bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Pre‐Conditioning with Copolymer‐Based Cell Membrane Stabilizer Poloxamer 188 on Adult Mouse Coronary Artery Endothelial Layer Integrity after Prolonged Hypoxia/Reoxygenation
Authors: Maknojia Insha H. , Hampton Matthew J. , Li Zhu , Barajas Matthew B. , Riess Matthias L. 
Published: May 2022

Prolonged ischemia followed by reperfusion can cause significant tissue damage and cell death. This ischemia/reperfusion (IR) injury can be alleviated by administration of Copolymer‐based Cell Membrane Stabilizers (CCMS), like the tri‐block Poloxamer (P)188, thought to be able to seal gaps in cell membranes and/or tight junctions and, thus, repair leaks attributable to severe cellular stress. We have previously tested post‐conditioning, i.e. the administration of P188 upon reperfusion/reoxygenation, to attenuate IR injury in various models. Here, we hypothesized that pre‐conditioning, i.e. its administration before simulated ischemia (hypoxia), has a protective effect on endothelial cell layer integrity as assessed by Trans‐Endothelial Electrical Resistance (TEER).Confluent adult mouse coronary artery endothelial cell (ECs) layers were pre‐treated with 0, 30, 100, 300, 1,000, or 2,000 µM P188 during plating of cells before simulated IR with a 24‐hour hypoxic followed by a 2‐hour reoxygenation (HR) period. EC monolayer barrier integrity was assessed by TEER.In contrast to mere post‐conditioning, pre‐conditioning dose‐dependently attenuated HR injury compared to control/normoxia (Fig 1) even after prolonged HR for 24+2 hrs.These results suggest that CCMS may indeed elicit its cardioprotective effect, in part, through protection of the EC layer. However, more research is needed to determine optimal dosage and timing for CCMS administration.
Targeting ADAM‐17 to Alleviate Cytokine Storm Associated with COVID‐19
Authors: Mattos Pereira Vitoria , Pradhanang Suyasha , Thakar Amit , Mansell John , Nair Sreejayan 
Published: May 2022

BackgroundAcute respiratory distress syndrome (ARDS) is the primary cause of mortality in critically ill subjects infected with the novel coronavirus (severe acute respiratory syndrome‐coronavirus‐2, or SARS‐CoV‐2). Given the elevated levels of circulating proinflammatory cytokines such as tumor necrosis factors (TNF)‐⍺ and interleukin (IL)‐6, it has been postulated that a “cytokine storm” is responsible for the COVID‐19 ARDS. A critical step in cytokine signaling is the shedding of membrane‐anchored cytokines and their receptors.ObjectiveTo determine the role of the metalloprotease/shreddase ADAM‐17 (A disinterring and metalloproteinase 17) in COVID‐19‐ mediated cytokine release from lung epithelial cells. We hypothesized pharmacological inhibition of ADAM‐17 attenuated COVID‐19‐induced release of TNF‐α in lung epithelial cells.Materials and MethodsHuman primary bronchial/tracheal epithelial cells (ATCC®, PS‐300‐010) were cultured to sub‐confluence and challenged with COVID‐19 Spike Coronavirus pseudovirus (SARS‐COV‐2 pseudo‐virus, MyBioSource), and phorbol‐12‐myristate‐13‐acetate (PMA, Sigma), which was used as a positive control in the presence or absence of the ADAM‐17 inhibitor TNF‐α Protease Inhibitor‐0 (TAPI‐0, Sigma). TNF‐α levels were determined in the cell media at different time points (2‐12h) following the challenge using an ELISA assay. Data are represented as mean ± SEM, and statistical significance was determined by using the Student’s t‐test.ResultsTreatment with both PMA (0.1 µM) and SARS‐COV‐2 pseudovirus (1x108pseudoviral particles), resulted in a robust increase of TNF‐α in the culture media 8 h post‐stimulation (3243.75± 55.3 and 1278.6 ± 357 pg/mL respectively, p <0.05, n = 4). Furthermore, treatment with TAPI‐0 resulted in a concentration‐dependent inhibition of the pseudovirus‐induced TNF‐α release by cultured bronchial epithelial cells, with a complete inhibition observed at 100 µM of TAPI‐0.ConclusionTargeting ADAM‐17 is a potential strategy to mitigated the cytokine storm associated with COVID‐19‐ARDS.
Cardiovascular Reactivity During a Fatiguing Exercise in Women: The Influence of Menopause Duration and Age
Authors: Anselmo Miguel F. , Stokes William , Lee Emma , Tahsin Chowdhury T. , Panigrahy Nisha , Glazos Aline , Reilly Cavan , Noven Marnie V. , Carter Jason R. , Keller‐Ross Manda L. 
Published: May 2022

IntroductionThe risk of hypertension (HTN) and cardiovascular disease (CVD) dramatically increases following menopause, and premature and early menopause (<45yr) substantially increase this risk. Greater blood pressure (BP) reactivity to stressors such as exercise can predict future risk of developing HTN. Thus, we aimed to determine if the time since menopause completion contribute to greater BP reactivity during a fatiguing hand grip (HG). We hypothesized that women who have been postmenopausal for a longer duration would exhibit greater cardiovascular reactivity to a fatiguing HG exercise.MethodsWomen who experienced early and typical‐age (>46yr) menopause completed two visits. Visit 1: Informed consent, medical and health questionnaires. Visit 2: Participants rested in a supine position. Three maximum voluntary contractions (MVC) of their left hand were completed followed by a baseline rest (BL) of 10 minutes. Following BL, a HG test at 30% of MVC was performed until time to task failure. Non‐invasive beat‐to‐beat BP and heart rate (HR) were recorded during BL and HG test. Systolic (SAP), diastolic (DAP), mean arterial (MAP), and HR were analyzed as BL averages and in 20‐30s intervals at start, 25%, 50%, 75%, and task failure. Repeated‐measures ANOVA and independent T‐tests were used for the statistical analysis; results are presented as mean±SD.ResultsEighteen postmenopausal females completed the study and were divided into two groups: 1) postmenopause ≤10yrs (PM≤10; 8±2 yrs; body mass index (BMI): 25±3 kg/m2, n=8) and 2) postmenopause ≥11 (PM≥11; 15±5 yrs; BMI: 25±5 kg/m2, n=10). The PM≥11 group was slightly older (64±3yrs) than the PM≤10 (60±4yrs, p=0.005). BL SAP was not different between PM≤10 (133±14mmHg) and PM≥11 (128.75±14mmHg; p=0.95). DAP (85±9 vs 81±10mmHg; p=0.68) and MAP (100±10 vs 96±9mmHG; p=0.40), were not different between PM≤10 and PM≥11, respectively. During the fatiguing contraction, SAP increases were exaggerated in PM≥11 compared with PM≤10 (Δ63±25 vs Δ40±16mmHg; group × time, p=0.03). Similarly, MAP increased more in PM≥11 compared with PM≤10 (Δ49±16 vs Δ31±10mmHg; group × time, p=0.04), but not DAP (group × time, p=0.1) or HR (group × time, p=0.08). When co‐varying for age, however, the interactions were no longer significant (p>0.05, for all). In addition, peak increases in SAP (p=0.04), MAP (p=0.01), and HR (p=0.02) were greater in PM≥11 compared with PM≤10, but these observations were no longer significant when co‐varying for age (p>0.05, for all).ConclusionNovel findings from this study suggest that although the duration of menopause influences BP reactivity during a moderate intensity fatiguing contraction, this greater increase may be explained by older age. This elevated response to submaximal exercise may contribute to the increased risk of HTN and CVD in postmenopausal women.
The Function and Surface Expression of the Organic Cation Transporter 1 (OCT1) is Affected by Free Cholesterol
Authors: Idowu Jessica Y. , Hagenbuch Bruno 
Published: May 2022

BackgroundThe function of the major renal organic cation transporter OCT2, is affected by plasma membrane cholesterol. OCT1, its liver counterpart, is important for the uptake of cationic drugs such as metformin into human hepatocytes. Fatty liver disease where free cholesterol accumulates in the liver can be a consequence of type 2 diabetes, which is treated with metformin. Thus, understanding how altered membrane cholesterol content impacts the function of OCT1 is important for the disposition of the drugs it transports.AimWe wanted to investigate to what extent the addition and the removal of free cholesterol from the plasma membrane would alter the function and expression of OCT1.MethodsHEK293 Flp‐In cells stably expressing OCT1 were used for all experiments. To add cholesterol, cells were treated with a cholesterol‐MCD complex in serum‐free media for 30 minutes. To remove cholesterol from the membrane cells were incubated for 30 minutes in serum‐free media containing methyl‐b‐cyclodextrin (MCD). Uptake of the radiolabeled model substrate MPP+ (1‐methyl‐4‐phenylpyridinium) was measured at 37°C. Michaelis‐Menten kinetics were determined under initial linear rate conditions (15 second uptake). OCT1 expression at the plasma membrane was determined using a surface biotinylation assay.ResultsTreatment of the cells with increasing concentrations of both cholesterol and MCD resulted in a concentration‐dependent decrease of OCT1 transport. Kinetics of OCT1‐mediated MPP+ uptake following cholesterol treatment demonstrated that the Km values increased from 35 ± 6.7 μM to 73 ± 10.2 μM while the Vmax values were essentially unchanged: 7.5 ± 0.6 pmol/mg x min and 8.4 ± 0.3 pmol/mg x min, respectively. As a consequence, the capacity of OCT1 decreased by 45%. Treatment of cells with MCD slightly decreased the Km value to 26 ± 4.9 μM while the Vmax value stayed the same at 7.7 ± 0.5 pmol/mg x min. With respect to surface expression, treatment with 20 mM MCD treatment decreased surface expression of OCT1 by about 65% while cholesterol treatment resulted in a 30% decrease.Summary and ConclusionBoth the addition and removal of free cholesterol from the plasma membrane impacts OCT1 transport and expression at the plasma membrane. These results suggest that drug disposition will be different in patients with either higher or lower free cholesterol in the liver, which can occur in fatty liver disease or type 2 diabetes.
Promoting the Resolution of Thromboinflammation
Authors: Gavins Felicity N. , Yadav Shreya , Ansari Junaid , Iffah Raayma 
Published: May 2022

BackgroundThrombosis is viewed as a multi‐step, multi‐cellular process driven by inflammatory stimuli and leukocyte (e.g. neutrophil) activation, and is a major pathophysiological contributor of stroke. Accumulating data supports the hypothesis that anti‐inflammatory therapies may limit thrombosis and anti‐thrombotic therapies may reduce vascular inflammation. One such target is the anti‐inflammatory and pro‐resolving endogenous mediator Annexin A1 (AnxA1). The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of stroke and other thromboinflammatory diseases. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in thrombosis.Material and MethodsWe used a well‐characterized light/dye thrombosis model, coupled with pharmacological and genetic approaches. Male and female adult wild type (WT, C57/BL6) or AnxA1 knock‐out (AnxA1‐/‐) mice were used. Fluorescence intravital microscopy (IVM) was used to monitor cerebral thrombosis, along with flow cytometry. Mice were treated with AnxA1 (1 µg/mouse) or saline vehicle 20 minutes before the onset of cerebral thrombus formation. Thrombogenesis and blood flow cessation times were quantified. (All animal experiments were approved by relevant institutional animal welfare panels and were conducted in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines and complied with ARRIVE [Animal Research: Reporting In Vivo Experiments]).ResultsIVM revealed that AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, via regulation of the key inflammation resolution receptor: FPR2/ALX, as evidenced by the co‐administration of the FPR2/ALX antagonist WRW4. Using AnxA1−/− mice, we found that a deletion of AnxA1 impacts thrombosis as shown by the accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) observed in both arterioles and venules of these mice compared to their WT counterparts (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p<0.05). Furthermore, we found that in an inflammatory backdrop (induced by the administration of lipopolysaccharide [LPS]), AnxA1 treatment was able to suppress both platelet αIIbβ3 and P‐selectin expression.ConclusionHerein, we found a previously unknown phenomenon that the anti‐inflammatory protein AnxA1 can modify a thromboinflammatory environment to promote resolution. These findings have far‐reaching applicability for drug discovery programs focused on resolution biologics targeting thromboinflammatory diseases such as cardiovascular disease and stroke.
Aberrant Regenerating‐Islet Derived (Reg) Gene Expression is an Early Indicator of Colonic Dysplasia in Heterogeneous Nuclear Ribonucleoprotein (Hnrnp I) Knockout Mice
Authors: Liang Siyuan , Xu Guanying , Pramana Abrory A. , Chen Hong , Pan Yuan‐Xiang 
Published: May 2022

ObjectivesReg is an evolutionarily conserved family of proteins with anti‐inflammatory and antimicrobial functions, is mainly involved in the defense of intestinal epithelial cells (IEC). Recent studies indicate that the attenuation of colitis depends on the functions of Reg3b. Here, knockout mice with an IEC‐specific ablation of the Hnrnp I gene (KO) were examined, which exhibited an increased risk of developing spontaneous colitis. The objective of this study is to examine the gene expression of the Reg genes in the KO mice.HypothesisWe hypothesize that epithelial ablation of Hnrnp I in the colon of the KO mice leads to early aberrant expression of Reg3b and Reg3g.MethodsSix weeks old WT and KO mice were sampled for body weight, colon weight and length. Whole colon was opened and jelly‐rolled from distal to proximal end for embedding. Total RNA was extracted for quantification of gene expression using qPCR. Colon tissue sections were examined by hematoxylin‐eosin staining (H&E) for colon morphology and colonic crypt length measurements. Colon localization of Reg proteins was analyzed using immunofluorescent staining. Results were analyzed using the Mann‐Whitney test. Spearman test was used to analyze the correlation. P less than 0.05 is considered significant.ResultsThe expression levels of Reg3b, Reg3g, and Il‐6 in KO mice were significantly lower than WT mice. Furthermore, expression levels of Reg3b and Reg3g are correlated in both WT and KO groups (R=0.95). It is worth mentioning that, in 30% of KO mice, the expression levels of Reg3b and Reg3g are significantly higher than the rest of the KO mice, exhibiting significant dichotomy. Histological analysis showed that the crypt lengths of KO mice were significantly longer than those in the WT mice both in the distal end and proximal colon. More importantly, the KO mice with elevated Reg gene expressions had longer crypt lengths in the distal end. Moreover, KO mice with elongated crypts also exhibited invasive dysplasia in the colon sections.ConclusionsOur results indicate that IECs‐specific Hnrnp I knockout abolished the expression of the Regfamily genes. Further investigations are warranted to determine the potentially detrimental effects from the diminished Reg3 to the overall protection against microbial invasion in the colon. The contradictory upregulation of Reg3b and Reg3g in a subset of the KO mice indicates the extreme responsiveness to the perturbation from the Hnrnp I knockout and may serve as an early indicator of colon dysplasia.
Targeting Resistance in Medulloblastoma
Authors: Telang Sucheta , Chang Simone 
Published: May 2022

Medulloblastoma is the most common malignant brain tumor of childhood. Although the current treatment protocols for this cancer have improved overall survival, they lead to devastating and lifelong sequelae and mortality remains high in recurrent and metastatic disease. Novel targeted therapies are crucial to improve survival in these children without treatment‐related toxicity.Medulloblastoma development is largely driven by deregulation of normal cerebellar proliferation wherein aberrant sonic hedgehog (Shh) pathway signaling plays a critical role. Activating mutations of Shh genes are frequent in medulloblastoma and promote their rapid growth. Poor outcomes in Shh‐driven tumors have prompted the evaluation of targeted agents against the Shh pathway but these have had limited success ‐ likely due to the upregulation of additional oncogenic pathways including the Ras/MAPK pathway and HIF‐1α. These pathways stimulate glycolysis in part by activating the 6‐phosphofructo‐2‐kinase/fructose‐2,6 bisphosphatase family of enzymes (PFKFB1‐4). The PFKFBs produce fructose‐2,6‐bisphosphate (F26BP), a potent activator of 6‐phosphofructo‐1‐kinase, a rate‐limiting step in glycolysis. We found that the PFKFB4 enzyme is highly expressed in a series of patient‐derived medulloblastomas and correlated significantly with expression of the Shh pathway effector Smoothened (SMO). Through HIF‐1α upregulation, we observed that hypoxia strongly induced PFKFB4 expression in Shh‐active medulloblastoma cells and that silencing PFKFB4 in these cells markedly suppressed F26BP and glycolytic flux to lactate and significantly decreased cell survival and proliferation, more prominently in hypoxia, indicating that PFKFB4 may be preferentially required for survival and growth under hypoxia. We found that co‐silencing PFKFB4 and Shh proteins significantly reduced cell survival and that co‐targeting PFKFB4 (with a novel small molecule inhibitor) and Shh effectors synergistically decreased cell viability. To mimic Shh antagonist resistance, we subjected Shh medulloblastoma cells to prolonged Shh inhibitor exposure and have found that these cells show increased proliferation and glycolysis and increased expression of PFKFB4 and are more sensitive to PFKFB4 inhibition. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option in aggressive, treatment‐resistant medulloblastoma and strongly support the examination of PFKFB4 inhibitors in these tumors.
A mechanism of TNK1 activation by C‐terminal gene truncation in human lymphomas
Authors: Ashworth Spencer W. , Egbert Christina M. , Lopez‐Palacios Tania , Chan Tsz‐Yin , Vaughan Alec , McCormack Katherine , Andersen Joshua 
Published: May 2022

Thirty‐eight‐negative kinase 1 (TNK1) is a poorly characterized non‐receptor tyrosine kinase (NRTK) first isolated from umbilical cord blood. TNK1 differs from most NRTKs in that it possesses a functional ubiquitin association (UBA) domain at its C‐terminus. We previously observed that the TNK1 UBA domain interacts with a variety of poly‐ubiquitin chains, is essential for full TNK1 activation, and facilitates the clustering of active TNK1 into ubiquitin‐rich condensates.1 Paradoxically, a genetic inversion that truncates the TNK1 C‐terminus (including the UBA domain) in human lymphoma hyperactivates the kinase and coverts it into an oncogenic driver. Our current research focuses on how deletion of the UBA, which is important for native TNK1 function, helps activate the kinase. Here we show that immediately adjacent to the UBA domain on TNK1 is an inhibitory 14‐3‐3 binding site, which is also truncated in lymphoma. Furthermore, point mutations that disrupt the 14‐3‐3 binding site are sufficient to convert TNK1 into a hyperactive oncogenic kinase. In addition, we show that although truncation of the UBA perturbs the normal function of TNK1, it also stabilizes TNK1 protein levels. Thus, the lymphoma‐associated truncations in TNK1 activate and stabilize the kinase, resulting in a highly expressed and active TNK1. Finally, we show that truncation of the 14‐3‐3 binding site and UBA domain of TNK1 is sufficient to transform pro‐B cells to growth factor‐independence and grow tumors in vivo. Thus, our data explain how genetic inversions convert TNK1 into an oncogenic driver in human cancers.1. Chan, TY., Egbert, C.M., Maxson, J.E. et al. TNK1 is a ubiquitin‐binding and 14‐3‐3‐regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021). https://doi.org/10.1038/s41467‐021‐25622‐3
Cross‐talk between Histone Modifications Modulate Chromatin Reader Activity of the ATAD2B Bromodomain
Authors: Phillips Margaret , Montgomery Cameron , Chang Sunsik , Boyson Samuel P. , Glass Karen C. 
Published: May 2022

The ATPase family, AAA domain‐containing protein 2 (ATAD2, or ANCCA) is an AAA nuclear co‐regulator protein containing two ATPase domains and a bromodomain. It’s closely related paralogue, ATAD2B (KIAA1240), also contains two ATPase domains and a bromodomain. The AAA ATPase domains are broadly associated with ATP driven molecular remodeling reactions while the bromodomains are evolutionarily conserved, chromatin ‘reader’ domains, known to regulate gene expression through recognition of histone post‐translational modifications (PTMs).Extensive studies on ATAD2 discuss its upregulation and correlation with poor prognosis in various cancers specially breast cancer and have successfully mapped the different histone interactions needed for targeting ATAD2 to the chromatin. Despite sharing high structural and sequence similarity with ATAD2, little is known about the unique role of ATAD2B. ATAD2B is expressed during neuronal differentiation and in tumor progression. However, limited information is available on how ATAD2B is involved or upregulated in various tumors specially in breast cancer. Our group has recently published a broad range of histone modifications recognized by the ATAD2B bromodomain. These modifications mostly include acetyllysine moieties on histone H4.Here, we present a more detailed structural and functional insight into how various histone post‐translational cross‐talk can modulate the chromatin ‘reader’ activity of ATAD2B bromodomain. Through ITC, solution NMR spectroscopy and X‐ray crystallography we demonstrate how combinations of PTMs can influence and regulate histone recognition by ATAD2B bromodomain. The studies also involve histone variants associated with breast cancer. These in‐vitro studies followed by in‐vivo analysis in breast cancer cells (MCF7) will further characterize the physiologically relevant, histone PTMs that are recognized by ATAD2B to direct it to the chromatin, thus playing a crucial role in activation of gene transcription.Through our study we highlight the importance of cross‐talk between various histone post‐translational modifications and how they may regulate the ATAD2B bromodomain activity. These combinations of PTMs can either promote or inhibit the affinity of ATAD2B to the chromatin and in turn regulate gene transcription. Understanding the molecular mechanisms driving these interactions will provide new insights for the development of therapeutic interventions for the treatment of various diseases.