Selected Cell Types Research Articles

Label-free live characterization of mesenchymal stem cell spheroids by biophysical properties measurement

Three-dimensional (3D) cell culture has become a consolidated method in the stem cell field, where mesenchymal stromal stem cells (MSCs) can be used to generate in vitro spheroid aggregates called MSC-Spheroids (MSph). MSph is a floating cluster of stem cells similar to those in literature are known as bone marrow-derived “mesenspheres”. Even though MSC properties are shared by MSph, depending on the cell type and their tissue source, the morphology and degree of compaction of the MSph can be variable, creating limitations in such a cell model. Thus, during culture, a variation in stem cell functionality and viability, in addition to the suitability for comparing MSph in some experimental protocols, can be affected by spheroid biophysical intrinsic properties like mass density. To investigate this limitation and provide a new method for researchers, MSph of seven different tissue sources were compared by combining mass density, weight, and size evaluations with viability assays for ATP measurement. MSph cultured in traditional static conditions showed decreased in viability over the days of culture, while mass density exhibited different trends among cell types. Additionally, treatment of MSph with a non-toxic concentration of a natural compound cell regulator, such as plumbagin, altered the mass density of a selected cell type, thereby confirming the efficacy of the biophysical approach in monitoring MSph variability post-treatment. The results encourage using MSph in the early days of culture after their formation to ensure viability and likely retention of the stem cell phenotype.

A novel “cells-on-particles” cytotoxicity testing platform in vitro: design, characterization, and validation against engineered nanoparticle aerosol

The presented research introduces the "Cells-on-Particles" integrated aerosol sampling and cytotoxicity testing in vitro platform, which allows for the direct assessment of the biological effects of captured aerosol particles on a selected cell type without the need for extraction or resuspension steps. By utilizing particles with unaltered chemical and physical properties, the method enables simple and fast screening of biological effects on specific cell types, making it a promising tool for assessing the cytotoxicity of particulate matter in ambient and occupational air. Platforms fabricated from cellulose acetate (CA) and poly[ε]caprolactone (PCL) were proven to be biocompatible and promoted the attachment and growth of the human bronchial epithelial cell line BEAS-2B. The PCL platforms were exposed to simulated occupational aerosols of silver, copper, and graphene oxide nanoparticles. Each nanoparticle type exhibited different and dose-dependent cytotoxic effects on cells, evidenced by reduced cell viability and distinct, particle type-dependent gene expression patterns. Notably, copper nanoparticles were identified as the most cytotoxic, and graphene oxide the least. Comparing the “Cells-on-Particles" and submerged exposure (“Particles-on-Cells") testing strategies, BEAS-2B cells responded to selected nanoparticles in a comparable manner, suggesting the developed testing system could be proposed for further evaluation with more complex environmental aerosols. Despite limitations, including particle agglomeration and the need for more replicates to address variability, the “Cells-on-Particles” platform enables effective detection of toxicity induced by relatively low levels of nanoparticles, demonstrating good sensitivity and a relatively simpler procedure compared to standard 2D cell exposure methods.

Transcranial focused ultrasound activates feedforward and feedback cortico-thalamo-cortical pathways by selectively activating excitatory neurons.

Transcranial focused ultrasound stimulation (tFUS) has been proven capable of altering focal neuronal activities and neural circuits non-invasively in both animals and humans. The abilities of tFUS for cell-type selection within the targeted area like somatosensory cortex have been shown to be parameter related. However, how neuronal subpopulations across neural pathways are affected, for example how tFUS affected neuronal connections between brain areas remains unclear. In this study, multi-site intracranial recordings were used to quantify the neuronal responses to tFUS stimulation at somatosensory cortex (S1), motor cortex (M1) and posterior medial thalamic nucleus (POm) of cortico-thalamo-cortical (CTC) pathway. We found that when targeting at S1 or POm, only regular spiking units (RSUs, putative excitatory neurons) responded to specific tFUS parameters (duty cycle: 6%-60% and pulse repetition frequency: 1500 and 3000 Hz ) during sonication. RSUs from the directly connected area (POm or S1) showed a synchronized response, which changed the directional correlation between RSUs from POm and S1. The tFUS induced excitation of RSUs activated the feedforward and feedback loops between cortex and thalamus, eliciting delayed neuronal responses of RSUs and delayed activities of fast spiking units (FSUs) by affecting local network. Our findings indicated that tFUS can modulate the CTC pathway through both feedforward and feedback loops, which could influence larger cortical areas including motor cortex.

Cellular and axonal transport phenotypes due to the C9ORF72 HRE in iPSC motor and sensory neurons

Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from patients with amyotrophic lateral sclerosis (ALS) and the C9ORF72 hexanucleotide repeat expansion (HRE) have multiple cellular phenotypes, but which of these accurately reflect the biology underlying the cell-specific vulnerability of ALS is uncertain. We therefore compared phenotypes due to the C9ORF72 HRE in MNs with sensory neurons (SNs), which are relatively spared in ALS. The iPSC models were able to partially reproduce the differential gene expression seen between adult SNs and MNs. We demonstrated that the typical hallmarks of C9ORF72-ALS, including RNA foci and dipeptide formation, as well as specific axonal transport defects, occurred equally in MNs and SNs, suggesting that these invitro phenotypes are not sufficient to explain the cell-type selectivity of ALS in isolation.

ERK3 is involved in regulating cardiac fibroblast function.

ERK3/MAPK6 activates MAP kinase-activated protein kinase (MK)-5 in selected cell types. Male MK5 haplodeficient mice show reduced hypertrophy and attenuated increase in Col1a1 mRNA in response to increased cardiac afterload. In addition, MK5 deficiency impairs cardiac fibroblast function. This study determined the effect of reduced ERK3 on cardiac hypertrophy following transverse aortic constriction (TAC) and fibroblast biology in male mice. Three weeks post-surgery, ERK3, but not ERK4 or p38α, co-immunoprecipitated with MK5 from both sham and TAC heart lysates. The increase in left ventricular mass and myocyte diameter was lower in TAC-ERK3+/- than TAC-ERK3+/+ hearts, whereas ERK3 haploinsufficiency did not alter systolic or diastolic function. Furthermore, the TAC-induced increase in Col1a1 mRNA abundance was diminished in ERK3+/- hearts. ERK3 immunoreactivity was detected in atrial and ventricular fibroblasts but not myocytes. In both quiescent fibroblasts and "activated" myofibroblasts isolated from adult mouse heart, siRNA-mediated knockdown of ERK3 reduced the TGF-β-induced increase in Col1a1 mRNA. In addition, intracellular type 1 collagen immunoreactivity was reduced following ERK3 depletion in quiescent fibroblasts but not myofibroblasts. Finally, knocking down ERK3 impaired motility in both atrial and ventricular myofibroblasts. These results suggest that ERK3 plays an important role in multiple aspects of cardiac fibroblast biology.

Single Cell Profiling Distinguishes Leukemia-Selective Chemotypes.

A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors - for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.

Radiation-induced bystander effect on the brain after fractionated spinal cord irradiation of aging rats

We investigated the influence of the so-called bystander effect on metabolic and histopathological changes in the rat brain after fractionated spinal cord irradiation. The study was initiated with adult Wistar male rats (n = 20) at the age of 9 months. The group designated to irradiation (n = 10) and the age-matched control animals (n = 10) were subjected to an initial measurement using in vivo proton magnetic resonance spectroscopy (1H MRS) and magnetic resonance imaging (MRI). After allowing the animals to survive until 12 months, they received fractionated spinal cord irradiation with a total dose of 24 Gy administered in 3 fractions (8 Gy per fraction) once a week on the same day for 3 consecutive weeks. 1H MRS and MRI of brain metabolites were performed in the hippocampus, corpus striatum, and olfactory bulb (OB) before irradiation (9-month-old rats) and subsequently 48 h (12-month-old) and 2 months (14-month-old) after the completion of irradiation. After the animals were sacrificed at the age of 14 months, brain tissue changes were investigated in two neurogenic regions: the hippocampal dentate gyrus (DG) and the rostral migratory stream (RMS). By comparing the group of 9-month-old rats and individuals measured 48 h (at the age of 12 months) after irradiation, we found a significant decrease in the ratio of total N-acetyl aspartate to total creatine (tNAA/tCr) and gamma-aminobutyric acid to tCr (GABA/tCr) in OB and hippocampus. A significant increase in myoinositol to tCr (mIns/tCr) in the OB persisted up to 14 months of age. Proton nuclear magnetic resonance (1H NMR)-based plasma metabolomics showed a significant increase in keto acids and decreased tyrosine and tricarboxylic cycle enzymes. Morphometric analysis of neurogenic regions of 14-month-old rats showed well-preserved stem cells, neuroblasts, and increased neurodegeneration. The radiation-induced bystander effect more significantly affected metabolite concentration than the distribution of selected cell types.

Abstract 3771: 3D correlated multi-scale cellular and molecular architecture of solid tumors

Abstract A solid tumor presents heterogeneous 3D structures at tissue, cell, and molecule levels. To develop advanced cancer diagnostics and therapies, it is important to understand in-depth cellular and molecular architecture of a tumor at multiple scales. Here we introduce novel optical microscopy methodologies for in-depth 3D spatial analysis of tumor tissues: 1) LED photobleaching-mediated 3D multiplex immunofluorescence (IF) microscopy, 2) Correlated 3D multi-resolution optical microscopy, and 3) 3D tissue region-based cell type-selective multi-omics. We built a high-power LED light irradiator with a broad emission spectrum (490-700 nm) that bleaches a broad wavelength of fluorescence signals simultaneously throughout an optically cleared, 400 µm-thick tumor tissue. Cyclic workflow involving IF staining, optical tissue clearing, 3D confocal microscopy, and LED photobleaching enables visualization of multiple cell types in the same whole 400 µm-thick tumor tissue after computational 3D image registration. The constructed multiplex image offers comprehensive 3D cellular landscape of a tumor. We also developed a method for tracking regions of interest (ROIs) in the middle of a large tumor tissue using a UV-activated visible dye in light sheet microscopy. Light sheet microscopy allows imaging an optically cleared large-sized tumor tissue at a low spatial, tissue resolution. To mark the ROI positions on a tumor, the dye is infused into agarose gel surrounding the tumor tissue and be activated and present purple-colored lines where are exposed to UV light sheets in light sheet microscopy. The tumor tissue was physically sectioned at the marked ROI positions using a vibratome after reversing tissue clearing process. Sequential IF staining and high-resolution confocal microscopy permit for visualizing detailed cellular compositions of the ROIs in the tumor. The tissue and cellular-resolution 3D images from light sheet and confocal microscopy are correlatively integrated to display ‘Google Earth’-like multi-scale view of a tumor tissue. To further analyze the ROIs of a tumor at molecular level, we have developed optical microscope-mediated fluorescence lithography methods that allow fluorescently labeling of a selected cell type in ROIs of an optically cleared 3D tumor tissue. After wash free from clearing solution and dissociation of the tumor into cells, fluorescence-marked, intact cells from the ROIs are collected through flow cell sorting. Proteins and mRNAs are extracted from the collected cells and sent to LC-MS/MS analysis and RNA sequencing. The spatial multi-omics method provides in-depth molecular information of selected cells in the ROIs of a tumor. We believe all these 3D microscopy methods will provide powerful spatial analysis tools for better understanding cellular and molecular architecture of solid tumors. Citation Format: Steve Seung-Young Lee, Yi-Chien Wu, Jingtian Zheng, Elie Abi Khalil, Samuel Wang, Alexander Lippert, Joshua Plank. 3D correlated multi-scale cellular and molecular architecture of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3771.

Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.

RatInABox, a toolkit for modelling locomotion and neuronal activity in continuous environments.

Generating synthetic locomotory and neural data is a useful yet cumbersome step commonly required to study theoretical models of the brain's role in spatial navigation. This process can be time consuming and, without a common framework, makes it difficult to reproduce or compare studies which each generate test data in different ways. In response, we present RatInABox, an open-source Python toolkit designed to model realistic rodent locomotion and generate synthetic neural data from spatially modulated cell types. This software provides users with (i) the ability to construct one- or two-dimensional environments with configurable barriers and visual cues, (ii) a physically realistic random motion model fitted to experimental data, (iii) rapid online calculation of neural data for many of the known self-location or velocity selective cell types in the hippocampal formation (including place cells, grid cells, boundary vector cells, head direction cells) and (iv) a framework for constructing custom cell types, multi-layer network models and data- or policy-controlled motion trajectories. The motion and neural models are spatially and temporally continuous as well as topographically sensitive to boundary conditions and walls. We demonstrate that out-of-the-box parameter settings replicate many aspects of rodent foraging behaviour such as velocity statistics and the tendency of rodents to over-explore walls. Numerous tutorial scripts are provided, including examples where RatInABox is used for decoding position from neural data or to solve a navigational reinforcement learning task. We hope this tool will significantly streamline computational research into the brain's role in navigation.

SGLT2 inhibition promotes glomerular repopulation by cells of renin lineage in experimental kidney disease.

Sodium glucose co-transporter-2 (SGLT2) inhibitors stimulate renal excretion of sodium and glucose and exert renal protective effects in patients with (non-)diabetic chronic kidney disease (CKD) and may as well protect against acute kidney injury (AKI). The mechanism behind this kidney protective effect remains unclear. Juxtaglomerular cells of renin lineage (CoRL) have been demonstrated to function as progenitors for multiple adult glomerular cell types in kidney disease. This study assesses the impact of SGLT2 inhibition on the repopulation of glomerular cells by CoRL and examines their phenotypic commitment. Experiments were performed in Ren1cre-tdTomato lineage-trace mice. Either 5/6 nephrectomy (5/6NX) modeling CKD or bilateral ischaemia reperfusion injury (bIRI) mimicking AKI was applied, while the SGLT2 inhibitor empagliflozin (10 mg/kg) was administered daily via oral gavage for 14 days. Both 5/6NX and bIRI-induced kidney injury increased the number of glomerular CoRL-derived cells. SGLT2 inhibition improved kidney function after 5/6NX, indicated by decreased blood creatinine and urea levels, but not after bIRI. In line with this, empagliflozin in 5/6NX animals resulted in less glomerulosclerosis, while it did not affect histopathological features in bIRI. Treatment with empagliflozin resulted in an increase in the number of CoRL-derived glomerular cells in both 5/6NX and bIRI conditions. Interestingly, SGLT2 inhibition led to more CoRL-derived podocytes in 5/6NX animals, whereas empagliflozin-treated bIRI mice presented with increased levels of parietal epithelial and mesangial cells derived from CoRL. We conclude that SGLT2 inhibition by empagliflozin promotes CoRL-mediated glomerular repopulation with selective CoRL-derived cell types depending on the type of experimental kidney injury. These findings suggest a previously unidentified mechanism that could contribute to the renoprotective effect of SGLT2 inhibitors.

Unveiling the anti-proliferative and pro-thermogenic activity of Staurosirella pinnata (Bacillariophyta) bioproducts

Biorefinery co-products obtained from biomass of the field isolated diatom Staurosirella pinnata have been investigated for their bioactivity on selected human and murine cell lines. Biomass grown in indoor photobioreactor, collected at the stationary phase, was subjected to methanolic extraction to produce a crude extract, sequentially fractionated (hydrophilic-hydrophobic gradient) to obtain six fractions (A4, A5, A7; B4, B5, B6). Fractions' bioactivity was tested on BRAFwt and BRAFV600E human melanoma cell lines, compared to normal keratinocytes. All fractions showed cytotoxic activity, but only fraction A4 showed a remarkable cell-type selectivity, with cell death induction up to 70 % on melanoma cells. Further characterization allowed to identify and purify four apocarotenoids from fraction A4, representing the first report of such class of compound s in mass cultivated diatoms. In addition, fraction A5 resulted to be a mixture of oxylipins and fraction B4 of purine nucleosides, with adenosine as main component. Residual biomass was further processed to obtain an enriched lipidic extract, characterized by a high content of poly-unsaturated fatty acids. Anti-diabetic and anti-obesity activities were assessed by evaluating the expression levels of key proteins involved into thermogenesis (UCP1), lipolysis (Perilipin, HSL660), and mitochondrial dynamics (pDRP1, VDAC), on murine white adipocyte cell line. Overall, the data here reported confirmed S. pinnata as a platform for the discovery and purification of bioactive compounds with biomedical potential, and the biorefinery pipeline allow their combined isolation, thus advancing the sustainability of diatom exploitation for the discovery and production of bioactive molecules.

In vivo human T cell engineering with enveloped delivery vehicles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Analysis of TiO2 nanoparticles accumulation in vitro

The physicochemical properties of titanium dioxide (TiO2) in nanoforms is often exploited as colorant in food, pharmaceuticals and other consumer products. However, the current evidence of potential hazards associated with titanium dioxide (TiO2) in nanoforms led to a ban of TiO2 as food additive in Europe. This regulatory decision has also an impact on thousands of pharmaceuticals. In the present study, we tested the internalisation, accumulation and resulting biological effects of different types of TiO2 nanomaterial in short and long-term vitro cultures. Even if we could demonstrate that all tested cell lines were able to take up and accumulate nanomaterial for a period of up to 30 days, the cellular responses using conventional in vitro tests were limited in all tested cell lines. Nevertheless, a transcriptomics study revealed that that the response to the accumulated material differed between two selected cell types. A keratinocyte like cell line reacted with a modified rate of keratinogenesis whereas the enterocyte like cell demonstrated mainly interactions with cell homeostasis. To further clarify possible harmful effects of TiO2, the study suggests analyzing cell/tissue type specific effects of TiO2.

Development of comprehensive approaches to characterizing CuII complexes: structures in solution and solid‐state, dynamic behavior, and bioactivity

AbstractCuII ternary systems with aromatic N‐donors (1,10‐phenanthroline, dipyrido‐[3,2‐f:2’,3’‐h]‐quinoxaline, and 2‐methyldipyrido‐[3,2‐f:2’,3’‐h]‐quinoxaline) and amino acids have been extensively studied in water solution under closely physiological conditions using several techniques, including potentiometric pH titration, mathematical modeling, nuclear magnetic relaxation, EPR, and DFT calculations. Based on the distribution diagrams of the complex species, single crystal syntheses of the ternary systems CuII‐aromatic N‐donor‐L‐amino acid were carried out. As a result, several crystal structures of the complexes with the desired copper‐to‐ligand ratio were established by X‐ray diffraction analysis. The most successful synthetic attempts were with L‐proline and L‐serine, resulting in the crystals with the desired copper(II) to ligand ratio. The developed approach made it possible to thoroughly describe systems with major proteinogenic amino acids in the L‐configuration. Numerous non‐covalent interactions such as H‐bonds, π‐π stacking, d‐π interactions, lone electron pair‐π interactions between CuII atom, conjugated aromatic ligand system, counterions, and water molecules were found to affect the stability and structure of the resulting complex particles. It has been found that in the CuII–N‐donor–amino acid systems there is an acceleration of ligand exchange by a factor of 2–25 compared to CuII‐amino acid systems, which is explained by taking into account steric effects, d‐π interactions, and the pseudo Jahn‐Teller effect. The investigated systems were tested for antitumor activity against MCF‐7, PC‐3, OVCAR‐4, SNB‐19, and M‐14 cell lines, and most samples, in particular, CuII–Phen–L‐His, CuII–MeDPQ−L‐Val and CuII–MeDPQ−L‐Ser, showed a therapeutic activity index greater than 2 for all selected cell types, which opens up prospects for their medical application.

Cellsig plug-in enhances CIBERSORTx signature selection for multidataset transcriptomes with sparse multilevel modelling.

The precise characterization of cell-type transcriptomes is pivotal to understanding cellular lineages, deconvolution of bulk transcriptomes, and clinical applications. Single-cell RNA sequencing resources like the Human Cell Atlas have revolutionised cell-type profiling. However, challenges persist due to data heterogeneity and discrepancies across different studies. One limitation of prevailing tools such as CIBERSORTx is their inability to address hierarchical data structures and handle nonoverlapping gene sets across samples, relying on filtering or imputation. Here, we present cellsig, a Bayesian sparse multilevel model designed to improve signature estimation by adjusting data for multilevel effects and modelling for gene-set sparsity. Our model is tailored to large-scale, heterogeneous pseudobulk and bulk RNA sequencing data collections with nonoverlapping gene sets. We tested the performances of cellsig on a novel curated Human Bulk Cell-type Catalogue, which harmonizes 1435 samples across 58 datasets. We show that cellsig significantly enhances cell-type marker gene ranking performance. This approach is valuable for cell-type signature selection, with implications for marker gene validation, single-cell annotation, and deconvolution benchmarks. Codes and the interactive app are available at https://github.com/stemangiola/cellsig; and the database is available at https://doi.org/10.5281/zenodo.7582421.

An ultra-compact promoter drives widespread neuronal expression in mouse and monkey brains

Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type selectivity. However, whether ultra-compact promoters can offer neuron-specific expression is unclear. Here, we report the development of an extremely short promoter that enables selective gene expression in neurons, but not glial cells, in the brain. The promoter sequence originates from the human CALM1 gene and is only 120bp in size. The CALM1 promoter (pCALM1) embedded in an adeno-associated virus (AAV) genome directed broad reporter expression in excitatory and inhibitory neurons in mouse and monkey brains. Moreover, pCALM1, when inserted into an all-in-one AAV vector expressing SpCas9 and sgRNA, drives constitutive and conditional invivo gene editing in neurons and elicits functional alterations. These data demonstrate the ability of pCALM1 to conduct restricted neuronal gene expression, illustrating the feasibility of ultra-miniature promoters for targeting brain-cell subtypes.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

Targeted drug delivery system inspired by macropinocytosis.

Macropinocytosis is a widely-observed and evolutionarily-conserved endocytic process found in the eukaryotic cells. In comparison to other endocytic routes, macropinocytosis allows for the internalization of greater quantities of fluid-phase drugs, making it an attractive avenue for drug delivery. Recent evidence showed that various drug delivery systems can be internalized through macropinocytosis. Utilizing macropinocytosis may therefore provide a new avenue for targeted intracellular delivery. In this review, we provide an overview into the origins and distinctive properties of macropinocytosis, summarize the roles of macropinocytosis under healthy and pathological settings. Furthermore, we highlight the biomimetic and synthetic drug delivery systems that employ macropinocytosis as the primary internalization mechanism. To facilitate the clinical applications of these drug delivery systems, additional research can be conducted to enhance the cell-type selectivity of macropinocytosis, the control of drug release at the target, and the prevention of potential toxicity. The rapidly emerging field of macropinocytosis-based targeted drug delivery and therapies holds great potential to drastically increase the efficiency and specificity of drug delivery.

EGFR stimulation enables IL-6 trans-signalling via iRhom2-dependent ADAM17 activation in mammary epithelial cells

The cytokine interleukin-6 (IL-6) has considerable pro-inflammatory properties and is a driver of many physiological and pathophysiological processes. Cellular responses to IL-6 are mediated by membrane-bound or soluble forms of the IL-6 receptor (IL-6R) complexed with the signal-transducing subunit gp130. While expression of the membrane-bound IL-6R is restricted to selected cell types, soluble IL-6R (sIL-6R) enables gp130 engagement on all cells, a process termed IL-6 trans-signalling and considered to be pro-inflammatory. sIL-6R is predominantly generated through proteolytic processing by the metalloproteinase ADAM17. ADAM17 also liberates ligands of the epidermal growth factor receptor (EGFR), which is a prerequisite for EGFR activation and results in stimulation of proliferative signals. Hyperactivation of EGFR mostly due to activating mutations drives cancer development. Here, we reveal an important link between overshooting EGFR signalling and the IL-6 trans-signalling pathway. In epithelial cells, EGFR activity induces not only IL-6 expression but also the proteolytic release of sIL-6R from the cell membrane by increasing ADAM17 surface activity. We find that this derives from the transcriptional upregulation of iRhom2, a crucial regulator of ADAM17 trafficking and activation, upon EGFR engagement, which results in increased surface localization of ADAM17. Also, phosphorylation of the EGFR-downstream mediator ERK mediates ADAM17 activity via interaction with iRhom2. In sum, our study reveals an unforeseen interplay between EGFR activation and IL-6 trans-signalling, which has been shown to be fundamental in inflammation and cancer.