bluuebunny/crossref_metadata_2025 huggingface dataset:
Financial protection and coping strategies in rural Uganda: an impact evaluation of community-based zero-interest healthcare loans
Authors: Nannini Maria , Biggeri Mario , Putoto Giovanni
Published: June 2021

In low- and middle-income countries, catastrophic health expenditures and economic hardship constitute a common risk for households’ welfare. Community health financing (CHF) represents a viable option to improve financial protection, but robust impact evaluations are needed to advance the debate concerning universal health coverage in informal settings. This study aims at assessing the impact of a CHF pilot programme and, specifically, of the initial phase involving zero-interest loans on health expenditures and coping strategies in a rural district of Uganda. The analysis relies on a panel household survey performed before and after the intervention and complemented by qualitative data obtained from structured focus group discussions. Exploiting an instrumental variable approach, we measured the causal effect of the intervention, and the main findings were then integrated with qualitative evidence on the heterogeneity of the programme’s impact across different household categories. We found that the intervention of zero-interest healthcare loans is effective in improving financial protection and longer-term welfare. Community perceptions suggested that the population excluded from the scheme is disadvantaged when facing unpredictable health costs. Among the enrolled members, the poorest seem to receive a greater benefit from the intervention. Overall, our study provides support for the positive role of community-based mechanisms to progress towards universal coverage and offers policy-relevant insights to timely design comprehensive health financing reforms.
Irregular Hodge Numbers for Rigid G2-Connections
Authors: Jakob Konstantin , Reiter Stefan
Published: June 2021

Certain rigid irregular $G_2$-connections constructed by the 1st-named author are related via pullbacks along a finite covering and Fourier transform to rigid local systems on a punctured projective line. This kind of property was first observed by Katz for hypergeometric connections and used by Sabbah and Yu to compute irregular Hodge filtrations for hypergeometric connections. This strategy can also be applied to the aforementioned $G_2$-connections and we compute jumping indices and dimensions for their irregular Hodge filtrations.
Illicit innovation and institutional folding: From purity to naturalness in the Bavarian brewing industry
Authors: Glückler Johannes , Eckhardt Yannick
Published: June 2021

We take an institutional perspective to examine how innovation thrives under conditions of resistance. Specifically, we conceive illicit innovation as a process of successive institutionalization of a new practice in the face of contrary law. In the German federal state of Bavaria, the global movement of craft-beer brewing collides with a regional jurisdiction that prohibits precisely these brewing practices and instead protects the traditional institution of purity-brewing (Reinheitsgebot). Grounded on an embedded qualitative case study of brewers and industry representatives, we build a theory of institutional folding of new norms and practices over established ones. This way, creative brewers have succeeded in legitimizing new practices of naturalness-brewing (Natürlichkeitsgebot). Whereas the legal resistance has stimulated brewers to create an original counter-institution, the illicit innovation has also begun to change the institutional context of the beer industry in Bavaria.
On the economic geography of climate change
Authors: Peri Giovanni , Robert-Nicoud Frédéric
Published: June 2021

Climate change is a defining challenge of our times. The five papers collected in this special issue provide foundations to well-informed policymaking by addressing two main themes of the economic geography of climate change. First, it brings effects that are heterogeneous across space. Some regions of the globe will lose more than others and some may even be better off as a result. Second, humans will have to adapt in order to survive. We emphasize how the lack of mobility could contribute to worsening the socioeconomic costs of climate change. This issue also considers alternative margins of adjustment, such as fertility, specialization and trade.
Opioid overprescription in adolescents and young adults undergoing hip arthroscopy
Authors: Hassan Mahad M , Rahman Omar F , Hussain Zaamin B , Burgess Stephanie L , Yen Yi-Meng , Kocher Mininder S
Published: June 2021

Few studies have examined factors related to the increased consumption of opioids after hip arthroscopy in adolescents and young adults. This study sought to determine prescription patterns following hip arthroscopy in this population, and to determine clinical or surgical factors associated with increased post-operative opioid use. Daily post-operative opioid intake was obtained from pain-control logbooks of adolescents and young adults who underwent hip arthroscopy between January 2017 and 2020. Study outcomes were defined as the median total number of opioid tablets consumed, total days opioids were consumed, mean daily opioid consumption and the ratio of opioids prescribed post-operatively to consumed. Clinical and surgical factors were analyzed to determine any association with opioid consumption. Fifty-eight (20%) patients returned completed logbooks. Most patients (73%) were prescribed 30 oxycodone tablets. The median number of tablets consumed was 7 (range 0–41) over a median duration of 7 days (range 1–22). The median ratio of tablets consumed to prescribed was 20%. Increasing patient age at surgery was associated with increased total number of tablets consumed (r = 0.28, P = 0.04) and to the ratio of tablets consumed to prescribed (r = 0.30, P = 0.03). Patients who were prescribed more than 30 tablets consumed on average 7.8 more tablets than patients prescribed fewer (P = 0.003). Patients who underwent regional anesthesia consumed tablets for longer compared with those who did not (median, 10 versus 4 days; P = 0.03). After undergoing hip arthroscopy, adolescents and young adult patients are commonly overprescribed opioids, consuming on average only one-fifth of the tablets prescribed.
Prosecute Little Fish at the ICC
Authors: Ferdinandusse Ward , Whiting Alex
Published: June 2021

A central aim of international criminal tribunals since Nuremberg has been to prosecute those most responsible, the ‘big fish’, leaving smaller cases to national jurisdictions. The authors, who have both academic backgrounds as well as experience prosecuting international crimes in both international and domestic courts, argue that the International Criminal Court (ICC) should change course and expand its focus to include more ‘little fish cases’, bringing more cases overall and initiating them more quickly. Particular features of the ICC, namely its global reach and its ambition to promote national prosecutions, compel this change in strategy. For normative, strategic, and practical reasons, the ICC should bring smaller fish cases to enhance its credibility, functioning, and legitimacy, and to spur prosecutions by national authorities.
Favoritism towards High-Status Clubs: Evidence from German Soccer
Authors: Bose* Paul , Feess Eberhard , Mueller Helge
Published: June 2021

Biases in legal decision-making are difficult to identify as type II errors (wrongful acquittals) are hardly observable and type I errors (wrongful convictions) are only observed for the subsample of subsequently exonerated convicts. Our data on the first German soccer league allow us to classify each referee decision accurately as correct, type I error or type II error. The potential bias we are interested in is favoritism toward clubs with higher long-term status, proxied by the ranking in the all-time table at the beginning of each session and by membership. Higher status clubs benefit largely from fewer type II errors. By contrast, the actual strength of clubs has no impact on referee decisions. We find no difference in type I errors and suggest anticipation of the bias as a potential explanation for the difference. We investigate several mechanisms potentially underlying our results; including career concerns and social pressure (JEL J00, M51, D81, D83).
History of the gas fuelling star formation in eagle galaxies
Authors: Scholz-Díaz Laura , Sánchez Almeida Jorge , Dalla Vecchia Claudio
Published: June 2021

Theory predicts that cosmological gas accretion plays a fundamental role fuelling star formation in galaxies. However, a detailed description of the accretion process to be used when interpreting observations is still lacking. Using the state-of-the-art cosmological hydrodynamical simulation eagle, we work out the chemical inhomogeneities arising in the disc of galaxies due to the randomness of the accretion process. In low-mass systems and outskirts of massive galaxies, low metallicity regions are associated with enhanced star-formation, a trend that reverses in the centres of massive galaxies. These predictions agree with the relation between surface density of star formation rate and metallicity observed in the local spiral galaxies from the MaNGA survey. Then, we analyse the origin of the gas that produce stars at two key epochs, z ≃ 0 and z ≃ 2. The main contribution comes from gas already in the galaxy about 1 Gyr before stars are formed, with a share from external gas that is larger at high redshift. The accreted gas may come from major and minor mergers, but also as gravitationally unbound gas and from mergers with dark galaxies (i.e. haloes where more than 95  per cent of the baryon mass is in gas). We give the relative contribution of these sources of gas as a function of stellar mass (8 ≤ log [M⋆/M⊙] ≤ 11). Even at z = 0, some low-mass galaxies form a significant fraction of their total stellar mass during the last Gyr from mergers with dark galaxies.
Tomato E8 Encodes a C-27 Hydroxylase in Metabolic Detoxification of α-Tomatine during Fruit Ripening
Authors: Akiyama Ryota , Nakayasu Masaru , Umemoto Naoyuki , Kato Junpei , Kobayashi Midori , Lee Hyoung Jae , Sugimoto Yukihiro , Iijima Yoko , Saito Kazuki , Muranaka Toshiya , Mizutani Masaharu
Published: June 2021

Tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid that contributes to the plant defense against pathogens and herbivores through its bitter taste and toxicity. It accumulates at high levels in all the plant tissues, especially in leaves and immature green fruits, whereas it decreases during fruit ripening through metabolic conversion to the nontoxic esculeoside A, which accumulates in the mature red fruit. This study aimed to identify the gene encoding a C-27 hydroxylase that is a key enzyme in the metabolic conversion of α-tomatine to esculeoside A. The E8 gene, encoding a 2-oxoglutalate-dependent dioxygenase, is well known as an inducible gene in response to ethylene during fruit ripening. The recombinant E8 was found to catalyze the C-27 hydroxylation of lycoperoside C to produce prosapogenin A and is designated as Sl27DOX. The ripe fruit of E8/Sl27DOX-silenced transgenic tomato plants accumulated lycoperoside C and exhibited decreased esculeoside A levels compared with the wild-type (WT) plants. Furthermore, E8/Sl27DOX deletion in tomato accessions resulted in higher lycoperoside C levels in ripe fruits than in WT plants. Thus, E8/Sl27DOX functions as a C-27 hydroxylase of lycoperoside C in the metabolic detoxification of α-tomatine during tomato fruit ripening, and the efficient detoxification by E8/27DOX may provide an advantage in the domestication of cultivated tomatoes.
Functionalizing Protein‐based Materials with Biomolecules
Authors: Bondos Sarah , Howell David , Tsai Shang‐Pu , Bayless Kayla
Published: June 2021

The development of materials with diverse mechanical and functional properties enables a broad range of applications. For materials composed of protein, well‐established molecular biology techniques can theoretically be used to alter the mechanical and functional properties of materials. However, repetition of amino acid sequences in the constituent proteins can complicate engineering the mechanical properties. Furthermore, most protein‐based materials are assembled under denaturing conditions, thus precluding incorporation of full‐length proteins in an active state. Our lab discovered that the Drosophila melanogaster transcription factor Ultrabithorax (Ubx) hierarchically self‐assembles into novel, ordered biomaterials, ranging from nanoscale fibrils to macroscale fibers, films, meshes. These materials are stabilized by specific dityrosine bonds that spontaneously form during assembly. The location of the participating tyrosines in unique sequence motifs enables the reliable tuning of the materials by mutagenesis to be either weaker or stronger. Because Ubx materials rapidly form in mild, aqueous buffers, a surprisingly wide variety of proteins can be incorporated via gene fusion without harming either materials assembly or the function of the appended protein. As one example, when Ubx is fused to angiogenic growth factors, the resulting fibers control cell signaling and cell behavior in vitro, and can instigate and guide blood vessel formation in vivo. Ubx materials provide an inexpensive and facile platform for customizing materials for a variety of applications.

Support or Funding Information

NSF

Ted Nash Long Life Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatiotemporal Phytochrome‐Dependent Regulation of Photosynthesis and Growth
Authors: Montgomery Beronda L.
Published: June 2021

Photosynthetic organisms depend upon light for carbon fixation and production of reductant. Thus, the ability of these organisms to adapt to changes in the photoenvironment is critical. Plants and other photosynthetic organisms have diverse mechanisms for light perception and exhibit numerous developmental, physiological, and metabolic photoresponses. Notably, light exposure results in distinct responses in specific plant tissues during photomorphogenesis. Light promotes growth of cotyledons and leaves, as well as development and elongation of roots, whereas light inhibits elongation of hypocotyls. Phytochromes are one of the most well‐known plant photoreceptors coordinating the aforementioned tissue‐ and organ‐specific plant responses. In prior molecular genetic studies, we used targeted phytochrome depletion to probe the physiological roles and biochemical mechanisms of spatial‐specific aspects of phytochrome signaling and the molecular bases of distinct aspects of photomorphogenesis. Localized pools of phytochromes regulate key factors that control hypocotyl elongation and coordinate nuclear and plastid genome expression in the development and differentiation of plastids required for the establishment of photosynthesis. The latter includes phytochrome‐dependent regulation of the expression of sigma (SIG) factor genes, which encode proteins involved in novel spatiotemporal aspects of photomorphogenesis and previously undescribed mechanistic aspects of coordinating expression of nuclear and plastid genomes. My laboratory is investigating specific roles of phytochromes and phytochrome‐dependent effectors in anterograde regulation of SIG expression. We are also identifying and characterizing factors that function downstream of SIG2 in the regulation of SIG2‐specific photomorphogenic and photosynthetic phenotypes. Results from our studies are significantly expanding our knowledge of the regulation of spatial‐specific phytochrome responses, which impacts distinct aspects of growth and development and our understanding of the light signaling mechanisms which coordinate gene expression in the nucleus and chloroplasts, thereby impacting the capacity for photosynthesis, other plastid‐related functions, and photomorphogenesis.

Support or Funding Information

Research in the lab of B.L.M. on phytochrome‐dependent signaling in plants is supported by the National Science Foundation (grant no. MCB‐1515002) and by infrastructure support from the Office of Science of the US Department of Energy(grant no. DE‐FG02‐91ER20021).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanosensitive TRPV4 Channel is Involved in Fibrotic Transdifferentiation of Retinal Pigment Epithelial Cells
Authors: Tamiya Shigeo , McDonald Kevin , Ueda Shunichiro
Published: June 2021

Sustained presence of myofibroblasts during wound healing can cause fibrosis. Within the eye, transdifferentiation of retinal pigment epithelial (RPE) cells to myofibroblasts have been implicated as a key event leading to fibrotic scarring of the retina. While TGF‐beta signaling has been shown to play key roles in this process, inhibition of this pathway can result in undesirable side‐effects by preventing physiological function of this multifaceted cytokine. Therefore, identification of other pathways/molecules that could potentially be targeted to prevent fibrotic scarring is warranted. In this study, roles of mechanosensitive channel TRPV4, which has been implicated to play roles in several fibrotic diseases, and its downstream effector MLCK in myofibroblast transdifferentiation of RPE cells as well as matrix contraction, a key phenomenon of retinal scarring, were examined.

Primary cultured porcine RPE cells were used to assess matrix contraction. Cells were cultured with media containing TGF‐beta on collagen matrices in the presence or absence of TRPV4 inhibitor HC‐067047 or MLCK inhibitor ML‐7. Collagen matrices with attached cells were released from 24 well plates and contraction was assessed as change in size of matrices. Cells on these matrices were then lysed to examine the expression of myofibroblast marker protein tropomyosin 1 (TPM1) as well as TRPV4 by western blot analyses.

TGF‐beta treatment significantly increased collagen matrix contraction by cultured RPE cells compared to untreated controls. TGF‐beta treatment did not significantly affect TRPV4 expression. Inhibitors for TRPV4 and MLCK significantly reduced matrix contraction as well as TPM1 expression. These data implicate that TRPV4 signaling pathway, while not directly regulated by TGF‐beta, is involved in myofibroblast transdifferentiation of RPE cells and subsequent fibrotic contraction of the matrix.

Support or Funding Information

Research to Prevent Blindness Unrestricted Grant, DoD Award No. W81XWH‐15‐1‐0298

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
What can protein methylation tell us about histones, ribosomes, translation factors and the biology of cancer and aging?
Authors: Clarke Steven G.
Published: June 2021

The chemical diversity of proteins is enhanced by posttranslational methylation of most of the 20 major ribosomally‐incorporated amino acids. The most abundant of these modifications occur at the side chains of lysine and arginine residues, although methylation is also known for histidine, glutamine, asparagine, glutamate, cysteine, and methionine residues, as well as for the alpha‐amino groups of several N‐terminal residues and the alpha‐carboxyl groups of leucine and isoprenylcysteine C‐terminal residues. Methylation reactions also can tag age‐damaged D‐aspartyl and L‐isoaspartyl residues for repair reactions.

Protein arginine methylation in mammalian cells is largely catalyzed by the nine members of the PRMT family. We have focused our work on the PRMT5 and PRMT9 species that catalyze symmetric dimethylarginine formation, as well as the PRMT7 species that only catalyzes monomethylarginine formation. PRMT9 appears to be specific for the modification of the SF3B2 splicing factor, whereas PRMT5 and PRMT7 appear to have many physiological substrates, including histones. The latter methylation reactions represent a crucial part of the histone code, and recognition of these methylated residues by protein interaction domains modulates transcription. We have recently explored crosstalk between PRMT5 and PRMT7 in the modification of histone H4. In in vitro experiments using a peptide corresponding to the N‐terminal 21 amino acid residues of histone H4, we showed that the activity of the PRMT5 enzyme at the arginine‐3 residue is greatly enhanced by PRMT7‐dependent methylation of arginine‐17 and/or arginine‐19. These experiments provide evidence that PRMTs can cooperate with each other to affect the final degree of protein modification and epigenetic control. Surprisingly, PRMT7 has very little activity in vitro at 37 °C; it is most active between 10 and 20 °C. This result suggests that PRMT7 may respond to cold stress in the organism.

Methylation reactions, including modification at arginine residues, can occur predominately on proteins of the translational apparatus. In the yeast Saccharomyces cerevisiae, some 51 of the approximately 70 methyltransferases modify rRNA (13 enzymes), mRNA (2 enzymes), tRNA (16 enzymes), translation factors (10 enzymes), and ribosomal proteins (10 enzymes). Of particular interest, we have shown that the Sfm1, Hpm1, Rkm3, and Rkm4 enzymes modify arginine, histidine, and lysine residues buried deep within the ribosome; these modified residues may function in protein–RNA interactions rather than protein–protein interactions.

Support or Funding Information

This work is funded by the National Science Foundation Grant MCB‐1714569.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Shape change throughout the body of the tongue during drinking in the striped skunk (Mephitis mephitis)
Authors: Olson Rachel A. , Montuelle Stéphane J. , Curtis Hannah E. , Williams Susan H.
Published: June 2021

In mammals, the tongue plays a central role in many oral behaviors, but it is particularly essential for feeding and drinking. During feeding, the tongue is used for ingestion, manipulation and positioning of food within the oral cavity, and swallowing. When mammals drink, they do so by lapping, licking, or sucking, and the tongue is vital to uptake and transport of fluid into and through the oral cavity and oropharynx. All of these behaviors are performed with only a partial reliance on a bony support system; instead the tongue engages in complex movements as a muscular hydrostat – where selective contraction of intrinsic and extrinsic muscle fibers produce movements and deformations of the tongue. The tongue's anatomical complexity and its location inside the oral cavity make it particularly difficult to visualize and study the movements that contribute to these and other oral behaviors. The objective of this study is to test the ability of marker‐based XROMM (X‐ray reconstruction of moving morphology) to track shape changes throughout the body of the tongue. The striped skunk (Mephitis mephitis), a mammalian omnivore belonging to the order Carnivora, was used as a model for rhythmic lapping while drinking a barium liquid. Data was collected using XROMM with markers implanted in the bones of the skull as well as in the body of the tongue. Tongue markers were used to calculate length changes throughout the body of the tongue. Timing and movement parameters are also registered to the gape cycle derived from the rigid body motions derived from skeletal markers. Based on an analysis of 150 cycles, during rhythmic drinking, the middle and posterior tongue underwent the most variation in length change, while the anterior tongue underwent the least amount of variation in length. In contrast, the posterior tongue underwent the least amount of variation in tongue width, as compared to the anterior and middle tongue. Maximum overall tongue length occurred 2.74±29.3 msec before maximum gape, whereas minimal overall tongue length occurred 20.24±7.38 msec before minimum gape. Based on these preliminary results, this method shows promise for producing quantitative data for studying tongue deformations within the oral cavity. Future studies incorporating spatial parameters of the tongue to the rest of the feeding apparatus across mammals will further our understanding of how the tongue functions during feeding and drinking behaviors.

Support or Funding Information

National Science Foundation Grant DBI‐0922988, National Science Foundation Grant IOS‐1456810

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Synthesis and Biochemical Use of N‐Hydroxyl‐N‐(5‐aminopentyl)succinamic acid (HSC) by the Enzyme FslA
Authors: Nguyen James , Hoffmann Katherine
Published: June 2021

Siderophores are small, high affinity iron chelators used by bacteria for resource gathering. Non‐Ribosomal Peptide Synthetases manufacture siderophores using a novel and under‐described chemistry: they create peptide bonds outside of a ribosome, in the process consuming ATP and a high‐energy adenylate intermediate. Beyond basic science, NIS synthetase have a novel fold and chemistry making it a good target for antibiotic design. Despite their interesting function, there are only nine relevant articles on PubMed database about NIS Synthetases. DesD is a model enzyme for this novel family, an enzyme from Streptomyces coelicolor that makes the siderophore desferrioxamine E. The Hoffmann Lab is pursuing a full biochemical characterization of DesD, which will require the appropriate techniques as well as access to the substrates in a pure, soluble form.

DesD is an iterative enzyme, meaning it catalyzes multiple bonds on the same substrate. Specifically, DesD catalyzes all of these events: the serial linking of three molecules of N‐Hydroxyl‐N‐(5‐aminopentyl)succinamic acid (HSC) into a linear trimer called desferrioxamine G, and then a macrocylization event linking the ends. The initial binding of two HSC molecules is a very different binding site occupancy than the binding of the linear trimer for the cyclization bond, and any biochemical characterization should quantify this binding and any differences in kinetics. Desferrioxamine G can be commercially bought to study binding of DesD. HSC however, cannot be bought, only synthesized. This project is concerned with the synthesis of HSC and then the use of it in binding and kinetic studies. While the synthesis has been published in the literature, we have made adjustments to improve yield. Additionally, we are optimizing purification via HPLC. This substrate will be used in thermodynamic binding studies via isothermal titration calorimetry and compared to current binding isotherms of the larger substrate. With the study of large substrate and small substrate thermodynamics and kinetics will contribute both to the understanding of NIS synthetases and of iterative enzymes more broadly.T

Support or Funding Information

This work was supported by a grant from the National Science Foundation (MCB ‐ 1716986) to K.M.H, and by California Lutheran University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intact Cell Assay to Measure Agonist‐ and GRK2‐Dependent Phosphorylation of the α2A‐Adrenergic Receptor
Authors: Farina Zachary , Hebert Aaron‐Michael , Weldin Erica , Varney Matthew , Sterne‐Marr Rachel
Published: June 2021

The α2A‐adrenergic receptor (α2A‐AR) is a G protein‐coupled receptor (GPCR) that is activated by the catecholamines epinephrine and norepinephrine. Its physiological roles include inhibition of insulin secretion from pancreatic beta cells, cardiovascular vasoconstriction, and the inhibition of lipolysis in adipocytes (among other functions). GPCRs are often phosphorylated in an agonist‐dependent fashion and thereby desensitized by GPCR kinases (GRKs). GRK2 phosphorylates the α2A‐AR in vitro and when co‐transfected with the receptor in monkey kidney (COS‐7) cells. GRK2 phosphorylation occurs at four consecutive serine residues in the third intracellular loop of the α2A‐AR (residues 296–299). We recently mapped sites on GRK2 that are necessary for phosphorylation of the β2‐adrenergic receptor and interaction with the α2A‐adrenergic receptor in intact cells. In order to test GRK2 mediated α2A‐AR phosphorylation, we generated an antibody that detects α2A‐AR phosphorylation at serines 296–299. We are developing an intact cell assay to assess residues on both GRK2 and the receptor that are important for α2A‐AR phosphorylation. This will give us a greater understanding of the regulation of this physiologically important receptor.

Support or Funding Information

This work was funded by the National Science Foundation MCB0744739 to Rachel Sterne‐Marr and the Siena College Center for Undergraduate Research and Creative Activity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating gibberellin phytohormone biosynthesis by plant‐associated bacteria
Authors: Peters Reuben J.
Published: June 2021

Gibberellins (GAs) are structurally complex diterpenoids that serve as important hormones in plants, with crucial roles in growth and development, but also in modulating defense. Beyond their endogenous production by vascular plants, GAs also are produced by plant‐associated microbes, both fungi and bacteria. We have recently elucidated the biosynthetic pathway by which bacteria produce GA, revealing convergent evolution, yet striking similarities in the underlying (bio)chemical transformations. We are currently engaged in mechanistic studies of the enzymes catalyzing the relevant complex reactions, including structure‐function analyses. In addition, we also are investigating the physiological function of such bacterial phytohormone production and have found two surprisingly distinct roles, which seem to depend on the nature of the plant‐bacteria relationship. In phytopathogens, production of bioactive GA seems to serve to increase virulence. By contrast, symbiotic rhizobia produce only the penultimate precursor, and seem to have co‐evolved with their leguminous host, which catalyze the final step to produce a bioactive GA that then assists symbiosis. Our most recent results will be discussed.

Support or Funding Information

This work was supported, in part, by grants from the USDA (NIFA‐AFRI‐2014‐67013‐21720) and NSF (CHE‐1609917).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding How the Protein Environment Accelerates Cofactor‐Free O2 Activation in Antibiotic Biosynthesis Monooxygenases (ABMs)
Authors: Ellis Emerald , Machovina Melodie M. , DuBois Jennifer L.
Published: June 2021

Molecular oxygen is a nontoxic, naturally abundant, thermodynamically powerful oxidant. Most enzyme‐catalyzed oxidation and oxygenation processes using O2 depend on small, tightly bound metal‐containing or organic cofactors, such as heme or flavin, to activate O2 and thereby accelerate its reaction with organic substrates. Antibiotic Biosynthesis Monooxygenases (ABMs) are a large family of small proteins with the unusual ability to catalyze direct reactions between their substrates and O2 without any cofactors. Prior studies of a representative ABM, Nogalamycin MonoOxygenase (NMO), suggested that its reaction proceeds by rate limiting electron transfer to form a superoxide/substrate radical pair. By comparing the rate and thermodynamic parameters for the catalyzed and uncatalyzed reactions, we sought to determine how the protein environment of NMO helps to catalyze the direct reaction between its substrate and O2. Marcus Theory relates the activation energy (ΔG‡, the difference in energy between the substrate and transition state) to the thermodynamic driving force (ΔG°, the difference in energy between substrate and product) and the reorganization energy (λ0, the energy required to reorganize all atomic nuclei in the reaction sphere without electron transfer): ΔG‡ =(ΔG° + λ0)2/4λ0. Prior work showed that for the flavin‐containing enzyme glucose oxidase, the reorganization energy was responsible for most of the difference in ΔG‡ between the enzymatic and uncatalyzed electron transfers that occur between flavin and O2. A single conserved positive charge was essential for lowering ΔG‡ and therefore accelerating the reaction. We hypothesized that λ0 might likewise be the major contributor to ΔΔG‡ (catalyzed versus uncatalyzed) in NMO, though these enzymes lack conserved positive charges. Here, we tested that hypothesis by measuring ΔG° (via spectroelectrochemical titration of the midpoint potentials) and ΔG‡ (Arrhenius plots) for uncatalyzed and NMO‐catalyzed oxygenation reactions, as well as for reactions catalyzed by two key NMO active site mutants. Our results suggest an unconventional means for accelerating reactions between O2 and certain classes of substrate that depends solely on a properly tuned protein environment.

Support or Funding Information

National Science Foundation, MCB1715176

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IL‐6 and Bile Acids are Skin‐Derived Factors that Regulate Whole‐Body Metabolism in SCD1 Deficient Mice
Authors: Dumas Sabrina N. , Guo Chang‐an , Ntambi James M.
Published: June 2021

Stearoyl‐CoA desaturase 1 (SCD1) is a lipogenic enzyme that catalyzes the rate limiting step in monounsaturated fatty acid synthesis by inserting a double bond at the delta‐9 position of long‐chain fatty acids. Specifically, SCD1 converts stearate (18:0) to oleate (18:1n9) and palmitate (16:0) to palmitoleate (16:1n7). Global deletion of SCD1 in mice (GKO) provides complete resistance to both high‐carbohydrate and high‐fat diet‐induced obesity. Furthermore, GKO mice also demonstrate improved insulin sensitivity and glucose clearance. Surprisingly, skin‐specific deletion of SCD1 also provides protection from high‐fat diet‐induced obesity and similar metabolic improvements to GKO mice. Histological analysis of SKO skin demonstrates dermal deviations that include 1) an increased number of hair follicles despite progressive hair‐loss, 2) increased macrophage infiltration and 3) differentially expressed IL‐6 that is localized around hair follicles. Co‐localization of macrophage and IL‐6 markers reveal that IL‐6 is not produced by macrophages but instead is produced by cells of the hair follicles. In plasma, IL‐6 protein levels are 25‐fold increased relative to controls, and downstream IL‐6‐mediated metabolic effects are increased at least 5‐fold in distal tissues such as white adipose tissue and liver. Another skin‐derived factor in SKO mice is bile acids, in that, genetic analysis of bile acid synthesis genes in the skin and liver of SKO mice show unexpected and divergent expression. Cyp7a1 the rate‐limiting bile acid synthesis gene is unchanged in SKO liver, but the extrahepatic equivalent Cyp7b1 is significantly increased in skin. In plasma, bile acid composition is not only increased but is also significantly skewed towards increased levels of tauro‐β‐muricholic acid which is indicative of increased extrahepatic bile acid synthesis in mice. Furthermore, the bile acid pool is also more hydrophilic which is a phenotype that is protective against the development of diseases of the metabolic syndrome. Together, changes in both IL‐6 and bile acid composition in SKO mice strongly suggest that skin metabolism is an important component of whole‐body metabolism, and this opens another avenue for the treatment of metabolic diseases.

Support or Funding Information

Sabrina Dumas was supported by USDA Hatch W2005

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modifications in MUC2 and OCLN Expression in Human Alveolar Adenocarcinoma Epithelial Cells Following E‐Cigarette Chemical Exposure
Authors: Wright Alexis , Mitchum Jessica , Daft Joseph G.
Published: June 2021

Electronic cigarettes (e‐cigarettes) have entered the market promoting a healthy and clean alternative to conventional tobacco cigarettes. Aggressive marketing has led to an increase in usage of e‐cigarettes among adults and adolescents. Despite the promotion of e‐cigarettes as an alternative for tobacco users, the chemical composition of these two products is very similar. Two of the predominant chemicals found in e‐cigarettes are formalin and methylbenzaldehyde, of which, formalin is also found in tobacco cigarettes. We hypothesize that exposure of the alveolar adenocarcinoma (A549) cells to these common e‐cigarette chemicals will alter the lung epithelial barrier as measured by occludin (OCLN) and mucin‐2 (MUC2) expression.

Mucin‐2 is a protein that secretes a protective mucus membrane and occludin is a tight junction protein, both contributing to a functional and protective lung epithelial barrier. Overexpression of MUC2 can lead to hypersecretion of mucus, while under expression of OCLN can cause an increase in epithelial permeability, representing a disruption of this barrier. A549 cells will be treated with physiological concentrations of formalin and methylbenzaldehyde to mimic e‐cigarette exposure. We will show that exposure to formalin and methylbenzaldehyde alters MUC2 and OCLN, indicating a disruption of the lung epithelial barrier. These results represent the negative health effects associated with e‐cigarette use, which contradicts the current advertisements marketing e‐cigarettes as a safe product.

Support or Funding Information

National Science Foundation Grant# 1458212

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A transient dopamine signal represents the value of avoidance in negative reinforcement
Authors: Roberts Jonte , Pultorak Katherine , Busch Brandon , Oleson Erik
Published: June 2021

The mesolimbic dopamine (DA) system has traditionally been examined under a reward based context. While accumulating evidence supports that DA release events represent the value of predicted rewards, there is little known about its role in avoidance. To investigate dopamine's role in the valuation of signaled operant avoidance, we developed an economics‐based shock avoidance task in which rats were given the opportunity to avoid (signaled by the presentation of a cue light) electrical foot‐shock across epochs wherein the unit price (response requirement over mA shock) to avoid or escape increases. After developing this task, we first used fast‐scan cyclic voltammetry (FSCV) to measure DA release events in vivo as the animals performed across a range of prices. We observed that the concentration of DA decreased with price, though both DA and avoidance were initially suppressed at session onset. To establish a causal role, we then used optogenetics to augment DA release at either the presentation of the cue signal or upon successful avoidance of the foot‐shock. Increasing release at the cue made avoidance more sensitive to price; whereas increasing release at successful avoidance made it less sensitive to price. We conclude that this is likely because the neural representation of the value to avoid is greater than predicted at successful avoidance, and worse than expected at the cue on trials in which foot‐shock is administered. We will next assess the role of the rostromedial tegmental nucleus (RMTg), a neural structure known to cause prediction errors, in the valuation of avoidance.

Support or Funding Information

Funding for the project was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBO and an institutional UROP to KJP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of residues in human melatonin type 2 receptor involved in signaling selectivity or general signal transmission using natural variants
Authors: Plouffe Bianca , Karamitri Angeliki , Flock Tilman , Gallion Jonathan M. , Bonnefond Amélie , Guillaume Jean‐Luc , Le Gouill Christian , Froguel Philippe , Lichtarge Olivier , Deupi Xavier , Jockers Ralf , Bouvier Michel
Published: June 2021

Melatonin is a neurohormone secreted by the pineal gland mainly regulating circadian rhythm and binding to 2 receptors: MT1 and MT2. A recent genome‐wide association studies revealed 40 non‐synonymous polymorphisms of MT2. All these variants are normally expressed at cell surface and 4 of them show loss of melatonin binding. Given that no crystal structure is available yet for MT2 and that variants are well distributed along MT2, we took advantage of these natural variants to delineate structural features controlling MT2 functionality. By monitoring β‐arrestin 2 recruitment, ERK phosphorylation, inhibition of cAMP production, Gαi1 and Gαz activation, we generated 40 signaling signatures and similar profiles were grouped together using non‐negative matrix factorization (NMF). A total of 8 different types of signatures (clusters) were obtained. Each cluster has unique signaling features and is distinctly spatially localized along MT2. While residues important for general signal transmission are located in transmembrane regions, the residues responsible for signal selectivity are almost exclusively localized in intracellular domains. Using computational homology modeling (PDB ID: 4Zwj and 3sn6), we proposed molecular mechanisms underlying general signal transmission but also Gα/β‐arrestin and Gαz/Gαi1 selectivities. Altogether, our findings represent a proof of principle that natural variants of a given receptor can be exploited and used as a platform to learn more about its structural properties.

Support or Funding Information

This work was supported by a grant from FRQS to MB, from FRM and ANR to RJ and from NIH, NSF and DARPA to OL. Postdoctoral fellowships from CIHR and from Diabetes Canada to BP, from FRM to AK and from ETH Zurich to TF also funded this work.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Site‐Specific Dueteration Decreases Cytrochrome P450 Production of 12‐Hydroxy‐Nevirapine through the Kinetic Isotope Effect
Authors: Heck Carley J. S. , Seneviratne Herana Kamal , Bumpus Namandjé N.
Published: June 2021

The non‐nucleoside reverse transcriptase inhibitor nevirapine (NVP), classified as an essential medication by the World Health Organization, has been shown to cause life‐threatening hepatotoxicity and skin rash in patients. The 12‐hydroxyNVP (12‐OHNVP) metabolite of NVP has been implicated in these toxicities and here we aimed to gain a detailed understanding of utility of introducing deuteriums at the 12‐position of NVP as an approach to decreasing the formation of this metabolite. To do so, an ultra‐high performance liquid chromatograph tandem high‐resolution mass spectrometry assay was developed and employed to detect 12‐OHNVP formation following incubation of NVP with human liver microsomes and individual cDNA‐expressed enzymes. Through this work, we found that formation of 12‐OHNVP was most abundant following incubation with cytochrome P450 (P450; CYP) 2C19 although each of the P450s tested, with the exception of CYP3A5, catalyzed the formation of 12‐OHNVP to some extent. In line with this, formation of 12‐OHNVP by human liver microsomes was not abrogated in the presence of any established small molecule inhibitors of P450 activity including ketoconazole (CYP3A inhibitor). In order to test whether the deuterium kinetic isotope effect could be employed to control 12‐OHNVP production by all P450s we investigated the impact of 12th position deuteration of NVP on the formation of this metabolite. Human liver microsomes were incubated with 10 μM NVP or 10 μM 12‐d3NVP for 30 minute and the production of the 12‐OHNVP was measured. The abundance of 12‐OHNVP was 6.8‐fold lower when human liver microsomes were incubated with 12‐d3NVP, as compared to undeuterated NVP incubations. Of note, no difference in the formation of other P450‐dependent metabolites of NVP was observed. In order to test the impact of deuterium kinetic isotope effects on the metabolism of NVP in a whole cell system, we measured metabolite levels in the medium of primary mouse hepatocytes treated with 10 μM NVP and 12‐d3NVP for 24 hours. A 4.7‐fold decrease was observed in 12‐OHNVP production in the medium of cells treated with deuterated NVP as compared to undeuterated NVP. As with the human liver microsome experiments, no significant difference was observed in the production of other P450‐dependent metabolites between treatments. These results demonstrate that dueteration at the 12th position can markedly decrease cytochrome P450 production of 12‐OHNVP through the kinetic isotope effect. The impact of this controlled metabolism on NVP toxicity will be a subject of further research. This work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.

Support or Funding Information

This work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.