bluuebunny/crossref_metadata_2025 huggingface dataset:
Biophysical Investigation of Gastrointestinal Fatty Acid Binding Proteins (FABPs) with Fatty Acid Ethanolamides (FAEs)
Authors: Lai May Poh , Katz Francine , Stark Ruth E.
Published: June 2021

Fatty acids are the precursors for lipids that make up cellular membranes and have significant roles in signal transduction pathways. In order for fatty acids to be shuttled inside the cytoplasm they bind to fatty acid binding proteins (FABPs). Much interest has been focused on a particular class of lipids known as the fatty acid ethanolamines (FAEs) due to the recent discovery that they are involved in regulating appetite, pain, and inflammation. In this present work, we explore the FAE interaction with the intestinal and liver fatty acid‐binding proteins (I‐ and L‐FABP) that are co‐expressed in the gastrointestinal system. Previously, a striking phenotypic divergence was observed in IFABP and LFABP knock out mice: IFABP−/− mice exhibited lean phenotype while the LFABP−/− mice exhibited obese phenotype after high fat diet. Our direct comparison will elucidate if IFABP and LFABP have distinct ligand preferences to the FAE ligands. Here, we adapt a fluorescence competition assay to characterize the affinity of the FAEs to I‐ and L‐FABP. From our preliminary work, we found that I‐FABP exhibits a lower Kd, i.e. tighter binding, to ligand in all cases examined. We also found that the functional group of the ligands has the most impact on binding affinity: specifically, we found the tightest protein binding for the ligand with the carboxyl end group followed by ligands with amide and then with glycerol ends. In addition, 2D 15N‐1H HSQC NMR was used to reveal significant backbone amide chemical shift changes in the presence of a subset of the ligands we investigated. Further analysis of the HSQC results is expected to identify the perturbed ligand‐binding locations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA‐based molecular tools for monitoring cellular activity
Authors: Söderberg Ola
Published: June 2021

Methods to determine levels of protein‐protein interactions are essential in analysis of cellular signaling activity. High‐resolution analysis of proteins or protein complexes requires either advanced microscopy, or molecular methods that combine distance dependence with signal amplification. By using DNA molecules as tools, we have over the years developed several molecular methods utilizing a variety of enzymes to modify and amplify the DNA molecules used in the methods. One such method is Proximity Ligation Assay (PLA) where pairs of antibodies equipped with DNA oligonucleotides (so‐called proximity probes) are used to target proteins. Proximal binding of such probes template the creation of a circular DNA molecule, which can be amplified using rolling circle amplification (RCA). The single‐stranded RCA product from a single recognition event will contain several hundreds of repetitive motifs that can be visualized by hybridization of fluorophore‐conjugated oligonucleotides.

Information on levels of both free and interacting proteins is important to determine if the interactions monitored are a result of a post‐translational protein modification, e.g. phosphorylations, that changes the conformation of the proteins and subsequently results in a different dissociation constant (KD), or if the amount of interactions reflects mere expression levels of the proteins. New more complex measurements are needed in order to model signaling pathway activity and information flow in single cells. We have hence further developed PLA to provided information on levels of both free and interacting proteins.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Delivery Method for Poly‐histidine Tagged Proteins and Peptides for Transient Protein Expression with Light Control via Hollow Gold Nanoshells: Successful delivery of CRISPR Cas9 and Apoptotic Peptide NuBCP with NIR light control.
Authors: Morgan Erin , Jain Piyush , Pearce Martin , Bhatia Sangeeta , Kolluri Siva , Reich Norbert
Published: June 2021

Intracellular delivery of biomolecules is hindered by the size and charge of the biomolecule. While a number of delivery methods are available for genetic material, few are available for larger biomolecules like peptides and proteins. The delivery of these biomolecules are often limited to viral transfections of the genetic material which can lead to off target gene editing due to prolonged expression of a protein within a cell. We have developed a delivery method for poly‐histidine tagged proteins and peptides of variable sizes and charges for transient protein expression with light control via hollow gold nanoshells (HGNs). The construct of our delivery system involves a thiolated DNA strand containing an NTA modification on the 3′ end attached to the gold surface. A his‐tagged protein or peptide of interest is attached to the HGN via copper‐NTA affinity. Endosomal uptake of these particles is mediated through an orthogonal strand containing a cell penetrating peptide (CPP). Protein release and endosomal disruption is achieved after irradiation with a focused femtosecond pulsed‐ laser of biologically benign near‐infrared light. With this technology we have successfully delivered multiple proteins of interest including the gene editing protein Cas9 for efficient knockout of GFP with light control as well as the Nur77 derived apoptotic peptide NuBCP which allowed for an increase in efficiency with our delivery method.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Plasmodium falciparum Glutamic Acid‐Rich Protein (PfGARP) in Recombinant Pichia pastoris for Malaria Vaccine Development
Authors: Martínez‐Muñiz Andrés Enrique
Published: June 2021

Malaria infections are common occurrences and one of the leading causes of death in developing countries. The World Health Organization reported 212 million malaria cases worldwide in 2016 which resulted in 429,000 deaths. The most affected group of individuals were children under the age of five which accounted for 70% of this death rate. There are no available vaccines for malaria, but the development of this type of treatment would provide a more cost‐effective and efficient way of battling this disease. Within populations affected by malaria, certain children and adolescents have developed protective immune responses against this disease. These protective immune responses decrease the intensity of the infection thus making the individual more resistant against the acquired disease. Through differential screening experiments, malarial‐protein antigens recognized by antibodies from resistant, but not susceptible individuals were discovered. The Plasmodium falciparum Glutamic Acid‐Rich Protein (PfGARP), an antigen that arrests the development of the parasite in its trophozoite stage was discovered using this method. The specific aim of this study was to express functional PfGARP in order to purify and characterize it as a vaccine candidate. The PfGARP gene was inserted into a plasmid vector that was then used to transform the expression system organism: Pichia pastoris. Individual colonies of recombinant P. pastoris were transferred from agar plates to liquid medium cultures. These were then analyzed for protein expression via Western blot every 24 hours after induction of protein expression. 94 colonies of recombinant P. pastoris were analyzed using this method and none showed expression of PfGARP. PCR analyses were then performed to determine the presence of the PfGARP gene in transformed P. pastoris. A set of vector‐specific primers was designed to amplify a region of 2242 base pairs which included the PfGARP gene in the middle of the region. Amplifications from DNA extracted from the recombinant cells showed a band size of roughly 1000 base pairs. Based on results from the Western blot and PCR analyses performed, it can be concluded that P. pastoris deletes a significant amount of the PfGARP gene's sequence and therefore is not a viable protein expression system for PfGARP.

Support or Funding Information

Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T34GM008419.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of botanicals and purified phenolic compounds on NFkB pathway in cultured human skin keratinocytes and fibroblasts
Authors: Higgins Jillian Lee , Kraus Aileen , Clark Matthew , Gallant Benjamin , Arruda Dominic , McTigue Christie , Ma Hang , Seeram Navindra P. , Guo Hao , Chen Hong‐duo , Cui Yali , Wan Yinsheng
Published: June 2021

Our previous studies have demonstrated that ultraviolet radiation‐induced skin photo‐aging is associated with collagen degradation, dehydration, and inflammation. While the cellular and molecular mechanisms through which UV induces skin aging are still being unraveled, active compounds, including those natural products, are being sought to attenuate UV‐induced skin cell damage and inflammation. Recent studies have shown that maple extracts having active ingredients may have beneficial effects for the development of cosmetics products. In this study, five samples including black cumin extracts (BCE), thymoquinone (TQ, a major component of black cumin), Maple syrup extracts (MSX), Maple leave extracts (MAP), gennalin A (GA), were investigated whether those abstracts or compounds have effects on cytokine‐induced activation of NFkB pathway, which is critical for UV‐induced skin inflammation and skin aging. First, we used MTT assay to determine the proper concentration in cultured skin keratinocytes. We found that 5 mg/ml is the concentration that does not affect cellular proliferation. Second, we treated both keratinocytes and fibroblasts with TNFa and IL1b (10 ng/ml) and measured the degradation of IkB and NFkB subunit, p65 translocation from cytoplasm to nucleus, using confocal microscope and Western blot analysis. The results showed that both TNFa and IL1b induce IkB degradation and p65 translocation in both types of skin cells, while fibroblasts are more responsive to TNFa and IL1b. Under UV radiation, keratinocytes are activated and release cytokines that further activate skin fibroblasts. Third, we tested the effects of all five extracts or compounds on TNFa and IL1b p65 translocation and IkB degradation by confocal and Western blot. The results showed that p65 translocation was inhibited after administering 10 mg/ml of the BCE, and inhibition improved as the dosage of BCE increased. IkB level was elevated at the following dosages of BCE 25 and 50 mg/ml. In addition, p65 translocation appeared to be inhibited exclusively at 50 mg/ml in fibroblast cells. IkB level was observed to be consistent as the dosage of BCE increased, suggesting that BCE may block IkB degradation in fibroblast cells, with a result of inhibition of p65 translocation. Other samples are still being further investigated. Taken all attained data together, we conclude that some of those tested extracts or compounds may be added to the cosmetics products to attenuate or prevent from UV‐induced skin cell damage and inflammation.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantifying HNF1A Regulated Gene Expression in Pancreatic Cancer
Authors: Alaniz Anthony A. , Abel Ethan V. , Simeone Diane M.
Published: June 2021

Hepatocyte nuclear factor 1A (HNF1A) is a transcription factor essential to normal pancreas and liver development and homeostasis. HNF1A has been shown to be upregulated in pancreatic cancer stem cells (CSC), a proposed key driver of pancreatic ductal adenocarcinoma (PDAC), relative to other pancreatic cancer cells. A CSC gene signature consisting of 50 upregulated genes, including HNF1A, was previously identified by a microarray. This signature predicts worse survival compared to HNF1A expression alone. This research aims to establish an HNF1A‐dependent gene signature that could potentially be used as a biomarker for pancreatic cancer. Using quantitative reverse transcriptase PCR, multiple CSC signature genes were shown to respond to HNF1A knockdown and overexpression. Responsive genes varied across cell lines, suggesting a complex pathway significantly affected by HNF1A transcriptional control.

Support or Funding Information

The work was supported by the Pancreatic Cancer Action Network‐AACR Pathway to Leadership Grant (16‐70‐25‐ABEL) and the American Cancer Society Postdoctoral Fellowship (127662‐PF‐15‐033‐01‐DDC) (to EVA) and the Gershenson Pancreatic Cancer Fund (DMS) and SKY Foundation (HC and DMS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can Less Be More: Evaluating the Synergistic Relationship between Chemotherapy Agents and NHE1 Inhibitors in Ovarian Cancer Cells
Authors: Larson Allie C. , Provost Joseph J. , Wallert Mark
Published: June 2021

Ovarian cancer is the seventh most prevalent cancer found in women, resulting in over 200,000 new cases and close to 140,000 deaths annually. In order to combat the risk of mortality associated with this disease, extensive chemotherapy regimes are employed. These measures often include the use of therapeutic agents such as paclitaxel, rucaparib, olaparib, and cisplatin. The mechanisms of action for each of these oncogenic therapeutics differs substantially. Paclitaxel stabilizes microtubule polymerization, effectively preventing mitotic completion. Rucaparib and olaparib both inhibit PARP, an enzyme associated with repairing damaged DNA. Cisplatin also hinders DNA repair. However, it does so by crosslinking the purine bases on DNA to promote cellular apoptosis. Although the use of such chemotherapy agents has been instrumental in reducing cancer‐related deaths, the dosage intensity of these cytostatics can procure adverse side effects in the form of hair loss, anemia, nausea, and vomiting. Because of this reality, it has become of interest to investigate methodologies for reducing the required chemotherapy drug dosage, while still maintaining the same rate of cancer cell death. The Na+‐H+ Exchanger Isoform 1 (NHE1) is a plasma membrane protein that exchanges 1 extracellular Na+ for 1 intracellular H+, thus playing a primary role in pH regulation. It is hypothesized that such a synergistic effect can be produced through the pairing of chemotherapy agents with NHE1 inhibitors. In cancer cells, NHE1 is often hyperactivated leading to the development of a pH inversion. This dysregulation contributes to increased cancer cell proliferation, migration and invasion. Inhibition of this transport protein typically results in intracellular acidification, a mechanism that supports the progression of apoptosis. Applying this inhibition, with an accompanying cytostatic, has thus interested researchers as a possible therapeutic approach towards mitigating neoplastic development. The potential of these drug‐inhibitor pairs will be measured primarily via flow cytometry. Through the addition of a fluorescent dye, this instrument is able to differentiate viable cells from compromised cells within a single population. Such analysis allows for a quantitative assessment of each inhibitor‐drug combination.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunohistochemical study of the epithelial‐to‐mesenchymal transition phenotype in nonsmall cell lung cancer
Authors: Rodriguez‐Lopez Xavier Luis , Perez‐Morales Jaileene , Santiago‐Cardona Pedro
Published: June 2021

Lung cancer is the leading cause of cancer death in both men and women in the United States. Lung cancer is divided into non‐small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for 80% of all primary lung cancers and subdivided into adenocarcinoma (ADC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). Due to late diagnosis, only 16% of diagnosed patients live beyond 5 years, but this survival rate increases if the cancer has not yet metastasized. Recent developments in lung cancer therapies depend on precise histological subtyping of NSCLC. Accurate discrimination of SCC from the remaining NSCLCs is crucial for understanding the outcome and metastatic dissemination of lung cancer. The epithelial‐to‐mesenchymal transition (EMT) has been associated with an early proclivity for metastasis in lung cancer. We want to identify if N‐cadherin and E‐cadherin expression in tumor samples can be of clinical value for discriminating between SCC and ADC and in predicting the development of lung cancer metastasis. We performed immunohistochemistry analysis in two tissue microarrays that contained 24 lung cancer patients. An EMT phenotype was defined as a low expression of E‐cadherin and high expression of N‐cadherin. An EMT phenotype was observed in 58% cases of SCC and carried prognostic utility for lymph node metastasis. The EMT phenotype observed in SCC can be suggested as potential markers of malignant transformation.

Support or Funding Information

R25GM096955, 2R25GM082406, 2U54CA163071‐06

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Src Family Kinase Tyrosine Phosphorylates Toll‐like Receptor 4 To Dissociate MyD88 And Mal/Tirap Suppressing LPS Induced Inflammatory Responses
Authors: Rhee Sang Hoon , Im Eunok , Mitchell Jonathon , Kim Su Jin
Published: June 2021

Src family kinases (SFKs) are a family of protein tyrosine kinases containing nine members: Src, Lyn, Fgr, Hck, Lck, Fyn, Blk, Yes, and Ylk. Although SFK activation is a major immediate signaling event in LPS/Toll‐like receptor 4 (TLR4) signaling, its precise role has remained elusive due to various contradictory results obtained from a certain SFK member‐deficient mice or cells. The observed inconsistencies may be due to the compensation or redundancy by other SFKs upon a SFK deficiency. The chemical rescuing approach was suggested to induce temporal and precise SFK activation in living cells, thereby limiting the chance of cellular adaption to a SFK‐deficient condition.

Using the rescuing approach, we demonstrate that restoring SFK activity not only induces tyrosine phosphorylation of TLR4, but also inhibits LPS‐induced NFkB and JNK1/2 activation and consequently suppresses LPS‐induced cytokine production. TLR4 normally recruits TIR domain‐containing adaptors in response to LPS, however, temporally restored SFK activation disrupts the LPS‐induced association of MyD88 and Mal/Tirap with TLR4. Additionally, using kinase‐dead SFK‐Lyn (Y397/508F) and constitutively active SFK‐Lyn (Y508F), we found that the kinase‐dead SFK inhibits TLR4 tyrosine phosphorylation with reduced binding affinity to TLR4, while the kinase‐active SFK strongly binds to TLR4 and promotes TLR4 tyrosine phosphorylation, suggesting that SFK kinase activity is required for TLR4 tyrosine phosphorylation and TLR4‐SFK interaction.

Together, our results demonstrate that SFK activation induces TLR4 tyrosine phosphorylation, consequently dissociating MyD88 and Mal/Tirap from TLR4 and inhibiting LPS‐induced inflammatory responses, suggesting a negative feedback loop regulated by SFK‐induced tyrosine phosphorylation in TLR4.

Support or Funding Information

Supported by a grant from Oakland University and the NIH (DK079015, S.H.R) and by Basic Science Research Program through the NRF of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03932222, E.I.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overcoming challenges in the diagnosis of Schistosoma mansoni infections using POC tests, recombinant protein and monoclonal antibody technologies.
Authors: Grenfell e Queiroz Rafaella Fortini , Harn Donald , Coelho Paulo Marcos
Published: June 2021

Control constraints of Schistosomiasis include the lack of diagnostic methods with high sensitivity. We develop a prospective study in southeast Brazil to standardize new sensitive and rapid diagnostic methods for Schistosoma mansoni infection. Currently, we are investigating 6 endemic areas (>1000 individuals with chronic infection) and 84 travelers infected in a freshwater pool (with acute infection). Sera, urine, feces and saliva samples were used for the standardization/validation of innovative methods, including acute, chronic and post‐treatment patients. Comparisons are performed with eggs in feces by 24 Kato‐Katz slides and 2 analysis of Saline Gradient and clinical symptoms. With our new point‐of‐care methods using a selected recombinant protein called CCAr e other markers, we were able to detect the disease early as 10 days post‐infection and more than 95% of positive cases from chronic and low endemicity areas were obtained. Plus, POC‐CCA® test was improved with a urine concentration step that turned its sensibility from 6% to 56%. Monoclonal antibody and recombinant protein technologies allowed a superior detection method when comparing it to the conventional methods. In conclusion, data showed 100% of sensitivity of chronic patients and 98% of acute patients.

Support or Funding Information

Fapemig, CNPq, FIOCRUZ, PDTIS (Brazil). Fulbright, NIH, University of Georgia (USA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Lytic Activity of Melittin‐nNOS Chimeric Antimicrobial Peptides
Authors: Fujii Maria , Stevens‐Truss Regina
Published: June 2021

Antibiotic resistance has become a growing problem in a world of prescription drug overuse and misuse. Antimicrobial peptides (AMPs) offer one possible solution. Melittin (Mel), isolated from bee venom, is a known AMP. It is active against a variety of microbes as well as cytotoxic towards eukaryotic cells. Previous work from our lab has demonstrated that the calmodulin‐binding sequence of the nNOS enzyme possesses selective activity against S. aureus. Our lab has been investigating the role that specific amino acid residues of Mel play in its lytic activities, and possibly what role these amino acids play in selectivity, by investigating the activities of a series of Mel‐nNOS chimeras. In this study, the chimeras were produced by replacing the middle 10 residues of one peptide with that of the other (nMel‐nNOS‐cMel and nnNOS‐Mel‐cnNOS). We also assessed the activity of Mel truncated peptides (nMel and cMel). All experimental peptides were tested for their ability to inhibit the growth of S. aureus and E. coli as well as their ability to lyse liposomes and red blood cells (RBCs). The results show that both truncated peptides were completely inactive. In addition, both chimeras were found to effectively cause liposome lysis at concentrations below 3 uM and have minimal effect on RBC lysis. Chimera nnNOS‐MelcnNOS was found to have IC50 values above 12 uM for both S. aureus and E. coli while chimera nMel‐nNOS‐cMel was found to be more selective for S. aureus (IC50 values between 4 uM and 8 uM), with IC50 for E. coli being greater than 16 uM. These results imply that the eight terminal residues of Mel are insufficient for bacterial growth inhibition. Further studies should focus on increasing the Mel to nNOS ratio of the chimeric AMPs.

Support or Funding Information

The Authors would like to acknowledge the American Society for Biochemistry and Molecular Biology, as well as the Kalamazoo College Chemistry Department and Faculty Development Committee for funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Membrane Composition on PLCβ and Gαq‐mediated Activation
Authors: Hudson Brianna N. , Hyun Seok‐Hee , Thompson David H. , Lyon Angeline M.
Published: June 2021

Phospholipase C β (PLCβ) enzymes are peripheral membrane proteins that are required for normal cardiovascular function and whose dysregulation results in cardiovascular disease. PLCβ hydrolyzes phosphatidylinositiol‐4,5‐bisphosphate (PIP2) to produce the potent second messengers inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG), which increase intracellular Ca2+ and activate protein kinase C. PLCβ has low basal activity, but is activated downstream of G protein‐coupled receptors (GPCRs) through direct interactions with the heterotrimeric G protein subunits Gαq and Gβγ. While G proteins stimulate activity, they are insufficient for full activation. This suggests that the membrane is required for full activation. However, the molecular basis for how the membrane and its properties contribute to PLCβ adsorption, activity, and Gαq‐dependent activation are not well understood. We seek to understand how the membrane composition and its surface charge regulate adsorption of PLCβ to the membrane and promote interfacial activation, and the role of Gαq in this process. Using a model membrane system, we are applying an innovative combination of atomic force microscopy, Raman spectroscopy and biochemical assays to begin understanding how the membrane itself and Gαq regulate PLCβ activation. These studies provide the first structure‐based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.

Support or Funding Information

American Heart Association Scientist Development Grant (16SDG29920017) to A.M.L.; Purdue Doctoral Fellowship to B.N.H.; Purdue Center for Cancer Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased levels of FOXM1 transcription factor in bronchioalveolar stem cells promote epithelial cell repopulation in injured mouse airway
Authors: Wang I‐Ching , Chiu Yi‐Shiuan , Chen Wei‐Hsin , Li Chien‐Cheng , Lee Yun‐Ching
Published: June 2021

Bronchioalveolar stem cells (BASCs) have been shown to maintain the bronchiolar and alveolar epithelial cell renewal upon lung injury. Previously we have shown Foxm1 transcription factors plays essential roles in cellular proliferation, migration, differentiation and tumorigenesis. Conditional deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibited lung maturation and caused respiratory failure after birth, while deletion of Foxm1 from adult lung airway epithelium reduced club cell proliferation after naphthalene‐induced lung injury. Here, we show that premature expression of transgenic FOXM1accelerated airway epithelial cell proliferation after naphthalene induced lung injury in CCSP‐rtTA/tetO‐FOXM1 mouse (C/FOXM1), which is associated with increased numbers of CC10 and Ki‐67 positive cells in terminal bronchiolar epithelium and bronchioalveolar duct junction (BADJ), the stem cell niche. Lineage tracing experiment showed that FOXM1 promotes proliferation of LacZ or Brainbow labelled naphthalene‐resistant CCSP expressing cells that differentiated into bronchiolar epithelium in healing processes. Co‐immunofluorescent staining showed an increased number of proSPCposCCSPpos BASCs were found in in BADJ of C/FOXM1 lungs, which consistent to result that an increased number of CD45neg CD31neg Sca‐1hi EpCAMhi bronchiolar stem cells in C/FoxM1 lungs by FASC analysis. Our results indicated that increased expression of FOXM1 contributes to the increases proliferation of lung stem/progenitor cells, and facilitates bronchiolar epithelium repair from acute lung injury.

Support or Funding Information

National Health Research Institute, Taiwan (NHRI‐EX106‐10309BC); Ministry of Science and Technology, Taiwan (MOST 105‐2628‐B‐007‐003‐MY3); and National Tsing Hua University, Taiwan (106J00X9V8) to I‐C.W.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Marijuana, Crack and Cocaine Consumption in Undergraduate Brazilian Students
Authors: Miranda Renato Pricoli , Olcerenko Bianca Giovanna Batista , Rebecchi Cassio , Taricano Ingrid Dragan
Published: June 2021

Drug addiction is one of the most prominent problems in many developing countries, including Brazil. Our study was aimed to investigate the relationship of the use of marijuana, the illicit drug with the biggest use prevalence in the world, with the use of crack/cocaine in undergraduate students from the city of São Paulo, covering samples from the north, south, east, west and center regions. The study sample comprised 907 undergraduate students. The group of respondents was chosen to represent undergraduate students of this city. Data on lifetime and recent frequency and intensity of use for alcohol, cannabis, amphetamines, ecstasy, LSD and cocaine was collected.

METHODOLOGY

The instrument used for data collection was Google FormsTM. The instant addition of responses to the data set makes this tool appealing for research purposes. Additionally, the respondents involved in the study have been regularly using Google FormsTM, and it was therefore considered the most appropriate method for data collection. The results of the survey was statistically analyzed. Within the various areas the group of students from medical, biology, nursing, engineering, architecture, psychology, and other courses, were chosen because their experience using these tools. Data on lifetime and recent frequency and intensity of use for alcohol, cannabis, amphetamines, ecstasy, LSD and cocaine are presented.

RESULTS

Participants already use illicit drugs. The greater consumption between the interviewed university population is marijuana, while the prevalence of cocaine use is 5% and crack 0.6%. According to the results obtained, 4.07% (37) of the participants had the use of marijuana associated with crack/cocaine, being that the marijuana/cocaine use relationship was 91.89% (34), the relationship of use marijuana/crack/cocaine was 8,11% (3), while the relationship of the use of marijuana associated only to the use of crack was not observed.

CONCLUSION

The relation of illicit drug use in undergraduate health area is the highest consumption indices. These results show some paradox, since the area in question is just formed by the professionals that will work with physical, psychological and social problems arising out of the use of drugs. This work was conducted in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration. CAAE(Br)67881517.1.0000.5511. All participants had full understanding and signed informed consent document.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Field Sobriety Test for Cannabis
Authors: Pabon Elisa , de Wit Harriet
Published: June 2021

Cannabis and its active ingredient delta‐9‐tetrahydrocannabinol (THC) have been shown to impair memory, reaction time and attention. However, it is difficult to assess these impairments in the nonlaboratory setting. We have developed a prototype for a phone application called Am I Stoned?. As a first step, we tested the app in a within‐subjects double‐blind placebo‐controlled study with THC (0, 7.5, 15 mg). Participants completed both iPhone‐based and standard computer tests of cognitive speed, reaction time, fine motor ability, and memory. As a secondary aim we also assessed participants' ability to estimate their performance impairment. Twenty‐four healthy experienced non‐daily cannabis users completed a laboratory study, which included three 4‐hour experimental sessions in which participants consumed a capsule containing THC (7.5, 15 mg) or placebo. They completed all tasks at both two and three hours after taking the capsule. Performance was impaired by THC in three of the four computer tasks, but only one of the iPhone tasks. It is likely that the computer tasks were more sensitive to THC impairment than the app task because the computer tasks were longer (15–20 min compared to 5–7 min), providing more opportunity to detect a drug effect. With regard to self‐assessments, subjects were in general accurate in their awareness of impairments on the tasks. In a follow‐up study we will improve the tasks used in the phone application to increase their sensitivity to THC impairment. This research is likely to lead to sensitive field tests that will allow users to objectively evaluate their ability to perform psychomotor or cognitive tasks. The research will also identify conditions under which individuals are or are not aware of their impaired state.

Support or Funding Information

This research was supported by the National Institute on Drug Abuse Grant DA02812 (HdW)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYP2J2 inhibition: the arcane mechanism of dronedarone‐induced proarrhythmia
Authors: Chan Eric Chun Yong Yong , Karkhanis Aneesh , Mehta Ashish , Shim Winston
Published: June 2021

Background

Like other Class III antiarrhythmic, dronedarone and amiodarone are potent human Ether‐à‐go‐go‐Related Gene (hERG) channel inhibitors.1–2 Although structurally and pharmacologically similar, dronedarone is known to worsen cardiac proarrhythmia3 and increase mortality in patients with permanent atrial fibrillation4 while amiodarone does not. Previously we demonstrated dronedarone is a more potent inhibitor of recombinant human cytochrome P4502J2 (CYP2J2)4 compared to amiodarone and yields negative perturbation of eicosatrienoic acid homeostasis5.

Objectives

We aimed to investigate and compare the in vitro proarrhythmic potential of dronedarone and amiodarone and CYP2J2 inhibition in a cellular model.

Methods

We first measured the expression of CYP2J2 in human induced pluripotent stem cell derived cardiomyocytes (hiPSC‐CMs) using quantitative PCR. The extent of CYP2J2 inhibition in hiPSC‐CMs was measured using astemizole as a probe substrate. The electrophysiological changes in terms of field potential duration (FPD) and beat rate variability (BRV) were measured using multielectrode array.

Results

CYP2J2 is abundantly expressed in hiPSC‐CMs. Unlike amiodarone, dronedarone potently inhibited CYP2J2 activity (97%) at 1 μM. Both dronedarone and amiodarone yielded concentration‐dependent increase in FPD. Dronedarone showed 17‐fold and 2‐fold higher BRV compared to control and amiodarone at unbound plasma drug concentrations. Higher extent of CYP2J2 inhibition correlates with higher BRV.

Conclusion

Dronedarone‐induced BRV corroborates its clinical proarrhythmia. For the first time, our study illuminates the currently arcane contribution of CYP2J2 inhibition to drug‐induced proarrhythmia.

Support or Funding Information

Singapore Ministry of Education Tier 1 Funding (R‐148‐000‐193‐112) provided to Eric Chun Yong Chan.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alkylated pyridyl amphiphiles display a pro‐apoptotic profile in cancerous cell lines dependent on hydrophobic chain length
Authors: Carrillo‐Alvarado Miguel Angel , Hernandez Evelyn Angel , Pardo Andrew Angel , Varela‐Ramirez Armando Angel , Noveron Juan Angel
Published: June 2021

Cationic lipids, particularly those with nitrogen containing heterocycles, such as a pyridinium headgroup, display various bactericidal, bacteriostatic and disinfectant activity as reported in various works, all dependent on the length of their respective alkyl chains. Two quaternary alkyl pyridinium amphiphiles, vis. 4‐amino‐1‐decyl (1) and 4‐amino‐1‐dodecyl (2), were evaluated for their cytotoxic activity in a human cervical cancer cell line. These two compounds were assessed for their cytotoxicity and the following CC50's, 15.68 μM and 4.58 μM respectively were established. 1 proves to be a poor candidate as an anti‐cancer therapeutic agent as its cytotoxicity induced necrotic death. At a low concentration, 2 displayed a pro‐apoptotic profile demonstrated by phosphatidylserine externalization suggesting induction via an extrinsic pathway since it did not disrupt mitochondrial polarity. Of both compounds, 2, acts as a cell cycle progression disruptor causing slight S‐phase arrest and a decrease in G2/M phase along with eliciting DNA fragmentation. An explicit difference of two carbons between both 1 and 2 warrants further investigation to elucidate alkyl‐chain length role in anti‐cancer drugs. Furthermore, a series of these cationic amphiphiles with different lengths for their hydrophobic moiety has been synthesized and will be studied on a wider panel of cancer cell lines and have their mechanism of death further elucidated.

Support or Funding Information

The Cytometry, Screening and Imaging Core Facility that was used in this work was supported by the National Institute on Minority Health and Health Disparities (NIMHD) through a Research Centers for Minority Institutions (RCMI) grants 8G12MD007592 and 5G12MD007592, a component of the National Institutes of Health (NIH).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Berberine on Insulin, GLP‐1R and DPP4 Gene Expression Levels in Streptozotocin Induced Diabetic Rats
Authors: Kilari Eswar Kumar , Swathi P
Published: June 2021

Berberine is a quaternary ammonium compound belongs to benzylisoquinoline alkaloids. Traditionally it was used in the treatment of diabetes and associated complications. The aim of the present study was to determine the effect of berberine on antihyperglycemia, in vivo antioxidant, hepatic and lipid biomarkers and gene expressions in streptozotocin induced diabetic rats. Albino Wistar rats were induced for diabetes by streptozotocin and divided into five groups as vehicle control (group I), disease control (group II), standard gliclazide (Group III), group IV, and V received Berberine 50 and 100 mg/kg body weight respectively orally once a day for 15 days in STZ induced diabetic rats. A significant increase in blood glucose, glycosylated hemoglobin (HbA1c), altered Lipid profile (Total cholesterol, triglycerides, HDL, LDL and VLDL) and elevated hepatic serum biomarker levels (AST, ALT, ALP, total & direct bilirubin) in STZ induced diabetic rats. The berberine showed significant reduction of blood glucose and HbA1c and elevation of insulin levels in STZ induced diabetic rats. The Berberine shown to normalize the lipid and hepatic biomarkers during 15 days treatment period in all groups of treatments in a dose dependent manner in STZ induced diabetic rats. Decreased levels of in vivo antioxidant enzymes SOD, CAT, GSH and GPx in both pancreas and ileum, decreased insulin & GLP‐1R and increased hepatic DPP4 mRNA gene expression levels in STZ induced diabetic rats. Treatment with Berberine shown to elevate the pancreatic and intestinal antioxidant enzymes and increased pancreatic insulin & GLP‐1R gene expression in ileum and decreased hepatic DPP4 in STZ induced diabetic rats. The pharmacological activity of the berberine might be due to the antioxidant property and by enhancing the incretin activity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of Notch signalling by soluble Dll4 decreases permeability via a cAMP/PKA‐dependent pathway
Authors: Bates David O. , Boardman Rachel , Pang Vincent , Leach Lopa , Machado Maria J.
Published: June 2021

The Notch ligand Delta like ligand 4 (Dll4), which is upregulated by VEGF during angiogenesis, is a key regulator of vessel morphogenesis, maturation and function. It has been widely described as a regulator of tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hyper‐sprouting, non‐functional and poorly perfused vessels, which suggests a role for Dll4 in the formation of mature, reactive, functional vessels, that have low permeability and are able to restrict fluid and solute exchange across vessel walls. We therefore tested the hypothesis that Dll4 plays a role in controlling transvascular fluid exchange. Using a recombinant protein expressing only the extracellular portion of Dll4 (soluble Dll4: sDll4), we were able to induce Notch signalling in endothelial cells (EC), resulting in an increased expression of VE‐Cadherin at intercellular junctions. sDll4 decreased permeability of FITC labelled albumin across EC monolayers and this effect was abrogated by co‐culture with DAPT. One of the known molecular effectors responsible for strengthening EC‐EC contacts is the cyclic AMP dependent protein kinase PKA, so we tested the effect of modulation of PKA on sDll4‐mediated reduction of permeability. Inhibition of PKA reversed the sDll4‐mediated reduction in permeability and reduced Hey‐1 expression. We showed that sDll4 caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon co‐perfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signalling, through Notch activation, acts upon intercellular adherens junctions through a cAMP/PKA pathway, to regulate endothelial barrier function.

Support or Funding Information

Supported the British Heart Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adverse Cardiac Effects Due to Cardiac Specific Disruption of the Nuclear Receptor Corepressor 1 (NCOR1)
Authors: Zhang Jie , Vatner Dorothy E. , Vatner Stephen F.
Published: June 2021

Disruption of the nuclear receptor corepressor 1 (NCOR1) knockout (KO), a transcriptional coregulatory, has been shown to exert only beneficial effects on disease processes, particularly in skeletal muscle, e.g., it improves exercise endurance, insulin sensitivity and reduces inflammation. The goal of this investigation was to determine if the cardiac specific NCOR1 KO also protects the heart against the stress of chronic pressure overload, induced by chronic thoracic aortic constriction. Baseline cardiac function, as reflected by left ventricular (LV) ejection fraction (EF), assessed echocardiographically, was similar in NCOR1 KO mice (62±2%) and their wild type (WT) littermates (68±1%). Surprisingly, after one week of pressure overload, induced by thoracic aortic banding, cardiac function deteriorated significantly more in NCOR1 KO than in their WT controls. LV EF was significantly less (31±7.4%), p<0.01, in NCOR1 KO than WT (59±3.1%) with higher LV end diastolic diameter (4.0±0.3 mm), compared to WT (3.4±0.2 mm). Survival rate was lower (60%) in NCOR1 KO with 1 week of pressure overload than WT, which all survived. At autopsy, lung/body weight, an index of heart failure, was also greater, p<0.01, in NCOR1 KO mice (10±0.7) vs. WT (6.5±0.4); confirmed by similar results for lung/tibial length. Histological analysis demonstrated greater fibrosis, reflecting greater cell death in NCOR1 KO compared with WT controls. Thus, the salutary features of other NCOR1 KOs could not be recapitulated in the heart with the stress of chronic pressure overload, where the cardiac specific NCOR1 KO actually fared worse with exacerbation of heart failure and mortality.

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of biliverdin reductase‐A (BVRA) promotes lipid accumulation and lipotoxicity in mouse proximal tubule cells
Authors: Adeosun Samuel O. , Gordon Daren M. , Weeks Mary‐Frances , Moore Kyle H. , Hall John E. , Hinds Terry D. , Stec David E.
Published: June 2021

Obesity and increased lipid availability have been implicated in the development and progression of chronic kidney disease. The proximal tubule cells of the kidney are the major site of lipid accumulation in the kidney, and thus are a major target for the toxic effect of excessive lipid accumulation –lipotoxicity. The aim of the current study was to test the hypothesis that the loss of biliverdin reductase‐A (BVRA) increases the susceptibility of mouse proximal tubule cells to excess lipid accumulation and lipotoxicity. We used CRISPR‐Cas9 technology to generate a BVRA knock out (BVRA‐KO) cell line from wildtype (WT) immortalized mouse MCT proximal tubule cells. We exposed the cells for 24 hrs to 400 μM Palmitic acid (PA) and then measured lipid accumulation and markers of lipotoxicity. Lipid accumulation was 83% (p=0.0033) and 54% higher (p=0.0002) in BVRA‐KO versus WT cells at baseline and following PA treatment respectively. This suggests that both de novo lipogenesis and fatty acid uptake were higher in BVRA‐KO versus WT cells. CD36, which play a major role in fatty acid uptake, was upregulated 7.8 folds in BVRA‐KO vs. WT cells (p=0.002). In addition, NGAL1 expression, Annexin‐v FITC staining, and LDH release were all significantly higher in BVRA‐KO cells vs. WT cells, demonstrating that BVRA‐KO cells are more sensitive to PA‐induced lipotoxicity than WT cells. Phosphorylation of AKT and BAD, which play important roles in cell survival and inhibition of apoptosis, were significantly reduced by PA, only in BVRA‐KO cells (52% and 39% reduction; p=0.0019 and 0.0061 respectively). These data demonstrate the protective role of BVRA in proximal tubule cells against saturated fatty acid‐induced lipotoxicity and suggest that activating BVRA may play a protective role in obesity‐induced kidney injury.

Support or Funding Information

This work was supported by grants from the National Heart, Lung and Blood Institute K01HL‐125445 (T.D.H.), PO1HL‐051971 (D.E.S.), HL088421 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM‐104357 (J.E.H.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.