Smart-RRBS for single-cell methylome and transcriptome analysis

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system

Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations6. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy

Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri

Abstract The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits such as nervous systems arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system, which with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range allows exploration of genomic changes both within sponges and in early animal evolution

Inositol 1,4,5- Trisphosphate Receptor Function in Drosophila Insulin Producing Cells

The Inositol 1,4,5- trisphosphate receptor (InsP3R) is an intracellular ligand gated channel that releases calcium from intracellular stores in response to extracellular signals. To identify and understand physiological processes and behavior that depends on the InsP3 signaling pathway at a systemic level, we are studying Drosophila mutants for the InsP3R (itpr) gene. Here, we show that growth defects precede larval lethality and both are a consequence of the inability to feed normally. Moreover, restoring InsP3R function in insulin producing cells (IPCs) in the larval brain rescues the feeding deficit, growth and lethality in the itpr mutants to a significant extent. We have previously demonstrated a critical requirement for InsP3R activity in neuronal cells, specifically in aminergic interneurons, for larval viability. Processes from the IPCs and aminergic domain are closely apposed in the third instar larval brain with no visible cellular overlap. Ubiquitous depletion of itpr by dsRNA results in feeding deficits leading to larval lethality similar to the itpr mutant phenotype. However, when itpr is depleted specifically in IPCs or aminergic neurons, the larvae are viable. These data support a model where InsP3R activity in non-overlapping neuronal domains independently rescues larval itpr phenotypes by non-cell autonomous mechanisms

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

A Differentiated Services API for Adaptive QoS Management in IP Networks

The introduction of quality of service in IP Networks brings solutions to real time applications requirements over Internet. Because of its simplicity, Differentiated Services architecture proposed by the Internet Engineering Task Force (IETF) is becoming the most employed solution for providing end-to-end QoS (Quality of Service) to network based applications. However, tuning QoS parameters in edge and core routers of a DiffServ domain for efficient resource utilization and stable network behavior is not an easy task. Because of the unpredictable and dynamic behavior of the Internet traffic, network entities need to take fast decisions and perform fast adaptations for the provision of adequate end-to-end QoS. This paper proposes an Application Programming Interface for near real time bandwidth allocation and parameters tuning of DiffServ queues in Cisco routers as a first step to address to the aforementioned problem