bluuebunny/crossref_metadata_2025_split huggingface dataset:
Participation of the pvd Gene Cluster in the Mechanism of Quorum Sensing and Virulence of Pseudomonas aeruginosa PAO1
Authors: Perez Sharel Pamela Diaz , Peña Aide Campos Espino , Perez Alma Laura Diaz , Alcántar Lorena Martínez , García Jesús Campos 
Published: April 2020

ObjectiveDetermination of the molecular mechanism involved in the virulence modulation of Pseudomonas aeruginosa PAO1 involving the pvdI and pvdL genes.MethodsSupernatants of the P. aeruginosa PAO1 strain and mutants pvdI, pvdJ, and pvdL were obtained from LB liquid medium after 24 h of growth. Evaluation of virulence factors: biofilm was determined by growth of P. aeruginosa strains in 96‐well plates using violet crystal staining. Rhamnolipids production was determined by quantification of rhamnose by the orcinol method. Survival assays were carried out in Caenorhabditis elegans using supernatants of PAO1 strains.AbstractPseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals and plants. This bacterium possesses a system of perception of bacterial quorum (quorum sensing, QS), which modulates several virulence factors. The LasI/LasR QS system modifies the production of the cyclodipeptides (CDPs) cyclo(L‐Pro‐L‐Val), cyclo(L‐Pro‐L‐Phe) and cyclo(L‐Pro‐L‐Tyr). Although CDPs are molecules with bioactive properties, low elements concern in the mechanism of biosynthesis in bacteria and their role in communication with host cells. In addition, it has been described that the CDPs production in P. aeruginosa mainly depends of the multi‐modular non‐ribosomal peptide synthetases (MM‐NRPS), whose products such as the aeruginaldehyde has been involved in the bacterial QS mechanisms. In this study, was found that levels of pyocyanin (a virulence factor in P. aeruginosa), it was increased in mutant strains in the pvdJ gene, which encodes for a NPRS. The pvd gene cluster is directly related to the pioverdin synthesis, which is a fluorescent peptide‐siderophore involved in the iron acquisition, being essential in the bacterial pathogenicity on the host. Therefore, is important to elucidate the mechanism by which the CDPs of P. aeruginosa PAO1 contribute to its pathogenesis and/or virulence. Production of several virulence factors in P. aeruginosa PAO1 with different single and double mutants in pvdI, pvdJ and pvdL genes were compared. Mutants in the pvdI, pvdL and pvdL/pvdI genes decrease 50% the production of biofilm respect to the WT strain. Likewise, when evaluating rhamnolipids amount, the production of rhamnolipids was significantly increased in the single mutants pvdJ and pvdL compared with the WT strain, but not modified in the double mutant pvdJ/pvdL. Survival in the C. elegans worms indicated that the pvdJ mutant increase the death of the nematode 40% than the WT. The results suggest that the pvd gene cluster was involved in the production of CDPs in P. aeruginosa PAO1 and may to regulate the production of virulence factors (biofilm and rhamnolipids), impacting its pathogenicity on the nematode in vivo model.
The Stability and Localization of Tetrahymena thermophila UBC9 Mutants
Authors: Meeks Kaylen , Forney James 
Published: April 2020

UBC9 is the sole E‐2 conjugating enzyme in the SUMOylation pathway, which is analogous to the ubiquitin pathway. SUMOylation can affect the localization of proteins or their ability to interact with other substrates. UBC9 is essential in most eukaryotes including the ciliate Tetrahymena thermophila. Tetrahymena thermophila are protozoa with two nuclei, a somatic macronucleus (MAC) and germline micronucleus (MIC). Transcription occurs in the macronucleus, but not in the micronucleus. Tetrahymena have a large increase in SUMOylation within the developing macronucleus during conjugation (sexual reproduction). In an effort to create UBC9 mutants with altered activity or cellular location, we designed site‐directed changes to the UBC9 gene. Some of these changes were analogous to mutations identified in yeast UBC9, others were directed at the N‐terminus that is conserved between Tetrahymena thermophila and other ciliates, but is substantially different from other eukaryotes. Plasmids containing YFP fusions of UBC9 with small deletions in the N‐ or C‐ terminus or single amino acid changes were transformed into Tetrahymena and viewed via fluorescence microscopy to determine localization. Constructs with the same mutations but N‐terminal FLAG tags were analyzed via Western blots to determine stability of the mutant proteins. Versions of UBC9‐YFP fusions with 13 amino acids removed at the N‐terminus were not detectable, or barely detectable by microscopy as compared with wild type UBC9‐YFP fusions. A YFP fusion with only 4 amino acids removed from the N‐terminus was visible with a similar intensity as wild type (full length) controls. In contrast, removal of 4 amino from the C‐terminus eliminated all signal from expression of a YFP‐fusion. The tagged versions of UBC9 with 13 and 4 amino acids of the protein removed at the N‐terminus produced a signal on a Western blot; while a UBC9 construct with 4 amino acids removed from the C‐terminus was not able to produce a signal on a Western blot. To our surprise, removing amino acids from the C‐terminus had a greater effect on protein stability than removing those from the N‐terminus. Analysis of two other mutations (R20A, R24D) predicted to create altered cellular location or stability of the protein showed small, if any differences from wild type UBC9 in the distribution between cytoplasm and nucleus. Additional mutations are being constructed in the C‐terminal region in an attempt to generated UBC9 with marginal stability that could be used for in vivo analysis of function.Support or Funding InformationDepartment of Biochemistry, Purdue University, West Lafayette, INCollege of Agriculture (Molecular Agriculture Summer Institute), Purdue University, West Lafayette, IN
Absorption, Distribution and Excretion of [14C] Zamicastat after Single Oral and Intravenous Administration in Rats and Dogs
Authors: Loureiro Ana I. , Araujo Francisca , Bonifacio Maria-Joao 
Published: April 2020

BackgroundZamicastat is a dopamine‐β‐hydroxylase inhibitor
(1), previously showed to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.ObjectivesThe purpose of this study was to characterize the absorption, distribution and elimination of 14C‐zamicastat in rat and dog, after single oral (p.o.) or intravenous (i.v.) administration.MethodsWistar rats and Beagle dogs were administered with 14C‐zamicastat at 30 mg/kg orally or intravenously with 2.5 mg/kg (rat) and 0.1 mg/kg (dog). Plasma, urine and faeces samples were collected at different time‐points to measure the plasma exposure and the recovery of total radioactivity up to 168 h post‐dose. Whole body phosphor imaging (WBPI) was evaluated in rat after p.o. administration of 30 mg/kg of 14C‐zamicastat. The extent of plasma protein binding was determined by equilibrium dialysis and blood cell binding was determined, using centrifugation, on pooled male samples from each species.ResultsAfter single oral dosing of 14C‐zamicastat, more than 95% of total radioactivity was recovered at five and seven days from rats and dogs, respectively. The radioactivity peaked in plasma between 6 to 8 h and 2 to 4 h post‐dosing in rats and dogs, respectively, followed by a slow elimination over time. Zamicastat related radioactivity was mainly recovered in faeces with very low levels (less than 20% of total radioactivity) excreted in urine in both species. Following i.v. administration more than 80% of radioactivity was recovered in both species mainly via feaces excretion. The WBPI study indicates that radioactivity was distributed throughout the body with the majority of tissues achieving a tissue:blood ratio greater than 2. Elimination of radioactivity progressed over time with most tissues reaching the low limit of quantification at 168h. The highest tissue:blood ratios were noted in thyroid gland, lung and harderian gland at, respectively, 168h, 8h and 8h post‐dosing. Protein binding data indicates that zamicastat is preferentially bound to plasma proteins in a concentration independent manner, with 98% and 96% bound to rat and dog plasma proteins, respectively.ConclusionZamicastat is a highly plasma protein bound compound after oral administration and it is extensively distributed to tissues. After oral administration to rats and dogs, radioactivity was almost completely recovered with most of zamicastat‐related radioactivity excreted through faeces.
Methamphetamine‐induced retinal hyperpermeability, hypoxia, and vascularization
Authors: Lee Minsup , Leskova Wendy , Pickett Haley , Eshaq Randa , Harris Norman 
Published: April 2020

PurposeMethamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation.Materials and methodsC57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot.ResultsThe level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P<0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P<0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P<0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment.ConclusionOur present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation.Support or Funding InformationThis work was supported by funding from the National Institute of Health (NIH) EY025632.
DNA‐Binding Properties of TBX5 Mutants Associated with Congenital Heart Defects
Authors: Cesar Brenda I. , Carrasquillo Emmanuel I. , Zazaboi Jasmin , Rodriguez Jose A. 
Published: April 2020

Congenital Heart Diseases (CHD) affects over 150,000 persons every year. CHDs are characterized by abnormal structural heart formation during embryonic development. Previous studies have identified several TBX5 mutations in CHD patients. TBX5 is a transcription factor (TF) that has been identified as an important regulator during heart development. TFs are sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. We selected 9 non‐synonymous TBX5 mutants discovered in CHD patients. The consequences on the interactions between these mutated cardiac TFs and DNA have not formerly been investigated. Evaluation of DNA‐recognition properties of TBX5 is fundamental for understanding its possible roles in controlling gene regulatory networks and could potentially help us know more about the role of TFs in the context of CHD. Our lab plans to determine the sequences our TFs prefer and how this specificity changes when it mutates. In order to test this, we have cloned TBX5 and its T‐Box domain that contain a His‐ and Strep‐Tag affinity tags that we will use for purifying after we overexpress it in competent E. coli. cells. We have also generated all 9 sequence‐verified TBX5 mutants using site‐directed mutagenesis. In site‐directed mutagenesis, mutations are introduced by the use of a PCR primer pair carrying the intended mismatch. We have also optimized experimental conditions for the overexpression and affinity‐purification of the TBX5 T‐Box domain. The DNA‐binding activity of TBX5 DNA binding domain T‐box has been confirmed through electrophoretic mobility shift assays (EMSA) using the TBX5 cognate site. This assay allows us to visualize DNA‐protein affinity through electrophoretic gel shifts. The mutation I54T shows more affinity towards the TBX5 cognate site and the mutation G80R shows no affinity towards it. Identifying the effects of non‐synonymous mutations on the DNA‐binding properties of TBX5 will contribute to our understanding of TBX5 role during heart development in congenital heart defects.Support or Funding InformationThis research was funded by SC1GM127231
A metformin‐methylglyoxal imidazolinone metabolite (IMZ) increases angiogenesis via HIF‐1 alpha activation in endothelial cells.
Authors: Nguyen Huong , Wang Jiemei , Monks Terrence 
Published: April 2020

Deficient angiogenesis exacerbates the outcome of patients with peripheral arterial diseases, leading to critical limb ischemia, non‐healing ulceration or even amputation in lower extremities. These events progress more rapidly in patients with diabetes mellitus. Therapeutic strategies aiming at improving angiogenesis have been intensively studied in clinical trials, but with limited benefit. We previously discovered that metformin, the first‐line medicine for diabetes, scavenges a biologically reactive endogenous dicarbonyl, methylglyoxal, to form a novel imidazolinone metabolite (IMZ). However, the impact of IMZ on endothelial cell function is unknown. We hypothesize that IMZ increases endothelial cell‐mediated angiogenesis by activating hypoxia inducible factor (HIF)‐1 alpha, engaging key angiogenesis pathway(s). We examined the effects of IMZ on endothelial cell angiogenesis using functional assays (aortic sprouting assay, network formation, migration and proliferation assay) in vitro and characterized IMZ’s potential signaling pathways regulating HIF‐1 alpha, using western blot analysis.Our findings revealed that 24‐hour treatment of IMZ at 1μM significantly increased endothelial sprouting, the formation of capillary‐like networks on matrix, and cellular migration in endothelial cells. These effects of IMZ occurred concomitantly with an upregulation of multiple pro‐angiogenic mediators, such as placenta and platelet‐derived growth factor, fibroblast growth factor 2 beta, vascular endothelial growth factor, angiopoietin 1, as revealed by protein arrays. Meanwhile, IMZ activated endothelial nitric oxide synthase, increased nitric oxide production, and engaged the PI3K/AKT and MAPK pathway in a concentration and time‐dependent manner. These pathways stabilize and upregulate HIF‐1 alpha expression in endothelial cells. Indeed, IMZ promoted HIF‐1 alpha expression as measured by western blot. More importantly, the effects of IMZ on angiogenesis were blunted by knockdown of HIF‐1 alpha with small interfering RNA. Collectively, our data uncovered, for the first time, the pro‐angiogenic effects of a metformin metabolite IMZ. Future studies will further characterize the therapeutic potential of IMZ in alleviating impaired angiogenesis under diabetic conditions.Support or Funding InformationNIH/NIDDK R01 DK119222 (Wang & Monks) and NIH/NIDDK R01 DK109036 (Wang)
Nuclear Receptors CAR, ERα and RORα Communicate via Phosphorylation to Differentially Regulate the SULT1E1 Gene Expression in Diabetic and Nondiabetic Human Livers
Authors: Fashe Muluneh Markos , Negishi Masahiko 
Published: April 2020

Studies showed that SULT1E1 is differentially expressed in nondiabetic and diabetic human as well as mouse livers. Here, we intended to investigate the molecular mechanism of the differential expression of SULT1E1 in diabetic and nondiabetic human livers. Utilizing chromatin immunoprecipitation (ChIP) in male human livers and coimmunoprecipitation (CoIP) assays, we report that nuclear receptors CAR, ERα and RORα communicated via phosphorylation of their respective DNA binding domains to differentially regulate the expression of SULT1E1 gene in nondiabetic and diabetic (type 2) male human livers. We studied the functional role of ERα and RORα with respect to SULT1E1 expression in human primary hepatocytes by treating the cells with siRNAs against ERα or RORα and SULT1E1 mRNAs were suppressed along with ERα or RORα mRNAs. Moreover, promoter analysis, electrophoretic mobility shift assays and cell‐based reporter assays in COS‐1 cells identified three DNA elements: ERα response element (h1E1‐ERE) and RORα response element (h1E1‐RORE) within the enhancer region and a TATA box within the proximal promoter that were critical for the regulation of SULT1E1 gene in human liver cells. ChIP assays using antibodies against ERα, p‐Ser212 ERα, RORα or p‐Ser100 RORα and primers targeting the proximal SULT1E1 promoter revealed that both receptors were phosphorylated and enriched on the proximal region of SULT1E1 gene promoter in nondiabetic but not in diabetic male human livers. Similar observations were also made in ChIP assays performed with antibody against CAR. Moreover, CoIP assay in HepG2 cells overexpressing ERα WT or its ERα S212D mutant and RORα WT, RORα S100A or RORα S100D displayed a stronger interaction between ERα S212D and RORα S100D and this interaction was further enhanced with CAR cotransfection. Altogether, phosphorylation driven interaction between nuclear receptors CAR, p‐Ser212 ERα and p‐Ser100 RORα was critical for a positive expression SULT1E1 gene in human livers and lack thereof might be responsible for the observed downregulation of SULT1E1 in diabetic livers.Support or Funding InformationNIEHS ‐ NIH Intramural Research Program
5‐HT2 Receptor Activation Differentially Modulates Linker Histone Kinetics and Histone Deacetylase Activity
Authors: Flanagan Thomas Wayne , Billac Gerald Bernard , Nichols Charles David 
Published: April 2020

IntroductionAlterations to chromatin dynamics and histone modification represent crucial processes metazoan cells employ to influence regulation and gene expression. As such, modification to the mobility of the linker histone, the chief component of chromatin compaction and higher‐order chromatin architecture, significantly impacts downstream transcriptional regulation and cellular processing. At present, the contributions of serotonergic signaling to not only heritable epigenetic modification but also higher‐order chromatin structure are a matter of intense research focus. Through both in vitro and in vivo assays, our lab has shown that serotonin 5‐HT2A receptor agonists (psychedelics) exhibit differential efficacy and potency in their ability to prevent inflammatory pathway activation. Modulation to both overall chromatin structure and epigenetic signaling may represent a potential mechanism that psychedelics employ to promote their long‐lasting, therapeutic effects.ObjectivesWe sought to evaluate the impact of chromatin compaction and histone acetylation on 5‐HT2A functional selectivity in human embryonic kidney cells stably expressing the 5‐HT2A receptor.MethodsWe utilized fluorescence recovery after photobleaching (FRAP) to probe the kinetics of the linker histone H1c transiently transfected into the aforementioned HEKH2A cell line. We concurrently measured histone deacetylase (HDAC) activity following exposure to numerous 5‐HT2A agonists.Results5‐HT itself minimally impacts H1c mobility, whereas compounds such as psilocin and the highly selective 5‐HT2 agonist (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI] significantly slow H1c kinetics. Interestingly, these changes to linker histone mobility do not correlate with putative epigenetic regulation, as 5‐HT2A compounds with anti‐inflammatory properties reduce histone deacetylase (HDAC) activity, whereas antagonism of the 5‐HT2A receptor induces HDAC activity.ConclusionTogether, this data illustrates that agonism of 5‐HT2A receptors induce unique changes to chromatin architecture and HDAC activity, a conceivable mechanism psychedelics might employ to promote their potent therapeutic properties. Translationally, while HDAC inhibitors have emerged as attractive agents in cancer therapy and asthma treatment, their utility is limited by off‐target toxicity. 5‐HT2A receptor activation represents a potentially more promising therapeutic strategy to modulate histone acetylation patterns for therapeutic effects.Support or Funding InformationSupport from this work was provided in part by the Eleusis Benefit Corporation and the LSU Foundation.
Investigation of Ceramide involvement in Diesel Exhaust‐Induced Astrocyte Apoptosis
Authors: Vettrus Noah , Morgan Connor , Johansen-Sallee Anthony 
Published: April 2020

Apoptosis is a complex process involving many different molecules that eventually leads to the programmed death of a cell. Ceramides are believed to function as a second messenger in this process; however, the full involvement of ceramides is under investigation. We are investigating a connection between airborne particulate matter and neurodegenerative processes, including cell death. The primary mission of our research is to better understand how ceramide levels change during apoptosis induced by exposure to diesel exhaust particulate matter (DEP). To track the changing levels of ceramides we performed total lipid extractions on our cell cultures at various timepoints after exposure to DEP. As we gain more understanding on this role of ceramides in apoptosis, we hope to make connections between Alzheimer’s disease and incorrectly functioning ceramide signaling networks. Our current results have indicated a change in ceramide levels during apoptosis at a long timepoint and we continue to work to understand how the ceramide signaling route works.Support or Funding InformationResearch reported in this publication was supported by grant number P20GM103443.