Jitao David Zhang

White-to-brown metabolic conversion of human adipocytes by JAK inhibition

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK–STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.

Effects of copper on CHO cells: Insights from gene expression analyses

Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20‐fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow‐up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0–7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0–3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA‐Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene—coding for a transcription factor that activates signaling pathways in response to environmental stimuli—satisfied the differential expression criteria (fold change ≥1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.

Highly sensitive amplicon-based transcript quantification by semiconductor sequencing

BackgroundIn clinical and basic research custom panels for transcript profiling are gaining importance because only project specific informative genes are interrogated. This approach reduces costs and complexity of data analysis and allows multiplexing of samples. Polymerase-chain-reaction (PCR) based TaqMan assays have high sensitivity but suffer from a limited dynamic range and sample throughput. Hence, there is a gap for a technology able to measure expression of large gene sets in multiple samples.ResultsWe have adapted a commercially available mRNA quantification assay (AmpliSeq-RNA) that measures mRNA abundance based on the frequency of PCR amplicons determined by high-throughput semiconductor sequencing. This approach allows for parallel, accurate quantification of about 1000 transcripts in multiple samples covering a dynamic range of five orders of magnitude. Using samples derived from a well-characterized stem cell differentiation model, we obtained a good correlation (r = 0.78) of transcript levels measured by AmpliSeq-RNA and DNA-microarrays. A significant portion of low abundant transcripts escapes detection by microarrays due to limited sensitivity. Standard quantitative RNA sequencing of the same samples confirms expression of low abundant genes with an overall correlation coefficient of r = 0.87. Based on digital AmpliSeq-RNA imaging we show switches of signaling cascades at four time points during differentiation of stem cells into cardiomyocytes.ConclusionsThe AmpliSeq-RNA technology adapted to high-throughput semiconductor sequencing allows robust transcript quantification based on amplicon frequency. Multiplexing of at least 900 parallel PCR reactions is feasible because sequencing-based quantification eliminates artefacts coming from off-target amplification. Using this approach, RNA quantification and detection of genetic variations can be performed in the same experiment.

Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

Detect tissue heterogeneity in gene expression data with BioQC

BackgroundGene expression data can be compromised by cells originating from other tissues than the target tissue of profiling. Failures in detecting such tissue heterogeneity have profound implications on data interpretation and reproducibility. A computational tool explicitly addressing the issue is warranted.ResultsWe introduce BioQC, a R/Bioconductor software package to detect tissue heterogeneity in gene expression data. To this end BioQC implements a computationally efficient Wilcoxon-Mann-Whitney test and provides more than 150 signatures of tissue-enriched genes derived from large-scale transcriptomics studies.Simulation experiments show that BioQC is both fast and sensitive in detecting tissue heterogeneity. In a case study with whole-organ profiling data, BioQC predicted contamination events that are confirmed by quantitative RT-PCR. Applied to transcriptomics data of the Genotype-Tissue Expression (GTEx) project, BioQC reveals clustering of samples and suggests that some samples likely suffer from tissue heterogeneity.ConclusionsOur experience with gene expression data indicates a prevalence of tissue heterogeneity that often goes unnoticed. BioQC addresses the issue by integrating prior knowledge with a scalable algorithm. We propose BioQC as a first-line tool to ensure quality and reproducibility of gene expression data.

Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model

BackgroundTuberous sclerosis complex (TSC) is a genetic disease characterized by benign tumor growths in multiple organs and neurological symptoms induced by mTOR hyperfunction. Because the molecular pathology is highly complex and the etiology poorly understood, we employed a defined human neuronal model with a single mTOR activating mutation to dissect the disease-relevant molecular responses driving the neuropathology and suggest new targets for treatment.MethodsWe investigate the disease phenotype of TSC by neural differentiation of a human stem cell model that had been deleted for TSC2 by genome editing. Comprehensive genomic analysis was performed by RNA sequencing and ribosome profiling to obtain a detailed genome-wide description of alterations on both the transcriptional and translational level. The molecular effect of mTOR inhibitors used in the clinic was monitored and comparison to published data from patient biopsies and mouse models highlights key pathogenic processes.ResultsTSC2-deficient neural stem cells showed severely reduced neuronal maturation and characteristics of astrogliosis instead. Transcriptome analysis indicated an active inflammatory response and increased metabolic activity, whereas at the level of translation ribosomal transcripts showed a 5’UTR motif-mediated increase in ribosome occupancy. Further, we observed enhanced protein synthesis rates of angiogenic growth factors. Treatment with mTOR inhibitors corrected translational alterations but transcriptional dysfunction persisted.ConclusionsOur results extend the understanding of the molecular pathophysiology of TSC brain lesions, and suggest phenotype-tailored pharmacological treatment strategies.

Antiviral innate immune activation in HIV infected adults negatively affects H1/IC31® induced vaccine-specific memory CD4+ T cells

ABSTRACT Tuberculosis (TB) remains a global health problem, with vaccination being a necessary strategy for disease containment and elimination. A TB vaccine should be safe and immunogenic as well as efficacious in all affected populations, including HIV-infected individuals. We investigated the induction and maintenance of vaccine-induced memory CD4+ T cells following vaccination with the subunit vaccine H1/IC31. H1/IC31 was inoculated twice on study days 0 and 56 among HIV-infected adults with CD4+ lymphocyte counts of >350 cells/mm3. Whole venous blood stimulation was conducted with the H1 protein, and memory CD4+ T cells were analyzed using intracellular cytokine staining and polychromatic flow cytometry. We identified high responders, intermediate responders, and nonresponders based on detection of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) expressing central (TCM) and effector memory CD4+ T cells (TEM) 182 days after the first immunization. Amplicon-based transcript quantification using next-generation sequencing was performed to identify differentially expressed genes that correlated with vaccine-induced immune responses. Genes implicated in resolution of inflammation discriminated the responders from the nonresponders 3 days after the first inoculation. The volunteers with higher expression levels of genes involved in antiviral innate immunity at baseline showed impaired H1-specific TCM and TEM maintenance 6 months after vaccination. Our study showed that in HIV-infected volunteers, expression levels of genes involved in the antiviral innate immune response affected long-term maintenance of H1/IC31 vaccine-induced cellular immunity. (The clinical trial was registered in the Pan African Clinical Trials Registry [PACTR] with the identifier PACTR201105000289276.)

Correction to: Detect tissue heterogeneity in gene expression data with BioQC

After the publication of this work [1], a mistake was noticed in the Eq. 1. Given an m  ×  n expression matrix with m genes and samples of n tissues, the correct definition of the Gini index for gene i is:

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.

Erratum to: Pathway reporter genes define molecular phenotypes of human cells

Background: The phenotype of a living cell is determined by its pattern of active signaling networks, giving rise to a “molecular phenotype” associated with differential gene expression. Digital amplicon based RNA quantification by sequencing is a useful technology for molecular phenotyping as a novel tool to characterize the state of biological systems. Results: We show here that the activity of signaling networks can be assessed based on a set of established key regulators and expression targets rather than the entire transcriptome. We compiled a panel of 917 human pathway reporter genes, representing 154 human signaling and metabolic networks for integrated knowledgeand data-driven understanding of biological processes. The reporter genes are significantly enriched for regulators and effectors covering a wide range of biological processes, and faithfully capture gene-level and pathway-level changes. We apply the approach to iPSC derived cardiomyocytes and primary human hepatocytes to describe changes in molecular phenotype during development or drug response. The reporter genes deliver an accurate pathway-centric view of the biological system under study, and identify known and novel modulation of signaling networks consistent with literature or experimental data. Conclusions: A panel of 917 pathway reporter genes is sufficient to describe changes in the molecular phenotype defined by 154 signaling cascades in various human cell types. AmpliSeq-RNA based digital transcript imaging enables simultaneous monitoring of the entire pathway reporter gene panel in up to 150 samples. We propose molecular phenotyping as a useful approach to understand diseases and drug action at the network level.