bluuebunny/crossref_metadata_2025_split huggingface dataset:
Old Drugs, New Synergy: Identifying Novel Synergistic Antibiotic Combinations to Combat MDR Bacterial Pathogens
Authors: Dillon Nicholas Allen , Nizet Victor 
Published: April 2020

Dwindling therapeutic options have necessitated a push to identify more efficacious antibiotic regimens for treating drug resistant bacterial pathogens. Acinetobacter baumannii, a member of the ESKAPE pathogens, is of special concern due to its high mortality rate and propensity for broad spectrum antibiotic resistance. We recently described hitherto unexplored azithromycin activity against MDR A. baumannii. To expand on those findings, we sought to compare the activity of azithromycin to minocycline, the current standard of care for MDR A. baumannii infections. We identified disparate media dependent susceptibility profiles for each drug that suggested the potential for an efficacious combination therapy. In combination, the two drugs were found to interact synergistically in standardized laboratory media, human whole blood, and in a murine MDR A. baumannii lung pneumoniae model. A synergistic interaction was a surprising finding as both azithromycin and minocycline are translation inhibitors and would be expected to only have an additive interaction. Synergy was found to be mediated through enhancing the kinetics of translation inhibition, yielding a combination that was faster, more potent, and longer lasting then either drug in monotherapy. The combination was found to have broad applicability as it was also synergistic against MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin‐resistant Staphylococcus aureus. Although initially found with azithromycin and minocycline, synergy was also uncovered with other macrolides and tetracyclines and may therefore be a class wide phenomenon. Interestingly, this synergistic interaction does not appear to necessitate bacterial sensitivity to the drugs in monotherapy. Considering both azithromycin and minocycline are already FDA approved, their inclusion in a combination therapy has the potential to have an immediate impact in the clinic for treating high‐priority MDR bacterial pathogen infections in humans.Support or Funding InformationNIAID U01AI124316
Optogenetic Multimerization of Dopamine Transporter Alters Its Uptake and Trafficking Kinetics
Authors: Ingram Shalonda Marie , Rana Tanu , Manson Ashley M. , Yayah Faisal , Goodwin Shawn 
Published: April 2020

The dopamine transporter (DAT) is essential for the reuptake of released neurotransmitter dopamine (DA) in the brain. DAT is one of the main targets for psychostimulants, leading to a disruption of DA homeostasis. Methamphetamine (METH), a psychostimulant, is a substrate for DAT and induces the formation of DAT (multimeric) complexes. The function of the DAT is not dependent on the multimer state, and the contribution of the multimerized state of DAT is unclear in the literature. We have developed a substrate‐independent, optogenetic model to test the functional significance of DAT multimers. Cry2‐mCh‐DAT (Cry2‐DAT) forms multimers upon blue light exposure. This construct has normal uptake activity under control conditions, and increased uptake function when multimerized with blue light pulses. Additionally, we found that blue light‐induced multimers of Cry2‐DAT restore the dopamine uptake to control levels after pretreatment with METH, and significantly increase the uptake after nomifensine pretreatment. We evaluated the mechanism of enhanced DAT uptake function by examining alterations in trafficking and conformational changes in DAT. We used endocytic trafficking inhibitors to determine which steps may affect the enhanced uptake after multimerization. We found that enhanced uptake of Cry2‐DAT was sensitive to Calphostin C treatment. We confirmed these findings with immunofluorescence staining and colocalization of Cry2‐DAT with Rab compartments. We could not detect any significant contribution of DAT conformational change to account for the increased uptake after DAT multimerization. These data will lead to a better understanding of the role of DAT multimers in the regulation of DA homeostasis in the presence and absence of conditions that disrupt DA homeostasis.Support or Funding InformationDA036420, MHD007593, CA163069, MHD007586, GM059994, D34HP16299‐10‐00
Proximal Tubule Transport Function Screening Assay Used to Optimize Function of Kidney Organoids
Authors: Baty Catherine J. , Hukriede Neil A. , Espiritu Eugenel , Przepiorski Aneta , Weisz Ora A. , Kleyman Thomas 
Published: April 2020

BackgroundThe mechanisms that mediate solute filtration, reabsorption, secretion and excretion in the kidney are well established. However, there are a paucity of data regarding the functional properties of tubular epithelia cells within organoids. The goal of our current work is to define specific function properties of tubular epithelia cells within kidney organoids.Study objectivesTo develop quantitative assays adapted to human kidney organoids to measure proximal tubule function and test whether they can be used to optimize function in developing kidney organoids.MethodsOrganoids were generated from induced pluripotent stem cells (hiPS) derived from human fibroblasts. 6‐carboxyfluorescein (6CF) was used as a fluorescent prototype of organic anion transport (OAT), and was loaded into live organoids (1 mM in OptimemTM, with orbital rotation for 45 min), to measure basolateral uptake using confocal microscopy with tile scanning to test organoid populations and allow quantitation of transport function. To mimic continuous fluid shear stress which occurs in the PT, one population of organoids were placed on an orbital shaker (at 146 or 74 rpm for at least 30 hrs) prior to loading with 6CF. Organoids from the same culture batch at d 14 of differentiation were divided into two treatment groups, static and shaken, and loaded with 6CF, and imaged by a blinded investigator. Quantitation of average maximum intensity of basolateral uptake was done on organoid stacks using LASX software.ResultsOrganoids derived from the LRP2:mtagBFP2 reporter line (available through the Rebuilding a Kidney consortium) confirmed the PT segments within organoids and showed colocalization with 6CF. Concurrent treatment with
probenecid (an OAT inhibitor) and 6CF inhibited uptake of 6CF into PT segments. Blinded quantitation of several organoid populations showed increased intensity of 6CF in PT segments subjected to chronic orbital shear. Assays demonstrated basolateral uptake and/or luminal secretion.ConclusionsKidney organoids subject to continuous fluid shear stress have increased basolateral anion transport capacity relative to static cultured controls. A screening assay for PT basolateral uptake, assumed to be OAT mediated, can be used to test and optimize PT function within developing human kidney organoids.Support or Funding InformationSupport from NIDDK: U01 DK107350RBK, 2 P30 DK079307‐07, R01 DK18726‐01.
Ozanimod Protects Against Hypoxia plus Glucose Deprivation‐Induced Morphological Changes and Improves Cell Health in Human Brain Vascular Smooth Muscle
Authors: Albrecht Anthony G. , Li Yu Jing , Wendt Trevor S. , Gonzales Rayna J. 
Published: April 2020

Vascular smooth muscle cells regulate arterial contractile tone, blood pressure, and flow in relation to specific metabolic demands during normal and pathophysiological (i.e. hypoxic or ischemic) conditions. The plasticity of vascular smooth muscle cells allows for phenotypic transitions from contractile to synthetic‐like dedifferentiated states during pathophysiological conditions which then can lead to the development and progression of vascular inflammation and blood vessel wall remodeling. Our previous studies demonstrated that hypoxia plus glucose deprivation (HGD), an in vitro model of ischemia, alters human vascular cell morphology and increases cell death. We further demonstrated that the selective sphingosine‐1‐phosphate receptor 1 (S1PR1) ligand, ozanimod, attenuated these HGD‐mediated responses. In this study, we characterized HGD mediated phenotypic transitions and assessed expression of proteins involved in contraction (myosin light chain kinase, MLCK) and structure formation (alpha‐smooth muscle actin, vimentin) following HGD exposure. We further investigated the effect of ozanimod HGD‐mediated phenotypic transitions by assessing protein levels and localization LC3B as an indicator of autophagocytosis. Using immunocytochemistry and standard immunoblotting, we determined the localization and expression levels of α‐smooth muscle actin, MLCK, vimentin, and LC3B protein during normoxia (21% O2, control) or HGD (1%O2) for 9 h in male human brain vascular smooth muscle cells (HBVSMC) at passage 6–7. We observed that LC3B protein, a vacuole marker associated with autophagy, is ubiquitously expressed in the cytoplasm of HBVSMC during normoxia. During HGD exposure, LC3B protein levels were increased and immunohistochemical staining demonstrated LC3B localization in what appears to be autophagic vacuole formation. Both responses were attenuated by ozanimod. Although alpha‐smooth muscle actin in HBVSMC was effective in visualization of morphological changes from spindle (contractile) to rhomboid (synthetic) shape following HGD, basal protein expression levels were not altered. Similarly, MLCK, an enzyme responsible for contractile protein phosphorylation, was not altered following HGD. Levels of the synthetic phenotype marker, vimentin, was basally expressed in HBVSMC, however, levels were not increased following HGD. In conclusion, although vacuole formation is prominent, indicating poor health, HBVSMCs retained a contractile phenotype during ischemic‐like conditions. Attenuation of HGD‐mediated LC3B via ozanimod suggests a novel mechanism associated with the beneficial effect of this selective S1PR1 ligand during ischemic‐like exposure.Support or Funding InformationAmerican Heart Association 19AIREA34480018 (RJG).
Biochemical Characterization of Receptor Activity‐Modifying Protein and Peptide Agonist Effects on G protein Coupling to the Calcitonin‐Like Receptor
Authors: Roehrkasse Amanda , Warner Margaret , Booe Jason , Pioszak Augen 
Published: April 2020

The vasoactive peptides calcitonin gene‐related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD) have demonstrated protective effects in disease processes that include ischemia/reperfusion injury and inflammatory bowel disease. The actions of these peptides are mediated by the calcitonin‐like receptor (CLR), which is a class B G protein‐coupled receptor (GPCR) that forms heterodimers with any one of three receptor activity‐modifying proteins (RAMPs 1/2/3). The RAMPs modulate its peptide binding preferences and may also alter signaling output, but the latter remains poorly understood. We hypothesized that different RAMP and peptide combinations stabilize distinct CLR conformations that alter transducer interactions. We adapted a native PAGE method as an inexpensive and efficient way to visualize detergent‐solubilized, fluorescently‐tagged RAMP:CLR complexes expressed in mammalian cells and to measure their interactions with transducer proteins. Addition of agonist peptides and the purified engineered minimal Gs protein (MiniGs) prior to solubilization resulted in agonist‐dependent coupling of MiniGs to each receptor visible as a quaternary complex. Using the MiniG chimeras MiniGs/i and MiniGs/q along with MiniGs we characterized the coupling preferences of each RAMP:CLR complex in the presence of each agonist peptide. For each of the three agonist peptides MiniGs coupled best to each RAMP:CLR complex, followed by MiniGs/q and finally MiniGs/i. Overall, all three MiniG proteins exhibited better coupling to the RAMP1:CLR complex consistent with RAMP‐dependent modulation of MiniG coupling. At each RAMP:CLR complex, we also observed peptide‐dependent effects with CGRP exhibiting distinct behavior from AM and AM2/IMD. This work demonstrates that the native gel assay is a powerful tool for screening and characterizing membrane protein stabilities and ligand interactions and supports our hypothesis that the RAMPs and agonist peptides alter CLR transducer interactions. Our findings may aid development of therapeutics that exploit the beneficial effects of these peptide hormones.Support or Funding InformationThis work was supported by grants NIH R01GM104251 (AAP) and NIH predoctoral MD/PhD fellowship 1F30 HL142232 (AMR).
The relation between aortic stiffness and retinal microvascular flow pulsatility is attenuated by higher vascular resistance in healthy adults
Authors: Holwerda Seth W. , Kardon Randy , Hashimoto Ryuya , Full Jan , Nellis Julie , DuBose Lyndsey , Fiedorowicz Jess , Pierce Gary 
Published: April 2020

Aortic stiffness is associated with increased risk of cerebrovascular disease and cognitive impairments with aging, although the mechanism that contribute to this link are not well‐defined. Increases in carotid artery flow pulsatility coincide with elevated aortic stiffness and may contribute to microvascular brain damage, but studies confirming augmented flow pulsatility in the downstream microvasculature have been limited. Given the ability to assess the retinal microvasculature non‐invasively in humans and evidence that disease‐ and age‐related alterations in the retinal microvasculature mirror changes in the cerebral microvasculature, we examined the hypothesis that age‐related increases in aortic stiffness would be correlated with higher retinal arteriole flow pulsatility (systolic flow – diastolic flow / mean flow). Among 33 healthy adults (age range: 25–60 years, 47% women), aortic stiffness was assessed by carotid‐femoral pulse wave velocity (CFPWV). Retinal arteriole flow pulsatility was quantified using laser speckle flowgraphy before and during a reduction in retinal vascular resistance via 30 s of diffuse luminance flicker. Multiple regression analysis including CFPWV, mean arterial pressure (MAP), and heart rate (HR) as independent variables demonstrated that lower MAP and HR, but not higher CFPWV, were significant determinants of greater flow pulsatility at baseline (CFPWV: β=0.10, P=0.548; MAP: β= −0.40, P=0.021; HR: β= −0.50, P=0.002; model R2=0.43, P=0.001). However, after reducing retinal vascular resistance (P<0.001) via 30s of diffuse luminance flicker, multiple regression analysis demonstrated that higher CFPWV was a significant determinant of greater retinal arteriole flow pulsatility (β=0.35, P=0.026) in addition to lower MAP (β= −0.54, P=0.001) and HR (β= −0.43, P=0.005) (model R2=0.51, P<0.001). In summary, no relation was observed between higher CFPWV and retinal arteriole flow pulsatility at rest, but a positive relation was observed during arteriole vasodilation. These findings suggest that greater vascular resistance may be an important compensatory mechanism to reduce transmission of flow pulsatility.Support or Funding InformationAHA 17POST33440101, T32 HL07121 NHLBI (P01HL014388), U54 TR001356 and AHA (13DG143400012).
TMEM163 (ZNT11) protein interacts with distinct ZNT efflux transporters
Authors: Escobar Adrian , Styrpejko Daniel , Ali Saima , Cuajungco Math P. 
Published: April 2020

Our recent work showed that TMEM163 is a zinc efflux transporter that belongs to the cation diffusion facilitator (CDF) family of proteins. We propose that the TMEM163 be now called ZNT11 as a new member of the Group 1 CDF family ZNT efflux proteins that include 10 mammalian zinc transporters, ZNT1‐ZNT10. Accordingly, we hypothesize that TMEM163/ZNT11 interacts with at least one of the ZNT proteins, namely ZNT1 and ZNT2, based on its structural and functional characteristics. To show that TMEM163/ZNT11 interacts with either ZNT1 or ZNT2, we cloned their cDNAs into mammalian expression constructs containing either Myc‐DDK or HA peptide tag. Using HEK‐293 cells, we individually transfected TMEM163/ZNT11, ZNT1, and ZNT2 constructs as negative controls, while we co‐transfected TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2. As a positive control, we co‐transfected HA‐tagged TMEM163/ZNT11 with a Myc‐DDK‐tagged TMEM163/ZNT11 construct in HEK‐293 cells, based on a previous report that its rodent counterpart exists as a homodimer. We then used a co‐immunoprecipitation assay using either anti‐HA‐ or anti‐DDK‐bound agarose beads to pull down one of the target proteins. Western blot analysis showed that TMEM163/ZNT11 physically bound ZNT1 or ZNT2 protein. To determine the functional relevance of the interaction, we performed zinc flux assays using two zinc‐specific fluorescence dyes, Fluozin‐3 (high affinity, membrane impermeable) and Newport Green (low affinity, membrane permeable) following single‐ and co‐transfection of TMEM163/ZNT11, ZNT1, and ZNT2 constructs in HeLa cells. Our results confirmed that homodimers of TMEM163/ZNT11, ZNT1, and ZNT2 proteins transport zinc out of the cells, but that the efflux activity of homodimer TMEM163/ZNT11 proteins varied slightly in magnitude when compared with heterodimers of TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2 proteins. These results suggest that the interaction between TMEM163/ZNT11 and distinct ZNT proteins is physiologically relevant and may serve to modify the transport activity of ZNT protein interactor. Overall, our investigations showed for the first time that TMEM163/ZNT11 forms functional heterodimers with ZNT1 and ZNT2 proteins. Thus, TMEM163/ZNT11 by itself, or in combination with one of these specific ZNT proteins, may play a crucial role in maintaining intracellular zinc homeostasis in specific cell types.Support or Funding InformationThis work is funded by NIH R15 NS101594.
Activation of Astrocytes in the Dorsomedial Striatum Facilitates Transition from Habitual to Goal‐Directed Reward‐Seeking Behavior
Authors: Kang Seungwoo , Hong Sa-Ik , Lee Jeyeon , Peyton Lee , Choi Sun , Kim Hyunjung , Chang Su-Youne , Choi Doo-Sup 
Published: April 2020

Habitual reward‐seeking behavior is a hallmark of addictive behaviors. Although habitual behavior could be reversed to goal‐directed behavior, this shifting is not well studied. Here, we identify a role of astrocyte activation in the dorsomedial striatum (DMS) on transition from habitual to goal‐directed reward‐seeking behavior. Utilizing the Inscopix endomicroscopy/fiber photometry in vivo calcium imaging and the cell‐type specific activation of designer receptors exclusively activated by designer drugs (DREADDs), we confirmed that GFAP‐driven activation of hM3Dq DREADDs increases intensity and frequency of calcium signaling in the DMS astrocytes of ALDH1L1‐GCaMP6s expressing mice. We found that this chemogenetic activation of the DMS astrocytes increase and decrease neuronal excitability of direct pathway Medium Spiny Neurons (dMSNs) and indirect pathway Medium Spiny Neurons (iMSNs), respectively. Specifically, it reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in the dMSNs, whereas it increased the amplitude of the sEPSCs and decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the iMSNs. The changes in synaptic events induced by astrocytic activation were significantly inhibited by either of the pretreatments of DPCPX (Adenosine A1R antagonist, 1 μM), PSB12379 (Inhibitor of ectonucleotidase, CD73, 10 μM), NBTI [Inhibitor of the astrocytic adenosine transporter, equilibrative nucleoside transporter 1 (ENT1, slc29a1), 10 μM], and genetic deletion of ENT1. These data suggest that astrocytic activation‐induced synaptic activities are, in part, adenosine‐dependent. In the evaluation tests of goal‐directed and habitual reward‐seeking, the chemogenetic activation of the DMS astrocytes shifted the habitual behavior to goal‐directed action in sucrose reward‐seeking for WT, but not ENT1 KO mice. The astrocytic activation‐induced shifting was rescued when ENT1 expression was normalized in the DMS astrocytes of the ENT1 KO mice. On the other hand, when mice were trained to have goal‐directed behavior, the astrocytic activation in the DMS did not change the reward‐seeking behavioral pattern. Together, our results indicate that astrocyte‐regulated adenosine signaling in the DMS determines goal‐directed and habitual behaviors by selectively regulating synapses in the MSNs. The interaction between astrocytes and neurons via adenosine signaling could be a potential therapeutic target for maladaptive reward‐seeking behaviors.Support or Funding InformationThis work was supported by the Samuel C. Johnson for Genomics of Addiction Program at Mayo Clinic, the Ulm Foundation, and National Institute on Alcohol Abuse and Alcoholism (K01 AA027773 to SK, R01 AA018779 to DSC).
Saving the Walking Dead: The delayed effects of radiation and mitigation by blocking the TGFβ‐1 pathway
Authors: Strandberg Stacey , Fleischmann Scott , Braun Makayla , Connelly Riley , DebBaruah Caroline , Fricker Ellen , Glaszcz Carly , Griffith-Topps Annabelle , Jenkins Ava , Jessick Madeline , Kuehn Claire , Kulju Tierney , Moews Alex , Rivas Jaylin , Schraufnagel Lauren , Strother Clarissa , Vanderhoef Kayla , Medhora Meetha , Narayanan Jayashree 
Published: April 2020

Fibrosis of the lungs in patients who have undergone radiation therapy is a silent killer, inducing dense scar tissue in injured areas. Transforming growth factor beta 1 (TGFβ‐1) plays a role in the development of fibrosis. Radiation fibrosis takes months to develop. Consequently, people who are exposed to radiation to their lungs can die suddenly from pulmonary fibrosis. Latent TGFβ‐1 is stored in the extracellular matrix and is activated by the binding of an AV integrin to the arginine, glycine, and aspartate (RGD) amino acid sequence in the prodomain. Inside of the TGFβ‐1 is a ring‐like structure containing a solvent. Two prodomain arms in the structure connect to form a crossed V‐shape surrounding the growth factor monomers. These arms connect to form a bowtie with disulphide links close to the RGD sequences. The arm domain is composed of two anti‐parallel, four‐stranded beta sheets which contain hydrophobic faces. Active TGFβ‐1 binds to receptors on the surface of cells which signal to other proteins called SMADs, by adding phosphates to the SMADs. This step is inhibited by the medicine currently being tested, IPW‐5371. To understand the long‐term effects of radiation and the structurefunction relationship role of TGFβ‐1, the Divine Savior Holy Angels High School SMART (Students Modeling A Research Topic) Team designed a model of TGFβ‐1 using 3D printing technology. Currently, research is being conducted into fibrosis from activated TGFβ‐1 due to radiation exposure and IPW’s ability to reduce delayed injury to many organs that are exposed to radiation.Support or Funding InformationNIAID award HHSN272201800012C to Innovation Pathways, Palo Alto and NIAID U01AI133594 to the Medical College of Wisconsin.