bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Background

The 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.

Aim

We 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.

Methods

HEK293 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.

Results

Treatment 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 Conclusion

Both 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

Background

Thrombosis 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 Methods

We 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]).

Results

IVM 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.

Conclusion

Herein, 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.
Perinatal nicotine exposure alters development of cardiac performance
Authors: Fregosi Ralph , Strom Joshua , Wollman Lila
Published: May 2022

Perinatal nicotine exposure alters development of brainstem neurons, including those involved in the control of breathing and cardiovascular function. Whether or not perinatal nicotine exposure alters development of cardiac performance is unknown. Pregnant rats were exposed to nicotine plus saccharin in drinking water beginning on the 4thday of gestation. Developing fetuses were exposed via the placental circulation, and exposure continued via breast milk after birth, up until the day of study. Plasma cotinine levels in the nicotine‐exposed pups ranged from 50‐100 ng/ml. Control neonates drank water with saccharin, but no nicotine. Animals aged postnatal day 10‐12 (P10‐P12) were lightly anesthetized with isoflurane, and cardiac function was assessed non‐invasively with echocardiography. Compared to controls (N=13), nicotine‐exposed pups (N=13) had reduced body weight (28.4 ± 1 vs. 21 ± 1 g, P=0.00032), significant reductions in stroke volume (39.1 ± 1 vs. 33.7 ± 1.7 μL/beat, P=0.027) and cardiac output (14.0 ± 0.8 vs. 12.4 ± 1 ml/min, P=0.011), and had significantly slower isovolumetric contraction (15 ± 1 vs. 22 ± 2 ms, P=0.005) and relaxation times (21 ± 1 vs. 33 ± 3 ms, P=0.04). There were also trends toward a lower heart rate (P=0.055), ejection fraction (P=0.065) and left ventricular wall thickness (P=0.059) in the nicotine exposed animals. These are the first data showing that perinatal nicotine exposure alters development of the heart’s pumping capacity.
Multiple pathways for Fig4 contributions to cellular homeostasis in yeast
Authors: Khan Imran , Reeves Hannah E. , Darbro Caroline S. , Hall Harrison S. , Strunk Bethany S.
Published: May 2022

Mutations in the lipid phosphatase Fig4 are associated with multiple neurodegenerative diseases including a rare and severe form of Charcot‐Marie‐Tooth disease known as type 4J (CMT4J). CMT4J has been attributed to dysregulation of the production and turnover of Fig4’s lipid substrate, the phosphoinositide signaling lipid, PI3,5P2 through disruption of its well characterized protein complex, the Fab1‐Vac14‐Fig4 complex. Fig4 is known to play roles in the cell independent of its catalytic function. Our work in yeast suggests that the presence of Fig4 in cells can confer a growth advantage under stress and that this growth advantage is conferred in a dominant manner, independent of Fig4 catalytic activity or its participation in the Fab1‐Vac14‐Fig4 complex. Importantly, we have found that Fig4 variants associated with multiple demyelinating diseases, including those involved in CMT4J, leukodystrophy and amyotrophic lateral sclerosis (ALS), display similar behavior. These data suggest that Fig4 influences cellular homeostasis, and perhaps human disease, through multiple pathways.
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

Objectives

Reg 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.

Hypothesis

We hypothesize that epithelial ablation of Hnrnp I in the colon of the KO mice leads to early aberrant expression of Reg3b and Reg3g.

Methods

Six 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.

Results

The 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.

Conclusions

Our 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.
Optogenetic inhibition of subcellular GαqGTP signaling
Authors: Ubeysinghe Sithurandi , Welivita Diniti , Karunarathne Ajith
Published: May 2022

Light‐sensitive G protein‐coupled receptors (GPCRs) such as opsins have been employed to activate some G protein pathways' signaling with subcellular spatial control. However, a limitation has been the lack of approaches to selectively control heterotrimeric G protein subunit signaling downstream of GPCRs with a user‐defined spatial and temporal control. For example, GNAQ is a cancer driver gene and encodes G protein alpha q (Gaq). Phospholipase C beta (PLC b) is a major downstream effector of Gaq that hydrolyze phosphatidylinositol‐4, 5‐bisphosphate (PIP2), generating inositol‐1, 4, 5‐triphosphate (IP3) and diacylglycerol (DAG). The subsequent signaling controls a wide array of physiological functions, including muscle contraction, opioid‐induced pain sensitivity, cell proliferation, and survival, and thus underlies many diseases such as heart diseases, cancer, and diabetes. To examine Gbg‐independent regulation of Gaq signaling, we engineered a series of highly efficient optogenetic inhibitors of GaqGTPsignaling that provides precise subcellular‐special and millisecond‐temporal control. Using these capabilities, we have uncovered several novel regulatory mechanisms in Gq‐pathway‐governed directional cell migration.
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
Rapamycin Supplementation: Effect on Longevity in Drosophila melanogaster
Authors: Jackson Lauren N. , Frye Caitlyn T. , Llivina Ginger E. , Mockett Robin J.
Published: May 2022

Target of rapamycin (TOR) regulates a number of cellular responses, including autophagy. The purpose of this study was to investigate the effect on life span of supplementation with rapamycin dissolved in either dimethylsulfoxide (DMSO) or ethanol, in male and female flies of three strains of Drosophila melanogaster: Oregon R, w1118, and y w. The hypothesis was that longevity would be decreased regardless of strain, sex or solvent, consistent with an unpublished prior study using y w males in this laboratory, or alternatively that it would be increased as in publications from other laboratories. Flies were exposed to food supplemented with 200 µM rapamycin dissolved in one of the two solvents before being mixed into a standard agar‐yeast‐cornmeal‐sucrose medium. Supplementation was continuous beginning 2 d posteclosion. The mean life span of Oregon R females was diminished by rapamycin in both ethanol and DMSO solvents, by 17% and 13%, respectively (P < 0.0005). Oregon R males also exhibited a shortened mean life span, which was significant in DMSO (8%, P = 0.008) but not ethanol (3%, P = 0.9). In contrast, w1118 female flies had a 4% increase in mean life span with rapamycin dissolved in ethanol (P < 0.0005) but not DMSO (1% increase, P = 0.2). Rapamycin decreased life span in w1118 males, but the effect was significant only in DMSO (22%, P = 0.002), not ethanol (10%, P = 0.6). It decreased longevity significantly in y wflies of both sexes on both solvents, by 9% in females and 7‐12% in males (all P < 0.0005 except male/DMSO P = 0.04). Notably, the life spans of control flies differed significantly between the solvents, being longer with ethanol for y w of both sexes and longer with DMSO for Oregon R males and w1118 males. In conclusion, these results show that effects of rapamycin on life span of Drosophila are sex‐, strain‐ and solvent‐dependent.
Temporal and Region‐Specific Tau Protein Hyper‐Phosphorylation in the Brain Coincides with the Development of Impaired Baroreflex Control of Arterial Pressure and Spatial Learning in Male Obese Zucker Rats
Authors: Das‐Earl Paromita , Schreihofer Derek , Sumien Nathalie , Schreihofer Ann M.
Published: May 2022

Obesity and other traits of metabolic syndrome (MetS) are recognized risk factors for the development of neurodegenerative diseases such as Alzheimer’s Disease (AD) and related dementias. Emerging evidence indicates pathophysiologies common to MetS and AD, such as obesity, hypertension, insulin resistance, and impaired glycemic control, occur with development of tauopathies and cognitive decline. Male obese Zucker rats (OZRs), a hyperphagic genetic model of MetS, develop these deleterious traits with predictable ages of onset and time courses. Juvenile OZRs display prominently elevated triglycerides and insulin compared to age‐matched lean Zucker rats (LZRs). By 12‐14 weeks of age although fasting glucose is still comparable in OZRs and LZRs, with access to food the OZRs exhibit chronic, marked hyperglycemia. At this age male OZRs also become hypertensive with impaired short‐term control of arterial pressure by baroreflexes, which are improved by treatments that normalize blood glucose. We hypothesized that young adult male OZRs would develop age‐dependent expression of markers of neurodegeneration in medulla and hippocampus coincident with deficits in functions related to these brain regions. Cohorts of age‐matched male OZRs and LZRs underwent cognitive testing using the Morris water maze (MWM). At 18‐19 weeks, the escape latencies and path lengths were comparable in male OZRs and LZRs (n=12/group). In contrast, at 28 weeks of age, OZRs (n=16) exhibited a greater than 2‐fold higher escape latency and significantly longer path lengths (p<0.05) in the acquisition and retention phases of the MWM compared to age‐matched LZRs (n=15), suggesting the emergence of impaired spatial learning ability in the OZRs. The 18‐19‐week‐old male OZRs exhibited tau hyper‐phosphorylation (S396 and S199,and S202) in the medulla, but the expressions of these markers in the hippocampus were comparable in OZRs and LZRs (n=6/group) at this age. In contrast, 27‐28‐week‐old OZRs (n=6) exhibited tau hyperphosphorylation at S396in hippocampus as well as medulla compared to LZRs (n=6). Our data suggest that changes in tau phosphorylation in male OZRs occur in a temporal and spatial manner coincident with changes in brain function in these regions. In male OZRs, earlier manifestation in the medulla occurred with impaired short‐term regulation of arterial pressure by the medulla. Later manifestation of tau hyperphosphorylation in hippocampus coincided with deficits in spatial learning. Understanding mechanisms by which these histological, physiological, and behavioral changes occur in male OZRs may provide important insights into connections between the development of MetS and AD‐related dementias in humans to yield improved early‐stage interventions.
Non‐Competitive Inhibition by a Substrate Mimetic of Human 5‐Lipoxygenase
Authors: Mathes Forge , Gilbert Nathaniel , Newcomer Marcia
Published: May 2022

Human 5‐lipoxygenase (5‐LOX) initiates the synthesis of the leukotrienes (LT) through a two‐step reaction: addition of molecular oxygen to arachidonic acid (AA) into 5–hydroperoxyeicosatetraenoic acid (5‐HPETE) and hydrolysis of 5‐HPETE into leukotriene A4. LTs are highly potent, pro‐inflammatory mediators, which have been implicated in asthmatic and allergic responses. 5‐LOX is mobilized in leukocytes by Ca2+ binding and targets to the membrane to receive substrate from its helper protein, 5‐lipoxygenase‐activating protein (FLAP). These allosteric partners are unnecessary in in vitro experiments but enhance activity in intact cells. The crystal structure of 5‐LOX was revealed in a “closed conformation” with the catalytic iron inaccessible to surface solvent. This closed active site is the result of a unique conformation of helix alpha 2 (Hα2) where two aromatic residues plug the putative substrate‐access portal. 5‐LOX has a disjointed Hα2 of 3 turns connected with multiple loop‐to‐helix transitions permitting this plugged conformation of the active site. For comparison, the structure of another human isoform, 15‐LOX‐2, has Hα2 in a fully elongated conformation of 6 turns and the catalytic iron solvent accessible for substrate entry. The detergent C8E4 was co‐crystallized with 15‐LOX‐2 in the active site, kinetically identified as a competitive inhibitor, and is an excellent substrate mimetic of AA. Initial kinetic studies of 5‐LOX with C8E4 have shown non‐competitive inhibition patterns. We detailed a non‐Michaelis‐Menton kinetic response of 5‐LOX to increasing amounts of substrate at the last EB meeting (https://doi.org/10.1096/fasebj.2020.34.s1.01876). We hypothesized that a ligand‐induced slow transition of the conformation of Hα2 causes the sigmoidal response to substrate by the monomeric enzyme. Kinetic cooperativity of monomeric enzymes with single substrate‐binding sites, while rare, is represented in highly regulated enzymes. We wanted to further elaborate on this model by performing inhibition kinetics of a substrate mimetic of AA to 5‐LOX. Non‐competitive inhibition of an active site binder is further evidence of a slow conformational change, as it indicates a two‐step inhibition mechanism with the slow transition to an inactive enzyme state. Understanding the inhibition kinetics of an enzyme that adopts multiple conformations and detailing the molecular determinants responsible for inhibitor binding could help delineate some of the discrepancies observed for different 5‐LOX inhibitor potencies in different cellular environments. Anti‐leukotriene therapies has proven to be a successful avenue for lowering inflammation in many disease states and more informed structure‐based drug design approach could hasten their development.
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.
The Horizontally Transferred Arginine Kinase in Myxococcus Xanthus Has Evolved Roles in Multiple Physiological Processes
Authors: MacLean Lillian , Arday Nate , Fannin Lindsey , Fraga Dean
Published: May 2022

Arginine kinase (AK) is a member of the phosphagen kinase family that is involved in energy homeostasis in a range of animal and protozoan species. However, recent studies have shown that some bacterial species have acquired AKs through horizontal gene transfer (HGT), raising the question as to what role they may play in bacterial systems. A recent study with one of these bacterial species, the soil bacteria Myxococcus xanthus, knocked out the endogenous AK to reveal several phenotypes, including diminished recovery from certain stressors and an unexpected role in the sporulation response induced by starvation conditions characteristic to the myxoccocales family. We have expanded upon these initial observations, identifying another novel phenotype in which the deletion strain is unexpectedly no longer recognized as kin by the non‐deletion strain. To further investigate how this AK has evolved in this system, we asked whether a heterologous AK could rescue the deletion strain. Our results will help us determine if the myxococcus AK has uniquely evolved after transmission and acquired novel traits not seen in other AKs.
Group Housing of Male Mice Causes a Significant Decrease in Day to Night Changes in Mean Arterial Pressure and are Indicative of the Development of Hypertension and Cardiovascular Disease
Authors: Brown Nina C. , Sampson Austin C. , Richardson Kimberlei , Lee Dexter L.
Published: May 2022

Changes in day to night time mean arterial pressure (MAP) predict hypertension and cardiovascular diseases. When there is a decrease in day ‐ night time MAP changes, the chances of hypertension and cardiovascular diseases increase significantly. Our hypothesis is that socialization of male mice causes a decrease in the day ‐ night time changes in MAP and heart rate (HR). Male mice, 6 – 8 weeks of age, were implanted with a biotelemetry device from Data Sciences (male mouse in a single cage) or TSE (multiple male mice in a single cage). Day and Night MAP for Data Science mice were 131 ± 0.5 and 157 ± 1.5 mmHg, respectively. Day and Night MAP for TSE mice were 114 ± 7.0 and 124 ± 3.6 mmHg, respectively. The change in day and night MAP were 26 ± 2.2 mmHg in Data Science and 8.4 ± 4.0 mmHg in TSE mice. Day and Night time HR for Data Science mice were 482 ± 6 bpm and 505 ± 5.4 bpm, respectively. The change in day and night time HR were 36 ± 6 bpm in Data Science and 51 ± 12 bpm in TSE mice. Data Science mice had a higher MAP than TSE mice, the change in day ‐ night time MAP was less than TSE mice. The change in day and night time HR was also less in Data Science mice. Therefore, socialization of mice decreases day to night change in MAP and is indicative of hypertension and cardiovascular diseases.
CT Visualization of Vasculature Channels in Modern and Fossil Sloth Ungual Claws
Authors: Jones Jade , Grass Andy
Published: May 2022

One of the interesting features of modern sloths is the vasculature patterns of their upper and lower limbs. Even though sloths are endothermic like other mammals, their mean body temperature tends to be lower, more variable, and more linked to the ambient temperature than other placental mammals. Like some other mammals from environments with temperature extremes, they possess rete arteriovenous bundles, which work as an effective heat exchange mechanism, where heat from distally flowing arterial blood is transferred to proximally flowing venous blood. This minimizes the heat loss from the extremities while still maintaining blood flow. The temperature gradient increases in the rete system when the animal’s core temperature decreases, which cools the limbs and preserves core body heat. When the body temperature rises the flow through the superficial venous system increases, which promotes heat loss and body cooling.

This begs the question of how this complex vasculature system may or may not extend to the distal phalanges and claws of the sloth, perhaps their most distinctive feature. One possibility could be that to work with the limb cooling function of the rete system, the distal phalanx and claw may be poorly vascularized so as not to increase chances for heat loss. However, considering the extensive and important use of their claws, they may require extensive vascularization to withstand the large amount of stress they are often under. This study uses Micro CT scans of sloth ungual phalanges to visualize their vascular channels. In both modern and extinct giant ground sloths, vascular networks through the bone of the ungual phalanx are quite extensive, and are very clearly linked to externally visible channels and impressions left by the proper digital arteries. In ground sloths these internal vascular channels quickly branch on either side and continue distally, letting off smaller branches as they go. These channels are rather large, even appearing to be artificial drill holes on one specimen that has the distal end broken off. Modern sloths show a similar pattern, but their vascular channels take up proportionally less space in the bone than in the giant ground sloths. This difference may be due extinct giant ground sloths’ more varied use of their claws. These complex patterns show that sloth claws require a significant blood supply, and may indicate that the rete system is effective enough that by the time the arterial blood gets to the distal phalanx it has been sufficiently cooled to not be a significant source of heat loss.
Anatomical variations of the maxillary sinus and their relationship with pathologies of surrounding structures: A literature review
Authors: Orellana Mathias I. , Ávila Roberto H. , Gold Marjorie H. , Cariseo Carolina , Valenzuela Juan J.
Published: May 2022

Introduction

Anatomically, the maxillary sinus (MS) and maxillary bone is a complex structure to study, due to its walls and its communication with different structures such as the respiratory system, upper teeth and the orbital cavity, specifically the floor of this cavity and the inferomedial region. The MS is a pneumatic cavity housed within the body of the mandible that communicates with the nasal cavity through the ostium at the middle nasal meatus.

Methods

This literature review seeks to describe the characteristics of the anatomical variations of the region of the maxillary sinus, The search process was carried out in the following databases; MEDLINE, SCIELO, WOS, CINHAL, SCOPUS and GOOGLE SCHOLAR, using as search terms; “Maxillary bone”, “Maxillary sinus”, “Paranasal sinus”, “Anatomical variations”, “Sinusitis” and “Clinical anatomy”.

Results

Different anatomical variations of the sinus and maxillary bone were found, being mainly the anterior and medial maxillary regions, those that presented the greatest number of variations in their walls or external and internal structures, from the clinical point of view, there are variations that can cause sinusitis, some types of tumors or affect neighboring structures that can be compromised by this variation.

Conclusion

The results obtained in this review show that MS is a complex anatomical structure for its study, which is why it is also certainly complex to be able to distinguish the presence of anatomical variations or pathological abnormalities. Therefore, the knowing the different variations and the possible clinical relationship of these can be very useful for the clinical practice of medical personnel who are dedicated to this region.
Assessing the synergistic effects of morphine and MP‐III‐024 co‐administration: enhanced antinociception with reduced side effects
Authors: Uribe Sara , DiDomenico Eric J. , Donegan Christa M. , Tapia Yulista , Conviser Jason , Nambiar Rohit R. , Juarez Amy A. , Side Christine M. , Aldaghma Danya , Poe Michael M. , Sharmin Dishary , Cook James M. , Keck Thomas M. , Fischer Bradford D.
Published: May 2022

Opioid analgesics are critical for acute and chronic pain management, but important side effects—including tolerance, constipation, respiratory depression, and abuse liability—limit their safety and utility. To provide patients with safer analgesic options, it is critically important to identify of new pharmacotherapeutic strategies to treat pain. Activation of µ‐opioid receptors (MORs) in central and peripheral nociceptive pathways mediates opioid analgesia and their critical side effects. Antinociception can also be achieved via selective enhancement of GABAergic signaling at ionotropic GABAA receptors. α2 and α3 subunit‐containing GABAA receptors (α2/α3GABAA), which are co‐expressed with MORs in dorsal horn spinal pathways important to nociceptive transmission, can be selectively targeted with novel imidazodiazepine positive allosteric modulators (PAMs), such as MP‐III‐024, which produces antinociceptive effects with limited behavioral disruption. MP‐III‐024 co‐administered with morphine produces synergistic antinociceptive and anti‐hyperalgesic effects. In this study, we evaluated whether MP‐III‐024/morphine co‐administration produces sub‐additive or synergistic effects in behavioral tests sensitive to morphine side effects. Herein we report that co‐administration of MP‐III‐024/morphine at a 0.94/1 ratio (synergistic in models of antinociception) produced sub‐additive effects in morphine‐induced hyperlocomotion and in measures of behavioral disruption in food‐maintained operant responding. Ongoing studies are evaluating the effects of MP‐III‐024/morphine co‐administration on tolerance in the hot plate test and in conditioned place preference. These experiments are the first comprehensive preclinical analyses of a dual MOR‐α2/α3GABAA pharmacotherapy strategy which may increase the therapeutic window between desirable opioid analgesic effects and side effects.
Insights into the effects of vibration on skeletal development and growth in teleosts
Authors: Franz‐Odendaal Tamara , Jeradi Shirine
Published: May 2022

Teleosts are superb models to study development and growth because their entire life history can be studied in a laboratory setting. The objective of this research is to understand the impacts of the external environment, namely whole body low frequency vibrations on skeletal development. We exposed zebrafish larvae at different stages of development to low frequency vibrations by placing them on a custom built vibration instrument for four days. Our results show age‐related and bone‐type specific effects. For example, some cartilages of the caudal fin were affected while others were not. This data suggests that chondrogenic cell progenitors are capable of sensing and reacting to mechanical stimuli early during development. Furthermore, we show that this effect is sox9‐independent. In summary, this data shows that there is a critical window during early caudal fin development that is susceptible to environmental influence. The plasticity of the skeleton to respond to and adapt to external influence is remarkable. These responses often have lasting impacts on the resulting phenotype and provides a hint at how development influences evolution.
Smooth Muscle LRRC8A Anion Channel Knockout Promotes Vasodilation and Protects Against TNFα‐induced Vascular Dysfunction
Authors: Choi Hyehun , Nguyen Hong N , Sah Rajan , Lamb Fred S.
Published: May 2022

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, is associated with cardiovascular risk. Volume‐regulated anion channels (VRACs) are activated by TNFα and are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E). LRRC8A is required for all VRACs. We previously determined that VRAC inhibitors or LRRC8A knockdown suppress the inflammatory response to TNFα in cultured vascular smooth muscle cells, however, the role of these channels in vasomotor function has not been studied. We hypothesized that loss of LRRC8A would prevent the impairment of vascular function by TNFα. We created vascular smooth muscle‐specific (Sm22alpha‐Cre) LRRC8A knockout (KO) mice and isolated mesenteric arteries from male mice for wire myograph recording. Vessel segments were incubated with TNFα (10 ng/mL) or vehicle for 2 days in tissue culture. Contractile responses to norepinephrine (NE) were unaltered in LRRC8A KO mice but enhanced by TNFα in wild type (WT) than in KO rings (WT+TNFα 284 ± 18.9 %, KO+TNFα 222 ± 7.1 % of KCl contraction, *p<0.05, n = 5‐6). In the vehicle‐treated group both endothelium‐dependent (acetylcholine, ACh) and ‐independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings contracted with phenylephrine (PE). Exposure to TNFα impaired relaxation to both ACh and SNP in WT vessels but had no effect on relaxation of LRRC8A KO vessels (black *p<0.05 compared to WT control, red *p<0.05 compared to TNFα‐treated, n = 6‐8). The enhanced relaxation responses of KO vessels were recapitulated in WT rings by 20 min of exposure to a VRAC inhibitor, carbenoxolone (CBX; 100 μM). Thus, in addition to its established role in TNFα signaling, LRRC8A anion channel activity modulates vascular reactivity with loss of function enhancing vasodilator function. VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of inflammation‐induced vascular dysfunction.
Toward Understanding the Role of an Active Site Network in LThDP Stabilization on DXPS
Authors: Bonett Eucolona , Freel Meyers Caren
Published: May 2022

Microbial metabolic pathways may provide novel, bacteria‐specific targets for antibiotic development, thus providing new strategies to combat the evolving conflict of antibiotic resistance. The bacterial metabolic enzyme 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXPS), which is absent in humans, catalyzes the thiamine diphosphate (ThDP)‐dependent formation of DXP from pyruvate and glyceraldehyde‐3‐phosphate (GAP). DXPS has emerged as an appealing target due to its essentiality in bacterial pathogens, as DXP feeds into the biosynthesis of isoprenoids, ThDP, and pyridoxal phosphate (PLP), positioning DXPS as a central player in bacterial metabolism. DXPS has a unique mechanism in ThDP enzymology, in which binding of a small molecule “trigger” is required to induce a conformational change from the closed to an open conformation, essential for decarboxylation of the first enzyme bound intermediate, C2α−lactyl‐ThDP (LThDP). GAP is one such trigger molecule, likely binding at a site that is distinct from its acceptor binding site. However, how DXPS stabilizes LThDP prior to GAP‐induced decarboxylation remains unknown. In this study, we propose an active site network connects key active site histidine residues to induce the closed conformation upon reaction with pyruvate, stabilizing the LThDP intermediate. E. coli Arginine 99 may be an essential component of this network. We found that artificial disruption of this network through substitution of R99 (R99A) 1) increases KmGAP three‐fold, 2) destabilizes LThDP (detected by Circular Dichroism), and 3) shifts the conformation to the open state believed to induce decarboxylation (detected by HDX‐MS and limited proteolysis). Overall, the results support the hypothesis that R99 serves to maintain the closed conformation by connecting the key histidine residues, providing preliminary evidence for the existence of an active site network that links catalysis to conformational dynamics. Targeting the unique gated mechanism of DXPS could prove to be an effective strategy for developing a selective antibiotic that has far reaching impacts on bacterial metabolism in a pathogenic state.
Skeletal Anatomic Reconstructionists (SkAR): The development of a student organization to enhance comparative anatomy and physiology education
Authors: Brundage Cord M.
Published: May 2022

Encouraging the development of discipline based student organizations is a high impact practice that that improves student success. An off shoot from the Animal Anatomy and Physiology curriculum at Cal Poly Pomona was the development of an undergraduate student group calling themselves the Skeletal Anatomic Reconstructionists (SkAR). The SkAR student group focused on the cleaning/preparation, articulation and display of vertebrate anatomic specimens, most often skeletons. The creation of a student club, also provided the students with access to campus resources and funding for student organizations. Members of the group maintained university dermestid beetle colonies, and conducted experiments to improve the care and efficiency of carcass skeletonization. Others focused on the anatomic identification of bone remnants and the rearticulation of those into specimens. Through this process, students not only became aware of anatomic differences between species, but they also explored the biomechanics of animal posture and balance. The finished specimens produced by SkAR were then put on display by the department and were used as teaching tools in the Animal Anatomy and Physiology. The educational value of this organization thereby benefits not only the students within SkAR, but generations of students in the department. Another topic tackled by the group was the creation of latex skin models to be used for suture skill practicing. Students were excited to learn and explore the integumentary system, because of extrinsic validation and tangible benefit they received for their efforts. This novel discipline‐based student organizations has the potential to inspire others in the biological sciences to create group projects that foster student engagement in the curriculum
Intermittent Hypoxia (IH) Impairs Hippocampal Oxygen Consumption and Neurophysiological Responses to Metabolic Challenge
Authors: Polo Ashleigh K. , Browe Brigitte , Piao Lin , Sharp Willard W. , Garcia Alfredo J.
Published: May 2022

IH is a consequence of several clinical conditions such as sleep apnea. Previous work has shown that IH impairs many neurophysiological functions such as hippocampal adult neurogenesis and NMDAr‐dependent synaptic plasticity. While IH produces hippocampal oxidative stress, IH‐dependent changes in hippocampal metabolism may also contribute to impaired neurophysiology. The objective of this study is to understand how IH impacts hippocampal physiology in response to metabolic challenge. We hypothesize that ten days of IH perturbs hippocampal synaptic transmission when metabolically challenged. In vitro recordings of field excitatory post‐synaptic potentials (fEPSPs) from hippocampal slices showed that glucose deprivation (i.e., 30mM Fructose aCSF with 95% O2) caused a complete loss of the fEPSP within 20min; whereas, the fEPSP in control tissue was reduced to 40% of baseline within the same period of time. This difference coincided with a reduction in both maximal and ATP‐linked O2 consumption rates (OCRs) in hippocampal tissue following IH. Despite the reductions in hippocampal OCRs, neither ATP/ADP ratio nor lactate concentrations were different between the two groups. These data indicate that while ATP/ADP levels following IH are sufficiently maintained under basal conditions, metabolic processes fail to support synaptic physiology during glucose deprivation. Our ongoing experiments will further delineate how IH impacts the contribution of glycolytic and oxidative metabolism in the hippocampus. These findings may better define how IH impacts hippocampal responses to metabolic stressors, which could contribute to impairing hippocampal physiology in conditions such as sleep apnea.