The Influence of Lipid Electric Charge on the Binding of Aß(1-42) Amyloid Peptide to Bilayers in the Liquid-Ordered State.

The amyloidogenic Aß peptides are widely considered as a pathogenic agent in Alzheimer's disease. Aß(1-42) would form aggregates of amyloid fibrils on the neuron plasma membranes, thus perturbing neuronal functionality. Conflicting data are available on the influence of bilayer order on Aß(1-42) binding to membranes. In the present study, a biophysical approach was used in which isothermal calorimetry and surface pressure measurements were applied to explore the interaction of Aß(1-42) in either monomeric, oligomeric, or fibrillar form with model membranes (bilayers or monolayers) in the liquid-ordered state that were either electrically neutral or negatively charged. In the latter case, this contained phosphatidic acid, cardiolipin, or ganglioside. The calorimetric studies showed that Aß(1-42) fibrils, oligomers, and monomers could bind and/or be inserted into bilayers, irrespective of electric charge, in the liquid-ordered state, except that monomers could not interact with electrically neutral bilayers. The monolayer studies in the Langmuir balance demonstrated that Aß(1-42) aggregation hindered peptide insertion into the monolayer, hindered insertion in the decreasing order of monomer > oligomer > fibril, and that lipid composition did not cause large differences in insertion, apart from a slight facilitation of monomer and oligomer insertion by gangliosides.

The N-terminal region of the ATG8 autophagy protein LC3C is essential for its membrane fusion properties.

Autophagy is a catabolic process in which a double-membrane organelle, the autophagosome (AP), engulfs cellular components that will be degraded in the lysosomes. ATG8 protein family members participate at various stages of AP formation. The present study compares the capacity to induce lipid-vesicle tethering and fusion of two ATG8 family members, LC3B and LC3C, with model membranes. LC3B is the most thoroughly studied ATG8 protein. It is generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. LC3C is less studied, but recent data have reported its implication in various processes, crucial to cellular homeostasis. The results in this paper show that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. As the N-terminus of LC3C is different from that of the other family members, various mutants of the N-terminal region of both LC3B and LC3C were designed, and their activities compared. It was concluded that the N-terminal region of LC3C was responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B. The results suggest a specialized function of LC3C in the AP expansion process.

Noninvasive Detection of Neuroendocrine Prostate Cancer through Targeted Cell-free DNA Methylation.

Castration-resistant prostate cancer (CRPC) is a heterogeneous disease associated with phenotypic subtypes that drive therapy response and outcome differences. Histologic transformation to castration-resistant neuroendocrine prostate cancer (CRPC-NE) is associated with distinct epigenetic alterations, including changes in DNA methylation. The current diagnosis of CRPC-NE is challenging and relies on metastatic biopsy. We developed a targeted DNA methylation assay to detect CRPC-NE using plasma cell-free DNA (cfDNA). The assay quantifies tumor content and provides a phenotype evidence score that captures diverse CRPC phenotypes, leveraging regions to inform transcriptional state. We tested the design in independent clinical cohorts (n = 222 plasma samples) and qualified it achieving an AUC > 0.93 for detecting pathology-confirmed CRPC-NE (n = 136). Methylation-defined cfDNA tumor content was associated with clinical outcomes in two prospective phase II clinical trials geared towards aggressive variant CRPC and CRPC-NE. These data support the application of targeted DNA methylation for CRPC-NE detection and patient stratification. SIGNIFICANCE: Neuroendocrine prostate cancer is an aggressive subtype of treatment-resistant prostate cancer. Early detection is important, but the diagnosis currently relies on metastatic biopsy. We describe the development and validation of a plasma cell-free DNA targeted methylation panel that can quantify tumor fraction and identify patients with neuroendocrine prostate cancer noninvasively. This article is featured in Selected Articles from This Issue, p. 384.

Ceramide regulation of autophagy: A biophysical approach.

Specific membrane lipids play unique roles in (macro)autophagy. Those include phosphatidylethanolamine, to which LC3/GABARAP autophagy proteins become covalently bound in the process, or cardiolipin, an important effector in mitochondrial autophagy (or mitophagy). Ceramide (Cer), or N-acyl sphingosine, is one of the simplest sphingolipids, known as a stress signal in the apoptotic pathway. Moreover, Cer is increasingly being recognized as an autophagy activator, although its mechanism of action is unclear. In the present review, the proposed Cer roles in autophagy are summarized, together with some biophysical properties of Cer in membranes. Possible pathways for Cer activation of autophagy are discussed, including specific protein binding of the lipid, and Cer-dependent perturbation of bilayer properties. Cer generation of lateral inhomogeneities (domain formation) is given special attention. Recent biophysical results, including fluorescence and atomic force microscopy data, show Cer-promoted enhanced binding of LC3/GABARAP to lipid bilayers. These observations could be interpreted in terms of the putative formation of Cer-rich nanodomains.

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers.

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.

Immunotherapy targeting different immune compartments in combination with radiation therapy induces regression of resistant tumors.

Radiation therapy (RT) increases tumor response to CTLA-4 inhibition (CTLA4i) in mice and in some patients, yet deep responses are rare. To identify rational combinations of immunotherapy to improve responses we use models of triple negative breast cancer highly resistant to immunotherapy in female mice. We find that CTLA4i promotes the expansion of CD4+ T helper cells, whereas RT enhances T cell clonality and enriches for CD8+ T cells with an exhausted phenotype. Combination therapy decreases regulatory CD4+ T cells and increases effector memory, early activation and precursor exhausted CD8+ T cells. A combined gene signature comprising these three CD8+ T cell clusters is associated with survival in patients. Here we show that targeting additional immune checkpoints expressed by intratumoral T cells, including PD1, is not effective, whereas CD40 agonist therapy recruits resistant tumors into responding to the combination of RT and CTLA4i, indicating the need to target different immune compartments.

Thermal insulation impact on overheating vulnerability reduction in Mediterranean dwellings.

Heat waves are expected to increase the use of air conditioning (AC), deriving in higher energy consumption. This research aims to determine whether thermal insulation is an effective retrofit strategy for tackling overheating. Four occupied dwellings in southern Spain were monitored: two houses built prior to any thermal criteria and two with current thermal standards. Thermal comfort is assessed considering adaptive models and user patterns for the operation of AC and natural ventilation. Results show that a high level of insulation combined with a proper use of night-time natural ventilation can increase thermal comfort hours under heat waves, lasting 2-5 times longer than in poorly-insulated houses and with up to 2 °C temperature difference at nights. Long-term effectiveness of insulation under extreme heat presents a better thermal performance, especially in intermediate floors. Yet, the activation of AC usually occurs with indoor temperatures of 27-31 °C, regardless of the envelope's solution.

Vesicle tethering and fusion promoted by LC3/GABARAP proteins is modulated by the ATG12-ATG5-ATG16L1 complex.

Recently, we have examined the membrane anchoring and subsequent lipidation of six members of the LC3/GABARAP protein family, together with their ability to promote membrane tethering and fusion. GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggested the existence of a lipidation threshold as a requisite for tethering and inter-vesicular lipid mixing. The presence of ATG12-ATG5-ATG16L1 (E3 in short) increased and accelerated LC3/GABARAP lipidation and subsequent vesicle tethering. However, E3 hampered LC3/GABARAP capacity to induce inter-vesicular lipid mixing and/or fusion. Our results suggest a model in which, together with the recently described inter-membrane lipid transfer mechanism, LC3/GABARAP could help in the phagophore expansion process through their ability to tether and fuse vesicles. The growing regions would be areas where the LC3/GABARAP proteins could be lipidated in the absence of E3, or else an independent regulatory mechanism would allow lipid/vesicle incorporation and phagophore growth when E3 was present.Abbreviations: Atg/ATG: autophagy-related protein (in yeast/human); E3: ATG12-ATG5-ATG16L1 complex; GABARAP: gamma-aminobutyric acid receptor associated protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3.

Lipids in Mitochondrial Macroautophagy: Phase Behavior of Bilayers Containing Cardiolipin and Ceramide.

Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction.

Multicenter phase 2 study of oral azacitidine (CC-486) plus CHOP as initial treatment for PTCL.

Peripheral T-cell lymphomas (PTCL) with T-follicular helper phenotype (PTCL-TFH) has recurrent mutations affecting epigenetic regulators, which may contribute to aberrant DNA methylation and chemoresistance. This phase 2 study evaluated oral azacitidine (CC-486) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as initial treatment for PTCL. CC-486 at 300 mg daily was administered for 7 days before C1 of CHOP, and for 14 days before CHOP C2-6. The primary end point was end-of-treatment complete response (CR). Secondary end points included safety and survival. Correlative studies assessed mutations, gene expression, and methylation in tumor samples. Grade 3 to 4 hematologic toxicities were mostly neutropenia (71%), with febrile neutropenia uncommon (14%). Nonhematologic toxicities included fatigue (14%) and gastrointestinal symptoms (5%). In 20 evaluable patients, CR was 75%, including 88.2% for PTCL-TFH (n = 17). The 2-year progression-free survival (PFS) was 65.8% for all and 69.2% for PTCL-TFH, whereas 2-year overall survival (OS) was 68.4% for all and 76.1% for PTCL-TFH. The frequencies of the TET2, RHOA, DNMT3A, and IDH2 mutations were 76.5%, 41.1%, 23.5%, and 23.5%, respectively, with TET2 mutations significantly associated with CR (P = .007), favorable PFS (P = .004) and OS (P = .015), and DNMT3A mutations associated with adverse PFS (P = .016). CC-486 priming contributed to the reprograming of the tumor microenvironment by upregulation of genes related to apoptosis (P < .01) and inflammation (P < .01). DNA methylation did not show significant shift. This safe and active regimen is being further evaluated in the ALLIANCE randomized study A051902 in CD30-negative PTCL. This trial was registered at www.clinicaltrials.gov as #NCT03542266.

Effect of ATG12-ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion.

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12-ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.

LC3/GABARAP binding to fluid membranes is potentiated by ceramide.

LC3/GABARAP constitute a macroautophagy/autophagy-related protein family derived from yeast Atg8. The involvement of specific lipids in LC3/GABARAP function is poorly understood. Exploring the interaction of LC3/GABARAP proteins with phosphatidylcholine- or sphingomyelin-based bilayers has revealed that cardiolipin is essential for the protein-bilayer interaction, and that ceramide markedly increases binding. Giant unilamellar vesicles examined under confocal fluorescence microscopy reveal that ceramide segregates laterally into very rigid domains, while GABARAP binds only the more fluid regions, suggesting that the enhancing role of ceramide is exerted by the minority of ceramide molecules dispersed in the fluid phase.Abbreviations: Atg8: autophagy-related 8; Cer: ceramide; CL: cardiolipin; eCer: egg ceramide; GABARAP: gamma-aminobutyric acid receptor associated protein; GUV: giant unilamellar vesicle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SM: sphingomyelin.

Detergentes: De los principios físicos a las aplicaciones biofarmacéuticas (o por qué prevenimos la covid-19 con agua y jabón / Detergents: From physical principles to biopharmaceutical applications (or why we fight covid-19 with toilet soap)

Los detergentes son anfifilos solubles que poseen la capacidad de solubilizar grasas, dando lugar a micelas mixtas lípido-detergente, que son solubles en agua. Los detergentes son ampliamente utilizados en las industrias alimentaria y de bebidas, textil, médica y farmacéutica, entre otras. En biología molecular, los detergentes son herramientas insustituibles en la solubilización de las membranas celulares y la posterior purificación de proteínas de membrana. La presente revisión resume cuatro décadas de investigación sobre detergentes en el laboratorio de los autores. Una introducción sobre los detergentes y las membranas va seguida por una descripción cuantitativa detallada del mecanismo de solubilización de la membrana por los detergentes, y por una discusión crítica del concepto de membranas resistentes a los detergentes en relación con la hipótesis de las balsas lipídicas (rafts). A continuación, se incluye una sección experimental que resume los principales resultados del grupo de los autores. Finalmente, se describen algunas aplicaciones biofarmacéuticas. Como ejemplo práctico, se discute el uso de jabón de tocador en la prevención de la COVID-19. (AU)

Detergents are soluble amphiphiles that possess the capacity to solubilize fats, giving rise to water-soluble, lipid-detergent mixed micelles. Detergents find an extensive use in food and drink, textile, medical and pharmaceutical industries, among others. In molecular biology, detergents are irreplaceable tools in the solubilization of cell membranes and subsequent membrane protein purification. The present review summarizes four decades of investigation on detergents in the authors’ laboratory. An introduction on detergents and membranes is followed by a detailed, quantitative description of the mechanism of membrane solubilization by detergents, and a critical discussion of the concept of detergent-resistant membranes as related to the lipid raft hypothesis. An experimental section follows, summarizing the main results in the authors’ group. Finally, some biopharmaceutical applications are described. As a working example, the use of toilet soap in the prevention of COVID-19 is discussed. (AU)

Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in Lithospermum officinale.

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

Transcriptional dynamics of Chitinophaga sp. strain R-73072-mediated alkannin/shikonin biosynthesis in Lithospermum officinale.

Plants are colonized by a wide range of bacteria, several of which are known to confer benefits to their hosts such as enhancing plant growth and the biosynthesis of secondary metabolites (SMs). Recently, it has been shown that Chitinophaga sp. strain R-73072 enhances the production of alkannin/shikonin, SMs of pharmaceutical and ecological importance. However, the mechanisms by which this bacterial strain increases these SMs in plants are not yet understood. To gain insight into these mechanisms, we analyzed the molecular responses of Lithospermum officinale, an alkannin/shikonin producing member of Boraginaceae, to inoculation with R-73072 in a gnotobiotic system using comparative transcriptomics and targeted metabolite profiling of root samples. We found that R-73072 modulated the expression of 1,328 genes, of which the majority appeared to be involved in plant defense and SMs biosynthesis including alkannin/shikonin derivatives. Importantly, bacterial inoculation induced the expression of genes that predominately participate in jasmonate and ethylene biosynthesis and signaling, suggesting an important role of these phytohormones in R-73072-mediated alkannin/shikonin biosynthesis. A detached leaf bioassay further showed that R-73072 confers systemic protection against Botrytis cinerea. Finally, R-73072-mediated coregulation of genes involved in plant defense and the enhanced production of alkannin/shikonin esters further suggest that these SMs could be important components of the plant defense machinery in alkannin/shikonin producing species.

Root-associated bacteria modulate the specialised metabolome of Lithospermum officinale L.

Bacteria influence plant growth and development and therefore are attractive resources for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of bacteria on the modulation of SM production in the medicinal plant Lithospermum officinale (Boraginaceae family) with a focus on the naphthoquinones alkannin/shikonin and their derivatives (A/Sd). The study was conducted in an in vitro cultivation system developed for that purpose, as well as in a greenhouse. Targeted and non-targeted metabolomics were performed, and expression of the gene PGT encoding for a key enzyme in the A/S biosynthesis pathway was evaluated with qPCR. Three strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of A/Sd in L. officinale in both systems, demonstrating the strength of our approach for screening A/Sd-inducing bacteria. The bacterial treatments altered other plant metabolites derived from the shikimate pathway as well. Our results demonstrate that bacteria influence the biosynthesis of A/Sd and interact with different metabolic pathways. This work highlights the potential of bacteria to increase the production of SM in medicinal plants and reveals new patterns in the metabolome regulation of L. officinale.

Phase behaviour of C18-N-acyl sphingolipids, the prevalent species in human brain.

Lipidomic analysis of the N-acyl components of sphingolipids in different mammalian tissues had revealed that brain tissue differed from all the other samples in that SM contained mainly C18:0 and C24:1N-acyl chains, and that the most abundant Cer species was C18:0. Only in the nervous system was C18:0 found in sizable proportions. The high levels of C18:0 and C16:0, respectively in brain and non-brain SM, were important because SM is by far the most abundant sphingolipid in the plasma membrane. In view of these observations, the present paper is devoted to a comparative study of the properties of C16:0 and C18:0 sphingolipids (SM and Cer) pure and in mixtures of increasing complexities, using differential scanning calorimetry, confocal microscopy of giant unilamellar vesicles, and correlative fluorescence microscopy and atomic force microscopy of supported lipid bilayers. Membrane rigidity was measured by force spectroscopy. It was found that in mixtures containing dioleoyl phosphatidylcholine, sphingomyelin and cholesterol, i.e. representing the lipids predominant in the outer monolayer of cell membranes, lateral inhomogeneities occurred, with the formation of rigid domains within a continuous fluid phase. Inclusion of saturated Cer in the system was always found to increase the rigidity of the segregated domains. C18:0-based sphingolipids exhibit hydrocarbon chain-length asymmetry, and some singularities observed with this N-acyl chain, e.g. complex calorimetric endotherms, could be attributed to this property. Moreover, C18:0-based sphingolipids, that are typical of the excitable cells, were less miscible with the fluid phase than their C16:0 counterparts. The results could be interpreted as suggesting that the predominance of C18:0 Cer in the nervous system would contribute to the tightness of its plasma membranes, thus facilitating maintenance of the ion gradients.

Ceramide enhances binding of LC3/GABARAP autophagy proteins to cardiolipin-containing membranes.

Macroautophagy, or autophagy, is a process in which cell macromolecules, or even organelles, are engulfed in a double-membrane vesicle, the autophagosome, and directed to a lysosome. Among autophagy-related proteins, LC3/GABARAP constitute a protein family derived from yeast Atg8. They play important roles in autophagosome formation, binding future cargo organelles and promoting autophagosome growth. The involvement of specific lipids in this process is poorly understood. The present study explores the interaction of LC3/GABARAP proteins with phospholipid monolayers and bilayers based on phosphatidylcholine or on sphingomyelin. Cardiolipin is found to be essential for the protein interaction with such bilayers, as measured through gradient centrifugation experiments, while ceramide markedly increases binding. Giant unilamellar vesicles examined under confocal fluorescence microscopy reveal that ceramide segregates laterally into very rigid domains, while GABARAP binds only the more fluid regions, suggesting that the enhancing role of ceramide is exerted by the minority of ceramide molecules dispersed in the fluid phase. Although in further autophagy steps the LC3/GABARAP proteins are covalently bound to a phospholipid, this is not the case in our system, thus it is proposed that the observed ceramide effects would correspond to very early stages in the process, such as cargo recognition.

Detection of infiltrating fibroblasts by single-cell transcriptomics in human kidney allografts.

We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune injury. We selected 3 biopsies of kidney cortex from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy, tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We did not study T-cell-mediated rejections. We generated 7217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches revealed by X and Y chromosome autosomal gene expression, we determined that in AK1 with fibrosis, 42 months after transplantation, more than half of the kidney allograft fibroblasts were recipient-derived and therefore likely migratory and graft infiltrative, whereas in AK2 without fibrosis, 84 months after transplantation, most fibroblasts were donor-organ-derived. Furthermore, AK1 was enriched for tubular progenitor cells overexpressing profibrotic extracellular matrix genes. AK2, eight months after successful treatment of rejection, contained plasmablast cells with high expression of immunoglobulins, endothelial cell elaboration of T cell chemoattractant cytokines, and persistent presence of cytotoxic T cells. In addition to these key findings, our analysis revealed unique cell types and states in the kidney. Altogether, single-cell transcriptomics yielded novel mechanistic insights, which could pave the way for individualizing the care of transplant recipients.

Autophagy protein LC3C binding to phospholipid and interaction with lipid membranes.

Autophagy is a process in which parts of the eukaryotic cell are selectively degraded in the lysosome. The materials to be catabolized are first surrounded by a double-membrane structure, the autophagosome. Autophagosome generation is a complex event, in which many proteins are involved. Among the latter, yeast Atg8 or its mammalian orthologues are essential in autophagosome membrane elongation, shaping and closure. A subfamily of the human Atg8 orthologues is formed by the proteins LC3A, LC3B, and LC3C. Previous studies suggest that, at variance with the other two, LC3C does not participate in cardiolipin-mediated mitophagy. The present study was devoted to exploring the binding of LC3C to lipid vesicles, bilayers and monolayers, and the ensuing protein-dependent perturbing effects, in the absence of the mitochondrial lipid cardiolipin. All Atg8 orthologues are covalently bound to a phospholipid prior to their involvement in autophagosome elongation. In our case, a mutant in the C-terminal amino acid, LC3C G126C, together with the use of a maleimide-derivatized phosphatidyl ethanolamine, ensured LC3C lipidation, up to 100% under certain conditions. Ultracentrifugation, surface pressure measurements, spectroscopic and cryo-electron microscopic techniques revealed that lipidated LC3C induced vesicle aggregation (5-fold faster in sonicated than in large unilamellar vesicles) and inter-vesicular lipid mixing (up to 82%), including inner-monolayer lipid mixing (up to 32%), consistent with in vitro partial vesicle fusion. LC3C was also able to cause the release of 80-90% vesicular aqueous contents. The data support the idea that LC3C would be able to help in autophagosome elongation/fusion in autophagy phenomena.