Search

Risk stratification of kidney cancer patients after nephrectomy may tailor surveillance intensity and selection for adjuvant therapy. Transcriptomic approaches are effective in predicting recurrence, but whether they add value to clinicopathologic models remains unclear.Data from patients with clear cell renal cell carcinoma (ccRCC) was downloaded from The Cancer Genome Atlas. Clinicopathologic variables were used to calculate SSIGN (stage, size, grade, and necrosis) scores. The 16 gene recurrence score (RS) signature was generated using RNA-seq data. Transcriptomic risk groups were calculated using the original thresholds. SSIGN groups were divided into low, intermediate, and high risk. Disease-free status was the primary endpoint assessed.SSIGN and RS were calculated for 428 patients with non-metastatic ccRCC. SSIGN low-, intermediate-, and high-risk groups demonstrated 2.7%, 15.2%, and 27.5%, 3-year recurrence risk, respectively. On multivariable analysis, the RS was associated with disease-free status (sub-distribution hazard ratio (sHR) 1.43 per 25 RS [95% CI (1.00-1.43)], p = 0.05). By risk groups, RS further risk stratified the SSIGN intermediate-risk group (sHR 2.22 [95% CI 1.10-4.50], p = 0.03). SSIGN intermediate-risk patients with low and high RS had a 3-year recurrence rate of 8.0% and 25.2%, respectively. Within this risk group, the area under the curve (AUC) at 3 years was 0.69 for SSIGN, 0.74 for RS, and 0.78 for their combination.Transcriptomic recurrence scores improve risk prediction even when controlling for clinicopathologic factors. Utility may be best suited for intermediate-risk patients who have heterogeneous outcomes and further refinement for clinical utility is warranted.

BACKGROUND & AIMSPrimary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multi-focal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear and therapeutic options for PSC patients are limited. We performed cell-free messenger RNA (cf-mRNA) next generation sequencing (RNA-Seq) to characterize the circulating transcriptome of PSC and non-invasively investigate potentially bioactive signals that are associated with PSC.METHODSSerum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with non-alcoholic fatty liver disease (NAFLD). Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were built using PSC dysregulated cf-mRNA genes.RESULTSDifferential expression analysis of the cf-mRNA transcriptomes of PSC and healthy controls resulted in identification of 1407 dysregulated genes. Furthermore, differentially expressed genes between PSC and the liver diseases (NAFL and Non-Alcohol Steatohepatitis (NASH)) or healthy controls shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, hepatic stellate cells and Kupffer cells, were highly abundant in cf-mRNA of subjects with PSC. Gene cluster analysis revealed that liver-specific genes dysregulated in PSC form a distinct cluster which corresponded to a subset of the PSC subject population. Finally, we developed a cf-mRNA classifier using liver-specific genes which discriminated PSC from healthy control subjects using gene transcripts of liver origin.CONCLUSIONSBlood-based whole transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in PSC subject sera which may be used to diagnose PSC patients. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for non-invasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.Graphical Abstract

Inflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signaling pathways of innate and adaptive immune systems. Therefore, there is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses. Here we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling to assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples. Considering that, both organspecific recruitment of immune cells and organ resident bespoke immune cells contributes to restoration of organ homeostasis, we also examined LPS-induced gene-expression dysregulation of multiple organs to shed light on organ crosstalk. Cf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. Lastly, we identified the dysregulation of liverspecific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this biological compartment. Collectively, using a preclinical model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses.Abstract Figure

The total number of nuclear pore complexes (NPCs) per nucleus varies greatly between different cell types and is known to change during cell differentiation and cell transformation. However, the underlying mechanisms that control how many nuclear transport channels are assembled into a given nuclear envelope remain unclear. Here, we report that depletion of the NPC basket protein Tpr, but not Nup153, dramatically increases the total NPC number in various cell types. This negative regulation of Tpr occurs via a phosphorylation cascade of extracellular signal-regulated kinase (ERK), the central kinase of the mitogen-activated protein kinase (MAPK) pathway. Tpr serves as a scaffold for ERK to phosphorylate the nucleoporin (Nup) Nup153, which is critical for early stages of NPC biogenesis. Our results reveal a critical role of the Nup Tpr in coordinating signal transduction pathways during cell proliferation and the dynamic organization of the nucleus.

Circulating cell free mRNA (cf-mRNA) holds great promise as a non-invasive diagnostic biomarker. However, the biological origin of cf-mRNA is still not well understood, limiting the clinical applications of this technology. Here, we use the bone marrow (BM) and pharmacologic manipulation of its resident cells as a window to study the origin of cf-mRNA. Using NGS-based profiling, we show that cf-mRNA is enriched in transcripts derived from the BM compared to circulating cells. Further, BM ablation experiments followed by hematopoietic stem cell transplants in cancer patients show that cf-mRNA levels reflect the transcriptional activity of BM resident hematopoietic lineages during marrow reconstitution. Finally, by stimulating specific BM cell populations in vivo using growth factor therapeutics (i.e. EPO, G-CSF), we show that cf-mRNA reveals dynamic functional changes in growing cell types, suggesting that, unlike other cell-free nucleic acids, cf-mRNA is secreted from living cells, rather than exclusively from apoptotic cells. Our results shed new light on the biology of cf-mRNA and demonstrate its potential applications in clinical practice.

Biomarkers of aging can be used to assess the health of individuals and to study aging and age-related diseases. We generate a large dataset of genome-wide RNA-seq profiles of human dermal fibroblasts from 133 people aged 1 to 94 years old to test whether signatures of aging are encoded within the transcriptome. We develop an ensemble machine learning method that predicts age to a median error of 4 years, outperforming previous methods used to predict age. The ensemble was further validated by testing it on ten progeria patients, and our method is the only one that predicts accelerated aging in these patients. Electronic supplementary material The online version of this article (10.1186/s13059-018-1599-6) contains supplementary material, which is available to authorized users.

The organization of the genome in the three-dimensional space of the nucleus is coupled with cell type-specific gene expression. However, how nuclear architecture influences transcription that governs cell identity remains unknown. Here, we show that nuclear pore complex (NPC) components Nup93 and Nup153 bind superenhancers (SE), regulatory structures that drive the expression of key genes that specify cell identity. We found that nucleoporin-associated SEs localize preferentially to the nuclear periphery, and absence of Nup153 and Nup93 results in dramatic transcriptional changes of SE-associated genes. Our results reveal a crucial role of NPC components in the regulation of cell type-specifying genes and highlight nuclear architecture as a regulatory layer of genome functions in cell fate.

Disponible material suplementario, DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase–primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus., Ministerio de Ciencia e Innovación (BFU2008-01344, BFU2011-23815 to G.M., FIS PI080291 to J.B., BFU2010-21467, CSD2007-00015 to J.M. and BFU2009-10085 to L.B.); Basque Government predoctoral fellowships (to J.S.B. and A.I.). Funding for open access charge: Spanish Ministry of Science and Innovation.

The six main minichromosome maintenance proteins (Mcm2–7), which presumably constitute the core of the replicative DNA helicase, are present in chromatin in large excess relative to the number of active replication forks. To evaluate the relevance of this apparent surplus of Mcm2–7 complexes in human cells, their levels were down-regulated by using RNA interference. Interestingly, cells continued to proliferate for several days after the acute (>90%) reduction of Mcm2–7 concentration. However, they became hypersensitive to DNA replication stress, accumulated DNA lesions, and eventually activated a checkpoint response that prevented mitotic division. When this checkpoint was abrogated by the addition of caffeine, cells quickly lost viability, and their karyotypes revealed striking chromosomal aberrations. Single-molecule analyses revealed that cells with a reduced concentration of Mcm2–7 complexes display normal fork progression but have lost the potential to activate “dormant” origins that serve a backup function during DNA replication. Our data show that the chromatin-bound “excess” Mcm2–7 complexes play an important role in maintaining genomic integrity under conditions of replicative stress.

Nuclear pore complexes (NPCs) are composed of several copies of ∼30 different proteins called nucleoporins (Nups). NPCs penetrate the nuclear envelope (NE) and regulate the nucleocytoplasmic trafficking of macromolecules. Beyond this vital role, NPC components influence genome functions in a transport-independent manner. Nups play an evolutionarily conserved role in gene expression regulation that, in metazoans, extends into the nuclear interior. Additionally, in proliferative cells, Nups play a crucial role in genome integrity maintenance and mitotic progression. Here we discuss genome-related functions of Nups and their impact on essential DNA metabolism processes such as transcription, chromosome duplication, and segregation.

11 páginas, 7 figuras -- PAGS nros. 2812-2822, Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication, This work was supported by the Spanish Ministry of Science and Innovation (J.M., A.L., and I.C.), Consolider Ingenio 2010 and Fundación Caja Madrid (J.M. and A.L.), Comunidad de Madrid (I.C.), and Institut National du Cancer and Association pour la Recherche sur le Cancer (E.S.). E.G. and V.C. were supported by the Fondation pour la Recherche Médicale and Institut National du Cancer, respectively. A.I. was supported by a fellowship from the Basque Government

The identity of the DNA helicase(s) involved in eukaryotic DNA replication is still a matter of debate, but the mini-chromosome maintenance (MCM) proteins are the chief candidate. Six conserved MCM proteins, Mcm2–7, are essential for the initiation and elongation stages of DNA replication, contain ATP binding pockets and can form a hexameric structure resembling that of known prokaryotic and viral helicases. However, biochemical proof of their presumed function has remained elusive. Several recent reports confirm that the MCM complex is part of the cellular machine responsible for the unwinding of DNA during S phase. In one of these reports, the helicase activity of Mcm2–7 is finally revealed, when they are purified in association with two partners: initiation factor Cdc45 and a four-subunit complex called GINS. The Cdc45-MCM-GINS complex could constitute the core of a larger macromolecular structure that has been termed the "replisome progression complex".