bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient modification of macrophages for activation their pro‐inflammatory functions
Authors: Poltavets Anastasiia , Vishnyakova Polina , Zatsepin Timofei , Fomina Mariia , Elchaninov Andrey , Fatkhudinov Timur
Published: May 2022

Macrophages are innate immune cells that play an important role in both physiological and pathological conditions. According to the concept of binary polarization, there are two the most polar states of macrophages: pro‐inflammatory and anti‐inflammatory macrophages. We proposed that targeted programming of macrophages derived from the specific monocyte subpopulation is perspective strategy for activation their inflammation properties that could be used in therapy of cancer or endometriosis. This work is devoted to the study of the effect of Indoleamine 2,3‐dioxygenase 1 (IDO1) and its inhibitor BIN1 on the shift in the polarization of macrophages into a pro‐inflammatory phenotype. We used magnetic sorting, differentiation, and further pro‐inflammatory polarization of CD14+ monocytes from the blood of healthy donors. We obtained data on the transcriptome and proteome and carried out a bioinformatic analysis to identify the most significant differences between two groups of cells – naive and pro‐inflammatory macrophages. IDO1 has been identified as an enzyme with immunomodulatory functions and is also involved in the catabolism of the essential amino acid L‐tryptophan. This enzyme is expressed in macrophages and its expression is induced by pro‐inflammatory cytokines. It was shown that the BIN1 protein is an inhibitor of IDO1 and can inhibit the growth of cancer cells. It was shown that upon the polarization of macrophages towards the pro‐inflammatory phenotype by LPS addition, the expression of IDO1 increases, while the expression of BIN1 decreases (p <0.05). To reveal the influence of the IDO1 and BIN1 genes on the shift of the polarization of macrophages towards the M1 phenotype, we decided to carry out knockdown by using siRNAs on the primary line of CD14+‐derived macrophages. The efficiency of transfection was assessed at different concentrations of siRNA ranged from 30 to 80%, but high concentrations of siRNA led to non‐specific activation of pro‐inflammatory phenotype. Expression analysis confirmed the silencing of the genes IDO1 and BIN1 using 5 nM siRNA. It was shown that knockdown of IDO1 and BIN1 leads to increased expression of pro‐inflammatory genes (CCR7 and TNFa) and decreased the level of anti‐inflammatory interleukin 10. Also, co‐cultivation CD14+ macrophages after knockdown of genes IDO1 and BIN1 didn’t influent on macrophage’s phagocytic functions. The proposed study has a scientific novelty since for the first time a transient modification of the primary line of macrophages from blood CD14 + was carried out and the influence of IDO1 and BIN1 on the activation of the pro‐ or anti‐inflammatory phenotype was shown. Directed programming of macrophages is a new and rapidly developing field.
NOS isoforms facilitate Ca2+‐cycling‐dependent and ‐independent cardiac contractility
Authors: Wang Ying , Zhao MeiMi , Rivera Gabriela , Xu Bing , Mir Vaseem M. , Reddy Raghu , Xiang Yang K.
Published: May 2022

Increasing biased G‐protein coupled receptor (GPCR) drugs are now under clinical investigation. More understanding of the biased β‐adrenergic receptor (β‐AR) signaling is urgently required for a better cardiovascular drug. Here we found that two nitric oxide synthase (NOS) isotypes, NOS1, and NOS3 coupled with β1ARs and transduce distinct β1AR‐cGMP signaling at sarcoplasmic reticulum and myofilament microdomain, respectively. Intriguingly, activation of NOS1 enhanced contractility and Ca2+ cycling, whereas NOS3 promoted contraction without affecting intracellular Ca2+. Quantitative proteomic revealed that NOS3 activation increased phosphorylation of myosin proteins including myosin binding protein C (MyBP‐C), myosin light chain kinase (MYLK) and myosin phosphatase target subunit 1 (MYPT1), to enhance contractility by Ca2+sensitization. Moreover, in heart failure, excessive adrenergic stimulation dissociated β1AR from NOS1 and impaired the NOS1‐cGMP signaling. Accordingly, stimulating NOS3 but NOS1 enhances systolic cardiac contraction in failing mice without rousing intracellular Ca2+. We propose NOS facilitate biased β1AR‐cGMP signaling via Ca2+‐dependent or independent mechanisms and define NOS3‐cGMP as specific therapeutic strategy targeting myofilament for heart failure.
Structure and function of DNA transposition assemblies involved in antibiotic resistance spreading
Authors: Barabas Orsolya , Smyshlyaev Georgy , Isbilir Buse , Khedkar Supriya , Rojas‐Cordova Carlos , Bateman Alex G. , Bork Peer
Published: May 2022

Transposons are mobile genetic elements that drive evolution and adaptation throughout the tree of life. In bacteria, they often transfer antibiotic resistance genes and contribute to the emergence of multidrug‐resistant pathogens.

In this talk, I will present our recent discoveries on a group of transposons that efficiently propagate antibiotic resistance genes across diverse microbial communities. Using a dedicated computational pipeline, we mapped the most wide‐spread transposons in bacterial genomes and characterized their diversity, genetic cargo, and transfer dynamics. Selected elements were experimentally reconstituted to describe their molecular mechanisms and biochemical pathways. We determined numerous high‐resolution crystal and cryo‐EM structures of the protein‐DNA assemblies involved in transposon movement. The structures capture various stages of transposition and reveal an intricate interplay between the core transposition machinery and host‐encoded proteins. Protein interactions shape transposon and genomic DNA in unique ways to cut, exchange and re‐join strands in a tightly regulated manner. Importantly, our data also explain how specific DNA distortion and cleavage mechanisms enable the transposons to move between diverse genomic sites, expanding gene transfer across many species.

These results shed new light onto the molecular strategies of antibiotic resistance carrying transposons and show how they evolved to effectively spread key genetic traits. Our insights also open new opportunities to develop strategies for blocking transposition and thereby help control resistance spreading.
SLC3A2 N‐glycosylation and alternate evolutionary trajectories for amino acid metabolism
Authors: Dennis James W. , Zhang Cunjie , Pawling Judy , Hesketh Geoff G. , Dransart Eselle , Shafaq‐Zahah Massiullah , Penn Linda Z. , Gingras Anne‐Claude , Johannes Ludger
Published: May 2022

Cancer and other proliferative diseases are driven by cooperative rewiring of growth signaling and metabolism, where posttranslational modifications to proteins play a critical role in maintaining the disease state. Most PTMs require metabolites as donor substrates, providing underappreciated link between metabolism and signaling. Protein N‐glycosylation and Golgi remodeling of N‐glycan on receptor kinases, T cell receptor, glucagon receptor, and glucose transporters provides feedback between signaling and metabolism. In turn, N‐glycosylation requires uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc), generated by the hexosamine biosynthesis pathway (HBP) from glucose, glutamine and acetyl‐CoA, where the flux of these substrates into UDP‐GlcNAc are in competition with glycolysis, glutaminolysis and fatty‐acid turnover. Thus, N‐glycan branching, a pathway of N‐acetylglucosaminyltransferases (MGAT1, 2, 4 and 5, avian 6) in the Golgi, is a sensor of central metabolism.

In a search for additional glycoproteins that link metabolism, N‐glycosylation and signaling, we identified SLC3A2 (4F2hc, CD98),an adaptor to amino acid (AA) transporters SLC7A5‐11, ‐13 which stabilizes cell surface residency. The SLC3A2*SLC7A5 exchanger imports essential AA that stimulate mTOR signaling and anabolic metabolism, while SLC3A2*SLC7A11 supports glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N‐glycans revealed stable site‐specific profiles of Golgi remodeling, with the exception of the conserved N365 where branching and poly‐N‐acetyllactosamine content were sensitive to the insertion of lost ancestral sites and to HBP. The N‐glycans are positioned to promote galectin‐mediated clustering with N‐glycosylated Na++/AA symporters and enhance diffusion‐limited flux between exchangers and AA /Na+ symporters. In a successful experiment of Nature, SLC3A2 has been deleted in Neoaves, a clade representing 95% of Ave species that are adapted for flapping flight, the most metabolically intense form of vertebrate locomotion. Neoaves have also acquired a unique N‐glycan branching specificity GnT‐VI (Mgat6, Q9DGD1), which may support an alternate clustering of N‐glycosylated transporters. KEAP1, and four SLC7A family exchangers are also absent, and two others have duplicated, consistent with a shift in metabolic imperatives of Neoaves. The fate of these genes in Primates, Neoaves and Naked mole rat highlights the tension between metabolic rates and oxidative stress that govern lifestyle and longevity.
The role of maternal factors in epigenetic programming of neurodevelopment
Authors: McGowan Patrick
Published: May 2022

Early life events are potent determinants of vulnerability and resistance to stressors. In many species, including rodents, the mother is the primary mediator of behavioral and physiological responses to stress in offspring during development. Emerging evidence indicates that epigenetic modifications in the brain of developing offspring are associated with the effects of maternal stress as well as individual neural, physiological, and behavioral responses to adversity. I will discuss research in my lab focused on identifying the relevant genomic targets (in the brain and periphery) of maternal stressors that exert long‐term ‘programming’ effects on stress responses. We have approached this question in several ways. First, we have studied ecologically important stressors applied during gestation. Second, we have investigated factors that co‐occur or interact with maternal care, including variations in ambient temperature and offspring genotype, that influence neurodevelopment and later‐life behavior. Third, we have explored direct exposure to maternal dietary stressors across the developmental period on offspring phenotype and genome‐wide epigenetic modifications in offspring brain. Through these investigations we will explore the significance of the maternal environment in neurodevelopment and the role of epigenetic mechanisms in important signaling pathways involved in susceptibility to stress‐related illness.
Protein Tyrosine Pseudophosphatase Signalling Mechanisms
Authors: Sharpe Hayley J. , Hay Iain M. , Deane Janet E.
Published: May 2022

Phosphatases are remarkably diverse, having evolved up to 10 distinct protein folds to mediate the removal of phosphate from substrates. Their functions are correspondingly wide ranging and a number are important clinical targets. As for kinases, several phosphatase classes comprise catalytically dead members. The classical protein tyrosine phosphatases (PTPs) are a subclass that utilise a cysteine nucleophile for catalysis and are critical for the control of cellular phosphotyrosine levels. Of the 49 human PTP domains, 19 possess sequence variants in key catalytic motifs defining them as pseudophosphatase domains. Strikingly, in all but two cases the catalytic cysteine remains intact. This is in contrast to other pseudophosphatases such as the pseudo‐dual specificity phosphatase (DUSP) MK‐Styx, which is inactivated by loss of its catalytic cysteine. We find that, like many pseudokinases, pseudoPTPs can mediate protein‐protein interactions, providing scaffolding functions, but also substrate recruitment to active PTP domains. Finally, given the striking conservation of cysteine residues in the pseudoPTPs, we have also explored a potential role for redox regulation in their signalling mechanisms.
Novel mechanisms in phosphoinositide turnover
Authors: Padinjat Raghu , Kumari Aastha , Ghosh Avishek , Kolay Sourav
Published: May 2022

The hydrolysis of phosphatidylinositol 4,5‐ bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K,dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2synthesis during phototransduction. The activity of dPIP5KLin vitrois enhanced by depletion of PNUT, a non‐redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of dPIP5KL rescues the effect of pnut depletion on the light response and PI(4,5)P2resynthesis during PLC signalling. Thus, our work defines a septin 7 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.
Structure‐Based Strategies for Rieske Oxygenase Catalysis
Authors: Bridwell‐Rabb Jennifer , Liu Jianxin , Tian Jiayi , Dill Zerick
Published: May 2022

Rieske Oxygenases are enzymes that showcase the remarkable ability to functionalize an inert C–H bond. These enzymes use a Rieske [2Fe‐2S] cluster and a mononuclear iron center to bind and activate molecular oxygen. Using these metallocenters, Rieske Oxygenases have been shown to catalyze a variety of reactions, including one, two, and sequential hydroxylation reactions. This chemistry is important to the biosynthetic pathways of multiple organisms and is thus attractive for industrially building and tailoring natural product scaffolds. Similarly, the chemistry of the Rieske Oxygenases is abundant in degradative pathways and therefore offers compelling strategies for degrading environmental pollutants. However, a traditional lack of structure–function information for this expansive class of enzymes has restricted our ability to capitalize on these important and diverse catalytic strategies. Therefore, our laboratory is working to understand the architectural features of these enzymes that allow them to control and facilitate catalysis. Here, we present our current understanding of how these enzymes selectively catalyze reactions and use structure to guide chemical outcome.
MALDI imaging mass spectrometry mapping of the glycocalyx
Authors: Drake Richard R. , Angel Peggi , Mehta Anand
Published: May 2022

The glycocalyx is a dense complex matrix of glycoproteins, glycosaminoglycans and glycolipids on the cell surface involved in maintaining cell integrity and interactions with the cellular microenvironment. Alterations to the glycan constituents of the glycocalyx are implicit in progression of diseases like cancer, but its overall complexity makes biomolecular analysis very challenging. Changes in cell surface protein glycosylation are common alterations that occur with tumor progression and reflect the clinical diagnostic detection of many cancer biomarkers like prostate specific antigen and CA19‐9. There is also an underlying metabolic connection to cancer glycosylation changes and the well described changes in tumor metabolism linked with glucose and glutamine utilization. Structurally, these potential tumor glycans are represented by multiple sialylated and fucosylated species including known tumor antigens such as sialyl Lewis X, sialyl Lewis A and Lewis Y motifs. Imaging mass spectrometry of clinical tissues and new enzymatic and chemical sample preparation strategies have provided new approaches for spatial characterization of the glycocalyx constituents. N‐glycan MALDI imaging MS allows direct histopathology localization of these glycans to tumor, stroma and immune regions. Peptide N‐glycosidase and other glycosidases are sprayed directly on tissues, and released glycans are detected spatially on a MALDI mass spectrometer. The method was developed to work on clinical formalin‐fixed paraffin embedded tissue slides readily obtainable from pathology services. Generally, 45‐60 N‐glycan species are detected in most tissues, and these numbers can increase to 100 or more in advanced cancer tissues. Example data from prostate, colon, and pancreatic cancer tissues will be presented, including new workflows that specifically target identification of isomeric species of branched fucosylated and sialylated N‐glycans. This includes combined use of immunohistochemistry staining of known carbohydrate tumor antigens prior to N‐glycan imaging, performed on the same slide. Strategies to use the tissue N‐glycan distribution maps for glycoproteomic and cancer biomarker analysis will also be presented. Additional glycosidases, other enzymes and other imaging modalities can also be incorporated into the established workflows to better define the complex organization of the tissue glycocalyx.
The role of rhomboid pseudoproteases in protecting the membrane proteome
Authors: Neal Sonya E.
Published: May 2022

Nearly one‐third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane where they are correctly folded, assembled, and then delivered to their final cellular destinations. In order to prevent the accumulation of misfolded membrane proteins, ER associated degradation (ERAD) moves these clients from the ER membrane to the cytosol; a process known as retrotranslocation. Our recent work in S. cerevisiae has revealed a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. We have sought to understand the mechanism associated with Dfm1’s actions and found that Dfm1’s conserved rhomboid residues are critical for membrane protein retrotranslocation. Specifically, we identified several retrotranslocation‐deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 has retained the lipid thinning functions of its rhomboid protease predecessors to facilitate in the removal of ER membrane substrates. We find this substrate engagement and lipid thinning feature is conserved in its human homolog, Derlin‐1. Utilizing interaction studies and molecular dynamic simulations, this work reveals that rhomboid pseudoprotease derlins employ novel mechanisms of substrate engagement and lipid thinning for catalyzing extraction of multi‐spanning membrane substrates.
Creative strategies to perform an inclusive faculty search
Authors: Corbett Anita H.
Published: May 2022

During the academic year 2020‐2021, the Biology Department at Emory University conducted a faculty search that was designed to continue the success of the department in building an inclusive department. There were several actions taken to ensure an equitable search process despite the challenges and biases inherent in search processes that were likely further exacerbated by a raging global pandemic.

The search was approved for two tenure track faculty positions with no formal limitation on the academic rank of the hires. The whole department invested in efforts to have a very broad search that would have the highest likelihood of identifying and recruiting outstanding scientists with a commitment to teaching who also showed evidence of actions toward the mission of diversity, equity, and inclusion.

For this search, we modified aspects of our traditional search in several ways that included the strategy for the search, the ad that described the position, the composition of the search committee, which was expanded to include trainee members, and aspects of the process (performed largely virtually due to COVID‐19). We were fortunate to have strong support from leadership at all levels from the Department to the Dean and Dean’s Office to allow us to make changes aligned with the goals of the search.

A successful search takes a great deal of time and effort dedicated from the search committee as well as all members of the department. The input from trainees, which was new to our process, was invaluable. We will seek to incorporate some of the lessons learned during this recent experience in future searches, with the caveat that we all hope to hold future searches in person. However, there are aspects of the virtual process that may enhance equity and, thus, should be considered for the future. Each search is unique with distinct challenges and opportunities, but there are concrete actions we can take to mitigate bias in the process.
Structural and mechanistic basis for protein glutamylation by the kinase fold
Authors: Tagliabracci Vincent , Osinski Adam , Black Miles , Pawlowski Krzysztof , Chen Zhe , Li Yang
Published: May 2022

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)‐dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo‐EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase‐like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl‐adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE‐ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non‐ribosomal amino acid ligations and reveal an unappreciated level of SidE‐family regulation.
Cell Surface Glycan Engineering to Examine Receptor Specificities of Influenza and Other Viruses
Authors: Boons Geart‐Jan
Published: May 2022

The transmission of viruses from animal reservoirs to humans poses great threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding how viruses of animal origin have adapted to replication in humans. In particular, it necessitates insight how viruses have evolved to binding to a cell surface component, or receptor, of the new host. We have developed glycan arrays and glycan cell surface engineering strategies to examine receptor specificities of viruses. The approaches were employed to examine receptor specificities of evolutionary distinct A/H3N2 influenza viruses. Recent A/H3N2 influenza viruses exhibit altered receptor specificities, and as a result they have lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Glycan array technology showed that these viruses have compensated for decreased binding of the prototypic human receptor by recognizing α2,6‐sialosides on extended LacNAc moieties. Erythrocyte glycomics showed an absence of extended glycans, providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installed functional receptors on erythrocytes, allowing antigenic characterization of recent A/H3N2 viruses and confirming the cocirculation of antigenically different viruses in humans. Computational studies of HAs in complex with sialosides having extended LacNAc moieties reveal that mutations distal to the RBD reoriented the Y159 side chain, resulting in an extended receptor binding site. The approach was extended to examining the receptor specificity of Lass Virus.
Mechanisms of membrane protein sorting
Authors: Shao Sichen (Susan)
Published: May 2022

Producing diverse membrane proteins requires multi‐step biosynthetic pathways with tightly integrated quality control mechanisms. Two biosynthetic challenges common to all membrane proteins are the need to localize to the appropriate cellular membrane and the need to insert each transmembrane segment (TM) in the correct topology. Using approaches that integrate cell biology, structural biology, and biochemistry, we examine mechanisms that ensure the fidelity of single‐spanning membrane protein localization and topogenesis at the endoplasmic reticulum (ER). We recently discovered that the ER‐resident P5A‐ATPase ATP13A1 (yeast Spf1) dislocates mislocalized mitochondrial tail‐anchored (TA) proteins. We leverage ATP13A1 as an experimental handle to investigate how mitochondrial membrane proteins misinsert into the ER. We also identify a role for ATP13A1 in the topogenesis of type II membrane proteins that should insert into the ER membrane with their N‐terminus in the cytosol. Without corrective protein quality control by ATP13A1, mislocalized TA proteins and misinserted type II proteins engage distinct ER‐associated degradation (ERAD) mechanisms. Our findings reveal­ how cells integrate counteracting ER activities with different specificities to fine tune membrane protein biogenesis.
Peroxisomal Quality Control in Arabidopsis
Authors: Bartel Bonnie
Published: May 2022

Peroxisomes are eukaryotic organelles that are essential for life in plants and metazoans. Peroxisomes sequester various oxidative reactions, including those acting fatty acid beta‐oxidation, photorespiration, and hormone biosynthesis, thereby improving metabolic efficiency while protecting cytosolic constituents from oxidative damage. Although our knowledge of peroxisome function and dysfunction is increasing, mechanistic understanding of how these critical organelles are dispose of individual proteins or are degraded in their entirety remains incomplete. We are studying the dynamics of this vital organelle in Arabidopsis thaliana; the peroxisomal functions, small size, and facile genetics of this reference plant allow straightforward impairment and enhancement of peroxisomal processes in an intact multicellular organism. Moreover, the relatively large size of plant peroxisomes (compared to yeast and mammalian peroxisomes) offers unique opportunities to view peroxisome dynamics using live‐cell imaging. Using forward‐genetic screens for Arabidopsis mutants defective in the biogenesis, function, or maintenance of peroxisomes, we have illuminated a variety of peroxisomal components, including a peroxisomal protease that prevents peroxisome degradation via autophagy.
Radical SAM Enzymes and the Unexplored Chemistry of RiPP Biosynthesis
Authors: Mitchell Douglas , Precord Timothy , Hudson Graham , Mahanta Nilkamal , Burkhart Brandon , Gerlt John
Published: May 2022

Radical S‐adenosylmethionine‐dependent (rSAM) enzymes comprise one of the largest and most functionally diverse protein superfamilies known. InterPro 87.0 (Nov‐2021) classifies >0.7M sequences in the UniProt database as containing a rSAM domain with the overwhelming majority having unknown and unpredictable functions. To accelerate the discovery of new enzymatic reactions, we have created an open‐access web resource, RadicalSAM.org, that applies modern genomic enzymology approaches to analyze the rSAM superfamily. This resource updates and considerably expands upon the archival Structure‐Function Linkage Database. RadicalSAM.org includes pre‐computed sequence similarity networks, domain architecture analysis, sequence logos, length histograms, multiple sequence alignments, hidden Markov models, genomic neighborhood diagrams, and taxonomical information for any desired grouping of rSAM proteins. Also included are relevant links to journal articles, UniProt, KEGG, Swiss‐Prot, PDB, and other external resources. This talk will briefly highlight examples of how we are leveraging insights provided by RadicalSAM.org, with our specific interests pertaining to the ribosomally synthesized and post‐translationally modified peptides (RiPPs).
Unraveling the secrets of radical SAM mechanisms
Authors: Broderick Joan B.
Published: May 2022

Radical SAM enzymes catalyze a remarkable diversity of biological radical reactions using a common radical initiation mechanism involving a [4Fe‐4S] cluster and SAM. The initial reductive cleavage of SAM leads to the formation of a catalytically central organometallic species (Ω) in which a SAM‐derived 5′‐deoxyadenosyl radical (5′‐dAdo•) is covalently bound through the 5′‐C to an iron of the [4Fe‐4S] cluster. Homolytic Fe‐C5′ bond cleavage then forms the 5′‐dAdo• radical intermediate, which abstracts a hydrogen atom from substrate. Efforts directed towards generating, trapping, and characterizing key radical intermediates during this radical initiation process will be presented and discussed. Further, recently‐discovered photochemistry in radical SAM enzymes, which can promote radical initiation in the absence of substrate, will be presented. This photochemistry has led to fundamental new understanding of the regioselectivity of reductive S‐C bond cleavage that is central to radical SAM mechanisms.
The glycocalyx in tumor progression and metastasis
Authors: Weaver Valerie M.
Published: May 2022

Tumors develop a desmoplastic response that creates a tissue microenvironment that is complicit in malignant transformation and metastasis. Tumor desmoplasia is characterized by fibrosis that is mediated by elevated extracellular matrix deposition, remodeling and cross‐linking that stiffen the tumor stroma. We have been studying how these aberrant cell and tissue level forces promote malignant transformation and drive tumor metastasis, and how they modulate tumor recurrence and treatment resistance in breast and pancreatic cancer and glioblastoma. We use two and three dimensional culture models with tuned extracellular matrix stiffness, as well as transgenic and syngeneic mouse models, human PDX models and human biospecimens, in which ECM crosslinking and stiffness and integrin mechanosignaling can be quantified and modified. Our studies have thus far revealed that the ECM in all tumors becomes progressively remodeled and stiffened by the resident stromal fibroblasts and revealed that this occurs prior to malignant transformation. The stromal‐fibroblast stiffened ECM disrupts tissue organization, promotes tumor cell growth, survival and invasion, induces angiogenesis, and promotes a pro‐tumor immunity that drives tumor aggression and metastasis. We recently found that the fibrotic stroma drives the metabolic rewiring of the tumor cells and determined that this associates with elevated expression of key enzymes that regulate the cellular glycoproteome. A stiffened fibrotic stroma stimulated sialoglycan expression through tension‐induced integrin signaling. The elevated mechanosignaling induced in the tumor cells by the stiffened stroma increased the level of secreted proteoglycans deposited at the tumor‐ECM interface where a high density of protumorigenic immune suppressive infiltrating myeloid cells were detected. The association between a stiff, fibrotic tumor stroma, elevated secreted proteoglycans and a pro tumor immune infiltrate was very predominant in the more aggressive human breast tumors. I will discuss the dynamic and reciprocal interplay between tissue tension, metabolic reprogramming and glycoprotein regulation and innate and acquired immunity in tumor aggression and progression.
Programmed epigenetic risk: Can stress exposures in utero predispose infants to obesity and metabolic diseases?
Authors: Boyle Kristen E.
Published: May 2022

Dr. Kristen Boyle is an Associate Professor in the Department of Pediatrics Nutrition Section studying the regulation of human stem cell metabolism and phenotype in the context of obesity and diabetes. During her early independent career Dr. Boyle began using primary infant mesenchymal stem cells, derived from umbilical cord tissue, to investigate stem cell phenotype and epigenetic signatures as a model for developmental programming in humans. She discovered that cells from infants born to mothers with obesity have epigenetic and metabolic perturbations similar to adults with established obesity and associated with greater child adiposity. Thus, infant stem cell metabolism may elucidate how some children are at increased risk for developing obesity and diabetes later in life, independent of postnatal lifestyle exposures. Dr. Boyle’s current research program focuses on using these infant cells to understand how various maternal stress exposures, from metabolic disease to environmental toxins, may impact offspring metabolic phenotypes through epigenetic mechanisms. Her work is supported by the American Diabetes Association and the NIDDK. The goal of this research is to help identify which maternal factors may most influence metabolic differences in the cells and children, and to determine which children may be most at risk for developing obesity or diabetes in the future so that we may develop better tools for obesity or diabetes prevention strategies.
Chronic stress, omics, and asthma
Authors: Celedon Juan C.
Published: May 2022

The U.S. has the highest rate of firearm‐related deaths of all economically developed nations. Current evidence suggests that exposure to violence (ETV) and violence‐related distress may cause or worsen asthma. Indeed, violence‐related distress may affect asthma pathogenesis through direct effects including abnormal responses to catecholamines and glucocorticoid therapy, and altered immune responses resulting in airway inflammation. Violence‐related distress may also affect asthma through indirect effects such as decreased physical activity and weight gain.

We previously showed that ETV is associated with asthma in a high‐risk population (Puerto Ricans), in whom ETV is also associated with methylation of a CpG site in the promoter of the gene for the pituitary adenylate cyclase activating polypeptide 1 type 1 receptor (ADCYAP1R1) in white blood cells. Moreover, we reported that such methylation is associated with childhood asthma in Puerto Ricans.

Based on previous results, we hypothesized that ETV and violence‐related distress would be linked to asthma through airway epithelial methylation of genes regulating autonomic, neuroendocrine, and immune responses. To examine this hypothesis, we carried out a epigenome‐wide association study (EWAS) of ETV or chronic stress measures and nasal (airway) epithelial methylation in 487 Puerto Rican participants (aged 9 to 20 years) in the Epigenetic Variation and Childhood Asthma in Puerto Ricans study [EVA‐PR]). We evaluated four measures of ETV and chronic stress in children (ETV scale, gun violence, and perceived stress) and their mothers (perceived stress). Linear regression was used for each of the EWASs, with CpGs as dependent variables and the violence or stress scale as a predictor, adjusting for age, sex, principal components, and latent factors. We then selected the top 100 CpGs (by P‐value) associated with each scale in EVA‐PR. Since no replication cohort had data on stress or violence, we conducted a meta‐analysis of the selected CpGs and atopic asthma using data from EVA‐PR and two other cohorts (Project Viva and PIAMA).

Three CpGs (in SNN, PTPRN2,and LINC01164) were associated with maternal stress or gun violence (P=1.28 to 3.36 ×10‐7), but not with atopic asthma, in EVA‐PR. In a meta‐analysis of three cohorts, which included the top CpGs associated with stress/violence measures in EVA‐PR, 12 CpGs (in STARD3NL, SLC35F4, TSR3, CDC42SE2, KLHL25, PLCB1, BUD13, OR2B3, GALR1, TMEM196, TEAD4 and ANAPC13) were associated with atopic asthma at FDR‐P< 0.05. PLCB1 has been associated with bronchodilator response and is differentially expressed in children with treatment‐resistant asthma compared with children with controlled persistent asthma or healthy controls.

Pending confirmation in longitudinal studies, our results suggest that nasal epithelial methylation markers associated with ETV and chronic stress may be linked to atopic asthma in youth.
Lipid Metabolism and Ferroptosis
Authors: Dixon Scott
Published: May 2022

Ferroptosis is an oxidative, iron‐dependent form of non‐apoptotic cell death that contributes to several forms of pathology and may be exploitable for cancer therapy. Ferroptosis can be induced using small molecule inhibitors of the plasma membrane cystine/glutamate antiporter system xc‐ or the glutathione‐dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4). The execution of ferroptosis requires membrane lipid peroxidation, but specific lipids and lipid metabolic enzymes that are involved in this process are only partly characterized. How ferroptosis sensitivity relates to other fundamental cellular processes such as the cell cycle is also unclear. We find that inhibition of the Rb‐E2F cell cycle pathway causes downregulation of specific lipid metabolic enzymes, leading to an increase in polyunsaturated phospholipid abundance. In turn, this metabolic alteration specifically enhances ferroptosis sensitivity. Drugs that inhibit Rb‐E2F pathway function synergize with a ferroptosis‐inducing small molecule to arrest tumor growth in vivo. These results suggest that ferroptosis sensitivity may be linked to cell cycle progression through specific alterations in membrane phospholipid composition.
A phosphoinositide cascade regulates a receptor recycling pathway
Authors: Weisman Lois S. , Srinivas Panapakkam Giridharan Sai , Luo Guangming , Rivero‐Rios Pilar , Steinfeld Noah , Tronchere Helene , Singla Amika , Burstein Ezra , Billadeau Daniel D. , Sutton Michael A.
Published: May 2022

Phosphatidylinositol polyphosphates (PIPs) are essential cellular regulators. Each PIP transiently appears at specific membranes to regulate the spatial and temporal recruitment of unique downstream effectors. Importantly, most PIPs serve as both signaling lipids, and as precursors for other PIPs. These precursor‐product relationships likely form cascades of pathways, which would provide a way for PIPs to coordinate events both spatially and temporally. Here we uncover a pathway on endosomes, which is likely governed by a cascade of three phosphoinositide lipids. The pathway is specific for the recycling of some receptors to the plasma membrane and is mediated by sorting‐nexin SNX17, and the Retriever, WASH and CCC complexes. We discover that PIKfyve and an upstream PI3‐kinase, VPS34 positively regulates this pathway. VPS34 produces PI3P, which provides the substrate for PIKfyve to generate PI3,5P2, which is then further converted to PI5P by myotubularin 3‐phosphatases. We show that PIKfyve regulates recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH and CCC complexes on endosomes. Importantly, PIKfyve inhibition causes a loss of Retriever and CCC from endosomes. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration. More broadly, these findings provide more general insights into mechanisms for how cells regulate the levels of some receptors at the cell surface.
Regulation of COPII‐mediated protein transport
Authors: Audhya Anjon
Published: May 2022

The export of newly synthesized membrane proteins from the endoplasmic reticulum (ER) requires the action of coat protein complex II (COPII), which is comprised of five core subunits (Sar1, Sec23, Sec24, Sec13, and Sec31) that co‐assemble to remodel membranes and generate transport carriers. Recent studies conducted in our lab demonstrate that COPII carrier biogenesis surprisingly continues in the absence of Sar1 GTPase activity, which was suggested previously to be indispensable for COPII‐mediated membrane reorganization. Specifically, we show that inhibition of Sar1 stimulates ER stress and triggers the formation of unconventional transport carriers that are decorated with other COPII components and remain capable of directing secretory protein trafficking out of the ER. Our studies suggest new mechanisms by which COPII drives membrane remodeling independently of Sar1.
Telomerase holoenzymes
Authors: Collins Kathleen
Published: May 2022

Telomerase is unusual genome replication machinery. The eukaryotic reverse transcriptase ribonucleoprotein (RNP) supplements telomeric simple‐sequence repeat tracts at chromosome ends by new repeat synthesis. Using each chromosome 3’ end as primer and an internal region of the integral telomerase RNA subunit as template, telomerase reiteratively reverse transcribes its short template into tandem single‐stranded telomeric repeats. Lagging‐strand DNA replication machinery and processing enzymes then create the functional telomere structure of duplex repeats with a 3’ overhang that can be single‐stranded, form alternative DNA structures such as G‐quadruplex, or invade the duplex region to form a displacment loop (D‐loop) of telomeric repeats termed the t‐loop.

Two and a half decades of research in the Collins lab led the way to a resolution for several puzzles that were facing the telomere and telomerase field since soon after telomerase discovery by Carol Greider and Elizabeth Blackburn. How do telomerase reverse transcriptase protein (TERT) and telomerase RNA co‐fold in a hierarchical, complex process of cellular telomerase RNP biogenesis, and how does this drastically limit the amount of telomerase made by even the most strongly telomerase‐positive human cancer and stem cells? How can a precisely defined region within telomerase RNA be used as template? How does the template‐product duplex unpair and reform in a different template register to enable processive repeat synthesis? How is telomerase brought to telomeres and how does each telomere control telomerase activity according to its individual length to generate telomere length homeostasis?

Human telomerase RNA (hTR) relies on proteins shared with H/ACA‐family small nucleolar RNAs for its processing, stability, and assembly with TERT. Inherited and spontaneous mutations in these H/ACA proteins cause disease syndromes by reducing telomerase function, as do subsequently identified mutations in hTR and TERT. Understanding of how these mutations compromise telomerase function has emerged from new high‐resolution structures of telomerase holoenzymes from the ciliate Tetrahymena thermophila and human cells, the two model systems of Collins lab research, and from understanding control of telomerase function at telomeres.
Beyond Diversity: Building a Culture of Inclusion in Science
Authors: Johnson Tracy L.
Published: May 2022

The future and vitality of the scientific enterprise is dependent upon maximizing access and participation by a diverse talent pool. After decades of discussion and numerous programs aimed at diversifying the research environment at the undergraduate, graduate and professional levels, individuals from Black, Indigenous, and Latinx populations are still woefully underrepresented in STEM. Many of our current strategies have focused on programs that prepare individuals for a scientific environment that is, at best, agnostic to diversity and is, at its worst, hostile. Given the slow pace of progress with respect to racial and ethnic diversity in the sciences, it is crucial that we critically assess our strategies to date and examine more closely the culture of the environment we are expecting individuals from diverse backgrounds to navigate. I will discuss the current state of the approaches to diversify STEM disciplines, provide examples of successful strategies, and explore ways to move beyond incremental change to systemic cultural transformation.
How One Eukaryote Invades and Co‐opts the Cells of Another: The Story of the Truly Audacious Toxoplasma gondii
Authors: Boothroyd John
Published: May 2022

For intracellular, infectious agents that live inside a vacuole there are many benefits, but maybe even more challenges. On the one hand, they can be protected from detection by innate immune sensors and buffered from some types of immune attack. On the other hand, this isolation represents a barrier to actively engaging with the host cell sitting all around their “bubble,” e.g., for feeding and active coopting of host cell functions. The single‐celled eukaryote, Toxoplasma gondii, is a remarkable example of an intracellular agent in that it can infect and grow within almost any mammalian or avian cell. Many studies have shown that despite growing within a parasitophorous vacuole (PV), Toxoplasma tachyzoites are in active dialogue with the host cell and that the parasites somehow introduce a large number of “effector” proteins into the infected cell cytoplasm; many of these eventually reach the host cell nucleus where they impact host gene expression. Toxoplasma effectors come in two flavors – ones that originate in the apical secretory organelles known as rhoptries and that are injected during invasion (ROPs); and a second set that are secreted into the PV from dense granules after invasion (GRAs). In my talk, I will first describe our recent efforts with cryo‐electron tomography to understand the machinery that enables this remarkable organism to invade almost any host cell and inject the first wave of effectors, the ROPS. I will then discuss how they modify the parasitophorous vacuole membrane (PVM) to facilitate translocation of GRAs across the PVM, involving a completely novel translocon system. I will end with how GRAs and ROPs impact gene expression within the infected host.
Methodological Approaches and Considerations for Generating Evidence that Informs the Science of Learning
Authors: Anderson Sarah J.
Published: May 2022

Educational neuroscience is field with the goal of applying what we know about the brain to improve education through improvements to teaching, learning, assessment, and curricular design. Applying neuroimaging and biometric methodology can help inform this field by understanding the mechanisms underlying a learner’s behaviours and cognitive processing. Advantages of these approaches include the ability to measure more acute or subtle changes that are not always indexed by overt behavioral changes. However, generating evidence that appropriately informs our understanding of the science of learning can be complex. Some of the challenges associated with traditional neuroscientific research protocols in educational research is that they have low ecological validity; neuroscientific tasks are typically short, decontextualized, and isolated while educational research tasks are lengthy, context relevant, and incorporate the complexity of the typical learning environment.

I will highlight approaches we have used on our lab to overcome methodological challenges to generate evidence that informs the science of learning. Discussion will focus on four key areas. First, meaningful study design will be addressed using an example of a novice‐expert design examining visual expertise. Second, the importance of appropriately combining multiple data sources (electroencephalography and eye‐tracking) will be discussed and a tool to support data collection will be shared. Third, a data analysis strategy that addresses the problems with time variability during cognitive tasks will be explored. Finally, one of the greatest challenges is to translate findings in a way that informs educational practice. In this case, an example of an evidence‐informed application of research generated through a cycle of basic and applied research will be followed to illustrate the cycle between research and practice in educational neuroscience. Together, this series will inform practical considerations and promote a shared discussion amongst our community for the advancement of educational neurosciences in anatomy education.