Adrenergic signaling controls early transcriptional programs during CD8+ T cell responses to viral infection.

Norepinephrine is a key sympathetic neurotransmitter, which acts to suppress CD8 + T cell cytokine secretion and lytic activity by signaling through the ß2-adrenergic receptor (ADRB2). Although ADRB2 signaling is considered generally immunosuppressive, its role in regulating the differentiation of effector T cells in response to infection has not been investigated. Using an adoptive transfer approach, we compared the expansion and differentiation of wild type (WT) to Adrb2-/- CD8 + T cells throughout the primary response to vesicular stomatitis virus (VSV) infection in vivo. We measured the dynamic changes in transcriptome profiles of antigen-specific CD8 + T cells as they responded to VSV. Within the first 7 days of infection, WT cells out-paced the expansion of Adrb2-/- cells, which correlated with reduced expression of IL-2 and the IL-2Rα in the absence of ADRB2. RNASeq analysis identified over 300 differentially expressed genes that were both temporally regulated following infection and selectively regulated in WT vs Adrb2-/- cells. These genes contributed to major transcriptional pathways including cytokine receptor activation, signaling in cancer, immune deficiency, and neurotransmitter pathways. By parsing genes within groups that were either induced or repressed over time in response to infection, we identified three main branches of genes that were differentially regulated by the ADRB2. These gene sets were predicted to be regulated by specific transcription factors involved in effector T cell development, such as Tbx21 and Eomes. Collectively, these data demonstrate a significant role for ADRB2 signaling in regulating key transcriptional pathways during CD8 + T cells responses to infection that may dramatically impact their functional capabilities and downstream memory cell development.

ß-Cleavage of the prion protein in the human eye: Implications for the spread of infectious prions and human ocular disorders.

Recently, we reported ß-cleavage of the prion protein (PrPC) in human ocular tissues. Here, we explored whether this is unique to the human eye, and its functional implications. A comparison of the cleavage pattern of PrPC in human ocular tissues with common nocturnal and diurnal animals revealed mainly ß-cleavage in humans, and mostly full-length PrPC in animal retinas. Soluble FL PrPC and N-terminal fragment (N2) released from ß-cleavage was observed in the aqueous and vitreous humor (AH & VH). Expression of human PrPC in ARPE-19 cells, a human retinal pigmented epithelial cell line, also showed ß-cleaved PrPC. Surprisingly, ß-cleavage was not altered by a variety of insults, including oxidative stress, suggesting a unique role of this cleavage in the human eye. It is likely that ß-cleaved C- or N-terminal fragments of PrPC protect from various insults unique to the human eye. On the contrary, ß-cleaved C-terminus of PrPC is susceptible to conversion to the pathological PrP-scrapie form, and includes the binding sites for ß1-integrin and amyloid-ß, molecules implicated in several ocular disorders. Considering the species and tissue-specific cleavage of PrPC, our data suggest re-evaluation of prion infectivity and other ocular disorders of the human eye conducted in mouse models.

Release of Iron-Loaded Ferritin in Sodium Iodate-Induced Model of Age Related Macular Degeneration: An In-Vitro and In-Vivo Study.

To evaluate the role of iron in sodium iodate (NaIO3)-induced model of age-related macular degeneration (AMD) in ARPE-19 cells in-vitro and in mouse models in-vivo. ARPE-19 cells, a human retinal pigment epithelial cell line, was exposed to 10 mM NaIO3 for 24 h, and the expression and localization of major iron modulating proteins was evaluated by Western blotting (WB) and immunostaining. Synthesis and maturation of cathepsin-D (cat-D), a lysosomal enzyme, was evaluated by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) and WB, respectively. For in-vivo studies, C57BL/6 mice were injected with 40 mg/kg mouse body weight of NaIO3 intraperitoneally, and their retina was evaluated after 3 weeks as above. NaIO3 induced a 10-fold increase in ferritin in ARPE-19 cells, which co-localized with LC3II, an autophagosomal marker, and LAMP-1, a lysosomal marker. A similar increase in ferritin was noted in retinal lysates and retinal sections of NaIO3-injected mice by WB and immunostaining. Impaired synthesis and maturation of cat-D was also noted. Accumulated ferritin was loaded with iron, and released from retinal pigmented epithelial (RPE) cells in Perls' and LAMP-1 positive vesicles. NaIO3 impairs lysosomal degradation of ferritin by decreasing the transcription and maturation of cat-D in RPE cells. Iron-loaded ferritin accumulates in lysosomes and is released in lysosomal membrane-enclosed vesicles to the extracellular milieu. Accumulation of ferritin in RPE cells and fusion of ferritin-containing vesicles with adjacent photoreceptor cells is likely to create an iron overload, compromising their viability. Moreover, reduced activity of cat-D is likely to promote accumulation of other cellular debris in lysosomal vesicles, contributing to AMD-like pathology.

Upregulation of brain hepcidin in prion diseases.

Accumulation of redox-active iron in human sporadic Creutzfeldt-Jakob disease (sCJD) brain tissue and scrapie-infected mouse brains has been demonstrated previously. Here, we explored whether upregulation of local hepcidin secreted within the brain is the underlying cause of iron accumulation and associated toxicity. Using scrapie-infected mouse brains, we demonstrate transcriptional upregulation of hepcidin relative to controls. As a result, ferroportin (Fpn), the downstream effector of hepcidin and the only known iron export protein was downregulated, and ferritin, an iron storage protein was upregulated, suggesting increased intracellular iron. A similar transcriptional and translational upregulation of hepcidin, and decreased expression of Fpn and an increase in ferritin expression was observed in sCJD brain tissue. Further evaluation in human neuroblastoma cells (M17) exposed to synthetic mini-hepcidin showed downregulation of Fpn, upregulation of ferritin, and an increase in reactive oxygen species (ROS), resulting in cytotoxicity in a dose-dependent manner. Similar effects were noted in primary neurons isolated from mouse brain. As in M17 cells, primary neurons accumulated ferritin and ROS, and showed toxicity at five times lower concentration of mini-hepcidin. These observations suggest that upregulation of brain hepcidin plays a significant role in iron accumulation and associated neurotoxicity in human and animal prion disorders.

Upregulation of Local Hepcidin Contributes to Iron Accumulation in Alzheimer's Disease Brains.

BACKGROUND: Accumulation of iron is a consistent feature of Alzheimer's disease (AD) brains. The underlying cause, however, remains debatable. OBJECTIVE: To explore whether local hepcidin synthesized by brain cells contributes to iron accumulation in AD brains. METHODS: Brain tissue from the cingulate cortex of 33 cases of AD pre-assigned to Braak stage I-VI, 6 cases of non-dementia, and 15 cases of non-AD dementia were analyzed for transcriptional upregulation of hepcidin by RT-qPCR and RT-PCR. Change in the expression of ferritin, ferroportin (Fpn), microglial activation marker Iba1, IL-6, and TGFß2 was determined by western blotting. Total tissue iron was determined by colorimetry. RESULTS: Significant transcriptional upregulation of hepcidin was observed in Braak stage III-VI relative to Braak stage I and II, non-AD dementia, and non-dementia samples. Ferritin was increased in Braak stage V, and a significant increase in tissue iron was evident in Braak stage III-VI. The expression of Iba1 and IL-6 was also increased in Braak stage III-VI relative to Braak stage I and II and non-AD dementia samples. Amyloid-ß plaques were absent in most Braak stage I and II samples, and present in Braak stage III-VI samples with few exceptions. CONCLUSION: These observations suggest that upregulation of brain hepcidin is mediated by IL-6, a known transcriptional activator of hepcidin. The consequent downregulation of Fpn on neuronal and other cells results in accumulation of iron in AD brains. The increase in hepcidin is disease-specific, and increases with disease progression, implicating AD-specific pathology in the accumulation of iron.

HERVs establish a distinct molecular subtype in stage II/III colorectal cancer with poor outcome.

Colorectal cancer (CRC) is one of the most lethal malignancies. The extreme heterogeneity in survival rate is driving the need for new prognostic biomarkers. Human endogenous retroviruses (hERVs) have been suggested to influence tumor progression, oncogenesis and elicit an immune response. We examined multiple next-generation sequencing (NGS)-derived biomarkers in 114 CRC patients with paired whole-exome and whole-transcriptome sequencing (WES and WTS, respectively). First, we demonstrate that the median expression of hERVs can serve as a potential biomarker for prognosis, relapse, and resistance to chemotherapy in stage II and III CRC. We show that hERV expression and CD8+ tumor-infiltrating T-lymphocytes (TILs) synergistically stratify overall and relapse-free survival (OS and RFS): the median OS of the CD8-/hERV+ subgroup was 29.8 months compared with 37.5 months for other subgroups (HR = 4.4, log-rank P < 0.001). Combing NGS-based biomarkers (hERV/CD8 status) with clinicopathological factors provided a better prediction of patient survival compared to clinicopathological factors alone. Moreover, we explored the association between genomic and transcriptomic features of tumors with high hERV expression and establish this subtype as distinct from previously described consensus molecular subtypes of CRC. Overall, our results underscore a previously unknown role for hERVs in leading to a more aggressive subtype of CRC.

Retinal Degeneration and Alzheimer's Disease: An Evolving Link.

Age-related macular degeneration (AMD) and glaucoma are degenerative conditions of the retina and a significant cause of irreversible blindness in developed countries. Alzheimer's disease (AD), the most common dementia of the elderly, is often associated with AMD and glaucoma. The cardinal features of AD include extracellular accumulation of amyloid ß (Aß) and intracellular deposits of hyper-phosphorylated tau (p-tau). Neuroinflammation and brain iron dyshomeostasis accompany Aß and p-tau deposits and, together, lead to progressive neuronal death and dementia. The accumulation of Aß and iron in drusen, the hallmark of AMD, and Aß and p-tau in retinal ganglion cells (RGC), the main retinal cell type implicated in glaucoma, and accompanying inflammation suggest overlapping pathology. Visual abnormalities are prominent in AD and are believed to develop before cognitive decline. Some are caused by degeneration of the visual cortex, while others are due to RGC loss or AMD-associated retinal degeneration. Here, we review recent information on Aß, p-tau, chronic inflammation, and iron dyshomeostasis as common pathogenic mechanisms linking the three degenerative conditions, and iron chelation as a common therapeutic option for these disorders. Additionally discussed is the role of prion protein, infamous for prion disorders, in Aß-mediated toxicity and, paradoxically, in neuroprotection.

Publisher Correction: Integrated multi-omics framework of the plant response to jasmonic acid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrated multi-omics framework of the plant response to jasmonic acid.

Understanding the systems-level actions of transcriptional responses to hormones provides insight into how the genome is reprogrammed in response to environmental stimuli. Here, we investigated the signalling pathway of the hormone jasmonic acid (JA), which controls a plethora of critically important processes in plants and is orchestrated by the transcription factor MYC2 and its closest relatives in Arabidopsis thaliana. We generated an integrated framework of the response to JA, which spans from the activity of master and secondary regulatory transcription factors, through gene expression outputs and alternative splicing, to protein abundance changes, protein phosphorylation and chromatin remodelling. We integrated time-series transcriptome analysis with (phospho)proteomic data to reconstruct gene regulatory network models. These enabled us to predict previously unknown points of crosstalk of JA to other signalling pathways and to identify new components of the JA regulatory mechanism, which we validated through targeted mutant analysis. These results provide a comprehensive understanding of how a plant hormone remodels cellular functions and plant behaviour, the general principles of which provide a framework for analyses of cross-regulation between other hormone and stress signalling pathways.

Development and Analytical Validation of a DNA Dual-Strand Approach for the US Food and Drug Administration-Approved Next-Generation Sequencing-Based Praxis Extended RAS Panel for Metastatic Colorectal Cancer Samples.

A next-generation sequencing method was developed that can distinguish single-stranded modifications from low-frequency somatic mutations present on both strands of DNA in formalin-fixed paraffin-embedded colorectal cancer samples. We applied this method for analytical validation of the Praxis Extended RAS Panel, a US Food and Drug Administration-approved companion diagnostic for panitumumab, on the Illumina MiSeqDx platform. With the use of the TruSeq amplicon workflow, both strands of DNA from the starting material were interrogated independently. Mutations were reported only if found on both strands; artifacts usually present on only one strand would not be reported. A total of 56 mutations were targeted within the KRAS and NRAS genes. A minimum read depth of 1800× per amplicon is required per sample but averaged >30,000× at maximum multiplexing levels. Analytical validation studies were performed to determine the simultaneous detection of mutations on both strands, reproducibility, assay detection level, precision of the assay across various factors, and the impact of interfering substances. In conclusion, this assay can clearly distinguish single-stranded artifacts from low-frequency mutations. Furthermore, the assay is accurate, precise, and reproducible, can achieve consistent detection of a mutation at 5% mutation frequency, exhibits minimal impact from tested interfering substances, and can simultaneously detect 56 mutations in a single run using 10 samples plus controls.

Prion protein modulates endothelial to mesenchyme-like transition in trabecular meshwork cells: Implications for primary open angle glaucoma.

Endothelial-to-mesenchyme-like transition (Endo-MT) of trabecular meshwork (TM) cells is known to be associated with primary open angle glaucoma (POAG). Here, we investigated whether the prion protein (PrPC), a neuronal protein known to modulate epithelial-to-mesenchymal transition in a variety of cell types, is expressed in the TM, and plays a similar role at this site. Using a combination of primary human TM cells and human, bovine, and PrP-knock-out (PrP-/-) mouse models, we demonstrate that PrPC is expressed in the TM of all three species, including endothelial cells lining the Schlemm's canal. Silencing of PrPC in primary human TM cells induces aggregation of ß1-integrin and upregulation of α-smooth muscle actin, fibronectin, collagen 1A, vimentin, and laminin, suggestive of transition to a mesenchyme-like phenotype. Remarkably, intraocular pressure is significantly elevated in PrP-/- mice relative to wild-type controls, suggesting reduced pliability of the extracellular matrix and increased resistance to aqueous outflow in the absence of PrPC. Since PrPC is cleaved by members of the disintegrin and matrix-metalloprotease family that are increased in the aqueous humor of POAG arising from a variety of conditions, it is likely that concomitant cleavage of PrPC exaggerates and confounds the pathology by inducing Endo-MT-like changes in the TM.

A transcription factor hierarchy defines an environmental stress response network.

Environmental stresses are universally encountered by microbes, plants, and animals. Yet systematic studies of stress-responsive transcription factor (TF) networks in multicellular organisms have been limited. The phytohormone abscisic acid (ABA) influences the expression of thousands of genes, allowing us to characterize complex stress-responsive regulatory networks. Using chromatin immunoprecipitation sequencing, we identified genome-wide targets of 21 ABA-related TFs to construct a comprehensive regulatory network in Arabidopsis thaliana Determinants of dynamic TF binding and a hierarchy among TFs were defined, illuminating the relationship between differential gene expression patterns and ABA pathway feedback regulation. By extrapolating regulatory characteristics of observed canonical ABA pathway components, we identified a new family of transcriptional regulators modulating ABA and salt responsiveness and demonstrated their utility to modulate plant resilience to osmotic stress.

cDREM: inferring dynamic combinatorial gene regulation.

Genes are often combinatorially regulated by multiple transcription factors (TFs). Such combinatorial regulation plays an important role in development and facilitates the ability of cells to respond to different stresses. While a number of approaches have utilized sequence and ChIP-based datasets to study combinational regulation, these have often ignored the combinational logic and the dynamics associated with such regulation. Here we present cDREM, a new method for reconstructing dynamic models of combinatorial regulation. cDREM integrates time series gene expression data with (static) protein interaction data. The method is based on a hidden Markov model and utilizes the sparse group Lasso to identify small subsets of combinatorially active TFs, their time of activation, and the logical function they implement. We tested cDREM on yeast and human data sets. Using yeast we show that the predicted combinatorial sets agree with other high throughput genomic datasets and improve upon prior methods developed to infer combinatorial regulation. Applying cDREM to study human response to flu, we were able to identify several combinatorial TF sets, some of which were known to regulate immune response while others represent novel combinations of important TFs.

SMARTS: reconstructing disease response networks from multiple individuals using time series gene expression data.

MOTIVATION: Current methods for reconstructing dynamic regulatory networks are focused on modeling a single response network using model organisms or cell lines. Unlike these models or cell lines, humans differ in their background expression profiles due to age, genetics and life factors. In addition, there are often differences in start and end times for time series human data and in the rate of progress based on the specific individual. Thus, new methods are required to integrate time series data from multiple individuals when modeling and constructing disease response networks. RESULTS: We developed Scalable Models for the Analysis of Regulation from Time Series (SMARTS), a method integrating static and time series data from multiple individuals to reconstruct condition-specific response networks in an unsupervised way. Using probabilistic graphical models, SMARTS iterates between reconstructing different regulatory networks and assigning individuals to these networks, taking into account varying individual start times and response rates. These models can be used to group different sets of patients and to identify transcription factors that differentiate the observed responses between these groups. We applied SMARTS to analyze human response to influenza and mouse brain development. In both cases, it was able to greatly improve baseline groupings while identifying key relevant TFs that differ between the groups. Several of these groupings and TFs are known to regulate the relevant processes while others represent novel hypotheses regarding immune response and development. AVAILABILITY AND IMPLEMENTATION: Software and Supplementary information are available at http://sb.cs.cmu.edu/smarts/. CONTACT: zivbj@cs.cmu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Breastfeeding trends in Cambodia, and the increased use of breast-milk substitute-why is it a danger?

A cross-sectional analysis of the Cambodia Demographic Health Surveys from 2000, 2005 and 2010 was conducted to observe the national trends in infant and young child feeding practices. The results showed that rates of exclusive breastfeeding among infants aged 0-5.9 months have increased substantially since 2000, concurrent with an increase in the rates of early initiation of breastfeeding and a reduction in the giving of pre-lacteal feeds. However, the proportion of infants being fed with breast-milk substitutes (BMS) during 0-5.9 months doubled in 5 years (3.4% to 7.0%) from 2000 to 2005, but then did not increase from 2005, likely due to extensive public health campaigns on exclusive breastfeeding. BMS use increased among children aged 6-23.9 months from 2000 to 2010 (4.8% to 9.3%). 26.1% of women delivering in a private clinic provided their child with breast-milk substitute at 0-5.9 months, which is five times more than women delivering in the public sector (5.1%), and the greatest increase in bottle use happened among the urban poor (5.8% to 21.7%). These findings are discussed with reference to the increased supply and marketing of BMS that is occurring in Cambodia.

Carbon catabolite repression correlates with the maintenance of near invariant molecular crowding in proliferating E. coli cells.

BACKGROUND: Carbon catabolite repression (CCR) is critical for optimal bacterial growth, and in bacterial (and yeast) cells it leads to their selective consumption of a single substrate from a complex environment. However, the root cause(s) for the development of this regulatory mechanism is unknown. Previously, a flux balance model (FBAwMC) of Escherichia coli metabolism that takes into account the crowded intracellular milieu of the bacterial cell correctly predicted selective glucose uptake in a medium containing five different carbon sources, suggesting that CCR may be an adaptive mechanism that ensures optimal bacterial metabolic network activity for growth. RESULTS: Here, we show that slowly growing E. coli cells do not display CCR in a mixed substrate culture and gradual activation of CCR correlates with an increasing rate of E. coli cell growth and proliferation. In contrast, CCR mutant cells do not achieve fast growth in mixed substrate culture, and display differences in their cell volume and density compared to wild-type cells. Analyses of transcriptome data from wt E. coli cells indicate the expected regulation of substrate uptake and metabolic pathway utilization upon growth rate change. We also find that forced transient increase of intracellular crowding or transient perturbation of CCR delay cell growth, the latter leading to associated cell density-and volume alterations. CONCLUSIONS: CCR is activated at an increased bacterial cell growth rate when it is required for optimal cell growth while intracellular macromolecular density is maintained within a narrow physiological range. In addition to CCR, there are likely to be other regulatory mechanisms of cell metabolism that have evolved to ensure optimal cell growth in the context of the fundamental biophysical constraint imposed by intracellular molecular crowding.

Matching experiments across species using expression values and textual information.

MOTIVATION: With the vast increase in the number of gene expression datasets deposited in public databases, novel techniques are required to analyze and mine this wealth of data. Similar to the way BLAST enables cross-species comparison of sequence data, tools that enable cross-species expression comparison will allow us to better utilize these datasets: cross-species expression comparison enables us to address questions in evolution and development, and further allows the identification of disease-related genes and pathways that play similar roles in humans and model organisms. Unlike sequence, which is static, expression data changes over time and under different conditions. Thus, a prerequisite for performing cross-species analysis is the ability to match experiments across species. RESULTS: To enable better cross-species comparisons, we developed methods for automatically identifying pairs of similar expression datasets across species. Our method uses a co-training algorithm to combine a model of expression similarity with a model of the text which accompanies the expression experiments. The co-training method outperforms previous methods based on expression similarity alone. Using expert analysis, we show that the new matches identified by our method indeed capture biological similarities across species. We then use the matched expression pairs between human and mouse to recover known and novel cycling genes as well as to identify genes with possible involvement in diabetes. By providing the ability to identify novel candidate genes in model organisms, our method opens the door to new models for studying diseases. AVAILABILITY: Source code and supplementary information is available at: www.andrew.cmu.edu/user/aaronwis/cotrain12.