bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic tools for study of the hypothalamus and thyroid in Zebrafish
Authors: Hutcheson David , Xie Yuanyuan , Duncan Robert , Dorsky Richard
Published: November 2023

The hypothalamus plays is a key regulatory role in a wide variety of animal behaviors including feeding, aggression, and mating. To further characterize the development and function of this CNS structure, we have begun to develop genetic tools with which to manipulate the hypothalamus in living zebrafish. Nkx2.4b is specifically expressed in the hypothalamus and thyroid during development, and is therefore an attractive target for genetic manipulation. First, we used TALENs to produce an Nkx2.4b−/− null. Damage caused by TALENs is typically repaired by non‐homologous end joining (NHEJ). We have obtained and bred several lines containing NHEJ in/dels that produce frameshifts in the 1st exon of Nkx2.4b and are predicted to be null alleles. Second, we are testing the ability of TALENS to mediate a targeted homologous recombination (HR). We created a plasmid containing Nkx2.4b genomic sequence flanking a GFP transgene, such that homologous recombination would create a “knock‐in knock‐out”. This plasmid was co‐injected along with Nkx2.4b TALENS RNAs. Initial results suggest we have successfully inserted our construct, both as a random transgene insertion as well as in a targeted manner to the Nkx2.4b locus. Confirmation by genetic mapping is underway. In nulls, the hypothalamus appears normal while the thyroid shows complete loss of both developmental and functional markers. Additionally, we seek to develop a homologous recombination vector containing a fluorophore‐2A‐CreERT2 transgene flanked by Nkx2.4b genomic sequence. This will allow equimolar expression of both a fluorescent protein as well as the CreERT2 protein. Using the same strategy we used to insert the GFP transgene into the genome, we will create a Cre driver suitable for temporally controlled manipulation of hypothalamic cells. This tool will enable a wide range of studies with the potential to greatly enhance our knowledge of hypothalamic development and function.

Support or Funding Information

R01 NS082645 ‐ Regulation of hypothalamic radial glia by Wnt signaling
Engineering a Putative Zinc Binding Site in SOD5
Authors: Romero Claudia Moreno , Peterson Ryan L. , Taylor Alexander B. , Holloway Stephen P. , Galaleldeen Ahmad , Culotta Valeria Cizewski , Hart P. John
Published: November 2023

Cu‐Zn Superoxide Dismutase (SOD1) is an enzyme that catalyzes the disproportionation of superoxide into molecular oxygen and hydrogen peroxide at nearly diffusion‐controlled rates. SOD5 represents a new class of Cu‐only superoxide dismutases that catalyzes the same reaction close to the same rate. SOD5 is upregulated during Candida albicans infection. Candida albicans is a diploid fungus that is a causal agent of oral and genital yeast infections in humans. Systemic infections of C. albicans can be lethal in immunocompromised individuals. Competent immune systems can cure the infections easily as the host macrophage engulfs the fungus and releases reactive oxygen species (ROS) that aid in killing it. SOD5 differs from SOD1 in that it is monomeric and possesses a copper binding site but lacks a zinc binding site. In this study, we mutated residues V101 and E110 to generate a putative zinc binding site. The double mutant, similar to wild‐type SOD5, is insoluble. The protein was expressed, denatured, refolded, metallated with copper and zinc, and crystallized. The structure of the double mutant reveals that mutating residues 101 and 110 is insufficient to create a functional zinc‐binding site.
Molecular Cloning and Expression of a Putative Oleate Hydratase Isozyme From Nocardia Cholesterolicum NRRL 5767
Authors: Huang Jenq‐Kuen , Alhmadi Hekmat B , Vanderway David R , Hoerner Cole A , Wen Lisa
Published: November 2023

Nocardia Cholesterolicum NRRL 5767 is well‐known for the ability to transform about 95% of added oleic acid to value‐added product, namely 10‐hydroxystearic acid (10‐HSA). Oleate hydratase is the enzyme catalyzes this conversion. The objective of this research was to clone, overexpress, and characterize oleate hydratase. Nocardia Cholesterolicum NRRL 5767 genome contains two annotated oleate hydratase. Here, we report cloning of an annotated oleate hydratase gene from the N. cholestericum genomic DNA by PCR. The gene was inserted into pET28a. Expression of functional recombinant oleate hydratase was achieved by IPTG induction. The ability to produce large quantities of the protein will permit characterization and evaluation of its suitability for use in immobilized enzyme for the production of 10‐HSA.

Support or Funding Information

Western Illinois University Foundation and the Department of Chemistry.
Optimization and Lipid Quantitation of Potential Algal Biofuels Feedstocks
Authors: Brad Samuel J. , Umbach Brynn E. , Gehlhausen Erin D. , Sweet Charles R.
Published: November 2023

This research investigates the lipid content and composition of extremophilic and estuarine microalgae for suitability as biofuel feedstocks. These are major considerations in optimizing algal biofuel production, as they affect important fuels characteristics including production yield, melting point, flash point, cetane number, and fuel value. In these studies the known feedstock alga Chlorella saccharophila and potential new feedstocks including Galdieria sulphuraria and cold‐tolerant estuarine algae isolated from the Severn River, MD were grown mixotrophically, under replete conditions and a variety of nutrient‐depleted growth conditions. The lipid content of these organisms was determined by spectroscopic quantitation techniques in comparison with dry‐weight percentage, and acyl content was characterized by GS‐MS of fatty acid‐methyl ester (FAME) derivatives. This work compares lipid yields, fatty acid profiles, and quantitation methods from a variety of organisms under various temperature and nutrient conditions, to examine their suitability as feedstocks and determine optimal feedstock production techniques. Observations include increased relative lipid yield in Galdieria under replete conditions compared to Chlorella and increased presence of unsaturated fatty acids in the lipids of cold‐tolerant estuarine algae.

Support or Funding Information

Research Corporation Cottrell College Science Award, DoD ONR/USNA Midshipman Research Fund, and DoD DTRA Service Academy Research Initiative
Substrate Synthesis and Inhibitor Design for Synthetase DesD from Streptomyces coelicolor
Authors: Smolarski Brittany A. , Adetyan Hasmik Jasmine , Kingsbury Jason S. , Hoffmann Katherine M
Published: November 2023

The increasing incidence of drug‐resistant microorganisms poses one of the most severe public health threats to modern society, sparking the need for entirely new classes of antibiotics. Because iron scavenging is vital for pathogen respiration and DNA synthesis, microbial siderophores synthesis represents a large class of previously understudied antiobiotic targets. Desferrioxamine siderophore synthetase DesD catalyzes the ATP‐dependent macrocyclization of ω‐aminocarboxylic acid‐derived cyclodimeric siderophores, and is a model enzyme for NIS synthetases, increasingly associated with bacterial virulence. With aid of x‐ray crystallography and isothermal titration calorimetry, the Hoffmann lab is working to characterize the structure and function of DesD.

Collaboration with the Kingsbury group has facilitated organic synthesis of substrates and substrate analogs towards this characterization of DesD. Optimal syntheses of DesD substrates and substrate analogs was successfully accomplished utilizing condition and catalyst screening, gas chromatograph assay, and standard Schlenk technique and glove box handling of compounds. Purity was confirmed using 1H NMR, 13C NMR, IR, and elemental analysis. We are currently employing these substrates in crystallographic structural work and binding studies of DesD to characterize the binding interactions. The utility of this work will be realized in the design and direct synthesis of inhibitors of DesD, which will then provide a basic framework for generation of a new class of antibiotics focused on siderophore inhibition.
Design of a Genetically Encoded 4Fe‐4S Cluster Sensor
Authors: Glynn Calina , Grossman John D , Perlstein Deborah
Published: November 2023

Iron sulfur cluster biosynthesis is essential in all organisms, but there are currently few cell‐based assays to monitor the cluster assembly and transfer process in vivo. Here we describe our work toward development of a genetically encoded [Fe4S4] cluster sensor that could be used to probe cluster dynamics in vivo. Our sensor design utilizes the cluster driven dimerization of the E. coli transcription factor Fumarate and Nitrate Reductase (FNR) to provide a FRET based readout of cluster binding levels. A fluorescent FNR was developed via genetic fusion of a SNAP‐tag to FNR's C terminus. The SNAP‐tag can be covalently labeled with a variety of fluorophores. We have purified the FNR‐SNAP fusion, verified it can be labeled with fluorophores, and determined that the fusion is monomeric in the absence of [4Fe4S]2+ cluster. With a cluster stabilizing mutant we have demonstrated that the fusion is capable of binding cluster. However, after fluorescent labeling of a mutant known to induce dimerization in the absence of cluster with fluorophores capable of a FRET interaction we were unable to detect a measurable FRET signal. Further analysis of the dimerization mutant implied that the SNAP tag in its current position interferes with the efficiency of dimerization. Current work is focused on minimizing the distance between labels via deletion of FNR's DNA binding domain.

Support or Funding Information

Boston University UROP
Lectin‐based methods for in vivo characterization of C‐mannosylated proteins
Authors: Kegley Nicholas Ryan , Naylor Shelley , Rakus John
Published: November 2023

Glycosylation functions in a variety of ways including ligand recognition and to ensure only proteins which are properly folded are transferred to the Golgi apparatus. Protein‐glycan structures resulting from glycosylation can be differentiated based on the nature of their sugar‐peptide bonds including O‐linked, N‐linked, and the less commonly detected C‐linked. C‐mannosylation is a form of glycolysis in which mannose is attached via a carbon‐carbon bond with the first tryptophan in trp‐x‐x‐trp sequences. Many proteins that are instrumental in the process of inflammation have been found to possess C‐mannosyltryptophan. Lectins are non‐immunological carbohydrate‐binding proteins. Galanthus nivalis lectin (GNL) has been shown to recognize C‐mannosyltryptophan in addition to α1,3‐ and α1,6‐mannose. Our objective is to use GNL pulldowns to isolate and characterize C‐mannosylated proteins from cells grown in culture. Prior PNGaseF treatment removes competing oligomannose structures allowing for C‐mannosyltryptophan‐specific elution of glycoproteins. We have applied this technique to probe C‐mannosylated proteins from B35 glioblastoma cells as well as lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages. GNL‐eluted proteins are lectin blotted with GNL (C‐linked glycosylation positive probe) and ConA (C‐linked glycosylation negative probe). Organization of the neocortex relies heavily on the regulation of neuronal migration. The migration of neurons is dependent on proteins with the C‐mannosylation modification for guidance cues. Proper neurite migration prevents neurological defects in humans. The understanding of the modification in neurite migration is significant because cell migration is an important step in the development of the nervous system. Our objective was to isolate and sequence via mass spectrometry C‐mannosylated proteins that may be involved in this guidance system. An interaction between C‐mannosylated proteins and heat shock protein 70 (Hsp70) has been found to increase the production of TNF‐α in macrophages, which can result in a strong inflammatory response, potentially leading to septicemia if unregulated. The goal of this project is to characterize the interaction between Hsc70 and this heretofore unidentified C‐mannosylated protein. Both LPS and non‐LPS treated cells are grown in parallel and collected at 0, 2, 24, and 48 hour time periods. GNL‐pulldowns were then used to isolate C‐mannosylated proteins and anti‐Hsc70 immunoprecipitation was utilized to isolate all Hsc70‐binding proteins.

Support or Funding Information

Marshall University College of Science,West Virginia Higher Education Policy Commission,West Virginia Space Grant Consortium
De Novo Synthesis from Tryptophan in the Absence of a QPRTase Homolog Contributes to NAD+ Biosynthesis in C. elegans
Authors: McReynolds Melanie R. , Wang Wenqing , Holleran Lauren , Hanna‐Rose Wendy
Published: November 2023

Nicotinamide adenine dinucleotide (NAD+) biosynthesis has proven to be an attractive and promising therapeutic target for influencing health‐span and obesity‐related phenotypes as well as tumor growth. It's a necessity to elucidate exactly how manipulating NAD+ biosynthetic pathways can lead to therapeutic benefits to fully utilize this target for drug discovery. The goal of my research is to understand how NAD+ homeostasis is maintained to support its core metabolic roles and its signaling and regulatory roles involving NAD+ consumers. It's been reported in the literature that C. elegans lack the de novo NAD+ biosynthetic pathway because quinolinic acid phosphoribosyltransferase (QPRTase) is not present in the genome. However, all genes coding for the key enzymes required for production of quinolinic acid (QA) from tryptophan are present in the C. elegans genome. Therefore, we hypothesized that de novo synthesis from tryptophan in C. elegans contributes to NAD+ biosynthesis. In order to investigate if de novo synthesis is indeed active in the absence of the QPRTase homolog, we first asked if QA gets incorporated into NAD+ in C. elegans. Using stable isotope/mass spectrometric flux analysis, I observed that label incorporated into QA was subsequently incorporated into NAD+ as well; supporting the notion de novo NAD+ synthesis is active. I further hypothesized that if QA were an endogenous precursor for NAD biosynthesis, then blocking this pathway would compromise NAD+ levels in vivo. Target metabolomics revealed a decrease in NAD+ and QA levels when this pathway was blocked. This data supports the hypothesis that this pathway is active and required for maintenance of global NAD+ levels. Next, I investigated if supplementation with QA could reverse a phenotype associated with NAD+ deficiency. Consistent with the previous results, supplementation with QA rescued the developmental phenotype and increased NAD+ levels in mutants that lack salvage NAD+ biosynthesis. This evidence suggests that boosting this pathway can lead to homeostatic mechanisms when salvage synthesis is blocked, further supporting its core role in maintaining NAD+ homeostasis. Finally, we've identified key candidate enzymes that may play the role of the missing QPRTase in C. elegans. Using genetics, I am currently investigating the role of these potential enzymes in NAD+ de novo synthesis. This data will outline the mechanisms by which tryptophan de novo synthesis is functioning in C. elegans. This work has implications for efforts to therapeutically target individual NAD+ biosynthetic pathways providing key insight into compensatory homeostatic mechanisms.

Support or Funding Information

This work was supported, in whole or in part, by National Institutes of Health Grant GM086786 (to W. H.‐R.).
GPR109a Signaling Alters the Hepatic Response to Fasting
Authors: Foy Caroline E , Hepler Chelsea , Renquist Benjamin J
Published: November 2023

Elevated hepatic ketogenesis and gluconeogenesis are integral to the metabolic adaptation to fasting. The increase in ketone body production allows for feedback inhibition of adipose tissue lipolysis through Gαi coupled GPR109a, the β‐OH butyrate receptor. To better understand the role of this feedback we investigated the hepatic metabolic response to fasting in sibling GPR109a +/+ and −/− mice that were injected every 2 hours for 8 hours with either saline (0.1 ml/10 g body weight) or niacin (100 mg/kg in 0.1 ml saline/10 g body weight), a non‐metabolizable GPR109a agonist. In wild type mice, niacin completely eliminated the rise in serum NEFAs during a fast and significantly (p<.05) inhibited PPARα mRNA upregulation. This muted response in PPARα is important, because hepatic PPARα is integral to the upregulation of ketogenic and gluconeogenic enzymes that support the metabolic demands of fasting. Accordingly, fasting serum β‐OH‐butyrate concentrations were suppressed by niacin (p<.01), indicating sub‐maximal ketogenesis. Interestingly, GPR109a null mice do not elevate serum NEFAs and less robustly increase hepatic PPARα mRNA during a fast. Yet, fasting increases G6Pase and PEPCK mRNA expression more robustly in GPR109a null than in wildtype mice. Given that G6Pase and PEPCK expression are regulated by cAMP response elements, further studies should examine the role of hepatic GPR109a Gαi signaling in antagonizing glucagon Gαs mediated transcription of these enzymes. These studies establish that adipose lipolysis is necessary to recruit a full‐strength hepatic adaptation to fasting, and that gluconeogenesis and ketogenesis are controlled by a complex coordinated fasting response including feedback through GPR109a.
Sequestration‐mediated Downregulation of De Novo Purine Biosynthesis by AMPK
Authors: Schmitt Danielle L , An Songon
Published: November 2023

Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We speculated that AMP‐activated protein kinase (AMPK), which downregulates ATP‐consuming biosynthetic pathways of lipids and carbohydrates, would be a regulator for de novo purine biosynthesis. To understand the functional role of AMPK in the regulation of de novo purine biosynthesis, we have investigated the impact of small molecule‐mediated AMPK activation on de novo purine biosynthesis under fluorescence single‐cell microscopy. While monitoring the subcellular localizations of green fluorescent protein‐tagged pathway enzymes in HeLa cells, we identified that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), was found to associate into cytoplasmic granules, thus self‐assemblies, after treatment with small‐molecule AMPK activators. In addition, subsequent biochemical studies revealed that the cellular levels of purine metabolites were decreased, whereas the ATP levels were conserved upon stimulation of AMPK‐promoted FGAMS self‐assembly. Collectively, we demonstrate that downregulation of de novo purine biosynthesis is controlled by AMPK‐associated signaling pathways through the spatial sequestration of FGAMS into the self‐assembly.

Support or Funding Information

University of Maryland, Baltimore County and NIH/NIGMS T32GMO66706 (K. Radtke)
Correlation between Tau Accumulation in Brain and Retina of Individuals with Late Stage Alzheimer's Disease
Authors: Bondarenko Vladimir , Skyba David
Published: November 2023

This study was aimed to determine if there is a correlation between tau accumulation in the brain and retina of individuals in the late stages of Alzheimer's disease. Characterizing the distribution and accumulation of tau proteins in the retina and brain may be important for early diagnosis of neurodegenerative diseases such as Alzheimer's disease.

We examined total tau protein accumulation and its aggregates in brain and retinal tissues from aged individuals with Alzheimer's disease.

Tissues from the prefrontal cortex, visual cortex and retinas of ten different cadavers were analyzed. We utilized cryosectioning to prepare tissue sections for immunostaining. Tissues obtained from individuals without a history of Alzheimer's disease or other types of dementia were used as negative controls. Detection of tau was performed using a primary antibody against human tau protein, followed by a universal secondary antibody.

Although tau accumulation was greater than controls for both brain tissues, the density was higher in the prefrontal cortex. We also found an increase in tau content in the retinas from individuals with Alzheimer's disease compared to that of individuals without neurodegenerative disorders.

These data demonstrate that neurodegenerative changes leading to accumulation of tau aggregates in the brain coincide with increased tau content in the retina. This raises the intriguing possibility that there may be a similar correlation in individuals during the early stages of Alzheimer's disease. Thus, future studies will concentrate on the accumulation of tau in the brain and retina of individuals during the earlier stages of Alzheimer's disease.

Support or Funding Information

Touro University Nevada internal research grant.
Hyperglycemia is exacerbated by dysregulation of hepatic SREBP1c‐CRY1 signaling pathway
Authors: KIM JAE BUM , CHOI WON GUN , JANG HAGOON , LEE GHA YOUNG , LEE GUNG , JEON YONG GEUN
Published: November 2023

SREBP1c is a key transcription factor of lipogenesis to store excess energy in postprandial state. Although SREBP1c appears to be involved in suppression of hepatic gluconeogenesis, the molecular mechanism(s) by which insulin‐activated SREBP1c could repress hepatic gluconeogenesis is not thoroughly understood. Here, we demonstrate that CRY1 activation by insulin‐induced SREBP1c led to decrease hepatic gluconeogenesis through FOXO1 degradation. In accordance with these, SREBP1c−/−and CRY1−/− mice showed higher blood glucose than wild type (WT) mice during pyruvate tolerance test, accompanied with enhanced expression of PEPCK and G6Pase genes. Moreover, CRY1 promoted degradation of nuclear FOXO1 by MDM2, one of the ubiquitin E3 ligases, by enhancing binding of FOXO1 and MDM2. Although hepatic SREBP1c failed to upregulate CRY1 expression in db/db mice, overexpression of CRY1 led to attenuate hyperglycemia through reduction of hepatic FOXO1 protein as well as gluconeogenic gene expression. Collectively, these data suggest that insulin‐activated SREBP1c downregulates hepatic gluconeogenesis through CRY1‐mediated FOXO1 degradation and thereby dysregulation of SREBP1c‐CRY1 signaling cascade in liver could confer hyperglycemia in diabetic animals.

Support or Funding Information

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT and Future Planning; 2011‐0018312)
Functional and Behavioral Consequences of Axial Stability in Primates and Other Mammals
Authors: Granatosky Michael Constantine
Published: November 2023

A stable thoracolumbar region found in some arboreal mammals and certain primates has been proposed as advantageous for maintaining stable back postures during antipronograde locomotion, which is any form of movement in which the forelimbs and/or hindlimbs are loaded “in tension.” However, little data exist testing the inferred link between osteological features cited as enhancing whole‐body stability and the frequency of antipronograde locomotion within an animal's locomotor repertoire. To fill this gap this study uses multivariate methods to compare vertebral morphology of primate and non‐primate mammals with varying levels of antipronograde locomotion within their locomotor repertoires. Overall patterns suggest that species that commonly use antipronograde locomotion exhibit thoracolumbar stability related to osteological features that reduce potential intervertebral spaces. As an additional test of the inferred link between axial rigidity and behavioral stability, this study contrasted the frequency of cantilevering and bridging of Caluromys philander and Loris tardigradus (species that commonly use antipronograde locomotion), to Monodelphis domestica and Cheirogaleus medius (species that rarely use antipronograde locomotion) on a raised horizontal pole and terminal branch experimental set‐up. We observed C. philander cantilevering and bridging significantly more often (55% of observed events) than M. domestica, which never cantilevered or crossed any arboreal gaps (P <0.001). No difference in cantilevering frequency was observed between L. tardigradus and C. medius, but the duration of cantilevering bouts were significantly greater in L. tardigradus (4.24 sec ± 2.11 vs. 2.35 sec ± 1.71; P < 0.01). These data suggest that bridging, cantilevering, and other antipronograde behaviors likely benefit from a stable trunk. However, disparate clustering of antipronograde species based on multivariate statistics suggests that no single adaptive suite characterizes the axial skeleton of these mammals. From this, we can infer that different functional solutions may have evolved to meet the similar mechanical challenges imposed by antipronograde positional behaviors.

Support or Funding Information

This research was support by the National Science Foundation's Graduate Research Fellowship Program and the Force and Motion Foundation.
Proteome‐wide structural analysis of PTM hotspots reveals large body of putative regulatory elements with predicted biological function
Authors: Dewhurst Henry , Torres Matthew
Published: November 2023

Post‐translational modifications (PTMs) have repeatedly been shown to play vital roles in regulation of protein behavior through modulation of protein‐protein binding and recognition site interactions, allosteric regulation, and conformational switching. SAPH‐ire (Structural Analysis of PTM Hotspots) has been used effectively to reveal novel PTM regulatory elements in G protein families (Dewhurst et al., 2015). Here, we proceed by applying SAPH‐ire to the comprehensive set of eukaryotic protein families containing experimental PTM and 3D structure data. This experiment captures 1,864 protein families and includes more than 60,756 unique PTM sites organized into 40,493 PTM hotspots, of which 2,659 possess known biological function (data obtained from PhosphoSitePlus), analyzed across 51,000 crystalized molecular structures representing 1,080 different species. This PTM data set represents the largest quantitative structural analysis of PTMs to date, yet less than 1/3 of the publically available eukaryotic PTM sites available at the time of investigation. PTM hotspots with a known biological function undergo up to 6‐fold enrichment using SAPH‐ire ranking methods – consistent with pilot studies (Dewhurst et al., 2015). Moreover, a preliminary survey revealed greater than 90% of top‐ranked hotspots lacking functional evidence in our input data could be linked to biological function by in‐depth literature searches. Thus, ~10% of PTM hotspots predicted to be biologically impactful have yet to be verified experimentally and represent putative regulatory elements. Taken together, these results highlight SAPH‐ire's value as a predictive tool for the identification of biologically important PTMs.

Support or Funding Information

This work was funded by the Georgia Tech School of Biology (M.T.) as well as the National Institutes of Health grant 4R00 GM094533‐03 (to M.T.).
Determinants of Gα12‐specific binding common to AKAP‐Lbc and p114RhoGEF
Authors: Garcia Ricardo Daniel , Towne Autumn Lynn , Cavagnini Kyle S. , Meigs Thomas E.
Published: November 2023

AKAP‐Lbc and p114RhoGEF are guanine nucleotide exchange factors (GEFs) that activate the small G protein Rho. These proteins lack a RGS‐homology (RH) domain that is found in a specific subgroup of RhoGEFs that serve as downstream effectors of heterotrimeric G proteins of the G12/13 family. AKAP‐Lbc plays a crucial role in cardiomyocyte fibrosis and cardiac development, and despite lacking an RH domain, is a binding partner of Gα12. In previous work, we identified a Gα12‐binding region near the C‐terminus of AKAP‐Lbc, and found this region to share close homology with a p114RhoGEF region that we also demonstrated to interact with Gα12. To identify determinants of Gα12 binding within these regions of AKAP‐Lbc and p114RhoGEF, we engineered charge‐substitution mutants at residues common to both RhoGEFs, and located several key amino acids that participate in Gα12 interaction. We also identified a mutant of Gα12 that shows impaired binding to both AKAP‐Lbc and p114RhoGEF, suggesting a common structural motif in the Gα protein for engaging these targets. In addition, we found AKAP‐Lbc and p114RhoGEF to preferentially bind Gα12 in comparison to Gα13, a finding that distinguishes these RhoGEFs from others reported as Gα13‐specific targets. Also, in cell culture assays measuring serum response factor (SRF) mediated transcriptional activation by mutationally activated G12/13, epitope‐tagged G protein binding domains of AKAP‐Lbc and p114RhoGEF selectively blocked Gα12 signaling to SRF. Furthermore, a dominant‐negative, constitutively GDP‐bound variant of Gα12 was more potent than dominant negative Gα13 in hindering serum‐induced p114RhoGEF stimulation of SRF signaling. These results define a Gα12‐selective binding region in AKAP‐Lbc and p114RhoGEF, and implicate p114RhoGEF as an effector of Gα12 mediated cell growth signaling.

Support or Funding Information

We acknowledge support from the GlaxoSmithKline and C.D. Spangler Foundations, UNC Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.
GPR161 and smAKAP Are Membrane‐bound PKA‐RI‐selective AKAP
Authors: Lu Tsan‐Wen , Ilouz Ronit , Stefan Eduard , Taylor Susab
Published: November 2023

A Kinase Anchoring Proteins (AKAPs) serve as scaffold proteins that target the PKA isoforms to distinct parts of the cell. The hallmark motif that defines an AKAP is an amphipathic helix that docks with high affinity (2–7 nM) onto the N‐terminal helical dimerization/docking domain of the PKA regulatory subunits. While most AKAPs are specific for RII subunits and target PKA, along with other signaling proteins, to channels, transporters and receptors, a few AKAPs are dual specific and can bind to both RI and RII subunits.

Some like the small membrane anchored AKAP (smAKAP) bind directly to membranes and are highly specific for RI subunits (Kd = 5 nM for RIs vs. 50 nM for RIIb). We recently discovered AKAP motif that is embedded in the C‐terminal tail of GPR161, an orphan G Protein‐Coupled receptor that is known to be associated with cilia. It is the first discovered G protein that has AKAP motif. This AKAP shows absolute high affinity selectivity for RI (Kd = 6 nM).

Structures of the smAKAP bound to the dimerization domain of RIa define the features that convey high affinity binding and specificity to the RI subunits and these features are conserved in the GPR161 AKAP based on peptide arrays. A unique feature of these RI‐specific and/or selective AKAPs is an extended amphipathic helix that caps the two disulfide bonds in the RI subunits. These disulfide bonds are specific feature of the RI subunits that allow them to function as redox sensors.

Support or Funding Information

NIH DKS4441 to SST
MicroRNA‐124 Negatively Regulates Nuclear Receptor Nur77
Authors: Farmer Alexa , Beard Jordan A , Takwi Apana , Wang Yue‐ming , Chen Taosheng
Published: November 2023

The nuclear receptor Nur77 is commonly upregulated in adult cancers and has oncogenic functions. Nur77 is an immediate‐early response gene that acts as a transcription factor to promote proliferation and protect the cell from apoptosis. Conversely, Nur77 can translocate to the mitochondria and induce apoptosis upon treatment with various cytotoxic agents. Because Nur77 is upregulated in cancer and may have a role in cancer progression, it is of interest to understand the mechanism controlling its expression. MicroRNAs (miRNAs) are responsible for inhibiting translation of their target genes by binding to the 3′UTR and either degrading the mRNA or preventing it from being translated into protein, thereby making these non‐coding endogenous RNAs vital regulators of every cellular process. Several miRNAs have been predicted to target Nur77; however, strong evidence showing the regulation of Nur77 by any miRNA is lacking. In this study we used a luciferase reporter assay containing the 3′UTR of Nur77 to screen 296 miRNAs and found that miR‐124, the most abundant miRNA in the brain with a role in promoting neuronal differentiation, caused the greatest reduction in luciferase activity. Interestingly, we discovered an inverse relationship in Daoy medulloblastoma cells and undifferentiated granule neuron precursors (GNPs) where Nur77 is upregulated and miR‐124 is downregulated. Further elevating Nur77 levels in Daoy cells by exogenous expression increased proliferation and viability, but knocking down Nur77 via siRNA resulted in the opposite phenotype. Importantly, exogenous expression of miR‐124 reduced Nur77 expression, cell viability, proliferation, and tumor spheroid size in 3D culture. In all, we have discovered miR‐124 to be downregulated in instances of medulloblastoma in which Nur77 is upregulated, resulting in a proliferative state that abets cancer progression. This study provides evidence for increasing miR‐124 expression as a potential therapy for cancers with elevated levels of Nur77.

Support or Funding Information

This work was supported by the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children's Research Hospital, and the National Institutes of Health [Grants RO1GM086415, RO1GM110034, & P30‐CA21765].
Local Aldosterone Synthesis in Skeletal Muscle
Authors: Chadwick Jessica Ann , Swagger Sarah , Lowe Jeovanna , Rafael‐Fortney Jill
Published: November 2023

OBJECTIVE

To characterize mineralocorticoid receptor (MR) regulation in normal vs. dystrophic skeletal muscle and elucidate the mechanism behind the efficacy of MR antagonists in dystrophic muscle.

BACKGROUND

Our lab has recently shown MR is present in skeletal muscle fibers and represents a novel therapeutic target for treating both the skeletal and cardiac muscle pathologies of Duchenne muscular dystrophy (DMD). However, the underlying mechanisms and functional role of MR activation in skeletal muscle is still unclear. Excessive MR activation is known to contribute to the pathogenesis of heart failure, presumably due to increased levels of its natural ligand aldosterone. However, clinical trials have shown that addition of an MR antagonist to standard‐of‐care regimens (ACE inhibitors like lisinopril) improves morbidity and mortality in patients, even when circulating aldosterone levels are within the physiological range. There is now a growing body of evidence that shows aldosterone, which was previously thought to only be synthesized in the adrenal cortex can also be synthesized and metabolized locally in extra‐adrenal tissues, including the heart, particularly in pathological states.

DESIGN/METHODS

Aldosterone synthase (CYP11B2) and other key enzymes in the aldosterone synthesis pathway were tested at the protein level in mouse skeletal muscles and myogenic cultures from normal and dystrophic models, and in normal human myogenic cultures using western blot analysis and immunofluorescence. Sequencing analysis of reverse transcribed PCR (RT‐PCR) of RNA from mouse skeletal muscle and human myotubes was used to confirm expression.

RESULTS

CYP11B2 is present in mouse skeletal muscle homogenates and up‐regulated in muscles from dystrophic mice (Figure 1). CYP11B2 does not appear to be coming from the skeletal muscle itself; immunofluorescence of dystrophic mouse muscles suggests this enzyme may be present in a subset of immune cells, which are present at much higher levels in dystrophic muscles (Figure 1). We have also found type 2 11‐beta hydroxysteroid dehydrogenase (11β‐HSD2), a key regulator of MR specificity, is present in normal human myotubes and is up‐regulated in dystrophic muscle (Figure 2).

CONCLUSIONS

CYP11B2 and 11β‐HSD2 levels are significantly increased in dystrophic muscle, suggesting MR activation is in excess of the physiological need. 11β‐HSD2 is expressed within the muscle fibers but CYP11B2 appears to be coming from a population of immune cells up‐regulated in dystrophic tissue due to increased inflammation and muscle degeneration.

Support or Funding Information

National Institutes of Health [R01 NS082868]
Regulation of Store‐Operated Calcium Channels by Sphingosine 1 Phosphate
Authors: El‐Shewy Hesham M. , Lee Mi Hye , Peterson Yuri K. , Luttrell Louis M.
Published: November 2023

Several studies have implicated sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) axis in mobilization of intracellular Ca2+ and defective vascular tone. We and other independent investigators demonstrated recently that SK1/S1P mediates transmembrane Ca2+ flux through store‐operated Ca2+ channel (SOC). SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. STIM function as Ca2+ sensors within the ER, and Orai proteins at PM. Depletion of Ca2+ from ER leads to oligomerization and translocation of STIM to bind Orai at the PM to activate Ca2+ flux. We found that S1P regulates SOC through yet undefined intracellular mechanism that does not involve activation of G protein‐coupled S1P receptors. Here, we have studied the mechanism by which SK/S1P mediates activation of SOC. Molecular modeling predicted the binding of S1P with STIM1 of SOC in monomer and dimer forms. Unlike biotin ceramide and biotin controls, only biotinylated S1P bound to endogenous STIM1 in the membrane fraction of cell lysates that was inhibited with competitive cold S1P suggesting specificity of S1P binding to STIM1. Intriguingly, direct mutation of the predicted S1P binding sites to STIM1 markedly inhibited angiotensinII (AngII) and intracellular S1P‐dependent Ca2+ flux without affecting Ca2+ release from intracellular stores. To examine the effect of SK1/S1P on STIM1/Orai1 assembly by BRET assay, we generated STIM1‐FlAsH and Orai1‐Rluc plasmids. We found that NET BRET values of AngII‐dependent stimulated HEK293 cells transiently transfected with STIM1‐FlAsH and Orai1‐Rluc were significantly inhibited by SK1 inhibitor. Functionally, transfecting STIM1 mutants in HEK293 cells inhibited cell proliferation compared to vehicle control. These findings suggest the requirement of intracellular S1P for activation of SOC via modulation of STIM1 and provide novel mechanistic and functional insights into pathophysiology of vascular dysfunction.
Dihydroxyacetone Phosphate Acyltransferase is a Peripheral Membrane Protein
Authors: Chilije Maxwell , Hoffarth Elesha , Bertolesi Gabriel , McFarlane Sarah , Zaremberg Vanina
Published: November 2023

Dihydroxyacetone phosphate acyltransferase(DHAPAT) catalyzes the initial acylation step in the synthesis of ether basedphospholipids in peroxisomes. DHAPAT adds an acyl group to dihydroxyacetonephosphate, a metabolic intermediate from glycolysis, to yield 1‐acyl‐dihydroxyacetonephosphate (1‐acyl‐DHAP). Ether lipids are abundant in the central nervoussystem and DHAPAT deficiency has been associated with many human disordersincluding cataracts and blindness. We will present results from studies using the tractable model organism Xenopuslaevis that also point to a role of DHAPAT in eye development. Analysis of DHAPAT expression by in situ hybridization indicated it is expressed in the lens epithelium and in the proliferative region of the ciliary marginal zone. Given its relevance and how little is known about DHAPAT, we have initiated biochemical studies aimed at fully characterizing this enzyme in terms of structure, function and regulation. Using a yeast heterologous expression system we have been able to overproduce functional X. laevis DHAPAT. A first purification scheme comprised of subcellular fractionation, protein solubilization with different detergents and ionic strength conditions followed by Ni‐NTA affinity chromatography has been developed. Our studies indicate that X laevis DHAPAT is a peripheral membrane protein that can associate‐dissociate from membranes. We are currently exploiting this novel feature for this kind of acyltransferases in order to setup the conditions to improve its purification with the goal of initiating structural studies.

Support or Funding Information

This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada to SM and VZ.
Structure‐function analysis of MntP and other manganese exporter proteins and their contribution to reactive oxygen species survival
Authors: Martinez Eli , Zeinert Rilee , Schmitz Jennifer , Draven Joseph , Waters Lauren
Published: November 2023

Manganese is an important trace nutrient involved in the detoxification of reactive oxygen species (ROS). Manganese also acts as an enzyme cofactor for superoxide dismutase and other enzymes. In bacteria, manganese and Mn‐SOD allow cells to withstand ROS created from internal metabolic processes and external conditions. Pathogenic bacteria require manganese to remain virulent in environments with reactive oxygen species, such as the host organism. Yet, manganese is toxic at high concentrations, so cells tightly regulate Mn levels. In E. coli, manganese homeostasis is maintained by the Mn importer, MntH, and the Mn exporter, MntP. MntP is a transmembrane exporter protein that we predict to traverse the membrane six times with the N‐ and C‐ termini located in the periplasm. Reporter protein fusions were used to study the orientation of MntP within the membrane. The reporter proteins used were alkaline phosphatase (PhoA) and green fluorescent protein (GFP). We created N‐ and C‐ terminal fusions of PhoA and GFP to MntP using Polymerase Incomplete Primer Extension cloning. Using an alkaline phosphatase assay, activity was seen with the C‐terminal fusion, which was confirmed to be oriented in the periplasm. The ability of the fusion proteins to export manganese was reduced, most notably with the N‐ terminal fusion, which was less than 50% active. Additional work on protein fusions is ongoing.

Currently, it is known that the ΔmntP leads to Mn sensitivity in cells, coupled with the accumulation of excess Mn. We investigated the relationship between MntP and reactive oxygen species to further characterize MntP. We have now showed that cells overproducing MntP, in comparison to cells lacking the protein, displayed sensitivity to hydrogen peroxide. This phenotype of MntP links the protein to reactive oxygen species resistance for the first time. Additionally, we have created mutations to MntP to further characterize the function of the protein. The new physiological relationship between MntP and reactive oxygen species is being used to study mutations to MntP. In addition to MntP, we have recently isolated new potential manganese exporter proteins. Mutations to potential manganese exporters, created by site‐directed mutagenesis, are being generated. These studies provides insight into the significance of manganese within bacterial cells. By better understanding MntP and other exporter proteins associated to manganese homeostasis, the virulence of pathogenic bacteria could be reduced.

Support or Funding Information

Research Corporation‐ #22658

TRIO grant from the United States Department of Education PR/Award Number P217A120210
Qualitative Methods: An Overview for Anatomy Education Research
Authors: Wasserman Jason Adam
Published: November 2023

This talk will provide a foundation in qualitative methodology that will 1) Inform the audience about the general uses and processes of qualitative research and 2) Highlight the general utility of qualitative study for research in anatomy and anatomy education. It begins by elucidating the limitations of deductive, quantitative approaches—those that first postulate a hypothesis that is then tested with data. These limitations include problems of operationalizing complex and multifactorial concepts, as well as the problems inherent to the prefabrication of hypotheses. The discussion of these limitations helps to outline the utility of inductive, qualitative approaches, which are then described. In particular, this talk focuses on utilizing methodological techniques that originate with data, rather than hypotheses, and then utilize rigorous narrative analytic schemes to produce sound qualitative findings. The talk concludes by elaborating various kinds of questions, germane to anatomy and anatomy education research, which qualitative inquiries are uniquely positioned to address. These include investigating complex patterns of social and emotional responses to cadaveric dissection and complex and iterative patterns of learning. In short, anatomy education must not only deal with promoting the learning of anatomy proper, but also with deeply emotional reactions to dissection, the social‐psychological development of students over time, issues of professional identity formation. Qualitative approaches hold advantages for studying the complexities and ambiguities inherent in these kinds of phenomena and therefore represent an important and underutilized methodology in anatomy education.

Support or Funding Information

None.
Effects of Helichrysum plicatum extract against Thallium(I) sulphate–induced reproductive damage in male rats
Authors: Omur Ali Dogan , Yildirim Betul Apaydın , Kucukler Sefa
Published: November 2023

In this study, 24 male Sprague Dawley rats aged eight weeks old and weighted 250–300 gr, were used in this experiment. The animals were obtained from Atatürk University Experimental Research Centre and housed in standard laboratory conditions. Commercial pellet chow and fresh drinking water were available ad libitum. Rats were divided into four groups. I. group (n= 6) referred as control group and physiological saline intraperitonally (IP) to the animals. II. group (n=6) referred as 10‐mg/kg Thallium Sulphate (TS) was injected IP to animals. III. Group (n=6) was referred as TS + HPE, 10‐mg/kg TS injected IP and 100 mg/kg HPE peros to animals. IV. group (n=6) was referred as HPE, 100 mg/kg HPE peros to animals. The treatment was performed for consecutive 8 days. The animals were tranquilized and sacrificed on 9ht day of study. Cauda epididymal semen samples and testes tissues were collected. Routine semen examinations were performed and oxidative stress levels of testicular tissues were assayed. Motility in TS group was significantly lower when compared to control and experimental groups. Head and tail abnormality in TS and HPE+TS group were statistically higher when compared to control and HPE groups. MDA level of TS group was significantly higher than in control and treatment groups. Glutathione peroxidase (GPx) and glutathione (GSH) levels in TS group were lower than the other groups. The lowest SOD level was in TS group while the highest level was in control group. MDA level was higher and CAT level was lower in TS group when compared to other groups.

Helichrysum plicatum extract is effective on mentioned spermatological and plasma /testis tissue biochemical parameters against Thallium(I) sulphate in terms of testicular toxicity. There are statistically significant differences between mentioned groups as can be seen in the above.

Support or Funding Information

Ataturk University Scientific Research Projects Coordination (Project No: 2013‐251)