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In this study in vitro and in vivo approaches were combined in order to investigate if the anti‐epileptic mechanism(s) of action of levetiracetam (LEV; Keppra®) may involve modulation of inhibitory neurotransmission. GABA‐ and glycine‐gated currents were studied in vitro using whole‐cell patch‐clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound‐susceptible mice. The effect of LEV was compared with reference anti‐epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. LEV contrasted the reference AEDs by an absence of any direct effect on glycine‐gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA‐gated currents. A similar action on GABA‐elicited currents was observed with the reference AEDs, except ethosuximide. These minor direct effects contrasted with a potent ability of LEV (EC50=1 – 10 μM) to reverse the inhibitory effects of the negative allosteric modulators zinc and β‐carbolines on both GABAA and glycine receptor‐mediated responses. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of β‐carbolines on GABA‐gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine‐gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. LEV (17 mg kg−1) produced a potent suppression of sound‐induced clonic convulsions in mice. This protective effect was significantly abolished by co‐administration of the β‐carboline FG 7142, from a dose of 5 mg kg−1. In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg−1) was without any effect on the protection afforded by LEV. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti‐epileptic mechanism(s) of action of LEV. British Journal of Pharmacology (2002) 136, 659–672; doi:10.1038/sj.bjp.0704766

HLA-G is considered to be an immune checkpoint molecule, a function that is closely linked to the structure and dynamics of the different HLA-G isoforms. Unfortunately, little is known about the structure and dynamics of these isoforms. For instance, there are only seven crystal structures of HLA-G molecules, being all related to a single isoform, and in some cases lacking important residues associated to the interaction with leukocyte receptors. In addition, they lack information on the dynamics of both membrane-bound HLA-G forms, and soluble forms. We took advantage of strategies to disclose the dynamic behavior of selected HLA-G forms, including the membrane-bound HLA-G1 molecule, soluble HLA-G1 dimer, and HLA-G5 isoform. Both the membrane-bound HLA-G1 molecule and the soluble HLA-G1 dimer were quite stable. Residues involved in the interaction with ILT2 and ILT4 receptors (α3 domain) were very close to the lipid bilayer in the complete HLA-G1 molecule, which might limit accessibility. On the other hand, these residues can be completely exposed in the soluble HLA-G1 dimer, due to the free rotation of the disulfide bridge (Cys42/Cys42). In fact, we speculate that this free rotation of each protomer (i.e., the chains composing the dimer) could enable alternative binding modes for ILT2/ILT4 receptors, which in turn could be associated with greater affinity of the soluble HLA-G1 dimer. Structural analysis of the HLA-G5 isoform demonstrated higher stability for the complex containing the peptide and coupled β2-microglobulin, while structures lacking such domains were significantly unstable. This study reports for the first time structural conformations for the HLA-G5 isoform and the dynamic behavior of HLA-G1 molecules under simulated biological conditions. All modeled structures were made available through GitHub (https://github.com/KavrakiLab/), enabling their use as templates for modeling other alleles and isoforms, as well as for other computational analyses to investigate key molecular interactions.

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient's own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide-ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide-MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC \"hot-spots\" for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our study provides further evidence that structural analyses of pMHC complexes can be used to assess the intrinsic likelihood of cross-reactivity among peptide-targets. Furthermore, we hypothesize that some apparent inconsistencies in reported cross-reactivities, such as a preferential directionality, might also be driven by particular structural features of the targeted pMHC complex. Finally, we explain why TCR-based immunotherapy provides a special context in which meaningful T-cell cross-reactivity predictions can be made.

Innate immune responses to cell damage-associated molecular patterns induce a controlled degree of inflammation, ideally avoiding the promotion of intense unwanted inflammatory adverse events. When released by damaged cells, Hsp70 can stimulate different responses that range from immune activation to immune suppression. The effects of Hsp70 are mediated through innate receptors expressed primarily by myeloid cells, such as dendritic cells (DCs). The regulatory innate receptors that bind to extracellular mouse Hsp70 (mHsp70) are not fully characterized, and neither are their potential interactions with activating innate receptors. Here, we show that extracellular mHsp70 interacts with a receptor complex formed by both inhibitory Siglec-E and activating lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) on DCs. We also find that this interaction takes place in lipid microdomains within the plasma membrane, and that Siglec-E acts as a negative regulator of LOX-1-mediated innate activation upon mHsp70 or oxidized LDL binding. Thus, Hsp70 can both bind to and modulate the interaction of inhibitory and activating innate receptors on the cell surface. These findings add another dimension of regulatory mechanism to indicate how self-molecules contribute to dampening of exacerbated inflammatory responses.

The development of the cerebral cortex requires coordinated regulation of proliferation, specification, migration and differentiation of cortical progenitors into functionally integrated neurons. The completion of the neurogenic program requires a dynamic interplay between cell intrinsic regulators and extrinsic cues, such as growth factor and neurotransmitters. We previously demonstrated a role for extrasynaptic glycine receptors (GlyRs) containing the α 2 subunit in cerebral cortical neurogenesis, revealing that endogenous GlyR activation promotes interneuron migration in the developing cortical wall. The proliferative compartment of the cortex comprises apical progenitors that give birth to neurons directly or indirectly through the generation of basal progenitors, which serve as amplification step to generate the bulk of cortical neurons. The present work shows that genetic inactivation of Glra2, the gene coding the α 2 subunit of GlyRs, disrupts dorsal cortical progenitor homeostasis with an impaired capability of apical progenitors to generate basal progenitors. This defect results in an overall reduction of projection neurons that settle in upper or deep layers of the cerebral cortex. Overall, the depletion of cortical neurons observed in Glra2 -knockout embryos leads to moderate microcephaly in newborn Glra2-knockout mice. Taken together, our findings support a contribution of GlyR α 2 to early processes in cerebral cortical neurogenesis that are required later for the proper development of cortical circuits.

Peptide-HLA class I (pHLA) complexes on the surface of tumor cells can be targeted by cytotoxic T-cells to eliminate tumors, and this is one of the bases for T-cell-based immunotherapies. However, there exist cases where therapeutic T-cells directed towards tumor pHLA complexes may also recognize pHLAs from healthy normal cells. The process where the same T-cell clone recognizes more than one pHLA is referred to as T-cell cross-reactivity and this process is driven mainly by features that make pHLAs similar to each other. T-cell cross-reactivity prediction is critical for designing T-cell-based cancer immunotherapies that are both effective and safe. Here we present PepSim, a novel score to predict T-cell cross-reactivity based on the structural and biochemical similarity of pHLAs. We show our method can accurately separate cross-reactive from non-crossreactive pHLAs in a diverse set of datasets including cancer, viral, and self-peptides. PepSim can be generalized to work on any dataset of class I peptide-HLAs and is freely available as a web server at pepsim.kavrakilab.org.

PurposeFor patients with oligometastatic/oligorecurrent/oligoprogressive lymph node metastases from PCa, metastases-directed therapy is an emerging strategy. The aim of this retrospective study was to evaluate the oncological outcome and pattern of recurrence in patients treated with stereotactic body radiation therapy (SBRT) to lymph node metastases.MethodsIn this multi-institutional analysis, patients with a maximum of five lymph node metastases from PCa treated with SBRT were included. Primary endpoints of the analysis were local control (LC), out-of-field nodal progression-free survival (NPFS), overall progression-free survival (PFS), and overall survival (OS).Results109 patients and 155 lymph node metastases were evaluated. Patients’ median age was 70.8 years (range 51–84) and median PSA before SBRT was 1.88 ng/ml (range 0.3–45.5 ng/ml). The dose delivered to the target ranged from 25 to 48 Gy in 4–7 fractions; median BED1.5 Gy was 198 Gy (range 108.3–432 Gy). With a median follow-up of 16 months, LC rates at 1 and 3 years were 93% and 86%, respectively. In-field progression of disease was observed in 11 (7%) lesions. One- and 3‑year NPFS was 59% and 29%, and median NPFS was 15 months. Rates of OS at 1 and 3 years were 100% and 95%. The median time to administration of a systemic treatment after SBRT was 7.8 months (1.7–54.8).ConclusionSBRT is an effective and well-tolerated treatment option in the management of lymph node metastases from PCa. Prospective trials are necessary to better select patients who benefit most from this ablative focal treatment and better define the recurrence patterns.

The Human Immunodeficiency Virus type 1 protease enzyme (HIV-1 PR) is one of the most important targets of antiretroviral therapy used in the treatment of AIDS patients. The success of protease-inhibitors (PIs), however, is often limited by the emergence of protease mutations that can confer resistance to a specific drug, or even to multiple PIs. In the present study, we used bioinformatics tools to evaluate the impact of the unusual mutations D30V and V32E over the dynamics of the PR-Nelfinavir complex, considering that codons involved in these mutations were previously related to major drug resistance to Nelfinavir. Both studied mutations presented structural features that indicate resistance to Nelfinavir, each one with a different impact over the interaction with the drug. The D30V mutation triggered a subtle change in the PR structure, which was also observed for the well-known Nelfinavir resistance mutation D30N, while the V32E exchange presented a much more dramatic impact over the PR flap dynamics. Moreover, our in silico approach was also able to describe different binding modes of the drug when bound to different proteases, identifying specific features of HIV-1 subtype B and subtype C proteases.

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to major obstetric pathologies, including placenta accreta spectrum, early onset preeclampsia, and fetal growth restriction. Characterization of the normal differentiation processes is, thus, essential for the analysis of these pathologies. Our gene expression analysis, employing purified human CTB and EVT cells, demonstrates a mechanism similar to the epithelial–mesenchymal transition (EMT), which underlies CTB–EVT differentiation. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome-wide hypomethylation in the transition to EVT. A small subgroup of genes undergoes gains of methylation (GOM), associated with differential gene expression (DE). Prominent in this GOM-DE group are genes involved in epithelial–mesenchymal plasticity (EMP). An exemplar is the transcription factor RUNX1, for which we demonstrate a functional role in regulating the migratory and invasive capacities of trophoblast cells. This analysis highlights epigenetically regulated genes acting to underpin the epithelial–mesenchymal plasticity characteristic of human trophoblast differentiation. Identification of these elements provides important information for the obstetric disorders in which these processes are dysregulated.

The onset of pelagic biomineralization was a milestone in the history of the long-term inorganic carbon cycle: as soon as calcareous nannofossils became major limestone producers, the pH and supersaturation state of the global ocean were stabilized (the so-called mid-Mesozoic revolution). But although it is known that calcareous nannofossils were abundant already by the end of the Triassic, no estimates exist on their contribution to hemipelagic carbonate sedimentation. With this work, we estimate the volume proportion of Prinsiosphaera, the dominant late Triassic calcareous nannofossil, in hemipelagic and pelagic carbonates of western Tethys. The investigated Upper Triassic lime mudstones are composed essentially of microspar and tests of calcareous nannofossils, plus minor bioclasts. Prinsiosphaera had become a significant component of lime mudstones since the late Norian, and was contributing up to ca. 60% of the carbonate by the late Rhaetian in periplatform environments with hemipelagic sedimentation. The increasing proportion of Prinsiosphaera in upper Rhaetian hemipelagic lime mudstones is paralleled by an increase of the δ13C of bulk carbonate. We interpreted this isotopic trend as related to the diagenesis of microspar, which incorporated respired organic carbon with a low δ13C when it formed during shallow burial. As the proportion of nannofossil tests increased, the contribution of microspar with low δ13C diminished, determining the isotopic trend. We suggest that a similar diagenetic effect may be observed in many Mesozoic limestones with a significant, but not yet dominant, proportion of calcareous plankton.