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The therapeutic application of drugs often faces challenges due to non-specific distribution, inadequate dosification and degradation, which limits their efficacy. Two primary strategies are employed to overcome these issues: the use of derivatives of the active substances and incorporation of those into porous materials. The latter, involving materials such as zeolites, metal-organic frameworks (MOFs), and hydrogels, has shown promising results in protecting the active ingredients from degradation and enabling a controlled release. This study focuses on supramolecular metal-organic frameworks (SMOFs), which leverage supramolecular interactions for enhanced pore size control. [Cu 6 Cr(μ-adeninato-κ N 3:κ N 9) 6 (μ 3 -OH) 6 (μ-OH 2 ) 6 ](SO 4 ) 1.5 · n H 2 O (Cu6Cr) was chosen for its flexible porous structure, water-stability, and paramagnetic properties. Magnetic sustentation studies showed that this compound was able to capture several drug molecules: 5-fluorouracil (5-FU), 5-aminosalicylic acid (5-ASA), 4-aminosalicylic acid (4-ASA) and theophylline (THEO). Their release follows a pseudo-first-order kinetics with desorption half-lives ranging from 2.2 to 4.7 hours. In this sense, a novel approach is proposed using bulkier raffinose and cholesterol as pore-blocking molecules. Cholesterol exhibited the best performance as a blocking molecule increasing the desorption half-life up to 8.2 hours. Cytotoxicity and RNA-seq transcriptomic assays carried out on human colorectal cancer cell cultures showed, on one hand, that the Cu6Cr porous material exhibits a proliferative effect, probably coming from the over-expression of MIR1248 and SUMO2 genes, and on the other hand, that there is a delay in the emergence of the cytotoxicity of 5-FU as expected for a slower release.

Multimorbidity (MM) is the co-occurrence of two or more chronic diseases. We provided a dynamic approach revealing the MM complexity constructing a multistep incidence-age model for all patients with MM between 2014 and 2021 in the Basque Health System, Spain. The multistep model, with eight steps for males and nine for females, is a very well-fitting representation of MM. To gain insight into the MM components, we modeled the 19 diseases used to calculate the Charlson Comorbidity Index (CCI). We observed that the CCI diseases formed a complex interaction network. Hierarchical clustering of the incidence-age profiles clustered the CCI diseases into low- and high-risk of dying pathologies. Diseases with a higher number of steps are better represented by a multistep model. Anatomically, diseases associated with the central nervous system have the highest number of steps, followed by those associated with the kidney, heart, peripheral vasulature, pancreas, joints, cerebral vasculature, lung, stomach, and liver., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Strong epigenetic pan-cancer biomarkers are required to meet several current, urgent clinical needs and to further improve the present chemotherapeutic standard. We have concentrated on the investigation of epigenetic alteration of the hTERT gene, which is frequently epigenetically dysregulated in a number of cancers in specific developmental stages. Distinct DNA methylation profiles were identified in our data on early urothelial cancer. An efficient EpihTERT assay could be developed utilizing suitable combinations with sequence-dependent thermodynamic parameters to distinguish between differentially methylated states. We infer from this data set, the epigenetic context, and the related literature that a CpG-rich, 2800 bp region, a prominent CpG island, surrounding the transcription start of the hTERT gene is the crucial epigenetic zone for the development of a potent biomarker. In order to accurately describe this region, we have named it "Acheron" (Ἀχέρων). In Greek mythology, this is the river of woe and misery and the path to the underworld. Exploitation of the DNA methylation profiles focused on this region, e.g., idiolocal normalized Methylation Specific PCR (IDLN-MSP), opens up a wide range of new possibilities for diagnosis, determination of prognosis, follow-up, and detection of residual disease. It may also have broad implications for the choice of chemotherapy.

Based on the impressive success of Car-T-cell therapy in the treatment of hematological malignancies, a broad application for solid tumors also appears promising. However, some important hurdles need to be overcome. One of these is certainly the identification of specific target antigens on cancer cells. Hypomethylation is a characteristic epigenetic aberration in many tumor entities. Genome-wide screenings for consistent DNA hypomethylations in tumors enable the identification of aberrantly upregulated transcripts, which might result in cell surface proteins. Thus, this approach provides a new perspective for the discovery of potential new Car-T-cell target antigens for almost every tumor entity. First, we focus on this approach as a possible treatment for prostate cancer.

Background: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro., Methods: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes., Results: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo., Conclusions: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment., (© 2024. The Author(s).)

Background: Frailty is a geriatric syndrome associated with negative health outcomes that represents a dynamic condition with a potential of reversibility after physical exercise interventions. Typically, inflammatory and senescence markers are increased in frail individuals. However, the impact that physical exercise exerts on inflammatory and senescence biomarkers remains unknown. We assessed the effect of physical intervention in old individuals and mice and determined the expression of inflammatory and senescence markers., Methods: Twelve elderly individuals were enrolled from a primary care setting to a 3-month intervention. Frailty was measured by SPPB and the expression of biomarkers by cytokine array and RT-qPCR. In addition, 12 aged C57BL/6 mice completed an intervention, and inflammation and senescence markers were studied., Results: The physical intervention improved the SPPB score, reducing frail and pre-frail individuals. This was correlated with a reduction in several pro-inflammatory biomarkers such as IL-6, CXCL-1, CXCL-10, IL-1β, IL-7, GM-CSF as well as p16 INK4a and p21 CIP1 senescence markers. Otherwise, the levels of anti-inflammatory biomarker IL-4 were significantly increased. Moreover, the physical intervention in mice also improved their functional capacity and restored the expression of inflammatory ( Il-1β , Cxcl-10 , Il-6 , and Cxcl-1 ) and senescence ( p21 Cip1 ) markers. Additionally, PLSDA and ROC curve analysis revealed CXCL-10 and IL-1β to be the biomarkers of functional improvement in both cohorts., Conclusions: Our results showed that a physical intervention improves physical frailty, and reverses inflammation and senescence biomarkers comprising CXCL-10 and IL-1β.

Cell-free extrachromosomal circular DNA (cf-eccDNA) has been proposed as a promising early biomarker for disease diagnosis, progression and drug response. Its established biomarker features are changes in the number and length distribution of cf-eccDNA. Another novel promising biomarker is a set of eccDNA excised from a panel of genes specific to a condition compared to a control. Deficiencies in two endonucleases that specifically target DNA, Dnase1 and Dnase1l3, are associated with systemic lupus erythematosus (SLE). To study the genic eccDNA profiles in the case of their deficiencies, we mapped sequenced eccDNA data from plasma, liver and buffy coat from Dnase1 and Dnase1l3 knockouts (KOs), and wild type controls in mouse. Next, we performed an eccDNA differential analysis between KO and control groups using our DifCir algorithm. We found a specific genic cf-eccDNA fingerprint of the Dnase1l3 group compared to the wild type controls involving 131 genes; 26% of them were associated with human chromosomal fragile sites (CFSs) and with a statistically significant enrichment of CFS-associated genes. We found six genes in common with the genic cf-eccDNA profile of SLE patients with DNASE1L3 deficiency, namely Rorb , Mvb12b , Osbpl10 , Fto , Tnik and Arhgap10 ; all of them were specific and present in all human plasma samples, and none of them were associated with CFSs. A not so distinctive genic cf-eccDNA difference involving only seven genes was observed in the case of the Dnase1 group compared to the wild type. In tissue-liver and buffy coat-we did not detect the same genic eccDNA difference observed in the plasma samples. These results point to a specific role of a set of genic eccDNA in plasma from DNase KOs, as well as a relation with CFS genes, confirming the promise of the genic cf-eccDNA in studying diseases and the need for further research on the relationship between eccDNA and CFSs.

Introduction: Bipolar disorder (BD) is a chronically progressive mental condition, associated with a reduced quality of life and greater disability. Patient admissions are preventable events with a considerable impact on global functioning and social adjustment. While machine learning (ML) approaches have proven prediction ability in other diseases, little is known about their utility to predict patient admissions in this pathology., Aim: To develop prediction models for hospital admission/readmission within 5 years of diagnosis in patients with BD using ML techniques., Methods: The study utilized data from patients diagnosed with BD in a major healthcare organization in Colombia. Candidate predictors were selected from Electronic Health Records (EHRs) and included sociodemographic and clinical variables. ML algorithms, including Decision Trees, Random Forests, Logistic Regressions, and Support Vector Machines, were used to predict patient admission or readmission. Survival models, including a penalized Cox Model and Random Survival Forest, were used to predict time to admission and first readmission. Model performance was evaluated using accuracy, precision, recall, F1 score, area under the receiver operating characteristic curve (AUC) and concordance index., Results: The admission dataset included 2,726 BD patients, with 354 admissions, while the readmission dataset included 352 patients, with almost half being readmitted. The best-performing model for predicting admission was the Random Forest, with an accuracy score of 0.951 and an AUC of 0.98. The variables with the greatest predictive power in the Recursive Feature Elimination (RFE) importance analysis were the number of psychiatric emergency visits, the number of outpatient follow-up appointments and age. Survival models showed similar results, with the Random Survival Forest performing best, achieving an AUC of 0.95. However, the prediction models for patient readmission had poorer performance, with the Random Forest model being again the best performer but with an AUC below 0.70., Conclusion: ML models, particularly the Random Forest model, outperformed traditional statistical techniques for admission prediction. However, readmission prediction models had poorer performance. This study demonstrates the potential of ML techniques in improving prediction accuracy for BD patient admissions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Palacios-Ariza, Morales-Mendoza, Murcia, Arias-Duarte, Lara-Castellanos, Cely-Jiménez, Rincón-Acuña, Araúzo-Bravo and McDouall.)

Background: Numerous genes, including SOD1, mutated in familial and sporadic amyotrophic lateral sclerosis (f/sALS) share a role in DNA damage and repair, emphasizing genome disintegration in ALS. One possible outcome of chromosomal instability and repair processes is extrachromosomal circular DNA (eccDNA) formation. Therefore, eccDNA might accumulate in f/sALS with yet unknown function., Methods: We combined rolling circle amplification with linear DNA digestion to purify eccDNA from the cervical spinal cord of 9 co-isogenic symptomatic hSOD1 G93A mutants and 10 controls, followed by deep short-read sequencing. We mapped the eccDNAs and performed differential analysis based on the split read signal of the eccDNAs, referred as DifCir, between the ALS and control specimens, to find differentially produced per gene circles (DPpGC) in the two groups. Compared were eccDNA abundances, length distributions and genic profiles. We further assessed proteome alterations in ALS by mass spectrometry, and matched the DPpGCs with differentially expressed proteins (DEPs) in ALS. Additionally, we aligned the ALS-specific DPpGCs to ALS risk gene databases., Results: We found a six-fold enrichment in the number of unique eccDNAs in the genotoxic ALS-model relative to controls. We uncovered a distinct genic circulome profile characterized by 225 up-DPpGCs, i.e., genes that produced more eccDNAs from distinct gene sequences in ALS than under control conditions. The inter-sample recurrence rate was at least 89% for the top 6 up-DPpGCs. ALS proteome analyses revealed 42 corresponding DEPs, of which 19 underlying genes were itemized for an ALS risk in GWAS databases. The up-DPpGCs and their DEP tandems mainly impart neuron-specific functions, and gene set enrichment analyses indicated an overrepresentation of the adenylate cyclase modulating G protein pathway., Conclusions: We prove, for the first time, a significant enrichment of eccDNA in the ALS-affected spinal cord. Our triple circulome, proteome and genome approach provide indication for a potential importance of certain eccDNAs in ALS neurodegeneration and a yet unconsidered role as ALS biomarkers. The related functional pathways might open up new targets for therapeutic intervention., (© 2023. Society of Chinese Bioscientists in America (SCBA).)

Background: Native bone marrow (BM) mesenchymal stem/stromal cells (BM-MSCs) participate in generating and shaping the skeleton and BM throughout the lifespan. Moreover, BM-MSCs regulate hematopoiesis by contributing to the hematopoietic stem cell niche in providing critical cytokines, chemokines and extracellular matrix components. However, BM-MSCs contain a heterogeneous cell population that remains ill-defined. Although studies on the taxonomy of native BM-MSCs in mice have just started to emerge, the taxonomy of native human BM-MSCs remains unelucidated., Methods: By using single-cell RNA sequencing (scRNA-seq), we aimed to define a proper taxonomy for native human BM non-hematopoietic subsets including endothelial cells (ECs) and mural cells (MCs) but with a focal point on MSCs. To this end, transcriptomic scRNA-seq data were generated from 5 distinct BM donors and were analyzed together with other transcriptomic data and with computational biology analyses at different levels to identify, characterize and classify distinct native cell subsets with relevant biomarkers., Results: We could ascribe novel specific biomarkers to ECs, MCs and MSCs. Unlike ECs and MCs, MSCs exhibited an adipogenic transcriptomic pattern while co-expressing genes related to hematopoiesis support and multilineage commitment potential. Furthermore, by a comparative analysis of scRNA-seq of BM cells from humans and mice, we identified core genes conserved in both species. Notably, we identified MARCKS, CXCL12, PDGFRA, and LEPR together with adipogenic factors as archetypal biomarkers of native MSCs within BM. In addition, our data suggest some complex gene nodes regulating critical biological functions of native BM-MSCs together with a preferential commitment toward an adipocyte lineage., Conclusions: Overall, our taxonomy for native BM non-hematopoietic compartment provides an explicit depiction of gene expression in human ECs, MCs and MSCs at single-cell resolution. This analysis helps enhance our understanding of the phenotype and the complexity of biological functions of native human BM-MSCs., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Inflammatory bowel diseases (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic multifactorial disorders which affect the gastrointestinal tract with variable extent. Despite extensive research, their etiology and exact pathogenesis are still unknown. Cell-free DNAs (cfDNAs) are defined as any DNA fragments which are free from the origin cell and able to circulate into the bloodstream with or without microvescicles. CfDNAs are now being increasingly studied in different human diseases, like cancer or inflammatory diseases. However, to date it is unclear how IBD etiology is linked to cfDNAs in plasma. Extrachromosomal circular DNA (eccDNA) are non-plasmidic, nuclear, circular and closed DNA molecules found in all eukaryotes tested. CfDNAs appear to play an important role in autoimmune diseases, inflammatory processes, and cancer; recently, interest has also grown in IBD, and their role in the pathogenesis of IBD has been suggested. We now suggest that eccDNAs also play a role in IBD. In this review, we have comprehensively collected available knowledge in literature regarding cfDNA, eccDNA, and structures involving them such as neutrophil extracellular traps and exosomes, and their role in IBD. Finally, we focused on old and novel potential molecular therapies and drug delivery systems, such as nanoparticles, for IBD treatment.

Cell-free (cf) extrachromosomal circular DNA (eccDNA) has a potential clinical application as a biomarker. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a complex immunological pathogenesis, associated with autoantibody synthesis. A previous study found that SLE patients with deoxyribonuclease 1-like 3 (DNASE1L3) deficiency exhibit changes in the frequency of short and long eccDNA in plasma compared to controls. Here, using the DifCir method for differential analysis of short-read sequenced purified eccDNA data based on the split-read signal of the eccDNA on circulomics data, we show that SLE patients with DNASE1L3 deficiency have a distinctive profile of eccDNA excised by gene regions compared to controls. Moreover, this profile is specific; cf-eccDNA from the top 93 genes is detected in all SLE with DNASE1L3 deficiency samples, and none in the control plasma. The top protein coding gene producing eccDNA-carrying gene fragments is the transcription factor BARX2, which is involved in skeletal muscle morphogenesis and connective tissue development. The top gene ontology terms are 'positive regulation of torc1 signaling' and 'chondrocyte development'. The top Harmonizome terms are 'lymphopenia', 'metabolic syndrome x', 'asthma', 'cardiovascular system disease', 'leukemia', and 'immune system disease'. Here, we show that gene associations of cf-eccDNA can serve as a biomarker in the autoimmune rheumatic diseases.

To bring new extrachromosomal circular DNA (eccDNA) enrichment technologies closer to the clinic, specifically for screening, early diagnosis, and monitoring of diseases or lifestyle conditions, it is paramount to identify the differential pattern of the genic eccDNA signal between two states. Current studies using short-read sequenced purified eccDNA data are based on absolute numbers of unique eccDNAs per sample or per gene, length distributions, or standard methods for RNA-seq differential analysis. Previous analyses of RNA-seq data found significant transcriptomics difference between sedentary and active life style skeletal muscle (SkM) in young people but very few in old. The first attempt using circulomics data from SkM and blood of aged lifelong sedentary and physically active males found no difference at eccDNA level. To improve the capability of finding differences between circulomics data groups, we designed a computational method to identify Differentially Produced per Gene Circles (DPpGCs) from short-read sequenced purified eccDNA data based on the circular junction, split-read signal, of the eccDNA, and implemented it into a software tool DifCir in Matlab. We employed DifCir to find to the distinctive features of the influence of the physical activity or inactivity in the aged SkM that would have remained undetected by transcriptomics methods. We mapped the data from tissue from SkM and blood from two groups of aged lifelong sedentary and physically active males using Circle_finder and subsequent merging and filtering, to find the number and length distribution of the unique eccDNA. Next, we used DifCir to find up-DPpGCs in the SkM of the sedentary and active groups. We assessed the functional enrichment of the DPpGCs using Disease Gene Network and Gene Set Enrichment Analysis. To find genes that produce eccDNA in a group without comparison with another group, we introduced a method to find Common PpGCs (CPpGCs) and used it to find CPpGCs in the SkM of the sedentary and active group. Finally, we found the eccDNA that carries whole genes. We discovered that the eccDNA in the SkM of the sedentary group is not statistically different from that of physically active aged men in terms of number and length distribution of eccDNA. In contrast, with DifCir we found distinctive gene-associated eccDNA fingerprints. We identified statistically significant up-DPpGCs in the two groups, with the top up-DPpGCs shed by the genes AGBL4 , RNF213 , DNAH7 , MED13 , and WWTR1 in the sedentary group, and ZBTB7C , TBCD , ITPR2 , and DDX11-AS1 in the active group. The up-DPpGCs in both groups carry mostly gene fragments rather than whole genes. Though the subtle transcriptomics difference, we found RYR1 to be both transcriptionally up-regulated and up-DPpGCs gene in sedentary SkM. DifCir emphasizes the high sensitivity of the circulome compared to the transcriptome to detect the molecular fingerprints of exercise in aged SkM. It allows efficient identification of gene hotspots that excise more eccDNA in a health state or disease compared to a control condition.

Aging is often associated with a loss of cell type identity that results in an increase in transcriptional noise in aged tissues. If this phenomenon reflects a fundamental property of aging remains an open question. Transcriptional changes at the cellular level are best detected by single-cell RNA sequencing (scRNAseq). However, the diverse computational methods used for the quantification of age-related loss of cellular identity have prevented reaching meaningful conclusions by direct comparison of existing scRNAseq datasets. To address these issues we created Decibel , a Python toolkit that implements side-to-side four commonly used methods for the quantification of age-related transcriptional noise in scRNAseq data. Additionally, we developed Scallop , a novel computational method for the quantification of membership of single cells to their assigned cell type cluster. Cells with a greater Scallop membership score are transcriptionally more stable. Application of these computational tools to seven aging datasets showed large variability between tissues and datasets, suggesting that increased transcriptional noise is not a universal hallmark of aging. To understand the source of apparent loss of cell type identity associated with aging, we analyzed cell type-specific changes in transcriptional noise and the changes in cell type composition of the mammalian lung. No robust pattern of cell type-specific transcriptional noise alteration was found across aging lung datasets. In contrast, age-associated changes in cell type composition of the lung were consistently found, particularly of immune cells. These results suggest that claims of increased transcriptional noise of aged tissues should be reformulated., Competing Interests: OI, AA, MA, AI No competing interests declared, (© 2022, Ibañez-Solé, Ascensión et al.)

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15 . These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS).

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of an accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from patients with DM1 and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which related to the accumulation of cellular senescence. The data from transcriptome analyses were corroborated in human myoblasts and blood samples, as well as in mouse and Drosophila models of the disease. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts, whereas the DM1 Drosophila model showed reduced longevity and impaired locomotor activity. Moreover, functional studies highlighted the impact of BMI1 and downstream p16INK4A/RB and ARF/p53/p21CIP pathways in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds Quercetin, Dasatinib, or Navitoclax reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in Drosophila in vivo. Our results identify the accumulation of senescence as part of DM1 pathophysiology and, therefore, demonstrate the efficacy of senolytic compounds in the preclinical setting.

Background: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5' of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC)., Methods: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail., Results: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT . In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated ( hTAPAS ), which represses hTERT ., Conclusions: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided., Competing Interests: The authors declare no conflict of interest.

The transplantation of pluripotent stem cell (PSC)-derived liver organoids has been studied to solve the current donor shortage. However, the differentiation of unintended cell populations, difficulty in generating multi-lineage organoids, and tumorigenicity of PSC-derived organoids are challenges. However, direct conversion technology has allowed for the generation lineage-restricted induced stem cells from somatic cells bypassing the pluripotent state, thereby eliminating tumorigenic risks. Here, liver assembloids (iHEAs) were generated by integrating induced endothelial cells (iECs) into the liver organoids (iHLOs) generated with induced hepatic stem cells (iHepSCs). Liver assembloids showed enhanced functional maturity compared to iHLOs in vitro and improved therapeutic effects on cholestatic liver fibrosis animals in vivo. Mechanistically, FN1 expressed from iECs led to the upregulation of Itgα5/β1 and Hnf4α in iHEAs and were correlated to the decreased expression of genes related to hepatic stellate cell activation such as Lox and Spp1 in the cholestatic liver fibrosis animals. In conclusion, our study demonstrates the possibility of generating transplantable iHEAs with directly converted cells, and our results evidence that integrating iECs allows iHEAs to have enhanced hepatic maturation compared to iHLOs.

Background: Feature selection is a relevant step in the analysis of single-cell RNA sequencing datasets. Most of the current feature selection methods are based on general univariate descriptors of the data such as the dispersion or the percentage of zeros. Despite the use of correction methods, the generality of these feature selection methods biases the genes selected towards highly expressed genes, instead of the genes defining the cell populations of the dataset., Results: Triku is a feature selection method that favors genes defining the main cell populations. It does so by selecting genes expressed by groups of cells that are close in the k-nearest neighbor graph. The expression of these genes is higher than the expected expression if the k-cells were chosen at random. Triku efficiently recovers cell populations present in artificial and biological benchmarking datasets, based on adjusted Rand index, normalized mutual information, supervised classification, and silhouette coefficient measurements. Additionally, gene sets selected by triku are more likely to be related to relevant Gene Ontology terms and contain fewer ribosomal and mitochondrial genes., Conclusion: Triku is developed in Python 3 and is available at https://github.com/alexmascension/triku., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Proprotein convertases or PCs are known to regulate the malignant phenotype of colon cancer cells by different mechanisms, but their effects on cancer stem cells (CSCs) have been less widely investigated. Here, we report that PCs expression is altered in colon CSCs, and the inhibition of their activity reduced colon CSCs growth, survival, and invasion in three-dimensional spheroid cultures. In vivo, repression of PCs activity by the general PC inhibitors α1-PDX, Spn4A, or decanoyl-RVKR-chloromethylketone (CMK) significantly reduced tumor expression levels of the stem cell markers LGR5 and NANOG that are associated with reduced tumor xenografts. Further analysis revealed that reduced tumor growth mediated by specific silencing of the convertase Furin in KRAS or BRAF mutated-induced colon tumors was associated with reduced expression of LGR5 and NANOG compared to wild-type KRAS and BRAF tumors. Analysis of various calcium regulator molecules revealed that while the calcium-transporting ATPase 4 (ATP2B4) is downregulated in all the Furin-silenced colon cancer cells, the Ca 2+ -mobilizing P2Y receptors, was specifically repressed in BRAF mutated cells and ORAI1 and CACNA1H in KRAS mutated cells. Taken together, our findings indicate that PCs play an important role in the malignant phenotype of colon CSCs and stem cell markers' expression and highlight PCs repression, particularly of Furin, to target colon tumors with KRAS or BRAF mutation.

Oct4 collaborates primarily with other transcriptional factors or coregulators to maintain pluripotency. However, how Oct4 exerts its function is still unclear. Here, we show that the Oct4 linker interface mediates competing yet balanced Oct4 protein interactions that are crucial for maintaining pluripotency. Oct4 linker mutant embryonic stem cells (ESCs) show decreased expression of self-renewal genes and increased expression of differentiation genes, resulting in impaired ESC self-renewal and early embryonic development. The linker mutation interrupts the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 is decreased. In contrast, interactions between Oct4 and Cbx1, Ctr9, and Cdc73 are increased, disrupting the epigenetic state of ESCs. Control of the expression level of Klf5, Cbx1, or Cdc73 rebalances the Oct4 interactome and rescues the pluripotency of linker mutant ESCs, indicating that such factors interact with Oct4 competitively. Thus, we provide previously unidentified molecular insights into how Oct4 maintains pluripotency.

Erythropoiesis and megakaryo-/thrombopoiesis occur in the bone marrow proceeding from common, even bipotent, progenitor cells. Recently, we have shown that protective vaccination accelerates extramedullary hepatic erythroblastosis in response to blood-stage malaria of Plasmodium chabaudi. Here, we investigated whether protective vaccination also accelerates extramedullary hepatic megakaryo-/thrombopoiesis. Female Balb/c mice were twice vaccinated with a non-infectious vaccine before infecting with 10 6 &nbsp; P. chabaudi -parasitized erythrocytes. Using gene expression microarrays and quantitative real-time PCR, transcripts of genes known to be expressed in the bone marrow by cells of the megakaryo-/thrombocytic lineage were compared in livers of vaccination-protected and unprotected mice on days 0, 1, 4, 8, and 11 p.i. Livers of vaccination-protected mice responded with expression of megakaryo-/thrombocytic genes faster to P. chabaudi than those of unvaccinated mice, evidenced at early patency on day 4 p.i. , when livers exhibited significantly higher levels of malaria-induced transcripts of the genes Selp and Pdgfb ( p -values < 0.0001), Gp5 ( p -value < 0.001), and Fli1 , Runx1 , Myb , Mpl , Gp1ba , Gp1bb , Gp6 , Gp9 , Pf4 , and Clec1b ( p -values < 0.01). Together with additionally analyzed genes known to be related to megakaryopoiesis, our data suggest that protective vaccination accelerates liver-intrinsic megakaryo-/thrombopoiesis in response to blood-stage malaria that presumably contributes to vaccination-induced survival of otherwise lethal blood-stage malaria.

Background: Previously, we collected age-stratified incidence data of 404 epidemiological datasets of 10 neurodegenerative diseases (NDs), namely Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Fronto Temporal Dementia (FTD), Dementia with Lewy Bodies (DLB), Parkinsonism (PDM), Parkinson's disease with Dementia (PDD), Creutzfeldt-Jakob disease (CJD), and Multiple Sclerosis (MS). We tested whether each ND follows a multistep model, found the number of steps necessary for the onset of each ND, found the number of common steps with other NDs and the number of specific steps of each ND, and built a parsimony tree of the genealogy of the NDs. The tree disclosed three groups of NDs: the stem NDs with less than 3 steps; the trunk NDs with 5-7 steps; and the crown NDs with more than 7 steps., Methods: We made a multidimensional reduction of the previously collected age-stratified incidence epidemiological data of the 10 NDs. We studied the general range of incidence of the 10 NDs using the age- and sex-stratified incidence data. First, we calculated the log of the incidence versus the log of the age for each ND. Next, we calculated the age intervals of the spread of the incidence of each ND. We calculated the regression of the steps obtained with the multistep model versus the age of incidence of the NDs., Results: We found that the number of steps of the NDs is inversely correlated with the age of incidence of the NDs, and calculated the number of years required for a single step for each ND. Based on these results, we extended the genealogy tree model of the NDs to account for the time needed for a ND step to occur., Conclusion: The extended genealogy tree disclosed three groups of NDs according to the estimated time needed for a step to occur: the stem ND, HD, with 32.5 years/step, the trunk NDs ALS, FTD, PD and CJD, with 6.7-13.7 years/step; and the crown NDs PDM, PDD, AD and DLB, with 2.3-3.8 years/step. Thus, the NDs cluster into three groups according to both the number of steps and the number of years for a step to occur., (© 2022. The Author(s).)

Epigenetic mechanisms are fundamentally important for cancer initiation and development. However, a survey of the literature reveals that, to date, they appear less comprehensively investigated in melanoma than in many other cancers, e.g., prostate, breast, and colon carcinoma. The aim of this review is to provide a short summary of epigenetic aspects of functional relevance for melanoma pathogenesis. In addition, some new perspectives from epigenetic research in other cancers with potential for melanoma diagnosis and therapy are introduced. For example, the PrimeEpiHit hypothesis in urothelial carcinoma, which, similarly to malignant melanoma, can also be triggered by a single exogenous noxa, states that one of the first steps for cancer initiation could be epigenetic changes in key genes of one-carbon metabolism. The application of such insights may contribute to further progress in the diagnosis and therapy of melanoma, a deadly type of cancer.

Skin is a complex and heterogeneous organ at the cellular level. This complexity is beginning to be understood through the application of single-cell genomics and computational tools. A large number of datasets that shed light on how the different human skin cell types interact in homeostasis-and what ceases to work in diverse dermatological diseases-have been generated and are publicly available. However, translation of these novel aspects to the clinic is lacking. This review aims to summarize the state-of-the-art of skin biology using single-cell technologies, with a special focus on skin pathologies and the translation of mechanistic findings to the clinic. The main implications of this review are to summarize the benefits and limitations of single-cell analysis and thus help translate the emerging insights from these novel techniques to the bedside.

Background & Aims: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis., Methods: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation., Results: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells., Conclusion: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver., Lay Summary: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis., Competing Interests: Conflict of interest MP, NH and FC are employees of CureVac AG, Tuebingen Germany, a publicly listed company developing mRNA-based vaccines, cancer immunotherapeutics and mRNA-based protein replacement therapies. All authors may hold shares or stock options in the company. MP, NH and FC are inventors on several patents on mRNA-related technology and use thereof. Other authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The tumor's physiology emerges from the dynamic interplay of numerous cell types, such as cancer cells, immune cells and stromal cells, within the tumor microenvironment. Immune and cancer cells compete for nutrients within the tumor microenvironment, leading to a metabolic battle between these cell populations. Tumor cells can reprogram their metabolism to meet the high demand of building blocks and ATP for proliferation, and to gain an advantage over the action of immune cells. The study of the metabolic reprogramming mechanisms underlying cancer requires the quantification of metabolic fluxes which can be estimated at the genome-scale with constraint-based or kinetic modeling. Constraint-based models use a set of linear constraints to simulate steady-state metabolic fluxes, whereas kinetic models can simulate both the transient behavior and steady-state values of cellular fluxes and concentrations. The integration of cell- or tissue-specific data enables the construction of context-specific models that reflect cell-type- or tissue-specific metabolic properties. While the available modeling frameworks enable limited modeling of the metabolic crosstalk between tumor and immune cells in the tumor stroma, future developments will likely involve new hybrid kinetic/stoichiometric formulations.

Background: The advent of single-cell RNA sequencing (scRNAseq) and additional single-cell omics technologies have provided scientists with unprecedented tools to explore biology at cellular resolution. However, reaching an appropriate number of good quality reads per cell and reasonable numbers of cells within each of the populations of interest are key to infer relevant conclusions about the underlying biology of the dataset. For these reasons, scRNAseq studies are constantly increasing the number of cells analysed and the granularity of the resultant transcriptomics analyses. Methods:  We aimed to identify previously described fibroblast subpopulations in healthy adult human skin by using the largest dataset published to date (528,253 sequenced cells) and an unsupervised population-matching algorithm. Results: Our reanalysis of this landmark resource demonstrates that a substantial proportion of cell transcriptomic signatures may be biased by cellular stress and response to hypoxic conditions. Conclusions: We postulate that careful design of experimental conditions is needed to avoid long processing times of biological samples. Additionally, computation of large datasets might undermine the extent of the analysis, possibly due to long processing times., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Ascensión AM et al.)

Background: The advent of single-cell RNA sequencing (scRNAseq) and additional single-cell omics technologies have provided scientists with unprecedented tools to explore biology at cellular resolution. However, reaching an appropriate number of good quality reads per cell and reasonable numbers of cells within each of the populations of interest are key to infer conclusions from otherwise limited analyses. For these reasons, scRNAseq studies are constantly increasing the number of cells analysed and the granularity of the resultant transcriptomics analyses. Methods:  We aimed to identify previously described fibroblast subpopulations in healthy adult human skin by using the largest dataset published to date (528,253 sequenced cells) and an unsupervised population-matching algorithm. Results: Our reanalysis of this landmark resource demonstrates that a substantial proportion of cell transcriptomic signatures may be biased by cellular stress and response to hypoxic conditions. Conclusions: We postulate that the "more is better" approach, currently prevalent in the scientific community, might undermine the extent of the analysis, possibly due to long computational processing times inherent to large datasets., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Ascensión AM et al.)

On the basis of their differential location within the dermis and of discrete changes in gene and protein expression, two major fibroblast subtypes (papillary and reticular) have traditionally been distinguished. In the last 3 years, a number of research groups have begun to address transcriptomic heterogeneity of human skin cells at the single-cell level by determining mRNA levels of expressed genes through single-cell RNA sequencing technologies. However, the outcome of single-cell RNA sequencing studies is thus far confusing. Very little overlap was found in fibroblast subpopulations, which also varied in number and composition in each dataset. After a careful reappraisal of the transcriptomic data of 13,823 human adult dermal fibroblasts that have been sequenced to date, we show that fibroblasts may robustly be assigned to three major types (axes A‒C), which in turn are composed of 10 major subtypes (clusters), which we denominated A1‒A4, B1 and B2, and C1‒C4. These computationally determined axes and clusters represent the major fibroblast types and subtypes in adult healthy human skin across different datasets, accounting for 92.5% of the sequenced fibroblasts. They thus may provide the basis to improve our understanding of dermal homeostasis and cellular function at the transcriptomic level., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer is a phenomenon broadly related to ageing in various ways such as cell cycle deregulation, metabolic defects or telomerases dysfunction as principal processes. Although the tumor cell is the main actor in cancer progression, it is not the only element of the disease. Cells and the matrix surrounding the tumor, called the tumor microenvironment (TME), play key roles in cancer progression. Phenotypic changes of the TME are indispensable for disease progression and a few of these transformations are produced by epigenetic changes including miRNA dysregulation. In this study, we found that a specific group of miRNAs in the liver TME produced by colon cancer called geromiRs, which are miRNAs related to the ageing process, are significantly downregulated. The three principal cell types involved in the liver TME, namely, liver sinusoidal endothelial cells, hepatic stellate (Ito) cells and Kupffer cells, were isolated from a murine hepatic metastasis model, and the miRNA and gene expression profiles were studied. From the 115 geromiRs and their associated hallmarks of aging, which we compiled from the literature, 75 were represented in the used microarrays, 26 out of them were downregulated in the TME cells during colon cancer colonization of the liver, and none of them were upregulated. The histone modification hallmark of the downregulated geromiRs is significantly enriched with the geromiRs miR-15a , miR-16 , miR-26a , miR-29a , miR-29b and miR-29c . We built a network of all of the geromiRs downregulated in the TME cells and their gene targets from the MirTarBase database, and we analyzed the expression of these geromiR gene targets in the TME. We found that Cercam and Spsb4 , identified as prognostic markers in a few cancer types, are associated with downregulated geromiRs and are upregulated in the TME cells.

Despite continuous exertion made, colon cancer still represents a major health problem and its incidence continues being high worldwide. There is growing evidence in support of the cancer stem cells (CSCs) being central in the initiation of this cancer, and CSCs have been the focus of various studies for the identification of new ways of treatment. Lately, the proprotein convertases (PCs) were reported to regulate the maturation and expression of various molecules involved in the malignant phenotype of colon cancer cells, however, the identity of the molecules regulated by these serine proteases in CSCs is unknown. In this study, we used the general PCs inhibitor, the Decanoyl-RVKR-chloromethylketone (Decanoyl-RVKR-CMK) that inhibits all the PCs found in the secretory pathway, and analyzed its effect on CSCs using RNA-seq analysis. Remarkably, from the only 9 up-regulated genes in the human SW620-derived sphere-forming cells, we identified 7 of the 11 human metallothioneins, all of them localized on chromosome 16, and zinc related proteins as downstream effectors of the PCs. The importance of these molecules in the regulation of cell proliferation, differentiation and chemoresistance, and their reported potential tumor suppressor role and loss in colon cancer patients associated with worse prognosis, suggests that targeting PCs in the control of the malignant phenotype of CSCs is a new potential therapeutic strategy in colon cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

High-throughput cell-data technologies such as single-cell RNA-seq create a demand for algorithms for automatic cell classification and characterization. There exist several cell classification ontologies with complementary information. However, one needs to merge them to synergistically combine their information. The main difficulty in merging is to match the ontologies since they use different naming conventions. Therefore, we developed an algorithm that merges ontologies by integrating the name matching between class label names with the structure mapping between the ontology elements based on graph convolution. Since the structure mapping is a time consuming process, we designed two methods to perform the graph convolution: vectorial structure matching and constraint-based structure matching. To perform the vectorial structure matching, we designed a general method to calculate the similarities between vectors of different lengths for different metrics. Additionally, we adapted the slower Blondel method to work for structure matching. We implemented our algorithms into FOntCell, a software module in Python for efficient automatic parallel-computed merging/fusion of ontologies in the same or similar knowledge domains. FOntCell can unify dispersed knowledge from one domain into a unique ontology in OWL format and iteratively reuse it to continuously adapt ontologies with new data endlessly produced by data-driven classification methods, such as of the Human Cell Atlas. To navigate easily across the merged ontologies, it generates HTML files with tabulated and graphic summaries, and interactive circular Directed Acyclic Graphs. We used FOntCell to merge the CELDA, LifeMap and LungMAP Human Anatomy cell ontologies into a comprehensive cell ontology. We compared FOntCell with tools used for the alignment of mouse and human anatomy ontologies task proposed by the Ontology Alignment Evaluation Initiative (OAEI) and found that the F β alignment accuracies of FOntCell are above the geometric mean of the other tools; more importantly, it outperforms significantly the best OAEI tools in cell ontology alignment in terms of F β alignment accuracies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past collaboration with one of the authors MA-B., (Copyright © 2021 Cabau-Laporta, Ascensión, Arrospide-Elgarresta, Gerovska and Araúzo-Bravo.)

Breast cancer is one of the most common pathologies diagnosed in the clinical practice. Despite major advancements in diagnostic approaches, there is no widely accepted biomarker in the clinical practice that can diagnose breast malignancy. Confirmatory diagnosis still relies on the pathological assessment of tissue biopsies by expert pathologists. Thus, there is an unmet need for new types of biomarkers and novel platform technologies that can be easily and robustly integrated into the clinic and that can assist pathologists. Herein, we show that nuclease activity associated to malignant tumors can be used as a novel biomarker in breast cancer, which can be detected via specific degradation of nucleic acid probes. In this study we have identified a set of three chemically modified nucleic acid probes that can diagnose malignancy in biopsy samples with high accuracy (89%), sensitivity (82%) and specificity (94%). This work represents a breakthrough for the potential clinical use of nuclease activity as biomarker, which can be detected via nucleic acids probes, for the clinical diagnosis of malignancy in breast tissue biopsies. This platform technology could be readily implemented into the clinic as adjunct to histopathological diagnostic.

Background: Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of cases are due to mutations in the COL1A1 or COL1A2 genes, which encode type I collagen. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes., Methods: To overcome the short-term effect of MSCs therapy, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs, administered in a 2.5-year period, in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in nonimmunosuppressed OI patients. The host response to MSCs was studied by analyzing the sera from OI patients, collected before, during, and after the cell therapy., Results: We first demonstrated that the sequential administration of MSCs was safe and improved the bone parameters and quality of life of OI patients along the cell treatment plus 2-year follow-up period. Moreover, the study of the mechanism of action indicated that MSCs therapy elicited a pro-osteogenic paracrine response in patients, especially noticeable in the patient affected by severe OI., Conclusions: Our results demonstrate the feasibility and potential of reiterative MSCs infusion for two pediatric OI and highlight the paracrine response shown by patients as a consequence of MSCs treatment., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

The role of natural killer (NK) cells in the liver as first-line post infectionem ( p.i. ) effectors against blood-stage malaria and their responsiveness to protective vaccination is poorly understood. Here, we investigate the effect of vaccination on NK cell-associated genes induced in the liver by blood-stage malaria of Plasmodium chabaudi. Female Balb/c mice were vaccinated at weeks 3 and 1 before being infected with 10 6 P. chabaudi -parasitized erythrocytes. Genes preferentially expressed by NK cells were investigated in livers of vaccination-protected and non-protected mice on days 0, 1, 4, 8, and 11 p.i. using microarrays, qRT-PCR, and chromosome landscape analysis. Blood-stage malaria induces expression of specific genes in the liver at different phases of infection, i.e., Itga1 in expanding liver-resident NK (lrNK) cells, Itga2 in immigrating conventional NK (cNK) cells; Eomes and Tbx21 encoding transcription factors; Ncr1, Tnfsf10, Prf1, Gzma, Gzmb, Gzmc, Gzmm, and Gzmk encoding cytolytic effectors; natural killer gene complex (NKC)-localized genes encoding the NK cell receptors KLRG1, KLRK1, KLRAs1, 2, 5, 7, KLRD1, KLRC1, KLRC3, as well as the three receptors KLRB1A, KLRB1C, KLRB1F and their potential ligands CLEC2D and CLEC2I. Vaccination enhances this malaria-induced expression of genes, but impairs Gzmm expression, accelerates decline of Tnfsf10 and Clec2d expression, whereas it accelerates increased expression of Clec2i , taking a very similar time course as that of genes encoding plasma membrane proteins of erythroblasts, whose malaria-induced extramedullary generation in the liver is known to be accelerated by vaccination. Collectively, vaccination reshapes the response of the liver NK cell compartment to blood-stage malaria. Particularly, the malaria-induced expansion of lrNK cells peaking on day 4 p.i. is highly significantly ( p < 0.0001) reduced by enhanced immigration of peripheral cNK cells, and KLRB1F:CLEC2I interactions between NK cells and erythroid cells facilitate extramedullary erythroblastosis in the liver, thus critically contributing to vaccination-induced survival of otherwise lethal blood-stage malaria of P. chabaudi .

While the central common feature of the neurodegenerative diseases (NDs) is the accumulation of misfolded proteins, they share other pathogenic mechanisms. However, we miss an explanation for the onset of the NDs. The mechanisms through which genetic mutations, present from conception are expressed only after several decades of life are unknown. We aim to find clues on the complexity of the disease onset trigger of the different NDs expressed in the number of steps of factors related to a disease. We collected brain autopsies on diseased patients with NDs, and found a dynamic increase of the ND multimorbidity with the advance of age. Together with the observation that the NDs accumulate multiple misfolded proteins, and the same misfolded proteins are involved in more than one ND, motivated us to propose a model for a genealogical tree of the NDs. To collect the dynamic data needed to build the tree, we used a Big-data approach that searched automatically epidemiological datasets for age-stratified incidence of NDs. Based on meta-analysis of over 400 datasets, we developed an algorithm that checks whether a ND follows a multistep model, finds the number of steps necessary for the onset of each ND, finds the number of common steps with other NDs and the number of specific steps of each ND, and builds with these findings a parsimony tree of the genealogy of the NDs. The tree discloses three types of NDs: the stem NDs with less than 3 steps; the trunk NDs with 5 to 6 steps; and the crown NDs with more than 7 steps. The tree provides a comprehensive understanding of the relationship across the different NDs, as well as a mathematical framework for dynamic adjustment of the genealogical tree of the NDs with the appearance of new epidemiological studies and the addition of new NDs to the model, thus setting the basis for the search for the identity and order of these steps. Understanding the complexity, or number of steps, of factors related to disease onset trigger is important prior deciding to study single factors for a multiple steps disease.

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student's t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1 . We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/β-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment.

Generation of autologous human motor neurons holds great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows generation of target cells from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic that are not scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequentially inducing POU5F1(OCT4) and LHX3 . Our strategy enables scalable production of pure iMNs because of the transient acquisition of proliferative iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited hallmarks of spinal motor neurons including transcriptional profiles, electrophysiological property, synaptic activity, and neuromuscular junction formation. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in rodent SCI model. Together, our advanced strategy will provide tools to acquire sufficient human iMNs that may represent a promising cell source for personalized cell therapy., Competing Interests: HL, HL, BL, DG, SP, HZ, MA, JK, YH, HS, JK No competing interests declared, (© 2020, Lee et al.)

Urothelial carcinoma (UC) is a common disease causing significant morbidity and mortality as well as considerable costs for health systems. Extensive aberrant methylation of DNA is broadly documented in early UC, contributing to genetic instability, altered gene expression and tumor progression. However the triggers initiating aberrant methylation are unknown. Recently we discovered that several genes encoding key enzymes of methyl group and polyamine metabolism, including Ornithine Decarboxylase 1 (ODC1), are affected by DNA methylation in early stage UC. In this study, we investigated the hypothesis that these epigenetic alterations act in a feed-forward fashion to promote aberrant DNA methylation in UC. We demonstrate that siRNA-mediated knockdown of ODC1 expression elicits genome-wide LINE-1 demethylation, induction of LINE-1 transcripts and double-strand DNA breaks and decreases viability in primary cultured uroepithelial cells. Similarly, following siRNA-mediated knockdown of ODC1, UC cells undergo double-strand DNA breaks and apoptosis. Collectively, our findings provide evidence that ODC1 gene hypermethylation could be a starting point for the onset of genome-wide epigenetic aberrations in urothelial carcinogenesis. Furthermore, LINE-1 induction enabled by ODC1 interference provides a new experimental model to study mechanisms and consequences of LINE-1 activation in the etiology and progression of UC as well as presumably other cancers.

Background: Vaccination induces survival of otherwise lethal blood-stage infections of the experimental malaria Plasmodium chabaudi. Blood-stage malaria induces extramedullary erythropoiesis in the liver. This study investigates how vaccination affects the course of malaria-induced expression of erythrocytic genes in the liver., Methods: Female Balb/c mice were vaccinated at week 3 and week 1 before challenging with 10 6 P. chabaudi-parasitized erythrocytes. The non-infectious vaccine consisted of erythrocyte ghosts isolated from P. chabaudi-infected erythrocytes. Gene expression microarrays and quantitative real-time PCR were used to compare mRNA expression of different erythrocytic genes in the liver of vaccination-protected and non-protected mice during infections on days 0, 1, 4, 8, and 11 p.i., Results: Global transcriptomics analyses reveal vaccination-induced modifications of malaria-induced increases in hepatic gene expression on days 4 and 11 p.i. On these days, vaccination also alters hepatic expression of the erythropoiesis-involved genes Ermap, Kel, Rhd, Rhag, Slc4a1, Gypa, Add2, Ank1, Epb4.1, Epb4.2, Epb4.9, Spta1, Sptb, Tmod1, Ahsp, Acyp1, Gata1, Gfi1b, Tal1, Klf1, Epor, and Cldn13. In vaccination-protected mice, expression of these genes, except Epb4.1, is significantly higher on day 4 p.i. than in un-protected non-vaccinated mice, reaches maximal expression at peak parasitaemia on day 8 p.i., and is slowed down or even decreased towards the end of crisis phase on day 11 p.i.. After day 1 p.i., Epor expression takes about the same course as that of the other erythroid genes. Hepatic expression of Epo, however, is delayed in both vaccinated and non-vaccinated mice for the first 4 days p.i. and is maximal at significantly higher levels in vaccinated mice on day 8 p.i., before declining towards the end of crisis phase on day 11 p.i., Conclusion: The present data indicate that vaccination accelerates malaria-induced erythroblastosis in the liver for 1-2 days. This may contribute to earlier replenishment of peripheral red blood cells by liver-derived reticulocytes, which may favour final survival of otherwise lethal blood-stage malaria, since reticulocytes are not preferred as host cells by P. chabaudi.

Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43 + hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34 + /CD45 + progenitors with high erythroid colony-forming potential and CD36 + erythroid precursors. A total of 1.5 × 10 7 cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA + erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.

In recent years, the macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) cytokines have been identified as opposing regulators of the inflammatory program. However, the two cytokines are simultaneously present in the inflammatory milieu, and it is not clear how cells integrate these signals. In order to understand the regulatory networks associated with the GM/M-CSF signaling axis, we analyzed DNA methylation in human monocytes. Our results indicate that GM-CSF induces activation of the inflammatory program and extensive DNA methylation changes, while M-CSF-polarized cells are in a less differentiated state. This inflammatory program is mediated via JAK2 associated with the GM-CSF receptor and the downstream extracellular signal-regulated (ERK) signaling. However, PI3K signaling is associated with a negative regulatory loop of the inflammatory program and M-CSF autocrine signaling in GM-CSF-polarized monocytes. Our findings describe the regulatory networks associated with the GM/M-CSF signaling axis and how they contribute to the establishment of the inflammatory program associated with monocyte activation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage., Competing Interests: The authors have declared that no competing interests exist.