FASEB Journal Research Articles

Withdrawn: A Controllable Protein Translation Strategy for Exploring Cellular Signal Transduction Based on Reading through Premature Termination Codons.

Withdrawal: Xiyao Cheng, Yongqi Huang, and Zhengding Su. A Controllable Protein Translation Strategy for Exploring Cellular Signal Transduction Based on Reading through Premature Termination Codons. The FASEB Journal. 34:s1. doi: 10.1096/fasebj.2020.34.s1.09195. The above abstract, published online on April 19, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, FASEB, and Wiley Periodicals, Inc. The abstract has been withdrawn at the request of the authors.

Experimental Biology 2020 Meeting Abstracts.

Withdrawal: Ivanoff, A., Einstein, G. and Tulp, O.L. (2020) A Review for Medical Students and Student Doctors on Detecting and Reporting Suspected Child Abuse and Neglect (SCAN) from Clinical and Forensic Observations. The FASEB Journal,34:S1. https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fasebj.2020.34.s1.07600/. The above abstract, published online on April 22, 2020 in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, FASEB, and Wiley Periodicals Inc. The withdrawal has been agreed due to a request made by the authors.

Withdrawn: Invisible State of MdmX and Design of its Inhibitors.

Withdrawal: Xiyao Cheng, Yongqi Huang, and Zhengding Su. Invisible State of MdmX and Design of its Inhibitors. The FASEB Journal. 34:s1. doi: 10.1096/fasebj.2020.34.s1.09193. The above abstract, published online on April 20, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, FASEB, and Wiley Periodicals, Inc. The abstract has been withdrawn at the request of the authors.

Unravelling Alzheimer's Disease: It's Not the Whole Story, but Aβ Still Matters.

In late 2016, we solicited a series of reviews covering the variety of processes that appeared to be involved in the pathogenesis of neurodegenerative disorders, particularly Alzheimer's disease (AD). These essays have appeared at regular intervals in The FASEB Journal. My instructions to the researchers were simply not to emphasize Aβ per se because there had been many reviews both supporting and questioning the etiologic role of Aβ in the late-onset, sporadic form of AD, and reciting either of those scientific positions would be redundant. My colleagues responded admirably, and I believe that their contributions have significantly informed readers' awareness of the current state of knowledge of AD. I have written my epilogue from the perspective of an investigator interested in the role of protein aggregation in human disease and as a physician who may be charged with making a diagnosis and prescribing treatment for a patient. We do not yet have etiology-based therapies of AD, but we continue to gain insight into the mechanisms responsible for synaptic loss and the consequent functional deterioration. A silver therapeutic bullet does not seem to be in the offing. It is more likely that an iterative approach will lead to the development of a group of treatments that are AD specific or applicable to various features of the entire class of neurodegenerative disorders. How and when those therapies succeed or fail will, in turn, provide additional insights into disease pathogenesis, which will inform the development of succeeding generations of therapeutics.-Buxbaum, J. N. Unravelling Alzheimer's disease: it's not the whole story, but Aβ still matters.

Matrix Metalloproteinase‐9 Release from Porphyromonas gingivalis Lipopolysaccharide (LPS)‐treated Rat Brain Microglia In Vitro

BackgroundThe putative association between periodontal disease (PD), a chronic inflammatory disease and neuroinflammation, a condition which involves brain microglia, remains under investigation. We have reported that Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), a bacterium implicated in periodontitis, activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) with concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Faseb Journal 32.1 Supplement: 702.2, 2018). We hypothesized that matrix metalloproteinase 9 (MMP‐9) release by Pg‐treated microglia might be detectable by both ELISA and visualized by confocal fluorescence imaging.MethodsP. gingivalis LPS (Pg LPS) 105 ng/mL from InvivoGen (San Diego, CA) and Escherichia coli LPS (Ec LPS) 026:B6 1 ng/mL from Difco Lab, Detroit, MI (positive control) were used to treat rat BMG for 18 hours at 35.9°C in vitro. MMP‐9 release was assessed with a rat MMP‐9 ELISA kit (R&D Systems). Confocal fluorescence imaging with a Nikon A1R laser confocal microscope was used to identify BMGs using a primary mouse anti‐rat CD11b/c antibody (Ab) (AbD SeroTec, Raleigh, NC), and secondary donkey anti‐mouse Ab (Thermo Fischer, Waltman, MA). MMP‐9 was visualized with a primary rabbit anti‐rat MMP‐9 polyclonal Ab, and secondary goat anti‐rabbit Ab, both from Abcam, Cambridge, MA.ResultsMMP‐9 presence in tissue culture supernates was detectable by ELISA in both Ec LPS and Pg LPS‐treated BMG. In contrast, MMP‐9 was observed by confocal fluorescence imaging only within Ec LPS‐treated BMG but not in Pg LPS‐treated BMG.ConclusionsBoth ELISA and confocal fluorescence imaging provide additional support for our working hypothesis, because Pg LPS stimulated BMGs to release MMP‐9. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize the putative role of Pg LPS in both periodontitis, neuroinflammation and neurodegeneration.Support or Funding InformationSupport by College of Dental Medicine, College of Graduate Studies, and Chicago College of Osteopathic Medicine, Midwestern University are gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interleukin‐22, a Pro‐inflammatory Cytokine, is Increased in the Serum of Canines with Inflammatory Bowel Disorder

Inflammatory Bowel Disease (IBD) is characterized by a chronic and/or recurrent inflammatory condition associated with adverse gastrointestinal signs including diarrhea, vomiting, and weight loss. Inflammation plays a key role in the initiation and continual progression of IBD. Interleukin 22 (IL‐22) is a pro‐inflammatory cytokine that is expressed by various cells of the immune system including dendritic cells, natural killer cells, and T cells. IL‐22 is a cytokine that can have either a helpful or a deleterious role in the intestine. The protective role of IL‐22 includes promoting mucosal barrier integrity and epithelial cell regeneration. Its deleterious actions may depend on the presence of other cytokines as well including IL‐23 and IL‐22 binding proteins. Regardless, a continually increased level in IL‐22 can increase associated gastrointestinal inflammatory processes. We tested the circulating levels of IL‐22 in the serum of dogs that were clinically diagnosed as having IBD following an endoscopy conducted by a veterinarian. Blood was collected from dogs with IBD (n=6; 2.2–13.1 yr) and control dogs (n=5; 2.2–11.3 yr) which did not show any signs of IBD. Blood was collected at two different time points (~1 year apart) from the same dogs. Using an ELISA technique, we found that the mean levels of serum IL‐22 from both time points were significantly higher in IBD (286.5 ± 69.32 pg/ml) when compared to controls (104.2 ± 17.8; p<0.05). The levels of IL‐22 in IBD dogs were higher than controls at both individual time points assessed as well. Our results identify IL‐22 as a promising biomarker of inflammatory bowel disorder in dogs and strategies to decrease a continual and elevated level of IL‐22 may be an effective way to reduce the adverse conditions associated with IBD. Further, an optimal anti‐inflammatory nutritional therapy may be a promising option to pursue as a therapeutic measure for IBD in caninesSupport or Funding InformationFunded by Hills PNCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Salt Restriction on Central and Peripheral Hemodynamics During Exercise in Essential Hypertension: A Systematic Investigation

BackgroundDietary salt restriction is a well‐established approach to lower blood pressure and reduce cardiovascular disease risk in hypertensive individuals. While high blood pressure increases the risk of cardiovascular events in hypertensive individuals during exercise, little is known regarding the effects of salt restriction on central and peripheral hemodynamic responses to exercise in individuals with hypertension.PurposeTherefore, this study sought to determine the impact of salt restriction on central and peripheral hemodynamic responses to handgrip and knee extension exercise, exercise modalities which circumvent the central limitations to whole body exercise.MethodsHypertensive patients (14M/8F, 51±10y, 173±11cm, 99±23kg) forwent antihypertensive medication use for at least 2 weeks prior to cross‐sectional enrollment on a 5‐d liberal salt (LS: 200mmol/d) followed by a 5‐d restricted salt (RS: 10mmol/d) diet. Patients were studied prior to and during: supine static intermittent handgrip (HG) exercise at 15, 30, 45% MVC (MVC: 20±4kg) and seated knee extension (KE) exercise at 40, 60, 80% KE Max (KE Max: 35±17W).ResultsSalt restriction lowered resting systolic blood pressure (SBP) (supine: −12±3mmHg, seated: −14±3mmHg, P<0.05), diastolic blood pressure (DBP) (supine: −3±2mmHg, seated: −5±1mmHg, P<0.05), and mean arterial pressure (MAP) (supine: −8±2mmHg, seated: −8±2mmHg, P<0.05). Similar reductions in SBP, DBP, and MAP were maintained during HG and KE exercise (all p<0.05). Despite lower blood pressure during exercise, peripheral blood flow and vascular conductance, were similar between groups during HG and KE exercise.ConclusionsSalt restriction appears to be an effective means to ameliorate blood pressure and reduce the risk of a cardiovascular event during exercise in hypertensive individuals without altering blood flow and vasodilatory ability during either arm or leg exercise.Support or Funding InformationVeteran Affairs Rehabilitation Research and Development Grant, 1KRX001215 CDA2, JD Trinity and Merit Grants, E1697R and E6910R, RS Richardson; American Heart Association Grant, 14SDG1850039, JD Trinity; National Heart, Lung, Blood Institute Grant, P01‐HL‐091830, RS Richardson.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 oncogenic G‐protein coupled receptor signaling pathways in Kras dependent pancreatic cancer cell lines

Pancreatic Ductal Adenocarcinoma (PDA) is the most common form of pancreatic cancer driven primarily by mutations in V‐Ki‐Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) gene. Its most common mutation, G12D, cause KRAS to remain constitutively active. KrasG12D expressing pancreatic acinar cells undergo transdifferentiation into ductal phenotype and form early tumorigenic lesions called Pancreatic Intraepithelial Neoplasm (PanIN). Coincidentally occurring inactivating mutations of tumor suppressor genes, such as Cyclin‐dependent Kinase Inhibitor 2A (Cdkn2a), Tumor Protein p53 (Tp53), and Mothers Against Decapentaplegic Homolog 4 (Smad4) trigger progression of PanINs through stages from 1A to 3, eventually leading to carcinoma in situ and metastasis.G‐protein Coupled Receptors (GPCR) are transmembrane receptors that transfer extracellular signals to intracellular heterotrimeric G‐proteins with Gα and Gβγ subunits. Upon activation, dissociated Gα and Gβγ subunits separately induce intracellular second messengers for key cellular responses involved in various aspects of development, metabolism, and cell motility. GPCR signaling is negatively regulated by intracellular Regulator of G‐protein Signaling (RGS) proteins that deactivate G‐protein by mediating re‐association of Gα and Gβγ subunits.Although GPCRs are the largest non‐antibiotic drug targets, their specific role in pancreatic cancer is not much known. GPCR signaling can influence RTK pathways in cross‐talk via various second messengers. The interplay between these two major classes of cellular pathways employing both heterotrimeric and monomeric G‐proteins has therefore potential to shed new light on pancreatic cancer research.We have previously identified Rgs16 expression as a marker of pancreatic tumor formation in Rgs16::GFP o/o; Ptf1aCre/+; Lox‐STOP‐Lox‐KrasG12D/+; Cdkn2af/f (Rgs16::GFP‐KIC) mice from the onset of earliest PanINs at two weeks of age (Dis. Model Mech. 8, 1201). Using ImageJ software to quantitatively determine eGFP expression in micrographs, we have developed a rapid in vivo assay (RIVA) where we represent pancreatic tumor burden by one month of age and compare outcomes of known and novel therapeutics following two week long drug regimens. We found that just like the recently developed drug, BGB324, targeting an important RTK, called Anexelekto (Axl), the anticoagulant warfarin was more effective in reducing tumor burden than standard chemotherapy alone.Currently, my research focus is centered on understanding active GPCR pathways that stimulate cell growth, survival, and migration by enhancement of Kras downstream signaling. My aim is comparison of the effects of general G‐protein signaling inhibition in Kras mutant and wild‐type cell lines, such as AsPC‐1 in contrast to BxPC‐3, on cell behavior and making connections to active GPCR signaling components.Support or Funding InformationSupported by NCI CA161624.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 Large Tumor Suppressor (LATS) and 14‐3‐3 Regulate Mixed Lineage Kinase 3 (MLK3) Subcellular Localization

Mixed Lineage Kinase 3 (MLK3) is a mitogen‐activated protein (MAP) kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways in response to cytokines and stress stimuli. MLK3 is important for ovarian cancer cell proliferation and invasion. However, few upstream regulators of MLK3 are known, and MLK3 subcellular localization in ovarian cells is not well understood. LATS (Large Tumor Suppressor), a Ser/Thr kinase, is activated by cell‐cell contact to inhibit proliferation, and also regulates apoptosis and mitotic exit. We observed that confluent ovarian epithelial cells had high levels of phosphorylated, activated LATS (p‐LATS). Furthermore, ovarian cancer cells had substantially lower amounts of p‐LATS in comparison to ovarian epithelial cells. Using confocal microscopy and cell fractionation, we found that MLK3 is both cytoplasmic and nuclear in ovarian cells. Co‐immunoprecipitation assays indicated that LATS interacts with MLK3 and promotes MLK3/14‐3‐3 association and MLK3 cytoplasmic localization; thereby linking the MAPK and Hippo signaling pathways. In conclusion, our results identify LATS as a novel regulator of MLK3 that affects MLK3 subcellular localization in ovarian epithelial cells.Support or Funding InformationR15‐CA199164This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Acute Antioxidant Administration on Inflammation and Vascular Function in Heart Failure with Preserved Ejection Fraction

BackgroundOne of the many unique features of heart failure with preserved ejection fraction (HFpEF) is the presence of multiple comorbidities, many of which are characterized by a pro‐inflammatory and pro‐oxidant state which may impair vascular function. This axis of inflammation, oxidative stress, and vascular function has been well described in other patient populations, whereby increases in reactive oxygen species lead to eNOS uncoupling, reduced nitric oxide (NO) bioavailability, and ultimately, impaired endothelium‐dependent vasodilation. Though there are many potential strategies for combating the damaging effects of inflammation and oxidative stress on peripheral vascular function, antioxidant (AO) administration has emerged as a simple, but effective, approach. However, no studies to date have evaluated the efficacy of AO administration to target peripheral vascular inflammation and dysfunction in patients with HFpEF.PurposeThe present study sought to evaluate the efficacy of an over‐the‐counter antioxidant (AO) cocktail (600mg alpha lipoic acid, 1,000mg vitamin C, and 600IU vitamin E) to acutely mitigate inflammation and oxidative stress, and subsequently improve vascular function, in patients with HFpEF.MethodsFlow mediated dilation (FMD) and reactive hyperemia (RH) were evaluated to assess conduit vessel and microvascular function, respectively, following administration of either placebo (PL) or AO in 16 HFpEF patients (73±10yrs) using a double‐blind, crossover design. Circulating biomarkers of inflammation (C‐reactive protein, CRP), oxidative stress (Malondialdehyde and Protein Carbonyl), free radical concentration (EPR Spectroscopy), antioxidant capacity, and nitric oxide (NO) bioavailability (plasma nitrate, NO3− and nitrite, NO2−) were also assessed.ResultsFMD improved following AO administration (PL: 3.49 ± 0.7%, AO: 5.83 ± 1.0%), while RH responses were similar between conditions (PL: 428 ± 51ml, AO: 425 ± 51ml). AO administration decreased CRP (PL: 4429 ± 705ng/ml, AO: 3664 ± 520ng/ml) and increased NO2− (PL: 182 ± 21nM, AO: 213 ± 24nM), but did not affect other biomarkers.ConclusionsThis study has identified the efficacy of an acute, over‐the‐counter dose of vitamins C, E, and alpha lipoic acid to mitigate vascular inflammation and improve conduit artery endothelium‐dependent vasodilation in patients with HFpEF, providing new insight into the mechanisms that govern peripheral vascular dysfunction in this patient group.Support or Funding InformationFunded in part by the National Institutes of Health (HL118313) and the U.S. Department of Veterans Affairs (RX001311, RX001697, CX001183).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of aqueous extract of Tamarindus indica Linn. (Leguminosae) pulp on basal and stimulated gastric acid secretion due to histamine, pentagastrin and carbachol in rats

Local folklore claims that Tamarindus indica Linn. (Leguminosae) has a gastric beneficial role in humans ( Pankaj et al., 2011). This study was carried out to examine the effects of tamarind on basal and stimulated gastric acid secretion (GAS) in the Wistar rats. Four groups of rats consisting of 6 rats (180 – 210 g) each were treated with aqueous extract of tamarind pulp (1.5 mg/kg, 2.5 mg/kg and 4 mg/kg) orally for 21 days. The ethical guidelines in accordance with NIH Guide for care and use of laboratory animals were followed in the handling of the animals during the studies. At the last day of the treatment, the rats were fasted for 24 hours, anaesthetized with ketamine and cannulated through the femoral vein. GAS (mEq/l−1) induced by histamine, pentagastrin and carbachol administration were assessed by using modified continuous perfusion technique method of Ghosh and Schild (1958) and its acidity determined by titration method. Data were expressed as mean ± SEM. Student's t‐test and one way ANOVA were used to analyze and determine levels of significance at p=0.05.The aqueous extract of tamarind significantly decreased (p<0.05) GAS induced by the histamine, pentagastrin and carbachol at small and medium doses, but not at higher doses over the 80 minute's period in all the treated groups. The gastroprotective action of tamarind may be multifactorial that may include anti‐secretory action.Support or Funding InformationThe financial support of Usmanu Danfodiyo University, Sokoto Nigeria is acknowledgedThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The BiosProject: An Erasmus+ European Action for Enhancing Bioinformatics and Computational Biology Knowledge, Skills and Literacy

A preliminary analysis performed by EMA, European Medical Association, confirmed the wide unmet needs in Europe regarding the availability of qualified health professional with expertise in bioinformatics. EMA addressed as a major need a strong link of bioinformatics and clinical activities, a greater medical literacy and awareness of the related opportunities with significant clinical correspondence. Nonetheless, this is a factor, and a gap, that weakens also the scientific credit of practising medical doctors vs. other health professionals and even vs. the patients, with a greater awareness of this topic. Healthcare is undergoing profound changes, driven by novel clinical skills and knowledge, Bioscience and technological advancements, demographic shifts, and changing lifestyles throughout Europe. The BioS project, an Erasmus+ action, is developing new innovative modular curricula that integrate the latest advancements in Computational Biology. The target is the Healthcare sector and transversal skills that can be immediately applied by medical doctors in clinical context.Healthcare professionals will become knowledgeable users who can understand the output from bioinformatics. This novel virtual learning environment is bringing together medical doctors, bioinformatics experts, educators and researchers, as well policy‐makers across Europe. On this platform, users can exchange experiences and follow virtual lessons. Differently from the already existing e‐learning Courses, BIOS is comprehensive of many facets of bioinformatics as a profession, included in the four modules: Introduction and preliminary knowledge; Computational Statistics for clinical doctors; Commercial personalized genomics services in patient care; Quality Improvement in Healthcare.The virtual learning platform and the educational resources will be available in 8 EU languages (EN, GR, PT, IT, ES, DE, FR, FI) to maximize the pool of possible users. The consortium includes12 Partners and one Leading Partner (Fig. 1). The flow‐chart is presented in Fig. 2.The dissemination strategy, itemized in fig. 3 and 4, is using Main Channels, i.e. Direct Mailing list – to EMA members (about 50.000), Direct Mailing list – to defined stakeholders (about 1500), Newsletters in EMA and BiosProject Webpages, Posts in Twitter BiosProject https://twitter.com/BiosProject1 and in Twitter EMA https://twitter.com/MedicalEmanet, BiosProject's channel https://youtu.be/pPt5eMN8qIA. The weekly accesses to Twitter BiosProject are currently around 1500/Week.Through the FaceBook and Linkedin groups, periodic information, 2–4 x year, is reaching about 4 million interested persons.The MOOC e‐learning course will be open source, credited by Erasmus, tutored in any institution or company which will assure essential and certified availability of expertise & facilities.Support or Funding InformationBioSProject is funded and approved in the European Framework of Erasmus+ /Sector Skills Alliances ProgrammeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

XRT: A reproducible and scalable training system that produces students capable of conducting novel molecular biology research

Many high‐school science education programs do not train students to be independent researchers that make novel contributions to the scientific community, which is arguably the primary goal of a scientist. There are many possible explanations for this paradox. Due to high student/teacher ratios, teachers are unable to give the individualized attention necessary to cultivate a scientist from a student. Class schedule, student motivation, and diverse intellectual support are also huge barriers. As a result, most high‐school programs struggle to establish a system that allows for university and graduate‐level laboratory education of a significant number of students.Here, we report a curriculum that trains students to design, conduct, manage, lead, and present novel research projects, but also a program that is self‐sustaining and scalable. The system produces a three‐tiered system of research students, each with an increasing set of laboratory and troubleshooting, goal‐setting, and project management skills. This management system is scaffolded. It can be completed at different speeds. These teams design projects that are novel and real. Trained teams come together and systematically create projects that can not only sustain themselves and pursue novel intellectual discovery, but also projects that are able to expand and adapt through multiple years and to a changing number of students, independent of staffing.Support or Funding InformationThis work was supported by The Nueva School.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Potential Specificity Code for B. subtilis SsbA and SsbB Quaternary Structures

Bacterial single‐stranded (ss) DNA‐binding proteins (SSBs) bind and protect ssDNA intermediates formed during genome maintenance reactions and help to recruit DNA replication and repair proteins to their sites of action through interactions mediated by their C‐terminal tails. While SSB has been studied extensively in E. coli, much less is known about the paralogous SSBs, SsbA and SsbB, that are found in many gram‐positive bacteria such as B. subtilis. Similarly to E. coli SSB, B. subtilis SsbA and SsbB both bind ssDNA through an oligosaccharide/oligonucleotide binding (OB) domain and function as homo‐tetramers. However, SsbB lacks the critical C‐terminal tail responsible for protein‐protein interactions, leading to differences in functionality. Although the OB domains of B. subtilis SsbA and SsbB are 63% identitical to one another, native gel analysis indicates that mixtures of SsbA and SsbB do not exchange monomers, but instead maintain strictly homo‐tetrameric quaternary structures. A recent crystal structure of B. subtilis SsbA revealed a unique hydrogen‐bonding interface between SsbA monomers that was not observed in the SsbB structure. We hypothesize that these interface differences could prevent SsbA/SsbB hetero‐tetramer formation. To test this hypothesis, we engineered a B. subtilis SsbA interface variant that change a key OB domain interface residue to the corresponding residue in SsbB (Asp74Ser). This variant failed to allow hetero‐tetramer formation. However, since Asp74 is only one residue in a more complex interaction network, we are now testing our hypothesis using a double variant of SsbA (Asp74Ser, Arg4Gln) that better mimics the SsbB interface network for mixed SsbA/SsbB tetramer formation. If a variant that is capable of forming mixed SsbA/SsbB tetramers is discovered, we plan to use it to test the effects of mixed‐tetramer formation in vivo and to further probe the functions of SSBs in gram‐positive bacteria.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of Epac1 or Epac2 compromises renal Na+ conservation by decreasing activity of the epithelial Na+ channel (ENaC)

Exchange proteins directly activated by cAMP (Epac1 and Epac2), along with protein kinase A (PKA), are major downstream effectors of the universal intracellular second messenger cAMP, which serves as a nexus of multiple endocrine inputs to regulate renal tubule water and electrolyte transport. We previously demonstrated that deletion of either Epac isoform, namely Epac1 or Epac2, diminishes urinary concentrating ability by reducing fluid reabsorption in the proximal tubule. Here, we employed balance studies in combination with direct electrophysiological assessment of Na+ influx in distal tubule segments from mice with targeted deletion of Epac1 or Epac2 to investigate the role of Epac cascade in tuning urinary Na+ excretion during variations in dietary salt intake. 24 h urinary Na+ levels were comparable in Epac WT and Epac isoform knockouts when animals were subjected to a regular salt intake (0.32% Na+). In contrast, we detected a delayed anti‐natriuretic response during the first 2 days of dietary sodium restriction (<0.01% Na+), which was more pronounced in Epac2 “−/− mice. Consistently, patch clamp analysis in freshly isolated split‐opened collecting ducts found indistinguishable activity (NPo) of the epithelial Na+ channel (ENaC) between Epac WT and Epac2 −/− mice on a regular salt intake: 0.37±0.03 and 0.36±0.05, respectively. Low salt diet greatly increased ENaC activity in Epac WT to 0.75±0.05 as expected, whereas this response was significantly blunted in Epac2 −/− mice (NPo = 0.48 ± 0.06). This was associated with a decrease in both number of active channels per patch and ENaC open probability. In summary, our results support a role of Epac signaling cascade (with a higher contribution of Epac2) in stimulation of ENaC activity by dietary salt restriction. We further propose that reduced ENaC‐mediated Na+ reabsorption at least partially contributes to impaired renal salt conservation in mice with deleted Epac isoforms.Support or Funding InformationNational Institutes of Health (NIH)‐National Institute of Diabetes and Digestive and Kidney Diseases Grants DK119170 (to O.P.), AHA Grant 17GRNT33660488 (to O.P.), ASN Ben J. Lipps Research Fellowship (to V.T.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anthropometric Evaluation of Body Composition and Foot Arches of African and European Women

Anthropometry is the branch of ergonomics that deals with body shape and size. The parts of the human body varies in size and in shape, thus, there is a need to take these variations into consideration whenever a product is designed for their use. The absence of the application of ergonomics principles could result musculoskeletal disorders. Evolutionary development of humans over the years has produced erect posture among humans necessary for their functionality and effective interaction with their immediate environment. The sole of the human foot has evolved to attain an arch and a three‐point support, which supports the body from either falling back or front. The foot is complex in structure and by function. It plays a major role in keeping the body in balance and steady. Body composition is the proportion of fat and fat‐free mass in the body. However, the fat and non‐essential body fat can be excessively stored in the body and may tend to be unhealthy to the human body and the foot. In this study we aim to preliminarily evaluate the relationship between body composition and anthropometric features of the foot, among women from African and European origin. A total of 150 feet of young women from African and European origin between the ages of 18 and 27 were recruited for the study. The required anthropometric parameters were measured. The shtriter index, a Russian method to determining height of foot arches was used, and body composition examined using BMI. The data were analysed using T‐test and regression analysis. The respective parameters for African and European women were measured: mean Height (H) of 159.6±0.54 and 164.3±0.69, (p<0.05), Body Mass Index (BMI) 24.5±0.40 and 21.1±0.25 (p<0.05), Foot Length (FL) 22.6±0.14 and 22.8±0.13 (p>0.05), Foot Width (FW) 8.3±0.07 and 8.4±0.05 (p>0.05), Length of Medial Longitudinal Arch (LMLA) 14.2±0.12 and 13.5±0.10 (p<0.05), and Width of Posterior Transverse Arch (WPTA) 3.4±0.13 and 2.5±0.12 (p<0.05). For African women, 51% of the total number of feet were FAF (Flat‐Arch Foot) and LAF (Low‐Arch Foot); 33% – VHAF (Very High‐Arched Foot) and HAF (High‐Arched Foot). European women recorded 18% – FAF and LAF; 74% – VHAF and HAF. The research revealed significance difference in the anthropometric parameters of the foot between women from the African and European origin. We speculate that the significant difference in the former is due to the significant difference in their body composition. Reasons for the significant difference in some of the anthropometric parameters of the foot were attributed to factors such as environment, genetics, and origin of race.Support or Funding InformationResearch supported by the Department of Human Anatomy at RUDN University, Moscow, RussiaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of NKCC2 Ubiquitination by the Skp1‐Cullin1‐FBox Complex in Thick Ascending Limbs

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. Nitric oxide and atrial natriuretic peptide decrease NaCl absorption in the thick ascending limb (TAL) by increasing the second messenger cyclic guanosine monophosphate (cGMP). We recently showed that NKCC2 is constitutively ubiquitinated, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification mediated by the ubiquitin‐activating enzyme (E1), the ubiquitin‐conjugating enzyme (E2), and the ubiquitin ligase (E3). The SCF (Skp1, Cullin‐1, F‐box protein) complex, is the largest family of E3 ubiquitin ligases in mammals. Therefore, we hypothesized that the SCF complex mediates the cGMP‐stimulated NKCC2 ubiquitination in rats TAL. To test our hypothesis we first tested whether we could inhibit neddylation of cullin‐1 with the inhibitor MLN4924 (1 μM). TALs samples were incubated for 60 minutes at 37°C in the presence or absence of the inhibitor, samples were lysed and we measured neddylated cullin‐1 by Western blot. As expected, neddylation of cullin‐1 was blunted in MLN4924 treated samples (baseline: 100%; MLN4924: 11.0 ± 3.5%, p<0.05). We then study whether inhibition of cullin‐1 inhibits the cGMP‐stimulated NKCC2 ubiquitination. TALs suspension from Sprague‐Dawley rats were aliquoted in 4 samples in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. Then aliquots were incubated at 37°C in the presence or absence of MLN4924 for 10 minutes, and treated with vehicle or db‐cGMP 500 μM at 37°C for an extra 50 minutes. We found that blocking neddylation of cullin‐1, inhibits the cGMP‐stimulated NKCC2 ubiquitination (baseline: 100%; db‐cGMP 500μM: 162.5 ± 10.3%; MLN4924: 75.1 ± 20.2%; MLN4924 + db‐cGMP 500μM: 96.8 ± 19.6%, p<0.05). To further test our hypothesis, we generated a peptide containing the conserved sequence of F‐Box proteins, called the F‐Box domain, to compete for the binding site between native F‐Box proteins to the SCF complex. We used a chariot to facilitate the delivery of the peptide into the TAL cells. We found that the F‐Box peptide binds to the SCF complex by the pull down of cullin‐1, and inhibits the cGMP‐stimulated NKCC2 ubiquitination (F‐Box peptide: 100%; F‐Box peptide + db‐cGMP 500μM: 89.8 ± 3.9%, n.s.). Our data indicates that blocking neddylation of cullin‐1 or impairment the binding of the native F‐Box proteins to the SCF complex inhibits the cGMP‐dependent increase in NKCC2 ubiquitination. We conclude that the cGMP‐dependent increase in NKCC2 ubiquitination is mediated by the SCF complex. To our knowledge, this is the first evidence pointing to the E3 ubiquitin ligase complex involved in the ubiquitination of NKCC2 in native TALs.Support or Funding InformationInstitutional Mentored Grant Program‐Henry Ford Health SystemThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA Therapeutic Strategies to Block VEGFR2 Expression and Angiogenesis in Glioblastoma Multiforme

Glioblastoma multiforme (GBM), a grade IV tumor of the central nervous system, is the most common malignant primary brain tumor, and has a median survival of only 14 months. Poor survival is due to a lack of efficacy in current therapies to pass the blood‐brain barrier (BBB). GBM tumors are characterized by angiogenesis, which is essential for tumor growth and survival. Endothelial cells form the walls of new blood vessels, bridging the gap between the growing tumor and the established vasculature. The membrane receptor that activates tumors to recruit endothelial cells to promote neo‐angiogenesis is vascular endothelial growth factor receptor 2 (VEGFR2). Changes in VEGFR2 expression to block its activation would inhibit the development of new blood vessels within the tumor microenvironment. We have designed novel therapies to bypass the BBB and deliver the genetic sequences of anti‐sense RNA molecules to alter the splicing pattern and expression of the VEGFR2 transcript. Critical splice sites, enhancers, silencers and intronic polyadenylation signals within the VEGFR2 pre‐mRNA transcript were analyzed using bioinformatics databases and RNA‐modeling tools. Based on this, nine different antisense sequences were generated to target and block these elements and were cloned into our therapeutic platform vector, pAAV‐U7‐smOPT. This vector directs the antisense RNA therapeutic to the spliceosome machinery. GBM cells were cultured and transfected with the therapeutic vectors, and the total protein and RNA was collected and analyzed. A172 and U87 GBM cells treated with antisense therapy revealed a greater than two‐fold reduction of VEGFR2. Current directions include direct delivery to the central nervous system using an adeno‐associated viral (AAV) gene therapy vector. In addition, we are isolating AAV‐exosomes with the potential to increase tropism toward the tumor microenvironment.Support or Funding InformationBristol‐Myers Squibb, Independent College Fund of New Jersey, Johnson and JohnsonThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multi‐therapeutic action of chronic treatment with tadalafil in cardiovascular alterations and erectile function of rats with heart failure

IntroductionHeart failure (HF) is the common endpoint of several cardiovascular diseases. HF and ED share similar pathophysiological mechanisms that contribute to the progression of both disorders, with emphasis on endothelial dysfunction and reduced nitric oxide (NO) bioavailability. Drugs mimic the effect exerted by NO, such as tadalafil, a phosphodiesterase type 5 (PDE5) inhibitor used in the ED treatment. Therefore, tadalafil may be a multi‐therapeutic agent in the treatment of cardiovascular alterations and erectile dysfunction associated with impaired of NO‐sGC‐cGMP signaling pathway? Thus, using the aortocaval fistula model (FAC) we investigated the contribution of tadalafil chronic treatment in the cardiac, vascular and erectile dysfunction of HF rats.MethodsHF was surgically induced through the FAC model, and after 8 weeks, these animals were submitted to cardiac function evaluation (echocardiogram) and subdivided into 3 groups: Sham, HF and HF/Tadalafil (5mg/day IP for more 4 weeks). Intracavernous pressure measurement (ICP) was used to evaluate erectile function in vivo. Response concentration curves were constructed to assess both vascular reactivity in response to phenylephrine (PE), acetylcholine and sodium nitroprusside (SNP), as well as the cavernosal muscle (CC) reactivity in response to PE and SNP. Electrical stimulation (EFS) were obtained to evaluate neurogenic contraction and nitrergic relaxation in CC rat.ResultsAfter 12 weeks, HF group presented both reduced of ejection and shortening fraction (p<0.05), as well present cardiac hypertrophy (P <0.05) when compared to the Sham group. Treatment with tadaladil restored the ejection and shortening fraction in the HF/Tadalafil (p<0.05), and reduced cardiac hypertrophy induced by HF. Moreover, HF animals presented an increase in the contractile vascular response mediated by PE when compared to Sham group (p<0.05), being this vascular hypercontractility restored by the tadalafil treatment (p<0.05). In addition, aorta of HF rats showed reduction of the ACh‐mediated relaxation (p<0.05) compared to the control, and this effect was restored by tadalafil treatment (p<0.05). Regarding erectile function, the data show that HF animals present a reduction of intracavernous pressure (p<0.05) compared to the Sham group, that was restored by tadalafil treatment (p<0.05). The contractile mechanism of CC, in response to PE and neurogenic electrical stimulation (p<0.05) were increased in HF rats, on the other side, the treatment with tadalafil improve the CC hypercontractility in these animals (p<0.05). In addition, tadalafil treatment also restored both SNP (p<0.05) and nitrergic (p<0.05) mediated relaxation in the CC of HF rats.ConclusionOur data show that the chronic treatment with tadalafil improved both vascular‐ and no‐vascular smooth muscle reactivity since exerting beneficial effects in the cardiovascular and erectile function in HF rats. Data suggesting that tadalafil acts is a multi‐therapeutic agent in HF rats.Support or Funding InformationFinancial suport: FAPESP: 2011/21095‐4 / CNPq: 423987/2016‐0This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction between dietary vitamin A, gut microbes, and host vitamin A status

Micronutrient deficiency during childhood, impairs microbiome development and establishment, ultimately affecting nutrient absorption and promoting inflammatory responses, especially in the gut, in a sort of vicious cycle1. Vitamin A is an essential micronutrient that regulates many critical biological functions, such as vision, reproduction and immunity, and that more generally contributes to the integrity of the epithelia by modulating cellular proliferation and differentiation 2. Vitamin A‐deficiency (VAD) is an overwhelming and still intractable public health problem3. The essential nutrient vitamin A regulates intestinal functions by modulating inflammation, gut immunity, and intestinal cell proliferation. Thus, the vitamin A status has a profound impact on intestinal health and, indeed, VAD is often associated with pathological conditions of the gut. On the other hand, a healthy intestine is critical to support a “healthy” microbiome which in turn helps maintaining proper intestinal functions in a feedback loop.To gain further understanding of the complex interplay between vitamin A, host physiology and microbiome, we used Lrat−/−Rbp−/−mice as a model of marginal VAD 4, when maintained on a vitamin A‐sufficient diet, and of severe VAD when deprived of dietary vitamin A. Due to the absence of lecithin:retinol acyltransferase (LRAT) and retinol‐binding protein (RBP), these mice cannot store and mobilize, respectively, hepatic vitamin A towards the periphery of the body. Thus, they rely exclusively on dietary vitamin A to support vitamin A‐dependent functions4. At 6 weeks of age, Lrat−/−Rbp−/− and WT mice were placed on either vitamin A‐sufficient or ‐deficient diet for 4 weeks and then returned to the initial regular chow regimen for 1 week. Various tissues, including intestine, and feces were collected at different time points throughout the experiment. We confirmed that VAD impairs intestinal vitamin A homeostasis and barrier functions (reduced antimicrobial defense and mucus production; increased oxidative stress; leaky gut). Genomic sequencing of the fecal 16S RNA showed that fecal dysbiosis is concomitant with the VAD status rather than the dietary vitamin A content per se, even though the degree of deficiency (marginal vs severe) did not further affect the dysbiosis. Instead, a preliminary analysis of microbial functionalities showed a significant separation of the microbial populations between vitamin A sufficient, marginal VAD and severe VAD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.