bluuebunny/crossref_metadata_2025_split huggingface dataset:
Granzyme B Disrupts Epithelial Barrier Function
Authors: Santacruz Stephanie , Parkinson Leigh , Granville David 
Published: November 2023

The epithelium functions as a barrier to the external environment, which is maintained in large part by cell junctions. Age‐related and chronic inflammatory conditions of the skin result in a loss of epithelial barrier integrity through the degradation of cell adhesion proteins. Granzyme B (GzmB) is a serine protease that accumulates in the extracellular milieu in a number of autoimmune skin conditions. As such, GzmB may contribute to the progressive loss of epithelial barrier function. We hypothesized that GzmB disrupts epithelial barrier function through the proteolytic cleavage of cell junction proteins. Human keratinocytes were treated with exogenous GzmB, in the presence or absence of a GzmB inhibitor. Subsequently, epithelial barrier function was assessed by Electric Cell‐substrate Impedance Sensing (ECIS) and immunofluorescence (confocal) microscopy was used to visualize E‐cadherin. GzmB treatment resulted in a loss of E‐cadherin staining on the cell membrane which was further supported by western blot analysis of cell supernatants and biochemical cleavage assays, where we observed a dose‐dependent increase in E‐cadherin fragmentation. E‐cadherin cleavage was markedly decreased when treated with a GzmB inhibitor. Moreover, ECIS analysis showed a noticeable decline in barrier function after GzmB treatment. In summary, GzmB contributes to a decline in epithelial barrier function in part through the proteolytic cleavage of E‐cadherin.Support or Funding InformationBC Lung Association
Wound Healing in a Model of Systemic Lupus Erythematosus may be Delayed by a Lack of Interleukin‐2
Authors: Doersch Karen Melissa , Newell‐Rogers M Karen 
Published: November 2023

ObjectiveThe purpose of this study is to examine the mechanism of delayed wound closure in a mouse model of Systemic Lupus Erythematosus (SLE).BackgroundSLE is a common and devastating autoimmune disease with symptoms that include skin changes and poor wound healing. SLE patients are frequently deficient in Interleukin‐2. Quercetin is known to alter the apoptosis‐verses‐proliferation balance and also to decrease Interleukin‐2 production by immune cells. This study uses quercetin to perturb wound healing in a mouse model of SLE and seeks to examine the Interleukin‐2‐dependent mechanism by which wounds in SLE patients fail to heal appropriately.HypothesisDecreased Interleukin‐2 delays and perturbs wound healing in SLE.MethodsFas‐deficient (LPR), and Fas‐Ligand mutant (GLD) mice served as the SLE model. These mice were wounded with an 8 mm biopsy punch, treated daily with intraperitoneal injections of quercetin and compared to wild‐type quercetin‐treated mice. Wounds were measured with calipers and lymphocyte frequencies and Interleukin‐2 receptor expression were measured by flow cytometry.ResultsQuercetin administration to LPR and GLD mice delayed wound healing compared with wild‐type mice. At day 10 post‐wounding, LPR and GLD mice had decreased percentages of both T‐helper and cytotoxic T lymphocytes in the draining lymph nodes of the wounds compared to wild‐type. This decrease in T‐cells may prevent adequate cytokine production at the wound site. Furthermore, CD4‐CD8‐ lymphocytes accumulate in LPR and GLD mice and express high levels of Interleukin‐2 receptor that can remove Interleukin‐2 from the wound environment. These cells appear to modulate the impaired wound closure.ConclusionsQuercetin impairs wound healing in SLE‐model mice, possibly in an Interleukin‐2 dependent manner. This study describes a novel model for understanding delayed wound healing in SLE and identifies Interleukin‐2 as a treatment target for wound healing in patients with SLE.Support or Funding InformationScott and White Foundation, Texas A&M Health Science Center College of Medicine
Soluble VE‐cadherin contributes to loss of VE‐cadherin‐mediated adhesion and endothelial barrier function
Authors: Flemming Sven , Burkard Natalie , Renschler Melanie , Vielmuth Franziska , Meir Michael , Schick Martin , Wunder Christian , Germer Christop‐Thomas , Spindler Volker , Waschke Jens , Schegel Nicolas 
Published: November 2023

Endothelial barrier breakdown in systemic inflammation and sepsis is a hallmark preceding organ failure and death in patients. Vascular endothelial (VE)‐cadherin‐mediated adhesion represents a crucial determinant of endothelial barrier integrity. Previously we demonstrated that under inflammatory conditions the disintegrin and metalloproteinase 10 (ADAM10) caused shedding of VE‐cadherin which resulted in the formation of soluble VE‐cadherin (sVE‐cadherin) in close temporal correlation of inflammation‐induced endothelial barrier disruption. Here we further characterized ADAM10‐induced VE‐cadherin shedding and tested whether recombinant sVE‐cadherin inferes VE‐cadherin mediated adhesion.First we identified possible cleavages sites of VE‐cadherin by mass spectrometry (MSM). Therefore, digestion of recombinant VE‐cadherin (rVE‐cadherin) with recombinant ADAM10 was performed. This led to the formation of sVE‐cadherin at 90 and 110kDa, as shown by coomassie stained gel an western blot, which correlated with several cleavage sites of the extracellular domain of VE‐cadherin. MSM showed only peptides up to 452 amino acids (aa) and one peptide at 503–530 aa, which also corresponds to the extracellular domains 1–5 of human VE‐cadherin. Additionally two other cleavage sites at 80–127aa and 160–175aa were observed. In the next step we performed atomic force microscopy measurements in a cell free sytem to test whether sVe‐cadherin blocked VE‐cadherin‐mediated adhesion. In these experiments both, sVE‐cadherin and rVE‐cadherin blocked VE‐cadherin‐mediated adhesion. Accordingly, application of rVE‐cadherin on Human dermal microvascular endothelial cells (HDMECs) resulted in a dose‐dependent loss of endothelial barrier functions as revealed by measurements of transendothelial electrical resistance (TER). In immunostaining of endothelial monolayers incubation with rVE‐cadherin led to intercellular gap formation.In summary our data demonstrate specific cleavage sites of VE‐cadherin by ADAM10. The resulting sVE‐cadherin fragments interfere VE‐cadherin mediated adhesion and thereby contribute to loss of endothelial barrier disruption. This may play a novel role in inflammation‐induced endothelial barrier breakdown.Support or Funding InformationThe present study was supported by grants from the German Research Foundation (DFG) to N.S. (Grant numbers: SCHL1962/2‐1 and SCHL 1962/4‐1).
Role of the Human Parainfluenza Virus Type 3 Fusion Machinery in Viral Growth in the Natural Host
Authors: Jusino Shirley , Palermo Laura M. , Moscona Anne 
Published: November 2023

Human parainfluenza virus type 3 (HPIV3) is a single‐stranded, negative‐sense RNA enveloped virus that causes severe respiratory diseases in children; including croup, bronchiolitis and pneumonia. HPIV3 infects its target cells by the coordinated action of the receptor binding protein hemagglutinin‐neuraminidase (HN) and the fusion (F) envelope glycoprotein, which together comprise the molecular fusion machinery. Previous studies from our lab show that viruses bearing HN‐F machinery that confer excellent growth in monolayer cultures cannot thrive in vivo. In turn, viruses that efficiently replicate in vivo‐ for example clinical isolates (CIs) ‐ bear HN‐F machinery that cannot cope with in vitro cell culture. Our aim is to characterize the fusion machinery properties of lung adapted HPIV3 virions. We hypothesized that circulating HPIV3 viruses (CIs) bear HN‐F fusion machineries well suited to the natural environment. In order to characterize the different functions of the CIs HNs and F HPIV3, viral RNA was extracted from CIs and the genomic sequences were analyzed by deep sequencing techniques. The cDNA encoding the CIs viral glycoproteins were inserted in expression vectors and assayed for expression levels by a biotinlyation assay. Additionally, neuraminidase activity was tested for all CIs HNs. Results were expressed as relative percentage of lab adapted ‐HPIV3 HN. Preliminary results suggest that HPIV3 CIs have a higher neuraminidase activity in comparison to lab adapted ‐HPIV3 HN and low fusogenicity. Overall, our results suggest that the fusion machinery of CIs is more stable and less readily activated than viruses adapted to growth in culture.Support or Funding InformationGrant: 5R01AI031917‐23Grant: NIH NIGMS R25GM096955
Identification of Protein Kinase R and Tissue Distribution in Channel Catfish
Authors: Ball Jenna , Vides Melissa , Kobayashi Yass 
Published: November 2023

Protein kinase R (PKR) is one enzyme responsible for the cessation of translation during stress exposure in mammals by inactivating the eukaryotic translation initiation factor 2 through phosphorylation. PKR is also considered a viral sensor as activity of PKR increases in response to the presence of double‐stranded RNA within the cell during viral infection. Consequently, activation of PKR leads to the inhibition of cell proliferation and apoptosis, which prevents generation of new viral particles. Channel catfish have been considered an excellent model fish species in basic and comparative physiological studies. However, little is known about the immune response of channel catfish against viral infection, and the underlying mechanism(s) associated with improved disease response has not been well‐defined. Given that PKR is an integral component of mammalian innate immunity, this study with PKR in channel catfish may serve as a potential model to investigate human immune response to viral infections. The objective of this study was to identify PKR mRNA in channel catfish and to examine the tissue distribution. The gene encoding the PKR was identified by comparing channel catfish EST database with zebra fish PKR mRNA. Expression of PKR mRNA was examined in the cDNA of brain, liver, muscle, spleen, trunk kidney, and heart using RT‐PCR. The PKR mRNA was detectable in all tissues examined at varying degrees. Nucleotide sequence of the amplicon (399 bp) was identical to the corresponding channel catfish EST (100%), but not with any other PKR mRNA. Currently, this study is investigating expression of PKR mRNA in relation to long‐term stress associated with food deprivation.Support or Funding InformationThis study is supported by the Kansas IDeA Network of Biomedical Research Excellence (Grant number 20GM103418).
Substitutions in the Proximity of the Active Site of Peptidylarginine Deiminase from P. gingivalis ‐ Is There a Correlation with Enzyme Activity?
Authors: Bereta Grzegorz , Gawron Katarzyna , Łazarz‐Bartyzel Katarzyna , Łazarz Mieczysław , Stożek Karol , Naskalska Antonina , Burmistrz Michał , Pyrć Krzysztof , Chomyszyn‐Gajewska Maria , Potempa Jan 
Published: November 2023

BackgroundPeriodontitis (PD) is a chronic inflammatory disease leading to tooth loss, which affects over 30 % of adults worldwide. A major causative agent of the disease is an anaerobic bacterium, Porphyromonas gingivalis (Pg). Recently, considerable interest has been focused on peptidylarginine deiminase (PPAD) from Pg, an enzyme which catalyzes conversion of arginine to citrulline. Our recent results imply PPAD activity as a critical factor in gingival cell infection and prostaglandin E2 induction. Those findings encouraged us to analyze polymorphisms of the PPAD gene and enzyme activity among Pg clinical strains.ObjectiveAnalysis of PPAD gene polymorphisms and enzyme activity in clinical isolates of Pg from patients with chronic periodontal disease (CPD), reference strains (RS) and healthy volunteers (HV).Material and methodsGingival crevicular fluid was collected from 9 CPD and 4 HV, seeded on blood agar and grown under anaerobic conditions. Pg was confirmed by 16S rRNA and PPAD PCR, followed by genomic DNA purification. Next, PPAD coding sequences were cloned into a pTZ57R vector, amplified and sequenced. The sequences from CPD and HV were aligned with the PPAD sequences deposited in the GenBank database, i.e. 2 of RS (ATCC33277, W83), 4 strains of PD patients (F0568, F0569, F0570, SJD2), and 5 other strains (F0185, F0566, JCVI SC001, W50, W4087). Further, PPAD activity from CPD, RS and HV was analyzed using colorimetric method.ResultsIn total, mutations were identified in 50 nucleotide positions in the PPAD sequence from all clinical isolates and those deposited in GenBank. One sequence was identical to the ATCC33277, but varied from the W83 strain, while 4 sequences were identical to the W83 strain. One isolate harboured 7 nucleotide substitutions, which resulted in a change of 3 amino acid residues in the proximity of the PPAD active site. This isolate displayed significantly increased PPAD activity in comparison to W83 strain. The identical nucleotide substitutions were noted in PPAD of strains from patients with PD found in USA and China.ConclusionsOur analyses shown variations in nucleotide and amino acid sequence of PPAD among clinical and reference strains. The presence of specific, identical amino acid substitutions in clinical isolates from Poland, USA and China in comparison to the RS along to the variation of enzyme activity may suggest an impact of PPAD on the virulence of Pg. This observation deserves further elucidation of functional and clinical significance of this mutation in the pathogenesis of PD.Support or Funding InformationNational Science Centre, Poland (2012/07/B/NZ6/03524, K.G.), Foundation for Polish Science (TEAM, DPS/424‐329/10, J.P.)
Efficacy Related Modulation of Behavioral Effects of Opioids During Chronic Buprenorphine Treatment in Squirrel Monkeys
Authors: Withey Sarah Louise , Paronis Carol Anne , Bergman Jack 
Published: November 2023

Opioid addiction is characterized as a chronic relapsing disorder in which renewed drug‐seeking behavior during abstinence can be provoked by exposure to an opioid or opioid‐associated cues. In laboratory subjects, drug‐seeking behavior similarly can be reinstated by priming with the drug or drug‐related stimuli. Buprenorphine (BUP), a partial agonist at the μ opioid receptor, is commonly prescribed for the management of opioid addiction but its ability to reduce reinstatement behavior in laboratory subjects is not well understood. In the present studies, squirrel monkeys (n=4) were trained to respond under a drug/food choice procedure (i.e., concurrent FR schedules of i.v. oxycodone and milk delivery). Next, the priming strength (i.e., % injection lever responding when the choice was between i.v. saline or milk) of different opioids was determined before and during chronic BUP treatment (0.1 or 0.32 mg/kg/day). Results thus far indicate that: 1) despite decreased potency, indicative of tolerance, full agonists (oxycodone, methadone) maintain priming strength during chronic treatment whereas 2) the priming strength of partial agonists (nalbuphine, butorphanol) is dramatically decreased. These results suggest a reduction in the ability of lower efficacy, but not high efficacy agonists to provoke relapse in BUP‐maintained people. The extent to which these findings may reflect efficacy‐related differences in other opioid endpoints (e.g. analgesia) during BUP maintenance remains to be determined.Support or Funding InformationFunded by NIH/NIDA: DA035857
Novel inhibitors of the soluble epoxide hydrolase block pain in multiple models
Authors: Wagner Karen , Lee Kin Sing Stephen , Hwang Sung Hee , Hammock Bruce D. 
Published: November 2023

The soluble epoxide hydrolase (sEH) is a master regulatory enzyme downstream in the cytochrome P450 (CYP450) branch of the arachidonic acid (ARA) cascade. The sEH enzyme is the principal route of degradation for the bioactive epoxidized metabolites of long chain fatty acids formed by CYP450 activity. The most studied of these are the epoxyeicosatrienoic acids (EETs) but the epoxidized metabolites of both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) the epoxydocosapentanoic acids (EDPs) and eicosatetraenoic acids (EEQs) respectively are known substrates of sEH. Targeting the sEH to inhibit the metabolism of these epoxy fatty acids (EpFAs) has been shown to be antiinflammatory, antihypertensive and to delay seizure. Important to the focus of this work, inhibiting sEH has also been efficacious against both inflammatory and neuropathic pain. Here we use newly designed inhibitors based on a N, N′ disubstituted urea pharmacophore guided by crystal structures which have exhibited improved drug‐like properties, pharmacokinetic profiles and potency in both equilibrium binding and drug residence time on the recombinant sEH of several species. This increased potency and druggability has transferred into better efficacy against modeled pain in vivo. We have tested the efficacy of these inhibitors in models of murine inflammatory pain and induced diabetic neuropathic pain. Newly we have extended this testing to the Akita mouse model which has a genetic mutation in the insulin receptor resulting in a loss of function and eventual type I diabetes. The inhibitors were potent and efficacious in these models using both withdrawal and operant nociceptive assays. Additionally, the inhibitors show synergy with both coxibs and fatty acid amide hydrolase inhibitors and the absence of any reward side effects which relate with addiction potential as seen with narcotics. Thus, targeting sEH is a promising strategy to alleviate pain and holds potential for impacting human health with translation to the clinic.Support or Funding InformationNIEHS R01 ES002710, NIEHS/Superfund P42 ES004699, NIH/NIDDK U24 DK097154, NIDDK R01DK090492, KW is supported by 5 T32L086350‐08.
Effects of MIA‐induced osteoarthritis on hind limb weight bearing and bone biology endpoints in rats with or without access to running wheels
Authors: Cone Katherine , Krivitsky Rebecca , Warner Emily , Imbert Ian , Allen Joshua , Henderson Terry , Rosen Clifford J. , Bilsky Edward J. , King Tamara , Stevenson Glenn W 
Published: November 2023

Our laboratories have been characterizing the interactions between exercise and pain‐like states in rodents. The present set of experiments characterized the effects of monosodium iodoacetate (MIA) on hind limb weight bearing in rats that were either sedentary or had access to running wheels. Rats were given intra‐articular saline or MIA (3.2 mg) into the left hind knee. Rats were divided into two groups. Group 1 had 24 hr voluntary access to running wheels for 42 consecutive days (21 days acquisition followed by 21 days post‐MIA). Group 2 had no access to running wheels. Weight bearing was measured on post‐MIA days 3, 7, and 21. In sedentary rats, MIA produced shifts onto the uninjured hind limb on PD 3, 7 only. In rats with access to running wheels, MIA produced weight asymmetry on PD 3, 7 and 21. To determine potential causal mechanisms behind the differences between sedentary and exercised rats, ex vivo microCT imaging analysis was performed to assess architectural changes to hind limb distal femur/proximal tibia and knee. Data indicated that MIA runners had greater lateral (but not medial) subchondral total volume loss relative to MIA non‐runners. Data for trabecular region are currently being analyzed. These results suggest that within the specific parameters of this study, voluntary wheel running exacerbated the effects of MIA‐induced OA on both behavioral and bone biology measures.Support or Funding InformationFunded by NIAMS/NIH grant AR054975‐02A1 to G.W.S.
The Lysophosphatidic Acid LPA1 Receptor as a Novel Molecular Target of Antidepressants in Neuronal Cells
Authors: Olianas Maria C. , Dedoni Simona , Onali Pierluigi 
Published: November 2023

Neuronal death and atrophy of brain limbic structures have been associated with stress and depression, and antidepressants have been reported to protect against neuronal cell loss induced by different neurotoxic insults. However, the molecular mechanisms mediating the neuroprotective effects of antidepressants have not been completely elucidated. Several studies have shown that the bioactive phospholipid lysophosphatidic acid (LPA) affects neurogenesis, synaptic plasticity and anxiety‐related behaviour through specific G‐protein coupled receptors, termed LPA1–6, which are expressed in the developing and adult brain. We have recently reported that different antidepressants induce growth factor receptor transactivation and cell proliferation in Chinese hamster ovary cells (Olianas et. al., Biochem. Pharmacol, 95, 311–323, 2015). In the present study, we show that in immortalized mouse hippocampal HT22 cells the tricyclic antidepressants amitriptyline and imipramine and the atypical antidepressants mianserin and mirtazapine trigger ERK1/2 phosphorylation and protect against apoptosis through the LPA1 receptor. ERK1/2 phosphorylation induced by acute exposure to the antidepressants or LPA was inhibited by cell pre‐treatment with pertussis toxin, indicating the involvement of G proteins of the Gi/o family. Antidepressants and LPA also promoted the phosphorylation of the transcription factor CREB, a downstream effector of ERK1/2 signaling. Cell treatment with either AM966, a selective LPA1 receptor antagonist, or Ki16425, which preferentially blocks LPA1 and LPA3 receptors, significantly inhibited ERK1/2 and CREB phosphorylation elicited by the antidepressants. HT22 hippocampal cells underwent apoptosis following serum deprivation. Antidepressants and LPA markedly attenuated neuronal apoptosis, as indexed by increased cell viability, decreased percentage of annexin V‐positive cells, inhibition of pro‐caspase activation and poly‐(ADP ribose) polymerase cleavage. AM966 and Ki16425 reduced the protective effects of antidepressants and LPA against HT22 hippocampal cell apoptosis. These data demonstrate the involvement of the LPA1 receptor in the neuroprotective action of different classes of antidepressants.Support or Funding InformationThe study was supported by Regione Autonoma della Sardegna, Italy, L.R. n. 7/2007‐CRP10810/2012.
Effects of Ketamine in the Forced Swim Test in Female Rats
Authors: Felger Rachel , Carreno Flavia , Frazer Alan 
Published: November 2023

Depression affects around 350 million people in the world, according to the World Health Organization. There are traditional pharmacological treatments that work for many, but not all who suffer. In fact, 10–30% of depression patients can be considered treatment resistant, as they do not improve significantly after treatment with traditional antidepressants. Ketamine, a noncompetitive N‐methyl‐D‐aspartate (NMDA) glutamate receptor antagonist, has been shown to produce antidepressant‐like effects in male rat models when administered at a low, sub‐anesthetic, dosage. Ovariectomized (OVX) and normal cycling female rats were used in this study to determine ketamine's effects on behavior in the Forced Swim Test (FST). Results show that OVX females exhibit decreased immobility in the first 24 hours after ketamine administration (p = 0.001), a typical antidepressant‐like effect, but this effect on immobility disappears by 7 days post‐treatment. Conversely, normal cycling females exhibit this antidepressant‐like effect overall at both the 24 hour and 7 day (p = 0.03) time period, and do not display an increase in locomotor activity during the first five minutes of the locomotor activity test. The results indicate the females respond in a similar manner to males, producing antidepressant‐like effects in both normally cycling and OVX females. However, further research is needed to clarify the mechanisms contributing to the shorter duration of response observed in the OVX rats.Support or Funding InformationDepartment of Veterans Affairs and ASPET