Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8(+) T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei

Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags. We prelabeled cells with cell tracking dye to easily distinguish CD8(+) T cell nuclei from APC nuclei by conventional flow cytometry. Using this approach, we observed clear digital activation of NFAT1 transcription factor in OT-I T cells stimulated with OVA peptide presented by bulk splenocytes. OVA concentration had discrete control over the fraction of the cells that translocated NFAT1, indicating that a distinct threshold amount of TCR signaling is required to switch on NFAT1 in naive T cells. This behavior was cell contact dependent and qualitatively more exact than the NFAT1 response in ionomycin-stimulated naive T cells. These data contribute to our understanding of the digital behavior of TCR signaling components documented in other studies and indicate how T cells might discriminate log-fold changes in Ag availability during an actual infection. Overall, these results highlight the potential of this coculture nuclei isolation protocol to address stimulation-dependent translocation of proteins in primary lymphocytes

Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes

Skeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers. We profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes. The muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 × 10-8), post-translational modification (P < 9.7 × 10-5) and developmental (P < 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation. We provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions

The Tec kinase ITK differentially optimizes NFAT, NF-κB, and MAPK signaling during early T cell activation to regulate graded gene induction [preprint]

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal kinase ITK simultaneously trigger many biochemically separate TCR signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, we examined Erk1/2 activation and NFAT, NF-κB translocation in naive OT-I CD8+ cell nuclei. We observed consistent digital activation of NFAT1 and Erk-MAPK, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength and was tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis also revealed genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells

The H alpha Galaxy Survey V. The star formation history of late-type galaxies

This study of 117 low-redshift Im and Sm galaxies investigates the star formation rates of late-type galaxies, to determine whether they are quasi-continuous or dominated by bursts with quiescent interludes. We analyse the distribution of star formation timescales (stellar masses/star formation rates) for the entire sample, and of gas depletion timescales for those galaxies with gas mass measurements. We find that, on average, the late-type galaxies studied could have produced their total stellar masses by an extrapolation of their current star formation activity over a period of just under a Hubble time. This is not the case for a comparison sample of earlier-type galaxies, even those with disk-dominated morphologies and similar total stellar masses to the late-type galaxies. The earlier-type galaxies are on average forming their stars more slowly at present than the average rate over their past histories. No totally quiescent Im or Sm galaxies are found, and although some evidence of intrinsic variation in the star formation rate with time is found, this is typically less than a factor of 2 increase or decrease relative to the mean level. The Im and Sm galaxies have extensive gas reservoirs and can maintain star formation at the current rate for more than another Hubble time. The average spatial distribution of star formation in the Im galaxies, and to a lesser extent the Sm galaxies, is very similar to that of the older stellar population traced by the red light.Comment: 8 pages, 7 figures, accepted for publication in Astronomy & Astrophysic

Surveillance of endemic human coronaviruses (HCoV-NL63, OC43 and 229E) associated with pneumonia in Kilifi, Kenya

Introduction: Human coronaviruses (HCoVs) circulate endemically in human populations, often with seasonal variation. We describe the long-term patterns of paediatric disease associated with three of these viruses, HCoV-NL63, OC43 and 229E, in coastal Kenya. Methods: Continuous surveillance of pneumonia admissions was conducted at the Kilifi county hospital (KCH) located in the northern coastal region of Kenya. Children aged &lt;5 years admitted to KCH with clinically defined syndromic severe or very severe pneumonia were recruited. Respiratory samples were taken and tested for 15 virus targets, using real-time polymerase chain reaction. Unadjusted odds ratios were used to estimate the association between demographic and clinical characteristics and HCoV positivity. Results: From 2007 to 2019, we observed 11,445 pneumonia admissions, of which 314 (3.9%) tested positive for at least one HCoV type. There were 129 (41.1%) OC43, 99 (31.5%) 229E, 74 (23.6%) NL63 positive cases and 12 (3.8%) cases of HCoV to HCoV coinfection.  Among HCoV positive cases, 47% (n=147) were coinfected with other respiratory virus pathogens. The majority of HCoV cases were among children aged &lt;1 year (66%, n=208), though there was no age-dependence in the proportion testing positive. HCoV-OC43 was predominant of the three HCoV types throughout the surveillance period. Evidence for seasonality was not identified. Conclusions: Overall, 4% of paediatric pneumonia admissions were associated with three endemic HCoVs, with a high proportion of cases co-occurring with another respiratory virus, with no clear seasonal pattern, and with the age-distribution of cases following that of pneumonia admissions (i.e. highest in infants). These observations suggest, at most, a small severe disease contribution of endemic HCoVs in this tropical setting and offer insight into the potential future burden and epidemiological characteristics of SARS-CoV-2.</ns4:p

Trade in the balance: reconciling trade and climate policy: report of the Working Group on Trade, Investment, and Climate Policy

This repository item contains a report published by the Working Group on Trade, Investment, and Climate Policy at The Frederick S. Pardee Center for the Study of the Longer-Range Future at Boston University, and the Global Economic Governance Initiative at Boston University.This report outlines the general tensions between the trade and investment regime and climate policy, and outlines a framework toward making trade and investment rules more climate friendly. Members of the working group have contributed short pieces addressing a range of issues related to the intersection of trade and climate policy. The first two are by natural scientists. Anthony Janetos discusses the need to address the effects of international trade on efforts to limit the increase in global annual temperature to no more than 2oC over preindustrial levels. James J. Corbett examines the failure of the Trans Pacific Partnership (TPP) and the Transatlantic Trade and Investment Partnership (TTIP) to adequately address the environmental implications of shipping and maritime transport. The next two pieces are by economists who examine economic aspects of the trade-climate linkage. Irene Monasterolo and Marco Raberto discuss the potential impacts of including fossil fuel subsidies reduction under the TTIP. Frank Ackerman explores the economic costs of efforts to promote convergence of regulatory standards between the United States and the European Union under the TTIP. The following two contributions are by legal scholars. Brooke Güven and Lise Johnson explore the potential for international investment treaties to redirect investment flows to support climate change mitigation and adaptation, particularly with regard to China and India. Matt Porterfield provides an overview of the ways in which both existing and proposed trade and investment agreements could have either “climate positive” or “climate negative” effects on mitigation policies. The final article is by Tao Hu, a former WTO trade and environment expert advisor for China and currently at the World Wildlife Fund, arguing that the definition of environmental goods and services’ under the WTO negotiations needs to be expanded to better incorporate climate change

Iron biochemistry is correlated with amyloid plaque morphology in an established mouse model of Alzheimer’s disease

A signature characteristic of Alzheimer's disease (AD) is aggregation of amyloid-beta (Aβ) fibrils in the brain. Nevertheless, the links between Aβ and AD pathology remain incompletely understood. It has been proposed that neurotoxicity arising from aggregation of the Aβ1-42 peptide can in part be explained by metal ion binding interactions. Using advanced X-ray microscopy techniques at sub-micron resolution, we investigated relationships between iron biochemistry and AD pathology in intact cortex from an established mouse model over-producing Aβ. We found a direct correlation of amyloid plaque morphology with iron, and evidence for the formation of an iron-amyloid complex. We also show that iron biomineral deposits in the cortical tissue contain the mineral magnetite, and provide evidence that Aβ-induced chemical reduction of iron could occur in vivo. Our observations point to the specific role of iron in amyloid deposition and AD pathology, and may impact development of iron-modifying therapeutics for AD