bluuebunny/crossref_metadata_2025_split huggingface dataset:
SRY‐box 9 (SOX9): A Novel Binding Partner of Cardiac Muscle‐specific A‐Kinase Anchoring Protein (mAKAP) Signalosome
Authors: Suryavanshi Santosh , Bark Steven , McConnell Bradley K 
Published: November 2023

BackgroundBy definition, A‐kinase anchoring proteins (AKAPs) bind to protein kinase A and various other signaling proteins at specific subcellular compartments to regulate downstream signal transduction. Heart specific muscle AKAP (mAKAP) is present on the nuclear envelope of cardiomyocytes and regulates cardiac hypertrophy by forming a signalosome of its binding partners. It was previously reported that hypoxia‐inducible factor 1 alpha (HIF‐1α) physically binds to mAKAP. Also, it was previously published that HIF‐1α physically interacts with SRY‐box 9 (SOX9). Chromatin immunoprecipitation (ChIP) studies showed that HIF‐1α binds to SOX9 promoter region. Interestingly, both mAKAP and SOX9 have been demonstrated to play significant role in cardiac differentiation and endocardial cushion formation, respectively. Hence, we were interested to know whether SOX9 is a part of mAKAP scaffold on the nuclear envelope.ObjectiveThe aim of the current work is to confirm that SOX9 unambiguously interacts with mAKAP; either directly or indirectly.MethodsAs HEK 293T cells express SOX9 endogenously and not mAKAP, we chose these cells for immunoprecipitation (IP) studies. Myc‐DDK tagged human mAKAP cDNA was purchased from Origene and overexpressed in HEK 293T cells using Lipofectamine 2000, according manufacturer's instructions. IP was performed using M2 affinity gels (Sigma) and immunoblotting was performed using mAKAP and SOX9 antibodies. HepG2 cell lysate was used as positive control for SOX9 expression. In another set of experiment, a Coomassie Blue stained gel was used for in‐gel digestion to perform MALDI‐TOF Mass Spectrometry, followed by data analysis.Summary of ResultsIP samples of mAKAP transfected HEK cells displayed SOX9 expression; whereas, untransfected cells did not show expression. mAKAP expression was used as a loading control and remained unchanged in all samples. Mass Spectrometric data validated our findings by demonstrating the expected mass peak for SOX9 in the IP samples.Conclusion and Future ImplicationsSOX9 was identified as a novel binding partner of mAKAP. As mAKAP and SOX9 already have a role in cardiac developmental biology, we suggest that mAKAP‐SOX9 interaction has substantial role in cardiac physiology and pathophysiology. The significance of this mAKAP‐SOX9 interaction is a part of our ongoing investigations.Support or Funding InformationR15HL124458 Scaffolding Protein Interaction Network Regulates Cardiac Cell Signaling
Regulation of neuroinflammatory cytokines by Angiotensin and Cannabinoid systems in SHR astrocytes
Authors: Haspula Dhanush , Clark Michelle 
Published: November 2023

ObjectiveTo understand the distinct regulatory roles of astroglial cannabinoid and Angiotensin systems, in altering neuroinflammatory states in hypertension.BackgroundNeuroinflammation in the brainstem is deemed as a major contributor to neurogenic hypertension. An imbalance in the levels of pro‐ and anti‐inflammatory cytokines, has been reported in the brains of spontaneously hypertensive rats (SHRs). These rats are a model of hypertension and Attention Deficit Hyperactivity Disorder (ADHD). Although cannabinoids exert potent neuroprotective and anti‐inflammatory effects, their role in regulation of cytokines in hypertension has not been investigated.MethodsAstrocytes were isolated from the brainstem and cerebellum of Wistar rats and Spontaneously Hypertensive Rat (SHRs) pups. treatments were done with 100 nM Angiotensin (Ang) II or 10 nM of the potent Cannabinoid Type 1 receptor (CB1R) agonist‐ACEA, both alone and in combination, for varying time periods, the mRNA and secreted protein levels of interleukin 1 (IL1) and IL10 were measured using qPCR and ELISA, respectively.ResultsBoth IL1 and IL10 basal levels were significantly higher in brainstem astrocytes, but not cerebellar astrocytes, of SHRs when compared to normotensive Wistar rat counterparts. Ang II treatment resulted in predominantly elevated IL1 and reduced IL10 levels in both brainstem and cerebellar astrocytes of SHRs and Wistar rats. However, only the Ang II‐mediated alterations in IL10 levels was significantly greater in brainstem astrocytes of SHRs when compared to Wistar rats. ACEA treatment on the other hand, was associated with a significant increase in IL10, and a slight reduction in IL1 levels, in both brainstem and cerebellar astrocytes of Wistar rats and SHRs. The effect of ACEA on IL10 was greater in brainstem astrocytes of Wistar rats when compared to SHRs. Co‐treatment of Ang II and ACEA resulted in a greater reduction of ACEA‐mediated increase in IL‐10 in brainstem astrocytes of SHR when compared to Wistar rats.ConclusionSignificant differences in basal cytokine levels in brainstem astroglial cultures from SHRs and Wistar rats, indicates a dysregulated neuroinflammatory state in brainstem astrocytes under hypertensive conditions. Greater IL10 elevation, by ACEA, in brainstem astrocytes of Wistar rats when compared to SHRs, is indicative of endocannabinoid hypofunction in hypertension. The inability of CB1R to elicit a protective role in brainstem astrocytes under hypertensive states, could well be an important facet in the etiology of essential hypertension.Support or Funding InformationNSU HPD: Grant # 335585
Regulation of the Functional Properties of Xeroderma Pigmentosum Complementation Group A (XPA) Protein through Lysine Acetylation
Authors: Njeri Catherine , Kaur Jasmeet , Turchi John , Balakrishnan Lata 
Published: November 2023

Nucleotide Excision Repair (NER) is the main repair pathway through which DNA damages occurring on account of ultraviolet (UV) or ionizing radiation (IR) are repaired. Xeroderma pigmentosum complementation group A (XPA) protein is a crucial part of the NER multi‐protein complex that plays an indispensable role in recognizing and binding the damaged DNA, as well as positioning other NER proteins optimally for efficient repair of the injury. Studies have shown that XPA can be dynamically acetylated and deacetylated impacting its involvement in the NER repair pathway. Deacetylation of XPA by SIRT1 enhances its interaction with the single strand DNA (ssDNA) binding protein, replication protein A (RPA). Our current research is focused on evaluating the effects of lysine acetylation on the structural and functional properties of XPA. Our mass spectrometry analysis revealed that XPA is in vitro acetylated at multiple sites by p300 and CBP acetyltransferases. We evaluated the effects of acetylation on the DNA binding properties of XPA protein using electrophoretic‐mobility shift assays (EMSA) and bio‐layer interferometry (BLI) technology. Interestingly, our results show that acetylation of XPA significantly lowers its binding affinity for cisplatin damaged DNA. Similar to reports in vivo, analysis of protein‐protein interactions between XPA and RPA, revealed that acetylation of XPA reduces its ability to interact with RPA. Our results suggest that acetylation of XPA negatively affects its role in recognizing, binding and consequently repairing damaged DNA, which in turn may impact subsequent recruitment of RPA. By application, our results present a mechanistic approach towards negatively modulating the effectiveness of the NER pathway, with the aim of improving the efficacy of current chemotherapeutics.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health GM0983289 (to LB)
G protein specificity of inhibitory adrenergic a2a receptor‐mediated modulation of synaptic transmission
Authors: Yim Yun Young , Betke Katherine , McDonald W. Hayes , Gilsbach Ralf , Chen Yunjia , Hyde Karren , Wang Qin , Hein Lutz , Hamm Heidi 
Published: November 2023

Modulation of neurotransmitter exocytosis by activated Gi/o‐type G‐protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is Gβγ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) complex interaction. There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR in vivo are not known. Presynaptic α2a‐adrenergic receptors (α2a‐ARs) in both adrenergic (auto α2a‐ARs) and non‐adrenergic neurons (hetero α2a‐ARs) inhibit neurotransmitter release and affect various physiological function such as anesthetic sparing and working memory enhancement. Here, we investigate whether auto α2a‐ARs in sympathetic neurons use the same Gbg subunits as hetero α2a‐ARs in other neuronal types to inhibit exocytosis by interacting with SNARE. Using several mice models including transgenic Flag‐α2a‐ARs, knock‐in HA‐α2a‐ARs, co‐immunoprecipitation, mass spectrometry analysis, we have determined the Gb and Gg subunits that interact with α2a‐ARs and SNARE complexes. So far, we find Gβ2 preferentially interacting with activated auto α2a‐ARs. We also see a basal Gβγ‐SNARE interaction and the 2 fold enhancement of this interaction upon the auto α2a‐ARs activation. Further understanding Gβγ specificity and Gβγ‐SNARE interaction may offer new insights into the normal functioning of the brain, as well as better understanding of disease progression.Support or Funding InformationThis work was supported by the National Institutes of Health (EY10291, MH101679, DK109204, and T32)
Glycosaminoglycans Inhibit Substance P‐ and IL‐33‐stimulated IL‐8 and TNF Release from Human‐Cultured Mast Cells
Authors: Gross Amanda Rae , Theoharides Theoharis C. 
Published: November 2023

Glycosaminoglycans (GAGs) are long, linear, highly negatively charged carbohydrate chains that are known for their functional role in connective tissue, joint lubrication, and anticoagulant activity. Mast cells (MC) are hematopoietic tissue immune cells, involved in allergic and inflammatory reactions, which contain high amounts of GAGs, such as chondroitin sulfate (CS) and heparin, in their numerous secretory granules. In fact, heparin is synthesized and stored exclusively in MC granules and is thought to electrostatically store biogenic amines, such as histamine. A few previous studies reported that CS and heparin can inhibit the release of histamine from activated connective tissue rat and canine MC, respectively. However, the inhibitory potential of GAGs on human MC remains unknown. Here we report that CS had no effect on MC degranulation, as measured by beta‐hexosaminidase release, from LAD2 human‐cultured MC stimulated by the neuropeptide substance P (SP; 2 μM for 1 hr); however, pre‐incubation with CS did inhibit SP (1 μM for 24 hrs)‐induced IL‐8 and TNF release in a dose‐dependent manner. Specifically, CS 100 μg/mL inhibited IL‐8 and TNF release from SP‐stimulated LAD2 MC by 32% and 40%, respectively. Moreover, dermatan sulfate (DS) 5 μg/mL and heparin 40 μg/mL inhibited IL‐8 and TNF release from LAD2 MC stimulated by the cytokine IL‐33 (10 ng/mL for 24 hrs) by approximately 32% (p<0.05) and 39% (p<0.05), respectively. Similarly to heparin, at 37.5 μg/mL CS inhibited IL‐8 and TNF release from IL‐33‐stimulated LAD2 MC by approximately 36% and 43%, respectively. Preliminary, ongoing studies indicate that DS has an inhibitory effect on TNF gene expression at 6 hrs after IL‐33 stimulation. These results are important because IL‐33 is known to be an alarmin involved in allergic inflammation. GAGs from the tissue microenvironment or released by MC, themselves, upon activation, could inhibit the release of mediators from MC in a paracrine or autocrine fashion. It would be interesting to investigate the amount of these GAGs present in certain conditions involving allergic inflammation of the skin, such as atopic dermatitis, cutaneous mastocytosis, and psoriasis. DS may be formulated for local application, alone or in conjunction with other anti‐allergic molecules for the topical treatment of these diseases.Support or Funding InformationSupport for this project comes from the Tufts University Department of Integrative Physiology and Pathobiology (IPP) Fiscal Year 2016 Research Seed Funds.
Joint application of N‐Acetylcysteine and Allopurinol reduces spinal cord TNF‐alpha expression and attenuates mechanical allodynia in diabetic Rats
Authors: Li Sisi , Yan Dan , Gu Pan , Zhu Qiqi , Cheung Chi Wai , Irwin Michael G , Yu Cong , Xia Zhengyuan 
Published: November 2023

Diabetic Neuropathy is a frequent and painful complication of diabetes that leads to progressive and length‐dependent Diabetic Peripheral Neuropathic Pain (DPNP). Studies have indicated the involvement of oxidative stress and the activation spinal pro‐inflammatory TNF‐alpha in the development of diabetic neuropathy. Joint application of the antioxidants N‐Acetylcysteine (NAC) and allopurinol (ALP) has been shown to confer superior protection against diabetic cardiomyopathy to their individual administration (Free Radic Biol Med. 2013; 63: 291–303). However, the potential effects and mechanism NAC and ALP combination on DPNP has not been explored. We hypothesized that NAC and ALP may reduce diabetes induced increase in central nervous system (CNS) TNF‐alpha expression and attenuate DPNP.Control or streptozotocin (STZ, 65mg/kg)‐induced diabetic Sprague Dawley rats (n=5 per group) received vehicle or NAC (1.5g/kg/day) plus ALP (100mg/kg/day) in drinking water for three weeks starting one week after STZ injection. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured weekly. At the end of the experiments (4 weeks after STZ injection), L3–L5 segments of spinal cord and somatosensory cortex were extracted for detecting TNF‐alpha protein expression by western blotting assay.The results showed that the thresholds for PWMT and PWTL were reduced significantly in diabetic rats respectively at 1 and 2 weeks after diabetic induction as compared to non‐diabetic control (all p<0.05), which was associated with significant increase of spinal cord TNF‐alpha protection expression (p<0.05 diabetic vs. control) and moderate increase in cortex TNF‐alpha protection expression (P>0.05). Joint application of NAC and ALP significantly attenuated the reduction of thresholds for PWMT starting at two weeks of treatment and onwards (P<0.01 diabetic vs. Control) that was associated significant reduction and normalization of spinal cord TNF‐alpha expression (P<0.05, NAC+ALP vs. diabetes; p>0.05, NAC+ALP vs. control). However, NAC and ALP combination did not affect diabetic induced reduction in the threshold for PWTL, nor did it affect cortex TNF‐alpha protein expression.It is concluded that reduction of spinal cord TNF‐alpha may represent a mechanism by which antioxidants NAC and ALP reduce mechanical allodynia in diabetic rats, and that diabetic thermal hyperalgesia may be mediated by mechanisms other than spinal cord TNF‐alpha at a relatively early stage of diabetes.
NHEJ and BER Concurrently Repair Oxidative DNA Damage via Initiation of APE1 in Rat Cortical Neurons
Authors: Yang Jenq‐Lin , Chen Yin‐Ping , Sun Shu‐Fang , Chen Shang‐Der 
Published: November 2023

Base excision repair (BER) is the primary mechanism to repair oxidative, alkylated, deaminated, and single‐strand break DNA damage in eukaryotic cells, which is also commonly suggested to be the major machinery repairing oxidative DNA damage in neurons due to highly active metabolism companied with reactive oxygen species production. Many previous studies have suggested that neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However two earlier studies observed that neurons could efficiently repair glutamate‐ and menadione‐induced oxidative DNA damage. The current study has shown that markers of DNA damage and double‐stranded break, γH2AX and phosphorylated p53 binding protein 1 (p53BP1), after menadione‐produced oxidative insults. Moreover, both γH2AX and p53BP1 appeared and highly co‐localized with Apurinic/apyrimidinic endonuclease 1 (APE1) respectively after the oxidative DNA lesion. In nuclei of oxidative insult‐treated neurons, foci of p53BP1 were robustly abolished by suppression of APE1 endonuclease activity. Inhibition of DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) prolonged the DSB repair after oxidative insults. APE1 plays a key role in BER mechanism, but our results also indicated APE1 diverting BER concurrently works with non‐homologous end join (NHEJ) to repair oxidized DNA. Findings of this study explain the neurons are able to repair their nuclear DNA efficiently to cope with highly oxidative burden. APE1 expression is inducible via CREB‐mediated signaling pathway, which has been reported by many studies. Accordingly, elevating APE1 will enhance repair efficiency in oxidative DNA by both BER and NHEJ, which is a potential strategy for prevention or therapeutic intervention in ischemic stroke and neurodegenerative diseases.Support or Funding InformationThis research is supported by Ministry of Science and Technology Taiwan [grant number 103‐2320‐B‐182A‐011‐MY3 and 103‐2314‐B‐182‐026‐MY2].
Effects of Chronic Pramipexole on AMPA Receptor Trafficking and Akt/GSK‐3β Signaling in a Rat Model of Parkinson's Disease
Authors: Kase Matthew Scott , Persons Amanda , Napier Celeste 
Published: November 2023

The D3 receptor (D3R)‐preferring agonist pramipexole (PPX) is an effective treatment for motor symptoms in Parkinson's disease (PD). However, behavioral addictions associated with limbic systems emerge following chronic PPX administration. The D3R is involved in the addiction process, and one D3R‐mediated signaling cascade involves the Akt/GSK‐3β pathway. We previously demonstrated in limbic brain regions that acute PPX dephosphorylated Akt, which prevented phosphorylation of constitutively active GSK‐3β. Un‐phosphorylated GSK‐3β strengthens glutamatergic synapses via insertion of AMPA receptors (AMPAR) in neuronal membranes; a phenomenon associated with addiction. However, the effects of chronic PPX on this pathway remain unknown. Because PPX is often used as a therapeutic in early‐stage PD, clarification of these neuronal processes is highly relevant to clinicians. Here, we tested the hypothesis that chronic PPX can increase AMPAR surface expression in limbic brain regions of PD‐like rats. The neurotoxin 6OHDA was injected bilaterally into the dorsolateral striatum of rats to model PD (4μg/2μl); sham‐lesioned controls were injected with ascorbic acid vehicle (2μl). Twenty‐one days post‐lesion, rats were subcutaneously implanted with osmotic minipumps containing vehicle or PPX (1.2 mg/kg/day). The forelimb akinesia task was used to confirm PD‐like motor deficits and PPX efficacy in lesioned rats. After 14 days of PPX treatment, brain tissue was harvested. Modified Western blot protocols determined surface and intracellular levels of GluA1 (an AMPAR subunit), as well as ratios of pAkt/Akt and pGSK3β/GSK3β. In the nucleus accumbens and ventral pallidum, there was no effect of chronic PPX on pAkt/Akt or pGSK/GSK‐3β. In the medial prefrontal cortex, the ratios of pAkt/Akt and pGSK‐3β/GSK‐3β were significantly increased in PPX‐treated, PD‐like rats (ANOVA; p=0.0161 and p=0.0119, respectively), but surface/intracellular GluA1 was unchanged. Thus, while acute PPX promoted AMPAR trafficking, AMPAR surface expression appeared to normalize with chronic PPX. In addition to AMPAR, adaptation of excitatory synapses can be regulated by GSK‐3β‐linked changes in NMDA receptors. Thus, it is possible that changes in NMDA receptors may contribute to PPX‐induced behavioral addictions. Future studies will make this determination.Support or Funding InformationWork supported by USPHSG #NS087559
Nanoceria Dissolution and Carboxylic Acid Stabilization in Aqueous Dispersions
Authors: Yokel Robert A , Hancock Matthew L , Grulke Eric A , Unrine Jason M , Graham Uschi M 
Published: November 2023

The objective was to determine whether carboxylic acids stabilize nanoceria in aqueous dispersions. Citric acid is used as a stabilizing agent for hydrothermal syntheses of nanoceria and for nanoceria dispersions. Nanoceria has been shown to have therapeutic potential for conditions with an oxidative stress/inflammation component including cancer, radiation damage, bacterial infection, sepsis, wounds, stroke‐induced ischemia, and retinal degeneration. It is used as an abrasive to prepare integrated circuits by chemical mechanical planarization and as a combustion catalyst in diesel fuel. Citrate‐coated nanoceria (average primary particle size ~ 4 nm (determined by electron microscopy; EM), hydrodynamic diameter of agglomerates in aqueous dispersions ~ 14 nm (determined by dynamic light scattering; DLS)) was incubated in multiple media for months. Nanoceria was introduced into 2 kD MWCO Slide‐A‐LyzerTM dialysis cassettes immersed in ~ 300 mOsm/L media. Samples from within and outside of the cassette were withdrawn for cerium quantitation by inductively coupled plasma mass spectrometry and high resolution electron microscopy (HR‐EM). Cerium quantitation, hydrodynamic diameter determination, and electron microscopic observations were repeatedly conducted. Nanoceria dissolution occurred at pH 4.5, shown by increased cerium in media surrounding the dialysis cassette and decreased primary particle size (1 to 3 nm, HR‐EM) within the cassette. Nanoceria agglomerates were ~ 10 to 15 nm in the presence of citric and malic acids and ammonium but grew to ~ 1 micron in media containing acetic, glutaric, tricarballylic, 3‐hydroxybutyric, adipic, or pimelic acid. In medium containing citrate, the nanoceria primary particle core retained its crystalline structure. Dissolution occurred at the nanoceria particle surface, which was rich in Ce(III); the antioxidant valence state. Cerium‐containing agglomerates (~ 10 nm comprised of 1 to 3 nm primary particles) were seen in the citric acid medium surrounding the dialysis cassette, suggesting diffusion of cerium through the dialysis cassette membrane followed by particle formation in the medium. In contrast, appreciable nanoceria dissolution did not occur in the presence of horseradish peroxidase and hydrogen peroxide at pH 6.2, a condition shown to degrade carbon nanotubes. By extension of the observations in acidic media, acidic environments, as found in phagolysosomes, may degrade nanoceria by dissolution. Carboxylic acids are known to form coordination complexes with metal ions. Formation of cerium‐chelate complexes after nanoceria dissolution may enable cerium redistribution within animals and uptake into plant roots. Ligands that can enable nanoceria dissolution in acidic environments greatly affect nanoceria's fate (dissolution rate and size).Support or Funding Information1R01GM109195