Crowdsourced benchmarking of taxonomic metagenome profilers: lessons learned from the sbv IMPROVER Microbiomics challenge.

BACKGROUND: Selection of optimal computational strategies for analyzing metagenomics data is a decisive step in determining the microbial composition of a sample, and this procedure is complex because of the numerous tools currently available. The aim of this research was to summarize the results of crowdsourced sbv IMPROVER Microbiomics Challenge designed to evaluate the performance of off-the-shelf metagenomics software as well as to investigate the robustness of these results by the extended post-challenge analysis. In total 21 off-the-shelf taxonomic metagenome profiling pipelines were benchmarked for their capacity to identify the microbiome composition at various taxon levels across 104 shotgun metagenomics datasets of bacterial genomes (representative of various microbiome samples) from public databases. Performance was determined by comparing predicted taxonomy profiles with the gold standard. RESULTS: Most taxonomic profilers performed homogeneously well at the phylum level but generated intermediate and heterogeneous scores at the genus and species levels, respectively. kmer-based pipelines using Kraken with and without Bracken or using CLARK-S performed best overall, but they exhibited lower precision than the two marker-gene-based methods MetaPhlAn and mOTU. Filtering out the 1% least abundance species-which were not reliably predicted-helped increase the performance of most profilers by increasing precision but at the cost of recall. However, the use of adaptive filtering thresholds determined from the sample's Shannon index increased the performance of most kmer-based profilers while mitigating the tradeoff between precision and recall. CONCLUSIONS: kmer-based metagenomic pipelines using Kraken/Bracken or CLARK-S performed most robustly across a large variety of microbiome datasets. Removing non-reliably predicted low-abundance species by using diversity-dependent adaptive filtering thresholds further enhanced the performance of these tools. This work demonstrates the applicability of computational pipelines for accurately determining taxonomic profiles in clinical and environmental contexts and exemplifies the power of crowdsourcing for unbiased evaluation.

Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor.

Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells.

LDL Receptor Pathway Regulation by miR-224 and miR-520d.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3'-untranslated region (3'-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3'-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr +/- mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

CDK8 Regulates Insulin Secretion and Mediates Postnatal and Stress-Induced Expression of Neuropeptides in Pancreatic ß Cells.

Cyclin-dependent kinases (CDKs) contribute to vital cellular processes including cell cycle regulation. Loss of CDKs is associated with impaired insulin secretion and ß cell survival; however, the function of CDK8 in ß cells remains elusive. Here, we report that genetic ablation of Cdk8 improves glucose tolerance by increasing insulin secretion. We identify OSBPL3 as a CDK8-dependent phosphoprotein, which acts as a negative regulator of insulin secretion in response to glucose. We also show that embryonic gene silencing of neuropeptide Y in ß cells is compromised in Cdk8-null mice, leading to continued expression into adulthood. Cdk8 ablation in ß cells aggravates apoptosis and induces de novo expression of neuropeptides upon oxidative stress. Moreover, pancreatic islets exposed to stress display augmented apoptosis in the presence of these same neuropeptides. Our results reveal critical roles for CDK8 in ß cell function and survival during metabolic stress that are in part mediated through de novo expression of neuropeptides.

Bile acids and gene regulation: from nuclear receptors to chromatin.

Transcription regulation by bile acids is far more complicated than it appeared at first when several groups began their investigations in the early '90. It has become clear now that bile acids regulate the transcription of genes involved in bile acid synthesis, transport and other metabolic pathways via multiple mechanisms that involve transcription factors, nuclear receptors, coregulators, chromatin and the related modifying enzyme complexes. At a first look this might seem surprising but if one considers the physical-chemical properties of these molecules it should be evident that, due to their detergent properties, bile acids may be harmful if they reach high concentrations in the liver and intestine. Therefore, living organisms have developed biochemical mechanisms that finely tune the concentration of bile acids according to the body needs and in response to environmental challenges. In this review, we will discuss the most recent evidences on the mechanisms through which bile acids regulate gene transcription, including the function of nuclear receptors and emphasizing the emerging role of chromatin and the associated modifying enzymes.

The pharmacological exploitation of cholesterol 7alpha-hydroxylase, the key enzyme in bile acid synthesis: from binding resins to chromatin remodelling to reduce plasma cholesterol.

Mammals dispose of cholesterol mainly through 7alpha-hydroxylated bile acids, and the enzyme catalyzing the 7alpha-hydroxylation, cholesterol 7alpha-hydroxylase (CYP7A1), has a deep impact on cholesterol homeostasis. In this review, we present the study of regulation of CYP7A1 as a good exemplification of the extraordinary contribution of molecular biology to the advancement of our understanding of metabolic pathways that has taken place in the last 2 decades. Since the cloning of the gene from different species, experimental evidence has accumulated, indicating that the enzyme is mainly regulated at the transcriptional level and that bile acids are the most important physiological inhibitors of CYP7A1 transcription. Multiple mechanisms are involved in the control of CYP7A1 transcription and a variety of transcription factors and nuclear receptors participate in sophisticated regulatory networks. A higher order of transcriptional regulation, stemming from the so-called histone code, also applies to CYP7A1, and recent findings clearly indicate that chromatin remodelling events have profound effects on its expression. CYP7A1 also acts as a sensor of signals coming from the gut, thus representing another line of defence against the toxic effects of bile acids and a downstream target of agents acting at the intestinal level. From the pharmacological point of view, bile acid binding resins were the first primitive approach targeting the negative feed-back regulation of CYP7A1 to reduce plasma cholesterol. In recent years, new drugs have been designed based on recent discoveries of the regulatory network, thus confirming the position of CYP7A1 as a focus for innovative pharmacological intervention.

Interrogating the microbiome: experimental and computational considerations in support of study reproducibility.

The microbiome is an important factor in human health and disease and is investigated to develop novel therapeutics. Metagenomics leverages advances in sequencing technologies and computational analysis to identify and quantify the microorganisms present in a sample. This field has, however, not yet reached maturity and the international metagenomics community, aware of the current limitations and of the necessity for standardization, has started investigating sources of variability in experimental and computational workflows. The first studies have already resulted in the identification of crucial steps and factors affecting metagenomics data quality, quantification and interpretation. This review summarizes experimental and computational considerations for interrogating the microbiome and establishing reproducible and robust analysis workflows.

The biological effects of long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product.

Cigarette smoking is the leading cause of preventable lung cancer (LC). Reduction of harmful constituents by heating rather than combusting tobacco may have the potential to reduce the risk of LC. We evaluated functional and molecular changes in human bronchial epithelial BEAS-2B cells following a 12-week exposure to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product (cMRTP) in comparison with those following exposure to TPM from the 3R4F reference cigarette. Endpoints linked to lung carcinogenesis were assessed. Four-week 3R4F TPM exposure resulted in crisis and epithelial to mesenchymal transition (EMT) accompanied by decreased barrier function and disrupted cell-to-cell contacts. By week eight, cells regained E-cadherin expression, suggesting that EMT was reversible. Increased levels of inflammatory mediators were noted in cells treated to 3R4F TPM but not in cells treated to the same or a five-fold higher concentration of cMRTP TPM. A 20-fold higher concentration of cMRTP TPM increased oxidative stress and DNA damage and caused reversible EMT. Anchorage-independent growth was observed in cells treated to 3R4F or a high concentration of cMRTP TPM. 3R4F TPM-derived clones were invasive, while cMRTP TPM-derived clones were not. Long-term exposure to TPM from the cMRTP had a lower biological impact on BEAS-2B cells compared with that of exposure to TPM from 3R4F.

Assessment of the impact of aerosol from a potential modified risk tobacco product compared with cigarette smoke on human organotypic oral epithelial cultures under different exposure regimens.

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm.

Comparative systems toxicology analysis of cigarette smoke and aerosol from a candidate modified risk tobacco product in organotypic human gingival epithelial cultures: A 3-day repeated exposure study.

Smoking is one of the major lifestyle-related risk factors for periodontal diseases. Modified risk tobacco products (MRTP) offer a promising alternative in the harm reduction strategy for adult smokers unable to quit. Using a systems toxicology approach, we investigated and compared the exposure effects of a reference cigarette (3R4F) and a heat-not-burn technology-based candidate MRTP, the Tobacco Heating System (THS) 2.2. Human gingival epithelial organotypic cultures were repeatedly exposed (3 days) for 28 min at two matching concentrations of cigarette smoke (CS) or THS2.2 aerosol. Results showed only minor histopathological alterations and minimal cytotoxicity upon THS2.2 aerosol exposure compared to CS (1% for THS2.2 aerosol vs. 30% for CS, at the high concentration). Among the 14 proinflammatory mediators analyzed, only 5 exhibited significant alterations with THS2.2 exposure compared with 11 upon CS exposure. Transcriptomic and metabolomic analysis indicated a general reduction of the impact in THS2.2 aerosol-exposed samples with respect to CS (∼79% lower biological impact for the high THS2.2 aerosol concentration compared to CS, and 13 metabolites significantly perturbed for THS2.2 vs. 181 for CS). This study indicates that exposure to THS2.2 aerosol had a lower impact on the pathophysiology of human gingival organotypic cultures than CS.

Chlamydia pneumoniae acute liver infection affects hepatic cholesterol and triglyceride metabolism in mice.

OBJECTIVE: Chlamydia pneumoniae has been linked to atherosclerosis, strictly associated with hyperlipidemia. The liver plays a central role in the regulation of lipid metabolism. Since in animal models C. pneumoniae can be found at hepatic level, this study aims to elucidate whether C. pneumoniae infection accelerates atherosclerosis by affecting lipid metabolism. METHODS: Thirty Balb/c mice were challenged intra-peritoneally with C. pneumoniae elementary bodies and thirty with Chlamydia trachomatis, serovar D. Thirty mice were injected with sucrose-phosphate-glutamate buffer, as negative controls. Seven days after infection, liver samples were examined both for presence of chlamydia and expression of genes involved in inflammation and lipid metabolism. RESULTS: C. pneumoniae was isolated from 26 liver homogenates, whereas C. trachomatis was never re-cultivated (P < 0.001). C. pneumoniae infected mice showed significantly increased serum cholesterol and triglycerides levels compared both with negative controls (P < 0.001 and P = 0.0197, respectively) and C. trachomatis infected mice (P < 0.001). Liver bile acids were significantly reduced in C. pneumoniae compared to controls and C. trachomatis infected mice. In C. pneumoniae infected livers, cholesterol 7α-hydroxylase (Cyp7a1) and low-density lipoprotein receptor (Ldlr) mRNA levels were reduced, while inducible degrader of the low-density lipoprotein receptor (Idol) expression was increased. Hypertriglyceridemia was associated to reduced expression of hepatic carnitine palmitoyltransferase-1a (Cpt1a) and medium chain acyl-Coenzyme A dehydrogenase (Acadm). Pro-inflammatory cytokines gene expression was increased compared to negative controls. Conversely, in C. trachomatis infected animals, normal serum lipid levels were associated with elevated pro-inflammatory cytokines gene expression, linked to only a mild disturbance of lipid regulatory genes. CONCLUSION: Our results indicate that C. pneumoniae mouse liver infection induces dyslipidemic effects with significant modifications of genes involved in lipid metabolism.

The LXR-Idol axis differentially regulates plasma LDL levels in primates and mice.

The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR receptor stability independent of SREBP and PCSK9, but its relevance to plasma lipid levels is unknown. Here we demonstrate that the effects of the LXR-IDOL axis are both tissue and species specific. In mice, LXR agonist induces Idol transcript levels in peripheral tissues but not in liver, and does not change plasma LDL levels. Accordingly, Idol-deficient mice exhibit elevated LDLR protein levels in peripheral tissues, but not in the liver. By contrast, LXR activation in cynomolgus monkeys induces hepatic IDOL expression, reduces LDLR protein levels, and raises plasma LDL levels. Knockdown of IDOL in monkeys with an antisense oligonucleotide blunts the effect of LXR agonist on LDL levels. These results implicate IDOL as a modulator of plasma lipid levels in primates and support further investigation into IDOL inhibition as a potential strategy for LDL lowering in humans.

Site-directed mutagenesis to study the role of specific amino acids in the ligand binding domain of PPARs.

The role of certain amino acids in the interactions of ligands with their cognate nuclear receptors is usually achieved by the resolution of the crystal structure of the receptor complexed with the ligand. As a complementary functional approach, site-directed mutagenesis, a technique broadly used in molecular biology, allows the assessment of the role of a specific amino acid in determining the interaction with a specific ligand. This method makes it possible to evaluate several mutations of a key amino acid for ligand binding and to determine the relationship between protein structure and ligand interaction. Here, we describe an application of this technique to evaluate different point mutations on the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in the absence or presence of chemically different ligands.

IDOL stimulates clathrin-independent endocytosis and multivesicular body-mediated lysosomal degradation of the low-density lipoprotein receptor.

The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.

Targeted disruption of the idol gene alters cellular regulation of the low-density lipoprotein receptor by sterols and liver x receptor agonists.

Previously, we identified the E3 ubiquitin ligase Idol (inducible degrader of the low-density lipoprotein [LDL] receptor [LDLR]) as a posttranscriptional regulator of the LDLR pathway. Idol stimulates LDLR degradation through ubiquitination of its C-terminal domain, thereby limiting cholesterol uptake. Here we report the generation and characterization of mouse embryonic stem cells homozygous for a null mutation in the Idol gene. Cells lacking Idol exhibit markedly elevated levels of the LDLR protein and increased rates of LDL uptake. Furthermore, despite an intact sterol responsive element-binding protein (SREBP) pathway, Idol-null cells exhibit an altered response to multiple regulators of sterol metabolism, including serum, oxysterols, and synthetic liver X receptor (LXR) agonists. The ability of oxysterols and lipoprotein-containing serum to suppress LDLR protein levels is reduced, and the time course of suppression is delayed, in cells lacking Idol. LXR ligands have no effect on LDLR levels in Idol-null cells, indicating that Idol is required for LXR-dependent inhibition of the LDLR pathway. In line with these results, the half-life of the LDLR protein is prolonged in the absence of Idol. Finally, the ability of statins and PCSK9 to alter LDLR levels is independent of, and additive with, the LXR-Idol pathway. These results demonstrate that the LXR-Idol pathway is an important contributor to feedback inhibition of the LDLR by sterols and a biological determinant of cellular LDL uptake.

Structural insight into peroxisome proliferator-activated receptor gamma binding of two ureidofibrate-like enantiomers by molecular dynamics, cofactor interaction analysis, and site-directed mutagenesis.

Molecular dynamics simulations were performed on two ureidofibrate-like enantiomers to gain insight into their different potency and efficacy against PPARgamma. The partial agonism of the S enantiomer seems to be due to its capability to stabilize different regions of the receptor allowing the interaction with both coactivators and corepressors as shown by fluorescence resonance energy transfer (FRET) assays. The recruitment of the corepressor N-CoR1 by the S enantiomer on two different responsive elements of PPARgamma regulated promoters was confirmed by chromatin immunoprecipitation assays. Cell-based transcription assays show that PPARgamma coactivator 1alpha (PGC-1alpha) and cAMP response element binding protein-binding protein (CBP) enhance the basal and ligand-stimulated receptor activity acting as coactivators of PPARgamma, whereas the receptor interacting protein 140 (RIP140) and the nuclear corepressor 1 (N-CoR1) repress the transcriptional activity of PPARgamma. We also tested the importance of the residue Q286 on the transcriptional activity of the receptor by site-directed mutagenesis and confirmed its key role in the stabilization of helix 12. Molecular modeling studies were performed to provide a molecular explanation for the different behavior of the mutants.

Crystal structure of the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain complexed with a novel partial agonist: a new region of the hydrophobic pocket could be exploited for drug design.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.

Insights in the regulation of cholesterol 7alpha-hydroxylase gene reveal a target for modulating bile acid synthesis.

UNLABELLED: The transcription of the gene (CYP7A1) encoding cholesterol 7alpha-hydroxylase, a key enzyme in cholesterol homeostasis, is repressed by bile acids via multiple mechanisms involving members of the nuclear receptor superfamily. Here, we describe a regulatory mechanism that can be exploited for modulating bile acid synthesis. By dissecting the mechanisms of CYP7A1 transcription, we found that bile acids stimulate the sequential recruitment of the histone deacetylases (HDACs) 7, 3, and 1, and of the corepressor SMRTalpha (silencing mediator of retinoid and thyroid receptors-alpha) and the nuclear corepressor. Bile acids, but not the farnesoid X receptor-selective agonist GW4064, increase the nuclear concentration of HDAC7, which promotes the assembly of a repressive complex that ultimately represses CYP7A1 transcription. Interestingly, despite its high basal expression level, small heterodimer partner (SHP) is associated with the CYP7A1 promoter only at a later stage of bile acid repression. Gene silencing with small interfering RNA confirms that HDAC7 is the key factor required for the repression of CYP7A1 transcription, whereas knockdown of SHP does not prevent the down-regulation of CYP7A1. Administration of the HDAC inhibitors valproic acid or trichostatin A to genetically hypercholesterolemic mice increases Cyp7a1 messenger RNA and bile acid synthesis and consequently markedly reduces total plasma and low-density lipoprotein cholesterol. CONCLUSION: By using a combination of molecular, cellular, and animal models, our study highlights the importance of HDACs in the feedback regulation of CYP7A1 transcription and identifies these enzymes as potential targets to modulate bile acid synthesis and for the treatment of hypercholesterolemia.