bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Glucose Transporter Proteins (GLUTs) in Human Placental Tissue after Normal vs. Diabetic Pregnancy
Authors: Szukiewicz Dariusz , Stanirowski Pawel Jan , Pyzlak Michal , Abdalla Nabil , Sawicki Wlodzimierz , Cendrowski Krzysztof
Published: November 2023

Altered expression of GLUTs in diabetic placenta has been suggested recently by independent authors, including the role of insulin therapy as a possible modulatory factor. In this study we examined comparatively the expression of GLUTs (GLUT‐1, GLUT‐4, and GLUT‐9) in the placental tissue obtained after normal healthy and diabetic pregnancies. Additionally, the effect of insulin therapy on the expression of selected GLUTs isoforms was analyzed. Placental samples derived from healthy gravidas after normal term pregnancies (N = 25) and obtained after diabetic pregnancies, including diet‐controlled gestational diabetes mellitus (GDMG1; N = 16), insulin‐controlled GDM (GDMG2; N = 6), and pre‐gestational DM (PGDM; N = 6). To visualize GLUT‐1, GLUT‐4, and GLUT‐9, standard immunohistochemical procedures were used with the respective anti‐GLUT antibodies. Thereafter, computer‐assisted quantitative morphometry was performed to identify immunostained GLUTs in 5‐μm paraffin‐embedded sections of the placental specimens, and the final results were adjusted for the mean microvascular density with the permitted margin of error not exceeding ± 5 %. Morphometric evaluation revealed a significant increase (p < 0.05) in the mean expression of GLUT‐4 and GLUT‐9 in insulin‐dependent diabetes (GDMG2 + PGDM) as compared to both, the control and GDMG1 groups. A significant increase in the mean GLUT‐1 expression was observed only in placental specimens obtained from PGDM group (p < 0.05). The differences in GLUTs expression between GDMG1 group and the healthy controls did not reach statistical significance. In conclusion, our results have confirmed the presence of GLUT‐1, GLUT‐4 and GLUT‐9 proteins in the trophoblast from both, uncomplicated and diabetic pregnancies. Interestingly, insulin therapy may significantly increase placental expression of GLUT‐4 and GLUT‐9 isoforms, and partially GLUT‐1, in diabetic gravidas classified as GDMG2 and PGDM.

Support or Funding Information

WUM grant: 2WA/PM23D/14
SGI‐1252, a TGF‐beta inhibitor, protects against diet‐induced obesity and insulin resistance in mice
Authors: Dallon Blake , Bikman Benjamin T.
Published: November 2023

Very recent research has revealed a muscle‐derived hormone called irisin, which has generated considerable industry interest given its anti‐obesity effect by increasing mitochondrial mass and, critically, uncoupling in distinct adipose tissue depots. By increasing mitochondrial uncoupling in adipocytes, the tissue catabolically uses nutrients for mild heat production, effectively increasing metabolic rate, lowering glucose and insulin, and, thereby, mitigating fat gain and protecting against diabetes onset. Inhibition of the TGFb pathway at muscle cells allows the production of irisin. Thus, we sought to exploit this phenomenon by the use of SGI‐1252, a TFGb inhibitor. In addition to differences in plasma irisin, mice treated with SGI‐1252 were leaner than placebo‐treated mice when fed a high‐fat, high‐sucrose diet. Moreover, UCP1 expression increased significantly in white adipose tissue and mitochondrial respiration was similarly increased in these adipose depots. Altogether, these findings suggest a potential therapeutic role for SGI‐1252 in the treatment of obesity and diabetes.
ErbB Receptors Signaling Promote Endothelial Phenotype of Human Left Ventricular Epicardial Highly Proliferative Cells
Authors: Ryzhov Sergey , Robich Michael P. , Rath Rutwik , Roberts Daniel J , Quinn Reed , Kramer Robert S , Sawyer Douglas B
Published: November 2023

Despite a long history of heart‐specific ErbB signaling research, the role of ErbB receptors in cardiac non‐myocyte cells is largely unknown. We recently have shown that neuregulin‐1b (NRG‐1b) prevented transition of CD105posCD31neg cells obtained from mouse heart into myfibroblasts. These cells can form single‐cell derived colonies with high proliferative potential and capability to differentiate into different cell types. The present study examines the expression of ErbB receptors on highly proliferative cells (HiPC) obtained from human LV epicardium and the role of ErbB receptor subtypes in the regulation of CD31, VEGFR2 and Tie2 endothelial markers expression. Left ventricular free wall epicardial biopsies from human subjects undergoing open heart surgery were used to obtain myocytes‐depleted cardiac cell suspension. These cells were seeded at low cell density to produce colonies of HiPC. We found that human HiPC are characterized by expression of all four ErbB receptors. The level of ErbB receptors varied among HiPC obtained from different subjects. ErbB2 and ErbB3 showed highest level of inter‐individual variability compared to ErbB1 and ErbB4. Flow cytometric analysis revealed strong expression of CD105 (endoglin), CD73 (ecto‐5′‐nucleotidase) and CD29 (integrin β1), and lack of CD34, CD117 (c‐kit), CD11b, and CD45 on HiPC. In addition, we detected expression of CD49f (integrin alpha 6), CD90 (Thy‐1) and CD31 (PECAM‐1) on cell surface of HiPC. A strong positive correlation was found between CD31/PECAM‐1 and ErbB2 but not ErbB1, 3 or 4. To determine the role of ErbB receptors, we incubated HiPC in the absence or presence of EGF to stimulate ErbB1, and NRG‐1b to stimulate ErbB3 and ErbB4 receptor activation. We found that both EGF and NRG‐1b ligands promoted CD31 cell surface expression on HiPC which express high level of ErbB2 (ErbB2hi). In contrast, no effects of ErbB ligands were seen in ErbB2low/− HiPC. Only EGF but not NRG‐1b increased VEGFR2 expression on HiPC. No difference was found between ErbB2hi and ErbB2low/− HiPC, indicating that ErbB1 receptors are solely responsible for the upregulation of cell surface expression of VEGFR2. In addition, both EGF and NRG‐1b induced up‐regulation of Tie2 on HiPC to a similar degree, irrespective of the level of ErbB2 expression. Thus our data identified a novel role of ErbB signaling in promotion of endothelial phenotype of human HiPC.

Support or Funding Information

Supported in part by Acorda therapeutics, Inc, U01 HL100398, and the Osher Cardiovascular Research Fund at Maine Medical Center
Phosphorylation of Plakoglobin at S665 is Essential for Positive Adhesiotropy in Cardiomyocytes
Authors: Schinner Camilla , Vielmuth Franziska , Radeva Mariya , Schmidt Andreas , Imhof Axel , Waschke Jens
Published: November 2023

Cardiomyocytes are coupled by intercalated discs (ICDs), which are composed of desmosomes, adherens junctions and gap junctions. Recently, we demonstrated sympathetic signaling to regulate ICDs and cardiomyocyte cohesive strength. Elevated cAMP levels increased adhesion mediated by the desmosomal adhesion molecule desmoglein 2 (Dsg2) paralleled with reorganization of the ICD, an effect we referred to as positive adhesiotropy. Our data indicated dependency of these effects on the plaque protein plakoglobin (Pg), but the underlying mechanisms are unknown. Mass spectroscopy of Pg reveled S665 as the only site with increased phosphorylation after cAMP elevation. A phosphodeficient Pg mutant for this site blocked both the increase of intercellular cohesion as well as the reorganization of Dsg2 positive junctions induced by adrenergic stimulation. Furthermore, the shift of Dsg2‐mediated binding events towards cell‐cell borders and the increase in Dsg2‐mediated binding forces were abrogated in cardiomyocytes expressing the phosphodeficient mutant compared to wild‐type Pg. In summary, we provide evidence for positive adhesiotropy to be dependent on Pg phosphorylation at S665. This mechanism may be of high medical relevance because impairment of Dsg2 and Pg by mutations are a major cause of arrhythmogenic cardiomyopathy.
Inhibition of HDAC, especially HDAC3, prevents diabetic cardiomyopathy in OVE26 mice via epigenetic inhibition of ERK1/2‐DUSP5 pathway
Authors: Xu Zheng , Cai Lu
Published: November 2023

Inhibition of histone deacetylase (HDAC) phenomenally protects against streptozotocin‐induced cardiomyopathy. To explore the mechanisms, male type 1 diabetes OVE26 mice and age‐matched wild type mice were given HDAC inhibitor (valproic acid, VPA), selective HDAC3 inhibitor (RGFP966) or vehicle for 3 months. These mice then were sacrificed immediately or 3 months after the end of 3‐month treatment (6 months). Cardiac function and pathological changes (hypertrophy, fibrosis, inflammation and oxidative damage) were assessed by echocardiography and histopathological examination. Both VPA and RGFP966 significantly prevented DCM, reflected by the prevention of diabetes‐induced cardiac dysfunction, hypertrophy and fibrosis, along with cardiac oxidative stress and inflammation. The protection from DCM was persistently observed even at the 3rd month after the end of 3‐month HDAC inhibition, suggesting the “preventive memory” of DCM by HDAC inhibition. Mechanistically phosphorylated ERK1/2 that is a well‐known initiator to cardiac hypertrophy was significantly increased, which was prevented by VPA and RGFP966. Meanwhile the DUSP5 (Dual Specificity Phosphatase 5), an ERK1/2 negatively regulating nuclear phosphatase, was oppositely changed. Chromatin immunoprecipitation assays showed the elevated histone H3 acetylation at DUSP5 gene promoter in both 3 months and 6 months. These findings suggest that diabetes increases HDAC3 activity to deacetylate DUSP5 for derepressing ERK1/2 inhibition, leading to its initiation of DCM pathogenesis, which is preventable by HDAC inhibition. The epigenetic mechanism responsible for the pathogenesis of DCM and its preventable by HDAC inhibition explains its preventive memory. Therefore, these results suggested the therapeutic potential of HDAC inhibition in treating DCM.
Naturally Occurring Mechanisms for the Resolution of Pulmonary Vascular and Right Ventricular Remodeling
Authors: Shults Nataliia V. , Suzuki Yuichiro
Published: November 2023

Pulmonary arterial hypertension (PAH) is a lethal disease and improved therapeutic strategies are needed. Increased pulmonary arterial pressure, due to vasoconstriction and vascular remodeling, causes right ventricle (RV) failure and death. The treatment of Sprague‐Dawley rats with the SU5416 injection and the exposure to chronic hypoxia for 3 weeks followed by maintenance in normoxia promotes progressive and severe PAH with pathologic features that resemble human PAH. At 5–17 weeks after the SU5416 injection, PAH is developed with pulmonary vascular remodeling as well as RV hypertrophy and fibrosis. The present study investigated subsequent events that occur in these PAH animals. At 35 weeks after the SU5416 injection, rats still maintained high RV pressure, but pulmonary vascular remodeling was significantly reduced. Metabolomics analysis revealed that lungs of normal rats and rats from the 35‐week time point had different molecular profiles. Despite the maintenance of high RV pressure, the fibrosis was resolved at 35‐weeks. Masson's trichrome stain and Western blotting monitoring collagen 1 determined 12% fibrosis in the RV at 17‐weeks, and this was decreased to 5% at 35‐weeks. The expression of a‐smooth muscle actin (a marker of myofibroblasts) was elevated at 17‐weeks compared to control, and this was normalized at 35‐weeks.

Conclusions

These results suggest that biological systems possess natural ways to resolve pulmonary and RV remodeling. The resolution of RV fibrosis appears to involve the reduction of myofibroblast‐dependent collagen synthesis. Understanding these endogenous mechanisms should help improve therapeutic strategies to treat PAH and RV failure.

Support or Funding Information

NIH
TNFalpha and IL‐1beta Synergically Decrease VEGFR2 and Increase VEGFR1 Protein Expression in Endothelial Cells
Authors: Yan Xiaocai , Managlia Elizabeth , De Plaen Isabelle G
Published: November 2023

Necrotizing enterocolitis (NEC) is the most serious gastrointestinal disease affecting premature infants. Among other inflammatory cytokines, TNFalpha and IL1beta are significantly increased in intestinal tissues during NEC induction in a neonatal mouse model. We previously found that intestinal villous endothelial cell (EC) proliferation and VEGFR2 expression are decreased, and mucosal vascular network development impaired during experimental NEC induction. Furthermore, inhibiting VEGFR2 kinase activity increased NEC severity. However, whether cytokines directly affect VEGFR2 expression in neonatal intestinal EC remains unknown. Here, we hypothesize that increased inflammatory cytokines may affect microvascular development by decreasing intestinal EC VEGFR2 signaling. To test our hypothesis, EC isolated from mouse neonatal intestine tissue and HUVEC were cultured in vitro and incubated with TNFalpha and/or IL1beta and VEGFR1 and 2 assessed by western blot. We found that: 1) TNFalpha treatment of both mouse EC and HUVEC for 12 hours dramatically decreased their VEGFR2 expression while inducing an increase in VEGFR1 expression. 2) IL‐1beta treatment for 12 hours decreased VEGFR2 expression without affecting VEGFR1 in both mouse EC and HUVEC. 3) When EC (both neonatal mouse intestinal EC and HUVEC) were incubated with IL1beta and TNFalpha together, there was a synergistic decrease in VEGFR2 expression and a synergistic increase in VEGFR1 protein expression. Other inflammatory cytokines found to be increased in NEC, such as IL6, IL10, CCL2 and CCL20 had no effect on HUVEC VEGFR1 and 2 expression. This suggests that TNFalpha and IL1beta produced during NEC induction may promote disease progression by impairing the development of the mucosal intestinal microvasculature, thus further affecting intestinal tissue perfusion.

Support or Funding Information

NIH grant RO1HD060876 and Ann & Robert H. Lurie Children's Hospital of Chicago Foundation
Improved triglyceride‐rich lipoprotein clearance in human apolipoprotein A‐II knockin rabbits
Authors: Koike Tomonari , Koike Yui , Yang Dongshan , Song Jun , Xu Jie , Fan Jianglin , Zhang Jifeng , Chen Y. Eugene
Published: November 2023

Apolipoprotein A‐II is a second major apolipoprotein in HDL particles, which account for 15–20 % of total HDL protein in human. Compared with apolipoprotein A‐I which is a most major apolipoproteins in HDL, physiological role of apoA‐II is not fully understood. We previously reported that overexpression of apoA‐II in rabbits significantly suppressed cholesterol‐rich diet induced atherosclerosis. Because rabbits are the animal of naturally deficient in apoA‐II, so it becomes a good animal model to study the functions of apoA‐II. To elucidate the role of apoA‐II independent from apoA‐I in HDL on lipid metabolism and atherogenesis, we generated a rabbit model in which the human apoA‐II was knocked in to rabbit apoA‐I locus by TALEN technology. The model was confirmed by genomic PCR, sequencing, RT‐PCR and proteomics. With this model, we found that the triglyceride levels are significantly lower in homozygote than that in wild type control. The results of lipoprotein analysis using ultracentrifugation and FPLC also show that TG‐rich lipoproteins were lower in homozygote than that of wild type. TGs were not increased in homozygote even after feeding of food. Small LDL and HDL2 particles were increased in homozygote rabbits. In addition, increased apoE was shown in HDL3 particle from homozygote. These results suggest that apoA‐II knock‐in has critical roles in the metabolism of TG‐rich lipoproteins. Roles of apoA‐II only HDLs from homozygote on cholesterol efflux and atherogenesis are under investigation.
Inhibition of the Receptor for Advanced Glycation End‐products (RAGE) protects from secondhand smoke (SHS) induced intrauterine growth restriction (IUGR) in mice
Authors: Lewis Joshua B , Mejia Camilo A , Jordan Clinton , Monson Troy D , Bodine Jared S , Dunaway Todd M , Egbert Kaleb M , Wright Tanner J , Ogden K Connor , Broberg Dallin S , Hall Parker D , Nelson Shawn M , Hirschi Kelsey M , Reynolds Paul R , Arroyo Juan A
Published: November 2023

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, or placental abnormalities, but studies investigating the effects of secondhand smoke (SHS) exposure and IUGR are limited. Receptors for Advanced Glycation End‐products (RAGE) are pro‐inflammatory transmembrane receptors increased by SHS in the placenta. We tested the hypothesis that inhibition of RAGE during SHS exposure protects from smoke‐induced IUGR. C57Bl6 mice were exposed to SHS or SHS + semi‐synthetic glycosaminoglycan ethers (SAGEs) known to inhibit RAGE signaling. RAGE −/− mice were also exposed to SHS. Trophoblast cells were treated with cigarette smoke extract (CSE) with or without SAGEs in order to address the effects of RAGE inhibition during trophoblast invasion in vitro. SHS treated mice demonstrated a significant reduction in fetal weight (7.35‐fold; p≤0.0001) and placental weight (1.13‐fold; p≤0.0001) compared with controls. RAGE −/− mice were completely protected from fetal and placental weight deviations when exposed to SHS. Mice co‐treated with SHS and SAGEs were protected from SHS‐induced fetal weights decreases (4.22‐fold; p≤0.0001). SHS treatment of C57Bl6 mice activated placental ERK (2.61‐fold, p≤0.05), JNK (1.79‐fold, p≤0.05), and p38 (2.44‐fold, p≤0.05) and the expression of inflammatory mediators including TNF‐α (1.34‐fold p≤0.05) and IL‐1β (1.03‐fold p≤0.05). SHS‐mediated activation of these molecules was reduced to basal levels when SAGE was co‐administered. Invasion of trophoblast cells decreased 92% (p<0.002) when treated with CSE and CSE‐mediated invasion was completely reversed by SAGEs. We conclude that inhibition of RAGE protects against fetal weight loss during SHS‐induced IUGR. Our results further suggest that there is a direct correlation between RAGE activation and the development of IUGR during SHS. These studies provide insight into tobacco‐mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
An experimental model of restoration of renal blood flow in murine 2‐Kidney 1‐Clip hypertension
Authors: Grande Joseph Peter , Osman Mazen , Kashyap Sonu , Hu Zeng
Published: November 2023

Renal artery stenosis (RAS) is a common public health problem, affecting up to 7% of individuals over 65 years of age. Optimal therapy for patients with RAS is controversial, as recent studies have indicated that revascularization via stenting fails to improve renal function or patient survival. Medical therapy is associated with high morbidity and mortality, primarily due to cardiovascular disease. However, morphologic correlates for reversibility following restoration of blood flow have not been adequately defined. The purpose of this study was to standardize an experimental model for the restoration of blood flow, employing a well characterized murine RAS model.

Total N=10 C57B/6J male mice were used. RAS was induced on the right renal artery by putting a polytetrafluoroethylene cuff (N=6) and sham surgery was performed without cuff placement (N=4). To determine the feasibly of restoring the blood flow to the stenotic kidney, cuff was taken off at day 7 following the RAS surgery. Blood pressure measurements were done using tail cuff method at baseline, before taking cuff off and then every week until harvest. MRI images were captured using an Avance DRX 700WB spectrometer with a vertical 16.4 T wide bore magnet connected with the imaging tools at baseline, 3 days after putting the cuff on or sham surgery, and at 3 weeks after cuff off. The animals were harvested at day 28 following surgery and kidney weights are obtained.

Baseline renal perfusion (1018±41.6 ml/100g/min) was significantly reduced (634.7±141.3 ml/100g/min, p=0.01) following cuff placement. Perfusion was restored to normal (955±59.8 ml/100g/min) at 28 days following cuff removal at 7 days after RAS. Similarly, volume of the stenotic kidney was significantly reduced (at day 3 after RAS 172±8.6 mm3) compared to baseline (206.3±4.4 mm3, p=0.0007). There was no significant difference in perfusion and volume after cuff off compared to baseline and sham groups. There was no significant difference in kidney weight between cuff‐off (167± 4.7 mg) and sham groups (163±9.95 mg). Also, there was no difference in heart weight between cuff off (150±3.7mg) and sham groups (148±6.7mg).

We conclude that this model will provide the basis for determining the latest time point associated with recovery of renal structure and potential histopathologic markers that predict preservation of renal structure following cuff removal. Also, this model would provide the basis for determining efficacy of additional therapies to target inflammation, etc. to widen the “therapeutic window” during which restoration of renal blood flow is beneficial.

Support or Funding Information

This project is funded by National Institutes of health, NIAID R01 AI100911.
Vascular endothelial cell responses to increased hydrostatic pressure
Authors: Chung Kyukwang , Patterson Eric , Lawendy Abdel , Fraser Douglas , Cepinskas Gediminas
Published: November 2023

Endothelial cell responses to mechanical forces such as shear stress and tensile stress have been a subject of intense investigations. However, there is a substantial lack of studies addressing the effects/mechanism of hydrostatic pressure on endothelial cell function. It is known that pathologies such as venous congestion, compartment syndrome etc., are closely associated to the increased intravascular and/or interstitial hydrostatic pressure.

In this study we examined vascular endothelial cell (HUVEC) responses to increased hydrostatic pressure (IHP). To this end, HUVEC were seeded into μ‐Slide IV (ibidi) channels and grown to confluence. Subsequently, one end of the channel was connected to the pressure monitor and the other end was connected to the cell culture medium reservoir, which was elevated to the height to induce 30mmHg hydrostatic pressure within the channel under conditions of “no flow”. HUVEC were exposed to the IHP (30mmHg) for 4 hrs in 5%CO2 cell culture incubator at 37°C. Control HUVEC were cultured at ambient pressure (0mmHg) under the same conditions.

The obtained results indicate that exposure of HUVEC to 30mmHg IHP results in: 1) prominent formation of F‐actin stress fibers (Phalloidin staining), 2) changes in VE‐cadherin junctional staining pattern, 3) induction of oxidative stress (DHR123 oxidation), 4) induction of apoptosis (caspase‐3 activity assay), and 5) increased neutrophil adhesion to HUVEC.

These results indicate that increased hydrostatic pressure per se is a critical factor contributing to endothelial cell activation and dysfunction.

Support or Funding Information

Supported by the grant from Lawson Health Research Institute (IRF‐28‐16)
A novel BET family bromodomain inhibitor NHWD‐870 represents a promising therapeutic agent for a broad spectrum of cancers
Authors: Yin Mingzhu , Wang Nenghui , Yan Qin
Published: November 2023

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. NHWD‐870 is a potent and selective BET family bromodomain inhibitor and only binds bromodomains of BRD2/3/4/T. NHWD‐870 exhibited robust single agent activity in cell viability assay across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to NHWD‐870 indicated that NHWD‐870 manifested diverse mechanisms of action in different cancer settings. These include: 1) inhibition of tumor cell growth by downregulating the PDGFRβ, MEK1/2 and STAT1/MYC signaling in tumor cells; 2) inhibition of tumor angiogenesis by decreasing PDGF production in tumor cells and the PDGFRβ and MEK1/2 signaling in endothelial cells. Consistent with its broad spectrum of activities in vitro, NHWD‐870 has potent tumor suppressive efficacies in xenograft mouse models of small cell lung cancer, triple negative breast cancer and ovarian cancer. These results support its further development for diverse solid tumor indications in the clinic.
miR‐16 Mediated MYB Gene Silencing Induces Fetal Hemoglobin Expression
Authors: Pounds Catherine R. , Takezaki Mayuko , Li Biaoru , Ward Christina , Lopez Nicole , Pace Betty S.
Published: November 2023

Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in the β‐globin gene affecting about 100,000 people in the United States and millions worldwide. Individuals living with this disease suffer from hemolytic anemia, progressive organ damage, and acute painful episodes. Fetal hemoglobin (HbF), a major genetic modifier of clinical presentation and progression of SCD, is of great interest due to its ability to inhibit sickle hemoglobin (HbS) polymerization and reducing erythrocyte damage. Variation in HbF levels among sickle cell patients is highly heritable and has been linked to the presence of single nucleotide polymorphisms (SNPs) in several genetic regulatory regions. In this study, DNA isolated from two cohorts of African American SCD patients with high and low HbF levels were analyzed by PCR‐based SNP analysis in the HBB, BCL11A, and HBS1L‐MYB (HMIP) gene regions. The presence of SNP alleles was then correlated with their clinical phenotype data to identify those associated with increased levels of HbF. Three SNPs in the BCL11A and HBB gene regions were identified in the high HbF patient cohort that may contribute to milder phenotypes in African Americans. To complement the SNP studies, genome wide microRNA (miRNA) data was generated which showed increased miR‐16 levels associated with high HbF. Furthermore, MYB, the negative regulator of γ‐globin expression, is silenced by miR‐16 through binding the 3′‐untranslated region. RT‐qPCR and western blot were used to measure γ‐globin mRNA and HbF protein levels respectively after transfection of miR‐16 mimic into KU812 cells. Preliminary data revealed dose dependent miR‐16 mediated MYB suppression and γ‐globin activation. The discovery of regulatory regions that modulate HbF expression and miRNA mediated suppression of negative regulators of γ‐globin, provides expanded understanding of gene regulation, which can be used to define novel treatment options for individuals with SCD.

Support or Funding Information

Sponsored by the Medical College of Georgia Medical Scholars Program
A Reductionist Approach to Understanding Membrane Fusion
Authors: Wickner William T.
Published: November 2023

Membrane fusion is essential for intracellular protein targeting, cell growth, neurotransmission, and hormone secretion. Since the mechanism of fusion is conserved from yeast to humans, we study fusion of the vacuole (lysosome) of the biochemically and genetically tractable yeast S. cerervisiae. Vacuole fusion requires phosphoinositides and non‐bilayer‐prone lipids, a Rab family GTPase, four SNARE proteins which form complexes together in cis (bound to the same membrane) or trans (bound to apposed membranes before fusion), the SNARE complex disassembly chaperone Sec18 (NSF), its cochaperone and fusion‐trigger protein Sec17, and a large, multisubunit complex termed HOPS (homotypic fusion and vacuole protein sorting). HOPS binds two membrane‐anchored Rabs to tether the organelle, catalyzes trans‐SNARE complex assembly through its Sec1/Munc18 (SM)‐homolog subunit, and directly associates with acidic lipids and phosphoinositides.

Each of these factors has been shown through genetics to be required for fusion in vivo, and in vitro for fusion of the isolated organelle. To understand the mechanism, we have reconstituted fusion with each of these purified, recombinant proteins and 8 defined lipids, revealing novel aspects of membrane fusion: 1. The lipid bilayer isn't just a passive player. Acidic lipids, non‐bilayer prone lipids, phosphoinositides, and the fluidity of the fatty acyl phase are each crucial. 2. Tethering is an essential step for fusion, even where trans‐SNARE complexes have formed. 3. SNARE complex assembly is catalyzed by SM proteins and associated proteins, 4. Sec17 (a‐SNAP) binds to the trans‐SNARE complex and inserts an apolar loop into the bilayer to trigger fusion. 5. A supercomplex of HOPS with trans‐SNARE complexes is found on the purified vacuole and can be reconstituted with purified components; we are seeking its precise function in the fusion cascade.

Support or Funding Information

This work was supported by NIH grant GM23377‐40
Kinesiological benefits of botulinum toxin type A combined with exercise on the functional recovery after spinal cord injury
Authors: Jin Yunho , Park Sookyoung , Hong Yonggeun
Published: November 2023

Spinal cord injury (SCI) is a result of a traumatic damage of the vertebral column. This injury interrupts the signal conduction from upper motor neuron to lower motor neuron and spasticity may occur. Spasticity is common motor dysfunction induced from upper motor neuron lesions. Botulinum toxin type A (BTA) can inhibit muscle contraction as well as acetylcholine release by without affection to acetylcholine synthesis. Treadmill exercise (Ex) is known to reduce body and fat mass resulting improvement of neuroplasticity. Therefore, the purpose of this study was to evaluate the effect of BTA combined with treadmill exercise after SCI. Experimental animals were divided into three groups; SCI, SCI+BTA, SCI+BTA+Ex. BTA was injected into the gastrocnemius of SCI+BTA and SCI+BTA+Ex rats. And, SCI+BTA+Ex rats were assigned to treadmill exercise. After 14 postoperative day, all animals were sacrificed to perform further analyses. SCI+BTA+Ex group showed the lowest mass of body weight and gastrocnemius which muscle subjected to injection of BTA directly. However, they showed the highest motor functioning scores. In addition, by light microscopy, several vacuoles were observed in the muscle fibers of SCI group rats, indicating delayed functional recovery. In this study, BTA is thought to have slight spasticity‐alleviating effect, and this effect is extended by combination of treadmill exercise.

Support or Funding Information

Funds: NRF‐2013R1A2A2A01067169 to Y.H., 2014R1A1A3051724 to S.P., KRIBB Research Initiative Program (KGM4611613 to Y.H.), Republic of Korea.
The immunomodulatory properties of adipose mesenchymal stem cell‐derived exosomes are induced by inflammatory cytokines
Authors: Domenis Rossana , Quaglia Sara , Cifù Adriana , Pistis Cinzia , Fabris Martina , Moretti Massimo , Vicario Annalisa , Niazi Kayvan R. , Curcio Francesco
Published: November 2023

Introduction

The predominant mechanism by which adipose mesenchymal stem cells (AMSCs) participate to tissue repair is through a paracrine activity and their communication with the inflammatory microenvironment is essential part of this process. This hypothesis has been strengthened by the recent discovery that stem cells release not only soluble factors but also extracellular vesicles, which elicit similar biological activity to the stem cells themselves. The most prominent of the extracellular vesicles are exosomes, that through the delivery and the transfer of protein, bio‐active lipid and nucleic acid cargo, are able to induce phenotypic and functional changes in the recipient cells that promote the activation of regenerative programs. The purpose of this study is to investigate the immunomodulatory properties of exosomes released from AMSCs under stimulation with inflammatory cytokines.

Methods

AMSCs were treated with different concentration of INFγ+TNFα for 48h and then proliferation/viability was measured by trypan blue exclusion count, expression of IDO enzyme was evaluated by flow cytometry and release of PGE2 by ELISA. Exosomes were isolated from AMSCs supernatants by polymer precipitation method (Exoquick). To evaluate their immune‐regulatory properties, CD14+ monocytes were treated, during their differentiation into M1 macrophages, with AMSCs‐derived exosomes. After treatment, macrophages were characterized for the expression of CD80 (M1 expression marker) or CD163 (M2 expression marker) by flow cytometry.

Results

Pro‐inflammatory INFγ+TNFα cytokines decreased AMSCs proliferation rate, while viability was not affected, and increased the expression of the immunosuppressive IDO enzyme and the release of PGE2 in dose‐dependent manner. Only exosomes released from cytokines‐treated cells were able to significantly reverse the M1 phenotype of macrophages indicating a polarization towards the M2 phenotype.

Conclusions

The treatment with inflammatory cytokines increase the immunosuppressive and anti‐inflammatory potential of AMSCs‐derived exosomes, which became able to shift macrophages from M1 to M2 phenotype. This suggests that the immunomodulatory properties of AMSCs‐derived exosomes may be not constitutive but are instead induced by the inflammatory microenvironment.
Ethnic variations in mercury exposure from seafood consumption and the risk of Kawasaki disease in young children
Authors: Portman Michael A. , Yeter Deniz , Kuo Ho‐Chang
Published: November 2023

Kawasaki disease (KD) is an acute febrile illness predominately affecting children <5 years that can result in serious cardiovascular complications such as coronary artery lesion formation. The cause of KD remains unknown 50 years after Kawasaki first described the illness in Japan, while KD has since replaced rheumatic fever as the leading cause of acquired heart disease for children in the developed world. KD has become the most commonly diagnosed pediatric vasculitis with the highest incidence rates of KD reported from East Asian countries such as Japan, Korea, and Taiwan. Even when residing in Western nations, East Asian children still have 10 to 20 times the risk of developing KD than children of other ethnicities. Speculation that genetic differences between ethnic groups account for the ethnic disparity in KD rates led to the discovery and confirmation of numerous susceptibility loci, although these risk alleles only appear to confer risk passively to the currently unknown environmental triggers of KD. Previously, we described how ITPKC susceptibility in KD patients would render these individuals vulnerable to mercury (Hg)‐induced autoimmunity through the induction of intracellular calcium release with downstream effects upon cell signaling, immune proliferation, and gene expression via the Ca2+/NFAT pathway. This led us to hypothesize that the ethnic disparities in KD risk may be accounted for by variations in Hg exposure rates, particularly from significant differences in total methylmercury (MeHg) intake from seafood. We examined two separate cohorts that were nationally representative of either KD hospitalization rates or toxic metal concentrations in the blood in our initial investigations. Our results demonstrated that increasing ethnic KD risk between Caucasian, Hispanic, Black, and Asian children was significantly associated with average blood levels and detection rates of Hg or cadmium (Cd) but not lead (Pb), manganese (Mn), or selenium (Se). In our most recent investigations using the same two datasets, we observed that ethnic KD risk was significantly associated with increasing consumption of total fish and seafood but not shellfish. Further examinations by Hg type demonstrated that MeHg from seafood is the primary source of blood Hg in children, while average blood MeHg levels but not inorganic or ethylmercury were significantly associated with increasing ethnic KD risk. Lastly, ethnic KD risk was significantly associated with molar ratios of Hg and Se in the blood that are indicative of seafood consumption. We conclude that Hg exposure particularly from seafood consumption may increase susceptibility for the development of KD in young children or trigger the disease, itself. However, our hypothesis requires determinations of blood Hg levels and cumulative Hg exposure such as from dietary seafood intake in KD patients, which are currently lacking and that we are presently examining.

Support or Funding Information

None
Quantitative Image Analysis of Traumatic Brain Injury Induced Aspiration Pneumonia Treatment
Authors: Howell Cody C. , Sandusky George E.
Published: November 2023

Infection of the patient is one of the greatest risks inherent to surgical transplants. While great effort is taken to minimize this risk; variables outside of surgeon control can still result in transference of disease from donor to patient. Such conditions are common in lung transplants from donors having suffered traumatic brain injury (TBI). Autonomic nervous system damage from injury prior to transplant can result in involuntary intake of food into the lungs, developing into aspiration pneumonia. Remaining undetected until complications arise in the recipient. This study evaluated TBI model mice treated with simulated transplant conditions. The control groups included: aspiration pneumonia, aspiration treated with phosphate buffered saline (PBS), TBI without aspiration, TBI with aspiration and no treatment, TBI with PBS, TBI with aspiration treated with PBS. Drug treatment groups of TBI with aspiration pneumonia were dosed with either BB5, or a proprietary complement inhibitor.

Mouse lung tissue was removed and fixed in 10% neutral buffered formalin (10% NBF) for 24 hours, processed into a paraffin block, cut via microtome and H&E stained. Slides were then examined and whole slide digital images were created and evaluated using quantitative imaging software. The software utilized for calculating the percentage of inflammatory cellular infiltrates across the groups used Red‐Green‐Blue color algorithms to classify stained tissue slides. Initial analysis with Aperio Imagescope was unable to classify the color difference between similarly stained inflammatory cells and alveoli septa in the lungs. Indica Lab's HALO software was used for the second round of analysis. Both drug treatment groups were determined to have a biologically significant reduction in aspiration pneumonia compared to the control groups in both analysis systems. Indica Labs' HALO software was more specific in differentiating inflammatory cells in this difficult analysis.
High‐precision pharmacokinetic measurements in anesthetized and awake behaving animals
Authors: Vieira Philip A. , Arroyo Netz , Shin Christina , Ploense Kyle , Plaxco Kevin , Kippin Tod
Published: November 2023

Objective

The development of technology capable of tracking the in vivo levels of drugs, metabolites, and biomarkers continuously and in real‐time would revolutionize our understanding of health and our ability to detect and treat disease. It would, for example, enable therapies guided by patient‐specific real‐time pharmacokinetics, opening a new dimension in personalized medicine.

Methods

In response we demonstrate here the ability of electrochemical aptamer‐based (E‐AB) sensors to support such measurements in vivo in living subjects.

Results

Specifically, we have used E‐AB sensors to perform multi‐hour measurements of specific molecules in the bloodstream of anesthetized and even awake, ambulatory rats, achieving clinically relevant detection limits, low‐ to sub‐micromolar precision, and second‐scale time resolution.

Conclusion

This work demonstrates the use of E‐AB sensors as a viable option for fast, real‐time measurement of drug targets as they interact with the body, suggesting the approach may prove a powerful new tool for the study of physiology, pharmacokinetics and pathology through the measurement of relevant biomarkers in conjunction with treatment.

Support or Funding Information

W.M. Keck Foundation
Circulating Concentrations of GDF8, but not GDF11, are Heritable when Evaluated by LC‐MS/MS
Authors: Bumgardner Sara A , Pazdro Robert
Published: November 2023

Growth differentiation factor 11 (GDF11) and GDF8/myostatin exhibit a high level of homology within their mature, active protein domains, which has led to a considerable degree of conflicting data. We previously demonstrated a high degree of narrow‐sense heritability for circulating GDF11 in mice utilizing antibody‐based methodologies, which correlated with and predicted the median lifespan of mice. In order to validate this data, we evaluated both serum GDF11 and GDF8/myostatin across four young‐adult mouse strains via LC‐MS/MS. We were unable to corroborate the narrow‐sense heritability of GDF11, but instead found that GDF8/myostatin is highly heritable and represents a large portion of the serum GDF11/8 phenotype. Additionally, the concentration of GDF11 as measured by LC‐MS/MS was significantly greater than that identified through antibody‐based methods. We hypothesize that these discrepancies could be due to variable assay specificity for total, latently‐bound, or active protein forms and/or assay sensitivity. Further research is needed to clarify the functional role and effect of natural genetic variation on circulating GDF11 and GDF8/myostatin.

Support or Funding Information

Research was funded by the University of Georgia Office of the Vice President for Research.
Kappa Opioid Receptor Signaling Contributes to Re‐escalation of Oxycodone Self‐administration Under Extended Access Conditions
Authors: Nguyen Jacques D , Kirson Dean , Grant Yanabel , Varodayan Florence P , Khom Sophia , Patel Reesha , Hedges David M , Roberto Marisa , Taffe Michael A
Published: November 2023

Prescription opioid abuse is a significant global problem, and opioid addiction is well‐characterized by compulsive drug seeking, motivational withdrawal and chronic relapse. The goal of this study was to investigate escalation of opioid intake with extended access to intravenous oxycodone self‐administration and re‐engagement of drug seeking (re‐escalation) following detoxification. Male Wistar rats were randomly assigned to short‐access (ShA, 1‐h) and long‐access (LgA, 12‐h) groups and trained to self‐administer oxycodone (0.15 mg/kg/infusion) using a fixed‐ratio 1 (FR1) response contingency and evaluated on a progressive‐ratio (PR) procedure incorporating dose‐substitution of oxycodone (0.06–0.3 mg/kg/infusion). Both groups underwent an abstinence period (4 weeks) and were tested for re‐escalation. Rats given LgA to oxycodone earned more infusions than rats trained with ShA during acquisition. PR test results demonstrated an increase in drug‐intake and breakpoints in rats assigned to LgA training relative to rats assigned to ShA training. All rats exhibited high levels of drug seeking upon re‐engagement; however, LgA rats failed to de‐escalate drug intake following the abstinence period. Previous findings show kappa opioid receptor (KOR) mediated disruption in GABAergic signaling in the central nucleus of the amygdala (CeA) is observed after acute (24‐h) withdrawal from escalated cocaine self‐administration. To investigate KOR dysregulation following acute oxycodone withdrawal, electrophysiological recordings were performed in CeA slices. The selective KOR agonist U‐50488 decreased GABAergic signaling in both ShA and LgA rats, and this effect was blocked by KOR antagonist nor‐binaltorphimine (nor‐BNI). Applied alone, norBNI increased GABAergic signaling, suggesting tonic KOR activity. These findings further support the conclusion that access duration differentially impacts the acquisition and maintenance of the self‐administration of oxycodone, and suggest that KOR‐mediated signaling during oxycodone dependence may be distinct from that of other drugs of abuse.

Support or Funding Information

NIH grant DA035281
Changes in Ventral Tegmental Area Serum‐ and Glucocorticoid‐Inducible Kinase 1 (SGK1) Catalytic Activity and Phosphorylation Alter Drug‐Related Behaviors
Authors: Doyle Marie A. , Bali Vedrana , Kaska Sophia , Cooper Sarah E. , Mazei‐Robison Michelle S.
Published: November 2023

Drugs of abuse are known to regulate activity of the mesolimbic dopamine system. Specifically, drug‐induced changes in ventral tegmental area (VTA) cellular activity and gene regulation contribute to behavioral outputs associated with addiction. Previous work from my lab has shown that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) phosphorylation and catalytic activity are increased by chronic, but not acute, administration of cocaine and morphine. However, the functional significance of these changes in drug‐related behaviors remains unclear. Thus, we have created novel viral constructs to overexpress SGK1 mutants in the VTA of adult mice, and I am assessing the impact of altered VTA SGK1 activity on drug reward via cocaine conditioned place preference (CPP) and voluntary morphine intake using a two‐bottle choice task. Intra‐VTA infusion of a catalytically inactive SGK1 mutant (K127Q) significantly decreased morphine intake and there was a similar non‐significant trend for decreased cocaine CPP, suggesting that decreasing VTA SGK1 activity is sufficient to decrease drug reward and intake. I am now extending these studies by investigating the role of SGK1 Ser78 in drug‐related behaviors, as phosphorylation of this site is increased by drugs of abuse. Intra‐VTA infusion SGK1 S78A, a mutant that prevents phosphorylation at S78, decreased morphine intake and preference. Conversely, mice with VTA overexpression of SGK1 S78D, a mutant that mimics phosphorylation, exhibited a trend for increased morphine intake and preference. These data suggest that alteration of VTA SGK S78 phosphorylation is sufficient to modulate drug reward and/or intake. To more fully understand the impact of VTA SGK1 activity on drug behaviors relevant to addiction, I am also now assessing effects on cocaine self‐administration. Additionally, future studies will employ cell type‐specific viral vectors to address if these behavioral changes are driven by SGK1 activity in VTA dopamine or GABA neurons. In summary, our goal is to characterize the role of VTA SGK1 activity in drug‐related behaviors in order to develop novel therapeutic strategies for addiction.

Support or Funding Information

T32 NS044928‐13 (NINDS), PhRMA Foundation Research Starter Grant, R01 DA039895‐01A1 (NIDA)
RGS Protein Regulation of Opioid Antinociception
Authors: Tai Sherrica , Senese Nicolas , Ingram Susan , Traynor John R
Published: November 2023

RGS proteins, or regulator of G protein signaling proteins, are negative modulators of G protein signaling downstream of G protein coupled receptors, including the mu‐opioid receptor (MOPR) and served to switch off signaling. We have previously shown that Gαo is the predominant Gα protein for mediating the effects of MOPR agonists‐mediated antinociception in the mouse. Consequently, male and female 129SvEv mice expressing a knock‐in RGS‐insensitive Gαo (RGSi‐Gαo) protein exhibit an increased antinociceptive response to MOPR agonists in the hot‐plate test. Here we show endogenous opioid peptide signaling is also enhanced in these RGSi‐Gαo mice. The enkephalinase inhibitor RB‐101, a compound that increases the life‐time of the endogenous opioid peptides, administered i.c.v. in wild type mice produced a naloxone‐reversible antinociception in the hot‐plate test, confirming a role for endogenous opioid peptides. This antinociceptive effect was significantly enhanced in the RGSi‐Gαo mice. Naloxone‐reversible antinociception is seen in rodent models following exposure to a stressor. A brief swim stress triggered antinociception in the RGSi‐Gαo mice but not in their wild‐type littermates, confirming RGS proteins are negatively regulating endogenous opioid signaling in the hot‐plate test. In contrast to the hot‐plate test there was a paradoxical inhibition, or no change, of opioid antinociception in the tail withdrawal test. The results show that the opioid peptide system is more active in mice expressing RGSi‐Gαo. However, the role of RGS proteins in negatively modulating MOPR signaling is not reflected as consistent inhibition of behavioral responses across models suggesting RGS proteins may be affecting other systems and/or acting on different intracellular substrates.

Support or Funding Information

Supported by DA035316 and DA035316‐03S1.