Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Fish transplantation as an ecological tool in boosting fish production: Tiga case study

The paper discusses the status of the Tiga Reservoir Fishery pre-and Clupeid transplantation. This was achieved by examining the species diversity, abundance and distribution with mitigating factors. It concludes with a verdict on the achievement of the transplantation exercise

Some ecological roles of Hippopotamus (Hippopotamus amphibious Linn. 1758) in fish production: possibilities for integrated fish-cum Agric production system

The common hippopotamus (Hippopotamus amphibious Linn. 1758) contributes to the productivity of aquatic systems where it lives. This paper reviews ecological roles of the hippo in this regard. Desk review of available literature information complemented with field observations were employed in the data collection. The ecological roles of the common hippopotamus being presented draw examples from East, West, Central and South African sub regions. The nutritional importance of the amphibious hippopotamus to rural communities was highlighted. In Southern Ethiopia, the Bodi, Bacha and Mura tribes eat hippo meat and this has led to severe hunting consequences on the wild populations of the animal. The important relationships between the hippopotamus and fish were presented. Hippopotamuses usually defecate in water and their excrements enrich the nutrients in the water resulting in favourable conditions for large fish populations. Some fish, including Labeo spp. were observed to feed on the micro-organisms and algae that grow on the skin of the hippotamus. A strong case was made for hippo-cum-fish integrated farm development in Nigeria based on ecological relationships so observed between the amphibious mammals and fish. This is one of the meeting points of fisheries and wildlife management that should be exploited for the benefits of the teeming Nigerian population

Limited resources? Let the habitat priority planner help!

In this time of scarce resources, coastal resource managers must find ways to prioritize conservation, land use, and restoration efforts. The Habitat Priority Planner (HPP) is a free geospatial tool created by the National Oceanic and Atmospheric Administration’s Coastal Services Center that has received wide praise for its ease of use and broad applicability to conservation strategic planning, restoration, climate change scenarios, and other natural resource management actions. Not a geographic information system (GIS) user? Don’t worry―this tool was designed to be used in a team setting. One intermediate-level GIS user can push the buttons to show quick results while a roomful of resource managers and stakeholders provide input criteria that determine the results. The Habitat Priority Planner is a toolbar for ESRI’s ArcGIS platform that is composed of three modules: Habitat Classification, Habitat Analysis, and Data Explorer. The tool calculates basic ecological statistics that are used to examine how habitats function within a landscape. The tool pre‐packages several common landscape metrics into a user‐friendly interface for intermediate GIS users. In addition, HPP allows the user to build queries interactively using a graphical interface for demonstrating criteria selections quickly in a visual manner that is useful in stakeholder interactions. Tool advocates and users include land trusts, conservation alliances, nonprofit organizations, and select National Estuarine Research Reserves and refuges of the U.S. Fish and Wildlife Service. Participants in this session will learn the basic requirements for HPP use and the multiple ways the HPP has been applied to geographies nationwide. (PDF contains 5 pages)

Toward a measure of social-ecological resilience for human communities

The purpose of this essay is to clarify the theoretical understanding of the concept of resilience in order to explore problems surrounding the empirical measurement and application of the concept, as well as to examine strategic examples of empirical measures and policy applications in the literature of several disciplines, fields, and professions. The examination of resilience occurs in two streams: one conceptual and one methodological. At the conceptual level, the focus will be on definitions, distinctions between resilience and related concepts, and the theoretical frameworks that underlie usage of the concept. At the empirical level, the examination of resilience will be centered on the methodological challenges associated with research on resilience as well as previous attempts to operationalize and measure resilience. (PDF contains 4 pages)

Function of microRNA-146a and NF-κB in physiologic and pathologic hematopoiesis

During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of the gene encoding miR-146a—expressed in all blood cell types—produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. Six-week-old deleted mice are normal, with no effect on cell numbers, but by 4 months bone marrow hypercellularity can be seen, and by 8 months marrow exhaustion is becoming evident. The ability of HSCs to replenish the entire hematopoietic repertoire in a myelo-ablated mouse also declines precipitously as miR-146a-deficient mice age. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. This chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of the signaling protein TRAF6 leading to excessive activity of the transcription factor NF-κB and overproduction of the cytokine IL-6. At the cellular level, transplant studies show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression.

Coastal resource planning system: Integrating evaluation of ecological integrity and ecosystem services valuation

Efficient and effective coastal management decisions rely on knowledge of the impact of human activities on ecosystem integrity, vulnerable species, and valued ecosystem services—collectively, human impact on environmental quality (EQ). Ecosystem-based management (EBM) is an emerging approach to address the dynamics and complexities of coupled social-ecological systems. EBM “is intended to directly address the long-term sustainable delivery of ecosystem services and the resilience of marine ecosystems to perturbations” (Rosenberg and Sandifer, 2009). The lack of a tool that integrates human choices with the ecological connections between contributing watersheds and nearshore areas, and that incorporates valuation of ecosystem services, is a critical missing piece needed for effective and efficient coastal management. To address the need for an integrative tool for evaluation of human impacts on ecosystems and their services, Battelle developed the EcoVal™ Environmental Quality Evaluation System. The EcoVal system is an updated (2009) version of the EQ Evaluation System for Water Resources developed by Battelle for the U.S. Bureau of Reclamation (Dee et al., 1972). The Battelle EQ evaluation system has a thirty-year history of providing a standard approach to evaluate watershed EQ. This paper describes the conceptual approach and methodology of the updated EcoVal system and its potential application to coastal ecosystems. (PDF contains 4 pages)

Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

The basque ecological debt: global socioecological impacts of a small open economy

4 p.

Edge function method applied to thin plates resting on Winkler foundation

The Edge Function method formerly developed by Quinlan⁽²⁵⁾ is applied to solve the problem of thin elastic plates resting on spring supported foundations subjected to lateral loads the method can be applied to plates of any convex polygonal shapes, however, since most plates are rectangular in shape, this specific class is investigated in this thesis. The method discussed can also be applied easily to other kinds of foundation models (e.g. springs connected to each other by a membrane) as long as the resulting differential equation is linear. In chapter VII, solution of a specific problem is compared with a known solution from literature. In chapter VIII, further comparisons are given. The problems of concentrated load on an edge and later on a corner of a plate as long as they are far away from other boundaries are also given in the chapter and generalized to other loading intensities and/or plates springs constants for Poisson's ratio equal to 0.2

The gated integration technique for the accurate measurement of the autocorrelation function of speckle intensities scattered from random phase screens

A new approach based on the gated integration technique is proposed for the accurate measurement of the autocorrelation function of speckle intensities scattered from a random phase screen. The Boxcar used for this technique in the acquisition of the speckle intensity data integrates the photoelectric signal during its sampling gate open, and it repeats the sampling by a preset number, in. The average analog of the in samplings output by the Boxcar enhances the signal-to-noise ratio by root m, because the repeated sampling and the average make the useful speckle signals stable, while the randomly varied photoelectric noise is suppressed by 1/ root m. In the experiment, we use an analog-to-digital converter module to synchronize all the actions such as the stepped movement of the phase screen, the repeated sampling, the readout of the averaged output of the Boxcar, etc. The experimental results show that speckle signals are better recovered from contaminated signals, and the autocorrelation function with the secondary maximum is obtained, indicating that the accuracy of the measurement of the autocorrelation function is greatly improved by the gated integration technique. (C) 2006 Elsevier Ltd. All rights reserved.

Exploitation of soil biota ecosystem services in agriculture: a bioeconomic approach

24 p.

Climate change impacts on the water services in Costa Rica: a production function for the hydroenergy sector

23 p.