In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQß-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQß eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.
Senescent cells are blocked in the cell cycle but remain metabolically active. These cells, once engaged in the senescence process, fail to initiate DNA replication. Due to the shortening of telomeres, replicative senescence can be triggered by a DNA damage response. Moreover, cells can also be induced to senesce by DNA damage in response to elevated reactive oxygen species (ROS), activation of oncogenes, cell-cell fusion or after ionizing radiation. There are multiple experimental ways to detect senescent cells directly or indirectly. Senescence-associated cellular traits (SA ß-Gal activity, increase in cell volume and lysosome content, appearance of γ-H2AX foci, increase of ROS and oxidative damage adducts, etc.) can be identified by numerous methods of detection (flow cytometry, confocal imaging, in situ staining, etc.). Here, we improved an existing flow cytometry protocol and further developed a new one specifically tailored to ionizing radiation-induced endothelial senescence. Thus, we have upgraded the Debacq-Chainiaux protocol and added improvements in this protocol (i) to better detect positive events (ii) to offer a compatibility to simultaneously analyze various intracellular molecules including phosphorylated signaling proteins and cytokines, whether related or not to senescence processes.
Cellular Senescence , Reactive Oxygen Species/metabolism , Cellular Senescence/genetics , Cells, Cultured , Phenotype
Background: An MRI is potentially hazardous for patients with retained ferromagnetic bullets. Recent studies have aimed to develop dual-energy computed tomography (DECT) as a screening tool for recognising highly ferromagnetic bullets. Inconsistent findings have been ascribed to inherent CT technology differences. Previous research demonstrated significant Hounsfield unit (HU) measurement variation among single-source CT machines. Objectives: This study investigated the theoretical dual-energy index (DEI) variation between DECT machines when evaluating the potential ferromagnetic properties within the same sample of ex vivo bullets and metal phantoms. Method: An experimental ex vivo study was conducted on eight metal phantoms and 10 unused bullets individually positioned in the same Perspex head phantom and scanned on two DECT machines. Two senior radiology registrars independently recorded the HU readings, and DEI values were calculated. Statistical analysis was performed using non-parametric methods for paired data, namely the Signed Rank Test. The DEI values based on mean HU readings between the DECT machines were compared. Results: Inter- and intra-reader agreement was not statistically significant. The metal phantoms had poor interscanner agreement, with an overlap of the ferromagnetic and non-ferromagnetic ranges. The bullets had good interscanner agreement, with a similar ferromagnetic to non-ferromagnetic relationship. Conclusion: The use of DEI values negates the previous assumption that significant interscanner variability exists among different DECT technologies while assessing highly attenuative ex vivo bullets. Contribution: This investigation demonstrated that even though HU readings may be variable, the implementation of the DEI equation translates this into comparable values with good interscanner agreement.
Rhythms are a common feature of brain activity. Across different types of rhythms, the phase has been proposed to have functional consequences, thus requiring its accurate specification from noisy data. Phase is conventionally specified using techniques that presume a frequency band-limited rhythm. However, in practice, observed brain rhythms are typically nonsinusoidal and amplitude modulated. How these features impact methods to estimate phase remains unclear. To address this, we consider three phase estimation methods, each with different underlying assumptions about the rhythm. We apply these methods to rhythms simulated with different generative mechanisms and demonstrate inconsistency in phase estimates across the different methods. We propose two improvements to the practice of phase estimation: (1) estimating confidence in the phase estimate, and (2) examining the consistency of phase estimates between two (or more) methods.
Brain , Electroencephalography , Uncertainty , Electroencephalography/methods
In a cross sectional study of 13,837 university students, we aimed to explore the association between attention deficit hyperactivity disorder (ADHD) symptoms and lifetime psychoactive substance use (LPSU) on a wide range of illicit substances. Logistic and Hurdel multivariable regressions were used. ADHD symptoms were significantly associated with the lifetime use of ketamine, magic mushrooms, poppers, and nine other psychoactive substances. There was an association between ADHD symptoms and both LPSU and truncated count of lifetime psychoactive substance use. High levels of ADHD symptoms are associated with the use of a large variety and multiple LPSU.
Rhythms are a common feature of brain activity. Across different types of rhythms, the phase has been proposed to have functional consequences, thus requiring its accurate specification from noisy data. Phase is conventionally specified using techniques that presume a frequency band-limited rhythm. However, in practice, observed brain rhythms are typically non-sinusoidal and amplitude modulated. How these features impact methods to estimate phase remains unclear. To address this, we consider three phase estimation methods, each with different underlying assumptions about the rhythm. We apply these methods to rhythms simulated with different generative mechanisms and demonstrate inconsistency in phase estimates across the different methods. We propose two improvements to the practice of phase estimation: (1) estimating confidence in the phase estimate, and (2) examining the consistency of phase estimates between two (or more) methods.
Radiation-induced toxicity of the digestive tract is a major clinical concern as many cancer survivors have received radiotherapy for tumours of the abdominopelvic area. The coordination and orchestration of a tissue's response to stress depend not only on the phenotype of the cells that make up the tissue but also on cell-cell interactions. The digestive system, i.e., the intestine/colon/rectum, is made up of a range of different cell populations: epithelial cells, stromal cells, i.e. endothelial cells and mesenchymal lineages, immune cells and nerve cells. Moreover, each of these populations is heterogeneous and presents very significant plasticity and differentiation states. The pathogenesis of radiation-induced digestive lesions is an integrated process that involves multiple cellular compartments interacting in a complex sequence of events. Understanding all the cellular events and communication networks that contribute to the tissue's response to stress is therefore a major conceptual and methodological scientific challenge. The study of heterogeneous populations of cells in a tissue is now possible thanks to "single cell' RNA sequencing and spatial transcriptomics techniques, which enable a comprehensive study of the transcriptomic profiles of individual cells in an integrated system. In addition, the mathematical and bioinformatics tools that are now available for the large-scale analysis of data allow the inference of cell-cell communication networks. Such approaches have become possible through advances in bioinformatics algorithms for the analysis and deciphering of interaction networks. Interactions influence the tissue regeneration process through expression of various molecules, including metabolites, integrins, junction proteins, ligands, receptors and proteins secreted into the extracellular space. The vascular network is viewed as a key player in the progression of digestive lesions, which are characterised by infiltration of a range of immune cells. A better characterisation of endothelium/immune cell interactions in suitable preclinical models, as well as in humans, may help to identify some promising therapeutic targets for the prediction, prevention or treatment of digestive toxicity after radiotherapy.
Neoplasms , Radiation Injuries , Humans , Endothelial Cells , Endothelium, Vascular/pathology , Neoplasms/pathology , Radiation Injuries/pathology , Phenotype
Patients with deep and extensive wounds need urgent skin coverage to re-establish the cutaneous barrier that prevents life-threatening infections and dehydration. However, the current clinically-available skin substitutes intended for permanent coverage are limited in number, and a trade-off between production time and quality must be made. Here, we report the use of decellularized self-assembled dermal matrices to reduce by half the manufacturing process time of clinical-grade skin substitutes. These decellularized matrices can be stored for over 18 months and recellularized with patients' cells in order to generate skin substitutes that show outstanding histological and mechanical properties in vitro. Once grafted in mice, these substitutes persist over weeks with high graft take, few contraction events, and high stem cell content. These next-generation skin substitutes constitute a substantial advancement in the treatment of major burn patients, combining, for the first time, high functionality, rapid manufacturability and easy handling for surgeons and healthcare practitioners. Future clinical trials will be conducted to assess the advantages of these substitutes over existing treatments. STATEMENT OF SIGNIFICANCE: The number of patients in need for organ transplantation is ever-growing and there is a shortage in tissue and organ donors. In this study, we show for the first time that we can preserve decellularized self-assembled tissues and keep them in storage. Then, in only three weeks we can use them to produce bilayered skin substitutes that have properties very close to those of the native human skin. These findings therefore represent a major step forward in the field of tissue engineering and organ transplantation, paving the way toward a universal off-the-shelf biomaterial for tissue reconstruction and surgery that will be beneficial for many clinicians and patients.
Skin, Artificial , Humans , Mice , Animals , Tissue Engineering , Skin/pathology , Skin Transplantation , Biocompatible Materials
PURPOSE: Although attention deficit hyperactivity disorder (ADHD) has been associated with illicit stimulants use, less is known about their prospective association in university students. We aimed to examine the association between ADHD symptoms at inclusion and illicit stimulants use following 1 year among university students. METHODS: The i-Share cohort recruited French students from February 2013 to July 2020. The study included 4270 participants. The Adult ADHD Self-Report Scale (ASRS) was used to evaluate ADHD symptoms at inclusion. Illicit stimulants use was assessed at inclusion and 1 year after inclusion. Multivariable logistic regressions were conducted to assess the association between ADHD symptoms at inclusion and illicit stimulants use following 1 year. RESULTS: High levels of ADHD symptoms at inclusion were associated with a greater probability of illicit stimulants use following 1 year (adjusted OR: 2.42 (1.51-3.8)). The adjusted odds ratio was 2.7 (1.08-7.84) among participants who had used illicit stimulant at least once (continuation) and 2.25 (1.04-4.37) among participants who had never used illicit stimulants at inclusion (initiation). CONCLUSION: High levels of ADHD symptoms are a feature that may promote both initiation and continuation of illicit stimulants use among university students. Our findings suggest that university students with high levels of ADHD symptoms may benefit from screening to help identify those at risk of illicit stimulants use.
Introduction: A highly efficacious and durable vaccine against malaria is an essential tool for global malaria eradication. One of the promising strategies to develop such a vaccine is to induce robust CD8+ T cell mediated immunity against malaria liver-stage parasites. Methods: Here we describe a novel malaria vaccine platform based on a secreted form of the heat shock protein, gp96-immunoglobulin, (gp96-Ig) to induce malaria antigen specific, memory CD8+ T cells. Gp96-Ig acts as an adjuvant to activate antigen presenting cells (APCs) and chaperone peptides/antigens to APCs for cross presentation to CD8+ T cells. Results: Our study shows that vaccination of mice and rhesus monkeys with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum CSP and AMA1 (PfCA) vaccine candidate antigens, induces liver-infiltrating, antigen specific, memory CD8+ T cell responses. The majority of the intrahepatic CSP and AMA1 specific CD8+ T cells expressed CD69 and CXCR3, the hallmark of tissue resident memory T cells (Trm). Also, we found intrahepatic, antigen-specific memory CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses in the liver. Discussion: Our novel gp96-Ig malaria vaccine strategy represents a unique approach to induce liver-homing, antigen-specific CD8+ T cells critical for Plasmodium liver-stage protection.
Malaria Vaccines , Malaria, Falciparum , Malaria , Humans , Heat-Shock Proteins/metabolism , HEK293 Cells , CD8-Positive T-Lymphocytes , Immunoglobulins/metabolism , Antigens, Protozoan , Malaria/prevention & control , Malaria/metabolism
The nutritional value, heavy metal content and lipid quality of five marine fishes from, Cameroon coast were be investigated. Fish samples from Ilisha africana, Sardinella, maderensis, Cyprinus carpio, Arius parkii and Ethmalosa fimbriata were collected at, the Douala sea port, carried to the laboratory, washed with distilled water and, processed. Proximal composition, minerals, lipid quality and heavy metal analyses, were performed using AOAC standard methods. Results show that proteins (18.43%), and lipids (3.69%) contents were higher in Ilisha africana. Cyprinus carpio had the, highest ash content (4.59%). Contents of minerals and heavy metals were found as, follows: P > Mg > K > Ca > Na > Fe > Zn > Cu > Mn and Hg > Pb > Cd > As. Oils extracted from C. carpio, A. parkii and E. fimbriata were semi-siccative while those of I. africana and S. maderensis were siccative. Thus, these fish species are good sources of proteins and, minerals that can be used for managing mineral deficiencies in humans and animals.
The long-term success of intraosseous transcutaneous amputation prostheses (ITAPs) mainly relies on dermal attachment of skin cells to the implant. Otherwise, bacteria can easily penetrate through the interface between the implant and the skin. Therefore, infection at this implant/skin interface remains a significant complication in orthopedic surgeries in which these prostheses are required. Two main strategies were investigated to prevent these potential infection problems which consist in either establishing a strong sealing at the skin/implant interface or on eradicating infections by killing bacteria. In this work, two adhesion peptides, either KRGDS or KYIGSR and one antimicrobial peptide, Magainin 2 (Mag 2), were covalently grafted via phosphonate anchor arms to the surface of the Ti6Al4V ELI (extra low interstitials) material, commonly used to manufacture ITAPs. X-ray photoelectron spectroscopy, contact angle, and confocal microscopy analyses enabled to validate the covalent and stable grafting of these three peptides. Dermal fibroblasts cultures on bare Ti6Al4V ELI surfaces and functionalized ones displayed a good cell adhesion and proliferation on all samples. However, cell spreading and viability appeared to be improved on grafted surfaces, especially for those conjugated with KRGDS and Mag 2. Moreover, the dermal sheet attachment, was significantly higher on surfaces functionalized with Mag 2 as compared to the other surfaces. Therefore, the surface functionalization with the antimicrobial peptide Mag 2 seems to be the best approach for the targeted application, as it could play a dual role, inducing a strong skin adhesion while limiting infections on Ti6Al4V ELI materials.
Prostheses and Implants , Titanium , Titanium/chemistry , Cell Adhesion , Peptides , Amputation, Surgical , Antimicrobial Peptides , Surface Properties
Temporal-lobe epilepsy in humans is often associated with widespread, synchronized neuron firing that co-occurs with traveling waves in local field potential. These traveling waves generate stochastic oscillations in a time series of microelectrode voltage, and previous work has deemed it informative for traveling-wave analysis to study the mean periodicity. This manuscript reveals that: a) mean voltage (i.e., traveling-wave periodicity) adequately explains the observed voltage periodicity only for a select few time intervals during seizure; and b) mean voltage has a 7 Hz cosine-series representation indicative of a nonlinear system response given alpha-rhythm input. The a) result implies that residual noise should be modelled explicitly, while b) motivates a departure from the conventional plane-wave modeling regime in source-localization efforts. The 7 Hz fundamental frequency is unsurprising given the relative transparency of the brain to 14 Hz alpha rhythms in neurophysiological diseases (14 Hz being a subharmonic frequency of the 7 Hz signal).
The self-assembled skin substitute (SASS) is an autologous bilayered skin substitute designed by our academic laboratory, the Laboratoire d'Organogenèse Expérimentale (LOEX) to offer definitive treatment for patients lacking donor sites (unwounded skin) to cover their burn wounds. This product shows skin-like attributes, such as an autologous dermal and epidermal layer, and is easily manipulable by the surgeon. Its development stems from the need for skin replacement in high total body surface area burned survivors presenting few donor sites for standard split-thickness skin grafting. This review aims to present the history, successes, challenges, and current therapeutic indications of this skin substitute. We review the product's development history, before discussing current production techniques, as well as clinical use. The progression observed since the initial SASS production technique described in 1999, up to the most recent technique expresses significant advances made in the technical aspect of our product, such as the reduction of the production time. We then explore the efficacy and benefits of SASS over existing skin substitutes and discuss the outcomes of a recent study focusing on the successful treatment of 14 patients. Moreover, an ongoing cross-Canada study is further assessing the product's safety and efficacy. The limitations and technical challenges of SASS are also discussed.
Burns , Skin, Artificial , Humans , Burns/surgery , Skin , Skin Transplantation/methods , Epidermis
MsbA is a member of the ATP-binding cassette (ABC) transporter family and harnesses the energy from adenosine triphosphate (ATP) binding and hydrolysis to flip lipopolysaccharide (LPS) across the cytoplasmic membrane in Gram-negative bacteria. MsbA is an essential component of the bacterial envelope biogenesis pathway and an attractive target for developing novel antibiotics against multidrug-resistant strains. Structural characterization of MsbA in different conformations provides crucial insights in understanding druggable pockets and mechanisms of inhibition of this transporter. Recent advances in membrane-mimetic environments and cryo-EM data acquisition and processing have enabled high-resolution imaging of MsbA in complex with its native LPS substrate. Despite these technical advances, MsbA remains a challenging target for cryo-EM analysis due to its small size and extraordinary conformational flexibility. Herein, we provide a protocol for the purification and incorporation of MsbA in lipid nanodiscs, cryo-EM sample preparation, and cryo-EM image processing. The method outlined here is generalizable to the study of other bacterial ABC transporters, including the LPS extractor LptB2FGC.
Escherichia coli Proteins , Lipopolysaccharides , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Cryoelectron Microscopy , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Lipopolysaccharides/metabolism
Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27-79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60-98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.
During human seizures organized waves of voltage activity rapidly sweep across the cortex. Two contradictory theories describe the source of these fast traveling waves: either a slowly advancing narrow region of multiunit activity (an ictal wavefront) or a fixed cortical location. Limited observations and different analyses prevent resolution of these incompatible theories. Here we address this disagreement by combining the methods and microelectrode array recordings (N=11 patients, 2 females, N=31 seizures) from previous human studies to analyze the traveling wave source. We find - inconsistent with both existing theories - a transient relationship between the ictal wavefront and traveling waves, and multiple stable directions of traveling waves in many seizures. Using a computational model that combines elements of both existing theories, we show that interactions between an ictal wavefront and fixed source reproduce the traveling wave dynamics observed in vivo We conclude that combining both existing theories can generate the diversity of ictal traveling waves.Significance StatementThe source of voltage discharges that propagate across cortex during human seizures remains unknown. Two candidate theories exist, each proposing a different discharge source. Support for each theory consists of observations from a small number of human subject recordings, analyzed with separately developed methods. How the different, limited data and different analysis methods impact the evidence for each theory is unclear. To resolve these differences, we combine the unique, human microelectrode array recordings collected separately for each theory and analyze these combined data with a unified approach. We show that neither existing theory adequately describes the data. We then propose a new theory that unifies existing proposals and successfully reproduces the voltage discharge dynamics observed in vivo.
OBJECTIVES: Caloric restriction (CR) has been shown to slow the aging processes in a number of preclinical studies and reduces expression of aging-associated biomarkers in human trials. We hypothesized that CR would lead to increased incidence of ketosis and that ketosis in CR individuals would alter the aging-protective effects of CR or biomarkers thereof. DESIGN/SETTING/PARTICIPANTS: We analyzed data from the "Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE, Phase 2)" Public Use Database available at calerie.duke.edu. In this study, non-obese adults between the ages of 21 and 50 were randomized to 25% CR or control (ad lib) diet groups and extensively monitored for two years. Given our focus on the effect of caloric restriction on ketosis, individuals with detectible ketones during the baseline visit (pre-randomization) and those with missing data for ketone testing were excluded from the analysis, leaving 71 control and 117 CR participants. MEASUREMENTS: We analyzed the incidence of ketosis as well as ketosis free survival in control and CR participants and assessed the effect of ketosis on a number of clinical lab values, functional assessments, and participant survey data related to aging biology. RESULTS: We report that CR was associated with modestly increased incidence of ketosis (4.4% in CR vs 1.9% in control), though CR-associated changes in T3, VO2, SUMPT-WT (weight normalized composite strength score - peak torque), physical functioning, and general health did not appear to be altered by the presence or absence of ketosis. Additional observations of interest include: 1) striking patterns of biomarker expression changes (MCP-1, TNFα, TGF-ß1, GH) in both the control and CR participants between the baseline visit and the 24-month post-randomization visit and 2) pro-growth/anti-inflammatory baseline (pre-randomization) biomarker expression profile in CR individuals that later test ketone positive relative to other CR individuals. CONCLUSIONS: CR modestly increases the incidence of ketosis in healthy adults, yet the increase in ketosis in CR patients did not significantly affect the aging-protective effects of CR. However, given the relatively small number of participants who were ketone positive, further investigation in larger study cohorts is still required for definitive conclusions.
Aging , Caloric Restriction , Diet , Humans , Incidence , Ketones
High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)-involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs-the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.
RNA , Riboswitch , Cryoelectron Microscopy , Ligands , RNA/genetics , Single Molecule Imaging
We show that the slow viscoelastic response of a foam is that of a power-law fluid with a terminal relaxation. Investigations of the foam mechanics in creep and recovery tests reveal that the power-law contribution is fully reversible, indicative of a delayed elastic response. We demonstrate how this contribution fully accounts for the non-Maxwellian features observed in all tests, probing the linear mechanical response function. The associated power-law spectrum is consistent with soft glassy rheology of systems with mechanical noise temperatures just above the glass transition [Fielding et al., J. Rheol. 44, 323 (2000)] and originates from a combination of superdiffusive bubble dynamics and stress diffusion, as recently evidenced in simulations of coarsening foam [Hwang et al., Nat. Mater. 15, 1031 (2016)].
