bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel drug inhibits exosome content loading and secretion in ovarian cancer cells
Authors: Vaughn Kiera , Lausen James , Bruns Matthew , Benbrook Doris , Hannafon Bethany
Published: April 2020

Ovarian cancer is the 5th leading cause of cancer deaths in women due to late stage diagnosis and limited treatment options. Exosomes are nanometer‐sized vesicles produced in multi‐vesicular bodies (MVBs) and released into the extracellular environment. Exosomes play a role in the progression of ovarian cancer by encapsulating and transferring DNA, RNA (including microRNAs), proteins, and lipids between cells and modifying the tumor microenvironment. Data from our laboratory shows that a novel drug binds to proteins involved in exosome secretion, transport, and content loading, including microRNA binding proteins. The goal of this research is to determine whether this novel drug, called tumor exosome inhibitor (TEXi), can prevent secretion and/or content loading of ovarian cancer exosomes. Using an exosome secretion assay (CD63‐nanoluciferase), nanoparticle tracking analysis, and western blot we found that TEXi inhibits exosome secretion in ovarian cancer cells. Ovarian cancer cells expressing CD63 tagged with GFP allowed for visualization of exosome production and co‐staining for lysosomes and mitochondria allowed for the visualization of the relationship between exosome production in MVBs and these other organelles. Treatment with TEXi followed by live and fixed‐cell imaging demonstrated that TEXi inhibits the secretion by promoting fusion of MVBs with the lysosome. Exosome microRNA secretion, measured by qRT‐PCR, was also reduced by TEXi treatment. In conclusion, TEXi can block exosome and microRNA release by promoting MVB lysosomal degradation. This study has identified a potential new strategy for the treatment of ovarian cancer by blocking the effect of exosomes on the tumor microenvironment.

Support or Funding Information

Native American Research Center for Health (NARCH), American Society of Investigative Pathology‐Summer Research Opportunity Program in Pathology (ASIP‐SROPP) and Oklahoma Center for the Advancement of Science and Technology (OCAST) HR17‐052.
Activity of NADH‐ and FADH2‐linked mitochondrial respiration are significantly decreased in two mice models of Parkinson’s disease
Authors: Soliz Jorge , Picón Marina Lorente , Doucet-Beaupré Hélène Hélène , Lévesque Martin , Laouafa Sofien
Published: April 2020

Parkinson’s disease (PD) is a complex neurodegenerative disorder, which etiology is still largely unknown. Overwhelming evidence indicates that mitochondrial dysfunction is a central factor in PD pathophysiology. Here we tested the hypothesis that the activity of complex I and II (NADH‐and FADH2‐linked mitochondrial respiration, respectively) in the substantia nigra are significantly decreased in PD. To test this hypothesis, we used two different mice models of PD: Mice that received a single unilateral intrastriatal injection of 1) 6‐OHDA (9μg ‐ a selective catecholaminergic neurotoxin), or 2) A53T‐α‐synuclein (5.62E12 GC/ml – a molecule that attenuates synaptic vesicle recycling and neurotransmitter release). Control groups were injected with vehicle (fluorescent protein). Mitochondrial oxygen consumption was evaluated by using our oxygraph‐2K system (O2k‐OROBOROS), in 2 mg weight of tissue permeabilized with saponin. Measurements were performed one week after treatment with 6‐OHDA, and five weeks after treatment with A53T‐α‐synuclein. Our preliminary results show that, compared to control animals, substrates of complex I (Pyruvate/Glutamate/Malate), in the presence of ADP (state 3 complex I), induced a reduced oxygen consumption in mice treated with 6‐OHDA,(50.65 ± 2 vs 78 ±3 pmol O2/s/mg tissue, p<0.001). Inline, compared to control animals, oxygen consumption in state 3 complex I, was significantly reduced in mice treated with α‐synuclein (31.05 ± 5 vs 43.56 ± 2 pmol O2/s/mg tissue, p<0.05). Moreover, oxygen consumption in the presence of substrates of the mitochondrial complex II (succinate – state 3‐complex II), was also significantly reduced after treatment with α‐synuclein (30.8 ± 4.5 vs 46.5 ± 5 pmol O2/s/mg tissue, p<0.05). No differences of oxygen consumption were found in the presence of PMG, but the absence of ADP (state 4 – mitochondrial membrane leak) with none of the treatments. We concluded that 6‐OHDA and α‐synuclein, two reliable models of PD, induce mitochondrial dysfunction by decreasing the activity of mitochondrial complexes I and II. Our results contribute to a better understanding of the mitochondrial dysfunction induced by PD, and open the avenue for alternative treatments aiming to avoid the degeneration of dopaminergic neurons in the substantia nigra.

Support or Funding Information

Jorge Soliz is supported by the “Fonds de recherche du Quebec‐Santé” (FRQ‐S; FQ121919)
Enhanced expression of the full‐length seven transmembrane mu opioid receptor by MOR‐1G, a truncated six transmembrane splice variants through heterodimerization
Authors: Zhang Tiffany , Xu Jin , Pan Ying-Xian
Published: April 2020

The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating an array of splice variants. One type of the splice variants are truncated variants that have six transmembrane (TM) domains lacking the first TM. Of five 6TM variants in mouse, MOR‐1G was quite abundant and conserved from rodent to humans. The 6TM variants are essential for analgesic actions of a novel type of analgesic drugs such as 3‐iodobenzoyl‐6β‐naltrexamide (IBNtxA) that is potent analgesic against a spectrum of pain models without many traditional opiate side effects. The 6TM variants also involve analgesia of delta and kappa opioids and α2‐adrenergic drugs. In the present study, we demonstrate a new function of MOR‐1G in enhancing expression of the full‐length 7TM mu opioid receptor, MOR‐1, using a Tet‐Off inducible system in Chinese Hamster Ovary cells. When co‐expressed, MOR‐1G had no effect on expression of MOR‐1 mRNA, but greatly increased MOR‐1 expression at protein level in a dose‐dependent manner determined by opioid receptor binding and [35S]GTPγS binding. Interestingly, co‐expression of MOR‐1 with MOR‐1G significantly altered mu binding profile for several mu agonists, including M6G, β‐endorphin and dynorphin A, determined by competition assay. We further show that MOR‐1G can physically interact with MOR‐1 through heterodimerization using co‐immunoprecipitation approach, providing a molecular mechanism of how MOR‐1G regulates expression and function of the full‐length 7TM mu opioid receptor.

Support or Funding Information

This work was supported by the National Institute on Drug Abuse (DA042888, DA046714 and DA007242), the Mayday Foundation, the Peter F. McManus Charitable Trust and a core grant from the National Cancer Institute to Memorial Sloan‐Kettering Cancer Center (CA08748).
Isolation and Purification of Functional Amyloid Proteins in Haloferax volcanii
Authors: Sawyer Erica , Kozodoy Lisa , Raimer Heather , Olendzenski Lorraine , Marano Nadia
Published: April 2020

Haloferax volcanii, an extremophile species of Archaea, is a microorganism that incorporates functional amyloids into its biofilms; these proteins are proposed to contribute to the structural stability of this matrix. We developed a protocol based on the procedure described by Jordal et al. (2009) to isolate these proteins via differential centrifugation. The presence of amyloid proteins was monitored and quantified throughout this process using Thioflavin T, a molecule that can selectively quantify amyloids by fluorescing when bound to their cross‐beta motif. These results showed that a four‐week growth period was ideal and resulted in the highest amyloid fiber yield. The final supernatant contained the majority of the amyloid protein and had percent recoveries ranging from 41.2% to 63.4%, while the final pellet had recoveries from 6.3%‐18.1%. To further improve the efficiency of this procedure, we are utilizing the detergent Triton X‐100 during the isolation process. Preliminary findings show that this detergent slightly increases the amyloid percent recovery. Isolated proteins will ultimately be purified using preparatory SDS‐PAGE for atomic force microscopy imaging, deaggregated into their monomeric forms using formic acid, and sequenced to gain further information about their structure.

Support or Funding Information

SLU Fellowship Program & Strading Research Fund
Determining the DNA Binding Preference of the Transcription Factor Optix in Heliconius Butterflies
Authors: Barreto Anthony Rafael Rivera , Martínez José Arcadio Rodríguez
Published: April 2020

Transcription factors (TFs) are sequence‐specific DNA binding proteins that determine cell state and have been shown to control cell differentiation. Evaluating comprehensive DNA specificity profiles of DNA binding proteins is a nontrivial challenge that hinders our ability to decipher gene regulatory networks. Optix is a homeodomain transcription factor that has been identified as a master regulator of red color patterning in the wings of Heliconius butterflies. However, the direct gene targets of optix and its role in final wing coloration remains to be determined. Because of this, we want to determine the intrinsic DNA‐binding preferences of optix. We have over‐expressed and purified full‐length Heliconius optix. To achieve our goal, we have used Cognate Site Identification by Systematic Evolution of Ligands by Exponential Enrichment with Parallel sequencing (SELEX‐seq). We are currently predicting optix possible binding sites, by using computational analysis. Our results will enable us to bioinformatically predict the genomic targets of optix and decipher its role in wing color patterning and development.

Support or Funding Information

NSF EPSCoR Research Infrastructure grant award 1736026

Research Initiative for Scientific Enhancement (RISE) 5R25GM061151‐18
Restoration of the Blood‐Spinal Cord Barrier after Spinal Cord Injury through 5‐HT1F Receptor Mediated Mitochondrial Biogenesis
Authors: Simmons Epiphani Ciara , Carroll Seth S. , Schnellmann Rick G.
Published: April 2020

Vascular and mitochondrial dysfunction are well‐established consequences of spinal cord injury (SCI). Evidence suggests mitigating these dysfunctions may be an effective approach in treating SCI. We propose to restore vascular and mitochondrial dysfunction following SCI through pharmacological induction of mitochondrial biogenesis (MB). We previously reported that 5‐hydroxytryptamine receptor 1F (5‐HT1F) agonism induces MB in the spinal cord and promotes locomotor recovery with drug initiation beginning at a clinically relevant time point of 8h post‐SCI. The goal of this study was to elucidate if MB induction with the newer selective 5‐HT1F receptor agonist lasmiditan can enhance restoration of the blood‐spinal cord barrier (BSCB) after SCI. C57BL/6J mice were subjected to moderate SCI using a force‐controlled impactor‐induced contusion model followed by daily administration of lasmiditan (0.1 mg/kg, i.p.) beginning 1h after injury. Integrity of the BSCB was assessed using Evans Blue dye extravasation. SCI increased dye extravasation at 1, 3, and 7d, and lasmiditan‐treated mice displayed reduced dye accumulation at 7d post‐SCI, suggesting accelerated BSCB recovery. Levels of tight junction proteins occludin and zonula occludens‐1 were decreased in vehicle‐treated mice and returned to sham levels with lasmiditan treatment by d7. These findings led us to investigate if lasmiditan induces MB in endothelial cells. Primary cultures of cerebral microvascular endothelial cells from C57BL6 mice were treated with lasmiditan. A Seahorse Biosciences flux analyzer was used to quantify FCCP‐uncoupled oxygen consumption rate (OCR), a marker of MB. Lasmiditan increased FCCP‐uncoupled OCR, nuclear‐ and mitochondrial‐encoded proteins (PGC‐1a, COX1, TFAM), and mitochondrial number, confirming MB. Lastly, treatment with lasmiditan enhanced endothelial branching morphogenesis and migration, indicating a role of the 5‐HT1F receptor activation in angiogenic pathways. Collectively, these data reveal that 5‐HT1F receptor agonist‐induced MB occurs in endothelial cells and may accelerate restoration of the BSCB after SCI.

Support or Funding Information

This study was supported by the National Institutes of Health National Institute of General Medical Sciences: GM084147 (R.G.S), and the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs: BX: 000851 (R.G.S.).
Structural Mechanism of Substrate Specificity in Human Cytidine Deaminase Family APOBEC3s
Authors: Hou Shurong , Nalivaika Ellen A. , Myint Wazo , Matsuo Hiroshi , Yilmaz Nese Kurt , Schiffer Celia A.
Published: April 2020

APOBEC3s proteins (A3s), a family of cytidine deaminases, protect the host cell from endogenous retro‐elements and exogenous viral infections by introducing hypermutations. However, ability to mutate genomic DNA makes A3s a potential cancer source, as has been reported for A3A, A3B and A3H. Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA‐bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using advanced molecular modeling followed by experimental mutational analysis and molecular dynamics (MD) simulations, we revealed the structural basis for DNA binding and relatively lower catalytic activity of A3B compared to the highly similar A3A. We identified Arg211 in loop 1 as the gatekeeper coordinating DNA, and critical residues for nucleotide specificity. We also identified a unique auto‐inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity. Recently, we investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive structural analysis of A3 domains in this project will reveal determinants of substrate specificity and shed light into the biological function of these enzymes, potentially leading to discover inhibitors to benefit cancer therapeutics. In addition, rational engineering of A3 domains to achieve varying catalytic efficiencies or distinct substrate specificity will be greatly beneficial for CPRISR–A3 base editing systems in gene therapy.

Support or Funding Information

This work is supported by the U.S. National Institutes of Health Institute of General Medical Sciences [R01GM118474].
Peripheral cannabinoid CB1 receptors control nutrient‐induced incretin secretion in vivo
Authors: Perez Pedro Antonio , Argueta Donovan , DiPatrizio Nicholas Vincent
Published: April 2020

Enteroendocrine cells along the intestinal epithelium secrete incretins and related hormones (i.e. GIP, GLP‐1, and CCK‐8) following ingestion of nutrients. Incretins bind and activate their respective receptors on cells located in – but not limited to – the endocrine pancreas, which enhances insulin secretion and maintains a tight window of circulating blood glucose. The ability to adequately regulate circulating glucose levels is lost in patients living with prediabetes and type 2 diabetes. The endocannabinoid system controls food intake and glucose homeostasis, and its activity becomes upregulated in the proximal small‐intestinal epithelium of Western‐diet induced obese mice. Cannabinoid subtype‐1 receptors (CB1Rs) are present in various incretin‐producing cells in the intestinal epithelium (e.g. K, L, I‐cells), yet their function in these cell types are poorly characterized. In the current investigation, we examined the ability for CB1Rs to control incretin secretion in vivo in male C57CL/6N mice. We administered the cannabinoid receptor agonist, WIN 55,212‐2 (WIN, 3 mg per kg), alone and in combination with the peripherally‐restricted neutral CB1R antagonist, AM6545 (10 mg per kg). Incretin secretion was then stimulated via corn oil gavage (0.5 mL 30 minutes after drug treatment) and circulating incretins were quantified by sensitive enzyme‐linked immunosorbent assays from plasma obtained 30 minutes later. Activating CB1Rs with WIN blunted corn‐oil induced secretion of GIP, active GLP‐1 and CCK‐8. Co‐administration of WIN with AM6545 completely reversed the inhibitory actions of WIN on incretin release. Collectively, our results suggest that CB1Rs in the small‐intestinal epithelium control nutrient induced incretin release. The functional consequences of these findings on glucose homeostasis are currently under investigation.

Support or Funding Information

Acknowledgements

Funded by the National Institutes of Health, USA (Grant no. DK119498, DK114978, and DA034009)
Modulations in Structure, Dynamics, and Allostery for Emerging Beta‐Lactamase Variants
Authors: Page Richard C.
Published: April 2020

Antibiotic resistance is a continual and growing threat to human health. The use, and overuse, of antibiotics increases the prevalence of antibiotic resistance observed in the clinic. The concomitant emergence of new beta‐lactamase variants, capable of hydrolyzing a broader library of beta‐lactam antibiotics, is particularly worrisome. The development of new antibiotics and of beta‐lactamase inhibitors for restoring efficacy of current generation antibiotics depends upon a strong understanding of the structures, dynamics, and substrate profiles for clinically‐relevant beta‐lactamases. Here we discuss ongoing biophysical, structural, and biochemical efforts using NMR, X‐ray crystallography, differential scanning fluorimetry, and a range of activity assays. These efforts are increasing our understanding of emerging beta‐lactamase variants and producing custom‐designed beta‐lactamase constructs to enable studies on fundamental mechanisms, stability, dynamics, and substrate interaction profiles.

Support or Funding Information

NIGMS R35 GM128595
HMGB1 Initiates Inflammasome Mediated Pyroptosis, Atrophy and Adverse Muscle Remodeling
Authors: Narasimhulu Chandrakala Aluganti , Singla Dinender K.
Published: April 2020

Diabetic cardiomyopathy and associated muscle toxicity is a major concern, therefore it is essential to understand the mechanism and pathophysiology of diabetic skeletal muscle which remains unknown. The current study is undertaken to investigate whether there is an upregulation of High Mobility Group‐B1 (HMGB1) that initiates inflammation induced pyroptosis and involves adverse muscle remodeling. C57BL/6J mice (10±2 weeks) were divided into 2‐groups (n=16/group; 8males and 8females); control (saline) and STZ treated (200mg/kg body weight) via i.p. injection. Diabetes was established by determining the increased levels of glucose in STZ animals. At Day‐42, muscle function was determined using Grip‐strength meter and Rotarod. Mice were euthanized and gastrocnemius (GM) muscle was harvested. Pyroptotic initiator HMGB1, activator TLR4, inflammasome marker (NLRP3), and pyroptotic markers (caspase1, IL1β, IL‐18 and GSDMD) were examined using immunohistochemistry, western blotting and RT‐PCR. Muscle atrophy and fibrosis was examined histologically. IL‐6 inflammatory cytokine levels were analyzed using ELISA. We observed a significant (p<0.05) increase in glucose levels, pyroptotic initiator HMGB1, inflammasome and pyroptotic markers in GM of STZ administered mice as compared to control. A significant (p<0.05) decrease in myofibrillar area was observed in diabetic mice. Atrophy was also confirmed by significant increase (p<0.05) in MuRF1 gene expression. Interstitial and vascular fibrosis was significantly (p<0.05) increased in STZ group versus control. Moreover, a significant decrease (p<0.05) in muscle functions were observed in diabetic animals compared to control. Furthermore, a significant (p<0.05) increase in plasma IL‐6 levels were observed in diabetic mice. In conclusion, our data suggest that diabetes involves a novel form of inflammation induced pyroptosis that leads to muscle dysfunction. Further therapeutic approaches need to be developed to target pyroptosis in diabetes.
Molecular and cellular insights into mechanosensation and volume regulation in the nervous system
Authors: Brohawn Stephen
Published: April 2020

The transduction of physical forces into cellular electrical signals is fundamental to a wide breadth of biology including the classic senses of touch, pain, hearing, and balance; less conspicuous sensations of proprioception, blood pressure, and osmolarity; and basic aspects of neural growth, signaling, and death. In this talk, I will discuss recent work aimed at understanding the basis of mechanosensation and volume regulation in the nervous system. First, I will present a model for the mechanism of membrane tension sensing and physiological roles of a mechanosensitive potassium channel based on a combination of structural, electrophysiological, and localization data. Second, I will present a model for ionic strength sensing and channel gating in a family of volume regulated anion channels based on structural and functional studies.

Support or Funding Information

New York Stem Cell Foundation ‐ Robertson Neuroscience Investigator Award, McKnight Scholar Award, Klingenstein‐Simons Fellowship Award, NIGMS New Innovator Award, Rose Hill Innovator Award
Exploring Hsp70 chaperone interactions with a full‐length substrate
Authors: Saini Sashrika , Clérico Eugenia M. , Meng Wenli , Gierasch Lila M.
Published: April 2020

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding and preventing proteins from aggregation (Clérico et al., 2015). Despite extensive research that reveals a selective promiscuity between the Hsp70 substrate‐binding domain (SBD) and client proteins, there is a paucity of data on the interaction of Hsp70s with large protein clients. To investigate the interaction between Hsp70s and a full‐length substrate, we chose to characterize the binding of the E. coli homolog,DnaK, to the precursor of alkaline phosphatase (proPhoA). proPhoA is an endogenous substrate of DnaK that is unfolded and soluble under reducing conditions, and contains at least five sequences that bind DnaK when presented as short peptides (Rüdiger et al., 1997; Wild et al., 1992). To simplify the experimental conditions we used truncated versions of proPhoA (~100 residues long) that contain 1 or 2 binding sites. We used biophysical techniques to reveal the affinity of binding, and NMR spectroscopy to elucidate the mode of chaperone binding to the proPhoA fragments. We found that there is a hierarchy of affinities among the 5 potential binding sites, and novel cryptic binding sites have been observed. In addition, data suggest there may be a role for residues beyond the canonical five‐residue core sequences. The mechanism of DnaK binding site selection on a substrate containing multiple potential sites and the structural basis of interaction between the chaperone and its respective binding sites on a full‐length substrate will be described.

Support or Funding Information

NIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/2021
Knockdown of Interleukin 17 Receptor A (IL‐17RA) in Paraventricular Nucleus of Hypothalamus Attenuates IL‐17‐induced Pressor Responses and Sympathetic Excitation
Authors: Yu Yang , Cao Yiling , Felder Robert B. , Wei Shun-Guang
Published: April 2020

Inflammation contributes significantly to cardiovascular and autonomic dysfunction in hypertension and heart failure. Over the years, the pro‐inflammatory cytokine interleukin (IL)‐17 (also known as IL‐17A) has been recognized as a critical inflammatory regulator bridging immune responses and tissue inflammation. Our previous studies have demonstrated that peripherally or centrally administrated IL‐17A dramatically elevates blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in rats, and that its receptors IL‐17RA are richly expressed in the brain. The present work sought to determine whether IL‐17RA in the hypothalamic paraventricular nucleus (PVN), a key cardiovascular/autonomic brain center, mediate hemodynamic and sympathetic responses to systemically administered IL‐17A. In urethane anesthetized male SD rats, Mean BP (MBP, mmHg), HR (beats/min) and RSNA (% change) were recorded before and for 6–8 hours after an intravenous (IV) injection of IL‐17A (500 ng). In age‐matched control rats, IV IL‐17A induced a large and long‐lasting increase (*p<0.001 vs. baseline) in MBP (16.2 ± 2.3*), HR (49.8 ± 5.1*) and RSNA (165.6 ± 13.2 %*), beginning within 120 mins and peaking 6–8 hours after the injection. However, in rats pretreated with bilateral PVN microinjections of an IL‐17RA siRNA AAV virus and studied one week later, the IV IL‐17A‐elicited pressor responses in MBP (10.1 ± 2.1*), HR (22.1 ± 4.0*) and RSNA (68.5 ± 7.8 %*) were significantly (p<0.05) attenuated. mRNA for tumor necrosis factor‐α (TNF‐α), IL‐1β, monocyte chemoattractant protein‐1 (MCP‐1) and IL‐17RA in the PVN was measured by real‐time qPCR. Knockdown efficiency of IL‐17RA siRNA was confirmed by a reduction (−55%) of IL‐17RA mRNA level in the PVN. Additionally, the mRNA levels of TNF‐α (1.12 ± 0.24 vs. 0.51 ± 0.11*), IL‐1β (1.05 ± 0.16 vs. 0.64 ± 0.09*), and MCP‐1 (1.01 ± 0.10 vs. 0.60 ± 0.10*) induced by IV IL‐17A were significantly reduced in the PVN in rats pretreated with IL‐17RA siRNA. These data indicate that systemically administered IL‐17A acts upon IL‐17RA in the PVN to increase the expression of inflammatory mediators that drive sympathetic excitation, suggesting that PVN plays an important role in mediating the effects of circulating IL‐17A on sympathetic drive and cardiovascular function in inflammatory conditions such as hypertension and heart failure. IL‐17RA in the PVN is a potential target for therapeutic intervention in these diseases.

Support or Funding Information

This research was supported by NIH/NHLBI RO1 HL139521.
Seasonal Cytotoxic Effects of Urban Watersheds in the Greater Houston Area
Authors: Abdullah-Smoot Dayana Attiya
Published: April 2020

Steadily rising populations are turning Houston, Texas to the urban concrete jungle it is today. With communities growing and searching for homes, the need for new construction and establishments only increases the amount of impermeable surfaces relying heavily on the native waterways. Houston is known for its complex network of waterways surrounding and interweaving throughout the city, which are referred to as bayous. They work as a storm water runoff system to help prevent flooding, but many communities use the watersheds for recreational purposes. Local patrons boat, fish and swim in the bayous, unaware of the chemicals and pollutants lurking in the water. Due to the extensive flood history in Houston and the surrounding areas, communities do not have to be near the bayou to be inundated with the polluted water. Heavy rain for only an hour or two can cause bayous to rise, streets flood and water can make the find its way into low‐lying homes. Current studies have shown that there are high concentrations of As, Hg, Cr, Pb and Zn in the water. We hypothesize that the metals and chemical compounds identified could cause the water to have deleterious effects on living organisms occupying the watersheds or using its resources. My study objectives include observing the cellular response when the cells are subjected to the bayou water samples collected during the summer and fall of 2018 to compare cellular viability and to determine which of the watersheds has the greatest over cytotoxic effects.

Water samples were collected in triplicates from Mustang Bayou, Dickinson Bayou, Horsepen Bayou, Houston Clear Lake, Cypress Creek and Cypress Creek Lake, which are six major waterways in the surrounding Houston area. The methods included buffering the water sample that will not be detrimental to the cells, then serial dilute the samples in media for varying concentrations (control or 0%, 12%, 25%, 50%, 100%) and tested the bayou water samples on the HT29 colon cells using a cytotoxicity assay and proliferation assays at 12, 24, and 48‐hour time points.

We observed a linear decrease in viable cells as the treatment time points of bayou water increased. Cytotoxic effects were observed at every time point no matter the concentration of bayou water in the sample and the highest overall cellular viability was observed in the fall 2018 samples, because the overall viability percentages were roughly 20% higher than summer samples for both the 12hr and 24hr treatments although the 48hr results stayed consistent. Future studies include treating FHC colon cells with the same bayou water concentrations to compare to the HT29 cellular response and investigate the cytotoxic cellular effect with media containing the chemical elements at the concentrations determined from the chemical analysis.

Support or Funding Information

This research was primarily supported by the National Science Foundation (NSF) through Texas Southern University (TSU) under the award numbers HRD‐1622993, BCS‐1831205, and HRD‐1829184
Loss of colonic epithelial NCOR1 aggravates experimental colitis chronicity
Authors: Boudreau Francois , Lecours Mia , Di Castro Ariane Cristina , Nicolas Vilcy Reyes , Jones Christine , Perreault Nathalie
Published: April 2020

The nuclear co‐repressor NCOR1 orchestrates the assembly of a large transcriptional repression complex that is involved in the negative regulation of pro‐inflammatory genes. Chronic intestinal inflammation is associated with sustained activation of pro‐inflammatory molecules. Since NCOR1 is considered as a potent repressor of pro‐inflammatory genes in macrophages, we aimed to investigate the role of intestinal epithelial NCOR1 during inflammatory stresses. Conditional deletion of Ncor1 in the whole intestinal epithelium was achieved by crossing Villin‐Cre and Ncor1loxP/loxP C57BL/6 mouse models. DSS‐induced colitis in NCOR1ΔIEC mice was more severe than control mice according to survival as well as clinical observations. A gene profiling analysis in the colon of non‐diseased NCOR1ΔIEC and control mice identified 85 unique and mapped transcripts being significantly modulated between NCOR1ΔIEC and control mice. An Ingenuity Pathway Analysis from these predicted target genes identified gastrointestinal disease (79 transcripts) as top disease and biofunction. Analysis of enriched targets in specific canonical pathways predicted an increase in the tryptophan degradation pathway (P = 3.2E‐02), a pathway recently demonstrated to be strongly relevant to inflammatory bowel disease severity. Indoleamine‐pyrrole 2,3‐dioxygenase (IDO1), that catalyzes the first and rate‐limiting step of tryptophan oxidation, was induced more than 7 times in the colon of NCOR1ΔIEC mice. IDO1 was also significantly more elevated in NCOR1ΔIEC mice when compared to control mice both subjected to chronic inflammation (3 cycles of DSS). Spontaneous induction of Ido1 gene expression was also confirmed in cultured ex vivo colon organoids deleted for Ncor1. In conclusion, our results highlight the critical role of NCOR1 to maintain intestinal inflammatory homeostasis during experimental colitis and uncover a novel function for NCOR1 in the regulation of Ido1 expression and potentially tryptophan metabolism.

Support or Funding Information

Canadian Institutes of Health Research
Secondary Analysis of Premature Respiratory Outcomes Program (PROP) Clinical Trial to Identify Correlations of Risk Factors for Respiratory Morbidity and Mortality in Premature Infants
Authors: Torres Juliet , Otero Jose Javier
Published: April 2020

Background

The Premature Respiratory Outcomes Program (PROP) was a collaborative, multi‐disciplinary clinical study that sought to analyze the factors that could predict future risk for respiratory morbidity and mortality for 835 premature infants with gestational ages 23 0/7 to 28 6/7 weeks. PROP investigators conducted physiologic challenges called Room Air Challenges (RAC) and Hypoxic Challenges Tests (HCT) to determine whether the premature infants could maintain their SpO2 in the target range of ≥90% and to assess pulmonary reserve in former premature infants without Bronchopulmonary Dysplasia(BPD) at 36 weeks’ premenstrual age (PMA). This project was funded by the National Institutes of Health (NIH) and the National Heart, Lung, and Blood Institute (NHLBI) for 5 years through a $18.5 million research initiative.

Objective

We are interested in understanding the relationship between brain imaging data and respiration outcomes, and infection or chorioamnionitis and respiration outcomes. In addition, the RAC and HCT provides the opportunity to analyze the outcomes of physiologic challenges in preterm infants compared to term infants undergoing the same challenges in studies that conducted preflight HCT.

Statement of the methods

We utilized machine learning tools such as random forest, support vector machines, gradient boosted machines, and logistic regression to generate predictive models of the aforementioned features.

Summary of results

Preliminary data analysis of key variables to be included in our future data analytics includes standard deviations for birth weight (gm) (235.98), the baby’s head circumference (cm) (83.05), APGAR for one minute (scored 1–10) (2.47), and APGAR 5 minutes (2.10).

Statement of conclusions

This PROP clinical study encapsulates an immense amount of multidisciplinary data from 835 premature infants, which is an extremely special population to include in research as approximately 1 out of every 9 births in the United States occurs prematurely.

Support or Funding Information

R01HL132355
Temporal RNA Integrity Analysis of Archived Spaceflight Biological Samples
Authors: Scott Ryan T. , Talburt Elizabeth Delgadillo , French Alison J. , Lopez Danielle , Polo San-Huei Lai , Boyko Valery , Dinh Marie T. , Rask Jon C. , Chakravarty Kaushik , Stewart Helen , Del Alto Martha E.
Published: April 2020

Spaceflight experiments have utilized model organisms for decades to assess effects of microgravity on biological systems. In many of those experiments, obtaining and assessing tissues from only a few biological systems are of interest to their Principal Investigators. To maximize scientific return from space flown experiments, the remaining tissues are captured, curated, and preserved in the Institutional Scientific Collection (ISC) at NASA Ames Research Center. The international scientific community is encouraged to request these available tissues through NASA’s ISC Biospecimen Sharing Program, and conduct research furthering insights into the field of space biology. The purpose of this study is to evaluate viability of tissues which have been archived for differing lengths of time within the NASA ISC. Garnering information from the NASA ISC tissues – such as downstream functional analysis for the generation of omics datasets – is partly dependent on the state of sample preservation. RNA integrity number (RIN) values were calculated for rodent liver tissues that were part of spaceflight scientific payloads. Rat livers were tested from Spacelab Life Sciences 1 (SLS‐1; 1991), as were mouse livers from Commercial Biomedical Test Module 3 (CBTM‐3; 2011), Rodent Research 1 (RR1; 2014), and Rodent Research 3 (RR3; 2016). It was found that mean RIN values from CBTM‐3, RR1, and RR3 were suitable for downstream functional analysis (RIN > 5), while the mean RIN value for SLS‐1 was not (RIN = 2.5 ± 0.1). This work to analyze RIN values of NASA ISC liver tissues will inform future investigators in their tissue and assay selections. This study lays the foundation for future efforts to understand the quality and viability of different tissues archived within the NASA ISC biospecimen repository.

Support or Funding Information

NASA Ames Research Center
Mouse Trophoblasts Can Provide Antiviral Protection to Embryonic Stem Cells
Authors: Fendereski Mona , Neupane Biswas , Bai Fengwei , Guo YanLin
Published: April 2020

The blastocyst is the preimplantation embryo that consists of two major components: the inner cell mass (ICM) and the trophectoderm, which give rise to the fetus and placenta, respectively. We have previously reported that embryonic stem cells (ESCs), the major component of the ICM of blastocyst, do not express type I interferons (IFNs) and lack response to inflammatory cytokines. One would assume that this could make ESCs susceptible to infection. Surprisingly ESCs are less susceptible to immunological cytotoxicity and are not hypersensitive to viral infection. These findings led to our hypothesis that the lack of antiviral responses could be a protective mechanism that allows ESCs to avoid negative impact of immune responses, but how ESCs deal with pathogen infection and immunological challenges is not known. In this study, we investigated IFN‐based antiviral innate immunity of the trophectoderm by using mouse trophoblast stem cells (TSCs) and their in vitro differentiated trophoblasts (TSC‐TBs). Unlike ESCs, our data indicated that both TSCs and TSC‐TBs have a functional IFN system as indicated by their ability to express IFNα, IFNβ, and IFNλ in respond to viral stimuli. They express IFN‐stimulated genes when treated with IFNα and IFNβ. We further showed that the conditioned medium from TSCs and TSC‐FBs stimulated with polyinosinic:polycytidylic acid (poly I:C), a synthetic viral RNA analog, contains antiviral activity that could inhibit infection of ESCs by Chikungunya and Zika virus, similar to the effect of purified IFNβ. These findings indicate that embryonic cell lineages in the blastocyst have different immunological properties at the same developmental stage. Although ESCs are deficient in producing IFNs, they can gain IFN antiviral protection from the trophectoderm via a paracrine signaling mechanism in the blastocyst. Therefore, lacking the production of IFN in ESCs could be a protective mechanism that allows ESCs to avoid the potential harmful impact of immune responses without compromising their antiviral innate immunity.

Support or Funding Information

This work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).
Low concentrations of nicotine increase frequency and decrease amplitude of respiratory bursts recorded from the hypoglossal nerve, with no change in amplitude of bursts recorded from the phrenic nerve, in the neonatal rat brainstem spinal cord preparation
Authors: Wollman Lila Buls , Flannigan Emily Gayle , Fregosi Ralph Frank
Published: April 2020

Nicotinic acetylcholine receptors (nAChRs) are expressed throughout the central respiratory network, including postsynaptically on pattern generating neurons and motor neurons, and presynaptically on both excitatory and inhibitory inputs to these neurons. Normally, specific populations of nAChRs are briefly activated by the endogenous neurotransmitter acetylcholine, which is then rapidly hydrolyzed by acetylcholinesterase. However, with active or passive nicotine exposure, low circulating concentrations of nicotine results in global and prolonged activation of nAChRs, the effects of which are largely unknown. Here, we used the brainstem spinal cord preparation and rhythmic brainstem slice from nicotine‐naïve neonatal rats on postnatal days 1–4 to study the effects of prolonged, global nAChR activation on respiratory motor output. In the brainstem spinal cord preparation, bath application of 250 nM nicotine for 30 minutes resulted in an increase in respiratory burst frequency (21 % above baseline) accompanied by a decrease in hypoglossal nerve amplitude (62 % below baseline), but no change in phrenic nerve amplitude. In the rhythmic brainstem slice, bath application of nicotine concentrations as low as 50 nM caused an increase in frequency (35 % above baseline) and a decrease in amplitude (33 % below baseline) of respiratory bursts recorded from the hypoglossal nerve. These results indicate that low concentrations of nicotine are sufficient to cause changes in the respiratory network, including profound inhibition of hypoglossal motor output.

Support or Funding Information

5R01HD071302‐07
Characterization of Amplified Malate Dehydrogenase 1 (MDH1) in the Context of Squamous Cell Lung Cancer
Authors: Alexander Sati Amrita , Weeks Joi , Hoang An , Sohl Christal
Published: April 2020

Cancer cells often reroute or stimulate their metabolism to support proliferation, which requires a change in the activity or regulation of key metabolic residues. Malate dehydrogenase 1 (MDH1) catalyzes the NADH‐dependent conversion of oxaloacetate to malate, which can help drive glycolysis through the production of NAD+. MDH1 is amplified in many squamous cell lung carcinomas, the second leading cause of cancer related death, and this amplification is associated with a poor prognosis of 50% reduced survival. The precise role of MDH1 in driving squamous cell lung cancer and the mechanisms that regulate this protein are not yet well understood.

Here, we develop a tool to examine the effect of MDH1 amplification in an in vitro model. We aim to examine MDH1 amplification in squamous cell lung carcinoma with an isogenic control. It has been previously shown that MDH1 methylation can suppress glutamine metabolism in pancreatic cancers, and that acetylation of MDH1 alters its activity. We will examine how MDH1 amplification affects squamous cell carcinomas by using biochemical kinetics and cellular methods. We have found that MDH1 amplification influences the production of metabolites in the TCA cycle. Through monoclonal single cell isolation, we will generate a squamous epithelial lung cancer cell line stably overexpressing MDH1. We hypothesize that MDH1 has cysteine residues that can be sensitive to oxidative stress which can alter the normal enzymatic activities of MDH1. Through site‐directed mutagenesis, we will mutate a cysteine residue to a serine residue at the 137 position to examine the possible effect of oxidative stress on MDH1. This residue has been shown to be sensitive to oxidation in the mouse form of MDH1. We will also heterologously express and purify MDH1 mutants and perform kinetic assays to examine the impact of amplified MDH1. By understanding the regulatory strategies of MDH1, we hope to identify new pathways that may be targeted therapeutically.

Support or Funding Information

This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).
Oxidative Susceptibility and Degradation of Calmodulin Cardiac Mutants
Authors: Moen Rebecca Jane , Julian Jordan , Isehak Imad , Thomas David Dale
Published: April 2020

Mutations within the ubiquitous calcium‐sensing protein calmodulin (CaM) have been linked to congenital arrhythmia disorders including long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In cardiomyocytes, CaM is critical for regulating excitation‐contraction coupling by binding calcium as well as binding a diverse set of targets including the ryanodine receptor (RyR2) to regulate intracellular calcium levels. The vertebrate CaM primary sequence contains nine methionine (Met) amino acid residues which are highly conserved. All nine Met are susceptible to oxidation to methionine sulfoxide (MetO). These methionine residues are solvent‐exposed upon binding calcium and facilitate CaM binding its targets. CaM also contains a potential PEST sequence, an internal signal for rapid proteolytic degradation. We have investigated the oxidative and proteolytic susceptibility of four calmodulinopathy mutants: D96V and D130G, which are linked to LQTS and N54I and N98S, which are associated with CPVT. The goal of this research is to establish a correlation of CaM disease mutants and oxidative susceptibility, with an emphasis on the compounding and detrimental functional and structural effects these two types of modifications may cause.

Support or Funding Information

This work is supported by a NIH grant AG26160 to DDT and a Minnesota State University, Mankato Undergraduate Research Center Foundation Grant to JJ.
Long OPA1 isoforms mediate a membrane potential‐dependent threshold for mitochondrial fusion in AC16 cardiomyocytes
Authors: Gilkerson Robert , Maynard Kristen , De La Torre Patrick , Keniry Megan
Published: April 2020

The bioenergetic function of mitochondria is linked to its organellar structural dynamics by optic atrophy‐1 (OPA1), a nuclear‐encoded protein that mediates fusion of the mitochondrial inner membrane. OPA1‐mediated inner membrane fusion is dependent on the transmembrane potential across the mitochondrial inner membrane (Δψm): long (L‐OPA1) isoforms accomplish fusion for an interconnected reticular morphology when Δψm is intact. Upon loss of Δψm, L‐OPA1 is cleaved by the OMA1 metalloprotease, causing accumulation of short S‐OPA1 and collapse of mitochondrial fusion to a fragmented morphology. We examined Δψm‐sensitive mitochondrial fusion in human AC16 cardiomyocytes treated with carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), an uncoupler of Δψm. Upon increasing titration with CCCP, mitochondrial fusion collapses at [CCCP] > 2 μM, as determined using blinded ImageJ quantification of mitochondrial morphology. Similarly, OPA1 Western blotting revealed that AC16s show a significant loss of fusion‐active L‐OPA1 at [CCCP] > 2 μM. Taken together, these findings indicate that L‐OPA1 maintains mitochondrial fusion, but that OMA1 is activated at [CCCP] > 2 μM, causing L‐OPA1 cleavage and collapse of mitochondrial fusion. Experiments in progress are examining OMA1 activation at this Δψm breakpoint, as well as impact of altered OPA1 and OMA1 expression.

Support or Funding Information

NIGMS 5SC3GM116669 (to R.G.)

NIGMS 1SC3GM132053 (to M.K.)

UTRGV Presidential Graduate Research Assistantship (to P. D.)