bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic dissection of endogenous siRNA involvement in the nonstop decay pathway in C. elegans
Authors: Mortezavi Evana , Wong Grace L , Youngman Elaine M
Published: June 2021

In Caenorhabditis elegans, endogenous small interfering RNAs known as 22G RNAs regulate expression of thousands of mRNAs and other transcripts in developing sperm and egg cells, and loss of 22G RNAs leads to reduced fertility. We are working to understand the molecular triggers that control which genes are targeted for regulation by 22G RNAs. Previous research in the Youngman lab identified a gene of unknown function, F43E2.6 (F43) that is targeted by 22G RNAs only in polymorphic wild strains of worms that lack a stop codon at F43. Conversely, when the stop codon is present, 22G RNA production is low. While it is well documented that mRNAs without a stop codon enter the nonstop decay pathway, resulting in destruction of the readthrough transcript, nonstop decay has not previously been linked to small silencing RNA pathways.

In addition to 22G RNAs, the F43 transcript is targeted by a Dicer product, and RDE‐1‐bound Dicer products trigger 22G RNA synthesis in the classical exogenous RNAi pathway. This leads to a critical question about how 22G RNA production is triggered at this locus: is it caused by the nonstop polymorphism, targeting by the Dicer product, or by both pathways acting in cooperation? Further, how does this novel biogenesis pathway integrate into known pathways for 22G RNA production in the C. elegans germline? To determine whether production of 22G RNAs at the F43 locus is independent of the Dicer product and dependent solely on the nonstop polymorphism, we first generated rde‐1; F43 nonstop(ns) homozygotes, which carry the nonstop allele in absence of the Dicer‐dependent RNAi pathway, and have completed deep sequencing of 22G RNAs from this strain. To further define the genetic requirements for production of 22G RNAs targeting this locus, we have sequenced 22G RNAs from strains that carry the nonstop polymorphism but lack either portions of the small RNA biogenesis machinery or of the known nonstop decay machinery (mut‐7; F43ns, prg‐1; F43ns, skih‐2; F43ns, and dom‐34; F43ns). Bioinformatic analyses to assess small RNA levels targeting F43 are underway and will be presented. Although it is well established that 22G RNAs serve to defend the genome against foreign or invasive nucleic acids, this work represents the first evidence that they may also serve to defend the transcriptome from the deleterious effects of low‐quality mRNAs, and suggests the intriguing possibility that quality control of mRNAs may play a role in animal fertility.

Support or Funding Information

NSF/MRI #1531855

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of protein‐protein interactions between isoforms of malate dehydrogenase and citrate synthase
Authors: Rementeria Gabriel , Berquist Blaine , Provost Joseph
Published: June 2021

Sequential enzymes involved in a metabolic pathway are often found in specific and dynamic protein‐protein interactions. The assembly of proteins into this metabolon allow the efficient transfer of substrate between adjacent proteins increasing the throughput and decreasing the potential for side metabolic reactions to occur between competing enzymes. One such pairing is the malate dehydrogenase (MDH) and citrate synthase (CS). In the forward direction of the tricarboxylic cycle, the oxidation of malate to OAA catalyzed by MDH is thermodynamically unfavorable. Coupling and potentially direct shuttling of product‐substrate affords the cycle to continue. The interaction site between mitochondrial MDH and CS has been partially identified via computational docking and cross‐linking studies. However, the domains and key residues of interaction and their impact on the actions and affinity of various isoforms of MDH and CS have not been identified. Using two biophysical techniques, protein thermal melts and surface plasminogen resonance, we have found both mitochondrial and cytosolic human MDH interact with CS (mitochondrial 3 fold greater than cytosolic), while plant MDH shows no interaction. Traditional pulldowns were used in addition to biophysical techniques. Using molecular crowding agents, we have identified the affinity and stoichiometry of interactions. This will allow us to probe the structures for key residues of interaction in the future.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Keto Acids React with Human Serum Albumin to Form Products Similar to Advanced Glycation Endproducts (SAGEs)
Authors: Cai Ang , Liu Weixi , Tang Xiaodan , Ma Hang , Seeram Navindra P. , Dain Joel A.
Published: June 2021

Diabetic ketoacidosis in children may contribute to cognitive decline. We report that in the model system, keto acids (KAs) can react with human serum albumin (HSA) and one of the effects is responsible for brain injury with children who have the diabetic ketoacidosis. KAs are a group of organic compounds containing functional groups including a carboxylic acid group and a ketone group. Some KAs, namely, pyruvic acid, α‐ketoglutaric acid, levulinic acid, and oxaloacetic acid, play significant roles in biological pathways related to carbohydrates metabolism. Although it is known that reduced derivatives of KAs including methylglyoxal are highly reactive to form advanced glycation endproducts (AGEs), the reactivity of KAs to form products similar to advanced glycation endproducts (SAGEs) remains unclear. Herein, we evaluated four keto acids including pyruvic acid, α‐ketoglutaric acid, levulinic acid, and oxaloacetic acid for their reactivity to form SAGEs with HSA. KAs were incubated with HSA at 37 °C for seven days and the formation of SAGEs were measured by fluorescent assay with an excitation and emission wavelength of 360 nm and 435 nm, respectively. Pyruvic acid and α‐ketoglutaric acid did not induce the formation of SAGEs while levulinic acid and oxaloacetic increased the formation of SAGEs compared to the control group. Further characterizations of KAs induced formation of SAGEs using a series of biophysical methods are warranted as they may provide critical insights of KAs induced formation of SAGEs and related physiological conditions. This result may contribute to understand brain injuries in diabetic ketoacidosis in children.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FLUORESCENCE SPECTROSCOPIC ANALYSIS OF TonB‐DEPENDENT TRANSPORT IN Klebsiella pneumoniae
Authors: Kumar Ashish , Chakravorty Somnath , Yang Taihao , Majumdar Aritri , Newton Sally , Klebba Phillip
Published: June 2021

Bacterial iron acquisition systems are potential targets for new antibiotics, because iron plays a central role in energy production, DNA synthesis, intermediate metabolism, nitrogen fixation, and oxygen detoxification. Bacteria actively acquire iron by secreting siderophores that chelate Fe3+, and then transporting the ferric siderophore complexes. Pathogenic K. pneumoniae produces at least four siderophores: enterobactin, glycosylated enterobactin, yersiniabactin, and aerobactin. Like all Gram‐negative species, K. pneumoniae obtains iron with TonB dependent transporters (TBDT) in its outer membrane (OM). For example, aided by the inner membrane protein TonB, that provides energy, the OM protein FepA actively transports ferric enterobactin (FeEnt). K. pneumoniae strain Kp52.145 contains three chromosomal and one plasmid‐mediated fepA homologs, that we individually cloned in pHSG575 and genetically engineered to encode Cys residues at strategic sites for attachment of maleimide fluorophores. Any of these FepA homologs efficiently functioned in the spectroscopic assay: FeEnt binding quenched light emissions from fluoresceinated FepA, but intensity recovered as the bacteria transported and depleted the ferric siderophore from solution. Furthermore, inhibitors that blocked the uptake of FeEnt prevented fluorescence recovery. This individualized, “species specific” method was sensitive and accurate, but genetic and biochemical manipulations of ESKAPE pathogens are potentially hazardous and technically difficult. So, we modified the approach to permit measurements of iron transport in clinical isolates without genetically engineering them. A TonB‐deficient E. coli strain with fluorescently labeled FepA in its OM binds, but cannot transport FeEnt, and thereby constitutes a “fluorescent decoy” (FD) sensor of [FeEnt]. When cohabiting an environment with ESKAPE bacteria (A. baumannii, P. aeruginosa, or E. cloacae), the E. coli sensor monitored their TonB‐dependent uptake of FeEnt. Such sensor strains are broadly applicable, in that they allow direct, in vivo measurements of the high‐affinity transport of virtually any ligand, in any organism. FD sensors also allowed high‐throughput screening of chemical libraries to find antagonists of TonB‐dependent iron uptake by K. pneumoniae or other bacteria. The increasing antibiotic resistance of Gram‐negative pathogens calls for novel agents against new microbial targets, and chemicals that block TonB action may thwart pathogenesis by preventing bacterial iron acquisition in humans and animals.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Med12 Mutations Promote Castration Resistant Prostate Cancer through Hyperactivated GLI3/SHH Signaling
Authors: Muthutkumar Soundharrya , Boyer Thomas , Burleson Marieke Oldenbroek
Published: June 2021

Prostate cancer is the highest diagnosed and second most common cause of cancer death among US men. The onset and progression of prostate cancer is critically dependent upon androgens through their interaction with the androgen receptor (AR). Accordingly, it has long been established that androgen deprivation therapy leads to prostate cancer regression. Though androgen deprivation therapies are initially highly successful, most patients eventually suffer from recurrence of the disease in the form of castration resistant prostate cancer (CRPC). Currently there is no effective treatment for CRPC thereby highlighting the compelling need to address resistance mechanisms for patients that suffer from this lethal disease. Sonic hedgehog (SHH) signaling is a stromal‐epithelial interacting pathway that is critical for prostate development and cell growth. SHH signaling is induced by androgen deprivation thereby leading to re‐activation of AR target genes to promote prostate cancer cell growth in the absence of androgens. Furthermore, AR interacts with GLI proteins, downstream effectors in the SHH pathway. Therefore, GLI3‐dependent SHH signaling in androgen deprived cells could promote re‐activation of AR target genes and consequently lead to CRPC. It is currently unknown how GLI3‐dependent SHH signaling is induced in CRPC. In this study we show that stabilized GLI3 leads to the induction of GLI3‐dependent SHH signaling in prostate cancers that harbor mutations in MED12. MED12 is an Xq13‐encoded 230 kDa subunit of the RNA pol II transcriptional Mediator that has been found to be mutated in ~5% of prostate cancers. Through stable knockdown and luciferase assays, we show that MED12 mutations disrupt a Mediator‐imposed constraint on GLI3‐dependent SHH signaling. Furthermore, through stable knockdown and xenograft studies, we show that stable GLI3 is critically dependent for CRPC growth. The results of our study therefore indicate that prostate cancer patients with MED12 mutations relapse to CRPC after androgen deprivation therapy by hyperactivating the GLI3 dependent SHH signaling pathway. Overall, this study provides critical insight into possible treatment strategies for prostate cancer patients with MED12 mutations by using the GLI3/SHH signaling axis as a therapeutic target.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lactobacillus paracasei PS23 modulated the age‐related inflammation in Senescence Accelerated Mouse Prone 8 (SAMP8) mice
Authors: Huang Hui‐Yu , Huang Ying‐Xu , Chen Yi‐Wen , Chou Nien‐Wei , Chen Yi‐Rong , Lee Hui‐Chun , Lee Yi‐Ju
Published: June 2021

Sarcopenia is a common impairment in the elderly population responsible for poor outcomes later in life, which is enhanced by inflammation. Only a few strategies have been reported to reduce sarcopenia. We investigated whether Lactobacillus paracasei PS23 (LPPS23) decelerates age‐related muscle loss and its inflammatory response; LPPS23 has been reported to delay some age‐related disorders. Female senescence‐accelerated mouse prone 8 (SAMP8) mice were divided into three groups (n = 6 each): non‐aging (16‐week‐old), control (28‐week‐old), and PS23 (28‐week‐old) groups. The control and PS23 groups were given saline and LPPS23, respectively. We evaluated the effects of LPPS23 by analyzing body weight and composition, reactive oxygen species, and IL‐6. The positive anti‐ROS effect of LPPS23 in the aged SAMP8 mice demonstrated that LPPS23 may protect muscle mitochondria against ROS damage, which may be associated with a lower level of sarcopenia in aged SAMP8 mice. Similar to LPPS23, some probiotics has been reported to have anti‐ROS properties; more probiotics may be also proofed to use for modulating sarcopenia in the future.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acrolein modifies protein functions and induces tissue damage at advanced age
Authors: Uemura Takeshi , Igarashi Kazuei
Published: June 2021

Acrolein (CH2=CHCHO) is a highly reactive unsaturated aldehyde generated mainly from oxidation of spermine, one of polyamines, in vivo. Acrolein causes strong oxidative stresses at sub microM concentration that is quite lower than reactive oxygen species (ROS). The activity of spermine oxidase, the enzyme involved in the generation of acrolein, is increased with age. Thus, acrolein plays important roles in tissue damage at advanced age such as brain stroke, dementia, renal failure and autoimmune diseases. As global society ages, it is required to prevent aging related diseases to maintain the quality of life (QOL) of the elderly. The objective of the presenting study is to identify the novel target of acrolein toxicity which can be a target for the prevention of age related disorders.

We have analyzed proteins modified with acrolein in cells and identified GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase), MMP‐9 (matrix metalloprotease 9), tubulin and GSTπ (glutathioneS‐transferase π). GAPDH was inactivated by acrolein modification that caused a decrease in cellular ATP content and induction of apoptosis through nuclear translocation. On the other hand, MMP‐9 was activated by acrolein to induce tissue damage in Sjogren's syndrome patients. Acrolein modification of tubulin inhibited polymerization of microtubules and disrupted cytoskeletal structures, then cell division and development of neuronal axons. GSTπ catalyzes conjugation of acrolein with glutathione in cells to neutralize it. GSTπ was modified and inactivated by acrolein to form polymer of proteins.

In conclusion, we have found that through modification of GAPDH, MMP‐9, tubulin and GSTπ proteins, acrolein caused tissue damage in age related disorders. Acrolein scavengers can prevent the modification of proteins and further tissue damage.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Berberine inhibits free fatty acid and LPS‐induced inflammation via modulating ER stress response in macrophages
Authors: Wang Yanyan , Yan Joanna , Chen Weidong , Zhou Huiping
Published: June 2021

Inflammation plays a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from many medicinal herbs, has been used to treat various diseases including liver diseases for hundreds of years. Our previous studies have shown that BBR inhibits high fat‐diet‐induced steatosis and inflammation in rat model. However, the underlying molecular mechanisms remain to be identified. This study was to identify the potential mechanism by which BBR inhibits free fatty acid (FFA) and LPS‐induced inflammatory response in mouse macrophages.

Methods

Mouse RAW2674.1 macrophages were treated with palmitic acid (PA) or LPS or PA+ LPS with or without different concentrations of BBR (0–10 μM) for different periods (0–24h). The mRNA and protein levels of IL‐1β, IL‐6, TNF‐α, MCP‐1, COX‐2, and ER stress genes (CHOP, ATF4, XBP‐1) were detected by real time RT‐PCR, Western blot and ELISA, respectively.

Results

BBR significantly inhibited PA‐ and LPS‐induced activation of ER stress and expression of proinflammatory cytokines (IL‐1β, IL‐6) and COX‐2. PA/LPS‐mediated activation of ERK1/2 was inhibited by BBR in a dose‐dependent manner.

Conclusion

BBR inhibits PA/LPS‐induced inflammatory responses through modulating ER stress‐mediated ERK1/2 activation in macrophages.

Support or Funding Information

This work was supported by VA Merit Award I01BX004033 and 1I01BX001390, VA Career Scientist Award IK6BX004477 and National Institutes of Health Grant R01 DK104893 and R01DK‐057543

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Relative Activity of Monomer and Dimer SecYEG Translocons
Authors: Strauss Alexa , Banerjee Tithi , Oliver Donald
Published: June 2021

The general secretory (Sec) pathway is the principal protein transport pathway in bacteria. Two key components of this system are the protein complex SecYEG, which forms a protein‐conducting channel within the plasma membrane, and the ATPase motor protein SecA, which drives the protein translocation process. Whether the SecYEG complex functions as a monomer or requires dimerization with a neighboring SecYEG complex for activity remains unclear. To address this important question, we have made use of both photobleaching and total internal reflection fluorescence (TIRF) microscopy to assess the fluorescently labeled SecY oligomeric state, as well as an in vitro protein translocation assay to assess Sec‐dependent protein transport activity. The key component of our system is the SecYEG proteoliposome, which is a lipid vesicle reconstituted solely with SecYEG protein. In this study, we reconstituted proteoliposomes at different lipid to SecYEG protein ratios to produce distinct vesicle populations containing either mostly SecYEG monomers or containing about half SecYEG monomers and half SecYEG dimers. Next, we utilized TIRF microscopy to determine the percent of singly or doubly reconstituted proteoliposomes existing within a given population based on the ratio of single or double photobleaching events. Finally, we optimized and performed an in vitro protein transport assay to assess the overall protein translocation activity of a given population. Initial findings from theses assays suggest that the monomer SecYEG proteoliposome population is more efficient at translocating protein compared to the population with substantial dimer content. Further studies will be required to fully confirm this finding. Ultimately, we hope to elucidate any differences in the protein translocation activity between these distinct populations in order to better understand the functionality of monomer and dimer SecYEG translocons in this universally conserved system.

Support or Funding Information

Funding generously provided by the NIH through the National Institute of General Medical Sciences (GM110552)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GLP‐1 Receptor Is Associated with Genes Involved in Fatty Acids Oxidation and White‐to‐Brown Fat Differentiation in Epicardial Adipose Tissue (EAT)
Authors: Romanelli Massimiliano Marco Corsi , Vianello Elena , Malavazos Alexis Elias , Tacchini Lorenza , Schmitz Gerd , Iacobellis Gianluca , Dozio Elena
Published: June 2021

Epicardial adipose tissue (EAT) is an important risk factor for cardiovascular diseases (CVD). Recent clinical evidence suggested that the potential beneficial effects of glucagon‐like peptide 1 (GLP‐1) analogs on CVD may deal with the reduction of EAT amount, possibly by targeting EAT GLP‐1 receptor (GLP‐1R), as recently discovered. Nevertheless, the role of EAT GLP‐1R and its interplay with EAT genes involved in adipogenesis and fatty acids metabolism are presently unknown.

EAT was collected from 17 patients with coronary artery disease (CAD) undergoing elective coronary artery bypass graft for microarray analysis of GLP‐1R and genes involved in fatty acid metabolism and adipogenesis. GLP‐1 plasma levels were measured by enzyme‐linked immunosorbent assay. EAT thickness was measured by echocardiography. The study has been performed in accordance with the Principles of Declaration of Helsinki.

EAT GLP‐1R was directly correlated with genes promoting beta‐oxidation and white‐to‐brown adipocyte differentiation, and inversely with pro‐adipogenic genes. Circulating GLP‐1 levels were higher in CAD than healthy subjects and in patients with greater ultrasound‐measured EAT thickness.

GLP‐1 and its analogs may target EAT GLP‐1R and therefore reduce local adipogenesis, improve fat utilization and induce brown fat differentiation. The increase in circulating GLP‐1 levels may be a compensatory mechanism to improve metabolic dysfunctions. As EAT lies in direct contiguity to the myocardium and coronary arteries, the beneficial effects of GLP‐1 activation may extent to the heart.

Support or Funding Information

The study was supported by funding from Fondazione E. A. Fiera Internazionale di Milano to Università degli Studi di Milano and Ricerca Corrente funding from Italian Ministry of Health to IRCCS Policlinico San Donato.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRISPR/Cas9‐Mediated Rescue of Shiverer Mice (MBPShi) via AAV9 Delivery across the Blood‐Brain Barrier
Authors: Ille Alexander , Lamont Hannah , Ruvio Stacy , Bernosky‐Smith Kimberly
Published: June 2021

The shiverer mouse is a genetically modified strain of M. musculus which exhibits ataxia, tremor, and seizure, among other symptoms, due to impaired CNS function. The strain is generated through deletion of a large portion of the Myelin Basic Protein (MBP) gene. Due to lack of MBP, the shiverer mouse presents with hypomyelination and the corresponding “shivering” phenotype. To date, shiverer mouse rescue has been accomplished through embryological stem‐cell implant, or gene delivery via zygote microinjection. However, these methods are limited to rescue of the subsequent generation and cannot be applied to a mouse of the current generation. Another obstacle to consider when exploring pathologies affecting the CNS is the ability of a therapeutic candidate to cross the blood‐brain barrier (BBB). Here we propose a novel method for the genetic rescue of current‐generation shiverer mice. This method involves CRISPR‐mediated insertion of a functional MBP transgene via the delivery of an AAV9 delivery mechanism. This involves a simple procedure of non‐specific intravenous injection of the mouse, relying on viral delivery across the BBB and bypassing the need for invasive surgical intervention.

Support or Funding Information

D'Youville College

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Activity Relationships of Selective Pyrimidine Agonists on α7‐nAChRs
Authors: Hernandez Gisela Andrea Camacho , Kaczanowska Katarzyna , Brandao Stefania , Stokes Clare , Papke Roger Lee , Taylor Palmer
Published: June 2021

Nicotinic acetylcholine receptors (nAChRs) are prototypic pentameric ligand‐gated ion channels (pLGIC), activated by the endogenous neurotransmitter acetylcholine. nAChRs are present in the peripheral and central nervous systems, where they modulate central, peripheral autonomic and neuromuscular functions. One of the most abundant neuronal nAChRs is the homopentameric α7. This subtype comprises of five identical α7 subunits with five binding sites, located between the subunit interfaces. The α7‐nAChR has a relatively low affinity for nicotine and acetylcholine, and a high permeability for Ca+2. Activation occurs on sub‐millisecond time scale; desensitization occurs upon prolonged application of agonist. nAChRs are found on presynaptic terminals in brain mediating the release of other neurotransmitters.

We previously synthesized a family of 4,6 substituted 2‐aminopyrimidines; our lead candidate, AC‐171A2, activates selectively the α7‐nAChRswith an EC50 of 70nM in the presence of PNU‐120596, diverging from classical agonist on binding interaction as well as chemical components. This compound has an EC50 of 360nM when fast activation was evaluated in a Xenopus oocyte system without PNU‐120596. When recovery of the receptor was assessed, AC‐171A2 allowed full recovery at 3mM concentration after 12 min. One of the goals of our current study is to better understand the molecular determinants of these ligands in these different time frames. We have developed an expanded library of compounds, based on previous leads, by combining the pyrimidine system with the di‐picolyl substitution at the 4‐position. To distinguish receptor interactions on nAChRs and related pentameric ligand‐gated ion channels, we employ HEK cells transfected with cDNA's encoding one of three requisite receptor subtypes: α7‐nAChR, α4β2‐nAChR and 5HT3AR, along with a fluorescent reporter, TN‐XXL. To assess fast activation of lead candidates, we use a Xenopus oocyte system expressing α7‐nAChR. This approach has enabled us to define the molecular determinants of the selective activation of α7‐nAChR's and offer more selective and potent lead candidates with EC50's extending to 30nM in the presence of PNU‐120596.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of TMEM16A Cl− Channel by Fungus‐Derived Penicillide in Airway Epithelial Cells
Authors: Yimnual Chantapol , Satitsri Saravut , Rukachaisirikul Vatcharin , Muanprasat Chatchai
Published: June 2021

Asthma represents one of the most common chronic diseases affecting around 300 million people with 250,000 annual deaths worldwide. It is characterized by airway inflammation, airway constriction, and mucus overproduction. The current treatments of asthma focus only on the first 2 characteristics without targeting on mucus overproduction. Transmembrane protein 16A (TMEM16A) is a Ca2+‐activated Cl− channel that, when activated, causes mucus production and secretion. Inhibition of TMEM16A activity effectively reduces mucus production along with its secretion. However, available TMEM16A inhibitors so far are synthetic compounds, or derived from plants which have limitation of large‐scale production. Therefore, this study aimed to identify novel TMEM16A inhibitor from fungal resources. A screening of >400 fungus‐derived natural compounds showed that N22, derivative of penicillide, potently inhibited ATP‐induced TMEM16A Cl− current with IC50 ~ 2 μM as analyzed by short‐circuit current measurement in human airway epithelial (Calu‐3) cell monolayers. Moreover, this inhibition was observed when TMEM16A is activated by Ionomycin (Ca2+ ionophore), or Eact (specific TMEM16A activator), suggesting that this inhibition was downstream to elevation of intracellular Ca2+ and might directly on TMEM16A molecule. As analyzed by MTT assays, inhibitory effect of N22 on TMEM16A‐mediated Cl− transport was not associated with cytotoxicity in Calu‐3 cells. Collectively, penicillides represent the first class of TMEM16A inhibitor from the fungi which may be beneficial for treatment of asthma resulting from mucus overproduction and secretion.

Support or Funding Information

This work was supported by the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation, Faculty of Science Mahidol University, Faculty of Graduate Studies Mahidol University and Medical Scholars Program, Mahidol University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing Inhibitors that Exploit ABCG2 and Cancer Dependencies to Improve Therapeutic Outcome
Authors: Fukuda Yu , Allcock Alice , Wijaya Juwina , Gose Tomoka , Lin Wenwei , Chen Taosheng , Schuetz John D.
Published: June 2021

Multidrug efflux transporters such as ABCG2 limit drug penetration into cancer cells and limit therapeutic effectiveness of many chemotherapeutic drugs. We have shown previously that inhibition of ABCG2, when combined with a conventional chemotherapy, improved treatment efficacy in murine models of acute myeloid leukemia and medulloblastoma (1,2). In this study, we sought to identify ABCG2 inhibitors that are also capable of targeting a cancer cell‐specific pathway. By combining these ABCG2 inhibitors with conventional chemotherapeutic drugs that are ABCG2 substrates, we anticipate synergistic cytotoxic effects that will produce superior treatment outcomes. A high‐throughput functional ABCG2 inhibition screen was performed using a library containing ~12,000 bioactive compounds including FDA‐approved drugs. Hit compounds were validated in a dose dependent manner, and compounds that were cytotoxic and had a low IC50 (< 1 μM) for ABCG2 inhibition were selected for further investigation. We identified kinase inhibitors (KIs) that have not been previously reported to be ABCG2 inhibitors and confirmed the direct interaction of one KI to ABCG2 using a membrane protein cellular thermal shift assay (CETSA). We investigated if the combination of a conventional cancer chemotherapeutic drug, mitoxantrone, and the KI produced an increase in efficacy against primary leukemic cells. In vitro testing of this combination on murine myeloid leukemia cells produced synergistic cell killing with an IC50 value ~10‐fold lower for each compound compared to the IC50 value for normal hematopoietic progenitors. Murine leukemia cells were then treated either singly or with the mitoxantrone‐KI combination ex vivo, followed by transplantation into lethally irradiated congenic recipient mice. The mice that received cells treated with mitoxantrone and the KI had a significant increase in their overall survival. We are extending our findings to other cancers that overexpress ABCG2 as this may be a viable therapeutic approach that improves treatment response in cancers that specifically express ABCG2 and are treated with ABCG2 substrates.

Support or Funding Information

This work was supported by NIH and by the ALSAC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of the Renal Lymphatic Circulation to the Development of Salt‐Sensitive Hypertension
Authors: Garner Brittney R , Stolarz Amanda J , Rusch Nancy J , Mu Shengyu
Published: June 2021

More than one billion people worldwide suffer from hypertension, which is a leading cause of stroke and cardiovascular disease. According to the National Heart, Lung, and Blood Institute (NHLBI) Working Group, an estimated 40% to 85% of hypertensive patients exhibit salt‐sensitivity, which increases the risk for cardiovascular events and mortality. Salt‐sensitive hypertension is known to be caused by an imbalance between sodium retention and excretion in the kidney. However, the mechanisms that enable the kidney to deliver excessive re‐absorbed salt and volume back to the systemic circulation remain obscure. Interestingly, recent studies indicate that the peripheral lymphatics act as a “salt sink” to store excessive salt and volume, thereby maintaining sodium homeostasis to regulate blood pressure. This finding led us to consider whether renal lymphatics contribute to excessive sodium retention in the kidney during the development of salt‐sensitive hypertension. To test this hypothesis, we initially evaluated lymph‐angiogenesis in the kidneys of rats with or without salt‐sensitive hypertension. Real‐time PCR, immunohistochemistry and Western blots revealed increased expression of the lymphatic‐specific marker VEGFR3 in the kidneys of Dahl salt‐sensitive (DSS) rats fed a high‐salt diet compared to either DSS rats fed regular chow or Dahl salt‐resistant (DSR) rats with or without high‐salt intake. To explore the impact of renal lymph‐angiogenesis on blood pressure, we surgically ligated the renal collecting lymph vessel (LV) in DSS rats. Ligation of the renal LV in DSS rats attenuated the increase in systolic blood pressure caused by a high‐salt diet. Notably, a growing body of evidence has implicated macrophages in lymph‐angiogenesis. Indeed, we also observed that a high‐salt diet promoted renal infiltration of macrophages, which stimulated renal lymph‐angiogenesis via upregulating VEGFR3 and its ligand VEGFC. Collectively, our data suggest that in response to a high‐salt diet, kidney‐infiltrated macrophages stimulate renal lymph‐angiogenesis, which appears to support the development of salt‐sensitive hypertension.

Support or Funding Information

Supported by NIH grants T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR), and a Medical Research Endowment Award (SM) from the Univ. of Arkansas for Medical Sciences.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Mineralocorticoid Receptor Mediates Cerebrovascular Dysfunction in Parenchymal Arterioles during Angiotensin II‐Hypertension
Authors: Diaz‐Otero Janice M , Yen Ting‐Chieh , Jaffe Iris Z , Jackson William F , Dorrance Anne M
Published: June 2021

Mineralocorticoid receptor (MR) activation causes cerebral parenchymal arteriole (PA) remodeling, impaired endothelium‐dependent dilation and cognitive dysfunction in hypertension. We used a mouse model of angiotensin II (AngII)‐induced hypertension to test the hypothesis that endothelial cell mineralocorticoid receptor (EC‐MR) activation impairs parenchymal arteriole (PA) transient receptor potential vanilloid 4 (TRPV4)‐mediated dilation and cognitive function. 16–18‐week‐old MR‐intact and endothelial mineralocorticoid receptor knockout (ECMRKO) mice were treated with AngII (800ng/kg/min) for 4 weeks; shams served as controls. Data are presented as means ± SEM; n=5–14 per group. EC‐MR deletion under control conditions did result in vascular changes. EC‐MR deletion prevented the impaired PA carbachol (CCh)‐mediated dilation in hypertension but not the increased myogenic tone. The TRPV4 inhibitor, GSK2193874 (10−7M) was used to confirm the importance of TRPV4 activation in CCh‐mediated dilation. TRPV4 inhibition blunted the CCh‐mediated dilation in all groups. Dilation in response to the TRPV4 agonist GSK1016790A (10−9–10−5mol/L) and IKCa/SKCa agonist NS309 (10−9–10−5mol/L) were also reduced in the hypertensive mice and this was corrected by EC‐MR deletion. Hypertensive mice had impaired cognitive function, but this was not corrected by EC‐MR deletion. Plasma protein oxidation was not altered by EC‐MR deletion or AngII treatment. The mRNA expression of markers for neuronal support and synapse proteins, synaptophysin (SYP), brain derived neurotrophic factor (BDNF) were not altered by AngII. However, the mRNA expression of doublecortin (DC) was reduced by AngII and EC‐MR deletion prevented this. Our data shows that EC‐MR signaling mediates PA endothelium‐dependent dilation but not cognitive function and suggests that the MR is a potential therapeutic target to improve cerebrovascular function during hypertension.

Support or Funding Information

R01‐HL‐137694‐01 and PO1‐HL‐070687 to WF Jackson and AM

Dorrance 5T32GM092715‐04 and F31NS090866 ‐ J.M. Diaz‐Otero

R01‐HL095590 ‐ I.Z. Jaffe. J.M.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serum Liver Enzymes and Oxidative Stress Biomarkers in Resveratrol Treated Cholesterol Diet Induced Type 2 Diabetes Mellitus in Rabbits
Authors: Abdulazeez Jimoh , Olusegun Ayo Joseph , Tanko Yusuf , Abubakar Ahmed , Bello Umar Muhammed , Ahmed Muhammed Kabir , Aliyu Mohammed
Published: June 2021

Fat accumulation in the body has been implicated in the increased generation of ROS and oxidative stress which enhances hepatocellular damage. Increase in serum liver enzymes have been widely used as an index of liver dysfunction and that higher ALT concentrations predicted development of type 2 diabetes. Thirty rabbits were randomly divided into six groups, each of five animals: Group 1 = control (C), Group 2 = cholesterol diet (CD) only, Group 3 = resveratrol 200 mg/kg (R200), Group 4 = resveratrol 400 mg/kg (R400), Group 5 = CD + R200 and group 6 = CD + R400. The preparations were administered for 8 weeks of experimental protocol. Blood glucose level was measured on week zero and the 8th week of the treatment. At the end of the study period, the rabbits were placed under light anesthesia (ketamine) to remain unconscious. Blood sample of about 3ml was drawn via cardiac puncture for serum extraction which were evaluated for serum concentration of liver enzymes (alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase ALP) and oxidative stress biomarkers (superoxide dismutase SOD, catalase CAT and glutathione peroxidase GPx). Blood glucose level was significantly higher in CD group compared to groups that received resveratrol supplement. Serum Liver enzymes activities showed significant (P < 0.05) decrease in ALT, AST and ALP levels in groups that were administered resveratrol with CD compared with CD alone. Significant decrease in antioxidant enzymes activities (SOD, CAT and GPx) in resveratrol treated groups (P < 0.05) were recorded when compared to CD alone group. In conclusion, the observed decreased in blood glucose level, liver enzymes and biomarkers of oxidative stress with resveratrol administration despite CD consumptions, elucidated the therapeutic potential of resveratrol.

Support or Funding Information

1. Self

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 lncRNA profile in control and ischemically injured kidneys of old mice
Authors: Hamar Péter , Kaucsár Tamás , Tod Pál , Róka Beáta , Godó Mária , Sárközy Henrietta , Kiss Norbert , Szénási Gábor
Published: June 2021

Aim

We investigated the influence of aging on the lncRNA profile after renal ischemia‐reperfusion (IR) injury in old mice.

Methods

The left renal pedicle of adult (9.4±0.3 months, n=7) and old (28.5±1.2 months, n=8) C57BL/6N mice was clamped for 20 min. The right kidney was left intact. Plasma urea and urine NGAL (uNGAL) were measured prior to and 7 days after reperfusion. On day 7 tubular injury was evaluated by histology (PAS, HE) and KIM1 mRNA (qPCR). Fibrosis (FN1) and senescence (p21) was analyzed with qPCR and long non‐coding RNA profile (90 lnc) with qPCR array.

Results

Older mice had higher baseline uNGAL (old: 285.7±93.8 ng/mg Crea vs. adult: 78.0±9.1 ng/mg Crea, p<0.05) and milder kidney injury (p<0.05). KIM1 (8.3x; p<0.001), FN1 (2x; p=0.05) and p21 (3.8x; p<0.05) mRNA levels were higher in their non‐ischemic kidneys relative to the adult group. Baseline plasma urea levels however, were similar (old: 75.9±10.2 mg/dl vs. adult: 57.6±5.3 mg/dl). Following IR tubular injury increased in both groups (p<0.05) with KIM1 (adult: 202x, old: 19.5x, p<0.0001) and FN1 (adult: 8.5x, old: 3x, p< 0.001) mRNA compared to the contralateral kidneys. uNGAL was higher after IR compared to baseline (old: 3817±1975 ng/mg Crea, p<0.0001; adult: 2833±1201 ng/mg Crea, p<0.001). p21 mRNA increased only in the ischemic kidneys (4.5x, p<0.01), compared to the contralateral control. The plasma urea concentration increased only in the old group following 7 days of reperfusion (140.7±25.6 mg/dl, p<0.01). From the 81 measureable lncRNAs 8 increased and 1 decreased only by IR. Old age only influenced 1 lncRNA expression. Further 12 lncRNA expression was influenced by both IR and old age.

Conclusion

Our results demonstrated significant tubular damage and decreased renal function in the kidneys of old mice, in accordance with the literature. We have also found that several lncRNAs were differentially expressed in old and adult mice in both the control and ischemic kidneys.

New National Excellence Program (Human Resources Department), OTKA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Embryonic Stem Cell‐Derived Exosomes Inhibit Doxorubicin‐Induced Pyroptosis in Cardiac Cells
Authors: Dargani Zahra Tavakoli , Singla Dinender K
Published: June 2021

Doxorubicin (Dox) is a potent antineoplastic drug used to treat various cancers. Unfortunately, Dox administration is limited due to induction of adverse cardiotoxicity (DIC), which is mediated through oxidative stress, ER stress, and inflammation. However, it is unknown whether Dox induces an inflammasomne‐mediated cell death, called “pyroptosis” in heart. Pyroptosis occurs via activation of TLR‐4 receptor and generation of NLRP3 inflammasome in response to damage‐associated pattern molecules (DAPMs). This would follow‐up with stimulation of caspase‐1 cascade and secretion of pro‐inflammatory cytokines from the dead cells. This study is undertaken to determine whether Dox generates pyroptosis in cardiac cell culture model. Furthermore, to elucidate the protective effects of embryonic stem cell‐derived exosomes (ES‐Exos) in inhibiting pyroptotic cell death. For this purpose, we designed a cell culture model using H9c2 cadiomyoblasts. To generate DIC, H9c2 were exposed to Dox (2 μM for 24 hrs) to induce pyroptosis and accordingly treated with exosomes (10 μg for additional 24 hrs). Cells were assigned into 4 groups: Control, Dox, Dox+ES‐Exos, and Dox+MEF‐Exos (as negative control). According to western blot analysis, immunohistochemical staining and ELISA data, Dox administration caused significant increase in expression of inflammasome markers, TLR‐4 and NLRP3, (p<0.05), pyroptotic markers, caspase‐1, IL1‐β, Caspase‐11, and gasdermin‐D, (p<0.05), as well as pro‐inflammatory cytokines, TNF‐α and IL‐6, (p<0.05) in H9c2 cells. Conversely, increased pyroptosis and inflammation post‐Dox treatment were attenuated significantly upon ES‐Exos treatment in H9c2 cells (p<0.05). However, MEF‐Exos treatment did not make significant changes vs Dox group (non‐significant vs Dox). Overall, our data shows Dox induces pyroptosis and inflammation within cardiac cells, which can be inhibited following treatment with ES‐exosomes. The significance of this study is a new mechanistic insight on the pathophysiological role of NLRP3 inflammasome‐mediated pyroptosis in Dox‐induced cardiotoxicity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alternative splicing of the clathrin heavy chain impacts skeletal muscle physiology and protects hearts from failure
Authors: Giudice Jimena , Black Adam J , Blue R. Eric , Engels Nichlas M
Published: June 2021

Alternative splicing is a posttranscriptional mechanism that produces multiple protein isoforms from a single gene. More than 90% of human genes undergo alternative splicing1,2. Coordinated splicing networks regulate organ development and tissue identity3 and genes encoding trafficking proteins are highly regulated by alternative splicing4. In particular, alternative splicing of the clathrin heavy chain (Cltc) gene regulates the expression of specific splice variants during striated muscle development5,6. CLTC is the main driver of clathrin mediated endocytosis; but also regulates the formation and maintenance of the costamere in skeletal muscles7. Nothing is known about the physiological impact of CLTC splicing, leading us to hypothesize that splicing regulation of CLTC contributes to heart and skeletal muscle physiology. We are using our new CRISPR mouse model where the endogenous Cltc splicing transition is blocked: homozygous Cltc‐ho mice express exclusively the fetal isoform throughout development while in wild type Cltc‐wt mice normal Cltc splicing transition takes place8.

We first characterized Cltc splicing patterns of in wild type animals during development in different tissues and demonstrated that exon 31 is skipped in neonates but included in adulthood exclusively in the heart, skeletal muscles, and brain. We observed the developmental regulation in human samples. Furthermore, splicing of Cltc gene reverted to neonatal patterns in mouse hearts after aortic constriction (TAC), a well‐established model of heart failure. Physiologically, Cltc‐ho mice exhibited a higher body and skeletal muscle weights than Cltc‐wt littermates that is accompanied by the downregulation of myostatin expression and the AMPK‐signaling pathway. Notably, hearts are similar in basal conditions; however, when TAC surgeries were performed in these animals, Cltc‐wt mice developed heart failure and hypertrophy whereas Cltc‐ho littermates were protected from cardiac dysfunction. Our ongoing experiments are investigating the different signaling pathways that control the observed phenotypes. Alternative splicing and membrane trafficking are fundamental cellular processes; however, we do not know how splicingtrafficking networks modulate tissue identity acquisition and maintenance, or cellular architecture. Therefore, the interaction between alternative splicing and trafficking is the knowledge gap inspiring our present and future work.

Support or Funding Information

Start‐up funds, Junior Faculty Development Award, Nutrition Obesity Research Center (NORC) Pilot and Feasibility Grant (P30DK056350) from The University of North Carolina at Chapel Hill (to JG).

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