Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely FcγR-independent manner. These Fc-silenced anti-CD40 antibodies induce an efficient upregulation of costimulatory receptors and cytokine release by dendritic cells. Finally, the most active identified anti-CD40 antibody shows activity in humanized mice. More importantly, there are no signs of obvious toxicities. These studies thus demonstrate the potent activation of antigen-presenting cells with anti-CD40 antibodies lacking FcγR-binding activity and open the possibility for an efficacious and safe combination therapy for cancer patients.

Whole-cortex in situ sequencing reveals input-dependent area identity.

The cerebral cortex is composed of neuronal types with diverse gene expression that are organized into specialized cortical areas. These areas, each with characteristic cytoarchitecture1,2, connectivity3,4 and neuronal activity5,6, are wired into modular networks3,4,7. However, it remains unclear whether these spatial organizations are reflected in neuronal transcriptomic signatures and how such signatures are established in development. Here we used BARseq, a high-throughput in situ sequencing technique, to interrogate the expression of 104 cell-type marker genes in 10.3 million cells, including 4,194,658 cortical neurons over nine mouse forebrain hemispheres, at cellular resolution. De novo clustering of gene expression in single neurons revealed transcriptomic types consistent with previous single-cell RNA sequencing studies8,9. The composition of transcriptomic types is highly predictive of cortical area identity. Moreover, areas with similar compositions of transcriptomic types, which we defined as cortical modules, overlap with areas that are highly connected, suggesting that the same modular organization is reflected in both transcriptomic signatures and connectivity. To explore how the transcriptomic profiles of cortical neurons depend on development, we assessed cell-type distributions after neonatal binocular enucleation. Notably, binocular enucleation caused the shifting of the cell-type compositional profiles of visual areas towards neighbouring cortical areas within the same module, suggesting that peripheral inputs sharpen the distinct transcriptomic identities of areas within cortical modules. Enabled by the high throughput, low cost and reproducibility of BARseq, our study provides a proof of principle for the use of large-scale in situ sequencing to both reveal brain-wide molecular architecture and understand its development.

The transcriptional legacy of developmental stochasticity.

Genetic and environmental variation are key contributors during organism development, but the influence of minor perturbations or noise is difficult to assess. This study focuses on the stochastic variation in allele-specific expression that persists through cell divisions in the nine-banded armadillo (Dasypus novemcinctus). We investigated the blood transcriptome of five wild monozygotic quadruplets over time to explore the influence of developmental stochasticity on gene expression. We identify an enduring signal of autosomal allelic variability that distinguishes individuals within a quadruplet despite their genetic similarity. This stochastic allelic variation, akin to X-inactivation but broader, provides insight into non-genetic influences on phenotype. The presence of stochastically canalized allelic signatures represents a novel axis for characterizing organismal variability, complementing traditional approaches based on genetic and environmental factors. We also developed a model to explain the inconsistent penetrance associated with these stochastically canalized allelic expressions. By elucidating mechanisms underlying the persistence of allele-specific expression, we enhance understanding of development's role in shaping organismal diversity.

Surface functionalization affects the retention and bio-distribution of orally administered mesoporous silica nanoparticles in a colitis mouse model.

Besides the many advantages of oral drug administration, challenges like premature drug degradation and limited bioavailability in the gastro-intestinal tract (GIT) remain. A prolonged residence time in the GIT is beneficial for enhancing the therapeutic outcome when treating diseases associated with an increased intestinal clearance rate, like inflammatory bowel disease (IBD). In this study, we synthesized rod-shaped mesoporous silica nanoparticles (MSNs) functionalized with polyethylene glycol (PEG) or hyaluronic acid (HA) and investigated their bio-distribution upon oral administration in vivo. The negatively charged, non-toxic particles showed different accumulation behavior over time in healthy mice and in mice with dextran sulfate sodium (DSS)-induced intestinal inflammation. PEGylated particles were shown to accumulate in the lower intestinal tract of healthy animals, whereas inflammation promoted retention of HA-functionalized particles in this area. Overall systemic absorption was low. However, some particles were detected in organs of mice with DSS-induced colitis, especially in the case of MSN-PEG. The in vivo findings were connected to surface chemistry-related differences in particle adhesion on Caco-2/Raji and mucus-producing Caco-2/Raji/HT29 cell co-culture epithelial models in vitro. While the particle adhesion behavior in vivo was mirrored in the in vitro results, this was not the case for the resorption results, suggesting that the in vitro model does not fully reflect the erosion of the inflamed epithelial tissue. Overall, our study demonstrates the possibility to modulate accumulation and retention of MSNs in the GIT of mice with and without inflammation through surface functionalization, which has important implications for the formulation of nanoparticle-based delivery systems for oral delivery applications.

Cell targeting and immunostimulatory properties of a novel Fcγ-receptor-independent agonistic anti-CD40 antibody in rhesus macaques.

Targeting CD40 by agonistic antibodies used as vaccine adjuvants or for cancer immunotherapy is a strategy to stimulate immune responses. The majority of studied agonistic anti-human CD40 antibodies require crosslinking of their Fc region to inhibitory FcγRIIb to induce immune stimulation although this has been associated with toxicity in previous studies. Here we introduce an agonistic anti-human CD40 monoclonal IgG1 antibody (MAB273) unique in its specificity to the CD40L binding site of CD40 but devoid of Fcγ-receptor binding. We demonstrate rapid binding of MAB273 to B cells and dendritic cells resulting in activation in vitro on human cells and in vivo in rhesus macaques. Dissemination of fluorescently labeled MAB273 after subcutaneous administration was found predominantly at the site of injection and specific draining lymph nodes. Phenotypic cell differentiation and upregulation of genes associated with immune activation were found in the targeted tissues. Antigen-specific T cell responses were enhanced by MAB273 when given in a prime-boost regimen and for boosting low preexisting responses. MAB273 may therefore be a promising immunostimulatory adjuvant that warrants future testing for therapeutic and prophylactic vaccination strategies.

Learning single-cell chromatin accessibility profiles using meta-analytic marker genes.

MOTIVATION: Single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) is a valuable resource to learn cis-regulatory elements such as cell-type specific enhancers and transcription factor binding sites. However, cell-type identification of scATAC-seq data is known to be challenging due to the heterogeneity derived from different protocols and the high dropout rate. RESULTS: In this study, we perform a systematic comparison of seven scATAC-seq datasets of mouse brain to benchmark the efficacy of neuronal cell-type annotation from gene sets. We find that redundant marker genes give a dramatic improvement for a sparse scATAC-seq annotation across the data collected from different studies. Interestingly, simple aggregation of such marker genes achieves performance comparable or higher than that of machine-learning classifiers, suggesting its potential for downstream applications. Based on our results, we reannotated all scATAC-seq data for detailed cell types using robust marker genes. Their meta scATAC-seq profiles are publicly available at https://gillisweb.cshl.edu/Meta_scATAC. Furthermore, we trained a deep neural network to predict chromatin accessibility from only DNA sequence and identified key motifs enriched for each neuronal subtype. Those predicted profiles are visualized together in our database as a valuable resource to explore cell-type specific epigenetic regulation in a sequence-dependent and -independent manner.

Antimicrobial Efficiency of Chitosan and Its Methylated Derivative against Lentilactobacillus parabuchneri Biofilms.

Antimicrobial materials are considered potential alternatives to prevent the development of biofilm-associated contaminations. Concerns regarding synthetic preservatives necessitate the development of innovative and safe natural antimicrobials. In the present study, we discuss the in situ infrared attenuated total reflection spectroscopy (IR-ATR) investigations of the selective antimicrobial efficiency of chitosan in controlling the growth of Lentilactobacillus parabuchneri biofilms. The protonated charges of chitosan were additionally amplified by structural modification via methylation, yielding quaternized derivative TMC (i.e., N, N, N-trimethyl chitosan). To evaluate antimicrobial effectiveness against L. parab. biofilms, IR-ATR spectroscopy provided information on molecular mechanisms and insights into chemical changes during real-time biofilm inhibition studies. The integrated fiberoptic oxygen microsensors enabled monitoring oxygen (O2) concentration gradients within biofilms, thereby confirming the metabolic oxygen depletion dropping from 4.5 to 0.7 mg L-1. IR studies revealed strong electrostatic interactions between chitosan/its water-soluble derivative and bacteria, indicating that a few hours were sufficient to affect biofilm disruption. The significant decrease in the IR bands is related to the characteristic spectral information of amide I, II, III, nucleic acid, and extracellular polymeric matrix (EPS) produced by L. parabuchneri biofilms. Cell clusters of biofilms, microcolonies, and destabilization of the EPS matrix after the addition of biopolymers were visualized using optical microscopy. In addition, scanning electron microscopy (SEM) of biofilms grown on polystyrene and stainless-steel surfaces was used to examine morphological changes, indicating the disintegration of the biofilm matrix into individual cells. Quantification of the total biofilm formation correlated with the CV assay results, indicating cell death and lysis. The electrostatic interactions between chitosan and the bacterial cell wall typically occur between protonated amino groups and negatively charged phospholipids, which promote permeabilization. Biofilm growth inhibition was assessed by a viability assay for a period of 72 h and in the range of low MIC values (varying 0.01-2%). These results support the potential of chitosan and TMC for bacterial growth prevention of the foodborne contaminant L. parabuchneri in the dairy industry and for further implementation in food packaging.

In situ monitoring of Lentilactobacillus parabuchneri biofilm formation via real-time infrared spectroscopy.

Foodborne pathogenic microorganisms form biofilms at abiotic surfaces, which is a particular challenge in food processing industries. The complexity of biofilm formation requires a fundamental understanding on the involved molecular mechanisms, which may then lead to efficient prevention strategies. In the present study, biogenic amine producing bacteria, i.e., Lentilactobacillus parabuchneri DSM 5987 strain isolated from cheese were studied in respect with biofilm formation, which is of substantial relevance given their contribution to the presence of histamine in dairy products. While scanning electron microscopy was used to investigate biofilm adhesion at stainless steel surfaces, in situ infrared attenuated total reflection spectroscopy (IR-ATR) using a custom flow-through assembly was used for real-time and non-destructive observations of biofilm formation during a period of several days. The spectral window of 1700-600 cm-1 provides access to vibrational signatures characteristic for identifying and tracking L. parabuchneri biofilm formation and maturation. Especially, the amide I and II bands, lactic acid produced as the biofilm matures, and a pronounced increase of bands characteristic for extracellular polymeric substances (EPS) provide molecular insight into biofilm formation, maturation, and changes in biofilm architecture. Finally, multivariate data evaluation strategies were applied facilitating the unambiguous classification of the observed biofilm changes via IR spectroscopic data.

A Carbon Nanodot Based Near-Infrared Photosensitizer with a Protein-Ruthenium Shell for Low-Power Photodynamic Applications.

Near-infrared (NIR) light-activated photosensitization represents an encouraging therapeutic method in photodynamic therapy, especially for deep tissue penetration. In this context, two-photon activation, i.e., utilization of photons with relatively low energy but high photon flux for populating a virtual intermediate state leading to an excited state, is attractive. This concept would be highly advantageous in photodynamic therapy due to its minimal side effects. Herein, we propose that the combination of plasma protein serum albumin (HSA) containing several Ru complexes and NIR two-photon excitable carbon nanodots (Cdots), termed HSA-Ru-Cdots, provides several attractive features for enhancing singlet oxygen formation within the mitochondria of cancer cells stimulated by two-photon excitation in the NIR region. HSA-Ru-Cdot features biocompatibility, water solubility, and photostability as well as uptake into cancer cells with an endosomal release, which is an essential feature for subcellular targeting of mitochondria. The NIR two-photon excitation induced visible emission of the Cdots allows fluorescence resonance energy transfer (FRET) to excite the metal-to-ligand charge transfer of the Ru moiety, and fluorescence-lifetime imaging microscopy (FLIM) has been applied to demonstrate FRET within the cells. The NIR two-photon excitation is indirectly transferred to the Ru complexes, which leads to the production of singlet oxygen within the mitochondria of cancer cells. Consequently, we observe the destruction of filamentous mitochondrial structures into spheroid aggregates within various cancer cell lines. Cell death is induced by the long-wavelength NIR light irradiation at 810 nm with a low power density (7 mW/cm2), which could be attractive for phototherapy applications where deeper tissue penetration is crucial.

Clostridium botulinum C3 Toxin for Selective Delivery of Cargo into Dendritic Cells and Macrophages.

The protein toxin C3bot from Clostridium botulinum is a mono-ADP-ribosyltransferase that selectively intoxicates monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (DCs) by cytosolic modification of Rho-A, -B, and -C. Here, we investigated the application of C3bot as well as its non-toxic variant C3botE174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3botE174Q facilitated the uptake of eGFP into early endosomes of human-monocyte-derived macrophages, as revealed by stimulated emission depletion (STED) super-resolution microscopy. The fusion of the cargo model peptide eGFP neither affected the cell-type selectivity (enhanced uptake into human macrophages ex vivo compared to lymphocytes) nor the cytosolic release of C3bot. Moreover, by cell fractionation, we demonstrated that C3bot and C3botE174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3botE174Q for covalent linkage of cargos via thiol-maleimide click chemistry. The functionality of this system was proven by loading small molecule fluorophores or an established reporter enzyme and investigating the cellular uptake and cytosolic release of cargo. Taken together, non-toxic C3botE174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.

Defining the extent of gene function using ROC curvature.

MOTIVATION: Interactions between proteins help us understand how genes are functionally related and how they contribute to phenotypes. Experiments provide imperfect 'ground truth' information about a small subset of potential interactions in a specific biological context, which can then be extended to the whole genome across different contexts, such as conditions, tissues or species, through machine learning methods. However, evaluating the performance of these methods remains a critical challenge. Here, we propose to evaluate the generalizability of gene characterizations through the shape of performance curves. RESULTS: We identify Functional Equivalence Classes (FECs), subsets of annotated and unannotated genes that jointly drive performance, by assessing the presence of straight lines in ROC curves built from gene-centric prediction tasks, such as function or interaction predictions. FECs are widespread across data types and methods, they can be used to evaluate the extent and context-specificity of functional annotations in a data-driven manner. For example, FECs suggest that B cell markers can be decomposed into shared primary markers (10-50 genes), and tissue-specific secondary markers (100-500 genes). In addition, FECs suggest the existence of functional modules that span a wide range of the genome, with marker sets spanning at most 5% of the genome and data-driven extensions of Gene Ontology sets spanning up to 40% of the genome. Simple to assess visually and statistically, the identification of FECs in performance curves paves the way for novel functional characterization and increased robustness in the definition of functional gene sets. AVAILABILITY AND IMPLEMENTATION: Code for analyses and figures is available at https://github.com/yexilein/pyroc. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human α-Defensin-6 Neutralizes Clostridioides difficile Toxins TcdA and TcdB by Direct Binding.

Rising incidences and mortalities have drawn attention to Clostridioides difficile infections (CDIs) in recent years. The main virulence factors of this bacterium are the exotoxins TcdA and TcdB, which glucosylate Rho-GTPases and thereby inhibit Rho/actin-mediated processes in cells. This results in cell rounding, gut barrier disruption and characteristic clinical symptoms. So far, treatment of CDIs is limited and mainly restricted to some antibiotics, often leading to a vicious circle of antibiotic-induced disease recurrence. Here, we demonstrate the protective effect of the human antimicrobial peptide α-defensin-6 against TcdA, TcdB and the combination of both toxins in vitro and in vivo and unravel the underlying molecular mechanism. The defensin prevented toxin-mediated glucosylation of Rho-GTPases in cells and protected human cells, model epithelial barriers as well as zebrafish embryos from toxic effects. In vitro analyses revealed direct binding to TcdB in an SPR approach and the rapid formation of TcdB/α-defensin-6 complexes, as analyzed with fluorescent TcdB by time-lapse microscopy. In conclusion, the results imply that α-defensin-6 rapidly sequesters the toxin into complexes, which prevents its cytotoxic activity. These findings extend the understanding of how human peptides neutralize bacterial protein toxins and might be a starting point for the development of novel therapeutic options against CDIs.

Implementation of Zebrafish Ontologies for Toxicology Screening.

Toxicological evaluation of chemicals using early-life stage zebrafish (Danio rerio) involves the observation and recording of altered phenotypes. Substantial variability has been observed among researchers in phenotypes reported from similar studies, as well as a lack of consistent data annotation, indicating a need for both terminological and data harmonization. When examined from a data science perspective, many of these apparent differences can be parsed into the same or similar endpoints whose measurements differ only in time, methodology, or nomenclature. Ontological knowledge structures can be leveraged to integrate diverse data sets across terminologies, scales, and modalities. Building on this premise, the National Toxicology Program's Systematic Evaluation of the Application of Zebrafish in Toxicology undertook a collaborative exercise to evaluate how the application of standardized phenotype terminology improved data consistency. To accomplish this, zebrafish researchers were asked to assess images of zebrafish larvae for morphological malformations in two surveys. In the first survey, researchers were asked to annotate observed malformations using their own terminology. In the second survey, researchers were asked to annotate the images from a list of terms and definitions from the Zebrafish Phenotype Ontology. Analysis of the results suggested that the use of ontology terms increased consistency and decreased ambiguity, but a larger study is needed to confirm. We conclude that utilizing a common data standard will not only reduce the heterogeneity of reported terms but increases agreement and repeatability between different laboratories. Thus, we advocate for the development of a zebrafish phenotype atlas to help laboratories create interoperable, computable data.

Erratum: How many markers are needed to robustly determine a cell's type?

[This corrects the article DOI: 10.1016/j.isci.2021.103292.].

The Pore-Forming Subunit C2IIa of the Binary Clostridium botulinum C2 Toxin Reduces the Chemotactic Translocation of Human Polymorphonuclear Leukocytes.

The binary C2 toxin of Clostridium (C.) botulinum consists of two non-linked proteins, the enzyme subunit C2I and the separate binding/transport subunit C2II. To exhibit toxic effects on mammalian cells, proteolytically activated C2II (C2IIa) forms barrel-shaped heptamers that bind to carbohydrate receptors which are present on all mammalian cell types. C2I binds to C2IIa and the toxin complexes are internalized via receptor-mediated endocytosis. In acidified endosomal vesicles, C2IIa heptamers change their conformation and insert as pores into endosomal membranes. These pores serve as translocation-channels for the subsequent transport of C2I from the endosomal lumen into the cytosol. There, C2I mono-ADP-ribosylates G-actin, which results in depolymerization of F-actin and cell rounding. Noteworthy, so far morphological changes in cells were only observed after incubation with the complete C2 toxin, i.e., C2IIa plus C2I, but not with the single subunits. Unexpectedly, we observed that the non-catalytic transport subunit C2IIa (but not C2II) alone induced morphological changes and actin alterations in primary human polymorphonuclear leukocytes (PMNs, alias neutrophils) from healthy donors ex vivo, but not macrophages, epithelial and endothelial cells, as detected by phase contrast microscopy and fluorescent microscopy of the actin cytoskeleton. This suggests a PMN selective mode of action for C2IIa. The cytotoxicity of C2IIa on PMNs was prevented by C2IIa pore blockers and treatment with C2IIa (but not C2II) rapidly induced Ca2+ influx in PMNs, suggesting that pore-formation by C2IIa in cell membranes of PMNs is crucial for this effect. In addition, incubation of primary human PMNs with C2IIa decreased their chemotaxis ex vivo through porous culture inserts and in co-culture with human endothelial cells which is closer to the physiological extravasation process. In conclusion, the results suggest that C2IIa is a PMN-selective inhibitor of chemotaxis. This provides new knowledge for a pathophysiological role of C2 toxin as a modulator of innate immune cells and makes C2IIa an attractive candidate for the development of novel pharmacological strategies to selectively down-modulate the excessive and detrimental PMN recruitment into organs after traumatic injuries.

Evaluation of a full-scale wastewater treatment plant with ozonation and different post-treatments using a broad range of in vitro and in vivo bioassays.

Micropollutants present in the effluent of wastewater treatment plants (WWTPs) after biological treatment are largely eliminated by effective advanced technologies such as ozonation. Discharge of contaminants into freshwater ecosystems can thus be minimized, while simultaneously protecting drinking water resources. However, ozonation can lead to reactive and potentially toxic transformation products. To remove these, the Swiss Federal Office for the Environment recommends additional "post-treatment" of ozonated WWTP effluent using sand filtration, but other treatments may be similarly effective. In this study, 48 h composite wastewater samples were collected before and after full-scale ozonation, and after post-treatments (full-scale sand filtration, pilot-scale fresh and pre-loaded granular activated carbon, and fixed and moving beds). Ecotoxicological tests were performed to quantify the changes in water quality following different treatment steps. These included standard in vitro bioassays for the detection of endocrine, genotoxic and mutagenic effects, as well as toxicity to green algae and bacteria, and flow-through in vivo bioassays using oligochaetes and early life stages of rainbow trout. Results show that ozonation reduced a number of ecotoxicological effects of biologically treated wastewater by 66 - 93%: It improved growth and photosynthesis of green algae, decreased toxicity to luminescent bacteria, reduced concentrations of hormonally active contaminants and significantly changed expression of biomarker genes in rainbow trout liver. Bioassay results showed that ozonation did not produce problematic levels of reaction products overall. Small increases in toxicity observed in a few samples were reduced or eliminated by post-treatments. However, only relatively fresh granular activated carbon (analyzed at 13,000 - 20,000 bed volumes) significantly reduced effects additionally (by up to 66%) compared to ozonation alone. Inhibition of algal photosynthesis, rainbow trout liver histopathology and biomarker gene expression proved to be sufficiently sensitive endpoints to detect the change in water quality achieved by post-treatment.

The Compound U18666A Inhibits the Intoxication of Cells by Clostridioides difficile Toxins TcdA and TcdB.

The intestinal pathogen Clostridioides (C.) difficile is a major cause of diarrhea both in hospitals and outpatient in industrialized countries. This bacterium produces two large exotoxins, toxin A (TcdA) and toxin B (TcdB), which are directly responsible for the onset of clinical symptoms of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and the severe, life-threatening pseudomembranous colitis. Both toxins are multidomain proteins and taken up into host eukaryotic cells via receptor-mediated endocytosis. Within the cell, TcdA and TcdB inactivate Rho and/or Ras protein family members by glucosylation, which eventually results in cell death. The cytotoxic mode of action of the toxins is the main reason for the disease. Thus, compounds capable of inhibiting the cellular uptake and/or mode-of-action of both toxins are of high therapeutic interest. Recently, we found that the sterol regulatory element-binding protein 2 (SREBP-2) pathway, which regulates cholesterol content in membranes, is crucial for the intoxication of cells by TcdA and TcdB. Furthermore, it has been shown that membrane cholesterol is required for TcdA- as well as TcdB-mediated pore formation in endosomal membranes, which is a key step during the translocation of the glucosyltransferase domain of both toxins from endocytic vesicles into the cytosol of host cells. In the current study, we demonstrate that intoxication by TcdA and TcdB is diminished in cultured cells preincubated with the compound U18666A, an established inhibitor of cholesterol biosynthesis and/or intracellular transport. U18666A-pretreated cells were also less sensitive against TcdA and TcdB variants from the epidemic NAP1/027 C. difficile strain. Our study corroborates the crucial role of membrane cholesterol for cell entry of TcdA and TcdB, thus providing a valuable basis for the development of novel antitoxin strategies in the context of CDADs.

How many markers are needed to robustly determine a cell's type?

Our understanding of cell types has advanced considerably with the publication of single-cell atlases. Marker genes play an essential role for experimental validation and computational analyses such as physiological characterization, annotation, and deconvolution. However, a framework for quantifying marker replicability and selecting replicable markers is currently lacking. Here, using high-quality data from the Brain Initiative Cell Census Network (BICCN), we systematically investigate marker replicability for 85 neuronal cell types. We show that, due to dataset-specific noise, we need to combine 5 datasets to obtain robust differentially expressed (DE) genes, particularly for rare populations and lowly expressed genes. We estimate that 10 to 200 meta-analytic markers provide optimal downstream performance and make available replicable marker lists for the 85 BICCN cell types. Replicable marker lists condense interpretable and generalizable information about cell types, opening avenues for downstream applications, including cell type annotation, selection of gene panels, and bulk data deconvolution.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.