bluuebunny/crossref_metadata_2025_split huggingface dataset:
Newer Concepts in Coronary Artery Anomalies, Based on Accurate In‐vivo Imaging (IVUS)
Authors: Angelini Paolo Enzo , Uribe Carlo e 
Published: November 2023

ObjectivesAn update on novel concepts in the understanding of coronary anomalies anatomy and their pathophysiology.MethodsA review of our Center's multiple investigations in these fields (80 publications in 26 years).ResultsAnatomic rare variants are frequent (up to 5.6% of a general population), and need to be properly recognized. At the end, Anomalous Coronary Artery Origin off an improper sinus of Valsalva (ACAOS) results to be the main culprit of sudden cardiac death in the young athletes and/or, in a few carriers, cause of important symptoms at any age. These anomalies were observed in a recent study of ours, based on an MRI prospective study, in 1.1% of a general schoolchildren population (that translates in about 2 million people in the US). This finding calls for the need to identify any predictive markers of the risk of sudden cardiac death (SCD). Stenosis of a coronary artery is the only known means of its dysfunction. Symptoms, participation in strenuous exercises, stress testing and intravascular ultrasounds imaging (when indicated to clarify its severity and to guide indications for interventions) seem to be the main indicators of risk. Still, SCD may be the most frequent first (and, unfortunately, the last) manifestation of ischemia, pointing to the necessity of prospective and effective screening, best done by a screening‐MRI protocol, especially in young athletes. The 2 main causes of ischemic spells in ACAOS are 1. Baseline stenosis, featuring phasic and exercise‐related changes in lateral compression, inside the aortic tunica media, or intermittent spontaneous spasm of distal coronary segments.ConclusionsCAAs are recently more frequently discovered by modern imaging, a fact that demands a prospective work up protocol to select the cases for preventive interventions. Integration of similar observation with developmental studies suggests the need to especially focus on ACAOS anomalies to better understand morphogenesis of the abnormal and the nature of functional impairment.Support or Funding InformationKinder Foundation and Texas Heart Institute
Intravital Microscopy Identifies Macrophages as Effector Cells for Monoclonal Antibody Therapy of Cancer
Authors: van Egmond Marjolein 
Published: November 2023

Monoclonal antibodies (mAbs) have become an important addition to chemo‐ and/or radiotherapy for the treatment of cancer. They can exert multiple effector functions that can lead to eradication of tumor in vitro, such as induction of apoptosis, growth inhibition, and initiation of complement‐dependent lysis. Furthermore, mAbs can recruit immune effector cells. To investigate how mAbs mediate tumor cell elimination in vivo, and to establish which effector cells are involved we performed live cell imaging as well as intravital microscopy. We show that antibody‐dependent phagocytosis (ADCP) by macrophages is a prominent mechanism to remove tumor cells from the circulation after mAb therapy. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells) even in the absence of mAbs. Furthermore, tumor cells were sampled by Kupffer cells, which was however not sufficient for elimination. By contrast, anti‐tumor mAb treatment resulted in fast ADCP of tumor cells by Kupffer cells, which was dependent on FcgRI and FcgRVI. Uptake was independent of reactive oxygen (ROS) or nitrogen (RNS) species production, and followed by degradation, which prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy was lost after depletion of Kupffer cells. As such, macrophages likely play an essential role in mAb‐mediated elimination of tumor cells. Therefore, by aiming to enhance macrophage recruitment and activity mAb therapeutical strategies of cancer patients may be optimized. Currently these strategies are investigated. Additionally, the role of macrophages in eliminating tumor cells in other tissues are studied.Support or Funding InformationThis project was financially supported by the Dutch Cancer Foundation (KWF) project VU2011–4931, and the Worldwide Cancer Research project 15‐1240.
Tracking the in vivo dynamics of antigenic variation In Trypanosoma brucei
Authors: Mugnier Monica , Cross George A.M. , Papavasiliou F. Nina 
Published: November 2023

Trypanosoma brucei changes its dense variant surface glycoprotein (VSG) surface coat to avoid detection by the immune system of its mammalian host. The dynamics of antigenic variation during T. brucei infection, a process that takes advantage of a genomic repertoire of over 2000 different VSG‐encoding genes, are poorly understood. How many variants appear over the course of an infection? Is there a pattern to VSG expression over time? Although some of these questions have been broached using Sanger sequencing of VSG cDNA, technical limitations have prevented a high‐resolution, quantitative study of VSG expression during T. brucei infection. We have developed a method, termed VSG‐seq, for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes. Based on de novo assembly, this approach can be used for the high‐resolution study of VSG expression in any strain of T. brucei, whether in the lab or in the field. Using VSG‐seq, we have studied the dynamics of VSG expression during chronic T. brucei infections in mice, and our experiments reveal more dynamic and nuanced host‐parasite interactions than previously expected. Nearly a quarter of the full‐length VSG repertoire is sampled within only four peaks of infection, and this level of diversity persists throughout infection. We can also detect the appearance and disappearance of mosaic VSGs using this method. Mosaic VSGs are novel variants which arise through recombination events within the parasite genome during infection, and our data hint at the mechanisms by which these mosaic VSGs form. We are now applying VSG‐seq to field isolates of T. brucei as well as to populations of parasites inhabiting extravascular spaces. Altogether, these results raise many new questions about the T. brucei host‐pathogen interaction; VSG‐seq will allow these questions to be addressed in their proper context, the natural host.Support or Funding InformationThis work has been supported in part by the NIH/National Institute of Allergy and Infectious Diseases (AI085973) to F.N.P., and by an NSF Graduate Research Fellowship (DGE‐1325261) to M.R.M.
A clinical candidate targeting dihydroorotate dehydrogenase for the treatment of malaria with blood and liver stage activity
Authors: Phillips Margaret A. , Charman Susan A. , Rathod Pradipsinh K , Burrows Jeremy , Rueckle Thomas 
Published: November 2023

Malaria is a deadly tropical disease that kills up to 0.6 million people yearly, mostly young children in Africa. Current drug therapies are threatened by drug resistance and the development of novel compounds that are active against drug resistant strains is essential to malarial control and eradication programs. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme for the biosynthesis of pyrimidines required for DNA and RNA synthesis in the parasite. We used a combination of high throughput screening and structure guided medicinal chemistry to optimize a triazolopyrimidine‐based series of DHODH inhibitors resulting in the selection of a clinical candidate for the treatment of malaria. We have shown that DHODH inhibitors have both blood and liver stage anti‐malarial activity, that they have potent activity in both in vitro and in vivo models of malaria and that they are active against a range of clinically drug resistant parasites. A compound called DSM265 was selected from the triazolopyrimidine series for clinical development. DSM265 was shown to have excellent safety during preclinical development. DSM265 has been shown to be a long acting compound with a pharmacokinetic profile that is expected to support single dose treatment or once weekly chemoprevention.Support or Funding InformationThis work was supported by funds from the United States National Institutes of Health grants, U01AI075594 (to MAP, PKR and SAC) and R01AI103947 (to MAP and PKR) and from Medicines for Malaria Venture (MMV).
Separated at Birth, the Evolution of Kinase Dynamics Over a Few Billion Years
Authors: Kern Dorothee 
Published: November 2023

I will present our recent exploration of the evolution of the fascinating rate acceleration of enzymes and the evolutionary origin of allostery. The secret of enzymes lies in their ability to partition energetic contributions among many atoms in a well‐coordinated style. To unravel these secrets, the evolution of protein dynamics in the family of protein kinases is spied on to provide a comprehensive description of enzyme catalysis in the form of an energy landscape. Ancestor resurrection combined with modern biophysical characterization combining NMR, fast kinetics and x‐ray crystallography shed light into evolution of protein function to create more sophisticated biomolecules. The evolution of energy landscapes offer a larger variety of signaling mechanisms and sophisticated enzyme catalysis compared to evolution of structure alone. Interestingly, our time‐travel delivered an additional unusual twist, the discovery of the molecular origin of the modern cancer drug Gleevec for the human tyrosine kinase Abl:Protein kinases are attractive drug targets against cancer due to their central role in cellular regulation, but the similarity of their active site has hampered the development of specific drugs. One of the major success stories has been Gleevec, a highly selective inhibitor of Abl kinase. The molecular mechanism underlying Gleevec's selectivity for Abl has not been solved since the discovery of the drug 20 years ago despite large efforts. Gleevec's selectivity is puzzling because the drug‐binding pocket with Gleevec bound is nearly identical between Abl and Src and other homologous kinases that have very weak affinity for the drug. I will present how the characterization of the evolution of kinase dynamics over 1 billion years reveals the mechanism responsible Gleevec's selectivity. The results simultaneously shed light on the longstanding paradox of the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve for catalysis. The resurrection of the tyrosine kinase ancestors explores a key event in the history of life, the development of multicellularity coinciding with the explosion of the kinome. Interestingly, gleevec takes advantage of evolutionary differences among the kinases “by accident” thereby leading to one of the most potent and specific cancer drugs to date.
Transformation In Biomedical Science Education For Future Physicians
Authors: Lieberman Steve 
Published: November 2023

Physician education in the US is undergoing a period of rapid change unmatched in the past 100 years. Advances in the science of learning and educational technology, rapid evolution of the American health care system, projected physician shortages, and rising student debt have spurred a new wave of curriculum reform at many medical schools as well as national initiatives by major organizations including the AAMC and the AMA. Structural changes include expansion of time for clinical education and shortening of the traditional 8‐year course of study. Pedagogical changes include use of active learning methods during faculty‐student interactions combined with independent (often online) acquisition of content, emphasis on the application of science knowledge to clinical problem‐solving rather than the accumulation of decontextualized knowledge, acquisition and demonstration of competence in patient care, and education in the delivery of health care and the health care system. Many of these changes are reinforced by national exams (MCAT, USMLE).Competency based education is diffusing from residency training to medical school and premedical programs, taking the form of entrustable professional activities (EPAs) at many medical schools. EPAs are units of clinical work (eg, developing a differential diagnosis) that learners can be entrusted to perform under progressively less supervision as they demonstrate increasing competence. Ubiquitous subsets of knowledge, skill, and attitudes (eg, biomedical and clinical science knowledge, communication) implicitly pervade the 13 Core EPAs for Entering Residency identified by an AAMC panel.In the University of Texas System, Transformation in Medical Education (TIME) is a student‐centered, clinically focused initiative to increase the effectiveness of medical education and shorten its duration. TIME consists of 4 scalable and generalizable pilot programs in which 11 UT premedical and medical programs are partnering to create 6–7 year programs built on the principles of competency based education (CBE), intentional professional identity development, and early clinical skills training to expand the time available for authentic clinical learning. The approach to CBE includes a set of collaboratively developed competencies and shared assessments of critical transition milestones (eg, biomedical science knowledge, clinical skills). The UTMB TIME curriculum features a novel 3‐year structure that fully integrates basic and clinical science education in the classroom and on modified clerkships that are organized by life cycle stages. An essential aspect is the Student Continuity of Practice Experience (SCOPE) in which students will provide care to their own panel of patients under supervision throughout medical school.Support or Funding InformationThe TIME initiative is supported by funding from the University of Texas Board of Regents.
Molecular Visualization of the Eukaryotic Transcription Initiation Process using Cryo‐EM
Authors: Nogales Eva , Louder Robert , He Yuan 
Published: November 2023

The critical step of transcription initiation requires the assembly of the general transcription factors into a pre‐initiation complex (PIC) that ensures the accurate loading of Pol II at the transcription start site, carries out the opening of the DNA duplex, and facilitates clearance of the promoter by Pol II. We are using single particle cryo‐EM to investigate the assembly, architecture and structural rearrangements of the human PIC during the process of transcription initiation. Our efforts have concentrated on studying, on one hand, the general transcription factor TFIID and its recognition of core promoter DNA, and on the other a simplified PIC using TATA‐binding protein (TBP) as a surrogate for TFIID.Using an in vitro reconstitution system we visualized the step by step assembly of the TBP‐based human PIC, ultimately containing TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH on promoter DNA. Our cryo‐EM structures provide pseudo‐atomic models that illuminate critical protein‐protein and protein‐DNA interactions with the PIC. Comparison of the closed PIC with the open complex has allowed us to shed light onto the process of promoter melting. The apparent movement of downstream DNA, together with the positioning of XPB, support the previously proposed translocase model for TFIIH. We propose that XPB would push and rotate the DNA with respect to a contact point of Pol II and the DNA, just upstream of the active site of XPB. These actions of XPB against the stably bound upstream DNA around the TATA box would in turn induce negative supercoiling near the transcription start site.TFIID plays a central role in the initiation of RNA polymerase II‐dependent transcription by nucleating PIC assembly at the core promoter. TFIID comprises TBP and 13 TBP‐associated factors (TAF1‐13), which specifically interact with a variety of core promoter DNA sequences. Using single‐particle cryo‐electron microscopy, we determined that TFIID exist in multiple conformational states. Two major conformers, which we refer to as the canonical and rearranged states, coexist in the apo complex. Only the rearranged state is capable of binding core promoter DNA. Recently we have determined the structure of human TFIID in complex with TFIIA and core promoter DNA at sub‐nanometer resolution. Our structure shows how all the super core promoter elements (TATA, INR, MTE and DPE) are contacted by subunits of TFIID. Together with the cryo‐EM reconstruction of a fully‐assembled human TAF‐less PIC, our structures provide novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.Support or Funding InformationThis work was funded by NIGMS and HHMI.
Insights On Mammalian Glycosylation Enzymes From High‐Throughput Expression Studies
Authors: Moremen Kelley , Meng L , Forouhar F. , Wang S. , Kadirvelraj R. , Moniz H. , Ramiah A. , Gao Z. , Seetharaman J. , Milaninia S. , Galay G. , Stuart M. , Steel J. , Labaer J. , Wood Z. , Tong L. , Jarvis D. 
Published: November 2023

Glycan structures on cell surface and secreted glycoproteins play critical roles in biological recognition and targeting events in animal systems. The enzymes that synthesize these structures reside in membranes of the secretory pathway and modify glycoproteins in transit to the cell surface. These enzymes are generally poorly understood largely because they are challenging targets for functional expression even in eukaryotic hosts. We have established library of expression constructs encoding all human glycosylation enzymes (target gene list of ~350 coding regions) as secreted catalytic domain fusion proteins for recombinant production in HEK293 cells and baculovirus‐infected insect cells. Comparison of expression levels in each host system indicates similar trends where most proteins are well‐expressed, but only a subset are efficiently secreted. This presentation will summarize our strategies for expression and downstream workflows for biochemical and structural studies. Discussion of resulting structures and insights into catalytic mechanisms and substrate recognition will focus on recently determined structures of sialyltransferases and fucosyltransferases coming from the mammalian expression platform.Support or Funding Information(supported by NIH grants P41GM103390 and P01GM107012).
Mechanistic and structural insights into a human carbohydrate degrading enzyme
Authors: Gloster Tracey , Alteen Matthew , Oehler Verena , Yang Lei , Wilson Iain , Vocadlo David 
Published: November 2023

Mammalian β‐hexosaminidases have emerged as playing essential roles in cellular physiology and disease. These enzymes are responsible for the cleavage of the monosaccharides N‐acetylglucosamine (GlcNAc) and N‐acetylgalactosamine (GalNAc) from cellular substrates. One of these β‐hexosaminidases, Hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this nucleocytoplasmically localized β‐hexosaminidase and its cellular function remains unknown. We will discuss a series of kinetic, mechanistic and structural investigations into HexD that have aided defining the precise catalytic mechanism of this enzyme. The fundamental insights gained from these studies will aid in the development of potent and selective probes for HexD, which will serve as useful tools to better understand the physiological role played by this enzyme.Support or Funding InformationT.M.G. and V.O. are supported by a Wellcome Trust Career Development Fellowship. D.J.V. thanks the Canada Research Chair program for support as a Canada Research Chair in Chemical Glycobiology. M.A. thanks the NSERC CREATE Collaborative Medicinal Chemistry Network in Epigenetics Training (ChemNET) for support. This work was supported by an NSERC Discovery and a CIHR Operating grant (MOP‐123341). I.N. was recipient of an “Aktion Austria‐Slovakia” scholarship from the Österreichischer Austauschdienst.
Impeding the formation of Transcription Initiation complex
Authors: Egly Jean‐Marc 
Published: November 2023

RNA synthesis is the result of a cascade of chronologically orchestrated events that requires several hundreds of proteins including RNA polymerase II, the general transcription factors, mediator, co‐activators, co‐repressors and chromatin remodelers are assembled around the promoter. Among these proteins necessary to initiate RNA synthesis, we recently identified the Nucleotide‐Excision‐Repair (NER) factors demonstrating their dual role in transcription and DNA repair. Mediator occupies a key role in protein coding genes expression in mediating the contacts between gene specific factors and the basal transcription machinery.Mutations in one of these factors results in rare genetic disorders: Xeroderma Pigmentosum (XP), Trichothiodystrophy (TTD), Cockayne Syndrome (CS), for TFIIH and NER factors; non‐syndromic mental retardation, Charcot‐Marie Tooth, Opitz‐Kaveggia … for Mediator.We show how each of the mutations in either NER factors or Mediator subunits specifically disturbs the protein/protein interactions network required for the accurate formation of the transcription complex at the promoter of activated genes.The NER proteins seem to be necessary at the promoter of activated genes in order to achieve an optimal 3D loop structure that requires DNA demethylation, formation of DNA cuts as well as histone posttranslational modifications. We also show how the Mediator mutations cause distinct expression of Immediate Early Genes (IEG) with as a consequence disruption of later genes expression.Our study shed light on the molecular mechanism underlying gene expression regulation at the transcription initiation level explaining at least partially some of the clinical features of the above‐mentioned genetic disorders.
ELONGIN AND THE ELONGIN A UBIQUITIN LIGASE COMPLEX IN TRANSCRIPTION AND THE RESPONSE TO DNA DAMAGE
Authors: Conaway Joan W , Weems Juston C. , Slaughter Brian D. , Unruh Jay R. , Conaway Ronald C. 
Published: November 2023

Elongin A performs dual functions in cells as a component of the Elongin ABC complex, a Pol II elongation factor, and as the substrate recognition subunit of an E3 ubiquitin ligase that works in concert with the ubiquitin ligase NEDD4 to target Pol II stalled at sites of DNA damage and other impediments for ubiquitination and degradation by the proteasome. Assembly of the Elongin A ubiquitin ligase proceeds via binding of the Elongin ABC complex to a heterodimer composed of Cullin family member CUL5 AND RING finger protein RBX. We recently discovered that assembly of the Elongin A ubiquitin ligase is a tightly regulated process induced by a wide variety of stimuli and signal transduction pathways. Whereas under normal conditions, the majority of Elongin A is present in cells in the Elongin ABC complex, assembly of the Elongin A ubiquitin ligase is induced by DNA damage and other stresses that lead to accumulation of stalled Pol II. Assembly of the Elongin A ubiquitin ligase is also induced by stimuli known to activate Elongin A‐dependent transcription, such as endoplasmic reticulum and nutrient stress and retinoic signaling. Furthermore, assembly of the Elongin A ubiquitin ligase can be induced by doxycycline in U2OS cells carrying multiple copies of a doxycycline‐inducible transgene, but not in parental U2OS cells, suggesting that transcription activation per se is sufficient to drive ligase assembly. These findings raise the intriguing possibility that the Elongin A ubiquitin ligase might play roles not only as part of a “fail‐safe” mechanism to remove stalled Pol II from genes, but also in normal transcription activation processes.Support or Funding InformationThis work was supported by National Institutes of Health Grant GM041628, a grant to the Stowers Institute from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation, and by the Stowers Institute for Medical Research.