The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an ...atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design.
This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific.
In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.
IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed ...tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1β, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1β and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis.
Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we ...studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.
Display omitted
•Single-cell and spatial sequencing reveals key features of renal cell carcinoma (RCC)•Intra-tumoral heterogeneity of CD8+ clonotypes dwarfs that from somatic mutations•RCC cells showing epithelial-mesenchymal transition (EMT) enrich at tumor edges•Macrophages expressing IL1B correlate with EMT and could have therapeutic importance
Li et al. use single-cell and spatial sequencing to examine the cellular features of kidney tumors and classify cancer cells according to function. They find a more invasive phenotype at the interface separating tumor with normal kidney, which appears to be driven by IL-1β signaling in macrophages.
The perylene derivative 1,7-dibromoperylene-3,4,9,10-tetracarboxylic tetrabutylester has been obtained in regioisomerically pure form, by employing a highly efficient, scalable, and robust synthesis ...starting from commercially available perylene-3,4,9,10-tetracarboxylic bisanhydride. Subsequently, this compound is utilized for the synthesis of extremely valuable and versatile regioisomerically pure intermediates, namely, 1,7-dibromoperylene-3,4,9,10-tetracarboxylic dibutylester monoanhydride, 1,7-dibromoperylene-3,4,9,10-tetracarboxylic bisanhydride, and 1,7-dibromoperylene monoimid monoanhydride. These compounds possess at least one anhydride functionality in addition to the 1,7 bromo substituents and thus allow for a virtually limitless attachment of substituents both at the “peri” and the “bay” positions. The intermediate 1,7-dibromoperylene monoimide monoanhydride is of special interest as it provides access to unsymmetrically imide-substituted 1,7-dibromoperylene derivatives, which are not accessible by previously known procedures. Finally, substitution of the 1,7 bromine atoms in the bay area by phenoxy groups, which is a generally applied reaction for 1,7-dibromoperylene bisimides, was proven to be equally effective for a 1,7-dibromoperylene tetraester and a 1,7-dibromoperylene diester monoimid.
We report herein a versatile and user-friendly synthetic methodology based on sequential functionalization that enables the synthesis of previously unknown perylene bisimide (PBI) dyes with up to ...five different substituents attached to the perylene core (e.g., compound 15). The key to the success of our strategy is a highly efficient regiospecific 7-mono- and 7,12-di-phenoxy bay substitution at the “imide-activated” 7- and 12-bay positions of 1,6,7,12-tetrachloroperylene monoimide diester 1. The facile subsequent conversion of the diester groups into an imide group resulted in novel PBIs (e.g., compound 14) with two phenoxy substituents specifically at the 7- and 12-bay positions. This conversion led to the activation of C-1 and C-6 bay positions, and thereafter, the remaining two chlorine atoms were substituted to obtain tetraphenoxy-PBI (compound 15) that has two different imide and three different bay substituents. The methodology provides excellent control over the functionalization pattern, which enables the synthesis of various regioisomeric pairs bearing the same bay substituents. Another important feature of this strategy is the high sensitivity of HOMO–LUMO energies and photoinduced charge transfer toward sequential functionalization. As a result, systematic fluorescence on–off switching has been demonstrated upon subsequent substitution with the electron-donating 4-methoxyphenoxy substituent.
In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation ...of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans.
Nucleophilic aromatic substitution reactions on 1,7-dibromoperylene-3,4,9,10-tetracarboxylic monoimide dibutylester, using phenol and pyrrolidine reagents, have been exploited to synthesize perylenes ...with four different substituents at the perylene core. The first substitution is always regiospecific at the imide-activated 7-position. A second substitution reaction does not always replace the bromine at C-1, but may replace a phenol substituent at the highly activated 7-position. Exploiting this reactivity pattern, a “mixed” 1,7-diphenoxy, 1,7-dipyrrolidinyl, and two 1-phenoxy-7-pyrrolidinyl derivatives have been synthesized.
A robust and scalable procedure to obtain pure 1,6,7,12-tetrachloroperylene-3,4,9,10-tetracarboxy bisanhydride, a highly valuable synthon, has been developed via synthesis of a novel intermediate ...compound 1,6,7,12-perylene-3,4,9,10-tetracarboxy tetrabutylester.
Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation. Oxidative damage to mitochondria is an early event in IR injury. Therefore, the uptake, ...safety, and efficacy of the mitochondria-targeted antioxidant MitoQ were investigated in models of transplant IR injury.
MitoQ uptake by warm and cooled pairs of pig and declined human kidneys was measured when preserved in cold static storage or by hypothermic machine perfusion. Pairs of pigs' kidneys were exposed to defined periods of warm and cold ischaemia, flushed and stored at 4°C with or without MitoQ (50 nmol/l to 250 µmol/l), followed by reperfusion with oxygenated autologous blood in an ex vivo normothermic perfusion (EVNP). Pairs of declined human kidneys were flushed and stored with or without MitoQ (5-100 µmol/l) at 4°C for 6 h and underwent EVNP with ABO group-matched blood.
Stable and concentration-dependent uptake of MitoQ was demonstrated for up to 24 h in pig and human kidneys. Total blood flow and urine output were significantly greater in pig kidneys treated with 50 µmol/l MitoQ compared with controls (P = 0.006 and P = 0.007 respectively). In proof-of-concept experiments, blood flow after 1 h of EVNP was significantly greater in human kidneys treated with 50 µmol/l MitoQ than in controls (P ≤ 0.001). Total urine output was numerically higher in the 50-µmol/l MitoQ group compared with the control, but the difference did not reach statistical significance (P = 0.054).
Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation.
We report the synthesis and excited-state dynamics of a series of five bichromophoric light-harvesting antenna systems, which are capable of efficient harvesting of solar energy in the spectral range ...of 350-580 nm. These antenna systems have been synthesized in a modular fashion by the covalent attachment of blue light absorbing naphthalene monoimide energy donors (
,
, and
) to green light absorbing perylene-3,4,9,10-tetracarboxylic acid derived energy acceptors, 1,7-perylene-3,4,9,10-tetracarboxylic tetrabutylester (
), 1,7-perylene-3,4,9,10-tetracarboxylic monoimide dibutylester (
), and 1,7-perylene-3,4,9,10-tetracarboxylic bisimide (
). The energy donors have been linked at the 1,7-bay-positions of the perylene derivatives, thus leaving the
positions free for further functionalization and device construction. A highly stable and rigid structure, with no electronic communication between the donor and acceptor components, has been realized
an all-aromatic non-conjugated phenoxy spacer between the constituent chromophores. The selection of donor naphthalene derivatives for attachment with perylene derivatives was based on the effective matching of their respective optical properties to achieve efficient excitation energy transfer (EET) by the Förster mechanism. A comprehensive study of the excited-state dynamics, in toluene, revealed quantitative and ultrafast (
1 ps) intramolecular EET from donor naphthalene chromophores to the acceptor perylenes in all the studied systems. Electron transfer from the donor naphthalene chromophores to the acceptor perylenes has not been observed, not even for antenna systems in which this process is thermodynamically allowed. Due to the combination of an efficient and fast energy transfer along with broad absorption in the visible region, these antenna systems are promising materials for solar-to-electric and solar-to-fuel devices.
PDF
