Inhibition of type I PRMTs reforms muscle stem cell identity enhancing their therapeutic capacity.

In skeletal muscle, muscle stem cells (MuSC) are the main cells responsible for regeneration upon injury. In diseased skeletal muscle, it would be therapeutically advantageous to replace defective MuSCs, or rejuvenate them with drugs to enhance their self-renewal and ensure long-term regenerative potential. One limitation of the replacement approach has been the inability to efficiently expand MuSCs ex vivo, while maintaining their stemness and engraftment abilities. Herein, we show that inhibition of type I protein arginine methyltransferases (PRMTs) with MS023 increases the proliferative capacity of ex vivo cultured MuSCs. Single cell RNA sequencing (scRNAseq) of ex vivo cultured MuSCs revealed the emergence of subpopulations in MS023-treated cells which are defined by elevated Pax7 expression and markers of MuSC quiescence, both features of enhanced self-renewal. Furthermore, the scRNAseq identified MS023-specific subpopulations to be metabolically altered with upregulated glycolysis and oxidative phosphorylation (OxPhos). Transplantation of MuSCs treated with MS023 had a better ability to repopulate the MuSC niche and contributed efficiently to muscle regeneration following injury. Interestingly, the preclinical mouse model of Duchenne muscular dystrophy had increased grip strength with MS023 treatment. Our findings show that inhibition of type I PRMTs increased the proliferation capabilities of MuSCs with altered cellular metabolism, while maintaining their stem-like properties such as self-renewal and engraftment potential.

Single-cell DNA sequencing-a potential dosimetric tool.

We hypothesised that single-cell whole-genome sequencing has the potential to detect mutational differences in the genomes of the cells that are irradiated with different doses of radiation and we set out to test our hypothesis using in silico and in vitro experiments. In this manuscript, we present our findings from a Monte Carlo single-cell irradiation simulation performed in TOPAS-nBio using a custom-built geometric nuclear deoxyribonucleic acid (DNA) model, which predicts a significant dose dependence of the number of cluster damages per cell as a function of radiation dose. We also present preliminary experimental results, obtained from single-cell whole-genome DNA sequencing analysis performed on cells irradiated with different doses of radiation, showing promising agreement with the simulation results.

Concerted epithelial and stromal changes during progression of Barrett's Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis.

Esophageal adenocarcinoma arises from Barrett's esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients' paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barrett's esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming.

Three-dimensional hydrogel structures as optical sensor arrays, for the detection of specific DNA sequences.

The fabrication and characterization of surface-attached PEG-diacrylate hydrogel structures and their application as sensing platforms for the detection of specific target sequences are reported. Hydrogel structures were formed by a photopolymerization process, using substrate-bound Eosin Y molecules for the production of free radicals. We have demonstrated that this fabrication process allows for control over hydrogel growth down to the micrometer scale. Confocal imaging revealed relatively large pore structures for 25% (v/v) PEG-diacrylate hydrogels, which appear to lie in tightly packed layers. Our data suggest that these pore structures decrease in size for hydrogels with increasing levels of PEG-diacrylate. Surface coverage values calculated for hydrogels immobilized with 21-mer DNA probe sequences were significantly higher compared to those previously reported for 2- and 3-dimensional sensing platforms, on the order of 10(16)molecules cm(-2). Used as sensing platforms in DNA hybridization assays, a detection limit of 3.9 nM was achieved for hybridization reactions between 21-mer probe and target sequences. The ability of these hydrogel sensing platforms to discriminate between wild-type and mutant allele sequences was also demonstrated, down to target concentrations of 1-2 nM. A reduction in the hybridization time down to a period of 15 min was also achieved, while still maintaining confident results, demonstrating the potential for future integration of these sensing platforms within Lab-on-Chip or diagnostic devices.

Animal models for arthritis: innovative tools for prevention and treatment.

The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive 'pathogenesis map' that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in 'omics' profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.

Single-cell analysis of childhood leukemia reveals a link between developmental states and ribosomal protein expression as a source of intra-individual heterogeneity.

Childhood acute lymphoblastic leukemia (cALL) is the most common pediatric cancer. It is characterized by bone marrow lymphoid precursors that acquire genetic alterations, resulting in disrupted maturation and uncontrollable proliferation. More than a dozen molecular subtypes of variable severity can be used to classify cALL cases. Modern therapy protocols currently cure 85-90% of cases, but other patients are refractory or will relapse and eventually succumb to their disease. To better understand intratumor heterogeneity in cALL patients, we investigated the nature and extent of transcriptional heterogeneity at the cellular level by sequencing the transcriptomes of 39,375 individual cells in eight patients (six B-ALL and two T-ALL) and three healthy pediatric controls. We observed intra-individual transcriptional clusters in five out of the eight patients. Using pseudotime maturation trajectories of healthy B and T cells, we obtained the predicted developmental state of each leukemia cell and observed distribution shifts within patients. We showed that the predicted developmental states of these cancer cells are inversely correlated with ribosomal protein expression levels, which could be a common contributor to intra-individual heterogeneity in cALL patients.

Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function.

BACKGROUND: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape" scale, and few if any have applied the newest nanopore technology. RESULTS: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. CONCLUSIONS: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

Analysis of FGGY as a risk factor for sporadic amyotrophic lateral sclerosis.

A genome-wide association study (GWAS) using pooled DNA samples from 386 sporadic ALS patients and 542 controls from the USA, identified genetic variation in FGGY (FLJ10986) as a risk factor, as well as 66 additional candidate SNPs. Considering the large number of hypotheses that are tested in GWAS, independent replication of associations is crucial for identifying true-positive genetic risk factors for disease. The primary aim of this study was to study the association between FGGY and sporadic ALS in large, homogeneous populations from northern Europe. Genotyping experiments were performed using Illumina Beadchips, Sequenom iPLEX assays and Taqman technology on large case-control series from The Netherlands, Belgium, Sweden and Ireland (total: 1883 sporadic ALS patients and 2063 controls). No significant association between sporadic ALS and the six previously reported associated SNPs in FGGY was observed: rs6700125 (p =0.56), rs6690993 (p =0.30), rs10493256 (p =0.68), rs6587852 (p =0.64), rs1470407 (p =0.28) and rs333662 (p =0.44). Screening of the additional candidate loci did not yield significant associations either, with the lowest p-value in joint analysis for rs7772593 (p =0.14). We concluded that common genetic variation in FGGY is not associated with susceptibility to sporadic ALS in genetically homogeneous populations from northern Europe.

Genome-wide analysis of androgen receptor binding and transcriptomic analysis in mesenchymal subsets during prostate development.

Prostate development is controlled by androgens, the eandrogen receptor (AR) and mesenchymal-epithelial signalling. We used chromatin immunoprecipitation sequencing (ChIP-seq) to define AR genomic binding in the male and female mesenchyme. Tissue- and single-cell-based transcriptional profiling was used to define mesenchymal AR target genes. We observed significant AR genomic binding in females and a strong enrichment at proximal promoters in both sexes. In males, there was greater AR binding to introns and intergenic regions as well as to classical AR binding motifs. In females, there was increased proximal promoter binding and involvement of cofactors. Comparison of AR-bound genes with transcriptomic data enabled the identification of novel sexually dimorphic AR target genes. We validated the dimorphic expression of AR target genes using published datasets and confirmed regulation by androgens using ex vivo organ cultures. AR targets showed variable expression in patients with androgen insensitivity syndrome. We examined AR function at single-cell resolution using single-cell RNA sequencing (scRNA-seq) in male and female mesenchyme. Surprisingly, both AR and target genes were distributed throughout cell subsets, with few positive cells within each subset. AR binding was weakly correlated with target gene expression.

Structural analysis and expression profile of a novel gene on chromosome 5q23 encoding a Golgi-associated protein with six splice variants, and involved within the 5q deletion of a Ph(-) CML patient.

We have identified a novel gene, upstream of the cytokine gene cluster region in 5q23-31, residing within one of the most common deleted segments associated with MDS. The novel gene exhibits significant alternative splicing generating at least six splice variants encoding four putative proline-rich protein isoforms, one of which is Golgi-associated. The gene is ubiquitously expressed and conserved among species with the C. elegans homologue being the most interesting, since it resides within an operon with two other genes, phospholipase D and dishevelled, a member of the Wnt pathway, suggesting a functional association. In addition, the novel gene and other key regulatory genes of the region, such IL3, Ril, AF5q31 and TCF-1, were found to be deleted in an atypical CML case, thus underscoring the significance of this subregion in the leukemogenesis process.

Phase I/II trial of bevacizumab and radiotherapy for locally advanced inoperable colorectal cancer: vasculature-independent radiosensitizing effect of bevacizumab.

PURPOSE: Anti-vascular endothelial growth factor therapy enhances the activity of radiotherapy in experimental models, and bevacizumab has therapeutic activity in patients with metastatic colorectal cancer. EXPERIMENTAL DESIGN: Twenty-two patients with locally advanced inoperable colorectal carcinomas (LA/I-CRC) were treated with conformal hypofractionated (3.4 Gy/fraction x 15) split-course accelerated radiotherapy (biological equivalent dose, 67.2 Gy) supported with amifostine, capecitabine (600 mg/m2 daily, 5 days/week), and bevacizumab (5 mg/kg every 2 weeks, five cycles). Biopsies from nine patients, performed before and 1 week after bevacizumab administration, were analyzed for changes in mRNA expression with Illumina gene arrays. RESULTS: No serious grade 3 chemotherapy-related side effects were recorded. There was low acute toxicity, with moist perineal desquamation noted in 2 of 22 patients, diarrhea grade 2 to 3 in 5 of 22 patients, and severe proctalgia in 2 of 22 patients. One patient died from Fournier's gangrene before treatment completion. Within a median follow-up of 18 months, two patients with preradiotheraphy direct involvement of adjacent organs expressed recto-vaginal/perineal fistula. Out of 19 evaluable cases, 13 (68.5%) showed complete response and 4 showed (21.1%) partial response. Fourteen patients are alive with no evidence of loco-regional relapse. In the gene array analysis, 30 known genes associated with transcription factors, DNA repair, and proliferation were downregulated by bevacizumab. DUSP1 gene was the most consistently downregulated transcript. CONCLUSIONS: The combination of radiotherapy with bevacizumab is feasible and results in a high rate of durable complete responses in patients with LA/I-CRC. Radiosensitization may occur through a direct effect on tumor cells followed by a wide scale suppression of transcription factors and genes involved in DNA repair and proliferation.