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Background: The commercially available 10x Genomics protocol to generate droplet-based single-cell RNA-seq (scRNA-seq) data is enjoying growing popularity among researchers. Fundamental to the analysis of such scRNA-seq data is the ability to cluster similar or same cells into non-overlapping groups. Many competing methods have been proposed for this task, but there is currently little guidance with regards to which method to use. Methods: Here we use one gold standard 10x Genomics dataset, generated from the mixture of three cell lines, as well as three silver standard 10x Genomics datasets generated from peripheral blood mononuclear cells to examine not only the accuracy but also robustness of a dozen methods. Results: We found that some methods, including Seurat and Cell Ranger, outperform other methods, although performance seems to be dependent on the complexity of the studied system. Furthermore, we found that solutions produced by different methods have little in common with each other. Conclusions: In light of this, we conclude that the choice of clustering tool crucially determines interpretation of scRNA-seq data generated by 10x Genomics. Hence practitioners and consumers should remain vigilant about the outcome of 10x Genomics scRNA-seq analysis.

Single cell RNA-sequencing (scRNA-seq) technology has undergone rapid development in recent years, leading to an explosion in the number of tailored data analysis methods. However, the current lack of gold-standard benchmark datasets makes it difficult for researchers to systematically compare the performance of the many methods available. Here, we generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create ‘pseudo cells’ from up to five distinct cancer cell lines. In total, 14 datasets were generated using both droplet and plate-based scRNA-seq protocols. We compared 3,913 combinations of data analysis methods for tasks ranging from normalization and imputation to clustering, trajectory analysis and data integration. Evaluation revealed pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps.A dataset made up of single cancer cells or their mixtures serves as a benchmark for testing almost 4,000 combinations of scRNA-seq data analysis methods.

The glioblastoma (GBM) microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly affect the immune system, but the mechanisms driving these interactions are not completely clear. Here, we demonstrate that the polyamine metabolite spermidine (SPD) was elevated in the GBM tumor microenvironment. Exogenous administration of SPD drove tumor aggressiveness in an immune-dependent manner in preclinical mouse models via reduction of CD8+ T cell frequency and reduced cytotoxic function. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in SPD synthesis, did not affect cancer cell growth in vitro but did result in extended survival. Furthermore, patients with GBM with a more favorable outcome had a significant reduction in SPD compared with patients with a poor prognosis. Our results demonstrate that SPD functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+ T cell numbers and function.

Glioblastoma, a rare, and highly lethal form of brain cancer, poses significant challenges in terms of therapeutic resistance, and poor survival rates for both adult and paediatric patients alike. Despite advancements in brain cancer research driven by a technological revolution, translating our understanding of glioblastoma pathogenesis into improved clinical outcomes remains a critical unmet need. This review emphasises the intricate role of receptor tyrosine kinase signalling pathways, epigenetic mechanisms, and metabolic functions in glioblastoma tumourigenesis and therapeutic resistance. We also discuss the extensive efforts over the past two decades that have explored targeted therapies against these pathways. Emerging therapeutic approaches, such as antibody-toxin conjugates or CAR T cell therapies, offer potential by specifically targeting proteins on the glioblastoma cell surface. Combination strategies incorporating protein-targeted therapy and immune-based therapies demonstrate great promise for future clinical research. Moreover, gaining insights into the role of cell-of-origin in glioblastoma treatment response holds the potential to advance precision medicine approaches. Addressing these challenges is crucial to improving outcomes for glioblastoma patients and moving towards more effective precision therapies.

Periventricular nodular heterotopia (PVNH) is a malformation of cortical development commonly associated with epilepsy. We exome sequenced 202 individuals with sporadic PVNH to identify novel genetic risk loci. We first performed a trio-based analysis and identified 219 de novo variants. Although no novel genes were implicated in this initial analysis, PVNH cases were found overall to have a significant excess of nonsynonymous de novo variants in intolerant genes (p = 3.27x10-7), suggesting a role for rare new alleles in genes yet to be associated with the condition. Using a gene-level collapsing analysis comparing cases and controls, we identified a genome-wide significant signal driven by four ultra-rare loss-of-function heterozygous variants in MAP1B, including one de novo variant. In at least one instance, the MAP1B variant was inherited from a parent with previously undiagnosed PVNH. The PVNH was frontally predominant and associated with perisylvian polymicrogyria. These results implicate MAP1B in PVNH. More broadly, our findings suggest that detrimental mutations likely arising in immediately preceding generations with incomplete penetrance may also be responsible for some apparently sporadic diseases.

RNA sequencing (RNA-seq) is widely used in biomedical research, advancing our understanding of gene expression across biological systems. Traditional methods require upstream RNA extraction from biological inputs, adding time and expense to workflows. We developed TIRE-seq (Turbocapture Integrated RNA Expression Sequencing) to address these challenges. TIRE-seq integrates mRNA purification directly into library preparation, eliminating the need for a separate extraction step. This streamlined approach reduces turnaround time, minimizes sample loss, and improves data quality. A comparative study with the widely used Prime-seq protocol demonstrates TIRE-seq’s superior sequencing efficiency with crude cell lysates as inputs. TIRE-seq’s utility was demonstrated across three biological applications. It captured transcriptional changes in stimulated human T cells, revealing activation-associated gene expression profiles. It also identified key genes driving murine dendritic cell differentiation, providing insights into lineage commitment. Lastly, TIRE-seq analyzed the dose-response and time-course effects of temozolomide on patient-derived neurospheres, identifying differentially expressed genes and enriched pathways linked to the drug’s mechanism of action. With its simplified workflow and high sequencing efficiency, TIRE-seq offers a cost-effective solution for large-scale gene expression studies across diverse biological systems.

Purpose In children and adolescents, preoperative planning for a semi-implantable bone conduction device (SIBCD) is crucial. The geometric changes of the new version of a common SIBCD should enable a higher rate of successful implantation due to its flatter actuator. Thus, this radioanatomic study compared the rate of successful implantation of both device versions at the traditional mastoidal localization and two alternative sites, retrosigmoidal, and parietal, and investigated parameters helping to estimate the feasibility. Methods A retrospective analysis of 136 CT scans of 0 to 20-year-old patients, evaluation of demographic parameters, radioanatomy, and assessment of head diameter was conducted. The feasibility was investigated for certain age groups at three implantation sites. Prediction of feasible implantation by means of different parameters was calculated. Results A significant higher implantation rate was observed with the new device for all three sites and age groups. The age group of 6–8 years ( n  = 19) had most striking differences with a 58.1% rate of successful implantation with the new device without spacer (80% with spacer) at the mastoidal localization, whereas none with the old implant. Head diameter was identified as the most predictive parameter regarding all implantation sites (mastoidal: p  = 0.030; retrosigmoidal: p  = 0.006; parietal: p  < 0.0001), age for the mastoidal ( p  < 0.0001) and retrosigmoidal ( p  < 0.0001), and gender for the parietal site ( p  = 0.001). Conclusion The geometric changes of the actuator lead to a higher rate of successful implantation in all age-groups and all three localizations with reducing the requirement for spacers. Parameters age and head diameter might aid in estimating the rate of successful implantation in young patients and may be a novel tool to assist in the decision-making process for a SIBCD.

Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography–mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

Background Selective neck dissection (SND) is the surgical treatment of choice in squamous cell carcinoma of the head and neck (HNSCC) with suspected or manifest metastases in the cervical lymph nodes. For SND to be successful, treated lymph node levels should be selected according to anatomic considerations and the extent of the disease. Aim of this study was to identify neck dissection levels that had an impact on individual prognosis. Methods We conducted a retrospective review of SND as part of primary treatment of HNSCC. Overall survival (OS) and regional control rates (RCR) were calculated for all patients treated at one academic tertiary referral center. Results 661 patients with HNSCC were included, 644 underwent ipsilateral and 319 contralateral SND. Average follow-up was 78.9 ± 106.4 months. 67 (10.1%) patients eventually developed nodal recurrence. Tumor sites were oral cavity (135), oropharynx (179), hypopharynx (118) and larynx (229). Tumor categories pT1–pT4a, and all clinical and pathological nodal categories were included. Multivariate analysis indicated improved OS rates for patients undergoing SND in ipsilateral levels I and V as well as level III contralaterally. Analysis for tumor origin showed that SND in ipsilateral level I showed significantly improved OS in HNSCC of the oral cavity. Conclusion The dissection of ipsilateral level I in oral cavity cancer was of particular relevance in our exploratory, retrospective analysis. To clarify the relevance for the determination of the extent of SND, this should be investigated prospectively in a more homogenous patient cohort.

Familial adult myoclonus epilepsy (FAME) is a rare autosomal dominant disorder characterized by adult onset, involuntary muscle jerks, cortical myoclonus and occasional seizures. FAME is genetically heterogeneous with more than 70 families reported worldwide and five potential disease loci. The efforts to identify potential causal variants have been unsuccessful in all but three families. To date, linkage analysis has been the main approach to find and narrow FAME critical regions. We propose an alternative method, pedigree free identity-by-descent (IBD) mapping, that infers regions of the genome between individuals that have been inherited from a common ancestor. IBD mapping provides an alternative to linkage analysis in the presence of allelic and locus heterogeneity by detecting clusters of individuals who share a common allele. Succeeding IBD mapping, gene prioritization based on gene co-expression analysis can be used to identify the most promising candidate genes. We performed an IBD analysis using high-density single nucleotide polymorphism (SNP) array data followed by gene prioritization on a FAME cohort of ten European families and one Australian/New Zealander family; eight of which had known disease loci. By identifying IBD regions common to multiple families, we were able to narrow the FAME2 locus to a 9.78 megabase interval within 2p11.2–q11.2. We provide additional evidence of a founder effect in four Italian families and allelic heterogeneity with at least four distinct founders responsible for FAME at the FAME2 locus. In addition, we suggest candidate disease genes using gene prioritization based on gene co-expression analysis.