Balancing accuracy and speed in the development of inhibitory control.

Inhibitory control develops rapidly and nonlinearly, making its accurate assessment challenging. This research investigated the developmental dynamics of accuracy and response latency in inhibitory control assessment of 3- to 6-year-old children in a longitudinal study (N = 431; 212 girls; Mage = 4.86 years, SD = 0.99) and a cross-sectional study (N = 135; 71 girls; Mage = 4.24 years, SD = 0.61). We employed a computerized Stroop task to measure inhibitory control, with fluid intelligence serving as a covariate. A growth curve analysis revealed that children who reached an accuracy threshold of 80% earlier demonstrated faster improvements in response latency. Both the cross-sectional and longitudinal findings demonstrated a positive association between response latency in the inhibitory control task and fluid intelligence, but only when participants had achieved and maintained high accuracy. These results suggest that researchers should consider response latency as an indicator of inhibitory control only in children who manage to respond accurately in an inhibitory control task.

Two-Stage Pedestrian Detection Model Using a New Classification Head for Domain Generalization.

Pedestrian detection based on deep learning methods have reached great success in the past few years with several possible real-world applications including autonomous driving, robotic navigation, and video surveillance. In this work, a new neural network two-stage pedestrian detector with a new custom classification head, adding the triplet loss function to the standard bounding box regression and classification losses, is presented. This aims to improve the domain generalization capabilities of existing pedestrian detectors, by explicitly maximizing inter-class distance and minimizing intra-class distance. Triplet loss is applied to the features generated by the region proposal network, aimed at clustering together pedestrian samples in the features space. We used Faster R-CNN and Cascade R-CNN with the HRNet backbone pre-trained on ImageNet, changing the standard classification head for Faster R-CNN, and changing one of the three heads for Cascade R-CNN. The best results were obtained using a progressive training pipeline, starting from a dataset that is further away from the target domain, and progressively fine-tuning on datasets closer to the target domain. We obtained state-of-the-art results, MR-2 of 9.9, 11.0, and 36.2 for the reasonable, small, and heavy subsets on the CityPersons benchmark with outstanding performance on the heavy subset, the most difficult one.

An end-to-end workflow for multiplexed image processing and analysis.

Multiplexed imaging enables the simultaneous spatial profiling of dozens of biological molecules in tissues at single-cell resolution. Extracting biologically relevant information, such as the spatial distribution of cell phenotypes from multiplexed tissue imaging data, involves a number of computational tasks, including image segmentation, feature extraction and spatially resolved single-cell analysis. Here, we present an end-to-end workflow for multiplexed tissue image processing and analysis that integrates previously developed computational tools to enable these tasks in a user-friendly and customizable fashion. For data quality assessment, we highlight the utility of napari-imc for interactively inspecting raw imaging data and the cytomapper R/Bioconductor package for image visualization in R. Raw data preprocessing, image segmentation and feature extraction are performed using the steinbock toolkit. We showcase two alternative approaches for segmenting cells on the basis of supervised pixel classification and pretrained deep learning models. The extracted single-cell data are then read, processed and analyzed in R. The protocol describes the use of community-established data containers, facilitating the application of R/Bioconductor packages for dimensionality reduction, single-cell visualization and phenotyping. We provide instructions for performing spatially resolved single-cell analysis, including community analysis, cellular neighborhood detection and cell-cell interaction testing using the imcRtools R/Bioconductor package. The workflow has been previously applied to imaging mass cytometry data, but can be easily adapted to other highly multiplexed imaging technologies. This protocol can be implemented by researchers with basic bioinformatics training, and the analysis of the provided dataset can be completed within 5-6 h. An extended version is available at https://bodenmillergroup.github.io/IMCDataAnalysis/ .

Circulating MicroRNAs as Biomarker for Vessel-Associated Retinal Diseases.

INTRODUCTION: Vessel-associated retinal diseases are a major cause of blindness and severe visual impairment. The identification of appropriate biomarkers is of great importance to better anticipate disease progression and establish more targeted treatment options. MicroRNAs (miRNAs) are short, single-stranded, noncoding ribonucleic acids that are involved in the posttranscriptional regulation of gene expression through hybridization with messenger RNA. The expression of certain miRNAs can be different in patients with pathological processes and can be used for the detection and differentiation of various diseases. In this study, we investigate to what extent previously in vitro identified miRNAs are present as cell-free circulating miRNAs in the serum and vitreous of human patients with and without vessel-associated retinal diseases. METHODS: Relative quantification by quantitative real-time polymerase chain reaction was used to analyze miRNA expression in patients with vessel-associated retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinal vein occlusion compared with control patients. RESULTS: In serum samples, miR-29a-3p and miR-192-5p showed increased expression in patients with neovascular AMD relative to control patients. Similarly, miR-335-5p, miR-192-5p, and miR-194-5p showed increased expression in serum from patients with proliferative DR. In vitreous samples, miR-100-5p was decreased in patients with proliferative DR. Differentially expressed miRNAs showed good diagnostic accuracy in receiver operating characteristic (ROC) and area under the ROC curve analysis. CONCLUSION: The miRNAs investigated in this study may have the potential to serve as biomarkers for vessel-associated retinal diseases. Combining multiple miRNAs may enhance the predictive power of the analysis.

Optimizing multiplexed imaging experimental design through tissue spatial segregation estimation.

Recent advances in multiplexed imaging methods allow simultaneous detection of dozens of proteins and hundreds of RNAs, enabling deep spatial characterization of both healthy and diseased tissues. Parameters for the design of optimal multiplex imaging studies, especially those estimating how much area has to be imaged to capture all cell phenotype clusters, are lacking. Here, using a spatial transcriptomic atlas of healthy and tumor human tissues, we developed a statistical framework that determines the number and area of fields of view necessary to accurately identify all cell phenotypes that are part of a tissue. Using this strategy on imaging mass cytometry data, we identified a measurement of tissue spatial segregation that enables optimal experimental design. This strategy will enable an improved design of multiplexed imaging studies.

Effects of Dermatan Sulfate from Marine Invertebrate Styela plicata in the Wound Healing Pathway: A Natural Resource Applied to Regenerative Therapy.

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.

Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma.

PURPOSE: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. EXPERIMENTAL DESIGN: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. RESULTS: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. CONCLUSIONS: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy.

Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13, suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7+ naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.

Cutaneous and systemic hyperinflammation drives maculopapular drug exanthema in severely ill COVID-19 patients.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has been associated with cutaneous findings, some being the result of drug hypersensitivity reactions such as maculopapular drug rashes (MDR). The aim of this study was to investigate whether COVID-19 may impact the development of the MDR. METHODS: Blood and skin samples from COVID-19 patients (based on a positive nasopharyngeal PCR) suffering from MDR (COVID-MDR), healthy controls, non-COVID-19-related patients with drug rash with eosinophilia and systemic symptoms (DRESS), and MDR were analyzed. We utilized imaging mass cytometry (IMC) to characterize the cellular infiltrate in skin biopsies. Furthermore, RNA sequencing transcriptome of skin biopsy samples and high-throughput multiplexed proteomic profiling of serum were performed. RESULTS: IMC revealed by clustering analyses a more prominent, phenotypically shifted cytotoxic CD8+ T cell population and highly activated monocyte/macrophage (Mo/Mac) clusters in COVID-MDR. The RNA sequencing transcriptome demonstrated a more robust cytotoxic response in COVID-MDR skin. However, severe acute respiratory syndrome coronavirus 2 was not detected in skin biopsies at the time point of MDR diagnosis. Serum proteomic profiling of COVID-MDR patients revealed upregulation of various inflammatory mediators (IL-4, IL-5, IL-6, TNF, and IFN-γ), eosinophil and Mo/Mac -attracting chemokines (MCP-2, MCP-3, MCP-4 and CCL11). Proteomics analyses demonstrated a massive systemic cytokine storm in COVID-MDR compared with the relatively milder cytokine storm observed in DRESS, while MDR did not exhibit such features. CONCLUSION: A systemic cytokine storm may promote activation of Mo/Mac and cytotoxic CD8+ T cells in severe COVID-19 patients, which in turn may impact the development of MDR.

Outcome of Repeat Pulmonary Metastasectomy.

Pulmonary metastasectomy is a well-established contribution to the cure of oligometastatic cancers, but its exact effectiveness is poorly understood. Here we report the outcomes of repeat pulmonary metastasectomy from a multicenter trial. This retrospective study included patients who underwent re-do metastasectomies between January 2010 and December 2014. The exclusion criterion was metastasectomy without curative intent. We reviewed medical files of 621 consecutive patients who underwent initial pulmonary metastasectomy. Of those, 64 patients underwent repeat metastasectomies, and these patients were included in the analysis. All the 64 patients underwent a second metastasectomy, later 35 of them underwent a third metastasectomy, 12 underwent a fourth metastasectomy, and 6 underwent a fifth metastasectomy. The total number of re-do metastasectomies was 181. The median overall survival among the patients undergoing re-do metastasectomy was 66.0 ± 3.8 months. Three and 5-year survival rates were 82.3% and 63.3%, respectively. The 5-year survival rates were 63.3% after the first, 50.9% after the second, 74.4% after the third, 83.3% after the fourth, and 60.0% after the fifth metastasectomy. We conclude that at the current stage of knowledge, there is an indication for repeat re-do metastasectomy with curative intent.

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

Hair eruption initiates and commensal skin microbiota aggravate adverse events of anti-EGFR therapy.

Epidermal growth factor receptor (EGFR)-targeted anticancer therapy induces stigmatizing skin toxicities affecting patients' quality of life and therapy adherence. The lack of mechanistic details underlying these adverse events hampers their management. We found that EGFR/ERK signaling is required in LRIG1-positive stem cells during de novo hair eruption to secure barrier integrity and prevent the invasion of commensal microbiota and inflammatory skin disease. EGFR-deficient epidermis is permissive for microbiota outgrowth and displays an atopic-like TH2-dominated signature. The opening of the follicular ostia during hair eruption allows invasion of commensal microbiota into the hair follicle, initiating an additional TH1 and TH17 response culminating in chronic folliculitis. Restoration of epidermal ERK signaling via prophylactic FGF7 treatment or transgenic SOS expression rescues the barrier defect in the absence of EGFR, highlighting a therapeutic anchor point. These data reveal that commensal skin microbiota provoke atopic-like inflammatory skin diseases by invading into the follicular opening of erupting hair.

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.

In-Depth Characterization of Monocyte-Derived Macrophages using a Mass Cytometry-Based Phagocytosis Assay.

Phagocytosis is a process in which target cells or particles are engulfed and taken up by other cells, typically professional phagocytes; this process is crucial in many physiological processes and disease states. The detection of targets for phagocytosis is directed by a complex repertoire of cell surface receptors. Pattern recognition receptors directly detect targets for binding and uptake, while opsonic and complement receptors detect objects coated by soluble factors. However, the importance of single and combinatorial surface marker expression across different phenotypes of professional phagocytes is not known. Here we developed a novel mass cytometry-based phagocytosis assay that enables the simultaneous detection of phagocytic events in combination with up to 40 other protein markers. We applied this assay to distinct monocyte derived macrophage (MDM) populations and found that prototypic M2-like MDMs phagocytose more E. coli than M1-like MDMs. Surface markers such as CD14, CD206, and CD163 rendered macrophages phagocytosis competent, but only CD209 directly correlated with the amount of particle uptake. Similarly, M2-like MDMs also phagocytosed more cancer cells than M1-like MDMs but, unlike M1-like MDMs, were insensitive to anti-CD47 opsonization. Our approach facilitates the simultaneous study of single-cell phenotypes, phagocytic activity, signaling and transcriptional events in complex cell mixtures.

Bringing together what belongs together: Optimizing murine infection models by using mouse-adapted Staphylococcus aureus strains.

Staphylococcus (S.) aureus is a leading cause of bacterial infection world-wide, and currently no vaccine is available for humans. Vaccine development relies heavily on clinically relevant infection models. However, the suitability of mice for S. aureus infection models has often been questioned, because experimental infection of mice with human-adapted S. aureus requires very high infection doses. Moreover, mice were not considered to be natural hosts of S. aureus. The latter has been disproven by our recent findings, showing that both laboratory mice, as well as wild small mammals including mice, voles, and shrews, are naturally colonized with S. aureus. Here, we investigated whether mouse-and vole-derived S. aureus strains show an enhanced virulence in mice as compared to the human-adapted strain Newman. Using a step-wise approach based on the bacterial genotype and in vitro assays for host adaptation, we selected the most promising candidates for murine infection models out of a total of 254 S. aureus isolates from laboratory mice as well as wild rodents and shrews. Four strains representing the clonal complexes (CC) 8, 49, and 88 (n = 2) were selected and compared to the human-adapted S. aureus strain Newman (CC8) in murine pneumonia and bacteremia models. Notably, a bank vole-derived CC49 strain, named DIP, was highly virulent in BALB/c mice in pneumonia and bacteremia models, whereas the other murine and vole strains showed virulence similar to or lower than that of Newman. At one tenth of the standard infection dose DIP induced disease severity, bacterial load and host cytokine and chemokine responses in the murine bacteremia model similar to that of Newman. In the pneumonia model, DIP was also more virulent than Newman but the effect was less pronounced. Whole genome sequencing data analysis identified a pore-forming toxin gene, lukF-PV(P83)/lukM, in DIP but not in the other tested S. aureus isolates. To conclude, the mouse-adapted S. aureus strain DIP allows a significant reduction of the inoculation dose in mice and is hence a promising tool to develop clinically more relevant infection models.

Inactivation of the Mouse L-Proline Transporter PROT Alters Glutamatergic Synapse Biochemistry and Perturbs Behaviors Required to Respond to Environmental Changes.

The endogenous neutral amino acid L-proline exhibits a variety of physiological and behavioral actions in the nervous system, highlighting the importance of accurately regulating its extracellular abundance. The L-proline transporter PROT (Slc6A7) is believed to control the spatial and temporal distribution of L-proline at glutamatergic synapses by rapid uptake of this amino acid into presynaptic terminals. Despite the importance of members of the Slc6 transporter family regulating neurotransmitter signaling and homeostasis in brain, evidence that PROT dysfunction supports risk for mental illness is lacking. Here we report the disruption of the PROT gene by homologous recombination. Mice defective in PROT displayed altered expression of glutamate transmission-related synaptic proteins in cortex and thalamus. PROT deficiency perturbed mouse behavior, such as reduced locomotor activity, decreased approach motivation and impaired memory extinction. Thus, our study demonstrates that PROT regulates behaviors that are needed to respond to environmental changes in vivo and suggests that PROT dysfunctions might contribute to mental disorders showing altered response choice following task contingency changes.

Simultaneous Multiplexed Imaging of mRNA and Proteins with Subcellular Resolution in Breast Cancer Tissue Samples by Mass Cytometry.

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.