxLDL.

Support or Funding Information

AHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik O Rahaman

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Variable Surface Display and Post‐Translational Regulation of the Fungal Adhesin Epa1p
Authors: Raposo Colin , McElroy Kyle A , Mirecki Jarett , Fuchs Stephen M
Published: June 2021

Infection by the fungal pathogen Candida glabrata is reliant on a collection of cell surface adhesins that play a variety of roles necessary for transmission, establishment, and cell proliferation. One particular adhesin, Epithelial Adhesin 1 (Epa1p), is responsible for binding to host tissue and biofilm formation, which is essential for fungal propagation. Epa1p structure consists of three domains: a C‐terminal GPI anchor for cell wall linkage, an N‐terminal intercellular binding domain responsible for epithelial cell binding, and a serine/threonine rich linker domain connecting these terminal domains. The linker domain contains a 40‐amino acid tandem repeat region which our lab has observed to be variable in repeat copy number between isolates from clinical sources. We expressed Epa1p with various repeat copy numbers in epa1Δ C. glabrata as well as in the related yeast, S. cerevisiae to determine how differences in repeat copy number affect Epa1p surface display and binding to human epithelial cells. Our initial data suggest that the processing of, as well as cell wall stability of, Epa1p is dependent upon the repeat copy number of Epa1p. We have also collected data, which suggest that Epa1p surface level is dependent on media growth conditions. Altogether our data demonstrate that Epa1p repeat copy number influences the properties of Epa1p and the adhesive characteristics of C. glabrata.

Support or Funding Information

1. Army Research Office grant W911NF1610175

2. Tufts Collaborates!

3. 2018 ASBMB Undergraduate Research Award

4. Tufts Summer Scholars

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FPT, a Novel 5‐HT7 and 5‐HT1A Partial Agonist, Treats Neuropsychiatric Symptoms Modeled in Fmr1 Knockout Mice
Authors: Armstrong Jessica , Mukherjee Munmun , Booth Raymond , Canal Clinton
Published: June 2021

Fragile X Syndrome (FXS) is the leading monogenetic cause of intellectual disabilities and autism. Seizures, severe anxiety, and sensory hypersensitivity are also common in FXS. The central serotonin system can modulate these symptoms, and certain serotonin receptors are functionally linked with proteins known to be altered in FXS, suggesting targeting them may be therapeutic for FXS. We are developing (S)‐5‐(2′‐Fluorophenyl)‐N, N‐dimethyl‐1,2,3,4‐tetrahydronaphthalen‐2‐amine (FPT), a partial agonist at both 5‐HT7 and 5‐HT1A receptors, as a treatment for FXS and autism. We previously reported that FPT corrects repetitive behaviors and enhances social interactions—core symptom domains in FXS and autism—in wild‐type (WT) mouse models. Herein, we report the results of tests assessing the efficacy of FPT to improve behavioral phenotypes in Fmr1 knockout (KO) mice, an etiologically valid genetic model of FXS. Fmr1 KO mice have seizures when exposed to a 120 dB alarm (audiogenic seizures, AGS). 73% of vehicle‐treated Fmr1 KO mice we tested had AGS (males and females, age P23–25, N=22), however, 0% of the Fmr1 KO mice pretreated with FPT (5.6 mg/kg) had AGS (N=20). This effect was very significant (P<0.0001). In addition, FPT significantly reduced marble burying (vehicle, N=11, FPT, N=10, P<0.001) and reduced rearing in Fmr1 KO mice (vehicle, N=12, FPT, N=12, P<0.05). These data corroborate our earlier studies that FPT corrects repetitive behaviors and may have anxiolytic properties. We next tested FPT's efficacy to promote social behavior. Mice were matched for genotype and sex and paired together for a 10 minute observation period. One mouse was given vehicle and the other was given FPT. Social behavior, scored by an observer blind to treatment, was defined as the number of approaches from one mouse to the other and was recorded for each mouse within the pair. FPT increased social behavior in both WT (P<0.005) and Fmr1 KO (P<0.05) mice. We did not observe overt behavioral toxicity (for example, hypolocomotion, flat body posture, tremor, or head weaving) in mice treated with FPT. Our data provide further evidence that FPT has activity in neurobehavioral assays suggesting anxiolytic and prosocial effects that extend from WT to Fmr1 KO mice.

Support or Funding Information

DOD W81XWH‐17‐1‐0329, FRAXA Research 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.
Crystal Structure of the C‐terminal Guanine Exchange Factor Module of Trio Reveals its Oncogenic Potential
Authors: Bandekar Sumit J. , Arang Nadia , Tully Ena S. , Tang Brittany A. , Barton Brenna L. , Li Sheng , Gutkind J. Silvio , Tesmer John J.G.
Published: June 2021

The C‐terminal guanine exchange factor module of Trio (TrioC) activates the small G protein RhoA, linking Gαq/11‐coupled 7TM receptors to cellular events including cytoskeletal rearrangements and gene transcription. This signal transduction pathway underlies the development of uveal melanoma, a fatal malignancy. Previous studies have shown that TrioC is regulated via autoinhibition relieved by the binding of Gαq/11. However, the structural determinants of the autoinhibited state have remained elusive. We determined the crystal structure of TrioC in its basal state, revealing the molecular basis of autoinhibition mediated by the pleckstrin homology (PH) domain via direct interactions with the Rho binding site on the Dbl homology domain (DH). We show that truncation of the PH domain or introduction of point mutations activates the TrioC module in nucleotide exchange assays by liberating the Rho binding site. This model is consistent with results obtained by hydrogen‐deuterium exchange mass spectrometry. Finally, we show that mutations in the PH domain found in cancer patients also hyper‐activate full length TrioC in cell based models. These mutations may thus represent the first known cancer drivers in Trio that operate in an analogous, yet Gαq/11‐independent manner.

Support or Funding Information

This work was supported by NIH Grants CA221289, HL122416, and HL071818 (to J.J.G.T.), 5T32GM007767‐38, 5T32GM007767‐39, and F31CA224804 (to S.J.B.), and 5T32GM007752‐39 (to N.A.). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐dependent responses to norditerpenoid alkaloids from Delphinium in Angus cattle
Authors: Green Ben , Welch Kevin , Pfister James , Davis Zane , Stonecipher Clint , Cook Daniel
Published: June 2021

Plants produce bioactive secondary chemical compounds which are not involved in metabolism, photosynthesis or reproduction. Norditerpenoid alkaloids produced by Delphinium spp. (larkspur) poison cattle grazing on rangelands of western North America. The objective of this study was to compare the physiological responses of yearling and two year old Angus heifers, steers, and bulls with a standardized dose of norditerpenoid alkaloids in the form of dried ground Delphinium barbeyi. Clinical signs of intoxication, including loss of muscle coordination and function were measured 24 h after oral dosing with larkspur by walking the cattle at a pace of 5–6 km/h for up to 40 min on an oval dirt track. The walk times of the yearling cattle were: 2.9 ± 1.2 minutes (30 heifers), 16.6 ± 1.9 minutes (60 steers), and 15.5 ± 2.4 minutes (33 bulls). Two‐year old heifers walked 6.7 ± 3.3 minutes (15 heifers). Serum alkaloid concentrations were measured immediately prior to walking, and deltaline concentrations averaged 266 ± 28, 131 ± 20, and 219 ± 28 ng/mL for all heifers, steers and bulls, respectively, and serum methyllycaconitine concentrations averaged 660 ± 46, 397 ± 32, and 612 ± 34 ng/mL for all heifers, steers and bulls, respectively. These results suggest that yearling and two year old Angus heifers are more susceptible to larkspur intoxication than bulls or steers.

Support or Funding Information

Supported by USDA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Next Generation Potent Cell‐active Inhibitors of the Oncogenic PTP4A3 Phosphatase
Authors: Blanco Isabella K. , Tasker Nikhil R. , Rastelli Etttore J. , Wipf Peter , Sharlow Elizabeth R. , Lazo John S.
Published: June 2021

High grade serous ovarian cancer and triple negative breast cancer respond poorly to existing drugs and, thus, demand new therapies. The protein tyrosine phosphatase PTP4A3 (Phosphatase of Regenerating Liver‐3, PRL‐3) is overexpressed in these diseases. Moreover, PTP4A3 promotes cancer cell migration and invasion and is believed to be the most oncogenic of all tyrosine phosphatases. We recently synthesized the novel iminothienopyridone, JMS‐053, and demonstrated that it is a specific, reversible, allosteric, and cell active small molecule PTP4A3 inhibitor with an in vitro IC50 for PTP4A3 of 30 nM. The water solubility of JMS‐053 is limited, however. Therefore, we synthesized twenty next generation analogs to improve aqueous solubility and to aid in determining the structure activity relationship for the chemotype, the PTP4A3 binding site, and the mechanism of inhibition. Seventeen analogs retained potent in vitro PTP4A3 inhibition with IC50 values <100 nM. Four compounds, EJR‐866‐36, EJR‐866‐68, EJR‐855‐81, and NRT‐892‐04, had in vitro PTP4A3 IC50values of <40 nM. EJR‐866‐75 and NRT‐870‐59 were computationally more drug‐like than JMS‐053. EJR‐855‐81, NRT‐870‐59, EJR‐866‐75, and NRT‐870‐59 were cytotoxic to human Hs578T breast cancer and OVCAR4 ovarian cancer 3D spheroid tumor cells grown in vitro with IC50 values below 20 mM. To further expand the mechanistic activity of this class of inhibitors, we have synthesized four third‐generation analogs of JMS‐053, which combine the iminothienopyridone motif with a chemical moiety capable of binding a E3 ubiquitin ligase that will accelerate the intracellular degradation of PTP4A3. One of these novel compounds, EJR‐887‐35, had a PTP4A3 in vitro IC50 value of 107 nM and retained cytotoxicity to Hs578T and OVCAR4 cells with IC50 values below 20 mM in 3D spheroid tumors. These results suggest the iminothienopyridone chemotype could be valuable for the development of the first potent, cell active PTP4A3 phosphatase inhibitors directed against breast and ovarian cancer.

Support or Funding Information

Department of Defense (Award W81XWH‐18‐1‐0011, BC170507), NIH Grant P30 CA044579 and S10 OD021723, and the Fiske Drug Discovery 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.
The Mechanism of Inhibition of the Undrugged Oncogenic Phosphatase PTP4A3 by a Novel Small Molecule JMS‐053
Authors: Garrott Sharon R. , Blanco Isabella K. , Tasker Nikhil R. , Rastelli Ettore J. , Sharlow Elizabeth R. , Wipf Peter , Lazo John S.
Published: June 2021

The oncogenic tyrosine phosphatase PTP4A3 is an attractive cancer therapeutic target because it is overexpressed in many types of cancer and promotes tumor metastasis, contributing to poor patient prognosis. We recently synthesized JMS‐053, which is a potent, selective, reversible, iminothienopyridinedione inhibitor of PTP4A3. We modeled possible interactions between JMS‐053 and PTP4A3 and the goal of our current work was to test the model and further define the crucial amino acids involved in the PTP4A3 inhibition by JMS‐053. We generated three PTP4A3 single amino acid mutants: C104S, K144I and C49S. The enzymatic activities of these phosphatases were determined in vitro in the absence and presence of JMS‐053, using the artificial substrate 6,8‐difluoro‐4‐methylumbelliferyl phosphate. The C104S mutant showed no catalytic activity, consistent with C104 being the catalytic cysteine. The K144I mutant retained full enzymatic activity. In contrast to our molecular modeling predictions, the K144I mutant was inhibited by JMS‐053 to the same extent as the wild type enzyme. We concluded K144 was not primarily involved in JMS‐053 inhibition. Tyrosine phosphatases are known to be inhibited by intramolecular disulfide bond formation and C49S has been reported to be the only cysteine involved with disulfide bond formation with the catalytic C104. The C49S mutant had reduced catalytic activity compared to the wild type PTP4A3 but JMS‐053 retained partial inhibition. Thus, C49‐C104 disulfide bond formation does not appear to be the primary mode of JMS‐053 inhibition, although we cannot completely exclude involvement of C49. We are now examining the oxidation state of C104 after JMS‐053 treatment and new analogs of JMS‐053. The information obtained from mapping the critical amino acids involved in PTP4A3 inhibition should assist in the design of the next generation phosphatase inhibitors.

Support or Funding Information

The Department of Defense (BC170507), the Fiske Drug Discovery Fund and the Ivy 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.
Phospholipids as Indicators of Docetaxel‐Resistant Castration‐Resistant Prostate Cancer
Authors: Ingram Lishann M , Mansoura Maryam , Cummings Brian S
Published: June 2021

The association of circulating lipids with clinical outcomes of drug‐resistant castration‐resistant prostate cancer (CRPC) is not fully understood. While it is known that increases in plasma lipids correlates to decreased survival, neither the mechanisms mediating alterations in these lipids nor the correlation of resistance to treatment are well characterized. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone sensitive, CRPC and drug‐resistant cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis. HPLC‐MS based metabolomic analysis identified 83 features that were significantly altered in the drug‐resistant cell lines as compared to non‐resistant control group. Within the 83 species, two were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer from previous studies. Principal component analysis (PCA) demonstrated that drug‐resistant and control cell‐lines were visually separated by these identified lipid biomarkers. Two out of the 83 lipid classes were identified as phosphatidylcholine (PC) 18:0/18:1 and PC 18:0/18:2), both of which had high level of abundance in Docetaxel‐resistant CRPC. An untargeted approach indicated that lipids corresponding to PC, phosphatidylglycerol (PG), phosphatidic acid (PA) and phosphatidylserine (PS) all had higher relative abundance in DR‐CRPC cells (DU145‐DR) compared to parent control (DU145). These data suggest that the lipidomic profiles of prostate cancer cells lines recapitulate lipidomic profiles in the plasma of prostate cancer patients. As such, these metastatic dependent and drug‐resistant cell lines may prove useful for understanding the molecular mechanisms mediating lipid changes in prostate cancer patients.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100) Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Potassium Channel Blocker, 4‐Aminopyridine, Attenuates Peripheral Nerve Injury‐induced Muscle Atrophy, Improves Intrinsic Muscle Force, and Suppresses the Expression of Muscle Atrophy‐related Transcription Factors in Mice
Authors: Talukder MA Hassan , Yue Li , Noble Mark , Dirksen Robert , Chakkalakal Joe , Elfar John C
Published: June 2021

4‐Aminopyridine (4‐AP), a broad‐spectrum potassium channel blocker and FDA‐approved drug for the symptomatic treatment of multiple sclerosis, is used to improve neuromuscular function in patients with diverse demyelinating disorders. The neurological benefits of 4‐AP are believed to result from increases in action potential duration, calcium influx, neurotransmitter release and synaptic transmission. Traumatic peripheral nerve injury (TPNI) represents a major health problem that often leads to significant functional impairment and permanent disability from the loss in axonal continuity, neuronal cell death, nerve demyelination, nerve conduction defects, muscle denervation, and muscle atrophy. We have recently repurposed the use of 4‐AP and demonstrated that systemic 4‐AP administration enhances global functional recovery of the affected limb, promotes remyelination of the nerve and improves the nerve conduction velocity in a mouse model of TPNI. While muscle atrophy occurs very rapidly following nerve injury, the effect 4‐AP on muscle atrophy and muscle contractile function is largely unknown. This study was designed to explore the possible beneficial effects of 4‐AP treatment in muscle atrophy, intrinsic muscle function, and muscle regeneration following acute sciatic nerve crush injury. Mice were assigned to moderate sciatic nerve crush injury and no‐injury groups and followed for 3, 7 and 14 days with/without 4‐AP (10 mg/daily, intraperitoneal injection) or saline treatment. Morphological, functional and transcriptional properties of skeletal muscle were assessed. In addition to improving in vivo global motor function as early as post‐operative day 3, 4‐AP treatment significantly attenuated muscle atrophy of the injured limb with increased muscle mass and muscle fiber area. 4‐AP also improved ex vivo intrinsic muscle contractile force 7 days post‐injury. Most importantly, the reduced muscle atrophy with 4‐AP treatment was concurrent with significantly reduced expression of atrophy‐related genes (myogenin, MuRF‐1, FoxO1, FoxO3) and increased expression of Pax7+ satellite cells and proliferating Ki67+ cells. These findings provide new insights into the beneficial effects of 4‐AP in nerve injury‐induced muscle atrophy and dysfunction and open a new window for further investigation.

Support or Funding Information

This work was supported by grants from the NIH (K08 AR060164‐01A) and DOD (W81XWH‐16‐1‐0725) in addition to institutional support from the University of Rochester and Pennsylvania State University Medical Centers.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Brain Endothelial Cell Bioenergetics via Extracellular Flux Analysis
Authors: McDonald Cade , Drewes Lester , Blankenheim Zachary
Published: June 2021

The neurovascular unit (NVU) is an important functional component in the central nervous system (CNS). The NVU consists of multiple cell types (endothelial cells, pericytes, astrocytes, others) working together as a restrictive blood‐tissue interface and functions to maintain homeostasis within nervous tissue in the CNS. A dysfunctional NVU is associated with many disease states, including Alzheimer's Disease, strokes, and tumors. The endothelial cell is an important barrier‐forming cell of the NVU because of its direct contact with the blood, its intercellular tight junctions, biotransforming enzymes, and asymmetric distribution of active and carrier‐mediated transporters. These properties are important for maintaining the neuro‐microenvironment and require an abundant supply of energy (ATP). Although energy metabolism has been studied in various tissues and cells, little is known about the energy producing pathways of brain endothelial cells. Therefore, using extracellular flux analysis, we characterized the bioenergetics of human brain microvascular endothelial cells (BMECs) in vitro. BMECs are primarily glycolytic and D‐glucose, pyruvate, and glutamine are preferred metabolic substrates. Inhibitors of monocarboxylate transporter (MCT1), mitochondrial pyruvate carrier, and glycolysis significantly alter ATP production, cellular respiration, and/or glycolytic rates. These findings contribute to our understanding of endothelial cell metabolic plasticity in normal and diseased brain as well as active angiogenesis during development or tumorigenesis. Understanding endothelial cell bioenergetics will be useful for future studies regarding development of therapies, that target endothelial cell energetics, for neurological disorders.

Support or Funding Information

Whiteside Foundation and the Department of Defense

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Daily acute, but not chronic, intermittent hypoxia enhances phrenic motor plasticity in chronic cervical spinal cord injury
Authors: Gonzalez‐Rothi Elisa J , Tadjalli Arash , Allen Latoya L , Ciesla Marissa C , Simon Alec , Asa Zachary , Smith Kristin , El‐Chami Mohamad , Holland Ashley , Santiago Juliet , Stefan Kelsey , Ross Ashley , Mitchell Gordon S
Published: June 2021

Daily acute intermittent hypoxia (dAIH; 3–15 hypoxic episodes per day) is a promising therapeutic strategy to improve respiratory function after incomplete spinal cord injury by inducing spinal respiratory motor plasticity. Conversely, chronic intermittent hypoxia (CIH; >100 episodes per day) elicits multisystem pathology and is a hallmark of moderate sleep apnea, a condition highly prevalent in individuals with cervical spinal injury. In intact rats, 7 days of dAIH preconditioning enhances phrenic motor facilitation whereas it is abolished by 7 days of CIH, an effect likely due to CIH‐induced neuroinflammation. Since the impact of prolonged CIH (> 1 wk) or dAIH on the capacity for phrenic motor facilitation with chronic spinal cord injury has not been explored, we compared the effect of 28 days of normoxia (21% O2; 8hrs/day), dAIH (10, 5 min episodes of 10.5% O2 with 5 min normoxic intervals/day) or CIH (2 min hypoxic episodes with 2 min intervals; 8 hrs/day) on phrenic output in rats with chronic C2 spinal hemisection (C2Hx). Neurophysiology experiments were conducted one day after the final hypoxic exposure; bilateral phrenic nerve activity and moderate AIH‐induced phrenic long‐term facilitation (pLTF) were assessed. Ipsilateral phrenic nerve activity during baseline conditions and maximal chemoreflex activation was: 1) reduced in all injury groups vs intact rats (p<0.0001 and p<0.009, respectively); and 2) unaffected by dAIH or CIH (p>0.94 and p>0.98, respectively). No group differences in contralateral phrenic nerve activity were observed in either condition (p>0.49 and p>0.18, respectively). dAIH enhanced ipsilateral pLTF vs. all other groups (p=0.01); in contrast, CIH had no effect on pLTF (p=0.43). In conclusion, neither dAIH nor CIH had any detectable impact on phrenic nerve activity in rats with chronic C2Hx, but dAIH enhanced the capacity for additional pLTF. Further work is needed to explore: 1) mechanisms of enhanced respiratory plasticity following dAIH; 2) if detrimental effects of CIH can be resolved by treatments that mitigate inflammation; and 3) how enhanced/restored phrenic motor plasticity can be harnessed to improve breathing ability in individuals with chronic cervical spinal cord injury.

Support or Funding Information

NIH: OT2OD023854, K12 HDO55929 (EGR), T32 HD055929 (LLA); Department of Defense: SCI160123 (EGR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gastrointestinal neuroimmune disruption in a mouse Gulf War Illness model
Authors: Hernandez‐Rivera Siomara , Grubišić Vladimir , Fried David , Gulbransen Brian D
Published: June 2021

Gulf War Illness (GWI) is a chronic disorder characterized by a spectrum of six symptoms that include gastrointestinal disorders. Exposure to the anti‐nerve gas drug pyridostigmine bromide (PB) is linked with the development of GWI, but the exact mechanisms remain unclear. We hypothesize that PB disrupts gut functions by creating persistent neuroinflammation within the enteric nervous system (ENS). We tested the effects of PB in vivo by exposing male and female mice to 9 μg/mL or 90 μg/mL PB for 7 and 30 days and subsequently assessing gut function using in vivo and ex vivo tests of colonic motility and barrier function. Neurochemistry of the ENS was assessed by immunohistochemistry and immune responses were studied using multiplex cytokine and chemokine arrays in the gut and brain. Acutely exposing whole mount preparations of myenteric plexus to PB drove calcium responses in enteric glia (181.0%, p<0.001). In vivo, exposure to PB acutely increased fecal pellet output (44.3%, p<0.05) and increased fecal fluid content (13.2%, p<0.05) in male mice, but decreased fecal pellet output (62.5%, p<0.05) and reduced fecal fluid content (18.1%, p<0.05) in females. Regardless of sex, PB treatment altered neuromuscular control, slower colonic bead expulsion (male 504.4%, p<0.001; female 275.8%, p<0.01), and reduced colon length (male 9.4%, p<0.05; female 9.9%, p<0.05). PB also drove enteric neurodegeneration in male mice (15.5% neuronal loss, p<0.05) and increased the proportion of excitatory enteric neurons (by 67.7% in 90 μg/mL group, p<0.05) in females. Despite having no effect on colonic permeability, exposure to PB caused major shifts in the expression of pro‐inflammatory cytokines and chemokines in the colon and brain that suggest immunosuppressive effects. Interestingly, immune disruption was still evident in the colon and brain of female animals at one month following exposure to PB. Our results show that the paradigm of PB exposure experienced by veterans of the Persian Gulf War contributes to long‐lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering functional anatomy of the colon in a sex‐dependent manner.

Support or Funding Information

Department of Defense (DOD) W81XWH1610631

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using dual labeling of thymidine analogs to investigate cell cycle dynamics in the regenerating salamander limb
Authors: Jeon Eun Kyung , Martinez Andy , Monaghan James R.
Published: June 2021

For almost two centuries, scientists have observed that most forms of regeneration fail to regrow in the absence of nerves, but at the cellular level, how the nerves regulate this phenomenon is still unknown. Understanding how cell cycle kinetics change when the nerve supply is cut would help us understand what role nerves play during regeneration. We used dual labeling of cells using thymidine analogs, 5‐bromo‐2′‐deoxyuridine (BrdU) and 5‐ethynyl‐2‐deoxyuridine (EdU), to quantify the changes in the cell cycle in the denervated regenerating limb of axolotl salamanders. Eight larval axolotls were denervated in their left limbs 12 days after amputation and injected with BrdU and EdU at 24 hour intervals. 72 hours after denervation, the limbs were collected and stained for DNA synthesis (S), mitosis (M), and cell death markers. The duration of S phase of the cell cycle shortened in the actively proliferating cells of denervated regenerating limb almost by half compared to those of the innervated regenerating limb. The total cell cycle lengths differed by about 10%, which suggests that the growth phases (G1 and G2) may have increased in length in the denervated limb. When the nerve supply was cut, less cells returned to cell cycle. Our results suggest that the nerves may regulate the duration of S phase. Future analysis of upregulated and downregulated proteins such as transcription factors during cell proliferation in denervated limb blastema may suggest what pathways are causing the observed changes in the cell cycle.

Support or Funding Information

This project was supported by Summer Scholars Independent Research Fellowship from the Office of Undergraduate Research and Fellowships and NSF grants 1558017 and 1656429.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome‐wide Discovery of Rare Riboswitches in Bacteria
Authors: Arachchilage Gayan Mirihana , Atilho Ruben , Stav Shira , Higgs Gadareth , Breaker Ronald
Published: June 2021

Riboswitches are noncoding RNAs (ncRNAs) that directly sense specific ligands and regulate various metabolic and physiological pathways. More than 40 different classes of riboswitches have been experimentally validated, and hundreds of additional novel riboswitch classes are predicted to remain to be discovered and analyzed. We are exploiting an improved computational pipeline that can be used to comprehensively uncover novel structured ncRNAs in bacteria, including rare riboswitches. Comprehensive analysis of long and GC‐rich intergenic regions in five different bacterial genomes yielded nearly 70 novel genetic element candidates, including 30 putative ncRNA motifs. Among these novel ncRNA classes, five are considered strong riboswitch candidates. One of the riboswitch candidates, called the thiS motif, has now been experimentally proven to regulate thiamin biosynthesis by directly sensing 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine pyrophosphate (HMP‐PP). The mechanistic studies of this HMP‐PP riboswitch demonstrate that the nascent RNA transcript undergoes a conformational change from an ‘on’ state to an ‘off’ state during the transcription process. HMP‐PP binding stabilizes the riboswitch aptamer in its ‘on’ conformation. Experimental validation of the HMP‐PP riboswitch also indicates that the existing bioinformatics pipeline can now be exploited to discover the vast majority of structured ncRNAs hidden among hundreds of sequenced bacterial genomes.

Support or Funding Information

High‐performance computing at the Yale Center for Research Computing is supported in part by Yale University and by NIH S10RR029676. R.M.A was supported by the National Science Foundation Graduate Research Fellowship Program (DGE1122492). This work was supported by the National Institutes of Health grants (GM022778, DE022340) to R.R.B. Research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Mechanism of Action of Membrane Fusion Protein IncA Using Molecular Dynamics Simulations
Authors: Greco Deanna L. , Bryer Alexander J. , Perilla Juan R.
Published: June 2021

Infecting millions of people each year, Chlamydia trachomatis is currently the leading cause of non‐congenital blindness and pelvic inflammatory disease. Incapable of replicating on its own, this bacterium survives by entering a host cell through endocytosis and subsequently modifying the vacuole in which it is enclosed to allow for replication. The newly formed replicative compartment, referred to as an inclusion, has numerous inclusion membrane proteins (Inc proteins) that promote growth and maintenance of the developing C. trachomatis bodies. One Inc in particular, known as IncA, exponentially enhances growth of C. trachomatis by initiating the homotypic fusion of multiple inclusions within a host cell. Although the structure and significance of IncA are known, its mechanism of action remains a mystery. The present research aims to elucidate the structure‐function relationship of IncA; offering insight into fusion protein functionality and potential targets for drug development. Analysis of IncA was accomplished by running Constant pH Molecular Dynamics (CpH MD) simulations with a refined focus on aspartic acids, glutamic acids, and histidines. Similar to conventional MD, CpH MD creates conditions that mimic the natural environment of IncA but with the additional capability to explore protonation states of residues. By running the CpH MD system across a range of pHs, the necessary data are generated to determine dissociation constants for specific residues. Combining proton dissociation with tests examining the solvent accessibility of IncA residues, results suggested that there is a correlation between accessibility of a residue and its proton dissociation. Further analysis of residue fluctuation at varying pHs shows that there appears to be a relationship between a residue's protonation state and the degree to which that residue fluctuated throughout a simulation. These findings demonstrate that the location of titratable residues influence the activity of IncA and furthermore, that IncA's titratable residues affect inclusion membrane fusion.

Support or Funding Information

National Science Foundation, Grant No. 1560325.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Molecular Mechanisms of Antibiotic Resistance in Acinetobacter baumannii
Authors: Whitehead Briana , Robinson Kyra , Alexander Heidi , Marshall Dana , Damo Steven
Published: June 2021

The threat of antibiotic resistance has increasingly become a global issue because of the gradual emergence of multi‐drug resistant (MDR) pathogens. Identifying mechanisms of resistance as well as characterization of attributes deemed beneficial for the survival of the pathogen is vital in contributing to the development of new therapeutics. Acinetobacter baumannii is a gram‐negative opportunistic, nosocomial MDR pathogen that primarily targets immunocompromised patients in medical care facilities around the world. From an assembled bank of clinical isolates of A. baumannii, there is a diverse spectrum of antibiotic resistance patterns, cell motility and biofilm formation represented. Initial RNA sequencing studies implicated the importance of the Gcn5‐related N‐acetyltransferases (GNATs) in facilitating A baumannii resistance to the aminoglycoside class of antibiotics. We aim to determine the role of GNAT proteins to antibiotic resistance in A. baumannii as well as identify which physiological processes are associated with antibiotic resistance. GNATs belong to a superfamily of enzymes that are found in all domains of life and are involved various functions including acetylation of a broad spectrum of substrates and, often times, conferring resistance to antibiotics. We performed structural and functional experiments to understand the mechanisms causing the resistant nature of A. baumannii. Preliminary crystallization studies of the enzyme GNAT 2199 yielded 4 initial conditions suitable for optimal crystal growth. We also conducted a series of enzyme activity assays which revealed that GNATs acetylate various clinically used aminoglycosides. seeking to reveal the natural aminoglycoside substrate for GNAT 2199. Furthermore, cell motility and biofilm assays were used to determine the physiological processes attributed to antibiotic resistance of A. baumannii isolates. Future work entails optimization of the crystallization conditions to produce samples suitable for X‐ray diffraction studies. This will allow us to develop GNAT superfamily structure‐function relationships as well as identify physiological processes of A. baumannii that contribute to antibiotic resistance. In total, this information will be useful for initiating new strategies to control the spread of this bacteria as well as developing new therapeutics to effectively combat this MDR pathogen.

Support or Funding Information

NSF No. HRD‐1547757

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