The effect of D-cycloserine on brain connectivity over a course of pulmonary rehabilitation - A randomised control trial with neuroimaging endpoints.
Combining traditional therapies such as pulmonary rehabilitation with brain-targeted drugs may offer new therapeutic opportunities for the treatment of chronic breathlessness. Recently, we asked whether D-cycloserine, a partial NMDA-receptor agonist which may enhance behavioural therapies, modifies the relationship between breathlessness related brain activity and breathlessness anxiety over pulmonary rehabilitation. However, whether any changes are supported by alterations to underlying brain structure remains unknown. Here we examine the effect of D-cycloserine over a course of pulmonary rehabilitation on the connectivity between key brain regions associated with the processing of breathlessness anxiety. 72 participants with mild-to-moderate COPD took part in a longitudinal study in parallel to their pulmonary rehabilitation course. Diffusion tensor brain imaging and clinical measures of respiratory function were collected at three time points (before, during and after pulmonary rehabilitation). Participants were assigned to 250mg of D-cycloserine or placebo, which they were administered with on four occasions in a randomised, double-blind procedure. Following the first four sessions of pulmonary rehabilitation (visit 2), during which D-cycloserine was administered, improvements in breathlessness anxiety were linked with increased insula-hippocampal structural connectivity in the D-cycloserine group when compared to the placebo group. No differences were found between the two groups following the completion of the full pulmonary rehabilitation course 4-6 weeks later (visit 3). The action of D-cycloserine on brain connectivity appears to be restricted to within a short time-window of its administration. This temporary boost of the brain connectivity of two key regions associated with the evaluation of how unpleasant an experience is may support the re-evaluation of breathlessness cues, illustrated improvements in breathlessness anxiety. Trial registration ClinicalTrials.gov (NCT01985750).
Evidence of focusing the MHC class I immunopeptidome by tapasin.
Major Histocompatibility Complex class I (MHC-I) molecules bind and present peptides to cytotoxic T cells, protecting against pathogens and cancer. MHC-I is highly polymorphic and each allotype is promiscuous, and capable of binding a unique and diverse repertoire of peptide ligands. The peptide editing chaperone tapasin optimizes this allotype specific repertoire of peptides, resulting in the selection of high affinity peptides. MHC-I allotypes differ in the extent they engage tapasin. This suggests that tapasin-dependent MHC-I allotypes should present a less diverse repertoire that is enriched in higher-affinity peptides, and which are present in higher abundance, than tapasin independent MHC-I allotypes, which should present a broader repertoire containing peptides with a lower average affinity. Experimental verification of this hypothesis has been confounded by the different peptide binding specificities of MHC-I allotypes. Here, we independently investigated the peptide focusing function of tapasin by introducing a point mutation into a tapasin independent MHC-I allotype that dramatically increased its tapasin dependence without substantially altering its peptide binding specificity. This allowed us to demonstrate ligand focusing by tapasin at both the repertoire level in cellulo, and by using an in vitro system in which tapasin was artificially tethered to MHC-I, at the individual peptide level. We found that tapasin had a greater influence on tapasin dependent MHC-I molecules, and that tapasin modulated peptide selection according to peptide-MHC-I complex stability, disfavoring short-lived peptide-MHC-I complexes. Thus, tapasin dependent MHC-I molecules experience greater tapasin filtering, resulting in less diverse MHC-I immunopeptidomes that are enriched in high affinity peptide-MHC-I complexes.
Strict advantage of complex quantum theory in a communication task
Standard formulations of quantum theory are based on complex numbers: Quantum states can be in superpositions, with weights given by complex probability amplitudes. Motivated by quantum theory promising a range of practical advantages over classical for a multitude of tasks, we investigate how the presence of complex amplitudes in quantum theory can yield operational advantages over counterpart real formulations. We identify a straightforward communication task for which complex quantum theory exhibits a provably lower communication cost than not just any classical approach, but also any approach based on real quantum theory. We certify the necessity of complex quantum theory for optimal approaches to the task through geometric properties of quantum state ensembles that witness the presence of basis-independent complexity. This substantiates a strict operational advantage of complex quantum theory. We discuss the relevance of this finding for quantum advantages in stochastic simulation.
Deep palaeoproteomic profiling of archaeological human brains.
Palaeoproteomics leverages the persistence, diversity, and biological import of ancient proteins to explore the past, and answer fundamental questions about phylogeny, environment, diet, and disease. These insights are largely gleaned from hard tissues like bone and teeth, as well-established protocols exist for extracting ancient proteins from mineralised tissues. No such method, however, exists for the soft tissues, which are underexplored in palaeoproteomics given permission for destructive analysis routinely depends on a proven methodology. Considering less than one-tenth of all human proteins are expressed in bone, compared to three-quarters in the internal organs, the amount of biological information presently inaccessible is substantial. We address this omission with an optimised LC-FAIMS-MS/MS workflow yielding the largest, most diverse palaeoproteome yet described. Using archaeological human brains, we test ten protocols with varied chemistries and find that urea lysis effectively disrupts preserved membrane regions to expose low-abundant, intracellular analytes. Further, we show that ion mobility spectrometry improves unique protein identification by as much as 40%, and represents a means of "cleaning" dirty archaeological samples. Our methodology will be useful for improving protein recovery from a range of ancient tissues and depositional environments.
Activity-Based Protein Profiling (ABPP) of Cellular DeISGylating Enzymes and Inhibitor Screening.
A detailed methodology platform is described for activity-based protein profiling (ABPP) of cellular deISGylating enzymes using a specific activity-based interferon-stimulated gene 15 (ISG15) probe. Manual and semi-automated workflows for medium- to high-throughput applications are outlined in this chapter, with western blotting and proteomics-based techniques as the main readouts. This methodology informs us of endogenous deISGylating enzyme expression and activity in a cellular context, including USP18, the type I interferon (IFN-I)-inducible deISGylase, and several constitutively expressed deubiquitinases (DUBs), such as USP5, USP14, USP16, and USP36, that exert cross-reactivity to ISG15. ISG15-ABPP also enables the identification and characterization of potent and selective deISGylating enzyme modulators.
A Novel Toolkit of SARS-CoV-2 Sub-Genomic Replicons for Efficient Antiviral Screening.
SARS-CoV-2 is classified as a containment level 3 (CL3) pathogen, limiting research access and antiviral testing. To address this, we developed a non-infectious viral surrogate system using reverse genetics to generate sub-genomic replicons. These replicons contained the nsp1 mutations K164A and H165A and had the spike, membrane, ORF6, and ORF7a coding sequences replaced with various reporter and selectable marker genes. Replicons based on the ancestral Wuhan Hu-1 strain and the Delta variant of concern were replication-competent in multiple cell lines, as assessed by Renilla luciferase activity, fluorescence, immunofluorescence staining, and single-molecule fluorescent in situ hybridization. Antiviral assays using transient replicon expression showed that remdesivir effectively inhibited both replicon and viral replication. Ritonavir and cobicistat inhibited Delta variant replicons similarly to wild-type virus but did not inhibit Wuhan Hu-1 replicon replication. To further investigate the impact of nsp1 mutations, we generated a recombinant SARS-CoV-2 virus carrying the K164A and H165A mutations. The virus exhibited attenuated replication across a range of mammalian cell lines, was restricted by the type I interferon response, and showed reduced cytopathic effects. These findings highlight the utility of sub-genomic replicons as reliable CL2-compatible surrogates for studying SARS-CoV-2 replication and drug activity mechanisms.
RIFINs displayed on malaria-infected erythrocytes bind KIR2DL1 and KIR2DS1.
Natural killer (NK) cells use inhibitory and activating immune receptors to differentiate between human cells and pathogens. Signalling by these receptors determines whether an NK cell becomes activated and destroys a target cell. In some cases, such as killer immunoglobulin-like receptors, immune receptors are found in pairs, with inhibitory and activating receptors containing nearly identical extracellular ligand-binding domains coupled to different intracellular signalling domains1. Previous studies showed that repetitive interspersed family (RIFIN) proteins, displayed on the surfaces of Plasmodium falciparum-infected erythrocytes, can bind to inhibitory immune receptors and dampen NK cell activation2,3, reducing parasite killing. However, no pathogen-derived ligand has been identified for any human activating receptor. Here we identified a clade of RIFINs that bind to inhibitory immune receptor KIR2DL1 more strongly than KIR2DL1 binds to the human ligand (MHC class I). This interaction mediates inhibitory signalling and suppresses the activation of KIR2DL1-expressing NK cells. We show that KIR2DL1-binding RIFINs are abundant in field-isolated strains from both Africa and Asia and reveal how the two RIFINs bind to KIR2DL1. The RIFIN binding surface of KIR2DL1 is conserved in the cognate activating immune receptor KIR2DS1. We find that KIR2DL1-binding RIFINs can also bind to KIR2DS1, resulting in the activation of KIR2DS1-expressing NK cells. This study demonstrates that activating killer immunoglobulin-like receptors can recruit NK cells to target a pathogen and reveals a potential role for activating immune receptors in controlling malaria infection.
Chromothripsis-associated chromosome 21 amplification orchestrates transformation to blast-phase MPN through targetable overexpression of DYRK1A
Abstract Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A, a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target.
Translational science at the undergraduate level: awakening talents to overcome the valley of death - case report
Abstract Background: In the biomedical field, translational science is the process of applying basic scientific knowledge to advance clinical research through the creation of new drugs, devices, medical procedures, preventive measures, and diagnostic kits. The Covid-19 pandemic exposed a shortage of professionals trained in translational research, essential for responding to global demands. To drive advancements, researchers must overcome the ‘valley of death’, a critical phase in clinical investigation. In response, CEVAP at São Paulo State University (UNESP), Botucatu, Brazil, has developed a strong 'knowledge industry' centered on Translational Science. As part of its research and innovation efforts, CEVAP has developed two biopharmaceuticals, the fibrin sealant and the apilic antivenom, which are currently in the final stage of development. In 2024, CEVAP began the first Brazilian Contract Development and Manufacturing Organization (CDMO) for developing and producing validated and qualified pilot-scale batches to generate clinical trial material. Case Presentation: The implementation of the optional undergraduate course in Translational Science marks a crucial step in strengthening the ‘knowledge industry’. The program, developed in collaboration with São Paulo’s three public universities (USP, UNESP, and UNICAMP), also involves an international partnership with the University of Oxford’s Department of Pediatrics and the Oxford Research Group LATAM. The successful launch of this course underscores its importance in interdisciplinary education and institutional collaboration. By bridging gaps between research and application, the program equips professionals to meet the growing demand for expertise in translational science. Given the project's success, it will transition into a one-year ‘Qualification in Translational Science’, available to students enrolled in São Paulo state universities. Conclusion: The preparation of these professionals will be strategic for transforming basic research into products for health, saving lives, and combating future pandemics that will emerge around the world.
Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse.
Ligation of T cell receptor (TCR) to peptide-MHC (pMHC) complexes initiates signaling leading to T cell activation and TCR ubiquitination. Ubiquitinated TCR is then either internalized by the T cell or released toward the antigen-presenting cell (APC) in extracellular vesicles. How these distinct fates are orchestrated is unknown. Here, we show that clathrin is first recruited to TCR microclusters by HRS and STAM2 to initiate release of TCR in extracellular vesicles through clathrin- and ESCRT-mediated ectocytosis directly from the plasma membrane. Subsequently, EPN1 recruits clathrin to remaining TCR microclusters to enable trans-endocytosis of pMHC-TCR conjugates from the APC. With these results, we demonstrate how clathrin governs bidirectional membrane exchange at the immunological synapse through two topologically opposite processes coordinated by the sequential recruitment of ecto- and endocytic adaptors. This provides a scaffold for direct two-way communication between T cells and APCs.
Androgens Profile in Blood Serum and Follicular Fluid of Women With Poor Ovarian Response During Controlled Ovarian Stimulation Reveals Differences Amongst POSEIDON Stratification Groups: A Pilot Study.
Patients with poor ovarian response (POR) to exogenous gonadotropins stimulation for assisted reproductive technology (ART) have decreased circulating androgens during spontaneous cycles. The Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) is a 4-tier stratification of women with POR to controlled ovarian stimulation (COH) based on age and biomarkers of ovarian reserve has been proposed to maximize the clinical management of this group for ART. The aim of the present study was to characterize the levels of androgens during COH in follicular fluid (FF) and serum in POSEIDON subgroups and compared them with women of normal ovarian response. Sixty nine consecutive patients undergoing ART were included and testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEA-S), estradiol, sex hormone-binding globulin (SHBG), and insulin-like growth factor 1 (IGF-1) were measured in serum and FF collected at the time of oocyte pick-up. The number of retrieved oocytes was registered for each patient for their allocation to the respective POSEIDON subgroup. The control group comprised 19 women and the POSEIDON group 1 (age < 35, normal ovarian reserve biomarkers) n = 14, group 2 (age ≥ 35, normal ovarian reserve biomarkers) n = 8, group 3 (age < 35, poor ovarian reserve biomarkers) n = 6 and group 4 (age ≥ 35, poor ovarian reserve biomarkers) n = 22. Serum levels of total testosterone, androstenedione and DHEA-S were not different in group 1 vs. control but significantly decreased in group 3 vs. control. DHEA-S in FF was also significantly decreased in group 3 vs. control. In addition, serum testosterone was decreased in groups 2 and 4 vs. control; and serum androstenedione and estradiol were reduced in group 4 vs. control. No differences were observed for estradiol, SHBG and IGF-1 in FF. Finally, a high correlation between serum and FF DHEA-S was observed when data from samples of all groups were pooled. Group 1 did not show hypoandrogenemia however group 3 had low levels of all measured androgens in serum and DHEA-S in FF. Such differences might help to better characterize and/or improve the clinical management of women with POR according to their respective POSEIDON stratification.
Percepciones y creencias sobre criopreservación embrionaria en mujeres y hombres que se realizan técnicas de reproducción asistida en Santiago, Chile.
Background and objetiveAlthough embryo cryopreservation is frequently used as part of assisted reproductive technology, quantitave information addressing how infertile couples live the experience of having cryopreserved embryos is lacking in Chile. The aim of this study is to examine men and women's perception and beliefs regarding their cryopreserved embryos, as well as their perspective on embryo donation and disposition. Methods: 153 women and men with frozen embryos from a public hospital, Instituto de Investigactiones Materno Infantil, and a private clinic, Clínica Las Condes, in Santiago, Chile, responded between May 2015 and May 2016 to an anonymous online survey addressing their perceptions and beliefs concerning their cryopreserved embryos.ResultsRespondents considered their frozen embryos to be equivalent to a child (53.2%) or a potential child (40.7%). Only 8% regard them as an organized group of cells. Over 60% of respondents disagree with destroying surplus embryos or using them for research. Participants from the public hospital are more willing to donate their embryos to another couple than those from the private center (61% vs 40%; P=0.016); 34% of respondents agreed to donate surplus embryos to same sex couples.ConclusionThis study reveals that Chilean couples are emotionally bound to their frozen embryos, and that discarding them is not an option. The results from this survey will help strengthen counseling for couples to enable them to make informed decisions regarding their surplus embryos.
[Prevalence of endometriosis in 287 women undergoing surgical sterilization in Santiago Chile].
BackgroundThe clinical manifestations of endometriosis are infertility, dysmenorrhea, sexuality disturbances, and chronic pelvic pain. It is the cause of 30 to 50% of infertility cases. In developed countries, the prevalence of endometriosis among women undergoing surgical sterilization is approximately 6%.AimTo determine the prevalence of endometriosis among women with proven fertility in Santiago de Chile.Material and methodsReview of surgical protocols of 287 women aged 25 to 49 years, subjected to a surgical sterilization between 2007 and 2011.ResultsEndometriosis was found in 14 of the 287 women (4.9%). In spite of being asymptomatic, five of the 14 women with endometriosis were classified as severe, due to the presence of at least one endometrioma. In order of frequency, the most commonly affected anatomical sites were the ovary, the peritoneum, the posterior cul-de-sac and uterosacral ligaments.ConclusionsOur findings are very similar to those found elsewhere and suggest that fertile women could better tolerate endometriosis than their infertile counterparts.