Jonathan L. Golob

Biphasic role for Wnt/β-catenin signaling in cardiac specification in zebrafish and embryonic stem cells

Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/β-catenin pathway negatively regulates cardiac differentiation, whereas studies with pluripotent embryonal carcinoma cells suggest that this pathway promotes cardiogenesis. This apparent contradiction led us to hypothesize that Wnt/β-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/β-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/β-catenin signaling before gastrulation promotes cardiac differentiation, whereas signaling during gastrulation inhibits heart formation. Early treatment of differentiating mouse embryonic stem (ES) cells with Wnt-3A stimulates mesoderm induction, activates a feedback loop that subsequently represses the Wnt pathway, and increases cardiac differentiation. Conversely, late activation of β-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the β-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/β-catenin signaling promotes cardiac differentiation at early developmental stages and inhibits it later. Control of this pathway may promote derivation of cardiomyocytes for basic research and cell therapy applications.

Continued maturation of thymic emigrants in the periphery

Developing thymocytes are selected for recognition of molecules encoded by the major histocompatibility complex, purged of self-reactive cells and committed to either the CD4 or CD8 lineage. The 1% of thymocytes that complete these tasks emigrate and join the population of peripheral lymphocytes. Whether T cell maturation is complete at the time of thymic exit has been a subject of debate. Using mice transgenic for green fluorescent protein driven by the recombination activating gene 2 promoter to identify recent thymic emigrants, we now show that T cell differentiation continues post-thymically, with progressive maturation of both surface phenotype and immune function. In addition, the relative contribution of CD4 and CD8 recent thymic emigrants was modulated as they entered the peripheral T cell pool. Thus, T cell maturation and subset contribution are both finalized in the lymphoid periphery.

Chromatin remodeling during mouse and human embryonic stem cell differentiation.

Embryonic stem cell (ESC) differentiation is an excellent model to study chromatin changes at developmentally regulated loci. Differentiating mouse and human ESCs increase genome‐wide acetylation (euchromatic) and tri‐methylation (heterochromatic) of lysine 9 on histone H3. The Oct4 locus is euchromatic when expressed in undifferentiated ESCs and heterochromatic after differentiation. Brachyury T, a mesoderm‐specific transcription factor, is not yet expressed in undifferentiated cells, where its locus has “bivalent” tri‐methyl lysine 4 and lysine 27 modifications. During directed differentiation to pre‐cardiac mesoderm, the activated brachyury locus has high levels of tri‐methyl lysine 4 (euchromatin), switching to heterochromatin after gene silencing. Thus, ESC differentiation is accompanied by genome‐wide commitment to euchromatin or heterochromatin. Undifferentiated hESCs bivalently modify the brachyury locus, activate it to euchromatin during mesoderm induction, and subsequently repress it to heterochromatin, demonstrating, to our knowledge, the first analysis of chromatin dynamics at a locus essential for mesoderm and endoderm differentiation. Developmental Dynamics 237:1389‐1398, 2008.

Evaluating the accuracy of amplicon-based microbiome computational pipelines on simulated human gut microbial communities

BackgroundMicrobiome studies commonly use 16S rRNA gene amplicon sequencing to characterize microbial communities. Errors introduced at multiple steps in this process can affect the interpretation of the data. Here we evaluate the accuracy of operational taxonomic unit (OTU) generation, taxonomic classification, alpha- and beta-diversity measures for different settings in QIIME, MOTHUR and a pplacer-based classification pipeline, using a novel software package: DECARD.ResultsIn-silico we generated 100 synthetic bacterial communities approximating human stool microbiomes to be used as a gold-standard for evaluating the colligative performance of microbiome analysis software. Our synthetic data closely matched the composition and complexity of actual healthy human stool microbiomes. Genus-level taxonomic classification was correctly done for only 50.4–74.8% of the source organisms. Miscall rates varied from 11.9 to 23.5%. Species-level classification was less successful, (6.9–18.9% correct); miscall rates were comparable to those of genus-level targets (12.5–26.2%). The degree of miscall varied by clade of organism, pipeline and specific settings used. OTU generation accuracy varied by strategy (closed, de novo or subsampling), reference database, algorithm and software implementation. Shannon diversity estimation accuracy correlated generally with OTU-generation accuracy. Beta-diversity estimates with Double Principle Coordinate Analysis (DPCoA) were more robust against errors introduced in processing than Weighted UniFrac. The settings suggested in the tutorials were among the worst performing in all outcomes tested.ConclusionsEven when using the same classification pipeline, the specific OTU-generation strategy, reference database and downstream analysis methods selection can have a dramatic effect on the accuracy of taxonomic classification, and alpha- and beta-diversity estimation. Even minor changes in settings adversely affected the accuracy of the results, bringing them far from the best-observed result. Thus, specific details of how a pipeline is used (including OTU generation strategy, reference sets, clustering algorithm and specific software implementation) should be specified in the methods section of all microbiome studies. Researchers should evaluate their chosen pipeline and settings to confirm it can adequately answer the research question rather than assuming the tutorial or standard-operating-procedure settings will be adequate or optimal.

Stool Microbiota at Neutrophil Recovery Is Predictive for Severe Acute Graft vs Host Disease After Hematopoietic Cell Transplantation

Low alpha diversity in stool microbiota at neutrophil recovery associates with increased severe graft-versus-host disease (GVHD), greater abundance of oral Actinobacteria or Firmicutes, and decreased abundance of intestinal Lachnospiraceae in stool microbiota at neutrophil recovery associated with increased severe GVHD.

Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3′ transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98–99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification.

Functional Analysis of Metagenomes by Likelihood Inference (FAMLI) Successfully Compensates for Multi-Mapping Short Reads from Metagenomic Samples

Abstract High-throughput sequencing can establish the functional capacity of a microbial community by cataloging the protein-coding sequences (CDS) present in the metagenome of the community. The relative performance of different computational methods for identifying CDS from whole-genome shotgun sequencing (WGS) is not fully established. Here we present an automated benchmarking workflow, using synthetic shotgun sequencing reads for which we know the true CDS content of the underlying communities, to determine the relative performance (sensitivity, positive predictive value or PPV, and computational efficiency) of different metagenome analysis tools for extracting the CDS content of a microbial community. Assembly-based methods are limited by coverage depth, with poor sensitivity for CDS atShort-read metagenomics use high-throughput sequencing to reveal the functional capability of microbial communities. The computational analysis of the raw short-read data is complicated by the shared functional domains in peptides, and the complex evolutionary origins of many peptides (including recombination and truncation), resulting in a given short read aligning equally well to many possible peptides. We find that a short read known to be from one peptide will on average align equally well to about 160 other peptide sequences not present in the sample. Here we describe an iterative algorithmic approach to successfully map reads to their true origin peptides and introduce a software package FAMLI that implements this algorithm. We demonstrate that FAMLI is able to identify peptides from a wide variety of metagenomes with a consistent precision of about 0.8, and recall of about 0.6, while retaining O(n) runtimes. This compares favorably to alternative approaches, including de novo assembly (that results in superior precision, but much inferior recall and much larger computational resource needs as compared to FAMLI), or hybrid taxonomic-identification approaches (that results in less consistent performance for extremely novel communities, as compared to FAMLI). Addressing the problem of short reads aligning equally well to hundreds of more peptides than are truly present in a sample is a key challenge any successful short-read-metagenomics software must address. We present an effective approach to mitigate this problem and improve the accuracy of functional metagenomic analysis. Author Summary Proteins are typically made up of modules that are shared with other proteins; a given short piece of protein can be shared with hundreds of other identified proteins. As a result, it is difficult to try to identify which proteins are present in a collection of microbes using short pieces of proteins. If this problem is not addressed, studies that rely on short stretches of peptides will falsely report how many proteins are present in a microbial community. We developed an algorithm to address this problem and wrote an open source software package called FAMLI that can successfully identify which proteins are present, while ignoring most of those not present.

Antibiotic Exposure Prior to Respiratory Viral Infection is Associated with Progression to Lower Respiratory Tract Disease in Allogeneic Hematopoietic Cell Transplant Recipients

Recent publications note an association between antibiotic exposure and respiratory viral infections (RVIs). Antibiotics affect microbiota and impair immune response against RVIs in mice, and low microbiome diversity is associated with pulmonary complications including viral lower respiratory tract disease (LRTD) in hematopoietic cell transplantation (HCT) recipients. In this study, we examined whether antibiotic exposure was associated with increased risk of disease progression in RVIs post-transplantation. We analyzed patients who underwent allogeneic HCT (June 2008 to February 2016) and had their first RVI due to parainfluenza virus (PIV), respiratory syncytial virus (RSV), or human metapneumovirus (MPV) during the initial 100 days post-transplantation. Antibiotic exposure in the 3 weeks before RVI onset was defined as (1) use of specific antibiotics versus none of these antibiotics and (2) number of antibiotic-days. Cox proportional hazards models were used to examine associations between antibiotic exposures and risk of viral disease progression to proven/probable/possible LRTD. Ninety HCT recipients (84 adults, 6 children) fulfilled study criteria; 33 progressed to LRTD. The number of antibiotic-days was associated with progression to LRTD after adjusting for neutropenia, steroid use, and either lymphopenia (hazard ratio, 1.41 [95% confidence interval, 1.04 to 1.92], P = .027) or monocytopenia (hazard ratio, 1.46 [95% confidence interval, 1.11 to 1.91], P = .006). Specific antibiotic classes was not associated with the outcome. Cumulative antibiotic exposure immediately before RVI onset is a risk factor for disease progression following PIV, RSV, and MPV infections post-transplantation. Larger cohort studies are needed to determine the impact of specific antibiotics or antibiotic classes on disease severity.

Underutilization of norovirus testing in hematopoietic cell transplant recipients at a large cancer center

HighlightsTesting patterns and incidence of norovirus are analyzed in transplant recipients.Norovirus testing is infrequently used despite clear seasonal variation.Targeted educational programs to enhance norovirus testing are needed. &NA; The development of a new laboratory‐developed norovirus assay provided an opportunity to assess testing patterns, incidence, and outcomes of norovirus among hematopoietic cell transplant (HCT) recipients. Clostridium difficile and norovirus tests from 1,393 HCT recipients were compared in these analyses. In this population of high‐risk patients, norovirus appeared to occur seasonally, but testing was infrequent despite a correlation with more severe disease when compared with patients with C difficile infection.